HK1258994B - Methods for treatment of diseases - Google Patents

Description

Methods for treating diseases

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to provisional patent application serial number 62/211,296, filed on August 28, 2015, the contents of which are incorporated herein by reference in their entirety.

Sequence Listing

This application contains a sequence listing, which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 31, 2012, is named 65292741.txt and is 18,884 bytes in size.

Field of the Invention

The present disclosure provides novel methods for treating diseases including acute myocardial infarction (AMI); gout; ischemia; stroke; traumatic brain injury; toxic shock and cardiac surgery complications using isolated and/or synthesized peptides with unexpected cytoprotective properties.

Background of the Invention

Acute myocardial infarction (AMI) remains a leading cause of morbidity and mortality in the United States and worldwide. Despite current strategies for early reperfusion, many patients die early during this period, and those who survive are at risk of later death from adverse cardiac remodeling, heart failure, and sudden death.

Many publications have highlighted the association between infarct size in AMI and the probability of developing heart failure in the following 2–5 years after AMI.

Patients who suffer an ST-segment elevation myocardial infarction (STEMI) are at particular risk for adverse cardiac remodeling, heart failure, and both in-hospital and long-term mortality. While significant improvements have been made in the treatment of STEMI, reductions in early mortality have been associated with an increased incidence of heart failure after STEMI. This may reflect a higher-risk patient population surviving the index event, as well as an aging population and the prevalence of hypertension and diabetes. Within 30 days of a STEMI, more than 20% of survivors are diagnosed with heart failure, a condition associated with significant morbidity, disability, and mortality.

Heart failure is indeed a major public health problem, affecting approximately 5 million Americans and adding 500,000 cases every year. Compared with other cardiovascular diseases, the incidence and prevalence of heart failure continue to improve, and heart failure is now the leading cause of hospitalization for people aged 65 years, a population segment that is also rapidly increasing. Although the survival rate after heart failure occurs has also improved, current therapy can slow down rather than stop the development of the disease. Being subject to the economic consequences of the progressive symptoms of functional capacity, dyspnea and fatigue, frequent hospitalization and loss of productivity and the limitation of increased medical care costs, heart failure imposes a significant burden on health care.

There is an urgent need to develop additional treatments to minimize infarct size and prevent heart failure after AMI. Current treatments for STEMI include rapid reperfusion of the ischemic myocardium by restoring coronary artery patency (i.e., angioplasty or fibrinolysis), prevention of re-embolism (i.e., antiplatelet agents and anticoagulants), and neurohormonal blockade (i.e., renin-angiotensin-aldosterone and adrenergic blockers). Although each of these interventions provides incremental benefits and significantly reduces morbidity and mortality, the incidence of heart failure after STEMI continues to increase, suggesting that current treatment paradigms are still missing one or more key pathophysiological mechanisms.

Therefore, identifying the mechanisms by which adverse cardiac remodeling and heart failure develop despite optimal treatment is a critical step in developing novel interventions with the ultimate goal of reducing the incidence, burden, and mortality of heart failure after STEMI.

Summary of the Invention

It has been unexpectedly discovered that C-terminal peptides from the serpin molecule (e.g., a short peptide, SP16 (SEQ ID NO: 1)) and variants and derivatives thereof act as effective cytoprotective agents in the treatment of conditions associated with cytokine storm and/or infarction, such as acute myocardial infarction (AMI). These peptides have previously been shown to have anti-inflammatory properties (see, for example, U.S. Patent 8,975,224), which makes their efficacy in treating conditions associated with cell death due to cytokine storm and/or infarction unexpected. In related conditions, although uncontrolled inflammation may contribute to the disease process, inflammation remains a key process required for recovery. This is evidenced by the fact that many attempts to modulate the inflammatory response as a treatment for, for example, acute myocardial infarction (AMI) have failed.

In contrast and unexpectedly, the peptides described herein: 1) allow for therapeutic modulation of inflammatory responses, 2) provide a cytoprotective effect, and 3) reduce infarct size. This combination of activities provides surprising and unexpected efficacy in treating conditions associated with cytokine storm and/or infarction. As described in the Examples herein, this surprising efficacy contrasts with the merely anti-inflammatory activity of, for example, IL-1 antagonists.

Therefore, peptide compositions, pharmaceutical compositions comprising C-terminal serpin peptides, and methods of using the peptides to treat conditions (including conditions associated with cytokine storm) and/or reduce infarction occurring in a variety of conditions (wherein cell protection is desired), such as conditions associated with ischemia-reperfusion injury and the risk of resulting pathology, are provided. The unexpected cell-protective properties of these peptides allow compositions comprising such peptides to be used for new indications and allow preventive intervention in conditions associated with, for example, ischemia-reperfusion injury.

In particular, a novel cytoprotective function of the SP16 peptide consisting of the amino acid sequence VKFNKPFVFLMIEQNTK (SEQ ID NO: 1), or SP163M VKFNKPFVFLNleIEQNTK, has been shown in a well-established animal model of acute myocardial infarction (AMI).

Thus, provided are novel uses of compositions comprising isolated peptides comprising, consisting essentially of, or consisting of the amino acid sequence X1-Z1-F-N-K-P-F-X2-Z2-X3-Z3-Q (SEQ ID NO: 2), wherein

X1 is V or L;

X2 is V, L, M or Nle;

X3 is M, Nle, I or V;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids; and

Z3 is any sequence of five amino acids, and wherein the isolated peptide consists of 37 or fewer amino acids.

The peptides can be modified to extend shelf life and/or bioavailability using one or more non-natural peptide bonds or amino acids, or by attachment to peptide functional groups such as polyethylene glycol (PEG).

The composition may further comprise a carrier, such as a pharmaceutically acceptable carrier.

In one aspect of any of the embodiments described herein is a method of: 1) treating a disease associated with a cytokine storm, 2) reducing infarct size, and/or 3) treating acute myocardial infarction (AMI), the method comprising administering to a human subject having the disease a pharmaceutical composition comprising a peptide selected from:

(a) a peptide comprising the amino acid sequence X1-Z1-F-N-K-P-F-X2-Z2-X3-Z3-Q (SEQ ID NO: 2), wherein

X1 is V or L;

X2 is V, L, Nle, or M;

X3 is M, Nle, I or V;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids; and

Z3 is any five amino acid sequence, and wherein the peptide comprises 37 or fewer amino acids;

(b) a peptide comprising the amino acid sequence VKFNKPFVFLMIEQNTK (SEQ ID NO: 1);

(c) essentially consisting of an amino acid sequence

A peptide consisting of X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3), wherein

X1 is V or L;

X2 is K or R;

X3 is V, L, Nle, or M;

X4 is M, Nle, I or V;

X5 is K or Q;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids; and

Z3 is a sequence of any five amino acids;

(d) a peptide consisting essentially of the amino acid sequence RFNRPFLR (SEQ ID NO: 4);

(e) a peptide consisting essentially of the amino acid sequence RRRFNRPFLRRR (SEQ ID NO: 8);

(f) a peptide consisting essentially of the amino acid sequence VKFNKPFVFLMIEQNTK (SEQ ID NO: 1);

(g) a peptide consisting essentially of the amino acid sequence FNRPFL (SEQ ID NO: 10); and

(h) A peptide comprising SEQ ID NO:57.

In some embodiments of any aspect described herein, the disease associated with cytokine storm is selected from acute myocardial infarction (AMI); gout; stroke; cardiac surgery complications; traumatic brain injury; acute respiratory distress syndrome (ARDS), sepsis, Ebola virus (Ebola), avian influenza, smallpox, and systemic inflammatory response syndrome (SIRS). In some embodiments of any aspect described herein, the human subject in need of reducing infarct size is a subject in need of treatment for a disease selected from the group consisting of acute myocardial infarction (AMI); ischemia; stroke; traumatic brain injury; and toxic shock.

In some embodiments of any of the aspects described herein, the pharmaceutical composition is an oral formulation.

In some embodiments of any aspect described herein, the peptide further comprises at least one second peptide or protein. In some embodiments of any aspect described herein, at least one second peptide or protein is attached to the peptide as a fusion peptide. In some embodiments of any aspect described herein, at least one second peptide or protein is an epitope tag or a half-life extender or both. In some embodiments of any aspect described herein, the peptide comprises one or more D-amino acids.

In some embodiments of any of the aspects described herein, the peptide causes a 75% reduction in serum TNF-α levels when administered to a human subject in an effective amount.

In some embodiments of any aspect described herein, the peptide consists of 35 or fewer amino acid residues. In some embodiments of any aspect described herein, the peptide consists of 22 or fewer amino acid residues. In some embodiments of any aspect described herein, the peptide consists of 21 or fewer amino acid residues.

In some embodiments of any of the aspects described herein, the composition further comprises a pharmaceutically acceptable carrier.

In some embodiments of any of the aspects described herein, the subject does not have, does not have symptoms of, or is not diagnosed with a condition selected from: type II diabetes, lupus, graft-versus-host disease, uveitis, eczema, psoriasis, cystic fibrosis, rheumatoid arthritis, acute radiation syndrome, burns, inflammatory bowel disease, type I diabetes, and hyperglycemia.

The peptides of the present invention can be used to reduce serum TNF-α levels in human subjects with pathologically elevated TNF-α levels. Therefore, the present invention provides methods or uses for reducing TNF-α levels in humans in need thereof, comprising administering the peptides of the present invention in a pharmaceutically acceptable carrier to a human subject. In certain embodiments, the isolated peptide, when administered to a human subject in an effective amount, results in a 50% or 75% reduction in serum TNF-α levels compared to levels prior to administration of the isolated polypeptide. In other embodiments, the isolated peptide further comprises at least one other protein. A combination of at least two proteins may be referred to as a fusion protein. The other protein may be selected from an epitope tag and a half-life extender. The peptide may comprise both an epitope tag and a half-life extender.

Also provided are compositions comprising an isolated peptide consisting essentially of or consisting of the amino acid sequence X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3), wherein

X1 is V or L;

X2 is K or R;

X3 is V, L, M or Nle;

X4 is M, Nle, I or V;

X5 is K or Q;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids;

Z3 is any sequence of five amino acids; and

wherein the isolated peptide, when administered to a human subject in an effective amount, causes a 75% reduction in serum TNF-α levels compared to the amount of TNF-α levels prior to administration of the peptide.

In certain embodiments, the isolated peptide comprises the amino acid sequence X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO:3), wherein X1, X2, X3, X4, X5, Z1, Z2, and Z3 are as defined above, and wherein the peptide consists of a maximum of 35, 22, or 21 amino acid residues.

In some aspects, the peptides described herein and throughout any embodiment of this specification further comprise at least one other protein. The combination of these at least two proteins may be referred to as a fusion protein. Specifically, the other protein may be selected from an epitope tag and a half-life extender. In some aspects of all embodiments of the invention, the isolated peptide may comprise both an epitope tag and a half-life extender.

The present disclosure also provides isolated peptides consisting essentially of or consisting of the amino acid sequences RFNRPFLR (SEQ ID NO: 4) and RFNKPFLR (SEQ ID NO: 5). In certain embodiments, the isolated peptides, when administered to a human subject in an effective amount, cause a 50% or 75% reduction in serum TNF-α levels compared to the amount of TNF-α levels prior to administration of the peptide.

In other aspects of all embodiments of the present invention, the isolated peptide is linked to another protein. The combination of these proteins can be referred to as a fusion protein. Specifically, the other protein can be selected from epitope tags and half-life extenders.

In some aspects of all embodiments of the invention, the isolated peptide consists of at most 100, 35, 22, 21, or 9 additional amino acids.

In other embodiments, the isolated peptide consists essentially of or consists of the amino acid sequence Z1-RFNRPFLR-Z2 (SEQ ID NO:6) and Z1-RFNKPFLR-Z2 (SEQ ID NO:7), wherein Z1 and Z2 are independently 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10 or 1 to 3, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 basic amino acids.

In some embodiments, the isolated peptide consists essentially of or consists of the amino acid sequence RRRFNRPFLRRR (SEQ ID NO: 8) and RRRFNKPFLRRR (SEQ ID NO: 9).

The present disclosure also provides compositions comprising isolated peptides consisting essentially of or consisting of the amino acid sequences FNRPFL (SEQ ID NO: 10) and FNKPFL (SEQ ID NO: 11).

The present disclosure also provides compositions comprising isolated or synthetic peptides consisting essentially of or consisting of any one or a combination of the following peptides: SP40, SP43, SP46, and SP49 as listed in Table B; and their use in the following methods: 1) treating diseases associated with cytokine storm, 2) reducing infarct size, and/or 3) treating acute myocardial infarction (AMI).

In some embodiments of any aspect described herein, the disease associated with cytokine storm is selected from acute myocardial infarction (AMI); gout; stroke; cardiac surgery complications; traumatic brain injury; acute respiratory distress syndrome (ARDS), sepsis, Ebola virus (Ebola), avian influenza, smallpox, and systemic inflammatory response syndrome (SIRS). In some embodiments of any aspect described herein, the human subject in need of reducing infarct size is a subject in need of treatment for a disease selected from the group consisting of acute myocardial infarction (AMI); ischemia; stroke; traumatic brain injury; and toxic shock.

In one embodiment, the present disclosure also provides a method for reducing serum TNF-α compared to the amount of TNF-α level before administering the peptide to the subject, the method comprising administering to the subject an effective amount of any one of the isolated peptides as defined above, thereby reducing serum TNF-α levels by at least 50%. In one embodiment, serum TNF-α levels are reduced by 75% compared to the amount of TNF-α level before administering the peptide. In other embodiments, the subject is a mammal. In some aspects of all embodiments of the present invention, the mammal is a human.

In some aspects of all embodiments of the invention, prior to administration of the peptide, the human has been diagnosed with a disease associated with a cytokine storm, AMI, or infarction. In some aspects of all embodiments of the invention, prior to administration of the peptide, the human has been diagnosed with acute myocardial infarction (AMI); gout, stroke, complications of cardiac surgery, traumatic brain injury, acute respiratory distress syndrome (ARDS), sepsis, Ebola, avian influenza, smallpox, systemic inflammatory response syndrome (SIRS), ischemia, and/or toxic shock.

In some aspects, the human has not undergone prior treatment with an alpha antitrypsin, such as alpha-1 -antitrypsin, prior to treatment with the peptides of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG1 is a table showing the sequences and homologies of serpin C-terminal peptides (SEQ ID NOs: 18-24, respectively, in order of appearance).

FIG2 is a table showing peptides derived from truncated proteins (SEQ ID NOs: 25-32, 1, 33-34, 1, 33, 38-51, 10, and 8, respectively, in order of appearance).

Figures 3A-3D show graphs and images demonstrating that AAT reduces infarct size in experimental acute myocardial infarction (AMI) in mice. Figure 3A shows a graph of the area at risk in the left ventricle. Figure 3B shows a graph of the infarct size (% of the left ventricle). Figures 3D and 3D show Masson's trichrome staining of mid-ventricular heart sections from representative control mice treated with albumin (Figure 3C) or AAT (Figure 3D) 7 days after AMI. The infarct scar is shown in blue. Alb = albumin.

Figures 4A-4C show graphs demonstrating the efficacy of SP163M in reducing myocardial infarct size (Figure 4A), lowering plasma levels of cardiac-specific troponin I (a biomarker of myocardial necrosis) (Figure 4B), which was also significantly reduced by SP16 treatment (Figure 4B), and preserving left ventricular systolic function, as measured by left ventricular fractional shortening using transthoracic echocardiography (Figure 4C).

Figures 5A and 5B are graphs demonstrating that SP163M administered 30' after reperfusion significantly reduced infarct size (Figure 5A) and preserved LV fractional shortening (Figure 5B).

Figures 6A and 6B are graphs demonstrating that SP163M administered 30' after reperfusion reduced infarct size (Figure 6A) and preserved LV fractional shortening (Figure 6B) in a dose-dependent manner.

Figure 7 is a graph demonstrating that SP163M does not affect cardiac contractility. It shows the results of measuring cardiac contractile function at rest (left panel) or after isoproterenol challenge (contraction preservation) (right panel).

FIG8 shows graphs and echocardiograms demonstrating that SP163M reduces infarct size and effectively treats AMI.

FIG9 shows a proposed model for the cytoprotective mechanism of action of SP16 and SP163M.

FIG10 shows graphs of flow cytometry demonstrating that SP16 increases LRP1 expression on Raw264.7 murine macrophages in a dose-dependent manner.

FIG11 shows a schematic diagram of the onset and resolution of the acute inflammatory response in acute myocardial infarction.

Figures 12A and 12B illustrate SP16 binding to LRP1. When serpins bind to inactivating proteases, they undergo a conformational change that exposes a short peptide (5-11 amino acids) containing a unique motif. Figure 12A provides a schematic (modified from Joslin G et al., J Biol Chem 1991 and Lillis A et al., Physiol Rev 2008) illustrating that SP16 contains the AAT pentapeptide FVFLM (amino acid residues 7-11 of SEQ ID NO: 1), which is responsible for binding to LRP1. Figure 12B provides evidence that SP163M, but not SP34, binds to LRP1 in vitro.

Figures 13A and 13B provide evidence that SP16 is an agonist of LRP1. SP163M inhibits NF-κB signaling induced by LPS (Figure 13A) or Gp96 (Figure 13B) in THP1-XBlue ^™ -MD2-CD14 cells, and treatment with an LRP1-blocking antibody or RAP, a cofactor that leads to LRP1 downregulation, limits SP16-related inhibition.

Figures 14A and 14B demonstrate that LRP1 mediates SP16-induced cardioprotection. Mice were pretreated with an LRP1 blocking antibody to test whether LRP1 mediates the cardioprotective signaling of SP163M and AAT. Treatment with the blocking antibody abolished the protective effects of both SP163M and plasma-derived AAT. N = 5-8 per group.

Detailed description

The present disclosure describes novel and unexpected uses of isolated peptides in the field of disease treatment and prevention, wherein the peptides provide a cytoprotective effect. Specifically, the present invention describes novel methods for treating diseases using isolated and/or synthesized peptides having unexpected cytoprotective properties, wherein cytoprotection is an essential component of treating the disease and/or preventing the progression of the condition, including, for example, acute myocardial infarction (AMI); gout; ischemia; stroke; traumatic brain injury; toxic shock, and complications of cardiac surgery.

Serine protease inhibitors (serpins) represent a large (>1000) family of protease inhibitors that are present in all branches of life and are involved in multiple physiological processes. In mammals such as humans, serpins are important for homeostasis, and although there is a certain level of promiscuity, each serpin has a cognate serine protease. For example, α-1-antitrypsin (AAT) and α-1-antichymotrypsin (ACT) inhibit inflammatory proteases such as elastase, while antithrombin inhibits thrombin and plays a role in blood coagulation.

In human diseases including COPD, thrombosis and serpin diseases (sclerosis and dementia), multiple specific AAT mutations have been identified. Currently, the FDA has approved a small amount of AAT preparations derived from human serum for the treatment of COPD. In this treatment method, AAT plays a role similar to that of a protease inhibitor of endogenous AAT.

AAT is the prototype serpin and has a common tertiary structure with other serpins. Serpins have an exposed ring of ~20 amino acids (aa), called the reactive central loop (RCL), which serves as a decoy for homologous proteases. Once protease binds to the RCL, it is trapped, partially unfolded, and destined to degrade. The cleavage of the RCL at its P1-P1 ' site drives the protease inactivation process and causes the release of a small C-terminal peptide from the serpin molecule.

Peptide fragments of serpin and derivatives thereof have been previously described and determined to possess anti-inflammatory properties (U.S. Pat. No. 8,975,224).

The data presented herein from the AMI model demonstrate that AAT-derived peptides, such as SP16, not only possess anti-inflammatory properties but also exhibit surprising cytoprotective and infarction-reducing activity. This cytoprotective and infarction-reducing activity was previously unknown and provides significant and unexpected advantages for treating certain diseases with the peptides described herein. This is demonstrated, for example, by the fact that SP16 exhibits surprising efficacy in treating AMI compared to compounds with only anti-inflammatory activity (e.g., IL-1 antagonists), providing activity not achievable through anti-inflammatory properties alone, resulting in a significantly improved therapeutic effect.

Without wishing to be bound by theory, it is hypothesized that SP16 mediates the cardioprotection described herein by interfering with inflammatory cell death (pyroptosis) caused by ischemia and reperfusion. Specifically, SP16 limits acute ischemia-reperfusion injury, limits acute inflammatory responses in the heart, reduces infarct size, prevents deleterious cardiac remodeling, and prevents heart failure. The proposed model of SP16 activity is shown in FIG9 .

This discovery has led us to provide methods for treating diseases associated with cytokine storm and/or reducing infarct size (e.g., treating AMI) by administering a molecule derived from the C-terminal peptide of a serpin molecule, wherein the molecule is administered in an amount effective to reduce infarct size or mitigate the cell-damaging effects of the cytokine storm, such as ischemia-reperfusion injury.

SP16 (SEQ ID NO: 1) and SP163M (SEQ ID NO: 57), derived from human α-antitrypsin, have been found to not only exhibit anti-inflammatory and immunomodulatory properties similar to those of the parent protein, α-1-antitrypsin, but also act as cytoprotective agents. SP16 appears to be a first-in-class peptide master switch for treating autoimmune, inflammatory, and metabolic diseases, and furthermore has unexpected effects as a cytoprotective agent. Without wishing to be bound by theory, the peptides of the present invention are expected to offer a good safety profile based on the good safety profile of the parent protein, α-1-antitrypsin. However, the peptides of the present invention are easier and therefore less expensive to produce because they are much smaller than the parent protein.

It has been shown previously that the C-terminal peptide derived from the cleavage of a serpin molecule caused by one of its homologous serine proteases has a biological function that is different from the protease inhibitor function of the parent (intact serpin molecule). For example, the C-terminal peptides from AAT, antichymotrypsin, and kallikrein binding proteins have varying degrees of anti-inflammatory effects. Based on our current research and unexpected results, we now believe that the therapeutic use of these peptides can be expanded from anti-inflammatory uses to cytoprotective uses. The new function allows for the prevention of treatments such as ischemia.

Previously known functions of serpins involve inhibiting the function of serine proteases. Some serpins inhibit other types of proteins, and several do not have an inhibitory function.

Serpins are a large family (>1,000) of serine protease inhibitors that are structurally similar but functionally diverse. They are involved in multiple physiological processes and are crucial for mammalian homeostasis. Mutations in the genes for individual serpins are implicated in diverse human diseases, including COPD, thrombosis, and emphysema.

Each serpin with inhibitory effect is responsible for blocking the activity of one or more proteins. Serpins bind to their target proteins, thereby preventing them from completing any further reactions. When bound to their targets, the structure of the serpins undergoes irreversible changes. Certain cells recognize when a serpin binds to its target and remove these attached proteins from the bloodstream.

Alpha-1-antitrypsin (AAT) is the prototypical serine protease inhibitor. PROLASTIN® (Talecris), ZEMAIRA® (Aventis Behring), and ARALAST® (Baxter) are human serum-derived AAT preparations approved by the FDA for the treatment of COPD. AAT is currently in clinical trials for the treatment of new-onset type 1 diabetes, graft-versus-host disease, and cystic fibrosis.

Researchers have identified at least 37 different serpin genes in humans. Based on our research, we propose that the isolation and synthesis of C-terminal fragments of serpins provide a new source of molecules with anti-inflammatory, cytoprotective, and infarction-reducing activities. Therefore, we believe that at least the C-terminal fragments of serpins listed in Table A are useful in the methods described herein.

The peptides described in U.S. Patent No. 8,975,224 are short, isolated or synthetic C-terminal peptides based on the specific definition of serpins and their variants and derivatives, which surprisingly have effective anti-inflammatory properties compared to native serpins and are of a much more useful size for therapeutic use. The isolated peptides are shown in Figures 1-2. Figure 1 shows the amino acid sequences of the C-terminal fragments of various serpins. Each peptide is indicated by SEQ ID NO: in column 2, immediately to the left of the peptide. Figure 2 shows truncations of the C-terminal fragments as shown in Figure 1, as well as their variants and derivatives. Similarly, each peptide is indicated by SEQ ID NO: in column 2, immediately to the left of the peptide.

Alanine screening has shown that the isolated, synthesized, or modified SP16 peptides shown in Table B below are particularly effective in reducing TNF-α levels in mouse models of inflammation. Specifically, within these particular fragments, the three N-terminal and two C-terminal amino acids appear to contribute to the peptide's anti-inflammatory properties, as replacing them appears to diminish the peptide's ability to reduce TNF-α levels in a mouse model of sepsis challenged with LPS (see, e.g., PCT/US13/20498; the entirety of which is incorporated herein by reference). Thus, in some aspects of all embodiments of the present invention, the peptide is selected from SP40, SP43, SP46, and SP49, whose peptide sequences are shown in Table B.

Table B shows peptides designated SP16; SP40; SP43; SP46; and SP49, which provide particularly good anti-inflammatory effects when administered to a mouse model of sepsis. SP16, SP163M, SP37, SP40, and/or SP51 bind to LRP1 and are contemplated to be cytoprotective as described herein. In some embodiments, SP16, SP163M, SP37, SP40, and/or SP51 can be used in the methods described herein.

According to some embodiments and aspects of the present invention, methods of providing cytoprotective therapy include administering to a subject, such as a human subject, an effective cytoprotective or infarct size reducing amount of any isolated peptide consisting of or consisting essentially of the sequences set forth in SEQ ID NOs: 8, 10, 19-34, and 38-49, which can be used in the methods described herein. Any peptide consisting of or consisting essentially of the sequences set forth in SEQ ID NOs: 8, 10, 19-34, and 38-49 can also be used to reduce TNF-α in a subject. In certain embodiments, the amount of TNF-α in serum is reduced by 50% or more, or 75% or more, compared to the amount of TNF-α in serum before administration of the peptide.

Table C below provides additional exemplary peptides that were used to reduce TNF-α levels in mice subjected to LPS challenge. Peptides having the ability to reduce TNF-α levels as described in PCT/US13/20498 (incorporated herein by reference in its entirety) are also contemplated for use in the present invention in compositions, pharmaceutical compositions, and methods of use and treatment of inflammatory conditions.

SP1 (SEQ ID NO:18); SP2 (SEQ ID NO:19); SP3 (SEQ ID NO:20); SP4 (SEQ ID NO:21); SP5 (SEQ ID NO:22); SP6 (SEQ ID NO:23); SP7 (SEQ ID NO:24); SP8 (SEQ ID NO:25); SP9 (SEQ ID NO:26); SP11 (SEQ ID NO:28); SP12 (SEQ ID NO:29); SP13 (SEQ ID NO:30); SP14 (SEQ ID NO:31); SP15 (SEQ ID NO:32); SP16 (SEQ ID NO:1); SP163M (SEQ ID NO:57) SP17 (SEQ ID NO:33); SP18 (SEQ ID NO:34).

The phrase "consisting essentially of..." is used herein to limit the scope of the peptide to the specified material amino acids and to include only additional amino acids or changes that do not materially affect the basic and novel properties of the invention, namely, the anti-inflammatory ability of the short isolated or synthetic peptide. This definition specifically excludes peptides having the entire serpin sequence, and this definition also specifically excludes any naturally occurring serpin peptide sequence that is equal to or greater than 37 amino acids.

Without wishing to be bound by theory, important amino acids that provide the core and possible modifications for, for example, the anti-inflammatory peptides made herein have also been identified. Also provided are isolated peptides encompassed by the formula shown below, and they can be used to reduce inflammation, treat cytokine storms, increase cell survival, and/or reduce infarct size.

It is known that human AAT, antichymotrypsin and kallikrein binding protein contain certain components with anti-inflammatory properties. However, these components have not been identified before. A new family of peptides derived from a human serpin has been determined using, for example, a mouse endotoxemia model (LPS-induced endotoxemia) that has an effective anti-inflammatory effect. Based on the efficacy of the peptide in the mouse inflammation model, the size of the peptide, and the safety profile of the parent protein, peptides based on AAT, peptides such as SP16, and their fragments and derivatives provide a novel and improved molecule for treating inflammation in the human body.

Formula I provides a composition comprising a peptide comprising, consisting essentially of, or consisting of the following amino acid sequences: X1-Z1-F-N-R-P-F-X2-Z2-X3-Z3-Q (SEQ ID NO: 35) and X1-Z1-F-N-K-P-F-X2-Z2-X3-Z3 (SEQ ID NO: 2), wherein

X1 is V or L;

X2 is V, L, M or Nle;

X3 is M or Nle, I or V;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids; and

Z3 is any five amino acid sequence, wherein the peptide comprises 37 or fewer amino acids.

In certain aspects of all embodiments, the isolated peptide, when administered to a human subject in an effective amount, causes a 50% or 75% reduction in serum TNF-α levels compared to the serum levels measured before administration of the isolated peptide. In some aspects of all embodiments of the invention, the peptide further comprises a fusion protein. The fusion protein can be selected from an epitope tag and a half-life extender or a combination thereof.

Formula II provides a composition comprising an isolated peptide comprising, consisting essentially of, or consisting of the following amino acid sequence: X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3), wherein

X1 is V or L;

X2 is K or R;

X3 is V, L, M or Nle;

X4 is M, Nle, I or V;

X5 is K or Q;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids;

Z3 is any five amino acid sequence; and wherein the isolated peptide, when administered to a human subject in an effective amount, causes a 75% reduction in serum TNF-α levels compared to serum levels prior to administration of the isolated peptide.

In certain embodiments, a peptide comprising the amino acid sequence X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3) as defined above comprises a maximum of 35, 22, or 21 amino acid residues. In certain aspects of all embodiments of the present invention, the peptide further comprises a fusion protein. Specifically, the fusion protein can be selected from an epitope tag and a half-life extender, or a combination thereof.

In some aspects of all methods and uses of the invention, the peptide is SP16.

In some aspects of all methods and uses of the invention, the peptide may comprise the peptide of SEQ ID NO: 57. In some aspects of all methods and uses of the invention, the peptide may consist essentially of the peptide of SEQ ID NO: 57.

Thus, the present invention also provides methods and uses involving isolated peptides consisting of or consisting essentially of the amino acid sequences RFNRPFLR (SEQ ID NO:4) and RFNKPFLR (SEQ ID NO:5), which can also be used to treat inflammation. In certain embodiments, the peptides, when administered to a human subject in an effective amount, cause a 50% or 75% reduction in serum TNF-α levels compared to serum TNF-α levels prior to administration of the isolated peptide. In some aspects of all embodiments of the present invention, the isolated peptide further comprises a fusion protein. Specifically, the fusion protein can be selected from an epitope tag and a half-life extender. In other embodiments, the isolated peptide comprises a maximum of 100, 35, 22, 21, 16, or 9 amino acids. In other embodiments, the isolated peptide comprises the amino acid sequence Z1-RFNRPFLR-Z2 (SEQ ID NO:6) and Z1-RFNKPFLR-Z2 (SEQ ID NO:7), wherein Z1 and Z2 are independently 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10 or 1 to 3, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 basic amino acids.

In some aspects of all embodiments of the invention, the isolated peptide consists essentially of, or consists of, the amino acid sequences RRRFNRPFLRRR (SEQ ID NO: 8) and RRRFNKPFLRRR (SEQ ID NO: 9). The disclosure also provides compositions comprising peptides consisting essentially of, or consisting of, the amino acid sequences FNRPFL (SEQ ID NO: 10) and FNKPFL (SEQ ID NO: 11).

In certain embodiments, the isolated peptides comprise five or more contiguous amino acids derived from the amino acid sequence FLMIEQNTK (SEQ ID NO: 36). These peptides can be used to reduce inflammation, enhance cell survival, treat cytokine storm, reduce infarct size, and/or lower TNF-α levels in a subject. In certain embodiments, the amount of TNF-α in serum is reduced compared to the amount of TNF-α in serum prior to administration of the isolated peptide.

In some aspects, the method of treatment further comprises analyzing TNF-α serum levels before and after administering the isolated peptide. If the reduction in TNF-α serum levels is less than 30%, the administering step can be repeated with the same dose as the first dose or a greater dose of the peptide than the first dose.

Fragments of any of the above peptides can vary in size. For example, the fragments can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 amino acids in length.

In addition to treating diseases associated with cytokine storms and/or reducing infarct size, the above-mentioned peptides can also, for example, treat AMI and reduce inflammation. The peptides have anti-inflammatory and immunomodulatory effects and, in addition, directly or indirectly, stimulate beta cell regeneration. In certain embodiments, these peptides reduce inflammation by reducing the activity or expression of TNF-α. The activity of TNF-α can be reduced by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%. The expression of TNF-α can be reduced by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%. When administered for treatment, the peptide composition generally also comprises a pharmaceutically acceptable solution or carrier.

The peptides described above can also be used to treat, prevent or improve the symptoms of several pathologies. Therefore, the present disclosure also provides a method for treating a disease associated with a cytokine storm or a method for reducing infarct size, the method comprising the step of administering any one of the peptides described herein or a combination thereof to a subject in need of treatment. In some aspects, the subject has not been treated with α-antitrypsin before the treatment. In some aspects, the method comprises the steps of detecting whether the individual has an increased serum TNF-α level, and if the subject has an increased serum TNF-α level, the peptide is administered to the subject, and if the subject does not have an increased serum TNF-α level, the peptide is not administered to the subject.

The present disclosure also enables methods for preventing the development of a disease associated with a cytokine storm or preventing an increase in infarct size, the methods comprising the steps of administering any one of the peptides or a combination thereof to a subject in need of prevention of a disease associated with a cytokine storm or prevention of an increase in infarct size. In some aspects, the subject has not been treated with α-antitrypsin prior to the treatment. In some aspects, the method comprises the steps of detecting whether the individual has an elevated serum TNF-α level, and administering the peptide to the subject if the subject has an elevated serum TNF-α level, and not administering the peptide to the subject if the subject does not have an elevated serum TNF-α level. In some aspects of all embodiments of the invention, the subject in need has not previously been treated for any condition with any peptide as described herein.

In some aspects of all embodiments of the present invention, the primary purpose of treatment is to treat or prevent the undesirable cell death and tissue degeneration associated with the damage following the effects of the following diseases: acute myocardial infarction (AMI); gout; stroke; complications of cardiac surgery; traumatic brain injury. Effective dosages in such applications are measured by determining the effect on preventing or ameliorating ischemia-reperfusion injury. The dosage is not necessarily the same as the dosage for treating inflammation alone. Effects can also be measured by determining, for example, the level of cell viability and/or cell death in specific organs or tissues.

For convenience, the definitions of certain terms used throughout the patent application (including the specification, examples, and appended claims) are used throughout the specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (the field to which this invention belongs).

The term "wild type" refers to a naturally occurring polynucleotide sequence encoding a protein or a portion thereof, or a protein sequence or a portion thereof, respectively, as it normally occurs in vivo.

The term "mutation" refers to any change in the genetic material of an organism, particularly a change in a wild-type polynucleotide sequence or a change in a wild-type protein sequence (i.e., a deletion, substitution, addition, or alteration). Although it is generally believed that changes in the genetic material result in changes in protein function, the term "mutation" refers to a change in the wild-type protein sequence, whether the change alters protein function (e.g., improves function, reduces function, or imparts new function), or whether the change has no effect on protein function (e.g., mutation or variation is silent). In this patent application, the terms mutation and polymorphism are used interchangeably herein.

Unless otherwise defined, the terms "polypeptide" and "protein" are used interchangeably to refer to isolated polymers of amino acid residues and are not limited by minimum length. Peptides, oligopeptides, dimers, multimers, and the like are also composed of linear arrays of amino acids linked by peptide bonds, and whether biologically produced and isolated from their natural environment, produced using recombinant techniques, or generally produced synthetically from naturally occurring amino acids.

In some aspects, the polypeptide or protein is a "modified polypeptide" comprising non-naturally occurring amino acids. In some aspects, the polypeptide comprises a combination of naturally occurring and non-naturally occurring amino acids, and in some embodiments, the peptide comprises only non-naturally occurring amino acids.

In some aspects of all embodiments of the invention, the peptides or modified peptides further comprise co-translational and post-translational (C-terminal peptide cleavage) modifications, such as disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., by furin or metalloproteinases), and the like, to the extent that such modifications do not affect the anti-inflammatory properties of the isolated peptides or their ability to improve glycemic control.

In some aspects of the present invention, the polypeptide is altered. The term "altered polypeptide" refers to a peptide comprising changes to the native sequence such as deletions, additions, and substitutions (properties are generally conservative, as known to those skilled in the art, such as alanine), as long as the protein maintains the desired activity, that is, maintains its anti-inflammatory activity, can improve glycemic control or reduce hyperglycemia. These modifications can be intentional, such as by site-directed mutagenesis, or can be accidental, such as by mutations in artificial hosts such as genetically engineered bacteria, yeast, or mammalian cells that produce the protein, or due to errors caused by PCR amplification or other recombinant DNA methods. Polypeptides or proteins are composed of linearly arranged amino acids connected by peptide bonds, but have a definite conformation relative to peptides. In contrast to peptides, proteins are generally composed of chains of 50 or more amino acids.

As used herein, the term "peptide" generally refers to an amino acid sequence consisting of a single chain of amino acids linked by peptide bonds. Unless otherwise defined, a peptide generally comprises at least two amino acid residues and is less than about 50 amino acids in length.

"Modified peptides" may include the incorporation of non-natural amino acids into the peptides of the present invention, including, in some cases, the incorporation of synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids into the peptide (or other components of the composition other than the protease recognition sequence), as desired. Peptides containing D-amino acids exhibit improved stability in vivo or in vitro compared to forms containing L-amino acids. Therefore, constructing peptides incorporating D-amino acids can be particularly useful when greater stability in vivo or within cells is desired or required. More specifically, D-peptides are resistant to endogenous peptidases and proteases, thereby providing better oral transepithelial and transdermal delivery of associated drugs and conjugates, improved bioavailability of membrane-persistent complexes (see below for details), and extended intravascular and interstitial life (when such properties are desired). The use of D-isomeric peptides can also facilitate transdermal and oral transepithelial delivery of associated drugs and other cargo molecules. In addition, D-peptides cannot be effectively processed for major histocompatibility complex class II-restricted presentation by T helper cells and are therefore less likely to elicit a humoral immune response in the whole organism. Peptide conjugates can thus be constructed using, for example, the D-isomer form of the cell penetrating peptide sequence, the L-isomer form of the cleavage site, and the D-isomer form of the therapeutic peptide. Thus, in some embodiments, the disclosed peptides comprise L and D amino acids, including no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 D-amino acids. In certain aspects, the peptides comprise more than 10 D-amino acids, and in certain aspects all amino acids of the peptides are D-amino acids.

In another aspect, the peptide or fragment or derivative thereof may be a "retro-flip peptide." A "retro-flip peptide" refers to a peptide having reversed peptide bonds at at least one site, i.e., the amino- and carboxyl-termini are reversed relative to the amino acid side chains. Thus, a retro-flip analog has reversed termini and reversed peptide bond orientations while generally maintaining the side chain topology of the native peptide sequence. Retro-flip peptides may comprise L-amino acids or D-amino acids, or a mixture of L- and D-amino acids, with all amino acids being D-isomers at most. Analogs of partial retro-flip peptides are polypeptides in which only a portion of the sequence is reversed and substituted with enantiomeric amino acid residues. Because the retro-flip portion of such an analog has reversed amino and carboxyl termini, the amino acid residues flanking the retro-flip portion are substituted with α-substituted gem-diaminomethane and malonate, respectively, having similar side chains. Retro-flip forms of cell-penetrating peptides have been found to be as effective as the native form in transmembrane transfer. The synthesis of retro-inverted peptide analogs is described in Bonelli, F. et al., Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A and Viscomi, G. C, J. Chem. Soc. Perkin Trans. 1:697-701 (1985); and U.S. Pat. No. 6,261,569, which are incorporated herein by reference in their entirety. A solid phase synthesis method for some retro-inverted peptide analogs has been described (EP 97994-B), which is also incorporated herein by reference in its entirety.

The terms "homology," "identity," and "similarity" refer to the degree of sequence similarity between two peptides or two optimally aligned nucleic acid molecules. Homology and identity can each be determined by comparing positions in respective sequences that can be aligned for comparison purposes. For example, it is based on the use of standard homology software, such as BLAST, version 2.2.14, at default positions. When equivalent positions in the compared sequences are occupied by identical bases or amino acids, the molecules are identical at that position; when equivalent sites are occupied by similar amino acid residues (e.g., similar in steric and/or electronic properties, such as conservative amino acid substitutions), the molecules are said to be homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of similar or identical amino acids at positions shared by the compared sequences, respectively. "Unrelated" or "non-identical" sequences have less than 40% identity, but preferably less than 25% identity, to the sequences disclosed herein.

As used herein, the term "sequence identity" refers to two polynucleotide or amino acid sequences that are identical over a comparison window (i.e., on a nucleotide-nucleotide or residue-residue basis). The term "percentage of sequence identity" is calculated as follows: two optimally aligned sequences within the comparison window are compared, the number of positions at which the same nucleic acid base (e.g., A, T, C, G, U, or I) or residue is present in the two sequences is determined to obtain the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to obtain the percentage of sequence identity.

As used herein, the term "substantially identical" refers to a characteristic of a polynucleotide or amino acid sequence that has at least 85% sequence identity, preferably at least 90% to 95% sequence identity, and more typically at least 99% sequence identity, over a comparison window of at least 18 nucleotide (6 amino acid) positions, typically over a window of at least 24-48 nucleotide (8-16 amino acid) positions, to a reference sequence, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that may contain deletions or additions totaling 20% or less of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence. When used to describe polypeptides, the term "similarity" is determined by comparing the amino acid sequence and conservative amino acid substitutions of one polypeptide to the sequence of a second polypeptide.

As used herein, the terms "homologous" or "homologs" are used interchangeably and, when used to describe polynucleotides or polypeptides, mean that two polynucleotides or polypeptides, or designated sequences thereof, are identical when optimally aligned and compared (e.g., using BLAST, version 2.2.14 (see herein) with default parameters for alignment), with appropriate nucleotide insertions or deletions or amino acid insertions or deletions in at least 70% of the nucleotides, typically from about 75% to 99%, more preferably at least about 98% to 99% of the nucleotides. As used herein, the terms "homologs" or "homologs" also refer to homology with respect to structure and/or function. With respect to sequence homology, sequences are homologs if they are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% identical, at least 97% identical, or at least 99% identical. The determination of homologs of the genes or peptides of the present invention can be readily determined by one skilled in the art.

The term "substantially homologous" refers to sequences that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Homologous sequences can be identical functional genes in different species. Homologs of the genes or peptides of the present invention can be readily determined by those skilled in the art.

For sequence comparison, usually a sequence is used as a reference sequence, and test sequence is compared with this reference sequence.When using a sequence comparison algorithm, test sequence and reference sequence are input into a computer, subsequence coordinates (if necessary), and sequence algorithm program parameters are specified.Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters specified.

Optimal alignment of sequences for comparison can be achieved by, for example, the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), incorporated herein by reference, the homology alignment algorithm of Needleman and Wunsch, J. MoI. Biol. 48:443-53 (1970), incorporated herein by reference, the search similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-48 (1988), incorporated herein by reference, by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection. (See generally, Ausubel et al. (eds.), Current Protocols in Molecular Biology, 4th ed., John Wiley and Sons, New York (1999)).

An example of an available algorithm is PILEUP. PILEUP uses progressive, pairwise alignments to create a multiple sequence alignment from a group of related sequences, displaying percentages of sequence identity. It also draws a tree or dendogram to display the clustering relationships used to create the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng and Doolittle (J. MoI. Evol. 25:351-60 (1987), which is incorporated herein by reference). The method used is similar to the method described by Higgins and Sharp (Comput. Appl. Biosci. 5:151-53 (1989), which is incorporated herein by reference). The program can align up to 300 sequences, with a maximum length of 5,000 nucleotides or amino acids per sequence. The multiple alignment process begins with a pairwise alignment of the two most similar sequences, generating a cluster of two aligned sequences. This cluster is then aligned with the next most related sequence or cluster of aligned sequences. The sequence of two clusters is contrasted by the simple extension of the paired comparison of two independent sequences.Finally, the comparison is realized by a series of progressive paired comparisons.This program is by specifying specific sequence and their amino acid or nucleotide coordinates for the sequence comparison region and running by specifying program parameters.For example, following parameters can be used to compare reference sequence and other test sequences, thereby determining the sequence identity percentage relationship: default gap weight (3.00), default gap length weight (0.10) and weighted end gap.

Another example of an algorithm suitable for determining percent sequence identity and percent sequence similarity is the BLAST algorithm, which is described by Altschul et al. (J. MoI. Biol. 215:403-410 (1990), which is incorporated herein by reference). (See also Zhang et al., Nucleic Acid Res. 26:3986-90 (1998); Altschul et al., Nucleic Acid Res. 25:3389-402 (1997), which are incorporated herein by reference). Software for performing BLAST analyses is publicly available through the Internet website of the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive threshold score T when aligned with a word of the same length in a database sequence. T is called the neighborhood word score threshold (Altschul et al. (1990), supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. These word hits are then extended as far as possible in both directions along each sequence as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is stopped when: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score reaches zero or below due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9 (1992), incorporated herein by reference) an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77 (1993), which is incorporated herein by reference). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a reference amino acid sequence if the smallest sum probability in a comparison of the test amino acid to the reference amino acid is less than about 0.1, more typically less than about 0.01, and most typically less than about 0.001.

As used herein, the term "variant" refers to a peptide or nucleic acid that differs from a polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, additions, substitutions, or side chain modifications, but retains one or more specific functions or biological activities of these naturally occurring molecules. Amino acid substitutions include changes in which amino acids are substituted with different naturally occurring or unconventional amino acid residues. Such substitutions can be classified as "conservative," in which case the amino acid residues contained in the polypeptide are replaced with another naturally occurring amino acid having similar properties in terms of polarity, side chain function, or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention can also be "non-conservative," in which amino acid residues present in a peptide are substituted with amino acids having different properties (e.g., naturally occurring amino acids from different groups) (e.g., charged amino acids or hydrophobic amino acids are substituted with alanine); or, wherein naturally occurring amino acids are substituted with unconventional amino acids. In some embodiments, amino acid substitutions are conservative. The term variant, when applied to polynucleotides or polypeptides, also encompasses polynucleotides or polypeptides that may have alterations in primary structure, secondary structure, or tertiary structure when compared to a reference polynucleotide or polypeptide, respectively, e.g., compared to a wild-type polynucleotide or polypeptide.

Variants may also be synthetic, recombinant, or chemically modified polynucleotides or polypeptides isolated or generated using methods known in the art. As described below, variants may comprise conservative or non-conservative amino acid changes. Alterations in a polynucleotide may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. Variants may also comprise insertions, deletions, or substitutions of amino acids, including insertions and substitutions of amino acids and other molecules not normally found in the peptide sequence underlying the variant, such as, but not limited to, insertions of ornithine, which do not normally occur in human proteins. The term "conservative substitution," when describing a polypeptide, refers to a change in the amino acid composition of a polypeptide that does not substantially alter the activity of the polypeptide. For example, a conservative substitution refers to the replacement of one amino acid residue with a different amino acid residue having similar chemical properties. Conservative amino acid substitutions include the substitution of leucine with isoleucine or valine, the substitution of aspartic acid with glutamic acid, or the substitution of threonine with serine.

"Conservative amino acid substitutions" result from replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as replacing leucine with isoleucine or valine, replacing aspartic acid with glutamic acid, or replacing threonine with serine. Thus, "conservative substitutions" of a particular amino acid sequence refer to replacing amino acids that are not critical for the activity of the polypeptide, or replacing amino acids with other amino acids having similar properties (e.g., acidity, basicity, positive or negative charge, polarity or non-polarity, etc.), such that even substitutions of critical amino acids do not reduce the activity of the peptide (i.e., the ability of the peptide to cross the blood-brain barrier (BBB)). Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company (1984), which is incorporated herein by reference in its entirety.) In some embodiments, individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small percentage of amino acids may also be considered "conservative substitutions" if the alteration does not reduce the activity of the peptide. Insertions or deletions are typically within the range of about 1 to 5 amino acids. The choice of a conservative amino acid can be made based on the position of the amino acid to be substituted in the peptide, for example, whether the amino acid is on the exterior of the peptide and exposed to the solvent, or whether it is internal and not exposed to the solvent.

In an alternative embodiment, the amino acid to be substituted for an existing amino acid can be selected based on the position of the existing amino acid, i.e., its exposure to the solvent (i.e., whether the amino acid is exposed to the solvent or is present on the outer surface of the peptide or polypeptide, as compared to an internally located amino acid that is not exposed to the solvent). The selection of such conservative amino acid substitutions is well known in the art, for example, as disclosed in Dordo et al., J. Mol Biol, 1999, 217, 721-739 and Taylor et al., J. Theor. Biol. 119 (1986); 205-218 and S. French and B. Robson, J. Mol. Evol. 19 (1983) 171. Thus, conservative amino acid substitutions can be selected for amino acids on the exterior of the protein or peptide (i.e., amino acids exposed to the solvent), for example, but not limited to, the following substitutions can be used: substitution of F for Y, substitution of S or K for T, substitution of A for P, substitution of D or Q for E, substitution of D or G for N, substitution of K for R, substitution of N or A for G, substitution of S or K for T, substitution of N or E for D, substitution of L or V for I, substitution of F for Y, substitution of S for T or A, substitution of K for R, substitution of N or A for G, substitution of K for R, substitution of S, K or P for A.

In alternative embodiments, conservative amino acid substitutions suitable for amino acids within the protein or peptide can also be selected, for example, suitable conservative substitutions can be used for amino acids within the protein or peptide (i.e., amino acids that are not exposed to solvents), for example, but not limited to, the following conservative substitutions can be used: where F replaces Y, A or S replaces T, L or V replaces I, Y replaces W, L replaces M, D replaces N, A replaces G, A or S replaces T, N replaces D, L or V replaces I, Y or L replaces F, A or T replaces S, and S, G, T or V replace A. In some embodiments, the term mutation also encompasses non-conservative amino acid substitutions.

As used herein, the term "derivative" refers to a peptide that has been chemically modified by techniques such as, but not limited to, ubiquitination, labeling, pegylation (derivation with polyethylene glycol), lipidation, glycosylation, or the addition of other molecules. A molecule is also a "derivative" of another molecule when it contains additional chemical moieties that are not normally part of the molecule. Such moieties can improve the solubility, absorption, biological half-life, etc. of the molecule. Alternatively, the moiety can reduce the toxicity of the molecule, eliminate or reduce any undesirable side effects of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences, 18th ed., A. R. Gennaro, Ed., Mack Publ., Easton, PA (1990), which is incorporated herein by reference in its entirety.

Thus, in certain aspects of all embodiments of the invention, the peptides of the invention comprise peptide derivatives, such as pegylated peptides.

The term "functional" when used in conjunction with "derivative" or "variant" refers to a peptide of the invention (as a functional derivative or functional variant of said entity or molecule) having a biological activity (functional or structural) substantially similar to that of the entity or molecule, i.e., anti-inflammatory activity in the context of the peptides described herein. The term functional derivative is intended to include fragments, analogs or chemical derivatives of a molecule.

The term "insertion" or "deletion" generally ranges from about 1 to 5 amino acids. The variation allowed can be determined experimentally by systematically generating insertions, deletions or substitutions of nucleotides in the sequence using recombinant DNA technology while simultaneously synthetically producing the peptide.

The term "substitution" when referring to a peptide refers to a change of an amino acid to a different entity, such as another amino acid or amino acid moiety. The substitution can be a conservative substitution or a non-conservative substitution.

"Analogs" of molecules such as peptides refer to molecules that are functionally similar to the complete molecule or its fragments. The term "analog" is also intended to include allelic species and induced variants. Analogs typically differ from naturally occurring peptides at one or several sites, often through conservative substitutions. Analogs typically exhibit at least 80 or 90% sequence identity with the native peptide. Some analogs also include non-natural amino acids or modifications of the N or C-terminal amino acids. Examples of non-natural amino acids include, but are not limited to, disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine. Fragments and analogs can be screened for preventive or therapeutic efficacy in transgenic animal models as described below.

"Covalently bonded" means attached directly or indirectly (eg, through a linker) via a covalent chemical bond. In some aspects of all embodiments of the invention, the fusion peptide is covalently bonded.

As used herein, the term "fusion protein" refers to a recombinant protein of two or more proteins. Fusion proteins can be produced, for example, by linking a nucleic acid sequence encoding one protein to a nucleic acid encoding another protein so that they form a single open reading frame that is translated into a single polypeptide containing all of the desired proteins in the cell. The order in which the proteins are arranged can vary. The fusion protein can include an epitope tag or a half-life extender. Epitope tags include biotin, FLAG tag, c-myc, hemagglutinin, His6 (SEQ ID NO:37), digoxigenin, FITC, Cy3, Cy5, green fluorescent protein, V5 epitope tag, GST, β-galactosidase, AU1, AU5, and avidin. Half-life extenders include Fc domains and serum albumin.

The terms "subject," "individual," and "patient" are used interchangeably herein and refer to an animal, such as a human or non-human animal (e.g., a mammal), to which a pharmaceutical composition as disclosed herein is provided for treatment, including prophylactic treatment. As used herein, the term "subject" refers to a human or non-human animal. The term "non-human animal" includes all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles, and the like. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or surrogate animal used as a disease model. Non-human mammals include mammals such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, and cows. In some aspects, the non-human animal is a companion animal such as a dog or cat.

"Treating" a disease or condition in a subject, or "treating" a patient suffering from a disease or condition, refers to subjecting an individual to drug therapy, e.g., administration of a drug, such that at least one symptom of the disease or condition is alleviated or stabilized. Typically, when the peptide is administered therapeutically as a treatment, it is administered to a subject exhibiting one or more symptoms of, e.g., AMI.

The term "prevention" is used in association with preventing symptoms or delaying symptom development from an asymptomatic state. Typically, when the peptide is administered prophylactically, it is administered to a subject who does not exhibit the near symptoms of a disease (e.g., AMI) as described herein. Typically, the subject is faced with the risk of developing an infarction or a disease associated with a cytokine storm, such as AMI, due to family history, laboratory results, genetic testing, or lifestyle. The peptide may also be administered within 10-30 minutes or 10-60 minutes after the onset of symptoms, such as AMI or a stroke, or after diagnosing the symptoms, or within 10 minutes to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, up to 2, 3, 4, or 5 days, to prevent symptom exacerbation. The peptide can also be administered within 10-30 minutes or 10-60 minutes after the injury and/or the indicator event, or within 10 minutes to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, up to 2, 3, 4, 5 days to prevent worsening of symptoms. In some embodiments of any aspect described herein, the peptide can be administered within 10-30 minutes, within 30-60 minutes, or within 10-60 minutes after the injury and/or the indicator event.

"Specifically binds" or "specific binding" refers to a compound or antibody that recognizes and binds to a desired polypeptide but does not substantially recognize and bind to other molecules in a sample (e.g., a biological sample), which naturally includes the polypeptides of the present invention. Specific binding can be characterized by a dissociation constant of at least about 1 x 10-6 M or less. In other embodiments, the dissociation constant is at least about 1 x 10-7 M, 1 x 10-8 M, or 1 x 10-9 M. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

By "isolated" is meant that the polypeptide has been separated from any natural environment, such as body fluids like blood, and from components which naturally accompany the peptide.

Isolated and "substantially pure" refer to polypeptides that have been isolated and purified, separated to at least some degree from components with which they are naturally associated. Generally, a polypeptide is substantially pure when it is at least about 60%, or at least about 70%, at least about 80%, at least about 90%, at least about 95%, or even at least about 99% free, by weight, from naturally associated proteins and naturally occurring molecules. For example, a substantially pure polypeptide can be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in cells that do not normally express the protein, or by chemical synthesis.

"Reduce" or "inhibit" as used in the context of, for example, TNF-α levels, refers to a decrease in the amount of protein in a biological sample, such as a blood or tissue sample, a cell, a cell extract, or a cell supernatant. For example, such a decrease can be caused by decreased RNA stability, transcription or translation, increased protein degradation, or RNA interference. Preferably, the decrease is at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 80%, or even at least about 90%, compared to a reference value.

In the context of the claims and this patent application, the term "reference value" generally refers to an abnormally high level of TNF-α present in an individual affected by or suffering from inflammation. A reference value is generally the amount of TNF-α in an individual before administration of the peptides of the present invention. In some aspects of all embodiments regarding glycemic control, the term "reference value" refers to a numerical value for measuring glycemic control in a subject. There are multiple tests that can be used to determine, for example, whether a human subject is affected by prediabetes. Such tests include, for example, an A1C test, a fasting plasma glucose test (FPG), and an oral glucose tolerance test (OGTT).

"Increase" in the expression or activity of a gene or protein refers to a positive change in the level or activity of a protein or nucleic acid in a cell, cell extract, or cell supernatant. For example, the increase can be due to increased RNA stability, transcription, or translation, or decreased protein degradation. Preferably, the increase is at least 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 100%, at least about 200%, or even about 500% or more above the expression or activity level under control conditions.

As used herein, the term "recombinant," when used to describe a nucleic acid molecule, refers to a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin that, due to its origin or manipulation, is not associated with all or part of the polynucleotide with which it is associated in nature. When applied to a protein or polypeptide, the term recombinant refers to a polypeptide produced by expression of a recombinant polynucleotide. When applied to a host cell, the term recombinant refers to a host cell into which a recombinant polynucleotide has been introduced. Recombinant is also used herein to refer to a substance (e.g., a cell, nucleic acid, protein, or vector) that has been modified by the introduction of heterologous material (e.g., a cell, nucleic acid, protein, or vector).

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked; a plasmid is one type of vector encompassed by the term "vector." The term "vector" generally refers to a nucleic acid sequence containing an origin of replication and other entities necessary for replication and/or maintenance in a host cell. Vectors capable of directing the expression of genes and/or nucleic acid sequences to which they are operably linked are referred to herein as "expression vectors." Generally, practical expression vectors are typically in the form of "plasmids," which refer to circular double-stranded DNA loops that, in their vector form, are not bound to chromosomes and typically contain entities for stable or transient expression of the encoding DNA. Other expression vectors that can be used in the methods disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, phage, or viral vectors, and such vectors can be integrated into the host genome or can replicate autonomously in specific cells. Vectors can be DNA or RNA vectors. Other forms of expression vectors known to those skilled in the art that provide equivalent functions, such as self-replicating extrachromosomal vectors or vectors that integrate into the host genome, can also be used. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors."

The term "viral vector" refers to a vector that utilizes a virus or a virus-associated vector as a nucleic acid construct that enters a cell. The construct can be integrated and packaged into a non-replicating, defective viral genome (such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV) or other, including retroviral vectors and lentiviral vectors) for infection or transduction into a cell. The vector may or may not be incorporated into the cell genome. If desired, the construct may include viral sequences for transfection. Alternatively, the construct may be incorporated into a vector capable of free replication, such as EPV and EBV vectors.

The article "a/an" is used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. By way of example, "an element" refers to one element or more than one element. Unless otherwise indicated in the operating examples or as otherwise noted, all numerals representing the amount of a component or reaction conditions used herein should be understood to be modified by the term "about" in all cases. When used together with a percentage, the term "about" may mean +1%. The present invention is further described in detail by the following examples, but the scope of the present invention should not be limited thereto.

It should be understood that the present invention is not limited by the specific methods, schemes and reagents described herein, and therefore can be varied. The terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the scope of the present invention, which is defined only by the claims. Other features and advantages of the present invention will be apparent from the following detailed description, drawings and claims.

The treatment method of the present invention

One aspect of the present invention relates to the use of peptides described herein and mutants, variants, analogs or derivatives thereof. Specifically, these methods involve administering any of the peptides described herein or their pharmaceutically acceptable modified forms in a pharmaceutically acceptable carrier to a subject, such as a mammal in need thereof, such as a human, i.e., a subject suffering from a disease associated with a cytokine storm; a subject in need of reducing infarct size; and/or a subject in need of treating AMI. The clinical descriptions of these diseases and conditions are well known. In some aspects, a person is first diagnosed with one or more symptoms of a disease before administering one or more peptides of the present invention. In some embodiments, the person has not previously been given AAT as a treatment for the symptoms.

As used herein, a disease "associated with a cytokine storm" is a disease in which a cytokine storm is a cause of the disease, a symptom of the disease, or a consequence of the pathogenesis of the disease. Non-limiting examples of diseases associated with a cytokine storm may include acute myocardial infarction (AMI); gout; stroke; complications of cardiac surgery; and/or traumatic brain injury.

The subject in need of reducing infarct size may be a subject with infarction. The subject in need of reducing infarct size may be a subject with or diagnosed with, for example, acute myocardial infarction (AMI); ischemia; stroke; traumatic brain injury; and/or toxic shock.

As used herein, "acute myocardial infarction" or "AMI" refers to a reduction or cessation of blood flow to the heart, which can lead to destruction of heart tissue through a variety of causes.

In some embodiments of any of the aspects described herein, the subject does not have a condition selected from, does not have symptoms of a condition selected from, and/or is not diagnosed with a condition selected from: type II diabetes, lupus, graft-versus-host disease, uveitis, eczema, psoriasis, cystic fibrosis, rheumatoid arthritis, acute radiation syndrome, burns, inflammatory bowel disease, type I diabetes, and hyperglycemia.

For example, SP16 peptides have been shown to significantly improve symptoms in preclinical mouse models established for human diseases including AMI. For example, SP16 has been shown to reduce infarct size.

Using well-established preclinical safety studies, evidence has been provided that the SP16 peptide can be safely administered. For example, SP16 has been shown to have no effect on cardiac contractility in an AMI model. Furthermore, the FastPatch assay has shown that the SP16 peptide does not affect hERG activity, and no hits have been identified for the SP16 peptide in human receptor panning studies (GenSEPExplorer).

Thus, in one aspect, a method for treating a subject having a disease associated with a cytokine storm; a subject in need of reducing infarct size; and/or a subject in need of treating AMI is provided, the method comprising administering to a human subject in need thereof a composition comprising at least one peptide of the present invention. In some aspects, the peptide comprises SP16.

It has been shown that the peptides of the present invention, such as SP16, are Toll-like receptor-2 agonists. Therefore, without wishing to be bound by theory, the peptides, such as SP16, are used as anti-inflammatory drugs, which work by promoting the distribution of anti-inflammatory cytokines. In addition, without wishing to be bound by theory, the peptides, such as SP16, can also be used as immunomodulators, which work by inducing the expansion of tolerogenic and protective T-regulatory cells (T-regs). In addition, without wishing to be bound by theory, compared to most currently available treatments that generally suppress the immune system (using general immunosuppressant treatments to expose the treated individual to infection risks), the peptides, such as SP16, can also downregulate the autoimmune response without inducing general immunosuppression, thereby providing excellent treatment for autoimmune diseases.

Because the peptides are derived from AAT, and based on results from in vivo and in vitro models, it is reasonable to expect that most of the therapeutic effects of AAT also apply to the peptides of the present invention (such as SP16). Specifically, AAT has been shown to modulate T-cell proliferation and NF-kappa-B activation; impair NK-target cell interactions; inhibit serine protease activation involved in EGFR/TLR-4 signaling in epithelial cells; participate in TNF-α-induced gene expression and apoptosis in endothelial cells; prevent Escherichia coli ( E. coli )-induced erythrocyte hemolysis and reduce circulating eosinophil counts; inhibit neutrophil chemotaxis, NADHP oxidase, and ANCA signaling; inhibit monocyte and macrophage cytokine release and CD14 expression, and inhibit mast cell histamine release; and modulate B-cell proliferation and cytokine production. In some aspects, the peptide is SP16, which may include one or more modifications commonly used to improve peptide bioavailability and/or shelf life, such as PEGylation.

Peptide optimization analysis was also performed using a TLR-2 assay, employing alanine scanning. Data were acquired using an experiment with an engineered TLR-2 indicator cell line (HEK-BLUE™ mTLR2, Invivogen). Cells were incubated with 20 μg/ml of the indicator peptide for 24 hours. Upon TLR2 activation, cells secrete alkaline phosphatase, which can be detected. The assay was performed in triplicate, and the average values were plotted. Peptide SP34 was a scrambled peptide control (yellow), and PAM (Pam3CSK4; red) was a positive control. See, for example, PCT/US13/20498, which is incorporated herein by reference in its entirety.

The following table provides the results of the pharmacokinetic profile analysis of SP16.

For this analysis, three normal rats were intravenously infused with 5 mg/kg of SP16, and SP16 plasma concentrations were measured at eight time points post-injection. SP16 levels were measured at each time point by LC/MS/MS, and these values were used to calculate the Cmax (2.5 μg/ml) and T1/2 (1.9 hours). The assay was performed by Apredica, Boston, MA. Therefore, SP16 was determined to have a half-life of 1.9 hours in normal rats.

The SP16 safety profile also includes hERG data. The hERG FastPatch analysis showed that SP16 did not inhibit hERG at doses up to 25 μM. This data predicts that SP16 will not pose cardiac safety concerns in humans. The study was conducted by Apredica, Boston, MA.

In addition, human receptor panning was also used for analysis. The GenSEP Explorer panel contains 111 in vitro assay targets that were carefully selected to evaluate the biological activity of drugs/chemicals. Assay categories include GPCRs, voltage-gated ion channels, ligand-gated ion channels, neurotransmitter transporters, nuclear receptors and steroids, as well as different groups of biochemical targets, including phosphodiesterases, kinases and other related enzymes. The study was performed by Caliper LifeSciences, and the results are summarized in the table below. It seems that SP16 has no effect on 111 human receptors, indicating that SP16 has an excellent human safety profile.

Based on the safety profile of SP16, it is reasonable to extrapolate that other peptide fragments provided herein will also be safe when administered to humans.

In one embodiment, the methods of treatment described herein also include selecting or diagnosing a subject with any of the above-mentioned conditions, such as a condition caused by inflammation, prior to administering a peptide as disclosed herein or a mutant, variant, analog, or derivative thereof, thereby treating a condition or malfunction, such as inflammation. Such selection is performed by a skilled artisan using a variety of available methods, such as assessing the symptoms described herein. For example, AMI can be diagnosed by, for example, an electrocardiogram, chest radiograph, or troponin levels. For example, the amount of TNF-α in a subject can be assessed to determine the degree of inflammation present in the subject.

In some aspects of all embodiments, C-reactive protein (CRP) can be used as a marker for inflammation or treatment efficacy. C-reactive protein (CRP) is used to detect inflammation if there is a high suspicion of tissue injury or infection somewhere in the body. CRP is used as a general marker for infection and inflammation and can be used to assess an individual for acute or chronic inflammatory conditions. High or elevated levels of CRP in the blood indicate the presence of inflammation. In individuals suspected of having a severe bacterial infection, high CRP indicates the presence of infection. In people with chronic inflammatory conditions, high levels of CRP indicate a disease flare or that treatment has not been effective. Normal concentrations in healthy human serum are typically below 10 mg/L, increasing slightly with age. Higher levels are seen in women in late pregnancy, mild inflammation and viral infections (10–40 mg/L), active inflammation, bacterial infections (40–200 mg/L), and severe bacterial infections and burns (>200 mg/L). In some aspects, the term "reference value" refers to the measured value of CRP when using CRP as a diagnostic test for inflammation.

Successful or effective treatment is demonstrated by alleviating one or more symptoms of illness or dysfunction as described herein. It is expected that a peptide as disclosed herein or its mutant, variant, analog or derivative will be administered to a subject in need thereof to prevent or delay the development of illness and body dysfunction as described herein (e.g., those resulting from infarction, cytokine storm, inflammation or autoimmune tissue destruction, or the illness alleviated by stimulating beta cell mass expansion in individuals with diabetes). The term "prevention" is used to refer to a situation in which the subject has not yet suffered from a specific illness through prevention, meaning that it has not yet been revealed in any perceptible form. Prevention encompasses the occurrence and/or severity of prevention or delay of symptoms (including situations in which the subject has already suffered from one or more symptoms of another illness). Prevention is generally carried out in subjects with a disease or body dysfunction. It is believed that such subjects need prevention. For example, a reduction in TNF-α levels compared to the level before administering the peptide of the present invention will be evidence of successful treatment.

In one embodiment, the prevention methods described herein also include selecting a subject at risk for a condition, such as those described herein that result from infarction, cytokine storm, inflammation, or autoimmune tissue destruction or body malfunction, before administering the peptide or its mutant, variant, analog, or derivative to the subject, thereby preventing the condition or malfunction. Such selection is performed by a skilled artisan using a variety of available methods. For example, risk factors may be assessed or a disease known to cause the condition or malfunction may be diagnosed, or a treatment or therapy known to cause the condition or malfunction may be performed. It is generally believed that subjects with a disease or injury or with a relevant family history (known to promote the condition) have an increased risk.

As used herein, the term "treat" refers to therapeutic measures where the goal is to prevent or delay the progression of a disease, such as reducing at least one effect or symptom of a disorder, disease, or condition associated with inflammation. Depending on the condition, treatment is generally "effective," as that term is defined herein, if one or more symptoms improve or if levels of clinical markers such as troponin, TNF-α, CRP, blood glucose, and/or HbA1c are within normal ranges or closer to normal reference values than abnormal values reflecting inflammation or poor glycemic control. Alternatively, treatment is "effective" if the progression of the disease is slowed, the manifestation of symptoms, or disease markers are reduced. That is, "treat" includes improving symptoms or markers, delaying progression, or delaying the worsening of at least one symptom that would be expected to worsen without treatment. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in the extent of the disease, stabilization of the condition (i.e., no worsening), delaying or slowing the progression of the disease, and remission or palliation of the condition. "Treatment" may also refer to prolonged survival compared to the expected survival if the patient had not received treatment. Those in need of treatment include patients suffering from one or more symptoms of infarction, cytokine storm, or inflammation, such as symptoms associated with AMI.

TNF-α levels can be assessed, for example, using a number of readily available commercial ELISA kits.

In some aspects, the present invention relates to a method for preventing cytokine storm, infarction, and/or AMI by administering the peptide to an individual who does not yet exhibit symptoms of cytokine storm, infarction, and/or AMI. For example, the peptide can be administered to an individual at high risk of developing AMI but who has not yet suffered AMI to help delay the development of AMI or prevent the development of AMI.

As used herein, the term "effective amount" refers to an amount of a pharmaceutical composition comprising one or more peptides as disclosed herein, or mutants, variants, analogs, or derivatives thereof, that is used to reduce at least one or more symptoms of a disease or condition, and relates to a sufficient amount of a pharmacological composition to provide the desired effect. As used herein, the phrase "therapeutically effective amount" refers to an amount of a composition that is sufficient to treat a condition, has a reasonable benefit/risk ratio, and can be used for any medical treatment. Thus, the term "therapeutically effective amount" refers to an amount of a composition as disclosed herein that, when administered to a typical subject suffering from, for example, AMI, is sufficient to achieve a therapeutic or prophylactic reduction in symptoms or clinical markers associated with elevated levels of inflammation, infarction, or cytokine storm. Typically, a reduction of more than 20% in disease markers, such as inflammatory markers (e.g., TNF-α), indicates effective treatment. In some cases, a reduction of more than 50% or more than 75% in TNF-α levels in a subject prior to administration of the peptides of the present invention indicates effective treatment.

A therapeutically or prophylactically significant reduction in symptoms is, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150% or more of a measured parameter compared to a control or untreated subject or the subject's state before administration of the peptide. Measured or measurable parameters include clinically detectable disease markers, such as increased or decreased levels of biomarkers such as TNF-α, and parameters related to clinically accepted symptom measures or markers of infarction, cytokine storm, and inflammation. However, it should be understood that the total daily dosage of the compositions and formulations disclosed herein will be determined by the attending physician within the scope of reasonable medical judgment. The exact amount required will vary according to a variety of factors, such as the type of disease to be treated, the sex, age, and weight of the subject.

For the treatment of subjects with diseases associated with cytokine storm; subjects in need of reduction of infarct size; and/or subjects in need of treatment for AMI, the term "therapeutically effective amount" refers to an amount that is safe and sufficient to delay the progression of one or more symptoms and result in a decrease in disease markers such as TNF-α or CRP concentrations. The therapeutically effective amount for a disease depends on the type of disease, the species being treated, the age and general condition of the subject, the mode of administration, and other factors. Therefore, it is impossible to specify a precise "effective amount." However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation. The therapeutic effect can be determined by one of ordinary skill in the art. For example, the therapeutic effect can be assessed in known animal models of inflammation (e.g., LPS models), AMI (e.g., isoproterenol models), autoimmune tissue destruction (e.g., CAIA models), or diabetes (e.g., db/db mouse models). When using experimental animal models, therapeutic efficacy is demonstrated when symptoms of cytokine storm or infarction are reduced relative to untreated animals.

In some embodiments of any aspect described herein, the subject does not have a condition selected from, does not have symptoms selected from, or is not diagnosed with a condition selected from: type II diabetes, lupus, graft-versus-host disease, uveitis, eczema, psoriasis, cystic fibrosis, rheumatoid arthritis, acute radiation syndrome, burns, inflammatory bowel disease, type I diabetes, and hyperglycemia. In some embodiments of any aspect described herein, the subject does not have elevated TNF-α levels. In some embodiments of any aspect described herein, the subject does not need to have reduced TNF-α levels.

As used herein, the terms "administering" and "introducing" are used interchangeably herein and refer to placing a therapeutic agent, such as one or more peptides or mutants, variants, analogs or derivatives thereof, as disclosed herein, into a subject by a method or route that results in delivery of such therapeutic agent to the desired site. The compound can be administered by any suitable route that results in effective treatment of the subject.

One or more peptides disclosed herein, or mutants, variants, analogs, or derivatives thereof, can be administered by any route known in the art or described herein, such as orally, parenterally (e.g., intravenously or intramuscularly), intraperitoneally, rectally, transdermally, nasally, vaginally, by inhalation, transdermally (patch), or ophthalmically. One or more peptides disclosed herein, or mutants, variants, analogs, or derivatives thereof, can be administered in any dose or dosage regimen. Pumps, such as those used for insulin administration, can also be utilized. In some embodiments, one or more peptides can be administered orally. As shown in FIG. 16 of U.S. Patent No. 8,975,224, which is incorporated herein by reference in its entirety, oral administration of SP16 has been shown to be effective compared to intraperitoneal injection of SP16 and dexamethasone.

dose

For the treatment methods of the present invention, it is not intended that the administration of one or more peptides or mutants, variants, analogs or derivatives thereof as disclosed herein be limited to a specific mode of administration, dosage, or frequency of administration; the present invention contemplates all modes of administration, including intramuscular, intravenous, intraperitoneal, intracapsular, intraarticular, intralesional, subcutaneous, or any other route of administration, which is sufficient to provide a dose sufficient to treat, for example, inflammation, infarction, or a condition associated with a cytokine storm. The therapeutic agent can be administered to the patient in a single dose or multiple doses. When multiple doses are administered, the doses can be spaced apart from each other by, for example, one hour, three hours, six hours, eight hours, one day, two days, one week, two weeks, or one month. For example, the therapeutic agent can be administered for 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It should be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition. For example, if a lower dose does not provide sufficient therapeutic activity, the dose of the therapeutic agent can be increased.

While the attending physician will ultimately determine the appropriate amount and administration schedule, a therapeutically effective amount of one or more peptides, or mutants, variants, analogs, or derivatives thereof, as disclosed herein, may be a dose of 0.0001, 0.01, 0.01, 0.1, 1, 5, 10, 25, 50, 100, 500, or 1,000 mg/kg or μg/kg. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test bioassays or systems.

The dosage for a particular patient or subject can be determined by one of ordinary skill in the art using conventional considerations (e.g., by suitable conventional pharmacological regimens). A physician may, for example, first prescribe a relatively low dose and subsequently increase the dose until a suitable response is obtained. The dose given to the patient is sufficient to achieve a beneficial therapeutic response, or, for example, to reduce symptoms, or other suitable activity, over time, in the patient, depending on the application. The dosage is determined by the following factors: the efficacy of the specific formulation, and the activity, stability, or serum half-life of one or more peptides as disclosed herein, or their mutants, variants, analogs, or derivatives, as well as the patient's condition, and the weight or body surface area of the patient to be treated. The dosage size is also determined by the presence or absence, nature, and extent of any adverse side effects given to a specific subject by a specific carrier, formulation, or the like. According to methods well known in the art, therapeutic compositions comprising one or more peptides as disclosed herein, or their mutants, variants, analogs, or derivatives are optionally tested in one or more suitable in vitro and/or in vivo disease animal models, such as inflammation or diabetes models, to confirm efficacy, tissue metabolism, and estimate dosage. Specifically, the dosage can be initially determined by activity, stability or other suitable measurements of treatment versus non-treatment (e.g., comparison of treatment versus non-treatment cells or animal models) using a relevant assay. The dosage rate is determined by the LD50 of the relevant formulation and/or observation of any side effects of one or more peptides as disclosed herein, or mutants, variants, analogs or derivatives thereof. Administration can be accomplished via a single dose or divided doses.

In determining the effective amount of one or more peptides as disclosed herein, or mutants, variants, analogs or derivatives thereof, which are administered to treat or prevent a disease, the physician assesses circulating plasma levels, formulation toxicity, and progression of the disease.

The efficacy and toxicity of a compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effect is the therapeutic index, and it can be expressed as the ratio LD50/ED50. Pharmaceutical compositions that exhibit high therapeutic indices are preferred.

These compounds can be administered to humans and other animals for therapeutic use by any suitable route of administration that is effective for small peptides, including oral, nasal (e.g., by spray), rectal, vaginal, parenteral, intracisternal, and topical, e.g., as powders, ointments, or drops, including buccal and sublingual administration.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, and is not toxic to the subject.

The selected dosage level will depend upon a variety of factors, including the activity of the specific compound employed in the present invention, or its ester, salt, or amide, the route of administration, the number of administrations, the rate of excretion of the specific compound employed, the duration of the treatment, other drugs, compounds and/or substances used in combination with the specific compound employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and the like, such factors being well known in the medical arts.

Pharmaceutical composition preparation – “Pharmaceutically acceptable carrier”

As disclosed herein, one or more peptides or mutants, variants, analogs or derivatives thereof can be administered in any suitable manner, which results in a protein concentration for the treatment of a disease. The compound can be included in any suitable carrier material in any suitable amount, and the amount generally present is 1-95% by weight of the total composition. The composition can be suitable for oral, parenteral (e.g., intravenous or intramuscular), intraperitoneal, rectal, skin, nasal, vaginal, inhalation, skin (patch), or eye administration. Therefore, the composition can be in the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels (including hydrogels), pastes, ointments, creams, plasters, immersions, osmotic delivery devices, suppositories, enemas, injections, implants, sprays, or aerosols. Pharmaceutical compositions can be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 20th edition, 2000, ed. A.R. Gennaro, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, herein incorporated by reference in their entireties).

Pharmaceutical compositions according to the present invention can be formulated to release the active compound either immediately upon administration or at any predetermined time or period of time after administration. The latter types of compositions are generally referred to as controlled-release formulations and include (i) formulations that produce a substantially constant concentration of the agent of the invention in vivo over an extended period of time; (ii) formulations that produce a substantially constant concentration of the agent of the invention in vivo over an extended period of time after a predetermined lag time; (iii) formulations that maintain the effect of the agent for a predetermined period of time by maintaining a relatively constant effective level of the agent in vivo while minimizing undesirable side effects associated with fluctuations in the plasma level of the agent (a sawtooth kinetic pattern); (iv) formulations that localize the effect of the agent, for example, by spatially placing the controlled-release composition near or within a diseased tissue or organ; (v) formulations that allow for convenient dosing, for example, by administering the composition once a week or once every two weeks; and (vi) formulations that target the effect of the agent by using carriers or chemical derivatives to deliver the therapeutic agent to specific target cell types. Administration of proteins in the form of controlled-release formulations is particularly preferred for compounds with a narrow absorption window in the gastrointestinal tract or a relatively short biological half-life.

Any of a number of strategies can be used to achieve controlled release, wherein the release rate is greater than the metabolic rate of the compound. In one example, controlled release is achieved by appropriate selection of different formulation parameters and ingredients, including, for example, different types of controlled release compositions and coatings. Thus, the protein is formulated into a pharmaceutical composition with suitable excipients that release the protein in a controlled manner upon administration. Examples include single or multi-unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, molecular complexes, microspheres, nanoparticles, patches, and liposomes.

As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. As used herein, the phrases "systemic administration," "administered systemically," "administered peripherally," and "administered peripherally" refer to administration of a therapeutic composition other than directly into a tumor, such that it enters the entire body of the animal and is thereby subjected to metabolism and other similar processes.

As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms: they are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment. As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition or carrier involved in maintaining the activity of the agent or carrying or transporting the agent from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. In addition to being the term "pharmaceutically acceptable" as defined herein, each carrier must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Pharmaceutical formulations contain one or more peptides or mutants, variants, analogs or derivatives thereof as disclosed herein together with one or more pharmaceutically acceptable ingredients. The carrier can be in the form of a solid, semi-solid or liquid diluent, cream or capsule. These pharmaceutical formulations are another object of the present invention. Typically, the amount of active compound is 0.1-95% by weight of the formulation, preferably 0.2-20% by weight of the formulation for parenteral use, and preferably 1% to 50% by weight of the formulation in the case of oral administration. In order to use the method of the present invention clinically, the targeted delivery composition of the present invention is formulated into a pharmaceutical composition or pharmaceutical preparation for parenteral administration, such as intravenous administration; mucosal administration, such as intranasal administration; enteral administration, such as oral administration; topical administration, such as transdermal administration; ocular administration, such as via corneal scarification or other modes of administration. The pharmaceutical composition comprises a combination of a compound of the present invention and one or more pharmaceutically acceptable ingredients. The carrier may be in the form of a solid, semisolid or liquid diluent, cream or capsule.

The term "pharmaceutically acceptable carrier" is intended to include all solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, suitable for the particular dosage form desired. Typically, such compounds are carried or transported from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its functional derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and other nontoxic, compatible substances used in pharmaceutical formulations.

As used herein, the term "pharmaceutical composition" refers to a composition or formulation that generally includes an excipient, such as a pharmaceutically acceptable carrier, that is conventional in the art and suitable for administration to a mammal, preferably a human or human cell. Such compositions can be specifically formulated for administration via one or more of a variety of routes, including but not limited to oral, ocular, parenteral, intravenous, intraarterial, subcutaneous, intranasal, sublingual, intraspinal, intracerebroventricular, and the like. In addition, compositions for topical (e.g., oral mucosa, respiratory mucosa) and/or oral administration are described herein and can be formed into solutions, suspensions, tablets, pills, capsules, sustained-release formulations, oral rinses, or powders, as are known in the art. Such compositions can also include stabilizers and preservatives. For example, carriers, stabilizers, and adjuvants are described in Remington: The Science and Practice of Pharmacy with Facts and Comparisons, 21st edition, University of the Sciences in Philadelphia (2005).

In certain embodiments, the compounds of the present invention may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. As used herein, the term "pharmaceutically acceptable salts, esters, amides, and prodrugs" refers to those carboxylates, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention that are suitable for contact with the patient's tissues within the scope of reasonable medical judgment, are free of excessive toxicity, irritation, allergic reactions, etc., are commensurate with a reasonable benefit/risk ratio, and are effective in the intended use of the compounds of the present invention. The term "salt" refers to relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compound, or can be prepared by reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus generated. These can include alkali and alkaline earth metal based cations such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like (see, e.g., Berge S. M. et al., (1977) J. Pharm. Sci. 66, 1, which is incorporated herein by reference).

The term "pharmaceutically acceptable ester" refers to relatively nontoxic esterification products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compound, or can be prepared by separately reacting the purified compound in its free acid form or at a hydroxyl group with a suitable esterifying agent. Carboxylic acids can be converted to esters by treatment with an alcohol in the presence of a catalyst. The term is also intended to include lower hydrocarbon groups that are capable of solvation under physiological conditions, such as alkyl esters, methyl, ethyl, and propyl esters.

As used herein, "pharmaceutically acceptable salts or prodrugs" are salts or prodrugs that are, within the scope of sound medical judgment, suitable for contact with the tissues of a subject without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use.

The term "prodrug" refers to a compound that is rapidly converted in vivo to produce a functionally active one or more peptides as disclosed herein, or mutants, variants, analogs, or derivatives thereof. A full discussion is provided in T. Higachi and V. Stella, "Pro-drugs as Novel Delivery Systems," A.C.S. Symposium Series, Vol. 14, and Bioreversible Carriers in: Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference. As used herein, a prodrug is a compound that, when administered in vivo, is metabolized or otherwise converted into a biologically, pharmaceutically, or therapeutically active form of the compound. Prodrugs of one or more peptides as disclosed herein, or mutants, variants, analogs, or derivatives thereof, can be designed to alter the metabolic stability or transport characteristics of one or more peptides as disclosed herein, or mutants, variants, analogs, or derivatives thereof, thereby eliminating side effects or toxicity, improving the flavor of the compound, or altering other characteristics or properties of the compound. Based on the knowledge of pharmacodynamic processes and drug metabolism in vivo, once the pharmaceutically active form of one or more peptides disclosed herein or their mutants, variants, analogs or derivatives is known, those skilled in the art of medicine are generally able to design prodrugs of the compounds (see, for example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, N.Y., pp. 388-392). Conventional methods for selecting and preparing suitable prodrugs are described, for example, in "Design of Prodrugs," ed. H. Bundgaard, Elsevier, 1985. Suitable examples of prodrugs include methyl esters, ethyl esters, and glycerol esters of the corresponding acids.

Parenteral compositions

The pharmaceutical composition can be administered parenterally in the following manner: injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) in a dosage form, formulation or via an appropriate delivery device or implant containing conventional non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulations.

Compositions for parenteral use can be provided in unit dosage form (e.g., single-dose ampoules) or in vials containing several doses, and suitable preservatives can be added to the vials (see below). The composition can be in the form of a solution, suspension, emulsion, infusion device, or delivery device for implantation, or it can be in the form of a dry powder that is reconstituted with water or other suitable carriers before use. In addition to the active agent, the composition can include a suitable parenteral acceptable carrier and/or excipient. The active agent can be incorporated into microspheres, microcapsules, nanoparticles, liposomes, etc. for controlled release. In addition, the composition can include a suspending agent, a solubilizing agent, a stabilizer, a pH adjusting agent, a tension adjusting agent, and/or a dispersing agent.

As described above, pharmaceutical compositions according to the present invention may be in a form suitable for sterile injection. To prepare such compositions, the appropriate active agent is dissolved or suspended in a parenterally acceptable liquid medium. Acceptable vehicles and solvents that may be used include: water; water adjusted to an appropriate pH by addition of hydrochloric acid, sodium hydroxide, or an appropriate buffer; 1,3-butanediol; Ringer's solution; dextrose solution; and isotonic sodium chloride solution. Aqueous formulations may also contain one or more preservatives (e.g., methylparaben, ethylparaben, or n-propylparaben). When a compound is only sparingly or slightly soluble in water, a solubility enhancer or solubilizer may be added, or the solvent may include 10-60% w/w propylene glycol, for example.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodium sulfite, etc.; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

The formulations of the present invention include those suitable for intravenous, oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral administration. The formulations can be conventionally provided in unit dosage form and can be prepared by any method well known in the pharmaceutical art. The amount of active ingredient that can be combined with a carrier material to prepare a single dosage form will generally be that amount of compound that produces a therapeutic effect.

The methods for preparing these formulations or compositions include the step of bringing into association the compound of the present invention with a carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound of the present invention with liquid carriers or finely divided solid carriers or both, and then shaping the product as desired.

Formulations of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, typically sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a lozenge (using an inert base such as gelatin and glycerin, or sucrose and acacia), and/or a mouthwash, etc., each containing a predetermined amount of a compound of the present invention as the active ingredient. The compounds of the present invention may also be administered as a bolus, electuary, or paste.

Pharmaceutical compositions of the present invention suitable for parenteral administration comprise one or more peptides as disclosed herein, or mutants, variants, analogs or derivatives thereof, in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers that can be used for pharmaceutical compositions comprising one or more peptides as disclosed herein, or mutants, variants, analogs or derivatives thereof, include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.

These compositions can also include adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of microbial effects can be ensured by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, etc. It can also be expected to include isotonic agents such as sugars, sodium chloride, etc. in the composition. In addition, the absorption of injectable drug forms can also be extended by including agents that prolong absorption, such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the drug, it is necessary to slow down the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using liquid dispersions of crystalline or amorphous substances with lower water solubility. Subsequently, the absorption rate of the drug depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. Alternatively, the absorption of a parenteral drug form can be delayed by dissolving or suspending the drug in an oil carrier.

Injectable depot formulations are prepared by forming a microencapsulated matrix of the subject compound in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the properties of the specific polymer used, the drug release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations can also be prepared by entrapping a drug, such as one or more peptides as disclosed herein, or mutants, variants, analogs, or derivatives thereof, in liposomes or microemulsions compatible with body tissues.

Regardless of the route of administration selected, the compounds of the present invention used in a suitable hydrated form and/or the pharmaceutical compositions of the present invention can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of ordinary skill in the art.

Controlled release of parenteral compositions

The controlled release parenteral composition can be in the form of an aqueous suspension, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, or emulsions. The composition can also be incorporated into a biocompatible carrier, liposome, nanoparticle, implant, or infusion device.

Materials used to prepare microspheres and/or microcapsules are, for example, biodegradable/bioerodible polymers such as polygalactose poly-(isobutylcyanoacrylate), poly(2-hydroxyethyl-L-glutamine), poly(lactic acid), polyglycolic acid, and mixtures thereof. Biocompatible carriers that can be used in the preparation of controlled-release parenteral formulations are carbohydrates (e.g., dextran), proteins (e.g., albumin), lipoproteins, or antibodies. Materials used for implants can be non-biodegradable (e.g., polydimethylsiloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid), or poly(orthoesters)), or combinations thereof.

Solid dosage forms for oral use

Formulations for oral use include tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients, and such formulations are known to those skilled in the art (e.g., U.S. Patents: 5,817,307; 5,824,300; 5,830,456; 5,846,526; 5,882,640; 5,910,304; 6,036,949; 6,036,949; and 6,372,218, which are incorporated herein by reference). For example, these excipients can be: inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch (including potato starch), calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate); granulating agents and disintegrants (e.g., cellulose derivatives (including microcrystalline cellulose), starch (including potato starch), cross-linked carboxymethyl cellulose sodium, alginates or alginic acid); binders (e.g., sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinyl pyrrolidone or polyethylene glycol); and lubricants, glidants and anti-adherents (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil or talc). Other pharmaceutically acceptable excipients can be colorants, flavorings, plasticizers, humectants, buffers, etc.

The tablet can be uncoated, or it can be coated by known technology, optionally to delay disintegration and absorption in the gastrointestinal tract, thereby providing a longer-term sustained effect. The coating can be suitable for releasing protein in a predetermined mode (for example, to obtain a controlled release formulation), or it can be adjusted to release the medicament (enteric coating) until after passing through the stomach. Coating can be sugar coating, film coating (for example, based on hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, acrylate copolymer, polyethylene glycol and/or polyvinyl pyrrolidone) or enteric coating (for example, based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac and/or ethylcellulose). In addition, time delay materials can be used, such as glyceryl monostearate or glyceryl distearate.

Solid tablet compositions may include a coating suitable for protecting the composition from unwanted chemical changes (e.g., chemical degradation prior to release of the active substance). The coating may be applied to the solid dosage form in a manner similar to that described in the above-mentioned Encyclopedia of Pharmaceutical Technology.

The compositions of the present invention can be blended in a tablet, or can be separated. In one example, the first agent is contained inside the tablet and the second agent is outside, so that the majority of the second agent is released before the first agent is released.

Formulations for oral administration may also be in the form of chewable tablets, or hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin or olive oil). Powders and granules may be prepared in a conventional manner using the ingredients mentioned above under tablets and capsules, for example, using a mixer, fluidized bed apparatus or spray drying equipment.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, lozenges, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate and/or any of the following: fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; dissolution retardants, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glyceryl monostearate; absorbents, such as kaolin and bentonite; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also contain buffering agents.Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar and high molecular weight polyethylene glycols.

Tablets may be prepared by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets may be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), a surfactant, or a dispersant. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Tablets and other solid dosage forms of the pharmaceutical compositions of the present invention, such as lozenges, capsules, pills, and granules, can optionally be scored or prepared using coatings and shells, such as enteric coatings and other coatings well-known in the pharmaceutical field. They can also be formulated using, for example, hydroxypropyl methylcellulose, other polymer matrices, liposomes, and/or microspheres in varying proportions to provide a desired release pattern, to provide a slow release or controlled release of the active ingredient. They can be sterilized, for example, by filtering through a filter that retains bacteria, or by incorporating a sterilizing agent into the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injection medium immediately before use. These compositions can also optionally contain an opacifier and can belong to this class of compositions, i.e., they only or preferably release the active ingredient in a delayed manner in a certain portion of the gastrointestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in a microencapsulated form together with one or more of the above-mentioned excipients. In one aspect, a solution of resolvin and/or protectin, or a precursor or analog thereof, can be administered as eye drops for ocular angiogenesis or as ear drops for the treatment of otitis.

Oral administration of peptides useful according to the invention has been shown to work as described, for example, in US Patent 8,975,224.

Oral administration of peptides has also been shown to be effective for other protein or peptide drugs. For example, oral administration of anti-CD3 antibodies has been shown to be effective in treating, for example, diabetes (Ishikawa et al. Diabetes. 2007 Aug; 56(8):2103-9. Epub 2007 Apr 24) and autoimmune encephalomyelitis (Ochi et al. Nat Med. 2006 Jun; 12(6):627-35. Epub 2006 May 21). Without wishing to be bound by theory, it is proposed that administration via the gut-associated lymphoid tissue (GALT) is possible. Thus, in some aspects of all embodiments of the present invention, the peptide formulation is an oral formulation, and the method is performed by orally administering the peptide.

Liquid dosage forms for oral administration of the compounds of this invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.

In addition to the active ingredient, the liquid dosage form may also contain inert diluents commonly used in the art, such as, for example, water or other solvents, cosolvents and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, colorants, fragrances and preservatives.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Dosage forms for topical or transdermal administration of one or more peptides as disclosed herein, or mutants, variants, analogs or derivatives thereof, include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers or propellants that may be required.

In addition to the active compound of the invention, ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. In addition to the active compound of the invention, powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may also contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of the compounds of the present invention (resolvins and/or protectins or their precursors or analogs) to the body. Such dosage forms can be prepared by dissolving or dispersing the compound in an appropriate medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of this flux can be controlled by providing a rate-controlling membrane or dispersing the active compound in a polymer matrix or gel. On the other hand, biodegradable or absorbable polymers can provide extended, often localized release of polypeptide agents. The potential beneficial effects of increased half-life or extended release of therapeutic agents are obvious. The potential beneficial effect of local release is the ability to achieve much higher local doses or concentrations over a longer period of time than with more extensive systemic administration, and it is also possible to avoid possible undesirable side effects that may occur with systemic administration.

The bioabsorbable polymer matrix suitable for delivering one or more peptides as disclosed herein or its mutant, variant, analog or derivative can be selected from a variety of bioabsorbable synthetic polymers, which are widely described in the literature. Such synthetic bioabsorbable, biocompatible polymers can release protein over several weeks or months, and they can include, for example, block copolymers (Polyactive™) of poly-α-hydroxy acids (such as polylactide, polyglycolide and their copolymers), polyanhydrides, polyorthoesters, polyethylene glycol and polybutylene terephthalate (α-hydroxy acids), tyrosine derivative polymers or poly(ester-amides). Suitable bioabsorbable polymers for the manufacture of drug delivery materials and implants are discussed in the following literature: for example, U.S. Patents 4,968,317, 5,618,563, etc., and " Biomedical Polymers " edited by S.W. Shalaby, Carl Hanser Verlag, Munich, Vienna, New York, 1994, and the multiple references cited in the above-mentioned publications. The particular bioabsorbable polymer that should be selected will depend on the particular patient being treated.

Gene therapy

One or more peptides as disclosed herein or mutants, variants, analogs or derivatives thereof can be effectively used for treatment by gene therapy. See generally, for example, U.S. Patent No. 5,399,346, which is incorporated herein by reference. The general principle is to introduce a polynucleotide into the target cells of the patient.

Entry into the cell is facilitated by appropriate techniques known in the art (e.g., providing the polynucleotide in the form of a suitable vector, or encapsulating the polynucleotide in liposomes).

The desired mode of gene therapy is to provide the polynucleotide in such a way that it replicates inside the cell, enhancing and prolonging the desired effect. Therefore, the polynucleotide is operably linked to a suitable promoter, for example, the natural promoter of the corresponding gene, a heterologous promoter that is inherently effective in liver, neurons, bone, muscle, skin, joint or cartilage cells, or a heterologous promoter that can be induced by a suitable agent.

Expression vectors compatible with eukaryotic cells, preferably expression vectors compatible with vertebrate cells, can be used to produce recombinant constructs expressing one or more peptides as disclosed herein, or mutants, variants, analogs or derivatives thereof (including fusion proteins with one or more peptides as disclosed herein, or mutants, variants, analogs or derivatives thereof). Eukaryotic cell expression vectors are well known in the art and can be obtained from a variety of commercial sources. Such vectors are generally provided containing restriction sites that facilitate the insertion of the desired DNA segment. Such vectors can be viral vectors, for example, adenoviruses, adeno-associated viruses, poxviruses such as orthopox (vaccinia and attenuated vaccinia), avipox viruses, lentiviruses, murine Moloney leukemia virus, and the like. Alternatively, plasmid expression vectors can be used.

Viral vector systems that can be used in the present invention include, but are not limited to: (a) adenoviral vectors; (b) retroviral vectors; (c) adeno-associated viral vectors; (d) herpes simplex viral vectors; (e) SV40 vectors; (f) polyoma viral vectors; (g) papilloma viral vectors; (h) picornaviral vectors; (i) poxvirus vectors, for example, orthopox, such as cowpox virus vectors or avian pox viruses (e.g., canarypox or fowlpox); and (j) helper-dependent adenovirus or gutless adenovirus. In a preferred embodiment, the vector is an adenovirus. Replication-defective viruses may also be advantageous.

The vector may or may not be incorporated into the cell genome. If desired, the construct may include viral sequences for transfection. Alternatively, the construct may incorporate vectors capable of free replication, such as EPV and EBV vectors.

"Operably linked" refers to a nucleic acid molecule being linked to one or more regulatory sequences (e.g., a promoter) in such a manner that the connection allows the nucleic acid molecule to express and/or secrete a product (e.g., a protein) when a suitable molecule (e.g., a transcriptional activator) binds to the regulatory sequence. In other words, the term "operably linked" as used herein refers to the functional relationship between a nucleic acid sequence and a regulatory sequence of nucleotides (e.g., a promoter, enhancer, transcription and translation termination sites, and other signal sequences). For example, the operative connection of a nucleic acid sequence (usually DNA) to a regulatory sequence or promoter region refers to the physical and functional relationship between the DNA and the regulatory sequence or promoter, such that transcription of such DNA is initiated by the regulatory sequence or promoter through an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA. In order to optimize expression and/or in vitro transcription, it may be necessary to modify the regulatory sequence for expression of the nucleic acid or DNA in the cell type in which it is expressed. The desirability or need for such modifications can be determined empirically. An operably linked polynucleotide to be expressed generally includes appropriate initiation signals (eg, ATG) and maintains the correct reading frame to allow expression of the polynucleotide sequence under the control of the expression control sequences and production of the desired polypeptide encoded by the polynucleotide sequence.

As used herein, the term "promoter" or "promoter region" or "promoter element" has been defined in this article and refers to a nucleic acid sequence segment that controls the transcription of a nucleic acid sequence to which it is operably linked, typically but not limited to DNA or RNA or their analogs. The promoter region contains specific sequences sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as a promoter. In addition, the promoter region contains sequences that regulate the recognition, binding and transcription initiation activity of RNA polymerase. These sequences can be cis-acting or can respond to trans-acting factors. The promoter can be constitutive or regulated, depending on the nature of the regulation.

The terms "regulatory sequence" and "regulatory element" are used interchangeably herein and refer to a nucleic acid segment that regulates the transcription of a nucleic acid sequence to which it is operably connected, and thus acts as a transcriptional regulator, typically but not limited to DNA or RNA or their analogs. A regulatory sequence regulates the expression of a gene and/or nucleic acid sequence to which it is operably connected. Regulatory sequences generally include "regulatory elements," which are nucleic acid sequences that serve as transcriptional binding domains and are recognized by the nucleic acid binding domains of transcription proteins and/or transcription factors, repressors, or enhancers. Typical regulatory sequences include, but are not limited to, transcription promoters, inducible promoters and transcriptional elements, optional operational sequences that control transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences that control transcription and/or translation termination. Nucleic acid sequences (such as initiation signals, enhancers, and promoters) that induce or control the transcription of a protein coding sequence to which it is operably connected are included in the term "regulatory element." In some examples, the transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence), which controls the expression of the recombinant gene in the cell type in which it is desired to express the gene. Should be understood that recombinant genes can be under the control of transcriptional regulatory sequences that are the same or different from the transcriptional regulatory sequences that control the transcription of the naturally occurring form of the protein. In some cases, promoter sequences are recognized by the cellular synthetic machinery or introduced synthetic machinery required to initiate transcription of a specific gene.

The regulatory sequence can be a single regulatory sequence or multiple regulatory sequences, or modified regulatory sequences, or fragments thereof. A modified regulatory sequence is a regulatory sequence in which the nucleic acid sequence has been altered or modified by some means (such as, but not limited to, mutation, methylation, etc.).

Regulatory sequences useful in the methods disclosed herein are promoter elements sufficient to render promoter-dependent gene expression controllable in a cell type-specific, tissue-specific manner, or inducible by external signals or agents (e.g., enhancers or repressors); such elements may be located in the 5' or 3' region or within an intron of a native gene.

As used herein, the term "tissue-specific promoter" refers to a nucleic acid sequence that acts as a promoter (i.e., regulates the expression of a selected nucleic acid sequence operably linked to the promoter) and selectively affects the expression of the selected nucleic acid sequence in specific tissue cells.

In some embodiments, it may be advantageous to direct the expression of one or more peptides as disclosed herein, or mutants, variants, analogs, or derivatives thereof, in a tissue- or cell-specific manner. Muscle-specific expression can be achieved, for example, using the skeletal muscle MKC promoter (disclosed in U.S. Patent Application WO 2007/100722, which is incorporated herein by reference), or other muscle-specific promoters, such as α-myosin heavy chain, myosin light chain-2 (which is skeletal muscle-specific (Shani et al., Nature, 314; 283-86, 1985), the gonadotropin-releasing hormone gene regulatory region activated in the hypothalamus (Mason et al., Science, 234; 1372-78, 1986), and the smooth muscle promoter SM22a, all of which are well known in the art.

The term "constitutively active promoter" refers to a promoter of a gene that is expressed at all times within a given cell. Exemplary promoters for use in mammalian cells include the cytomegalovirus (CMV) promoter, and exemplary promoters for use in prokaryotic cells include the bacteriophage T7 promoter and the T3 promoter, among others. The term "inducible promoter" refers to a promoter of a gene that is expressed in response to a given signal (e.g., an increase or decrease in an agent). Non-limiting examples of inducible promoters are "tet-on" and "tet-off" promoters, or promoters that are regulated in specific tissue types.

In a specific embodiment, a viral vector comprising a nucleic acid sequence encoding one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217: 581-599 (1993)). These retroviral vectors contain the components required for correct packaging of the viral genome and integration into host cell DNA. More detailed information about retroviral vectors can be found in Boesen et al., Biotherapy 6: 291-302 (1994), which describes the use of retroviral vectors to deliver the mdrl gene to hematopoietic stem cells to make stem cells more resistant to chemotherapy. Other references describing the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

The manufacture of recombinant retroviral vectors carrying a gene of interest is typically achieved in two steps. First, a sequence encoding one or more peptides as disclosed herein, or mutants, variants, analogs, or derivatives thereof, is inserted into a retroviral vector comprising sequences required for the effective expression of a metabolic regulator (including promoter elements and/or enhancer elements that can be provided by viral long terminal repeats (LTRs) or by internal promoters/enhancers and associated splicing signals); and sequences required for the effective packaging of viral RNA into infectious virions (e.g., packaging signals (Psi), tRNA primer binding sites (-PBS), 3' regulatory sequences (+PBS) required for reverse transcription, and viral LTRs). The LTR contains sequences required for viral genomic RNA binding, reverse transcriptase, and integrase function, as well as sequences directed to the expression of genomic RNA to be packaged into viral particles.

After constructing the recombinant retroviral vector, the vector DNA is introduced into a packaging cell line. The packaging cell line provides the viral proteins (e.g., virally encoded core (gag), polymerase (pol), and envelope (env) proteins) required to package the viral genomic RNA into virus particles with the desired host range in trans. The host range is partially controlled by the type of envelope gene product expressed on the surface of the virus particle. The packaging cell line can express ecotrophic, amphotropic, or xenotropic envelope gene products. Alternatively, the packaging cell line can lack sequences encoding viral envelope (env) proteins. In this case, the packaging cell line can package the viral genome into particles lacking membrane-bound proteins (e.g., env proteins). In order to produce virus particles containing membrane-bound proteins that allow the virus to enter cells, the packaging cell line containing the retroviral sequence can be transfected with sequences encoding membrane-bound proteins (e.g., the G protein of vesicular stomatitis virus (VSV)). The transfected packaging cells can then produce viral particles containing the membrane-bound proteins expressed by the transfected packaging cell line; these viral particles containing viral genomic RNA derived from one virus encapsidated by the envelope proteins of another virus are considered pseudotyped viral particles.

Adenovirus is another viral vector that can be used for gene therapy. Adenovirus is a particularly attractive medium for delivering genes to respiratory epithelial cells. Adenovirus naturally infects respiratory epithelial cells and causes mild disease there. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenovirus has the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), provide a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994), demonstrated the use of adenoviral vectors to transfer genes to respiratory epithelial cells in rhesus monkeys. Another preferred viral vector is a poxvirus, such as vaccinia virus, for example, attenuated vaccinia (such as modified vaccinia virus Ankara (MVA) or NYVAC), avian pox virus, such as fowlpox or canarypox. Other examples of the use of adenovirus in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO 94/12649; and Wang et al., Gene Therapy 2:775-783 (1995). In another embodiment, a lentiviral vector is used, such as the HIV-based vectors described in U.S. Patents 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference. The use of adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); and U.S. Pat. No. 5,436,146, which are incorporated herein by reference).

Another approach to gene therapy involves transferring genes to cells in tissue culture through methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. Typically, the transfer method involves transferring a selectable marker to the cells. The cells are then screened to isolate cells that have taken up and expressed the transferred gene. Those cells are then delivered to the patient.

U.S. Patent No. 5,676,954 (incorporated herein by reference) reports the injection of genetic material complexed with cationic liposome carriers into mice. U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and International Publication No. WO 94/9469 (incorporated herein by reference) provide cationic lipids for transfecting DNA into cells and mammals. U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and International Publication No. WO 94/9469 (incorporated herein by reference) provide methods for delivering DNA-cationic lipid complexes into mammals. Such cationic lipid complexes or nanoparticles can also be used to deliver proteins.

The gene or nucleic acid sequence can be introduced into the target cell by any suitable method. For example, a construct of one or more peptides disclosed herein, or mutants, variants, analogs or derivatives thereof, can be introduced into the cell by transfection (e.g., calcium phosphate or DEAE-dextran mediated transfection), lipofection, electroporation, microinjection (e.g., by direct injection of naked DNA), gene gun, infection with a viral vector containing a muscle-related transgene, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, nuclear transfer, and the like. Nucleic acids encoding one or more peptides as disclosed herein, or mutants, variants, analogs or derivatives thereof, can be introduced into cells by electroporation (see, e.g., Wong and Neumann, Biochem. Biophys. Res. Commun. 107:584-87 (1982)) and gene guns (e.g., gene gun; Johnston and Tang, Methods Cell Biol. 43 Pt A:353-65 (1994); Fynan et al., Proc. Natl. Acad. Sci. USA 90:11478-82 (1993)).

In certain embodiments, a gene or nucleic acid sequence encoding one or more peptides as disclosed herein, or mutants, variants, analogs or derivatives thereof, can be introduced into target cells by transfection or lipofection. Suitable reagents for transfection or lipofection include, for example, calcium phosphate, DEAE-dextran, liposomes, lipfectamine, DIMRIE C, Superfect, and Effectin (Qiagen), unifectin, maxifectin, DOTMA, DOGS (Transfectam; dioctadecylamide glycyl spermine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP (1,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyldioctadecyl ammonium bromide), DHDEAB (N, N-bis-n-hexadecyl-N, N-dihydroxyethylammonium bromide), HDEAB (N-n-hexadecyl-N, N-dihydroethylammonium bromide), polybrene, polyethyleneimine (PEI), etc. (See, e.g., Banerjee et al., Med. Chem. 42:4292-99 (1999); Godbey et al., Gene Ther. 6:1380-88 (1999); Kichler et al., Gene Ther. 5:855-60 (1998); Birchaa et al., J. Pharm. 183:195-207 (1999), which are incorporated herein by reference in their entireties).

Methods known in the art for therapeutic delivery of agents (e.g., proteins and/or nucleic acids) can be used to deliver polypeptides or nucleic acids encoding one or more peptides as disclosed herein, or mutants, variants, analogs or derivatives thereof, such as cell transfection, gene therapy, direct administration with a delivery vehicle or pharmaceutically acceptable carrier, indirect delivery by providing recombinant cells containing a nucleic acid encoding the targeted fusion polypeptide of the invention.

Various delivery systems are known and can be used to directly administer therapeutic polypeptides (e.g., one or more peptides as disclosed herein, or mutants, variants, analogs, or derivatives thereof) and/or nucleic acids encoding one or more peptides as disclosed herein, or mutants, variants, analogs, or derivatives thereof, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, and receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432). The method of introduction can be enteral or parenteral, and includes, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary, intranasal, intraocular, epidural, and oral routes. The agent can be administered by any convenient route (e.g., by infusion or bolus injection, by absorption through epithelial lining or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.)) and can be administered with other biologically active agents. Administration can be systemic or local.

In a specific embodiment, it is desirable to administer the pharmaceutical compositions of the present invention topically to the area in need of treatment; this can be achieved, for example, but not limited to, by local infusion during surgery; topical application, for example, by injection, by means of a catheter, or by means of an implant (the implant being a porous material, a non-porous material, or a gel-like material, including membranes (such as silicone rubber membranes), fibers, or commercial skin substitutes).

In another embodiment, the active agent can be delivered in a vesicle, particularly a liposome (see Langer (1990) Science 249: 1527-1533). In another embodiment, the active agent can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer (1990), supra). In another embodiment, a polymeric material can be used (see Howard et al. (1989) J. Neurosurg. 71: 105).

Thus, a wide variety of gene transfer/gene therapy vectors and constructs are known in the art. These vectors are readily adapted for use in the methods of the present invention. By inserting an operably linked polypeptide-encoding nucleic acid segment into a selected expression/delivery vector using appropriate manipulation of recombinant DNA/molecular biology techniques, a wide variety of equivalent vectors for practicing the methods described herein can be generated.

Other implementation plans

From the foregoing, it will be apparent that various changes and modifications can be made to the invention described herein to adapt it to various usages and conditions. Such embodiments also fall within the scope of the appended claims.

The present disclosure also contemplates an article of manufacture that is a labeled container for providing one or more peptides as disclosed herein, or mutants, variants, analogs, or derivatives thereof. The article of manufacture comprises packaging material and a pharmaceutical agent of one or more peptides as disclosed herein, or mutants, variants, analogs, or derivatives thereof, contained in the packaging material.

The pharmaceutical agent in the article of manufacture is any composition of the present invention suitable for providing one or more peptides as disclosed herein or mutants, variants, analogs or derivatives thereof, and formulated in a pharmaceutically acceptable form as described herein according to the disclosed instructions. Thus, the composition may comprise one or more peptides as disclosed herein or mutants, variants, analogs or derivatives thereof, or a DNA molecule capable of expressing such a peptide.

The article of manufacture contains a unit dose or multiple doses of an amount of the agent sufficient for treating the conditions described herein. The packaging material includes a label indicating the use of the agent contained therein.

The label may also include instructions for use and related information that may be required by the market. The packaging material may include a container for storing the pharmaceutical preparation.

As used herein, the term packaging material refers to materials such as glass, plastic, paper, foil, etc., which are capable of holding a medicament in a fixed device. Thus, for example, the packaging material can be a plastic or glass vial, laminated shell, and similar containers that are used to hold a pharmaceutical composition containing a medicament.

In a preferred embodiment, the packaging material includes a label, which is a tangible representation describing the contents of the article and the use of the pharmaceutical agent contained therein.

Example

Example 1 - Treatment of Acute Myocardial Infarction Using SP163M Peptide

The SP16 peptide is a short fragment of the circulating serum protein α-1-antitrypsin (AAT). The US FDA has approved the use of three α1-antitrypsin products derived from human plasma: Prolastin, Zemaira, and Aralast.

As described herein, the SP163M peptide incorporates the reported anti-inflammatory properties of the parent AAT. In pilot clinical studies conducted at Virginia Commonwealth University (VCU), AAT has been shown to safely and effectively block the inflammatory response following acute myocardial infarction (AMI). Furthermore, SP163M has demonstrated equivalent or superior anti-inflammatory activity in preclinical models of AMI. We collaborated with VCU researchers to rapidly develop SP16 for the treatment of AMI, a major unmet medical need in the United States and worldwide.

Acute myocardial infarction (AMI) remains a leading cause of morbidity and mortality in the United States and worldwide. Despite current strategies for early reperfusion, many patients die early during this period, and those who survive face the risk of later death from adverse cardiac remodeling, heart failure, and sudden death. Multiple publications have highlighted the association between infarct size in AMI and the probability of developing heart failure in the following 2-5 years after AMI.

Patients who suffer an ST-segment elevation myocardial infarction (STEMI) are at particular risk for adverse cardiac remodeling, heart failure, and both in-hospital and long-term mortality. Although significant improvements have been made in the treatment of STEMI, reductions in early mortality have been associated with an increased incidence of post-STEMI heart failure. ¹ This may reflect a higher-risk patient population surviving the index event, as well as an aging population and the prevalence of hypertension and diabetes. Within 30 days of STEMI, more than 20% of survivors are diagnosed with heart failure, a condition associated with significant morbidity, disability, and mortality.

Heart failure is indeed a major public health problem, affecting approximately 5 million Americans and adding 500,000 cases every year. Compared with other cardiovascular diseases, the incidence and prevalence of heart failure continue to improve, and heart failure is now the leading cause of hospitalization for people aged 65 years, a population segment that is also rapidly increasing. Although the survival rate after heart failure occurs has also improved, current therapy can slow down rather than stop the development of the disease. Due to the economic consequences of the progressive symptoms of functional capacity, dyspnea and fatigue, frequent hospitalizations and loss of productivity and the limitations of increased medical care costs, heart failure imposes a significant burden on health care.

There is an urgent need to develop additional treatments to minimize infarct size and prevent heart failure after AMI. ² Current treatments for STEMI include rapid reperfusion of the ischemic myocardium by restoring coronary artery patency (i.e., angioplasty or fibrinolysis), prevention of reembolism (i.e., antiplatelet agents and anticoagulants), and neurohormonal blockade (i.e., renin-angiotensin-aldosterone and adrenergic blockers). While each of these interventions provides incremental benefits and significantly reduces morbidity and mortality, the incidence of heart failure after STEMI continues to increase, suggesting that the current treatment paradigm still misses one or more key pathophysiological mechanisms.

Therefore, identifying the mechanisms by which adverse cardiac remodeling and heart failure develop despite optimal treatment is a critical step in developing novel interventions with the ultimate goal of reducing the incidence, burden, and mortality of heart failure after STEMI.

There is a close interaction between inflammation, adverse cardiac remodeling, and heart failure following acute myocardial infarction (AMI). Acute myocardial ischemia and infarction trigger a robust inflammatory response within the myocardium. ³ Leukocytes infiltrate the damaged myocardium to coordinate tissue repair and infarct healing, leading to neovascularization and reparative fibrosis. Thus, inflammation is essential for infarct healing, but uncontrolled inflammation leads to further damage to the heart and nonfunctional healing, which underlie adverse cardiac remodeling and heart failure. In experimental animal models, the extent of the inflammatory response determines adverse cardiac remodeling, independent of infarct size. ³ In patients with AMI, the intensity of the inflammatory response is reflected in the levels of circulating biomarkers that predict adverse cardiac remodeling, heart failure, and death. ⁴ C-reactive protein, an acute-phase reactant, is increased in AMI and predicts outcome.

Therefore, modulation of the inflammatory response is an object of the present invention. Although previous attempts to modulate the inflammatory response have failed, we propose a new and significantly different approach for modulating the inflammatory response using the SP163M peptide, a 17-mer modified derivative of alpha-1 anti-trypsin (AAT). ⁵ AAT is a naturally occurring anti-inflammatory protein abundant in plasma that provides potent cytoprotective effects in endothelial cells, cardiomyocytes, and fibroblasts. ⁵

In an experimental model of acute myocardial infarction (AMI), administration of AAT at the onset of ischemia or during reperfusion resulted in significantly smaller infarct size and more favorable cardiac healing and remodeling (Figures 3A-3D) ⁶ .

Although more than one mechanism may be involved in the improvement of cardiac function by AAT during AMI, inhibition ^of interleukin-1 (IL-1) production has been proposed.6-7 This is relevant because IL-1 inhibition has been shown to be safe and effective in limiting myocardial injury during AMI in experimental studies and in two small pilot clinical trials completed at the ^VCU.8-10

To examine the effects of SP163M in AMI, the peptide was tested in the same mouse model previously used for AAT. Briefly, adult male ICR mice underwent coronary artery ligation to induce myocardial ischemia for 30 minutes, followed by reperfusion. A 100 μg dose of SP16 or its equivalent volume of a 0.9% NaCl solution was provided during reperfusion. SP16 significantly reduced myocardial infarct size by >50% (Figure 4A), measured as the percentage of left ventricular infarction over the entire left ventricle (appears white when stained with triphenyltetrazolium chloride), while viable myocardium appeared bright red. Plasma levels of cardiac-specific troponin I, a biomarker of myocardial necrosis, were also significantly reduced by SP163M treatment (Figure 4B). The cardioprotective effect of SP16 translated into preservation of left ventricular systolic function, as measured by transthoracic echocardiography as left ventricular fractional shortening (Figure 4C).

Protective effect of SP163M on experimental acute myocardial infarction in mice

Ten-week-old male ICR (CD1) mice underwent 30-minute surgical coronary artery ligation followed by 24-hour reperfusion to simulate an acute myocardial infarction (AMI) scenario treated with reperfusion therapy. A subgroup of mice underwent a sham surgery, in which the coronary arteries were identified and isolated but not ligated.

SP163M active peptide or control solution vehicle was intraperitoneally administered at a dose of 100 μg/mouse during reperfusion.

Infarct size was measured by triphenyltetrazolium chloride (TTC)/Evans blue staining for pathological blood analysis and by measuring plasma troponin I levels at 24 hours. After re-ligation of the coronary artery knot, Evans blue was infused retrogradely through the aorta to identify non-risk myocardium (blue). TTC was used to stain viable myocardium (red).

Echocardiography is used to assess left ventricular (LV) fractional shortening as a measure of systolic function in vivo.

Each group included at least 6 mice.

Protective effect of SP163M administered within 30 minutes of reperfusion in experimental AMI in mice

The option of delaying treatment with SP163M for an additional 30 minutes after reperfusion was examined. A control peptide, SP34, which has 16/17 identical amino acids to SP163M but in scrambled order, was also tested.

Ten-week-old male ICR (CD1) mice underwent 30-minute surgical coronary artery ligation followed by 24-hour reperfusion to simulate the AMI scenario treated with reperfusion therapy.

SP163M active peptide or SP34 control peptide was administered intraperitoneally at a dose of 100 μg/mouse at the time of reperfusion or after a 30’ delay to simulate the clinical scenario in which a delay may occur between reperfusion and drug treatment.

Infarct size was measured using TTC/Evans blue staining. Echocardiography was used to assess left ventricular (LV) fractional shortening as a measure of in vivo systolic function. Each group included at least six mice.

SP163M administered 30' after reperfusion significantly reduced infarct size (Figure 5A) and preserved LV fractional shortening (Figure 5B).

SP163M provides a dose-dependent reduction in infarct size in experimental AMI in mice

The cardioprotective capacity of SP163M at doses of 10 µg to 100 µg was compared. The results are shown in Figure 6 , which demonstrates that even a 90% dose reduction of SP163M was effective, as measured by infarct size and LV systolic function (Figures 6A and 6B , respectively).

Ten-week-old ICR mice underwent surgical coronary artery ligation for 30 minutes, followed by 24-hour reperfusion. SP16 was administered at two different doses (10 or 100 μg) with a 30-minute delay. Control peptide SP34 (100 μg) or vehicle solution was used as a control.

Infarct size was measured using TTC/Evans blue staining. Left ventricular (LV) fractional shortening was assessed by transthoracic echocardiography. Each group included at least six mice.

SP163M administered 30' after reperfusion reduced infarct size in a dose-dependent manner (Figure 6A) and preserved LV fractional shortening (Figure 6B).

SP163M has no effect on cardiac contractility in healthy mice

To test the effect of SP163M on cardiac contractility, the following study was performed. The SP163M peptide showed no effect on cardiac contractility, which increases the safety of this peptide drug (Figure 7). SP34 is a peptide with a scrambled sequence of SP16 and was used as a control peptide.

Two-week-old ICR mice were treated with SP163M (100 µg/mouse, intraperitoneally), control peptide SP34 (100 µg/mouse), or vehicle solution. Echocardiography was used to assess left ventricular (LV) fractional shortening at baseline and after 180 minutes.

Preservation of cardiac contractility was tested by isoproterenol challenge (10 ng/kg, intraperitoneal) and the increase in LV fractional shortening was measured within 5' of treatment. Each group included at least 6 mice.

SP163M did not alter cardiac contractile function at rest ( FIG. 7 , left panel) or after isoproterenol challenge (contraction preservation) ( FIG. 7 , right panel).

discuss

In addition to its anti-inflammatory effects, SP163M is also cardioprotective. It reduces infarct size by saving cardiac cells from death during reperfusion. IL1 antagonists have been tested in AMI and have shown significant anti-inflammatory effects, but do not reduce infarct size (no cardioprotective effect). The cytoprotective effects of SP163M shown in AMI that reduces infarction can also be applied to other injuries that cause cell death, such as other ischemic conditions, stroke, traumatic brain injury, and toxic shock. It is contemplated herein that SP163M may allow the treatment of acute myocardial infarction, gout, stroke, complications associated with cardiac surgery, traumatic brain injury, or any other disease associated with a "cytokine storm."

References

1. Velagaleti RS, Pencina MJ, Murabito JM, et al., Long-term trends in theincidence of heart failure after myocardial infarction.Circulation 2008;118:2057-2062

2. Eapen ZJ, Tang WH, Felker GM et al., Defining heart failure end points in ST-elevation myocardial infarction trials: integrating past experiences to chart a path forward. Circ Cardiovasc Qual Outcomes 2012;5:594-600

3. Mezzaroma E, Toldo S, Farkas D, et al. The inflammasome promotes adversecardiac remodeling following acute myocardial infarction in the mouse. ProcNatl Acadi Scie USA 2011;108:19725-19730

4. Roubille F, Samri A, Cornillet L, et al. Routinely-feasible multiple biomarkers score to predict prognosis after revascularized STEMI. Eur J InternMed 2010;21:131-136

5. Lewis EC. Expanding the clinical indications for α(1)-antitrypsintherapy. Mol Med 2012;18:957-70.

6. Toldo S, Seropian IM, Mezzaroma E, et al. Alpha-1 antitrypsin inhibitscaspase-1 and protects from acute myocardial ischemia-reperfusion injury. JMol Cell Cardiol 2011;51:244-51.

7. Pott GB, Chan ED, Dinarello CA, Shapiro L. Alpha-1-antitrypsin is an endogenous inhibitor of proinflammatory cytokine production in wholeblood. J Leukoc Biol. 2009;85:886-95.

8. Abbate A, Salloum FN, Vecile E, et al. Anakinra, a recombinant humaninterleukin-1 receptor antagonist, inhibits apoptosis in experimental acute myocardial infarction. Circulation 2008;117:2670-2683

9. Abbate A, Kontos MC, Grizzard JD, et al. Interleukin-1 blockade with anakinra to prevent adverse cardiac remodeling after acute myocardialinfarction (Virginia Commonwealth University Anakinra Remodeling Trial [VCU-ART] Pilot Study). Am J Cardiol 2010; 15:1371- 1377

10. Abbate A, Van Tassell BW, Biondi-Zoccai GG, Kontos MC, Grizzard JD, Spillman DW, Oddi C, Roberts CS, Melchior RD, Mueller GH, Abouzaki NA, Rengel LR, Varma A, Gambill ML, Falcao RA, Voelkel NF, Dinarello CA, Vetrovec GW. Effects ofInterleukin-1 Blockade with Anakinra on Adverse Cardiac Remodeling and HeartFailure Following Acute Myocardial Infarction (from the VCU-ART2 PilotStudy). Am J Cardiol 2013 – in press.

Example 2

Met can be replaced with Nle to improve peptide stability, for example in the peptide of SEQ ID NO:57 -Ac-Val-Lys-Phe-Asn-Lys-Pro-Phe-Val-Phe- Leu -Nle-Ile-Glu-Gln-Asn-Thr-Lys-NH2 (SEQ ID NO:57). Methionine can be oxidized.

Example 3 - LRP1 mediates SP16-induced cardioprotection

SP163M was demonstrated herein to bind to LRP1 (low-density lipoprotein receptor-related protein) and to be an agonist of LRP1 (as shown for the parent AAT). Blocking LRP1 was also shown to abrogate the cardioprotective effects of SP163M and AAT.

introduce

LRP1 is a receptor responsible for the plasma clearance of SEC (a serpin complex that accumulates during inflammatory responses). Therefore, LRP1 functions to prevent and interrupt inflammation. LRP1 may act by directly regulating key inflammatory pathways, such as NFkB, IRF-3, and AP-1 (e.g., ERK p42/p44, p38, and JNK).

In murine peritoneal macrophages, LRP1 downregulates LPS-driven inflammatory responses through intracellular interaction with IRF-3. Furthermore, LRP1 regulates inflammatory cytokines, affecting phagocytosis and cell migration, as well as Treg distribution. Serpins, including peptides such as SEQ ID NOs: 1 and 57, can bind to clusters II or IV of LRP1 via highly conserved pentapeptides. See Figure 12A. Thus, SP163M can induce a cascade of reactions that downregulate inflammatory responses through cell signaling, cytokine regulation, and the reduction of CD4 T cells and Th17 cells.

Regulation of LRP1 is involved in a variety of inflammatory and autoimmune diseases. Diseases such as neurodegenerative diseases, atherosclerosis, and type II diabetes. Acute myocardial ischemia and reperfusion lead to myocardial necrosis and inflammation, which can lead to tissue injury and heart failure if not treated. Ischemic injury is a hallmark of AMI. Reperfusion, although beneficial in reducing the total infarct size, is associated with more damage. The inflammatory response to ischemia-reperfusion injury, although promoting infarct healing, induces further loss of viable myocardium, leading to the formation of dysfunctional scars. Large infarct size, strong inflammatory response, and the formation of dysfunctional scars all represent the basis of heart failure after AMI. On the other hand, rapid resolution of the inflammatory response is associated with more favorable healing and a reduced incidence of heart failure.

While not wishing to be bound by theory, it is proposed that the inflammatory response to tissue damage following AMI results in the recruitment of plasma serpins at the site of injury, which inhibit leukocyte serine proteases and signal resolution of the inflammatory response via LRP1. Therefore, it is proposed that administration of exogenous plasma-derived serpins in the early stages of AMI will result in inhibition of inflammatory damage via LRP1, leading to a cardioprotective effect and more rapid resolution of inflammation. Furthermore, it is proposed that administration of a synthetic small peptide derived from the C3 terminus of serpins will provide a strong LRP1-mediated cardioprotective signal without inhibiting plasma serine proteases. See Figure 11.

SP163M increases LRP1 expression in murine macrophages

SP163M was demonstrated herein to increase LRP1 expression in Raw264.7 murine macrophages in a dose-dependent manner (Figure 10). 0.2 x 10^6 macrophages were seeded into 24-well plates and grown to 70% confluence. Cells were treated with vehicle (red), 50 μg/ml SP16 (purple), 100 μg/ml SP16 (green), or 200 μg/ml SP16 (blue), a scrambled control (orange), or COG133 (ApoE peptide) (pink) for 3 hours and then harvested with cold PBS 5 mM EDTA. Samples were labeled with anti-LRP1 (abcam ab92544) and stained with Alexa Fluor™ 488 rabbit secondary antibody (Invitrogen) and subsequently analyzed by flow cytometry (Guava easyCyte ^™ flow cytometer). Mean fluorescence intensity (MFI) normalized to the control is shown. (>10,000 events were collected for each sample).

SP163M binds to LRP1

An in vitro assay was performed to examine the binding of SP163M to LRP1. To perform the assay, recombinant LRP1 protein (1 µg/ml) (R&D Systems, Minneapolis, MN) was incubated in the presence of biotin-SP163M (1-5 µg/ml) at 4°C on ice for 1 hour. This mixture was then incubated with 1 mg/ml M280 Streptavidin Dynabeads (Invitrogen, Carlsbad, CA) at 4°C for 1.5 hours. The Dynabeads were washed with PBS + 0.1% Tween 20 and boiled in SDS loading buffer for 5 minutes. The supernatant was separated on a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane. The membrane was probed with anti-LRP1 (LRP-1 Cluster II Affinity Purified Polyclonal Ab, R&D, Minneapolis, MN) and incubated overnight at 4°C. The membrane was then washed three times with TBS-T and incubated with an HRP-conjugated anti-goat secondary antibody diluted 1:10,000 in 5% milk TBS-T. Western blots were visualized by chemiluminescence using the SuperSignal West Femto Maximum Sensitivity Substrate Kit (ThermoScientific) and the Bio-Rad Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA).

Figure 12B shows that LRP1 binds to SP163M at concentrations of 1 μg and 5 μg. Meanwhile, LRP1 does not bind to the control SP34 peptide.

SP163M is an LRP1 agonist

THP1-XBlue™-MD2-CD14 cells (InvivoGen, San Diego, CA) were used in an in vitro assay to examine whether SP163M is an agonist of LRP1. THP1-XBlue™-MD2-CD14 cells were maintained in RPMI 1640 medium (Thermo Scientific) supplemented with 10% heat-inactivated FBS, 1% Pen-Strep, 100 μg/ml Normocin™, 200 μg/ml Zeocin™, and 250 μg/ml G418. The cell line was maintained at 37°C in 5% CO2 according to the manufacturer's instructions.

THP1-XBlue™-MD2-CD14 cells were seeded at 1 x 10 ⁵ cells/well in a 96-well cell culture plate. The cells were treated with peptide or scrambled control peptide (100 μg/ml) for 30 minutes, followed by the addition of LPS (5 ng/ml) or GP96 (100 pM). After incubation at 37°C for 18-24 hours, 20 μl of supernatant was transferred to 180 μl of QUANTI-Blue™ SEAP Detection Medium. NF-κB-inducible SEAP levels were measured by absorbance. For LRP1 blocking experiments, anti-LRP1 antibodies (125 μg/ml) (clone 5A6, Molecular Innovations, Novi, MI) or receptor-associated protein (RAP) (1 μM) (Molecular Innovations) were used for 30 minutes before the addition of the peptide.

As shown in Figure 13, SP163M inhibited NF-κB signaling induced by LPS (Figure 13A) and GP96 (Figure 13B). Treatment with LRP antibodies or RAP (a cofactor that leads to LRP1 downregulation) limited SP163M-related inhibition in cells treated with LPS and GP96.

LRP1 mediates SP163M-induced cardioprotection

To examine whether LRP1 mediates the cardioprotective effects of SP163M, groups of mice were treated with an LRP1 blocking antibody before experimental AMI. The resulting infarct size and LV systolic function were measured.

Adult hybrid male CD1 mice (10 weeks old) supplied by Harlan Sprague Dawley (Indianapolis, IN) were used for this study. All surgeries were performed by a single operator (S.T.) skilled in coronary artery ligation in mice. Experimental AMI was induced by transient coronary ligation of the left anterior descending coronary artery for 30 minutes to induce anterior wall and apical ischemia (shown in grayish white), followed by reperfusion and resulting in approximately 15% infarction of the left ventricle [1].

At the time of surgery, mice were deeply anesthetized with sodium pentobarbital (70-100 mg/kg), intubated and placed in the right lateral decubitus position. Left thoracotomy was performed, followed by pericardiectomy and ligation of the proximal left coronary artery, followed by reperfusion. After closing the thoracic access, mice recovered for up to 1 week with no restriction on food and water. Mice that showed ischemia only during surgery and involving the entire apex of the heart were randomly assigned to different treated groups by a researcher (A.G.M), who did not participate in judging the target endpoint. The experiment was conducted under the Guidelines for Laboratory Animals in Biomedical Research, published by the National Institutes of Health (revised in 2011). The study protocol was approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University.

Mice pretreated with LRP1-blocking antibodies were given the antibodies 16 hours before surgery to allow sufficient time for the antibodies to bind to their target receptors. Mice were randomly assigned to different postoperative treatment groups, where postoperative treatment was provided as a single intraperitoneal dose immediately after reperfusion. The postoperative treatment groups received: SP163M 100 μg, an equivalent volume of vehicle to SP163M, LRP1-blocking antibody (AB) clone 5A6 3 mg/kg (Molecular Innovations), LRP1-AB and SP163M 100 μg, plasma-derived alpha-1 antitrypsin (AAT) 60 mg/kg (Aralast NP, Baxter, Deerfield, IL, USA), LRP1-AB, and AAT. Each group included 5 to 8 mice.

Infarct size was measured as described previously (reported in Figure 14A) [1]. A subgroup of mice was sacrificed 24 hours after surgery, and the hearts were rapidly removed and placed on a Langendorff apparatus, where the coronary arteries were perfused antegradely with phosphate-buffered saline (PBS) 1X pH 7.4 containing heparin (40 U/ml). After washing out the blood, 10% triphenyltetrazolium chloride (Sigma Aldrich) in PBS 1X was perfused, followed by ligation with a band and perfusion with 1% phthalocyanine blue dye (Quantum Ink, Louisville, KI, USA). 5 mM adenosine in PBS 1X was bolus-infused into the aorta until most of the heart turned blue. Subsequently, the heart was removed from the Langendorff apparatus, frozen, and cut into six transverse sections of uniform thickness (approximately 1 mm) from the apex to the base of the heart. Infarcted tissue (visible as white) and viable tissue (bright red) were measured by computer morphometry using ImagePro Plus 6.0 software (Media Cybernetics, Silver Spring, MD). Infarct size is reported as a % of the left ventricle.

LV systolic function was also determined (reported in Figure 14B). Mice were lightly anesthetized with sodium pentobarbital (30-50 mg/kg) and underwent baseline (before surgery) transthoracic echocardiography at 24 hours. Echocardiography was performed using a Vevo770 imaging system (VisualSonics Inc, Toronto, Ontario, Canada) and a 30-MHz probe [1, 2]. The heart was visualized in B-mode from the parasternal short axis and apical views. Left ventricular (LV) end-diastolic and end-systolic areas in B-mode and LV end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD) in M-mode were measured. LV fractional shortening (FS) and LV ejection fraction (EF) were calculated. A subgroup of mice underwent repeated echocardiography at 7 days to measure infarct size (number of unpowered segments [2]) and left ventricular systolic function.

All results are expressed as mean ± standard error. Comparisons between 3 or more groups were performed using analysis of variance (ANOVA), followed by a T-test for unpaired data to compare each treatment group with the corresponding control. Kaplan Meyer survival curves for SP16 and vehicle after LPS challenge were compared using the log-rank test. All analyses were performed using the Statistical Package for Social Sciences (SPSS, Version 22.0, IBM Inc., New York, NY). P values of <0.05 were considered significant.

Results: Treatment of mice with blocking LRP1 antibodies abolished the protective effects of both SP163M and plasma-derived AAT.

References

1. Toldo S, Seropian IM, Mezzaroma E, Van Tassell BW, Salloum FN, Lewis EC, Voelkel N, Dinarello CA, Abbate A: Alpha-1 antitrypsin inhibitscaspase-1 and protects from acute myocardial ischemia-reperfusion injury. Journal of molecular and cellular cardiology 2011, 51(2):244-251.

2. Seropian IM, Abbate A, Toldo S, Harrington J, Smithson L, OckailiR, Mezzaroma E, Damilano F, Hirsch E, Van Tassell BW: PharmacologicInhibition of Phosphoinositide 3-Kinase Gamma (PI3Kγ) Promotes InfarctResorption and Prevents Adverse Cardiac Remodeling After MyocardialInfarction in Mice. Journal of cardiovascular pharmacology 2010, 56(6):651-658.

Claims (3)

1. Use of a pharmaceutical composition in the preparation of a medicament for treating acute myocardial infarction (AMI), said pharmaceutical composition comprising a peptide consisting of the amino acid sequence VKFNKPFVFL[Nle]IEQNTK (SEQ ID NO:57). 2. The use according to claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier. 3. The use according to claim 1 or 2, wherein the composition is a parenteral preparation.