WO2022003673A1 - Analogues de l'humanine et leurs utilisations - Google Patents

Analogues de l'humanine et leurs utilisations Download PDF

Info

Publication number
WO2022003673A1
WO2022003673A1 PCT/IL2021/050774 IL2021050774W WO2022003673A1 WO 2022003673 A1 WO2022003673 A1 WO 2022003673A1 IL 2021050774 W IL2021050774 W IL 2021050774W WO 2022003673 A1 WO2022003673 A1 WO 2022003673A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
amino acid
seq
disease
disorder
Prior art date
Application number
PCT/IL2021/050774
Other languages
English (en)
Inventor
Chaim Gilon
Amnon Hoffman
Philip Lazarovici
Dan GILON
Original Assignee
Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
Hadasit Medical Research Services & Development Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd., Hadasit Medical Research Services & Development Ltd. filed Critical Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
Publication of WO2022003673A1 publication Critical patent/WO2022003673A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • 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).
  • ATP cellular energy
  • Stroke Cerebrovascular insult-CVI
  • CVI cerebrovascular insult-CVI
  • This oxygen and glucose deprivation (OGD) ischemic event can result in pathological irreversible consequences, eventually leading to impaired neurological functions and morbidity.
  • OGD oxygen and glucose deprivation
  • 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 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.
  • 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.
  • ROS reactive oxygen species
  • HN Humanin
  • MDP 24-amino acid mitochondrial-derived peptide
  • 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).
  • HNG Humanin G
  • HN Humanin G
  • 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
  • ADNF activity-dependent neuroprotective protein
  • 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.
  • ADNF I and ADNF III also referred to as ADNP
  • analogs, subsequences such as NAP and SAL
  • D-amino acid versions either wholly D-amino acid peptides or mixed D- and L-amino acid peptides
  • 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.
  • the present invention provides novel peptide analogs of Humanin (HN) having improved therapeutic properties, pharmaceutical compositions comprising said analogs, and therapeutic uses thereof.
  • HN Humanin
  • 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.
  • 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.
  • 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.
  • c(D-Serl4-HN) that comprises the amino acid sequence MAPAGASRLLLLTsEIDLPVKRRA (SEQ ID NO: 2)
  • 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.
  • 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).
  • X3 is D-Ser (s).
  • the peptide is cyclic.
  • X3 is D-Ser (s) and the peptide is cyclic.
  • 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.
  • X3 is Gly (G).
  • X3 is Gly (G) and the peptide is linear.
  • X2 is Pro (P).
  • 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).
  • 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.
  • the peptide comprises the amino acid sequence NAPVSIPQPAGASRLLLLTGEIDLX4 (SEQ ID NO: 8), wherein X 4 is absent or Proline. In some specific embodiments, X4 is proline and the peptide comprises the amino acid sequence NAPVSIPQPAGASRLLLLTGEIDLP (SEQ ID NO: 9).
  • the peptide comprises at least one amino acid residue that allows labeling.
  • said amino acid residue is Tyrosine (Tyr, Y).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • Xi is absent or Glutamine (Gin, Q);
  • X2 is absent or Proline (Pro, P);
  • X3 is D-Serine (DSer, s).
  • X4 is absent or is selected from Proline (Pro, P) and a sequence of 2-6 amino acid residues comprising Proline (Pro, P);
  • 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.
  • 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.
  • X2 is Proline.
  • X4 is Proline.
  • X2 and X4 are both Proline.
  • the cyclic peptide comprises the amino acid sequence PAGASRLLLLTsEIDLP (SEQ ID NO: 3).
  • the cyclic peptide consists of the amino acid sequence set forth in SEQ ID NO: 3.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cyclic peptide is a backbone cyclic peptide.
  • 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.
  • the bond used for cyclization is a urea bond.
  • the backbone cyclic peptide comprises the amino acid sequence set forth in SEQ ID NO: 4 and has a structure according to Formula (I):
  • 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.
  • the present invention provides a prodrug of the peptides or peptide analogs of the invention in all embodiments thereof, including linear and cyclic forms.
  • the present invention provides a pharmaceutical composition comprising the peptide or the prodrug of the invention in all embodiments thereof.
  • the present invention provides a pharmaceutical composition
  • 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).
  • the present invention provides a pharmaceutical composition
  • 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.
  • the pharmaceutical composition comprises at least one peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4.
  • the pharmaceutical composition comprises a peptide comprising the amino acid sequence set forth in SEQ ID NO:l.
  • the pharmaceutical composition comprises at least one peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-4.
  • 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.
  • 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.
  • each peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-5 or of an analog thereof.
  • the term "distinct peptides" refers to peptides differing in at least one amino acid.
  • the pharmaceutical composition comprises a prodrug of the peptide of the invention in all embodiments thereof.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • 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.
  • 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 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.
  • 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
  • X4 is absent or is selected from Proline (Pro, P) and a 2-6 amino acid sequence comprising
  • Proline 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.
  • 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.
  • 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.
  • an ischemia related disease or disorder a neurodegenerative disease or disorder
  • a cardiovascular disease or disorder a chronic obstructive pulmonary disease or disorder.
  • the disease, disorder or condition is selected from the group consisting of cerebral ischemic reperfusion, myocardial ischemic reperfusion and anthracycline- induced cardiomyopathy.
  • cerebral ischemic reperfusion cerebral ischemic reperfusion
  • myocardial ischemic reperfusion myocardial ischemic reperfusion
  • anthracycline- induced cardiomyopathy Each possibility represents a separate embodiment of the present invention.
  • the disease, disorder or condition is anthracycline-induced cardiomyopathy.
  • the disease, disorder or condition is a chemotherapy- induced cardiomyopathy.
  • the condition is a Doxorubicin-induced cardiomyopathy.
  • 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- NH 2 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 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.
  • 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 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 PI 3 K/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-NH 2 (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-NH 2 (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.
  • 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 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).
  • 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.
  • HN Humanin
  • 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 (G 14 ) HN17 (PAGASRLLLLTGEIDLP (SEQ ID NO: 16), and the 8-amino acid sequence NAPVSIPQ (SEQ ID NO: 7), denoted NAP derived from ADNF.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 (which
  • 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.
  • 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.
  • 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.
  • acyl moieties e.g., alkanoyl or carbocyclic aroyl groups
  • O-acyl derivatives of free hydroxyl group e.g., that of seryl or threonyl residues
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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).
  • 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
  • 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, Xi is Glutamine (Gin, Q), and when X3 is D-Ser, the peptide is cyclic.
  • 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,
  • Each possibility represents a separate embodiment of the invention.
  • 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.
  • linear refers to peptides that have a wholly linear sequence of amino acids with no cross-linking between non-adjacent amino acids.
  • X2 in SEQ ID NO: 5 is Proline (Pro, P).
  • 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).
  • 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).
  • 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.
  • 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.
  • the peptide comprises the amino acid sequence
  • NAPVSIPQPAGASRLLLLTGEIDLP (SEQ ID NO: 9).
  • the peptide comprises an amino acid residue that allows chemical labeling.
  • the amino acid residue is Tyrosine (Tyr, Y).
  • said amino acid residue is conjugated to the N-terminus of the peptide.
  • the peptide comprises or consists of the amino acid sequence
  • 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.
  • detectable marker 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.
  • the peptide comprises a modified C-terminus and/or a modified N- terminus.
  • the C-terminus of the peptide is amidated.
  • the N-terminus of the peptide is acetylated.
  • the C-terminus of the peptide is amidated and the N-terminus of the peptide is acetylated.
  • 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).
  • X4 is absent or is selected from Proline (Pro, P) and a 2-6 amino acid sequence comprising
  • the peptide is a cyclic peptide. In some embodiments, the peptide comprises at least one cyclization.
  • 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.
  • 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).
  • X4 is the amino acid sequence PVKRRA (SEQ ID NO: 11).
  • X4 is the amino acid sequence PVKRR (SEQ ID NO: 12).
  • X4 IS the amino acid sequence PVKR (SEQ ID NO: 13).
  • X4 is the amino acid sequence PVK (SEQ ID NO: 14).
  • 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.
  • the cyclic peptide comprises the amino acid sequence set forth in SEQ ID NO: 4 and has a structure according to Formula (I):
  • 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 peptide of the invention further comprises a capping moiety coupled to the N-terminus.
  • the peptide further comprises at least one capping moiety coupled to the C -terminus.
  • the at least one capping moiety increases the solubility, permeability or stability of the peptide.
  • the at least one capping moiety increases the stability of the peptide under physiological conditions.
  • 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.
  • 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.
  • the peptides of the invention are synthesized according to solid phase peptide synthesis (SPPS) principles, utilizing standard Fmoc (9 -fluorenylmethoxy carbonyl) chemistry protocols.
  • SPPS solid phase peptide synthesis
  • 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.
  • 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-chlorophospho
  • 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.
  • coupling reagents such as PyBOP (Benzotriazole-l-yl--oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
  • 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.
  • UNCA'S pre-formed urethane-protected N- carboxy anhydrides
  • 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.
  • 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.
  • salts refers to both salts of carboxyl groups and to acid addition salts of amino or guanido groups of the peptide molecule.
  • pharmaceutically acceptable means suitable for administration to a subject, e.g., a human.
  • 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.
  • 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 ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ - ⁇ -functionalized or an C“- ⁇ - functionalized derivative of amino acid residue, with a moiety selected from the group consisting of: another N ⁇ - ⁇ -functionalized or an C ⁇ - ⁇ -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.
  • 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.
  • 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.
  • 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.
  • the cyclic peptide comprises at least one N ⁇ -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” indicates a N ⁇ - ⁇ -functionalized or a C ⁇ - ⁇ -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.
  • the peptide sequence is cyclized by covalently connecting one N ⁇ - ⁇ -functionalized derivative of amino acid residue added to the sequence, or a substituted amino acid residue in the sequence, with another N ⁇ - ⁇ -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.
  • 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.
  • the peptides of the invention further comprise a permeability enhancing moiety.
  • 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.
  • DLPs drug-like properties
  • 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.
  • the peptides of the present invention can be chemically modified or administered as prodrugs.
  • said prodrugs have improved oral bioavailabilty compared to the parental peptides.
  • 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.
  • prodrug 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.
  • 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.
  • a compound of the invention, or its salt form or a stereochemically isomeric form can be combined with a pharmaceutically acceptable carrier.
  • 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.
  • media such as water, glycols, oils, alcohols can be used in liquid preparations such as suspensions, syrups, elixirs, and solutions.
  • 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;
  • 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.
  • the compositions may additionally comprise an antimicrobial preservative such as benzyl alcohol.
  • the composition may additionally comprise a conventionally known antioxidant such as ascorbyl palmitate, butylhydroxyanisole, butylhydroxy toluene, propyl gallate and/or tocopherol.
  • 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.
  • 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.
  • 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.
  • 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.
  • the carrier can include a penetration enhancing agent or a wetting agent.
  • 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.
  • 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.
  • peptides are less suitable for oral administration due to susceptibility to digestion by gastric acids or intestinal enzymes.
  • the route of administration of the peptides of the invention is oral administration.
  • the peptides and cyclic peptides of the invention are modified as prodrugs and administered orally.
  • topical administration comprises ocular administration.
  • 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.
  • 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.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • 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.
  • physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants for example polyethylene glycol are generally known in the art.
  • Dragee cores are provided with suitable coatings.
  • 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.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • 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.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • 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.
  • compositions for parenteral administration include aqueous solutions of the active ingredients in water-soluble form.
  • 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).
  • 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.
  • 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.
  • the pharmaceutical compositions of the invention are formulated for local administration.
  • the compositions may be formulated for topical, subcutaneous or transdermal administration.
  • the pharmaceutical composition for topical and/or transdermal administration is formulated as an ointment, cream, lotion, or spray.
  • 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.
  • 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.
  • 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.
  • 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.
  • mitochondrial function includes any cellular activity carried out by mitochondria.
  • 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.
  • mitochondrial functionality can be assessed in vitro by e.g., monitoring a mitochondrial parameter such as mitochondrial membrane potential (MMP), O 2 consumption, ATP production and mitochondrial Ca 2+ uptake.
  • MMP mitochondrial membrane potential
  • 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.
  • 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.
  • terapéuticaally effective amount means an amount of a compound effective to prevent, alleviate or ameliorate symptoms of a disease of the subject being treated.
  • mitochondrial dysfunction includes any failure or deficiency of mitochondria or mitochondrial genes to carry out a mitochondrial function.
  • mitochondrial dysfunction is due to an oxidative stress related process such as hypoxia/ischemia/reperfusion injury.
  • 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.
  • associated with mitochondrial dysfunction 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.
  • associated with mitochondrial dysfunction refers to diseases, disorders or conditions in which improvement of mitochondria function may results in a clinical beneficial effect.
  • 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.
  • 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.
  • 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.
  • the disease, disorder or condition is an ischemia related disease or disorder.
  • 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.
  • 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
  • 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.
  • the ischemia related disease or disorder is an ischemic stroke.
  • the ischemic stroke is a focal or global ischemic stroke.
  • 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.
  • tPA tissue plasminogen activator
  • Activase recombinant tissue plasminogen activator
  • 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.
  • renewal of blood flow does not prevent the neuronal cell death which is expanding around the ischemic core.
  • the ischemia related disease or disorder is related to a myocardial ischemia.
  • 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).
  • the myocardial ischemia related disease or disorder is acute myocardial infarction (AMI) ("heart attack").
  • AMI acute myocardial infarction
  • 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).
  • 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.
  • the disease, disorder or condition is a neurodegenerative disease or disorder.
  • 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.
  • ALS amyotrophic lateral sclerosis
  • MELAS encephalopathy, lactic acidosis, stroke
  • MERFF myoclonic epilepsy with ragged red fibers
  • epilepsy Parkinson's disease
  • Alzheimer's disease Alzheimer's disease
  • Huntington's Disease Huntington's Disease
  • the neurodegenerative disease or disorder is Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • 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.
  • HN Humanin
  • AD Alzheimer's disease
  • Humanin 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.
  • the neurodegenerative disease or disorder is amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • 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.
  • ROS reactive oxygen species
  • Humanin peptide was shown to exhibit neuroprotective activity against toxicity by familial ALS-related mutant superoxide dismutase (SOD1).
  • the disease, disorder or condition is a cardiovascular disease or disorder and or cardiomyopathy.
  • 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.
  • the disease, disorder or condition is cardiomyopathy.
  • the cardiomyopathy is Anthracycline-induced cardiomyopathy (AIC).
  • AIC Anthracycline-induced cardiomyopathy
  • the histological pathophysiology of AIC is characterized by myocardial damage due to proteolysis, necrosis, apoptosis, and fibrosis.
  • Dox Doxorubicin
  • mitochondria play a key role in the pathogenesis of AIC (M. Alessandra, et al, Frontiers in cardiovascular medicine 7 (2020)).
  • 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.
  • the disease, disorder or condition is an age-related disease or disorder.
  • 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.
  • MDPs Mitochondrial-Derived Peptides
  • the disease, disorder or condition is an inflammatory and/or fibrotic disease or disorder.
  • 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.
  • 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.
  • the peptides of the invention are neuroprotective agents. In some embodiments, the peptides of the invention are cardioprotective agents.
  • neuroprotective 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.
  • 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.
  • 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.
  • cardioprotective refers to the ability to protect myocardium during ischemia.
  • 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.
  • the peptides of the invention inhibit Erkl/2 phosphorylation.
  • the peptides of the invention inhibit Erkl/2 hyperphosphorylation in cells under oxidative stress.
  • Protein kinase B 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.
  • the peptides of the invention stimulate Akt phosphorylation.
  • the pharmaceutical composition of the invention can be administered by any acceptable route.
  • the administration is enteral or parenteral administration.
  • administration is selected from oral, intra-articular, intravenous, intramuscular, subcutaneous, topical, transdermal, intradermal, intrathecal, and intranasal administration.
  • topical administration comprises ocular administration.
  • 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.
  • the methods of treatment further comprise treating with an additional therapy.
  • 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.
  • treatment with the compounds of the invention can be combined with a thrombolytic drug.
  • the treatment in treating brain ischemic stroke, can further comprise the use of recombinant tissue plasminogen activator (tPA), also called alteplase (Activase).
  • tPA tissue plasminogen activator
  • Activase recombinant tissue plasminogen activator
  • 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).
  • 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.
  • 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 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 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.
  • 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.
  • 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.
  • 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 ⁇ g/ml of streptomycin, supplemented with 10% fetal bovine serum (FBS) in 75 cm 2 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.
  • 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 ⁇ g/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)
  • SH-SY5Y human neuroblastoma cell cultures (ATCC, CRL-2266) were maintained to reach over 80% confluence (in DMEM/F12 + glutamaxTM 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.
  • Rat pheochromocytoma, PC12 cells were propagated in 25 cm 2 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 ⁇ g/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.
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • HS horse serum
  • streptomycin 10,000 U/mlin and 100 ⁇ g/ml streptomycin
  • PC12 cells (0.2 xlO 6 cells/well) were applied on 12-wells plates, pre-coated with 200 ⁇ g/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).
  • NGF nerve growth factor
  • ischemic insult represents therefore a combination of both OGD and reperfusion phases.
  • Serum withdrawal insult To induce trophic factors withdrawal (serum starvation) cell death insult, lxlO 6 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.
  • H9c2 or C2C12 myoblast cell suspensions were applied to 6- or 24-well plates (0.5 x 10 6 cells/ml), coated with 200 ⁇ g/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.
  • 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.
  • 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.
  • Hoechst 33342 staining assay labels nuclear DNA and allows visualization of the nucleus in the interphase and chromosomes in the mitotic living cells.
  • myoblasts were plated into 24 wells (2.5 x 10 4 cells/well). After treatments, the cells were fixed with 4% formaldehyde for 20 min at 25 °C. Subsequently, 5 ⁇ g/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.
  • 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 MgCI 2 , 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).
  • 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 CaCI 2 , 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.
  • Rhod-2AM a sensor for mitochondrial calcium level
  • 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).
  • 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.
  • 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
  • the secondary reactions were performed with the respective secondary antibody.
  • 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).
  • 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.
  • 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.
  • Example 1 Humanin (HN) analogs design, synthesis and characterization
  • 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 (G 14 ) 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.
  • 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).
  • HN analog was synthesized according to the flow chart represented in Figure IB.
  • a corresponding linear peptide, denoted (D-Derl4)HN-NH 2 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
  • HNG17-NH 2 Abbreviated-HNG17-NH 2 , also referred to herein as HNG17 or HNG-17.
  • the molecular weight corresponds only to the amino acid residues of SEQ ID NO: 4, not including the bridge.
  • Example 2 HUJInin peptide protects PC12 neuronal cells from OGD/R- and serum starvation -induced cell death
  • the ability of the synthetic novel peptide HUJInin to enhance the neuroprotective effects of (G 14 )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.
  • NGF nerve growth factor
  • OGD/R ischemic oxygen-glucose-deprivation-reoxygenation
  • Example 3 HUJInin peptide inhibits Erk 1/2 phosphorylation and stimulates AKT phosphorylation in PC12 neuronal cells under OGD/R or serum starvation conditions
  • 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).
  • 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).
  • 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.
  • 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
  • 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.
  • 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.
  • 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-NH 2 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.
  • 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.
  • the linear (D-Serl4)HN-NH 2 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 H 2 0 2 -induced loss of MMP.
  • MMP mitochondrial membrane potential
  • Example 8 HUJInin and c(D-Serl4-HN) peptides protect H9c2 myoblast cells from Doxorubicin induced apoptotic cell death
  • 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.
  • Dox doxorubicin
  • 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.
  • treatment with c(D-Serl4)HN significantly reduced DOX-induced apoptosis by approximately 56%.
  • 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).
  • Dox doxorubicin
  • 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.
  • Example 9 HUJinin and c(D-Serl4-HN) peptides protect C2C12 skeletal myoblast cells from Doxorubicin-induced apoptotic cell death
  • 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 (2016). 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.
  • Example 10 Evaluation of the neuroprotective and cardioprotective effect of HN analogs in vivo
  • HUJinin c(D-Serl4-HN) 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.
  • 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.
  • HUJinin c(D-Serl4-HN) 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.
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Neurosurgery (AREA)
  • Toxicology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychiatry (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Urology & Nephrology (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne de nouveaux analogues peptidiques de l'humanine, une composition pharmaceutique comprenant ces peptides et leurs utilisations pour le traitement de maladies, de troubles ou d'états associés à un dysfonctionnement mitochondrial, tels que, entre autres, l'accident ischémique cérébral et la cardiotoxicité induite par la doxorubicine.
PCT/IL2021/050774 2020-06-30 2021-06-24 Analogues de l'humanine et leurs utilisations WO2022003673A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202062705496P 2020-06-30 2020-06-30
US62/705,496 2020-06-30

Publications (1)

Publication Number Publication Date
WO2022003673A1 true WO2022003673A1 (fr) 2022-01-06

Family

ID=77168349

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2021/050774 WO2022003673A1 (fr) 2020-06-30 2021-06-24 Analogues de l'humanine et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2022003673A1 (fr)

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995033765A1 (fr) 1994-06-08 1995-12-14 Peptor Ltd. Analogues peptidiques cyclises par le squelette, a conformation restreinte
WO1997009344A2 (fr) 1995-08-29 1997-03-13 Peptor Limited Banques de nouveaux analogues de peptides a squelette cyclise sur le squelette
WO1998004583A1 (fr) 1996-07-31 1998-02-05 Peptor Ltd. Analogues de la somatostatine cyclises par squelette a contrainte de conformation
WO1999031121A2 (fr) 1997-12-15 1999-06-24 Peptor Ltd. Unites structurales ramifiees pour synthetiser des peptides cycliques
WO1999065508A1 (fr) 1998-06-19 1999-12-23 Peptor Ltd. Analogues de la somatostatine cyclises par squelette a contrainte de conformation
WO2000002898A1 (fr) 1998-07-12 2000-01-20 Peptor Ltd. Procedes de couplage d'acides amines a l'aide de bis-(trichloromethyl) carbonate
US6051554A (en) 1995-06-07 2000-04-18 Peptor Limited Conformationally constrained backbone cyclized somatostatin analogs
WO2000065467A1 (fr) 1999-04-23 2000-11-02 Peptor Ltd. Procedes d'identification de pharmacophore contenant des molecules provenant d'une banque virtuelle
US6407059B1 (en) 1994-06-08 2002-06-18 Peptor Limited Conformationally constrained backbone cyclized peptide analogs
WO2002062819A2 (fr) 2001-02-05 2002-08-15 Peptor Ltd. Analogues de somatostatine radiomarques et cyclises par squelette
US20050233413A1 (en) 2002-05-16 2005-10-20 Ikuo Nishimoto Neuroprotective polypeptides and methods of use
US7452867B2 (en) 2005-03-23 2008-11-18 Ramot At Tel-Aviv University, Ltd. Use of ADNF polypeptides for treating peripheral neurotoxicity
US8076449B2 (en) 2004-04-08 2011-12-13 Tomohiro Chiba Therapeutic agents of colivelin for neurodegenerative diseases
WO2019058365A1 (fr) 2017-09-19 2019-03-28 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Promédicaments de somatostatine
WO2019058367A1 (fr) 2017-09-19 2019-03-28 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Promédicaments lipophiles à base de peptides
WO2019058374A1 (fr) 2017-09-19 2019-03-28 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Peptides cycliques n-méthylés et promédicaments de ceux-ci

Patent Citations (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6117974A (en) 1991-10-02 2000-09-12 Peptor Limited Libraries of backbone-cyclized peptidomimetics
US5883293A (en) 1994-06-08 1999-03-16 Peptor Ltd. Conformationally constrained backbone cyclized peptide analogs
US5811392A (en) 1994-06-08 1998-09-22 Yissum Research Development Co. Of The Hebrew University Conformationally constrained backbone cyclized peptide analogs
US6265375B1 (en) 1994-06-08 2001-07-24 Yissum Research Development Co. Of The Hebrew University Conformationally constrained backbone cyclized peptide analogs
US5874529A (en) 1994-06-08 1999-02-23 Peptor Ltd. Conformationally constrained backbone cyclized peptide analogs
WO1995033765A1 (fr) 1994-06-08 1995-12-14 Peptor Ltd. Analogues peptidiques cyclises par le squelette, a conformation restreinte
US6407059B1 (en) 1994-06-08 2002-06-18 Peptor Limited Conformationally constrained backbone cyclized peptide analogs
US6051554A (en) 1995-06-07 2000-04-18 Peptor Limited Conformationally constrained backbone cyclized somatostatin analogs
WO1997009344A2 (fr) 1995-08-29 1997-03-13 Peptor Limited Banques de nouveaux analogues de peptides a squelette cyclise sur le squelette
WO1998004583A1 (fr) 1996-07-31 1998-02-05 Peptor Ltd. Analogues de la somatostatine cyclises par squelette a contrainte de conformation
US6355613B1 (en) 1996-07-31 2002-03-12 Peptor Limited Conformationally constrained backbone cyclized somatostatin analogs
WO1999031121A2 (fr) 1997-12-15 1999-06-24 Peptor Ltd. Unites structurales ramifiees pour synthetiser des peptides cycliques
WO1999065508A1 (fr) 1998-06-19 1999-12-23 Peptor Ltd. Analogues de la somatostatine cyclises par squelette a contrainte de conformation
WO2000002898A1 (fr) 1998-07-12 2000-01-20 Peptor Ltd. Procedes de couplage d'acides amines a l'aide de bis-(trichloromethyl) carbonate
US6512092B2 (en) 1998-07-12 2003-01-28 Peptor Ltd. Processes for coupling amino acids using bis-(trichloromethyl) carbonate
WO2000065467A1 (fr) 1999-04-23 2000-11-02 Peptor Ltd. Procedes d'identification de pharmacophore contenant des molecules provenant d'une banque virtuelle
WO2002062819A2 (fr) 2001-02-05 2002-08-15 Peptor Ltd. Analogues de somatostatine radiomarques et cyclises par squelette
US20050233413A1 (en) 2002-05-16 2005-10-20 Ikuo Nishimoto Neuroprotective polypeptides and methods of use
US8076449B2 (en) 2004-04-08 2011-12-13 Tomohiro Chiba Therapeutic agents of colivelin for neurodegenerative diseases
US7452867B2 (en) 2005-03-23 2008-11-18 Ramot At Tel-Aviv University, Ltd. Use of ADNF polypeptides for treating peripheral neurotoxicity
WO2019058374A1 (fr) 2017-09-19 2019-03-28 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Peptides cycliques n-méthylés et promédicaments de ceux-ci
WO2019058365A1 (fr) 2017-09-19 2019-03-28 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Promédicaments de somatostatine
WO2019058367A1 (fr) 2017-09-19 2019-03-28 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Promédicaments lipophiles à base de peptides

Non-Patent Citations (44)

* Cited by examiner, † Cited by third party
Title
"Solid-Phase Synthesis: A Practical Guide", 2000, CRC PRESS
A. LAHIANI ET AL., ACS CHEM. NEUROSCI, vol. 7, 2016, pages 1452 - 1462
A. LAHIANI ET AL., ACS CHEM. NEUROSCI., vol. 7, 2016, pages 1452 - 1462
A. LAHIANI ET AL., BIOCHIM. BIOPHYS. ACTA - MOL. CELL RES., vol. 1853, 2015, pages 422 - 430
A. LAHIANI ET AL., BRAIN SCI., vol. 8, 2018
ARAKAWA T ET AL: "A Rescue Factor for Alzheimer's Diseases: Discovery, Activity, Structure, and Mechanism", CURRENT MEDICINAL CHEMISTRY, BENTHAM, NL, vol. 15, 1 September 2008 (2008-09-01), pages 2086 - 2098, XP002612055, ISSN: 0929-8673, DOI: 10.2174/092986708785747616 *
B. KALYANARAMAN, REDOX BIOL., vol. 29, 2020, pages 101394
B. YANG ET AL., EXPERT OPIN. THER. TARGETS, vol. 17, 2013, pages 255 - 63
BASSAN M ET AL: "COMPLETE SEQUENCE OF A NOVEL PROTEIN CONTAINING A FEMTOMOLAR- ACTIVITY-DEPENDENT NEUROPROTECTIVE PEPTIDE", JOURNAL OF NEUROCHEMISTRY, WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 72, no. 3, 1 March 1999 (1999-03-01), pages 1283 - 1293, XP000882112, ISSN: 0022-3042, DOI: 10.1046/J.1471-4159.1999.0721283.X *
CHIBA TOMOHIRO ET AL: "Development of a femtomolar-acting humanin derivative named colivelin by attaching activity-dependent neurotrophic factor to its N terminus: characterization of colivelin-mediated neuroprotection against Alzheimer's disease-relevant insults in vitro and in vivo", THE JOURNAL OF NEUROSCIENCE, SOCIETY FOR NEUROSCIENCE, US, vol. 25, no. 44, 2 November 2005 (2005-11-02), pages 10252 - 10261, XP002532185, ISSN: 0270-6474, DOI: 10.1523/JNEUROSCI.3348.05.2005 *
D. MUKHERJEEC.G. PATIL, WORLD NEUROSURG, vol. 76, 2011, pages 85 - 90
E. ARCHER-LAHLOU ET AL., PHYSIOL. REP., vol. 6, 2018
E. KAISER ET AL., ANAL. BIOCHEM., vol. 34, 1970, pages 595 - 598
FINGL ET AL., THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 1975
G. GAO ET AL., NEUROL. RES., vol. 39, 2017, pages 895 - 903
GILON CHAIM ET AL: "Novel humanin analogs confer neuroprotection and myoprotection to neuronal and myoblast cell cultures exposed to ischemia-like and doxorubicin-induced cell death insults", PEPTIDES, ELSEVIER, AMSTERDAM, NL, vol. 134, 1 September 2020 (2020-09-01), XP086385895, ISSN: 0196-9781, [retrieved on 20200901], DOI: 10.1016/J.PEPTIDES.2020.170399 *
H. WANG ET AL., J. MOL. NEUROSCI., vol. 55, 2015, pages 931 - 40
HOANG, P. T. ET AL., METABOLISM: CLINICAL AND EXPERIMENTAL, vol. 59, no. 3, 2010, pages 343
HUMPHREY ET AL., CHEM. REV., 1997, pages 2243 - 2266
I. GOZES, CURR. PHARM. DES, vol. 17, 2011, pages 1040 - 1044
J. V. MCGOWAN ET AL., CARDIOVASC. DRUGS THER., vol. 31, 2017, pages 63 - 75
J.A. HILLION ET AL., J. CEREB. BLOOD FLOW METAB., vol. 26, 2006, pages 1323 - 1331
K. CHANDRA ET AL., ORG. BIOMOL. CHEM., vol. 12, 2014, pages 1879 - 1884
KHANNAHID AKHTAR ET AL., THE INDIAN JOURNAL OF MEDICAL RESEARCH, vol. 141, no. 1, 2015, pages 13
LAMBERT ET AL.: "reviewed variety of peptide cyclization methodologies", J. CHEM. SOC. PERKIN TRANS., vol. 1, 2001, pages 471 - 484
LAZAROVICI ET AL., J MOL NEUROSCI., vol. 48, no. 3, 2012, pages 526 - 40
M. ALESSANDRA ET AL., FRONTIERS IN CARDIOVASCULAR MEDICINE, vol. 7, 2020
M. KOSTIC ET AL., CELL REP., vol. 25, 2018, pages 3465 - 3475
N. ALSANOUSI, BIOCHEM. BIOPHYS. RES. COMMUN., vol. 477, 2016, pages 647 - 653
N.L. ROSIN ET AL., AM. J. PATHOL., vol. 185, 2015, pages 631 - 42
PILLAI ET AL., CURR. OPIN. CHEM. BIOL., vol. 5, 2001, pages 447
R. TABAKMAN ET AL., J. MOL. NEUROSCI., vol. 22, 2004, pages 237 - 249
R. WANG ET AL., NEUROSCIENCE, vol. 286, 2015, pages 242 - 250
R.H. MUZUMDAR ET AL., ARTERIOSCLER. THROMB. VASE. BIOL., vol. 30, 2010, pages 1940 - 1948
S.T. CHARUNUNTAKORN ET AL., CARDIOVASC. THER., vol. 34, 2016, pages 107 - 114
SIMPLFCIO ET AL., MOLECULES, vol. 13, no. 3, 2008, pages 519 - 547
T. VOJKOVSKY, PEPT. RES., vol. 8, 1995, pages 236 - 237
THUMMASORN ET AL., CARDIOVASCULAR THERAPEUTICS, vol. 34, 2016, pages 404 - 414
WU DONGDONG ET AL: "Protective roles of bioactive peptides during ischemia-reperfusion injury: From bench to bedside", LIFE SCIENCE, PERGAMON PRESS, OXFORD, GB, vol. 180, 17 May 2017 (2017-05-17), pages 83 - 92, XP085059766, ISSN: 0024-3205, DOI: 10.1016/J.LFS.2017.05.014 *
X. XINGSHUN ET AL., STROKE, vol. 37, no. 10, 2006, pages 2613 - 2619
X. XU ET AL., BRAIN RES., vol. 1227, 2008, pages 12 - 8
XU X ET AL: "Humanin is a novel neuroprotective agent against stroke", STROKE; A JOURNAL OF CEREBRAL CIRCULATION NOV 2004, AMERICAN HEART ASSOCIATION, DALLAS, TX, vol. 37, no. 10, 1 October 2006 (2006-10-01), pages 2613 - 2619, XP002713880, ISSN: 1524-4628, [retrieved on 20060907], DOI: 10.1161/?01.STR.0000242772.94277.1F *
Y. YANG ET AL., BIOMED. PHARMACOTHER, vol. 117, 2019, pages 109075
Z. ZHENG ET AL., DRUG NEWS PERSPECT., vol. 16, 2003, pages 497 - 503

Similar Documents

Publication Publication Date Title
Bourgault et al. Novel stable PACAP analogs with potent activity towards the PAC1 receptor
US20210206817A1 (en) Metabolically Stable Apelin Analogs in the Treatment of Disease Mediated by the Apelin Receptor
Kaneko et al. Drastic neuronal loss in vivo by β-amyloid racemized at Ser26 residue: conversion of non-toxic [D-Ser26] β-amyloid 1–40 to toxic and proteinase-resistant fragments
JP5247708B2 (ja) 抗糖尿病化合物
JP5707014B2 (ja) グルタミニル、及びグルタミン酸シクラーゼのエフェクターの使用
JP5270687B2 (ja) グルカゴンアンタゴニスト活性及びｇｌｐ−１アゴニスト活性を有するペプチド模倣体
US9902754B2 (en) Modified peptides as potent inhibitors of the PSD-95/NMDA receptor interaction
JP2009507844A (ja) 安定化ｇｌｐ−１類似体
US10683333B2 (en) Islet amyloid polypeptides with improved solubility
US20060211606A1 (en) Peptides
CA2584263A1 (fr) Analogues de peptides modifies chimiquement
Gilon et al. Novel humanin analogs confer neuroprotection and myoprotection to neuronal and myoblast cell cultures exposed to ischemia-like and doxorubicin-induced cell death insults
KR102230368B1 (ko) 아실화 옥신토모듈린 펩타이드 유사체
JP2013505230A (ja) 酸化ストレス関連障害を処置するためのペプチド
WO2022003673A1 (fr) Analogues de l'humanine et leurs utilisations
WO2011144714A1 (fr) Peptides kissorphine pour l'utilisation dans le traitement de la maladie d'alzheimer, la maladie de creutzfeldt-jakob ou le diabète sucré
AU2012272550A1 (en) Prevention and treatment of acute inflammatory conditions
Sidorova et al. [MeArg1, NLe10]-apelin-12: Optimization of solid-phase synthesis and evaluation of biological properties in vitro and in vivo
CN117940444A (zh) 胱天蛋白酶-2抑制剂化合物
CA2601227A1 (fr) Peptides de facteur de croissance mecano et leur utilisation
AU2018314833B2 (en) Novel compounds activating the NRF2 pathway
US20190085040A1 (en) Peptide modulators of specific calcineurin protein-protein interactions
US20200299332A1 (en) Neuroprotective peptide
CN116554263A (zh) Sirt1激动剂及其在相关疾病治疗中的应用
CN117279887A (zh) 抑制剂及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21749346

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21749346

Country of ref document: EP

Kind code of ref document: A1