WO2022003673A1

WO2022003673A1 - Humanin analogs and uses thereof

Info

Publication number: WO2022003673A1
Application number: PCT/IL2021/050774
Authority: WO
Inventors: Chaim Gilon; Amnon Hoffman; Philip Lazarovici; Dan GILON
Original assignee: Hadasit Medical Research Services and Development Co; Yissum Research Development Co of Hebrew University of Jerusalem
Current assignee: Hadasit Medical Research Services and Development Co; Yissum Research Development Co of Hebrew University of Jerusalem
Priority date: 2020-06-30
Filing date: 2021-06-24
Publication date: 2022-01-06
Anticipated expiration: 2022-12-30

Abstract

The present invention provides novel peptide analogs of Humanin, pharmaceutical composition comprising the peptides and uses thereof for treating diseases, disorders or conditions associated with mitochondrial dysfunction, such as but not limited to ischemic stroke and doxorubicin-induced cardiotoxicity.

Description

HUMANIN ANALOGS AND USES THEREOF

FIELD OF INVENTION

The present invention relates to novel Humanin analogs and uses thereof for treating diseases, disorders or conditions associated with mitochondrial dysfunction, such as but not limited to ischemic stroke and doxorubicin-induced cardiotoxicity.

BACKGROUND OF THE INVENTION

Mitochondrial dysfunction is associated with a wide range of human diseases and disorders, such as neurodegenerative disorders, cardiovascular disorders, neurometabolic diseases, cancer and obesity. Impaired oxidative phosphorylation leading to a decrease in cellular energy (ATP) production is a significant factor involved in these disorders (Khan, Nahid Akhtar, et al, The Indian journal of medical research 141.1 (2015): 13).

Stroke (cerebrovascular insult-CVI) is defined as a neuropathological disease, accounting for 5.5 million deaths annually worldwide (D. Mukherjee, C.G. Patil, World Neurosurg. 76 (2011) S85-90) which occurs when the blood flow, which supplies the brain with oxygen and essential nutrients such as glucose, as well as certain growth factors, is partially or entirely perturbed. This oxygen and glucose deprivation (OGD) ischemic event can result in pathological irreversible consequences, eventually leading to impaired neurological functions and morbidity. Within the focal “core” region, many of the cells, neurons in particular, die by necrosis making rescue attempts almost impossible. However, expansion of the damage extending beyond the core region to a greater area, also defined as “penumbra”, can lead to a secondary stage of neuronal cell death. The reason for damage in this particular region stems paradoxically from the restoration of blood circulation (reperfusion) and resupply of oxygen and glucose. This ischemia-reperfusion-injury (IRI) stroke process accelerates apo-necrotic neuronal cell death through a variety of post-ischemic neuropathological responses (Z. Zheng, et al, Drug News Perspect. 16 (2003) 497-503). Neuroprotection, aiming to minimize the cascade of events leading to cerebral ischemia cell death, in particular the penumbra, is defined as a pharmacological strategy that antagonizes biochemical and molecular events that, if left untreated, would eventuate in irreversible neuronal cell death.

Doxorubicin (Dox), a member of the anthracycline family of anti-cancer cytotoxic drugs is an ingredient of various chemotherapy drug protocols used in oncology for treating a wide range of tumors, e.g., lymphoma, leukemia and breast cancer. However, Dox treatment is associated with some life-threatening side effects including cardiotoxicity and, late onset of congestive heart failure often limits its clinical applications (J. V. McGowan, et al, Cardiovasc. Drugs Ther. 31 (2017) 63-75). Dox treatment results in subclinical, progressive, irreversible cardiotoxicity causing significant morbidity and mortality in cancer patients. Dox induces myocardial pathology by increasing reactive oxygen species (ROS) production and causing mitochondrial dysfunction (B. Kalyanaraman, Redox Biol. 29 (2020) 101394). Although a few drugs have been used to reduce doxorubicin cardiotoxicity, no effective treatment for established doxorubicin cardiomyopathy is presently available.

Humanin (HN), a 24-amino acid mitochondrial-derived peptide (MDP), is a metabolic regulator of human body, and plays a cytoprotective role in maintaining mitochondrial function and cell viability under different physiological insults (Y. Yang, et al., Biomed. Pharmacother. 117 (2019) 109075). HN has neuroprotective effects in stroke (X. Xu, et al, Stroke. 37 (2006) 2613- 9) and cardioprotection in myocardial infarction or doxorubicin (Dox)-induced cardiotoxicity (N.L. Rosin, et al, Am. J. Pathol. 185 (2015) 631-42). Studies have also shown that HN protects heart against brain and cardiac ischemia/reperfusion-induced cardiomyopathy in rodents by decreasing mitochondrial ROS levels and reducing mitochondrial dysfunction (X. Xu, et al, Brain Res. 1227 (2008) 12-8; and S. Thummasorn, et al, Cardiovasc. Ther. 34 (2016) 404M44).

Humanin G (HNG) is an HN analog with a single amino acid substitution of serine to glycine at position 14, that was found to have an improved activity compared to HN (X. Xingshun, et al, Stroke 37.10 (2006) 2613-2619). In particular, it was shown that HNG has substantially greater cytoprotective action than HN, and has neuroprotective effect in experimental stroke (G. Gao, et al, Neurol. Res. 39 (2017) 895-903) and cardioprotective effect in myocardial ischemia and reperfusion injury in mice (R.H. Muzumdar, et al, Arterioscler. Thromb. Vase. Biol. 30 (2010) 1940-1948). A shorter, 17-amino acid peptide denoted AGA-(C8R)HNG17, was also discovered having a higher cytoprotective activity than HNG (A. Fumio, et al, Int. J. Biol. Macromol. 43.2 (2008) 88-93).

NAP peptide (also known as davunetide) is an 8-amino-acid peptide that was identified as the smallest active sequence motif of activity-dependent neuroprotective protein (ADNF), that exhibits potent neuroprotection in vitro and in vivo (I. Gozes, Curr. Pharm. Des. 17 (2011) 1040- 1044). US 7,452,867 relates to the use of ADNF polypeptides in the treatment of neurotoxicity induced by chemical agents or by disease processes. The ADNF polypeptides include ADNF I and ADNF III (also referred to as ADNP) polypeptides, analogs, subsequences such as NAP and SAL, and D-amino acid versions (either wholly D-amino acid peptides or mixed D- and L-amino acid peptides), and combinations thereof which contain their respective active core sites.

US 2005/0233413 provides polypeptides derived from Humanin, which contain one or more D-amino acids or phosphorylated amino acids, or amino acids that form a multimer. The HN derivatives are useful in protecting neuronal cells from cytotoxicity related to neurodegenerative diseases.

US 8,076,449 relates to a pharmaceutical composition for the treatment and/or prevention of a neurodegenerative disease, wherein the pharmaceutical composition comprises a polypeptide having an activity that inhibits neuronal cell death associated with neurodegenerative disease, or analogs, derivatives or salts thereof.

There remains an unmet clinical need for additional and improved drug candidates for the treatment of diseases and disorders associated with mitochondrial dysfunction, e.g., ischemic stroke and drug-induced cardiotoxicity.

SUMMARY OF THE INVENTION

The present invention provides novel peptide analogs of Humanin (HN) having improved therapeutic properties, pharmaceutical compositions comprising said analogs, and therapeutic uses thereof.

The present invention is based in part on the finding that the newly designed HN analogs, denoted HUJInin and c(D-Serl4-HN), are effective in improving mitochondrial functions of cells exposed to oxidative stress, and possess significant neuroprotective and myoprotective properties as demonstrated in several in vitro insult models. In particular, the novel peptide analogs conferred a significant, dose-dependent neuroprotection against oxygen-glucose-deprivation-reoxygenation (OGD/R)- and serum starvation-induced insults in neuronal cell cultures, and a robust myoprotection against apo-necrotic cell death insults in myoblast cell cultures. Without wishing to be bound by any theory or mechanism of action, it is hypothesized that the neuroprotective and myoprotective properties of the peptides can be at least partly attributed to their ability to improve mitochondrial function. The present invention is further based in part on the finding that the peptide analog HUJInin, having the amino acid sequence YNAPVSIPQPAGASRLLLLTGEIDLP (SEQ ID NO: 1), which is a conjugate of the HN analog AGA-(C8R) HNG17 (PAGASRLLLLTGEIDLP, SEQ ID NO: 16), and the 8-amino acid sequence NAPVSIPQ (SEQ ID NO: 7, denoted NAP), showed at least similar protective effects, as compared to each of the parental compounds, with the advantage of stimulating Akt survival kinase and decreasing Erk hyperphosphorylation cytotoxic activity. Furthermore, in some assays an enhanced protective effect was obtained with the peptide conjugate.

The present invention is further based in part on the surprising finding that the cyclic peptide analog denoted c(D-Serl4-HN) that comprises the amino acid sequence MAPAGASRLLLLTsEIDLPVKRRA (SEQ ID NO: 2), not only maintained the neuroprotective and myoprotective properties despite cyclization, but also showed some improved properties as compared to the corresponding linear analog.

Furthermore, the design of the linear and cyclic peptides of the invention advantageously confers enhanced drug like properties such as increased metabolic stability and cell membrane permeability.

The peptides and peptide analogs of the present invention are therefore useful in neuroprotection, myoprotection, and for the treatment of diseases, disorders or conditions associated with mitochondrial dysfunction.

According to one aspect, the present invention provides a peptide of 15-40 amino acid residues, or an analog thereof, the peptide comprising the sequence X1X2AGASRLLLLTX3EIDLX4 (SEQ ID NO: 5), wherein:

Xi is absent or Glutamine (Gin, Q);

X2 is absent or Proline (Pro, P);

X3 is selected from Glycine (Gly, G) and D-Serine (DSer, s); and

X4 is absent or is selected from Proline (Pro, P) and a sequence of 2-6 amino acid residues comprising Proline (Pro, P); with the proviso that when X3 is Gly (G), Xi is Gin (Q).

In some embodiments, X3 is D-Ser (s).

In some embodiments the peptide is cyclic. In yet other embodiments, X3 is D-Ser (s) and the peptide is cyclic. According to an additional aspect, the present invention provides a peptide of 15-40 amino acid residues, or an analog thereof, the peptide comprising the sequence set forth in SEQ ID NO: 5, wherein:

Xi is absent or Glutamine (Gin, Q);

X2 is absent or Proline (Pro, P);

X3 is selected from Glycine (Gly, G) and D-Serine (DSer, s); and

X4 is absent or is selected from Proline (Pro, P) and a sequence of 2-6 amino acid residues comprising Proline (Pro, P); with the proviso that when X3 is Gly (G), Xi is Gin (Q), and when X3 is DSer (s), the peptide is cyclic.

In some embodiments, X3 is Gly (G).

In some embodiments, X3 is Gly (G) and the peptide is linear.

In some embodiments, X2 is Pro (P).

In some embodiments, the peptide further comprises the amino acid sequence NAPVSIP (SEQ ID NO: 6). In some embodiments, the peptide comprises the amino acid sequence NAPVSIPQ (SEQ ID NO: 7).

In some embodiments, the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7 is conjugated to the N-terminus of the amino acid of the sequence set forth in SEQ ID NO: 5, namely the carboxy terminal residue P or Q of SEQ ID NO: 6 or 7 respectively, is coupled to the terminal amine of Q, P or A residues.

In some related embodiments, the peptide comprises the amino acid sequence NAPVSIPQPAGASRLLLLTGEIDLX4 (SEQ ID NO: 8), wherein X₄is absent or Proline. In some specific embodiments, X4 is proline and the peptide comprises the amino acid sequence NAPVSIPQPAGASRLLLLTGEIDLP (SEQ ID NO: 9).

In some embodiments, the peptide comprises at least one amino acid residue that allows labeling. In some embodiments, said amino acid residue is Tyrosine (Tyr, Y).

In some specific embodiments, the peptide comprises the amino acid sequence YNAPVSIPQPAG ASRLLLLTGEIDLP (SEQ ID NO: 1). In further specific embodiments, the peptide consists of the amino acid sequence set forth in SEQ ID NO: 1.

In some embodiments, the peptide comprises a modified C-terminus. In some specific embodiments, the C-terminus of the peptide is ami dated. In some embodiments, the peptide comprises a modified N-terminus. In specific embodiments, the N-terminus of the peptide is acetylated.

In some embodiments, the peptide comprises the amino acid sequence YNAPVSIPQPAG ASRLLLLTGEIDLP (SEQ ID NO: 1), wherein the C-terminus of the peptide is amidated and the N-terminus of the peptide is acetylated.

In some embodiments, the peptide consists of the amino acid sequence set forth in SEQ ID NO: 1, wherein the C-terminus of the peptide is amidated and the N-terminus of the peptide is acetylated to form Ac-YNAPVSIPQPAGASRLLLLTGEIDLP-NPb (SEQ ID NO: 1).

In some embodiments, the present invention provides a cyclic peptide of 15-40 amino acid residues, or an analog thereof, the peptide comprising the sequence AGASRLLLLTsEIDL (SEQ ID NO: 4).

In some embodiments, there is provided a cyclic peptide of 15-40 amino acid residues, or an analog thereof, the peptide comprising the sequence X1X2AGASRLLLLTX3EIDLX4 (SEQ ID NO: 5), wherein:

Xi is absent or Glutamine (Gin, Q);

X2 is absent or Proline (Pro, P);

X3 is D-Serine (DSer, s); and

X4 is absent or is selected from Proline (Pro, P) and a sequence of 2-6 amino acid residues comprising Proline (Pro, P);

Any cyclization type or technology may be utilized for the cyclic peptides and analogs of the present invention. In some embodiments, the peptide is cyclized via a cyclization type selected from: an end-to-end (C-terminal to N-terminal) cyclization, a backbone to end cyclization, a backbone-to-backbone cyclization, a side-chain to end, or a side-chain to side-chain cyclization. Each possibility represents a separate embodiment of the present invention.

In some embodiments of the cyclic peptides, X2 is Proline. In some embodiments, X4 is Proline. In some embodiment, X2 and X4 are both Proline. In related embodiments, the cyclic peptide comprises the amino acid sequence PAGASRLLLLTsEIDLP (SEQ ID NO: 3). In further embodiments, the cyclic peptide consists of the amino acid sequence set forth in SEQ ID NO: 3.

In some embodiments, X4 is a 2-6 amino acid sequence comprising Proline (Pro, P). In some embodiments, X4 is the amino acid sequence PVKRRA (SEQ ID NO: 11). In some embodiments, the cyclic peptide comprises the amino acid sequence MAPAGASRLLLLTsEIDLPVKRRA (SEQ ID NO: 2). In specific embodiments, the cyclic peptide consists of the amino acid sequence set forth in SEQ ID NO: 2.

In some embodiments, the cyclic peptide is cyclized by a formation of an amide bond between the N-terminal and the C-terminal amino acid residues, directly or through a spacer or a linker.

In some embodiments, the cyclic peptide consists of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3, wherein cyclization is formed by an amide bond between the N- terminal and the C-terminal amino acid residues. In some embodiments, the cyclic peptide consists of the amino acid sequence set forth in SEQ ID NO: 2, wherein cyclization is formed by an amide bond between the N-terminal and the C-terminal amino acid residues.

In some embodiments, the cyclic peptide comprises a cyclization type selected from: an end- to-end (C-terminal to N-terminal) cyclization, a backbone to end cyclization, a backbone-to- backbone cyclization, a side-chain to end, or a side-chain to side-chain cyclization. Each possibility represents a separate embodiment of the present invention.

In some embodiments, the cyclic peptide is a backbone cyclic peptide.

According to some embodiments, the backbone cyclic peptide is cyclized via a bond selected from the group consisting of: urea bond, thiourea bond, amide bond, disulfide bond and guanidino group, namely the cyclization bridge is selected from the group consisting of: urea bridge, thiourea bridge and guanidino bridge. Each possibility represents a separate embodiment of the present invention. According to some particular embodiments, the bond used for cyclization is a urea bond.

In some embodiments, the backbone cyclic peptide comprises the amino acid sequence set forth in SEQ ID NO: 4 and has a structure according to Formula (I):

Formula (I) wherein:

X- Y -Z is a bridge selected from urea bridge, thiourea bridge, amide bridge, disulfide bridge and guanidino group;

Ri and R2 are independently selected from a hydrogen and the side chain of an amino acid; and m and n are each independently an integer of between 2 to 6.

According to some aspects and embodiments, the present invention provides a prodrug of the peptides or peptide analogs of the invention in all embodiments thereof, including linear and cyclic forms.

According to some aspects, the present invention provides a pharmaceutical composition comprising the peptide or the prodrug of the invention in all embodiments thereof.

According to some aspects, the present invention provides a pharmaceutical composition comprising a peptide of 15-40 amino acid residues, or an analog thereof, the peptide comprising the sequence X1X2AGASRLLLLTX3EIDLX4 (SEQ ID NO: 5), wherein:

Xi is absent or Glutamine (Gin, Q);

X2 is absent or Proline (Pro, P);

X3 is selected from Glycine (Gly, G) and D-Serine (DSer, s); and

X4 is absent or is selected from Proline (Pro, P) and a 2-6 amino acid sequence comprising Proline (Pro, P); with the proviso that when X3 is Gly (G), Xi is Gin (Q).

According to some aspects, the present invention provides a pharmaceutical composition comprising a peptide of 15-40 amino acid residues, or an analog thereof, the peptide comprising the sequence set forth in SEQ ID NO: 5, wherein:

Xi is absent or Glutamine (Gin, Q);

X2 is absent or Proline (Pro, P);

X3 is selected from Glycine (Gly, G) and D-Serine (DSer, s); and

X4 is absent or is selected from Proline (Pro, P) and a 2-6 amino acid sequence comprising Proline (Pro, P); with the proviso that when X3 is Gly (G), Xi is Gin (Q), and when X3 is DSer (s), the peptide is cyclic. In some embodiments, the pharmaceutical composition comprises at least one peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4. In specific embodiments, the pharmaceutical composition comprises a peptide comprising the amino acid sequence set forth in SEQ ID NO:l. In other embodiments, the pharmaceutical composition comprises at least one peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-4. In further embodiments, the pharmaceutical composition comprises at least one cyclic peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-4. Each possibility represents a separate embodiment of the present invention.

In some embodiments, the pharmaceutical composition comprises a combination of at least two distinct peptides, each peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-5 or of an analog thereof. As used herein, the term "distinct peptides" refers to peptides differing in at least one amino acid.

In some embodiments, the pharmaceutical composition comprises a prodrug of the peptide of the invention in all embodiments thereof.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition is formulated in a form suitable for an administration route selected from oral (per os), intravenous, intramuscular, subcutaneous, intrathecal and intranasal administration or for administration by inhalation. In some embodiments, the pharmaceutical composition is formulated in a form suitable for an administration route selected from oral, intravenous, intramuscular, subcutaneous intrathecal and intranasal administration. In some embodiments, the pharmaceutical composition is formulated in a form suitable for an administration route selected from intravenous, intramuscular, subcutaneous, intrathecal and intranasal administration. Each possibility represents a separate embodiment of the present invention.

According to some aspects, the present invention provides a pharmaceutical composition for use in treating a disease, disorder or condition associated with mitochondrial dysfunction, the pharmaceutical composition comprising a peptide of 15-40 amino acid residues, comprising the sequence X1X2AGASRLLLLTX3EIDLX4 (SEQ ID NO: 5), or an analog thereof, wherein:

Xi is absent or Glutamine (Gin, Q); X2 is absent or Proline (Pro, P);

X3 is selected from Glycine (Gly, G) and D-Serine (DSer, s); and

X4 is absent or is selected from Proline (Pro, P) and a 2-6 amino acid sequence comprising

Proline (Pro, P); with the proviso that when X3 is Gly (G), Xi is Gin (Q), and when X3 is DSer (s), the peptide is cyclic.

According to other aspects, the present invention provides a method of treating a disease, disorder or condition associated with mitochondrial dysfunction, the method comprising administering to a subject in need thereof a pharmaceutical composition comprising a peptide of 15-40 amino acid residues, or an analog thereof, wherein the peptide comprises the sequence X1X2AGASRLLLLTX3EIDLX4 (SEQ ID NO: 5), wherein:

Xi is absent or Glutamine (Gin, Q);

X2 is absent or Proline (Pro, P);

X3 is selected from Glycine (Gly, G) and D-Serine (DSer, s); and

In some embodiments, the pharmaceutical composition is administered by a route selected from oral, intravenous, intramuscular, subcutaneous, intrathecal and intranasal administration or by inhalation. In some embodiments, the pharmaceutical composition is administered by a route selected from oral, intravenous, intramuscular, subcutaneous, intrathecal and intranasal administration. In some embodiments, the pharmaceutical composition is administered by a route selected from intravenous, intramuscular, subcutaneous, intrathecal and intranasal administration. Each possibility represents a separate embodiment of the present invention.

In some embodiments, the disease, disorder or condition which is associated with mitochondrial dysfunction is selected from the group consisting of an ischemia related disease or disorder, a neurodegenerative disease or disorder, and a cardiovascular disease or disorder. Each possibility represents a separate embodiment of the present invention.

In some embodiments, the disease, disorder or condition is selected from the group consisting of cerebral ischemic reperfusion, myocardial ischemic reperfusion and anthracycline- induced cardiomyopathy. Each possibility represents a separate embodiment of the present invention.

In some embodiments, the disease, disorder or condition is anthracycline-induced cardiomyopathy. In specific embodiments, the disease, disorder or condition is a chemotherapy- induced cardiomyopathy. In other specific embodiments, the condition is a Doxorubicin-induced cardiomyopathy.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A represents the structure of the HN analog c(D-Serl4)HN (SEQ ID NO: 2).

Figure IB depicts a schematic representation of the solid phase synthesis of c(D-Serl4)HN (SEQ ID NO: 2). Fmoc, fluourenylmetyloxycarbonyl; HFIP, hexaflouroisopropanol; TPPA, triphenylphosphoryl azide; TFA, triflouroacetic acid; TIS, triisopropylsilane; CTC, chloro- tritylchloride polystyrene resin.

Figures 2A-2B show HPLC (Figure 2A) and MS (Figure 2B) chemical characterizations of HNG17 peptide (SEQ ID NO: 16).

Figures 2C-2D show HPLC (Figure 2C) and MS (Figure 2D) chemical characterizations of NAP- NH₂ peptide (SEQ ID NO: 7).

Figures 2E-2F show HPLC (Figure 2E) and MS (Figure 2F) chemical characterizations of HUJInin peptide (SEQ ID NO: 1).

Figures 2G-2H show HPLC (Figure 2G) and MS (Figure 2H) chemical characterizations of c(D- Serl4)HN peptide (SEQ ID NO: 2).

Figure 3A shows a dose-dependent effect of NAP (SEQ ID NO: 7), HNG17 (SEQ ID NO: 16) and HUJInin (MRPV; SEQ ID NO: 1) on oxygen-glucose-deprivation-reoxygenation (OGD/R)- induced cell death in PC 12 catecholaminergic neuronal cultures, compared to untreated cells (negative control) and cells treated with Tempol (positive control). Cell death is expressed by % LDH release. Results presented as mean ± SE (n = 30); *p < 0.01 vs control; **p < 0.01 vs 0.5 mM of the same peptide group.

Figure 3B shows a time-dependent effect of NAP (SEQ ID NO: 7), HNG17 (SEQ ID NO: 16) and HUJInin (SEQ ID NO: 1; MRPV, 10 mM) against serum deprivation induced cell death in undifferentiated PC 12 cells, compared to untreated cells (control). Cell death is expressed by % LDH release. Results presented as mean ± SE (n = 30); *p < 0.01 vs untreated group; **p < 0.01 vs respective control group.

Figure 4A shows SDS-polyacrylamide gel electrophoresis and immunoblotting analysis of Erk 1/2 phosphorylation in PC 12 cell cultures under OGD insult with or without treating with NAP (SEQ ID NO: 7), HNG17 (SEQ ID NO: 16), HUJInin (MRPV; SEQ ID NO: 1), or the specific MEK/Erkl/2 inhibitor PD98059, compared to normoxia conditions (control). Immunodetection was performed using primary antibodies against phospho-Erkl/2 (p-Erk, top) or pan Erkl/2 (pan- Erk, bottom).

Figure 4B represents quantification of the phosphorylation of the protein kinase Erkl/2 in PC12 cells lysates from OGD and normoxia (control) groups, as determined by densitometric analysis of the immunoblots presented in Figure 4A. Results are shown as mean ± SE, and represent three independent experiments. *p < 0.01 vs control normoxia; **p < 0.01 vs 4 h OGD.

Figures 5A and 5C show SDS-polyacrylamide gel electrophoresis and immunoblotting analysis of AKT phosphorylation in PC 12 cell cultures under OGD insult (Figure 5A) and its quantification (Figure 5C), with or without treating with HUJInin (MRPV; SEQ ID NO: 1) in the absence or presence of the PI3K/AKT inhibitor LY294002. Immunodetection was performed using primary antibodies against phospho- (p-) or pan- Akt. (5C) Results are shown as the mean ± SE, and represent three independent experiments. C- *p < 0.01 vs OGD ; **p < 0.01 vs HUJInin.

Figures 5B and 5D represent SDS-polyacrylamide gel electrophoresis and immunoblotting analysis of AKT phosphorylation in PC 12 cell cultures (Figure 5B) and its quantification (Figure 5D) upon serum starvation, with or without treating with HUJInin (MRPV; SEQ ID NO: 1) in the absence or presence of the PI3K/AKT inhibitor LY294002. (5D) Results are shown as the mean ± SE, and represent three independent experiments. *p < 0.01 vs serum **p < 0.01 vs serum starvation; ***p < 0.01 vs HUJInin.

Figure 6A shows a dose-dependent effect of the cyclic HN analog c(D-Serl4-HN) (SEQ ID NO: 2) against OGD/R induced cell death in PC 12 (white columns) and SH-SY5Y (black column) cells, compared to untreated cells (control, left columns). Cell death is expressed by % LDH release out of total LDH. Results presented as mean ± SE (n = 18); *p < 0.01 vs respective control; **p < 0.01 vs respective 0.5 mM.

Figure 6B shows SDS-polyacrylamide gel electrophoresis and immunoblotting analysis of Erkl/2 phosphorylation in PC 12 cell cultures under OGD insult (for 4 hours) with or without treating with 5 or 10 mM c(D-Serl4-HN) (SEQ ID NO: 2), compared to normoxia conditions (control). Immunodetection was performed using primary antibodies against phospho-Erkl/2 (p-Erk, top) or pan Erkl/2 (pan-Erk, bottom).

Figure 6C shows SDS-polyacrylamide gel electrophoresis and immunoblotting analysis of AKT phosphorylation in PC 12 cell cultures under exposure to OGD conditions for 1 or 4 hours, with or without treating with c(D-Serl4-HN) (SEQ ID NO: 2), in the absence or presence of the PI₃K/AKT inhibitor LY294002. Immunodetection was performed using primary antibodies against phospho- (p-) or pan- Akt.

Figures 7A and 7B show SDS-polyacrylamide gel electrophoresis and immunoblotting analysis of Erk 1/2 (Figure 7A) and AKT (Figure 7B) phosphorylation in PC12 cell cultures under normoxia with or without treating with NAP (SEQ ID NO: 7), HNG17 (SEQ ID NO: 16), HUJInin (SEQ ID NO: 1), or c(D-Serl4)HN (SEQ ID NO: 2). Immunodetection was performed using primary antibodies against phospho- (p-) or pan- Erkl/2 and Akt.

Figures 7C and 7D represent quantification of the phosphorylation of Erk 1/2 (Figure 7C) and AKT (Figure 7D) in PC12 cells treated with NAP (SEQ ID NO: 7), HNG17 (SEQ ID NO: 16), HUJInin (SEQ ID NO: 1), or c(D-Serl4)HN (SEQ ID NO: 2), compared to untreated cells (control), as determined by densitometric analysis of the immunoblots presented in Figures 7A and 7B. Results are shown as mean ± SE, and represent three independent experiments. *p < 0.01 vs control. Figures 8A-8C represent the effect of the HN analogs HUJInin (SEQ ID NO: 1), c(D-Serl4-HN) (SEQ ID NO: 2, cyclic), and (D-Serl4)HN-NH₂ (SEQ ID NO: 2, linear) on the basal and the physiological ATP-induced response of mitochondrial calcium in human neuroblastoma SH- SY5Y cells. Figure 8A shows representative fluorescent traces of [Ca2+]mito transients in Rhod2-AM loaded cells triggered by 100 mM ATP, in the presence or absence of the tested HN analogs at a concentration of 5 pM. Figures 8B and 8C show quantification of the Basal [Ca2+]mito level (Figure 8B) and [Ca2+]mito amplitudes (Figure 8C).

Figure 8D and 8E represent the effect of the HN analogs HUJInin (SEQ ID NO: 1), c(D-Serl4- HN) (SEQ ID NO: 2, cyclic), and (D-Serl4)HN-NH₂ (SEQ ID NO: 2, linear) on the mitochondrial membrane potential of human neuroblastoma SH-SY5Y cells. Figure 8D shows representative fluorescent traces of mitochondrial membrane potential records in TMRM pre-loaded cells triggered by 1 pM FCCP and treated (or untreated - control) with 5 pM of the tested HN analogs. Black traces show the records before incubation with 0.4 mM H2O2 and the gray traces show thereafter records. Figure 8E shows quantification of membrane potential (MP) divided by minimal membrane potential (MMP). *p < 0.01 vs before H2O2 respective group; **p < 0.01 vs control after H2O2 group.

Figure 9 shows a dose-dependent effect of HUJInin (SEQ ID NO: 1) and c(D-Serl4)HN (SEQ ID NO: 2) against Dox-induced necrotic cell death in H9c2 myoblast cells, compared to untreated cells (negative control) and cells treated with Tempol (positive control). Cell death is expressed by % LDH release out of total LDH. Results presented as mean ± SE (n = 18); *p < 0.01 vs control; **p < 0.01 vs HUJInin 10 pM.

Figures 10A shows Hoechst staining of H9c2 cells that were untreated (control, upper left panel); treated with Dox (upper right panel); treated with Dox + c(D- Serl4-HN) (bottom left panel); or treated with Dox + DMSO (bottom right panel).

Figure 10B shows the percentage of apoptotic cells calculated using NIH Image J software, based on the Hoechst staining assay. Results represent as mean ± SD of three separate experiments *p < 0.01 vs control group; **p < 0.01 vs Dox (untreated); ***p < 0.01 vs Dox + c(D-Serl4-HN).

Figure 11A depicts western blots of cultures extracts expressing troponin T (left panel) and phosphorylated form Troponin- 1 (right panel). Figure 11B shows the expression of Caspase-3 protein, measured by western blotting with specific antibody, in cultures treated with 1 mM doxorubicin (Dox) for 24 hours24 h in the absence and presence of 5 mM HN analogs.

Figure 11C represents Caspase-3 enzymatic activity of cultures treated with 1 pM Dox for 24 h, in the absence and presence of different concentrations of HN analogs; *p < 0.01 vs control group; **p < 0.01 vs Dox .

Figure 12 shows the effect of HUJInin (MRPV; SEQ ID NO: 1) and c(D-Serl4)HN (SEQ ID NO: 2) against Dox-induced necrotic cell death in C2C12 myoblast cells, compared to untreated cells. Cell death is expressed by % LDH release out of total LDH. Results presented as mean ± SE (n = 18); *p < 0.01 vs control; **p < 0.01 vs Dox (untreated).

Figure 13A represents the structure of the HN analog c(D-Serl4)HN17 (SEQ ID NO: 3).

Figure 13B depicts a schematic representation of the solid phase synthesis of the cyclic peptide denoted c(D-Serl4)HN17 (SEQ ID NO: 3). Fmoc, fluourenylmetyloxy carbonyl; HFIP, hexaflouroisopropanol; TPPA, triphenylphosphoryl azide; TFA, triflouroacetic acid; TIS, triisopropylsilane; CTC, chloro-tritylchloride polystyrene resin.

Figure 14 depicts a schematic representation of the solid phase synthesis of the cyclic peptide denoted c(sl4-HN17(m-n)) (Formula (I), SEQ ID NO: 4).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel analogs of Humanin (HN) peptide that can be useful for the treatment of various diseases associated with mitochondrial dysfunction.

The present invention is based in part on the design of a novel linear peptide conjugate denoted HUJInin that combines the sequence of the HN analog (G¹⁴) HN17 (PAGASRLLLLTGEIDLP (SEQ ID NO: 16), and the 8-amino acid sequence NAPVSIPQ (SEQ ID NO: 7), denoted NAP derived from ADNF. It was found that the novel peptide conjugate conferred a significant, dose-dependent, neuroprotective effect against OGD/R- and serum starvation-induced cell death, and a robust cardioprotective effect against Doxorubicin-induced apoptotic cell death. Furthermore, it was shown that this peptide improved the mitochondrial functions of cells exposed to oxidative stress. In some assays an enhanced effect was obtained with this peptide conjugate compared to each of the parental compounds individually.

The present invention is further based in part on the design of a novel cyclic HN analog denoted c(D-Serl4-HN), having the amino acid sequence MAPAGASRLLLLTsEIDLPVKRRA (SEQ ID NO: 2,). This peptide also showed significant neuroprotective and cardioprotective effects against OGD/R- and serum starvation induced cell death in neuronal cell cultures, and against Doxorubicin induced cell death in myoblast cultures, and was also found beneficial in improving mitochondrial function of cells exposed to oxidative stress.

It was further found that both HUJInin and c(D-Serl4-HN) inhibited Erk 1/2 phosphorylation and stimulated AKT phosphorylation in neuronal cell cultures under OGD/R or serum starvation conditions. Without wishing to be bound by any theory or mechanism, it is hypothesized that activation of AKT phosphorylation and/or inhibition of Erk 1/2 phosphorylation contribute to the protective effect of the peptides of the invention.

Peptide analogs that include backbone cyclization and/or D-amino acids and or that are designed as pro-drugs by attaching specific moieties known in the art, have improved therapeutic properties such as metabolic stability, bioavailability, and improved pharmacokinetic and pharmacodynamic.

Peptides

As used herein "peptide" indicates a sequence of amino acids linked by peptide bonds. Peptides according to some embodiments of the present invention consist of 4-100, 5-70, 6-60, 7- 55, 7-50, 8-45, 10-40, 15-40 or 15-30 amino acids. Each possibility represents a separate embodiment of the present invention.

The term “amino acid” refers to compounds, which have an amino group and a carboxylic acid group, preferably in a 1,2- 1,3-, or 1,4- substitution pattern on a carbon backbone. Alpha- Amino acids are most preferred, and include the 20 natural amino acids (which are E-amino acids except for glycine) which are found in proteins, the corresponding D-amino acids, the corresponding N-methyl amino acids (methylated amino acids), side chain modified amino acids, the biosynthetically available amino acids which are not found in proteins (e.g., 4-hydroxy -proline, 5-hydroxy-lysine, citrulline, ornithine, canavanine, djenkolic acid, b-cyanolanine), and synthetically derived a-amino acids, such as amino-isobutyric acid, norleucine, norvaline, homocysteine and homoserine b- Alanine and g-amino butyric acid are examples of 1,3 and 1,4- amino acids, respectively, and many others as well known to the art.

Some of the amino acids used in this invention are those, which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and either sequential, divergent or convergent synthetic approaches to the peptide sequence are useful in this invention. Natural coded amino acids and their derivatives are represented by one-letter codes or three-letter codes according to IUPAC conventions. When there is no indication, the L isomer was used. The D isomers are indicated by “D” before the residue abbreviation or by a small letter in one-letter code, for example “s”, DSer and D-Ser designate the residue D-Serine.

Analogs and derivatives of the peptides are also within the scope of the present application.

The term “derivatives" of the peptides as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C- terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the peptide and do not confer toxic properties on compositions containing it. The term “derivatives” encompass also cyclic and backbone -cyclic peptides based on the amino acid sequences of the invention.

These derivatives may include, for example, aliphatic esters of the carboxyl groups, amides of the carboxyl groups produced by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed by reaction with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups), or O-acyl derivatives of free hydroxyl group (e.g., that of seryl or threonyl residues) formed by reaction with acyl moieties.

The term “analog” indicates a molecule which has the amino acid sequence according to the invention except for one or more amino acid changes. According to some embodiments, the analog comprises substitutions, deletions or additions of 1 or 2 amino acids. According to some embodiments, an analog comprises one or two conservative amino acid substitutions.

In some embodiments, an analog has at least about 70% identity to the sequence of the peptide of the invention, for example at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 99% identity to the sequence of the peptide of the invention. As used herein, the term "about", when referring to a measurable value is meant to encompass variations of +/-10%, more preferably +/- 5%, even more preferably +/- 1%, and still more preferably +/-0.1% from the specified value.

Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain e.g. aliphatic, aromatic, positively charged, negatively charged. One of skill will recognize that individual substitutions, deletions or additions to peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K), Histidine (H);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (L), Tyrosine (Y), Tryptophan (W).

Analogs according to the present invention may comprise also peptidomimetics. “Peptidomimetic” means that a peptide according to the invention is modified in such a way that it includes at least one non-coded residue or non-peptidic bond. Such modifications include, e.g., alkylation and more specific methylation of one or more residues, insertion of or replacement of natural amino acid by non-natural amino acids, replacement of an amide bond with another covalent bond. A peptidomimetic according to the present invention may optionally comprise at least one bond which is an amide -replacement bond such as urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond. The design of appropriate "analogs" may be computer assisted. Analogs are included in the invention as long as they remain pharmaceutically acceptable and their activity is not damaged. "Peptides" of the invention also include modified peptides (with amino acid substitutions, both conservative and non-conservative as described below) that have the same or improved activity as a wild-type or unmodified peptide.

Prodrugs comprising the peptides or peptide analogs of the present invention are also included within the scope of the present invention. The prodrugs may include, according to soe embodiments, derivatization of or conjugation to a functionalized amino acid of the peptide sequence, including but not limited to Y, S, R, T, E, D, K, H, W and combinations thereof.

Glycosylation of an amino acid residue of any of the peptides disclosed is also included in the scope of the invention.

Any Acyl group in the peptide sequence may be replaced with any alkyl or hetroalkyl group.

Any NH2 moiety may be replaced with another group, including OH, H, SH, glycosyl etc.

These optional modifications may be present alone or in combination with any additional modification to the sequence, including deletion, addition and substitution on 1-5 amnio acids in a given peptide sequence of 15-40 amino acids.

According to one aspect, the present invention provides a peptide comprising the sequence X1X2AGASRLLLLTX3EIDLX4 (SEQ ID NO: 5), wherein:

Xi is absent or Glutamine (Gin, Q);

X2 is absent or Proline (Pro, P);

X3 is selected from Glycine (Gly, G) and D-Serine (DSer, s); and

Proline (Pro, P); with the proviso that when X3 is Gly (G), Xi is Gin (Q).

According to additional aspect, the present invention provides a peptide comprising the sequence set forth in SEQ ID NO: 5, wherein:

Xi is absent or Glutamine (Gin, Q);

X2 is absent or Proline (Pro, P);

X3 is selected from Glycine (Gly, G) and D-Serine (D-Ser, s); and

Proline (Pro, P); provided that when X3 is Gly, Xi is Glutamine (Gin, Q), and when X3 is D-Ser, the peptide is cyclic.

According to some embodiments, the peptide is of 15-100 amino acids, 15-90 amino acids, 15-80 amino acids, 15-70 amino acids, 15-60 amino acids, 15-55 amino acids, 15-50 amino acids, 15-45 amino acids, 15-40 amino acids, 15-35 amino acids, 15-30 amino acids, 15-28 amino acids,

15-26 amino acids, 15-25 amino acids, 15-20 amino acids, 16-40 amino acids, 16-35 amino acids,

16-30 amino acids, 16-28 amino acids, 16-26 amino acids, 16-25 amino acids, 16-20 amino acids, 18-40 amino acids, 18-35 amino acids, 18-30 amino acids, 18-28 amino acids, 18-26 amino acids, 18-25 amino acids, 18-20 amino acids, 20-40 amino acids, 20-35 amino acids, 20-30 amino acids, 20-28 amino acids, 20-26 amino acids, 20-25 amino acids, 25-40 amino acids, 25-35 amino acids, 25-30 amino acids or 25-28 amino acids. Each possibility represents a separate embodiment of the invention.

In some embodiments, X3 in SEQ ID NO: 5 is Glycine (Gly, G). In some embodiments, X3 is glycine and the peptide is linear. In other embodiments, the peptide comprises at least one cyclization.

As used herein, the term "linear" refers to peptides that have a wholly linear sequence of amino acids with no cross-linking between non-adjacent amino acids.

In some embodiments, X2 in SEQ ID NO: 5 is Proline (Pro, P).

In some embodiments, Xi is Gin (Q), X2 is Pro (P), and X3 is Gly (G). In some embodiments, Xi is Gin (Q), X2 is Pro (P), X3 is Gly (G) and X4 is Pro (P). In some embodiments, the peptide comprises the amino sequence QPAGASRLLLLTGEIDLP (SEQ ID NO: 10).

In some embodiments, the peptide further comprises the amino acid sequence NAPVSIP (SEQ ID NO: 6). According to further embodiments, the peptide comprises the amino acid sequence NAPVSIPQ (SEQ ID NO: 7).

In some embodiments, the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7 is conjugated directly or indirectly through a linker or spacer to the N-terminus of the peptide set forth in SEQ ID NO: 5. In some embodiments, the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7 is conjugated directly to the N-terminus of the peptide set forth in SEQ ID NO: 5, via an amide bond, namely the carboxy terminal residue P or Q of SEQ ID NO: 6 or 7 respectively, is coupled to the terminal amine of Q, P or A residues of SEQ ID NO: 5. In some related embodiments, the peptide comprises the amino acid sequence

NAPVSIPQPAGASRLLLLTGEIDLP (SEQ ID NO: 9).

In some embodiments, the peptide comprises an amino acid residue that allows chemical labeling. In some embodiments, the amino acid residue is Tyrosine (Tyr, Y). In some embodiments, said amino acid residue is conjugated to the N-terminus of the peptide. In some embodiments, the peptide comprises or consists of the amino acid sequence

YNAPVSIPQPAG ASRLLLLTGEIDLP (SEQ ID NO: 1).

The terms "labeling" and “chemical labeling”, as used interchangeably herein, denote an attachment or incorporation of one or more detectable markers into an amino acid residue or a peptide. The term “detectable marker”, as used herein, refers to any atom or compound that comprises one or more appropriate chemical substances which directly or indirectly generate a detectable compound or signal in a chemical or physical reaction. In some embodiments, the detectable marker is radioactive.

In some embodiments, the peptide comprises a modified C-terminus and/or a modified N- terminus. In some embodiments, the C-terminus of the peptide is amidated. In some embodiments, the N-terminus of the peptide is acetylated. In some embodiments, the C-terminus of the peptide is amidated and the N-terminus of the peptide is acetylated.

In some embodiments, the peptide of the invention comprises the sequence set forth in SEQ ID NO: 5, wherein:

Xi is absent or Glutamine (Gin, Q);

X2 is absent or Proline (Pro, P);

X3 is D-Serine (DSer, s); and

Proline (Pro, P).

In some embodiments, the peptide is a cyclic peptide. In some embodiments, the peptide comprises at least one cyclization.

In some embodiments, the peptide comprises the sequence AGASRLLLLTsEIDL (SEQ ID NO: 4). In some embodiments, the peptide is a cyclic peptide comprising the sequence set forth in SEQ ID NO: 4. In some embodiments, the peptide comprises or consists of the amino acid sequence PAGASRLLLLTsEIDLP (SEQ ID NO: 3). In some embodiments, the peptide is a cyclic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3.

In some embodiments, the peptide comprises the sequence set forth in SEQ ID NO: 5 wherein X4 is a 2-6 amino acid sequence comprising Proline (Pro, P). In some embodiments, X4 is the amino acid sequence PVKRRA (SEQ ID NO: 11). In some embodiments, X4 is the amino acid sequence PVKRR (SEQ ID NO: 12). In some embodiments, X4IS the amino acid sequence PVKR (SEQ ID NO: 13). In some embodiments, X4 is the amino acid sequence PVK (SEQ ID NO: 14).

In some embodiments, the peptide comprises or consists of the amino acid sequence MAPAGASRLLLLTsEIDLPVKRRA (SEQ ID NO: 2). In some embodiments, the peptide is a cyclic peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2.

In some embodiments, the cyclic peptide comprises the amino acid sequence set forth in SEQ ID NO: 4 and has a structure according to Formula (I):

Formula (I) wherein:

X- Y -Z is a bridge selected from urea bridge, thiourea bridge, amide bridge, disulfide bridge and guanidino group; Ri and R2 are independently selected from hydrogen and the side chain of an amino acid; and m and n are each independently an integer of between 2 to 6.

The different possibilities of the substituents X, Y and Z in the bridge X-Y -Z of Formula (I) are presented in Table 1. Table 1. Types of X-Y-Z bridges in formula (I)

The various possibilities of combinations of m and n variables are specified in Table 2.

Table 2. Permutations of ring size according to m & n

According to some embodiments, the peptide of the invention further comprises a capping moiety coupled to the N-terminus. According to other embodiments, the peptide further comprises at least one capping moiety coupled to the C -terminus. According to some embodiments, the at least one capping moiety increases the solubility, permeability or stability of the peptide. According to some embodiments, the at least one capping moiety increases the stability of the peptide under physiological conditions.

Any suitable capping moiety known in the art can be used. In some embodiments, the capping moiety comprises an acyl group. In some embodiments, the capping moiety is an acetyl group. In some embodiments, the capping moiety is a primary or secondary amine (-NH2 or -NHR, wherein R is an organic moiety, such as an alkyl group). According to some specific embodiments, the capping moiety is selected from acetyl, amine, alkyl and heteroalkyl. Each possibility represents a separate embodiment of the present invention.

The procedures utilized to construct peptide compounds of the present invention generally rely on the known principles of peptide synthesis. However, it will be appreciated that accommodation of the procedures to the specific sequences of the present invention may be required. Examples of several synthetic procedures are given below.

Solid phase peptide synthesis procedures are well known in the art and further described in "Solid-Phase Synthesis: A Practical Guide", Ed. Steven A. Kates and Fernando Albericio, CRC Press; 1st Edition (2000). A skilled artesian may synthesize any of the peptides of the present invention by using an automated peptide synthesizer using standard chemistry such as, for example, t-Boc or Fmoc chemistry. The methods of synthesis can include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, classical solution synthesis.

In particular embodiments, the peptides of the invention are synthesized according to solid phase peptide synthesis (SPPS) principles, utilizing standard Fmoc (9 -fluorenylmethoxy carbonyl) chemistry protocols.

Coupling of the amino acids in solid phase peptide chemistry can be achieved by means of a coupling agent such as but not limited to dicyclohexycarbodiimide (DCC), bis(2-oxo-3-- oxazolidinyl) phosphinic chloride (BOP-C1), benzotriazolyl-N-oxytrisdimethyl- aminophosphonium hexafluoro phosphate (BOP), 1-oxo-l-chlorophospholane (Cpt-Cl), hydroxybenzotriazole (HOBT), or mixtures thereof. The use of additional coupling reagents including, but not limited to: coupling reagents such as PyBOP (Benzotriazole-l-yl--oxy-tris-pyrrolidino-phosphonium hexafluorophosphate), PyBrOP (Bromo-tris-pyrrolidino-phosphonium hexafluoro-phosphate), HBTU (2-(lH- Benzotriazole-l-yl)-l, 1,3,3- tetramethyluronium hexafluoro-phosphate), TBTU (2-(lH- Benzotriazole-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate), may be also utilized for synthesizing the peptide compounds of the present invention.

Additional coupling chemistries may be used, such as pre-formed urethane-protected N- carboxy anhydrides (UNCA'S), pre-formed acyl halides most preferably acyl chlorides.

Such coupling may take place at room temperature and also at elevated temperatures, in solvents such as toluene, DCM (dichloromethane), DMF (dimethylformamide), DMA (dimethylacetamide), NMP (N-methyl pyrrolidinone), dioxane, tetrahydrofuran, diglyme and 1,3 dichloropropane, or mixtures of the above.

Synthetic peptides can be purified by preparative high performance liquid chromatography and the composition of which can be confirmed via amino acid sequencing by methods known to one skilled in the art. Some of the peptides of the invention, which include only natural amino acids, may be prepared using recombinant DNA techniques known in the art.

The peptides of the invention can be used in the form of pharmaceutically acceptable salts. As used herein the term “salts” refers to both salts of carboxyl groups and to acid addition salts of amino or guanido groups of the peptide molecule. The term "pharmaceutically acceptable" means suitable for administration to a subject, e.g., a human. For example, the term “pharmaceutically acceptable” can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Pharmaceutically acceptable salts include those salts formed with free amino groups such as salts derived from non-toxic inorganic or organic acids such as acetic acid, citric acid or oxalic acid and the like, and those salts formed with free carboxyl groups such as salts derived from non-toxic inorganic or organic bases such as sodium, calcium, potassium, ammonium, calcium, ferric or zinc, isopropylamine, triethylamine, procaine, and the like.

The peptides of the present invention can comprise at least one cyclization. According to some embodiments, the peptides comprise at least two cyclizations. Thus, the peptides of the present invention may be monocyclic, bi-cyclic, tri-cyclic or even tetra-cyclic (with four cyclizations). Each possibility represents a separate embodiment of the present invention. In some embodiments, the cyclization of the peptide is selected from the group consisting of side-chain to side-chain (e.g. disulfide bond between two Cysteine residues); end to end; backbone to backbone; backbone to end.

Cyclization of peptides has been shown to be a useful approach in developing diagnostically and therapeutically useful peptidic and peptidomimetic agents. Cyclization of peptides reduces the conformational freedom of these flexible, linear molecules, and often results in higher receptor binding affinities by reducing unfavorable en tropic effects. Because of the more constrained structural framework, these agents are more selective in their affinity to specific receptor cavities. By the same reasoning, structurally constrained cyclic peptides confer greater stability against the action of proteolytic enzymes (Humphrey, et al, 1997, Chem. Rev., 2243-2266).

Methods for cyclization can be classified into cyclization by the formation of an amide bond between the N-terminal and the C-terminal amino acid residues, and cyclizations involving the side chains of individual amino acids. The latter method includes the formation of disulfide bridges between two ω-thio amino acid residues (cysteine, homocysteine), the formation of lactam bridges between glutamic/aspartic acid and lysine residues, the formation of lactone or thiolactone bridges between amino acid residues containing carboxyl, hydroxyl or mercapto functional groups, the formation of thioether or ether bridges between the amino acids containing hydroxyl or mercapto functional groups and other special methods. Lambert, et al, reviewed variety of peptide cyclization methodologies (J. Chem. Soc. Perkin Trans., 2001, 1:471-484).

Backbone cyclization is a general method by which conformational constraint is imposed on peptides. In backbone cyclization, atoms in the peptide backbone (N and/or C) are interconnected covalently to form a ring. Backbone cyclized analogs are peptide analogs cyclized via bridging groups attached to the alpha nitrogens or alpha carbonyl of amino acids. In general, the procedures utilized to construct such peptide analogs from their building units rely on the known principles of peptide synthesis; most conveniently, the procedures can be performed according to the known principles of solid phase peptide synthesis. During solid phase synthesis of a backbone cyclized peptide the protected building unit is coupled to the N-terminus of the peptide chain or to the peptide resin in a similar procedure to the coupling of other amino acids. After completion of the peptide assembly, the protective group is removed from the building unit’ s functional group and the cyclization is accomplished by coupling the building unit’s functional group and a second functional group selected from a second building unit, a side chain of an amino acid residue of the peptide sequence, and an N-terminal amino acid residue.

Backbone cyclization is achieved by covalently connecting at least one amino acid residue in the helix sequence, which was substituted with an N^α-ω-functionalized or an C“-ω- functionalized derivative of amino acid residue, with a moiety selected from the group consisting of: another N^α-ω-functionalized or an C^α-ω-functionalized derivative of amino acid residue, with the side chain of an amino acid in the peptide’s sequence, or with one of the peptide terminals. Any covalent bond may be used to connect the anchoring positions of the peptide sequence using backbone cyclization.

As used interchangeably herein, the terms "backbone cyclic peptide" and “backbone cyclized peptide”, “backbone cyclic derivative” refer to a sequence of amino acid residues wherein at least one nitrogen or carbon of the peptide backbone is joined to a moiety selected from another such nitrogen or carbon, to a side chain or to one of the termini of the peptide. It should therefore be understood that a peptide having a structure according to Formula (I) is a backbone cyclic peptide. However, it is to be understood that the stmctures provided herein are non-limiting examples. The exact position and nature of the connecting building units, as well as the chemical bridge type can be modified in order to further improve the activity of the compounds.

The uniqueness of the backbone cyclic peptide approach provides both utilization of the right sequence in its specific active conformation while preventing peptidases from the biological fluids in the surrounding vicinity to degrade these compounds. The size and chemistry of the bridge is the key for obtaining these achievements of the novel molecules.

In general, the procedures utilized to construct backbone cyclic molecules and their building units rely on the known principles of peptide synthesis and peptidomimetic synthesis; most conveniently, the procedures can be performed according to the known principles of solid phase peptide synthesis. Some of the methods used for producing backbone cyclized peptides and their building units are disclosed in US Patent Nos.: 5,811,392; 5,874,529; 5,883,293; 6,051,554; 6,117,974; 6,265,375, 6,355613, 6,407059, 6,512092 and international applications WO 95/33765; WO 97/09344; WO 98/04583; WO 99/31121; WO 99/65508; WO 00/02898; WO 00/65467 and WO 02/062819.

According to some embodiments, the cyclic peptide comprises at least one N^α-w- functionalized derivative of amino acid residue (building unit, BU). According to some embodiments, the cyclic peptide comprises at least two BUs. According to some embodiments, the cyclic peptide is a backbone cyclized peptide. According to some embodiments, the peptides are cyclized via backbone cyclization with N-alkylation.

A "building unit" (BU) indicates a N^α-ω-functionalized or a C^α-ω-functionalized derivative of amino acids. Use of such building units permits different length and type of linkers and different types of moieties to be attached to the scaffold. This enables flexible design and easiness of production using conventional and modified solid-phase peptide synthesis methods known in the art.

According to some embodiments, the peptide sequence is cyclized by covalently connecting one N^α-ω-functionalized derivative of amino acid residue added to the sequence, or a substituted amino acid residue in the sequence, with another N^α-ω-functionalized derivative of amino acid residue in the sequence.

Any covalent bond may be used to connect the anchoring positions of the peptide sequence using backbone cyclization.

According to some embodiments, the backbone cyclic peptide comprises at least one modified terminal, including but not limited to an ami dated C-terminus and an acylated N- terminus.

According to some embodiments, the peptides of the invention further comprise a permeability enhancing moiety.

As used herein, “permeability" refers to the ability of an agent or substance to penetrate, pervade, or diffuse through a barrier, membrane, or a skin layer. A “cell permeability moiety”, a “permeability enhancing moiety” or a “cell-penetration moiety” refers to any molecule known in the art which is able to facilitate or enhance penetration of molecules through membranes. Non- limitative examples include: hydrophobic moieties such as lipids, fatty acids, steroids and bulky aromatic or aliphatic compounds; hydrophilic moieties such as Arginine residues or guanidino- containing moieties; moieties which may have cell-membrane receptors or carriers, such as steroids, vitamins and sugars, natural and non-natural amino acids and transporter peptides.

The compounds/peptides disclosed herein may have chiral centers. All enantiomeric, diastereomeric, and racemic forms are included in the present invention. Many geometric isomers of double bonds and the like can also be present in the compounds disclosed herein, and all such stable isomers are contemplated in the present invention. The compounds/peptides disclosed herein may have N-Methylated peptide bond and any other peptide bond surrogates replacing one or more peptide bonds.

A popular method that intends to improve the drug-like properties (DLPs) of peptides is the prodrug approach. In this approach, the prodrug is a poorly active or inactive compound containing the parental drug that undergoes some in vivo biotransformation through chemical or enzymatic cleavage. The method attempts to deliver of the active compound to its target overcoming pharmacokinetic, pharmacodynamic and toxicology challenges without permanently altering the pharmacological properties of the parental drug.

According to some embodiments, the peptides of the present invention can be chemically modified or administered as prodrugs. According to some embodiments, said prodrugs have improved oral bioavailabilty compared to the parental peptides. According to some embodiments, said prodrugs are suitable for oral administration. Modification of peptides for in vivo use is well known in the art. For example, Simplicio et al. (Molecules, 2008, 13(3), 519-547) review the published strategies for the production of prodrugs of amines. Processes for the preparation of peptide -based prodrugs are also disclosed in WO 2019/058367, WO 2019/058365 and WO 2019/058374.

The term “prodrug” as used herein refers to an inactive or relatively less active form of an active agent that becomes active through one or more metabolic processes in a subject. Accordingly, “prodrug” is a precursor substance that is chemically or biochemically metabolized after administration to function as an effective drug.

The compounds/peptides/prodrugs of the present invention can be formulated into various pharmaceutical forms for purposes of administration. Pharmaceutical composition of interest may comprise at least one additive selected from a disintegrating agent, binder, flavoring agent, preservative, colorant and a mixture thereof, as detailed for example in "Handbook of Pharmaceutical Excipients"; Ed. A. H. Kibbe, 3rd Ed., American Pharmaceutical Association, USA. For example, a compound of the invention, or its salt form or a stereochemically isomeric form, can be combined with a pharmaceutically acceptable carrier. Such a carrier can depend on the route of administration, such as enteral or parenteral injection. A "carrier" as used herein refers to a non-toxic solid, semisolid or liquid filler, diluent, vehicle, excipient, solubilizing agent, encapsulating material or formulation auxiliary of any conventional type, and encompasses all of the components of the composition other than the active pharmaceutical ingredient. The carrier may contain additional agents such as wetting or emulsifying agents, or pH buffering agents. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary. For example, in preparing the compositions in oral dosage form, media such as water, glycols, oils, alcohols can be used in liquid preparations such as suspensions, syrups, elixirs, and solutions. Alternatively, solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents can be used, for example, in powders, pills, capsules or tablets.

The pharmaceutically acceptable excipient(s) useful in the composition of the present invention are selected from but not limited to a group of excipients generally known to persons skilled in the art e.g. diluents such as lactose (Pharmatose DCL 21), starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, dibasic calcium phosphate, sucrose-based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, inositol, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, and bentonite; disintegrants; binders; fillers; bulking agent; organic acid(s); colorants; stabilizers; preservatives; lubricants; glidants/antiadherants; chelating agents; vehicles; bulking agents; stabilizers; preservatives; hydrophilic polymers; solubility enhancing agents such as glycerin, various grades of polyethylene oxides, transcutol and glycofiirol; tonicity adjusting agents; pH adjusting agents; antioxidants; osmotic agents; chelating agents; viscosifying agents; wetting agents; emulsifying agents; acids; sugar alcohol; reducing sugars; non-reducing sugars and the like, used either alone or in combination thereof. The disintegrants useful in the present invention include but not limited to starch or its derivatives, partially pregelatinized maize starch (Starch 1500®), croscarmellose sodium, sodium starch glycollate, clays, celluloses, alginates, pregelatinized corn starch, crospovidone, gums and the like used either alone or in combination thereof. The lubricants useful in the present invention include but not limited to talc, magnesium stearate, calcium stearate, sodium stearate, stearic acid, hydrogenated vegetable oil, glyceryl behenate, glyceryl behapate, waxes, Stearowet, boric acid, sodium benzoate, sodium acetate, sodium chloride, DL-leucine, polyethylene glycols, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate and the like used either alone or in combination thereof. The anti- adherents or glidants useful in the present invention are selected from but not limited to a group comprising talc, corn starch, DLleucine, sodium lauryl sulfate, and magnesium, calcium and sodium stearates, and the like or mixtures thereof. In another embodiment of the present invention, the compositions may additionally comprise an antimicrobial preservative such as benzyl alcohol. In an embodiment of the present invention, the composition may additionally comprise a conventionally known antioxidant such as ascorbyl palmitate, butylhydroxyanisole, butylhydroxy toluene, propyl gallate and/or tocopherol. In another embodiment, the dosage form of the present invention additionally comprises at least one wetting agent(s) such as a surfactant selected from a group comprising anionic surfactants, cationic surfactants, non-ionic surfactants, zwitterionic surfactants, or mixtures thereof. The wetting agents are selected from but not limited to a group comprising oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodium lauryl sulfate and the like, or mixtures thereof. In yet another embodiment, the dosage form of the present invention additionally comprises at least one complexing agent such as cyclodextrin selected from a group comprising but not limited to alpha- cyclodextrin, beta-cyclodextrin, betahydroxy-cyclodextrin, gammacyclodextrin, and hydroxypropyl beta-cyclodextrin, or the like. In yet another embodiment, the dosage form of the present invention additionally comprises of lipid(s) selected from but not limited to glyceryl behenate such as Compritol® AT0888, Compritol® ATO 5, and the like; hydrogenated vegetable oil such as hydrogenated castor oil e.g. Lubritab®; glyceryl palmitostearate such as Precirol® ATO 5 and the like, or mixtures thereof used either alone or in combination thereof. It will be appreciated that any given excipient may serve more than one function in the compositions according to the present invention.

For parenteral compositions, the carrier can comprise sterile water. Other ingredients may be included to aid in solubility. Injectable solutions can be prepared where the carrier includes a saline solution, glucose solution or mixture of both. Injectable suspensions can also be prepared. In addition, solid preparations that are converted to liquid form shortly before use can be made. For percutaneous administration, the carrier can include a penetration enhancing agent or a wetting agent.

It can be advantageous to formulate the compositions of the invention in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form” refers to physically discrete units suitable as unitary dosages, each unit containing a pre-determined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the chosen carrier.

Apart from other considerations, the fact that the novel active ingredients of the invention are peptides, peptide analogs or peptidomimetics, dictates that the formulation be suitable for delivery of these types of compounds. Although in general peptides are less suitable for oral administration due to susceptibility to digestion by gastric acids or intestinal enzymes. According to some embodiments, the route of administration of the peptides of the invention is oral administration. According to some embodiments, the peptides and cyclic peptides of the invention are modified as prodrugs and administered orally.

Other routes of administration are intra-articular, intravenous, intramuscular, subcutaneous, topical, transdermal, intradermal, or intrathecal. According to some embodiments, topical administration comprises ocular administration.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

For injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants for example polyethylene glycol are generally known in the art.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the variants for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the peptide and a suitable powder base such as lactose or starch.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredients in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable natural or synthetic carriers are well known in the art (Pillai et al, 2001, Curr. Opin. Chem. Biol. 5, 447). Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds, to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The compounds of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

In some embodiments, the pharmaceutical compositions of the invention are formulated for local administration. In some embodiments, the compositions may be formulated for topical, subcutaneous or transdermal administration. According to some embodiments, the pharmaceutical composition for topical and/or transdermal administration is formulated as an ointment, cream, lotion, or spray. For example, the pharmaceutical composition may be formulated in a cream for topical application to the skin (e.g., for alopecia), in a powder for chaffing (e.g., for dermatitis), in a liquid, in a dry formulation, and the like. Other formulations will be readily apparent to one skilled in the art. Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prevent, alleviate or ameliorate symptoms of a disease of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

Therapeutic use

Mitochondria are the primary energy source of cells and play a role in a number of important cellular functions, including inter alia ATP production, oxidative energy metabolism, amino acid biosynthesis, fatty acid oxidation, steroid metabolism, and apoptosis. Mitochondrial membrane polarization is essential for ATP production and maintenance of the calcium level.

According to one aspect, the peptides, derivatives or analogs thereof are provided for use in improving mitochondrial function of cells. According to further embodiments, the peptides, derivatives or analogs thereof are provided for use in improving mitochondrial function of cells under oxidative stress. According to some embodiments, the peptides, derivatives or analogs thereof are provided for use in improving mitochondrial function of cells in a subject in need thereof.

The term "mitochondrial function" as used herein includes any cellular activity carried out by mitochondria. The term “improving mitochondrial function” refers to either restoring at least one indicator of mitochondrial function to a normal level in cells having impaired mitochondrial function, or increasing at least one indicator of mitochondrial function to a level beyond normal levels in cells having normal mitochondrial function.

Measurable indicators for evaluating mitochondrial function are known in the art. For example, mitochondrial functionality can be assessed in vitro by e.g., monitoring a mitochondrial parameter such as mitochondrial membrane potential (MMP), O₂ consumption, ATP production and mitochondrial Ca²⁺ uptake.

In one aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the peptide of the invention in all embodiments thereof, for use in treating a disease, disorders or condition associated with mitochondrial dysfunction, or a disease, disorder or condition that may benefit from improving mitochondrial function. In another aspect, the present invention provides a method for treating a disease, disorders or condition associated with mitochondrial dysfunction, or a disease, disorder or condition that may benefit from improving mitochondrial function, the method comprising administering a therapeutically effective amount of the pharmaceutical composition described hereinabove in all embodiments thereof.

As used herein, the term “therapeutically effective amount” means an amount of a compound effective to prevent, alleviate or ameliorate symptoms of a disease of the subject being treated.

The term “Mitochondrial dysfunction” includes any failure or deficiency of mitochondria or mitochondrial genes to carry out a mitochondrial function. Typically, mitochondrial dysfunction is due to an oxidative stress related process such as hypoxia/ischemia/reperfusion injury. In some embodiments, mitochondrial dysfunction is associated with cellular calcium overload. Under pathological conditions of cellular calcium overload, particularly in association with oxidative stress (e.g., in stroke), mitochondrial calcium uptake triggers pathological states that lead to cell death.

The term "associated with mitochondrial dysfunction" as used herein refers to diseases, disorders or conditions that are caused by mitochondrial dysfunction as well as diseases, disorders or conditions that are not caused by mitochondrial dysfunction, but involve mitochondrial dysfunction as one of the manifestations of the disease, disorder or condition. In some embodiments, "associated with mitochondrial dysfunction" refers to diseases, disorders or conditions in which improvement of mitochondria function may results in a clinical beneficial effect.

In some embodiments, the disease, disorder or condition associated with mitochondrial dysfunction is selected from the group consisting of a genetic disease or disorder, an ischemia related disease or disorder, a neurodegenerative disease or disorder, a cancer, a cardiovascular disease or disorder, cardiomyopathy, an autoimmune disease, an inflammatory disease, a fibrotic disease, an age-related disease or disorder, and a disease or associated with complications of birth. In some embodiments, the disease, disorder or condition is selected from the group consisting of an ischemia related disease or disorder, a neurodegenerative disease or disorder, a cancer, a cardiovascular disease or disorder, an autoimmune disease, an inflammatory disease, a fibrotic disease, and an aging disease or disorder. In further embodiments, the disease, disorder or condition is selected from the group consisting of an ischemia related disease or disorder, a neurodegenerative disease or disorder, or a cardiovascular disease or disorder. Each possibility represents a separate embodiment of the present invention.

In some embodiments, the disease, disorder or condition is an ischemia related disease or disorder. In some embodiments, the ischemia related disease or disorder is selected from the group consisting of ischemic stroke, cerebral ischemic reperfusion, hypoxia ischemic encephalopathy, acute coronary syndrome, myocardial ischemia and reperfusion, a myocardial infarction, a liver ischemia-reperfusion injury, an ischemic injury-compartmental syndrome, a blood vessel blockage, wound healing (e.g., an acute wound or a chronic wound; a cut, laceration, compression wound, bum wound (e.g., chemical, heat or flame, wind, or sun bum), or a wound resulting from a medical or surgical intervention), spinal cord injury, sickle cell disease, and reperfusion injury of a transplanted organ. Each possibility represents a separate embodiment of the present invention.

In some embodiments, the ischemia related disease or disorder is a cerebral ischemic reperfusion. Ischemia-reperfusion injury is a common feature of ischemic stroke, which occurs when blood supply is restored after a period of ischemia. Mitochondrial dysfunction has been regarded as one of the hallmarks of ischemia-reperfusion induced neuronal death. In some embodiments, the ischemia related disease or disorder is an ischemic stroke. In some embodiments, the ischemic stroke is a focal or global ischemic stroke. In some embodiments, the ischemic stroke is a focal ischemic stroke. Brain injury after focal ischemia evolves along two different pathophysiologies, depending on the severity of the primary flow reduction and the dynamics of post-ischemic recirculation. In permanent and gradually reversed focal ischemia as after thromboembolic occlusion, primary core injury is irreversible but the expansion of the core into the penumbra can be alleviated by hemodynamic and drugs. Such alleviation can only be achieved within 2-4 hours after the onset of ischemia because untreated core injury expands to near maximum size during this interval. In promptly reversed transient ischemia as after mechanical vascular occlusion, primary core injury may recover but a secondary delayed injury (aponecrosis) evolves after a free interval of as long as 6 to 12 hours.

Today, there are no drugs in the clinic that target and confer neuroprotection to the penumbra of the brain ischemic stroke core. In the clinic the approach is indirect, using thrombolytic drugs in order to renew the blood flow to the stroke area. An IV injection of recombinant tissue plasminogen activator (tPA), also called alteplase (Activase) is the gold standard treatment for ischemic stroke. An injection of tPA is usually given through a vein in the arm with the first three hours. Sometimes, tPA can be given up to 4-6 hours after the starting of the stroke symptoms. However, renewal of blood flow does not prevent the neuronal cell death which is expanding around the ischemic core.

In some embodiments, the ischemia related disease or disorder is related to a myocardial ischemia. In some embodiments, the myocardial ischemia related disease or disorder is coronary heart disease (CHD). Coronary heart disease (CHD), also called coronary artery disease or ischemic heart disease, is characterized by an inadequate supply of oxygen-rich blood to the heart muscle (myocardium) because of narrowing or blocking of a coronary artery by fatty plaques (atherosclerosis).

In some embodiments, the myocardial ischemia related disease or disorder is acute myocardial infarction (AMI) ("heart attack"). AMI is an irreversible damage of myocardial tissue caused by prolonged ischemia and hypoxia. This most commonly occurs when a coronary artery becomes occluded following the rupture of an atherosclerotic plaque, which then leads to the formation of a blood clot (coronary thrombosis).

In some embodiments, the myocardial ischemia related disease or disorder is stable angina or unstable angina. Coronary artery disease is almost always due to atheromatous narrowing and subsequent occlusion of the vessel. A mature plaque is composed of two constituents, each associated with a particular cell population. The lipid core is mainly released from necrotic “foam cells” — monocyte derived macrophages, which migrate into the intima and ingest lipids. The connective tissue matrix is derived from smooth muscle cells, which migrate from the media into the intima, where they proliferate and change their phenotype to form a fibrous capsule around the lipid core. When a plaque produces a >50% diameter stenosis (or >75% reduction in cross sectional area), reduced blood flow through the coronary artery, causing hypoxia/ischemia, during exertion may lead to angina. Acute coronary events usually arise when thrombus formation follows disruption of a plaque. Intimal injury causes denudation of the thrombogenic matrix or lipid pool and triggers thrombus formation. In acute myocardial infarction, occlusion is more complete than in unstable angina, where arterial occlusion is usually subtotal. Downstream embolism of thrombus may also produce micro infarcts. In some embodiments, the disease, disorder or condition is a neurodegenerative disease or disorder. In some embodiments, the neurodegenerative disease or disorder is selected from the group consisting of dementia, Friedrich's ataxia, amyotrophic lateral sclerosis (ALS), mitochondrial myopathy, MELAS (encephalopathy, lactic acidosis, stroke), myoclonic epilepsy with ragged red fibers (MERFF), epilepsy, Parkinson's disease, Alzheimer's disease, and Huntington's Disease. Each possibility represents a separate embodiment of the present invention.

In some embodiments, the neurodegenerative disease or disorder is Alzheimer's disease (AD). Oxidative stress is one of the mechanisms that have been associated with Alzheimer's disease b-amyloid’s aggregates are believed to be the principal component of senile plaques which have been found in the brains of AD patients. The abnormal deposition of b-amyloid in the brain tissue leads to a high diversity of neurotoxic mechanisms, including mitochondrial deficits, oxidative stress, and excitotoxicity by interactions with putative Ab-binding receptors. These neurotoxic effects cause the neuronal necrotic and apoptotic cell death and synaptic dysfunction, which affect memory. Humanin (HN) was originally found in the occipital lobe of an autopsied Alzheimer's disease (AD) patient. Humanin was shown to inhibit neuronal cell death induced by enforced expression of familial AD-related genes. Humanin also protected neurons from being killed by toxic amyloid betas in vitro. In addition, neuronal dysfunction-associated dementia of mice caused by muscarinic receptor antagonists and intracranially injected toxic amyloid betas was ameliorated by Humanin therapy. Without wishing to be bound by any theory or mechanism, it is hypothesized that the HN analogs of the invention may inhibit the progression of AD-related dementia by inhibiting neuronal cell death and dysfunction, a protective effect that may be partially attributed to improving mitochondrial function of neuronal cells.

In some embodiments, the neurodegenerative disease or disorder is amyotrophic lateral sclerosis (ALS). The pathology of ALS is degeneration and loss of motor neurons in the anterior horns and motor nuclei of spinal cord, associated with gliosis, microglial activation, and cytoplasmic deposits of TDP-43, ubiquitin, and SOD1. Because of loss of lower motor neurons, muscles undergo denervation and atrophy. Possible pathogenic mechanisms underlying motor neuron degeneration include RNA toxicity, excitotoxicity, disruption of proteostasis, defective axonal transport, oxidative stress, and mitochondrial dysfunction. ALS associated mitochondrial dysfunction occurs at multiple levels, including defective oxidative phosphorylation, production of reactive oxygen species (ROS), impaired calcium buffering capacity and defective mitochondrial dynamics. Humanin peptide was shown to exhibit neuroprotective activity against toxicity by familial ALS-related mutant superoxide dismutase (SOD1).

In some embodiments, the disease, disorder or condition is a cardiovascular disease or disorder and or cardiomyopathy. In some embodiments, the cardiovascular disease or disorder is selected from the group consisting of coronary heart disease, myocardial infarction, atherosclerosis, high blood pressure, cardiac arrest, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, congestive heart failure, arrhythmia, stroke, deep vein thrombosis, and pulmonary embolism.

In some embodiments, the disease, disorder or condition is cardiomyopathy. In further embodiments, the cardiomyopathy is Anthracycline-induced cardiomyopathy (AIC). The histological pathophysiology of AIC is characterized by myocardial damage due to proteolysis, necrosis, apoptosis, and fibrosis. For example, Doxorubicin (Dox) causes increased cardiac apoptosis and fibrosis leading to adverse effects on cardiac function in cancer patients, thereby limiting its effectiveness in chemotherapy. It was found that mitochondria play a key role in the pathogenesis of AIC (M. Alessandra, et al, Frontiers in cardiovascular medicine 7 (2020)). In some embodiments, the AIC is Doxorubicin-induced cardiomyopathy. In some embodiments, the AIC is Daunorubicin-induced cardiomyopathy. In some embodiments, the AIC is Epirubicin- induced cardiomyopathy. In some embodiments, the AIC is Idarubicin-induced cardiomyopathy.

In some embodiments, the disease, disorder or condition is an age-related disease or disorder. In some embodiments, the age-related disease or disorder is age-related macular degeneration (AMD). AMD pathology is characterized by degeneration involving the retinal photoreceptors, retinal pigment epithelium, and Bruch's membrane, as well as, in some cases, alterations in choroidal capillaries. Substantive evidence demonstrates the contribution of mitochondrial dysfunction in the etiology and pathogenesis of AMD. Recently, extensive characterization of Mitochondrial-Derived Peptides (MDPs) including Humanin has revealed their cytoprotective role in several eye diseases, including AMD.

In some embodiments, the disease, disorder or condition is an inflammatory and/or fibrotic disease or disorder. In some embodiments, the inflammatory and/or fibrotic disease or disorder is atherosclerosis. From a clinical standpoint, a spectrum of acute coronary events may follow atherosclerotic plaque rupture. The severity of the resulting coronary event appears to be related to the change in blood flow around the site of plaque disruption. In those cases where blood flow is essentially unaffected, plaque rupture may result only in asymptomatic progression of the atherosclerotic lesion. If blood flow is reduced (hypoxia), a change in the pattern of angina may result, producing unstable angina. If complete vessel occlusion follows plaque rupture (ischemia), acute MI results.

In some embodiments, the disease, disorder or condition is diabetes type 1. Type 1 diabetes is the result of a chronic inflammatory process that causes elimination of insulin-producing beta cells, resulting in insulin deficiency and hyperglycemia. Pancreatic beta-cell apoptosis is important in the pathogenesis and potential treatment of type 1 diabetes mellitus. Humanin was shown to reduce apoptosis induced by cytokine treatment. It was shown that Humanin treatment decreases cytokine-induced apoptosis in beta-cells in vitro and improved glucose tolerance and onset of diabetes in NOD mice in vivo (Hoang, P. T., et al. Metabolism: clinical and experimental 59.3 (2010): 343). It is therefore suggested that the HN analogs of the present invention may be useful for islet protection and survival in a spectrum of diabetes-related therapeutics.

In some embodiments, the peptides of the invention are neuroprotective agents. In some embodiments, the peptides of the invention are cardioprotective agents.

The term “neuroprotective” as used herein refers to a capability of maintaining the survival and activity of neuronal cells, or maintaining or even recovering their neuronal functions, or relieving or alleviating one or more factors that may lead to neuronal damage. The term “neuroprotective” may encompass preventing the neuronal cells from being damaged in a subject and/or treating the neuronal damage after its emergence in the subject.

The term "cardioprotective" refers to a capability of preventing or attenuating myocardial dysfunction (i.e. cardiomyopathy and / or congestive heart failure). A cardioprotective agent may, for example, prevent or reduce damage caused by oxidative stress. In some embodiments, the term "cardioprotective" refers to the ability to protect myocardium during ischemia.

In this regard, the term “preventing” includes reducing the severity/intensity of, or initiation of, the cell damage.

Extracellular signal-regulated protein kinase (ERK)l/2 is a mitogen-activated protein kinase (MAPK) family protein with typical cascade signaling characteristics and serves an important role in signal transduction pathways and the function of transcription factors. The ERK cascade functions in cellular proliferation, differentiation, and survival. In some embodiments, the peptides of the invention inhibit Erkl/2 phosphorylation. In some embodiments, the peptides of the invention inhibit Erkl/2 hyperphosphorylation in cells under oxidative stress.

Protein kinase B (PKB), also known as Akt, is a serine/threonine-specific protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. In some embodiments, the peptides of the invention stimulate Akt phosphorylation.

The pharmaceutical composition of the invention can be administered by any acceptable route. In some embodiments, the administration is enteral or parenteral administration.

In some embodiments, administration is selected from oral, intra-articular, intravenous, intramuscular, subcutaneous, topical, transdermal, intradermal, intrathecal, and intranasal administration. According to some embodiments, topical administration comprises ocular administration. In some embodiments, administration is by inhalation.

The precise dosage and frequency of administration depends on the particular compound of the invention being used, as well as the particular condition being treated, the severity of the condition, the age, weight, and general physical condition of the subject being treated, as well as other medication being taken by the subject, as is well known to those skilled in the art. It is also known that the effective daily amount can be lowered or increased depending on the response of the subject or the evaluation of the prescribing physician.

In some embodiments, the methods of treatment further comprise treating with an additional therapy. For example, the combination of a compound of the invention with another therapeutic agent can be used. Such combination can be used simultaneously, sequentially or separately.

For example, treatment with the compounds of the invention can be combined with a thrombolytic drug. For example, in treating brain ischemic stroke, the treatment can further comprise the use of recombinant tissue plasminogen activator (tPA), also called alteplase (Activase). The delivery by infusion or iv injection of Humanin analog of the invention together with tPA can achieve both thrombolysis and neuroprotection, leading to improved clinical outcomes, lower healthcare resource use and lower treatment costs.

Toxicity and therapeutic efficacy of the peptides described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 (the concentration which provides 50% inhibition) and the FD50 (lethal dose causing death in 50 % of the tested animals) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (e.g. Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

The term “compound/s of the invention” encompasses all the peptides, polypeptides peptide analogs and peptide derivatives of the invention, as well as pharmaceutically acceptable salts thereof, or prodrugs thereof.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES Materials and Methods

1. Materials

Methanol, ethanol, trifluoroacetic acid, phenol, triisopropylsilane, dimethylformamide, Hoechst 33342, 4-Hydroxy-2, 2, 6, 6-tetramethylpiperidine-l-oxyl (Tempol), and Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) were purchased from Aldrich-Sigma Chemical Co, (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM) with either low or high glucose, fetal calf serum, horse serum, penicillin, streptomycin, pymvate, non- essential amino acids and collagen type 1 were all purchased from Beit Ha'emek (Afula, Israel). ATCC-formulated MEM (Cat # 11095-080 with L-Glutamate), F12 and Tetramethylrhodamine- methyl ester perchlorate (TMRM) were purchased from Invitrogen-ThermoFisher Scientific Co. (Carlsbad, CA, USA). Anti-Akt (pan) antibody, anti-phospho-Akt (Ser473) antibody, anti-p44/42 MAPK (Erkl/2) antibody and anti-phospho-p44/42 MAPK (Erkl/2) (Thr202/ Tyr204) antibody were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The monoclonal antibodies: anti-myogenin (5FD), anti-MyoD (G-l), anti-GAPDH (0411), troponin I (C-4), p- troponin I-C (1G11) and caspase-3 pl7 (B-4) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-fibronectin monoclonal antibody (F14) was purchased from Abeam Co. (Burlingame, CA, USA). Horseradish-peroxidase conjugated anti-rabbit and anti mouse secondary antibodies were obtained from Jackson ImmunoResearch, West Grove, PA, USA. MEK inhibitor PD 98059 and PI₃K inhibitor LY 294002 were purchased from Promega (Madison, WI, USA). Rhod-2AM was purchased from Biotium (Fremont, CA, USA). PNGF was purchased from Alomone Labs (Jerusalem, Israel). Doxorubicin hydrochloride (Dox, clinical grade, Pfizer Co.) was a kind gift from the pharmacy of the Hadassah Hebrew University Medical Center. Other chemicals and reagents were of analytical grade.

2. Solid Phase Peptide Synthesis ( SPPS )

Peptides were synthesized by microwave assisted solid-phase peptide synthesis applying Fmoc chemistry. Peptides were synthesized using Fmoc-RINKAMIDE-MBHA resin (loading 0.71 mmol/g). Peptide chains were elongated in sequential cycles of deprotection and coupling. Deprotection was performed with 20% piperidine in DMF (2X20min) and for coupling 5 equivalents of the protected amino acid derivatives during the first coupling were used, in one of the following mixtures: Fmoc-AA/ HBTU/ HOBt/ DIPEA, molar ratio 1:1:1 :2; Fmoc-AA/ HATU/ HO At/ DIPEA, molar ratio 1:1: 1:2. Each coupling and deprotection were followed by Kaiser (E. Kaiser, et al, Anal. Biochem. 34 (1970) 595-598) or chloranil tests (T. Vojkovsky, Pept. Res. 8 (1995) 236-237) to confirm coupling completion. Consequently, if needed the coupling was repeated. After the attachment of the last amino acid residue and removal of the Fmoc protecting group, the N-terminal free amine was acetylated according to a procedure described before (K. Chandra, et al, Org. Biomol. Chem. 12 (2014) 1879-1884). In short, peptidyl resin was treated with a mixture of malonic acid/HBTU/DIPEA in DMF (molar ratio 1:1.2: 1.5) for about 1 h. The completion of the reaction was monitored by the Kaiser and chloranil tests. After successful acetylation step, the peptide was cleaved from the resin simultaneously with the side chain deprotection in a one-step procedure. Thereafter, the dried resin was suspended in a cold mixture of TFA/H20/PhOH/TIPS (88:5:5:2, v/v/v/v) and stirred for 3 hours in room temperature. The crude peptide was purified by RP-HPLC (Merck-Hitachi, Japan), using a XSELECT CSH 130Prep preparative RP column (C18, 19 x 150 mm, 5 pm). The solvent systems were TDW (0.1% TFA) (A) and ACN (0.1% TFA) (B). The purity of the synthesized peptides was evaluated by RP-HPLC using Agilent ZORBAX RX-C18 analytical RP column (C18, 4.6 xl50 mm, 5 pm) or Agilent ZORBAX 300SB-C18 (C18, 4.6 x 250 mm, 5um) and was carried out on a Merck-Hitachi HPLC, monitored at 226 nm. The solvent systems were TDW 0.1% TFA in 100% H20 (A) and 0.1% TFA in 80% ACN (B). The mass spectrometry analyzes were performed using ESI-MS on LCQ Fleet Ion Trap mass spectrometer (Thermo Scientific). c(D-Serl4)HN was synthesized on CTC- resin by Shanghai Yaxian Chemical Co., Ltd Moyu Road, Jiading, Shanghai (Website: http://www.yaxianchemical.com), according to the scheme presented in Figure 1.

3. Myoblast cell cultures

The H9c2 cell line (ATTC, Manassas, VA; catalog # CRL - 1446), a clone originally derived from embryonic rat heart tissue, was cultured in DMEM with low D-glucose (1000 mg/1), L-glutamine (584 mg/1), sodium pyruvate (1 mM) , 10% FBS 100 U/ml of penicillin and 100 μg/ml of streptomycin, supplemented with 10% fetal bovine serum (FBS) in 75 cm² tissue culture flasks, at 37°C, in a humidified atmosphere of 5% CO2. Medium was changed every 2 - 3 days, and cells sub-cultured once they reached 80% confluence. Skeletal muscles, low passage (< 15) C2C12 mouse myoblast cell line was grown in DMEM with low D-glucose (1000 mg/1), supplemented with 20% FBS, 2 mM L-glutamine 100 μg/ml streptomycin, and 10,000 U/ml penicillin (Beit Haemek, Afula, Israel). The cultures were maintained in an incubator at 37 °C, in a humidified atmosphere of 6% CO2. The cultures at intermediate confluence were split at a 1:10 ratio, twice a week. The morphology of both myoblast clones cells was evaluated by an inverted phase contrast light microscope at a magnification of XI 00 and the photographs were collected with Nicon® digital camera (Nicon® Eclipse TS-100, Japan)

4. SH-SY5Y neuroblastoma cell cultures

SH-SY5Y human neuroblastoma cell cultures (ATCC, CRL-2266) were maintained to reach over 80% confluence (in DMEM/F12 + glutamax™ medium containing 10% fetal bovine serum (FBS), 4% non-essential amino acids, 1% penicillin/streptomycin, 4.5 g/1 glucose, 0.1% amphotericin B, and sodium pyruvate. Cell cultures were maintained at 37 °C, in 5% CO2 / 95% O2 normoxic conditions. Medium was exchanged every 3 days during cell growth, and cultures were passed when confluent once or twice per week. Even though this cell line can form floating clusters of neuroblasts, mostly of these cells grow as adherent cells in culture under present conditions. Within 3-4 days, 60-80% confluent cultures were obtained and used for sub-culturing after detachment with 0.05% trypsin-EDTA (for 75 cm² flask) for 5 minutes, at 37°C. The cells (lxlO⁶ cells/ml) in fresh culture media were applied to the experimental tissue culture plates and prior experiments were incubated at 37°C in 5% CO2 incubator. Viable cells were preserved in 10% DMSO containing growth medium in a liquid nitrogen container.

5. PCI 2 cell cultures

Rat pheochromocytoma, PC12 cells, were propagated in 25 cm² flasks in growth medium composed of Dulbecco's modified Eagle's medium (DMEM) supplemented with 7% fetal calf serum (FCS), 7% horse serum (HS), 10,000 U/ml pencillin and 100 μg/ml streptomycin, as previously described (A. Lahiani, et al., ACS Chem. Neurosci. 7 (2016) 1452-1462). The medium was replaced every second day and cells were grown at 37°C, in a humidified atmosphere of 6% CO2.

6. Oxygen-glucose-deprivation insult using PC 12 cells

PC12 cells (0.2 xlO⁶ cells/well) were applied on 12-wells plates, pre-coated with 200 μg/ml collagen type-I and grown for either 2 days (undifferentiated cells) or 7 days in the presence of 50 ng/ml of nerve growth factor (NGF) to induce neuronal differentiation (A. Lahiani, et al., Biochim. Biophys. Acta - Mol. Cell Res. 1853 (2015) 422-430). On the day of the experiment, cell medium was replaced to glucose-free DMEM (hypoglycemic insult) and the cultures were introduced into an ischemic chamber with oxygen level below 1% (anoxic insult) for 4 hours at 37°C under oxygen and glucose deprivation (OGD, ischemic phase) as previously described (A. Lahiani, et al., ACS Chem. Neurosci. 7 (2016) 1452-1462). In order to mimic in vivo reperfusion conditions (simulating renewal of blood supply), at the end of the OGD insult, 4.5 mg/ml glucose was added and cultures were incubated for additional 18 hours under normoxic conditions (R, reperfusion/reoxygenation phase) to complete the ischemic insult. Operationally, ischemic insult represents therefore a combination of both OGD and reperfusion phases. Control cultures were maintained under regular atmospheric conditions (normoxia) in the presence of 6% CO2. Addition of peptides was performed one hour prior to OGD and they were present during all duration of the experiment. At the end of the reperfusion phase, necrotic cell death was measured as detailed below. All experiments (n=5-7) were carried out under good laboratory practice conditions using a GMP grade C clean room, regulated according to IS07 requirements (10,000 particles /m³).

7. Serum withdrawal insult To induce trophic factors withdrawal (serum starvation) cell death insult, lxlO⁶ PC12 cell cultures were washed with free medium twice and grown in serum free medium for 72 hr followed by treatment with peptides for up to 96 hours.

8. Doxorubicin-induced cell death insult

H9c2 or C2C12 myoblast cell suspensions were applied to 6- or 24-well plates (0.5 x 10⁶ cells/ml), coated with 200 μg/ml collagen type -I and allowed to attach for 1 day. The experiment was initiated by supplementation of cultures with different concentrations of Humanin analogs or Tempol (a membrane -permeable radical scavenger and metal-independent, superoxide dismutase- mimetic with neuroprotective effects) for one hour before initiation of the cell death assay with 1 mM doxorubicin (Dox), treatment continuing for 24 hours. The peptides were present in culture media during the whole 24 hr period. At the end of the experiment, the cell culture media was evaluated for necrotic cell death measuring LDH release and the cell culture ’extracts were evaluated for apoptotic cell death measuring caspase-3 activity.

9. Determination of cell death by lactate dehydrogenase (LDH) release

Necrotic cell death was evaluated by measuring the leakage of LDH into the medium as previously described (A. Lahiani, et al., ACS Chem. Neurosci. 7 (2016) 1452-1462). LDH activity was determined at 340 nm using a spectrofluorimeter (TECAN, SPECTRA Fluor PLUS, Salzburg, Austria). Basal LDH release was measured in monolayer cultures maintained under normoxic conditions. Under OGD insult, LDH release representing cell death, was expressed as percent of total LDH released into the medium upon subtracting the basal values of LDH release. Total LDH (extracellular + intracellular) was obtained by freezing and thawing the cultures. The neuroprotective effect, defined as the percent decrease in LDH release in the presence of peptides was normalized to untreated ischemic cultures and is calculated according to the formula: Cell death (%) = (LDH (ischemia - basal) / (LDH total) X 100

10. Hoechst 33342 staining for immunofluorescence

Hoechst 33342 staining assay labels nuclear DNA and allows visualization of the nucleus in the interphase and chromosomes in the mitotic living cells. In brief, myoblasts were plated into 24 wells (2.5 x 10⁴ cells/well). After treatments, the cells were fixed with 4% formaldehyde for 20 min at 25 °C. Subsequently, 5 μg/mL Hoechst 33342 dye solution (50 pl/well) was used to stain the cells for 10 min. After two washings, cell nuclei were visualized by EVOS FL Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). Apoptotic dying cells were identified as the cells with blue fragmented, condensed nuclei, and the percentage of apoptotic cells was calculated from total number of cell population using NIH Image J software.

11. Caspase 3 assay

Cellular extracts were collected using trypsin and centrifuged twice at 1 ,000g at 4°C during 5 min. The pellet was suspended in buffer (20 mM HEPES/NaOH pH 7.5, 250 mM sucrose, 10 mM KC1, 2 mM MgCI₂, 1 mM EDTA) supplemented with 2 mM DTT, 100 mM PMSF and protease inhibitor cocktail. Protein concentration was determined by the Bradford assay. To measure caspase 3-like activity we used the colorimetric Caspase-3 Assay Kit, (Sigma-Aldrich). Aliquots of cell extracts containing 25 μg protein were incubated in the reaction buffer, containing 25 mM HEPES (pH=7.5), 10% sucrose, 10 mM DTT, 0.1% CHAPS and 100 mM of caspase substrate Ac-DEDV-pNA, for 2h at 37°C. Caspase-3 activity was determined by following the detection of the chromophore p-nitroanilide (p-Na) after cleavage from labeled substrate Ac- DEDV. The assay was calibrated with known concentrations of p-nitroanilide (p-NA). The optical density of the solution was determined in a spectrophotometer at 405 nm. All samples and standards were loaded in sixplicates.

12. Mitochondrial calcium fluorescence imaging

SH-S Y 5 Y neuroblastoma cells grown on coverslips were loaded with 1 mM of Rhod-2AM (a sensor for mitochondrial calcium level) in 0.1% BSA at 37°C for 30 min in Ringer buffer (126 mM NaCl, 5.4 mM KC1, 0.8 mM MgCb, 20 mM HEPES, 1.8 mM CaCI₂, 15 mM glucose, pH adjusted to 7.4 with NaOH). After this loading incubation, unincorporated dye was removed by washing the coverslips twice in fresh Ringer buffer and incubated for additional 30 min to allow the deesterification of the dye. During [Ca²⁺]mito recording, the cells were perfused with Ca²⁺ free, Ringer’s solution supplemented with 0.1% BSA and containing 100 mM ATP to induce calcium signaling in the presence or absence of tested peptides at a concentration of 5 mM. Rhod-2 was excited at 552 nm wavelength light and imaged with a 570-nm-long pass filter. All the fluorescent [Ca2+]mito signals were normalized to the averaged baseline signal (F/F0) obtained at the beginning of the measurements. The amplitude of [Ca2+]mito was calculated from each graph (summarizing an individual experiment) by a linear fit of the change in the fluorescence following initiation of apparent influx, typically for 15 s, as previously described (M. Kostic, et al., Cell Rep. 25 (2018) 3465-3475). In oxidative stress experiments the dye was present all the time, cells were perfused with Ringer’s solution supplemented with 0.1% BSA and after reaching steady baseline, 0.4 mM H2O2 was added to induce the depolarization of the mitochondrial membrane in the presence or absence of tested peptides at a concentration of 5 mM. TMRM was excited at 548 nm wavelength light and imaged with a 574 nm-long pass filter. All the fluorescent membrane potential signals were normalized to the averaged baseline signal (F/F0) obtained at the beginning of the measurements. The membrane potential was calculated by dividing the baseline records on the minimal value obtained by addition of 1 mM FCCP from each graph (summarizing an individual experiment) as previously described (M. Kostic, et al., Cell Rep. 25 (2018) 3465-3475).

13. Phosphorylation evaluation, SDS polyacrylamide gel electrophoresis and immunoblotting Equal amounts of protein (40 μg) were heated for 3 min at 95 °C in Laemmli buffer (Bio- Rad) and loaded on 12% polyacrylamide gels, separated by SDS-PAGE (100 V for 1.5 h), transferred to nitrocellulose membranes (Whatman, Germany) at 28 V over night at 4 °C. To minimize nonspecific binding, the membranes were incubated for 1 h at room temperature (RT) with 5% non-fat powdered milk (Bio-Rad) in Tris-buffered saline containing 0.1% Tween-20 (TBST). For phosphorylation experiments the immunodetection was performed using primary antibodies against phospho- or pan Erkl/2 and Akt at a dilution of 1:1000. The membranes were incubated with primary antibodies against phosphorylated form of a protein at 4 °C overnight. Following overnight incubation, the membranes were washed with TBST five times and then incubated for 1 h at RT with a horseradish peroxidase conjugated goat anti-rabbit secondary antibody at a dilution of 1:10,000. The blots were visualized using an ECL reagent (Pierce, Rockford, IL, USA). For measuring protein expression the primary antibodies were used at the following dilutions: anti-fibronectin 1:1000, anti-myogenin 1:500, anti-myoD 1:500, Anti- GAPDH 1:1000, phospho-specific and pan-anti Erkl/2, Akt 1:1000 and anti-caspase-3 at 1:3000. With each antibody the secondary reactions were performed with the respective secondary antibody. Thereafter, the membranes were washed and incubated for 30 min at RT in Restore Western Blot Stripping Buffer (Pierce, Rockford, IL, USA), and then incubated with the respective anti-pan. Films were exposed from 1-5 min, developed and scanned in a flatbed scanner (Epson, Long Beach, CA, USA). Densitometric analysis was performed with TINA software package (version 2.07d, Raytest Isotopenmessgeraete, Straiibenhardt, Germany). For each band, the densitometric values were obtained for the phospho and pan antibodies. The background of each film was subtracted and the relative density of the bands of phosphorylated Erk and Akt was divided by the density of the respective band of pan-protein (H. Wang, et al, J. Mol. Neurosci. 55 (2015) 931-40).

14. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated using the Tri-reagent method (Molecular Research Center. Inc. Cincinnati, OH, USA) according to the protocol recommended by the manufacturer. For reverse transcription, 1 μg of total RNA was used with the Reverse Transcription kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. Then RT-PCR reaction was performed using GoTaq Green Master Mix (Promega, Madison, WI, USA). The reactions were carried out with 0.1 μg cDNA and 50 pmol each of sense and antisense primers in a final volume of 25 pi. The following mouse-specific primer pairs, purchased from Syntezza Co. (Jerusalem, Israel), were used: myoD (5'-GCT ACG ACA CCG CCT ACT ACA-3' (SEQ ID NO: 17) and 5'-GGG TCT GGG TTC CCT GTT C-3' (SEQ ID NO: 18); myogenin (5'-TGA ATG CAA CTC CCA CAG C- 3' (SEQ ID NO: 19) and 5'-CAC CCA GCC TGA CAG ACA A-3' (SEQ ID NO: 20); b-actin (5'- TCA TGA AGT GTG ACG TTG ACA TCC GT-3' (SEQ ID NO: 21) and 5'-CTT AGA AGC ATT TGC GGT GCA CGA TG-3' (SEQ ID NO: 22). PCR conditions were calibrated for each pair of primers. The PCR reactions started by denaturation of the mixture for 2-3 min at 94-95 °C followed by 35 cycles of 30-60 seconds at 91-95 °C, 1 min at 58-65 °C, 1-2 min at 68-72 °C each, and a final extension step of 5-7 min at 68-72 °C. b-actin was used as a normalization control and its PCR reaction started with denaturation for 2 min at 94 °C followed by 35 amplification cycles of 30 seconds at 94 °C, 1 min at 65 °C, 2 min at 68 °C, and a final extension step of 7 min at 68 °C. The PCR reactions were performed using Master cycler gradient instrument in the linear range of each primer reaction (Eppendorf, Germany). PCR products were separated by electrophoresis (100 V for 40 min) on an agarose gel (2%) containing ethidium bromide for UV visualization.

15. Statistical analysis

Unless otherwise stated, each experiment was performed 3-6 times, each in sextuplicate wells. The results are presented as mean ±S.E. Statistical comparisons between experimental groups were determined by using analysis of variance program (ANOVA) followed by Dunnett's multiple comparison test, p value of 0.01 and 0.05 were considered significant for all comparisons.

Example 1: Humanin (HN) analogs design, synthesis and characterization

Two novel analogs of Humanin peptide, denoted HUJInin and c(D-Serl4)HN were designed and synthesized.

HUJInin peptide (also denoted MRPV), having the amino acid sequence Ac- YNAPVSIPQPAG ASRLLLLTGEIDLP-NH2 (SEQ ID NO: 1), is a peptide conjugate combining the two peptides PAGASRLLLLTGEIDLP (SEQ ID NO: 16, denoted (G¹⁴) HN17 or AGA-(C8R) HNG 17), and NAPVSIPQ (SEQ ID NO: 7, denoted NAP). HUJInin peptide was designed with blocked amino and carboxy terminals (by acetyl and amide groups, respectively), in order to enhance metabolic stability by preventing degradation by amino- and carboxyl- peptidases. It should be understood that "Ac-" indicates an acetylated N- terminus, and "-NH2" indicates an ami dated C-terminus. The amino acid Tyr (Y) was added at the amino terminal to allow future chemical labelling for imaging and radioactive binding studies.

HUJInin, (G¹⁴)HN17-NH₂ and NAP-NH2 peptides (presented in Table 3 below) were synthesized by microwave assisted solid-phase peptide synthesis applying Fmoc chemistry and purified by Prep-HPLC to > 95% homogeneity.

The second HN analog was designed based on the conformation of the known linear peptide Humanin D-Serl4, determined by NMR which indicated a bent around position 14 and a close proximity of the amino and carboxy terminals (N. Alsanousi, el al, Biochem. Biophys. Res. Commun. 477 (2016) 647-653). Based on this, a cyclic D-S14-HN analog comprising the amino acid sequence MAPAGASRLLLLT sEIDLPVKRRA (SEQ ID NO: 2), denoted c(D-Serl4)HN or Protectolev), was designed in which the N and C terminals are covalently connected by an amide bond (Figure 1A). This HN analog was synthesized according to the flow chart represented in Figure IB. A corresponding linear peptide, denoted (D-Derl4)HN-NH₂ was also synthesized, comprising the sequence set forth in SEQ ID NO: 2, with an ami dated C-terminus. Table 3 summarizes the various HN peptide analogs details. The pure peptides were characterized by analytical HPLC/MS (Figure 2A-2H). Table 3. selected peptides and peptide analogs

* Abbreviated-HNG17-NH₂, also referred to herein as HNG17 or HNG-17.

** Also termed (D-Serl4)AGA-(C8R)HN-NH2.

*** A cyclized version of SEQ ID NO: 2, also termed c(D-Serl4-AGA-(C8R)HN. # Cyclic peptide according to Formula (I)

## The molecular weight corresponds only to the amino acid residues of SEQ ID NO: 4, not including the bridge.

It should be understood that in the peptide sequences listed above, "Ac-" indicates an acetylated N- terminus, "-NH2" indicates an amidated C-terminus, "c(" indicates that the peptide is cyclic, and "14" indicates that the amino acid (G or s) corresponds to position 14 in the original Humanin peptide.

Example 2: HUJInin peptide protects PC12 neuronal cells from OGD/R- and serum starvation -induced cell death First, the ability of the synthetic novel peptide HUJInin to enhance the neuroprotective effects of (G¹⁴)HN17-NH2 was investigated in in vitro OGD/R and serum starvations neuronal models.

PC 12 catecholaminergic neuronal cultures, differentiated with nerve growth factor (NGF) for 7 days, were subjected to ischemic oxygen-glucose-deprivation-reoxygenation (OGD/R) insult at day 8 in culture. One hour before the start of the ischemic experiment, cells were treated with Tempol (1.5 mM), or different concentrations of the NAP, HNG17 or HUJInin. OGD/R-induced necrotic cell death was evaluated by LDH release assay as described in the Methods section.

As can be seen in Figure 3 A, OGD/R-induced necrotic cell death caused an insult of 55 ± 5 % (control). The cell membrane permeable, radical scavenger and superoxide dismutase-mimetic Tempol, used as positive control, conferred about 50% neuroprotection. Remarkably, HUJInin peptide significantly increased neuronal viability in a dose-dependent fashion, similarly to the parental compounds (Figure 3A).

In the other insult model investigated, undifferentiated PC 12 cells were transferred to serum free medium in the absence (control) or presence of 10 mM of NAP, HNG17 or HUJInin. The time course of the neuroprotective effect of HN analogs was investigated by comparing to control untreated cultures. As can be seen in Figure 3B, after 48-96 hours treatment with 10 m of HUJInin, a range of 25-40% neuroprotective effect was obtained. Interestingly, the protective effect of HUJInin peptide after 48-96 hours treatment was higher than the effect of the parental compounds NAP and HNG-17, suggesting that the conjugation of the peptides conferred improved properties.

Example 3: HUJInin peptide inhibits Erk 1/2 phosphorylation and stimulates AKT phosphorylation in PC12 neuronal cells under OGD/R or serum starvation conditions

It was previously reported that serum starvation (R. Wang, et al., Neuroscience. 286 (2015) 242-250) and ischemic insults modulated Erk 1/2 (R. Tabakman, et al, J. Mol. Neurosci. 22 (2004) 237-249) and Akt (J.A. Hillion, et al, J. Cereb. Blood Flow Metab. 26 (2006) 1323-1331) phosphorylations in PC 12 cell model. Therefore, the effect of HN analogs on Erk 1/2 and/or AKT phosphorylation was investigated in PC 12 cultures exposed to normoxia, OGD or serum starvation insults. The activation of these kinases was validated using the specific MEK/Erk 1/2 inhibitor PD98059 and PI3K/AKT inhibitor LY294002, respectively.

Figures 4A and 4B present the effect of HN synthetic analogs on the phosphorylation of Erk 1/2 in PC 12 cell cultures exposed to OGD insult. PC 12 cells were treated with 10 mM peptides or 30 mM PD 98059, during the whole OGD insult. Immunodetection was performed using primary antibodies against phospho- or pan Erkl/2 (p-Erk and pan-Erk; Figure 4A). The phosphorylation of Erk 1/2 of PC 12 cells lysates from OGD and normoxia (control) groups was determined by densitometry analysis of the immunoblots and was expressed as fold increase over normoxia level (Figure 4B). The results indicated that 10 mM of HUJInin inhibited by 60-70% OGD-induced Erkl/2 phosphorylation, similar to the inhibition observed with 30 mM of PD98059. Considering that Erk 1/2 exert deleterious effects leading to acceleration of the post-ischemia neuronal apoptosis (A. Lahiani, et al, Brain Sci. 8 (2018). doi:10.3390/brainsci8020032) and since (G¹⁴) HN17-NH2 decreased Erkl/2 activity in mice with stroke (X. Xu, et al, Stroke. 37 (2006) 2613- 9), HUJinin-induced Erk 1/2 inhibition may provide an explanation to its neuroprotective effects.

Figures 5A-5D demonstrate the effect of HUJInin on the phosphorylation of AKT in the presence or absence of PI3K inhibitor, in PC 12 cell cultures exposed to OGD (Figures 5A and 5C) and serum starvation (Figures 5B and 5D) insults. PC12 cells were treated with 10 mM HUJInin in the absence or presence of 10 mM LY294002, during the whole insult. The phosphorylation of AKT (Figures 5A and 5B) of PC12 cells lysates was further analyzed by densitometric analysis of the immunoblots and was expressed as ratio between p-AKT and pan-AKT (Figures 5C and 5D). The results indicate that HUJinin significantly stimulated Akt phosphorylation during both OGD and serum starvation insults, an effect abrogated by FY294002. Since PI3K/AKT signaling pathway is an important contributor to neuronal survival, it is hypothesized that HUJinin-induced activation of AKT may represent another mechanism contributing to neuroprotection.

Example 4: c(D-Serl4-HN) peptide protects PC12 and SH-SY5Y neuronal cells from OGD/R- induced cell death

The neuroprotective properties of the synthetic novel peptide c(D-Serl4-HN) were investigated in vitro. PC 12 and SH-SY5Y cells were applied to 12- well plates and treated with different concentrations of c(D-Serl4-HN) or left untreated (control) one hour before exposure to OGD insult, which was carried out for 4 h followed by 18 h reperfusion. Aliquots from the culture media were taken for FDH release measurements as described in the Methods section. Cell death is expressed by % FDH release out of total FDH. As can be seen in Figure 6A, treatment with c(D- Serl4)HN humanin cyclic analog has led to a significant dose-dependent decrease in % FDH release in both PC 12 and SH-SY5Y cells exposed to OGD/R conditions, compared to the control untreated groups, indicating the neuroprotective effect of this HN analog. Example 5: c(D-Serl4-HN) peptide inhibits Erk 1/2 phosphorylation and stimulates AKT phosphorylation in PC12 neuronal cells under OGD/R conditions

The effect of c(D-Serl4-HN) on the phosphorylation of Erk 1/2 and AKT was evaluated in PC 12 cultures under normoxia or OGD conditions. PC 12 cells under OGD/R insult were treated with 5 mM or 10 pM c(D-Serl4-HN). 10 pM of the PI₃K/AKT inhibitor LY294002 was used to validate AKT activation.

As can be seen in Figures 6B and 6C, similarly to HUJInin, the cyclic HN analog c(D-Serl4- HN) also attenuated Erk 1/2 phosphorylation (Figure 6B) and stimulated AKT phosphorylation (Figure 6C) under OGD/R conditions.

Example 6: The effect of HUJInin and c(D-Serl4-HN) on Erk 1/2 and AKT phosphorylation in PC 12 neuronal cells under normoxia

The effect of HN synthetic analogs on Erk 1/2 and AKT phosphorylation in PC 12 cells was further investigated under normoxia conditions (Figures 7A-7D). PC12 cells were treated for 20 min with 10 mM of NAP, HNG17, HUJInin or c(D-Serl4-HN), or left untreated (control). The phosphorylation of Erk 1/2 and AKT was measured from the same PC 12 cells lysates (20 micrograms of protein/sample) and determined by densitometry analysis of the immunoblots. Interestingly, under normoxia physiological conditions, HNG17, HUJInin and c(D-Serl4-HN), but not NAP, activated to different degrees the phosphorylation of both Erk 1/2 (Figures 7A and 7C) and AKT (Figurs 7B and 7D), with the highest activation obtained with HUJInin. These results which are opposite to the HNG17, HUJInin and c(D-Serl4-HN) effects on Erk 1/2 in cell cultures exposed to ischemic pathologic insults are explained by the complex physiological role of Erk 1/2 in the different cells including neurons, in both cell culture and in animal models. Erk 1/2 was reported to be activated in response to various insults through divergent mechanism involving the Ras/MEK pathway to maintain neuronal survival, growth, proliferation and differentiation. However, Erkl/2 signaling also mediates apoptosis and performs complex cross talk with PI3K/AKT survival pathway under oxidative stressed enriched environments. HUJInin can differentiate between these situations and therefore, appropriate as a disease modifying agent. Example 7: HUJInin and c(D-Serl4-HN) peptides improve mitochondrial function in SH- SY5Y neuronal cells exposed to oxidative stress

HN is known as having a cytoprotective role in maintaining mitochondrial function and cell viability (Y. Yang, et al., Biomed. Pharmacother. 117 (2019). doi:10.1016/j.biopha.2019.109075). Therefore, the effect of the novel synthetic HN analogs on the basal and the physiological ATP- induced response of mitochondrial calcium and on the mitochondrial membrane potential was evaluated in human neuroblastoma SH-SY5Y neuronal cells exposed to pathological H2O2- oxidative stress.

Figures 8A-8C present the effect of the HN analogs HUJInin, c(D-Serl4-HN), and (D- Serl4)HN-NH₂ on the basal and the physiological ATP-induced response of mitochondrial calcium. Cells were perfused with Ringer’s solution supplemented with 0.1 % BSA and containing 100 mM ATP to induce calcium signaling in the presence or absence of the tested peptides at a concentration of 5 pM. As can be seen, both HUJinin and c(D-Serl4)HN decreased the basal mitochondria calcium level (Figure 8B) and increased ATP-induced mitochondrial calcium amplitude (Figure 8C) compared to the control group (no peptide), with a more significant effect observed with the HUJinin analog. In contrast, the linear (D-Serl4)HN-NH₂ peptide showed no significant effect.

Figure 8D presents the mitochondrial membrane potential (MMP) records in cells incubated with 5 pM of the HN analogs HUJinin, c(D-Serl4-HN), and (D-Serl4)HN-NH2, before and after oxidative stress induced by addition of 0.4 mM H2O2. Quantification of membrane potential (MP) divided by minimal membrane potential (MMP) is presented in Figure 8E. The results indicate that HN analogs rescued treated cells from the H₂0₂-induced loss of MMP. Interestingly, HUJinin was found to reverse oxidative stress-induced loss of the mitochondrial membrane potential, while the cyclic analog c(D-Serl4-HN) at the same concentration showed lower effect, but yet mitigated the oxidative stress-induced pathological decrease of the mitochondrial membrane potential. These findings correlate with the results presented in Figures 8A-8C showing a more significant effect with the HUJinin analog.

The results suggest that the HN analogs HUJinin and c(D-Serl4)HN, and particularly HUJinin, are beneficial in improving mitochondrial functions. It is hypothesized that improving the mitochondrial functions may represent a third mechanism contributing to HUJinin- and c(D- Serl4)HN- induced neuroprotective effects. Overall, the data presented in Examples 2-7 establish the neuroprotective effects of the HN analogs HUJinin and c(D-Serl4)HN in OGD/R-, serum starvation- and H202-induced PC12 and/or SH-SY5Y cell death and suggest that inhibition of MEK/Erkl/2, activation of PI3K/AKT signaling pathways and maintenance of mitochondrial properties may mediate in part their neuroprotective effects. Cumulatively, these findings demonstrate the strong potential of HUJinin and c(D- Serl4)HN as neuroprotective agents in particular for treating ischemic stroke.

Example 8: HUJInin and c(D-Serl4-HN) peptides protect H9c2 myoblast cells from Doxorubicin induced apoptotic cell death

Evidence from in vitro, in vivo and clinical studies suggests that HN may exert potential cardioprotective effects against oxidative stress and cellular apoptosis (S.T. Charununtakorn, et al., Cardiovasc. Ther. 34 (2016) 107-114). Therefore, the cardioprotective effect of the novel synthetic HN analogs on doxorubicin (Dox)-induced cardiotoxicity was investigated in myoblast cell culture models.

H9c2 myoblast cells were initially treated with different concentrations of HUJinin, c(D- Serl4)HN or Tempol, or left untreated (control), for one hour before initiation of cell death assay with 1 mM doxorubicin (Dox) treatment for 24 h. At the end of the experiment, the culture media was evaluated for necrotic cell death by measuring LDH release.

As can be seen in Figure 9, Dox treatment induced a significant cell death (control) as reflected by high LDH release. Remarkably, when the cells were preincubated with both HN synthetic analogs for 1 hour before co-incubation with DOX for another 24 hours, cell death was significantly reduced compared to the control group. Interestingly, c(D-Serl4)HN conferred more potent myoprotection than HUJinin, reducing cell death by 28% and 40 % at concentrations of 10 and 40 mM, respectively.

To further evaluate the cytoprotective effects of c(D-Serl4)HN, apoptotic cell death was measured in H9c2 myoblast cells using nuclear DNA Hoechst staining. The cultures were treated with 5 pM cyclic peptide or 0.01 % DMSO for one hour before initiation of the cell death assay with 1 pM doxorubicin (Dox) treatment for 24 h, and then the cells were stained with Hoechst 33342. Apoptotic dying cells were identified as cells with blue fragmented, condensed nuclei (Figure 10 A), and the percentage of apoptotic cells was calculated from total number of cell population. As can be seen in Figure 10B, treatment with c(D-Serl4)HN significantly reduced DOX-induced apoptosis by approximately 56%.

The anti-apoptotic, myoprotective effects of the synthetic HN analogs on H9c2 myoblast cells was further investigated using caspase-3 assay. Caspase-3 is the major effector protease involved in apoptotic pathways and inhibitors of caspase-3 have been shown to hold great promises for apoptosis interruption in heart tissues (B. Yang, et al., Expert Opin. Ther. Targets. 17 (2013) 255-63). First, by using western blotting (Figure 11 A), it was possible to confirm that the cultures used in the experiments have increased troponin-T (left panel) and phosphorylated-Troponin-I (P- troponin) (right panel), two recognized cardiac markers. H9c2 cells were treated with 1 mM doxorubicin (Dox) for 24 hours in the absence and presence of 5 mM HN analogs HUJInin and c(D-Serl4-HN). The expression of caspase-3 protein was examined in triplicate groups by western blot analysis in cell lysates. As can be seen in Figure 11B, 1 pM DOX exposure for 24 hours induced a strong level of caspase-3 protein expression compared to the control group. Remarkably, preincubation for 1 hour with 5 pM HUJinin and c(D-Serl4)HN, followed by co-incubation with 1 pM DOX for 24 hours caused a 75% reduction in caspase-3 expression, compared with DOX alone group. Caspase-3 enzymatic activity evaluated in cultures treated with 1 pM Dox for 24 h, in the absence and presence of different concentrations of HN analogs. As can be seen in Figure llC, caspase-3 activity was increased by 6 fold in H9c2 myoblast cell cultures exposed to DOX for 24 hours, while HUJinin and c(D-Serl4)HN significantly decreased this activity in a dose- response fashion.

Example 9: HUJinin and c(D-Serl4-HN) peptides protect C2C12 skeletal myoblast cells from Doxorubicin-induced apoptotic cell death

Since doxorubicin causes skeletal muscle wasting in cancer patients and atrophy in C2C12 skeletal myoblast cell culture model (E. Archer-Lahlou, et al, Physiol. Rep. 6 (2018). doi: 10.14814/phy2.13726), the myoprotective effects of synthetic HN analogs was investigated in this cellular mode. C2C12 cell cultures were supplemented with 10 pM HUJinin or with different concentrations of c(D-Serl4-HN) (5, 10 and 40 pM), or left untreated for one hour before initiation of the cell death assay with 1 pM doxorubicin (Dox) treatment for 24 h. Cultures without Dox and peptide treatment were used as control. At the end of the experiment, the culture media was evaluated for necrotic cell death by measuring LDH release. As can be seen in Figure 12, treatment with DOX induced necrotic cell death expressed by 75±3 % LDH release, while HUJinin and c(D- Serl4)HN significantly decreased this DOX-induced cell death. A dose dependent protective effect of c(D-Serl4)HN was observed.

Overall, the results presented in Examples 8-9 show a robust, significant, myoprotective effect of the synthetic HN analogs HUJinin and c(D-Serl4)HN against apo-necrotic cell death insults.

Example 10: Evaluation of the neuroprotective and cardioprotective effect of HN analogs in vivo

The neuroprotective effect of HUJinin c(D-Serl4-HN) is studied in a rat stroke model of middle cerebral artery occlusion (MCAO), as described in Lazarovici et al., J Mol Neurosci. 2012; 48(3):526-40. In brief, rats are subjected to 2 h of right MCAO. followed by 48 h of reoxygenation in the presence or absence of HUJinin or c(D-Serl4-HN) injected i.v. at a dose of XXX/kg into the tail vein. Thereafter, brains are removed for different ELISA, western blotting, RT-PCR, and immunohistochemistry evaluations.

The neuroprotective effect of HUJinin c(D-Serl4-HN) is studied in myocardial I/R model in rats, as described in Thummasorn et al, Cardiovascular Therapeutics 2016; 34: 404-414.

Example 11: Design and synthesis of additional Humanin analogs

In light of the positive results achieved with HUJinin and c(D-Serl4)HN, additional HN analogs are developed and synthesized. c(D-Serl4)HN17 (also denoted Protectolev II) is a cyclic peptide comprising the sequence PAGASRLLLLTsEIDLP (SEQ ID NO: 3), in which the N and C terminals are covalently connected by an amide bond (Figure 13A). c(D-Serl4)HN17 is synthesized by solid-phase peptide synthesis applying Fmoc chemistry according to the schematic flow chart presented in Figure 13B.

Additional cyclic peptides denoted c(sl4-HN17(m-n)), comprising the sequence AGASRLLLLTsEIDL (SEQ ID NO: 4) and having a structure according to Formula (I) below, are synthesized as shown in the flow chart presented in Figure 14.

Formula (I), m,n=2-6

The neuroprotective and cardioprotective effect of the synthesized peptide, as well as their effect on mitochondrial function are tested based on the experiments described in Examples 2-10.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. A peptide of 15-40 amino acid residues, or an analog thereof, the peptide comprising the sequence X1X2AGASRLLLLTX3EIDLX4 (SEQ ID NO: 5), wherein:

Xi is absent or Glutamine (Gin, Q);

X2 is absent or Proline (Pro, P);

X3 is selected from Glycine (Gly, G) and D-Serine (DSer, s); and

X4 is absent or is selected from Proline (Pro, P) and a 2-6 amino acid sequence comprising Proline (Pro, P); provided that when X3 is Gly (G), Xi is Gin (Q), and when X3 is DSer (s), the peptide is cyclic.

2. The peptide according to claim 1, wherein X3 is Glycine (Gly, G).

3. The peptide according to claim 2, wherein X2 is Proline (Pro, P).

4. The peptide according to claim 2 or claim 3, further comprising the amino acid sequence NAPVSIP (SEQ ID NO: 6).

5. The peptide according to any one of claims 2-4, wherein X4 is Proline (Pro, P).

6. The peptide according to claim 5, comprising the sequence NAPVSIPQPAGASRLLLLTGEIDLP (SEQ ID NO: 9).

7. The peptide according to any one of claims 2 to 6, further comprising an amino acid residue that allows chemical labeling.

8. The peptide according to claim 7, wherein said amino acid residue is Tyrosine (Tyr , Y).

9. The peptide according to claim 8, comprising the amino acid sequence YNAPVSIPQPAGASRLLLLTGEIDLP (SEQ ID NO: 1).

10. The peptide according to any one of claims 2-9, wherein the C-terminus is ami dated.

11. The peptide according to any one of claims 2-10, wherein the N-terminus is acetylated.

12. The peptide according to any one of claims 9-11, consisting of the amino acid sequence Ac- YNAPVSIPQPAGASRLLLLTGEIDLP-NH2 (SEQ ID NO: 1).

13. The peptide according to claim 1, wherein X3 is DSer (s) and the peptide is cyclic.

14. The peptide according to claim 13, wherein the cyclization is selected from an end to end cyclization, a backbone to end cyclization, a backbone to backbone cyclization and a side- chain to side-chain cyclization.

15. The peptide according to claim 14, comprising the sequence AGASRLLLLTsEIDL (SEQ ID NO: 4).

16. The peptide according to any one of claims 13 to 15, comprising the amino acid sequence P AGASRLLLLT sEIDLP (SEQ ID NO: 3).

17. The peptide according to claim 16, consisting of the amino acid sequence

P AGASRLLLLT sEIDLP (SEQ ID NO: 3)

18. The peptide according to claim 16, comprising the amino acid sequence

MAPAGASRLLLLTsEIDLPVKRRA (SEQ ID NO: 2).

19. The peptide according to claim 18, consisting of the amino acid sequence

MAPAGASRLLLLTsEIDLPVKRRA (SEQ ID NO: 2).

20. The peptide according to any one of claims 13-19, wherein the peptide is cyclized by a formation of an amide bond between the N-terminal and the C-terminal amino acid residues.

21. The peptide according to claim 15, having a structure according to Lormula (I):

wherein:

Ri and R₂ are independently selected from hydrogen and the side chain of an amino acid; and m and n are each independently an integer of between 2 to 6.

22. A prodrug of the peptide according to any one of claims 1 to 21.

23. A pharmaceutical composition comprising the peptide or the prodrug according to any one of claims 1 to 22.

24. The pharmaceutical composition according to claim 23, formulated in a form suitable for an administration route selected from oral, intravenous, intramuscular, subcutaneous, intrathecal and intranasal administration.

25. The pharmaceutical composition according to claims 23 or 24, for use in treating a disease, disorder or condition associated with mitochondrial dysfunction.

26. The pharmaceutical composition for use according to claim 25, wherein the disease, disorder or condition is selected from the group consisting of an ischemia related disease or disorder, a neurodegenerative disease or disorder, and a cardiovascular disease or disorder.

27. The pharmaceutical composition for use according to claims 25 or 26, wherein the disease, disorder or condition is selected from the group consisting of cerebral ischemic reperfusion, myocardial ischemic reperfusion and anthracycline-induced cardiomyopathy.

28. The pharmaceutical composition for use according to claim 27, wherein the anthracycline is Doxorubicin.

29. A method of treating a disease, disorder or condition associated with mitochondrial dysfunction, the method comprising administering the pharmaceutical composition of claims 23 or 24 to a subject in need thereof.

30. The method according to claim 29, wherein the disease, disorder or condition is selected from the group consisting of an ischemia related disease or disorder, a neurodegenerative disease or disorder, and a cardiovascular disease or disorder.

31. The method according to claims 29 or 30, wherein the disease, disorder or condition is selected from the group consisting of cerebral ischemic reperfusion, myocardial ischemic reperfusion and anthracycline-induced cardiomyopathy.

32. The method according to claim 31 , wherein the anthracycline is Doxorubicin.

33. The method according to any one of claims 29 to 32, wherein the pharmaceutical composition is administered by a route selected from oral, intravenous, intramuscular, subcutaneous, intrathecal and intranasal administration.