阅读说明：本技术 用于心脏保护的经修饰的轴突生长诱向因子-1肽和组合物 (Modified axon growth-attractant factor-1 peptides and compositions for cardioprotection ) 是由 才华 于 2019-12-11 设计创作，主要内容包括：本文公开了经修饰的轴突生长诱向因子-1肽及其组合物,以及使用其赋予心脏保护作用的方法。在一些实施方案中,本发明提供了使用一种或多种经修饰的轴突生长诱向因子-1肽及其组合物的方法。在一些实施方案中,本发明提供了刺激、增加或增强内皮细胞产生一氧化氮的方法,其包括向内皮细胞施用一种或多种经修饰的轴突生长诱向因子-1肽或其组合物。(Disclosed herein are modified axon growth-inducing factor-1 peptides and compositions thereof, and methods of using the same to confer cardioprotective effects. In some embodiments, the present invention provides methods of using one or more modified neurite growth attractant factor-1 peptides and compositions thereof. In some embodiments, the present invention provides methods of stimulating, increasing, or enhancing nitric oxide production by endothelial cells, comprising administering to the endothelial cells one or more modified neurite growth attractant factor-1 peptides, or compositions thereof.)

1. A peptide comprising, consisting essentially of, or consisting of SEQ ID NO 1 as follows:

X1-X2-X3-C-X4-X5-X6-X7-T-X8-G(SEQ ID NO:1)

wherein

X1 is Ala, Asn, Cys, Ser, or Thr, preferably X1 is Cys, Ser, or Thr, wherein when X1 is Cys, it is covalently or non-covalently linked via a disulfide bond to a cysteine residue at the fourth amino acid position, or covalently or non-covalently linked to an oxirane compound;

x2 is present or absent, and if present, X2 is Ala, Ile, Leu, Met, Phe, Pro, Trp, or Val, preferably X2 is Leu or Pro;

x3 is present or absent, and if present, X3 is Asn, Asp, Cys, Gln, Glu, Gly, Ser, Thr, or Tyr, preferably X3 is Asn or Asp;

x4 is Arg, His or Lys, preferably X4 is Arg or Lys;

x5 is Arg, Asp, Glu, His, Lys, Phe, Trp or Tyr, preferably X5 is Asp or His;

x6 is Asn, Cys, Gln, Gly, Ser, Thr, or Tyr, preferably X6 is Asn or Gly;

x7 is present or absent and, if present, X7 is Ile, Thr or Val, preferably X7 is Val; and is

X8 is present or absent, and if present, X8 is Ala, Ile, Leu, Met, Phe, Pro, Trp, or Val, preferably X8 is Ala; and is

Wherein X2, X3, or both X2 and X3 are present.

2. The peptide according to claim 1, wherein the oxirane compound is polyethylene glycol (PEG), polyethylene oxide (PEO) and Polyoxyethylene (POE), methoxypolyethylene glycol (MPEG) or monomethoxypolyethylene glycol (mPEG), preferably the oxirane compound is mini-PEG.

3. The peptide of claim 1 or claim 2, wherein the peptide comprises, consists essentially of, or consists of: 2,3,4, 5,6 or 7.

4. The peptide of any one of claims 1-3, wherein the peptide is about 8-60, about 8-55, about 8-50, about 8-45, about 8-40, about 8-35, about 8-30, about 8-25, about 8-20, about 8-15, about 8-12, about 8-11, about 9-60, about 9-55, about 9-50, about 9-45, about 9-40, about 9-35, about 9-30, about 9-25, about 9-20, about 9-15, about 9-12, or 9-11 amino acid residues in length.

5. The peptide of any one of claims 1-3, wherein the peptide is 8, 9, 10, or 11 amino acid residues in length.

6. A composition comprising one or more peptides according to any one of claims 1-5.

7. A method of stimulating, increasing or enhancing nitric oxide production by endothelial cells, the method comprising administering one or more peptides of claims 1-5 or the composition of claim 6 to the endothelial cells.

8. A method of stimulating or inducing phosphorylation of ERK1/2 and/or eNOS in endothelial cells, the method comprising administering to the endothelial cells one or more peptides of claims 1-5 or a composition of claim 6.

9. A method of treating, inhibiting or reducing damage to a tissue or organ having endothelial cells, which are vascular endothelial cells, the method comprising stimulating, increasing or enhancing nitric oxide production by the endothelial cells and/or stimulating or inducing phosphorylation of ERK1/2, eNOS or both in the endothelial cells by administering one or more peptides according to claims 1-5 or a composition according to claim 6 to the endothelial cells before, during and/or after the damage.

10. The method of any one of claims 7-9, wherein the administration to the endothelial cells is in vivo.

11. The method of claim 10, wherein the injury is caused by superoxide generation, ischemia/reperfusion, or myocardial infarction.

12. The method of claim 11, wherein the tissue is cardiac tissue or the organ is a heart.

13. The method of claim 12, wherein the injury is caused by a myocardial infarction and the administration reduces infarct size in the heart.

14. A method of treating, inhibiting, or reducing ischemia/reperfusion injury of a heart of a subject, the method comprising administering to the subject a therapeutically effective amount of one or more peptides of claims 1-5 or a composition of claim 6, thereby treating, inhibiting, or reducing the ischemia/reperfusion injury.

15. A method of reducing or reducing the infarct size in a heart of a subject resulting from ischemia/reperfusion injury, the method comprising administering to the subject a therapeutically effective amount of one or more peptides of claims 1-5 or a composition of claim 6, thereby reducing or reducing the infarct size.

1. Field of the invention

The present invention relates generally to modified axon growth-inducing factor-1 peptides, compositions thereof, and methods of use thereof.

2. Correlation technique

Axon growth-inducing factors and their receptors are well known in the art, as exemplified in: US 5,565,331; US 6,096,866; US 6,017,714; US 6,309,638; US 6,670,451; and US 8,168,593; and US20060019896 and US 20060025335.

Axon growth-inducing factor-1 is a secreted molecule that is well known to play a clear role in directing vertebrate commissural axons in neuronal development. See Kennedy et al (1994) Cell 78: 425-35; serafini et al (1994) Cell 78: 409-24; and Serafini et al (1996) Cell 87: 1001-14. Recent studies have further demonstrated that neurite growth-inducing factor-1 plays a key role in endothelial cell proliferation, migration, and angiogenic signaling, in addition to playing a key role in epithelial cell morphogenesis. See Park et al (2004) PNAS USA 101: 16210-5; carmeliet et al (2005) Nature 436: 193-; nguyen et al (2006) PNAS USA 103: 6530-5; wilson et al (2006) Science 313: 640-4; liu et al (2004) Curr Biol 14: 897-905. At least eight neurite growth attractant factor receptors have been characterized in mammalian neurons, vascular systems, and other cell types. These include deletions in colorectal cancer (DCC), UNC5A, B, C, D, neogenin, α 6 β 4 and α 3 β 1 integrins. See Tessier-LaVigne et al (1996) Science 274: 1123-33; huber et al (2003) Annu Rev Neurosci 26: 509-63; ciruli et al (2007) Nat Rev Mol Cell Biol 8: 296-306; and Yebra et al (2003) Dev Cell 5: 695-. Binding of axon growth attractant factor-1 to DCC mediates attractive growth of axons, as well as positive angiogenic signaling in endothelial cells. In contrast, the UNC5B receptor appears to be repulsive, mediating cellular effects such as filopodia retraction, particularly in developing capillaries. See Lu et al (2004) Nature 432: 179-86; and Larrive et al (2007) Genes Dev 21: 2433-47.

Background

Disclosure of Invention

In some embodiments, the invention provides modified neurite growth attractant factor-1 peptides and compositions thereof, as described herein.

In some embodiments, the present invention provides methods of using one or more modified neurite growth attractant factor-1 peptides and compositions thereof. In some embodiments, the present invention provides methods of stimulating, increasing, or enhancing nitric oxide production by endothelial cells, comprising administering to the endothelial cells one or more modified neurite growth attractant factor-1 peptides, or compositions thereof. In some embodiments, the present invention provides methods of stimulating or inducing phosphorylation of ERK1/2 and/or eNOS in endothelial cells, comprising administering to the endothelial cells one or more modified axotrophin-1 peptides or compositions thereof. In some embodiments, the present invention provides methods of treating, inhibiting, or reducing injury to a tissue or organ having endothelial cells, comprising stimulating, increasing, or enhancing nitric oxide production by the endothelial cells and/or stimulating or inducing phosphorylation of ERK1/2, eNOS, or both in the endothelial cells by administering one or more modified axotrophic factor-1 peptides or compositions thereof to the endothelial cells before, during, and/or after the injury. In some embodiments, the endothelial cells are vascular endothelial cells. In some methods according to the invention, the administration to endothelial cells is in vivo. In some embodiments, the injury is caused by superoxide generation, ischemia/reperfusion, or myocardial infarction. In some embodiments, the tissue is cardiac tissue. In some embodiments, the organ is a heart. In some embodiments, the injury is caused by a myocardial infarction, and the administration reduces the infarct size of the heart. In some embodiments, the invention relates to treating or inhibiting restenosis in a subject, comprising administering to the subject a therapeutically effective amount of one or more modified axogenes attractant factor-1 peptides before, during, or after endothelial cell injury in blood vessels. In some embodiments, the present invention provides methods of treating, inhibiting, or reducing ischemia/reperfusion injury in an organ (e.g., heart) in a subject, comprising administering to the subject a therapeutically effective amount of one or more modified axon growth-inducing factor-1 peptides or compositions thereof, thereby treating, inhibiting, or reducing the ischemia/reperfusion injury. In some embodiments, the present invention provides methods of reducing or decreasing the infarct size in a heart of a subject resulting from ischemia/reperfusion injury, comprising administering to the subject a therapeutically effective amount of one or more modified axon growth attractant factor-1 peptides or compositions thereof, thereby reducing or decreasing the infarct size. In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is an animal model, e.g., a mouse. In some embodiments, the subject is a human. In some embodiments, the subject treated with one or more modified axon growth-inducing factor-1 peptides or compositions according to the invention is a subject in need thereof. Subjects in need thereof include those who may benefit from stimulation, increased or enhanced nitric oxide production, those who may benefit from stimulation or induction of phosphorylation of ERK1/2 and/or eNOS, those with or likely to have tissue or organ damage caused by increased production of reactive oxygen species or oxidative stress, ischemia/reperfusion or myocardial infarction, and those who will be exposed to or will likely be exposed to increased production of superoxide, ischemia/reperfusion conditions or myocardial infarction.

In some embodiments, the invention provides artificial packages, e.g., kits, comprising one or more modified neurite growth attractant factor-1 peptides or compositions thereof. In some embodiments, the artificial package further comprises a drug delivery device. In some embodiments, the present invention provides a device comprising one or more modified neurite growth attractant factor-1 peptides or compositions thereof.

Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description serve to explain the principles of the invention.

Detailed Description

Previously, it was found that axon growth-inducing factor-1 and axon growth-inducing factor-1 peptide fragments exhibited cardioprotective activity when administered to a subject. See PCT/US2011/038277, PCT/US2015/023248 and Li&Cai (2015) Am J Physiol Cell Physiol 309: C100-106, which is incorporated herein by reference in its entirety. Prior art axon growth attractant factor-1 peptide fragments are referred to herein as "prior art peptides". When delivered to the heart, the prior art peptides activate the protective pathway opened by axon growth attractant factor-1, i.e., the DCC-dependent activation of ERK1/2 and eNOS. ERK1/2 and eNOS_s1177(1177 residues for human/mouse and 1179 residues for bovine) phosphorylation was increased in a time-dependent manner in cultured endothelial cells by prior art peptides, which is believed to increase Nitric Oxide (NO) production for cardioprotective effect.

As disclosed herein, the present inventors further modified the prior art peptides and determined their cardioprotective efficacy against ischemia reperfusion (I/R) injury. Analysis of post I/R infarct size indicated a significant reduction in myocardial damage following treatment with the modified neurite-inducing factor-1 peptide. In fact, the modified neurite growth attractant factor-1 peptide reduced infarct size to a significantly greater extent-the extent of infarct size reduction increased by about 14% to 40% compared to prior art peptides.

Accordingly, the present invention provides modified axon growth-inducing factor-1 peptides and compositions and methods thereof. As used herein, a "modified axon growth attractant factor-1 peptide" refers to a peptide or protein comprising, consisting essentially of, or consisting of SEQ ID NO:1 as follows:

X1-X2-X3-C-X4-X5-X6-X7-T-X8-G(SEQ ID NO:1)

wherein

X1 is Ala, Asn, Cys, Ser, or Thr, preferably X1 is Cys, Ser, or Thr, wherein when X1 is Cys, it is covalently or non-covalently linked (e.g., attached) to a cysteine residue at the fourth amino acid position via a disulfide bond, or covalently or non-covalently linked (e.g., attached) to an oxirane compound;

x2 is present or absent, and if present, X2 is Ala, Asp, Ile, Leu, Met, Phe, Pro, Trp, or Val, preferably X2 is Leu or Pro;

x3 is present or absent, and if present, X3 is Asn, Arg, Asp, Cys, gin, Glu, Gly, Ser, Thr, or Tyr, preferably X3 is Asn or Asp;

x4 is Arg, His or Lys, preferably X4 is Arg or Lys;

x5 is Arg, Asp, Glu, His, Lys, Phe, Trp or Tyr, preferably X5 is Asn, Asp or His;

x6 is Asn, Cys, Gln, Gly, Ser, Thr, Tyr, or Val, preferably X6 is Asn or Gly;

x7 is present or absent and, if present, X7 is Asn, Gly, His, Ile, Thr, or Val, preferably X7 is Val; and is

X8 is present or absent, and if present, X8 is Ala, Asn, lie, Leu, Met, Phe, Pro, Thr, Trp, or Val, preferably X8 is Ala; and is

Wherein X2, X3, or both X2 and X3 are present.

In some embodiments, the oxirane compound is polyethylene glycol (PEG), polyethylene oxide (PEO), and Polyoxyethylene (POE), methoxypolyethylene glycol (MPEG), or monomethoxypolyethylene glycol (MPEG), or diethylene glycol (mini-PEG), preferably the oxirane compound is mini-PEG.

In some embodiments, the modified neurite growth attractant factor-1 peptide is about 8-60, about 8-55, about 8-50, about 8-45, about 8-40, about 8-35, about 8-30, about 8-25, about 8-20, about 8-15, about 8-12, 8-11, about 9-60, about 9-55, about 9-50, about 9-45, about 9-40, about 9-35, about 9-30, about 9-25, about 9-20, about 9-15, about 9-12, or 9-11 amino acid residues in length. In some embodiments, the modified axon growth attractant factor-1 peptide is 8, 9, 10, or 11 amino acid residues in length.

As used herein, a peptide "comprising" a given sequence means that the peptide may include additional amino acid residues, amino acid isomers, and/or amino acid analogs at the N-terminus, the C-terminus, or both. These additional residues may or may not alter the activity or function of a given sequence, i.e., a peptide having these additional residues, isomers, or analogs may have a different activity or function than the given sequence itself (without these additional residues, isomers, or analogs). As used herein, a peptide "consisting essentially of" a given sequence "means that the peptide may include additional amino acid residues, amino acid isomers, and/or amino acid analogs at the N-terminus, C-terminus, or both, so long as they do not substantially alter the function or activity of the given sequence, i.e., a peptide having these additional residues, isomers, or analogs has substantially similar activity and function as the given sequence itself. As used herein, a peptide "consisting of" a given sequence "means that the peptide does not include additional amino acid residues, amino acid isomers, and/or amino acid analogs at the N-terminus and C-terminus.

In some embodiments, the modified axon growth attractant factor-1 peptide may be isolated. As used herein, an "isolated" compound refers to a compound that is isolated from its natural environment. For example, an isolated peptide is a peptide that does not have the natural amino acids corresponding to the N-terminal, C-terminal, or both flanks of the full-length polypeptide. For example, an isolated Vl-9aa peptide refers to a peptide having amino acid residues of Vl (aa 304 to 312) that may have an unnatural amino acid at its N-terminus, C-terminus, or both, but does not have a proline amino acid residue after the 9 th amino acid residue at its C-terminus, or does not have a valine amino acid residue before the cysteine amino acid residue at its N-terminus, or both. As another example, an isolated peptide can be a peptide that is immobilized to a substrate that is not naturally associated with the peptide. As a further example, an isolated peptide may be a peptide linked to another molecule (e.g., a PEG compound, such as mPEG) that is not naturally associated with the peptide.

In some embodiments, the modified axon growth attractant factor-1 peptide can include one or more natural amino acids, unnatural amino acids, or a combination thereof. The amino acid residues of the peptide may be the D-isomer, the L-isomer, or both. The peptide may be composed of alpha-amino acids, beta-amino acids, natural amino acids, unnatural amino acids, amino acid analogs, or combinations thereof. Amino acid analogs include beta-amino acids and amino acids in which the amino group or the carboxyl group is replaced with a similarly reactive group (e.g., a primary amine is replaced with a secondary or tertiary amine, or a carboxyl group is replaced with an ester).

Examples of β -amino acid analogs include cyclic β -amino acid analogs; beta-alanine; i- β -phenylalanine; i-l,2,3, 4-tetrahydro-isoquinoline-3-acetic acid; 1-3-amino-4- (1-naphthyl) -butyric acid; i-3-amino-4- (2, 4-dichlorophenyl) butanoic acid; i-3-amino-4- (2-chlorophenyl) -butyric acid; 1-3-amino-4- (2-cyanophenyl) -butyric acid; 1-3-amino-4- (2-fluorophenyl) -butyric acid; 1-3-amino-4- (2-furyl) -butyric acid; 1-3-amino-4- (2-methylphenyl) -butyric acid; i-3-amino-4- (2-naphthyl) -butyric acid; 1-3-amino-4- (2-thienyl) -butyric acid; 1-3-amino-4- (2-trifluoromethylphenyl) -butyric acid; i-3-amino-4- (3, 4-dichlorophenyl) butanoic acid; i-3-amino-4- (3, 4-difluorophenyl) butanoic acid; 1-3-amino-4- (3-benzothienyl) -butyric acid; i-3-amino-4- (3-chlorophenyl) -butyric acid; 1-3-amino-4- (3-cyanophenyl) -butyric acid; 1-3-amino-4- (3-fluorophenyl) -butyric acid; 1-3-amino-4- (3-methylphenyl) -butyric acid; 1-3-amino-4- (3-pyridinyl) -butyric acid; 1-3-amino-4- (3-thienyl) -butyric acid; 1-3-amino-4- (3-trifluoromethylphenyl) -butyric acid; i-3-amino-4- (4-bromophenyl) -butyric acid; i-3-amino-4- (4-chlorophenyl) -butyric acid; 1-3-amino-4- (4-cyanophenyl) -butyric acid; 1-3-amino-4- (4-fluorophenyl) -butyric acid; 1-3-amino-4- (4-iodophenyl) -butyric acid; 1-3-amino-4- (4-methylphenyl) -butyric acid; 1-3-amino-4- (4-nitrophenyl) -butyric acid; 1-3-amino-4- (4-pyridinyl) -butyric acid; 1-3-amino-4- (4-trifluoromethylphenyl) -butyric acid; i-3-amino-4-pentafluoro-phenylbutyric acid; i-3-amino-5-hexenoic acid; i-3-amino-5-hexynoic acid; i-3-amino-5-phenylpentanoic acid; i-3-amino-6-phenyl-5-hexenoic acid; (S) -1,2,3, 4-tetrahydro-isoquinoline-3-acetic acid; (S) -3-amino-4- (1-naphthyl) -butyric acid; (S) -3-amino-4- (2, 4-dichlorophenyl) butanoic acid; (S) -3-amino-4- (2-chlorophenyl) -butyric acid; (S) -3-amino-4- (2-cyanophenyl) -butyric acid; (S) -3-amino-4- (2-fluorophenyl) -butyric acid; (S) -3-amino-4- (2-furyl) -butyric acid; (S) -3-amino-4- (2-methylphenyl) -butyric acid; (S) -3-amino-4- (2-naphthyl) -butyric acid; (S) -3-amino-4- (2-thienyl) -butyric acid; (S) -3-amino-4- (2-trifluoromethylphenyl) -butyric acid; (S) -3-amino-4- (3, 4-dichlorophenyl) butanoic acid; (S) -3-amino-4- (3, 4-difluorophenyl) butanoic acid; (S) -3-amino-4- (3-benzothienyl) -butyric acid; (S) -3-amino-4- (3-chlorophenyl) -butyric acid; (S) -3-amino-4- (3-cyanophenyl) -butyric acid; (S) -3-amino-4- (3-fluorophenyl) -butyric acid; (S) -3-amino-4- (3-methylphenyl) -butyric acid; (S) -3-amino-4- (3-pyridyl) -butyric acid; (S) -3-amino-4- (3-thienyl) -butyric acid; (S) -3-amino-4- (3-trifluoromethylphenyl) -butyric acid; (S) -3-amino-4- (4-bromophenyl) -butyric acid; (S) -3-amino-4- (4-chlorophenyl) butanoic acid; (S) -3-amino-4- (4-cyanophenyl) -butyric acid; (S) -3-amino-4- (4-fluorophenyl) butanoic acid; (S) -3-amino-4- (4-iodophenyl) -butyric acid; (S) -3-amino-4- (4-methylphenyl) -butyric acid; (S) -3-amino-4- (4-nitrophenyl) -butyric acid; (S) -3-amino-4- (4-pyridyl) -butyric acid; (S) -3-amino-4- (4-trifluoromethylphenyl) -butyric acid; (S) -3-amino-4-pentafluoro-phenylbutyric acid; (S) -3-amino-5-hexenoic acid; (S) -3-amino-5-hexynoic acid; (S) -3-amino-5-phenylpentanoic acid; (S) -3-amino-6-phenyl-5-hexenoic acid; 1,2,5, 6-tetrahydropyridine-3-carboxylic acid; 1,2,5, 6-tetrahydropyridine-4-carboxylic acid; 3-amino-3- (2-chlorophenyl) -propionic acid; 3-amino-3- (2-thienyl) -propionic acid; 3-amino-3- (3-bromophenyl) -propionic acid; 3-amino-3- (4-chlorophenyl) -propionic acid; 3-amino-3- (4-methoxyphenyl) -propionic acid; 3-amino-4, 4, 4-trifluoro-butyric acid; 3-aminoadipic acid; d- β -phenylalanine; beta-leucine; l- β -homoalanine; l- β -homoaspartic acid γ -benzyl ester; l- β -homoglutamic acid δ -benzyl ester; l- β -homoisoleucine; l- β -homoleucine; l- β -homomethionine; l- β -homophenylalanine; l- β -homoproline; l- β -homotryptophan; l- β -homovaline; L-N ω -benzyloxycarbonyl- β -homolysine; n ω -L- β -homoarginine; O-benzyl-L- β -homoproline; O-benzyl-L- β -homoserine; O-benzyl-L- β -homothreonine; O-benzyl-L- β -homotyrosine; gamma-trityl-L-beta-homoasparagine; i- β -phenylalanine; l- β -homoaspartic acid γ -tert-butyl ester; delta-tert-butyl L-beta-homoglutamate; L-N ω - β -homolysine; n δ -trityl-L- β -homoglutamine; n ω -2,2,4,6, 7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L- β -homoarginine; O-tert-butyl-L- β -homohydroxy-proline; O-tert-butyl-L- β -homoserine; O-tert-butyl-L- β -homothreonine; O-tert-butyl-L- β -homotyrosine; 2-aminocyclopentanecarboxylic acid; and 2-aminocyclohexanecarboxylic acid.

Examples of amino acid analogs of alanine, valine, glycine and leucine include α -methoxyglycine; α -allyl-L-alanine; α -aminoisobutyric acid; alpha-methyl-leucine; β - (1-naphthyl) -D-alanine; β - (1-naphthyl) -L-alanine; β - (2-naphthyl) -D-alanine; β - (2-naphthyl) -L-alanine; β - (2-pyridyl) -D-alanine; beta- (2-pyridyl) -L-alanine; β - (2-thienyl) -D-alanine; beta- (2-thienyl) -L-alanine; β - (3-benzothienyl) -D-alanine; beta- (3-benzothienyl) -L-alanine; beta- (3-pyridyl) -D-alanine; beta- (3-pyridyl) -L-alanine; β - (4-pyridyl) -D-alanine; beta- (4-pyridyl) -L-alanine; beta-chloro-L-alanine; beta-cyano-L-alanine; beta-cyclohexyl-D-alanine; beta-cyclohexyl-L-alanine; beta-cyclopenten-1-yl-alanine; beta-cyclopentyl-alanine; β -cyclopropyl-L-Ala-oh, dicyclohexylammonium salt; beta-tert-butyl-D-alanine; beta-tert-butyl-L-alanine; gamma-aminobutyric acid; l- α, β -diaminopropionic acid; 2, 4-dinitro-phenylglycine; 2, 5-dihydro-D-phenylglycine; 2-amino-4, 4, 4-trifluorobutanoic acid; 2-fluoro-phenylglycine; 3-amino-4, 4, 4-trifluoro-butyric acid; 3-fluoro-valine; 4,4, 4-trifluoro-valine; 4, 5-dehydro-L-leu-oh, dicyclohexylammonium salt; 4-fluoro-D-phenylglycine; 4-fluoro-L-phenylglycine; 4-hydroxy-D-phenylglycine; 5,5, 5-trifluoro-leucine; 6-aminocaproic acid; cyclopentyl-D-Gly-oh, dicyclohexylammonium salt; cyclopentyl-Gly-oh, dicyclohexylammonium salt; d- α, β -diaminopropionic acid; d- α -aminobutyric acid; d- α -tert-butylglycine; d- (2-thienyl) glycine; d- (3-thienyl) glycine; d-2-aminocaproic acid; d-2-indanylglycine; d-allylglycine-dicyclohexylammonium salt; d-cyclohexylglycine; d-norvaline; d-phenylglycine; beta-aminobutyric acid; beta-aminoisobutyric acid; (2-bromophenyl) glycine; (2-methoxyphenyl) glycine; (2-methylphenyl) glycine; (2-thiazolyl) glycine; (2-thienyl) glycine; 2-amino-3- (dimethylamino) -propionic acid; l- α, β -diaminopropionic acid; l-alpha-aminobutyric acid; l- α -tert-butylglycine; l- (3-thienyl) glycine; l-2-amino-3- (dimethylamino) -propionic acid; dicyclohexyl-ammonium salt of L-2-aminocaproic acid; l-2-indanylglycine; dicyclohexylammonium salt of L-allylglycine; l-cyclohexylglycine; l-phenylglycine; l-propargylglycine; l-norvaline; n- α -aminomethyl-L-alanine; d- α, γ -diaminobutyric acid; l-alpha, gamma-diaminobutyric acid; beta-cyclopropyl-L-alanine; (N- β - (2, 4-dinitrophenyl)) -L- α, β -diaminopropionic acid; (N- β -l- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) ethyl) -D- α, β -diaminopropionic acid; (N- β -1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) ethyl) -L- α, β -diaminopropionic acid; (N- β -4-methyltrityl) -L- α, β -diaminopropionic acid; (N- β -allyloxycarbonyl) -L- α, β -diaminopropionic acid; (N- γ -1- (4, 4-dimethyl-2, 6-dioxocyclohex-l-ylidene) ethyl) -D- α, γ -diaminobutyric acid; (N- γ -1- (4, 4-dimethyl-2, 6-dioxocyclohex-L-ylidene) ethyl) -L- α, γ -diaminobutyric acid; (N- γ -4-methyltrityl) -D- α, γ -diaminobutyric acid; (N- γ -4-methyltrityl) -L- α, γ -diaminobutyric acid; (N- γ -allyloxycarbonyl) -L- α, γ -diaminobutyric acid; d- α, γ -diaminobutyric acid; 4, 5-dehydro-L-leucine; cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH; d-allylglycine; d-high cyclohexylalanine; l-1-pyrenylalanine; l-2-aminocaproic acid; l-allylglycine; l-homocyclohexylalanine; and N- (2-hydroxy-4-methoxy-Bzl) -Gly-OH.

Examples of amino acid analogs of arginine and lysine include citrulline; l-2-amino-3-guanidinopropionic acid; l-2-amino-3-ureidopropionic acid; l-citrulline; lys (Me) 2-OH; lys (N3) -OH; n δ -benzyloxycarbonyl-L-ornithine; n ω -nitro-D-arginine; n ω -nitro-L-arginine; alpha-methyl-ornithine; 2, 6-diaminopimelic acid; l-ornithine; (N δ -1- (4, 4-dimethyl-2, 6-dioxo-cyclohexen-1-ylidene) ethyl) -D-ornithine; (N δ -1- (4, 4-dimethyl-2, 6-dioxo-cyclohexen-1-ylidene) ethyl) -L-ornithine; (N δ -4-methyltrityl) -D-ornithine; (N δ -4-methyltrityl) -L-ornithine; d-ornithine; l-ornithine; arg (Me) (Pbf) -OH; arg (Me)2-OH (asymmetric); arg (Me)2-OH (symmetrical); lys (ivDde) -OH; lys (me) 2-oh.hcl; lys (Me3) -OH chloride; n ω -nitro-D-arginine; and N ω -nitro-L-arginine.

Examples of amino acid analogs of aspartic acid and glutamic acid include α -methyl-D-aspartic acid; alpha-methyl-glutamic acid; alpha-methyl-L-aspartic acid; gamma-methylene-glutamic acid; (N- γ -ethyl) -L-glutamine; [ N- α - (4-aminobenzoyl) ] -L-glutamic acid; 2, 6-diaminopimelic acid; l- α -amino suberic acid; d-2-aminoadipic acid; d- α -amino suberic acid; alpha-aminopimelic acid; iminodiacetic acid; l-2-aminoadipic acid; threo- β -methyl-aspartic acid; gamma-carboxy-D-glutamic acid gamma, gamma-di-tert-butyl ester; gamma-carboxy-L-glutamic acid gamma, gamma-di-tert-butyl ester; glu (Oall) -OH; L-Asu (OtBu) -OH; and pyroglutamic acid.

Examples of amino acid analogs of cysteine and methionine include Cys (farnesyl) -OH, Cys (farnesyl) -Ome, α -methyl-methionine, Cys (2-hydroxyethyl) -OH, Cys (3-aminopropyl) -OH, 2-amino-4- (ethylthio) butyric acid, buthionine sulfoximine, ethionine, methionine methanesulfonyl chloride, selenomethionine, cysteine, [2- (4-pyridyl) ethyl ] -DL-penicillamine, [2- (4-pyridyl) ethyl ] -L-cysteine, 4-methoxybenzyl-D-penicillamine, 4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine, and methionine, 4-methylbenzyl-L-penicillamine, benzyl-D-cysteine, benzyl-L-cysteine, benzyl-DL-homocysteine, carbamoyl-L-cysteine, carboxyethyl-L-cysteine, carboxymethyl-L-cysteine, diphenylmethyl-L-cysteine, ethyl-L-cysteine, methyl-L-cysteine, tert-butyl-D-cysteine, trityl-L-homocysteine, trityl-D-penicillamine, cystathionine, homocystine, L-homocystine, (2-aminoethyl) -L-cysteine, selenium-L-cystine, cystathionine, beta-cysteine, beta-form factor, beta-, Cys (StBu) -OH and acetamidomethyl-D-penicillamine.

Examples of amino acid analogs of phenylalanine and tyrosine include beta-methyl-phenylalanine, beta-hydroxyphenylalanine, alpha-methyl-3-methoxy-DL-phenylalanine, alpha-methyl-D-phenylalanine, alpha-methyl-L-phenylalanine, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, 2, 4-dichloro-phenylalanine, 2- (trifluoromethyl) -D-phenylalanine, 2- (trifluoromethyl) -L-phenylalanine, 2-bromo-D-phenylalanine, 2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine, 2-chloro-L-phenylalanine, beta-hydroxyphenylalanine, alpha-methyl-3-methoxy-DL-phenylalanine, alpha-methyl-D-phenylalanine, alpha-methyl-L-phenylalanine, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, 2, 4-dichloro-phenylalanine, 2- (trifluoromethyl) -D-phenylalanine, 2- (trifluoromethyl) -L-phenylalanine, 2-bromo-D-phenylalanine, 2-chloro-L-phenylalanine, beta-D-phenylalanine, beta-D-phenylalanine, 2-D-phenylalanine, and a, 2-cyano-D-phenylalanine, 2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine, 2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine, 2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine, 2-nitro-L-phenylalanine, 2,4, 5-trihydroxy-phenylalanine, 3,4, 5-trifluoro-D-phenylalanine, 3,4, 5-trifluoro-L-phenylalanine, 3, 4-dichloro-D-phenylalanine, 3, 4-dichloro-L-phenylalanine, 2-fluoro-L-phenylalanine, 2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine, 2-hydroxy-L-phenylalanine, 2,4, 5-trifluoro-D-phenylalanine, 3, 4-dichloro-D-phenylalanine, 2-methyl-L-phenylalanine, 2-methyl-D-phenylalanine, 3, 4-methyl-phenylalanine, 2-methyl-L-phenylalanine, 2-methyl-L-phenylalanine, 2-phenyl alanine, 2-methyl-4, 2-D-phenylalanine, 4,5, 3,5, and a, 3, 4-difluoro-D-phenylalanine, 3, 4-difluoro-L-phenylalanine, 3, 4-dihydroxy-L-phenylalanine, 3, 4-dimethoxy-L-phenylalanine, 3,5,3' -triiodo-L-thyronine, 3, 5-diiodo-D-tyrosine, 3, 5-diiodo-L-thyronine, 3- (trifluoromethyl) -D-phenylalanine, 3- (trifluoromethyl) -L-phenylalanine, 3-amino-L-tyrosine, 3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine, 3-dihydroxy-L-phenylalanine, 3-hydroxy-L-phenylalanine, 3, 4-hydroxy-L-phenylalanine, 3, 5-hydroxy-L-tyrosine, 3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine, 3-hydroxy-L-thyronine, 3-hydroxy-L-thyronine, 3, 4-hydroxy-L-thyronine, 3,5,3, 4, 3,4, L-D, L-phenylalanine, L-or a, 3-chloro-D-phenylalanine, 3-chloro-L-tyrosine, 3-cyano-D-phenylalanine, 3-cyano-L-phenylalanine, 3-fluoro-D-phenylalanine, 3-fluoro-L-phenylalanine, 3-fluoro-tyrosine, 3-iodo-D-phenylalanine, 3-iodo-L-tyrosine, 3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine, 3-methyl-L-phenylalanine, 3-nitro-D-phenylalanine, 3-D-tyrosine, and its derivatives, 3-nitro-L-phenylalanine, 3-nitro-L-tyrosine, 4- (trifluoromethyl) -D-phenylalanine, 4- (trifluoromethyl) -L-phenylalanine, 4-amino-D-phenylalanine, 4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine, 4-benzoyl-L-phenylalanine, 4-bis (2-chloroethyl) amino-L-phenylalanine, 4-bromo-D-phenylalanine, 4-bromo-L-phenylalanine, 4-chloro-D-phenylalanine, 4-chloro-L-phenylalanine, 4-nitro-L-phenylalanine, 4- (trifluoromethyl) -D-phenylalanine, 4- (trifluoromethyl) -L-phenylalanine, 4- (4-amino-D-phenylalanine, 4-bromo-L-phenylalanine, 4-D-phenylalanine, 4-bromo-L-phenylalanine, 4-L-phenylalanine, 4-D-L-phenylalanine, 4-D-phenylalanine, and a pharmaceutically acceptable salt thereof, 4-cyano-D-phenylalanine, 4-cyano-L-phenylalanine, 4-fluoro-D-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-D-phenylalanine, 4-iodo-L-phenylalanine, homophenylalanine, thyroxine, 3-diphenylalanine, thyronine, ethyl-tyrosine and methyl-tyrosine.

Examples of amino acid analogues of proline include 3, 4-dehydro-proline, 4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid and trans-4-fluoro-proline.

Examples of amino acid analogs of serine and threonine include 3-amino-2-hydroxy-5-methylhexanoic acid, 2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutyric acid, 2-amino-3-methoxybutyric acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutyric acid, and α -methylserine.

Examples of amino acid analogs of tryptophan include alpha-methyl-tryptophan; β - (3-benzothienyl) -D-alanine; beta- (3-benzothienyl) -L-alanine; 1-methyl-tryptophan; 4-methyl-tryptophan; 5-benzyloxy-tryptophan; 5-bromo-tryptophan; 5-chloro-tryptophan; 5-fluoro-tryptophan; 5-hydroxy-tryptophan; 5-hydroxy-L-tryptophan; 5-methoxy-tryptophan; 5-methoxy-L-tryptophan; 5-methyl-tryptophan; 6-bromo-tryptophan; 6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan; 6-methyl-tryptophan; 7-benzyloxy-tryptophan; 7-bromo-tryptophan; 7-methyl-tryptophan; d-1,2,3, 4-tetrahydro-norharman-3-carboxylic acid; 6-methoxy-1, 2,3, 4-tetrahydro norharman-1-carboxylic acid; 7-azatryptophan; l-1,2,3, 4-tetrahydro-norharman-3-carboxylic acid; 5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.

In some embodiments, the modified axon growth attractant factor-1 peptide may comprise one or more non-essential amino acids. A non-essential amino acid residue can be a residue that can be altered from the wild-type sequence of a polypeptide without abolishing or substantially altering its essential biological or biochemical activity (e.g., receptor binding or activation).

In some embodiments, the modified axon growth attractant factor-1 peptide may comprise one or more conservative amino acid substitutions. In some embodiments, a conservative amino acid substitution is a substitution in which an amino acid residue is replaced with an amino acid residue having a side chain. Amino acids with basic side chains include Arg, His and Lys, amino acids with acidic side chains include Asp and Glu, amino acids with uncharged polar side chains include Asn, Cys, gin, Gly, Ser, Thr and Tyr, amino acids with nonpolar side chains include Ala, Ile, Leu, Met, Phe, Pro, Trp and Val, amino acids with I-branched side chains include Ile, Thr and Val, and amino acids with aromatic side chains include His, Phe, Trp and Tyr. In some embodiments, the conservative amino acid substitution is a very highly conservative substitution, a highly conservative substitution, or a conservative substitution as shown in the following table:

the modified neurite growth-inducing factor-1 peptides are superior to full-length neurite growth-inducing factor-1 proteins because they are shorter in size and, therefore, may 1) be conveniently and successfully produced in large quantities, 2) be more affordable, 3) activate protective pathways with smaller or reduced side effects compared to full-length neurite growth-inducing factor-1, 4) be more easily delivered to a target organ of a subject, and 5) be more stable and active during treatment to have greater protective effects. The modified neurite growth attractant factor-1 peptide is superior to prior art peptides in that the modified neurite growth attractant factor-1 peptide exhibits superior activity over prior art peptides.

Based on the sequence similarities and differences between the modified neurite growth attractant factor-1 peptides exemplified herein and the prior art peptides, it is believed that substitution of the first amino acid residue (X1 of SEQ ID NO:1, as described herein, and the first cysteine, amino acid residue at position 1 of the "core sequence" of the prior art peptides) is responsible for conferring superior activity to the modified neurite growth attractant factor-1 peptide. The superior activity conferred by the X1 amino acid position is unexpected because serine is considered by those skilled in the art to be a very highly conservative substitution of cysteine. Thus, one skilled in the art would expect that modification of the prior art peptide V1 to have a serine instead of a cysteine at amino acid position 1 would result in substantially similar activity. However, as shown in table 3, the replacement of cysteine by serine resulted in an increase in infarct size reduction of nearly 30% ((71-55)/55-29%).

Because the modified neurite growth attractant factor-1 peptides are demonstrated to significantly reduce infarct size in cardiac tissue, one or more of the modified neurite growth attractant factor-1 peptides can be used to treat acute myocardial infarction in a subject. In some embodiments, one or more modified axon growth-inducing factor-1 peptides can be used to treat, inhibit, or reduce ischemia/reperfusion injury in cardiac tissue of a subject. As used herein, the term "ischemia/reperfusion injury" (I/R injury) refers to an injury to an organ (e.g., heart) that results from placing the organ in an ischemic state, such as due to a thromboembolic event, surgery, or cardiac arrest. Clinically relevant conditions include occlusion of coronary arteries/branches that occurs during myocardial infarction (ischemia). Treatment with the Percutaneous Transluminal Coronary Angioplasty (PTCA) procedure produces a reperfusion condition known to cause additional injury, which however can be protected by pharmacological post-conditioning (administration of the modified axon growth-inducing factor-1 peptide during the reperfusion phase). Thus, in some embodiments, the invention also relates to acute treatment of myocardial infarction by administering one or more modified axotrophin-1 peptides (e.g., intravenously) alone or in combination with a PTCA/drug eluting stent. In some embodiments, the modified neurite growth attractant factor-1 peptide may be used to reduce or inhibit infarct size in cardiac tissue and/or to treat, inhibit or reduce damage to cardiac tissue caused by myocardial infarction.

Because the modified neurite growth attractant factor-1 peptide reduces infarct size in heart tissue like prior art peptides, it is expected that the modified neurite growth attractant factor-1 peptide will induce ERK1/2, eNOS, in addition to a reduction in size that is significantly greater than that observed for prior art peptides_s1177And/or eNOS_s1179And induces nitric oxide production to at least the same extent as the prior art peptides. Thus, in some embodiments, one or more modified axon growth attractant factor-1 peptides are used to induce ERK1/2, eNOS, in a subject_s1177And/or eNOS_s1179Phosphorylation of (2). Similarly, it is believed that the modified neurite growth attractant factor-1 peptide can induce nitric oxide production to at least the same extent as prior art peptides. Thus, in some embodiments, one or more modified neurite growth attractant factor-1 peptides are used to increase nitric oxide production in a subject.

Peptide compositions and delivery

Administration of one or more modified axon growth-inducing factor-1 peptides can be accomplished by direct administration, or by administration of one or more nucleic acid molecules encoding one or more modified axon growth-inducing factor-1 peptides.

In some embodiments, a therapeutically effective amount of one or more peptide fragments according to the present invention is administered to a subject. As used herein, a "therapeutically effective amount" refers to an amount that can be used to treat, alleviate, ameliorate, prevent or inhibit a given disease or disorder (such as I/R injury or symptoms thereof) in a subject as compared to a control (such as a placebo). For example, in some embodiments, a therapeutically effective amount is an amount that has a beneficial effect on the signs and/or symptoms of I/R damage to cardiac tissue in a subject. In some embodiments, a therapeutically effective amount is an amount that inhibits or reduces the signs and/or symptoms of I/R injury as compared to a control. Signs and symptoms of I/R damage to cardiac tissue are known in the art and include sudden chest pain (usually radiating to the left arm or neck), shortness of breath, nausea, vomiting, palpitations, sweating, and anxiety. In some embodiments, a therapeutically effective amount is an amount sufficient to increase phosphorylation of ERK1/2, eNOS, or both, and/or increase nitric oxide production in a subject, as compared to a control. In some embodiments, a therapeutically effective amount is an amount sufficient to activate the subject's cardioprotective mechanism as compared to a control. In some embodiments, a therapeutically effective amount of one or more modified axon growth attractant factor-1 peptides is within the following range: about 1ng/kg to about 100mg/kg body weight, about 0.001mg/kg to about 100mg/kg body weight, about 0.01mg/kg to about 10mg/kg body weight, about 0.01mg/kg to about 5mg/kg body weight, about 0.01mg/kg to about 3mg/kg body weight, about 0.01mg/kg to about 2mg/kg body weight, about 0.01mg/kg to about 1mg/kg body weight, or about 0.01mg/kg to about 0.5mg/kg body weight. In some embodiments, one or more modified axon growth-inducing factor-1 peptides in an amount of about 5mg/kg to about 10mg/kg body weight are administered to a subject at or immediately after ischemia and reperfusion or I/R injury in the subject. In some embodiments, one or more modified axotrophin-1 peptides are administered daily for a given period of time, e.g., about 4 weeks, at about 1ng/kg body weight to about 25ng/kg body weight, preferably about 10ng/kg body weight to about 20ng/kg body weight, and more preferably about 15ng/kg body weight to treat, prevent, or inhibit restenosis following angioplasty.

It should be noted that treatment of a subject with a therapeutically effective amount may be administered as a single dose or as a series of several doses. The dosage for treatment may be increased or decreased over the course of a given treatment. The optimal dosage for a given set of conditions and a given subject can be determined by one skilled in the art using art-recognized dosimetric assays and/or diagnostic assays. Dosimetry assays and/or diagnostic assays can be used to monitor and adjust dosages during the course of treatment. In some embodiments, one or more axon growth-inducing factor-1 compounds are administered in the form of a composition.

In some embodiments, these compositions comprise, consist essentially of, or consist of one or more modified axon growth-inducing factor-1 peptides. As used herein, a composition "comprising" one or more modified axon growth-inducing factor-1 peptides means that the composition may comprise other compounds, including proteins (e.g., prior art peptides) that are not modified axon growth-inducing factor-1 peptides. As used herein, a composition "consisting essentially of" one or more modified axon growth attractant factor-1 peptides means that the composition may comprise proteins other than the modified axon growth attractant factor-1 peptides, so long as the additional proteins do not substantially alter the activity or function of the modified axon growth attractant factor-1 peptides contained in the composition. As used herein, a composition "consisting of" one or more modified axon growth-inducing factor-1 peptides means that the composition does not comprise a protein other than the one or more modified axon growth-inducing factor-1 peptides. Compositions comprised of one or more modified axon growth attractant factor-1 peptides can include components other than proteins, e.g., pharmaceutically acceptable carriers, surfactants, preservatives, and the like. In some embodiments, a composition consisting of one or more modified axon growth attractant factor-1 peptides may comprise trace contaminants, which may include peptide contaminants, e.g., smaller fragments of the one or more modified axon growth attractant factor-1 peptides, which may result from, e.g., synthesis, subsequent processing, storage conditions, and/or protein degradation of the one or more modified axon growth attractant factor-1 peptides.

In some embodiments, these compositions may comprise, consist essentially of, or consist of one or more purified modified axon growth-inducing factor-1 peptides. As used herein, a "purified" modified axon growth attractant factor-1 peptide means that an amount of the macromolecular components that are naturally associated with the modified axon growth attractant factor-1 peptide have been removed from the modified axon growth attractant factor-1 peptide. As used herein, a composition comprising, consisting essentially of, or consisting of one or more purified modified axon growth attractant factor-1 peptides means that the composition does not include an amount of macromolecular components naturally associated with the one or more modified axon growth attractant factor-1 peptides and/or reagents for synthesizing the modified axon growth attractant factor-1 peptides. In some embodiments, the amount removed from the one or more modified neurite outgrowth attractant factor-1 peptides (or not present in the composition) is at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the macromolecular components and/or agents. In some embodiments, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the macromolecular component with which the one or more modified axotrophin-1 peptides are naturally associated and/or the agent used to synthesize the one or more modified axotrophin-1 peptides is removed from the composition. In some embodiments, the compositions of the invention consist only of one or more modified axon growth-inducing factor-1 peptides (e.g., one or more modified axon growth-inducing factor-1 peptides in solid or crystalline form).

In some embodiments, a composition according to the invention comprises one or more modified axon growth-inducing factor-1 peptides and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that is added to the composition by hand, which is generally non-toxic to the intended recipient and does not significantly inhibit the activity of the one or more modified neurite growth attractant factor-1 peptides contained in the composition. In some embodiments, a composition according to the present invention may comprise one or more excipients, diluents, adjuvants, preservatives, solubilizers, buffers, thickeners, gelling agents, foaming agents, surfactants, binders, suspending agents, disintegrants, wetting agents, solvents, plasticizers, fillers, colorants, dispersants, flavorants, and/or the like as is known in the art.

Compositions according to the invention typically comprise about 0.1% to 99% of one or more modified neurite-growth-inducing factor-1 peptides. In some embodiments, a composition according to the invention comprises one or more modified axon growth-inducing factor-1 peptides and one or more prior art peptides. In some embodiments, a composition according to the invention comprises one or more modified axon growth-inducing factor-1 peptides and full-length axon growth-inducing factor-1. In some embodiments, the composition is a synergistic composition, e.g., a composition comprising a modified axon growth attractant factor-1 peptide according to the invention and a second protein, which may be a second modified axon growth attractant factor-1 peptide according to the invention, or e.g., a full length axon growth attractant factor-1 protein or a prior art peptide, in synergistic amounts.

In some embodiments, one or more modified axon growth attractant factor-1 peptides are included in the compositions of the invention in the form of a free acid or a free base. In some embodiments, the one or more modified axon growth attractant factor-1 peptides are included in the composition in the form of a pharmaceutically acceptable salt (such as an acid addition salt or a base addition salt). Pharmaceutically acceptable salts refer to any salt form of the one or more modified axon growth-inducing factor-1 peptides that is generally non-toxic to the intended recipient and does not significantly inhibit the activity of the one or more modified axon growth-inducing factor-1 peptides or other active agents contained in the composition. In some embodiments, the one or more modified axon growth-inducing factor-1 peptides are provided in the form of a hydrate or a prodrug.

Compositions comprising one or more modified axon growth-inducing factor-1 peptides can be administered by systemic routes and/or by local routes. Suitable routes of administration illustratively include intravenous, oral, buccal, parenteral, intrathecal, intracerebroventricular, intraperitoneal, intracardiac, intraarterial, intravesical, transocular, intraocular, rectal, vaginal, subcutaneous, intradermal, transdermal, intramuscular, topical, intranasal, and transmucosal. In some embodiments, one or more modified axon growth-inducing factor-1 peptides and compositions thereof are administered intravenously or by intraventricular injection, for example, during angioplasty for acute MI treatment or open heart surgery.

In some embodiments, modified axon growth attractant factor-1 peptides and compositions according to the invention can be modified using methods and compositions known in the art to improve their biological half-life, stability, efficacy, bioavailability, bioactivity, or a combination of these properties.

For example, in some embodiments, a modified axon growth attractant factor-1 peptide can be cyclized to produce a cyclic peptide that is resistant to proteolytic degradation. Cyclization between the side chains or termini of the peptide sequence can be effected by disulfide bonds, lanthionines, two-carbon, hydrazine, or lactam bridges using methods known in the art.

In some embodiments, the modified neurite growth-inducing factor-1 peptide may be conjugated to a molecule such as vitamin B12, a lipid or an oxirane compound, e.g., polyethylene glycol (PEG), polyethylene oxide (PEO) and Polyoxyethylene (POE), methoxypolyethylene glycol (MPEG), monomethoxypolyethylene glycol (MPEG), diethylene glycol (mini-PEG), and the like. The oxirane compounds can be further functionalized with, for example, amine-binding terminal functional groups such as N-hydroxysuccinimide esters, N-hydroxysuccinimide carbonates, and aliphatic aldehydes, or thiol-binding groups such as maleimides, pyridyl disulfides, and vinyl sulfonates. Since amino groups (alpha-amino and epsilon-lysine amino) and cysteine residues are well suited for conjugation, the modified neurite growth attractant factor-1 peptide may further comprise one or more amino acid residues for conjugation to an ethylene oxide molecule or a carrier compound known in the art. The pharmacokinetic and pharmacodynamic properties of the conjugated peptide can be further modified by using specific linkers. For example, propyl and pentyl linkers can be used to provide conjugates with a relaxed conformation, while phenyl linkers can be used to provide a more compact conformation and shielding domains adjacent the C-terminus. Notably, the dense conformation is generally more effective in maintaining biological activity, extending plasma half-life, reducing proteolytic sensitivity and immunogenicity relative to the loose conformation.

In some embodiments, the modified neurite growth attractant factor-1 peptide may be hyperglycosylated using methods known in the art (e.g., in situ chemical reactions or site-directed mutagenesis). Hyperglycosylation can result in N-linked or O-linked glycosylation of proteins. The clearance rate of a given modified neurite growth attractant factor-1 peptide can be optimized by selecting a particular carbohydrate. For example, polysialic acid (PSA) is available in different sizes and its clearance depends on the type of polymer and the molecular size. Thus, for example, PSA having a high molecular weight may be suitable for delivery of a low molecular weight modified axogenes attractant factor-1 peptide, while PSA having a low molecular weight may be suitable for delivery of a high molecular weight modified axogenes attractant factor-1 peptide. The type of carbohydrate can be used to target the modified neurite growth attractant factor-1 peptide to a particular tissue or cell. For example, a modified axon growth-inducing factor-1 peptide conjugated to mannose can be recognized by mannose-specific lectins (e.g., mannose receptors and mannan-binding proteins) and taken up by the liver. In some embodiments, the modified axon growth attractant factor-1 peptides can be hyperglycosylated to improve their physical and chemical stability under different environmental conditions, e.g., to inhibit inactivation under stress conditions and reduce aggregation caused by production conditions and storage conditions.

In some embodiments, drug delivery systems, such as microparticles, nanoparticles (particles having a size in the range of 10nm to 1000 nm), nanoemulsions, liposomes, and the like, can be used to provide protection against sensitive proteins, extended release, reduced frequency of administration, improved patient compliance, and control of plasma levels. Various natural or synthetic microparticles and nanoparticles may be used, which may be biodegradable polymers and/or biocompatible polymers. The microparticles and nanoparticles may be made of lipids, polymers and/or metals. The polymeric microparticles and nanoparticles may be made from natural or synthetic polymers such as starch, alginate, collagen, chitosan, Polycaprolactone (PCL), polylactic acid (PLA), poly (lactide-co-glycolide) (PLGA), and the like. In some embodiments, the nanoparticle is a Solid Lipid Nanoparticle (SLN), a carbon nanotube, a nanosphere, a nanocapsule, or the like. In some embodiments, the polymer is hydrophilic. In some embodiments, the polymer is a thiolated polymer.

Since the rate and extent of drug release from microparticles and nanoparticles may depend on the composition and method of manufacture of the polymer, a given composition and method of manufacture, e.g., spray drying, lyophilization, micro extrusion, and double emulsion, may be selected to impart a desired drug release profile. Since peptide fragments incorporated into or onto microparticles or nanoparticles may be susceptible to denaturation at the aqueous-organic interface during formulation development, different stabilizing excipients and compositions may be used to prevent aggregation and denaturation. For example, PEG and sugars (e.g., PEG (MW 5000) and maltose) can be added to the composition along with alpha-chymotrypsin to reduce aggregation and denaturation. In addition, chemically modified peptide fragments, for example, conjugated peptide fragments and hyperglycosylated peptide fragments, may be employed.

Protein stability may also be achieved by the chosen manufacturing method. For example, to prevent degradation at the water-organic interface, a so-called "water-organic" interface may be usedNon-aqueous methods of the art. Solid-state peptide fragments may also be encapsulated using a solid-in-water-in-oil (s/o/w) method, e.g., spray-dried or spray-freeze-dried peptide fragments or peptide-loaded solid nanoparticles may be encapsulated in microspheres using the s/o/w method. Hydrophobic Ion Pairing (HIP) complexation can be used to enhance protein stability and improveHigh encapsulation efficiency of micro-and nano-particles. In Hydrophobic Ion Pairing (HIP) complexation, an ionizable functional group of a peptide is complexed with an ion pairing agent (e.g., a surfactant or polymer) containing an oppositely charged functional group, resulting in the formation of a HIP complex in which hydrophilic protein molecules are present in the form of a hydrophobic complex.

In some embodiments, liposomes of synthetic or natural origin and of various sizes (e.g., 20nm to several hundred microns) can be used to deliver peptide fragments. Depending on the preparation method, liposomes can be small unilamellar vesicles (25nm to 50nm), large unilamellar vesicles (100nm to 200nm), large unilamellar vesicles (1 μm to 2 μm), and multilamellar vesicles (MLV; 1 μm to 2 μm). The delivered peptide fragments can be encapsulated into liposomes or adsorbed on a surface. The size and surface characteristics of the liposomes can be optimized to achieve the desired results. For example, unilamellar and multilamellar liposomes provide sustained release hours to days after intravascular administration. Prolonged drug release can be via multivesicular liposomes (also known as multivesicular liposomes)Technology). Unlike ULV and MLV, multivesicular liposomes are composed of multiple non-concentric aqueous chambers surrounded by a network of lipid layers, where the lipid layer network confers an increased level of stability and longer duration of drug release. Liposomes can be further modified to achieve desired results. For example, liposomes may be pegylated or have other surface modifications in order to interfere with the recognition and uptake of the reticuloendothelial system and provide increased circulation time.

Exemplary liposomes suitable for use according to the invention include multilamellar vesicles (MLVs), oligolamellar vesicles (OLVs), Unilamellar Vesicles (UV), Small Unilamellar Vesicles (SUVs), medium-sized unilamellar vesicles (MUVs), Large Unilamellar Vesicles (LUVs), Giant Unilamellar Vesicles (GUVs), multivesicular vesicles (MW), unilamellar or oligolamellar vesicles (REVs) prepared by reverse-phase evaporation, multilamellar vesicles (MLV-REVs) prepared by reverse-phase evaporation, stable multilamellar vesicles (SPLV), frozen and thawed MLVs (fatmlv), Vesicles (VETs) prepared by extrusion methods, vesicles (FPVs) prepared by french press filtration, FUVs (DRVs) prepared by fusion, dehydrated-rehydrated vesicles (DRVs) and vesicles (BSVs).

Liposomes can contain additional lipids, for example, carrier lipids including palmitoyl phosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), Phosphatidic Acid (PA), Phosphatidylglycerol (PG), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), Distearoylphosphatidylcholine (DSPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylglycerol (DPPG), Distearoylphosphatidylglycerol (DSPG), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidic acid (DPPA); dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), Dipalmitoylphosphatidylserine (DPPS), Dimyristoylphosphatidylserine (DMPS), Distearoylphosphatidylserine (DSPS), Dipalmitoylphosphatidylethanolamine (DPPE), Dimyristoylphosphatidylethanolamine (DMPE), Distearoylphosphatidylethanolamine (DSPE), and the like, or combinations thereof. In some embodiments, the liposome further comprises a sterol (e.g., cholesterol).

In some embodiments, micelles may be used to deliver modified axon growth-inducing factor-1 peptides. Phospholipids such as DSPE-PEG, copolymehzed systems PEG-PE, PLA-PEG and hyperbranched poly ([ amine-ester ] -co- [ d, l-lactide ]) and polyion complexes can be used to enhance stability and pharmacokinetic properties.

Thermosensitive gels may be used to deliver the modified neurite growth attractant factor-1 peptide. Thermally reversible block copolymers comprising PEG, PCL, PLA, poly (glycolide), PLGA, poly (N-isopropylacrylamide), polyethylene oxide, chitosan, and the like can be used to provide controlled release of peptide fragments. Examples of thermosensitive gels include PLGA-PEG-PLGA triblock copolymer gels and Pluronic F-127(PF 127). Polyelectrolyte complexes and/or pegylation can be used to provide sustained release of proteins from the gel. Microparticles and/or nanoparticles may also be used in combination with the gel to provide sustained drug delivery.

The modified neurite growth attractant factor-1 peptide may be chemically synthesized or recombinantly expressed in a cellular or acellular system. The synthetic method comprises liquid phase synthesis, solid phase synthesis and microwave-assisted peptide synthesis. Peptide fragments can be modified by: acylation, alkylation, amidation, arginylation, polyglutamylation, polysacchariylation, butyrylation, gamma-carboxylation, glycosylation, malonylation, hydroxylation, iodination, nucleotide addition (e.g., ADP-ribosylation), oxidation, phosphorylation, adenylylation, propionylation, S-glutathionylation, S-nitrosylation, succinylation, sulfation, glycation, palmitoylation, myristoylation, prenylation, or prenylation (e.g., farnesylation or geranylgeranylation), glycosylphosphatidylinositol, lipoylation, flavin moiety (e.g., FMN or FAD) attachment, heme C attachment, phosphopantetheinylation, retinoid Schiff base formation, diphtheria amide formation, ethanolamine phosphoglycerol attachment, carboxyputrescine lysine residue (hypusine) formation, biotinylation, pegylation, gamma-carboxylation, glycosylation, malonylation, glycosylation, prenylation, or a, ISG, SUMU, ubiquitination, bacteroid, prokaryotic ubiquitination, citrullination, deamidation, eliminatation (elimidation), carbamylation, or combinations thereof.

Compositions comprising one or more modified axon growth attractant factor-1 peptides can be subjected to one or more cycles of purification steps or concentration steps known in the art to remove impurities and/or to concentrate peptide fragments. Thus, in some embodiments, the present invention provides peptide compositions having a purity and/or composition that does not occur in nature. In some cases, the peptide composition is at most 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure peptide fragment. In some cases, the peptide composition is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure peptide fragment. In some cases, the composition is free of impurities. In some cases, the amount of peptide fragments in the peptide composition is up to 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% by weight of the total composition. In some cases, the amount of peptide fragments in the peptide composition is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% by weight of the total composition.

The compositions of the present invention include pharmaceutical compositions comprising one or more modified neurite-growth-inducing factor-1 peptides. The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a subject. Pharmaceutical compositions generally comprise an effective amount of an active agent, e.g., one or more modified axon growth-inducing factor-1 peptides according to the invention, and a pharmaceutically acceptable carrier. The term "effective amount" refers to a dose or amount sufficient to produce the desired result. The desired result may include objective or subjective improvement of the recipient of the dose or amount, e.g., long-term survival, effective prevention of a disease state, and the like. In some embodiments, an "effective amount" is less than a therapeutically effective amount. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more axon growth-inducing factor-1 compounds. The pharmaceutical composition according to the invention may further comprise one or more supplements.

One or more modified axon growth attractant factor-1 peptides according to the invention may be administered to a subject, preferably in the form of a pharmaceutical composition. Preferably, the subject is a mammal, more preferably, the subject is a human. Preferred pharmaceutical compositions are those comprising at least one modified neurite-1 peptide in a therapeutically effective amount and a pharmaceutically acceptable vehicle.

The pharmaceutical compositions of the present invention may be formulated for the intended route of delivery using methods known in the art, including intravenous, intramuscular, intraperitoneal, subcutaneous, intraocular, intrathecal, intraarticular, intrasynovial, cerebral cistern, intrahepatic, intralesional injection, intracranial injection, infusion and/or inhalation routes of administration. The pharmaceutical composition according to the invention may comprise one or more of: pH buffered solutions, adjuvants (e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents), liposomal formulations, nanoparticles, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. The compositions and formulations of the present invention may be optimized to improve stability and efficacy using methods known in the art. See, e.g., Carra et al (2007) Vaccine 25: 4149-.

The compositions of the invention may be administered to a subject by any suitable route, including oral, transdermal, subcutaneous, intranasal, inhalation, intramuscular, and intravascular administration. It will be appreciated that the preferred route of administration and pharmaceutical formulation will vary with the condition and age of the subject, the nature of the condition to be treated, the desired therapeutic effect and the particular modified axon growth-inducing factor-1 peptide used.

As used herein, "pharmaceutically acceptable vehicle" or "pharmaceutically acceptable carrier" are used interchangeably and refer to solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with drug administration and that comply with applicable standards and regulations (e.g., pharmacopoeia standards for drug administration set forth in the united states pharmacopoeia and national formulary (USP-NF) books). Thus, for example, unsterilized water is excluded as a pharmaceutically acceptable carrier at least for intravenous administration. Pharmaceutically acceptable vehicles include those known in the art. See, e.g., Remington, The Science and Practice of pharmacy, 20 th edition, (2000) Lippincott Williams & Wilkins.

The pharmaceutical compositions of the present invention may be provided in dosage unit form. As used herein, "dosage unit form" refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit comprising a predetermined amount of one or more modified axon growth attractant factor-1 peptides calculated to produce the desired therapeutic effect, and a required pharmaceutically acceptable carrier. The specification for the dosage unit forms of the invention is dictated by and directly dependent on the unique characteristics of a given modified neurite-inducing factor-1 peptide and the desired therapeutic effect to be achieved, as well as limitations inherent in the art in compounding such active compounds for treating an individual.

Toxicity and therapeutic efficacy of modified neurite growth attractant factor-1 peptides and compositions thereof according to the invention can be determined using cell cultures and/or experimental animals and pharmaceutical protocols in the art. For example, lethal dose LC can be determined by methods in the art₅₀(dose lethal to 50% of the population, expressed as concentration x time of exposure) or LD₅₀(dose lethal to 50% of the population), and ED₅₀(a therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as LD₅₀/ED₅₀The ratio of. Modified axon growth attractant factor-1 peptides exhibiting a large therapeutic index are preferred. Although modified neurite growth attractant factor-1 peptides can be used which cause toxic side effects, care should be taken to design a delivery system that targets such compounds to the site of treatment to minimize potential damage to uninfected cells and thereby reduce side effects.

Data obtained from cell culture assays and animal studies can be used to formulate a series of doses for use in humans. Preferred dosages provide for inclusion of ED₅₀In a series of circulating concentrations, there was little or no toxicity. The dosage may vary depending upon the dosage form employed and the route of administration utilized. Therapeutically effective amounts and dosages of one or more modified neurite growth attractant factor-1 peptides according to the invention can be estimated initially from cell culture assays. The dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC as determined in cell culture₅₀(i.e., the concentration of test compound that achieves half-maximal inhibition of symptoms). This information can be used to more accurately determine useful doses in humans. For example, levels in plasma can be measured by high performance liquid chromatography. In addition, the dosage appropriate for a given subject may be determined by an attending physician or qualified medical practitioner based on various clinical factors.

The following examples are intended to illustrate the invention without limiting it.

The modified neurite growth attractant factor-1 peptide induces unexpectedly superior cardioprotective effects against ischemia/reperfusion injury

Axon growth attractant factor-1 and prior art peptides V1, V2 and V3 are in the heart via DCC/ERKl/2/eNOS_s1177Cardioprotective substances with/NO signalling. When in ischemia/reperfusion injuryThe infarct size of hearts perfused with V1, V2, or V3 was significantly reduced when previously administered (control I/R: 39.3. + -. 0.3% vs I/R + V1: 14.6. + -. 2.3% vs I/R + V2: 23.0. + -. 2.8% vs I/R + V3: 18.8. + -. 0.8%, p)<0.001). Thus, the V1, V2 and V3 prior art peptides are very effective in inducing cardioprotective effects against ischemia/reperfusion injury. In addition, all three prior art peptides also induced a potent cardioprotective effect against I/R injury when administered after ischemia/reperfusion injury (control I/R: 37.6. + -. 1.3% vs I/R + V1: 17.6. + -. 3.2% vs I/R + V2: 20.6. + -. 1.7% vs I/R + V3: 15.8. + -. 2.0%, p<0.001)。

Fragments of the prior art peptides (e.g., Vl-9aa, V2-10aa, and V3-11aa) also significantly reduced infarct size compared to the control (control I/R: 37.6 + -1.3% vs I/R + Vl-9 aa: 16.8 + -2.2%; I/R + V2-10 aa: 18.6 + -1.7%; I/R + V3-11 aa: 16.7 + -3.0%, p < 0.001%). See table 1:

TABLE 1

Thus, the ability of the modified neurite growth attractant factor-1 peptide to reduce infarct size was similarly measured and the results are summarized in table 2:

TABLE 2

Comparison of the percent reduction in infarct size conferred by the modified axotrophin-1 peptide with the percent reduction in infarct size conferred by the closest prior art peptide demonstrates that the activity of the modified axotrophin-1 peptide is significantly superior to that of the prior art peptide, as shown in table 3:

TABLE 3

Comparison	Improvement in MI reduction%
		V1P vs Vl-9aa	(77-55)/55＝40％
V2P vs V2-10aa	(58-51)/51＝14％
		V3P vs V3-llaa	(70-58)/70＝25％
V1S vs Vl-9aa	(71-55)/55＝29％
		V1T vs Vl-9aa	(64-55)/55＝16％
V1C vs Vl-9aa	(74-55)/55＝35％

Accordingly, in some embodiments, the present invention provides one or more modified axon growth-inducing factor-1 peptides that may be used to treat acute myocardial infarction in a subject. In some embodiments, the invention relates to treating myocardial infarction, reducing or inhibiting infarct size, and/or reducing or inhibiting I/R damage in a subject comprising administering to the subject a therapeutically effective amount of one or more modified neurite growth inducing factor-1 peptides before, during or after myocardial infarction or ischemia or reperfusion.

The modified axr-1 peptide is expected to exhibit a robust inhibitory effect on neointimal formation following endothelial injury, mimicking clinical restenosis following PCTA in a manner similar to the full-length axr-1 peptide. In addition, the modified neurite growth attractant factor-1 peptides are expected to attenuate restenosis by enhancing Endothelial Progenitor Cell (EPC) function to enhance re-endothelialization at the site of trauma (e.g., coronary endothelial cell injury caused by angioplasty procedures) and increasing nitric oxide production to inhibit proliferation and migration of Vascular Smooth Muscle Cells (VSMCs). Thus, in some embodiments, the invention relates to treating or inhibiting restenosis in a subject, comprising administering to the subject a therapeutically effective amount of one or more modified axogenes attractant factor-1 peptides before, during, or after endothelial cell injury in blood vessels.

Peptide sequences

The following is the amino acid sequence of the modified neurite growth attractant factor-1 peptide exemplified herein:

V1P：(mini-PEG)-CDCRHNTAG(SEQ ID NO:2)

V2P：(mini-PEG)-CLNCRHNTAG(SEQ ID NO:3)

V3P：(mini-PEG)-CPCKDGVTIGIT(SEQ ID NO:4)

V1S：SDCRHNTAG(SEQ ID NO:5)

V1T：TDCRHNTAG(SEQ ID NO:6)

V1C：CDCRHNTAG (SEQ ID NO:7) in which cysteine residues are linked by a disulfide bond

The following are the amino acid sequences of the prior art peptides exemplified herein:

V1：CKCNGHAARCVRDRDDSLVCDCRHNTAGPECDRCKPFHYDRPWQRATAREANEC(SEQ ID NO:8)

V2：CNCNLHARRCRFNMELYKLSGRKSGGVCLNCRHNTAGRHCHYCKEGYYRDMGKPITHRKAC(SEQ ID NO:9)

V3：CDCHPVGAAGKTCNQTTGQCPCKDGVTGITCNRCAKGYQQSRSPIAPC(SEQ ID NO:10)

Vl-9aa：CDCRHNTAG(SEQ ID NO:11)

V2-10aa：CLNCRHNTAG(SEQ ID NO:12)

V3-11aa：CPCKDGVTGIT(SEQ ID NO:13)

negative control V3-16 aa: HPVGAAGKTCNQTTGQ (SEQ ID NO:14)

Full length axon growth inducible factor-1 human sequence accession number GI 148613884.

Materials and methods

Various methods disclosed in PCT/US2011/038277 and PCT/US2015/023248 may be employed. Because the experiments herein show that the exemplified modified axon growth attractant factor-1 peptides exhibit superior activity over prior art peptides, the modified axon growth attractant factor-1 peptides can be used to replace or supplement therapeutic treatments that employ axon growth attractant factor-1 or other axon growth attractant factor-1 derivatives (such as prior art peptides).

Material

Purified mouse neurite growth-inducing factor-1 was purchased from R&D Systems (Minneapolis, MN, USA). Peptide fragments V1 (285-338 amino acids of human axon growth-inducing factor-1), V2(341-401aa), V3(404-451aa), Vl-9aa (304-312aa), V2-10aa (368-377aa), V3-16aa (407-422aa) and V3-11aa (423-433aa) were synthesized by GenicBioLimited (Shanghai, China). For phosphorylation of ERK1/2, ERK1/2 and eNOS_s1179Polyclonal antibodies with specificity were obtained from Cell Signaling Technology (Danvers, MA, USA). Monoclonal antibodies to eNOS were purchased from BD Biosciences (San lose, CA, USA).

Cell culture

Bovine aortic endothelial cells (BAEC, Cell Systems, Kirkland, WA, USA) were cultured in medium 199 containing 10% Fetal Bovine Serum (FBS) as described previously. See chaluppky and Cai, PNAS USA 2005; 9056-; and Nguyen and Cai, PNAS USA 2006; 103:6530-6535. One day after confluence, cells were starved overnight in media containing 5% FBS, then stimulated with axon growth attractant factor-1 protein or different peptide fragments, and harvested at different time points.

Western blot

For western blotting, approximately 20 μ g to 40 μ g of protein was separated by 10% SDS-PAGE, transferred to nitrocellulose membrane, and purified with phosphorylated ERK1/2, ERK1/2, eNOS and eNOS using methods known in the art_s1179(1:1,000) antibody was detected.See, for example, Gao et al, Journal of Molecular and Cellular diagnostics.2009; 47:752-760.

Langen doff (Langen Dorff) perfusion

Male C57BL/6J mice (8 to 12 weeks old) were obtained from Charles River Laboratories (Wilmington, MA, USA). Immediately after anesthesia with intraperitoneal injection of pentobarbital (60mg/kg), mouse hearts were harvested and the aorta cannulated with a 20-gauge stainless steel blunt-ended needle and transferred to a Raddedoff apparatus and immediately perfused retrograde with modified Krebs-Henseleit buffer (KHB) for 30 minutes as previously described. See Bouhidel et al Front Biosci (Landmark Ed) 2014; 566-; 48:1060-1070. The heart was then pre-perfused for 45 minutes with or without axon growth-inducing factor-1 (100ng/ml) or a different peptide fragment at the same molar concentration of 1.47nmol/L as axon growth-inducing factor-1, before undergoing an ischemia/reperfusion (I/R) injury (20 minutes global ischemia followed by 60 minutes reperfusion with or without axon growth-inducing factor-1 or a different peptide fragment). The hearts were then harvested for analysis of infarct size. For post-conditioning treatment with peptide fragments, the heart underwent KHB perfusion for 40 minutes, global ischemia for 20 minutes, and reperfusion with different peptide fragments for 60 minutes.

Infarct size analysis

At the end of the I/R protocol, the heart was sectioned perpendicular to the long axis of the heart at 1mm intervals and stained with 1% triphenyltetrazolium chloride (TTC) in PBS for 10 minutes at room temperature. After washing once with PBS, sections of the heart were fixed in 10% formalin overnight. The heart slices were then digitally photographed using NIH Image 1.62 for area measurement. Infarct size is expressed as the ratio of infarct to risk zone (risk zone is the entire ventricular volume in this global ischemia model).

Statistical analysis

Densitometric data of western blots were obtained by the software Image J. The grouping data was analyzed by the software Gradpad Prism 6. All values are expressed as mean ± SEM. Comparisons were performed on two or more groups using one-way ANOVA analysis and Newman-Keuls test as post hoc tests. Statistical significance was set at p < 0.05.

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood in the art.

As used herein, the terms "subject," "patient," and "individual" are used interchangeably to refer to both human and non-human animals. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, as well as other veterinary subjects and test animals. In some embodiments of the invention, the subject is a mammal. In some embodiments of the invention, the subject is a human.

As used herein, the term "diagnosis" refers to the physical and proactive step of informing (i.e., verbally communicating or by writing (on, for example, paper or electronic media)) another party (e.g., a patient) of a diagnosis. Similarly, "providing a prognosis" refers to the physical and proactive step of informing (i.e., verbally communicating or by writing (on, for example, paper or electronic media)) another party (e.g., a patient) of the prognosis.

The use of the singular may include the plural unless specifically stated otherwise. As used in the specification and the appended claims, the singular forms "a," "an," and "the" may include plural referents unless the context clearly dictates otherwise.

As used herein, "and/or" means "and" or ". For example, "A and/or B" means "A, B or both A and B", "A, B, C and/or D" means "A, B, C, D, or a combination thereof", and the "A, B, C, D, or a combination thereof" means any subset of A, B, C and D, e.g., a single member subset (e.g., A or B or C or D), a two member subset (e.g., A and B; A and C; etc.), or a three member subset (e.g., A, B and C; or A, B and D; etc.), or all four members (e.g., A, B, C and D).

As used herein, the phrase "one or more of … …," e.g., "one or more of A, B and/or C" means "one or more of a," one or more of B, "" one or more of C, "" one or more of a and one or more of B, "" one or more of B and one or more of C, "" one or more of a and one or more of C, "and" one or more of a, one or more of B, and one or more of C.

The phrase "comprising, consisting essentially of, or consisting of a" is used as a tool to avoid page and translation cost overruns, and means that in some embodiments, a given thing in question: comprises, consists essentially of, or consists of A. For example, the sentence "in some embodiments, a composition comprises, consists essentially of, or consists of a" should be interpreted as if written as three separate sentences: "in some embodiments, the composition comprises a. In some embodiments, the composition consists essentially of a. In some embodiments, the composition consists of a. "

Similarly, a sentence reciting a string of alternatives should be interpreted as if a string of sentences were provided, such that each given alternative is provided by itself in the sentence. For example, the sentence "in some embodiments, the composition comprises A, B or C" should be interpreted as if written as three separate sentences: "in some embodiments, the composition comprises a. In some embodiments, the composition comprises B. In some embodiments, the composition comprises C. "as another example, the sentence" in some embodiments, the composition comprises at least A, B or C "should be interpreted as if written as three separate sentences: "in some embodiments, the composition comprises at least a. In some embodiments, the composition comprises at least B. In some embodiments, the composition comprises at least C. "

As used herein, the terms "protein," "polypeptide," "peptide," and "peptide fragment" are used interchangeably to refer to two or more natural and/or unnatural amino acids that are linked together, and are referred to herein in the sequences and formulae using the single letter amino acid designation. As used herein, "aa" is an abbreviation for "amino acid". For example, "9 aa" of "Vl-9 aa" means that the peptide is 9 amino acid residues in length.

To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as shown herein, but is limited only by the following claims.

Sequence listing

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