HK1241715B - Therapeutic dosing of a neuregulin or a subsequence thereof for treatment or prophylaxis of heart failure - Google Patents

Description

Therapeutic administration of neuregulin or its subsequences for treating or preventing heart failure

This application is a divisional application of the invention patent application No. 200980136079.8 filed on July 17, 2009.

【Technical field】

The field of the invention relates to treating heart failure. More particularly, the invention is directed to improved dosing regimens that maintain and/or enhance the therapeutic benefit of administering a neuregulin, such as glial growth factor 2 (GGF2), or a fragment thereof, while minimizing any potential side effects.

[Background Technology]

A fundamental challenge associated with administering medications to patients in need is the relationship between tolerability and efficacy. The therapeutic index is the range between the effective dose of a substance that can be administered to a patient and the dose at which undesirable side effects are detected in the patient. Generally speaking, the greater the difference between the effective dose and the dose at which side effects begin, the more benign the substance is and the more likely it is to be tolerated by the patient.

Heart failure, particularly congestive heart failure (CHF), is one of the leading causes of death in industrialized countries. Factors that underlie congestive heart failure include hypertension, ischemic heart disease, exposure to cardiotoxic compounds (e.g., anthracyclines), radiation exposure, physical trauma, and genetic defects associated with an increased risk of heart failure. Thus, CHF is often caused by increased workload on the heart due to hypertension, damage to the myocardium from chronic ischemia, myocardial infarction, viral disease, chemical toxicity, radiation, and other diseases (e.g., scleroderma). These conditions lead to a progressive decrease in the heart's pumping ability. Initially, the increased workload caused by hypertension or loss of contractile tissue induces compensatory cardiomyocyte hypertrophy and left ventricular wall thickening, thereby enhancing contractility and maintaining cardiac function. Over time, however, the left ventricle dilates, the systolic pump function deteriorates, cardiomyocytes undergo apoptotic cell death, and myocardial function progressively deteriorates.

Neuregulins (NRGs) and NRG receptors comprise a growth factor-receptor tyrosine kinase system involved in intercellular signaling in nerves, muscles, epithelia, and other tissues involved in organogenesis and cell development (Lemke, Mol. Cell. Neurosci. 7:247-262, 1996 and Burden et al., Neuron 18:847-855, 1997). The NRG family consists of four genes encoding various ligands containing epidermal growth factor (EGF)-like, immunoglobulin (Ig), and other recognition domains. Various secreted and membrane-bound isoforms function as ligands in this signal transduction system. The receptors for NRG ligands in humans are all members of the EGF receptor (EGFR) family, and include EGFR (or ErbB1), ErbB2, ErbB3, and ErbB4, also known as HER1 to HER4 (Meyer et al., Development 124:3575-3586, 1997; Orr-Urtreger et al., Proc. Natl. Acad. Sci. USA 90:1867-71, 1993; Marchionni et al., Nature 362:312-8, 1993; Chen et al., J. Comp. Neurol. 349:389-400, 1994; Corfas et al., Neuron 14:103-115, 1995; Meyer et al., Proc. Natl. Acad. Sci. USA 91:1064-1068, 1994; and Pinkas-Kramarski et al., Oncogene 15:2803-2815, 1997).

Four NRG genes, NRG-1, NRG-2, NRG-3, and NRG-4, are located at different chromosomal loci (Pinkas-Kramarski et al., Proc. Natl. Acad. Sci. USA 91:9387-91, 1994; Carraway et al., Nature 387:512-516, 1997; Chang et al., Nature 387:509-511, 1997; and Zhang et al., Proc. Natl. Acad. Sci. USA 94:9562-9567, 1997), and collectively encode a variety of NRG proteins. The gene product of NRG-1, for example, includes a group of approximately 15 different structurally-related isoforms (Lemke, Mol. Cell. Neurosci. 7:247-262, 1996 and Peles and Yarden, BioEssays 15:815-824, 1993). The first identified isoforms of NRG-1 include Neu differentiation factor (NDF; Peles et al., Cell 69, 205-216, 1992 and Wen et al., Cell 69, 559-572, 1992), nerve growth factor (HRG; Holmes et al., Science 256: 1205-1210, 1992), acetylcholine receptor inducing activity (ARIA; Falls et al., Cell 72: 801-815, 1993), and glial growth factors GGF1, GGF2, and GGF3 (Marchionni et al. Nature 362: 312-8, 1993).

The NRG-2 gene was identified by homology cloning (Chang et al., Nature 387:509-512, 1997; Carraway et al., Nature 387:512-516, 1997; and Higashiyama et al., J. Biochem. 122:675-680, 1997) and genomic methods (Busfield et al., Mol. Cell. Biol. 17:4007-4014, 1997). The NRG-2 cDNA is also known as neural- and thymus-derived ErbB kinase activator (NTAK; Genbank Accession No. AB005060), neuregulin-1 (Don-1), and cerebellar-derived growth factor (CDGF; PCT Application WO 97/09425). Experimental evidence suggests that cells expressing ErbB4 or a combination of ErbB2/ErbB4 may display a particularly robust response to NRG-2 (Pinkas-Kramarski et al., Mol. Cell. Biol. 18:6090-6101, 1998). The NRG-3 gene product (Zhang et al., supra) is also known to bind to and activate the ErbB4 receptor (Hijazi et al., Int. J. Oncol. 13:1061-1067, 1998).

An EGF-like domain is present in the core of all NRG forms and is required for binding and activation of ErbB receptors. The deduced amino acid sequences of the EGF-like domains encoded in the three genes are approximately 30-40% identical (alignment). Furthermore, NRG-1 and NRG-2 appear to have at least two subtypes of EGF-like domains, which may confer distinct biological activities and tissue-specific potentials.

Cellular responses to NRG are mediated by the NRG receptor tyrosine kinases EGFR, ErbB2, ErbB3, and ErbB4 of the epidermal growth factor receptor family. High-affinity binding of all NRGs is primarily mediated through either ErbB3 or ErbB4. Binding of NRG ligands leads to dimerization with other ErbB subunits and transactivation through phosphorylation of specific tyrosine residues. In certain experimental settings, almost all combinations of ErbB receptors appear to be able to form dimers in response to binding of NRG-1 isoforms. However, ErbB2 appears to be the preferred dimerization partner that may play an important role in stabilizing the ligand-receptor complex. ErbB2 does not bind ligands on its own, but must heterologously pair with one of the other receptor subtypes. ErbB3 does have tyrosine kinase activity, but is a target for phosphorylation by other receptors. Expression of NRG-1, ErbB2, and ErbB4 is known to be necessary for trabeculation of the ventricular myocardium during mouse development.

Neuregulin stimulates compensatory hypertrophic growth and inhibits apoptosis in cardiomyocytes undergoing physiological stress. Based on these observations, administration of neuregulin is useful for preventing, minimizing, or reversing congestive heart disease caused by underlying factors such as hypertension, ischemic heart disease, and cardiotoxicity. See, for example, U.S. Patent No. (USPN) 6,635,249, which is incorporated herein in its entirety.

Given the high prevalence of heart failure in the general population, there remains an unmet need to prevent or minimize the progression of this disease (eg, by inhibiting the loss of cardiac function or by improving cardiac function).

Summary of the invention

The present invention includes a method for treating or preventing heart failure in a mammal. The method is based on the surprising discovery that the therapeutic benefits of peptides comprising epidermal growth factor-like (EGF-like) domains can be achieved by a dosage regimen that does not maintain steady-state neuregulin administration (e.g., by administering a therapeutically effective amount of the peptide to a mammal at an administration interval of 48, 72, 96 or more hours). Thus, the present method requires intermittent or discontinuous administration (every 48 to 96 hours, or even longer intervals) of a peptide comprising an EGF-like domain to a mammal, wherein the EGF-like domain is encoded by a neuregulin gene, and wherein the administration of the peptide is in an amount effective for treating or preventing heart failure in a mammal. The dosage regimen that does not maintain steady-state concentrations of neuregulin administration is as effective as a more frequent dosage regimen, yet without the inconvenience, cost, or side effects of more frequent administration. As used herein, the term intermittent or discontinuous administration includes a dosing regimen at intervals of at least 48 hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, or any combination or increment thereof, so long as the interval/regime is at least 48 hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months. As used herein, the term intermittent or discontinuous administration includes dosing regimens at intervals of no less than 48 hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, or any combination or increment thereof, so long as the intervals/regime are no less than 48 hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months.

According to the present invention, intermittent or discontinuous administration of a peptide containing an EGF-like domain (wherein the EGF-like domain is encoded by a neuregulin gene) to a mammal is directed to a dosage regimen that achieves a narrow steady-state concentration of the administered peptide that is not maintained, thereby reducing the probability that the mammal will experience adverse side effects that may result from maintaining supraphysiological levels of the administered peptide for an extended period of time. For example, side effects associated with supraphysiological levels of exogenously administered NRG include: nerve sheath hyperplasia, breast hyperplasia, kidney disease, oligospermia, elevated liver enzymes, changes in heart valves, and skin changes at the injection site.

In a preferred embodiment, the present invention is directed to an intermittent dosage regimen that causes or allows fluctuations in serum levels of a peptide comprising an EGF-like domain encoded by a neuregulin gene, thereby reducing the potential for adverse side effects associated with more frequent administration of the peptide. The intermittent dosage regimen of the present invention thereby confers therapeutic advantages to mammals, but does not maintain steady-state therapeutic levels of a peptide comprising an EGF-like domain encoded by a neuregulin gene. As would be appreciated by one of ordinary skill in the art, there are various embodiments of the present invention to achieve intermittent administration; the benefits of these embodiments can be stated in various ways, for example, the administration does not maintain steady-state therapeutic levels of the peptide, the administration reduces the potential for adverse side effects associated with more frequent administration of NRG peptides, etc.

In certain embodiments of the invention, the neuregulin protein may be a gene comprising, consisting essentially of, or consisting of NRG-1, NRG-2, NRG-3, or NRG-4, a gene product, or a corresponding subsequence or fragment thereof. In a preferred embodiment, the NRG subsequence or fragment of the invention comprises an epidermal growth factor-like (EGF-like) domain or a homolog thereof. As recognized by one of ordinary skill in the art, homologs of peptides with EGF-like domains are determined by finding structural homology or by performing homologous peptides in functional assays as with EGF-like peptides (e.g., by binding to and activating ErbB receptors). Preferably, the fragment is at least 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 amino acids in length. The neuregulin peptides of the present invention can, in turn, be encoded by any of these neuregulin genes (or subsequences thereof). In more specific embodiments, the peptide used in the method is recombinant human GGF2 or a fragment or subsequence thereof. See Figures 8A to 8D for the amino acid and nucleic acid sequences of full-length human GGF2.

In one aspect of the invention, suitable mammals include, but are not limited to, mice, rats, rabbits, dogs, monkeys or pigs. In one embodiment of the invention, the mammal is a human.

In other embodiments of the invention, heart failure can result from hypertension, ischemic heart disease, exposure to cardiotoxic compounds (e.g., cocaine, alcohol, anti-ErbB2 antibodies or anti-HER antibodies, such as or anthracyclines, such as doxorubicin or daunomycin), myocarditis, thyroid disease, viral infection, gingivitis, drug abuse, alcohol abuse, pericarditis, atherosclerosis, vascular disease, hypertrophic cardiomyopathy, acute myocardial infarction or prior myocardial infarction, left ventricular systolic dysfunction, coronary artery bypass surgery, starvation, radiation exposure, eating disorders, or genetic defects.

In another embodiment of the invention, an anti-ErbB2 or anti-HER2 antibody is administered, for example, to a mammal before, during, or after administration of an anthracycline.

In other embodiments of the present invention, the peptide is administered before, during, or after exposure to a cardiotoxic compound; or before or after diagnosis of congestive heart failure in the mammal. The methods of the present invention may be performed after the subject mammal has experienced compensatory cardiac hypertrophy; the methods of the present invention include methods where the results of the methods are intended to maintain left ventricular hypertrophy, arrest the progression of myocardial thinning, or inhibit cardiomyocyte apoptosis. In the methods of the present invention, the peptide may comprise, consist essentially of, or consist of an EGF-like domain encoded by a neuregulin gene. The peptide of the present invention is administered before, during, or after exposure to a cardiotoxic compound. In another embodiment, the peptide containing an EGF-like domain is administered during two or all three of these periods. According to the present invention, the peptide containing an EGF-like domain encoded by a neuregulin gene is administered at intervals of 48 to 96 hours. In one embodiment of the present invention, the peptide containing an EGF-like domain encoded by a neuregulin gene is GGF2. In yet another embodiment of the present invention, the peptide is administered before or after diagnosis of congestive heart failure in the mammal. In yet another embodiment of the invention, the peptide is administered to a mammal that has undergone compensatory cardiac hypertrophy. In other specific embodiments of the invention, administration of the peptide maintains left ventricular hypertrophy, prevents progression of myocardial thinning, and/or inhibits cardiomyocyte apoptosis.

Embodiments of the present invention include the following: a method for treating heart failure in a mammal, the method comprising exogenously administering to the mammal a peptide comprising an epidermal growth factor-like (EGF-like) domain, wherein the administration at the intervals described reduces adverse side effects associated with the administration of the exogenous peptide in the mammal. A method for treating heart failure in a mammal, the method comprising exogenously administering to the mammal a peptide comprising an epidermal growth factor-like (EGF-like) domain, wherein the EGF-like domain is encoded by a neuregulin (NRG)-1 gene, and the exogenous peptide is administered at intervals of at least 48 hours in an amount therapeutically effective for treating heart failure in the mammal, wherein the administration at the intervals described does not maintain steady-state levels of the exogenous peptide in the mammal. A method for treating heart failure in a mammal, the method comprising exogenously administering to the mammal a peptide comprising an epidermal growth factor-like (EGF-like) domain or a homolog thereof, and administering the exogenous peptide in a therapeutically effective amount for treating heart failure in the mammal at intervals of at least or not less than 48 hours, wherein the administration at said intervals allows for intra-dose fluctuations in the serum concentration of the exogenous peptide in the mammal relative to a baseline or pre-administration level.

The term adverse or harmful side effects herein refers to unintended and undesirable results of medical treatment. With respect to the present invention, adverse or harmful side effects caused by the administration of exogenous peptides may include any one or more of the following: nerve sheath hyperplasia, breast hyperplasia, kidney disease, and skin changes at the injection site.

The term "intra-dose fluctuation of the serum concentration of the exogenous peptide in the mammal relative to the pre-administration level" herein refers to the difference between serum concentration levels before administration of a dose of the exogenous peptide.

The term "steady-state level" herein refers to a level of an exogenous agent (e.g., peptide) sufficient to achieve a balance between administration and elimination (within fluctuations between subsequent doses). "Maintaining steady-state therapeutic levels" refers to maintaining the concentration of the exogenous agent at a level sufficient to confer a therapeutic benefit to the subject or patient.

【Brief Description of the Drawings】

Figure 1 shows histograms depicting cardiac function as exemplified by changes in ejection fraction and fractional shortening. Rats were treated intravenously (iv) daily (q days) with 0.625 mg/kg of GGF2 or an equimolar amount of an EGF-like fragment (fragment; EGF-id), as indicated.

Figure 2 shows line graphs depicting cardiac function as shown by changes in ejection fraction and fractional shortening. Rats were treated intravenously with 0.625 mg/kg or 3.25 mg/kg of GGF2 for q day as indicated.

Figure 3 shows a line graph depicting cardiac function as shown by a significant improvement in end-systolic volume during the treatment period. Rats were treated intravenously with 0.625 mg/kg or 3.25 mg/kg of GGF2 for q days as indicated.

Figure 4 shows line graphs depicting cardiac function as indicated by changes in ejection fraction and fractional shortening. Rats were treated intravenously (iv) with 3.25 mg/kg of GGF2 q24, 48 or 96 hours as indicated.

Figure 5 shows a line graph depicting cardiac function as shown by changes in ejection fraction by echocardiography. Rats were treated intravenously (iv) with vehicle or 3.25 mg/kg of GGF2 (with or without BSA) as indicated.

Figure 6 shows a line graph depicting the half-life of recombinant human GGF2 (rhGGF2) following intravenous administration.

Figure 7 shows a line graph depicting the half-life of recombinant human GGF2 (rhGGF2) following subcutaneous administration.

8A-D show the nucleic acid and amino acid sequences of full-length GGF2. The nucleic acid sequence is designated SEQ ID NO: 1 and the amino acid sequence is designated SEQ ID NO: 2.

Figure 9 shows the nucleic acid and amino acid sequences of epidermal growth factor-like (EGFL) domain 1. The nucleic acid sequence of EGFL domain 1 is designated herein as SEQ ID NO: 3 and the amino acid sequence of EGFL domain 1 is designated herein as SEQ ID NO: 4.

Figure 10 shows the nucleic acid and amino acid sequences of epidermal growth factor-like (EGFL) domain 2. The nucleic acid sequence of EGFL domain 2 is designated herein as SEQ ID NO: 5 and the amino acid sequence of EGFL domain 2 is designated herein as SEQ ID NO: 6.

Figure 11 shows the nucleic acid and amino acid sequences of epidermal growth factor-like (EGFL) domain 3. The nucleic acid sequence of EGFL domain 3 is designated herein as SEQ ID NO: 7 and the amino acid sequence of EGFL domain 3 is designated herein as SEQ ID NO: 8.

Figure 12 shows the nucleic acid and amino acid sequences of epidermal growth factor-like (EGFL) domain 4. The nucleic acid sequence of EGFL domain 4 is designated herein as SEQ ID NO: 9 and the amino acid sequence of EGFL domain 4 is designated herein as SEQ ID NO: 10.

Figure 13 shows the nucleic acid and amino acid sequences of epidermal growth factor-like (EGFL) domain 5. The nucleic acid sequence of EGFL domain 5 is designated herein as SEQ ID NO: 11 and the amino acid sequence of EGFL domain 5 is designated herein as SEQ ID NO: 12.

Figure 14 shows the nucleic acid and amino acid sequences of epidermal growth factor-like (EGFL) domain 6. The nucleic acid sequence of EGFL domain 6 is designated herein as SEQ ID NO: 13 and the amino acid sequence of EGFL domain 6 is designated herein as SEQ ID NO: 14.

Figure 15 shows the amino acid sequence of a polypeptide comprising an epidermal growth factor-like (EGFL) domain, designated herein as SEQ ID NO: 21.

FIG. 16 shows schematic diagrams of the pSV-AHSG vector and the pCMGGF2 vector containing the coding sequence of human GGF2.

Figure 17 shows a schematic diagram of the placement of the GGF2 coding sequence after the EBV BMLF-1 interfering sequence (MIS) to enhance transcription.

[Detailed description of the invention]

The present inventors have made the surprising discovery that discontinuous or intermittent administration of neuregulin at appropriately spaced time intervals delivers therapeutically effective amounts of neuregulin to a patient in need thereof and that such treatment regimens are useful for preventing, prophylaxis, amelioration, minimization, treatment or reversal of heart disease, such as congestive heart failure.

While conventional wisdom and development practice dictate that dosage regimens be designed to maintain the narrowest range of steady-state concentrations, the inventors herein demonstrate that dosage regimens for neuregulin administration that do not maintain a narrow steady-state concentration are as effective as more frequent dosage regimens. Indeed, the inventors demonstrate that neuregulin administered at intervals of at least 48 hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, or any combination or increment thereof, is as effective as daily dosing for treating heart failure as long as the interval/regimen is at least 48 hours.

To assess the pharmacokinetics of exogenous NRG, the inventors showed that the half-life of neuregulin is 4-8 hours when delivered intravenously and 11-15 hours when delivered subcutaneously. See, for example, Tables 1 and 2 and Figures 6 and 7. Dosing on a schedule as infrequent as every fourth day will, therefore, not maintain any detectable levels for at least 3 days between doses. Based on these findings, prior to the present invention, it would not have been predicted that such peak/trough ratios would be associated with consistent therapeutic benefit. Notably, compounds with half-lives of this magnitude are typically administered according to frequent dosing schedules (e.g., one dose per day or multiple doses per day). Indeed, based on the pharmacokinetic data available for GGF2, traditional development would predict that optimal treatment would involve daily subcutaneous dosing.

In accordance with conventional wisdom and ongoing practice, other medical treatments for CHF are generally administered on at least a daily basis. This cyclical nature of the regimen is considered necessary because CHF is a chronic disease, often caused by impaired contraction and/or relaxation of the heart, rather than an acute condition. In people with weakened hearts that contribute to impaired relaxation and CHF, medical treatments include medications that block the production or action of specific neurohormones (e.g., angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin receptor blockers (ARBs), aldosterone antagonists, and beta-adrenergic receptor blockers). These and other medications are now the standard of care in chronic CHF because they have been shown to result in improved symptoms, life expectancy, and/or reduced hospitalizations. In cases of acute exacerbations or chronic symptoms, patients are often treated with inotropic agents (e.g., dobutamine, digoxin) to increase cardiac contractility, along with vasodilators (e.g., nitrates, nesiritide) and/or diuretics (e.g., furosemide) to reduce congestion. Patients with hypertension and congestive heart failure are treated with one or more antihypertensive agents (eg, beta-blockers, ACE-inhibitors and ARBs, nitrates (isosorbide dinitrate), hydralazine, and calcium channel blockers).

Thus, despite typical practice with respect to the treatment of CHF, the present inventors have demonstrated that a novel dosing regimen results in effective treatment of CHF while avoiding undesirable side effects. While not wishing to be bound by theory, it is possible that the neuregulin treatment enhances the heart's pumping capacity by stimulating cardiomyocyte hypertrophy and partially or completely inhibiting further deterioration of the heart by inhibiting cardiomyocyte apoptosis.

As additional background, the rationale for dosing is to determine effective circulating concentrations and design a dosage regimen to maintain those levels. Pharmacokinetic (PK) and pharmacodynamic (PD) studies are combined to predict the dosage regimen for a particular drug that will maintain steady-state levels. Typical plans aim to minimize the difference between Cmax and Cmin, thereby reducing side effects.

Drugs are described by their 'therapeutic index,' which is the ratio of the toxic dose or circulating level divided by the effective dose or circulating concentration. When the therapeutic index is large, there is a wide safe range over which the effective dose can be administered without approaching toxic levels. When adverse effects are achieved at concentrations too close to the effective concentration, the therapeutic index is described as narrow, and the drug is difficult to administer safely.

When developing a dosage regimen, PK/PD data is combined with knowledge of the therapeutic index to design the dose and frequency of administration to maintain a concentration of the compound in the patient (e.g., human) above the effective concentration and below the toxic concentration. If an effective concentration of the drug cannot be maintained without inducing unsafe effects, the drug will fail during development. Additional commentary related to drug development can be found in various references, including: Pharmacokinetics in Drug Development: Clinical Study Design and Analysis (2004, Peter Bonate and Danny Howard, eds.), which is incorporated herein in its entirety.

Neuregulins are growth factors related to epidermal growth factor that bind to erbB receptors. They have been shown to improve cardiac function in multiple models of heart failure, cardiotoxicity, and ischemia. They have also been shown to protect the nervous system in models of stroke, spinal cord injury, exposure to neurological agents, peripheral nerve injury, and chemical toxicity.

However, maintaining excessive levels of exogenously supplied neuregulin has been shown to have adverse effects, including nerve sheath hyperplasia, breast hyperplasia, and kidney disease. These effects were observed after daily subcutaneous administration of neuregulin. See, for example, Table 10.

As described herein, subcutaneous administration was explored due to its extended half-life compared to intravenous administration, and it was originally believed that maintaining a constant level of ligand would be advantageous. Developing a dosage regimen to reduce these effects would significantly enhance the ability of neuregulin to be used as a therapeutic agent, and the present invention is also directed to this. Demonstrating that less frequent dosing without maintaining a constant level would also enable this development.

Neuregulins: As described above, the peptides encoded by the NRG-1, NRG-2, NRG-3, and NRG-4 genes possess an EGF-like domain that enables them to bind to and activate ErbB receptors. Holmes et al. (Science 256:1205-1210, 1992) showed that the EGF-like domain alone is sufficient to bind to and activate the p185erbB2 receptor. Thus, any peptide product encoded by the NRG-1, NRG-2, or NRG-3 gene, or any neuregulin-like peptide, for example, a peptide having an EGF-like domain encoded by a neuregulin gene or cDNA (e.g., an EGF-like domain containing the NRG-1 peptide subdomains C-C/D or C-C/D', as described in U.S. Patent Nos. 5,530,109, 5,716,930, and 7,037,888; or an EGF-like domain as disclosed in WO 97/09425) can be used in the methods of the present invention to prevent or treat congestive heart failure. The contents of each of U.S. Patent Nos. 5,530,109; 5,716,930; 7,037,888; and WO 97/09425 are incorporated herein in their entirety.

Risk factors: Risk factors that increase an individual's likelihood of developing congestive heart failure are well known. These include, but are not limited to, smoking, obesity, hypertension, ischemic heart disease, vascular disease, coronary artery bypass surgery, myocardial infarction, left ventricular systolic dysfunction, exposure to cardiotoxic compounds (alcohol, drugs (e.g., cocaine), and anthracycline antibiotics (e.g., doxorubicin and daunorubicin)), viral infections, pericarditis, myocarditis, gingivitis, thyroid disease, radiation exposure, genetic defects known to increase the risk of heart failure (e.g., as described in Bachinski and Roberts, Cardiol. Clin. 16:603-610, 1998; Siu et al., Circulation 8:1022-1026, 1999; and Arbustini et al., Heart 80:548-558, 1998), starvation, eating disorders (e.g., anorexia and bulimia), family history of heart failure, and cardiac hypertrophy.

According to the present invention, neuregulin can be administered intermittently to achieve prevention, for example, by preventing or reducing the rate of progression of congestive heart disease in those identified as at risk. For example, administration of neuregulin to patients with early compensatory hypertrophy allows for maintenance of the hypertrophic state and prevents progression to heart failure. In addition, cardioprotective neuregulin treatment can be given to those identified as at risk before the development of compensatory hypertrophy.

Administration of neuregulin to cancer patients before and during anthracycline chemotherapy or anthracycline/anti-ErbB2 (anti-HER2) antibody (e.g., ) combination therapy can prevent the patient's cardiomyocytes from undergoing apoptosis, thereby preserving cardiac function. Patients who have already suffered cardiomyocyte loss also benefit from neuregulin treatment because the remaining myocardial tissue responds to neuregulin exposure by exhibiting hypertrophic growth and increased contractility.

Treatment: Neuregulin and peptides containing an EGF-like domain encoded by the neuregulin gene and a pharmaceutically acceptable diluent, carrier, or excipient can be administered to a patient or an experimental animal. The composition of the present invention can be provided in unit dosage form.

Conventional pharmaceutical practices are used to provide suitable formulations or compositions and to administer the compositions to patients or experimental animals. Although intravenous administration is preferred, any appropriate route of administration may be employed, for example, parenteral, subcutaneous, intramuscular, transdermal, intracardial, intraperitoneal, intranasal, aerosol, oral, or topical (e.g., by application of an adhesive patch with a formulation that crosses the dermis and enters the bloodstream).

Therapeutic preparations can take the form of liquid solutions or suspensions; for oral administration, preparations can take the form of tablets or capsules; and for intranasal preparations, in the form of powders, nasal drops, or aerosols.

Methods for preparing formulations well known in the art are described, for example, in "Remington's Pharmaceutical Sciences". Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols, such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Other potentially useful parenteral delivery systems for administering the molecules of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, such as lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, and deoxycholate, or may be oily solutions for administration as nasal drops or as a gel.

As a further aspect of the present invention, there is provided the use of the present compound as a medicament, in particular for the treatment or prevention of the aforementioned conditions and diseases. Also provided herein is the use of the present compound in the preparation of a medicament for the treatment or prevention of one of the aforementioned conditions and diseases.

For intravenous injection, the dosage level varies from about 0.001 mg/kg, 0.01 mg/kg to at least 10 mg/kg, at regular intervals of at least about every 24, 36, 48 hours to about every 96 hours, and especially every 48, 72, or 96 hours or more, as described herein. In a specific embodiment, the intravenous dosage level varies from about 0.1 mg/kg to about 10 mg/kg, at regular intervals of about every 48 hours to about every 96 hours, and especially every 48, 72, or 96 hours or more, as described herein. In another specific embodiment, the intravenous dosage level varies from about 1 mg/kg to about 10 mg/kg, at regular intervals of about every 48 hours to about every 96 hours, and especially every 48, 72, or 96 hours or more, as described herein. In yet another specific embodiment, the intravenous dosage level varies from about 0.01 mg/kg to about 1 mg/kg, at regular intervals of from about every 48 hours to about every 96 hours, and particularly every 48, 72, or 96 hours or more, as described herein. In yet another specific embodiment, the intravenous dosage level varies from about 0.1 mg/kg to about 1 mg/kg, at regular intervals of from about every 48 hours to about every 96 hours, and particularly every 48, 72, or 96 hours or more, as described herein.

For subcutaneous injection, the dosage level varies from about 0.01 mg/kg to at least 10 mg/kg, at regular intervals from about every 48 hours to about every 96 hours, and particularly every 48, 72, or 96 hours or more, as described herein. In a specific embodiment, the injection dosage level varies from about 0.1 mg/kg to about 10 mg/kg, at regular intervals from about every 48 hours to about every 96 hours or more, and particularly every 48, 72, or 96 hours, as described herein. In another specific embodiment, the injection dosage level varies from about 1 mg/kg to about 10 mg/kg, at regular intervals from about every 48 hours to about every 96 hours or more, and particularly every 48, 72, or 96 hours, as described herein.

In yet another specific embodiment, the injected dose level varies from about 0.01 mg/kg to about 1 mg/kg, at regular intervals from about every 48 hours to about every 96 hours or more as described herein, and particularly every 48, 72, or 96 hours. In yet another specific embodiment, the injected dose level varies from about 0.1 mg/kg to about 1 mg/kg, at regular intervals from about every 48 hours to about every 96 hours or more as described herein, and particularly every 48, 72, or 96 hours.

Transdermal doses are generally selected to provide similar or lower blood levels than achieved using injected doses.

The compounds of the present invention can be administered as the sole active agent, or they can be administered in combination with other agents, including other compounds that exhibit the same or similar therapeutic activity and are determined to be safe and effective for administration in combination. Other such compounds useful in treating CHF include: brain natriuretic peptide (BNP), drugs that block the formation or action of specific neurohormones (e.g., angiotensin-converting enzyme inhibitors (ACE-inhibitors), angiotensin receptor antagonists (ARBs), aldosterone antagonists, and beta-adrenergic receptor blockers), inotropic agents that increase cardiac contractility (e.g., dobutamine, digoxin), vasodilators that reduce congestion (e.g., nitrates, nesiritide) and/or diuretics (e.g., furosemide), and one or more antihypertensive agents (e.g., beta-blockers, ACE-inhibitors, and ARBs), nitrates (isosorbide dinitrate), hydralazine, and calcium channel blockers.

As described above, medical interventions involving drug therapy require the selection of an appropriate drug and its delivery in an adequate dosage regimen. An adequate dosage regimen involves adequate dosage, route, frequency, and duration of treatment. The fundamental goal of drug therapy is to achieve optimal drug concentrations at the site of action so that the treated patient overcomes the pathological process that requires treatment. Broadly speaking, a basic knowledge of the principles of drug management assists in the selection of an appropriate dosage regimen. However, therapeutic drug monitoring (TDM) can be used here as a supplemental tool to assist the attending physician in determining an effective and safe dosage regimen for a selected drug for medical treatment of an individual patient.

Target Concentration and Therapeutic Window: The definition of optimal drug concentration varies depending on the pharmacodynamic characteristics of a particular drug. Optimal therapy for time-dependent antibiotics (e.g., penicillin), for example, is associated with achieving a ratio of peak concentration to MIC (minimum inhibitory concentration) of 2 to 4 and a time above the MIC equal to 75% of the dosing interval. For concentration-dependent antibiotics (e.g., gentamicin), efficacy is associated with achieving a peak concentration to MIC ratio of approximately 8 to 10. Regardless of the nuances associated with administration of a particular drug, drug therapy aims to achieve target plasma concentrations (which often reflect the concentration at the site of action) within the limits of a "therapeutic window" that has been previously determined based on the drug's pharmacokinetic, pharmacodynamic, and toxicity characteristics in the target species. The width of this window varies for different drugs and species. When the difference between the minimum effective concentration and the minimum toxic concentration is small (2- to 4-fold), the therapeutic window is considered narrow. Conversely, when there is a large difference between the effective and toxic concentrations, the drug is considered to have a wide therapeutic window. An example of a drug with a narrow therapeutic window is digoxin, where the difference between the average effective and toxic concentrations is 2- or 3-fold. On the other hand, amoxicillin has a wide therapeutic range, and overdose of patients is not usually associated with toxicity problems.

Survival in Drug Response: Significant survival is common in healthy subjects of the same species with corresponding drug responses. Furthermore, disease states have the potential to affect organ systems and functions (e.g., kidney, liver, water content) that can in turn affect drug response. This, in turn, contributes to increased variability in drug response among diseased individuals administered drugs. However, another related issue involves administering more than one drug at a time that results in pharmacokinetic interactions that can lead to altered responses to one or both drugs. In summary, physiological (e.g., age), pathological (e.g., disease effects), and pharmacological (e.g., drug interactions) factors can alter drug disposition in animals. The resulting increased survival in individuals can lead to therapeutic failure or toxicity of drugs with narrow therapeutic indices.

The patient population that will benefit from the treatment regimen of the present invention is quite diverse, for example, patients with impaired renal function are good candidates because continuous levels of protein therapeutics are often associated with renal glomerular deposits. Therefore, the practicality of the therapeutic regimen of not maintaining constant plasma levels as described in the present invention will be very beneficial for patients with mild renal function to which any reduction in existing function may be harmful. Similarly, as described herein, short-term and intermittent exposure to therapeutic agents (e.g., GGF2) can be beneficial for patients with tumor types that respond to chronic and continuous stimulation of growth factors. Other patients that can specifically benefit from intermittent treatment as described herein are patients with Schwannoma and other peripheral neuropathies. This is an advantage of the present invention, that intermittent administration can have significant advantages in not maintaining continuous side effect-related stimulation of each tissue.

The appropriate timing of blood sampling for the purpose of determining serum drug levels, as well as the interpretation of reported levels, requires consideration of the pharmacokinetic properties of the drug being measured. Some terms used in the discussion of these properties are defined in the following paragraphs.

Half-life: The time required for the serum concentration present at the beginning of the interval to decrease by 50%. Knowing the approximate half-life is essential to the clinician as he or she determines the optimal dosing schedule for oral agents, intra-dose fluctuations in serum concentrations, and the time required to reach steady state.

In brief, numerous pharmacokinetic studies have been conducted on GGF2. The typical half-life of GGF2 is between 4 and 8 hours for intravenous (iv) administration, whereas the half-life of GGF2 administered subcutaneously (sc) is between 11 and 15 hours. Cmax, AUC, Tmax, and T1/2 are shown in Tables 1 and 2 below. When the half-life was too long to be accurately determined by these methods, several doses were used instead of one.

Table 1 and Table 2

Appendix 7

Mean pharmacokinetics of 125I-rhGGF2-derived radioactivity in plasma of male Sprague-Dawley rats following a single intravenous or subcutaneous dose of 125I-rhGGF2

Mean pharmacokinetics of 125I-rhGGF2-derived radioactivity in plasma of male Sprague-Dawley rats following a single intravenous or subcutaneous dose of 125I-rhGGF2

Appendix 9

For intravenous and subcutaneous administration, the plasma concentrations after administration are shown in Figures 6 and 7, respectively. As shown in Figures 6 and 7, Cmax refers to the maximum plasma concentration (the maximum concentration measured in plasma at any time after administration); AUCinf refers to the area under the concentration versus time curve for time infinity (this method is used to predict the detection limit of the assay); AUC0-t refers to the area under the plasma concentration (the time curve from the start of the timing to the last measurable concentration); AUC by any method refers to an estimate of the total exposure to the animal; and Tmax refers to the median time of maximum plasma concentration.

As demonstrated by the tables and figures, it is not possible to maintain steady state therapeutic levels by dosing every fourth day, every other day, or every day. After one day and even long before, levels are not measurable, as reflected by the data shown in Table 11.

*Derived from data obtained from plasma GGF2 concentrations measured by ELISA. Data reported are mean ± SD.

Steady State: Steady state serum concentrations are those values that are returned to with each dose and represent the state of equilibrium between the amount of drug administered and the amount eliminated in a given time interval. During chronic dosing of any drug, the two main determinants of its average steady state serum concentration are the rate of drug administration and the total clearance of the drug in a particular patient.

Peak serum concentration: The point of maximum concentration on the serum concentration-versus-time curve. The exact time of peak serum concentration is difficult to predict because it represents a complex relationship between input and output rates.

Trough serum concentration: The minimum serum concentration found during the dosing interval. Trough concentrations ideally occur in the period immediately prior to the next dose.

Absorption: The process by which a drug enters the body. Intravascularly administered drugs are generally absorbed, but extravascular administration produces varying degrees and rates of absorption. The relationship between absorption rate and elimination rate is the primary determinant of drug concentration in the bloodstream.

Distribution: Diffusion of systemically available drug from the intravascular space to the extravascular fluid and tissues and thus to target receptor sites.

Therapeutic range: The range of serum drug concentrations associated with a high degree of efficacy and a low risk of dose-related toxicity. The therapeutic range is a statistical concept: it is the concentration range associated with a therapeutic response in the majority of patients. Consequently, some patients exhibit a therapeutic response at serum levels below the lower limit of the range, while others require serum levels above the upper limit for therapeutic benefit.

Correct timing of sample collection is important, as drug therapy is often adjusted based on serum concentration measurements. Absorption and distribution phases should be complete, and steady-state concentrations should be achieved before samples are drawn. Levels obtained before steady-state concentrations exist may be falsely low; increasing the dose based on these results may result in toxic concentrations. Furthermore, consistent sampling timing is important when performing comparative measurements.

The timing of blood sampling relative to dose is critical for the correct interpretation of serum concentration results. The choice of sample withdrawal time relative to drug administration should be based on the pharmacokinetic properties of the drug, its dosage form, and the clinical reason for measuring the sample (e.g., efficacy assessment or elucidation of possible drug-induced toxicity). For routine serum level monitoring of drugs with short half-lives, steady-state peak and trough samples can be collected to characterize serum concentration profiles; for drugs with long half-lives, steady-state trough samples alone are usually sufficient.

"Congestive heart failure" refers to impaired heart function that renders the heart unable to maintain normal blood output at rest or during exercise, or unable to maintain normal cardiac output under normal cardiac filling pressures. A left ventricular ejection fraction of about 40% or less indicates congestive heart failure (for comparison, an ejection fraction of about 60% is normal). Patients with congestive heart failure display well-known clinical symptoms and signs, such as shortness of breath, pleural effusion, fatigue at rest or during exercise, systolic dysfunction, and edema. Congestive heart failure is readily diagnosed by well-known methods (see, e.g., "Consensus recommendations for the management of chronic heart failure." Am. J. Cardiol., 83(2A): 1A-38-A, 1999).

Relative severity and disease progression are assessed using well-known methods, such as physical examination, echocardiography, radionuclide imaging, invasive hemodynamic monitoring, magnetic resonance angiography, and exercise bicycle testing coupled with oxygen uptake studies.

"Ischemic heart disease" refers to any condition caused by an imbalance between the myocardium's need for oxygen and its adequate supply. Most cases of ischemic heart disease result from narrowing of the coronary arteries, as occurs in atherosclerosis or other vascular disorders.

"Myocardial infarction" refers to the process in which ischemic disease results in an area of myocardium being replaced by scar tissue.

"Cardiotoxic" refers to compounds that reduce cardiac function by directly or indirectly damaging or killing cardiomyocytes.

"Hypertension" refers to blood pressure that is considered by a medical professional (eg, a physician or nurse) to be higher than normal and carries an increased risk of developing congestive heart failure.

"Treatment" refers to the administration of a neuregulin or neuregulin-like peptide that, during the treatment period, slows or inhibits the progression of congestive heart failure in a statistically significant manner relative to the progression of the disease that would occur in the absence of treatment. Well-known markers (e.g., left ventricular ejection fraction, exercise performance, and other clinical tests listed above), as well as survival rates and hospitalization rates can be used to assess disease progression. Whether treatment slows or inhibits disease progression in a statistically significant manner can be determined by methods well known in the art (see, e.g., SOLVD Investigators, N. Engl. J. Med. 327: 685-691, 1992 and Cohn et al., N. Engl. J Med. 339: 1810-1816, 1998).

"Prevention" refers to minimizing or partially or completely inhibiting the development of congestive heart failure in a mammal at risk of developing congestive heart failure (as specified in "Consensus recommendations for the management of chronic heart failure." Am. J. Cardiol., 83(2A): 1A-38-A, 1999). It is determined whether congestive heart failure is minimized or prevented by administering a neuregulin or neuregulin-like peptide prepared by known methods, such as those described in SOLVD Investigators, supra, and Cohn et al., supra.

The term "therapeutically effective amount" is intended to mean an amount of a drug or pharmaceutical agent that elicits a biological or medical response in a tissue, system, animal, or human as observed by a researcher, veterinarian, medical doctor, or other clinician. A therapeutic change is a change in a biochemical characteristic measured in the direction of expected alleviation of a disease or resolution of a condition. More specifically, a "therapeutically effective amount" is an amount sufficient to reduce symptoms associated with a medical condition or infirmity, to normalize bodily function in a disease or disorder that results in impairment of a specific bodily function, or to provide improvement in one or more clinically measured disease parameters.

The term "prophylactically effective amount" is intended to mean that amount of a pharmaceutical agent that will prevent or reduce the risk of occurrence of a biological or medical event observed by a researcher, veterinarian, medical doctor or other clinician.

The term "therapeutic window" is intended to mean the range of dosages between the minimum amount that achieves any therapeutic change and the maximum amount that results in a response immediately preceding toxicity to the patient.

"At risk for congestive heart failure" refers to individuals who smoke, are obese (i.e., 20% or more over their ideal weight), have been or will be exposed to cardiotoxic compounds (e.g., anthracyclines), or have (or have had) hypertension, ischemic heart disease, myocardial infarction, a genetic defect known to increase the risk of heart failure, a family history of heart failure, cardiac hypertrophy, hypertrophic cardiomyopathy, left ventricular systolic dysfunction, coronary artery bypass surgery, vascular disease, atherosclerosis, alcoholism, pericarditis, viral infection, gingivitis, or an eating disorder (e.g., anorexia nervosa or bulimia nervosa), or are addicted to alcohol or cocaine.

"Reducing the progression of myocardial thinning" refers to maintaining ventricular cardiomyocyte hypertrophy such that ventricular wall thickness is maintained or increased.

"Inhibition of myocardial apoptosis" means that neuregulin treatment inhibits myocardial cell death by at least 10%, more preferably at least 15%, even more preferably at least 25%, even more preferably at least 50%, even more preferably at least 75%, and most preferably at least 90% compared to untreated myocardial cells.

"Neuregulin" or "NRG" refers to a peptide encoded by an NRG-1, NRG-2, or NRG-3 gene or nucleic acid (eg, cDNA) and that binds and activates an ErbB2, ErbB3, or ErbB4 receptor, or a combination thereof.

"Neuregulin-1," "NRG-1," "nerve growth factor," "GGF2," or "p185erbB2 ligand" refers to a peptide that binds to the ErbB2 receptor when paired with another receptor (ErbB1, ErbB3, or ErbB4), and is encoded by the p185erbB2 ligand gene, as described in U.S. Patent No. 5,530,109; U.S. Patent No. 5,716,930; and U.S. Patent No. 7,037,888, each of which is incorporated herein by reference in its entirety.

"Neuregulin-like peptide" refers to a peptide having an EGF-like domain encoded by the neuregulin gene and binding to and activating ErbB2, ErbB3, ErbB4, or a combination thereof.

"Epidermal growth factor-like domain" or "EGF-like domain" refers to a domain that binds and activates ErbB2, ErbB3, ErbB4, or a combination thereof, and as disclosed in Holmes et al., Science 256:1205-1210, 1992; U.S. Patent No. 5,530,109; U.S. Patent No. 5,716,930; U.S. Patent No. 7,037,888; Hijazi et al., Int. J. Oncol. 13:1061-1067, 1998; Chang et al., Nature 387:509-512, 1997; Carraway et al., Nature 387:512-516, 1997; Higashiyama et al., J Biochem. 122:675-680, 1997; and WO The EGF receptor-binding domain of 97/09425 has structural similarity to a peptide motif encoded by the NRG-1, NRG-2, or NRG-3 genes. See Figures 9-14 for the nucleic acid and amino acid sequences corresponding to EGFL domains 1-6 encoded by the NRG-1 gene.

"Anti-ErbB2 antibody" or "anti-HER2 antibody" refers to an antibody that specifically binds to the extracellular domain of the ErbB2 (also known as HER2 in humans) receptor and prevents the initiation of ErbB2 (HER2)-dependent signaling through neuregulin binding.

"Transformed cell" refers to a cell (or cell progeny) into which a DNA molecule encoding neuregulin or a peptide having a neuregulin EGF-like domain has been introduced using recombinant DNA technology or known gene therapy techniques.

"Promoter" refers to the minimal sequence sufficient to direct transcription. Also included in the present invention are promoter elements sufficient to render promoter-dependent gene expression controllable based on cell type or physiological state (e.g., hypoxic versus normoxic conditions), or inducible by external signals or agents; such elements may be located in the 5' or 3' or internal regions of the native gene.

"Operably linked" refers to a nucleic acid (eg, cDNA) encoding a peptide and one or more regulatory sequences that are linked in such a way that gene expression is achieved when an appropriate molecule (eg, a transcriptional activator protein) binds to the regulatory sequences.

An "expression vector" refers to a genetically engineered plasmid or virus derived from, for example, a bacteriophage, adenovirus, retrovirus, poxvirus, herpesvirus, or artificial chromosome, that is used to transfer a peptide (e.g., neuregulin) coding sequence operably linked to a promoter to a host cell such that the encoded peptide or peptides are expressed in the host cell.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The discussion of documents, acts, materials, devices, articles and the like included in this specification is intended only to provide a background to the present invention. It is not intended to suggest or represent that any or all of these forms of invention are prior art or were common general knowledge in the field relevant to the present invention before the priority date of the respective claims of this application.

[Other implementation methods]

While the invention has been described in conjunction with particular embodiments thereof, it will be appreciated that it is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention which follow generally from the principles of the invention and which come within known or customary practice in the art to which the invention pertains and which may be applied to the essential features set forth above and which fall within the scope of the appended claims.

The following examples will assist those skilled in the art to better understand the present invention and its principles and advantages. It is hoped that these examples are for the purpose of illustrating the present invention and not for limiting its scope.

[Example]

As shown herein above, neuregulins are a family of growth factors that are structurally related to epidermal growth factor (EGF) and are essential for normal heart development. Evidence suggests that neuregulins are potential therapeutic agents for the treatment of heart diseases, including heart failure, myocardial infarction, chemotherapeutic agent toxicity, and viral myocarditis.

The studies described herein were used to define dosing in the left anterior descending (LAD) artery ligation model of congestive heart failure in rats. Multiple neuregulin splice variants were cloned and generated. A neuregulin fragment consisting of an EGF-like domain (EGF-ld) from a previous report (Liu et al., 2006) was compared with a full-length neuregulin called glial growth factor 2 (GGF2) and an EGF-like structure with an Ig domain (EGF-Ig). Male and female Sprague-Dawley rats underwent LAD artery ligation. Seven days after ligation, the rats were treated daily with neuregulin intravenously (iv). Cardiac function was monitored by echocardiography.

The first study compared 10 days of administration of equimolar amounts of EGF-1d or GGF2 (for GGF2, this was calculated as 0.0625 and 0.325 mg/kg). GGF2 treatment resulted in significantly (p < 0.05) greater improvements in ejection fraction (EF) and fractional shortening (FS) compared to EGF-1d at the end of the administration period. The second study compared 20 days of administration of GGF2 at equimolar concentrations of EGF-1d and EGF-Ig. GGF2 treatment resulted in significantly improved EF, FS, and LVESD (p < 0.01). The improvements in cardiac physiology with EGF-1d or EGF-Ig were not maintained over this period. The third study compared 20 days of administration of GGF2 (3.25 mg/kg) daily (q 24 hours), every other day (q 48 hours), and every fourth day (q 96 hours). All three GGF2 treatment regimens resulted in significant improvements in cardiac physiology, including EF, ESV, and EDV, and the effects were maintained 10 days after the end of the administration. The studies presented herein identify GGF2 as a key neuregulin compound and establish an optimal dosing regimen for its administration.

As indicated herein, this study established the relative efficacy of GGF2 compared to published neuregulin fragments (Liu et al., 2006), initiated dose ranging and dose frequency studies, and determined whether the BSA excipient was required as previously reported.

Methods and Materials

Cloning, Expression, and Purification of the IgEGF (Ig154Y) Domain of GGF2 (EGF-Ig)

DNA: The IgEGF domain was amplified from pre-existing GGF2 cDNA and cloned into the pet15b vector (Novagen cat# 69661-3) using the Nde1 and BamH1 restriction sites. The resulting protein was 21.89 kDa + a ~3 kDa His tag (~25 kDa).

DNA sequence of IgEgf pet 15 clone: The underlined sequences are the primers used for amplification. The sequences in bold are the cloning sites (Nde1 and BamH1) used to insert the sequence into the pet vector.

The final protein translated from the pet15b vector is shown below. The vector portion is underlined.

Protein expression: The clones were transformed into Bl21 cells for protein expression using the overnight expression auto-induction system (Novagen) in LB medium at 25°C for 24 hours.

[Protein Refolding] Adapted from Novagen Protein Refolding Kit, 70123-3.

[Protein purification] His TRAP column - according to the manufacturer's instructions

[Western Blot] Protein expression was assessed by Western blot. The resulting His-tagged band ran at approximately 25 kD.

A 4-20% standard gel (Biorad) was used for protein resolution, followed by transfer to Protran nitrocellulose paper (0.1 μm pore size from Schliecher and Schull). The blot was blocked in 5% milk in TBS-T (0.1%). A 1:1000 dilution of the primary antibody (anti-EGF human NRG1-α/HRG1-α affinity purified polyclonal AbCat #AF-296-NA from R&D Systems) in 5% milk in TBS-T was incubated for 1 hour at room temperature (also overnight at 4°C). A rabbit anti-goat HRP secondary antibody was used at a 1:10,000 dilution in 5% milk in TBS-T for 1 hour at room temperature. All washes were performed in TBS-T.

[Purification process of Ig154Y] The culture was grown in the overnight expression automatic induction system 1 from Novagen (cat#71300-4) at 25° C. The culture was centrifuged and the pellet was extracted, dissolved and refolded to obtain Ig154Y before purification could take place.

【Substances used for extraction, solubilization and refolding】

10× Wash Buffer: 200 mM Tris-HCl, pH 7.5, 100 mM EDTA, 10% Triton X-100

10× Solubilization Buffer: 500 mM CAPS, pH 11.0

50× dialysis buffer: 1M Tris-HCl, pH 8.5

30% N-lauryl sarcosine - added as powder (Sigma 61739-5G)

1M DTT

Reduced glutathione (Novagen 3541)

Oxidized glutathione (Novagen 3542)

A. Cell Lysis and Preparation of Inclusion Bodies

- Thaw the cell pellet and resuspend in 30 ml 1x wash buffer.

- Protease inhibitors (25 μl of 10×/50 ml), DNase (200 μl of 1 mg/ml/50 ml) and MgCl ₂ (500 μl of 1 M/50 ml) were added to the suspension.

- Cells were lysed by sonication with cooling on ice.

- Inclusion bodies were collected by centrifugation at 10,000 x g for 12 minutes after sonication.

- Remove the supernatant and resuspend the pellet thoroughly in 30 ml of 1× wash buffer.

-Repeat step 4.

- Resuspend the pellet thoroughly in 30 ml of 1× wash buffer.

-Inclusion bodies were collected by centrifugation at 10,000 x g for 10 minutes.

B. Solubilization and Refolding

- From the wet weight of the inclusion bodies to be processed, calculate the amount of 1x solubilization buffer necessary to resuspend the inclusion bodies at a concentration of 10-15 mg/ml. If the calculated volume is greater than 250 ml, use 250 ml.

- Prepare the calculated volume of 1× Solubilization Buffer supplemented with 0.3% N-laurylsarcosine (up to 2% can be used if needed in further optimization) (300 mg/100 mL buffer) and 1 mM DTT at room temperature.

- Add the calculated amount of 1× Solubilization Buffer from step 2 to the inclusion bodies and mix gently. Large fragments can be broken up by repeated pipetting.

- Incubate in a refrigerator shaker at 25°C, 50-100 rpm for 4-5 hours (or longer if needed for further optimization).

- Clarify by centrifugation at 10,000 x g for 10 minutes at room temperature.

- Transfer the supernatant containing soluble proteins to a clean tube.

C. Dialysis Process for Protein Refolding

Prepare the required volume of buffer for dialysis of the dissolved protein. Dialysis should be performed with at least 2 buffer changes 50 times greater than the sample volume. Dilute 50× dialysis buffer to 1× the desired volume and supplement with 0.1 mM DTT.

- Dialyze at 4°C for at least 4 hours. Change buffer and continue dialysis for an additional 4 or more hours.

- Prepare additional dialysis buffer as determined in step 1, but omit the DTT.

- Dialysis was continued by 2 additional changes (4 hr each) with dialysis buffer lacking DTT.

D. Redox refolding buffer that promotes disulfide bond formation

Prepare a dialysis buffer containing 1 mM reduced glutathione (1.2 g/4 L) and 0.2 mM oxidized glutathione (0.48 g/4 L) in 1× dialysis buffer. The volume should be 25 times greater than the volume of the dissolved protein sample. Cool to 4°C.

- Dialyze the refolded protein from step 1 overnight at 4°C.

Substances used for purification

The entire process was carried out at 4°C.

Chemicals

Tromethamine hydrochloride (Sigma T5941-500G)

Sodium chloride 5M solution (Sigma S6546-4L)

Sodium hydroxide 10N (JT Baker 5674-02)

Imidazole (JT Baker N811-06)

A. Purification on a HISPrep FF 16/10 column-20 ml (GE Healthcare)

Buffer A: 20 mM Tris-HCl + 500 mM NaCl pH 7.5

Buffer B: Buffer + 500 mM Imidazole pH 7.5

Column equilibration: Buffer A-5CV, Buffer B-5CV, Buffer A-10CV

20 ml of sample was loaded and run on a 20 ml column at 0.5 ml/min each time.

Wash the column with 5 CV of buffer A

The column was eluted with 5 CV of 280 mM imidazole.

Clean with 10 CV of 100% buffer B.

Equilibrate with 15 CV of buffer A

Analysis of fractions by SDS-PAGE and silver staining

Pooled fractions with Ig154Y

B. His-tag removal

Removal of the His-tag was performed using a thrombin cleavage capture kit from Novagen (Cat# 69022-3). Based on previous testing, optimal conditions were thrombin at 0.005 U/μl per 10 μg of Ig154Y protein for 4 hours at room temperature. After a 4-hour incubation, 16 μl of streptavidin agarose slurry per unit of thrombin was added. The sample was shaken at room temperature for 30 minutes. Ig154Y was recovered by spin filtration or sterile filtration (depending on the volume).

Complete cleavage was confirmed by EGF and anti-His Western blotting.

C.Ig154Y concentration

Use Millipore Centriprep 3000MWCO 15ml concentrator (Ultracel YM-3, 4320) to adjust to the desired concentration

D. Storage in final buffer

Stored in 20 mM Tris + 500 mM NaCl pH 7.5 and 1× PBS + 0.2% BSA.

Cloning, expression, and purification of 156Q (EGF-Id) [NRG1b2 EGF domain (156Q)]

DNA: The NRG1b2 EGF domain was cloned from human brain cDNA and cloned into the pet 15b vector (Novagen cat# 69661-3) using the Nde1 and BamH1 restriction sites. The resulting protein is 6.92 kDa + ~3 kDa His tag (= 9.35 kDa).

DNA sequence of NRG1b2 EGF PET 15 clone

The underlined sequences are cloning sites (Nde1 and BamH1)

The final translated protein from the pET15b vector is shown below. The EGF domain is highlighted in green.

Calculated pI/Mw: 7.69/9349.58

Protein expression

The clones were transformed into Bl21 cells for protein expression using the overnight expression autoinduction system (Novagen) in LB medium at 25°C for 24 hours. Expression was mainly in insoluble inclusion bodies.

[Protein Refolding] Adapted from Novagen Protein Refolding Kit, 70123-3.

Protein Purification: The protein was loaded onto a DEAE anion exchange column at 2.5 ml/min. The EGF-Id fragment remained in the flow-through, while contaminants were bound and eluted with higher salts. The loading and wash buffers were 50 mM Tris pH 7.9, and the elution buffer was 50 mM Tris pH 7.9 with 1 M NaCl. The flow-throughs were combined and concentrated using a Centriprep YM-3 from Millipore.

[Western Blot] Protein expression was assessed by Western blot. The resulting band was approximately 10 kD.

A 4-20% standard gel (Biorad) was used for protein resolution, followed by transfer to Protran nitrocellulose paper (0.1 μm pore size from Schliecher and Schull). The blot was blocked in 5% milk in TBS-T (0.1%). A 1:1000 dilution of the primary antibody (anti-EGF human NRG1-α/HRG1-α affinity purified polyclonal Ab Cat# AF-296-NA from R&D Systems) in 5% milk in TBS-T was incubated for 1 hour at room temperature (also overnight at 4°C). A rabbit anti-goat HRP secondary antibody was used at a 1:10,000 dilution in 5% milk in TBS-T for 1 hour at room temperature. All washes were performed in TBS-T.

Purification process of NRG-156Q

The culture was grown at 25°C in the Overnight Expression Auto-Induction System 1 from Novagen (cat# 71300-4). Very little soluble NRG-156Q (EGF-Id) was present. The culture was centrifuged and the precipitate was extracted, dissolved, and refolded to obtain NRG-156Q before purification could be performed.

【Substances used for extraction, solubilization and refolding】

10× Wash Buffer: 200 mM Tris-HCl, pH 7.5, 100 mM EDTA, 10% Triton X-100

10× Solubilization Buffer: 500 mM CAPS, pH 11.0

50× dialysis buffer: 1M Tris-HCl, pH 8.5

30% N-lauryl sarcosine - added as a powder (Sigma 61739-5G)

1M DTT

Reduced glutathione (Novagen 3541)

Oxidized glutathione (Novagen 3542)

A. Cell Lysis and Preparation of Inclusion Bodies

- Thaw and resuspend the cell pellet in 30 ml of 1x Wash Buffer. Resuspend as needed.

- Protease inhibitors (25 μl of 10×/50 ml), DNase (200 μl of 1 mg/ml/50 ml) and MgCl2 (500 μl of 1 M/50 ml) were added to the suspension.

- Lyse cells by sonication.

a. Keep cells cool on ice throughout this step.

b. Using a square tip, sonicate at level 6, 10X for 30 seconds, until the suspension becomes less viscous. Cool the suspension on ice for 60 seconds between sonications. Keep the volume in a 50 ml conical tube no higher than 40 ml while sonicating.

- When complete, each suspension was transferred to a 250 ml retort centrifuge bottle for use with an F-16/250 rotor.

-Inclusion bodies were collected by centrifugation at 10,000 xg for 12 minutes.

- Remove the supernatant (retain the sample for soluble protein analysis) and resuspend the pellet thoroughly in 30 ml of 1× wash buffer.

- Repeat centrifugation as in step 4 and retain the pellet.

- Again, resuspend the pellet thoroughly in 30 ml of 1× Wash Buffer.

- Collect the inclusion bodies by centrifugation at 10,000 x g for 10 minutes. Decant the supernatant and remove the last traces of liquid by tapping the inverted tube on a paper towel.

B. Solubilization and Refolding

- From the wet weight of the inclusion bodies to be processed, calculate the amount of 1x solubilization buffer necessary to resuspend the inclusion bodies at a concentration of 10-15 mg/ml. If the calculated volume is greater than 250 ml, use 250 ml.

- Prepare the calculated volume of 1× Solubilization Buffer supplemented with 0.3% N-laurylsarcosine (up to 2% can be used if needed in further optimization) (300 mg/100 mL buffer) and 1 mM DTT at room temperature.

- Add the calculated amount of 1× Solubilization Buffer from step 2 to the inclusion bodies and mix gently. Large fragments can be broken up by repeated pipetting.

- Incubate in a refrigerator shaker at 25°C, 50-100 rpm for 4-5 hours.

- Clarify by centrifugation at 10,000 xg for 10 minutes at room temperature.

C. Dialysis Process for Protein Refolding

- Prepare the required volume of buffer for dialysis of the dissolved protein. Dialysis should be performed using at least 2 buffer changes of 50 times the volume of the sample.

- Dilute 50X Dialysis Buffer to 1X at the desired volume and supplement with 0.1 mM DTT.

- Dialyze at 4°C for at least 4 hours. Change buffer and continue dialysis for an additional 4 or more hours.

- Prepare additional dialysis buffer as determined in step 1, but omit the DTT.

- Dialysis was continued through 2 additional changes (4 hours each) with dialysis buffer lacking DTT.

D. Redox refolding buffer that promotes disulfide bond formation

Prepare a dialysis buffer containing 1 mM reduced glutathione (1.2 g/4 L) and 0.2 mM oxidized glutathione (0.48 g/4 L) in 1× dialysis buffer. The volume should be 25 times greater than the volume of the dissolved protein sample. Cool to 4°C.

- Dialyze the refolded protein from step 1 overnight at 4°C.

Substances used for purification

The entire process was carried out at 4°C.

Chemicals

Tromethamine hydrochloride (Sigma T5941-500G)

Sodium chloride 5M solution (Sigma S6546~4L)

Sodium hydroxide 10N (JT Baker 5674-02)

E. Purification on a DEAE HiPrep 16/10 anion column-20 ml (GE Healthcare)

Buffer A: 50 mM Tris-HCl pH 8.0

Buffer B: 50 mM Tris-HCl with 1 M NaCl pH 8.0

Column equilibration: Buffer A-5CV, Buffer B-5CV, Buffer A-10CV

- 50 ml of sample were loaded per run on a 20 ml column at 2.0 ml/min (NRG-156 (EGF-Id) in flow-through).

- Wash the 20 ml column with 5 CV of buffer A

The 20 ml column was run with a 5 CV gradient to 100% B. This was to elute the lower influent.

- Clean with 10 CV of 100% buffer B.

- Equilibrate with 15 CV of buffer A

- Analysis of fractions by SDS-PAGE and silver staining

- Pool fractions with NRG-156Q (10 kDa)

F. Concentration of NRG-156 (EGF-Id)

- Concentrate using Millipore Centriprep 3000MWCO 15ml concentrator (Ultracel YM-3, 4320)

- Concentrations were determined using a modified Lowry protein assay.

【G.His-tag removal】

Removal of the His-tag was performed using the Thrombin Cleavage Capture Kit from Novagen (Cat# 69022-3). Based on previous testing, optimal conditions were thrombin at 0.005 U/μl per 10 μg of NRG-156Q (EGF-Id) protein for 4 hours at room temperature. After a 4-hour incubation, 16 μl of streptavidin agarose slurry per unit of thrombin was added. The sample was shaken at room temperature for 30 minutes. NRG-156Q was recovered by spin filtration or sterile filtration (depending on the volume). Complete cleavage was confirmed by EGF and anti-His protein blotting.

H. Storage in Final Buffer

Store at 4°C in 1X PBS with 0.2% BSA.

Expression and purification of GGF2

For cloning and background information on GGF2, see USPN 5,530,109. Cell lines are described in USPN 6,051,401. The entire contents of each of USPN 5,530,109 and USPN 6,051,401 are incorporated herein by reference in their entirety.

[CHO-(Alpha2HSG)-GGF Cell Line] This cell line is designed to produce sufficient amounts of fetuin (human alpha2HSG) to support high production rates of rhGGF2 in serum-free conditions.

Cho (dhfr-) cells were transfected with the expression vector (pSV-AHSG) shown in Figure 16. Stable cells were grown under ampicillin selection. The designated cell line ( ^{dhfr- /} α2HSGP) was then transfected with the pCMGGF2 vector shown in Figure 16, containing the coding sequence for human GGF2, using the cationic lipid DMRIE-C reagent (Life Technologies #10459-014).

Stable and high-producing cell lines were derived using methotrexate (100 nM, 200 nM, 400 nM, 1 μM) at 4-6 week intervals using standard protocols. Cells were gradually weaned from serum-containing medium. Clones were isolated by standard limiting dilution methods. Details of the culture medium requirements are provided in the aforementioned report.

To enhance transcription, the GGF2 coding sequence was placed after the EBV BMLF-1 interfering sequence (MIS). See the schematic diagram in Figure 17.

MIS sequence (SEQ ID NO: 20)

CGAT[AACTAGCAGCATTTCCTCCAACGAGGATCCCGCAG

( GTAAGAAGCTACACCGGCCAGTGGCCGGGGCC

CGATAACTAGCAGCATTTCCTCCAACGAGGATCCCGCAG(GTAAGAAGCTACACCGGCC

AGTGGCCGGGGCC

GTGGAGCCGGGGGCATCCGGTGCCTGAGACAG A G G TGCTCAAGGCAGTCTCCACCTTTT

GTCTCCCCTCTGCAG)AGAGCCACATTCTGGAA]GT

GGF2 coding sequence (SEQ ID NO: 1)

GGF2 protein sequence (SEQ ID NO: 2)

MRWRRAPRRSGRPGPRAQRPGSAARSSPPLPLLPLLLLLGTAALAPGAAAGNEAAPAGASVCYSSPPSVGSVQELAQRAAVVIEGKVHPQRRQQGALDRKAAAAAGEAGAWGGDREPPAAGPRALGPPAEEPLLAANGTVPSWPTAPVPSAGEPGEEAPYLVKVHQVWAVKAGGLKKDSLLTVRLGTWGHPAFPSCGRLKEDSRYIFFMEP DANSTSRAPAAFRASFPPLETGRNLKKEVSRVLCKRCALPPQLKEMKSQESAAGSKLVLRCETSSEYSSLRFKWFKNGNELNRKNKPQNIKIQKKPGKSELRINK ASLADSGEYMCKVISKLGNDSASANITIVESNATSTSTTGTSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYSTSTPFLSLPE

GGF2 production: Thaw one bottle of GGF2 at 2.2×10 ⁶ cells/mL into 100 ml of Acorda Medium 1 (see Table 3) and expand until sufficient numbers are reached to seed production vessels. Cells are seeded in 2 liter vented roller bottles at 1.0×10 ⁵ cells/mL in production medium Acorda Medium 2 (see Table 4). The roller bottles are maintained at 37°C for 5 days and then reduced to 27°C for 26 days. The roller bottles are monitored for cell count and overall appearance, but they are not fed. Once viability is below 10%, the cells are spun out and the conditioned medium is harvested and sterile filtered.

Table 3: Culture Medium 1

project supplier Item No. Final concentration CD-CHO Invitrogen 10743-029 - Remove 50ml and add the following ingredients Sigma F-0518 1×(10ml/L) L-Glutamine Cellgro 25-005-CI 4mM (20ml/L) recombinant human insulin Sigma I-9278 290U/L(1ml/L) Non-essential amino acids Cellgro 25-025-CI 1×(10ml/L) Peptone 4 type soybean-HySoy Sigma P0521 Powder prepared in 20×CD-CHO (50ml/L) Gentamycin Invitrogen 15750-078 100 μg (2 ml/L)

Table 4: Medium 2

project supplier Item No. Final concentration CD-CHO Invitrogen 10743-029 50% (-50ml first) HyQ SFX-CHO HyClone SH30187.02 50% (-50ml first) Sigma F-0518 1×(10ml/L) L-Glutamine Cellgro 25-005-CI 4mM (20ml/L) recombinant human insulin Sigma I-9278 290U/L(1ml/L) Non-essential amino acids Cellgro 25-025-CI 1×(10ml/L) Peptone 4 type soybean-HySoy Sigma P0521 Powder prepared in 20×CD-CHO (50ml/L) Gentamycin Invitrogen 15750-078 100 μg (2 ml/L)

GGF2 purification process

The entire process was carried out at 4°C.

Chemicals:

Sodium acetate

Glacial acetic acid (for pH adjustment)

10N NaOH (for pH adjustment)

NaCl

sodium sulfate

L-Arginine (JT Baker cat#: 2066-06)

Mannitol (JT Baker cat#: 2553-01)

Raw materials: Conditioned medium supernatant. Adjust pH to 6.5.

Step 1

Capture-cation exchange chromatography

HiPrep SP 16/10(Amersham Biosciences)

Column equilibration: Buffer A-5CV, Buffer B-5CV, Buffer 15% B-5CV

Buffer A: 20 mM sodium acetate, pH 6.0

Buffer B: 20 mM sodium acetate, pH 6.0, 1 M NaCl

Load the sample using a continuous load at 2 ml/min overnight (if possible). A combination with continuous load is preferred.

Maximum volume of starting sample: 5mg GGF2/ml culture medium

Flow rate: 3ml/min

First wash: 15% B, 10CV

Second wash: 35% B, 10CV

GGF2 elution: 60% B, 8CV

Column wash: 100% B, 8CV

Step 2

Purification-gel filtration chromatography

Polyacrylamide dextran S200 26/60

Elution buffer: 20 mM sodium acetate, 100 mM sodium sulfate, 1% mannitol, 10 mM L-arginine, pH 6.5

Buffer conductivity:

Sample: SP GGF2 eluted and concentrated to AU280 1.0

Flow rate: 1.3 ml/min

Peak elution: ~0.36CV from injection

Step 3

DNA and endotoxin removal - filtration through Intercept Q membrane.

Pre-equilibration buffer: 20 mM sodium acetate, 100 mM sodium sulfate, 1% mannitol, 10 mM L-arginine, pH 6.5

Collection and circulation

Step 4

Final formulation and sample preparation

Add an additional 90 mM L-arginine to the sample

concentrate

Sterile filtration

The vehicle/control article used herein was 0.2% bovine serum albumin (BSA), 0.1 M sodium phosphate, pH 7.6.

The rat strains [Crl: (SD)/MYOINFARCT] and naive Sprague Dawley were used in this study. These strains were obtained from Charles River Laboratories. Test animals were approximately 6-7 weeks old upon arrival and weighed approximately 160-200 g at the time of surgery. Actual ranges may vary and are reported in the data.

All naive Sprague Dawley animals were accepted for study and assigned to Group 1. Animals deemed suitable for study were weighed prior to handling.

All animals receiving [Crl:/MYOINFARCT] were randomized to treatment groups (Groups 2-5) using a simple randomization process based on calculated ejection fraction from echocardiography performed 7 days after the surgical procedure at Charles River Laboratories. Simple randomization was performed so that each treatment group (Groups 2-5) consisted of the applicable number of animals, resulting in approximately equal group mean ejection fractions (±3%) across Groups 2-5.

All animals in Groups 2 to 6 were acclimated to the Charles River laboratory environment according to the laboratory's standard operating procedures. Animals were then randomized to treatment groups. All naive animals in Group 1 were acclimated for approximately 24 hours after receipt prior to their initial echocardiographic examination.

Animals are housed individually in suspended, stainless steel, or wire mesh type cages. Solid-bottom cages are generally not used because rodents are coprophagous and ingest feces containing excreted test article and metabolites, or ingest the bedding itself, which can confound the interpretation of toxicity studies.

Fluorescent illumination was provided for approximately 12 hours per day via an automatic timer. The dark cycle was occasionally interrupted due to research-related activities. Temperature and humidity were monitored and recorded daily and maintained, as far as possible, between 64 and 79°F and 30 and 70%, respectively.

The basal diet was a laboratory block diet certified by Rodent Diet #5002, PMI Nutrition International, Inc. This diet was available ad libitum unless otherwise specified. The batch number used is identified in the study records. All animals were provided with tap water ad libitum via an automated water system unless otherwise specified.

Study Design

Table 5: GGF2 vs. EGF-ld fragments (Liu et al., 2006)

Administer the drug starting on the 7th day after LAD for 10 days

Table 6: Higher doses of GGF2 compared to EGF-1d and EGF-Ig

The drug was administered starting 7 days after LAD for 20 days and then washed out for 10 days.

Table 7: GGF2 Dosage Frequency

TA 1 - test article 1; M = male; F = female.

Table 8: GGF2 with and without BSA

【Administration of test and control articles】

【Route of administration】

Test and control articles were administered by intravenous injection. Animals assigned to Group 1 were not treated with vehicle or test article; these animals served as untreated, age-matched controls. Administration frequency, duration, and dose were as described in Tables 5-8. The dose volume was approximately 1 ml/kg.

【Test article administration】

Test and control articles were administered via the tail vein. Individual doses were based on most recent body weight. Doses were administered by bolus injection unless otherwise indicated by the sponsor.

Preparation of the test system

[Surgical Procedure - Left Anterior Descending Artery Ligation]

The surgical procedure was performed at Charles River Laboratories as described in the Charles River Laboratories Surgical Competence Reference Paper, Vol. 13, No. 1, 2005. Briefly, a craniocaudal incision was made through the skin and chest muscles in the chest, slightly to the left of the sternum. The third and fourth ribs were transected, and the intercostal muscles were blunt dissected. The chest cavity was rapidly entered, and the pericardium was completely opened. The heart was removed through the incision. The pulmonary cone and left atrial appendage were identified. A 5-0 silk suture was placed under the left anterior descending coronary artery using a small curved needle. A bandage was applied, and the heart was placed back into the chest. The air in the chest cavity was gently squeezed out and the chest wall and skin incisions were closed. The animal was allowed to regain consciousness using positive pressure ventilation and placed in an oxygen-enriched environment.

【Post-operative recovery】

Short-term postoperative monitoring and administration of appropriate analgesics were performed by Charles River Laboratories as described in the Charles River Laboratories Surgical Competence Reference Paper, Vol. 13, No. 1, 2005.

Long-term postoperative monitoring was performed to assess the animals for signs of pain or infection. The incision site was observed daily for 7 days after receipt of the animals. Supplemental pain management and antimicrobial therapy were administered as needed.

Table 9. Planned medication and dosage

*-ECHO procedure day designated by animal group assignment as shown below.

Antemortem Research Assessment

【Cage side observation】

All animals were observed at least twice daily for morbidity, mortality, injuries, and food and water availability. Any animal in poor health was identified for further monitoring and possible euthanasia.

【weight】

Body weight was measured and recorded at least once per week before randomization and during the study.

Food consumption

Food consumption was not measured, but loss of appetite was recorded.

Echocardiography

Echocardiography was performed on all animals assigned to Group 1 on days 1, 12, 22, and 32 after receipt (Day 0). Echocardiography was performed on all animals assigned to Groups 2-5 on days 7, 18, 28, and 38 after the surgical procedure (Day 0) performed at Charles River Laboratories.

For echocardiography, each animal was anesthetized and its chest hair was clipped according to Table 5. Coupling gel was applied to the echocardiographic transducer, and images were obtained to measure cardiac function at multiple levels. Images were obtained for each animal in the short-axis view (at the mid-papillary level, or other depending on the location of the infarct area observed by echocardiography).

Echocardiographic parameters

ECHO images of the left ventricle are taken at the mid-papillary muscle level, or at other locations depending on the location of the infarct area as observed by echocardiography. M-mode and 2-D images are recorded and stored on CD and/or MOD. Parameters measured using ECHO include: intraventricular septal wall thickness (diastole); unit = cm; intraventricular septal wall thickness (systole); unit = cm; left ventricular internal dimensions (diastole); unit = cm; left ventricular internal dimensions (systole); unit = cm; left ventricular papillary wall thickness (diastole); unit = cm; left ventricular papillary wall thickness (systole); unit = cm; end-diastolic volume; unit = mL; end-systolic volume; unit = mL; ejection fraction; reported as a percentage; stroke volume; unit = ml; and % fractional shortening; reported as a percentage.

Euthanasia

【Moribundity】

Any moribund animals were euthanized for humane reasons as specified by the testing facility's standard operating procedures. All euthanized animals that were moribund or found dead underwent routine necropsies.

Euthanasia Methods

Euthanasia is performed by injection of saturated potassium chloride into a large vein, followed by an approved method of ensuring death, such as exsanguination.

【Final Disposal】

All surviving animals in the study were euthanized at the time of their scheduled necropsy or, if necessary, at the time of moribundity.

Study 1 - Treatment of rats with GGF2 at 0.625 mg/kg intravenously for 1 day resulted in significant improvement in cardiac function as shown herein by changes in ejection fraction and fractional shortening. EGF-1d fragment did not result in the same degree of improvement. See Table 5.

Study 2 - Treatment of rats with GGF2 at 0.625 and 3.25 mg/kg intravenously for 1 day resulted in significant improvements in cardiac function as shown herein by changes in ejection fraction and fractional shortening. Significant improvements were also seen in end-systolic and end-diastolic volumes during treatment. See Table 6.

Study 3 Results - Treatment of rats with GGF at 23.25 mg/kg intravenously every 24, 48, or 96 hours resulted in significant improvements in cardiac function as demonstrated herein by changes in ejection fraction and fractional shortening. Significant improvements were also seen in end-systolic and end-diastolic volumes during the treatment period. See Table 7.

Previous reports (Liu et al) have shown that carrier proteins (such as BSA) are required for optimal neuregulin stability and activity. GGF2 exhibits stability without the need for a carrier (such as BSA). This experiment was designed to test whether GGF2 is stable and active in a therapeutic regimen without BSA. After 10 days of treatment, both BSA-containing and BSA-free GGF2 formulations resulted in improvements in ejection fraction compared to vehicle controls similar to those seen in previous studies. Therefore, from this study, it is clear that GGF2 formulations for CHF treatment do not require BSA or other carrier proteins. See Table 8.

Table 10: Pathological findings

Application Sciatic nerve sheath hyperplasia (NSH) Breast NSH Injection site/skin changes Cardiac effects Subcutaneous every day ++ ++ ++ + Daily intravenous + + + +/- Intravenous injection at 48-hour intervals +/- - - +/-- intravenously at 96-hour intervals - - - -

++ often present; + present; +/- occasionally observed, - rare or not observed

As shown in Table 10, intermittent administration of GGF2 reduces the side effects associated with supranormal levels of exogenously administered GGF2. The inventors have found that this finding is true regardless of whether GGF2 is administered intravenously or subcutaneously.

Proliferative and cardiac effects were sometimes observed with every-other-day dosing. We did not observe these with less frequent dosing.

Several publications and patent documents are referenced in this application in order to more fully describe the prior art to which this invention pertains. All publications and patent applications mentioned in this specification are herein incorporated by reference as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

This manual also includes the following:

1. A method for treating or preventing heart failure in a mammal, comprising:

Providing peptides comprising an epidermal growth factor-like (EGF-like) domain;

A therapeutically effective amount of the peptide is administered to the mammal at intervals of at least 48 hours, wherein the therapeutically effective amount is effective for treating or preventing heart failure in the mammal.

2. The method of embodiment 1, wherein the administering is performed every 48 hours.

3. The method of embodiment 1, wherein the administering is performed every 96 hours.

4. The method of embodiment 1, wherein the administration is performed on a schedule selected from the group consisting of: every 4 days, every week, every 10 days, every 14 days, every month, every 2 months, every 3 months, or every 4 months.

5. The method of embodiment 1, wherein the mammal is a human.

6. The method of embodiment 1, wherein the peptide is recombinant human GGF2.

7. The method of embodiment, wherein the peptide is:

SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ.

8. The method of embodiment, wherein the peptide is:

SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELY.

9. The method of embodiment 1, wherein the peptide is encoded by a neuregulin (NRG)-1 gene, a neuregulin (NRG)-2 gene, a neuregulin (NRG)-3 gene, or a neuregulin (NRG)-4 gene.

Sequence Listing

<110> CAGGIANO, ANTHONY

IACI, JENNIFER

GANGULY, ANINDITA

PARRY, TOM

<120> Therapeutic administration of neuregulin or its subsequence for treating or preventing heart failure

<130> ACOR.P0040WO

<140> PCT/US2009/004130

<141> 2009-07-17

<150> 61/135,171

<151> 2008-07-17

<160> 33

<170> PatentIn version 3.5

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ggaattcctttttttttttttttttttctt nntttttttttgcccttata cctcttcgcc 60

tttctgtggt tccatccact tcttccccct cctcctccca taaacaactc tcctacccct 120

gcacccccaa taaataaata aaaggaggag ggcaaggggg gaggaggagg agtggtgctg 180

cgaggggaag gaaaagggag gcagcgcgag aagagccggg cagagtccga accgacagcc 240

agaagcccgc acgcacctcg cacc atg aga tgg cga cgc gcc ccg cgc cgc 291

Met Arg Trp Arg Arg Ala Pro Arg Arg

1 5

tcc ggg cgt ccc ggc ccc cgg gcc cag cgc ccc ggc tcc gcc gcc cgc 339

Ser Gly Arg Pro Gly Pro Arg Ala Gln Arg Pro Gly Ser Ala Ala Arg

10 15 20 25

tcg tcg ccg ccg ctg ccg ctg ctg cca cta ctg ctg ctg ctg ggg acc 387

Ser Ser Pro Pro Leu Pro Leu Leu Pro Leu Leu Leu Leu Leu Gly Thr

30 35 40

gcg gcc ctg gcg ccg ggg gcg gcg gcc ggc aac gag gcg gct ccc gcg 435

Ala Ala Leu Ala Pro Gly Ala Ala Ala Gly Asn Glu Ala Ala Pro Ala

45 50 55

ggg gcc tcg gtg tgc tac tgc tcc ccg ccc agc gtg gga tcg gtg cag 483

Gly Ala Ser Val Cys Tyr Cys Ser Pro Pro Ser Val Gly Ser Val Gln

60 65 70

gag cta gct cag cgc gcc gcg gtg gtg atc gag gga aag gtg cac ccg 531

Glu Leu Ala Gln Arg Ala Ala Val Val Ile Glu Gly Lys Val His Pro

75 80 85

cag cgg cgg cag cag ggg gca ctc gac agg aag gcg gcg gcg gcg gcg 579

Gln Arg Arg Gln Gln Gly Ala Leu Asp Arg Lys Ala Ala Ala Ala Ala

90 95 100 105

ggc gag gca ggg gcg tgg ggc ggc gat cgc gag ccg cca gcc gcg ggc 627

Gly Glu Ala Gly Ala Trp Gly Gly Asp Arg Glu Pro Pro Ala Ala Gly

110 115 120

cca cgg gcg ctg ggg ccg ccc gcc gag gag ccg ctg ctc gcc gcc aac 675

Pro Arg Ala Leu Gly Pro Pro Ala Glu Glu Pro Leu Leu Ala Ala Asn

125 130 135

ggg acc gtg ccc tct tgg ccc acc gcc ccg gtg ccc agc gcc ggc gag 723

Gly Thr Val Pro Ser Trp Pro Thr Ala Pro Val Pro Ser Ala Gly Glu

140 145 150

ccc ggg gag gag gcg ccc tat ctg gtg aag gtg cac cag gtg tgg gcg 771

Pro Gly Glu Glu Ala Pro Tyr Leu Val Lys Val His Gln Val Trp Ala

155 160 165

gtg aaa gcc ggg ggc ttg aag aag gac tcg ctg ctc acc gtg cgc ctg 819

Val Lys Ala Gly Gly Leu Lys Lys Asp Ser Leu Leu Thr Val Arg Leu

170 175 180 185

ggg acc tgg ggc cac ccc gcc ttc ccc tcc tgc ggg agg ctc aag gag 867

Gly Thr Trp Gly His Pro Ala Phe Pro Ser Cys Gly Arg Leu Lys Glu

190 195 200

gac agc agg tac atc ttc ttc atg gag ccc gac gcc aac agc acc agc 915

Asp Ser Arg Tyr Ile Phe Phe Met Glu Pro Asp Ala Asn Ser Thr Ser

205 210 215

cgc gcg ccg gcc gcc ttc cga gcc tct ttc ccc cct ctg gag acg ggc 963

Arg Ala Pro Ala Ala Phe Arg Ala Ser Phe Pro Pro Leu Glu Thr Gly

220 225 230

cgg aac ctc aag aag gag gtc agc cgg gtg ctg tgc aag cgg tgc gcc 1011

Arg Asn Leu Lys Lys Glu Val Ser Arg Val Leu Cys Lys Arg Cys Ala

235 240 245

ttg cct ccc caa ttg aaa gag atg aaa agc cag gaa tcg gct gca ggt 1059

Leu Pro Pro Gln Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly

250 255 260 265

tcc aaa cta gtc ctt cgg tgt gaa acc agt tct gaa tac tcc tct ctc 1107

Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu

270 275 280

aga ttc aag tgg ttc aag aat ggg aat gaa ttg aat cga aaa aac aaa 1155

Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys

285 290 295

cca caa aat atc aag ata caa aaa aag cca ggg aag tca gaa ctt cgc 1203

Pro Gln Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg

300 305 310

att aac aaa gca tca ctg gct gat tct gga gag tat atg tgc aaa gtg 1251

Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val

315 320 325

atc agc aaa tta gga aat gac agt gcc tct gcc aat atc acc atc gtg 1299

Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val

330 335 340 345

gaa tca aac gct aca tct aca tcc acc act ggg aca agc cat ctt gta 1347

Glu Ser Asn Ala Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Val

350 355 360

aaa tgt gcg gag aag gag aaa act ttc tgt gtg aat gga ggg gag tgc 1395

Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys

365 370 375

ttc atg gtg aaa gac ctt tca aac ccc tcg aga tac ttg tgc aag tgc 1443

Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys

380 385 390

cca aat gag ttt act ggt gat cgc tgc caa aac tac gta atg gcc agc 1491

Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser

395 400 405

ttc tac agt acg tcc act ccc ttt ctg tct ctg cct gaa taggagcatg 1540

Phe Tyr Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro Glu

410 415 420

ctcagttggt gctgctttct tgttgctgca tctcccctca gattccacct agagctagat 1600

gtgtcttacc agatctaata ttgactgcct ctgcctgtcg catgagaaca ttaacaaaag 1660

caattgtatt acttcctctg ttcgcgacta gttggctctg agatactaat aggtgtgtga 1720

ggctccggat gtttctggaa ttgatattga atgatgtgat acaaattgat agtcaatatc 1780

aagcagtgaa atatgataat aaaggcattt caaagtctca cttttattga taaaataaaa 1840

atcattctac tgaacagtcc atcttcttta tacaatgacc acatcctgaa aagggtgttg 1900

ctaagctgta accgatatgc acttgaaatg atggtaagtt aattttgatt cagaatgtgt 1960

tatttgtcac aaataaacat aataaaagga aaaaaaaaaa aaa 2003

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Met Arg Trp Arg Arg Ala Pro Arg Arg Ser Gly Arg Pro Gly Pro Arg

1 5 10 15

Ala Gln Arg Pro Gly Ser Ala Ala Arg Ser Ser Pro Pro Leu Pro Leu

20 25 30

Leu Pro Leu Leu Leu Leu Leu Gly Thr Ala Ala Leu Ala Pro Gly Ala

35 40 45

Ala Ala Gly Asn Glu Ala Ala Pro Ala Gly Ala Ser Val Cys Tyr Cys

50 55 60

Ser Pro Pro Ser Val Gly Ser Val Gln Glu Leu Ala Gln Arg Ala Ala

65 70 75 80

Val Val Ile Glu Gly Lys Val His Pro Gln Arg Arg Gln Gln Gly Ala

85 90 95

Leu Asp Arg Lys Ala Ala Ala Ala Ala Gly Glu Ala Gly Ala Trp Gly

100 105 110

Gly Asp Arg Glu Pro Pro Ala Ala Gly Pro Arg Ala Leu Gly Pro Pro

115 120 125

Ala Glu Glu Pro Leu Leu Ala Ala Asn Gly Thr Val Pro Ser Trp Pro

130 135 140

Thr Ala Pro Val Pro Ser Ala Gly Glu Pro Gly Glu Glu Ala Pro Tyr

145 150 155 160

Leu Val Lys Val His Gln Val Trp Ala Val Lys Ala Gly Gly Leu Lys

165 170 175

Lys Asp Ser Leu Leu Thr Val Arg Leu Gly Thr Trp Gly His Pro Ala

180 185 190

Phe Pro Ser Cys Gly Arg Leu Lys Glu Asp Ser Arg Tyr Ile Phe Phe

195 200 205

Met Glu Pro Asp Ala Asn Ser Thr Ser Arg Ala Pro Ala Ala Phe Arg

210 215 220

Ala Ser Phe Pro Pro Leu Glu Thr Gly Arg Asn Leu Lys Lys Glu Val

225 230 235 240

Ser Arg Val Leu Cys Lys Arg Cys Ala Leu Pro Pro Gln Leu Lys Glu

245 250 255

Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys Leu Val Leu Arg Cys

260 265 270

Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe Lys Trp Phe Lys Asn

275 280 285

Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Gln Asn Ile Lys Ile Gln

290 295 300

Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn Lys Ala Ser Leu Ala

305 310 315 320

Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp

325 330 335

Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn Ala Thr Ser Thr

340 345 350

Ser Thr Thr Gly Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys

355 360 365

Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser

370 375 380

Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp

385 390 395 400

Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pro

405 410 415

Phe Leu Ser Leu Pro Glu

420

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<222> (1)..(195)

<400> 3

agc cat ctt gtc aag tgt gca gag aag gag aaa act ttc tgt gtg aat 48

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

gga ggc gag tgc ttc atg gtg aaa gac ctt tca aat ccc tca aga tac 96

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

ttg tgc aag tgc cca aat gag ttt act ggt gat cgc tgc caa aac tac 144

Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr

35 40 45

gta atg gcc agc ttc tac agt acg tcc act ccc ttt ctg tct ctg cct 192

Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro

50 55 60

gaa tag 198

Glu

65

<210> 4

<211> 65

<212> PRT

<213> Homo sapiens

<400> 4

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr

35 40 45

Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro

50 55 60

Glu

65

<210> 5

<211> 192

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1)..(192)

<400> 5

agc cat ctt gtc aag tgt gca gag aag gag aaa act ttc tgt gtg aat 48

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

gga ggc gag tgc ttc atg gtg aaa gac ctt tca aat ccc tca aga tac 96

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

ttg tgc aag tgc caa cct gga ttc act gga gcg aga tgt act gag aat 144

Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn

35 40 45

gtg ccc atg aaa gtc caa acc caa gaa aaa gcg gag gag ctc tac taa 192

Val Pro Met Lys Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr

50 55 60

<210> 6

<211> 63

<212> PRT

<213> Homo sapiens

<400> 6

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn

35 40 45

Val Pro Met Lys Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr

50 55 60

<210> 7

<211> 183

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1)..(183)

<400> 7

agc cat ctt gtc aag tgt gca gag aag gag aaa act ttc tgt gtg aat 48

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

gga ggc gag tgc ttc atg gtg aaa gac ctt tca aat ccc tca aga tac 96

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

ttg tgc aag tgc cca aat gag ttt act ggt gat cgc tgc caa aac tac 144

Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr

35 40 45

gta atg gcc agc ttc tac aaa gcg gag gag ctc tac taa 183

Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr

50 55 60

<210> 8

<211> 60

<212> PRT

<213> Homo sapiens

<400> 8

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr

35 40 45

Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr

50 55 60

<210> 9

<211> 210

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1)..(210)

<400> 9

agc cat ctt gtc aag tgt gca gag aag gag aaa act ttc tgt gtg aat 48

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

gga ggc gag tgc ttc atg gtg aaa gac ctt tca aat ccc tca aga tac 96

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

ttg tgc aag tgc cca aat gag ttt act ggt gat cgc tgc caa aac tac 144

Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr

35 40 45

gta atg gcc agc ttc tac aag cat ctt ggg att gaa ttt atg gag aaa 192

Val Met Ala Ser Phe Tyr Lys His Leu Gly Ile Glu Phe Met Glu Lys

50 55 60

gcg gag gag ctc tac taa 210

Ala Glu Glu Leu Tyr

65

<210> 10

<211> 69

<212> PRT

<213> Homo sapiens

<400> 10

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr

35 40 45

Val Met Ala Ser Phe Tyr Lys His Leu Gly Ile Glu Phe Met Glu Lys

50 55 60

Ala Glu Glu Leu Tyr

65

<210> 11

<211> 267

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1)..(267)

<400> 11

agc cat ctt gtc aag tgt gca gag aag gag aaa act ttc tgt gtg aat 48

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

gga ggc gag tgc ttc atg gtg aaa gac ctt tca aat ccc tca aga tac 96

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

ttg tgc aag tgc caa cct gga ttc act gga gcg aga tgt act gag aat 144

Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn

35 40 45

gtg ccc atg aaa gtc caa acc caa gaa aag tgc cca aat gag ttt act 192

Val Pro Met Lys Val Gln Thr Gln Glu Lys Cys Pro Asn Glu Phe Thr

50 55 60

ggt gat cgc tgc caa aac tac gta atg gcc agc ttc tac agt acg tcc 240

Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Ser Thr Ser

65 70 75 80

act ccc ttt ctg tct ctg cct gaa tag 267

Thr Pro Phe Leu Ser Leu Pro Glu

85

<210> 12

<211> 88

<212> PRT

<213> Homo sapiens

<400> 12

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn

35 40 45

Val Pro Met Lys Val Gln Thr Gln Glu Lys Cys Pro Asn Glu Phe Thr

50 55 60

Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Ser Thr Ser

65 70 75 80

Thr Pro Phe Leu Ser Leu Pro Glu

85

<210> 13

<211> 252

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1)..(252)

<400> 13

agc cat ctt gtc aag tgt gca gag aag gag aaa act ttc tgt gtg aat 48

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

gga ggc gag tgc ttc atg gtg aaa gac ctt tca aat ccc tca aga tac 96

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

ttg tgc aag tgc caa cct gga ttc act gga gcg aga tgt act gag aat 144

Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn

35 40 45

gtg ccc atg aaa gtc caa acc caa gaa aag tgc cca aat gag ttt act 192

Val Pro Met Lys Val Gln Thr Gln Glu Lys Cys Pro Asn Glu Phe Thr

50 55 60

ggt gat cgc tgc caa aac tac gta atg gcc agc ttc tac aaa gcg gag 240

Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu

65 70 75 80

gag ctc tac taa 252

Glu Leu Tyr

<210> 14

<211> 83

<212> PRT

<213> Homo sapiens

<400> 14

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn

35 40 45

Val Pro Met Lys Val Gln Thr Gln Glu Lys Cys Pro Asn Glu Phe Thr

50 55 60

Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu

65 70 75 80

Glu Leu Tyr

<210> 15

<211> 498

<212> DNA

<213> Homo sapiens

<400> 15

catatgttgc ctccccaatt gaaagagatg aaaagccagg aatcggctgc aggttccaaa 60

ctagtccttc ggtgtgaaac cagttctgaa tactcctctc tcagattcaa gtggttcaag 120

aatgggaatg aattgaatcg aaaaaacaaa ccacaaaata tcaagataca aaaaaagcca 180

gggaagtcag aacttcgcat taacaaagca tcactggctg attctggaga gtatatgtgc 240

aaagtgatca gcaaattagg aaatgacagt gcctctgcca atatcaccat cgtggaatca 300

aacgctacat ctacatccac cactgggaca agccatcttg taaaatgtgc ggagaaggag 360

aaaactttct gtgtgaatgg aggggagtgc ttcatggtga aagacctttc aaacccctcg 420

agatacttgt gcaagtgccc aaatgagttt actggtgatc gctgccaaaa ctacgtaatg 480

gccagcttct acggatcc 498

<210> 16

<211> 162

<212> PRT

<213> Homo sapiens

<400> 16

Leu Pro Pro Gln Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly

1 5 10 15

Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu

20 25 30

Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys

35 40 45

Pro Gln Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg

50 55 60

Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val

65 70 75 80

Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val

85 90 95

Glu Ser Asn Ala Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Val

100 105 110

Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys

115 120 125

Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys

130 135 140

Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser

145 150 155 160

Phe Tyr

<210> 17

<211> 198

<212> PRT

<213> Homo sapiens

<400> 17

Met Gly Gly Ser His His His His His Gly Met Ala Ser Met Thr

1 5 10 15

Gly Gly Thr Ala Asn Gly Val Gly Asp Leu Tyr Asp Asp Asp Asp Lys

20 25 30

Val Pro Gly Ser Leu Pro Pro Gln Leu Lys Glu Met Lys Ser Gln Glu

35 40 45

Ser Ala Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu

50 55 60

Tyr Ser Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn

65 70 75 80

Arg Lys Asn Lys Pro Gln Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys

85 90 95

Ser Glu Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr

100 105 110

Met Cys Lys Val Ile Ser Lys Leu Glu Asn Asp Ser Ala Ser Ala Asn

115 120 125

Ile Thr Ile Val Glu Ser Asn Ala Thr Ser Thr Ser Thr Thr Gly Thr

130 135 140

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

145 150 155 160

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

165 170 175

Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr

180 185 190

Val Met Ala Ser Phe Tyr

195

<210> 18

<211> 198

<212> DNA

<213> Homo sapiens

<400> 18

catatgagcc atcttgtaaa atgtgcggag aaggagaaaa ctttctgtgt gaatggaggg 60

gagtgcttca tggtgaaaga cctttcaaac ccctcgagat acttgtgcaa gtgcccaaat 120

gagtttactg gtgatcgctg ccaaaactac gtaatggcca gcttctacaa ggcggaggag 180

ctgtaccagt aaggatcc 198

<210> 19

<211> 82

<212> PRT

<213> Homo sapiens

<400> 19

Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys

20 25 30

Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser

35 40 45

Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp

50 55 60

Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu

65 70 75 80

Tyr Gln

<210> 20

<211> 236

<212> DNA

<213> Homo sapiens

<400> 20

cgataactag cagcatttcc tccaacgagg atcccgcagg taagaagcta caccggccag 60

tggccggggc ccgataacta gcagcatttc ctccaacgag gatcccgcag gtaagaagct 120

acaccggcca gtggccgggg ccgtggagcc gggggcatcc ggtgcctgag acagaggtgc 180

tcaaggcagt ctccaccttt tgtctcccct ctgcagagag ccacattctg gaagtt 236

<210> 21

<211> 60

<212> PRT

<213> Homo sapiens

<400> 21

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr

35 40 45

Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr

50 55 60

<210> 22

<211> 61

<212> PRT

<213> Homo sapiens

<400> 22

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn

1 5 10 15

Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr

20 25 30

Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr

35 40 45

Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln

50 55 60

<210> 23

<211> 1269

<212> DNA

<213> Homo sapiens

<400> 23

atgagatggc gacgcgcccc gcgccgctcc gggcgtcccg gcccccgggc ccagcgcccc 60

ggctccgccg cccgctcgtc gccgccgctg ccgctgctgc cactactgct gctgctgggg 120

accgcggccc tggcgccggg ggcggcggcc ggcaacgagg cggctcccgc gggggcctcg 180

gtgtgctact cgtccccgcc cagcgtggga tcggtgcagg agctagctca gcgcgccgcg 240

gtggtgatcg agggaaaggt gcacccgcag cggcggcagc aggggggcact cgacaggaag 300

gcggcggcgg cggcgggcga ggcaggggcg tggggcggcg atcgcgagcc gccagccgcg 360

ggcccacggg cgctggggcc gcccgccgag gagccgctgc tcgccgccaa cgggaccgtg 420

ccctcttggc ccaccgcccc ggtgcccagc gccggcgagc ccggggagga ggcgccctat 480

ctggtgaagg tgcaccaggt gtgggcggtg aaagccgggg gcttgaagaa ggactcgctg 540

ctcaccgtgc gcctggggac ctggggccac cccgccttcc cctcctgcgg gaggctcaag 600

gaggacagca ggtacatctt cttcatggag cccgacgcca acagcaccag ccgcgcgccg 660

gccgccttcc gagcctcttt cccccctctg gagacgggcc ggaacctcaa gaaggaggtc 720

agccgggtgc tgtgcaagcg gtgcgccttg cctccccaat tgaaagagat gaaaagccag 780

gaatcggctg caggttccaa actagtcctt cggtgtgaaa ccagttctga atactcctct 840

ctcagattca agtggttcaa gaatgggaat gaattgaatc gaaaaaacaa accacaaaat 900

atcaagatac aaaaaaagcc agggaagtca gaacttcgca ttaacaaagc atcactggct 960

gattctggag agtatatgtg caaagtgatc agcaaattag gaaatgacag tgcctctgcc 1020

aatatcacca tcgtggaatc aaacgctaca tctacatcca ccactgggac aagccatctt 1080

gtaaaatgtg cggagaagga gaaaactttc tgtgtgaatg gaggggagtg cttcatggtg 1140

aaagaccttt caaacccctc gagatacttg tgcaagtgcc caaatgagtt tactggtgat 1200

cgctgccaaa actacgtaat ggccagcttc tacagtacgt ccactccctt tctgtctctg 1260

cctgaatag 1269

<210> 24

<211> 422

<212> PRT

<213> Homo sapiens

<400> 24

Met Arg Trp Arg Arg Ala Pro Arg Arg Ser Gly Arg Pro Gly Pro Arg

1 5 10 15

Ala Gln Arg Pro Gly Ser Ala Ala Arg Ser Ser Pro Pro Leu Pro Leu

20 25 30

Leu Pro Leu Leu Leu Leu Leu Gly Thr Ala Ala Leu Ala Pro Gly Ala

35 40 45

Ala Ala Gly Asn Glu Ala Ala Pro Ala Gly Ala Ser Val Cys Tyr Ser

50 55 60

Ser Pro Pro Ser Val Gly Ser Val Gln Glu Leu Ala Gln Arg Ala Ala

65 70 75 80

Val Val Ile Glu Gly Lys Val His Pro Gln Arg Arg Gln Gln Gly Ala

85 90 95

Leu Asp Arg Lys Ala Ala Ala Ala Ala Gly Glu Ala Gly Ala Trp Gly

100 105 110

Gly Asp Arg Glu Pro Pro Ala Ala Gly Pro Arg Ala Leu Gly Pro Pro

115 120 125

Ala Glu Glu Pro Leu Leu Ala Ala Asn Gly Thr Val Pro Ser Trp Pro

130 135 140

Thr Ala Pro Val Pro Ser Ala Gly Glu Pro Gly Glu Glu Ala Pro Tyr

145 150 155 160

Leu Val Lys Val His Gln Val Trp Ala Val Lys Ala Gly Gly Leu Lys

165 170 175

Lys Asp Ser Leu Leu Thr Val Arg Leu Gly Thr Trp Gly His Pro Ala

180 185 190

Phe Pro Ser Cys Gly Arg Leu Lys Glu Asp Ser Arg Tyr Ile Phe Phe

195 200 205

Met Glu Pro Asp Ala Asn Ser Thr Ser Arg Ala Pro Ala Ala Phe Arg

210 215 220

Ala Ser Phe Pro Pro Leu Glu Thr Gly Arg Asn Leu Lys Lys Glu Val

225 230 235 240

Ser Arg Val Leu Cys Lys Arg Cys Ala Leu Pro Pro Gln Leu Lys Glu

245 250 255

Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys Leu Val Leu Arg Cys

260 265 270

Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe Lys Trp Phe Lys Asn

275 280 285

Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Gln Asn Ile Lys Ile Gln

290 295 300

Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn Lys Ala Ser Leu Ala

305 310 315 320

Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp

325 330 335

Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn Ala Thr Ser Thr

340 345 350

Ser Thr Thr Gly Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys

355 360 365

Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser

370 375 380

Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp

385 390 395 400

Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pro

405 410 415

Phe Leu Ser Leu Pro Glu

420

<210> 25

<211> 11

<212> PRT

<213> Homo sapiens

<400> 25

Val Cys Leu Leu Thr Val Ala Ala Leu Pro Pro

1 5 10

<210> 26

<211> 15

<212> PRT

<213> Homo sapiens

<400> 26

Ala Ser Pro Val Ser Val Gly Ser Val Gln Glu Leu Val Gln Arg

1 5 10 15

<210> 27

<211> 8

<212> PRT

<213> Homo sapiens

<400> 27

Trp Phe Val Val Ile Glu Gly Lys

1 5

<210> 28

<211> 9

<212> PRT

<213> Homo sapiens

<400> 28

Lys Val His Glu Val Trp Ala Ala Lys

1 5

<210> 29

<211> 7

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<400> 29

Asp Leu Leu Leu Xaa Val Leu

1 5

<210> 30

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa can be any naturally occurring amino acid

<400> 30

Gly Ala Trp Gly Pro Pro Ala Phe Pro Val Xaa Tyr

1 5 10

<210> 31

<211> 13

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa can be any naturally occurring amino acid

<400> 31

Tyr Ile Phe Phe Met Glu Pro Glu Ala Xaa Ser Ser Gly

1 5 10

<210> 32

<211> 4

<212> PRT

<213> Homo sapiens

<400> 32

Leu Val Leu Arg

1

<210> 33

<211> 13

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa can be any naturally occurring amino acid

<400> 33

Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Xaa Lys

1 5 10

Claims (14)

1. Use of a recombinant peptide comprising an epidermal growth factor-like (EGF-like) domain for the preparation of a composition of a carrier-free protein for the treatment or prevention of heart failure in mammals, wherein the composition is suitable for administration at least once every 48 hours. 2. The use of claim 1, wherein the composition is suitable for application every 48 hours. 3. The use of claim 1, wherein the composition is suitable for application every 96 hours. 4. The use of claim 1, wherein the composition is suitable for application every 5 days, every 6 days, weekly, every 10 days, every 14 days, monthly, every 2 months, every 3 months, or every 4 months. 5. The use of claim 1, wherein the mammal is a human. 6. The use of claim 1, wherein the EGF-like domain is the EGF domain of glial growth factor 2 (GGF2) comprising the amino acid sequence of SEQ ID NO:4. 7. The use of claim 1, wherein the EGF-like domain is an EGF domain of GGF2 comprising the amino acid sequence of SEQ ID NO:2. 8. The use of claim 1, wherein the peptide comprises a fragment of recombinant human GGF2. 9. The use of claim 8, wherein the fragment is at least 85 amino acids long. 10. The use of claim 8, wherein the composition comprises 1 mg/kg to 10 mg/kg of GGF2 peptide. 11. Use of any of the preceding claims, wherein the composition comprises 0.01 mg/kg to about 1 mg/kg of GGF2 peptide. 12. Use of any of the preceding claims, wherein the composition comprises 0.1 mg/kg to about 1 mg/kg of GGF2 peptide. 13. Use of any of the preceding claims, wherein the composition comprises 3.25 mg/kg of GGF2 peptide. 14. Use of any of the preceding claims, wherein the composition comprises 0.625 mg/kg of GGF2 peptide.