EA046847B1 - COMPOUNDS BASED ON NEUREGULIN-4 AND METHODS OF THEIR APPLICATION - Google Patents

Description

The present invention relates to neuregulin (NRG) 4-based compounds and methods of treatment with NRG4 compounds. The NRG4 compounds of the present invention preferably actively bind human epidermal growth factor receptor (HER) 4 and have high activity due to said binding. Preferred NRG4 compounds may be suitable for improving cardiac function and/or treating diseases in which HER4 binding and resulting activity plays a significant role, such as heart failure (HF), including HF with reduced ejection fraction (HFrEF).

There is an unmet need in medicine to develop new and improved treatments for heart failure. Currently available therapies are designed to slow disease progression and relieve symptoms and rely on hemodynamic changes to reduce the load on the diseased heart. These therapies include agents designed to: (a) reduce heart rate, such as beta blockers and ivabradine; (b) blood pressure lowerers such as angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), mineralocorticoid receptor antagonists (MRAs) and sacubitril/valsartan (ENTRESTO®); and/or (c) treating or preventing volume overload, such as diuretics and MRAs. These treatments, however, do not directly treat the heart and have practical limitations, such as the need for dose titration and monitoring for hypotension. In addition, even with these available treatment options, all patients with HF, even those with mild symptoms, are at high risk of death. See, for example, Ahmed A, A propensity matched study of New York Heart Association class and natural history end points in heart failure, AM. J. CARDIOL. 2007;99(4):549-553. Accordingly, new and improved treatments for HF are required.

Research and development efforts have been conducted to identify new treatment options. For example, WO 2014/187342 describes the prevention, treatment or delay of the onset of HF through the sustained release of NRG, which is said to include NRG1, NRG2, NRG3 and NRG4. This publication also describes a clinical study examining long-term administration of NRG-1 to patients with chronic heart failure.

However, there remains a need to develop treatment options with increased efficacy and reduced risk of toxicity compared to existing therapies. There is a need for therapies that improve long-term outcomes, including improved survival and reduced hospitalizations. There is also a need for therapies that improve cardiac function and have the potential to modify or reverse disease through mechanisms other than hemodynamic changes. There is also a need for therapies with improved tolerability, including, in particular, the treatment of patients with comorbidities such as hypotension, hypokalemia and renal dysfunction. There is also a need for therapies that improve the quality of life (QOL) of patients with advanced disease. The present invention is aimed at satisfying one or more of these critical unmet needs.

Accordingly, the present invention provides NRG4 compounds having selective HER4 binding activity. The NRG4 compounds of the present invention bind HER3 weakly or not at all, resulting in a reduced risk of toxicity and/or tolerability issues associated with HER3.

The present invention also provides methods of using NRG4 compounds to treat or prevent cardiovascular diseases (CVD) and related conditions, including, in particular, HF. Preferred NRG4 compounds and methods of the present invention reduce the risk of CVD-related death or HF-related hospitalization, reduce the risk of myocardial infarction (MI) or stroke, reduce the likelihood of needing a left ventricular assist device (LVAD) or heart transplantation, improve cardiac performance. function and structure and/or alleviate symptoms and physical limitations associated with HF, resulting in improved quality of life. Preferred NRG4 compounds and methods can be used in combination with standard of care (SoC) without the need for titration or monitoring and do not increase hypotension, impair renal function, cause electrolyte imbalances, contribute to liver dysfunction, increase the likelihood of tumors and/or or do not lead to persistent hypospermia.

The present invention also provides NRG4 compounds containing a modification of the D residue at position 1 to a G residue and up to five additional modifications compared to the human NRG4 amino acid sequence (SEQ ID NO: 1).

The present invention also provides NRG4 compounds containing the formula:

X0GHEEPCGX8SHKSFCLNGGLCYX22IPTX26PSPFCRCVX35NYTGARCEX44VFL;

where: X ₀ is selected from the group consisting of T, PT, MPT, S, GS, GGS, GGGS and (GGGGXX)n, where XX is Q, A, E or S, and n=1-5 (SEQ ID NO:5), or absent; X ₈ represents E or

- 1 046847

R; X ₂₂ represents Q or V; X ₂₆ represents F or I; X35 represents E or A; and X ₄₄ is H, K or E (SEQ ID NO:2).

In another aspect, the present invention provides a method of treating or preventing HFpEF in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an NRG4 compound over a 24-96 hour period.

In another embodiment, the present invention provides the NRG4 compound of the present invention for use in therapy.

In another embodiment, the present invention provides a compound NRG4 for use in the treatment or prevention of HFpEF, wherein the compound NRG4 is administered within 24-96

h.

In another embodiment, the present invention provides an NRG4 compound for use in the manufacture of a medicament for treating or preventing HFpEF, wherein the NRG4 compound is administered over a 24-96 hour period.

In another embodiment, the present invention provides a pharmaceutical composition comprising an NRG4 compound of the present invention and a pharmaceutically acceptable carrier, diluent or excipient.

In another embodiment, the present invention provides a pump device containing the NRG4 compound of the present invention. In certain embodiments, the pump is a patch pump.

NRG4 is one of four members of the NRG family of proteins, which are themselves part of a larger family of proteins called epidermal growth factor (EGF) proteins. The EGF family of proteins includes EGF itself, as well as other proteins called EGF-like proteins, which share structural features with EGF. EGF-like proteins include NRG1, NRG2, NRG3 and NRG4, as well as heparin binding EGF-like growth factor (HB-EGF), transforming growth factor-a, amphiregulin (AR), epiregulin (EPR), epigen, betacellulin (BTC) and different isoforms of these proteins. Although these ligands are extracellular membrane proteins, various cellular stimuli lead to activation of the cellular metalloprotease, resulting in the proteolytic release of the EGF domain of the protein, which then freely binds the receptor and initiates signal transduction. The different members of the EGF family of proteins have a wide variety of developmental effects, including effects associated with both normal development and the pathology of a variety of diseases, including cancer. These effects are mediated by interactions with a subfamily of four receptor tyrosine kinases called the ErbB receptor family: EGFR (ErbB-1), HER2 (ErbB-2), HER3 (ErbB-3), HER4 (ErbB-4), and with isoforms of these receptors. Members of the NRG family, as such, have different effects mediated, in particular, by interactions with EGFR, HER3 and HER4. Although HER2 does not bind NRG family members, HER2 enhances downstream signaling by forming heterodimers with EGFR, HER3, and HER4 upon attachment of ligands. Thus, reference herein to HER3 (or HER3/2 or HER2/3) or HER4 (or HER4/2 or HER2/4) activity should be interpreted as activity determined by downstream signaling following binding of a ligand, such as a family element NRG, with either HER3 or HER4, and subsequent heterodimerization of the specified associated receptor with HER2.

As noted above, different members of the NRG family have different effects mediated by different receptor affinity and activity profiles. For example, NRG1 compounds interact with the extracellular domains of HER3 and HER4 and are involved in the normal development of many organs as well as in the pathogenesis of cancer. HER4 binding and resulting activity has been associated with improvements in cardiac function and structure, while HER3 binding and resulting activity has been associated with cancer, nausea, diarrhea and hepatotoxicity. See, for example, O'Shea S, Johnson K, Clark R, Sliwkowski MX, Erickson SL. Effects of in vivo heregulin betal treatment in wild type and ErbB gene-targeted mice depend on receptor levels and pregnancy. Am J Pathol. 2001;158(5):1871-80; MendesFerreira P, De Keulenaer GW, Leite-Moreira AF, Bras-Silva C. Therapeutic potential of neuregulin-1 in cardiovascular disease. DRUG DISCOV Today. 2013;18(17-18):836-42. Review. NRG4, on the other hand, binds and activates HER4 but exhibits little or no binding or activity toward HER3.

NRG4 is expressed as a 115 amino acid precursor protein having the sequence shown in SEQ ID NO:3: MPTDHEEPCG PSHKSFCLNG GLCYVIPTIP SPFCRCVENY TGARCEEVFL PGSSIQTKSN

LFEAFVALAV LVTLIIGAFY FLCRKGHFQR ASSVQYDINL VETSSTSAHH SHEQH (SEQ ID NO:3).

The expressed protein is a transmembrane protein having a transmembrane region, a cytoplasmic region, and an extracellular region that includes an EGF region that is proteolytically released in response to cellular stimuli. The NRG4 compounds of the present invention contain the expressed protein region described above (SEQ ID NO:3),

- 2 046847 containing amino acids numbers 4-50, which is the extracellular region of the expressed protein, such as shown below in SEQ ID NO:1:

DHEEPCGPSH KSFCLNGGLC YVIPTISPPF CRCVENYTGA RCEEVFL (SEQ id do.C

For the avoidance of conflict, the numbering of any amino acid modifications given herein in the context of the NRG4 compounds of the present invention is based on the sequence set forth in SEQ ID NO:1 above.

As used herein, the term NRG4 compound means a protein or peptide that contains all or part of the amino acid sequence of SEQ ID NO:1, or a variant or conjugate thereof. As used herein, the term variant refers to an amino acid sequence containing one or more modifications in the amino acid sequence of a human protein. These modifications include substitution of one or more amino acids in the native human sequence with another natural or non-naturally occurring amino acid and/or deletion or addition of one or more amino acids in the native human sequence. As used herein, the term conjugate refers to a protein or peptide that contains all or part of SEQ ID NO:1 or a variant thereof, and which is attached to the protein, peptide or other chemical moiety by a covalent bond. Said attachment fragments include, for example, the Fc regions of IgG, human albumin, glycine-rich peptides, or fatty acid fragments. In addition, unless expressly stated otherwise, it should be understood that when an NRG4 compound is described in an embodiment or claimed in a claim, the compound may be in free or salt form.

The present invention provides NRG4 compounds that contain the formula SEQ ID NO:1 with one or more modifications and have higher affinity for HER4 and HER4 binding activity than native human NRG4, and which have at least the same binding selectivity of the HER4 receptor over the HER3 receptor as native human NRG4. Affinity and activity for the HER4 and HER3 receptors can be determined by methods known in the art, including those described below in the examples section. In certain embodiments, the NRG4 compounds of the present invention have a HER4 binding activity that is at least 50% of the maximum activity of the native human NRG1 EGF domain. In certain embodiments, the NRG4 compounds of the present invention have HER4 binding activity that is up to 90% of the maximum activity of the native human NRG1 EGF domain.

As used herein, when describing certain NRG4 compounds, the term does not have HER3 binding activity refers to compounds whose HER3 binding activity is no greater than that of native NRG4 when tested in in vitro studies such as those described in the Examples below.

The NRG4 compounds of the present invention contain one or more modifications in the amino acid sequence set forth above in SEQ ID NO:1, including at least one substitution of D at position 1 with G (ie, D1G). The D1G substitution was found to significantly improve binding to HER4 without negatively affecting selectivity over HER3, including NRG4 compounds that contain additional modifications compared to the wild-type NRG4 amino acid sequence. Other amino acid modifications that have been found to have beneficial effects—for example, in terms of HER4 binding and/or selectivity over HER3—in combination with DIG in certain preferred embodiments of the present invention include modifications at one or more of positions 8, 22, 26, 35 and 44, including, in particular, one or more of the following: P8E, V22Q, I26F, E35A, E44H and E44K. Particularly preferred variants of the NRG4 compounds of the present invention are triple substitution variants having a combination of three amino acid substitutions selected from the following list: D1G/I26F/E44K; D1G/I26F/P8E; D1G/I26F/E44H; and D1G/V22Q/E44K. The most preferred embodiment of the NRG4 compounds of the present invention contains the following three amino acid substitutions: D1G/V22Q/E44K, as shown in SEQ ID NO:4:

IGHEEPCGPSHKSFCLNGGLCYQIPTIPSPFCRCVENYTGARCEKVFL (SEQ ID NO:4)

The NRG4 compounds of the present invention may also include, in certain embodiments, one or more amino acids at the C- or N-termini of the sequence set forth in SEQ ID NO:1 above, which may be present in the full-length expressed protein set forth in SEQ ID NO:3. provided that inclusion of said amino acids does not result in the elimination of superior binding and activity for HER4 or selectivity for HER3 compared to the native form.

The NRG4 compounds of the present invention may also contain, in certain embodiments, one or more additional amino acids or other chemical fragments

- 3,046,847 ments that are not present in the full-length protein shown in SEQ ID NO:3. For example, certain NRG4 compounds of the present invention are conjugated to a protein, glycine-rich peptide, and/or other chemical moiety by a covalent bond. In certain embodiments, the NRG4 compounds of the present invention comprise a glycine-rich peptide containing the sequence (OGGGX/.)ii, wherein X/ is Q, A, E, or S, and n=1-5 (SEQ ID NO:5) . In certain embodiments, the NRG4 compounds of the present invention contain the sequence set forth in SEQ ID NO:5, wherein X/ is S, and n=1, at the N terminus.

In certain embodiments, the NRG4 compounds of the present invention contain the formula:

X0GHEEPCGX8SHKSFCLNGGLCYX22IPTX26PSPFCRCVX35NYTGARCEX44VFL;

where: X ₀ is selected from the group consisting of T, PT, MPT, S, GS, GGS, GGGS and (GGGGX/)n, where X/ is Q, A, E or S, and n=1-5 ( SEQ ID NO:5), or missing; X ₈ represents E or P; X ₂₂ represents Q or V; X ₂₆ represents F or I; X ₃₅ represents E or A; and X ₄₄ is H, K or E (SEQ ID NO:2).

In certain embodiments, X ₈ represents P; X ₂₂ represents V; X ₂₆ represents I; X ₃₅ represents E; and X4 ₄ is E. In other embodiments, X ₈ is E; X ₂₂ represents V; X ₂₆ represents I; X ₃₅ represents E; and X4 ₄ is E. In other embodiments, X ₈ is P; X ₂₂ represents Q; X ₂₆ represents I; X ₃₅ represents E; and X4 ₄ is E. In other embodiments, X ₈ is P; X ₂₂ represents V; X ₂₆ represents F; X ₃₅ represents E; and X4 ₄ is E. In other embodiments, X ₈ is P; X ₂₂ represents V; X ₂₆ represents I; X ₃₅ represents A; and X4 ₄ is H or E. In other embodiments, X ₈ is P; X ₂₂ represents V; X ₂₆ represents F; X ₃₅ represents E; and X ₄₄ is K. In other embodiments, X ₈ is P; X ₂₂ represents V; X ₂₆ represents F; X ₃₅ represents E; and X44 is H. In other embodiments, X8 is E; X22 represents V; X ₂₆ represents F; X ₃₅ represents E; and X4 ₄ is E. In preferred embodiments, X ₈ is P; X ₂₂ represents Q; X ₂₆ represents I; X ₃₅ represents E; and X4 ₄ represents K.

In certain embodiments, the NRG4 compounds of the present invention comprise an IgG Fc region. As used herein, the term IgG Fc region refers to the constant region of a human IgG antibody fragment. In particular, the Fc region includes the CH2 and CH3 domains of the antibody constant region and may also include part or all of the hinge region. The IgG Fc regions contained in certain NRG4 compounds of the present invention may contain constant region fragments of either one heavy chain of an IgG antibody or two heavy chains that are associated with each other through non-covalent interactions and disulfide bonds. As used herein, the term IgG Fc region also includes forms of said antibody fragments that are modified, extended and/or truncated, for example, to alter the properties or characteristics of the NRG4 compound, such as activity and/or pharmacokinetics. In the Fc regions of human IgG, certain embodiments of the present invention may also have the hinge region partially or completely removed to facilitate disulfide-mediated Fc dimerization. The Fc regions of IgG in certain embodiments of the present invention contain dimers of two heavy chains having different amino acid sequences, which can be prepared by methods known in the art. See, for example, Lewis SM, et al. Nat. Biotechnol. 32(2): 191-8 (2014); Carter, J. Immunol. Methods, 248(1-2):7-15 (2001); Ridgway, J.B. et al. Protein Eng. 9(7):617-2 (1996).

One of the preferred Fc regions of IgG is a dimer of the following two amino acid chains:

ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ GO NO:6); And

ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ GO NO:7).

Another preferred Fc region of IgG is a dimer of the following two amino acid chains:

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ECPPCPAPPVAGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG KLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG (SEQ ID NO:8); And

ECPPCPAPPVAGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG KLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG (SEQ ID NO:9).

In certain embodiments in which the NRG4 compounds of the present invention contain an IgG Fc region, said IgG Fc region is attached via a linker. In certain embodiments, the linker is a peptide linker comprising the sequence set forth above in SEQ ID NO:5 wherein XX is Q, A, E, or S, and n=1-5. In certain embodiments, the linker comprises the sequence SEQ ID NO:5, wherein XX is S, and n is 3. In certain embodiments in which the NRG4 compounds of the present invention comprise an IgG Fc region linked by a peptide linker, N- the terminal residue of the peptide linker is directly fused to the C-terminal residue of one chain of the Fc region of IgG, and the N-terminal residue of the NRG4 agonist is directly fused to the C-terminal residue of the peptide linker.

Incorporation of IgG Fc regions into certain NRG4 compounds of the present invention may provide manufacturing and/or administration advantages. With regard to production, these compounds can be quickly produced by biological expression methods known in the art, such as those described below in the examples. Rapid production by these methods makes all phases of research and development efficient, including, in particular, the production of NRG4 variants for binding and/or activity screening. With respect to administration, NRG4 compounds that contain the Fc region of IgG may have long-lasting pharmacokinetic profiles, including serum levels over a period of up to approximately 14 days.

However, despite these benefits, it has also been found that long-term exposure to NRG4, including persistence in serum for more than approximately 8 days, may pose risks of cardiotoxicity, as noted in the examples below. It has also been found that the risk of toxicity can be reduced, although not eliminated, in all species by producing compounds (eg, using modified Fc regions) with shorter pharmacokinetic half-lives, including persistence in serum for approximately 8 days.

It has also been found that providing shorter exposure to NRG4 compounds compared to the 8-14 day duration of exposure described above allows for sufficient and long-lasting effectiveness of NRG4 compounds without the risks of cardiotoxicity described in the paragraphs above. Examples of said duration of exposure are from about 1 to about 7 days, preferably 2, 3 or 4 days. Thus, in certain embodiments, the present invention provides a method of treating or preventing HFpEF, wherein the NRG4 compound is administered in an amount sufficient to provide a therapeutically effective serum concentration for about 24 to about 168 hours. In certain embodiments, the NRG4 compound is administered in an amount sufficient to provide a therapeutically effective serum concentration for about 24 to about 96 hours. In certain embodiments, the NRG4 compound is administered in an amount sufficient to provide a therapeutically effective serum concentration for about 24 hours. In certain embodiments, the compound NRG4 is administered in an amount sufficient to provide a therapeutically effective serum concentration for about 48 hours. In certain embodiments, the NRG4 compound is administered in an amount sufficient to provide a therapeutically effective serum concentration for about 72 hours. In certain embodiments, the NRG4 compound is administered in an amount sufficient to provide a therapeutically effective serum concentration for approximately 96 hours.

The duration of exposure described in the previous paragraph can be achieved either by providing a single bolus injection of NRG4 compounds having a pharmacokinetic profile that allows persistence in serum for the periods of time described in the previous paragraph, or by continuous administration (e.g., using a pump) rapidly clearing compound NRG4 for the periods of time described in the previous paragraph.

As used herein, the term long-term effectiveness refers to improvements in conditions associated with heart failure that persist beyond the period of time during which the NRG4 compound is administered. For example, in certain embodiments in which the NRG4 compound is administered in an amount sufficient to provide a therapeutically effective serum concentration for 72 hours, the effectiveness of the treatment regimen can be maintained for a period of time from about 6 days (~2X the duration of administration) to at

- 5 046847 approximately 4 months (~30X from the duration of administration). The duration of persistence of efficacy (i.e., the period of time over which a clinical benefit is observed) relative to the duration of administration (i.e., the period of time over which a therapeutically effective serum concentration is achieved) in certain embodiments is defined herein as the ratio effectiveness: introduction. In certain embodiments, the potency:administration ratio is from about 2 to about 50. In certain embodiments, the potency:administration ratio is from about 20 to about 45. In certain embodiments, the potency:administration ratio is about 22, about 30, or about 45.

This sustained efficacy allows the NRG4 compound to be administered relatively infrequently in an amount sufficient to provide a therapeutically effective serum concentration for the desired period of time. In certain embodiments, NRG4 compounds are administered once every six months. In certain embodiments, NRG4 compounds are administered no more than once per week. In other embodiments, NRG4 compounds are administered twice a month. In other embodiments, NRG4 compounds are administered once a month. In certain preferred embodiments, NRG4 compounds are administered quarterly. In other preferred embodiments, NRG4 compounds are administered once every six months.

As used herein, the term treat or treat includes limiting, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder. Treatment of heart failure or HF EFV according to the present invention may be reflected by one or more of a number of parameters relevant to heart failure, including, for example: an increase in left ventricular ejection fraction (LVEF), an increase in left ventricular end-systolic volume (LVESV), a decrease left ventricular end-diastolic pressure (LVEDP), reduced risk of death from CVD and/or hospitalization for heart failure, reduced risk of all-cause death, reduced risk of myocardial infarction (MI), reduced risk of stroke, reduced risk of requiring left ventricular assist device implantation (PLV) and/or heart transplantation, relief of symptoms and physical limitations of heart failure and/or improvement in quality of life (QoL). Certain beneficial effects of treatment according to embodiments of the present invention can be achieved after treatment for at least 1 month. Certain beneficial effects of treatment according to embodiments of the present invention can be achieved after treatment for at least 6 months. Certain beneficial effects of treatment according to embodiments of the present invention can be achieved after treatment for at least 1 year.

In certain embodiments, administration of the NRG4 compounds of the present invention results in a significant increase in LVEF. In certain embodiments, administration of the NRG4 compounds of the present invention results in an increase in LVEF by at least 5%. In certain embodiments, administration of the NRG4 compounds of the present invention results in an increase in LVEF of at least 5% after 6 months of treatment. In certain embodiments, administration of the NRG4 compounds of the present invention results in an increase in LVEF of at least 5% after 1 year of treatment. In certain embodiments, administration of the NRG4 compounds of the present invention results in an increase in LVEF of at least 5% after 1 year of treatment. In certain embodiments, administration of the NRG4 compounds of the present invention results in a significant reduction in LVEF after 1 year of treatment. In certain embodiments, administration of the NRG4 compounds of the present invention results in a significant reduction in global longitudinal strain (GLS). In certain embodiments, administration of the NRG4 compounds of the present invention results in a reduction in GPA of at least 3.5%. In certain embodiments, administration of the NRG4 compounds of the present invention results in a reduction in the risk of CV death and/or HF hospitalization by at least 15%. In certain embodiments, administration of the NRG4 compounds of the present invention results in a significant reduction in the risk of one or more of all-cause death, MI, stroke, LVAD implantation, or heart transplantation. In certain embodiments, administration of the NRG4 compounds of the present invention results in significant relief of symptoms and physical limitations of heart failure and/or improvement in quality of life.

In addition, as noted above, administration of NRG4 compounds according to certain embodiments of the invention allows for improvements in parameters associated with heart failure, such as those described above, without increasing safety risks. Thus, in preferred embodiments, administration of the NRG4 compounds of the present invention does not result in increased safety risks such as increased hypotension; deterioration of renal function; electrolyte imbalance; liver dysfunction; the occurrence of tumors or persistent hypospermia.

As used herein, the term therapeutically effective amount refers to the amount

- 6 046847 or the dose of the NRG4 compound that provides the desired effect in the patient. For NRG4 compounds having long-lasting pharmacokinetic profiles, the dosage may be an amount administered in one or more doses. For fast-acting compounds administered over a period of time, e.g., by 24-96 hour infusion as described above, the dose or amount may be expressed as the total weight of the NRG4 compound administered over a specified period of time, e.g., total weight in mg, or as the rate at which the NRG4 compound is administered over a specified period of time, for example, mg/min, or as the plasma concentration of the NRG4 compound during the administration period. The effective amount can be readily determined by those skilled in the art by known methods and by observing results obtained under similar conditions. In certain embodiments, quarterly dosages may fall within a dosage range sufficient to provide plasma concentrations of from about 3 nM to about 100 nM during the administration period.

The NRG4 compounds of the present invention are typically administered parenterally, for example, intravenously (IV), subcutaneously (SC), or intraperitoneally (IP). Thus, in certain embodiments of the present invention, the NRG4 compounds are administered intravenously. In other embodiments of the present invention, NRG4 compounds are administered intraperitoneally. In other embodiments, NRG4 compounds are administered subcutaneously.

To control the duration of exposure to optimize and prolong efficacy with sufficient margin before cardiotoxicity occurs, parenteral administration of NRG4 compounds is preferably done by continuous or intermittent infusion. For example, many types of infusion pumps are used to infuse a wide range of drugs, and these pumps can be used to administer the NRG4 compounds of the present invention. More recently, patch pumps have been developed that are smaller than traditional devices and are attached directly to the patient's skin with minimal disruption to the patient's daily activities. Thus, in certain embodiments, NRG4 compounds are administered using an infusion pump or patch pump. Preferably, NRG4 compounds are administered using a patch pump.

The present invention also includes new intermediates and methods suitable for preparing the NRG4 compounds of the present invention. The NRG4 intermediates and compounds of the present invention can be prepared by a variety of methods known in the art, including methods using chemical synthesis or biological expression, such as those described below in the examples section.

With regard to chemical synthesis, the specific synthetic steps in each of the described methods can be combined in different ways to obtain the NRG4 compounds of the present invention. The reagents and starting materials are readily available to those of ordinary skill in the art.

With regard to biological expression, the compounds can be expressed in a host cell after operably linking the sequences to an expression control sequence. The compounds can be readily produced in mammalian cells such as CHO, NSO, HEK293, BHK or COS cells; in bacterial cells such as E. coli, Bacillus subtilis or Pseudomonas fluorescens; in insect cells or in fungal or yeast cells that are cultured by methods known in the art. Vectors containing polynucleotide sequences of interest can be transferred into a host cell by well known methods that vary depending on the type of host cell. Various methods for purifying proteins can be used, and these methods are known in the art.

As noted above, all patients with HF, even those with mild symptoms, are at high risk of death. Thus, references herein to a patient in need of treatment for heart failure (HF) may refer to a wide range of individuals suffering from HF, including individuals with a wide range of disease severity, such as those described below. The New York Heart Association (NYHA) has proposed a classification scheme for the degree or severity of HF, which is summarized in Table 1 below. 1:

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Table 1

Class NYHA Symptoms I There are no restrictions on physical activity. Ordinary physical activity does not cause excessive fatigue, rapid heartbeat, or shortness of breath (difficulty breathing). II Slight limitation of physical activity. Comfort at rest. Normal physical activity causes fatigue, rapid heartbeat, and shortness of breath (difficulty breathing). III Marked limitation of physical activity. Comfort at rest. Light exercise causes fatigue, palpitations, or shortness of breath. IV Inability to perform any physical exercise without discomfort. Symptoms of heart failure at rest. When performing any physical exercise, the discomfort increases.

In certain embodiments, the patient in need of treatment suffers from NYHA class II-IV heart failure. In certain embodiments, the patient in need of treatment suffers from NYHA Class II heart failure. In certain embodiments, the patient in need of treatment suffers from NYHA class III heart failure. In certain embodiments, the patient in need of treatment suffers from NYHA class IV heart failure. In certain embodiments, the patient in need of treatment suffers from NYHA class II-III heart failure.

As noted above, existing therapeutic treatment options for heart failure, including the current standard of care, alleviate symptoms and slow disease progression through hemodynamic mechanisms—for example, by reducing blood pressure, heart rate, and/or plasma volume—to reduce the workload on the diseased heart. The NRG4 compounds of the present invention, in contrast, exert their action through a different mechanism, namely through selective binding of HER4 and the resulting activity, which directly improves cardiomyocyte survival and cellular metabolism and promotes myocardial repair. Due to these different mechanisms of action, the NRG4 compounds of the present invention can be administered in addition to an existing SoC without titration or monitoring. Thus, in certain embodiments, the NRG4 compounds of the present invention may be administered in combination with one or more additional treatments for heart failure. In certain embodiments, one or more additional treatments for heart failure are selected from the group consisting of beta blockers, ACE inhibitors, ARBs, MRAs, diuretics, ivabradine, and sacubitril/valsartan (ENTRESTO®). In certain embodiments, the NRG4 compounds of the present invention may be administered in combination with SGLT2 inhibitors or sGC activators.

The NRG4 compounds of the present invention may also be used in the treatment of other diseases or conditions, including, but not limited to, heart failure with preserved ejection fraction (HFpEF), other cardiac disorders or conditions, Parkinson's disease, Alzheimer's disease, irritable bowel syndrome, bone disorders systems, kidney disease, diabetes, metabolic disease, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

Additional embodiments are described below.

An NRG4 compound containing a modification of the D residue at position 1 with its replacement by a G residue and up to five additional modifications compared to the amino acid sequence of human NRG4 (SEQ ID NO: 1).

NRG4 connection according to the previous embodiment, having no more than 4 additional modifications. In one embodiment, the NRG4 connection has no more than 3 additional modifications. In one embodiment, the NRG4 connection has no more than 2 additional modifications.

The NRG4 compound according to any of the embodiments described above, wherein said compound includes at least one modification at a position selected from the group consisting of 8, 22, 26, 35 and 44.

A compound containing the formula:

X0GHEEPCGX8SHKSFCLNGGLCYX22IPTX26PSPFCRCVX35NYTGARCEX44VFL;

where: X ₀ is selected from the group consisting of T, PT, MPT, S, GS, GGS, GGGS and (GGGGXX)n, where XX is Q, A, E or S, and n=1-5 (SEQ ID NO:5), or absent; X ₈ represents E or P; X ₂₂ represents Q or V; X ₂₆ represents F or I; X ₃₅ represents E or A; and X ₄₄ is H, K or E (SEQ ID NO:2).

The connection according to the above embodiment, characterized in that X ₈ is

- 8 046847 is P; X ₂₂ represents V; X ₂₆ represents I; X35 represents E; and X44 represents E.

A connection according to any of the embodiments described above, characterized in that X ₈ represents E; X ₂₂ represents V; X ₂₆ represents I; X ₃₅ represents E; and X ₄₄ represents E.

A connection according to any of the embodiments described above, characterized in that X ₈ represents P; X ₂₂ represents Q; X ₂₆ represents I; X35 represents E; and X ₄₄ represents E.

A connection according to any of the embodiments described above, characterized in that X ₈ represents P; X ₂₂ represents V; X ₂₆ represents F; X35 represents E; and X44 represents E.

A connection according to any of the embodiments described above, characterized in that X8 is P; X ₂₂ represents V; X ₂₆ represents I; X35 represents A; and X ₄₄ is H or E.

A connection according to any of the embodiments described above, characterized in that X ₈ represents P; X ₂₂ represents V; X ₂₆ represents F; X ₃₅ represents E; and X ₄₄ represents K.

A connection according to any of the embodiments described above, characterized in that X ₈ represents P; X ₂₂ represents V; X ₂₆ represents F; X ₃₅ represents E; and X ₄₄ represents H.

A connection according to any of the embodiments described above, characterized in that X8 is E; X22 represents V; X26 represents F; X35 represents E; and X44 represents E.

A connection according to any of the embodiments described above, characterized in that X8 is P; X22 represents Q; X26 represents I; X35 represents E; and X ₄₄ represents K.

The compound according to the preceding embodiment, characterized in that X0 is SEQ ID NO:5, where XX is S, and n=1.

A compound containing the amino acid sequence of SEQ ID NO:4.

A compound containing the amino acid sequence of SEQ ID NO:10.

A compound containing the amino acid sequence of SEQ ID NO:11.

A compound containing the amino acid sequence of SEQ ID NO:12.

A compound containing the amino acid sequence of SEQ ID NO:13.

A compound containing the amino acid sequence of SEQ ID NO:14.

A compound containing the amino acid sequence of SEQ ID NO:15.

A compound containing the amino acid sequence of SEQ ID NO:16.

A compound containing the amino acid sequence of SEQ ID NO:17.

A compound containing the amino acid sequence of SEQ ID NO:18.

A compound containing the amino acid sequence of SEQ ID NO:19.

A compound consisting of the amino acid sequence SEQ ID NO:18.

A compound according to any of the preceding embodiments, characterized in that said compound is attached to a protein, peptide, or other chemical moiety by a covalent bond. A compound according to any of the preceding embodiments, characterized in that the compound is attached to one or more of the Fc region of an IgG, human albumin, a glycine-rich peptide, or a fatty acid moiety. A compound according to any of the preceding embodiments, characterized in that said compound is attached to the Fc region of an IgG. The compound of the preceding embodiment, wherein the Fc region of the IgG comprises a dimer of either SEQ ID NO:6 and SEQ ID NO:7 or SEQ ID NO:8 and SEQ ID NO:9. The compound according to the preceding embodiments, characterized in that X0 is SEQ ID NO:5, where XX is S, and n=3.

A compound according to any of the above embodiments, wherein said compound has an HER4 binding activity that is greater than that of native human NRG4.

A compound according to any of the above embodiments, wherein said compound has an HER4 binding activity that is at least 50% of the maximum activity of native human NRG1.

A compound according to any of the above embodiments, wherein said compound has an HER4 binding activity that is at least 70% of the maximum activity of native human NRG1.

A compound according to any of the above embodiments, characterized in that said compound has an HER4 binding activity of

- 9 046847 at least 90% of the maximum activity of native human NRG1.

A compound according to any of the above embodiments, characterized in that said compound does not have HER3 binding activity.

A pharmaceutical composition containing a compound according to any of the above embodiments.

A pump-type device comprising a connection according to any of the above embodiments.

A method of treating or preventing heart failure in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an NRG4 compound. In one embodiment, the NRG4 connection is a connection according to any of the above embodiments.

Compound NRG4 for use in the treatment or prevention of HFpEF. In one embodiment, the NRG4 connection is a connection according to any of the above embodiments.

Compound NRG4 for use in the preparation of a medicament for the treatment or prevention of HFpEF. In one embodiment, the NRG4 connection is a connection according to any of the above embodiments.

In one embodiment, the compound is administered over 24-168 hours. In one embodiment, the compound is administered over 24-96 hours. In one embodiment, the compound is administered over 24, 48, 72, or 96 hours. In one embodiment implementation, the compound is administered within 96 hours.

In one embodiment, the compound is administered no more than once a month. In one embodiment, the compound is administered no more than once per quarter (Q3M). In one embodiment, the compound is administered by Q3M. In one embodiment, the compound is administered Q3M over 96 hours. In one embodiment, the compound is administered once every six months.

In one embodiment, the potency:administration ratio is from about 2 to about 50. In one embodiment, the potency:administration ratio is from about 20 to about 45. In one embodiment, the potency:administration ratio is about 22, about 30, or approximately 45.

In one embodiment, treatment is continued for at least 6 months.

In one embodiment, treatment is continued for at least 1 year.

In one embodiment, the compound results in a significant increase in LVEF. In one embodiment, the compound results in an increase in LVEF by at least 5%. In one embodiment, the compound results in an improvement in LVEF by at least 5% after 1 year of treatment.

In one embodiment, the compound results in a significant increase in LSV. In one embodiment, the compound results in an improvement in LVEF by at least 5%. In one embodiment, the compound results in an improvement in LVEF by at least 5% after 1 year of treatment.

In one embodiment, the compound results in a significant reduction in LVEF. In one embodiment, the compound results in a significant reduction in LVEDV after 1 year of treatment.

In one embodiment, the connection results in a significant reduction in global longitudinal strain (GLA). In one embodiment, the connection results in a reduction in GPA of at least 3.5%.

In one embodiment, the compound results in a significant increase in left ventricular end-diastolic volume (LVEDV).

In one embodiment, the compound results in a significant reduction in the risk of death from CVD and/or hospitalization for HF.

In one embodiment, the compound results in a reduction in the risk of death from CVD by at least 15%.

In one embodiment, the compound results in a reduction in the risk of hospitalization for HF by at least 15%.

In one embodiment, the compound results in a significant reduction in the risk of one or more of all-cause death, MI, stroke, LVAD implantation, or heart transplantation.

In one embodiment, the compound results in significant relief of symptoms and physical limitations of heart failure and/or improvement in quality of life.

In one embodiment, the compound does not result in: increased hypotension; deterioration of renal function; imbalance of electrolytes; liver dysfunction; the occurrence of tumors or persistent hypospermia.

In one embodiment, the compound is administered parenterally.

In one embodiment, the compound is administered intravenously, subcutaneously, or intraperitoneally.

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In one embodiment, the compound is administered using an infusion pump.

In one embodiment, the compound is administered using a patch pump.

In one embodiment, a therapeutically effective amount is an amount of compound that provides a serum concentration of from about 3 nM to about 100 nM.

In one embodiment, the compound is administered to a patient with NYHA class II-IV heart failure.

In one embodiment, the compound is administered to a patient with NYHA class II-III heart failure.

In one embodiment, the compound is administered simultaneously or sequentially in combination with one or more additional treatments for heart failure. In one embodiment, the one or more additional treatments for heart failure are selected from the group consisting of beta blockers, ACE inhibitors, ARBs, MRAs, diuretics, ivabradine, and sacubitril/valsartan (ENTRESTO®). In one embodiment, the compound is administered simultaneously or sequentially in combination with an SGLT2 inhibitor and/or an sGC activator.

In one embodiment, the NRG4 compound administered is an NRG4 compound of the present invention.

The invention is further illustrated in the following examples, which should not be construed as limiting.

Receive suggested connections.

Examples of NRG4 compounds according to the present invention are described below in table. 2.

table 2

Components of proposed NRG4 connections

Example Modifications NRG4 N-terminal AAs/linker No. sequences 1 DIG SEQ Ш NO:5, where Χλ represents S and η is equal to 3 SEQIDNO:10 2 D1G/P8E SEQ Ш ΝΟ:5, where Χλ represents S and η is equal to 3 SEQ GO NO: AND 3 D1G/V22Q SEQ Ш ΝΟ:5, where Χλ represents S and η is equal to 3 SEQ GO NO: 12 4 D1G/I26F SEQ ID NO: 5 where Χλ is S and η is 3 SEQ GO NO: 13 5 D1G/E35A SEQ Ш ΝΟ:5, where Χλ represents S and η is equal to 3 SEQ GO NO: 14 6 D1G/I26F/E44K SEQ Ш ΝΟ:5, where Χλ represents S and η is equal to 3 SEQ ID NO: 15 7 D1G/I26F/E44H SEQ Ш ΝΟ:5, where Χλ represents S and η is equal to 3 SEQ ID NO: 16 8 D1G/I26F/P8E SEQ Ш ΝΟ:5, where Χλ represents S and η is equal to 3 SEQ ID NO: 17 9 D1G/V22Q/E44K SEQ Ш ΝΟ:5, where Χλ represents S and η is equal to 1 SEQ ID NO: 18 10 D1G/V22Q/E44K SEQ Ш ΝΟ:5, where Χλ represents S and η is equal to 3 SEQ ID NO: 19 AND D1G/V22Q/E44K SEQ Ш ΝΟ:5, where Χλ represents S and η is equal to 3 SEQ ID NO: 19

The complete amino acid sequences of the proposed compounds, excluding any IgG Fc region sequences, are indicated as SEQ ID NO listed in the fourth column. Examples 1-8 and 10-11 additionally contain Fc regions of IgG. The Fc regions of the IgG in Examples 1-8 and 10 contain a dimer of SEQ ID NO:6 and SEQ ID NO:7, wherein the N-terminal amino acid in the sequence shown in the third column of the table above is fused to the C-terminal amino acid in SEQ ID NO :7. The Fc region of the IgG in Example 11 contains a dimer of SEQ ID NO:8 and SEQ ID NO:9, wherein the N-terminal amino acid in the sequence shown in the third column of the above table is fused to the C-terminal amino acid in SEQ ID NO:9.

Biological expression.

Examples of NRG4 compounds containing Fc regions were produced in a mammalian cell expression system using the CHO GSKO cell line. GS gene knockout provides narrow and stringent selection conditions by eliminating background endogenous GS activity, which may promote the survival of low-producing or unproductive cells under selection conditions. The genes encoding the knob chain and the hole chain in the Fc fusion of the present invention can be subcloned into separate expression plasmids containing glutamine synthetase (GS) for co-transfection, or both chains can be subcloned

- 11 046847 baths into one expression plasmid containing GS. Alternatively, the protruding strands and recessed strands in different ratios can be combined in a single expression plasmid containing GS if the relative expression levels of each strand are not required to be altered. The cDNA sequence encoding the overhanging strand or the recessed strand is hybridized in frame with a signal peptide coding sequence, which may be a murine kappa chain leader sequence, to enhance secretion of the target product into the cell culture medium. Expression is regulated by the cytomegalovirus (CMV) viral promoter.

Transient or stable transfection of GSKO CHO cells can be carried out. For stable transfection, CHO GSKO cells were transfected by electroporation using appropriate amounts of recombinant overhang and recessed chain expression plasmids, and maintained the transfected cells in suspension culture at sufficient cell density. Transfected cells were selected by growing in glutamine-free, 25 μM methionine sulfoximine (MSX) serum-free medium and incubating at 3237°C and 5-7% CO ₂ . Fc-fusion proteins were secreted into the medium from CHO cells.

Proteins were purified by either: (1) protein A affinity chromatography followed by cation exchange or hydrophobic interaction chromatography (or other suitable methods); or (2) multimodal chromatography and subsequent hydrophobic interaction chromatography (or other suitable methods).

For purification using Protein A chromatography, proteins from the collected media were first applied to MabSelect SuRe Protein A resin (GE Healthcare). The resin was then quickly washed with a running buffer such as phosphate buffered saline (PBS), pH 7.4, or a running buffer containing Tris to remove nonspecifically bound material. The protein was then eluted from the resin with a low pH solution such as 20 mM acetic acid/5 mM citric acid. Fractions containing the Fc-fusion protein were pooled. If necessary, increase the pH by adding a base such as 0.1 M Tris, pH 8.0. At this stage, the Fc fusion proteins can be used for binding/activity screening, or, if desired, they can be further purified by hydrophobic interaction chromatography using resins such as phenyl-Sepharose HP. Fc-fusion proteins can be eluted from a phenyl-Sepharose HP column with a gradient of 500 mM to 0 mM sodium sulfate in 10 mM sodium phosphate, pH 7, using 10 column volumes. Fc-fusion proteins can be further purified by gel permeation chromatography on a Superdex 200 column (GE Healthcare) eluting isocratically in PBS, pH 7.4, or in buffer substituted with the desired buffer. Examples 1-8 and 10 were purified as described.

For purification using multimodal chromatography, proteins from the collected media were first applied to CaptoMMC resin (GE Healthcare). The resin was then quickly washed with 100 mM citrate, pH 5.0 (buffer A), followed by an 80%/20% wash solution of buffer A and 25 mM sodium phosphate, 1 M sodium chloride, pH 7.5 (buffer B). The target protein was then eluted from the resin using 70% buffer B. Fc fusion proteins can be further purified by hydrophobic interaction chromatography using resins such as phenyl-Sepharose HP (GE Healthcare). Fc fusion proteins can be eluted from a phenyl Sepharose HP column with a linear gradient from 800 mM to 0 mM sodium sulfate in 20 mM Tris, pH 8. Fc fusion proteins can be further purified by ion exchange chromatography on a Sepharose Q column (GE Healthcare) . Fc-fusion proteins can be eluted from the column with a gradient of 0 M to 1 M sodium chloride in 20 mM Tris, pH 8. The fractions were pooled and the buffer exchanged to the desired storage buffer. Example 11 was purified as described.

Chemical synthesis.

The NRG4 compounds of the present invention, which are peptides lacking the Fc region of IgG (eg, Example 9 in Table 2 above), can also be prepared by solid phase peptide synthesis using the Fmoc/t-Bu strategy. Peptides were synthesized using a SymphonyX automated peptide synthesizer (PTI Protein Technologies Inc.).

Fmoc-L-Leu-Wang resin (0.3-0.8 mmol/gram, 200-400 mesh, Chem-Impex) was used for peptide synthesis. Removal of Fmoc protecting groups was carried out using a 20% (v/v) solution of piperidine in DMF. Amino acid coupling was performed using 10 equivalents of Fmoamino acid, 0.9 M diisopropylcarbodiimide (DIC) and 0.9 M Oxyma (molar ratio 1:1:1) in DMF for 2 h at 25°C.

DMF washing steps were performed after each coupling and deprotection step. Additional details are given in the table below. 3.

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Table 3

Fmoc deprotection protocol and combinations

Stage Solvent/Procedure Mixing time Replays 1 DMF 00:01:00 2 2 25% piperidine/DMF 00:10:00 3 3 DMF 00:01:00 1 4 DMF 00:01:00 5 5 Methylene chloride 00:01:00 1 6 Methylene chloride 00:00:02 1 7 Reagent (amino acid) 00:00:01 1 8 0.9 M Ohusha in DMF 00:00:01 1 9 0.9 M DIC in DXM 03:00:00 1 10 DMF 00:01:00 3

All amino acids used in the main sequence are L amino acids: Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(Otbu)-OH, Fmoc-Glu(Otbu)-OH, Fmoc-PheOH, Fmoc -Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, FmocThr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc -Val-OH, Fmoc-Trp(Boc)-OH.

After completion of the peptide chain extension on the resin described above, the last Fmoc group was removed and the resin was washed with methylene chloride, followed by a peptide cleavage treatment. Add 1.5 ml of a solution of water, triisopropylsilane, thioanisole, 1,2-ethanedithiol (1:1:1) in 13 ml of trifluoroacetic acid (TFA) and add the resulting cleavage cocktail to the resin and stir for 2.5 hours in the reaction syringe. Transfer the TFA solution containing the cleaved peptide into a 50 ml conical tube containing cold diethyl ether. The solution containing the precipitated peptide was centrifuged until a thick precipitate was obtained, and the ether was decanted. Precipitation in ether was repeated twice to wash away the residual cocktail, after which re-stacking was carried out.

The crude peptide was dissolved in water and acetonitrile was added if necessary. The concentration of the crude peptide was adjusted to 2.5-3 mg/ml (200 ml) in acetonitrile and water. Refolding was then carried out directly in the sample. Refolding buffer was prepared by adding oxidized glutathione and reduced glutathione at a ratio of GSSG:GSH (2 mM:1 mM) into 0.1 M Tris buffer, pH 8. Crude peptide solution was added to the refolding buffer to achieve a concentration of 0.13 -0.15 mg/ml (20-fold dilution) and kept the mixture for refolding at 4°C without stirring. After 1 day, the mixture was quenched for re-laying by adding TFA to a 0.2% concentration, bringing the pH to 3.0. After quenching, the solution was filtered and purified.

The peptide solution was placed on an HPLC column and then equilibrated using buffer A before starting gradient elution. Buffer A: 0.1% TFA in water; buffer B: 100% acetonitrile. Gradient: 10% B to 25% B over 100 min. Flow rate: 18 ml/min; detection at 220 nm (Waters 2489 detector). Column: Waters Symmetry Prep C18 Column, 7µm, 19x300mm (Waters Part, Cat# WAT066245). Fractions were automatically monitored by a UV detector and collected using a Waters Fraction Collector III.

The collected fractions containing the target peptide were pooled and lyophilized.

In vitro studies.

HER4/NRG-Fc binding assay to screen NRG4 variants for improved binding.

A binding assay was performed to high-throughput screen variants of NRG4 Fc fusion proteins against the HER4 receptor to identify affinity variants with the potential to enhance receptor activation and signal transduction. ELISA plates were coated with 2 μg/mL tagged anti-His antibody in PBS to promote standardized capture of the HER4 receptor extracellular domain (ECD) construct with a C-terminal His tag after blocking the plate with 1% BSA/PBS/0.1% Tween 20 (HER4 ECD at a concentration of 5 μg/ml). Saturating amounts of crude NRG4-Fc variants were incubated in expression medium (initial concentration 10 μg/ml, serial dilutions in 1% BSA/PBS/0.1% Tween 20) with the captured receptor for 1 h. After washing, bound NRG4-Fc variant using a secondary detection reagent of an anti-human Fc-specific antibody alkaline phosphatase conjugate to quantify binding. The duration of incubation of the variant to bind to the receptor or the duration of subsequent washing steps was varied to further identify binding variants by affinity based on association and dissociation rates, respectively. This screening method shows that the DIG modification, such as included in Example 1, has high affinity and has the potential to enhance receptor activation and signal transduction, as supported by the data shown in Table 1 below. 4.

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Table 4

Binding to the extracellular domain of HER4

Dose No. Example 1 Wild type NRG1 Wild type NRG4 dose (mcg/ml) O.P. 405 dose (µg/ml) O.P. 405 dose (mcg/ml) O.P. 405 1 10.65 1.485 10 1,501 17 0.651 2 5.325 1.438 5 1.439 8.5 0.597 3 2.6625 1.4 2.5 1,399 4.25 0.485 4 1.33125 1.332 1.25 1.31 2.125 0.347 5 0.665625 1.217 0.625 1.27 1.0625 0.224 6 0.332813 1,081 0.3125 1.022 0.53125 0.154 7 0.166406 0.865 0.15625 0.8 0.265625 0.111 8 0.083203 0.607 0.078125 0.514 0.132813 0.088 9 0.041602 0.404 0.039063 0.316 0.066406 0.071 10 0.020801 0.235 0.019531 0.179 0.033203 0.07 AND 0.0104 0.136 0.009766 0.114 0.016602 0.066 12 0.0052 0.096 0.004883 0.084 0.008301 0.066

As can be seen from table. 4, Example 1, which includes the DIG modification, has a similar HER4 binding profile compared to wild-type NRG1 at similar doses and a significantly improved HER4 binding profile compared to wild-type NRG4 at similar doses in this study.

Preparation of human and rat CHO HER2-4 and HER2-3 cell lines.

A cellular assay was performed using CHO-Ki cells overexpressing human or rat HER receptors to determine the activity of NRG4 compounds. CHO-Ki cells (ATCC) were grown in DMEM-F12 3:1 with 5% FBS and 20 mM HEPES, 40 μg/ml L-proline, 1x antibiotics and diluted 1:5 every 2-3 days using TripLE Express (Gibco) . Cells were transfected with plasmid DNA encoding HER receptor pairs (hHER2-3, hHER2-4, rHER2-3, and rHER2-4) using Fugene 6 (Promega) according to the manufacturer's instructions. HER2 was included in the study because this receptor does not bind a ligand but is the preferred dimerization partner of HER receptors for downstream signaling. Transfected cells were selected using puromycin (12 μg/ml) and hygromycin (1 mg/ml) for 3-4 weeks and clonal lines were obtained by limiting dilution cloning in 96-well plates. Clonal lines were selected for proper expression of the HER2, HER3 or HER4 genes by the Taqman method. Proper receptor expression in the lines was confirmed by phospho- _{ERK 1/2} ^responses induced by NRG1. Clones were grown, harvested, aliquoted into cryovials, and then frozen in liquid nitrogen for long-term storage.

Molecular screening for human HER2-HER4 mediated ERK 1/2 phosphorylation in vitro.

A phospho-ERK1/2 activity assay was performed to determine the activity of NRG4 compounds. The activity of Examples 1-8 and 10 was analyzed relative to wild-type human NRG1 and/or human NRG4 in studies that measured stimulation of human HER2-HER4-mediated ERK 1/2 phosphorylation. The study included the CHO-K^ line stably expressing human HER2 and HER4. CHO-hHER2-hHER4 cells (clone 1H6) were grown in DMEM: F12(3:1), 5% FBS, 40 μg/ml L-proline, 10 μg/ml puromycin and 1 mg/ml hygromycin at 37°C using the traditional method. , 5% CO2. Cells were washed twice with 1X PBS, dissociated in 0.05% trypsin/0.53 μM EDTA, and collected by centrifugation at 300xg for 10 min. The cells were resuspended in maintenance medium. 20,000 cells/well were seeded in 30 μl in poly-O-lysine-coated 384-well cell culture plates (Greiner, cat. no. 781946). The plates were covered with manufacturer's caps and placed in a tissue culture incubator maintained at 37°C at 5% CO ₂ , 80%+ humidity and allowed to adhere. After 24 h, the media was removed from the plates and replaced with 30 μl of serum-free starvation medium (DMEM with 0.1% BSA low glucose) using a Biomek FX liquid handler. The plates were returned to the incubator for 24 hours.

hFcNRG4 variants transiently expressed as 293F supernatants were prepared in starvation medium in a 384-well clear plate (Greiner, cat. no. 781185) using a 1:8 serial dilution to obtain 4 spots (average dose range 250 pM to 140,000 pM ). The medium was removed from the cell culture plates and 30 μl/well of the resulting variants was transferred into the plates using a Biomek FX multi-channel (stamp) device for working with liquids. The plates were sealed with foil and stimulated for 15 min at room temperature on an orbital shaker. The cells were then washed once with 50 μl of cold PBS. 30 μL of lysis buffer was added to the cells and mixed for 10 min at RT on an orbital shaker.

ERK 1/2 phosphorylation was detected using either the AlphaScreen SureFire phospho-ERK1/2 (T202/Y204) kit (Perkin Elmer, cat. no. TGRES) or the AlphaLISA® Assay Kit

- 14 046847

SureFire® Ultrax p ERK 1/2 with AlphaScreen Protein A IgG Detection Kit (Protein-A) (PerkinElmer, cat. no. 6760617). After lysing the Cho-Κι cells, 4 μl of lysate was transferred to a 384-well Alpha Proxiplate (Perkin Elmer, cat. no. 6008280) using a Biomek FX 384-channel pipette. 4 μl of positive and negative control lysates (Perkin Elmer, TGRES-L) were added to the available wells. 5 μl/well of acceptor mixture (containing anti-Phospho-Thr202/Thr204 antibodies and protein A-conjugated acceptor beads) was added to the plate using Formulatrix Mantis. The plate was sealed, centrifuged for 1 min at 300xg and incubated for 2 h at room temperature on an orbital shaker. 2 μl/well of donor mixture (containing streptavidin-coated donor beads and biotinylated ERK1/2 distant epitope antibodies) was added using Mantis. The plate was closed, centrifuged for 1 min at 300xg and incubated for 2 h at room temperature on an orbital shaker, after which it was analyzed on a Perkin Elmer Envision 2103 Multilabel Reader (HTS Alpha mode, excitation duration: 40 ms, total measurement duration: 130 ms).

Each sample was plotted against the dose response of parent hFcNRG4, which was included in each plate in duplicate, using XLFit. A graph was built from the obtained data in GraphPad Prism using a four-parameter substitution and the EC50 activity values were calculated from them. The results are presented in table. 5 and 6.

Table 5

Activity of examples 1-5 compared to wild-type NRG4

Sample EC50 (nM) Wild type NRG4 2.53 Example 1 0.77 + 0.16 Example 2 2.77 + 0.39 Example 3 0.29 ±0.01 Example 4 0.53 + 0.05 Example 5 0.84 + 0.27

Data for Examples 1-5 are the average of two experiments, and standard deviation is also indicated.

As can be seen from the data given in table. 5, and consistent with the increased affinity for HER4, in this study the DIG modification also significantly increased activity against HER4 and HER2 compared to wild-type NRG4, including examples that contained additional amino acid modifications.

Table 6

Activity of Examples 6-8 and 10 compared to wild-type NRG1 and NRG4

Sample EC50 (nM) Wild type NRG1 0.3192 Wild type NRG4 1.2980 Example 6 0.3947 Example 7 0.5629 Example 8 0.4033 Example 10 0.2448

As can be seen from the data given in table. 6, in this study, Examples 6-8 and 10, each of which included the D1G modification and additional modifications, show increased activity against HER4 and HER2 compared to wild-type NRG4 and similar activity compared to wild-type NRG1.

Human and rat HER2-4 or HER2-3 studies using phospho-ERK 1/2 (Thr202/Tyr204) to test purified proteins.

Phospho-NIK1-2 activity assays were performed using CHO-K1 cells overexpressing human or rat HER receptors to determine the activity and selectivity of purified NRG4 compounds. CHO-K1 cell lines expressing human or rat HER receptors were cultured in selection medium (DMEM-F12 3:1 with 5% FBS and 20 mM HEPES, 40 μg/ml Lproline, 1x antibiotics, 12 μg/ml puromycin, 1 mg /ml hygromycin B). On day -1 (the day before phospho-NIK1-2 assay), cells were washed once with PBS, detached with enzyme-free cell dissociation solution (GIBCO, cat. no. 13151-014) and resuspended in plating medium (DMEM- F12 3:1 with 20 mM HEPES, 1x antibiotics, 0.2% FBS). Cells were plated in a 96-well poly-O-lysine coated plate (BD, cat. no. 354640) at a density of 10,000 cells in 0.1 ml per well. Cells were cultured in a tissue culture incubator at 37°C, 5% CO ₂ overnight. On day 1 (pERK1/2 assay day), plates were incubated at room temperature for 30 min. Deleted

- 15 046847 nutrient medium, then 50 μl of ligand at different concentrations diluted in PBS-20 mM HEPES-0.005% BSA was added. Native human NRG1 and NRG4 peptides were purchased from Reprokine (Tampa, FL). The duration of stimulation was 30 min (with the exception of the hHER2-4 1H6 line, for which stimulation was carried out for 15 min). Upon completion of stimulation, ligands were removed and 70 μl of lysis buffer (prepared according to the manufacturer's recipe, Perkin Elmer, cat. no. ALSU-PERKA10K) was added. The plates were incubated at room temperature for 10 minutes on a plate shaker, stirring at 350 rpm, then in ice for 1 hour without shaking. Transfer 30 μl of cell lysates to a 96-well white Optiplate (Perkin Elmer, cat. no. 6002290) for phospho-ERK1/2 assay. Briefly, 7.5 μL of acceptor beads were added to 30 μl of lysates in the Optiplate. Cover the plate with aluminum foil and incubate at room temperature for 1 hour on a plate shaker, stirring at 350 rpm. Then 7.5 µl of donor beads were added and the plate was covered with aluminum foil and incubated at room temperature for 2 hours on a plate shaker, stirring at 350 rpm, or at 4°C overnight (warmed the plate at room temperature for 2 hours before analysis). The Alpha signal was read using Envision equipment (a tablet reader compatible with Alpha technology). Donor beads and acceptor beads were prepared according to the study protocol. Data shown were obtained using either the AlphaScreen SureFire p-ERK1/2 (Thr202/Tyr204) Assay Kit (Cat# TGRES50k) or the AlphaLISA® SureFire® Ultra™ p-ERK 1/2 (Thr202/Tyr204) Assay Kit ( Perkin Elmer, cat. no. ALSU-PERKA10K). The activity of the NRG1 peptide was similar in both types of studies. The raw data obtained on Envision equipment was imported into GraphPad Prism software (version 7). The EC50 value was obtained from a four-parameter dose-response curve with a variable slope. Data were obtained as % of maximum NRG1 activity as the ratio of the average maximum baseline value for compound NRG4 to the average maximum baseline value for native human NRG1, multiplied by 100. The data are shown below in table. 7-9. N values reflect the number of independent studies performed.

Table 7

Human HER2/HER4 Study

Sample EC50 (nM) % of maximal NRG1 activity wild type NRG1 0.67 ± 0.07 (N=9) 100 Wild type NRG4 90.09 ±33.21 (N=8) 75 ± 4 (N=8) Example 9 0.20 ±0.01 (N=ll) 91 ±3 (N=ll) Example 10 0.16 ± 0.02 (N=8) 82 ± 4 (N=8) Example I 0.55 ±0.11 (N=5) 79 ± 2 (N=5)

Table 8

Rat HER2/HER4 study

Sample EC50 (nM) % of maximal NRG1 activity wild type NRG1 0.29 ± 0.05 (N=4) 100 Example 9 0.17 ± 0.02 (N=4) 87 ± 4 (N=4) Example 10 0.12 ± 0.04 (N=2) 88 ± 7 (N=2) Example I 0.17 ± 0.06 (N=2) 96 ± 2 (N=2)

As can be seen from table. 7, wild-type human NRG1 peptide exhibits strong activity at human HER4 and HER2 receptors, with wild-type human NRG4 peptide acting as a weak partial agonist. Examples 9-11 demonstrate high activity, and the EC50 values correspond to slightly higher potency compared to wild-type human NRG1, and the maximum activity is % greater than the activity of the wild-type human NRG4 peptide and similar to that of human NRG1. As can be seen from table. 8, similar results were obtained for rat HER4 HER2 receptors, there is a slight increase in efficiency compared to human NRG1 and maximum activity similar to that of human NRG1. In conclusion, the example NRG4 compounds shown demonstrate phospho-ERK1/2 activity at both human and rat HER4 HER2 receptors that is superior to native human NRG4 and similar to that of native human NRG1.

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Table 9

Human and Rat HER2/HER3 Study

Human HER2/HER3 Study Rat HER2/HER3 study Sample EC50 (nM) EC50 (nM) Wild type NRG1 0.83 + 0.20 (N=6) 32 + 14 (N=2) Wild type NRG4 No activity up to 1000hM(N=1) BUT Example 9 No activity up to 3000 nM (N=6) No activity up to 3000 nM (N=4) Example 10 No activity up to 1000 nM (N=3) No activity up to 100 nM (N=2) Example I No activity up to 1000hM(N=1) No activity up to 1000 nM (N=2)

As can be seen from Table 9, the wild-type human NRG1 peptide demonstrates high activity against the human HER3 HER2 receptors, while the wild-type human NRG4 peptide and all examples do not show activity against this pair of receptors, which indicates selectivity that differs from the selectivity in relation to the HER4 HER2 receptor pair. In conclusion, the exemplary NRG4 compounds did not demonstrate phospho-ERK1/2 activity at HER3 HER2 receptors, thus indicating that the HER4 and HER2 receptor selectivity of the wild-type NRG4 peptide is retained.

In vivo studies.

Effect of example 9 on cardiac function and structure in a rat model of MI.

Two non-clinical pharmacological efficacy studies were conducted in a rat model of heart failure with reduced ejection fraction (HFrEF) to examine the plasma concentration and duration of exposure of Example 9. Both studies measured the effects on cardiac function and structure in male Sprague-Dawley rats with surgically induced myocardial infarction.

Both studies used similar methods. Male SpragueDawley rats with surgically induced myocardial infarction were acquired, anesthetized, and ventilated with positive pressure ventilation during the procedure. An incision was made between the fourth and fifth ribs, allowing access to the heart. Permanent ligation of the left coronary artery was performed. Sham-operated animals underwent the same procedure, except that a silk suture was placed around the left coronary artery, which was not tightened. All rats were housed in individual cages in a temperature- and humidity-controlled room and maintained on a 12-h light/dark cycle. Two to three weeks after surgery, transthoracic echocardiography of rats was performed to determine ejection fraction (EF%) and left ventricular size (LVD) using a Vevo 2100 ultrasound system. Rats were randomly assigned to treatment groups according to EF% and LVD.

Measured EF% and LVR values were expressed as mean ± standard error (SE). Statistical analyzes were performed using JMP® 13 software (SAS Institute, Inc.; Cary, NC) and Dunnett's test was used to statistically compare treatment groups. P<0.05 was taken as an acceptable level of statistical significance.

Design of a non-clinical efficacy study to determine plasma concentrations of Example 9. At an infusion rate of 10 μl/h, Example 9 or vehicle (1X Dulbecco's phosphate buffer saline [DPBS]) was administered continuously subcutaneously for 4 days using Alzet® pumps (model 2ML1; Alzet® osmotic pumps; Cupertino, CA). The infusion pump dosages of Example 9 were 0.22, 0.73, 2.18 and 7.28 mg/kg/day. On day 4, blood samples were collected to determine plasma concentrations and the infusion pump was removed. Seven days after the start of administration, cardiac function (EF%) and structure (LV) were assessed in all animals. Fourteen days after the start of administration, EF% and LVD were assessed in the vehicle group and the two highest drug dose groups.

Results. Administration of Example 9 demonstrated a dose-dependent improvement in cardiac function (EF%) when administered over 96 hours using an osmotic pump (Table 10). The highest dose groups of 2.18 and 7.28 mg/kg/day showed improvements in cardiac function within 2 weeks of infusion initiation (Table 10). The indicated doses provide steady-state plasma concentrations of 29.62 and 72.24 nM, respectively (Table 11). For all animals with MI, after treatment, a smaller increase in LVR was shown compared to the group in which the vehicle was administered (Table 10).

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Table 10

Effect of 96-hour infusion on EF in a rat model of HFpEF

Ejection fraction Baseline 7 days after the start of administration 14 days after the start of administration Dose (mg/kg/day) AVERAGE S.E. AVERAGE S.E. AVERAGE S.E. Imitation, N=3 70.2 6.42 68.1 4.64 BUT BUT Carrier, N=7 41.7 1.97 39.6 1.92 39.6 1.73 0.22, N=5 41.6 2.57 43.9 2.04 BUT BUT 0.73, N=7 42.3 1.42 45.8 2.09 But But 2.18, N=7 41.4 1.90 48.9* 1.72 45.5 1.61 7.28, N=7 41D 1.06 49.8* 2.10 48.0* 2.08 Left ventricular diameter (end-diastolic) Baseline 7 days after the start of administration 14 days after the start of administration Dose (mg/kg/day) AVERAGE S.E. AVERAGE S.E. AVERAGE S.E. Imitation, N=3 7.21 0.51 7.49 0.76 BUT BUT Carrier, N=7 7.56 0.35 7.94 0.26 7.90 0.25 0.22, N=5 7.51 0.42 7.53 0.57 BUT BUT 0.73, N=7 7.55 0.27 7.83 0.30 But But 2.18, N=7 7.50 0.34 7.68 0.24 7.75 0.19 7.28, N=7 7.60 0.29 7.52 0.31 7.67 0.19

Abbreviations: n=number of animals; ND = not determined; SE = standard error. *p<0.05 compared to vehicle.

Table 11

Plasma levels in rats after 96-hour infusion

Dose (mg/kg/day) Average (nM) Standard deviation 0.22 2.59 0.71 0.73 9.94 1.59 2.18 29.62 4.86 7.28 72.24 13.66

Non-clinical efficacy study design to determine infusion duration. At an infusion rate of 10 μl/hour, PC 2.18 mg/kg/day of Example 9 or vehicle (DPBS) was administered over 48, 72, or 96 hours using Alzet® pumps. After completion of this infusion phase, blood samples were collected to determine plasma concentrations and the infusion pump was removed. Seven days after the start of administration, cardiac function (EF%) and structure (LV) were assessed in all animals.

Results. As can be seen from table. 12, this study for administration of Example 9 demonstrated an infusion duration-dependent improvement in cardiac function when administered over a period of 48 to 96 hours using the Alzet pump. Infusion of Example 9 for 72 hours and 96 hours resulted in a significant increase in EF on day 7 after initiation of administration. All animals with MI showed a smaller increase in LV RV after treatment compared to the vehicle group.

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Table 12

Effect of different infusion durations of Example 9 on cardiac function

Ejection fraction Baseline 7 days after the start of administration Duration (hours) AVERAGE S.E. AVERAGE S.E. Carrier, N=8 40.8 2.80 39.2 2.78 48, N=8 40.6 1.26 42.6 1.35 72, N=9 41.0 1.96 44.6* 1.69 96, N=9 41.8 1.77 46.2* 2.19 Left ventricular diameter (end-diastolic) Baseline 7 days after the start of administration Duration (hours) AVERAGE S.E. AVERAGE S.E. Carrier, N=8 7.49 0.20 8.10 0.24 48, N=8 7.52 0.29 7.68 0.30 72, N=9 7.55 0.21 7.87 0.20 96, N=9 7.44 0.16 7.70 0.22

Abbreviations: n=number of animals; ND = not determined; SE = standard error, *p < 0.05 compared with vehicle.

Table 13

Plasma levels in a rat model of myocardial infarction after infusion of Example 9

Duration of infusion (hours) Average (nM) Standard deviation 48 40.13 6.37 72 47.28 17.85 96 31.42 9.86

Data from non-clinical studies showed improvement in LVEF in a rat model of HFpEF when plasma concentrations of Example 9 were maintained between 3 and 100 nM over a period of 72 to 96 hours.

Effect of example 10 on cardiac function and structure in a rat model of MI.

A non-clinical pharmacological efficacy study was conducted in a rat model of heart failure with reduced ejection fraction (HFrEF) to evaluate changes in cardiac function and structure after administration of captopril, administration of Example 10, or combination treatment.

Ways. Male Sprague-Dawley rats with surgically induced myocardial infarction were acquired, anesthetized, and ventilated with positive pressure ventilation during the procedure. An incision was made between the fourth and fifth ribs, allowing access to the heart. Permanent ligation of the left coronary artery was performed. All rats were housed in individual cages in a temperature- and humidity-controlled room and maintained on a 12-h light/dark cycle. Two to three weeks after surgery, transthoracic echocardiography of rats was performed to determine ejection fraction (EF%) and left ventricular size (LVD) using a Vevo 2100 ultrasound system. Rats were randomly assigned to treatment groups according to EF% and LVD.

Measured EF% and LVR values were expressed as mean ± standard error (SE). Statistical analyzes were performed using JMP® 13 software (SAS Institute, Inc.; Cary, NC) and Dunnett's test was used to statistically compare treatment groups. P<0.05 was taken as an acceptable level of statistical significance.

Study design. Three weeks after surgical induction of MI, animals were randomly assigned to 2 treatment groups (placebo or captopril, 2 g/l in drinking water, ad libitum) according to EF% and LVD. Three weeks and four weeks after the start of administration, cardiac function (EF%) and structure (LV) were assessed by transthoracic echocardiography. Animals were further randomized into additional groups based on indicators of cardiac function (EF%) and structure (LVH): placebo, example 10, captopril and example 10+captopril. Animals in the Example 10 treatment groups received one injection at week 4 after the ^2nd randomization. Cardiac function (EF%) and structure (LV) were assessed at weeks 5 and 6.

Results. The results are presented in table. 14.

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Table 14

Effect of captopril, a single injection of Example 10, or combination therapy on EF in a rat model of heart failure with reduced ejection fraction

Ejection fraction Baseline 3 week 4 week 5 week week 6 SR S.E. SR S.E. SR S.E. SR S.E. SR S.E. Carrier 38.2 1.42 32.8 1.55 Carrier 33.4 2.49 30.0 2.70 29.9 2.4 3 Captopril 39.1 1.30 38.4 2.43 Captopril 36.1 2.66 36.2 3.23 34.6 3.0 9 Example 10 33.3 2.06 46.5* 3.68 46.0* 2.4 6 Example 10 + captopril 35.8 1.96 47.3* 1.97 46.7* 2.4 5 Left ventricular diameter (end-diastolic) Baseline 3 week 4 week 5 week week 6 SR S.E. SR S.E. SR S.E. SR S.E. SR S.E. Carrier 8.75 0.25 9.00 0.18 Carrier 9.25 0.44 10.16 0.46 9.92 0.40 Captopril 8.66 0.16 8.57 0.14 Captopril 8.64 0.29 8.64* 0.24 8.71* 0.21 Example 10 9.33 0.24 9.35 0.37 9.01 0.25 Example 10+ captopril 8.82 0.12 8.51* 0.23 8.41* 0.28

Abbreviations: ME = mean, SE = standard error. *p<0.05 compared to vehicle.

As can be seen from table. 14, although administration of captopril slowed the decline in cardiac function after 6 weeks of treatment (not statistically significant), administration of Example 10 significantly improved cardiac function (EF%) and reduced LV dilatation after a single dose.

Effect of example 11 on cardiac function and structure in a rat model of MI.

A non-clinical pharmacological efficacy study was conducted in a rat model of heart failure with reduced ejection fraction (HFrEF) to determine the dose-response relationship for Example 11.

Way. Male Sprague-Dawley rats with surgically induced myocardial infarction were purchased from Charles River. Briefly, rats were anesthetized and ventilated with positive pressure during the procedure. An incision was made between the fourth and fifth ribs, allowing access to the heart. Permanent ligation of the left coronary artery was performed. Sham-operated animals underwent the same procedure, except that a silk suture was placed around the left coronary artery, which was not tightened. All rats were housed in individual cages in a temperature- and humidity-controlled room and maintained on a 12-h light/dark cycle. Three weeks after surgery, transthoracic echocardiography of rats was performed to determine ejection fraction (EF%) and left ventricular dimensions (LVD) using a Vevo 2100 ultrasound system. Rats were randomly assigned to treatment groups according to EF% and LVD.

Measured EF% and LVR values were expressed as mean ± standard error (SE). Statistical analyzes were performed using JMP® 13 software (SAS Institute, Inc.; Cary, NC) and Dunnett's test was used to statistically compare treatment groups. P<0.05 was taken as an acceptable level of statistical significance.

Study design. Two to three weeks after surgical induction of MI, animals were assigned to treatment groups and given one injection of vehicle (DPBS) or one of 3 dosages (0.3, 1, 3 mg/kg) of Example 11. After seven days, 14 days and 21 the day after the start of administration, cardiac function (EF%) and structure (LVH) were assessed in all animals.

Results. The results are presented in table. 15.

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Table 15

Effect of Example 11 on EF in a rat model of heart failure with reduced ejection fraction

Ejection fraction Baseline 7 days after administration 14 days after administration 21 days after administration Dose AVERAGE S.E. AVERAGE S.E. AVERAGE S.E. AVERAGE S.E. Imitation, N=5 73.7 2.2 4 72.5 1.76 71.8 0.81 71.9 3.6 0 Carrier, N=8 45.0 1.5 0 41.2 2.90 38.4 1.98 37.8 1.9 8 0.3 mg/kg, N=8 45.4 2.4 6 50.1* 3.22 42.6 1.43 36.4 1.4 3 1 mg/kg, N=8 46.6 1.8 6 52.4* 2.61 42.2 2.77 39.7 2.7 7 3 mg/kg, N=8 45.8 1.4 8 54.7* 2,I 54.0* 2.49 39.6 2.4 9

Abbreviations: n=number of animals; SE = standard error. *p<0.05 compared to vehicle.

As can be seen from table. 15, administration of Example 11 demonstrated a dose-dependent improvement in cardiac function (EF). The high dose group showed improvement in cardiac function within 2 weeks of treatment. There were no differences in LVR increase between groups over the course of the study (data not shown).

Effect of examples 9-11 on cardiotoxicity.

Toxicological studies of Examples 9-11 were conducted in rats and/or cynomolgus monkeys. Example 10 was administered to rats as a single PC 0.3 mg/kg injection, resulting in detectable plasma concentrations for periods greater than 168 hours. The rats were sacrificed and tissue examination revealed cardiac degeneration and necrosis. Example 11 was administered to rats in the form of one PC 30 mg/kg injection; plasma concentrations were undetectable after 96 hours. Rats were sacrificed and tissue examination revealed no evidence of cardiac degeneration and/or necrosis. Example 11 was administered as a single 3 mg/kg dose to male and female monkeys by PC bolus injection. In contrast to the pharmacokinetics in rats, single injections in monkeys resulted in more than 168 hours being required to clear detectable plasma concentrations. The monkeys were sacrificed and tissue examination revealed cardiac degeneration and necrosis. Example 9, which had a half-life of less than an hour after PC administration in rats, was administered at doses that provided plasma concentrations in the range of 3-300 nM for 96-168 hours using subcutaneously (SC) implanted pumps. The rats were sacrificed, and tissue examination revealed no signs of cardiac degeneration and/or necrosis. Example 9, which had a half-life of less than 5 hours after PC administration to monkeys, was administered at doses that provided plasma concentrations in the range of 30-1000 nM over 96 hours to male and female monkeys by surgical insertion of PC or intravenous (IV) catheter. The monkeys were sacrificed and tissue examination revealed no signs of cardiotoxicity and/or necrosis. The findings indicate that NRG4 compounds can be administered without cardiotoxicity by limiting the duration of exposure to the NRG4 compound.

Sequences.

SEQ ID NO:1 - NRG4 human

DHEEPCGPSH KSFCLNGGLC YVIPTISPPF CRCVENYTGA RCEEVFL

SEQ ID NO:2 - NRG4 connection

X0GHEEPCGX8SHKSFCLNGGLCYX22IPTX26PSPFCRCVX35NYTGARCEX44VFL where: X0 is selected from the group consisting of T, PT, MPT, S, GS, GGS, GGGS and (GGGGXL)n, and

Χ _λ is Q, A, E or S, and n=1-5 (SEQ ID NO:5), or absent;

X ₈ represents E or P;

X ₂₂ represents Q or V;

X ₂₆ represents F or I;

X ₃₅ represents E or A; And

X44 represents H, K or E.

SEQ ID NO:3 - complete sequence of expressed human NRG4

MPTDHEEPCGPSHKSFCLNG GLCYVIPTIP SPFCRCVENY TGARCEEVFL PGSSIQTKSN LFEAFVALAV LVTLIIGAFY FLCRKGHFQR ASSVQYDINL VETSSTSAHH SHEQH

SEQ ID NO:4 - NRG4 connection

GHEEPCGPSH KSFCLNGGLC YQIPTIPSPF CRCVENYTGA RCEKVFL

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SEQ ID NO:5 - glycine-rich peptide or linker (GGGGXZ)n where: X/. is Q, A, E or S; and n is 1-5.

SEQ ID NO:6 - IgG Fc region

ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO:7 - IgG Fc region

ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEO ID NO:8 - Fc region IgG

ECPPCPAPPVAGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG KLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG

SEQ ID NO:9 - IgG Fc region

ECPPCPAPPVAGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG KLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG

SEO ID NO:10 - NRG4 connection

GGGGSGGGGSGGGGS GHEEPCGPSH KSFCLNGGLC YVIPTISPPF CRCVENYTGA RCEEVFL

SEO ID NO: 11 - NRG4 connection

GGGGSGGGGSGGGGS GHEEPCGESH KSFCLNGGLC YVIPTISPPF CRCVENYTGA RCEEVFL

SEO ID NO:12 - NRG4 connection

GGGGSGGGGSGGGGS GHEEPCGPSH KSFCLNGGLC YQIPTIPSPF CRCVENYTGA RCEEVFL

SEO ID NO: 13 - NRG4 connection

GGGGSGGGGSGGGGS GHEEPCGPSH KSFCLNGGLC YVIPTFPSPF CRCVENYTGA RCEEVFL

SEO ID NO:14 - NRG4 connection

GGGGSGGGGSGGGGSGHEEPCGPSHKSFCLNGGLCYVIPTIPSPFCRCVANYTGA RCEEVFL

SEO ID NO:15 - NRG4 connection

GGGGSGGGGSGGGGSGHEEPCGPSHKSFCLNGGLCYVIPTFPSPFCRCVENYTGARCEK VFL

SEO ID NO:16 - NRG4 connection

GGGGSGGGGSGGGGSGHEEPCGPSHKSFCLNGGLCYVIPTFPSPFCRCVENYTGARCEH VFL

SEO ID NO:17 - NRG4 connection

GGGGSGGGGSGGGGSGHEEPCGESHKSFCLNGGLCYVIPTFPSPFCRCVENYTGARCEE VFL

SEO ID NO:18 - NRG4 connection

GGGGSGHEEPCGPSHKSFCLNGGLCYQIPTIPSPFCRCVENYTGARCEKVFL

SEQ ID NO:19 - NRG4 connection

GGGGSGGGGSGGGGSGHEEPCGPSHKSFCLNGGLCYQIPTIPSPFCRCVENYTGARCEK VFL

SEO ID NO:20

GGGS

Claims (39)

1. An NRG4 compound containing a modification of the D residue at position 1 with its replacement by a G residue and up to five additional modifications compared to the amino acid sequence of human NRG4 (SEQ ID NO:1). 2. A compound containing the formula: X ₀ GHEEPCGX ₈ SHKSFCLNGGLCYX ₂₂ IPTX ₂₆ PSPFCRCVX ₃₅ NYTGARCEX ₄₄ VFL, where: - 22 046847 X ₀ is selected from the group consisting of GGGS and (GGGGXjn. wherein Χ _λ represents S, and n=1-3 (SEQ ID NO:5), or absent; X ₈ represents E or P; X22 represents Q or V; X ₂₆ represents F or I; X ₃₅ represents E or A; And X ₄₄ is H, K or E (SEQ ID NO:2). 3. The connection according to claim 2, characterized in that X8 represents P; X ₂₂ represents V; X ₂₆ represents I; X ₃₅ represents E; and X ₄₄ represents E. 4. The connection according to claim 2, characterized in that X8 represents E; X ₂₂ represents V; X ₂₆ represents I; X ₃₅ represents E; and X ₄₄ represents E. 5. The connection according to claim 2, characterized in that X8 represents P; X ₂₂ represents Q; X26 represents I; X35 represents E; and X44 represents E. 6. The connection according to claim 2, characterized in that X8 represents P; X ₂₂ represents V; X ₂₆ represents F; X ₃₅ represents E; and X ₄₄ represents E. 7. The connection according to claim 2, characterized in that X8 represents P; X ₂₂ represents V; X ₂₆ represents I; X ₃₅ represents A; and X ₄₄ is H or E. 8. The connection according to claim 2, characterized in that X8 represents P; X ₂₂ represents V; X ₂₆ represents F; X ₃₅ represents E; and X ₄₄ represents K. 9. The connection according to claim 2, characterized in that X8 represents P; X ₂₂ represents V; X26 represents F; X35 represents E; and X44 represents H. 10. The connection according to claim 2, characterized in that X8 represents E; X ₂₂ represents V; X26 represents F; X35 represents E; and X44 represents E. 11. The connection according to claim 2, characterized in that X8 represents P; X ₂₂ represents Q; X26 represents I; X35 represents E; and X44 represents K. 12. The compound according to any one of claims 2 to 11, additionally containing the Fc region of IgG. 13. The compound according to claim 12, characterized in that X0 represents SEQ ID NO:5, where X _a represents S, and n=3, and the Fc region of the IgG contains a dimer of SEQ ID NO:6 and SEQ ID NO:7 wherein the N-terminal amino acid in X ₀ is fused to the C-terminal amino acid in SEQ ID NO:7. 14. The compound according to claim 12, characterized in that X0 represents SEQ ID NO:5, where Χ _λ represents S, and n=3, and the Fc region of the IgG contains a dimer of SEQ ID NO:8 and SEQ ID NO:9 wherein the N-terminal amino acid in X ₀ is fused to the C-terminal amino acid in SEQ ID NO:9. 15. A compound according to any one of claims 2 to 11, characterized in that X ₀ represents SEQ ID NO:5, where X _a represents S, and n=1. 16. A compound according to any one of claims 2 to 11, characterized in that said compound contains an amino acid sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO :12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. 17. A compound consisting of the amino acid sequence of SEQ ID NO:18. 18. The compound according to any one of claims 1 to 17, characterized in that said compound has an activity due to HER4 binding that exceeds the activity of native human NRG4 and is at least 70% of the maximum activity of native human NRG1. 19. A compound according to any one of claims 1 to 18, characterized in that said compound does not have HER3 binding activity. 20. A pharmaceutical composition containing a compound according to any one of claims 1 to 19 and a pharmaceutically acceptable carrier, diluent or excipient. 21. A device in the form of a pump, containing a connection according to any one of claims 1-19. 22. A method of treating or preventing heart failure with reduced ejection fraction (HFrEF) in a patient in need thereof, comprising administering to said patient an NRG4 compound according to any one of claims 1 to 19 in an amount sufficient to provide a therapeutically effective concentration of said NRG4 compound in serum for 24-96 hours. 23. The method of claim 22, wherein said efficacy:administration ratio is from about 2 to about 50. 24. The method according to any one of claims 22, 23, characterized in that said NRG4 compound is administered quarterly (Q3M) by continuous subcutaneous infusion over 24-96 hours. 25. Method according to any one of claims 22 to 24, characterized in that said NRG4 compound is administered using a patch pump. 26. The method according to any one of claims 22-25, characterized in that the treatment is continued for at least 1 year. 27. The method according to any one of claims 22-26, characterized in that said method leads to a significant - 23 046847 significant increase in left ventricular ejection fraction (LVEF). 28. The method according to any one of claims 22-27, characterized in that said method leads to a significant reduction in global longitudinal strain (GLD). 29. The method according to any one of claims 22-28, characterized in that said method leads to a significant increase in left ventricular end-diastolic volume (LVEDV). 30. The method according to any one of claims 22-29, characterized in that said method leads to a significant reduction in the risk of death from cardiovascular disease. 31. The method according to any one of claims 22-30, characterized in that said method leads to a significant reduction in the risk of hospitalization associated with heart failure (HF). 32. The method according to any one of claims 22 to 31, characterized in that said patient suffers from New York Heart Association (NYHA) class II or III heart failure. 33. The method according to any one of claims 22-32, characterized in that said NRG4 compound is an NRG4 compound according to any one of claims 1-19. 34. Use of the NRG4 compound according to any one of claims 1 to 19 for the treatment or prevention of HFpEF. 35. Use according to claim 34, characterized in that said NRG4 compound is administered by Q3M by continuous subcutaneous infusion over 24-96 hours. 36. Use according to any one of claims 34, 35, characterized in that said efficiency:administration ratio is from about 2 to about 50. 37. Use according to any one of claims 34 to 36, characterized in that said NRG4 compound is administered using a patch pump. 38. Use according to any one of claims 34-37, characterized in that said patient suffers from NYHA class II or III heart failure. 39. Use of an NRG4 compound according to any one of claims 1 to 19 for the treatment or prevention of HFpEF, characterized in that said NRG4 compound is administered over a period of 48 to 96 hours.