Use of an integrin α5β1 inhibitor in the treatment of pulmonary hyperfunction 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 α5β1, including administering an integrin α5β1 inhibitor to a subject. Also provided herein are methods for 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 regulates complex cell adhesion and signaling via cell surface integrin receptors (fibronectin-binding integrins such as α5β1) during tissue development, remodeling, and in diseases such as hypertension and heart failure. Heart failure (HF) is a debilitating disease in which abnormal heart function leads to inadequate low perfusion of body tissues and organs. Hypertension is a cause of various adverse effects and has a high morbidity and mortality rate, 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 increased by pulmonary vascular vasoconstriction and remodeling. Vasoconstriction and vascular remodeling increase the workload on the right side of the heart, causing right ventricular hypertrophy, fibrosis, and ultimately heart failure.

[0002] Current treatments include vasodilators that target Ca channels or endothelin receptors. There is a need for new approaches to the treatment of pulmonary hypertension, PAH, heart failure, and related diseases.

Summary of the Invention

[0003] The present invention is based, in part, on the discovery that integrin signaling can promote apoptosis resistance, which promotes cell growth and contributes to vascular remodeling of pulmonary arterioles and arteries (PA). In the right ventricle (RV), integrin signaling can contribute to maladaptive hypertrophy and fibrosis, leading to RV 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 heart or lung disease in a subject, comprising administering an integrin α5β1 inhibitor. As described herein, a5b1 inhibition not only maintains cardiac output but also prevents RV adaptation. Accordingly, the present invention includes, but is not limited to, treating hypertension or heart disorders such as heart failure, RV failure (e.g., RV failure due to RV volume overload caused by a septal defect or valvular regurgitation), RV pressure overload due to other WHO groups of pulmonary hypertension, or outflow obstruction such as pulmonary artery stenosis, and RV cardiomyopathy due to myocardial infarction, arrhythmia, or fibrosis.

[0005] The present invention provides a method of treating a disease in which α5βl function is involved using an integrin α5β1 inhibitor (e.g., small molecule compounds and antibodies). In one aspect, the present invention provides a method of treating a disease associated with an increase in the 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) group.

[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 a World Health Organization (WHO) classification system. In some embodiments, the disease is characterized by a WHO functional class 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 present 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, 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-binding 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 antagonizes integrin binding to OS2966.

[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 antagonizes 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 antagonizes integrin binding with anti-α5β1 antibody clone 339.1.

[0021] In some embodiments, the antibody is integrin α5β1 antibody 3C5 or 5B11. In some embodiments, the antibody is an integrin α5β1 antibody that antagonizes integrin binding with an antibody selected from 3C5 and 5B11. 3C5 and 5B11 are integrin α5β1 antibodies described in International Publication No. WO 2010 / 072740A2, 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 antagonizes integrin binding with an anti-integrin α5 (CD49e) antibody, clone mAb16.

[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.

Chemical formula

[0028] In some embodiments, R 2 is methyl or ethyl.

[0029] In some embodiments, R 3 is methyl, ethyl, isopropyl, or cyclopropyl.

[0030] In some embodiments, R 1 is hydrogen.

[0031] In some embodiments, the compound of formula (I) is selected from the group consisting of the following.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0032] In some embodiments, the integrin α5β1 inhibitor is a compound of formula (II).

Chemical formula

[0033] In some embodiments, the integrin α5β1 inhibitor is administered by inhalation, orally, intravenously, subcutaneously, intranasally, transdermally, intraperitoneally, intramuscularly, or intraperitoneally. In some embodiments, the integrin α5β1 inhibitor is administered via inhalation.

[0034] In some embodiments, the treatment methods described herein further comprise administering to the subject one or more additional therapies. In some embodiments, the treatment method comprises administering an integrin α5β1 inhibitor in combination with one or more additional therapies. In some embodiments, the treatment method further comprises administering to the subject an integrin α5β1 inhibitor and a second therapy. In some embodiments, the treatment method further comprises administering to the subject an integrin α5β1 inhibitor and two additional therapies.

[0035] 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.

[0036] 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 thromboendarterectomy, and / or atrial septostomy.

[0037] In some embodiments, the additional therapy is macitentan and / or tadalafil.

[0038] In some embodiments, the additional therapy is an SGLT2 inhibitor. In some embodiments, the SGLT2 inhibitor is dapagliflozin.

[0039] In some embodiments, administration of an integrin α5β1 inhibitor reduces the proliferation and / or survival of pulmonary artery smooth muscle cells (PASMCs), pulmonary arteries, right ventricular fibroblasts (RVFbs), vascular fibroblasts, exogenous fibroblasts, cardiomyocytes, and / or endothelial cells.

[0040] In some embodiments, the method comprises administering an integrin α5β1 inhibitor to modulate the level of a biomarker in a subject. In some embodiments, the biomarker is the N-terminal fragment (NT) of proBNP (NT-pro-BNP), TNFα, IFNγ, IL-6, IL-8, and IL-10. In some embodiments, the biomarker is the N-terminal fragment (NT) of brain natriuretic peptide (BNP) or proBNP (NT-proBNP).

[0041] In some embodiments, the treatment method described herein comprises administering an integrin α5β1 inhibitor in a dose of 1 mg to 1000 mg.

[0042] In some embodiments, the integrin α5β1 inhibitor is administered daily. In some embodiments, the integrin α5β1 inhibitor is administered twice a day.

[0043] In some embodiments, the present invention provides an integrin α5β1 inhibitor for use in the treatment of pulmonary hypertension in a subject in need thereof, comprising administering the integrin α5β1 inhibitor and a pharmaceutical excipient to the subject.

[0044] In some embodiments, the present invention provides an integrin α5β1 inhibitor for use in the treatment of pulmonary arterial hypertension (PAH) in a subject in need thereof, comprising administering the integrin α5β1 inhibitor and a pharmaceutical excipient to the subject.

[0045] In some embodiments, the present invention provides an integrin α5β1 inhibitor for use in the treatment of right ventricular failure in a subject in need thereof, comprising administering the integrin α5β1 inhibitor and a pharmaceutical excipient to the subject.

[0046] Definitions For the present invention to be more readily understood, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout this specification.

[0047] "Active agent" and "therapeutic agent" mean a molecule (e.g., a small molecule compound, peptide, antibody or antibody fragment, etc.) that has a prophylactic or therapeutic effect on a disease or disease state. An active agent can refer to not only a single active agent but also a combination of two or more different active agents.

[0048] "Reduction" and "improvement" mean the process by which the severity of the signs or symptoms of a disorder decreases. Importantly, the signs or symptoms can be reduced without being eliminated. A therapeutically effective dose is expected to reduce the severity of the disease and alleviate and improve the signs or symptoms of the disease.

[0049] As used herein, the term "affinity" refers to the characteristics of the 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 represented as the 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, greater than about 10 -6 M, greater than about 10 -5 M, or greater than about 10 -4 M of K D ).

[0050] As used herein, the term "about" or "approximately" when applied to one or more values of interest refers to a value similar to the recited reference value. In certain embodiments, the term "about" or "approximately" refers to a range of values that fall within any of (greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the recited reference value, unless otherwise indicated or otherwise apparent from the context (except when such number exceeds 100% of the possible values).

[0051] As used herein, the term "antibody" refers to a polypeptide comprising at least one immunoglobulin variable region, e.g., an amino acid sequence that provides an immunoglobulin variable domain or an immunoglobulin variable domain sequence. For example, an antibody can comprise a heavy chain (H) variable region sequence (abbreviated herein as VH), and a light chain (L) variable region sequence (abbreviated herein as VL). In another embodiment, an antibody comprises two heavy chain (H) chain variable regions and two light chain (L) chain variable regions. An antibody typically comprises three complementarity determining regions (abbreviated, CDR) in the light chain of the immunoglobulin, and three complementarity determining regions (CDR) in the heavy chain of the immunoglobulin. The three CDRs in the light chain of the immunoglobulin are referred to as CDR1, CDR2, and CDR3, respectively, from the N-terminal side. The three CDRs in the heavy chain of the immunoglobulin are also referred to as CDR1, CDR2, and CDR3, respectively, from the N-terminal side. "CDR" can be identified according to the Kabat, Chothia definitions, the accumulation of both Kabat and Chothia, the AbM definition, the contact definition, and / or the conformation definition, or any method of CDR determination known in the art. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single domain antibodies, Fab, F(ab')2, Fd, Fv, and dAb fragments), as well as intact antibodies, e.g., intact immunoglobulins of the IgA, IgG, IgE, IgD, IgM types (and their subtypes). The light chain of the immunoglobulin can be of the κ or λ type.

[0052] "Combination therapy" and "combined therapy" mean the administration of a first active agent and at least a second different active agent as part of a specific treatment regimen intended to produce a beneficial effect from the co-action of at least two active agents. The beneficial effects of the combination can include, but are not limited to, pharmacokinetic or pharmacodynamic co-actions resulting from the combination of therapeutic agents. The administration of the combined therapeutic agents can be carried out over a defined period (e.g., minutes, hours, days, or weeks depending on the selected combination). In some embodiments, the integrin α5β1 inhibitor is administered via inhalation. The combination therapy is not intended to include the administration of two or more different therapeutic agents as part of a separate monotherapy regimen that incidentally and optionally results in the combination therapy of the present invention. The combination therapy includes the sequential administration of at least two different therapeutic agents, each therapeutic agent being administered at a different time point, as well as the administration of at least two different therapeutic agents substantially simultaneously. Substantially simultaneous administration can be achieved, for example, by administering a single capsule having a fixed ratio of each therapeutic agent to the subject, or by administering each therapeutic agent in a separate capsule. The sequential or substantially simultaneous administration of each therapeutic agent can be affected by any suitable route, including but not limited to oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The two different therapeutic agents can be administered by the same route or by different routes. For example, the first therapeutic agent of a selected combination can be administered by intravenous injection, while the second therapy 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 stated, the sequence in which the therapeutic agents are administered is not important.

[0053] Combination therapy also includes administration of the different therapeutic agents described above, further combined with other biologically active ingredients and non-drug therapies (e.g., surgery or physical therapy). When combination therapy includes non-drug therapy, the non-drug therapy can be carried out at any appropriate time as long as a beneficial effect from the co-action of the combination of the therapeutic agent and the non-drug therapy is achieved. For example, when appropriately, beneficial effects can still be achieved even when the non-drug therapy is removed in time from the administration of the therapeutic agent, perhaps even in days or weeks.

[0054] A compound means a molecule and includes not only a particular molecular entity but also, when the compound is an active agent or a drug, active metabolites, amides, conjugates, esters, hydrates, polymorphs, prodrugs, salts, solvates, and other such derivatives, deuterated analogs and analogs containing radioactive atoms or other labeled moieties, as well as pharmaceutically acceptable pharmacologically active analogs of related compounds, including but not limited to these. In some embodiments, the compound is a small molecule compound.

[0055] "Dosage form" means any form of a pharmaceutical composition for administration to a subject (typically a human or an animal for veterinary purposes suffering from a disease or condition to be treated). "Dosage" refers to the amount of the active agent. A unit dosage form refers to a dosage form containing a fixed amount of the active agent. A single tablet or capsule is a unit dosage form. Multiple unit dosage forms can be administered to provide a therapeutically effective dosage. Dosage forms can include combinations of dosage forms.

[0056] "Effective amount" and "therapeutically effective amount" refer to a non-toxic but sufficient amount of an active agent to achieve the desired therapeutic effect.

[0057] "Integrin inhibitor" or "Integrin α5s1 inhibitor" or "VLA5 inhibitor" refers to a molecule that can bind to integrin α5 (ITGA5) and has α5s1 integrin and / or ITGA5 inhibitory activity. In some embodiments, inhibiting the activity includes inhibiting the binding of α5s1 integrin and / or ITGA5 to smooth muscle cells, fibroblasts, stellate cells, myofibroblasts, pericytes, and / or other cells of mesenchymal origin. In some embodiments, inhibiting the activity includes inhibiting the migration of smooth muscle cells, fibroblasts, stellate cells, myofibroblasts, pericytes, and / or other cells of mesenchymal origin. In some embodiments, inhibiting the activity includes inhibiting the differentiation of smooth muscle cells, fibroblasts, stellate cells, myofibroblasts, pericytes, and / or other cells of mesenchymal origin. In some embodiments, inhibiting the activity includes inhibiting extracellular matrix synthesis and / or deposition.

[0058] As used herein, "K" D " is intended herein to mean the dissociation constant, and the dissociation constant is the ratio of K d to K a (i.e., K d / K a ) and is expressed as molar concentration (M). The K D value can be determined using methods well established in the art, for example, by using surface plasmon resonance or by using a biosensor system such as a Biacore® system.

[0059] As used herein, "pulmonary arterial hypertension (PAH)" refers to a rare disease in which normally low pulmonary artery pressure is increased due to vasoconstriction and pulmonary vascular remodeling. This in turn increases the workload on the right side of the heart, causing right heart hypertrophy, fibrosis, and ultimately heart failure.

[0060] As used herein, "peptide" refers to a peptide or polypeptide containing a plurality of amino acids. The terms "peptide" and "polypeptide" are used interchangeably. An amino acid sequence or a variant thereof can be part of a larger peptide, i.e., part of a peptide extended at the N-terminus and / or C-terminus by one or more additional amino acids. The amino acid sequence of a peptide of the present invention or a variant thereof may also be modified at the N-terminus and / or C-terminus, preferably by including N-terminal and / or C-terminal extension groups. Alternatively, the amino acid sequence or a variant thereof extends at the N-terminus and / or C-terminus.

[0061] "Pharmaceutically acceptable" means not biologically undesirable, i.e., it can be incorporated into a pharmaceutical composition administered to a patient without causing undesirable biological effects or without undergoing an adverse interaction with other components of the composition in which it is contained. When the term pharmaceutically acceptable is used to refer to a pharmaceutical carrier or excipient, it is implied that the carrier or excipient meets the criteria of the required toxicity and manufacturing tests or is included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration.

[0062] "Pharmaceutically acceptable salts" means derivatives of the active agent produced by making an acid or its base salt. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral salts or organic salts of basic residues such as amines, and alkali salts or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts include salts formed when the acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, an alkaline earth ion, or an aluminum ion, or when coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. Pharmaceutically acceptable salts include solvated forms (solvates) or crystalline forms (polymorphs) of the same salt as defined herein.

[0063] As in the case of pharmacologically active derivatives or analogs, "pharmacologically active" (or "active") refers to a derivative or analog having a type of pharmacological activity substantially equivalent to that of the parent compound.

[0064] "Prevent" and "preventing" mean completely avoiding the onset of clinically apparent disease progression or delaying the onset of the pre-clinically apparent stage of a disease in an at-risk individual. Prevention includes prophylactic treatment of patients at risk of developing a disease.

[0065] As used herein, "selectively bind", "binds selectively", or "binds specifically" refers to a preferential association of the binding moiety with the target, with respect to the binding moiety and the target, and not with respect to entities that are not the target. Some degree of non-specific binding may occur between the binding moiety and non-targets. In some embodiments, when the binding between the binding moiety and the target is more than 2-fold, more than 5-fold, more than 10-fold, or more than 100-fold compared to the binding of the binding moiety and non-targets, the binding moiety binds selectively to the target. In some embodiments, the binding affinity is less than about 10 -5 M, less than about 10-6 less than M, about 10 -7 less than M, about 10 -8 less than M, or about 10 -9 When less than M, the binding moiety binds selectively to the target.

[0066] "Sign" means a sign of a disease and includes conditions observable by a physician, nurse, or other healthcare provider.

[0067] As used herein, "small molecule" refers to a molecule having a relatively low molecular weight, whether occurring naturally or produced artificially (e.g., via chemical synthesis). Preferred small molecules are biologically active in that they produce a local or systemic effect in an animal, preferably a mammal, more preferably a human. In certain preferred embodiments, the small molecule is a drug, and the small molecule is referred to as a drug molecule, or a drug, or a therapeutic agent. The small molecule can have a molecular weight of about 5 kDa or less. In other embodiments, the drug molecule has a molecular weight of about 1.5 kDa or less.

[0068] As used herein, the term "subject" means any subject for which diagnosis, prognosis, or therapy is desirable. For example, the subject can be a mammal, such as a human or non-human primate (e.g., ape, monkey, orangutan, or chimpanzee), dog, cat, guinea pig, rabbit, rat, mouse, horse, cow, or bull.

[0069] "Subject in need thereof" refers to a human or other mammal suitable for treatment with an active agent. A subject in need thereof can have a disease or an increased risk compared to the general population that develops the disease.

[0070] "Symptom" means a sign or other indication of a disease, illness, or injury. Symptoms can be felt or noticed by the individual experiencing them or by others, including non-medical professionals.

[0071] As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic molecule (e.g., an integrin α5β1 inhibitor described herein) that provides a therapeutic effect to a subject at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject shows or feels an effect). In particular, a "therapeutically effective amount" is an amount of a therapeutic molecule or composition effective to produce a detectable therapeutic or prophylactic effect for treating, ameliorating or preventing a particular disease or condition, or by improving the symptoms associated with the disease, by preventing or delaying the onset of the disease, and / or by reducing the severity or frequency of the symptoms of the disease. A therapeutically effective amount may be administered in a dosing regimen that includes multiple unit doses. For any particular therapeutic molecule, the therapeutically effective amount (and / or the appropriate unit dose within an effective dosing regimen) can vary, for example, depending on the route of administration, in combination with other pharmaceuticals. Also, the therapeutically effective amount (and / or unit dose) specific to any particular subject depends on various factors including the disorder being treated and the severity of the disorder; the activity of the particular pharmaceutical agent employed; the particular composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and / or rate of excretion or metabolism of the particular therapeutic molecule employed; the duration of the treatment; and like factors well known in the medical arts.

[0072] "Treatment" and "treating" describe the management and care of a patient for the purpose of combating a disease, condition, or disorder, and includes the administration of an active agent to reduce the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder.

[0073] "Pulmonary hypertension (PH)" includes diseases that share the defining element of mean pulmonary artery pressure ≥ 25 mmHg. PH is classified and divided into five groups and five classes 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

[0074] The drawings are for illustrative purposes only and not for limitation.

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[0075] The present invention provides methods and compositions for treating diseases associated with increased expression or activity of integrin α5β1, including administering an integrin α5β1 inhibitor. In one aspect, the present invention provides a method for treating heart or lung diseases in a subject, including administering an integrin α5β1 inhibitor. In some embodiments, the disease is characterized by a World Health Organization (WHO) group.

[0076] 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.

[0077] 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 classes 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 an increase in the expression or activity of integrin α5β1 is heart failure or right ventricular failure.

[0078] Due to the limitations of current treatments for pulmonary hypertension, there remains a great interest and need for additional or alternative therapies to treat, stabilize, prevent, and / or delay pulmonary hypertension. To obtain more efficient and / or less toxic drugs for the treatment of pulmonary hypertension, various processes have been developed. However, these processes still exhibit severe side effects, and the resulting agents often show short half-lives and low bioavailability.

[0079] The present invention provides, inter alia, methods and compositions for treating diseases associated with an increase in the 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 adhesion are mediated through several protein families including integrins, selectins, cadherins, and immunoglobulins, and promote various normal cell functions such as proliferation, migration, differentiation, or survival. Cell adhesion is also important in various pathological conditions, and thus pharmacological disruption of cell adhesion interactions can provide a mechanism for therapeutic intervention. Members of the integrin superfamily of adhesion molecules play important roles in acute and chronic disease states such as cancer, inflammatory diseases, stroke, and neurodegenerative disorders. Thus, integrins represent a complex biological area.

[0080] The integrin superfamily of cell surface receptors is formed from several structurally and functionally related surface glycoproteins, and each receptor exists as one of 20 heterodimers of non-covalently associated α and β subunits. In mammals, at least 18 different α and 8 different β subunits have been identified, which are known to form more than 24 different receptors. Each integrin specifically interacts with defined extracellular ligands, including extracellular matrix proteins such as fibronectin, vitronectin, collagen, and cell surface molecules such as VCAM, ICAM, and PECAM, via a linear 25-amino acid adhesion motif.

[0081] Integrin α5β1 (α5β1 or α5β1) is composed of α5 (α5 or α5) and β1 (β1 or β1) subunits. The α5 subunit forms a specific dimer with the β1 subunit and is widely expressed in most tissues. Integrin α5β1 mediates cell adhesion almost exclusively through its interaction with fibronectin and binds via a short arginine-glycine-aspartic acid (RGD) adhesion motif. However, endothelial cells and platelets can bind to fibrin via α5β1. The interaction of α5β1 with fibronectin plays an important role in pathophysiological angiogenesis and vascular integrity. Endothelial cells express various integrins, but α5β1 is important for the survival of endothelial cells on provisional substrates in vitro, suppressing apoptosis and promoting proliferation. α5β1 expression is upregulated in the tumor vasculature and in patients with pulmonary hypertension. Consistent with the important functional role of receptor-ligand engagement, the α5β1 ligand fibronectin is also upregulated in tumor tissue and during wound healing.

[0082] As demonstrated herein, inhibition of integrin α5β1 by blocking the activity of α5β1, or inhibition of α5β1 fibronectin binding, is effective in the prevention and treatment of pulmonary hypertension, PAH, heart failure, and right ventricular failure. The present invention provides various compounds (e.g., small molecule compounds and antibodies) that inhibit their interaction. Such compounds are generally referred to herein as "integrin a5b1 inhibitors".

[0083] Various aspects of the invention are described in detail in the following sections. The use of the sections is not meant to limit the invention. Each section can be applied to any aspect of the invention. In this application, the use of "or" means "and / or" unless otherwise stated.

[0084] Pulmonary hypertension Pulmonary hypertension (PH) is a syndrome characterized by an increase in pulmonary artery pressure. PH is defined hemodynamically as an assessment of systolic pulmonary artery pressure greater than 30 mmHg or mean pulmonary artery pressure greater than 25 mmHg. See Zaiman et al., Am. J. Respir. Cell Mol. Biol. 33:425-31 (2005). Further, PH damages both large and small pulmonary arteries as a result of the increased pressure. The walls of the smallest blood vessels thicken and are unable to normally move oxygen and carbon dioxide between the blood and the lungs. Over time, pulmonary hypertension leads to pulmonary artery thickening and narrowing of the blood flow pathways. When pulmonary hypertension develops, the right side of the heart works harder to compensate, but the increased effort causes the heart to enlarge and thicken. Proliferation of smooth muscle and endothelial cells that normally exist in a quiescent state results in vascular remodeling with loss of the lumen of the pulmonary vasculature. This causes a progressive increase in pulmonary pressure as blood is pumped through the reduced lumen area. Since blood tends to pool in the ventricles and legs, an enlarged right ventricle places people at risk for pulmonary embolism. If blood clots form in the pooled blood, they may eventually move to and clog the lungs. The progressive increase in pressure also places additional workload on the right ventricle and ultimately causes heart failure, leading to early death in these patients.

[0085] As a result of PH, various pathological changes occur in the pulmonary artery. Persistent vasoconstriction and pulmonary vascular structural remodeling are the main features of PH. Pulmonary vascular smooth muscle cells undergo a phenotypic switch from a normal contractile phenotype to a synthetic phenotype that leads to cell growth and matrix deposition. Histological examination of tissue specimens from patients with pulmonary hypertension reveals intimal thickening as well as smooth muscle cell hypertrophy, especially in vessels with a diameter of less than 100 µm. Furthermore, abnormal smooth muscle cells often overexpress endothelin and serotonin transporters, which are likely to play a role in the development of PH.

[0086] The most common symptom of pulmonary hypertension is initially shortness of breath during exercise. Some people may experience lightheadedness or fatigue during exercise, and angina-like chest pain is common. General weakness is another problem because body tissues are not receiving enough oxygen. Other symptoms such as cough or wheezing can be caused by underlying lung diseases. Edema, especially in the legs, can occur because fluid leaks from veins into tissues, which may indicate the development of pulmonary fibrosis. Some people with pulmonary hypertension have connective tissue disorders, especially scleroderma. When people have both pulmonary hypertension and a connective tissue disorder, Raynaud's phenomenon often develops years before the symptoms of pulmonary hypertension appear.

[0087] Treatment of some types of pulmonary hypertension often targets the underlying lung disease. Currently, treatment options available to individuals suffering from PH-targeted cell dysfunction result in vascular constriction. Therapies such as prostanoids, phosphodiesterase-5 inhibitors, and endothelin receptor antagonists act primarily by causing vasodilation of the pulmonary vasculature. Vasodilators such as calcium channel blockers, nitric oxide, and prostacyclin are often useful in pulmonary hypertension associated with scleroderma, chronic liver disease, and HIV infection. In contrast, these agents have not been proven effective in people with pulmonary hypertension due to underlying lung disease. For most people with idiopathic pulmonary hypertension, vasodilators such as prostacyclin significantly reduce the blood pressure in the pulmonary artery. Prostacyclin administered intravenously through a catheter surgically implanted in the skin improves quality of life, increases survival, and reduces the urgency of lung transplantation. Unfortunately, many patients either respond poorly to these therapies or lose their response over time. The only option remaining at that point is single or double lung transplantation to treat PH. Although there is some evidence that available therapies have secondary effects on vascular remodeling, there are currently no therapies that target abnormal cell proliferation in PAH.

[0088] In some embodiments, the pulmonary hypertension is pulmonary venous hypertension (PVH). In some embodiments, the PVH is due to left heart failure. In some embodiments, the pulmonary hypertension is pulmonary hypertension associated with a respiratory disorder and / or hypoxia. In some embodiments, the pulmonary hypertension is pulmonary hypertension due to chronic thromboembolic disease and / or embolic disease. In some embodiments, the pulmonary hypertension is miscellaneous pulmonary hypertension. In some embodiments, the miscellaneous pulmonary hypertension is associated with sarcoidosis, eosinophilic granuloma, histiocytosis X, lymphangioleiomyomatosis, or compression of the pulmonary vessels (e.g., adenopathy, tumor, or fibrosing mediastinitis). In some embodiments, the pulmonary hypertension is associated with chronic obstructive pulmonary disease (COPD). In some embodiments, the pulmonary hypertension is associated with pulmonary fibrosis. In some embodiments, the pulmonary hypertension is associated with cardiac fibrosis. In some embodiments, the pulmonary hypertension is early pulmonary hypertension or advanced pulmonary hypertension.

[0089] In some embodiments, the subject has pulmonary venous hypertension (PVH). In some embodiments, the PVH is due to left heart failure. In some embodiments, the subject has a respiratory disorder and / or hypoxia. In some embodiments, the subject has chronic thromboembolic and / or embolic disease. In some embodiments, the subject has sarcoidosis, eosinophilic granuloma, histiocytosis X, lymphangioleiomyomatosis, or compression of the pulmonary vessels (e.g., due to adenopathy, tumor, or fibrosing mediastinitis). In some embodiments, the subject has chronic obstructive pulmonary disease (COPD). In some embodiments, the subject has pulmonary fibrosis. In some embodiments, the subject has cardiac fibrosis. In some embodiments, the subject has early pulmonary hypertension or progressive pulmonary hypertension.

[0090] In some embodiments, one or more symptoms of pulmonary hypertension are improved. In some embodiments, the progression of pulmonary hypertension is delayed. In some embodiments, pulmonary hypertension is prevented. In some embodiments, the treatment methods provided herein reduce pulmonary pressure. In some embodiments, the treatment methods provided herein inhibit and / or reduce abnormal cell proliferation in the pulmonary artery.

[0091] In some embodiments, pulmonary hypertension is characterized by the World Health Organization (WHO) group.

[0092] In some embodiments, 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.

[0093] 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 class based on cardiac function. In some embodiments, pulmonary hypertension is characterized as 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.

[0094] In some embodiments, the subject is suffering from pulmonary hypertension characterized by the World Health Organization (WHO) group.

[0095] In some embodiments, the subject is suffering from 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.

[0096] In some embodiments, the subject is suffering from a disease characterized by the World Health Organization (WHO) classification system. In some embodiments, the subject is suffering from a disease characterized by the WHO functional class based on cardiac function. In some embodiments, the subject is suffering from pulmonary hypertension classified as 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.

[0097] In some embodiments, pulmonary hypertension is associated with pulmonary capillary hemangiomatosis.

[0098] Pulmonary arterial hypertension (PAH) In one aspect, the present invention provides a method of treating pulmonary arterial hypertension (PAH) comprising administering an integrin α5β1 inhibitor (e.g., the small molecule compounds and antibodies disclosed herein). PAH is characterized by right ventricular overload and a progressive increase in pulmonary vascular resistance that ultimately leads to heart failure. PAH results in progressive occlusion and decreased compliance of the pulmonary artery (PA), leading to right ventricular (RV) failure and early death. Similar to cancer cells, pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs) exhibit exaggerated proliferation and resistance to apoptosis in response to increased PA stiffness caused by extracellular matrix (ECM) remodeling. Integrin signaling promotes resistance to proliferation and apoptosis in PAH-PASMCs and PAH-PAECs, contributing to PA vascular remodeling, while in the RV, maladaptive hypertrophy, and fibrosis, it can 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 and prevent RV dysfunction in PAH.

[0099] PAH is a chronic disorder involving all layers of the pulmonary vasculature. Vasoconstriction, structural changes in the pulmonary vascular wall (vascular remodeling), and thrombosis contribute to the increased pulmonary vascular resistance in PAH. Structural and functional changes in the endothelium lead to endothelial dysfunction. Increased levels of vasoconstrictor factors (e.g., endothelin) and decreased vasodilatory capacity (e.g., decreased prostacyclin) result in vasoconstriction and increased pulmonary vascular resistance. Current treatments aimed at addressing vasoconstriction can slow the progression of PAH or improve clinical symptoms over a limited time, but have not been shown to substantially reduce overall PAH morbidity and mortality. The underlying structural changes in the pulmonary vasculature (vascular remodeling) are not affected by these treatments.

[0100] Vascular remodeling resulting from PAH is characterized by proliferative and obstructive changes involving many cell types, including endothelial cells, smooth muscle cells, and fibroblasts. Vascular remodeling can manifest itself, for example, as medial thickening of the pulmonary vessels due to smooth muscle cell hyperplasia and hypertrophy, formation of neomembranes made from smooth muscle cells and / or myofibroblasts, and / or formation of a reticular lesion consisting of local proliferation of endothelial cells, smooth muscle cells, lymphocytes, and mast cells. Vascular remodeling leads to occlusion of the vascular lumen, which results in pulmonary hypertension. There is a need for therapies that address the proliferative patterns of PAH.

[0101] In some embodiments, the pulmonary hypertension is pulmonary arterial hypertension (PAH). In some variations, the PAH is idiopathic PAH. In some variations, the PAH is familial PAH. In some variations, the PAH is associated with persistent pulmonary hypertension of the newborn. In some variations, the PAH is associated with pulmonary veno-occlusive disease.

[0102] In some embodiments, the pulmonary hypertension is associated with a lung disease. In some embodiments, the lung disease is idiopathic pulmonary fibrosis (IPF) or interstitial pneumonia (IIP). IPF is a type of idiopathic interstitial pneumonia (IIP), which in turn is a type of interstitial lung disease (also known as diffuse parenchymal lung disease (DPLD)). Interstitial lung disease pertains to alveolar epithelium, pulmonary capillary endothelium, basement membrane, perivascular and perilymphatic tissues. 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 diseases such as scleroderma and rheumatoid arthritis.

[0103] Accordingly, in some embodiments, the subject has a lung disease such as idiopathic pulmonary fibrosis (IPF) or interstitial pneumonia (IIP). In some embodiments, the subject has 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 has sarcoidosis, hypersensitivity pneumonitis, pulmonary Langerhans cell histiocytosis, asbestosis, or a collagen vascular disease such as scleroderma or rheumatoid arthritis.

[0104] Pulmonary fibrosis is the formation or development of excessive fibrous connective tissue in the lungs.

[0105] Heart failure (HF) In one aspect, the invention provides a method of treating heart failure (HF), comprising administering an integrin α5β1 inhibitor (e.g., the small molecule compounds and antibodies disclosed herein). Heart failure refers to any condition characterized by the heart's inability to pump sufficient blood supply to the body. It refers to a physiological state in which cardiac output is insufficient to meet the body's needs or can only be achieved at higher filling pressures. There are many underlying causes of HF, including myocardial infarction, coronary artery disease, valvular disease, hypertension, and myocarditis. Chronic heart failure is associated with neurohormonal activation and changes in autonomic regulation. These compensatory neurohormonal mechanisms provide valuable support to the heart under normal physiological circumstances but also play a fundamental role in the development and subsequent progression of HF.

[0106] For example, one of the body's main compensatory mechanisms for reducing blood flow in the HF is to increase the amount of salt and water retained by the kidneys. Instead of being excreted via urine, retaining salt and water increases the amount of blood in the bloodstream and helps maintain blood pressure. However, the more blood there is, the more the myocardium, especially the ventricles, also expands. As the heart chambers expand, the wall thickness decreases, the heart's contraction weakens, causing a downward spiral of cardiac function. Another compensatory mechanism is vasoconstriction of the arterial system, which helps raise blood pressure and maintain proper perfusion, thus increasing the load the heart has to pump against.

[0107] In low ejection fraction (EF) heart failure, the high pressure in the heart results from attempts to maintain the high pressure necessary for proper peripheral perfusion. However, when the heart weakens due to such high pressure, the disorder worsens. The pressure in the left atrium can exceed 25 mmHg, at which stage fluid from the blood flowing through the pulmonary circulation system leaks or flows out from the pulmonary capillaries into the pulmonary interstitial space and into the alveoli, causing pulmonary congestion and, if left untreated, a syndrome of acute pulmonary edema and death.

[0108] Right ventricular failure In one aspect, the present invention provides a method of treating right ventricle (RV) failure comprising administering an integrin α5β1 inhibitor (e.g., small molecule compounds and antibodies disclosed herein). In some embodiments, the RV disorder is due to RV volume overload. In some embodiments, the RV volume excess is due to a septal defect or valvular regurgitation. In some embodiments, the RV pressure overload is due to pulmonary hypertension or other WHO groups of outflow obstruction. In some embodiments, the RV failure is due to pulmonary artery stenosis.

[0109] In one aspect, the present invention provides a method of treating RV cardiomyopathy comprising administering an integrin α5β1 inhibitor (e.g., small molecule compounds and antibodies disclosed herein). In some embodiments, the RV cardiomyopathy is due to myocardial infarction, arrhythmia, or fibrosis.

[0110] 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 improvement of hypertrophy and / or fibrosis. In some embodiments, administration of an α5β1 inhibitor prevents hypertrophy and / or fibrosis.

[0111] Integrin inhibitor Integrins are a family of transmembrane receptor glycoproteins that mediate cell-cell and cell-matrix interactions. Integrins are heterodimers having two different chains, an α subunit and a β subunit. In mammals, 18 α subunits and 8 β subunits have been described.

[0112] Integrin α5s1 is composed of the subunit ITGA5 (integrin α5) and integrin s1. Some integrins bind to fibronectin. Integrin α5s1 is selective for fibronectin because it requires both the 9th and 10th type II repeats of fibronectin (FNIII-9 and FNIII-10) for interaction. Expression of α5s1 integrin is mainly in the vascular system and connective tissue. Expression is significantly enhanced in tumor blood vessels but also markedly enhanced in the tumor cells themselves of many types of cancer, including colon, breast, ovarian, lung, and brain tumors. This is further expressed to varying degrees in many cell types, including fibroblasts, hematopoietic cells, immune cells, smooth muscle cells, and epithelial cells. High expression of α5s1 integrin has also been observed in fibrotic tissues such as pulmonary fibrosis.

[0113] In the tissue, normal fibroblasts are present in a low population of only 4-5%. However, during fibrosis, they can proliferate and account for up to 80-90% of the organ mass. Myofibroblasts in the fibrous tissue produce large amounts of extracellular matrix proteins that scar and render the tissue non-functional. Inhibition of myofibroblasts can counter these processes. Integrins promote cell proliferation, survival, hypertrophic growth, and fibrosis. As described herein, integrin inhibition can regulate these major factors leading to the progression of pulmonary hypertension (e.g., PAH).

[0114] The present invention provides a method of treating a disease associated with an increase in the expression or activity of integrin α5β1, comprising administering an integrin α5β1 inhibitor.

[0115] In one aspect, the present invention provides an integrin α5β1 inhibitor for use in the treatment of a disease associated with an increase in the expression or activity of integrin α5β1 in a subject in need thereof, comprising administering the integrin α5β1 inhibitor and a pharmaceutical excipient to the subject.

[0116] In some embodiments, the disease is characterized by a World Health Organization (WHO) group.

[0117] 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.

[0118] In some embodiments, the disease is characterized by a World Health Organization (WHO) classification system. In some embodiments, the disease is characterized by a WHO functional class 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.

[0119] In some embodiments, the disease is heart failure or right ventricular failure.

[0120] In one aspect, the present invention provides a method for treating pulmonary arterial hypertension (PAH) in a subject, comprising administering an integrin α5β1 inhibitor.

[0121] Integrin α5β1 small molecule inhibitor In some embodiments, the integrin α5β1 inhibitor is a small molecule compound that binds to integrin α5β1.

[0122] In some embodiments, the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin α5.

[0123] In some embodiments, the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin β1.

[0124] In some embodiments, the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin α5β1.

[0125] In some embodiments, the integrin α5β1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof,

Chemical formula

[0126] In some embodiments, R 2 is methyl or ethyl.

[0127] In some embodiments, R 3 is methyl, ethyl, isopropyl, or cyclopropyl.

[0128] In some embodiments, R 1 is hydrogen.

[0129] In some embodiments, the compound of formula (I) is selected from the group consisting of:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0130] In some embodiments, the integrin α5β1 inhibitor is a compound that is SMi.

Chemical formula

[0131] In some embodiments, the integrin α5β1 inhibitor is a dual inhibitor of α5β1 and αvβ1 (e.g., MRT).

[0132] Integrin α5β1 antibody inhibitor In some embodiments, the integrin α5β1 inhibitor is a 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-binding Fab (s-Fab), antibody, or a combination thereof.

[0133] In some embodiments, the integrin α5β1 inhibitor is an antibody.

[0134] In some embodiments, the integrin α5β1 inhibitor is an antibody that specifically binds to integrin α5.

[0135] In some embodiments, the integrin α5β1 inhibitor is an antibody that specifically binds to integrin β1.

[0136] In some embodiments, the integrin α5β1 inhibitor is an antibody that specifically binds to integrin α5β1.

[0137] In some embodiments, the antibody is an integrin α5β1 antibody selected from the group consisting of volociximab (M200), PF-04605412, and MINT1526A.

[0138] In some embodiments, the antibody is the integrin α5β1 antibody volociximab (M200).

[0139] The antibody volociximab (M200) comprises the heavy chain amino acid sequence of SEQ ID NO: 1. QVQLKESGPGLVAPSQSLSITCTISGFSLTDYGVHWVRQPPGKGLEWLVVIWSDGSSTYNSALKSRMTIRKDNSKSQVFLIMNSLQTDDSAMYYCARHGTYYGMTTTGDALDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 1)

[0140] The antibody vorociximab (M200) comprises the light chain amino acid sequence of SEQ ID NO: 2. QIVLTQSPAIMSASLGERVTMTCTASSSVSSNYLHWYQQKPGSAPNLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYLRSPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2)

[0141] In some embodiments, the antibody is the integrin α5β1 antibody M200. In some embodiments, the antibody is an integrin α5β1 antibody that antagonizes and / or binds to the same epitope and integrin binding as M200.

[0142] In some embodiments, the antibody is the integrin α5β1 antibody PF-04605412. In some embodiments, the antibody is an integrin α5β1 antibody that antagonizes and / or binds to the same epitope and integrin binding as PF-04605412.

[0143] In some embodiments, the antibody is the integrin α5β1 antibody described in International Publication No. WO 2009 / 100110A1, which is incorporated herein by reference. In some embodiments, the antibody is an integrin α5β1 antibody that antagonizes integrin binding and / or binds to the same epitope as the integrin α5β1 antibody described in International Publication No. WO 2009 / 100110A1.

[0144] In some embodiments, the antibody is the integrin α5β1 antibody 22B5. In some embodiments, the antibody is an integrin α5β1 antibody that antagonizes and / or binds to the same epitope and integrin binding as 22B5.

[0145] In some embodiments, the antibody is the integrin α5β1 antibody 24C7. In some embodiments, the antibody is an integrin α5β1 antibody that antagonizes and / or binds to the same epitope and integrin binding as 24C7.

[0146] In some embodiments, the antibody is the integrin α5β1 antibody 1D9. In some embodiments, the antibody is an integrin α5β1 antibody that antagonizes and / or binds to the same epitope and integrin binding as 1D9.

[0147] In some embodiments, the antibody is the integrin α5β1 antibody 2D2. In some embodiments, the antibody is an integrin α5β1 antibody that antagonizes and / or binds to the same epitope and integrin binding as 2D2.

[0148] 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.

[0149] Exemplary integrin α5β1 antibodies are described in International Publication No. WO 2010 / 111254 A1, which is incorporated herein by reference. In some embodiments, the antibody is an integrin α5β1 antibody that antagonizes integrin binding and / or binds to the same epitope as the integrin α5β1 antibodies described in International Publication No. WO 2010 / 111254 A1.

[0150] In some embodiments, the anti-α5β1 antibody comprises a VL domain comprising CDR-L1 comprising TL-S / T-S / P / T-Q / N-H-F / S-T / I-Y-K / T-I-G / D / S, CDR-L2 comprising L / I-N / T-S-D / H / S-G / S-S / L / T-H / Y-N / K / Q / I-K / T-G / A-D / S / V, and CDR-L3 comprising G / A-S / A / Y-S / Y-Y-S / A / Y-S / Y / T-GY-V / I, and a VH domain comprising CDR-H1 comprising GFTFS-N / A-RW-I / V-Y, CDR-H2 comprising GIKTKP-N / A / T-I / R-YAT-E / Q-YADSVKG, and CDR-H3 comprising L / V-TG-M / K-R / K-YFDY.

[0151] In some embodiments, the integrin α5β1 antibody antagonizes integrin binding and / or binds to the same epitope as an antibody comprising a VL domain comprising CDR-L1 comprising TL-S / T-S / P / T-Q / N-H-F / S-T / I-Y-K / T-I-G / D / S, CDR-L2 comprising L / I-N / T-S-D / H / S-G / S-S / L / T-H / Y-N / K / Q / I-K / T-G / A-D / S / V, and CDR-L3 comprising G / A-S / A / Y-S / Y-Y-S / A / Y-S / Y / T-GY-V / I, and a VH domain comprising CDR-H1 comprising GFTFS-N / A-RW-I / V-Y, CDR-H2 comprising GIKTKP-N / A / T-I / R-YAT-E / Q-YADSVKG, and CDR-H3 comprising L / V-TG-M / K-R / K-YFDY.

[0152] In some embodiments, the integrin α5β1 antibody comprises a VL domain comprising CDR-L1 comprising TLSSQHSTYTI, CDR-L2 comprising LNSDSSHNKGSGIPD, and CDR-L3 comprising AAYYAYGYV, and a VH domain comprising CDR-H1 comprising GFTFSARWIY, CDR-H2 comprising GIKTKPAIYATEYADSVKGRFT, and CDR-H3 comprising LTGMKYFDY.

[0153] In some embodiments, the integrin α5β1 antibody antagonizes integrin binding and / or binds to the same epitope as an antibody comprising a VL domain comprising CDR-L1 comprising TLSSQHSTYTI, CDR-L2 comprising LNSDSSHNKGSGIPD, and CDR-L3 comprising AAYYAYGYV, and a VH domain comprising CDR-H1 comprising GFTFSARWIY, CDR-H2 comprising GIKTKPAIYATEYADSVKGRFT, and CDR-H3 comprising LTGMKYFDY.

[0154] In some embodiments, the anti-α5β1 antibody comprises a VL domain comprising CDR-L1 containing TLSSQHSTYTIG, CDR-L2 LNSDSSHNKGS, and CDR-L3 containing AAYYAYGYV, and a VH domain comprising CDR-H1 containing the residue GFTFSARWIY, CDR-H2 containing the residue GIKTKPAIYATEYADSVKG, and CDR-H3 containing the residue LTGMKYFDY.

[0155] In some embodiments, the integrin α5β1 antibody competes for integrin binding and / or binds to the same epitope and antagonizes integrin binding as an anti-α5β1 antibody comprising a VL domain comprising CDR-L1 containing TLSSQHSTYTIG, CDR-L2 LNSDSSHNKGS, and CDR-L3 containing AAYYAYGYV, and a VH domain comprising CDR-H1 containing the residue GFTFSARWIY, CDR-H2 containing the residue GIKTKPAIYATEYADSVKG, and CDR-H3 containing the residue LTGMKYFDY.

[0156] In some embodiments, the integrin α5β1 antibody is selected from the antibodies described in Table 1. In some embodiments, the integrin α5β1 antibody competes for integrin binding and / or binding to the same epitope as the anti-α5β1 antibody described in Table 1.

Table 1

[0157] In some embodiments, the antibody is integrin α5β1 antibody 3C5 or 5B11. In some embodiments, the antibody is an integrin α5β1 antibody that antagonizes integrin binding with an antibody selected from 3C5 and 5B11. 3C5 and 5B11 are integrin α5β1 antibodies described in International Publication No. WO 2010 / 072740 A2, which is incorporated herein by reference.

[0158] In some embodiments, the antibody is an integrin α5β1 antibody that antagonizes integrin binding and is 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).

[0159] 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.

[0160] In some embodiments, the α5β1 integrin inhibitor is selected from Table 2.

Table 2

[0161] Method of treatment The present invention provides a method of treating a heart or lung 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 an increase in the 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) group.

[0162] 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.

[0163] 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 class 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.

[0164] 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).

[0165] In some embodiments, the patient has persistent / recurrent chronic thromboembolic pulmonary hypertension (CTEPH) (WHO group 4) after surgical treatment or inoperable CTEPH. In some embodiments, the patient has pulmonary arterial hypertension (PAH) (WHO group 1).

[0166] In some embodiments, the treatment is administered to improve exercise capacity and WHO functional class. In some embodiments, the treatment is administered to improve exercise capacity, improve the WHO functional class, and delay clinical deterioration.

[0167] 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.

[0168] In one aspect, the present invention provides a method of treating pulmonary arterial hypertension (PAH) in a subject, comprising administering an integrin α5β1 inhibitor.

[0169] In some embodiments, the integrin α5β1 inhibitor is administered orally, intravenously, subcutaneously, intranasally, transdermally, intraperitoneally, intramuscularly, or by inhalation.

[0170] Also provided is a method for the treatment of a subject suffering from fibrosis or a fibrosis-related disorder, the method comprising administering to the subject a therapeutically effective amount of an integrin α5β1 inhibitor according to the present invention. As used herein, the term fibrosis refers to a condition characterized by the deposition of extracellular matrix components in the skin or organs including the lung, kidney, heart, liver, skin, and joints, resulting in scar tissue. This term also refers to the process of scar tissue formation.

[0171] In some embodiments, the fibrosis-related disorder is a disorder or condition that can result from fibrosis or is related to fibrosis. In some embodiments, the fibrosis and / or fibrosis-related disorder is selected from the group consisting of renal fibrosis, hepatic fibrosis, cirrhosis, pulmonary fibrosis, dermal fibrosis, biliary 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, intervertebral disc hernia and other spinal cord injuries, fibroma, fibromyalgia, arthritis, restenosis. Pulmonary fibrosis includes idiopathic pulmonary fibrosis and scleroderma pulmonary fibrosis.

[0172] Therapeutic dose In some embodiments, a method for treating a disease associated with an increase in the 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, a method for treating a heart or lung disease in a subject 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 the World Health Organization (WHO) group as discussed above.

[0173] 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.

[0174] Cardiac function parameters Various endpoint parameters can be evaluated to determine the effectiveness of the treatment of the present invention, for example, Α5Β1 level, 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 (6 MWD), brain natriuretic peptide (BNP) level, diffusion of pulmonary gas volume (DLCO), and death or survival. See Chung et al. Phest (2010), 138(6):1383-1394.

[0175] PVR is commonly used as an endpoint parameter for determining the effectiveness of treatment for PAH. The PVR of a subject exceeding 240 dyn·sec / cm 5 is an indicator of mild PAH. The PVR of a subject of 600-800 dyn·sec / cm 5 is an indicator of moderate to severe PAH. After treatment using the methods and compositions of the present invention, a decrease in PVR in a subject of 130 dyn·sec / cm 5 or more indicates effective treatment. For example, administration of an Α5Β1 inhibitor to a subject having PAH that results in a decrease in PVR of 180-350 dyn·sec / cm 5 indicates effective treatment.

[0176] Mean pulmonary artery pressure (PAP) is also used as an endpoint parameter to determine the effectiveness of PAH treatment. Subjects without PAH have a mean PAP in the range of about 15 - 24 mmHg. Subjects with mild PAH have a mean PAP of about 25 - 30 mmHg (e.g., > 25 mmHg at rest or 30 mmHg during exercise). Subjects with severe PAH have a PAP of greater than 30 mmHg, e.g., 40 - 70 mmHg or 60 - 70 mmHg. After treatment, a decrease in PAP of greater than 1.5 mmHg indicates effective treatment. In some embodiments, treatment results in a decrease in PAP of greater than 5, 10, 20, 40, or 50 mmHg, indicating effective treatment.

[0177] Cardiac index (CI) is also used as an endpoint parameter to determine the effectiveness of PAH treatment. A low or decreasing 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 greater than 0.3 L / min / m2 indicates effective treatment.

[0178] Mean pulmonary capillary wedge pressure (PCWP) can be used as an endpoint parameter to determine the effectiveness of PAH treatment. A mean PCWP of 18 mmHg or less (e.g., 10 mmHg or less) indicates a subject with PAH. After treatment, an increase in mean PCWP of greater than 0.5 mmHg indicates effective treatment.

[0179] Right atrial pressure (RAP) is also used as an endpoint parameter to determine the effectiveness of PAH treatment. Subjects not suffering from PAH have a normal RAP of 0 - 8 mmHg. An RAP of 8 mmHg or more indicates PAH. Subjects suffering from severe PAH have an RAP of about 20 mmHg. After treatment, a decrease of greater than 0.5 mmHg indicates effective treatment.

[0180] The 6-minute walking distance (6MWD) is used as an endpoint parameter to determine the effectiveness of PAH treatment. The average 6MWD in CTD-PAH patients is about 300m. After treatment, an increase in 6MWD of 25m or more, or an increase exceeding 10%, indicates an effective treatment. For example, after treatment, a 6MWD of 1000m or more indicates an effective treatment.

[0181] Brain natriuretic peptide (BNP) is used as an endpoint parameter to determine the effectiveness of PAH treatment. BNP is a sensitivity marker for the deterioration of heart failure and a predictor of mortality in PAH patients. Normal levels of BNP are <100 pg / mL, for example, 30 - 90 pg / mL. Higher levels of BNP indicate deterioration of heart failure. BNP levels of about 100 - 200 pg / mL, for example, 160 pg / mL or more, indicate early heart failure. BNP levels of about 200 - 1000 pg / mL indicate actual heart failure. The average BNP level in CTD-PAH patients is about 430 pg / mL. After treatment, a decrease in BNP level indicates an effective treatment.

[0182] N-terminal pro-brain natriuretic peptide (NT-proBNP): A reproducible and non-invasive parameter is useful for patients with PAH. BNP is produced in the heart ventricles and increases in PPH / IPAH. BNP levels are closely related to dysfunction in PPH / IPAH patients and have been shown to parallel pulmonary hemodynamic changes and the degree of right heart failure. BNP levels correlate longitudinally with functional evaluations performed over the course of a study. Plasma NT-pro-BNP is measured by a sandwich immunoassay using a polyclonal antibody that recognizes an epitope located in the N-terminal segment (1 - 76) of Pro-BNP (1 - 108) (Elecsys analyzer, Roche Diagnostics, Mannheim, Germany).

[0183] Diffusion of pulmonary volume (DLCO), or the diffusing capacity of CO, is also used as an endpoint parameter to determine the effectiveness of PAH treatment. DLCO measures the ability of carbon monoxide (CO) to diffuse across the membrane. Subjects without PAH have a normal DLCO of greater than 80%. Subjects with PAH have an abnormal DLC of less than 80%, less than 65%, or less than 45%. Any increase in DLCO% after treatment indicates effective treatment.

[0184] In some embodiments, administration of an integrin α5β1 inhibitor modulates the level of a biomarker in a subject, and the biomarker is selected from the group consisting of survivin, PCNA, Ki67, and annexin V.

[0185] SoC and combination therapy As described herein, a method of treating a disease associated with an increase in the expression or activity of integrin α5β1, comprising administering an integrin α5β1 inhibitor, may include a combination therapy in which a patient in need of treatment is administered an integrin α5β1 inhibitor in combination with one or more drugs approved for the treatment of PH, PAH, heart failure, or right ventricular failure.

[0186] Approved drugs currently used for the treatment of PH, PAH, heart failure, and right ventricular failure in the United States or the European Union (EU) include orally administered PDE-5 inhibitors, sildenafil (Revatio) and tadalafil (Adeirca), dual endothelin-1A receptor antagonists (ERA), bosentan (Tracleer), ambrisentan (Letairis (in the United States), Volibris (internationally)). Patients with more advanced disease often receive a prostacyclin or prostacyclin analog, such as iloprost (Ventavis) or treprostinil (Tyvaso), as multiple daily inhalations, epoprostenol (Flolan / Veletri) or treprostinil (Remodulin) as continuous intravenous infusions, or treprostinil as continuous subcutaneous infusions. Intravenous injection of sildenafil is currently prescribed and is approved for patients who are temporarily unable to take oral sildenafil. Inhaled nitric oxide (INOmax) is approved for the neonatal form of PAH, i.e., persistent pulmonary hypertension of the newborn (PPHN). Thus, according to the present invention, combination therapy of any of these drugs and an integrin α5β1 inhibitor is useful for the treatment of PAH or disorders disclosed herein.

[0187] 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.

[0188] In some embodiments, the second 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 thromboendarterectomy, and / or atrial septostomy.

[0189] In some embodiments, the second therapy is macitentan and / or tadalafil.

[0190] Flolan (a prostacyclin analogue) is an approved therapy for PAH but is very cumbersome, inconvenient to use (intravenous), and has inherent safety concerns. As a result, Flolan is typically reserved for patients with severe functional status or rapidly progressive PAH. Patients must compound the drug several times a day in a sterile state. The drug is available as a lyophilized preparation that needs to be dissolved in an alkaline buffer. Due to its short half-life (3 - 5 minutes) and stability (8 hours at room temperature), Flolan must be maintained in a cooled state while being administered by continuous infusion through a central venous catheter via a portable pump (CADD pump, Smith’s Medical MD, St. Paul, Minn.) worn around the waist. In 2008, the FDA also approved a new continuous intravenous formulation of epoprostenol that is stable at room temperature for up to 24 hours after dilution and can be stored at refrigerator temperature for up to 5 days prior to use (GeneraMedix Inc., Liberty corner, N.J.). In 2009, GeneraMedix Inc. sold this formulation to Actelion and began selling the drug (under the brand name Veletri) in April 2010. In late 2010, the Veletri label was expanded to allow preparation of the drug for up to 7 days at refrigerator temperature or up to 48 hours at room temperature prior to use. Thus, in one embodiment of the present invention, the integrin α5β1 inhibitor is administered in combination with epoprostenol in any of its approved forms for treating PAH.

[0191] Remodulin (continuous subcutaneous injection form of a prostacyclin analog) was not generally used as initial therapy because of its cost, route of delivery, and limited efficacy. In 2004, the FDA and Health Canada approved an intravenous formulation of Remodulin for patients with PAH classes II-IV diseases for which the subcutaneous form was not acceptable. In early 2006, the FDA expanded the Remodulin label to include patients who require transition from Flolan. In 2009, United Therapeutics received FDA approval for an inhaled formulation of treprostinil (Tyvaso). Accordingly, in one embodiment of the present invention, an integrin α5β1 inhibitor is administered in combination with treprostinil to treat PAH.

[0192] Ventavis (iloprost), a prostacyclin analog administered via inhalation, is also commercially available in some member states of the EU as Ilomedin as an intravenous formulation. The label for inhaled iloprost in the EU is limited to patients with idiopathic PAH and functional class III symptoms. In contrast, the US label is broader, being for patients with PAH (regardless of etiology) as well as class III or IV symptoms. Administration 6-9 times a day is required. Accordingly, in one embodiment of the present invention, an integrin α5β1 inhibitor is administered in combination with iloprost in any of its approved forms to treat PAH.

[0193] In 2001, non-selective ERA Tracleer (bosentan) became the first oral PAH therapy and was only available in the United States through a special centralized access program due to significant risks of liver injury (reversible), teratogenicity, testicular atrophy, and male sterility. 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 first indicated for PAH patients and patients with moderate or severe functional status (WHO classes III, IV). In 2008 (EU) and 2009 (US), the indication was expanded to patients with mild symptoms (functional class II). Thus, in one embodiment of the present invention, the integrin α5β1 inhibitor is administered in any of its approved forms in combination with bosentan for the treatment of PAH.

[0194] Ambrisentan is a marketed oral selective ERA receptor antagonist that improves exercise capacity and delays clinical worsening for once-daily treatment of patients with WHO class II or III symptoms by BMS Sciences in the United States (Letairis) and GlaxoSmithKline in other regions (Volibris). Similar to bosentan, ambrisentan has class effects of teratogenicity, testicular injury, decreased male fertility, and anemia. Thus, in one embodiment of the present invention, the integrin α5β1 inhibitor is administered in any of its approved forms in combination with ambrisentan for the treatment of PAH.

[0195] The oral PDE-5 inhibitor Levatio (sildenafil) is approved in the United States for the treatment of PAH (WHO Group I) to improve exercise capacity and delay clinical worsening at a dose of 20 mg three times a day, regardless of functional class or etiology. The EU label is associated with idiopathic or collagen vascular disease and is limited to improvement of exercise capacity in PAH patients who are in functional class III status. In 2009, the FDA approved an intravenous form of Revatio, which is given as an injection (10 mg three times a day), for patients who cannot take the oral formulation. In May 2010, the EU approved Revatio as an oral suspension (composed of 20 mg tablets) for the treatment of pediatric patients 1 to 17 years of age with PAH. Thus, in one embodiment of the present invention, the integrin α5β1 inhibitor is administered in combination with sildenafil in any of its approved forms for the treatment of PAH.

[0196] The oral PDE-5 inhibitor Adeirca (tadalafil) 40 mg once daily has been shown (Packet Insert) to improve exercise capacity in patients with PAH (WHO Group I) in the United States, regardless of etiology or functional class. The EU label is limited to patients with functional class II and III status. Tadalafil has a long half-life (35 hours) in patients with PAH (US prescribing information) and has also shown benefit in patients with PAH on combination bosentan.

[0197] Accordingly, a method of treating a patient can involve administering at least one additional active agent, i.e., in addition to an integrin a5b1 inhibitor. The additional active agent can 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), an agent for treating diseases associated with hyperactive B cells or dysfunctional B cells such as rituximab, and / or a type V phosphodiesterase (PDE5) inhibitor.

[0198] When combination therapy is involved in the method of the present invention, i.e., when a secondary agent such as a vasodilator is co-administered with an integrin a5b1 inhibitor, the agents can be administered separately, at the same or different times, or in a single composition. Accordingly, the present invention provides a novel pharmaceutical formulation in which an integrin a5b1 inhibitor is combined with one of the above active agents and a unit dosage form of their formulations.

[0199] In the combination therapy of the present invention, each agent can be administered in an immediate release mode or a controlled release mode. When the additional active agent is a vasodilator, for example, any dosage form containing both active agents, i.e., both an integrin a5b1 inhibitor and a vasodilator, can provide either immediate release or controlled release of the vasodilator and immediate release or controlled release of the integrin a5b1 inhibitor.

[0200] As a general example, the combination dosage form of the present invention for once-daily administration comprises an integrin α5β1 inhibitor in the range of about 1 mg to about 1000 mg, in a controlled-release (e.g., sustained-release) or immediate-release form, and either an immediate-release form of sildenafil or a controlled-release form of sildenafil, and the additional active agent is present in an amount providing the weight ratio of the integrin α5β1 inhibitor to sildenafil, or the weight ratio of the integrin α5β1 inhibitor to sildenafil, as defined above. In other formulations of the present invention, two or more additional active agents, which may or may not be in the same class of agents (e.g., vasodilators), may be present in combination with the integrin α5β1 inhibitor. In such cases, the effective amount of any or each additional active agent present is generally reduced as compared to the amount required when using only a single additive.

[0201] The additional active agent may also be a type V phosphodiesterase inhibitor, as described above, and may be administered with the integrin α5β1 inhibitor, or with both the integrin α5β1 inhibitor and a vasodilator. Examples of type V phosphodiesterase inhibitors include, but are not limited to, avanafil, sildenafil, tadalafil, zaprinast, dipyridamole, vardenafil, and acid addition, or other pharmaceutically acceptable salts. Sildenafil is an excellent example. In an exemplary embodiment, the integrin α5β1 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 integrin α5β1 inhibitor compound is as presented above for the monotherapy regimen.

[0202] In one embodiment, the vasodilator is selected from sildenafil, avanafil, tadalafil, zaprinast, dipyridamole, vardenafil, bosentan, and pharmaceutically acceptable salts thereof.

[0203] The additional active agent may also be, as described above, an endothelin receptor antagonist, for example, bosentan, sitaxentan, or ambrisentan, and bosentan is an exemplary active agent.

[0204] The pharmaceutical composition of the present invention is a pharmaceutical preparation containing an active agent formulated in a manner compatible with its intended route of administration. Various routes are contemplated, including but not limited to oral, pulmonary, inhalation, sublingual, intranasal, parenteral, intradermal, transdermal, topical, transmucosal, subcutaneous, intravenous, intramuscular, intraperitoneal, intraoral, rectal, etc. As used herein, the term "parenteral" is intended to include subcutaneous, intravenous, and intramuscular injections.

[0205] Generally, the pharmaceutical preparation of the present invention is prepared for oral administration and is in an immediate-release form suitable for once-daily (QD) dosing. Certain preparations are suitable for intranasal administration to patients.

[0206] A specific pharmaceutical preparation of the present invention comprises an integrin α5β1 inhibitor or a salt thereof, and one or more pharmaceutically acceptable (approved by state or federal regulatory authorities for use in humans or listed in the United States Pharmacopeia, European Pharmacopeia) excipients or carriers. As used herein, the term excipient or carrier broadly refers to a biologically inert substance used in combination with the active agent of the formulation. Excipients can be used, for example, as solubilizing agents, stabilizers, diluents, inert carriers, preservatives, binders, coating agents, flavoring agents, or coloring agents. Preferably, at least one excipient is selected to provide the formulation with one or more beneficial physical properties such as an increase in the stability and / or solubility of the active agent(s). The integrin α5β1 inhibitor or a salt thereof described herein is an exemplary active agent suitable for use in the formulations of the present invention.

[0207] Examples of suitable excipients include certain inert proteins such as albumin, hydrophilic polymers such as polyvinylpyrrolidone, and amino acids such as aspartic acid (alternatively, it may be called aspartate), glutamic acid (alternatively, it may be called glutamate), lysine, arginine glycine, and histidine, fatty acids and phospholipids such as alkyl sulfonates and caprylates; surfactants such as sodium dodecyl sulfate and polysorbate, nonionic surfactants such as TWEEN® , PLURONICS® or polyethylene glycol (PEG), carbohydrates such as glucose, sucrose, mannose, maltose, trehalose, and dextrin, cyclodextrin, polyols such as mannitol and sorbitol, chelating agents such as EDTA, and salt-forming counterions such as sodium.

[0208] The solution or suspension used for delivery may contain the following components. Sterile diluents such as water for injection, physiological saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, polysorbate, tocopherol polyethylene glycol succinate (TPGS), or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium sulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetate, citrate or phosphate, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. These preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0209] In some embodiments, the pharmaceutical preparation of the present invention contains a plurality of liposomes or microparticles containing an integrin α5β1 inhibitor activator. In various embodiments, the pharmaceutical preparation of the integrin α5β1 inhibitor is a powder containing solid particles (e.g., liposomes or microparticles) suitable for administration via inhalation. The solid particles include an active agent, a carrier, optionally a surfactant, and optionally additional recipients. The powder may be prepared by any convenient method. An example of a preparation method is spray-drying a solution containing the active agent (and other components) onto a powder containing a carrier compound. Another example is freeze-drying a solution containing all of the components of the final powder.

[0210] Suitable liposomes for use in the present preparation 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, and the weight ratio is about 5:10:1. In some embodiments, the liposome preparation contains about 0.1-25%, for example, 0.1%, 1%, 5%, 10%, or 20% (w / w) of phospholipids such as dipalmitoyl phosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the liposome preparation contains 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 preparation contains about 10-35% of an amino acid such as L-leucine.

[0211] Suitable microparticles for use in the formulations of the present invention are known in the art. For example, the microparticles are formed of one or more hydrophilic polymers such as polyvinylpyrrolidone (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 wt% polyvinyl alcohol (e.g., PVA-30), about 5-15 wt% PVP (e.g., PVP-10), 1-5% poloxamer (e.g., Poloxamer-188), 0-10% L-leucine, and about 0.5-10 wt% integrin α5β1 inhibitor compound (e.g., 5%). In some embodiments, the formulation is suitable for administration via the airway.

[0212] Pharmaceutical formulations of integrin α5β1 inhibitors useful in the methods of the present invention can be prepared as liquids for oral administration or in solid forms such as powders, tablets, pills, or capsules. The liquid formulations of the present invention can take the form of suspensions, solutions, or emulsions in an oily vehicle or an aqueous vehicle and can contain formulation agents such as suspending agents, stabilizers, and / or dispersing agents. In one embodiment, the formulation is an aqueous solution. In another embodiment, the final formulation is lyophilized. In some embodiments, the integrin α5β1 inhibitor is formulated for inhalation.

[0213] In various embodiments, the formulations of the present invention contain integrin α5β1 inhibitor at a concentration of 0.25 wt% to 100 wt%, or 0.25 wt% in 50 wt%, or 0.8 wt% to 25 wt%, or 1 wt% to 10 wt%, or 1.5 wt% to 5 wt%. In certain embodiments, the integrin α5β1 inhibitor compound is formulated at a concentration of about 0.5 wt% to about 5 wt%. In certain embodiments, the integrin α5β1 inhibitor compound is formulated at a concentration of about 0.25 wt% to about 10 wt%.

[0214] The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with a solid or liquid formulation of an integrin α5β1 inhibitor. In certain embodiments, the formulation is a powder formulation of an integrin α5β1 inhibitor. In various embodiments, the integrin α5β1 inhibitor is formulated at a concentration of at least about 0.5% by weight, and the formulation is suitable for delivery via inhalation to a human.

[0215] The present invention also provides the use of an integrin α5β1 inhibitor in the manufacture of a medicament for treating PAH in a subject in need thereof, or in the disorders disclosed herein. Generally, the pharmaceutical formulation is sterile.

[0216] Generally, a dosage form, e.g., an inhalable dosage form, provides a sustained release, i.e., a stepwise release, of the compound of the present invention, e.g., an integrin α5β1 inhibitor, from the dosage form to the body of the patient over a long period of time, typically providing a substantially constant blood level of the drug over a period ranging from about 4 to about 12 hours, typically ranging from about 6 to about 10 hours. In particularly preferred embodiments, there is a very gradual increase in the blood level of the drug after nasal administration of a dosage form containing the compound of the present invention, e.g., an integrin α5β1 inhibitor, such that the peak blood level is not reached until at least 4 - 6 hours have elapsed and the rate of increase of the blood level drug is substantially linear. In addition, in preferred embodiments, there is an equally gradual decrease in the blood level at the end of the sustained release period.

[0217] The pharmaceutical composition of the present invention is preferably formulated for inhalation, e.g., as a solution in physiological saline, as a dry powder, or as an aerosol, although other modes of administration are also suitable. For example, administration may be by means of an implanted depot, transmucosal, e.g., via the rectum or vagina, sublingual, oral, parenteral, transdermal, preferably using a suppository containing excipients such as suppository wax in addition to the active agent. Transmucosal administration also includes, for example, transurethral administration as described in U.S. Pat. Nos. 5,242,391, 5,474,535, and 5,773,020 to Place et al.

[0218] Depending on the intended mode of administration, the pharmaceutical preparation may be a solid, semi-solid, or liquid, such as, for example, tablets, capsules, caplets, liquids, suspensions, emulsions, suppositories, granules, pellets, beads, powders, etc., and preferably may be a unit dosage form suitable for single administration of an exact dosage. Suitable pharmaceutical compositions and dosage forms are known to those in the pharmaceutical preparation field and can be prepared using conventional methods described in relevant texts and literature, such as Remington: The Science and Practice of Pharmacy (Easton, Pa.: Mack Publishing Co., 1995). For compounds that are orally active, oral dosage forms are generally preferred and include tablets, capsules, caplets, solutions, suspensions, and syrups, and may contain multiple granules, beads, powders, or pellets, which may or may not be encapsulated. Preferred oral dosage forms are tablets and capsules.

[0219] In embodiments, it may be particularly advantageous to formulate the compositions of the present invention in unit dosage forms for ease of administration and uniformity of dosage. As used herein, the term unit dosage form refers to physically discrete units suitable as a single dosage for the individual to be treated. That is, the composition is formulated into discrete dosage units, each containing a predetermined unit dose of the active agent calculated to provide the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of the unit dosage forms of the present invention depend on the unique characteristics of the active agent to be delivered. The dosage can be further determined by reference to the usual dosages and methods of administration of the components. In some cases, two or more individual dosage units combined provide a therapeutically effective amount of the active agent. For example, it should be noted that two tablets or capsules taken together may provide a therapeutically effective dose of an integrin α5β1 inhibitor, such that the unit dose in each tablet or capsule is about 50% of the therapeutically effective amount.

[0220] Tablets may be manufactured using standard tablet processing procedures and equipment. Direct compression and granulation techniques are preferred. In addition to the active agent, tablets generally contain inert pharmaceutically acceptable carrier materials such as binders, lubricants, disintegrants, fillers, stabilizers, surfactants, colorants, and the like.

[0221] Capsules are another oral dosage form of the compounds of the present invention, for example, for integrin α5β1 inhibitors that are orally active, in which case the active agent-containing composition may be encapsulated in liquid or solid form (including particles such as granules, beads, powders, or pellets). Suitable capsules may be either hard or soft, with gelatin capsules being preferred and generally made of gelatin, starch, or cellulose-based materials. Two-piece hard gelatin capsules are preferably sealed with a gelatin band or the like. See, for example, Remington. "The Science and Practice of Pharmacy," cited earlier herein, describes materials and methods for preparing encapsulated pharmaceuticals.

[0222] Oral dosage forms may be formulated to provide controlled release of the compounds of the present invention, such as integrin α5β1 inhibitors, if tablets, capsules, caplets, or microparticles are desired. In a preferred embodiment, the formulation is a controlled release oral dosage form.

[0223] Generally, as understood by those skilled in the art, sustained-release dosage forms are formulated by dispersing an active agent within a matrix of a material that is gradually hydrolyzable, such as a hydrophilic polymer, or by coating a solid pharmaceutical dosage form with such a material. Hydrophilic polymers useful for providing a sustained-release coating or substrate include, by way of example, cellulose-based polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, cellulose acetate, sodium carboxymethylcellulose, and acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, alkyl esters of acrylic acid, alkyl esters of methacrylic acid, etc., for example, 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.

[0224] Formulations according to the invention for parenteral administration include sterile aqueous and non-aqueous solutions, suspensions, and emulsions. Aqueous injection solutions contain the active agent 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, and the like. Parenteral formulations may also contain adjuvants such as solubilizing agents, preservatives, wetting agents, emulsifying agents, dispersing agents, and stabilizers. Aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and dextran. Injectable formulations are sterilized by incorporation of a sterilizing agent, filtration through a bacteria-retaining filter, irradiation, or heat. They can also be manufactured using a sterile injectable medium. The active pharmaceutical agent may also be in a dry, for example, lyophilized form, which can be rehydrated with a suitable vehicle immediately prior to administration by injection.

[0225] The active agent may also be administered through the skin using conventional transdermal drug delivery systems, and the active agent is contained within a laminated structure that functions as a drug delivery device affixed to the skin. In such structures, the drug composition is contained within a layer beneath the upper backing layer, or reservoir. The laminated structure may include a single reservoir or multiple reservoirs. In one embodiment, the reservoir includes a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Alternatively, the drug-containing reservoir and the skin contact adhesive exist as separate and distinct layers, with the adhesive beneath the reservoir, which may in this case be the aforementioned polymeric matrix, or a liquid or hydrogel reservoir, or take some other form. The transdermal drug delivery system may further include a skin penetration enhancer.

[0226] In addition to the formulations described above, the active agent may be formulated as a depot preparation for the controlled release, preferably sustained release over a long period of time, of the active agent. These sustained release dosage forms are generally administered by implantation (e.g., subcutaneously or intramuscularly, or by intramuscular injection).

[0227] The specific compounds or active agents of the present invention can further form salts. All of these forms are also contemplated within the scope of the claimed invention.

[0228] The compounds of the present invention can also be prepared as esters, for example, pharmaceutically acceptable esters. For example, carboxylic acid functional groups in the compounds can be converted to their corresponding esters, such as methyl, ethyl, or other esters. Also, alcohol groups in the compounds can be converted to their corresponding esters, such as acetates, propionates, or other esters.

[0229] Certain compounds of the present invention may also be prepared as prodrugs, for example, pharmaceutically acceptable prodrugs. The terms "pro-drug" and "prodrug" are used interchangeably herein and refer to any compound that releases an active parent drug in vivo. Since prodrugs are known to enhance many desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacture, etc.), the compounds of the present invention may be delivered in prodrug form. Accordingly, the present invention is intended to cover prodrugs of the claimed compounds, methods of delivering them, and compositions containing them. A prodrug is intended to include any covalent carrier that releases the active parent drug of the present invention in vivo when such prodrug is administered to a subject. The prodrugs of the present invention are prepared by modifying the functional groups present in the compounds such that the modification is cleaved to the parent compound in routine operations or in vivo. Examples of prodrugs include, but are not limited to, esters (e.g., acetates, dialkylaminoacetates, formates, phosphates, sulfates and benzoate derivatives) and carbamates (e.g., N, N-dimethylaminocarbonyl of a hydroxy functional group), esters (e.g., ethyl ester, morpholinoethanol ester of a carboxyl functional group), N-acyl derivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases and enamino ketones of an amino functional group, 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, p 1-92, Elesevier, New York-Oxford (1985).

[0230] Examples of prodrugs include, but are not limited to, esters (e.g., acetates, dialkylaminoacetates, formates, phosphates, sulfates and benzoate derivatives) and carbamates (e.g., N, N-dimethylaminocarbonyl of a hydroxy functional group), esters (e.g., ethyl ester, morpholinoethanol ester of a carboxyl functional group), N-acyl derivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases and enamino ketones of an amino functional group, 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, p 1-92, Elesevier, New York-Oxford (1985).

[0231] The dosing regimen utilizing the compound is selected according to various factors including the type, species, age, weight, sex and medical condition of the patient; the severity of the medical condition to be treated; the route of administration; the renal and hepatic functions of the patient; and the specific compound or its salt employed. A physician or veterinarian of ordinary skill can readily determine and prescribe an effective amount of the agent necessary for the prevention, management, or arrest of the medical condition.

[0232] In some embodiments, the composition is suitable for inhalation. In one embodiment, the composition is an inhalable formulation for treating PAH or the disorders disclosed herein.

[0233] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an integrin α5β1 inhibitor and a plurality of particles, wherein the plurality of particles are a plurality of liposomes comprising a plurality of microparticles comprising 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE) or 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 the disorders disclosed herein.

[0234] Composition The pharmaceutical compositions described herein can be administered in a variety of different ways. By way of example, it includes administering a pharmaceutical composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, transdermal, intrathecal, and intracranial methods, which contains a peptide or multimer according to the present invention, preferably a peptide according to the present invention. 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.

[0235] 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 physiological saline. Examples of excipients that can be incorporated into tablets, capsules, etc. are binders such as tragacanth gum, acacia, corn starch, or gelatin, excipients such as microcrystalline cellulose, disintegrants such as corn starch, pregelatinized starch, alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose, or saccharin, and flavoring agents such as peppermint, wintergreen, or cherry. When the dosage unit form is a capsule, in addition to the above types of substances, it may also contain a liquid carrier such as, for example, a fatty oil. Various other materials may be present as coatings or otherwise to modify the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar, or both. Syrups or elixirs may contain the active compound, sucrose as a sweetener, methyl and propyl parabens as preservatives, dyes, and flavoring agents such as cherry or orange flavor. The pharmaceutical composition according to the present invention is preferably suitable for human use.

[0236] The sterile composition for injection can be formulated according to conventional pharmaceutical practice by dissolving or suspending the integrin α5β1 inhibitor of the present invention in an injectable vehicle such as water or a natural vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, or a synthetic fatty vehicle such as ethyl oleate. Buffers, preservatives, antioxidants, etc. may also be incorporated.

[0237] The composition for topical administration can also be formulated according to conventional pharmacy. As used herein, topical administration refers to the application to a body surface such as the skin or mucosa for locally treating a condition resulting from a microbial or parasitic infection. Examples of formulations suitable for topical administration include, but are not limited to, creams, gels, ointments, lotions, foams, suspensions, sprays, aerosols, powder aerosols. The topical agent can be an epidermis, which means it is applied directly to the skin. The topical agent can also be inhaled, for example, for application to the mucosal epithelium of the airway, or for application to the surface of tissues other than the skin such as eye drops applied to the conjunctiva or ear drops placed in the ear. The pharmaceutical composition formulated for topical administration preferably contains at least one pharmaceutical excipient suitable for topical application such as an emulsifier, a diluent, a wetting agent, a preservative, a pH adjuster, and / or water.

Example

[0238] The following examples illustrate some of the preferred ways of making and practicing 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.

[0239] Example 1: The integrin α5β1 inhibitor inhibits the proliferation of hPASMC on fibronectin This example shows the in vitro effect of the integrin α5β1 inhibitor on hPASMC in vitro. The RNA expression of human integrins expressed in pulmonary artery smooth muscle cells derived from PAH patients was determined using the NanoString detection integrin target (Figure 1).

[0240] The antibody integrin α5β1 inhibitor was evaluated to determine whether an integrin α5β1 inhibitor (e.g., P1D6 antibody or M200 antibody) could impair proliferation. Cells were seeded at 3000 cells / well in 96-well plates coated with fibronectin. The next day, integrin α5β1 inhibitor treatment was added and proliferation was monitored for up to 5 days. Anti-integrin a5b1 antibody, integrin-expressing hPASMC treated with M200 (Figure 2A) and P1D6 (Figure 2B) showed a decrease in proliferation. Selective inhibition of a5b1 resulted in a reduction of approximately 70% in hPASMC proliferation.

[0241] As shown in Figure 3, the proliferation of hPASMC treated with P1D6 was reduced only in fibronectin-coated plates, suggesting that the inhibition of proliferation by blocking a5b1 is fibronectin-dependent.

[0242] A dose-response experiment using P1D6 was performed to determine the range of cell specificity of P1D6. PAH-PASMC 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 at 48, 72, and 120 hours were evaluated. Figures 4A - 4C show exemplary Western blot analyses measuring the expression levels of p-FAK (Figure 4B) and PCNA (Figure 4C) in PAH-PASMC 72 hours after exposure to P1D6 on fibronectin-coated plates.

[0243] The integrin α5β1 inhibitors (MRT, SMi, P1D6, or M200) were evaluated for proliferation and apoptosis on Fb-coated plates. Figures 5A - 5C show exemplary proliferation (Ki67) and apoptosis (annexin V) of PAH-PASMC treated with integrin α5β1 inhibitors (MRT, SMi, P1D6, or M200).

[0244] 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 ITGα 5, ITGα V, ITGβ1, p-FAK, MCM2, PLK1, p-ERK, ERK, PCNA, and survivin in hPASMCs (Figures 6A - 6B). Figures 7A - 7C, SMi treatment inhibited proliferation and increased apoptosis in a dose-dependent manner.

[0245] Primary rat cardiomyocytes were treated with 50 μM, 100 μM, or 200 μM of phenylephrine (PE). The expression of ITGα 5, ITGα V, ITGβ1, ITGβ3, ITGβ5 was determined by Western blot (Figure 21A). Cells treated with 100 μM of PE were simultaneously exposed to 0.25 μM, 1 μM, and 4 μM of the integrin α5β1 inhibitor SMi. As shown in Figures 21B and 21C, inhibition of α5β1 prevents hypertrophy of PE-induced adult rat cardiomyocytes.

[0246] PAH patient samples were evaluated for the expression of fibronectin-binding integrins. As shown in Figures 20A - 20D, the expression of specific integrins changes significantly in dissected PA, isolated PASMCs, isolated PAECs, and non-compensated RV from PAH patients compared to controls. Human PAH-RV Fbs were treated with 0.25 μM, 1 μM, and 4 μM of the integrin α5β1 inhibitor SMi. Pharmacological inhibition by SMi showed that integrin α5β1 decreases the proliferation and activation of PAH-RV Fbs in a dose-dependent manner. (Figures 22A and 22B).

[0247] Example 2: In Vivo Efficacy of Integrin α5β1 Inhibitor in the SuGen-Hypoxia Animal Model This example demonstrates the efficacy of an integrin α5β1 inhibitor alone and in combination with standard of care (SoC) therapy in a Sugen / Hypoxia (SuHx)-induced PAH rat model. Pharmacological inhibition of the broad spectrum of α5β1 improves hemodynamics and vascular remodeling in established PAH alone or in combination with standard of care in Su / Hx rats.

[0248] Animals were injected with 20 mg / kg of Sugen 5416 on day 0 and subjected to hypoxia (10% oxygen) for 3 weeks. Electrocardiogram examinations were performed at week 3 and at the start of treatment with the integrin α5β1 inhibitor at week 5. Right heart catheterization was performed at the end of week 5. Tissues were harvested for analysis.

[0249] As shown in FIG. 8 by exemplary histological staining for EVG, α SMA, PCNA, and C3C, SMi demonstrated vascular remodeling in Sugen / Hypoxia (SuHx)-induced PAH rats. Treatment with SMi and / or the vasodilator SoC (e.g., Macitentan (Maci) and Tadalafil (Tada)) improved cardiac output (FIG. 9A), stroke volume (FIG. 9B), thickness of the medial cell wall (FIG. 9C), vascular remodeling (FIG. 9D), proliferation (FIG. 9E), and apoptosis (FIG. 9F) were measured. The dotted line indicates the disease window.

[0250] As shown in FIGS. 10A - 10C, treatment with SMi and / or SoC (e.g., Macitentan (Maci) and Tadalafil (Tada)) decreased hypertrophy and fibrosis in SuHx rats. Integrin α5β1 inhibition improves cardiac function in SuHx animals

[0251] To determine the selectivity of SMi for α5β1, SuHx animals were prepared as described above and treated with the dual α5β1 / αvβ1 inhibitor (MRT). Animals were divided into the following groups: 1) Non - SuHx vehicle PO BID (n = 5) 2) SuHx + vehicle PO BID (PBS) (n = 10) 3) SuHx + MRT 60 mpk PO BID (n = 10) 4) SuHx + macitentan 1 mg / Kg + tadalafil 10 mg / Kg daily (n = 10) 5) SuHx + MRT 60 mpk PO BID + macitentan 1 mg / Kg + tadalafil 10 mg / Kg daily (n = 10)

[0252] Evaluate the improvement of cardiac function and vascular remodeling in MRT-treated SuHx rats as indicated by EVG staining, medial wall thickness, mean pulmonary artery pressure, cardiac output, and changes in right ventricular sectional area.

[0253] Integrin α5β1 antibody inhibitor in a SuHx (SuHx) mouse model of pulmonary arterial hypertension (PAH) 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 is 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 hereby incorporated by reference in its entirety.

[0254] Mice were injected with 20 mg / kg of Sugen 5416 (vascular endothelial growth factor receptor inhibitor) on days 0, 7, and 14 and subjected to a hypoxic condition (10% oxygen) for 3 weeks. Electrocardiogram examinations were performed at the end of the 3rd week (e.g., day 21) and the 5th week, at the start of treatment with the integrin α5β1 inhibitor. Right heart catheterization was performed at the end of the 5th week. Tissues were collected for analysis as summarized in Figure 24A.

[0255] Anti-integrin α5β1 antibody-treated SuHx rats were evaluated for improved cardiac function and vascular remodeling as shown by EVG staining, medial cell wall thickness, mean pulmonary artery pressure, cardiac output, and right ventricular fractional area change. Cardiac function, including improvement in cardiac output and stroke volume, was measured. Other characteristics, including the thickness of the media cell wall, vascular remodeling, proliferation, and apoptosis, were measured.

[0256] Mice were divided into the following treatment and control groups: · Control group (n = 5) · Vehicle PO BID (n = 10) · SMi 100 mpk PO BID (n = 10) · Macitentan 15 mpk PO BID (n = 15) · IgG isotype control 10 mpk IP (3× / week) (n = 15) · 339.1 10 mpk IP (3× / week) (n = 15) · 339.1 10 mpk IP + macitentan 15 mpk PO BID (n = 15)

[0257] In Figures 24B - 24G, the anti-α5β1 mouse antibody (antibody 339.1) phenotypically analyzed the cardiac and vascular improvements of SMi. These results confirm that α5β1 plays an important role in PAH and that PAH can be treated using integrin α5β1 inhibitors.

[0258] The robust effect of the α5β1 inhibitor in the right ventricle of SuHx animals was demonstrated by reduction of RVSP and TAPSE, improvement of cardiomyocyte hypertrophy, and reduction of cardiac fibrosis. Treatment with the α5β1 inhibitor resulted in a significant increase in cardiac output, suggesting a functional improvement of the right heart.

[0259] Example 3: In Vivo Efficacy of Integrin α5β1 Inhibitors in MCT Studies This example shows the efficacy of compound SMi treatment in a monocrotaline (MCT) rat model.

[0260] MCT animals were divided into test groups as follows: (1) Non-MCT vehicle, orally twice a day (BID) (n = 6) (2) MCT + PBS vehicle, orally twice a day (BID) (n = 8) (3) MCT + SMi at 100 mpk, orally BID (n = 8)

[0261] MCT (60 mg / kg) was administered by subcutaneous injection. The animals were exposed to vehicle or SMi treatment for 2 weeks. Analyses of blood, RV, and lungs were performed. Cells were evaluated for the expression of EVG, αSMA, PCNA, and C3C. As shown in Figure 11, animals treated with SMi showed improved vascular remodeling in the monocrotaline (MCT) rat pulmonary hypertension model. Figures 12A - 12D show exemplary results of treatment with SMi in MCT rats. Occlusion score (Figure 12A), vascular remodeling (Figure 12B), apoptosis (Figure 12C), and proliferation (Figure 12D) were measured.

[0262] To test the effect of SMi on cardiac function, MCT animals with cardiac defects were treated with (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. In this study, the pulmonary hypertension phenotype was not induced (MCT causes both vascular and cardiac injury). Treatment with SMi, vehicle, or sildenafil was administered twice a day from day 14 to day 27. Echocardiogram monitoring of disease progression was performed on all animals on day 0, day 14, and the surgical day (day 28). Blood samples were collected on day 0, day 14, and day 28.

[0263] The maximum velocity of the pulmonary artery, cardiac output, stroke volume, thickness of the anterior wall of the right ventricle, and pulmonary artery acceleration time were determined. Using an echocardiogram (model Vivid E9, XDclear Ultrasound, GE Healthcare, Illinois, United States) connected to a 13.0 MHz linear transducer, the maximum velocity of the pulmonary artery (Vmax), arterial pulmonary velocity time integral (VTI), pulmonary artery diameter, heart rate, thickness of the anterior wall of the right ventricle, and pulmonary artery acceleration time were measured. The data were used to calculate cardiac output and stroke volume as follows.

[0264] Cardiac output was calculated using the following formula (JF Lewis et al. 1984): Cardiac output = heart rate × VTI ((pulmonary artery diameter 2 × 3.1416) / 4)

[0265] Stroke volume was calculated using the following formula. Stroke volume = cardiac output / heart rate

[0266] In this MCT model, a significant increase in cardiac output by SMi was observed, suggesting dissociation of cardiac benefit from the effect on pulmonary resistance. Treatment with SMi brought about a direct improvement in the heart independent of the reverse remodeling effect in the pulmonary arterioles due to a5b1 inhibition.

[0267] Combination therapy of integrin α5β1 inhibitor and SoC in MCT animals MCT animals were treated daily with SMi alone or in combination with SoC therapy (macitentan (1 mg / kg) + tadalafil (10 mg / kg)). MCT animals were divided into test groups as follows: (1) Non-MCT vehicle twice daily orally (BID) (n = 6) (2) MCT + PBS vehicle twice daily orally (BID) (n = 8) (3) MCT + SMi at 100 mpk orally BID (n = 8) (4) Oral BID + SoC at 100mpk (MCT + SMi in macitentan (1mg / kg) + tadalafil (10mg / kg), n = 8).

[0268] MCT (60mg / kg) was administered by subcutaneous injection on day 0. From week 3, animals were exposed to vehicle or SMi treatment for 2 weeks. (Figure 23A) Analyses of blood, RV, and lung were performed. Exemplary histology and tissue analysis showed improvement in hypertrophy and right ventricle (RV) fibrosis with SMi alone or in combination with SoC (Figures 15A - 15C). Right heart catheterization was performed and animals were evaluated for RVSP (mmHg), mPAP (mmHg), and CO (ml / min) as shown in Figure 23B. As shown in Figure 23C, MCT - lung - engorged animals treated with SMi alone or in combination with Maci (1mg / kg) + Tada (10mg / kg) showed improvement in vascular remodeling.

[0269] Integrin α5β1 inhibition improves cardiac function in MCT animals 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).

[0270] Figures 14A - 14E, both SMi - treated MCT rats and MRT - treated MCT rats showed improvement in cardiac function vascular remodeling demonstrated by EVG staining (Figure 14A), medial cell wall thickness (Figure 14B), mean pulmonary artery pressure (Figure 14C), cardiac output (Figure 14D) and change in fractional area of the right ventricle (Figure 14E). Figure 13 shows exemplary pharmacokinetics of SMi and MRT. These data suggest that integrin α5β1 inhibition is involved in the improvement of cardiac function in MCT animals.

[0271] The protein levels of α5β1 integrin in human and mouse tissues were measured, and it was confirmed that α5β1 is highly expressed in the hearts of humans and mice (Figures 25A and 25B). In cultured cardiomyocytes stimulated with phenylephrine, an α-1 adrenergic receptor agonist that blocks α5β1, cardiomyocyte hypertrophy was reduced. Furthermore, in patient-derived cardiac fibroblasts, α5β1 blockade decreased fibroblast activation. These experiments in the in vitro system further demonstrate the direct pharmacological effects of α5β1 inhibition in cardiac cells.

[0272] Integrin α5β1 antibody inhibitor in MCT animals MCT animals are prepared as described above and treated with an integrin α5β1 antibody inhibitor. Anti-integrin α5β1 antibody-treated MCT rats are evaluated for improved cardiac function vascular remodeling as shown by EVG staining, thickness of the medial cell wall, mean pulmonary artery pressure, cardiac output, and change in right ventricular fractional area. Cardiac function parameters are measured including improvement in cardiac output, stroke volume, medial cell wall thickness, vascular remodeling, proliferation, and apoptosis.

[0273] Example 4: In vivo efficacy of integrin α5β1 inhibitor in PAB This example shows the efficacy of compound SMi in the right ventricle in a pulmonary artery banding (PAB) rat model. The animals were divided into three test groups as follows. (1) Control group / sham (n = 5) (2) PAB + PBS vehicle twice daily orally (BID) (n = 10) (3) PAB + SMi at 100 mpk orally BID (n = 10)

[0274] Pulmonary artery stenosis was surgically performed in rats on day 0. Starting from 4 weeks after surgery, the animals were exposed to vehicle or SMi treatment. Echocardiography was performed once a week from 10 weeks after surgery until 10 weeks in total. After a total of 10 weeks, the animals underwent RV catheterization. Blood, RV, and lung analyses were performed. Exemplary histology and tissue analysis showed improvement in hypertrophy and right ventricle (RV) fibrosis in a pulmonary artery banding (PAB) rat model treated with SMi (Figures 16A - 16D). Representative echocardiogram images of (PAB) rats treated with SMi are shown in Figure 17.

[0275] Hemodynamic and tissue harvest analysis showed that integrin α5β1 inhibition by SMi directly improved cardiac function over time compared to vehicle control in the RV PAB model. Cardiac output (Figure 18A), stroke volume (Figure 18B), tricuspid annular plane systolic excursion (TAPSE) (Figure 18C), S wave (Figure 18D), right ventricular fractional area change (RVFAC) (Figure 18E), RVEDD (Figure 18F), cardiac index (CI) (Figure 18G), pressure gradient (PG) (Figure 18H).

[0276] Right heart catheterization PAB rats were subjected to right heart catheterization (RHC) (using standard techniques while the subject was in a steady state). Exemplary cardiac parameters of right heart catheterization in PAB rats treated with SMi are shown in Figures 19A - 19E. Treatment with the integrin α5β1 inhibitor, SMi, improved cardiac output, stroke volume, RVSP, and RVEPD.

[0277] Integrin α5β1 antibody inhibitor in PAB animals PAB rats were prepared as described above and treated with an integrin α5β1 antibody inhibitor. Anti - integrin α5β1 antibody - treated PAB rats were evaluated for improvement in cardiac function vascular remodeling as shown by EVG staining, medial cell wall thickness, mean pulmonary artery pressure, cardiac output, and right ventricular fractional area change. Cardiac function parameters are measured including improvement in cardiac output, stroke volume, medial cell wall thickness, vascular remodeling, proliferation, and apoptosis.

[0278] Example 5: Characterization of integrin α5β1 inhibitors This example shows a method for screening and characterizing integrin α5β1 inhibitors.

[0279] Cell adhesion assay (CAA) A 96-well assay plate was coated with 0.625 μg / ml of purified recombinant human fibronectin consisting of domains 9 and 10, fused to glutathione S-transferase, and then blocked with 1% bovine serum albumin. Cells expressing rat α5β1 were incubated with test samples, which were prepared in serial dilution series in a 96-well deep well plate. 0.1 ml of the assay mixture consisted of 100,000 cells, antibody sample, 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 a 15-minute pre-incubation period, 0.1 ml of the assay mixture was transferred to the 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 four-parameter non-linear regression analysis.

[0280] As shown in Table 3, a5b1 antibody inhibitors (e.g., P1D6 and M200) show nanomolar range affinity in blocking cell adhesion.

Table 3

[0281] Solid phase (SP) assay The antibody activity was measured through binding competition with purified fibronectin isolated from rat plasma using a solid-phase (SP) assay. A 384-well assay plate was coated with 2 μg / ml fibronectin and then blocked with 1% bovine serum albumin. In a separate 384-well plate, 2 nM His-tagged rat α5β1 protein was incubated with test samples for 1 hour at room temperature in 50 mM HEPES pH 7.3, 150 mM sodium chloride, 0.5% bovine serum albumin, 1 mM magnesium chloride, 1 mM calcium chloride, and 0.05% Tween 20. 20 μl of the assay mixture was transferred to the fibronectin-coated assay plate and incubated for 1 hour at room temperature. Unbound components were removed by 3 rounds of washing using a BioTek 405 / TS plate washer. 20 μl of anti-6X Histag antibody conjugated to horseradish peroxidase was added and incubated for 1 hour at room temperature, and unbound antibody was removed by 3 rounds of washing. The amount of His-tagged rat α5β1 protein bound to the anti-6X Histag antibody was quantified using a Quantablue Fluorogenic substrate (Thermo Fisher), and the concentration-response curve was analyzed for IC50 values using 4-parameter non-linear regression analysis.

[0282] Ligand Binding Assay (LBA) To measure the potency of a sample against α5β1 in a cell-based ligand binding assay (LBA), cells expressing rat α5β1 were incubated with 10 μl of the test sample for 15 minutes 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 viability dye. 5 μl of 75 nM fibronectin fluorescently labeled with Dylight 650 in 50 mM HEPES pH 7.3, 150 mM sodium chloride, and 1% bovine serum albumin, isolated from human plasma, was added to the cells. The samples were incubated for 45 minutes at room temperature, 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 via flow cytometry. Dead cells were excluded from subsequent analysis based on staining with the 780 fixable viability dye. The median fluorescence intensity of Dylight 650 was determined for each sample, and the concentration-response curve was analyzed for IC50 values using four-parameter non-linear regression analysis. [Table 4]

[0283] Enumeration of Embodiments Embodiment 1: A method of treating pulmonary arterial hypertension (PAH) in a subject, the method comprising administering an integrin α5β1 inhibitor.

[0284] Embodiment 2: A method of treating a disease associated with an increase in the expression or activity of integrin α5β1, the method comprising administering an integrin α5β1 inhibitor.

[0285] Embodiment 3: A method of treating a heart disease or a lung disease, the method comprising administering an integrin α5β1 inhibitor.

[0286] Embodiment 4: The method according to any one of 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, or WHO class IV pulmonary hypertension.

[0287] Embodiment 5: The method according to any one of Embodiments 1 to 3, wherein the disease is cardiac fibrosis, heart failure, or right ventricular failure, and the method according to Embodiment 3.

[0288] Embodiment 6: The method according to Embodiment 5, wherein the disease is cardiac fibrosis.

[0289] Embodiment 7: The method according to Embodiment 5, wherein the disease is heart failure.

[0290] Embodiment 8: The method according to Embodiment 5, wherein the disease is right ventricular failure.

[0291] Embodiment 9: The method according to Embodiment 3, wherein the disease is a heart disease.

[0292] Embodiment 10: The method according to Embodiment 3, wherein the disease is a lung disease.

[0293] Embodiment 11: The method according to any one of the preceding embodiments, wherein the integrin α5β1 inhibitor is a 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-binding Fab (s-Fab), an antibody, or a combination thereof.

[0294] Embodiment 12: The method according to any one of the preceding embodiments, wherein the integrin α5β1 inhibitor is an antibody.

[0295] 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.

[0296] Embodiment 14: The method according to any one of Embodiments 1 to 12, wherein the integrin α5β1 inhibitor is an antibody that specifically binds to integrin β1.

[0297] 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.

[0298] Embodiment 16: The method according to Embodiment 15, wherein the antibody is an integrin α5β1 antibody selected from the group consisting of volociximab (M200), PF-04605412, and MINT1526A.

[0299] Embodiment 17: The method according to Embodiment 16, wherein the antibody is volociximab (M200).

[0300] Embodiment 18: The method according to Embodiment 16, wherein the antibody is PF-04605412.

[0301] Embodiment 19: The method according to Embodiment 16, wherein the antibody is MINT1526A.

[0302] Embodiment 20: The method according to Embodiment 11, wherein the integrin α5β1 inhibitor is 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 and an integrin α5β1 antibody that antagonizes integrin binding.

[0303] Embodiment 21: The method according to any one of Embodiments 1 to 10, wherein the integrin α5β1 inhibitor is a small molecule compound that binds to integrin α5β1.

[0304] Embodiment 22: The method according to any one of Embodiments 1 to 10, wherein the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin α5.

[0305] Embodiment 23: The method according to any one of Embodiments 1 to 10, wherein the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin β1.

[0306] Embodiment 24: The method according to any one of Embodiments 1 to 10, wherein the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin α5β1.

[0307] Embodiment 25: The integrin α5β1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof,

Chemical formula

[0308] Embodiment 26: The method according to Embodiment 25, wherein R 2 is methyl or ethyl.

[0309] Embodiment 27: The method according to Embodiment 25 or 26, wherein R 3 is methyl, ethyl, isopropyl, or cyclopropyl.

[0310] Embodiment 28: R 1 The method according to any one of Embodiments 25 to 27, wherein R is hydrogen.

[0311] Embodiment 29: The compound of formula (I) is

Chem.

Chem.

Chem.

Chem.

[0312] Embodiment 30: The method according to any one of the preceding embodiments, wherein the integrin α5β1 inhibitor is administered orally, intravenously, subcutaneously, intranasally, transdermally, intraperitoneally, intramuscularly, or intraperitoneally.

[0313] Embodiment 31: The method according to any one of the preceding embodiments, further comprising administering a second therapy to the subject.

[0314] Embodiment 32: The method according to any one 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, prostanoids, prostanoid receptor agonists, soluble guanylate cyclase stimulators, and / or surgery.

[0315] Embodiment 33: The method according to Embodiment 32, wherein the second therapy is a prostanoid.

[0316] Embodiment 34: The method according to Embodiment 33, wherein the prostanoid is epoprostenol or treprostinil.

[0317] Embodiment 35: The method according to embodiment 32, wherein the second therapy is an endothelin receptor antagonist.

[0318] Embodiment 36: The method according to embodiment 35, wherein the endothelin receptor is bosentan, ambrisentan, macitentan.

[0319] Embodiment 37: The method according to embodiment 32, wherein the second therapy is a phosphodiesterase type 5 (PDE-5) inhibitor.

[0320] Embodiment 38: The method according to embodiment 37, wherein the phosphodiesterase type 5 (PDE-5) inhibitor is sildenafil or tadalafil.

[0321] Embodiment 39: The method according to embodiment 32, wherein the second therapy is a soluble guanylate cyclase (sGC) stimulator.

[0322] Embodiment 40: The method according to embodiment 39, wherein the soluble guanylate cyclase (sGC) stimulator is riociguat.

[0323] Embodiment 41: The method according to embodiment 31 or 32, wherein the second 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 thromboendarterectomy, and / or atrial septostomy.

[0324] Embodiment 42: The method according to embodiment 31, wherein the second therapy is macitentan and / or tadalafil.

[0325] Embodiment 43: The method according to any one of the preceding embodiments, wherein the subject has previously received treatment for pulmonary hypertension.

[0326] Embodiment 44: The method according to embodiment 43, wherein the previously received treatment for pulmonary hypertension is selected from the group consisting of anticoagulants, diuretics, digitalis glycosides, calcium channel blockers, endothelin receptor antagonists, phosphodiesterase 5 (PDE5) inhibitors, prostaglandins, prostanoid receptor agonists, soluble guanylate cyclase stimulators, and / or surgery.

[0327] Embodiment 45: The method according to any one of the preceding embodiments, wherein administering an integrin α5β1 inhibitor reduces the proliferation and / or survival of PASMCs, fibroblasts, right ventricular fibroblasts (RVFbs), vascular fibroblasts, exogenous fibroblasts, cardiomyocytes, and / or endothelial cells.

[0328] Embodiment 46: The method according to any one of the preceding embodiments, wherein administering an integrin α5β1 inhibitor results in an improvement in mean pulmonary artery pressure (mPAP).

[0329] Embodiment 47: The method according to any one of the preceding embodiments, wherein administering an integrin α5β1 inhibitor results in an improvement in pulmonary vascular resistance (PVR).

[0330] Embodiment 48: The method according to any one of the preceding embodiments, wherein administering an integrin α5β1 inhibitor results in an improvement in systemic vascular resistance (SVR).

[0331] Embodiment 49: The method according to any one of the preceding embodiments, wherein administering an integrin α5β1 inhibitor results in an improvement in right atrial pressure (RAP).

[0332] Embodiment 50: The method according to any one of the preceding embodiments, wherein administering an integrin α5β1 inhibitor results in an improvement in cardiac output (CO).

[0333] Embodiment 51: The method according to any one of the preceding embodiments, wherein administering an integrin α5β1 inhibitor results in an improvement in heart rate (HR).

[0334] Embodiment 52: The method according to any one of the preceding embodiments, wherein an α5β1 inhibitor is administered to improve motor ability and delay the clinical worsening of the disease.

[0335] Embodiment 53: The method according to any one of the preceding embodiments, wherein an α5β1 inhibitor and a second therapy are administered to improve motor ability and delay the clinical worsening of the disease.

[0336] Embodiment 54: The method according to any one of the preceding embodiments, comprising administering an integrin α5β1 inhibitor to regulate the level of a biomarker in a subject, wherein the biomarker is the N-terminal fragment (NT) of brain natriuretic peptide (BNP) or pro-BNP (NT-proBNP), TNFα, IFNγ, IL-6, IL-8, or IL-10.

[0337] Embodiment 55: The method according to any one of the preceding embodiments, further comprising administering the integrin α5β1 inhibitor at a dose of 1 mg to 1000 mg.

[0338] Embodiment 56: The method according to any one of the preceding embodiments, wherein the integrin α5β1 inhibitor is administered daily.

[0339] Embodiment 57: An integrin α5β1 inhibitor for use in the treatment of pulmonary arterial hypertension (PAH) in a subject in need of treatment, comprising administering the integrin α5β1 inhibitor and a pharmaceutical excipient to the subject.

[0340] Equivalents and ranges One of ordinary skill 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 as set forth in the following claims.

Claims

1. A pharmaceutical composition for use in treating a cardiac or pulmonary disease in a subject, the pharmaceutical composition comprising an integrin α5β1 inhibitor.

2. A pharmaceutical composition used to treat pulmonary arterial hypertension (PAH) in a subject, the pharmaceutical composition comprising an integrin α5β1 inhibitor.

3. 2. The pharmaceutical composition of claim 1, 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, or WHO Class IV pulmonary hypertension.

4. 2. The pharmaceutical composition of claim 1, wherein the disease is cardiac fibrosis, heart failure, or right ventricular failure.

5. 3. The pharmaceutical composition of claim 1 or 2, 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-binding Fab (s-Fab), an antibody, or a combination thereof.

6. The pharmaceutical composition 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 pharmaceutical composition 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 pharmaceutical composition of claim 1 or 2, wherein the integrin α5β1 inhibitor is a small molecule compound that binds to integrin α5β1.

9. The integrin α5β1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 During the ceremony, R １ is hydrogen or OMe, R ２ But C １－４ C optionally substituted with alkoxy １－４ is alkyl, R ３ But C １－４ Alkyl or C ３－５ The pharmaceutical composition of claim 8, wherein the aryl group is cycloalkyl.

10. R ２ The pharmaceutical composition of claim 9, wherein is methyl or ethyl.

11. R ３ 10. The pharmaceutical composition of claim 9, wherein is methyl, ethyl, isopropyl, or cyclopropyl.

12. The compound of formula (I) 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 The pharmaceutical composition of claim 9, wherein the compound is selected from the group consisting of:

13. The pharmaceutical composition described in claim 1 or 2, wherein the integrin α5β1 inhibitor is administered orally, intravenously, subcutaneously, intranasally, transdermally, intraperitoneally, intramuscularly, or intraperitoneally.

14. The pharmaceutical composition described in claim 1 or 2, wherein the pharmaceutical composition is intended to be administered to the subject in combination with a second therapy.

15. 15. The pharmaceutical composition 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, prostanoids, prostanoid receptor agonists, soluble guanylate cyclase stimulators, and / or surgery.

16. 16. The pharmaceutical composition of claim 15, wherein the second therapy is selected from the group consisting of oxygen, warfarin, furosemide, bumetanide, bendroflumethiazide, metolazone, spironolactone, amiloride, digoxin, nifedipine, diltiazem, nicardipine, amlodipine, ambrisentan, bosentan, macitentan, sildenafil, tadalafil, epolostenol, iloprost, treprostinil, riociguat, selexipag, surgery, pulmonary thromboendarterectomy, and / or atrial septostomy.

17. The pharmaceutical composition described in claim 1 or 2, wherein administration of the integrin α5β1 inhibitor can reduce the proliferation and / or survival of PASMCs, fibroblasts, right ventricular fibroblasts (RVFb), vascular fibroblasts, exogenous fibroblasts, cardiomyocytes, and / or endothelial cells.

18. The pharmaceutical composition of claim 1 or 2, wherein the integrin α5β1 inhibitor is administered to regulate the level of a biomarker in the subject, and the biomarker is brain natriuretic peptide (BNP) or the N-terminal fragment (NT) of pro-BNP (NT-proBNP), TNFα, IFNγ, IL-6, IL-8 or IL-10.

19. The pharmaceutical composition described in claim 1 or 2, wherein the integrin α5β1 inhibitor is administered at a dose of 1 mg to 1000 mg.

20. The pharmaceutical composition of claim 1 or 2, further comprising a pharmaceutical excipient.

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