Pro-angiogenic peptides and peptide conjugates

Abstract

Short bioactive sequences derived from the 2.sup.nd loop of the 7-transmembranal receptor of endothelial differentiation gene 3 (EDG3) useful in stimulation of angiogenesis, and peptide conjugates comprising a permeability enhancing moiety, are provided. Also provided are pharmaceutical compositions comprising the peptides and methods of use in conditions were insufficient blood-supply occurs, or which are associated with endothelia dysfunction such as peripheral vascular diseases, coronary artery diseases, cerebrovascular diseases, diabetes and delayed wound healing, pulmonary disease, eye diseases and pathological condition related to severe infection.

Claims

1. A peptide consisting of 4-5 amino acids, the peptide having the sequence Nle-RPY, wherein Nle is norleucine, R is selected from the group consisting of an arginine residue, an homoarginine residue (hArg), an N-methyl arginine residue (NMeArg), a citruline residue, and a 2-amino-3-guanidinopropionic acid residue; P is selected from the group consisting of a proline residue, an hydroxyproline residue, and a thiazolidine-carboxylate residue, and Y is selected from the group consisting of: a tyrosine residue, a 3,5 diiodo tyrosine residue (35dITyr), a 3,5 diBromo tyrosine residue (35dBTyr), and an homotyrosine residue.

2. A peptide conjugate comprising a peptide according to claim 1 and a moiety capable of increasing permeability, wherein the peptide-conjugate is according to Formula I:
i. ZXRPYB(Formula I) wherein Z designates a moiety capable of increasing permeability covalently connected via a direct bond or via a linker; X designates Nle, R is selected from the group consisting of an arginine residue, an homoarginine residue (hArg), an N-methyl arginine residue (NMeArg), a citruline residue, and a 2-amino-3-guanidinopropionic acid residue; P is selected from the group consisting of a proline residue, an hydroxyproline residue, and a thiazolidine-carboxylate residue, and Y is selected from the group consisting of: a tyrosine residue, a 3,5 diiodo tyrosine residue (35dITyr), a 3,5 diBromo tyrosine residue (35dBTyr), and an homotyrosine residue, and B designates a terminal carboxy acid, amide, ester or alcohol group.

3. The peptide conjugate of claim 2 wherein Z designates Myristoyl-glycine (Myr-Gly), X is a norleucine (Nle) residue, R is an arginine residue or an homoarginine residue (hArg), P is a proline residue or 4-hydroxy-Proline residue (4HyP), Y is a tyrosine residue or a (3,5-diiodo)tyrosine (35dITyr) residue and B is terminal designates a terminal carboxy acid, amide, ester or alcohol group.

4. The peptide conjugate according to claim 2 comprising a peptide selected from the group consisting of: TABLE-US-00012 Gly-Nle-Arg-Pro-Tyr-NH2; (SEQIDNO:6) Gly-Nle-Arg-Pro-35dITyr-NH2; (SEQIDNO:7) Gly-Nle-hArg-Pro-Tyr-NH2; (SEQIDNO:8) Gly-Nle-hArg-Pro-35dITyr-NH2; (SEQIDNO:9) and Gly-Nle-hArg-4HyP-Tyr. (SEQIDNO:10)

5. The peptide conjugate of claim 2 selected from the group consisting of: TABLE-US-00013 Myr-Gly-Nle-Arg-Pro-Tyr-NH2; (SEQIDNO:12) Myr-Gly-Nle-Arg-Pro-35dITyr-NH2; (SEQIDNO:13) Myr-Gly-Nle-hArg-Pro-Tyr-NH2; (SEQIDNO:14) Myr-Gly-Nle-hArg-Pro-35dITyr-NH2, (SEQIDNO:15) and Myr-Gly-Nle-hArg-4HyP-Tyr. (SEQIDNO:16)

6. A pharmaceutical composition comprising as an active ingredient a peptide according to claim 1, and a pharmaceutically acceptable carrier.

7. A pharmaceutical composition comprising as an active ingredient a peptide conjugate according to claim 2 and a pharmaceutically acceptable carrier.

8. A method of prevention or treatment a condition in which insufficient blood-supply occurs, a condition which is associated with endothelia dysfunction, or a condition mediates through S1P receptor, comprising administering to a subject in need thereof a pharmaceutical composition according to claim 7.

9. The method of claim 8 wherein the condition is selected from the group consisting of: peripheral vascular disease, myocardial ischemia, tissue graft, coronary artery diseases, stroke, diabetes, pancreatic islet transplantation, and delayed wound healing, pulmonary disease, eye disease, bone loss and pathological condition related to severe infection.

10. The method according to claim 9 wherein the disease is selected from the group consisting of: acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and ventilation induced ling injury (VILI); age-related macular disease (AMD); and sepsis.

11. A stent comprising at least one peptide conjugate according claim 2.

12. A method of prevention or treatment of myocardial infection or coronary artery disease, comprising administering to a subject in need thereof a stent according to claim 11.

13. The peptide of claim 1 wherein R is an arginine residue or an homoarginine residue (hArg), P is a proline residue or 4-hydroxy-Proline residue (4HyP), and Y is a tyrosine residue or a (3,5-diiodo)tyrosine (35dITyr) residue.

14. A peptide conjugate consisting of Myr-Gly-Nle-Arg-Pro-Tyr-NH2 (SEQ ID NO: 12).

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1A-1C show an induction of capillary formation in aortic ring assay by the peptides of SEQ ID NOs: 13 (FIG. 1A), 14 (FIG. 1B), and 12 (FIG. 1C), as determined by aortic ring capillary length.

(2) FIGS. 2A-C show an induction of capillary formation in aortic ring assay by the peptides of SEQ ID NOs: 13 (FIG. 1A), 14 (FIG. 1B), and 12 (FIG. 1C), as determined by aortic ring capillary width.

(3) FIG. 3 shows the effects of compound of SEQ ID NO: 9 vs. compound of SEQ ID NO: 1 on perfusion (LDI) in ischemic leg model in mice.

(4) FIG. 4 represents synergistic effect of peptides of SEQ ID NOs: 13 and 14 with VEGF in aortic ring assay.

(5) FIG. 5 depicts in-vivo results which demonstrate the ability of peptide SEQ ID NO: 12 (GPS-725.017) to rescue mice from death in a septic-shock model. C57BL mice (15 for each group) were injected s.c. once a day, for 5 days, with either vehicle or 20 mg/Kg of GPS-725.017 (SEQ ID NO: 12).

DETAILED DESCRIPTION OF THE INVENTION

(6) As a result of major advances in organic chemistry and in molecular biology, many bioactive peptides can now be prepared in quantities sufficient for pharmacological and clinical use. Thus in the last few years new methods have been established for the treatment and diagnosis of illnesses in which peptides have been implicated. However, the use of peptides as therapeutic and diagnostic agents is limited by the following factors: a) tissue penetration; b) low metabolic stability towards proteolysis in the gastrointestinal tract and in serum; c) poor absorption after oral ingestion, in particular due to their relatively high molecular mass or the lack of specific transport systems or both; d) rapid excretion through the liver and kidneys; and e) undesired side effects in non-target organ systems, since peptide receptors can be widely distributed in an organism. The present invention provides bioactive peptides that: a) have improved tissue penetration due to their small size and the optional permeability moiety; b) have improved metabolic stability due to the blocked terminals and to incorporation of non-natural amino acids; c) have improved absorption due to their low molecular mass and the optional permeability moiety; d) are easier and cheaper to produce due to their low molecular mass and short sequence; and e) are expected to be selective to the EDG3 receptor from which they derive and therefore are expected to have less side effects.

(7) As presented in table 1, previously disclosed peptides derived from EDG3 are at least 6 amino acids long and contain at least 5 residues which are identical or homologous to the native EDG3 sequence. The shortest known peptide is the peptide R002L106 (725-106, SEQ ID NO: 2) which contain in four of its six positions residues identical to those of the native sequence and one which is a conservative substitution (asparagine instead of aspartic acid) of the residue present in the native sequence. Some of the EDG3 derived sequences of the present invention, are only 4 amino acids long and contain in no more than three positions residues identical or homologous to the native sequence. It is unexpected that these sequences which are at least 30% shorter are more active than longer sequences which have higher homology to the native sequence from which they are derived.

(8) TABLE-US-00005 TABLE 1 Native EDG3 SEQ ID No. Ile Lys Met Arg Pro Tyr Asp Ala Asn Lys Arg 17 A. Previously disclosed sequences R002L103 Met Arg Pro Tyr Asp Ala Asn Lys Arg 18 R002L106 Nle Arg Pro Tyr Asn Ala 2 (725-106) B. New sequences Nle Arg Pro Tyr 19 Nle Arg Pro 35dITyr 20 Nle hArg Pro Tyr 21 Nle hArg Pro 35dITyr 22 Nle hArg 4HyP Tyr 23 Nle is a norleucine residue, hArg designates an homoarginine residue, 4HyP designates a 4-hydroxyproline residue, and 35dITyr designates a 3,5-diiodotyrosine residue.

(9) According to a specific embodiment of the present invention, the new sequences presented in part B of table 1 serves as the basis for novel molecules disclosed in the present application, further containing a cell-permeability moiety coupled through an optional spacer to the peptide, preferably to the peptide's N-terminus.

(10) Without wishing to be bound to a theory, the peptides of the present invention, comprising sequences sharing only three amino acids homology with the sequence of the EDG3 receptor, mimic the effects of sphingosine-1-phosphate (S1P), the natural ligand of S1P3 (EDG3) by triggering a Gi-dependent signal transduction while inhibiting G12/13 signaling. It is hypothesized that the G-protein inhibitor Pertusis toxin inhibits angiogenesis induced by the peptides of the present invention.

(11) It is now shown by ex-vivo (sprouting of blood vessels from mouse aortic rings embedded in collagen matrix) and in-vivo experiments (mouse ischemic hind limb assay), that peptides according to the invention induce the formation of multi-layered and mature vessels. The results demonstrate that these peptides synergize with endogenous protein angiogenic factors (like VEGF,) and stimulate the formation of mature and stable blood vessels in contrast to VEGF tested alone under the same conditions. Since the levels of protein angiogenic factors like VEGF increase at the ischemic regions, it is provided that administration of the peptide of the invention as a monotherapy will yield the desirable angiogenic effect in situ, by synergizing with locally secreted endogenous factors.

(12) In addition, it is now shown that peptides according to the invention, prevents blood-vessel leakiness in-vivo. The results demonstrate that these peptides enhance blood vessel integrity and inhibit macromolecules escape to the interstitial fluid.

(13) The peptides of the present invention are preferably synthesized using conventional synthesis techniques known in the art, e.g., by chemical synthesis techniques including peptidomimetic methodologies. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. Solid phase peptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). A skilled artesian may synthesize any of the peptides of the present invention by using an automated peptide synthesizer using standard chemistry such as, for example, t-Boc or Fmoc chemistry. Synthetic peptides can be purified by preparative high performance liquid chromatography (Creighton T. 1983, Proteins, structures and molecular principles. WH Freeman and Co. N.Y.), and the composition of which can be confirmed via amino acid sequencing. Some of the peptides of the invention, which include only natural, namely genetically encoded, amino acids, may further be prepared using recombinant DNA techniques known in the art. The conjugation of the peptidic and permeability moieties may be performed using any methods known in the art, either by solid phase or solution phase chemistry. Some of the preferred compounds of the present invention may conveniently be prepared using solution phase synthesis methods. Other methods known in the art to prepare compounds like those of the present invention can be used and are comprised in the scope of the present invention.

(14) Cyclic versions of the peptides disclosed herein are also within the scope of the present invention. Cyclization of peptides may take place by any means known in the art, for example through free amino and carboxylic groups present in the peptide sequence, or through amino acids or moieties added for cyclization. Non limiting examples of cyclization types are: side chain to side chain cyclization, C-to-N terminal cyclization, side chain to terminal cyclization, and any type of backbone cyclization incorporating at least one N.sup.--substituted amino acid residue/s as described for example in WO 95/33765.

(15) The permeability-enhancing moiety of the conjugates of the present invention may be connected to any position in the peptide moiety, directly or through a spacer. According to a specific embodiment, the cell-permeability moiety is connected to the amino terminus of the peptide moiety. The optional connective spacer may be of varied lengths and conformations comprising any suitable chemistry including but not limited to amine, amide, carbamate, thioether, oxyether, sulfonamide bond and the like. Non-limiting examples for such spacers include amino acids, sulfone amide derivatives, amino thiol derivatives and amino alcohol derivatives.

Definitions

(16) For convenience and clarity certain terms employed in the specification, examples and claims are described herein.

(17) The term peptide as used herein is meant to encompass natural (genetically encoded), non-natural and/or chemically modified amino acid residues, each residue being characterized by having an amino and a carboxy terminus, connected one to the other by peptide or non-peptide bonds. The amino acid residues are represented throughout the specification and claims by either one or three-letter codes, as is commonly known in the art. The peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.

(18) The amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and either sequential, divergent or convergent synthetic approaches to the peptide sequence are useful in this invention. Natural coded amino acids and their derivatives are represented by three-letter codes according to IUPAC conventions. When there is no indication, either the L or D isomers may be used.

(19) Conservative substitution of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions includes replacement of one amino acid with another having the same type of functional group or side chain e.g. aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, affinity to the target protein, metabolic stability, penetration into the central nervous system, targeting to specific cell populations and the like. One of skill will recognize that individual substitutions, deletions or additions to peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a conservatively modified variant where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

(20) The following six groups each contain amino acids that are conservative substitutions for one another.

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

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

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

(24) 4) Arginine (R), Lysine (K);

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

(26) 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(27) Also included within the scope of the invention are salts of the peptides, analogs, and chemical derivatives of the peptides of the invention.

(28) As used herein the term salts refers to both salts of carboxyl groups and to acid addition salts of amino or guanido groups of the peptide molecule. Salts of carboxyl groups may be formed by means known in the art and include inorganic salts, for example sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as salts formed for example with amines such as triethanolamine, piperidine, procaine, and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, acetic acid or oxalic acid. Salts describe here also ionic components added to the peptide solution to enhance hydrogel formation and/or mineralization of calcium minerals.

(29) A chemical derivative as used herein refers to peptides containing one or more chemical moieties not normally a part of the peptide molecule such as esters and amides of free carboxy groups, acyl and alkyl derivatives of free amino groups, phospho esters and ethers of free hydroxy groups. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Preferred chemical derivatives include peptides that have been phosphorylated, C-termini amidated or N-termini acetylated.

(30) Functional derivatives of the peptides of the invention as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the peptide, do not confer toxic properties on compositions containing it and do not adversely affect the antigenic properties thereof. These derivatives may, for example, include aliphatic esters of the carboxyl groups, amides of the carboxyl groups produced by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed by reaction with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group (for example that of seryl or threonyl residues) formed by reaction with acyl moieties.

(31) The term peptide analog indicates molecule which has the amino acid sequence according to the invention except for one or more amino acid changes or one or more modification/replacement of an amide bond. Peptide analogs include amino acid substitutions and/or additions with natural or non-natural amino acid residues, and chemical modifications which do not occur in nature. Peptide analogs include peptide mimetics. A peptide mimetic or peptidomimetic means that a peptide according to the invention is modified in such a way that it includes at least one non-coded residue or non-peptidic bond. Such modifications include, e.g., alkylation and more specific methylation of one or more residues, insertion of or replacement of natural amino acid by non-natural amino acids, replacement of an amide bond with other covalent bond. A peptidomimetic according to the present invention may optionally comprises at least one bond which is an amide-replacement bond such as urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond. The design of appropriate analogs may be computer assisted. Additional peptide analogs according to the present invention comprise a specific peptide or peptide analog sequence in a reversed order, namely, the amino acids are coupled in the peptide sequence in a reverse order to the amino acids order which appears in the native protein or in a specific peptide or analog identified as active. Whether completely or partially non-peptide, peptidomimetics according to this invention provide a spatial arrangement of chemical moieties that closely resembles the three-dimensional arrangement of groups in the peptide on which the peptidomimetic is based. As a result of this similar active-site structure, the peptidomimetic has effects on biological systems, which are similar to the biological activity of the peptide.

(32) A modified amino acid residue is an amino acid residue in which any group or bond was modified by deletion, addition, or replacement with a different group or bond, as long as the functionality of the amino acid residue is preserved or if functionality changed (for example replacement of tyrosine with substituted phenylalanine) as long as the modification did not impair the activity of the peptide containing the modified residue.

(33) A peptide conjugate according to the present invention, denotes a molecule comprising a sequence of a blood-vessel promoting peptide to which another moiety, either peptidic or non peptidic, is covalently bound, directly or via a linker.

(34) The term linker denotes a chemical moiety, a direct chemical bond of any type, or a spacer whose purpose is to link, covalently, a cell-permeability moiety and a peptide or peptidomimetic. The spacer may be used to allow distance between the permeability-enhancing moiety and the peptide.

(35) Permeability refers to the ability of an agent or substance to penetrate, pervade, or diffuse through a barrier, membrane, or a skin layer. A cell permeability or a cell-penetration moiety refers to any molecule known in the art which is able to facilitate or enhance penetration of molecules through membranes. Non-limitative examples include: hydrophobic moieties such as lipids, fatty acids, steroids and bulky aromatic or aliphatic compounds; moieties which may have cell-membrane receptors or carriers, such as steroids, vitamins and sugars, natural and non-natural amino acids, transporter peptides, nanoparticles and liposomes.

(36) The hydrophobic moiety according to the invention may preferably comprise a lipid moiety or an amino acid moiety. According to a specific embodiment the hydrophobic moiety is selected from the group consisting of: phospholipids, steroids, sphingosines, ceramides, octyl-glycine, 2-cyclohexylalanine, benzolylphenylalanine, propionoyl (C.sub.3); butanoyl (C.sub.4); pentanoyl (C.sub.5); caproyl (C.sub.6); heptanoyl (C.sub.7); capryloyl (C.sub.8); nonanoyl (C.sub.9); capryl (C.sub.10); undecanoyl (C.sub.11); lauroyl (C.sub.12); tridecanoyl (C.sub.13); myristoyl (C.sub.14); pentadecanoyl (C.sub.15); palmitoyl (C.sub.16); phtanoyl ((CH.sub.3).sub.4); heptadecanoyl (C.sub.17); stearoyl (C.sub.18); nonadecanoyl (C.sub.19); arachidoyl (C.sub.20); heniecosanoyl (C.sub.21); behenoyl (C.sub.22); trucisanoyl (C.sub.23); and lignoceroyl (C.sub.24); wherein said hydrophobic moiety is attached to said chimeric polypeptide with amide bonds, sulfhydryls, amines, alcohols, phenolic groups, or carbon-carbon bonds.

(37) Other examples for lipidic moieties which may be used according to the present invention: Lipofectamine, Transfectace, Transfectam, Cytofectin, DMRIE, DLRIE, GAP-DLRIE, DOTAP, DOPE, DMEAP, DODMP, DOPC, DDAB, DOSPA, EDLPC, EDMPC, DPH, TMADPH, CTAB, lysyl-PE, DC-Cho, -alanyl cholesterol; DCGS, DPPES, DCPE, DMAP, DMPE, DOGS, DOHME, DPEPC, Pluronic, Tween, BRIJ, plasmalogen, phosphatidylethanolamine, phosphatidylcholine, glycerol-3-ethylphosphatidylcholine, dimethyl ammonium propane, trimethyl ammonium propane, diethylammonium propane, triethylammonium propane, dimethyldioctadecylammonium bromide, a sphingolipid, sphingomyelin, a lysolipid, a glycolipid, a sulfatide, a glycosphingolipid, cholesterol, cholesterol ester, cholesterol salt, oil, N-succinyldioleoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol, 1,3-dipalmitoyl-2-succinylglycerol, 1,2-dipalmitoyl-sn-3-succinylglycerol, 1-hexadecyl-2-palmitoylglycerophosphatidylethanolamine, palmitoylhomocystiene, N,N-Bis (dodecyaminocarbonylmethylene)-N,N-bis((-N,N,N-trimethylammoniumethyl-aminocarbonylmethylene)ethylenediamine tetraiodide; N,N-Bis(hexadecylaminocarbonylmethylene)-N,N,N-tris((-N,N,N-trimethylammonium-ethylaminocarbonylmethylenediethylenetri amine hexaiodide; N,N-Bis(dodecylaminocarbonylmethylene)-N,N-bis((-N,N,N-trimethylammonium ethylaminocarbonylmethylene)cyclohexylene-1,4-diamine tetraiodide; 1,7,7-tetra-((-N,N,N,N-tetramethylammoniumethylamino-carbonylmethylene)-3-hexadecylaminocarbonyl-methylene-1,3,7-triaazaheptane heptaiodide; N,N,N,N-tetra((-N,N,N-trimethylammonium-ethylaminocarbonylmethylene)-N-(1,2-dioleoylglycero-3-phosphoethanolamino carbonylmethylene)diethylenetriamine tetraiodide; dioleoylphosphatidylethanolamine, a fatty acid, a lysolipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, a sphingolipid, a glycolipid, a glucolipid, a sulfatide, a glycosphingolipid, phosphatidic acid, palmitic acid, stearic acid, arachidonic acid, oleic acid, a lipid bearing a polymer, a lipid bearing a sulfonated saccharide, cholesterol, tocopherol hemisuccinate, a lipid with an ether-linked fatty acid, a lipid with an ester-linked fatty acid, a polymerized lipid, diacetyl phosphate, stearylamine, cardiolipin, a phospholipid with a fatty acid of 6-8 carbons in length, a phospholipid with asymmetric acyl chains, 6-(5-cholesten-3b-yloxy)-1-thio-b-D-galactopyranoside, digalactosyldiglyceride, 6-(5-cholesten-3b-yloxy)hexyl-6-amino-6-deoxy-1-thio-b-D-galactopyranoside, 6-(5-cholesten-3b-yloxy)hexyl-6-amino-6-deoxyl-1-thio-a-D-mannopyranoside, 12-(((7-diethylamino-coumarin-3-yl)carbonyl)methylamino)-octadecanoic acid; N-[12-(((7-diethylaminocoumarin-3-yl)carbonyl)methyl-amino) octadecanoyl]-2-aminopalmitic acid; cholesteryl)4-trimethyl-ammonio)butanoate; N-succinyldioleoyl-phosphatidylethanolamine; 1,2-dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinyl-glycerol; 1,3-dipalmitoyl-2-succinylglycerol, 1-hexadecyl-2-palmitoylglycero-phosphoethanolamine, and palmitoylhomocysteine.

(38) The term blood vessel growth refers both to the processes of angiogenesis and arteriogenesis.

(39) The term angiogenesis (also referred to at times as neovascularization) is a general term used to denote the growth of new blood vessels both in normal and pathological conditions.

(40) The term physiologically acceptable carrier or diluent or excipient refers to an aqueous or non-aqueous fluid that is well suited for pharmaceutical preparations. Furthermore, the term a pharmaceutically acceptable carrier or excipient refers to at least one carrier or excipient and includes mixtures of carriers and or excipients. The term therapeutic refers to any pharmaceutical, drug or prophylactic agent which may be used in the treatment (including the prevention, diagnosis, alleviation, or cure) of a malady, affliction, disease or injury in a patient.

(41) Pharmacology

(42) Apart from other considerations, the fact that the novel active ingredients of the invention are peptides, peptide analogs or peptidomimetics, dictates that the formulation be suitable for delivery of these types of compounds. Although in general peptides are less suitable for oral administration due to susceptibility to digestion by gastric acids or intestinal enzymes novel methods are being used, in order to design and provide metabolically stable and oral bioavailable peptidomimetic analogs.

(43) The pharmaceutical composition of this invention may be administered by any suitable means, such as orally, topically, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, intraarticulary, intralesionally, by inhalation or parenterally, and are specifically formulated for the administration route. Ordinarily, parenteral administration or administration by inhalation will be preferred and the compositions will be formulated accordingly.

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

(45) Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

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

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

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

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

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

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

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

(53) In case of myocardial ischemia and coronary artery diseases, a preferred method for the prevention and/or treatment of such conditions would be the incorporation of the therapeutic peptide into a drug-eluting stent. Stents are being widely used to treat such conditions and the coating of stents with drug-containable polymers is well known to a person versed in the art. The local release of the therapeutic peptide according to the present invention, via a drug-eluting stent, might be advantageous due to the high concentration achieved at the desired site of the myocardium while the systemic concentration can become negligible.

(54) Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prevent, alleviate or ameliorate symptoms of a disease of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

(55) Toxicity and therapeutic efficacy of the peptides described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 (the concentration which provides 50% inhibition) and the LD50 (lethal dose causing death in 50% of the tested animals) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (e.g. Fingl, et al., 1975, in The Pharmacological Basis of Therapeutics, Ch. 1 p. 1).

(56) The preferred doses for administration of such pharmaceutical compositions range from about 0.1 g/kg to about 20 mg/kg body weight. Preferably, the amount of the active ingredient is in the range of from about 10 to 5000 g/kg.

(57) Depending on the severity and responsiveness of the condition to be treated, dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and all other relevant factors.

(58) In certain embodiments, peptide delivery can be enhanced by the use of protective excipients. This is typically accomplished either by complexing the peptide with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the polypeptide in an appropriately resistant carrier such as a liposome. Means of protecting polypeptides for oral delivery are well known in the art (see, e.g., U.S. Pat. No. 5,391,377 describing lipid compositions for oral delivery of therapeutic agents).

(59) Elevated serum half-life can be maintained by the use of sustained-release protein packaging systems. Such sustained release systems are well known to those of skill in the art. In one preferred embodiment, the ProLease biodegradable microsphere delivery system for proteins and peptides (Tracy, 1998, Biotechnol. Prog. 14, 108; Johnson et al., 1996, Nature Med. 2, 795; Herbert et al., 1998, Pharmaceut. Res. 15, 357) a dry powder composed of biodegradable polymeric microspheres containing the protein in a polymer matrix that can be compounded as a dry formulation with or without other agents.

(60) In certain embodiments, dosage forms of the compositions of the present invention include, but are not limited to, biodegradable injectable depot systems such as, PLGA based injectable depot systems; non-PLGA based injectable depot systems, and injectable biodegradable gels or dispersions. Each possibility represents a separate embodiment of the invention. The term biodegradable as used herein refers to a component which erodes or degrades at its surfaces over time due, at least in part, to contact with substances found in the surrounding tissue fluids, or by cellular action. In particular, the biodegradable component is a polymer such as, but not limited to, lactic acid-based polymers such as polylactides e.g. poly (D,L-lactide) i.e. PLA; glycolic acid-based polymers such as polyglycolides (PGA) e.g. Lactel from Durect; poly (D,L-lactide-co-glycolide) i.e. PLGA, (Resomer RG-504, Resomer RG-502, Resomer RG-504H, Resomer RG-502H, Resomer RG-504S, Resomer RG-502S, from Boehringer, Lactel from Durect); polycaprolactones such as Poly(e-caprolactone) i.e. PCL (Lactel from Durect); polyanhydrides; poly(sebacic acid) SA; poly(ricenolic acid) RA; poly(fumaric acid), FA; poly(fatty acid dimmer), FAD; poly(terephthalic acid), TA; poly(isophthalic acid), IPA; poly(p-{carboxyphenoxy}) methane), CPM; poly(p-{carboxyphenoxy}propane), CPP; poly(p-{carboxyphenoxy}hexane)s CPH; polyamines, polyurethanes, polyesteramides, polyorthoesters {CHDM: cis/trans-cyclohexyl dimethanol, HD:1,6-hexanediol. DETOU: (3,9-diethylidene-2,4,8, 10-tetraoxaspiro undecane)}; polydioxanones; polyhydroxybutyrates; polyalkylene oxalates; polyamides; polyesteramides; polyurethanes; polyacetals; polyketals; polycarbonates; polyorthocarbonates; polysiloxanes; polyphosphazenes; succinates; hyaluronic acid; poly(malic acid); poly(amino acids); polyhydroxyvalerates; polyalkylene succinates; polyvinylpyrrolidone; polystyrene; synthetic cellulose esters; polyacrylic acids; polybutyric acid; triblock copolymers (PLGA-PEG-PLGA), triblock copolymers (PEG-PLGA-PEG), poly (N-isopropylacrylamide) (PNIPAAm), poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) tri-block copolymers (PEO-PPO-PEO), poly valeric acid; polyethylene glycol; polyhydroxyalkylcellulose; chitin; chitosan; polyorthoesters and copolymers, terpolymers; lipids such as cholesterol, lecithin; poly(glutamic acid-co-ethyl glutamate) and the like, or mixtures thereof.

(61) In some embodiments, the compositions of the present invention comprise a biodegradable polymer selected from, but not limited to, PLGA, PLA, PGA, polycaprolactone, polyhydroxybutyrate, polyorthoesters, polyalkaneanhydrides, gelatin, collagen, oxidized cellulose, polyphosphazene and the like. Each possibility represents a separate embodiment.

(62) Depot Systems

(63) The parenteral route by intravenous (IV), intramuscular (IM), or subcutaneous (SC) injection is the most common and effective form of delivery for small as well as large molecular weight drugs. However, pain, discomfort and inconvenience due to needle sticks makes this mode of drug delivery the least preferred by patients. Therefore, any drug delivery technology that can at a minimum reduce the total number of injections is preferred. Such reductions in frequency of drug dosing in practice may be achieved through the use of injectable depot formulations that are capable of releasing drugs in a slow but predictable manner and consequently improve compliance. For most drugs, depending on the dose, it may be possible to reduce the injection frequency from daily to once or twice monthly or even longer (6 months). In addition to improving patient comfort, less frequent injections of drugs in the form of depot formulations smoothes out the plasma concentration-time profile by eliminating the hills and valleys. Such smoothing out of plasma profiles has the potential to not only boost the therapeutic benefit in most cases, but also to reduce any unwanted events, such as immunogenicity.

(64) Microparticles, implants and gels are the most common forms of biodegradable polymeric devices used in practice for prolonging the release of drugs in the body. Microparticles are suspended in an aqueous media right before injection and one can load as much as 40% solids in suspensions. Implant/rod formulations are delivered to SC/IM tissue with the aid of special needles in the dry state without the need for an aqueous media. This feature of rods/implants allows for higher masses of formulation, as well as drug content to be delivered. Further, in the rods/implants, the initial burst problems are minimized due to much smaller area in implants compared to the microparticles. Besides biodegradable systems, there are non-biodegradable implants and infusion pumps that can be worn outside the body. Non-biodegradable implants require a doctor's visit not only for implanting the device into the SC/IM tissue but also to remove them after the drug release period.

(65) Injectable compositions containing microparticle preparations are particularly susceptible to problems. Microparticle suspensions may contain as much as 40% solids as compared with 0.5-5% solids in other types of injectable suspensions. Further, microparticles used in injectable depot products, range in size up to about 250 m (average, 60-100 nm), as compared with a particle size of less than 5 m recommended for IM or SC administration. The higher concentrations of solids, as well as the larger solid particle size require larger size of needle (around 18-21 gauge) for injection. Overall, despite the infrequent uses of larger and uncomfortable needles, patients still prefer less frequently administered dosage forms over everyday drug injections with a smaller needle.

(66) Biodegradable polyesters of poly(lactic acid) (PLA) and copolymers of lactide and glycolide referred to as poly(lactide-co-glycolide) (PLGA) are the most common polymers used in biodegradable dosage forms. PLA is hydrophobic molecule and PLGA degrades faster than PLA because of the presence of more hydrophilic glycolide groups. These biocompatible polymers undergo random, non-enzymatic, hydrolytic cleavage of the ester linkages to form lactic acid and glycolic acid, which are normal metabolic compounds in the body. Resorbable sutures, clips and implants are the earliest applications of these polymers. Southern Research Institute developed the first synthetic, resorbable suture (Dexon) in 1970. The first patent describing the use of PLGA polymers in a sustained release dosage form appeared in 1973 (U.S. Pat. No. 3,773,919).

(67) Today, PLGA polymers are commercially available from multiple suppliers; Alkermes (Medisorb polymers), Absorbable Polymers International [formerly Birmingham Polymers (a Division of Durect)], Purac and Boehringer Ingelheim. Besides PLGA and PLA, natural cellulosic polymers such as starch, starch derivatives, dextran and non-PLGA synthetic polymers are also being explored as biodegradable polymers in such systems.

(68) Therapeutic Uses

(69) According to the principles of the present invention the peptides of the invention are useful in indications were insufficient blood-supply occurs, adequate blood vessels' growth and circulation is not properly restored, and there is a risk for tissue death due to insufficient blood flow or in cases of endothelia dysfunction. Typically, these conditions are for example peripheral vascular disease, myocardial ischemia, tissue graft diseases, coronary artery disease, stroke, and delayed wound healing (for example in ulcer lesions), pulmonary disease including acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and ventilation induced ling injury (VILI), eye diseases including age-related macular disease (AMD) and pathological condition related to severe infection such as for example sepsis

(70) The peptides of the present invention are also useful for the treatment of diabetes and additional disease states which involve damage to pancreatic beta cells, or require the regeneration of these cells. Pancreatic islet vasculature has been shown to be a key determinant of the regenerative capacity of pancreatic beta cells. Among others, Brissova et al. (Diabetes 55:2974-2985, 2006) demonstrate that angiogenic factors and their receptors are differentially expressed in the adult pancreas, and reduced VEGF-A expression by pancreatic beta cells results in abnormal islet vascularization and a pre-diabetic phenotype, leading to a reduced insulin output into the vascular system. Angiogenic factors like VEGF are major regulators of pancreatic islet vascularization and function, and are furthermore required for revascularization of transplanted pancreatic islets. Thus, the peptides of this invention can be utilized for the treatment of diabetes in general, or for pancreatic islet transplantation in particular.

(71) Although the present invention has been described with respect to various specific embodiments thereof in order to illustrate it, such specifically disclosed embodiments should not be considered limiting. Many other specific embodiments will occur to those skilled in the art based upon applicants' disclosure herein, and applicants propose to be bound only by the spirit and scope of their invention as defined in the appended claims.

EXAMPLES

(72) The following examples demonstrate the activity of the peptides of the present invention in stimulation of angiogenesis ex-vivo and in-vivo.

Example 1: Peptide Synthesis

(73) The peptides were synthesized by conventional solid phase synthesis methods, using either tBoc or Fmoc chemistry.

Example 2: Aortic Ring Assay Ex-Vivo

(74) Thoracic aortas were dissected from 7- to 8-week-old male SABRA mice. The aortas were immediately transferred to Petri dishes containing BIO-MPM-1. The adventitia and small vessels around the aorta were carefully removed under a dissecting microscope, and transverse cuts of 0.75 mm were made. The resulting aortic rings were extensively rinsed and incubated overnight in BIO-MPM-1 containing penicillin-streptomycin. Subsequently, the rings were embedded in 50 l collagen mix (7 parts collagen, 1 part 10 Eagle minimal essential medium, MEM, and 2 parts 0.15 M sodium bicarbonate) in 96-well plates (Nunc, Rochester, N.Y.). Solidification of the collagen solution was achieved by raising the pH to neutral and the temperature to 37 C. 150 l BIO-MPM-1 containing penicillin-streptomycin and the tested factors or peptides) was added to the embedded rings, and the plates were incubated at 37 C. in a humidified 10% CO2 atmosphere. Medium containing factors and/or peptides was refreshed at days 4 and 8. After two weeks, the rings were fixed with 4% formaldehyde for 24 hours, followed by staining with crystal violet (0.02%) dissolved in ethanol and extensive washing to remove excessive stain. The effect of factors and peptides was examined in 4 wells (4 rings of mouse aortic rings) per assay. Micrographs of representative rings were taken using a digital camera (Nikon, Tokyo, Japan). Morphometric analysis of sprouting was performed manually using Image-Pro 4.5 software (Media Cybernetics, Silver Spring, Md.) according to Nissanov et al. (Lab Invest. 1995; 73:734-739). Nonstained rings were subjected to paraffin sections and hematoxylin and eosin (H&E) staining.

(75) Table 2 summarizes the results of several compounds tested in several aortic ring assays. As demonstrated also in FIGS. 1A-1C and FIGS. 2A-2C, peptides AB-017 (SEQ ID NO: 12), AB-033 (SEQ ID NO: 13) and AB-034 (SEQ ID NO: 14) induce massive capillary formation in this assay. FIGS. 1A-1C represents aortic ring capillary length and FIGS. 2A-2C aortic ring capillary width. In the table: K-Flu is a Lysine residue coupled with a fluorescein moiety, 4BrPhe is 4-bromo-Phenylalanine, Dod is Dodecanoyl, Dec designates Decanoyl, Oct is Octanoyl, and Hex is Hexanoyl.

(76) TABLE-US-00006 TABLE 2 Capillary length Peptide Z 1 2 3 4 5 6 7 B 10 M 10 M 20 M SEQ ID NO: 725-106 Myr-Gly Nle Arg Pro Tyr Asn Ala NH.sub.2 7,000 4,600 2 AB-018 Myr-Gly Nle Arg Pro Tyr Asn NH.sub.2 12,600 10,200 24 AB-017 Myr-Gly Nle Arg Pro Tyr NH.sub.2 15,300 15,300 12 AB-016 Myr-Gly Arg Pro Tyr NH.sub.2 8,700 14,500 11 AB-015 Acetyl-Gly- Arg Pro Tyr NH.sub.2 1,000 1,700 25 AB-014 Arg Pro Tyr NH.sub.2 2,500 9,300 AB-013 Myr-Gly Nle Arg Pro Tyr Asn DAla NH.sub.2 8700 900 26 AB-011 Myr-Gly Nle Arg Pro Tyr Asn Ala K-Flu NH.sub.2 2500 5000 27 vehicle 1,700 1,700 AB-017 Myr-Gly Nle Arg Pro Tyr 1,720 3,100 11,200 12 AB-033 Myr-Gly Nle Arg Pro 35dIY 3,570 3,800 13 AB-034 Myr-Gly Nle hArg Pro Tyr 9,570 9,700 10,780 14 AB-031 Myr-Gly Nle Arg Pro 4BrPhe 1,840 28 vehicle 490 0 490 AB-017 Myr-Gly Nle Arg Pro Tyr 3,100 3,770 12 AB-020 Dod-Gly Nle Arg Pro Tyr 1,900 5,600 2,700 29 AB-021 Dec-Gly Nle Arg Pro Tyr 590 3,940 3,200 30 AB-022 Oct-Gly Nle Arg Pro Tyr 380 2,170 1,700 31 AB-023 Hex-Gly Nle Arg Pro Tyr 430 1,700 320 32 vehicle 0.0 1,370 0.0 AB-017 Myr-Gly Nle Arg Pro Tyr 1,720 11,200 12 AB-033 Myr-Gly Nle Arg Pro 35dIY 3,570 3,800 1013 AB-034 Myr-Gly Nle hArg Pro Tyr 9,570 10,780 14 vehicle 490 490

Example 3: Efficacy StudyMouse Hind Limb Ischemia/Angiogenesis Model

(77) The object of this study was to determine whether administration of the test items, peptides AB-017 (SEQ ID NO: 12) and 725-106 (positive control, SEQ ID NO: 2) can enhance angiogenesis, subsequently inducing clinical and motor function improvement in a hind limb ischemia model. The principle of the test is based on changes in perfusion rate, clinical signs and histological parameters (necrosis) following administration of the test compounds.

(78) The mouse is the chosen species for the study, and historically has been used for the hind limb ischemia models, in the assessment and evaluation of potential pro-angiogenesis properties of tested compound. The mouse hind limb ischemia model is an initial step providing early information about efficacy of this strategy.

(79) Each tested group included 4-7 mice per group. Intraperitoneal (i.p) injection is the preferred route of administration of the test items in this model and DMSO/PEG is used as vehicle to prepare the test item solution.

(80) Tested peptides were dissolved in 100% DMSO. Thereafter Polyethylene glycol (PEG) 400 is added to the solution. Sterile water was used to dilute to a final concentration of 0.5% DMSO/10% PEG 400/H.sub.2O to result 2 mg/ml. The solution is prepared and divided into marked aliquots and stored at 70 C.

(81) Species/Strain: Mice/Balb/c male8 weeks

(82) Source: Harlan Laboratories, Israel

(83) Initial Body weight: Approximately 25 g at study initiation. The minimum and maximum weights of the group are within a range of 20% of group mean weight.

(84) Animals handling is performed according to guidelines of the National Institute of Health (NIH) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

(85) Animals were housed under standard laboratory conditions, air conditioned and filtered (HEPA F6/6) with adequate fresh air supply (31 air changes/hour). Animals were kept in a climate controlled environment. Temperatures range is between 20-24 C. and RH is between 30-70% with 12 hours light and 12 hours dark cycle. Animals were housed in polyethylene cages (7/cage) measuring 353015 cm, with stainless steel top grill facilitating pelleted food and drinking water in plastic bottle; bedding: steam sterilized clean paddy husk. Bedding material is changed along with the cage at least twice a week.
Diet: Animals are fed ad libitum a commercial rodent diet (Teklad Certified Global 18%
Protein Diet cat #: 106S8216). Animals have free access to drinking water obtained from the municipality supply.
Surgical Procedure:
Anesthesia: Isoflurane 1.5-3% in a mixture of O.sub.2/N.sub.2O, under spontaneous respiration. All four limbs of the animal were taped down in order to facilitate the animal for surgery. Both hind legs of the animals are shaved and disinfected. On the operated leg, the right leg in all animals, an incision was made from the inguinal area to the proximity of the knee. The femoral vein/artery/nerve were exposed. The artery is dissected free from the vein, nerve and surrounding fascia. Distal to its bifurcation with the profound femoral artery, using 6-0 silk suture the artery is ligated twice, and thereafter transected between these two points (see prox-A-group model appendix I). The surgical wound is closed with 6-0 silk and the mouse is returned to its cage. The left leg is untreated control in all animals.
Exclusion criteria: Only mice, whose mean flux measurement post surgery is 10% and 50% of the pre surgical measurement, are incorporated into the study. Mice whose mean flux measurement post surgery out of this range were excluded from the study.

(86) $\mathrm{Calculation}\text{:}\mathrm{Mean}\mathrm{Flux}=\frac{\left(\mathrm{Pre}\mathrm{Treated}-0.5\right)/\left(\mathrm{Pre}\mathrm{Control}-0.5\right)}{\left(\mathrm{Post}\mathrm{Treated}-0.5\right)/\left(\mathrm{Post}\mathrm{Control}-0.5\right)}$
Drug administration: All animals were dosed by IP administration at a dose volume of 10 ml/kg.

(87) TABLE-US-00007 TABLE 3 Treatment scheme: Group Dose Level No. Treatment mg/kg Frequency of Dosing 1M Vehicle NA Twice weekly for 4 weeks 2M AB-017 20 (First application on the 3M 725-106 20 day following surgery) Positive control
Tests and Evaluation:
Body weight: Body weights were measured prior to surgery, prior to each dosing, and prior to necropsy.
Blood flow measurement: Using a Laser Doppler apparatus (Perimed PeriScan PIM II System, Technical manual available at test facility) Blood flow was measured (operated leg & control leg) while the legs are in their natural resting position at the following time points: Before surgery (after shaving) & Post surgery (Day 0); Day 1; Day 8; Day 15; Day 22; and Day 29. Each time point for each mouse consists of three measurements.
Blood flow was measured, additionally, in first 10 animals at Pre before shaving.
Macroscopic evaluation of ischemic severity: Two weeks after the operation and at study end, the ischemic limb was macroscopically evaluated by using a 5 point graded morphological scale for necrotic area; Grade 0: No change; Grade 1: Toe necrosis (limited to); Grade 2: Foot necrosis (extending to a dorsum pedis); Grade 3: Knee necrosis (extending to knee); Grade 4: Total necrosis (total hind-limb loss), according to Tokai. J. et al. (Exp. Clin. Med., 31, 3, pp. 128-132, 2006).
Assessment of limb motor function damage: Two weeks after the operation and at study end assessment of limb motor function was performed using the following 4 point scale: Grade 0: Flexing the toes to resist gentle traction of the tail; Grade 1: plantar flexion; Grade 2: No dragging but no plantar flexion; Grade 3: Dragging of foot (Rutherford et al., J. Vascular Surgery; 1997, 26, 517-538). Termination muscle weight & tissue fixation: At study termination Day 29, animals are sacrificed. The following muscles were dissected free of the leg, weighted and fixed in formalin until retrieved by the Sponsor: Anterior Tibial, Gastrocnemius, and Soleus.
Results
Data compilation: Data was summarized in a graphical form showing perfusion of the operated leg for 4 weeks (FIG. 3, Numerical results are given as means and standard errors), and in a tabular form (Table 4) showing for each group the number of animals, scores of leg function and scores of ischemic condition of the leg at day 15. Only animals whose leg perfusion was decreased >50% post operation were included in this analysis.
Ischemic Score
4: necrosis extending to a thigh (total hind-limb loss)
3: necrosis extending to a crus (knee loss)
2: necrosis extending to a dorsum pedis (foot loss)
1: necrosis limiting to toes (toes loss)
0: absence of necrosis
Function Score
3: dragging of foot
2: no dragging but no plantar flexion
1: plantar flexion
0: flexing the toes to resist gentle traction of the tail (Rutherford et al., 1997).
FIG. 3 and Table 4 summarize the results of treatments with compounds AB-017 (SEQ ID NO: 12), and 725-106 (positive control, SEQ ID NO: 2) as compared to vehicle control in the hind limb ischemia model in vivo. As can be seen, in all parameters of perfusion (FIG. 3), leg function and leg necrosis (Table 4) treatment with AB-017 leads to a significant improvement of the ischemic leg as compared to both the vehicle control and the longer peptide 725-106.

(88) TABLE-US-00008 TABLE 4 Hind limb ischemia Functional Treatment Mouse # evaluation at day 15 evaluation at day 15 Vehicle 39 2.5 3.0 43 1.0 1.0 19 3.0 NA 30 2.3 3.0 82 0.0 0.0 Average 1.8 AB-107 46 2.0 3.0 38 2.0 3.0 42 0.0 2.0 78 0.0 0.0 15 1.0 0.0 28 0.0 0.0 66 0.0 0.0 Average 0.7 106 44 2.0 3.0 (Positive 40 3.0 NA control) 33 2.5 3.0 16 2.0 3.0 Average 2.4

Example 4: In Vivo EfficacyAngiogenesis Model in Mice

(89) The aim of this study was to test the pro-angiogenic activity of the peptides in the mouse myocardial infarction model and to determine whether administration of peptides can induce blood vessel growth in this model and whether, as a result, improvement in clinical function of the ischemic heart can be demonstrated. The principle of the test is based on changes in perfusion rate, clinical signs and histological parameters following administration of the test peptides. Study end points include measurement of ECG and measurement of myocardial damage.

(90) The mouse is the chosen species for the study, and historically has been used for the myocardial infarction studies, in the assessment and evaluation of potential pro-angiogenesis properties of tested compound. The mice myocardial infarction ischemia model is usually an initial step providing early information about efficacy of this strategy.

(91) A total of 30 mice were utilized. Each tested group included 10 mice. Intraperitoneal (i.p) injection is the preferred route of administration of the tested compounds in this model and DMSO/PEG is used as vehicle to prepare the test item solution.

(92) Materials and Methods

(93) Tested peptides were dissolved in 100% DMSO. Thereafter Polyethylene glycol (PEG) 400 is added to the solution. Sterile water is use to dilute to a final concentration of 0.5% DMSO/10% PEG 400/H2O to result 2 mg/ml. The solution is prepared and divided into marked aliquots and stored at 70 C.

(94) Species/Strain: Mice/F1 hybrid CB6F1 (C57BL/6JBALBc)

(95) Gender/Number/Age: Male/30 (3 groups)/8 weeks

(96) Source: Harlan Laboratories, Israel

(97) Body weight: Approximately 25 g at study initiation. The minimum and maximum weights of the group will not exceed 20% of group mean weight.

(98) Acclimation period: Minimum 5 days.

(99) Identification: By mice accession number, ear notching and cage cards.

(100) Animals handling was according to the National Institute of Health (NIH) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

(101) Animals were housed in polyethylene cages (4/cage) measuring 353015 cm, with stainless steel top grill having facilities for pelleted food and drinking water in glass bottle; Animals are housed under standard laboratory conditions, air conditioned and filtered (HEPA F6/6) with adequate fresh air supply (31 air changes/hour). Animals were kept in a climate controlled environment. Temperatures range is between 20-24 C. and RH is between 30-70% with 12 hours light and 12 hours dark cycle. Steam sterilized clean paddy husk (Harlan, Sani-chip cat#:2018SC+F) are used and bedding material is changed along with the cage at least twice a week. Animals were fed ad libitum free-feeding a commercial rodent diet (Teklad Certified Global 18% Protein Diet cat#: 106S8216). Animals had free access to tap drinking water. Reasonably expected contaminants in food and water supplies should not have the potential to influence the outcomes of this test. Animals were randomized using a computer generated randomization program Research Randomizer and divided into 3 groups of 10 animals.

(102) Experimental Design and Conditions

(103) The animal was anesthetized with Pentobarbital sodium (70 mg/kg). The landmark for the incision is the left armpit. Lidocaine, 0.1 ml of 0.1% solution, was injected subcutaneously. An oblique 8-mm incision was made 2 mm away from the left sternal border toward the left armpit (1-2 mm below it). The operator tried to visualize the superficial thoracic vein that runs under the skin at the lateral corner of the incision. Both layers of thoracic muscles were cut, taking caution to avoid the vein; through the thin and semitransparent chest wall, the ribs and inflating lung are visible. The 4.sup.th intercostal space represents the area between those ribs where the lowest part of the lung is observed. The chest cavity was then opened taking caution not to damage the lung. The chest retractor is inserted and opened gently to spread the wound 8-10 mm in width. The heart partially covered by the lung was then visualized. The pericardium was gently picked up with curved and straight forceps, pulled apart, and placed behind the arms of the retractor. This maneuver pushes the lung up slightly, mobilizing it and providing better exposure of the heart. The left anterior descending coronary artery (LAD) was then visualized as a pulsating bright red spike, running in the midst of the heart wall from underneath of the left atrium toward the apex. If the LAD artery cannot be visualized, the left atrium can be lifted so that the origin of the LAD artery from the aorta is located. The LAD artery was ligated 1-2 mm below the tip of the left auricle in its normal position, which induces roughly 40-50% ischemia of the left ventricular (LV). Once the site of ligation has been determined, the curved forceps were used to gently press on the artery a little below the subsequent ligation (this enhance the view of the artery and stabilize the heart). Occlusion was confirmed by the change of color (becoming pale) of the anterior wall of the LV. The retractor was removed, and the lungs were reinflated by shutting off the ventilator outflow. The chest cavity was closed by bringing together the 4th and the 5th ribs with one or two 6-0 nylon sutures (with pressure applied to the chest wall to reduce the volume of free air). The muscles and skin were closed layer by layer with 6-0 absorbable and nylon sutures, respectively. The duration of the whole procedure took about 12-15 min.

(104) Drug Administration:

(105) First dosing was done by IP administration of 20 mg/kg of the tested compound in 10 ml/kg solution, approximately 45-120 min after induction of myocardial infarction only on recovered animals, spontaneous breathing animals and thereafter twice weekly.

(106) TABLE-US-00009 TABLE 5 Groups allocation Group # and Dose Level No. animals Treatment g/kg 1M (n = 10) Vehicle N/A 2M (n = 10) AB-017 (SEQ ID NO: 12) 20 mg/kg 3M (n = 10) AB-034 (SEQ ID NO: 14) 20 mg/kg
Tests and Evaluation:
Body weight: Body weight was measured on study day prior to dosing at each dosing day.
ECG measurements: The inductions of myocardial infarction are confirmed by an ECG ST segment elevation after LAD artery occlusion (before and immediately after occlusion). Thereafter, ECG measurements are made once a week after cardiac infarction by a BIOPAC system.
LDH measurement: Blood (150 l) is collected from each mouse at the conclusion of 24 hours and is stored for 1 to 2 hours at room temperature. Serum is obtained by centrifugation at 2,000 rpm for 10 min and stored at 70 C. LDH was measured with a standard Elisa Kit.
Termination: At study termination 4 weeks after operation the animals were sacrificed and gross pathology was performed. The hearts were excised and cannulated through the ascending aorta with a 23-gauge needle for sequential perfusion with 2 to 3 mL 37 C. 0.9% sodium chloride and with 3 to 4 mL 37 C. 1.0% TTC in phosphate buffer (pH 7.4). The hearts were then perfused with 2 to 3 ml of 10%/o Phthalo Blue (Heubach Ltd) to delineate nonischemic myocardium. Hearts were weighed and photographed from both sides with a digital camera. Image J was used to digitally planimeter the borders of the entire heart, the nonischemic area, and the infracted area on both sides of each slice. The sizes of the nonischemic area and ischemic area (area at risk) were calculated as size in mm.sup.2 or as percentages of the total area multiplied by the total weight of the heart.

Example 5: Whole-Mount Immune Staining of Aortic Rings

(107) Rings embedded and grown in collagen (as described in Aortic ring assay) were fixed with 1% paraformaldehyde in PBS for 30 minutes at room temperature and left overnight in PBS. Blocking and permeabilization of the fixed rings was achieved by incubation with PBS containing 1% BSA and 0.01% Triton-X 100 for 8 hours at 4 C. The rings were incubated with FITC-conjugated anti alpha smooth muscle actin antibody (ab8211, diluted 1:200; Abcam, Cambridge, England) in PBS containing 1% BSA for 8 hours. After removal of excess unbound antibody by several washes with PBS during the next 8 to 10 hours, the samples were visualized using a fluorescent microscope (Axiovert 200M; Zeiss).

Example 6: Corneal Pocket In-Vivo Assay

(108) The corneal pocket assay tests blood vessel formation from the limbus in a mouse eye towards a pellet containing the tested material/s, inserted to the cornea of the eye.

(109) Angiogenic responses were examined as described by Kenyon et al. (Invest Ophthalmol Vis Sci. 1996; 37:1625-1632). Briefly, pellets were prepared by dissolving sucralfate (500 mg/mL) in 20 mM sodium citrate, 1 mM EDTA, and 9% sucrose solution, and 20 l of this solution was added to 5 mg of the tested peptide, or 40 g of FGF, VEGF or CSF. Single pellets contained approximately 1:450 of these amounts. Following addition of 20 L Hydron polymer in ethanol (120 mg/mL), the mixtures were applied to a 1515-mm.sup.2 piece of synthetic mesh (Sefar America, Kansas City, Mo.) and coated by Hydron. The pellets were allowed to air dry, and fibers of the mesh were pulled apart. Corneal micropockets were created in both eyes of anesthetized C57BL/6 mice (8-10 weeks old). Incisions were made after local administration of lidocaine, using a Von-Graefe cataract knife. Pellets (0.20.20.3 mm) were impregnated into the corneal micropockets at a distance of 1 to 2 mm from the vascularized limbus. In case of peptide and protein combinations, 2 pellets were inserted to the same incision. Polymyxin-B-neomycin-bacitracin (Bamyxin) ophthalmic ointment was applied to the eyes to prevent infection. Responses were recorded and photographed after 8 days using stereoscopy. Vessel development was monitored twice weekly until full microscope, and transverse cuts of 0.5 mm were made. The resulting aortic vessel regression occurred. Each peptide and factor tested in this assay was examined in 4 animals (4-6 eyes).

Example 6: Synergistic Effect

(110) The aortic ring assay was repeated in the presence of 10 M concentration of peptides AB-033 (SEQ ID NO: 13) and AB-034 (SEQ ID NO: 14) together with the pro-angiogenetic protein VEGF. The results shown in Table 6 and in FIG. 4 demonstrate a synergistic effect of treatment with the peptides together with VEGF.

(111) TABLE-US-00010 TABLE 6 Average Treatment capillary length SE Fold vs vehicle Vehicle 1369 244 1.0 VEGF 3221 508 2.4 AB-033 4941 392 3.6 AB-033 + VEGF 13402 1591 9.8 AB-034 3062 558 2.2 AB-034 + VEGF 7818 1995 5.7

Example 7: Additional Peptides

(112) Table 7 lists additional peptides which are peptide analogs of the peptides disclosed above. These peptides are being synthesized and tested for stimulation of angiogenesis.

(113) TABLE-US-00011 TABLE 7 Modification Peptide Z 1 2 3 4 B SEQ ID NO: Arg.sup.2 AB-017 Myr-Gly Nle Arg Pro Tyr NH.sub.2 12 AB-066 Myr-Gly Nle X Pro Tyr NH.sub.2 33 AB-067 Myr-Gly Nle Cit Pro Tyr NH.sub.2 34 AB-0 Myr-Gly Nle Lys Pro Tyr NH.sub.2 35 AB-0 Myr-Gly Nle Gln Pro Tyr NH.sub.2 36 spacer AB-0 Myr-Ala Nle hArg Pro Tyr NH.sub.2 37 AB-0 Myr-Ala Nle Arg Pro Tyr NH.sub.2 38 Z AB-0 Cho-Gly Nle hArg Pro Tyr OH 39 AB-0 Cho-Gly Nle Arg Pro Tyr OH 40 B AB-0 Myr-Gly Nle hArg Pro Tyr OR 41 AB-0 Myr-Gly Nle Arg Pro Tyr OR 42 N- AB-0 Myr-Gly NMeNle Arg Pro Tyr NH.sub.2 43 methylation AB-0 Myr-Gly Nle NMeArg Pro Tyr NH.sub.2 44 AB-061 Myr-Gly NMeNle hArg Pro Tyr NH.sub.2 45 Pro.sup.3 AB-068 Myr-Gly Nle hArg Z1 Tyr NH.sub.2 46 AB-0 Myr-Gly Nle Arg Z1 Tyr NH.sub.2 47 AB-064 Myr-Gly Nle hArg 4HyP Tyr NH.sub.2 48 AB-0 Myr-Gly Nle hArg 3HyP Tyr NH.sub.2 49 AB-0 Myr-Gly Nle Arg 4HyP Tyr NH.sub.2 50 AB-0 Myr-Gly Nle Arg 3HyP Tyr NH.sub.2 51 AB-065 Myr-Gly Nle hArg Z2 Tyr NH.sub.2 52 AB-0 Myr-Gly Nle Arg Z2 Tyr NH.sub.2 53 Tyr.sup.4 AB-032 Myr-Gly Nle Arg Pro 4NF NH.sub.2 54 AB-0 Myr-Gly Nle hArg Pro 4NF NH.sub.2 55 AB-070 Myr-Gly Nle hArg Pro 35dIY NH.sub.2 56
Myr is a myristoyl, Cho is a cholesteryl, X is an 2-amino-3-guanidinopropionic acid, hArg is homoarginine, Cit is a citruline, R is a lower alkyl group, Z1 is (S)-thiazolidine-2-carboxylic acid, Z2 is (2S,3aS,7aS) Octahydro-1H-indole-2-carboxylic acid, NMeArg is N-methyl arginine, NMeNIe is N-methyl Norleucine, 4HyP is (4-hydroxy)proline, 3HyP is (3-hydroxy)proline, 4NF is (4-NH.sub.2) Phenylalanine, and 35dIY is (3,5-diiodo)tyrosine.

Example 8: Inhibition of Blood-Vessel Leakiness

(114) The peptide of SEQ ID NO: 12 (Myr-Gly-Nle-Arg-Pro-Tyr-NH.sub.2), or vehicle (10% PEG-400, 90% DDW), were injected i.p. to mice, in an amount of 20 or 40 mg/Kg. 5 min, 10 min, 1 hr or 4 hrs later, 0.2 ml per 25 g mouse (40 mg/Kg) of 5 mg/ml Evans-Blue solution in double distilled water (DDW) was injected i.p. The experiment was terminated after 30 min and skull was opened to view the brain surface (Neurosurgery) and assess the amount of Evans-Blue leaking from blood vessels.

(115) Principle of peripheral vessels staining: Evans-Blue; it is a poly-anionic compound having a M.W. of 960 Dalton, that binds tightly to albumin and being used to track vessel-permeability. In case of blood-vessel leakiness, the Evans-Blue-albumin complex escapes from the blood-stream into the surrounding interstitial fluid with the result of blue coloring of the extremities and the bluish appearance at the brain surface.

(116) In a specific experiment, 40 mg/kg of peptide of SEQ ID NO: 12 or vehicle were injected, each to 2 mice, i.p. 5 min before color injection. Evans-blue, at 40 mg/kg was then injected i.p. and 30 min later Neurosurgery was performed and the escape of the color from the blood stream was monitored as manifested by the bluish appearance of the brain surrounding tissue. The results clearly demonstrate that while Evans-Blue was leaking from blood-vessels of control mice (Vehicle-injected), as evident by the brain's bluish color, no color was leaking from vessels of animals pre-treated with peptide SEQ ID NO: 12.

(117) In an additional experiment, 20 mg/kg of peptide of SEQ ID NO: 12 or vehicle (10%/PEG-400, 90% DDW) were injected i.p. into 2 mice, 10 min before color injection (Evans-blue, 40 mg/kg i.p. for 30 minutes. The results clearly demonstrate that while Evans-Blue was leaking from blood-vessels of control mice (Vehicle-injected), as evident by the brain's bluish color and the bluish color of the paws, no color was leaking from vessels of animals pre-treated with peptide SEQ ID NO: 12, neither at the brain, nor at the periphery.

(118) In another experiment, 40 mg/kg of peptide SEQ ID NO: 12 were injected to mice, i.p. 1 or 4 hours before color injection (Evans-blue, 40 mg/kg i.p. for 30 minutes). Control mice received i.p. injection of vehicle (10%/PEG-4 i.p. 00, 90% DDW) 1 hour before color injection. The results clearly demonstrate that while Evans-Blue was leaking from blood-vessels of control mice (Vehicle-injected), as evident by the brain's bluish color, no color was leaking from vessels of animals pre-treated with peptide SEQ ID NO: 12, 1 hr before Evans-Blue injection, while the injection of the peptide 4 hrs before Evans-Blue gave only a partial protection. i.e. the appearance of faint-bluish color.

Example 9: In-Vivo Septic Shock Model

(119) The polymicrobial septic mouse model of severe sepsis (Belikoff et al. The Journal of Immunology, 2011, 186: 2444-2453), was induced in wild-type (WT) C57BL mice using cecum ligation and puncture (CLP) approach. In summary, mice were anesthetized, a 1.5-cm, longitudinal incision in the lower right quadrant of the abdomen was performed and the cecum was exposed. The distal two third of the cecum was ligated with 4-0 silk suture and punctured doubly with an 18-gauge needle. The cecum was then replaced into the peritoneal cavity and the incision was closed with surgical staples. After that, the mice were resuscitated with sterile saline injected subcutaneously and allowed free access to food and water after awaking.

(120) Following this procedure, mice were then injected s.c. once a day, for 5 days, with either vehicle or with 20 mg/Kg of GPS-725.017 (SEQ ID NO: 12).

(121) The results depicted in FIG. 5 demonstrate that the ability of peptide SEQ ID NO: 12 (denoted GPS-725.017 in the chart) to rescue mice from death in an established septic-shock in-vivo model.

(122) The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.