WO2018218273A1 - Method of treating hypertension and kidney disease - Google Patents

Abstract

The present disclosure provides a method of treating hypertension in a subject suffering from or at risk of developing type 1 diabetes, the method comprising administering to the subject an agent that down-regulates the functional level of activin or up-regulates the functional level of follistatin. The present disclosure also provides a method of treating glomerular hyperfiltration in a subject, the method comprising administering to the subject an agent that down-regulates the functional level of activin or up-regulates the functional level of follistatin.

Description

METHOD OF TREATING HYPERTENSION AND KIDNEY DISEASE

RELATED APPLICATION DATA

The present application claims priority from Australian Patent Application No. 2017902023 entitled "Method of treating hypertension and kidney disease" filed on 29 May 2017, the entire contents of which are hereby incorporated by reference.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

FIELD

The present disclosure relates to methods for treating or preventing hypertension and/or glomerular hyperfiltration in a subject.

BACKGROUND

Glomerular hyperfiltration is a characteristic early feature of diabetic kidney disease defined by an increase in the glomerular filtration rate (GFR). Glomerular hyperfiltration also occurs in various other kidney diseases, in which the increase in GFR may be present at the level of single nephrons and associated with an overall normal or reduced total GFR (as may occur when some kidney scarring is already present). Usually associated with intraglomerular hypertension, glomerular hyperfiltration is thought to contribute to the progression of chronic kidney disease, leading to albuminuria (loss of excess amounts of albumin in the urine) and glomerular scarring.

Hypertension, or high blood pressure, is a long term medical condition in which the blood pressure is persistently elevated. Long term high blood pressure is a major risk factor for coronary artery disease, stroke, heart failure, peripheral vascular disease, vision loss and chronic kidney disease.

A reduction in blood pressure by at least 5mmHg has been shown to reduce the risk of stroke by up to 34% and ischaemic heart disease by 21%, as well as the likelihood of dementia, heart failure and mortality from cardiovascular disease. The first line of treatment for hypertension is lifestyle changes. These changes include maintaining normal body weight, reducing dietary sodium intake, engaging in regular aerobic physical activity, limiting alcohol consumption and increasing consumption of fruit and vegetables. Anti-hypertensive medications are a class of drugs that are used to treat hypertension when lifestyle modification is insufficient. Common therapies include thiazide-diuretics, calcium channel blockers, angiotensin converting enzyme inhibitors and angiotensin receptor blockers as common first line medications. However, resistant hypertension can persist in some patients despite treatment with three antihypertensive medications belonging to different drug classes. Low adherence to treatment is an important cause of resistant hypertension.

It will be apparent to the skilled person from the foregoing that there is a need in the art to develop therapeutics for treating hypertension and/or glomerular hyperfiltration.

SUMMARY

In producing the present invention, the inventor studied the effects of attenuating members of the TGFP signalling pathway in an accepted mouse model of type 1 diabetes (e.g., Akita mouse model). The inventor studied the effect of modulating the functional levels of activin and follistatin by enhancing functional levels of follistatin (e.g., by administration of a follistatin protein). The present inventor found that, surprisingly, down-regulation of the functional level of activin or up-regulation of the functional level of follistatin has a beneficial effect in the animal model of type 1 diabetes. For example, the inventor was able to treat hypertension associated with type 1 diabetes as well as treat glomerular hyperfiltration.

The inventor found that modulating the functional levels of activin and/or follistatin decreases or prevents mesangial extracellular matrix deposition, abnormal thickening of the glomerular basement membrane and albuminuria.

The findings by the inventor provide the basis for methods for treating hypertension in a subject suffering from or at risk of developing type 1 diabetes by antagonising activin activity or enhancing follistatin activity in a subject.

For example, the present disclosure provides a method for treating hypertension in a subject suffering from or at risk of developing type 1 diabetes by administration of an agent that down-regulates the functional level of activin or an agent that up-regulates the functional level of follistatin.

The findings by the inventor also provide the basis for methods for treating glomerular hyperfiltration in a subject by administration of an agent that down- regulates the functional level of activin or an agent that up-regulates the functional level of follistatin. For example, the present disclosure provides a method of treating hypertension in a subject suffering from or at risk of developing type 1 diabetes, the method comprising administering to the subject an agent that down-regulates the functional level of activin or up-regulates the functional level of follistatin. In one example, the subject is suffering from glomerular hyperfiltration.

The present disclosure also provides a method of treating glomerular hyperfiltration in a subject, the method comprising administering to the subject an agent that down-regulates the functional level of activin or up-regulates the functional level of follistatin.

In one example, the subject is suffering from or at risk of developing hypertension and/or type 1 diabetes.

In one example, the subject is suffering from hypertension.

In one example, the subject is at risk of developing hypertension. For example, the subject is pre-hypertensive or suffering from pre-hypertension.

In one example, the subject is suffering from type 1 diabetes.

In one example, the subject is at risk of developing type 1 diabetes. For example, the subject suffers from a symptom associated with type 1 diabetes.

In one example of the present disclosure, the subject is suffering from or at risk of developing microalbuminuria, macroalbuminuria, diabetic nephropathy, hypertensive kidney disease, polycystic kidney disease, diabetic kidney disease, chronic kidney disease associated with obesity and/or chronic kidney disease associated with renal mass reduction. In one example, the glomerulosclerosis is related to increased glomerular pressure and hyperfiltration at either the single nephron and/or the whole kidney level.

In one example, the subject is suffering from or at risk of developing glomerulosclerosis. For example, the glomerulosclerosis is secondary focal glomerulosclosis (i.e., chronic kidney disease secondary to a reduction in renal mass which may occur after loss of functional kidney mass from any insult).

In one example, the subject is suffering from or at risk of developing albuminuria. For example, the albuminuria is microalbuminuria. In another example, the albuminuria is macroalbuminuria.

In one example, the subject is suffering from or at risk of developing kidney disease. For example, the kidney disease is hypertensive kidney disease, polycystic kidney disease, diabetic kidney disease and/or chronic kidney disease associated with obesity. In one example, the agent is administered in an amount effective to have one or more of the following effects:

a) reduce hypertension;

b) reduce or prevent glomerular sclerosis;

c) reduce or prevent microalbuminuria or macroalbuminuria;

d) reduce or prevent diabetic nephropathy;

e) reduce or prevent mesangial extracellular matrix deposition and/or abnormal thickening of the glomerular basement membrane;

f) reduce or prevent glomerular collagen deposits;

g) reduce or prevent glomerular mesangial expansion; and/or

h) normalize glomerular filtration rate.

In one example, the hypertension is intra-glomerular hypertension.

In one example, the agent that down-regulates the functional level of activin or up-regulates the functional level of foUistatin is foUistatin or a functional fragment thereof, or an inhibitor of activin.

In one example, the agent is foUistatin or a functional fragment thereof. For example, the foUistatin is FS315 or FS288.

In one example, the agent is an inhibitor of activin. For example, the inhibitor of activin is selected from the group consisting of:

a) an activin antagonist selected from the group consisting of inhibin, an activin βο subunit, an a subunit of inhibin, an antibody directed to activin, a nonfunctional activin mutant, a non-functional activin receptor mutant, a modified activin pro-domain and a soluble activin receptor; or

b) a non-pro teinaceous molecule selected from a group consisting of an activin antisense oligonucleotide, a short hairpin RNA (shRNA), a siRNA, an interfering RNA (RNAi), a ribozyme, a microRNA, a microRNA adapted shRNA (shRNAmir) and a DNAzyme which downregulates the transcription or translation of the activin gene.

In one example, the activin is activin A or activin B. For example, the activin is activin A (i.e., βΑ - βΑ). In another example, the activin is activin B (i.e., βΒ - βΒ).

In one example, the functional level of foUistatin is upregulated by increasing the transcription or translation of foUistatin.

In one example, the agent is administered systemically. In one example, the agent is administered parenterally, such as subcutaneously or intravenously.

In one example, the subject is a mammal, for example a primate, such as a human. Methods of treatment described herein can additionally comprise administering a further compound to treat or prevent (or delay progression of the complications of) type 1 diabetes. Exemplary compounds are described herein.

The present disclosure also provides for use of an agent in the manufacture of a medicament for treating hypertension in a subject suffering from or at risk of developing type 1 diabetes, wherein the agent down-regulates the functional level of activin or up-regulates the functional level of foUistatin.

The present disclosure further provides for use of an agent in the manufacture of a medicament for treating glomerular hyperfiltration or glomerular hypertension in a subject, wherein the agent down-regulates the functional level of activin or up-regulates the functional level of foUistatin.

The present disclosure also provides a kit comprising an agent that down- regulates the functional level of activin or up-regulates the functional level of foUistatin packaged with instructions for use in the treatment of hypertension in a subject suffering from or at risk of developing type 1 diabetes.

The present disclosure also provides a kit comprising an agent that down- regulates the functional level of activin or up-regulates the functional level of foUistatin packaged with instructions for use in the treatment of glomerular hyperfiltration.

Exemplary agents are described herein and are to be taken to apply mutatis mutandis to the examples of the disclosure set out in the previous four paragraphs.

KEY TO SEQUENCE LISTING

SEQ ID NO: 1 is an amino acid sequence of human foUistatin.

SEQ ID NO: 2 is an amino acid sequence of FST315.

SEQ ID NO: 3 is an amino acid sequence of FST288.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 comprises graphical representations showing body mass composition of Akita diabetic mice (DM) compared to wild-type (WT) controls. (A) Adiposity, (B) lean mass and (C) lean mass to adiposity ratio.

Figure 2 comprises graphical representations showing markers of kidney disease. (A) Glomerular filtration rate (GFR) and (B) Albuminuria in Akita diabetic mice (DM) and WT controls, untreated or treated with foUistatin at low and high doses

(FST LD and FST HD).

Figure 3 comprises graphical representations showing histological assessment of diabetic kidneys. (A) Quantitative analysis of glomerular basement membrane thickness. (B) Quantitative analysis of collagen deposition (as assessed by PSR staining).

Figure 4 comprises graphical representations showing (A) systolic and (B) diastolic blood pressure measurements in WT and Akita mice (DM) 4, 8 and 12 weeks after treatment with follistatin at low and high doses (FST LD and FST HD).

Figure 5 comprises graphical representations showing effects of activin A or follistatin on vascular contractility. Mouse aortic rings cultured in activin A (AA) or follistatin (FST) for 24h and tested for their contractility in response to phenylephrine (A) or relaxation in response to carbachol (B). The graphs display results from three independent experiments. The effects of short-term incubation with activin A (AA) on contraction (C) and relaxation (D) of resistance mesenteric vessels obtained from normal rats (WKY) were tested.

Figure 6 comprises graphical representations showing effects of follistatin administration on vascular reactivity in a 5/6 nephrectomy model of impaired renal function and hypertension. Mouse aortic rings were removed from animals that had undergone 5/6 nephrectomy or sham operations and were treated with either vehicle or FST over 12 weeks and tested for their relaxation in response to carbachol (A) or contractility in response to phenylephrine (B). DETAILED DESCRIPTION

General

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure. Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

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

Unless otherwise indicated, the techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, JUL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

Any discussion of a protein or antibody herein will be understood to include any variants of the protein or antibody produced during manufacturing and/or storage. For example, during manufacturing or storage a protein can be deamidated (e.g., at an asparagine or a glutamine residue) and/or have altered glycosylation and/or have a glutamine residue converted to pyroglutamate and/or have a N-terminal or C-terminal residue removed or "clipped" and/or have part or all of a signal sequence incompletely processed and, as a consequence, remain at the terminus of the protein. It is understood that a composition comprising a particular amino acid sequence may be a heterogeneous mixture of the stated or encoded sequence and/or variants of that stated or encoded sequence.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and

Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. As used herein, the term "derived from" shall be taken to indicate that a specified integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.

Further, as used herein the singular forms of "a", "and" and "the" include plural referents unless the context clearly dictates otherwise.

Selected Definitions

"FoUistatin" is a glycoprotein that primarily functions to bind and neutralize some members of the TGF-β superfamily. For the purposes of nomenclature only and not limitation exemplary sequences of human foUistatin are set out in NCBI Reference Sequence AAH04107 and in SEQ ID NO: 1. It should also be understood that the term "foUistatin" includes any isoform (including FST288, FST300 and FST315) which may arise from alternative splicing of foUistatin mRNA or mutant or polymorphic forms of foUistatin. It should still further be understood to extend to any protein encoded by the foUistatin gene, any subunit polypeptide, such as precursor forms which may be generated, and any foUistatin protein, whether existing as a monomer, multimer or fusion protein. FoUistatin 288 (FST288) and FST315 are two major isoforms arising from the alternatively spliced mRNAs. The term "FST315" refers to 315 amino acid mature foUistatin and is the most abundant and the sole form found in plasma. For the purposes of nomenclature only and not limitation exemplary sequences of human FST315 are set out in NCBI Reference AAA35851 and in SEQ ID NO: 2. The term "FST288" refers to the 288 amino acid length foUistatin. For the purposes of nomenclature only and not limitation exemplary sequences of FST288 are set out in NCBI Reference ALC04452 and in SEQ ID NO: 3. Additional sequences of foUistatin can be determined using sequences provided herein and/or in publically available databases and/or determined using standard techniques (e.g., as described in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989 Reference to a "functional fragment" of foUistatin should be understood as a reference to a fragment of foUistatin which exhibits foUistatin functionality.

As used herein, the term "activin" refers to a homodimer or heterodimer of inhibin β subunits, which may be βΑ or βΒ subunits. Accordingly, the term as used herein encompasses activin A (βΑ - βΑ), activin AB (βΑ - βΒ) and activin B (βΒ - βΒ).

The term "activin A" refers to a dimeric protein which comprises two activin βΑ subunits (i.e., βΑ - βΑ). The term "activin βΑ subunit" is also interchangeably referred to as "activin βΑ". It should also be understood that the term "activin A" includes reference to a dimer comprising any isoforms which may arise from alternative splicing of activin βΑ mRNA or mutant or polymorphic forms of activin βΑ including all precursor, proprotein or intermediate forms thereof. Reference to activin A should also be understood to extend to any activin A protein, whether existing as a dimer, multimer or fusion protein.

The term "activin B" refers to a homodimer of activin comprising two activin βΒ subunits (i.e., βΒ - βΒ). The term "activin βΒ subunit" is also interchangeably referred to as "activin βΒ". It should be understood to include reference to any isoforms which may arise from alternative splicing of activin βΒ mRNA or mutant or polymorphic forms of activin βΒ. Reference to "activin βΒ" is not intended to be limiting and should be read as including reference to all forms of activin βΒ including any protein encoded by the activin βΒ subunit gene, any subunit polypeptide such as precursor forms which may be generated, and any βΒ protein, whether existing as a monomer, multimer or fusion protein. Multimeric protein forms of activin βΒ include for example the homodimeric activin B (βΒ - βΒ) or the heterodimeric activin AB (βΑ - βΒ), activin BC (βΒ - βC), activin BD (βΒ - βϋ) or activin BE (βΒ - βΕ) proteins.

The term "down-regulating" or "down-regulate(s)" should be understood to mean preventing, reducing or otherwise inhibiting one or more aspects of the functional level of activin. In the context of the present disclosure, this term should be understood to mean down-regulation of the transcription or translation of activin by regulation of the nucleic acid or protein.

The term "up-regulating" or "up-regulate(s)" as used herein should be understood to mean increasing, enhancing or promoting one or more aspects of the functional level of follistatin. In the context of the present disclosure, this term should be understood to mean up-regulation of the transcription or translation of follistatin by regulation of the nucleic acid or protein.

As used herein, the term "functional level" refers to the level of functionality of the protein. This will most often be assessed by reference to the absolute level in the subject. In some instances, the absolute levels may change only marginally but the functionality is significantly altered.

"Hypertension" refers to a subject (e.g., a human subject) having a systolic pressure of 140 mm Hg or higher and/or a diastolic pressure of 90 mm Hg or higher. In some examples of a method or use described herein, a subject is pre -hypertensive, e.g., having a systolic pressure of about 120-139 mm Hg or higher and/or a diastolic pressure of 80-89 mm Hg or higher. The term "normal blood pressure" or "normalised blood pressure" refers to a human resting blood pressure of approximately 120 mmHg systolic, and 80 mmHg diastolic, abbreviated " 120/80 mmHg".

As used herein, the term "intra-glomerular hypertension" refers to the sustained elevated increase in pressure within the glomerular capillaries within the renal corpuscle of the kidney.

The term "glomerular hyperfiltration" as used herein refers to an absolute increase in the production of pro-urine by the glomeruli of the kidney or an increase in the glomerular filtration rate (abbreviated to GFR). Generally, a "normal" human GFR is considered to be in the range of about 90 to 120 mL/min/1.73m , a reduced GFR is below 90 mL/min/1.73m 2 and a GFR of between 125 and 175 mL/min/1.73m 2 is considered hyperfiltration. The term "normalize" in the context of the present invention refers to reducing or decreasing the glomerular filtration rate towards a normal GFR.

The term "nephropathy" shall be understood to mean damage to or disease of a kidney. This term encompasses all clinical-pathological changes in the kidney which may result in kidney fibrosis and/or glomerular diseases (e.g. glomerulosclerosis, glomerulonephritis) and/or chronic renal insufficiency, and can cause end stage renal disease and/or renal failure. Exemplary nephropathies include hypertensive nephropathy, diabetic nephropathy, and other types of nephropathy such as analgesic nephropathy, immune-mediated glomerulopathies (e.g., IgA nephropathy or Berger's disease, lupus nephritis), ischemic nephropathy, HIV-associated nephropathy, membranous nephropathy, glomerulonephritis, glomerulosclerosis, radiocontrast media-induced nephropathy, toxic nephropathy, analgesic-induced nephrotoxicity, cisplatin nephropathy, transplant nephropathy, and other forms of glomerular abnormality or injury; glomerular capillary injury (tubular fibrosis). In some examples, the terms "nephropathy" or "nephropathies" refers specifically to a disorder or disease where there is either the presence of proteins (i.e., proteinuria), such as albumin, in the urine of a subject and/or the presence of renal insufficiency. Nephropathy is often diagnosed based on the presence of albumin in the urine (microalbuminuria or macroalbuminuria), increased blood urea nitrogen levels (e.g., levels above 20mg/dL) and/or increased serum creatinine levels (e.g., levels above 1.3mg/dL for males and l.lmg/dL for females).

"Diabetic nephropathy" is a clinically well-defined pathology characterized by proteinuria, hypertension, edema and renal insufficiency. Characteristic aspects of diabetic nephropathy include glomerulosclerosis, modification of the vascular structure, and tubulointerstitial disease. The first clinical evidence of diabetic nephropathy is often the presence of albuminuria in the urine, e.g. microalbuminuria or macroalbuminuria. Diabetic nephropathy is typically characterized by the following: 1) glomerulosclerosis, 2) modification of the vascular structure, mainly in the small arterioles and 3) tubulointerstitial disease. The most characteristic aspect of diabetic nephropathy is the glomerular injury, detectable by the enlargement of the mesangium and by the thickening of the basement membrane, which when advanced leads to diffuse scarring of the whole glomerulus. The first clinical evidence of diabetic nephropathy is the presence of albuminuria or proteinuria.

The term "diabetic kidney disease" is used herein to refer to any damage or disease caused by or associated with diabetes that reduces the function of the kidneys in removing waste products and excess fluid from the body.

By "microalbuminuria" is meant the presence of 30-300mg albumin per 24 hours of urine collection and/or 30-300mg/L albumin in a single sample. Generally, both of the foregoing should be measured in at least two of three samples over a two to three month period. Microalbuminuria can also be defined by a ratio of albumin to creatinine (ACR) of >3.5mg/mmol for females or >2.5 mg/mmol for males or between 30-300μ albumin/mg creatinine. Albumin levels can be assessed using, for example, commercially available dipsticks (e.g., comprising bromophenol blue as an indicator).

The term "macroalbuminuria" means the presence of amounts of albumin higher (or higher ACR) than is observed in microalbuminuria.

The term "proteinuria" means the amount of total protein in urine is about >30mg/dL or a protein/creatinine ratio greater than 45 mg/mmol.

The term "fibrosis" refers to abnormal accumulation of fibrous tissue. Fibrosis can result from various injuries or diseases. Fibrosis typically involves the abnormal production, accumulation, or deposition of extracellular matrix components, including overproduction and increased deposition of, for example, collagen and fibronectin. As used herein, the terms "kidney fibrosis" or "renal fibrosis" or "fibrosis of the kidney" refer to diseases or disorders associated with the overproduction or abnormal deposition of extracellular matrix components, particularly collagen, leading to the degradation or impairment of kidney function.

The term "nephritis" will be understood to mean inflammation of a kidney. In the context of the present disclosure, nephritis encompasses a subset of nephropathy characterized by inflammation in a kidney. The inflammation can involve glomeruli, tubules, or interstitial tissue surrounding the glomeruli and tubules. Generally, nephritis is either glomerulonephritis (i.e., inflammation of the glomeruli) or interstitial nephritis (i.e., inflammation of the interstitial spaces between renal tubules). The term "glomerulonephritis" encompasses a class of kidney diseases, which can be broken into sub-class of proliferative diseases and non-proliferative diseases. As the names suggest, "proliferative" diseases include forms of glomerulonephritis in which there is a significant increase in the number of cells in the glomerulus, while "non-proliferative" diseases include forms of glomerulonephritis in which such an increase in cell numbers is not present. Exemplary proliferative diseases include IgA nephropathy, post-infectious glomerulonephritis, membranoproliferative glomerulonephritis and rapidly progressive glomerulonephritis. Exemplary nonproliferative diseases include minimal change disease, focal segment glomerulosclerosis think basement membrane disease and membranous glomerulonephritis .

As used herein, the term "treat" or "treatment" or "treating" shall be understood to mean administering a therapeutically effective amount of an agent that downregulates the functional level of activin or upregulates the functional level of follistatin and reducing or inhibiting hypertension or glomerular hyperfiltration such that the subject is no longer clinically diagnosed with the condition.

As used herein, the term "prevent" or "preventing" or "prevention" shall be taken to mean administering a prophylactically effective amount of an agent that downregulates the functional level of activin or upregulates the functional level of follistatin and stopping or hindering or delaying the development or progression of glomerular hyperfiltration.

As used herein, the term "therapeutically effective amount" shall be taken to mean a sufficient quantity of an agent that downregulates the functional level of activin or upregulates the functional level of follistatin to treat hypertension and/or glomerular hyperfiltration, i.e., such that the subject no longer satisfies the clinical criteria for hypertension and/or glomerular hyperfiltration. For example, the blood pressure of a hypertensive subject is reduced to a point where the subject is no longer hypertensive (e.g., they may be normal).

As used herein, the term "prophylactically effective amount" shall be taken to mean a sufficient quantity of an agent that downregulates the functional level of activin or upregulates the functional level of follistatin to prevent or inhibit or delay the onset of glomerular hyperfiltration, e.g., preventing a subject from developing the clinical criteria for a diagnosis of glomerular hyperfiltration.

As used herein, a subject "at risk" of developing a disease or condition or relapse thereof or relapsing may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment according to the present disclosure. "At risk" denotes that a subject has one or more risk factors, which are measurable parameters that correlate with development of the disease or condition, as known in the art and/or described herein.

The term "recombinant" shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising an antibody variable region, this term does not encompass an antibody naturally-occurring within a subject's body that is the product of natural recombination that occurs during B cell maturation. However, if such an antibody is isolated, it is to be considered an isolated protein comprising an antibody variable region. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

The term "protein" shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

The term "polypeptide" or "polypeptide chain" will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

The skilled artisan will be aware that an "antibody" is generally considered to be a protein that comprises a variable region made up of a plurality of polypeptide chains, e.g., a polypeptide comprising a light chain variable region (V_L) and a polypeptide comprising a heavy chain variable region (V_H). An antibody also generally comprises constant domains, some of which can be arranged into a constant region, which includes a constant fragment or fragment crystallizable (Fc), in the case of a heavy chain. A V_H and a V_L interact to form a Fv comprising an antigen binding region that is capable of specifically binding to one or a few closely related antigens. Generally, a light chain from mammals is either a κ light chain or a λ light chain and a heavy chain from mammals is α, δ, ε, γ, or μ. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGi, IgG₂, IgG₃, IgG₄, IgAi and IgA₂) or subclass. The term "antibody" also encompasses humanized antibodies, primatized antibodies, human antibodies, synhumanized antibodies and chimeric antibodies. As used herein, "variable region" refers to the portions of the light and/or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). Exemplary variable regions comprise three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. In the case of a protein derived from an IgNAR, the protein may lack a CDR2. V_H refers to the variable region of the heavy chain. V_L refers to the variable region of the light chain.

As used herein, the term "complementarity determining regions" (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3. The amino acid positions assigned to CDRs and FRs can be defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 or other numbering systems in the performance of this disclosure, e.g., the canonical numbering system of Chothia and Lesk J. Mol Biol. 196: 901-917, 1987; Chothia et al. Nature 342, 877-883, 1989; and/or Al-Lazikani et al, J Mol Biol 273: 927-948, 1997; the IMGT numbering system of Lefranc et al., Devel. And Compar. Immunol., 27: 55-77, 2003; or the AHO numbering system of Honnegher and Pliikthun J. Mol. Biol, 309: 657-670, 2001.

"Framework regions" (FRs) are those variable domain residues other than the CDR residues.

As used herein, the term "binds" in reference to the interaction of a protein or an antigen binding site thereof with another protein or antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the protein or antigen. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds to epitope "A", the presence of a molecule containing epitope "A" (or free, unlabeled "A"), in a reaction containing labeled "A" and the protein, will reduce the amount of labeled "A" bound to the antibody.

As used herein, the term "specifically binds" or "binds specifically" shall be taken to mean that a protein of the disclosure reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with another protein, a particular antigen or cell expressing same than it does with alternative proteins, antigens or cells. For example, a protein binds to activin with materially greater affinity (e.g., 5 fold or 10 fold or 20 fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200 fold) than it does to other members of the TGF-β superfamily or to antigens commonly recognized by polyreactive natural antibodies (i.e., by naturally occurring antibodies known to bind a variety of antigens naturally found in humans). Generally, but not necessarily, reference to binding means specific binding, and each term shall be understood to provide explicit support for the other term.

As used herein, the term "inhibitor of activin" or "inhibits activin signalling" will be understood to mean an agent that inhibits activin signalling or specifically inhibits activin signalling and does not significantly or detectably inhibit signalling by one or more other structurally related proteins of the TGF-β superfamily, e.g., anti- Mullerian hormone, bone morphogenic proteins and growth differentiation factors.

As used herein, the term "subject" shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human. Treatment of Hypertension and/or Glomerular Hyperfiltration

The present disclosure provides methods for treating hypertension by administering an agent that down-regulates the functional level of activin or up- regulates the functional level of follistatin. In one example the subject suffers from glomerular hyperfiltration, e.g., as measured by enhanced creatinine clearance rate (e.g., above 125 mL/min/1.73m .). In one example, a method of the disclosure reduces glomerular filtration rate.

The present disclosure also provides methods for treating glomerular hyperfiltration by administering an agent that down-regulates the functional level of activin or up-regulates the functional level of follistatin.

In one example, the subject suffers from hypertension. In one example, a method of the disclosure is effective in lowering a subject's systolic and/or diastolic blood pressure by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mm Hg or more.

In one example, a method of the disclosure is effective in reducing the blood pressure of a hypertensive subject to normal blood pressure.

In one example, a method of the disclosure is effective in normalising blood pressure in a hypertensive subject.

In one example, the subject is suffering from or at risk of developing type 1 diabetes. For example, a subject suffering from type 1 diabetes has a clinically accepted marker of type 1 diabetes, such as:

· Fasting plasma glucose of greater than or equal to 7mmol/L or 126mg/dl. • Casual plasma glucose (taken at any time of the day) of greater than or equal to 1 l.lmmol/L or 200 mg/dl with the symptoms of diabetes.

• Oral glucose tolerance test (OGTT) value of greater than or equal to 1 l.lmmol/L or 200 mg/dl measured at a two-hour interval. The OGTT is given over a two or three-hour time span.

• Positive urine test for ketones.

• Positive test for islet cell autoantibodies.

In one example, the subject suffers from type 1 diabetes.

In one example, the subject is at risk of developing type 1 diabetes. For example, the subject is suffering from one or more symptoms of type 1 diabetes and/or has one or more known risk factors for type 1 diabetes.

Symptoms of type 1 diabetes will be apparent to the skilled person and include, for example:

• Increased thirst;

• Frequent urination;

• Bedwetting in children who previously didn't wet the bed during the night;

• Extreme hunger;

• Unintended weight loss;

• Irritability and other mood changes;

• Fatigue and weakness;

• Blurred vision; and/or

• In females, a vaginal yeast infection.

Risk factors for type 1 diabetes will be apparent to the skilled person and include, for example:

• Family history (e.g., parent or sibling with type 1 diabetes);

• Presence of islet cell autoantibodies in the blood;

• Presence of insulin autoantibodies in the blood; and/or

• Race (i.e., risk is increased in white subjects as compared to black, Asian or Hispanic individuals).

In one example, the subject suffers from diabetic nephropathy. For example, the subject suffers from nephropathy associated with type 1 diabetes.

In one example, the subject is at risk of developing diabetic nephropathy. For example, the subject is at risk of developing nephropathy associated with type 1 diabetes.

In one example, the subject suffers from diabetic kidney disease. For example, the subject suffers from diabetic kidney disease associated with type 1 diabetes. In another example, the subject is at risk of developing diabetic kidney disease. For example, the subject is at risk of developing diabetic kidney disease associated with type 1 diabetes.

In one example, the subject suffers from microalbuminuria. In accordance with this example, treatment according to the present disclosure may reduce the microalbuminuria (e.g., to less than about 30mg albumin per 24 hours of urine collection and/or 30mg/L albumin in a single sample and/or an ACR of less than 3.5mg/mmol for females or less than 2.5 mg/mmol for males or less than about 30μg albumin/mg creatinine).

In one example, the subject suffers from macroalbuminuria. For example, treatment according to the present disclosure may reduce the macroalbuminuria (e.g., to microalbuminuria or less).

In another example, treatment according to the present disclosure prevents or slows progression of microalbuminuria to macroalbuminuria.

In one example, the subject suffers from or is at risk of developing hypertensive kidney disease, diabetic kidney disease, chronic kidney disease secondary to a reduction in renal mass (secondary focal glomerulosclerosis), polycystic kidney disease and chronic kidney disease associated with obesity.

In one example, performing a method described herein according to any example of the disclosure results in enhancement of a clinical response and/or delayed disease progression.

By "clinical response" is meant an improvement in the symptoms of disease. The clinical response may be achieved within a certain time frame, for example, within or at about 8 weeks from the start of treatment with, or from the initial administration. Clinical response may also be sustained for a period of time, such as for >24 weeks, or >48 weeks.

Quantitative assessment of renal function and parameters of renal dysfunction are well known in the art and can be found, for example, in Levey (Am J Kidney Dis. 22(1):207-214, 1993). Examples of assays for the determination of renal function/dysfunction are: serum creatinine level; creatinine clearance rate; cystatin C clearance rate, 24-hour urinary creatinine clearance, 24-hour urinary protein secretion; Glomerular filtration rate (GFR); urinary albumin creatinine ratio (ACR); albumin excretion rate (AER); and renal biopsy. Agents that up-regulate the functional level of foUistatin

Methods of the present disclosure comprise administering an agent that up- regulates the functional level of foUistatin in the subject, to treat hypertension and/or to treat glomerular hyperfiltration in a subject.

In one example, the agent up-regulates the functional level of foUistatin to treat hypertension in a subject suffering from or at risk of developing type 1 diabetes.

In a further example, the agent up-regulates the functional level of foUistatin to treat glomerular hyperfiltration in a subject

Agents suitable for up-regulating the functional level of foUistatin are known in the art, or exemplified herein.

FoUistatin and Functional Fragments Thereof

In one example, the agent that up-regulates the level of foUistatin is foUistatin or functional fragment thereof.

The foUistatin or functional fragment thereof for use in the present disclosure is in the form of a protein, such as a recombinant or human protein.

Forms of foUistatin and functional fragments thereof suitable for use in the present disclosure will be apparent to the skilled person and include, for example:

(i) Wild-type foUistatin (FS), comprising an N-terminal domain (ND) followed by three foUistatin domains (FSDl, FSD2 and FSD3) with a heparin -binding sequence located in FSDl (amino acid sequence positions 72-86), and isoforms thereof (i.e., FS315 and FS288).

(ii) Wild-type follistatin-like 3 protein (FSTL3), which is also known as follistatin-related gene product (FLRG) and follistatin-related protein (FSRP), comprising an N-terminal domain (N3D) followed by two follistatin-like 3 domains (FS3D1 and FS3D2), and isoforms thereof.

(iii) FoUistatin analogue having the structure ND-FSD1-FSD2 (i.e. wild-type minus FSD3).

(iv) Analogues of (i) and (iii) above with FSDl substituted by FSDl ', where FSDl ' represents FSDl with heparin-binding site removed.

(v) Analogues of (i) and (iii) above with FSDl substituted by FSDl*, where FSDl* represents FSDl with sequence prior to and including the heparin-binding sequence removed.

(vi) Hybrid forms of (i) and (iii) above where at least one of the domains is substituted by a corresponding FSTL3 domain N3D, FS3D1 and FS3D2. (vii) Hybrid forms of (ii) above where at least one of the domains is substituted by a corresponding F S domain D, FSD1, FSD1 ', FSD1 * and F SD2.

(viii) Any of the above proteins modified by one or more deletions, insertions and/or mutations in ND, N3D, FSD1, FSD1 ', FSD1 *, FS3D1, FSD2, FS3D2, and FSD3.

(ix) Genetically modified or codon optimized forms of follistatin.

In one example, the follistatin is an isoform of wild-type follistatin. For example, the follistatin is FS315. In another example, the follistatin is FS288. Agents that down-regulate the functional level of activin

Methods of the present disclosure comprise administering an agent that down- regulates the functional level of activin in the subject, to treat hypertension and/or to treat glomerular hyperfiltration in a subject.

In one example, the agent down-regulates the functional level of activin to treat hypertension in a subject suffering from or at risk of developing type 1 diabetes.

In a further example, the agent down-regulates the functional level of activin to treat glomerular hyperfiltration in a subject

Agents suitable for down-regulating the functional level of activin are known in the art, or exemplified herein.

Activin Inhibitors

Activins bind to the cell surface transmembrane receptor Type II and initiate a cascade reaction that leads to the recruitment, phosphorylation and activation of the Type 1 activin receptor and ultimately phosphorylation of SMAD2 and SMAD3.

In one example of any method described herein, the agent down-regulates the functional level of activin. For example, the agent down-regulates the functional level of activin A. In another example, the agent down-regulates the functional level of activin B.

In one example, the agent that down-regulates the functional level of activin is an inhibitor of activin (i.e., an activin inhibitor).

Agents suitable for down-regulating the functional level of activin will be apparent to the skilled person, or are described herein and include, but are not limited to, inhibin, the activin βο subunit, the a subunit of inhibin, an antibody directed to activin, a non-functional activin mutant, a non-functional activin receptor mutant, a modified activin pro-domain and a soluble activin receptor. Inhibin

Inhibin binds to β-glycan and inhibits the actions of activin via its receptor. Inhibin plays a role in the downregulation of follicle stimulating hormone (FSH) synthesis and inhibits FSH secretion. Inhibin is a dimer wherein the first component is a beta subunit similar or identical to the beta subunit in activin. However, in contrast to activin, the second component of the inhibin dimer is a more distantly-related alpha subunit.

In one example, the activin antagonist for use in the present disclosure is any agent that upregulates the expression or functioning of the a subunit of inhibin. In one example, the activin antagonist is the a subunit of inhibin. For example, the a subunit can dimerise with the 3 subunits of activin to form inhibin, thereby effectively down- regulating the functional level of activin.

Activin mutants

In one example, the agent that down-regulates the functional level of activin is an activin mutant. For example, the activin mutant is a non -functional activin mutant.

In another example, the activin mutant is a modified activin pro-domain.

Activin mutants which inhibit native activin from binding to its receptor will be apparent to the skilled person and include for example mutants in the finger (M91E, I105E, M108A) and wrist (activin A/activin C chimera, S60P, I63P) regions of activin-

A (as described in Harrison et al, 2004 (J. Biol. Chem. 279:28036-28044).

Modified activin pro-domains will be apparent to the skilled person and are described in Chen, J. L., K. L. Walton, et al. (2015). "Development of novel activin- targeted therapeutics." Molecular therapy 23(3): 434-444.

Proteins Comprising Antibody Variable Regions

In one example, the agent that down-regulates the functional level of activin is an activin inhibitor comprising an antibody variable region, e.g., an antibody or an antibody fragment that binds to activin and neutralizes activin signalling.

In one example, the antibody variable region binds specifically to activin.

Suitable antibodies and proteins comprising variable regions thereof are known in the art. For example, anti-activin antibodies and fragments thereof are described in Poulaki et al, 2004 (Am. J. Pathol 164:1293-1302)

In one example, the anti-activin antibody or fragment thereof is an antibody that competitively binds to activin and inhibits the binding of activin to an activin receptor. For example, the anti-activin antibody or fragment thereof inhibits the binding of activin to an activin type 1 receptor (e.g., ACVR1, ACVR1B and/or ACVR1C). In another example, the anti-activin antibody or fragment thereof inhibits the binding of activin to an activin type 2 receptor (e.g., ACVR2A and/or ACVR2B).

In one example, the antibody or fragment thereof is an antibody that competitively binds to an activin receptor and inhibits the binding of activin to the activin receptor. For example, the antibody binds to ACVR2B. Suitable antibodies will be apparent to the skilled person and include, for example, bimagrumab (BYM338; Novartis). Soluble Activin Receptor

In one example, the agent that down-regulates the functional level of activin is a soluble activin receptor. For example, the soluble activin receptor acts as a competitive inhibitor "ligand trap".

In one example, the soluble activin receptor is a soluble activin type IIA receptor.

In another example, the soluble activin receptor is a soluble activin type IIA receptor fusion protein. For example, a humanized fusion protein comprising of the extracellular domain of activin receptor type IIA and the human IgGl Fc domain (sActRIIA-hFc). Suitable sActRIIA-hFc fusion proteins will be apparent to the skilled person and include, for example, Sotatercept (ACE-011; Acceleron Pharma Inc.).

In one example, the soluble activin receptor is a soluble activin type IIB receptor.

In another example, the soluble activin receptor is a soluble activin type IIB receptor fusion protein. For example, a humanized fusion protein comprising of the extracellular domain of activin receptor type IIB and the human IgGl Fc domain (sActRIIB-hFc). Suitable sActRIIB-hFc fusion proteins will be apparent to the skilled person and include, for example, ACE-031 (Acceleron), ACE-083 (Acceleron) and STM-434 (Atara Biotherapeutics). Nucleic Acid-Based Agents

In one example, the method of the present disclosure involves administration of a non-pro teinaceous (i.e., nucleic acid) molecule that reduces the functional level of activin. Suitable agents will be apparent to the skilled person and include, for example, an antisense oligonucleotide, a short hairpin RNA (shRNA), siRNA, an interfering RNA (RNAi), a ribozyme, a microRNA and a DNAzyme. Antisense Oligonucleotides

The agent of the present disclosure may be an antisense oligonucleotide or antisense nucleic acid.

The terms "antisense oligonucleotide" or "antisense nucleic acid" shall be taken to mean a DNA or RNA or derivative thereof (e.g., LNA or PNA), or combination thereof that is complementary to at least a portion of a specific mRNA molecule encoding a polypeptide as described herein in any example of the disclosure and capable of interfering with a post-transcriptional event such as mRNA translation. The use of antisense methods is known in the art (see for example, Hartmann and Endres (editors), Manual of Antisense Methodology, Kluwer (1999)).

An antisense nucleic acid of the disclosure will hybridize to a target nucleic acid under physiological conditions. Antisense nucleic acids include sequences that correspond to structural genes or coding regions or to sequences that effect control over gene expression or splicing. For example, the antisense nucleic acid may correspond to the targeted coding region of a nucleic acid, or the 5 '-untranslated region (UTR) or the 3'-UTR or combination of these. It may be complementary in part to intron sequences, which may be spliced out during or after transcription, for example only to exon sequences of the target gene. The length of the antisense sequence should be at least 19 contiguous nucleotides, for example, at least 50 nucleotides, such as at least 100, 200, 500 or 1000 nucleotides of a nucleic acid. The full-length sequence complementary to the entire gene transcript may be used. The length can be 100-2000 nucleotides. The degree of identity of the antisense sequence to the targeted transcript should be at least 90%, for example, 95-100%. Catalytic Nucleic Acid

The term "catalytic nucleic acid" refers to a DNA molecule or DNA-containing molecule (also known in the art as a "deoxyribozyme" or "DNAzyme") or a RNA or RNA-containing molecule (also known as a "ribozyme" or "RNAzyme") which specifically recognizes a distinct substrate and catalyzes the chemical modification of this substrate. The nucleic acid bases in the catalytic nucleic acid can be bases A, C, G, T (and U for RNA).

Typically, the catalytic nucleic acid contains an antisense sequence for specific recognition of a target nucleic acid, and a nucleic acid cleaving enzymatic activity (also referred to herein as the "catalytic domain"). The types of ribozymes that are useful in this disclosure are a hammerhead ribozyme and a hairpin ribozyme. RNA Interference

RNA interference (RNAi) is useful for specifically inhibiting the production of a particular protein. Without being limited by theory, this technology relies on the presence of dsRNA molecules that contain a sequence that is essentially identical to the mRNA of the gene of interest or part thereof. Conveniently, the dsRNA can be produced from a single promoter in a recombinant vector host cell, where the sense and anti-sense sequences are flanked by an unrelated sequence which enables the sense and anti-sense sequences to hybridize to form the dsRNA molecule with the unrelated sequence forming a loop structure. The design and production of suitable dsRNA molecules for the present disclosure is well within the capacity of a person skilled in the art.

The length of the sense and antisense sequences that hybridize should each be at least 19 contiguous nucleotides, such as at least 30 or 50 nucleotides, for example at least 100, 200, 500 or 1000 nucleotides. The full-length sequence corresponding to the entire gene transcript may be used. The lengths can be 100-2000 nucleotides. The degree of identity of the sense and antisense sequences to the targeted transcript should be at least 85%, for example, at least 90% such as, 95-100%.

Exemplary small interfering RNA ("siRNA") molecules comprise a nucleotide sequence that is identical to about 19-21 contiguous nucleotides of the target mRNA. For example, the siRNA sequence commences with the dinucleotide AA, comprises a GC-content of about 30-70% (for example, 30-60%, such as 40-60% for example about 45%-55%), and does not have a high percentage identity to any nucleotide sequence other than the target in the genome of the subject in which it is to be introduced, for example as determined by standard BLAST search.

Assessing Therapeutic Efficacy

Agents that down-regulate the functional level of activin or up-regulate the functional level of follistatin are assayed to assess the ability of the agent to treat hypertension and/or glomerular hyperfiltration as described herein.

Various in vivo and in vitro assays are available, or described herein. For example, an agent is assessed for its ability to reduce glomerular filtration rate, using a method described herein.

In vivo animal models

In one example, an animal model is used to assess therapeutic efficacy of the agent. Examples of animal models of type 1 diabetes include the Akita or Ins2 (Akita) mouse (monogenic model of type 1 diabetes), RIP-LCMV-GP (virus -induced) mouse, non-obese diabetic (NOD mouse), biobreeding (BB or BB-DP) rat, alloxan or stre tozotocin (STZ) treated mice.

In one example, the animal model of type 1 diabetes is treated with an agent for a period of time and the effect of the agent on fasting serum glucose levels, albumin-to- creatinine ratios, glomerular filtration rate and kidney morphology is assessed.

In one example, an animal model of renal impairment and/or hypertension is used to assess therapeutic efficacy of the agent. For example, animals are subjected to 5/6 nephrectomy and the effect of the agent on vascular contractility and reactivity is assessed.

Albumin-to-creatinine ratio

In one example, the therapeutic efficacy of an agent is determined by assessing the albumin-to-creatinine ratio.

For example, urine is collected following administration of the agent. Albumin- to-creatinine ratio is determined using commercially available kits (e.g., Albuwell M, Exocell kit for urine albumin and Crystal Chem for creatinine) according to manufacturer's instructions. An agent that decreases the albumin-to-creatinine ratio is considered suitable for use in the methods described herein.

Glomerular filtration rate

In one example, the therapeutic efficacy of an agent is determined by assessing glomerular filtration rate.

For example, glomerular filtration rate is assessed following administration of the agent by determining clearance of fluorescein isothiocyanate (FITC) -labeled sinistrin. A 5% FITC-sinistrin solution is injected into the subject, after which blood is collected. Plasma fluorescence is assessed using a fluorometer (Gemini EM, Molecular Devices) at 485 nm excitation and 538nm emission. An agent that normalizes the glomerular filtration rate is considered suitable for use in the methods described herein.

Blood pressure

In one example, the therapeutic efficacy of an agent is determined by assessing the effect on systolic and diastolic blood pressure.

For example, blood pressure is measured every 4 weeks by cuff volume pressure recording. An agent that decreases the systolic and/or diastolic blood pressure is considered suitable for use in the methods described herein. Vascular reactivity

In one example, the therapeutic efficacy of an agent is determined by assessing the effect on vascular contractility and relaxation.

For example, aortas or mesenteric arteries are dissected from the animal (e.g., mouse or rat), cut into ring segments and cultured medium supplemented with an agent (e.g., Activin A or FST) for 24h. Rings are then mounted on a small vessel wire style myograph (Radnoti LLC) in HBSS, maintained at 37°C and bubbled with 100% oxygen. Contractility is tested with cumulative doses of phenylephrine followed by carbachol. An agent that increases the contractile response to phenylephrine and/or attenuates the response to carbachol is considered suitable for use in the methods described herein.

Pharmaceutical Compositions and Methods of Treatment

An agent that down-regulates the functional level of activin or up-regulates the functional level of follistatin (syn. active ingredient) is useful for parenteral, topical, oral, or local administration, aerosol administration, or transdermal administration, for prophylactic or for therapeutic treatment. In one example, the agent is administered parenterally, such as subcutaneously or intravenously.

Formulation of an agent to be administered will vary according to the route of administration and formulation (e.g., solution, emulsion, capsule) selected. An appropriate pharmaceutical composition comprising an agent to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. A variety of appropriate aqueous carriers are known to the skilled artisan, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The agent can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.

The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.

The dosage ranges for the administration of the agent of the disclosure are those large enough to produce the desired effect. For example, the composition comprises a therapeutically or prophylactically effective amount of the agent.

As used herein, the term "effective amount" shall be taken to mean a sufficient quantity of the agent to down-regulate (i.e., inhibit/reduce) the functional level of activin or up-regulate (i.e., increase/enhance) the functional level of follistatin in a subject. The skilled artisan will be aware that such an amount will vary depending on, for example, the agent and/or the particular subject and/or the type and/or the severity of hypertension and/or glomerular hyperfiltration being treated. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, e.g., weight or number of agents.

As used herein, the term "therapeutically effective amount" shall be taken to mean a sufficient quantity of agent to reduce or inhibit one or more symptoms of hypertension and/or glomerular hyperfiltration.

As used herein, the term "prophylactically effective amount" shall be taken to mean a sufficient quantity of compound to prevent or inhibit or delay the onset of one or more detectable symptoms of hypertension and/or glomerular hyperfiltration.

In one example, the agent is administered in an amount effective to have one or more of the following effects:

· Reduce hypertension;

• Reduce or prevent glomerular sclerosis;

• Reduce or prevent diabetic nephropathy

• Reduce or prevent mesangial extracellular matrix deposition and/or abnormal thickening of the glomerular basement membrane;

· Reduce or prevent glomerular mesangial expansion;

• Reduce glomerular filtration rate

• Reduce or prevent glomerular collagen deposits; and/or

• Reduce or prevent microalbuminuria or macroalbuminuria.

The dosage should not be so large as to cause adverse side effects, such as hyper viscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.

A method of the present disclosure may also include co-administration of an agent according to the disclosure together with the administration of another therapeutically effective agent for the prevention or treatment of a renal disorder or complication, nephropathy (e.g. diabetic nephropathy), diabetes (e.g., type 1 diabetes) and/or obesity.

In one example, the agent of the disclosure is used in combination with at least one additional known compound which is currently being used or is in development for preventing or treating type 1 diabetes or type 1 diabetic kidney disease. For example, the agent is used in combination with insulin. Examples of insulin include, but are not limited to all naturally-occurring, synthetic and modified forms of insulin, such as insulin of human, bovine or porcine origin; insulin suspended in, for example, isophane or zinc and derivatives such as insulin glulisine, insulin lispro, insulin lispro protamine, insulin glargine, insulin detemir or insulin aspart.

In one example, the agent of the disclosure is used in combination with at least one additional known compound which is currently being used or in development for preventing or treating renal disorder such as nephropathy, or an associated disorder or complication. Examples of such known compounds include but are not limited to: ACE inhibitor drugs (e.g. captopril (Capoten™), enalapril (Innovace™), fosinopril (Staril™), lisinopril (Zestril™), perindopril (Coversyl™), quinapril (Accupro™), trandanalopril (Gopten™), lotensin, moexipril, ramipril); RAS blockers; angiotensin receptor blockers (ARBs) (e.g. Olmesartan, Irbesartan, Losartan, Valsartan, candesartan, eprosartan, telmisartan, etc); protein kinase C (PKC) inhibitors (e.g. ruboxistaurin); inhibitors of AGE-dependent pathways (e.g. aminoguanidine, ALT-946, pyrodoxamine (pyrododorin), OPB-9295, alagebrium); anti-inflammatory agents (e.g. clyclooxigenase-2 inhibitors, mycophenolate mophetil, mizoribine, pentoxifylline), GAGs (e.g. sulodexide (U.S. Pat. No. 5,496,807)); pyridoxamine (U.S. Pat. No. 7,030,146); endothelin antagonists (e.g. SPP 301), PPAR-gamma antagonists and other compounds like amifostine (used for cisplatin nephropathy), captopril (used for diabetic nephropathy), sodium thiosulfate (used for cisplatin nephropathy).

Additionally, the methods of the disclosure may also include co-administration of at least one other therapeutic agent for the treatment of another disease directly or indirectly related to type 1 diabetes and/or nephropathy, including but not limited to: dyslipidemia, hypertension, obesity, neuropathy, and/or retinopathy, etc. Additional examples of agents that can be co-administered with the compound(s) according to the invention are corticosteroids; immunosuppressive medications; antibiotics; antihypertensive and diuretic medications (such as ACE-inhibitors); lipid lowering agents such as bile sequestrant resins, cholestyramine, colestipol, nicotinic acid, and more particularly drugs and medications used to reduce cholesterol and triglycerides (e.g. fibrates (e.g. Gemfibrozil™) and HMG-CoA inhibitors such as Lovastatin™, Atorvastatin™, Fluvastatin™, Lescol™), Lipitor™, Mevacor™), Pravachol™, Pravastatin™, Simvastatin™, Zocor™, Cerivastatin™), etc); compounds that inhibit intestinal absorption of lipids (e.g. ezetiminde); nicotinic acid; and Vitamin D.

As will be apparent from the foregoing, the present disclosure provides methods of concomitant therapeutic treatment of a subject, comprising administering to a subject in need thereof an effective amount of a first agent and a second agent, wherein said first agent down-regulates the functional level of activin or up-regulates the functional level of follistatin, and the second agent is for the prevention or treatment of nephropathy, diabetic nephropathy, type 1 diabetes, hyperlipidemia or obesity.

As used herein, the term "concomitant" as in the phrase "concomitant therapeutic treatment" includes administering a first agent in the presence of a second agent. A concomitant therapeutic treatment method includes methods in which the first, second, third or additional agents are co-administered. A concomitant therapeutic treatment method also includes methods in which the first or additional agents are administered in the presence of a second or additional agents, wherein the second or additional agents, for example, may have been previously administered. A concomitant therapeutic treatment method may be executed step- wise by different actors. For example, one actor may administer to a subject a first agent and as a second actor may administer to the subject a second agent and the administering steps may be executed at the same time, or nearly the same time, or at distant times, so long as the first agent (and/or additional agents) are after administration in the presence of the second agent (and/or additional agents). The actor and the subject may be the same entity (e.g. a human).

In one example, the disclosure also provides a method for treating hypertension in a subject suffering from type 1 diabetes, the method comprising administering to the subject a first pharmaceutical composition comprising an agent of the disclosure and a second pharmaceutical composition comprising one or more additional compounds.

In another example, the disclosure provides a method for treating glomerular hyperfiltration in a subject, the method comprising administering to the subject a first pharmaceutical composition comprising an agent of the disclosure and a second pharmaceutical composition comprising one or more additional compounds. Kits

Another example of the disclosure provides kits containing an agent useful for the treatment of any method described herein.

In one example, the kit comprises (a) a container comprising an agent that down-regulates the functional level of activin or up-regulates the functional level of follistatin as described herein, optionally in a pharmaceutically acceptable carrier or diluent; and (b) a package insert with instructions for treating hypertension and/or glomerular hyperfiltration in a subject.

In accordance with this example of the disclosure, the package insert is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds or contains a composition that is effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the agent that down- regulates the functional level of activin or up-regulates the functional level of follistatin. The label or package insert indicates that the composition is used for treating a subject eligible for treatment, e.g., one having or predisposed to hypertension and/or glomerular hyperfiltration, with specific guidance regarding dosing amounts and intervals of compound and any other medicament being provided. The kit may further comprise an additional container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and/or dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The kit optionally further comprises a container comprising a second medicament, wherein the agent that down-regulates the functional level of activin or up-regulates the functional level of follistatin is a first medicament, and which article further comprises instructions on the package insert for treating the subject with the second medicament, in an effective amount. The second medicament may be any of those set forth above.

The present disclosure includes the following non-limiting examples. EXAMPLES

Example 1: Follistatin treatment decreases body weight and adiposity in diabetic mice

Six week old diabetic Akita (C57BL/6-Ins2Akita/J) mice were randomized to one of the following groups: untreated saline control (DM; n=12), low-dose follistatin (FST LD; 3 ug in saline intraperitoneally (IP) every other day; n=13), and high-dose follistatin (FST HD; 3 ug in saline IP daily; n=12). Wild-type mice were used for parallel non-diabetic studies (n=5-8 per group). Mice were followed for 12 weeks.

Follistatin treatment was associated with a small decrease in body weight (DM vs DM + FST HD) (Table 1) as well as a decrease in adiposity in both FST groups (Figure 1A). The lean mass/adipose ratio was increased with FST treatment, most likely due to the reduced adiposity as there was no FST dose-response relationship seen with lean mass (Figure IB and 1C).

* p<0.05 vs con; ^A p<0.05 vs DM; † p<0.05 vs FST LD; J p<0.05 vs FST HD. Statistical analyses were performed with SPSS22 for Windows using one-way ANOVA, with Tukey's HSD for post-hoc analysis. A P-value < 0.05 (two-tailed) was considered significant. Data are presented as the mean + standard error of the mean.

Example 2: Follistatin treatment decreases glomerular filtration rate and albuminuria but does not affect kidney weight in diabetic mice

Urine was collected at endpoint (i.e., at 12 weeks) from mice treated as described above for measurement of the albumin-to-creatinine ratio. Albumin-to- creatinine ratio was determined using commercially available kits (Albuwell M, Exocell kit for urine albumin and Crystal Chem for creatinine) according to manufacturer's instructions. Glomerular filtration rate (GFR) was assessed by clearance of fluorescein isothiocyanate (FITC) -labelled sinistrin (Fresenius Kabi Linz, Austria). A 5% FITC- sinistrin solution was injected retro-orbitally, after which blood was collected from the saphenous vein at 3, 7, 10, 15, 35, 55 and 75 minutes. Plasma fluorescence was assessed using a fluorometer (Gemini EM, Molecular Devices) at 485 nm excitation and 538nm emission.

Following GFR assessment, mice were perfused with cold PBS and organs harvested. Kidney weights were obtained of both kidneys and averaged to obtain an average kidney weight.

Diabetic kidneys showed the expected hypertrophy. FST treatment did not affect either control or diabetic kidney weights (Table 1). Hyperfiltration was seen in diabetic kidneys, as shown by the increased glomerular filtration rate (GFR) (Figure 2A). Although this was not affected by FST LD, FST HD normalized GFR to levels seen in wild-type (WT) mice. While FST HD also tended to decrease GFR in WT mice, this was not statistically significant (Figure 2A).

Albuminuria, another characteristic hallmark of diabetic kidney disease, was significantly increased in diabetic mice (Figure 2B). FST dose-dependently decreased albuminuria, as assessed by the albumin-to-creatinine ratio (ACR), with FST HD normalizing albumin excretion to that seen in WT mice (Figure 2B).

Example 3: Follistatin treatment decreases glomerular basement membrane thickening and attenuates collagen matrix deposition in diabetic mice

The earliest pathologic hallmark of diabetic kidney disease is thickening of the glomerular basement membrane (GBM). To assess GBM thickening and collagen matrix deposition, kidneys were collected as described above. Formalin-fixed sections (4μιη) were stained with Picrosirius Red (PSR, Polysciences) to assess collagens I and III. Positive staining was quantified using ImagePro 6.2 from 5 different fields at a magnification of 20x.

A small piece of cortex was taken for electron microscopy (EM), fixed in 0.2M glutaraldehyde/O.lM sodium cacodylate, pH7.4 and samples processed by the McMaster University EM facility. Basement membrane thickness was assessed on peripheral loops photographed randomly at 10,000x magnification, with calculation of the harmonic mean of measurements at 80-100 points crossing a grid from 1-2 glomeruli.

As seen in Figure 3A, the increase in GBM thickening observed in kidneys from DM was normalized by FST LD. The increased collagen matrix accumulation seen in diabetic kidneys, as measured by Picrosirius Red (PSR), was also significantly attenuated by FST LD (Figure 3B).

Example 4: Follistatin treatment reduces systolic and diastolic blood pressure in normal and diabetic mice

Blood pressure was measured every 4 weeks by tail cuff volume pressure recording (Coda 2, Kent Scientific).

A significant increase in blood pressure was not observed in diabetic mice, however both systolic and diastolic blood pressure was dose-dependently reduced by FST in both diabetic and control groups (Figures 4A and 4B).

Example 5: Follistatin and activin A modulate vascular reactivity in vitro

Aortas were dissected from wild-type C57BL/6 mice, cleaned of adventitia and cut into 3mm ring segments after which they were cultured in DMEM medium with 10% FBS supplemented with either Activin A (50ng/ml) or FST (300ng/ml) for 24h. Rings were then mounted on a small vessel wire style myograph (Radnoti LLC) in HBSS, maintained at 37°C and bubbled with 100% oxygen. After equilibration, contraction was initially tested with KC1. After washing, responses were then tested to cumulative doses of phenylephrine followed by carbachol. In a separate set of experiments, first order mesenteric arteries were dissected from WKY rats and similarly mounted in the myograph in HBSS, and contraction/relaxation responses tested after incubation for 15 minutes with Activin A (50ng/ml).

Activin A tended to increase the contractile response to phenylephrine and attenuate the relaxation response to carbachol, while FST attenuated the contractile response to phenylephrine (Figures 5A and 5B). In mesenteric resistance arteries exposed acutely to activin A (15 minutes), a similar increase in contractile and relaxation responses to phenylephrine and carbachol respectively was observed (Figures 5C and 5D). These data support a direct effect of both activin A and FST on vascular reactivity which may help to explain the observed effects of FST on lowering blood pressure in mice. Example 6: Follistatin modulates vascular reactivity in vivo

Superior mesenteric arteries were dissected from mice that had undergone 5/6 nephrectomy or sham operations and were treated with either vehicle or FST (3 μg, ip, every second day for 12 weeks). 3mm ring segments were placed into HBSS and contractility and relaxation tested as previously described above.

Arteries from 5/6 nephrectomy mice had a reduced relaxation response to carbacholine, which was improved by treatment with FST (Figure 6A). FST treatment also attenuated the contractile response to phenylephrine in both sham and 5/6 nephrectomy mice (Figure 6B). These data support a direct effect of FST on vascular reactivity in vivo in a mouse model of renal impairment and hypertension.

Claims

A method of treating hypertension in a subject suffering from or at risk of developing type 1 diabetes, the method comprising administering to the subject an agent that down-regulates the functional level of activin or up-regulates the functional level of follistatin.

The method of claim 1, wherein the subject is suffering from glomerular hyperfiltration.

A method of treating glomerular hyperfiltration in a subject, the method comprising administering to the subject an agent that down-regulates the functional level of activin or up-regulates the functional level of follistatin.

The method of claim 3, wherein the subject is suffering from or at risk of developing hypertension and/or type 1 diabetes.

The method of any one of claims 1 to 4, wherein the subject is suffering from or at risk of developing glomerulosclerosis, microalbuminuria, macroalbuminuria, diabetic nephropathy, hypertensive kidney disease, diabetic kidney disease, chronic kidney disease secondary to a reduction in renal mass (secondary focal glomerulosclerosis), polycystic kidney disease and/or chronic kidney disease associated with obesity.

The method of any one of claims 1 to 5, wherein the agent is administered in an amount effective to have one or more of the following effects:

a) reduce hypertension;

b) reduce or prevent glomerular sclerosis;

c) reduce or prevent microalbuminuria or macroalbuminuria;

d) reduce or prevent diabetic nephropathy;

e) reduce or prevent mesangial extracellular matrix deposition and/or abnormal thickening of the glomerular basement membrane;

f) reduce or prevent glomerular collagen deposits;

g) reduce or prevent glomerular mesangial expansion; and/or

h) reduce glomerular filtration rate.

7. The method of any one of claims 1 to 6, wherein the hypertension is intra- glomerular hypertension.

8. The method of any one of claims 1 to 7, wherein the agent is follistatin or a functional fragment thereof, or an inhibitor of activin.

9. The method of claim 8, wherein the inhibitor of activin is selected from the group consisting of:

a) an activin antagonist selected from the group consisting of inhibin, the activin βο subunit, the a subunit of inhibin, an antibody directed to activin, a nonfunctional activin mutant, a non-functional activin receptor mutant, a modified activin pro-domain and a soluble activin receptor; or

b) a non-pro teinaceous molecule selected from a group consisting of an activin antisense oligonucleotide, a short hairpin RNA (shRNA), a siRNA, an interfering RNA (RNAi), a ribozyme, a microRNA, a microRNA adapted shRNA (shRNAmir) and a DNAzyme which downregulates the transcription or translation of the activin gene.

10. The method of any one of claims 1 to 9, wherein the activin is activin A or activin B.

11. The method of any one of claims 1 to 10, wherein the functional level of follistatin is upregulated by increasing the transcription or translation of follistatin. 12. The method of any one of claims 1 to 11, wherein the follistatin is FS315 or FS288.

13. The method of any one of claims 1 to 12, wherein the agent is administered systemically.

14. Use of an agent in the manufacture of a medicament for treating hypertension in a subject suffering from or at risk of developing type 1 diabetes, wherein the agent down-regulates the functional level of activin or up-regulates the functional level of follistatin. Use of an agent in the manufacture of a medicament for treating glomerular hyperfiltration in a subject, wherein the agent down-regulates the functional level of activin or up-regulates the functional level of follistatin.