WO2015035405A1 - Myostatin antagonist for treatment of pew in esrd patients - Google Patents

Abstract

Disclosed are methods of treating or modulating protein energy wasting (PEW) in an ESRD patient by administration of a myostatin antagonist.

Description

Title

[0001] Myostatin antagonist for treatment of PEW in ESRD patients.

Cross Reference To Related Applications

[0002] This application claims the benefit of U.S. Provisional application number 61/875,609 filed on September 9, 2013, which is hereby incorporated in its entirety by reference.

Statement Regarding Federally Sponsored Research Or Development

[0003] Not applicable.

Sequence Listing

[0004] The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 9, 2014, is named 26100PCT_sequencelisting.txt, and is 3000 bytes in size.

Background Of The Invention

Field of the invention

[0005] The invention relates to compositions and methods of treatment for protein energy wasting in end stage renal disease patients.

Description of the Related Art

[0006] End Stage Renal Disease (ESRD) has become an increasingly important global public health concern with the current estimated hemodialysis population of more than 2 million people predicted to increase by 7% annually (Jha et al. 2012). The major risk factors for chronic kidney disease (CKD) and subsequent ESRD, such as diabetes, hypertension, obesity, cardiovascular disease and smoking, are becoming common throughout the world.

[0007] ESRD patients typically undergo Maintenance Hemodialysis (MHD). Patients on MHD have high rates of morbidity and mortality; in the United States (US), approximately one in five will die < 1 year after commencing dialysis (US Renal Data System 2012). In addition, quality of life for patients on hemodialysis is impaired by a low tolerance for physical activity, low endurance and impaired muscle strength (Storer et al. 2005; Lewis et al. 2012). The risk for hospitalization and death are increased in those with low muscle mass and low serum albumin levels (Kalantar-Zadeh et al. 2001 ; Kalantar-Zadeh et al. 2004; Huang et al. 2010; Lopes et al. 2010; Noori et al. 2010).

[0008] As described in more detail below, this syndrome of muscle wasting and poor nutritional state in ESRD patients is termed Protein Energy Wasting (PEW) (Fouque et al. 2008). Although nutrition supplements are often administered according to consensus guidelines (Tkizler et al. 2013), the metabolic and catabolic alterations of dialysis and underlying uremia render increased food intake alone unable to correct the pathophysiology of PEW (Carrero et al. 2013). PEW increases the risk for infections, cardiovascular disease complications, frailty, depression and mortality.

[0009] Patients on hemodialysis also receive medications to treat complications of renal disease such as anemia, hypercholesterolemia, opportunistic infections, and abnormal calcium-phosphorus homeostasis. While there are many clinical benefits to the use of these agents, their ability to improve patient survival has not yet been demonstrated in clinical trials. For example, correction of anemia to normal levels using erythropoiesis-stimulating agents has been reported to modestly increase, rather than decrease, rates of mortality, cardiovascular events and vascular access thrombosis (Skali et al. 201 1). In one recent study, reduction of cholesterol levels using HMG Co-A reductase inhibitors decreased the rate of major atherosclerotic events in patients with advanced CKD, but offered no significant survival benefit in patients on maintenance dialysis (Baigent et al. 2011), and no significant effect on fatal or nonfatal cardiovascular events in another study (Fellstrom et al 2009;

Baigent et al. 201 1). Similarly, correction of hyperparathyroidism using calcimimetics did not significantly reduce the risk of death or major cardiovascular events in patients with moderate-to-severe secondary hyperparathyroidism who were undergoing dialysis

(Investigators et al. 2012). Reports also conflict as to whether dose of dialysis or dialysis flux rate affect outcomes, as one study showed no differences in mortality for high vs. low dose or for high vs. low flux rate (Eknoyan et al. 2002), while another showed that all-cause mortality was significantly decreased with the use of high efficiency, post-dilution, online

hemodiafiltration in place of conventional hemodialysis (Maduell et al. 2,013).

[0010] Patients on MHD who are able to gain muscle mass, even in the context of overall weight loss over time, have a survival benefit relative to those who gain overall weight while losing muscle mass (Kalantar-Zadeh et al. 2010). Additional therapies with the potential ability to increase muscle mass and improve multiple outcomes, including survival, and are urgently needed for ESRD patients including those on maintenance hemodialysis.

[0011] At this time, no products are approved by the Food and Drug Administration (FDA) for the treatment of PEW in MHD. A variety of agents approved for other indications, as well as nutritional/exercise interventions, have been investigated as potential treatments for patients with ESRD and PEW. Examples include human growth hormone (hGH) and hGH analogs; ghrelin analogs, anabolic steroids, exercise intervention and nutritional supplements. This growing body of literature highlights the unmet need for effective treatment for this condition.

[0012] As described in more detail below, Compound 745 is a fusion protein comprising the human IgGl Fc region and a myostatin-neutralizing bioactive peptide. Myostatin antagonists, including the peptibody Compound 745 and the murine surrogate of Compound 745, are described in International patent application nos. PCT/US2003/040781 filed on December 19, 2003 (WO/2004/058988) and PCT/US2006/046546 filed on December 6, 2006 (WO/2007/067616).

[0013] Myostatin is a transforming growth factor-β (TGF-β) family member that is expressed mainly in skeletal muscle and acts as a negative regulator of muscle growth (Roth et al. 2004; MePherron 2010). Myostatin is upregulated in skeletal muscle of uremic rodents (Sun et al. 2006; Zhang et al. 201 1 ), and has been reported to be upregulated in some (Verzola et al. 201 1) but not all studies ( opple et al. 2006) of patients with CKD.

[0014] The murine surrogate of Compound 745 increased skeletal muscle mass and muscle strength in a variety of additional preclinical models, including normal mice, immune-deficient mice, MDX mice (Duchenne muscular dystrophy model), colon-26 tumor- bearing mice (cancer cachexia model), hind limb suspended mice (muscle disuse/atrophy model) and orchiectomized mice (androgen-deficiency model) (WO/2004/058988 and WO/2007/067616). In the 5/6* nephrectomy mouse model of ESRD, treatment with a murine surrogate of Compound 745 reversed loss of body weight, increased skeletal muscle mass, and reduced inflammation (Zhang et al. 2011 Given that the human condition of protein energy wasting is complex, encompassing a syndrome of malnutrition inflammation and muscle wasting despite treatment with dialysis that corrects uremic, it could not be assumed that 5/6th nephrectomized mice (which do not receive dialysis) would fully model the human condition.

[0015] Compound 745 was used in a clinical study treating cachexia in prostate cancer patients. The patients received 3 mg/kg weekly SC or IV Compound 745. The weekly 3 mg/kg SC dose was associated with statistically significant changes in efficacy endpoints (i.e., increases in lean body mass, decreased fat mass, and increased lower extremity muscle size) compared with placebo (Padhi et al. 2014). However, cachexia resulting from cancer is different from PEW, as treatment with androgenic hormones are sometime effective with cancer related cachexia but are ineffective with PEW. Summary Of The Invention

[0016] Disclosed herein are methods of treating protein energy wasting (PEW) in a human subject with End-Stage Renal Disease (ESRD) in need thereof by administering a therapeutically effective amount of Compound 745, an anti-myostatin peptibody consisting of two identical polypeptide chains each consisting of SEQ ID NO: l . In some embodiments, the human subject has a serum albumin level < 3.8 g/dL, e.g., has a serum albumin level < 3.8 g/dL at any time within 60 days prior to start of treatment. In some embodiments, the human subject has a body mass index (BMI) of < 28 kg/m². In some embodiments, the method also includes determining a body mass index and/or a serum albumin level of the human subject before treatment.

[0017] Typically the human subject is undergoing hemodialysis, e.g., maintenance hemodialysis (MHD), e.g., is undergoing MHD > 3 times per week. The method can include administering a nutritional supplement comprising > 10 grams of protein once daily to the human subject during at least a portion of the span of the treatment.

[0018] In some embodiments, Compound 745 is administered in a liquid pharmaceutical composition, e.g., administered in a liquid pharmaceutical composition of 10 mM sodium acetate, 9% (w/v) sucrose, and 0.004% (w/v) polysorbate 20, pH 4.75. In some

embodiments, Compound 745 is administered by intravenous (IV) infusion. Compound 745 can be produced by, e.g., the expressing the Compound 745 as insoluble inclusion bodies in E. coli cells; harvesting the cells; lysing the cells; solubilizing the inclusion bodies; refolding, concentrating, and chromatographically purifying the Compound 745.

[0019] In the methods described herein, Compound 745 is administered at a dose that is therapeutically effective, typically at a dose of 0.1 to 10 mg/kg. In some embodiments, Compound 745 is administered at a dose of 1.0 mg/kg or 2.0 mg/kg or 3.0 mg/kg or 6.0 mg/kg or 10 mg/kg. Compound 745 can be administered, for example, once weekly for , e.g., 12 weeks. In some embodiments, Compound 745 is administered once weekly at a loading dose for a first time period followed by a maintenance dose for a second time period, the loading dose greater than the maintenance dose, e.g., once weekly at a dose of 3 mg/kg for 3 weeks then 1 mg/kg for 9 weeks or at a dose of 6 mg/kg for 3 weeks then 2 mg/kg for 9 weeks.

[0020] In one embodiment, the method results in an increase in lean body mass (LBM) in the human subject as determined by, e.g., Dual-energy X-ray absorptiometry (DXA). Other clinical endpoints include but are not limited to an increase in muscle cross sectional area by CT, an increase in appendicular lean mass (ALM), an increase in physical function by the Stair climbing power test (SCPT) and an increase in distance on the 6-minute walk test (6MWT), an increase in quality of life using at least one of three Patient Reported Outcome (PRO tools) (the Kidney Dialysis Quality of Life™ (KDQOL™)-36 short form questionnaire, the Functional Assessment of Chronic Illness Therapy -Fatigue (FACIT -Fatigue) scale, and the Functional Assessment of Anorexia/Cachexia Treatment (FAACT) anorexia/cachexia subscale). The method of treatment can also result in, for example, a change in at least one biomarker selected from the group consisting of a serum myostatin, C-reactive protein, interleukin-6 (IL-6), tumor necrosis factor- a (TNF-a), adiponectin, leptin, resistin, serum lipid, and serum albumin.

[0021] Also described herein is a kit for treatment of protein energy wasting (PEW) in a human subject with End-Stage Renal Disease (ESRD) comprising a container comprising Compound 745 and instructions for use.

Brief Description Of The Figures

[0022] Figure 1 is a graph showing plasma profiles in a first cohort of patients after administration of Compound 745. The concentration versus time plots show higher plasma concentrations at Week 12 (squares and triangles) compared to Week 1 (circles). For Week 1, the plot shows the median values from 8 subjects.

[0023] Figure 2 is a graph showing pharmacokinetic modeling, with a simulated plasma concentration-time profile of Compound 745 administered as 3 weekly loading I.V. infusions of 6 mg/kg followed thereafter by weekly maintenance iv infusions of 2 mg/kg.

Detailed Description

Definitions

[0024] Effective amount. The term "effective amount" refers to an amount of a composition an amount that is effective to ameliorate a symptom of a disease, e.g., PEW in ESRD patients. An effective amount can be a therapeutically effective amount or a prophylactically effective amount. An effective amount of a pharmaceutical composition will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication, the route of administration, the treatment regimen (number and timing of doses) and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient.

[0025] End stage renal disease (ESRD). As described in more detail herein, end-stage kidney disease is a condition when the kidneys are no longer able to work at a level needed for day-to-day life. The most common causes of ESRD in the U.S. are diabetes and high blood pressure. ESRD almost always comes after chronic kidney disease.

[0026] Myostatin. Myostatin, a growth factor also known as GDF-8, is a member of the TGF-β family. Myostatin known to be a negative regulator of skeletal muscle tissue.

Myostatin is synthesized as an inactive preproprotein which is activated by proteolyic cleavage (Zimmers et al, supra (2002)). The precurser protein is cleaved to produce an NH2- terminal inactive prodomain and an approximately 109 amino acid COOH-terminal protein in the form of a homodimer of about 25 kDa, which is the mature, active form (Zimmers et al, supra (2002)). It is now believed that the mature dimer circulates in the blood as an inactive latent complex bound to the propeptide (Zimmers et al, supra (2002)). Full-length myostatin refers to the full-length human preproprotein sequence described in McPherron et al. PNAS USA 94, 12457 (1997), as well as related full-length polypeptides including allelic variants and interspecies homologs (McPherron et al. supra (1997)). As used herein the term

"myostatin" or "mature myostatin" refers to the mature, biologically active COOH-terminal polypeptide, in monomer, dimer, multimeric form or other form. "Myostatin" or "mature myostatin" also refers to fragments of the biologically active mature myostatin, as well as related polypeptides including allelic variants, splice variants, and fusion peptides and polypeptides. Myostatin may or may not include additional terminal residues such as targeting sequences, or methionine and lysine residues and /or tag or fusion protein sequences, depending on how it is prepared.

[0027] Protein energy wasting (PEW). As described in more detail below, PEW is a syndrome of muscle wasting and poor nutritional status in ESRD patients.

[0028] Subject. The term "subject" or "individual" is a mammal. In one embodiment, a subject is a human.

[0029] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS

Abbreviation Definition Explanation

AUCo-72 Area under the serum concentration-time curve from time 0 to 72 hours after dosing

AUCo.l₆₈ Area under the serum concentration-time curve from time 0 to 168 hours after dosing

AUCo-, Area under the serum concentration-time curve from time 0 to time tau (dosing interval)

BMI Body mass index

BMP Bone morphogenic protein

BUN Blood urea nitrogen

CI Confidence interval

CKD Chronic kidney disease

CL Clearance

c Maximum observed serum concentration

CRF Case report form

C P C-reactive protein

CT Computed tomography

CTCAE Common Terminology Criteria for Adverse Events

DLT Dose limiting toxicity

DOPPS Dialysis Outcomes and Practice Patterns Study

DXA Dual-energy X-ray absorptiometry

ECG Electroc ardiogram

eCRF Electronic case report form

EOS End of study

ESRD End stage renal disease

FAACT Functional Assessment of Anorexia/Cachexia Treatment

FACIT Functional Assessment of Chronic Illness Therapy

FDA Food and Drug Administration (US)

FSH Follicle-stimulating hormone

GCP Good clinical practice

GDF Growth differentiation factor

HbAlc Hemoglobin Ale / glycated hemoglobin

HCG Human chorionic gonadotropin

hGH Human growth hormone

HOMA Homeostatic model assessment

HR Hazard ratio

ICF Informed consent form

ICH International Conference on Harmonisation

IL-6 Interleukin-6 Abbreviation Definition Explanation

INR International normalized ratio

IR Insulin resistance

IRB Institutional Review Board

ISRNM International Society of Renal and Nutrition and Metabolism

IUD Intrauterine device

IV Intravenous(ly)

KDQOL™ Kidney Dialysis Quality of Life™

LBM Lean body mass

mCCI Modified Charlson comorbidity index

MDX Mouse model of Duchenne muscular dystrophy

MedDRA Medical Dictionary for Regulatory Affairs

MHD Maintenance hemodialysis

MRI Magnetic resonance imaging

MRT Mean residence time

MTD Maximum tolerated dose

Mu-S Murine surrogate of Compound 745

MUAC Mid-upper arm circumference

MUAMC Mid-upper arm muscle circumference

NOAEL No observed adverse effect level

nPCR Normalized protein catabolic rate

PDW Post-dialysis weight

PEW Protein energy wasting

P Pharmacokinetic( s)

PRO Patient-reported outcomes

PT Prothrombin time

PTT Partial thromboplastin time

QTcF Calculated (Fredericia) measure of time between the start of the Q wave and the end of the T wave

SAE Serious adverse event

SC Subcutaneous

SD Standard deviation

SE Standard error

SCPT Stair climbing power test

SOP Standard operating procedure

SUSAR Suspected unexpected serious adverse reactions

tl/2 Terminal phase half-life

TEAE Treatment-emergent adverse event

TGF-β Transforming growth factor -β Abbreviation Definition Explanation

tmax Time of maximum observed serum concentration

TNF-a Tumor necrosis factor-a

TSF Tricep skin fold

ULN Upper limit of normal

v_d Volume of distribution

v_ss Volume of distribution at steady state

Protein energy wasting in ESRD patients

[0030] Disclosed herein are methods for treating protein energy wasting in ESRD patients. Protein energy wasting, or PEW, is a syndrome of muscle wasting and poor nutritional status in ESRD patients leading to low muscle mass, low serum albumin levels, impaired quality of life, low tolerance for physical activity, low endurance, impaired muscle strength, increased risk for hospitalization, prolonged hospitalization/poor outcomes, increased risk for infections, cardiovascular disease complications, frailty, depression and mortality. PEW is not only associated with muscle breakdown and loss of physical function but also high degrees of inflammation and uremia (high blood levels nitrogen) uncorrected by dialysis which may play a role in a decrease in platelet blotting.

[0031] The nature of PEW is different from other muscle wasting syndromes such as cachexia resulting from cancer, as treatment with androgenic hormones have not worked for PEW. Additional differences include that cancer cachexia is typically a result of inadequate nutritional intake (anorexia), whereas PEW can result even in the face of protein intake that would be adequate for an unaffected person. In PEW this may be because abnormal proteolysis of the muscle is a key factor (Proteolytic mechanisms, not malnutrition, cause loss of muscle mass in kidney failure. Mitch WE J Ren Nutr. 2006 Jul; 16(3):208-1 1.) that can outweigh nutrition. In addition, PEW manifests a large component of inflammation, whereas cachexia is not always associated with inflammation

Serum albumin levels

[0032] In some embodiments, the ESRD patient having PEW is selected for treatment by measurement of blood serum albumin levels. Serum albumin levels can be determined by any method well known to one of skill in the art and/or any method typically performed by a clinical laboratory. Examples include but are not limited to bromocresol green methods and bromocresol purple methods. In some embodiments, the method of treatment includes determining the serum albumin level of the human subject. [0033] In some embodiments, the human subject selected for treatment has a blood serum albumin level of less than or equal to 3.8 g/dL. In other embodiments, the human subject selected for treatment has a blood serum albumin level of less than or equal to 3.7 or 3.6 or 3.5 g/dL. In some embodiments, the human subject has a serum albumin level < 3.8 g/dL at any time within 60 days prior to start of treatment.

Body mass index

[0034] In some embodiments, the human subject undergoing the method of treatment has a low body mass index (BMI). BMI is determined by dividing weight (in kg) by the square of height (in meters). In one embodiment, the human subject has a BMI of < 28 kg/m², based on a post-dialysis weight and a height of the human subject. In other embodiments, the human subject has a BMI of <=25 or a BMI of <=23. In some embodiments, the method of treatment includes determining the BMI of the human subject.

Hemodialysis

[0035] In some embodiments, the ESRD patient treated with Compound 745 is undergoing hemodialysis. In some embodiments, the ESRD patient is undergoing maintenance hemodialysis (MHD), e.g., MHD greater than or equal to 3 times per week. In other embodiments, the ESRD patient is undergoing MHD 2, 3, 4, 5, or 6 times per week. In other embodiments, the ESRD patient is undergoing daily hemodialysis, e.g., 2 hours 6 days a week. In other embodiments, the ESRD patient is undergoing nocturnal hemodialysis, e.g., three to six nights per week and between 6 and 10 hours per session while the patient sleeps. In other embodiments, the ESRD patient is undergoing peritoneal dialysis.

[0036] In some embodiments, the ESRD patient treated with Compound 745 is undergoing hemodialysis and is taking a nutritional supplement having at least 10 grams of protein per day. Examples of nutritional supplements include but are not limited to nutritional bars, yoghurt, and the like.

Clinical endpoints

[0037] The methods of treatment disclosed herein results in an improvement in at least one clinical endpoint in the ESRD patient, e.g., at least one improvement in a measurement of a parameter related to PEW. In one embodiment, the method results in an increase in lean body mass (LBM) in the human subject. Other clinical endpoints include but are not limited to an increase in LBM as determined by Dual-energy X-ray absorptiometry (DXA), an increase in muscle cross sectional area as determined by CT, an increase in appendicular lean mass (ALM), an increase in physical function by the Stair climbing power test (SCPT), an increase in distance on the and 6-minute walk test (6MWT), an increase in quality of life using at least one of three Patient Reported Outcome (PRO tools) (the Kidney Dialysis Quality of Life™ (KDQOL™)-36 short form questionnaire, the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) scale, and the Functional Assessment of Anorexia/Cachexia Treatment (FAACT) anorexia/cachexia subscale). Other clinical endpoints include a reduction in hospitalizations, fractures, and/or cardiovascular events. Additional clinical endpoints that can result from the methods of treatment are described below in the examples.

[0038] In other embodiments, the methods of treatment results in a change in at least one biomarker selected from the group consisting of a serum myostatin, C-reactive protein, interleukin-6 (IL-6), tumor necrosis factor- a (TNF-a), adiponectin, leptin, resistin, serum lipid, hemoglobin, and serum albumin. Additional biomarkers are described below in the examples.

[0039] Assays and methods for measuring clinical endpoints can be any that are well know to one of skill in the art and/or are used in a clinical laboratory setting. In some embodiments, the assays and methods for measuring clinical endpoints are those described in the examples below.

[0040] The clinical endpoints may be measured in absolute values or as a percent change over baseline. The changes will typically be statistically significant, e.g., a two-sided one- sample t-test at the alpha = 0.05 level of significance. In most cases, analysis of variance (ANOVA) or analysis of covariance (ANCOVA) models are used.

Compound 745

[0041] The methods of treating PEW in ESRD patients include administration of the myostatin antagonist Compound 745.

[0042] Compound 745 is an anti-myostatin peptibody. A peptibody represents the component peptide (the "pepti-") and the Fc portion of an immunoglobulin in an overall structure that resembles an antibody (the "-body"). In this format, the peptide "warhead" interacts with myostatin and inhibits signaling through its receptor. The second domain, the Fc component, stabilizes the complex in the body, allows for endothelial cell trancytosis and recycling through FcRnl and extends residence time into a therapeutically useful range.

[0043] Compound 745 consists of 2 identical polypeptide chains, which are covalently linked through disulfide bonds. The N-terminal portion of each chain consists of the human IgGl Fc sequence which is fused at the C-terminus via a glycine (five glycines plus AQ) linker to an anti-myostatin peptide. Each polypeptide chain consists of 255 amino acids beginning with the amino acid methionine and ending with glutamic acid. There are 3

42 102 148 206 242 249 intrachain disulfide links between residues Cys -Cys , Cys -Cys , and Cys -Cys on

7 7

each polypeptide chain and 2 interchain disulfide links between Cys _chainl-Cys _chain2, and

10 10

Cy^s chaini'Cys _chain2- The 510 amino acids that constitute the Compound 745 molecule yield a theoretical molecular mass of 57,099 daltons. As a microbially expressed protein,

Compound 745 is not glycosylated.

[0044] The 255 residue amino acid sequence of each polypeptide chain (SEQ ID NO: 1) of Compound 745 is shown below. The plain font portion of the sequence indicates the IgGl Fc sequence. The bold font portion indicates the five glycine plus AQ linker sequence

(GGGGGAQ; SEQ ID NO:2). The bold and italic portion of the sequence indicates the anti- myostatin peptide (LADHG QCIR WPWMCP PEG WE; SEQ ID NO:3).

Compound 745 Sequence (amino acid)

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKGG GGGAQLADHG QCIRWPWMCP PEGWE

(SEQ ID NO:l)

Pharmaceutical compositions

[0045] The methods of treatment described herein include administering an effective amount of Compound 745. Compound 745 is typically formulated in a pharmaceutical composition. These pharmaceutical compositions can comprise, in addition to Compound 745, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, and intraperitoneal routes.

[0046] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant.

[0047] Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included. [0048] In the methods of the invention, the pharmaceutical composition may be administered in a solution. A pharmaceutical composition may be administered in an unbuffered solution, e.g., in saline or in water. Alternatively, the pharmaceutical composition may also be administered in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution can be adjusted such that it is suitable for administering to a subject.

[0049] In some embodiments, the buffer solution further comprises an agent for controlling the osmolarity of the solution, such that the osmolarity is kept at a desired value, e.g., at the physiologic values of the human plasma. Solutes which can be added to the buffer solution to control the osmolarity include, but are not limited to, proteins, peptides, amino acids, non-metabolized polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the agent for controlling the osmolarity of the solution is a salt. In certain embodiments, the agent for controlling the osmolarity of the solution is sodium chloride or potassium chloride or sodium acetate.

[0050] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the pharmaceutical composition will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives can be included, as required.

[0051] Other components of a liquid pharmaceutical composition include detergents and the like.

[0052] In some embodiments, Compound 745 is in a liquid pharmaceutical composition suitable for IV injection, for example, 10 mM sodium acetate, 9% (w/v) sucrose, and 0.004% (w/v) polysorbate 20, pH 4.75 .

[0053] In some embodiments, Compound 745 is provided to the administrator (e.g., treating medical staff) in a lyophilized form. The lyophilized form is then resuspended in an appropriate solution before administration.

[0054] Compound 745, can be administered alone or in combination with other treatments, either simultaneously or sequentially. Administration methods, dosages and treatment regimens

[0055] The methods of treatment include administration of Compound 745 in an

"effective amount", this being sufficient to show benefit to the ESRD patient. The actual amount, e.g., dose, administered, and rate and time-course of administration, will depend on the nature and severity of the PEW being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0056] Administration can be oral, intravenous, cutaneous or subcutaneous. In one embodiment, Compound 745 is administered via intravenous infusion. In other

embodiments, Compound 745 is administered subcutaneous ly or intraperitoneally.

[0057] Dosage can depend on the variables listed herein. In some embodiments, the dose is between 0.1 to 100 mg/kg or 0.5 to 50 mg/kg or 0.5 to 10 mg/kg or 0.1 to 10 mg/kg or 1.0 to 10 mg/kg or 1.0 to 6.0 mg/kg. In some embodiments, the dose is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0058] Compound 745 can be administered once daily, or can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the

pharmaceutical composition contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the pharmaceutical composition over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of pharmaceutical composition at a particular site. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0059] In other embodiments, a single dose of the pharmaceutical compositions can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals. In some embodiments of the invention, a single dose of the pharmaceutical compositions is administered once per week. In other embodiments of the invention, a single dose of the pharmaceutical compositions is administered bi-monthly. In one embodiment, Compound 745 is administered once weekly.

[0060] In some embodiments, Compound 745 is administered once weekly at a loading dose for a first time period followed by a maintenance dose for a second time period, the loading dose greater than the maintenance dose. For example, Compound 745 can be administered once weekly at a dose of 3 mg/kg for 3 weeks then 1 mg/kg for 9 weeks or at a dose of 6 mg/kg for 3 weeks then 2 mg/kg for 9 weeks.

Kits

[0061] Also described herein are kits for treatment of protein energy wasting (PEW) in a human subject with End-Stage Renal Disease (ESRD) comprising a vial comprising Compound 745 and instructions for use. Compound 745 can be in any suitable

pharmaceutical composition as described herein, e.g., a liquid suspension suitable for IV infusion. Alternatively, Compound 745 can be in a lyophilized state suitable for re- suspension before use. The instructions for use can include storage instructions, patient selection, dosages, administration methods, time periods for use, clinical endpoints, and the like.

Polypeptides

[0062] The method of treatment described herein include administration of a peptibody, Compound 745 which consists of two identical polypeptides with the sequence SEQ ID NO: l .

[0063] In some embodiments, the compositions used in the method of the invention include a polypeptide that is less than 100% identical to an amino acid sequence disclosed herein. In some embodiments, the polypeptide is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or between 99 and 100% identical to a sequence disclosed herein.

[0064] The term "percent identical" in the context of two or more amino acid or nucleic acid sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent identity can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0065] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0066] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al, infra).

[0067] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information

(www.ncbi.nlm.nih.gov/).

Variants

[0068] The compositions described herein also encompass variants of the polypeptides described herein. As used herein, the term "variants" refers to polypeptides having one or more amino acid residues inserted, deleted or substituted into the original amino acid sequence and which retain at least a portion of the function of the polypeptide described herein. As used herein, fragments of the polypeptides are included within the definition of "variants". It is understood that any given peptide or peptibody may contain one or two or all three types of variants. Insertional and substitutional variants may contain natural amino acids, as well as non-naturally occurring amino acids or both. Variants can include, e.g., polypeptides that include a leader or signal sequence; polypeptides with additional amino terminal residues, e.g., Metl or Lys 2; polypeptides with expression tags, e.g., histidine tags; and polypeptides expressed as fusion proteins. [0069] Variants of the polypeptides described herein can include amino acid

substitutions. Stereoisomers (e.g., D-amino acids) of the twenty conventional (naturally occurring) amino acids, non-naturally occurring amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of non-naturally occurring amino acids include, for example: aminoadipic acid, beta-alanine, beta- aminopropionic acid, aminobutyric acid, piperidinic acid, aminocaprioic acid,

aminoheptanoic acid, aminoisobutyric acid, aminopimelic acid, diaminobutyric acid, desmosine, diaminopimelic acid, diaminopropionic acid, N-ethylglycine, N-ethylaspargine, hyroxylysine, allo-hydroxylysine, hydroxyproline, isodesmosine, allo-isoleucine, N- methylglycine, sarcosine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, orithine, 4-hydroxyproline, γ-carboxyglutamate, ε-Ν,Ν,Ν-trimethyllysine, ε-Ν-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5 -hydroxy lysine, σ-Ν-methylarginine, and other similar amino acids and amino acids (e.g., 4-hydroxyproline).

[0070] Naturally occurring residues may be divided into (overlapping) classes based on common side chain properties:

1) neutral hydrophobic: Met, Ala, Val, Leu, He, Pro, Trp, Met, Phe;

2) neutral polar: Cys, Ser, Thr, Asn, Gin, Tyr, Gly;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

[0071] Substitution with naturally occurring amino acids can be conservative or non- conservative. Conservative amino acid substitutions involve exchanging a member of one of the above classes for another member of the same class. Conservative changes may encompass unconventional amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include

peptidomimetics and other reversed or inverted forms of amino acid moieties. Examples

[0072] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0073] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al, Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);

Remington 's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3^rd Ed. (Plenum Press) Vols A and B(1992).

Example 1: Compound 745.

Expression and Preparation

[0074] Compound 745 was expressed as insoluble inclusion bodies by fermentation of E coli (e.g., pAMG -Fc fusion constructs in E. coli strain 2596) as described in International patent application nos. PCT/US2003/040781 filed on December 19, 2003 (WO/2004/058988) and PCT/US2006/046546 filed on December 6, 2006 (WO/ 2007/067616).

[0075] Typical fermentation proceeded for 12 to 16 hours post induction, followed by cell harvest with a disk-stack centrifuge. Lysing the cells with high-pressure homogenization isolated the inclusion bodies. After wash and centrifugation, the resulting double-washed inclusion body slurry (DWIBs) was stored at -30° ± 10°C until purification.

[0076] Following solubilization of the DWIBs, Compound 745 was refolded in a solution containing urea, glycerol, arginine, and the redox pair cysteine/cystamine. After refolding, the product was concentrated and the refold reagents removed by means of an ultrafiltration and diafiltration (UF/DF) process. The diafiltered product was acidified, followed by

clarification. The product was subsequently purified through 3 different chromatography steps: 2 anion-exchange (Q Sepharose Fast Flow) columns, one operated in flow-through mode and one in bind and elute mode, and a HIC (Butyl Sepharose Fast Flow) column. The product was then further concentrated and diafiltered into formulation buffer with a UF/DF process. The formulated product was then filtered through a 0.2 μιη filter into bulk containers and frozen at -30° ± 10°C.

[0077] An additional chromatography step was added to the purification process for the clinical drug substance process to remove host cell-related impurities.

Formulation

[0078] The final dosage formulation for Compound 745 at 30 mg/mL was 10 mM sodium acetate, 9% (w/v) sucrose, 0.004% (w/v) polysorbate 20, pH 4.75.

Drug Supply and Storage

[0079] Compound 745 was stored in a non- frost- free freezer set between -20° and -70°C. A range of -20 (+5)°C to -70 (-10)°C is acceptable to accommodate fluctuations in the temperature of the freezer. Exposure to higher temperatures and vigorous shaking of the vial should be avoided because these conditions may lead to loss of Compound 745 potency and structural integrity.

Example 2: Treatment of End-Stage Renal Disease with Protein Energy Wasting

[0080] PEW in ESRD patients is treated by administration of the anti-myostatin peptibody Compound 745. Typically the patient has a serum albumin level < 3.8 g/dL, e.g., has a serum albumin level < 3.8 g/dL at any time within 60 days prior to start of treatment. In addition, the patient may have a body mass index (BMI) of < 28 kg/m². Typically the patient is undergoing maintenance hemodialysis (MHD) > 3 times per week. Compound 745 is administered in a liquid pharmaceutical composition of 10 mM sodium acetate, 9% (w/v) sucrose, and 0.004% (w/v) polysorbate 20, pH 4.75 and is administered by intravenous (IV) infusion.

[0081] Compound 745 is administered at a dose that is therapeutically effective once weekly for 12 weeks: 3 mg/kg once weekly for 12 weeks; once weekly at a dose of 3 mg/kg for 3 weeks then 1 mg/kg for 9 weeks; or at a dose of 6 mg/kg for 3 weeks then 2 mg/kg for 9 weeks.

[0082] The method results in an increase in lean body mass (LBM) in the patient as determined by, e.g., Dual-energy X-ray absorptiometry (DXA). Other clinical endpoints include an increase in muscle cross sectional area by CT, an increase in appendicular lean mass (ALM), an increase in physical function by the Stair climbing power test (SCPT) and an increase in distance on the 6-minute walk test (6MWT), an increase in quality of life using at least one of three Patient Reported Outcome (PRO tools) (the Kidney Dialysis Quality of Life™ (KDQOL™)-36 short form questionnaire, the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) scale, and the Functional Assessment of

Anorexia/Cachexia Treatment (FAACT) anorexia/cachexia subscale). The method also results in a change in at least one biomarker selected from the group consisting of a serum myostatin, C-reactive protein, interleukin-6 (IL-6), tumor necrosis factor- a (TNF-a), adiponectin, leptin, resistin, serum lipid, and serum albumin.

Patient selection

[0083] Eligible patients are men and women between 18-85 years old who have ESRD; received outpatient maintenance hemodialysis for at least 6 months prior to start of treatment; received adequate dialysis with Kt/V > 1.2 on two occasions within 12 weeks prior to enrollment; undergone dialysis > 3 times per week, on average; hypoalbuminemia and low BMI defined as serum albumin level < 3.8 g/dL and body mass index (BMI) < 28 kg/m²; and a life expectancy of > 6 months.

Exclusion Criteria

[0084] A patient can be (but may not necessarily be) excluded from treatment for at least one of the following reasons: Presence of an indwelling central vascular catheter; current medical condition (such as severe neuropathy, limb amputation, use of a walking frame or other conditions), that, in the judgment of the medical professional, would interfere with the subject's ability to perform tests, such as stair climbing, that are required for study endpoints; non-healing ulcers/sores > 1 cm in diameter for > 4 months, or history of ankle/brachial index < 0.9; active infection requiring hospitalization or treatment with IV antibiotics within 4 weeks of start of treatment or oral antibiotics within 2 weeks of start of treatment; major surgery within 3 months prior to start of treatment, minor surgery within 4 weeks of start of treatment, or dialysis access revision/angioplasty/replacement within 2 weeks of start of treatment; history of a renal transplant, whether or not functional, within 2 years of start of treatment (however, if the graft has been removed the patient will be considered eligible, regardless of when the original transplant occurred) or plans to undergo renal transplantation within 6 months of start of treatment; current treatment with appetite stimulants, including anabolic steroids or growth hormone (male subjects may receive physiologic testosterone replacement if they are hypogonadal— the dose must be stable for 3 months prior to screening and not expected to change during treatment); dysphagia, aspiration risk, or difficulty swallowing food or liquid; myocardial infarction or unstable angina within 6 months of start of treatment, or congestive heart failure (New York Heart Association Class III or higher) at the time of screening; elevated liver function tests, including total bilirubin > 1.5 x the upper limit of normal (ULN; unless subject has documented Gilbert's disease), aspartate aminotransferase (AST) or alanine aminotransferase (ALT) > 3.0 x ULN; platelet count < 80 x 109/L (80,000/mm3); hemoglobin < 8.5 g/dL (85 g/L; 5.3 mmol/L); white blood cells count < 3 x 109/L (3000/mm3); positive hepatitis B surface antigen (HBsAg) or hepatitis C antibody with abnormal liver function test results not acceptable to the investigator and the sponsor; HIV infection or AIDS; history of neoplasia, except non- melanoma skin cancers, with a 30% probability of recurrence within 12 months; prior treatment with any investigational product 4 weeks or 5 half-lives (whichever is longer), of start of treatment. Prior treatment with any investigational device within 4 weeks.; if female, pregnant, by positive human chorionic gonadotropin (HCG) test; if HCG test is

indeterminate, confirm using follicle-stimulating hormone (FSH) test; if female, currently breast feeding; if female and of childbearing potential, or male with a female partner of childbearing potential, unwilling to use a highly effective method of contraception (i.e., one that results in pregnancy less than 1% per year) when used consistently and correctly, such as implants, injectables, combined oral contraceptives, some intrauterine contraceptive devices (IUDs), sexual abstinence, or a vasectomized partner; men unwilling to use a highly effective contraceptive measure during their participation in this study with female partners and/or unwilling to refrain from donating sperm while undergoing treatment with Compound 745 and for three months after the last dose administration; presence of ascites or pleural effusion requiring medical intervention; presence of portal-venous shunt device or history of extensive hepatic resection (more than one segment)

Required Nutritional Supplements

[0085] Subjects take one oral nutritional supplement containing > 10 grams of protein daily while undergoing s treatment 8. The supplement may be any brand of oral bar or supplement containing the required amount of protein.

Administration of Compound 745

[0086] Compound 745 is administered by IV infusion once weekly for 12 weeks. Three protocols are used: 3 mg/kg once weekly for 12 weeks; once weekly at a dose of 3 mg/kg for 3 weeks then 1 mg/kg for 9 weeks; or at a dose of 6 mg/kg for 3 weeks then 2 mg/kg for 9 weeks.

[0087] The drug is administered following dialysis. Infusion is performed through the venous fistula needle, post-dialysis run, IV push at a rate of 6mL/min. Clinical endpoints; efficacy assessments

[0088] After treatment, clinical endpoints are measure, e.g., efficacy assessments are made. To summarize and as described in more detail below, this can include measurement of LBM by DXA, muscle cross sectional area by CT, muscle anthropometry, physical function by the SCPT and 6-minute walk test (6MWT), levels of biomarkers (cytokines, lipids, and myostatin) over the course of treatment, and changes in quality of life using three PRO tools (the KDQOL™-36 short form questionnaire, the Functional Assessment of Chronic Illness Therapy-Fatigue [FACIT -Fatigue] scale, and the Functional Assessment of

Anorexia/Cachexia Treatment [FAACT] anorexia/cachexia subscale). Efficacy assessments are done throughout the 12 weeks of treatment and 8 weeks of post-treatment monitoring. In general, assessments are done either at the time of Compound 745 administration or on other days in the same week, as needed for convenience. Muscle function tests (SCPT and 6MWT), however, should be done on non-dialysis days due to expected subject fatigue on dialysis days) and the DXA/CT scans should be performed within 30 hours of completion of the last dialysis, due to the potential of edema build-up to confound the scan analysis.

[0089] Pharmacokinetics : Compound 745 levels in pharmacokinetic blood samples are determined by a central laboratory. The following parameters are estimated from blood levels using standard noncompartmental pharmacokinetic methods: terminal half-life, clearance, mean residence time, volume of distribution, and volume of steady state.

[0090] Historically -based Assessments: Historical information includes general medical history, diabetes history, dialysis history, dialysis vintage, history of decrease in PDW, and type of vascular access for dialysis (arteriovenous fistula versus arteriovenous graft) and baseline score on the mCCI.

[0091] Normalized Protein Catabolic Rate (nPCR): The protein catabolic rate (PCR) quantifies protein catabolism and can be derived from the interdialytic increase in the rate of urea generation. The nPCR, calculated by dividing the PCR by the subject's weight, is correlated to dietary protein intake and studies have shown that moderate protein intake results in positive nitrogen balance without causing a need for increase in dialysis dose (Grupe et al. 1983). PCR is estimated according to the method of Randerson (Keshaviah et al. 1991) using the BUN and nPCR is calculated based on the PCR and the most recent PDW (Uribarri et al. 1997), using the following equations: PCR (g/day) = 5.02 x (BUN in mg/min + 3.12), and nPCR = PCR/PDW. The nPCR is calculated automatically on the eCRF based on the reported values for BUN and PDW. [0092] Muscle Function Tests: Two muscle function tests, the SCPT and the 6MWT, are used. Both the SCPT and 6MWT are performed on non-dialysis days due to expected subject fatigue on dialysis days (Majchrzak et al. 2005).

[0093] Myostatin, Cytokine, and Adipokine Biomarkers: As Compound 745 inhibits myostatin, serum myostatin levels are monitored to determine whether serum myostatin levels are differentially affected in subjects treated with Compound 745 compared with subjects treated with placebo. Also, levels of cytokines such as C-reactive protein, interleukin-6, and tumor necrosis factor alpha, as well as levels of adipokines such as adiponectin, leptin, and resistin are monitored to determine whether cytokine and adipokine levels are differentially affected in subjects treated with Compound 745 compared with subjects treated with placebo.

[0094] Serum Lipids: Levels of serum lipids are monitored in patients to determine if serum lipid levels are differentially affected in subjects treated with Compound 745 compared with subjects treated with placebo.

[0095] Serum Albumin: Serum albumin levels below 4.0 g/dL are correlated with increased mortality in patients with ESRD. Thus, serum albumin levels are monitored to determine if serum albumin levels are differentially affected in subjects treated with

Compound 745 compared with subjects treated with placebo.

[0096] Lean Body Mass (LBM): LBM, appendicular lean mass (ALM), and fat body mass is determined by using dual energy X-ray absorptiometry.

Efficacy Assessments

Post-Dialysis Weight

PDW is the subject's measured weight at the end of dialysis.

Normalized Protein Catabolic Rate (nPCR)

[0097] The protein catabolic rate (PCR) quantifies protein catabolism and can be derived from the interdialytic increase in the rate of urea generation. The nPCR, calculated by dividing the PCR by the subject's weight, is correlated to dietary protein intake and studies have shown that moderate protein intake results in positive nitrogen balance without causing a need for increase in dialysis dose (Grape et al. 1983). In this method of treatment, PCR should be documented using the value provided by the local laboratory. Every effort should be used to maintain the same laboratory for baseline and on-study treatment nPCR results to avoid variability. In the event that nPCR is not reported by the local laboratory, nPCR will be estimated according to the following equation (Jindal et al. 1988):

[0098] nPCR = 0.22 + (0.036 x intradialytic rise in BUN x 24)/(intradialytic interval)

[0099] For example, with a pre-dialysis BUN of 80 and post-dialysis BUN of 20, and an intradialytic interval of 44 hours (e.g., there is an interval of 44 hours from the end of one dialysis until the beginning of the next), then the nPCR is calculated to be 1.40 g/kg/day.

Body Composition

[00100] LBM, ALM, and fat body mass are determined by Dula energy X-Ray

Absorptiometry (DXA) using standard procedures well known to one of skill in the art, including those disclosed in Senver et al (1995) Am J Nephrol 15: 105-110.

Thigh Muscle Cross-sectional Area

[00101] Thigh muscle cross-sectional area is determined by Computed Tomography (CT) using standard procedures well known to one of skill in the art, including those disclosed in Kaizu et al (2003) American Journal of Kindey Diseses 42:295-302.

Muscle Function Tests

[00102] Two muscle function tests, the SCPT and the 6MWT, are used. Both the SCPT and 6MWT must be done on non-dialysis days due to expected subject fatigue on dialysis days (Majchrzak et al 2005).

Stair Climbing Power Test

[00103] The SCPT measures the power used to climb vertical steps as quickly and safely as possible, without running, and is calculated using the subject's weight, the time required for climbing, and the distance covered) (Bean et al. 2007). Timing of the test is facilitated by the use of photoelectric step switch pads that are interfaced to a timer that automatically records the interval between each of the subject's steps. Power is calculated using the following equations:

1. Velocity (v) = total vertical distance (d) / time required to climb the 10 steps (t)

2. Force = mass of the subject (m) x acceleration due to gravity (9.81 m/sec²)

3. Power = (Force) ^χ (v)

Six-minute Walk Test

[00104] The 6MWT measures the distance the subject covers when walking on a flat surface over a 6-minute time period (Guyatt et al. 1984; Hamilton et al. 2000). The 6MWT is performed in accordance with guidelines from the American Thoracic Society (ATS) regarding use of the 6MWT for evaluation of patients with heart or lung disease (Brooks et at. 2003). These guidelines are largely appropriate and adaptable for patients with ESRD, who may have concurrent heart or lung morbidities and have an overall reduced level of physical activity.

Patient-Reported Outcomes (PROs)

[00105] If PROs are assessed on the same day as a physical function test (6MWT or SCPT), the PRO tool (KDQOL™-36, FAACT anorexia cachexia scale, or FACIT-fatigue scale) should be administered prior to the first physical function test.

Kidney Disease Quality of Life Questionnaire

[00106] The KDQOL™-36 is 36-item survey that has been validated in patients on hemodialysis (Korevaar et al. 2002).

Functional Assessment Anorexia and Cachexia Treatment - Anorexia/Cachexia Subscale

[00107] The FAACT anorexia/cachexia subscale for anorexia and cachexia is quality-of life instrument comprising 12 questions about appetite and feelings toward food and eating. This instrument has been validated in patients with cancer (Ribaudo et al. 2000; Chang et al. 2005).

Functional Assessment of Chronic Illness Therapy-Fatigue Scale

[00108] The FACIT-Fatigue scale is quality-of life instrument comprising 13 questions about general fatigue and its effects on daily life. FACIT-Fatigue has been validated in patients with rheumatoid arthritis (Cella et al. 2005), chronic immune thrombocytopenia (Signorovitch et at. 2011 . paroxysmal nocturnal haemoglobinuria (Weitz et at. 2013), and systemic lupus erythematosus (Lai et al. 201 1 ).

Medical Resource Utilization

[00109] Utilization of healthcare resources and the duration of use is determined. The following resources are captured: Hospitalization; In the event of hospitalization, admission to the ICU; Visits to the emergency room; Use of mobility assistive devices (walker, cane, wheelchair); Other healthcare visits.

Biomarkers

Myostatin, Cytokines and Adipokines

[00110] Assays for myostatin, CRP, IL-6, TNF-a, adiponectin, leptin and resistin are performed using standard assays by a clinical laboratory.. Serum Albumin

[00111] Serum albumin levels below 4.0 g/dL are correlated with increased mortality in patients with ESRD (Lopes et al. 2010; Mazairac et al. 2011 ; Molnar et al. 201 1). Serum albumin will be measured as part of the standard clinical chemistry panel, and every effort should be made to standardize the time of collection so that albumin is consistently assessed either pre-dialysis or post-dialysis. Serum albumin is measured using standard methods including but limited to a bromocresol green or a bromocresol purple assay.

Example 3: Preliminary Pharmacokinetics of Compound 745

[00112] Using the methods described in Example 2, ESRD patients ("Cohort 1A" n=8, randomized 3: 1 active:placebo) were administered Compound 745 (or placebo) once weekly at a dose of 3mg/kg weekly for 12 weeks.

[00113] Plasma concentrations of Compound 745 are displayed in Figure 1 (FIG. 1). Preliminary PK data following initial dosing of all subjects in Cohort 1A (n=8) demonstrated a C_max of 63.1 ± 9.8 μg/mL, a half-life of 243 ± 261 hours, and an AUC₀-_t of 2480 ± 128 hxμg/mL in Week 1. PK data from 2 subjects were available at Week 12, following 12 weekly doses, which showed a mean C_max of 110 μg/mL, a mean half-life of 321 hours, and a mean AUCo-t of 22400 hxμg/mL. The steady-state Week 12 C_max and exposure exceeded pre- study expectations for the 3 mg/kg weekly dose. Because of the lower than expected clearance, exposure increased 9-fold, based on a comparison of Week 1 and Week 12 AUC values, with a continuing rise in AUC observed over the 12 weeks..

[00114] Compound 745 exposure is higher and the half-life longer than expected based on prior clinical studies in subjects without renal impairment. These data are consistent with a recent report in the literature (Wu and Sun, 2014) indicating that other peptibodies structurally similar to Compound 745 undergo renal clearance, and have decreased clearance and a prolonged half-life in renally-impaired patients.

[00115] As shown in Figure 2 (FIG. 2) PK modeling based on Cohort 1A data support the use of a loading dose in order to optimize achievement of steady state as illustrated for 3 loading doses of 6 mg/kg administered once weekly followed by 2 mg/kg once weekly as a maintenance regimen. Therefore, as described in Example 2, patients receive 3 weekly loading doses of 6 mg/kg Compound 745, followed thereafter by weekly maintenance doses of 2 mg/kg. This dosing regimen will result in an exposure similar to that of Cohort 1A and is expected to establish steady state exposure after approximately 3 weeks of dosing. [00116] Patients can also be administered Compound 745 as follows: 3 mg/kg Compound 745 weekly for 3 (loading) doses, followed thereafter by weekly 1 mg/kg (maintenance) doses. PK modeling indicates that this dosing schema in subjects with ESRD is expected to produce steady-state C_max as shown in Table 1. A weekly 3 mg/kg SC dose was associated with statistically significant changes in efficacy endpoints (i.e., increases in lean body mass, decreased fat mass, and increased lower extremity muscle size) compared with placebo (Padhi et al 2014).

Table 1 Simulated Pharmacokinetic Parameters for Compound 745

[00117] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[00118] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

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Claims

1. A method of treating protein energy wasting (PEW) in a human subject with End- Stage Renal Disease (ESRD) in need thereof comprising administering a therapeutically effective amount of Compound 745, an anti-myostatin peptibody consisting of two identical polypeptide chains each consisting of SEQ ID NO: l, wherein: the human subject has a serum albumin level < 3.8 g/dL and is undergoing maintenance hemodialysis (MHD);

Compound 745 is in a liquid pharmaceutical composition comprising 10 mM sodium acetate, 9% (w/v) sucrose, and 0.004% (w/v) polysorbate 20, pH 4.75.

Compound 745 is administered intravenously once weekly at a dose of 1 mg/kg to 10 mg/kg, optionally once weekly at a loading dose for a first time period followed by a maintenance dose for a second time period, the loading dose greater than the maintenance dose; and the method results a statistically significant increase in lean body mass (LBM) as measured by Dual-energy X-ray absorptiometry (DXA).

2. A method of treating protein energy wasting (PEW) in a human subject with End- Stage Renal Disease (ESRD) in need thereof comprising administering a therapeutically effective amount of Compound 745, an anti-myostatin peptibody consisting of two identical polypeptide chains each consisting of SEQ ID NO: l .

3. The method of claim 2, wherein the human subject has a serum albumin level < 3.8 g/dL, the serum albumin level determined by a bromocresol green or a bromocresol purple assay.

4. The method of claim 2, wherein the human subject has a serum albumin level < 3.8 g/dL at any time within 60 days prior to start of treatment, the serum albumin level determined by a bromocresol green or a bromocresol purple assay.

5. The method of any of the above claims, wherein the human subject has a body mass index (BMI) of < 28 kg/m2, based on a post-dialysis weight and a height of the human subject.

6. The method of any of the above claims, further comprising determining a body mass index and/or a serum albumin level of the human subject before treatment.

7. The method of any of the above claims, wherein the human subject is undergoing maintenance hemodialysis (MHD).

8. The method of any of the above claims, wherein the human subject is undergoing MHD > 3 times per week.

9. The method of any of the above claims, further comprising administering a nutritional supplement comprising > 10 grams of protein once daily to the human subject during at least a portion of the span of the treatment.

10. The method of any of the above claims, wherein Compound 745 is in a liquid pharmaceutical composition comprising 10 mM sodium acetate, 9% (w/v) sucrose, and 0.004% (w/v) polysorbate 20, pH 4.75.

11. The method of any of the above claims, wherein Compound 745 is administered by intravenous (IV) infusion.

12. The method of any of the above claims, wherein Compound 745 is produced by the method of expressing the Compound 745 as insoluble inclusion bodies in E. coli cells;

harvesting the cells; lysing the cells; solubilizing the inclusion bodies; refolding,

concentrating, and chromatographically purifying the Compound 745.

13. The method of any of the above claims, wherein Compound 745 is administered at a dose of 0.1 mg/kg-10 mg/kg.

14. The method of any of the above claims, wherein Compound 745 is administered at a dose of 1.0 mg/kg-10 mg/kg or 1.0 mg/kg or 2.0 mg/kg or 3.0 mg/kg or 6.0 mg/kg or 10 mg/kg.

15. The method of any of the above claims, wherein Compound 745 is administered once weekly.

16. The method of any of the above claims, wherein Compound 745 is administered once weekly at a loading dose for a first time period followed by a maintenance dose for a second time period, the loading dose greater than the maintenance dose.

17. The method of any of the above claims, wherein Compound 745 is administered once weekly at a dose of 3 mg kg for 3 weeks then 1 mg/kg for 9 weeks or at a dose of 6 mg/kg for 3 weeks then 2 mg/kg for 9 weeks.

18. The method of any of the above claims, wherein the method results in an increase in lean body mass (LBM) in the human subject.

19. The method of any of the above claims 1, wherein the method results in at least one clinical endpoint in the human subject, the endpoint selected from the group consisting of: an increase in LBM by Dual-energy X-ray absorptiometry (DXA), an increase in muscle cross sectional area by Computed tomography (CT), an increase in appendicular lean mass (ALM) by DXA, an increase in physical function by the Stair climbing power test (SCPT), an increase in distance on the 6-minute walk test (6MWT), an increase in quality of life using at least one of three Patient Reported Outcome (PRO tools) (the Kidney Dialysis Quality of Life™ (KDQOL™)-36 short form questionnaire, the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) scale, and the Functional Assessment of

Anorexia/Cachexia Treatment (FAACT) anorexia/cachexia subscale).

20. The method of any of the above claims, wherein the method results in a change in at least one biomarker selected from the group consisting of a serum myostatin, C-reactive protein, interleukin-6 (IL-6), tumor necrosis factor- a (TNF-a), adiponectin, leptin, resistin, serum lipid, and serum albumin.

21. A kit for treatment of protein energy wasting (PEW) in a human subject with End- Stage Renal Disease (ESRD) comprising a container comprising Compound 745 and instructions for use.

22. Compound 745 for use in the treatment of PEW in an ESRD patient undergoing MHD.

23. Use of Compound 745 for the treatment of PEW in an ESRD patient undergoing MHD.

24. Use of Compound 745 for the manufacture of a medicament for treatment of PEW in an ESRD patient undergoing MHD.

25. Method for manufacturing a medicament intended for treatment of PEW in an ESRD patient undergoing MHD, characterised in that Compound 745 is used.

26. The use of claims 22-25, wherein the ESRD patient has a serum albumin level < 3.8 g dL.

27. The use of claims 22-25, wherein Compound 745 is administered intravenously once weekly at a dose of 1 mg kg to 10 mg/kg, optionally once weekly at a loading dose for a first time period followed by a maintenance dose for a second time period, the loading dose greater than the maintenance dose.

28. The use of claims 22-25, wherein treatment results in a statistically significant increase in lean body mass (LBM) as measured by Dual-energy X-ray absorptiometry (DXA).