Tissue protective peptides and uses thereof

Abstract

The present invention is directed to novel tissue protective peptides. The tissue protective peptides of the invention may bind to a tissue protective receptor complex. In particular, the present invention is drawn to tissue protective peptides derived from or sharing consensus sequences with portions of cytokine receptor ligands, including Erythropoietin (EPO), that are not involved in the binding of the ligand to the receptor complex, e.g., to the EPO receptor homodimer. Accordingly, the tissue protective peptides of the invention are derived from the amino acid sequences of regions of cytokine receptor ligands that are generally located on or within the region of the ligand protein that is opposite of the receptor complex, i.e., are generally derived from amino acid sequences of regions of the ligand protein that face away from the receptor complex while the ligand is bound to the receptor. The invention is further directed to the consensus sequences for use in engineering a synthetic tissue protective peptide. These tissue protective peptides also include fragments, chimeras, as well as peptides designed to mimic the spatial localization of key amino acid residues within the tissue protective receptor ligands, e.g., EPO. The invention further encompasses methods for treating or preventing a disease or disorder using tissue protective peptides of the current invention. The invention also encompasses methods for enhancing excitable tissue function using tissue protective peptides of the current invention.

What is claimed is: 
     
         1 . An isolated polypeptide consisting of a sequence of no more than 30 amino acids and comprising the amino acid motif:
 (a) H 1 -N 1 -(X) n -N 2 -H 2 , wherein n is 0-5;   (b) H 1 -N 1 -(X) n -N 2 -L 1 , wherein n is 0-5; or   (c) L 1 -N 1 -(X) n -N 2 -H 1 , wherein n is 0-5;   and wherein H 1  and H 2  are hydrophobic amino acids, N 1  and N 2  are negatively charged amino acids, X is any amino acid, and L 1  is a polar amino acid;   
       and wherein said polypeptide has cellular protective activity in a responsive cell, tissue or organ. 
     
     
         2 . An isolated polypeptide consisting of a sequence of no more than 30 amino acids and comprising the amino acid motif:
 (a) H 1 -N 1 -(L) n -P 1 -H 2 , wherein n is 0-1; or   (b) H 1 -P 1 -(L) n -N 1 -H 2 , wherein n is 0-1;   and wherein H 1  and H 2  are hydrophobic amino acids, N 1  is a negatively charged amino acid, L 1  is a polar amino acid, and P 1  is a positively charged amino acid;   
       and wherein said polypeptide has cellular protective activity in a responsive cell, tissue or organ. 
     
     
         3 . An isolated polypeptide comprising:
 (i) an amino acid sequence having less than 90% sequence identity with any portion of SEQ ID NO:1 having the same number of amino acid residues as said polypeptide; and   (ii) the amino acid motif:
 (a) H 1 -N 1 -(X) n -N 2 -H 2 , wherein n is 0-5; 
 (b) H 1 -N 1 -(X) n -N 2 -L 1 , wherein n is 0-5; or 
 (c) L 1 -N 1 -(X) n -N 2 -H 1 , wherein n is 0-5; 
 and wherein H 1  and H 2  are hydrophobic amino acids, N 1  and N 2  are negatively charged amino acids, X is any amino acid, and L 1  is a polar amino acid; 
   
       and wherein said polypeptide has cellular protective activity in a responsive cell, tissue or organ. 
     
     
         4 . An isolated polypeptide comprising:
 (i) an amino acid sequence having less than 90% sequence identity with any portion of SEQ ID NO:1 having the same number of amino acid residues as said polypeptide; and   (ii) the amino acid motif:
 (a) H 1 -N 1 -(L) n -P 1 -H 2 , wherein n is 0-1; or 
 (b) H 1 -P 1 -(L) n -N 1 -H 2 , wherein n is 0-1; 
 and wherein H 1  and H 2  are hydrophobic amino acids, N 1  is a negatively charged amino acid, L 1  is a polar amino acid, and P 1  is a positively charged amino acid; 
   
       and wherein said polypeptide has cellular protective activity in a responsive cell, tissue or organ. 
     
     
         5 . The isolated polypeptide of  claim 1  or  3 , wherein the distance between N 1  and N 2  as a result of the tertiary structure of said polypeptide is between about 3 Å to about 5 Å. 
     
     
         6 . The isolated polypeptide of  claim 5 , wherein said distance is between about 4 Å to about 5 Å. 
     
     
         7 . The isolated polypeptide of  claim 6 , wherein said distance is between about 4.4 Å to about 4.8 Å. 
     
     
         8 . The isolated polypeptide of  claim 2  or  4 , wherein the distance between N 1  and P 2  as a result of the tertiary structure of said polypeptide is between about 3 Å to about 5 Å. 
     
     
         9 . The isolated polypeptide of  claim 8 , wherein said distance is between about 4 Å to about 5 Å. 
     
     
         10 . The isolated polypeptide of  claim 9 , wherein said distance is between about 4.4 Å to about 4.8 Å. 
     
     
         11 . An isolated polypeptide consisting of a sequence of no more than 30 amino acids and comprising the amino acid motif:
 (a) H 1 N 1 N 2 H 2 ;   (b) H 1 N 1 N 2 L; or   (c)   that is formed as a result of the of the tertiary structure of said polypeptide such that the distance between the carbonyl carbons of N 1  and N 2  is about 3 Å to about 5 Å, wherein H 1  and H 2  are hydrophobic amino acids, N 1  and N 2  are negatively charged amino acids, and L 1  is a polar amino acid;   
       and wherein said polypeptide has cellular protective activity in a responsive cell, tissue or organ. 
     
     
         12 . An isolated polypeptide consisting of a sequence of no more than 30 amino acids and comprising the amino acid motif:
 (a) H 1 N 1 (L) n PiH 2 , wherein n is 0-1; or   (b) H 1 P 1 (L) n N 1 H 2 , wherein n is 0-1;   that is formed as a result of the of the tertiary structure of said polypeptide such that the distance between the carbonyl carbons of N 1  and P 2  is about 3 Å to about 5 Å, wherein H 1  and H 2  are hydrophobic amino acids, N 1  is a negatively charged amino acid, L 1  is a polar amino acid, and P 1  is a positively charged amino acid;   
       and wherein said polypeptide has cellular protective activity in a responsive cell, tissue or organ. 
     
     
         13 . An isolated polypeptide comprising:
 (i) an amino acid sequence having less than 90% sequence identity with any portion of SEQ ID NO:1 having the same number of amino acid residues as said polypeptide; and   (ii) the amino acid motif:
 (a) H 1 N 1 N 2 H 2 ; 
 (b) H 1 N 1 N 2 L 1 ; or 
 (c) 
 that is formed as a result of the of the tertiary structure of said polypeptide such that the distance between the carbonyl carbons of N 1  and N 2  is about 3 Å to about 5 Å, wherein H 1  and H 2  are hydrophobic amino acids, N 1  and N 2  are negatively charged amino acids, and L 1  is a polar amino acid; 
   
       and wherein said polypeptide has cellular protective activity in a responsive cell, tissue or organ. 
     
     
         14 . An isolated polypeptide comprising:
 (i) an amino acid sequence having less than 90% sequence identity with any portion of SEQ ID NO:1 having the same number of amino acid residues as said polypeptide; and   (ii) the amino acid motif:
 (a) H 1 N 1 (L) n PiH 2 , wherein n is 0-1; or 
 (b) H 1 P 1 (L) n N 1 H 2 , wherein n is 0-1; 
 that is formed as a result of the of the tertiary structure of said polypeptide such that the distance between the carbonyl carbons of N 1  and P 2  is between about 3 Å and about 5 Å, wherein H 1  and H 2  are hydrophobic amino acids, N 1  is a negatively charged amino acid, L 1  is a polar amino acid, and P 1  is a positively charged amino acid; 
   
       and wherein said polypeptide has cellular protective activity in a responsive cell, tissue or organ. 
     
     
         15 . The isolated polypeptide of any one of  claims 1 ,  3 , and  5 - 7 , wherein said amino acid motif is (a) and wherein H 1  and H 2  are the same hydrophobic amino acid. 
     
     
         16 . The isolated polypeptide of  claim 11  or  13 , wherein said amino acid motif is (a) and wherein H 1  and H 2  are the same hydrophobic amino acid. 
     
     
         17 . The isolated polypeptide of any one of  claims 1 ,  3 , and  5 - 7 , wherein said amino acid motif is (a) and wherein H 1  and H 2  are different hydrophobic amino acids. 
     
     
         18 . The isolated polypeptide of  claim 11  or  13 , wherein said amino acid motif is (a) and wherein H 1  and H 2  are different hydrophobic amino acids. 
     
     
         19 . The isolated polypeptide of any one of  claims 2 ,  4 , and  8 - 10 , wherein said amino acid motif is (a) or (b) and wherein H 1  and H 2  are the same hydrophobic amino acid. 
     
     
         20 . The isolated polypeptide of  claim 12  or  14 , wherein said amino acid motif is (a) or (b) and wherein H 1  and H 2  are the same hydrophobic amino acid. 
     
     
         21 . The isolated polypeptide of any one of  claims 2 ,  4 , and  8 - 10 , wherein said amino acid motif is (a) or (b) and wherein H 1  and H 2  are different hydrophobic amino acids. 
     
     
         22 . The isolated polypeptide of  claim 12  or  14 , wherein said amino acid motif is (a) or (b) and wherein H 1  and H 2  are different hydrophobic amino acids. 
     
     
         23 . The isolated polypeptide of any one of  claims 1 ,  3 , and  5 - 7 , wherein said amino acid motif is (a), (b), or (c) and wherein N 1  and N 2  are the same negatively charged amino acid. 
     
     
         24 . The isolated polypeptide of  claim 11  or  13 , wherein said amino acid motif is (a), (b), or (c) and wherein N 1  and N 2  are the same negatively charged amino acid. 
     
     
         25 . The isolated polypeptide of any one of  claims 1 ,  3 , and  5 - 7 , wherein said amino acid motif is (a), (b), or (c) and wherein N 1  and N 2  are different negatively charged amino acids. 
     
     
         26 . The isolated polypeptide of  claim 11  or  13 , wherein said amino acid motif is (a), (b), or (c) and wherein N 1  and N 2  are different negatively charged amino acids. 
     
     
         27 . The isolated polypeptide of any one of  claims 1 - 26 , wherein said peptide is derived from a type 1 cytokine. 
     
     
         28 . The isolated peptide of  claim 27 , wherein the type 1 cytokine is granulocyte-macrophage colony stimulating factor, interleukin-3, Thrombopoietin, Ciliary Neurotrophic Factor or Leukemia Inhibitory Factor. 
     
     
         29 . The isolated polypeptide of any one of  claims 1 - 28 , wherein said polypeptide further comprises at least one other of the following amino acids motifs:
 (a) H 1 -N 1 -(X) n -N 2 -H 2 , wherein n is 0-5;   (b) H 1 -N 1 -(X) n -N 2 -L 1 , wherein n is 0-5;   (c) L 1 -N 1 -(X) n -N 2 -H 1 , wherein n is 0-5   (d) H 1 -N 1 -(L) n -P 1 -H 2 , wherein n is 0-1;   (e) H 1 -P 1 -(L) n -N 1 -H 2 , wherein n is 0-1   (f) H 1 N 1 N 2 H 2 ;   (g) H 1 N 1 N 2 L 1 ;   (h) L 1 N 1 N 2 H 1      (i) H 1 N 1 (L) n PiH 2 , wherein n is 0-1; or   (j) H 1 P 1 (L) n N 1 H 2 , wherein n is 0-1;   
       wherein motif (f), (g), or (h) is formed as a result of the of the tertiary structure of said polypeptide such that the distance between the carbonyl carbons of N 1  and N 2  is about 3 Å to about 5 Å; wherein motif (i) or (j) is formed as a result of the of the tertiary structure of said polypeptide such that the distance between the carbonyl carbons of N 1  and P 2  is about 3 Å to about 5 Å; and wherein H 1  and H 2  are hydrophobic amino acids, N 1  and N 2  are negatively charged amino acids, X is any amino acid, L 1  is a polar amino acid, and P 1  is a positively charged amino acid. 
     
     
         30 . The isolated polypeptide of  claim 29 , wherein at least two of said amino acid motifs are different. 
     
     
         31 . The isolated polypeptide of any one of  claims 1 - 29 , wherein said polypeptide is chimeric peptide further comprising an amphipathic peptide helix. 
     
     
         32 . The isolated polypeptide of  claim 31 , wherein said amphipathic peptide helix comprises the amino acid sequence ALSILVLLQAGS (SEQ ID NO:48); VALLPCPPCRA (SEQ ID NO:49); NAIIKNAYKKG (SEQ ID NO:50); GSWQRSLQDTE (SEQ ID NO:51); GGSAARPAPP (SEQ ID NO:52); NALAENDTPYY (SEQ ID NO:53); GALAEAYPSKP (SEQ ID NO:54); GCSSQHWSYGL (SEQ ID NO:55); VMIVMLAICFL (SEQ ID NO:56); LRRYINMLTRP (SEQ ID NO:28); or LALSILVLYQA (SEQ ID NO:57). 
     
     
         33 . The isolated polypeptide of any one of  claims 1 - 30 , wherein said polypeptide does not increase hemoglobin in the recipient. 
     
     
         34 . The isolated polypeptide of any one of  claims 1 - 31 , wherein the cellular protective activity is protecting, maintaining, enhancing or restoring the function and/or viability of said cell, tissue or organ. 
     
     
         35 . The isolated polypeptide of any one of  claims 1 - 32 , wherein said polypeptide has cellular protective activity in neuronal, bone, eye, adipose, connective, hair, teeth, mucosal, pancreas, endocrine, ear, epithelial, skin, muscle, heart, lung, liver, kidney, intestine, adrenal, capillary, endothelial, testes, ovary, or endometrial cells or tissues. 
     
     
         36 . The isolated polypeptide of any one of  claims 1 - 32  wherein said polypeptide has cellular protective activity in a stem cell. 
     
     
         37 . The isolated polypeptide of claim any one of  claims 1 - 32 , wherein said polypeptide has cellular protective activity in excitable tissue. 
     
     
         38 . The isolated peptide of  claim 35 , wherein said excitable tissue is central nervous system tissue, peripheral nervous system tissue, cardiac tissue or retinal tissue. 
     
     
         39 . The isolated peptide of any one of  claims 1 - 36 , wherein said peptide is capable of traversing an endothelial cell barrier. 
     
     
         40 . The isolated peptide of  claim 37 , wherein the endothelial cell barrier comprises the blood-brain barrier, the blood-eye barrier, the blood-testes barrier, blood-ovary barrier, blood-nerve or the blood-spinal cord barrier. 
     
     
         41 . A pharmaceutical composition comprising the isolated polypeptide of any of  claims 1 - 38  and a pharmaceutically acceptable carrier. 
     
     
         42 . The pharmaceutical composition of  claim 39 , wherein said composition is formulated for oral, intranasal, ocular, inhalational, transdermal, rectal, sublingual or parenteral administration. 
     
     
         43 . The pharmaceutical composition of  claim 39 , wherein said composition is formulated as a perfusate solution. 
     
     
         44 . A method for protecting, maintaining or enhancing the viability of a responsive cell, tissue or organ isolated from a mammalian body comprising exposing said cell, tissue or organ to a pharmaceutical composition comprising exposing said cell, tissue or organ to the pharmaceutical composition of  claim 39 ,  40 , or  41 . 
     
     
         45 . Use of an isolated peptide of any one of  claims 1 - 38  for the preparation of a pharmaceutical composition for the protection against and/or prevention of a tissue injury, for the restoration of, or for the rejuvenation of tissue and/or tissue function in a subject in need thereof. 
     
     
         46 . Use of an isolated peptide of any one of  claims 1 - 38  for the preparation of a pharmaceutical composition for the prevention, therapeutic treatment or prophylactic treatment in a subject in need thereof of a cardiovascular disease, cardiopulmonary disease, respiratory disease, kidney disease, disease of the urinary system, disease of the reproductive system, done disease, skin disease, gastrointestinal disease, endocrine abnormality, metabolic abnormality, cognitive dysfunction, or a disease or disorders of the central or peripheral nervous system. 
     
     
         47 . The use of  claim 43  or  44  wherein the subject is a mammal. 
     
     
         48 . The use of  claim 45  wherein the mammal is a human. 
     
     
         49 . A method for facilitating the transcytosis of a molecule across an endothelial cell barrier in a subject in need thereof comprising administration to said subject a composition comprising said molecule in association with an isolated peptide of any one of  claims 1 - 38 . 
     
     
         50 . The method of  claim 47 , wherein said association is a labile covalent bond, a stable covalent bond, or a non-covalent association with a binding site for said molecule. 
     
     
         51 . An isolated nucleic acid comprising the nucleotide sequence encoding the isolated peptide of any one of  claims 1 - 38 . 
     
     
         52 . A vector comprising the nucleic acid of  claim 49 . 
     
     
         53 . The vector of  claim 50  which is an expression vector. 
     
     
         54 . A host cell containing the expression vector of  claim 51 . 
     
     
         55 . A method of recombinantly producing the isolated peptide of any one of  claims 1 - 38 , said method comprising i) culturing in a medium the host cell of  claim 52 , under conditions suitable for the expression of said peptide, and ii) recovery and isolation of said peptide from said medium. 
     
     
         56 . The isolated peptide of  claim 2 , wherein said peptide comprises the amino acid sequence peptide A (APPRLICDSRVLERYLLEAKEAE, SEQ ID NO:32), peptide C (NITVPDTKVNFYAWKRMEVG, SEQ ID NO:29), peptide D (QQAVEVWQGLALLSEAVLRGQALLV, SEQ ID NO:30), peptide E (GCAEHCSLNENITVPDTKVN, SEQ ID NO:31), peptide F (RYLLUNITTGC, SEQ ID NO:33), peptide G (QEQLERALNSS, SEQ ID NO:40), peptide I (CSLNENIQEQLERALNSS, SEQ ID NO:43), peptide J (QEQLERALNSSLRRYINMLTRTR, SEQ ID NO:41), peptide K (WEHVNAIQEARRLL, SEQ ID NO:35), or peptide L (KIRSDLTALTESYVKH, SEQ ID NO:37). 
     
     
         57 . The isolated peptide of any one of  claims 1 - 38 , wherein said at least one cellular protective activity is neuroprotection, which activity is evaluated in vitro by a method comprising:
 (a) contacting a test culture of primary hippocampal neurons with N-methyl-D-aspartate and said peptide; and   (b) determining the cell viability at 48 hours post said contact, such that if the cell viability determined in step (b) is greater than that of a control culture in the absence of said peptide, the peptide possesses cellular protective activity.