Peptides and use of same for pain treatment

Abstract

The present invention is directed to a dual agonist of cannabinoid receptor 2 and of opioid receptor, composition comprising same, and methods of using same in the treatment of pain, in a subject in need thereof.

Description

PEPTIDES AND USE OF SAME FOR PAIN TREATMENTREFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (ARL-P-048-PCT.xml; size: 11,723 bytes; and date of creation: March 17, 2025) is herein incorporated by reference in its entirety.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority of IL Patent Application No. 311899, titled ‘PEPTIDES AND USE OF SAME FOR PAIN TREATMENT’, filed 2 April 2024, the contents of which are incorporated herein by reference in their entirety.FIELD OF INVENTION

[0003] The present invention is in the field of peptide engineering, and pain treatment.BACKGROUND OF THE INVENTION

[0004] According to the NIH, approximately 25 million American adults suffer from chronic pain that is present daily. Prescription opioids have been the mainstay of pain management however, opioid misuse, addiction, and overdose have reached crisis levels in the United States. Enkephalins are small peptides produced endogenously and potent analgesics via opioid receptors (OR) activation. Leucine-Enkephalin (Leu-Enk) is considered a promising alternative for pain treatment and cause less side effects associated with alkaloid opioids such as morphine. However, the enkephalins have a poor pharmacokinetic profile and cannot be used as analgesics, as they are very sensitive to enzymatic degradation. Almost all endogenous opioid peptides contain the YGGFL or YGGFM peptide sequence, where the 1stTyr and the 4thPhe are necessary pharmacophores. However, there is a group of endogenous opioid peptides, endomorphins, including endomorphin- 1 (YPWF-NH2) and endomorphin-2 (YPFF- NH2), which have a different sequence. Both endomorphins lack the common enkephalin motif (YGGF) shared by other opioid peptides. The peptide sequence of these peptides is in close resemblance to that of another endogenous peptide, the short Osteogenic Growth Peptide (sOGP, YGFGG), recently discovered to be a cannabinoidreceptor 2 (CB2) agonist. CB2 receptors have been previously shown to modulate acute pain, chronic inflammatory pain, post-surgical pain, cancer pain and pain associated with nerve injury. Multifunctional opioid compounds have been previously proposed as a strategy for drug development. The mixed p-and K- opioid receptor agonist and the mixed opioid / nociception receptor agonists exhibited powerful analgesia.

[0005] In view of high pain prevalence and the opioid crisis, there is still great need for pain treatment alternatives to opioids.

[0006] Provided herein is a peptidomimetic agent being a mixed or dual CB2-OR agonist, that is required for antinociception free of undesired side- or adverse-effects.SUMMARY OF THE INVENTION

[0007] The present invention is directed to a dual agonist and use of same for reduction and / or treatment of pain, or a disease or condition inducing, promoting, or associated therewith, in a subject in need thereof.

[0008] According to the first aspect, there is provided a peptide being a dual agonist of cannabinoid receptor 2 (CB2) and of opioid receptor (OR).

[0009] According to another aspect, there is provided a composition comprising the peptide of the invention, and a pharmaceutically acceptable carrier.

[0010] According to the first aspect, there is provided a composition comprising a therapeutically effective amount of a dual peptide agonist of CB2 and OR, or a precursor peptide comprising thereof, for use in the treatment of pain in a subject in need thereof.[Oi l] In some embodiments, the peptide is a precursor peptide comprising the dual agonist of CB2 and of OR.

[0012] In some embodiments, the peptide is of 5 to 7 amino acids.

[0013] In some embodiments, the peptide is characterized by increased stability compared to a control.

[0014] In some embodiments, stability is in-blood or in-plasma stability.

[0015] In some embodiments, the stability is determined as the half-life of the peptide in plasma and / or in blood at a temperature ranging between 30 °C and 40 °C.

[0016] In some embodiments, the peptide is a cyclic peptide.

[0017] In some embodiments, the peptide comprises an amino acid sequence selected from the group consisting of: XiGFGG (SEQ ID NO: 1), YX2FGG (SEQ ID NO: 2), YGYGG (SEQ ID NO: 3), YSFPG (SEQ ID NO: 4), X3AFGG (SEQ ID NO: 5), YAX4GG (SEQ ID NO: 6), YAX5GG (SEQ ID NO: 7), and any combination thereof, wherein: Xi is L-DOPA; X2is D-2,4-diaminobutyric acid (DA2bu); X3is 2,6-dimethyl- tyrosine (Dmt); X4is D-l -naphthylalanine (D-l-Nal); and Xs is meta- aminobenzoic acid (mABA).

[0018] In some embodiments, the amino acid at position 2 of any one of SEQ ID Nos: 4-7 is a D amino acid.

[0019] In some embodiments, SEQ ID NO: 2 is a cyclic peptide.

[0020] In some embodiments, the DA2bu of SEQ ID NO: 2 is bound to a carboxyl group of the C-terminal glycine residue of SEQ ID NO: 2, thereby forming the cyclic peptide.

[0021] In some embodiments, the peptide excludes SEQ ID Nos: 8-10.

[0022] In some embodiments, the composition is formulated for injection or systemic administration.

[0023] In some embodiments, the composition is for use in the treatment of pain in a subject in need thereof.

[0024] In some embodiments, the pain is acute pain or chronic pain.

[0025] In some embodiments, the pain is a symptom of or induced by: inflammation, post-surgery, cancer, nerve injury or damage, or any combination thereof.

[0026] In some embodiments, the peptide comprises the amino acid sequence: XIX2X3GG (SEQ ID NO: 11) or a retro-verso isoform thereof, wherein: Xi is Tyrosine or an analog thereof, X2is a naturally or non-naturally occurring aliphatic amino acid, X3is a naturally or non-naturally occurring aromatic amino acid.

[0027] In some embodiments, the Tyrosine analog is L-DOPA or Dmt.

[0028] In some embodiments, X2is Glycine, DA2bu, or Alanine.

[0029] In some embodiments, X3is Phenylalanine, Tyrosine, or D-l-Nal.

[0030] In some embodiments, the amino acid at position 2 or 3 of the peptide is a D amino acid.

[0031] In some embodiments, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 1-3, 5-6, and 8-10.

[0032] In some embodiments, the retro verso isoform of said peptide is SEQ ID NO: 9.

[0033] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0034] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0035] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Fig. 1 includes a graph showing the effect of YGFGG (SEQ ID NO: 8) and derivatives thereof (SEQ ID Nos: 1-3, and 9-10) on lipopolysaccharide (LPS)-induced nitric oxide (NO) excretion from RAW macrophages cell line.

[0037] Fig. 2 includes a vertical bar graph showing the effect of YGFGG (SEQ ID NO: 8) and derivatives thereof (SEQ ID Nos: 1-3, and 9-10) on MC3T3 osteoblast cell line proliferation as a marker of CB2 activity, reflected as nitric excretion.

[0038] Fig. 3 includes a vertical graph showing the effect of YGFGG (SEQ ID NO: 8) on inflammatory pain. The CB2 antagonist SR144528 and OR antagonist naloxone, were used as controls showing that the YGFGG (SEQ ID NO: 8), provides a dual CB2-OR dependent analgesic effect.

[0039] Figs. 4A-4C include vertical bar graphs showing the effect of YGFGG (SEQ ID NO: 8) on diabetic neuropathic pain induced by Streptozocin (STZ) and chemotherapy- induced pain induced by paclitaxel (PTX). Pain perception was assessed by Von Frei filaments (4A), hot plate (4B), and acetone cooling test (4C). YGFGG (SEQ ID NO: 8) was found to be active in all tests in the STZ-induced pain model, and in the Von Frei and hot plate tests in the PTX-induced pain model.

[0040] Figs. 5A-5C include vertical bar graphs showing the effect of YGFGG (SEQ ID NO: 8) and synthetic derivatives thereof (SEQ ID Nos: 9-10) on colon length (5A), weight loss (5B), and tumor necrosis factor alpha (TNFa) levels (5C), in dextran sulphate sodium (DSS)-induced colitis in mice.DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention is directed to a peptide and compositions comprising same for use in methods of for ameliorating or treating pain or diseases associated therewith, in a subject in need thereof.Peptides

[0042] According to the first aspect, there is provided a cannabinoid receptor 2 (CB2) agonist, an opioid receptor (OR) agonist, or a dual agonist of both. In some embodiments, the agonist comprises a peptide or peptidaceous (or proteinaceous, e.g., made of or comprises amino acid(s)) agonist. In some embodiments, the agonist is capable of binding to cannabinoid receptor 2 (CB2). In some embodiments, the agonist is capable of binding opioid receptor (OR). In some embodiments, the agonist is capable of binding to CB2 and to OR.

[0043] According to some embodiments, there is provided a precursor peptide or a prodrug, comprising the peptide of the invention. In some embodiments, the peptide of the invention comprises or is a precursor peptide comprising the dual agonist of CB2 and of OR. In some embodiments, a precursor peptide is capable of undergoing biochemical modification so as to obtain the peptide of the invention. In some embodiments, a biochemical modification comprises cleavage, such as by a protease / peptidase. In some embodiments, biochemical modification comprises cleavage by a chemical (e.g., chemical digestion). In some embodiments, a precursor comprises 6 to 20 amino acids, 8 to 20 amino acids, 10 to 20 amino acids, 12 to 20 aminoacids, 15 to 20 amino acids, or 8 to 16 amino acids. Each possibility represents a separate embodiment of the invention.

[0044] According to another aspect, there is provided a peptide comprising the amino acid sequence XiGFGG (SEQ ID NO: 1), Xi is L-DOPA.

[0045] According to another aspect, there is provided a peptide comprising the amino acid sequence YX2FGG (SEQ ID NO: 2), X2 is D-2,4-diaminobutyric acid (DA2bu).

[0046] According to another aspect, there is provided a peptide comprising the amino acid sequence YGYGG (SEQ ID NO: 3).

[0047] According to another aspect, there is provided a peptide comprising the amino acid sequence YSFPG (SEQ ID NO: 4).

[0048] According to another aspect, there is provided a peptide comprising the amino acid sequence X3AFGG (SEQ ID NO: 5), X3 is 2,6-dimethyl-tyrosine (Dmt).

[0049] According to another aspect, there is provided a peptide comprising the amino acid sequence YAX4GG (SEQ ID NO: 6), X4 is D-l -naphthylalanine (D-l-Nal).

[0050] According to another aspect, there is provided a peptide comprising the amino acid sequence YAX5GG (SEQ ID NO: 7), X5 is meta-aminobenzoic acid (mABA).

[0051] According to another aspect, there is provided a combination comprising at least two peptides comprising amino acid sequences selected from SEQ ID Nos: 1-7.

[0052] In some embodiments, the combination comprises 2-3, 2-4, 2-5, 2-6, 2-7, 3-4, 3- 5, 3-6, 3-7, 4-5, 4-6, 4-7, 5-6, 5-7, or 6-7 peptides comprising amino acid sequences selected from SEQ ID Nos: 1-7.

[0053] In some embodiments, the peptide is of 5 to 7 amino acids.

[0054] In some embodiments, the peptide comprises up to 5, up to 6, up to 7, up to 8, up to 9, up to 10 amino acids, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the peptide comprises 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-9, 8-10, or 9-10 amin acids. Each possibility represents a separate embodiment of the invention.

[0055] In some embodiments, the peptide comprises at least 1 D amino acid. In some embodiments, the peptide comprises at least 2 D amino acids. In some embodiments, the peptides comprises 1-4, 1-3, 1-2, 2-4, or 2-3 D amino acids.

[0056] As used herein, the term "D amino acid" refers to an amino acid wherein the stereogenic carbon alpha to the amino group has the D -configuration.

[0057] In some embodiments, the amino acid at position 2 of a peptide comprising SEQ ID NO: 4 is a D amino acid.

[0058] In some embodiments, the amino acid at position 2 of a peptide comprising SEQ ID NO: 5 is a D amino acid.

[0059] In some embodiments, the amino acid at position 2 of a peptide comprising SEQ ID NO: 6 is a D amino acid.

[0060] In some embodiments, the amino acid at position 2 of a peptide comprising SEQ ID NO: 7 is a D amino acid.

[0061] In some embodiments, a peptide of the invention is linear or cyclic. In some embodiments, the peptide is a cyclic peptide.

[0062] In some embodiments, a peptide comprising SEQ ID NO: 2 is a cyclic peptide. In some embodiments, SEQ ID NO: 2 is a cyclic peptide. In some embodiments, DA2bu of SEQ ID NO: 2 is bound to a carboxyl group of the C-terminal glycine residue of SEQ ID NO: 2 (e.g., thereby forming a cyclic peptide).

[0063] In some embodiments, the peptide excludes SEQ ID Nos: 8-10.

[0064] In some embodiments, the peptide is characterized by increased stability. In some embodiments, stability is in-blood stability. In some embodiments, stability is in-plasma stability. In some embodiments, stability is in-blood stability and in-plasma stability.

[0065] In some embodiments, stability is determined as the half-life of the peptide. In some embodiments, half-life is biological half-life. In some embodiments, half-life is in plasma and / or in blood. In some embodiments, half-life is at a temperature ranging between 30 °C and 40 °C. In some embodiments, half-life is in plasma and / or in blood at a temperature ranging between 30 °C and 40 °C. In some embodiments, a temperature ranging between 30 °C and 40 °C comprises between 30 °C and 40 °C, 31 °C and 40 °C, 32 °C and 40 °C, 33 °C and 40 °C, 35 °C and 40 °C, 37 °C and 40 °C, 38 °C and 40 °C, 30 °C and 38 °C, 32 °C and 38 °C, 34 °C and 39 °C, 33 °C and 39 °C, 35 °C and 38 °C, or 36 °C and 38 °C. Each possibility represents a separate embodiment of the invention.

[0066] In some embodiments, an increased half-life comprises a half-life of at least: 7 min, 10 min, 20 min, 30 min, 45 min, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 12 hr, 16 hr, 18 hr, 24hr, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, an increased half-life comprises a half-life of : 5-60 min, 10-120 min, 20-240 min, 6-200 min, 45-480 min, 1-2 hr, 1-3 hr, 2-4 hr, 6-10 hr, 8-16 hr, 12-20 hr, 16-24 hr, 18-36 hr, 24-72 hr, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0067] In some embodiments, increased is compared to a control. In some embodiments, a control comprises a control peptide. In some embodiments, a control peptide does not include SEQ ID Nos: 1-7, and 9-10. In some embodiments, a control peptide comprises or consists of SEQ ID NO: 8.

[0068] In some embodiments, increased is at least: 5%, 10%, 20%, 50%, 100%, 250%, 500%, 650%, 750%, 900%, or 1,000% greater compared to a control, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, increased is by 1-50%, 10-100%, 50-250%, 25-500%, 30-650%, 100-700%, 150-750%, 200-850%, 250-900%, 300-1,000%, or 200-1,200% compared to control. Each possibility represents a separate embodiment of the invention.

[0069] In some embodiments, the peptide is capable of binding to cannabinoid receptor 2 (CB2). In some embodiments, the peptide is capable of binding opioid receptor (OR). In some embodiments, the peptide is capable of binding to CB2 and to OR. In some embodiments, the peptide is a CB2 agonist. In some embodiments, the peptide is OR agonist. In some, the peptide is CB2 and OR agonist (e.g., a "dual agonist").

[0070] In some embodiments, a peptide of the invention is capable of binding to CB2, OR, or both, in vitro, in vivo, ex vivo, or any combination thereof.

[0071] As used herein, "control" encompasses any baseline to which the binding and / or agonistic activity affinity of a peptide of the invention to CB2, OR, or both is compared to. In some embodiments, a control is a peptide having no CB2 binding and / or agonistic activity. In some embodiments, a control is a peptide having no OR binding and / or agonistic activity. In some embodiments, a control is a peptide having no CB2 and OR binding and / or agonistic activity. In some embodiments, a control is a peptide having low or reduced stability. In some embodiments, a control is a peptide having low or reduced in blood and / or in plasma stability. In some embodiments, a control is a peptide having in blood and / or in plasma stability, e.g., half-life, of not more than 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, or 10 min, or any value and range therebetween. Eachpossibility represents a separate embodiment of the invention. In some embodiments, a control is a peptide having in blood and / or in plasma stability, e.g., half-life, of 1- 10 min, 2-9 min, or 4-8 min. Each possibility represents a separate embodiment of the invention. In some embodiments, a control is a peptide of the invention in its linear form. In some embodiments, a control is a peptide of the invention which is initially inactivated, such as by antibody neutralization, enzymatic digestion, denaturation, or other methodologies known in the art of protein inactivation, prior to incubation in an environment comprising CB2, OR, or both, ex vivo, in vitro or in vivo.

[0072] The present invention encompasses derivatives of the peptide of the invention. The term "derivative" or "chemical derivative" includes any chemical derivative of the peptide having one or more residues chemically derivatized by reaction of side chains or functional groups. Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acid residues. For example: 4-hydroxyproline may be substituted for proline; 5 -hydroxy lysine may be substituted for lysine; 3 -methylhistidine may be substituted for histidine; homoserine may be substituted or serine; and ornithine (O) may be substituted for lysine.

[0073] In addition, a peptide derivative can differ from the natural sequence of the peptide of the invention by chemical modifications including, but are not limited to, terminal-NH2 acylation, acetylation, or thioglycolic acid amidation, and by terminal- carboxlyamidation, e.g., with ammonia, methylamine, and the like. Peptides can be either linear, cyclic, or branched and the like, having any conformation, which can be achieved using methods known in the art.

[0074] As used herein, the terms “peptide”, "polypeptide" and "protein" are used interchangeably, and refer to a polymer of amino acid residues.

[0075] The term "amino acid" as used herein means an organic compound containing both a basic amino group and an acidic carboxyl group. Included within this term are naturally occurring amino acids, modified, unusual, non-naturally occurring amino acids, as well as amino acids which are known to occur biologically in free or combined form but usually do not occur in proteins. Included within this term are modified and unusual amino acids, such as those disclosed in, for example, Roberts and Vellaccio (1983) The Peptides. 5: 342-429. Modified, unusual or non-naturally occurring amino acids include, but are not limited to, D-amino acids, hydroxylysine, 4-hydroxyproline, N-Cbz-protected aminovaleric acid (Nva), ornithine (O), aminooctanoic acid (Aoc), 2,4- diaminobutyric acid (Abu), homoarginine, norleucine (Nle), N-methylaminobutyric acid (MeB), 2-naphthylalanine (2Np), aminoheptanoic acid (Ahp), phenylglycine, [3- phenylproline, tert-leucine, 4-aminocyclohexylalanine (Cha), N-methyl-norleucine, 3,4- dehydroproline, N,N-dimethylaminoglycine, N-methylaminoglycine, 4- aminopipetdine-4-carboxylic acid, 6-aminocaproic acid, trans-4- (aminomethyl) - cyclohexanecarboxylic acid, 2-, 3-, and 4- (aminomethyl) - benzoic acid, 1- aminocyclopentanecarboxylic acid, 1 -aminocyclopropanecarboxylic acid, cyanopropionic acid, 2-benzyl-5- aminopentanoic acid, Norvaline (Nva), 4-O-methyl- threonine (TMe), 5-O-methyl-homoserine (hSM), tert-butyl- alanine (tBu), cyclopentylalanine (Cpa), 2-amino-isobutyric acid (Aib), N-methyl-glycine (MeG), N-methyl- alanine (MeA), N-methyl-phenylalanine (MeF), 2-thienyl-alanine (2Th), 3-thienyl- alanine (3Th), O-methyl-tyrosine (YMe), 3-Benzothienyl-alanine (Bzt) and D-alanine (DAI).

[0076] The term "amino acid residue" as used herein refers to the portion of an amino acid that is present in a peptide.

[0077] The term "peptide bond" means a covalent amide linkage formed by loss of a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid.

[0078] The terms "peptide", "polypeptide" and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogs peptoids and semi-peptoids or any combination thereof. In another embodiment, the terms “peptide”, "polypeptide" and "protein" apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analog of a corresponding naturally occurring amino acid.

[0079] One of skill in the art will recognize that individual substitutions, deletions or additions to a peptide, 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 similar charge, size, and / or hydrophobicity characteristics, such as, for example, substitution of a glutamic acid (E) to an aspartic acid (D).

[0080] As used herein, the phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite function as specified herein.

[0081] The peptide derivatives according to the principles of the present invention can also include side chain bond modifications, including but not limited to -CH2-NH-, - CH2-S-, -CH2-S=0, OC-NH-, -CH2-O-, -CH2-CH2-, S=C-NH-, and -CH=CH-, and backbone modifications such as modified peptide bonds. Peptide bonds (-CO-NH-) within the peptide can be substituted, for example, by N-methylated bonds (-N(CH3)- CO-); ester bonds (-C(R)H-C-O-O-C(R)H-N); ketomethylene bonds (-CO-CH2-); a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl group, e.g., methyl; carba bonds (-CH2- NH-); hydroxyethylene bonds (-CH(OH)-CH2-); thioamide bonds (-CS-NH); olefmic double bonds (-CH=CH-); and peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. These modifications can occur at one or more of the bonds along the peptide chain and even at several (e.g., 2-3) at the same time.

[0082] The invention further includes peptides and derivatives thereof, which can contain one or more D-isomer forms of amino acids. Production of retro-inverso D- amino acid peptides where at least one amino acid and perhaps all amino acids are D- amino acids is well known in the art. When all of the amino acids in the peptide are D- amino acids, and the N- and C-terminals of the molecule are reversed, the result is a molecule having the same structural groups being at the same positions as in the L-amino acid form of the molecule. However, the molecule is more stable to proteolytic degradation and is therefore useful in many of the applications recited herein. Diastereomeric peptides may be highly advantageous over all L- or all D-amino acid peptides having the same amino acid sequence because of their higher water solubility, lower immunogenicity, and lower susceptibility to proteolytic degradation. The term "diastereomeric peptide" as used herein refers to a peptide comprising both L-amino acidresidues and D-amino acid residues. The number and position of D-amino acid residues in a diastereomeric peptide of the preset invention may be variable so long as the peptide is capable of displaying the function of disclosed chimera of the invention.

[0083] In some embodiments, the peptide of the invention is a diastereomeric peptide.

[0084] According to one embodiment, the peptide of the invention may be synthesized or prepared by any method and / or technique known in the art for peptide synthesis. According to another embodiment, the polypeptide may be synthesized by a solid phase peptide synthesis method of Merrifield (see J. Am. Chem. Soc, 85:2149, 1964). According to another embodiment, the polypeptide of the invention can be synthesized using standard solution methods, which are well known in the art (see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer- Verlag, 1984).

[0085] In general, the synthesis methods comprise sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain bound to a suitable resin. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support (resin) or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions conductive for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups are removed sequentially or concurrently, and the peptide chain, if synthesized by the solid phase method, is cleaved from the solid support to afford the final peptide.

[0086] In the solid phase peptide synthesis method, the alpha-amino group of the amino acid is protected by an acid or base sensitive group. Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation, while being readily removable without destruction of the growing peptide chain. Suitable protecting groups are t-butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, (alpha, alpha)-dimethyl-3 ,5 dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2- cyano-t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. In the solid phase peptide synthesis method, the C-terminal amino acid is attached to a suitablesolid support. Suitable solid supports useful for the above synthesis are those materials, which are inert to the reagents and reaction conditions of the stepwise condensationdeprotection reactions, as well as being insoluble in the solvent media used. Suitable solid supports are chloromethylpolystyrene-divinylbenzene polymer, hydroxymethyl- polystyrene-divinylbenzene polymer, and the like. The coupling reaction is accomplished in a solvent such as ethanol, acetonitrile, N,N-dimethylformamide (DMF), and the like. The coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer as is well known in the art.

[0087] In another embodiment, a peptide of the invention may be synthesized such that one or more of the bonds, which link the amino acid residues of the peptide are nonpeptide bonds. In another embodiment, the non-peptide bonds include, but are not limited to, imino, ester, hydrazide, semicarbazide, and azo bonds, which can be formed by reactions well known to one skilled in the art.

[0088] Recombinant technology may be used to express the peptide of the invention, and is well known in the art. In one embodiment, the peptide of the invention can also be synthesized using in vitro expression systems. In vitro synthesis methods are well known in the art and the components of the system are commercially available.Compositions and methods of sung same

[0089] According to another aspect, there is provided a composition comprising the peptide of the invention, and a pharmaceutically acceptable carrier.

[0090] In some embodiments, the composition is a pharmaceutical composition.

[0091] In some embodiments, the composition is formulated for injection or systemic administration.

[0092] In some embodiments, the pharmaceutical composition facilitates administration of a compound to an organism. According to another embodiment, the invention provides a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of the peptide of the invention.

[0093] In another embodiment, the pharmaceutical composition of the invention may be formulated in the form of a pharmaceutically acceptable salt of the peptide of the present invention or an analog, or a derivative thereof. In another embodiment, a pharmaceutically acceptable salt includes salts formed with free amino groups such assalts derived from non-toxic inorganic or organic acids such as hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those salts formed with free carboxyl groups such as salts derived from non-toxic inorganic or organic bases such as sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, and the like.

[0094] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic compound (e.g., a peptide of the invention) is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates, or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0095] As used herein, the term "pharmaceutically acceptable" means suitable for administration to a subject, e.g., a human. For example, the term "pharmaceutically acceptable" can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0096] In another embodiment, the composition of the invention takes the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, gels, creams, ointments, foams, pastes, sustained-release formulations, and the like. In another embodiment, the composition of the invention can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gumtragacanth or gelatin. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in: Remington's Pharmaceutical Sciences" by E.W. Martin, the contents of which are hereby incorporated by reference herein. Such compositions will contain a therapeutically effective amount of the polypeptide of the invention, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0097] According to an embodiment of the invention, a pharmaceutical composition contains 0.1-95% of the peptide of the invention, a derivative, or an analog thereof. According to another embodiment of the invention, a pharmaceutical composition contains 1-70% of the peptide. According to another embodiment of the invention, the composition or formulation to be administered may contain a quantity or a plurality of peptides, according to embodiments of the invention in an amount effective to treat the condition or disease of the subject being treated.

[0098] As used herein, the term "plurality" refers to any integer being equal to greater than 2.

[0099] An embodiment of the invention relates to a peptide of the invention, presented in unit dosage form, and prepared by any of the methods well known in the art of pharmacy. In an embodiment of the invention, the unit dosage form is in the form of a tablet, capsule, lozenge, wafer, patch, ampoule, vial, or pre- filled syringe. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the nature of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in-vitro or in-vivo animal model test bioassays or systems.

[0100] According to one embodiment, the composition of the invention is administered in the form of a pharmaceutical composition comprising at least one of the active component of this invention (the peptide) together with a pharmaceutically acceptable carrier or diluent. In another embodiment, the composition of this invention can be administered either individually or together in any conventional oral, parenteral, ortransdermal dosage form. In some embodiments, the pharmaceutical composition further comprises at least one analgesic agent. In some embodiments, the pharmaceutical composition is adopted for combined administration with an anti-pain therapy such as analgesic drug.

[0101] As used herein, the terms “administering”, “administration”, and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect.

[0102] Depending on the location of the tissue of interest, the peptide of the invention can be administered in any manner suitable for the provision of the peptides to cells within the tissue of interest. Thus, for example, a composition containing the peptide of the invention can be introduced, for example, into the systemic circulation, which will distribute the peptide to the tissue of interest. Alternatively, a composition can be applied topically to the tissue of interest (e.g., injected, or pumped as a continuous infusion, or as a bolus within a tissue, applied to all or a portion of the surface of the skin, etc.).

[0103] In some embodiments, the pharmaceutical composition comprising the peptide is administered via oral, rectal, vaginal, topical, nasal, ophthalmic, transdermal, subcutaneous, intramuscular, intraperitoneal, or intravenous routes of administration. The route of administration of the pharmaceutical composition will depend on the disease or condition treated. Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other mode of injection as known in the art. Although the bioavailability of peptide administered by other routes can be lower than when administered via parenteral injection, by using appropriate formulations it is envisaged that it will be possible to administer the composition of the invention via transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular modes of treatment. In addition, it may be desirable to introduce the pharmaceutical composition of the invention by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer.

[0104] For topical application, a peptide of the invention, derivative, analog or a fragment thereof can be combined with a pharmaceutically acceptable carrier so that aneffective dosage is delivered, based on the desired activity. The carrier can be in the form of, for example, and not by way of limitation, an ointment, cream, gel, paste, foam, aerosol, suppository, pad, or gelled stick.

[0105] For oral applications, the pharmaceutical composition may be in the form of tablets or capsules, which can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate; or a glidant such as colloidal silicon dioxide. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier such as fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents. The tablets of the invention can further be film coated.

[0106] For purposes of parenteral administration, solutions in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal injection purposes.

[0107] According to some embodiments, the peptide of the invention, can be delivered in a controlled release system. In another embodiment, an infusion pump can be used to administer the peptide such as the one that is used, for example, for delivering insulin or chemotherapy to specific organs or tumors. In another embodiment, the peptide of the invention is administered in combination with a biodegradable, biocompatible polymeric implant, which releases the peptide over a controlled period of time at a selected site. Examples of preferred polymeric materials include, but are not limited to, polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers and blends thereof (See, Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla., the contents of which are hereby incorporated by reference in their entirety). In yet another embodiment, a controlled release system can be placed in proximity to a therapeutic target, thus requiring only a fraction of the systemic dose.

[0108] The presently described peptide may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use with the presently described peptide are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0109] In one embodiment, depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is affected or diminution of the disease state is achieved.

[0110] In some embodiments, the peptide is administered in a therapeutically safe and effective amount. As used herein, the term “safe and effective amount” refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit / risk ratio when used in the presently described manner. In another embodiment, a therapeutically effective amount of the peptide is the amount of the polypeptide necessary for the in vivo, in vitro, or ex vivo measurable expected biological effect. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005). In some embodiments, preparation of effective amount or dose can be estimated initially from invitro assays. In one embodiment, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

[0111] In one embodiment, toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. In one embodiment, the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. In one embodiment, the dosages vary depending upon the dosage form employed and the route of administration utilized. In one embodiment, the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.l].

[0112] Pharmaceutical compositions containing the presently described peptide as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990). See also, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa. (2005).

[0113] In one embodiment, compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier are prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0114] In one embodiment, the composition of the invention is presented in a pack or dispenser device, such as an FDA approved kit, which contains one or more-unit dosages forms containing the active ingredient. In one embodiment, the pack, for example, comprises metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, in one embodiment, is labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.

[0115] According to another aspect, there is provided a method for treating pain in a subject in need thereof.

[0116] According to another aspect, there is provided a composition for use in the treatment of pain in a subject in need thereof. In some embodiments, the composition is for use in the treatment of pain in a subject diagnosed therewith.

[0117] In some embodiments, pain comprises acute pain or chronic pain.

[0118] In some embodiments, pain comprises pain induced by chemotherapy.

[0119] In some embodiments, pain comprises pain induced by inflammation, or a disease associated therewith. In some embodiments, a disease associated with inflammation comprises metabolic syndrome. In some embodiments, a disease associated with inflammation comprises an inflammatory bowel disease (IBD), such as Chron's disease and / or ulcerative colitis. In some embodiments, a disease associated with inflammation comprises diabetes.

[0120] In some embodiments, pain is a symptom of or induced by: inflammation, postsurgery, cancer, nerve injury or damage, or any combination thereof.

[0121] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a dual CB2 OR agonist, a precursor peptide comprising thereof, or a composition comprising thereof. In some embodiments, the composition comprises a therapeutically effective amount of a dual CB2 OR of the invention.

[0122] In some embodiments, the agonist is a peptide agonist. In some embodiments, the dual CB2 OR agonist is a peptide dual agonist.

[0123] In some embodiments, the peptide comprises the amino acid sequence: X1X2X3GG (SEQ ID NO: 11) or a retro-verso isoform thereof, wherein Xi is Tyrosine or an analog thereof, X2 is a naturally or non-naturally occurring aliphatic amino acid, X3 is a naturally or non-naturally occurring aromatic amino acid.

[0124] Tyrosine analogs include para-substituted tyrosines, ortho-substituted tyrosines, and meta substituted tyrosines. In some embodiments, the substituted tyrosine comprises an acetyl group, a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C6-C20 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O-methyl group, apolyether group, a nitro group, or the like. In addition, multiply substituted aryl rings are also contemplated.

[0125] In some embodiments, Tyrosine analog is L-DOPA or Dmt.

[0126] As used herein the term "aliphatic amino acid" encompasses any amino acid containing an aliphatic side chain functional group.

[0127] As used herein, the term "aliphatic amino acid" encompasses any amino acid being non-polar, hydrophobic, or both.

[0128] In some embodiments, a naturally or non-naturally occurring aliphatic amino acid comprises Glycine, Proline, Leucine , Isoleucine, Valine DA2bu, or Alanine.

[0129] In some embodiments, a naturally or non-naturally occurring aliphatic amino acid comprises Glycine, DA2bu, or Alanine.

[0130] In some embodiments, naturally or non-naturally occurring aromatic amino acid comprises Phenylalanine, Tyrosine, or D-l-Nal.

[0131] In some embodiments, the amino acid at position 2, 3, or both, of the peptide is a D amino acid.

[0132] In some embodiments, the peptide comprises an amino acid sequence selected from SEQ ID Nos: 1-3, 5-6, and 8-10.

[0133] In some embodiments, a retro verso isoform of the peptide is SEQ ID NO: 9.

[0134] According to another aspect, there is provided a composition comprising a therapeutically effective amount of a dual CB2 OR agonist, for use in the treatment of an inflammatory disease, or a symptom associated therewith.

[0135] According to another aspect, there is provided method for treating an inflammatory disease, or a symptom associated therewith, in a subject in need thereof.

[0136] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a composition comprising a dual CB2 OR agonist.

[0137] In some embodiments, an inflammatory disease comprises inflammatory bowel disease (IBD). In some embodiments, a symptom comprises: colon shortening, reduced body weight or increased weight loss, increased TNFa blood levels, or any combination thereof, in the subject.

[0138] In some embodiments, a treated subject is characterized by reduced colon shortening, increased body weight or reduced weight loss, reduced blood TNFa levels, or any combination thereof.

[0139] Methods for determining colon shortening, body weight, and TNFa levels are common and would be apparent to one of skill in the art, such as, but not limited, to the methods exemplified herein.

[0140] In some embodiments, increased, reduced, or both, is compared to a control subject. In some embodiments, a control subject is a healthy subject. In some embodiments, a control subject is not treated with a peptide of the invention, a composition comprising same ,or both.

[0141] The terms "inhibiting", "reducing" and "decreasing" are interchangeable.

[0142] The term "subject" as used herein refers to an animal, more particularly to nonhuman mammals and human organism. Non-human animal subjects may also include prenatal forms of animals, such as, e.g., embryos or fetuses. Non-limiting examples of non-human animals include: horse, cow, camel, goat, sheep, dog, cat, non-human primate, mouse, rat, rabbit, hamster, guinea pig, pig. In one embodiment, the subject is a human. Human subjects may also include fetuses. In one embodiment, a subject in need thereof is a subject afflicted with and / or at risk of being afflicted with a condition associated with increased cell proliferation, deubiquitination activity, or combination thereof.

[0143] As used herein, the terms “treatment” or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life. In some embodiments, treatment comprises prevention. In some embodiments, treatment comprises prevention and treatment.

[0144] As used herein, the term “prevention” of a disease, disorder, or condition encompasses the delay, prevention, suppression, or inhibition of the onset of a disease, disorder, or condition. As used in accordance with the presently described subject matter, the term "prevention" relates to a process of prophylaxis in which a subject is exposedto the presently described peptides prior to the induction or onset of the disease / disorder process. This could be done where an individual has a genetic pedigree indicating a predisposition toward occurrence of the disease / disorder to be prevented. For example, this might be true of an individual whose ancestors show a predisposition toward certain types of, for example, pain. The term "suppression" is used to describe a condition wherein the disease / disorder process has already begun but obvious symptoms of the condition have yet to be realized. Thus, the cells of an individual may have the disease / disorder, but no outside signs of the disease / disorder have yet been clinically recognized. In either case, the term prophylaxis can be applied to encompass both prevention and suppression. Conversely, the term "treatment" refers to the clinical application of active agents to combat an already existing condition whose clinical presentation has already been realized in a patient.

[0145] As used herein, the term "condition" includes anatomic and physiological deviations from the normal that constitute an impairment of the normal state of the living animal or one of its parts, that interrupts or modifies the performance of the bodily functions.

[0146] Any concentration ranges, percentage range, or ratio range recited herein are to be understood to include concentrations, percentages, or ratios of any integer within that range and fractions thereof, such as one tenth and one hundredth of an integer, unless otherwise indicated.

[0147] Any number range recited herein relating to any physical feature, such as polymer subunits, size, or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated.

[0148] As used herein, the terms “subject” or “individual” or “animal” or “patient” or “mammal,” refers to any subject, particularly a mammalian subject, for whom therapy is desired, for example, a human.

[0149] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” ratherthan the exclusive or, and indicates at least one of, or any combination of items it conjoins.

[0150] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a”, “an” and “at least one” are used interchangeably in this application.

[0151] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0152] The term "about" refers to ±10%.

[0153] In the description and claims of the present application, each of the verbs, “comprise”, “include”, and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.

[0154] Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0155] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0156] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, forbrevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub -combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.EXAMPLES

[0157] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

[0158] The inventors have synthesized a few peptides, some assayed on a previously described osteogenic model, and some novel, e.g., SEQ ID Nos: 1-3, and 9-10. SEQ ID NO: 9 is a peptide with retro-inverso sequence of SEQ ID NO: 8. Generally, non-related retro-inverso peptides have been shown to be more stable, while retaining or even improving the biological activity, compared to their "parent" peptide. In SEQ ID NO: 1, the 1stamino acid, tyrosine, is replaced by the structurally similar L-DOPA. In SEQ ID NO: 10, the 2ndamino acid is D-alanine, which may turn the molecule to be more activeand stable. In SEQ ID NO: 2, the 2ndamino acid is replaced by D-2,4-diaminobutyric acid, which is then cyclized with the COOH of the terminal glycine, to obtain a cyclic peptide, which may be more stable and more active. SEQ ID NO: 3 has a replacement of the 3rdphenylalanine with tyrosine.

[0159] YGFGG (SEQ ID NO: 8) is the parent peptide, and is characterized by the structure:

[0160] GGfGy (SEQ ID NO: 9) is a retro-inverso isoform of SEQ ID NO: 8, comprising a D-Phenylalanine (indicated by a lowercase 'f ), a D-Tyrosine (indicated by a lowercase 'y'), and is characterized by the structure:

[0161] [L-DOPA]GFGG (SEQ ID NO: 1) comprises L-DOPA instead of the 1stTyrosine (of the parent peptide), and is characterized by the structure:

[0162] YaFGG (SEQ ID NO: 10) comprises a D-Alanine (indicated by a lowercase 'a') instead of the 2ndGlycine (of the parent peptide), and is characterized by the structure:

[0163] Y[DA2bu]GFGG (SEQ ID NO: 2) is a cyclic peptide comprising D-2,4- diaminobutyric acid (DA2bu) that is covalently bound to the 'COOH' group of the terminal Glycine, and is characterized by the structure:

[0165] All derivatives significantly inhibited nitric oxide (NO) excretion (with P values of less than 0.01). In this regard, SEQ ID NO: 10 produced an effect that was even significantly stronger (P=0.0055) than that of the parent peptide YGFGG (SEQ ID NO: 8) (Fig. 1).

[0166] Further, the inventors tested the effect of YGFGG (SEQ ID NO: 8) and its modified peptides (SEQ ID NO: 1-3, and 9-10) on MC3T3 osteoblasts proliferation, as a marker of CB2 activity. In this regard, while all tested peptides were found to have a positive activity, the activity of SEQ ID Nos: 9 and 10 even exceed that of the parent peptide YGFGG (SEQ ID NO: 8) (Fig. 2).

[0167] The inventors have further synthesized 4 additional new derivatives.

[0168] YsFPG (SEQ ID NO: 4) comprises a D-Serine (indicated by a lowercase 's') and a substitution of the 4thGlycine (of the parent peptide) with a Proline.

[0169] [Dmt]aFGG (SEQ ID NO: 5) comprises a 2,6-dimethyl-tyrosine (Dmt) instead of the 1stTyrosine (of the parent peptide), and a D-Alanine (indicated by a lowercase 'a') instead of the 2ndGlycine (of the parent peptide).

[0170] Ya[D-l-Nal]GG (SEQ ID NO: 6) comprises a D-Alanine (indicated by a lowercase 'a') instead of the 2ndGlycine (of the parent peptide), and a D-l- naphthylalanine (D-l-Nal) instead of phenylalanine (of the parent peptide).

[0171] Ya[mABA]GG (SEQ ID NO: 7) comprises a D-Alanine (indicated by a lowercase 'a') instead of the 2ndGlycine (of the parent peptide), and meta- aminobenzoic acid (mABA) instead of phenylalanine (of the parent peptide).

[0172] In a preliminary stability tests, all peptides derivatives were found to have increased stability over the parent peptide.

[0173] Further, the inventors found that the parent peptide, YGFGG (SEQ ID NO: 8) has therapeutic effect relevant for treatment of diseases with inflammatory origin. The synthetic derivatives, SEQ ID Nos: 2, and 9-10, indeed show that a retro-inverso peptide, and / or addition of D amino acids, yield peptides with comparable and even improved activity compared to a parent peptide.

[0174] Both CB2 and OR are G protein coupled receptors (GPCR) coupled to Gi / o, thus their activation inhibits adenylyl cyclase and lowers cAMP levels. The inventors used cells transfected with each receptor separately to assess activation of receptors by the novel pep tidomime tics disclosed herein. HEK-OR (OR expressing HEK cells) and HEK-CB2 (CB2 expressing HEK cells) cells were treated with peptides of the invention or control before adding forskolin at a concentration of 10 pM to stimulate adenylyl cyclase. Samples were analyzed using a colorimetric cAMP ELISA kit. Most of the peptidomimetics of the invention have shown activity with half-maximal inhibitory concentration (IC50) ranging from high picomolar to high nanomolar range.

[0175] Stability analysis of the peptidomimetics of the invention was performed in mouse serum. The peptidomimetics were incubated in mouse serum at a temperature of 37 °C. Aliquots of the mixture were sampled at 0, 5, 15, 60, 300 and 600 min after incubation started, proteins were denaturized, and extraction and analysis by reverse phase (RP)-HPLC took place. The half-life (ti / 2) in mouse plasma was calculated using linear regression after log transformation. The half-life of YGFGG (SEQ ID NO: 8) inmouse serum was 7.52 min. SEQ ID NO: 10 has a prolonged half-life of 192.74 min. Of notion are the half-lives of SEQ ID Nos: 2 and 9, standing at more than 24 h.Pain models in-vivo

[0176] To assess the mechanism of the involvement of the peptidomimetics of the invention in antinociception, the inventors assessed their influence on inflammatory pain, using paclitaxel (PTX) model of chemotherapy-induced pain, and streptozocin (STZ) model of diabetic neuropathic pain. The sensory readouts for nociceptive measurements are further delineated herein below.Von Frey filament test

[0177] The mechanical threshold force in grams was defined as the lowest force required to obtain a paw retraction response.Hot Plate

[0178] Hot plate (53 °C) assay was used, and time taken by the animal to the first response was used as a surrogate for pain perception. A cutoff time of 20 s was selected to prevent any harm to the animal.Acetone Cooling Test

[0179] In the cold allodynia test, 20 pL of acetone was applied on the center of the plantar surface of the hind paw; the onset of licking or shaking the hind paw was measured, observed for 2 min.Zymozan model of inflammatory pain

[0180] Inflammation was induced by injection of a suspension of zymosan A into the sub-plantar surface of the hind paw of the mice. This was followed immediately by an injection of the herein disclosed peptidomimetics. Paw swelling was measured by calibrated caliper and pain perception was assessed by Von Frei filaments after 2, 6 and 24 h. Blood was collected after 24 h for analysis of TNFa serum levels using a mouse TNFa ELISA. YGFGG (SEQ ID NO: 8) and other peptides were able to reduce zymosan-induced inflammatory pain (Fig. 3). This effect was reduced by either the OR antagonist, naloxone, or the CB2 antagonist, SR144528, and completely prevented by their combination (Fig. 3). This result unequivocally shows that SEQ ID NO: 8, and the herein disclosed peptidomimetics, being derivative thereof, are dual CB2-OR agonists.Paclitaxel model of chemotherapy -induced pain

[0181] Paclitaxel (PTX) was intraperitoneally administered to mice every other day (days 0, 2, 4, and 6) with a total dose of 8 mg / kg (2 mg / kg / day). Pain perception was assessed by Von Frei filaments, hot plate, and acetone cooling test. Nociceptive tests were performed on day 7, while mice were treated with vehicle or the peptidomimetics of the invention a day before the tests. The activity of YGFGG (SEQ ID NO: 8) was found to be statistically significant over the controls in the Von Frei filament test and the hot plate test (Figs. 4A-4B).Streptozocin model of diabetic neuropathic pain

[0182] Diabetes is induced in Institute of Cancer Research (ICR) mice by a single intraperitoneal (i.p.) injection of streptozocin (STZ) (dose of 150 mg / kg).

[0183] Nociceptive tests was performed 2 weeks after STZ injection, while mice were treated with vehicle or the peptidomimetics of the invention a day before the tests. In a preliminary test, the inventors showed that YGFGG (SEQ ID NO: 8) was active in the Von Frei filament, hot plate, and acetone cooling tests (Figs. 4A-4C).

[0184] Thereafter, the inventors compared the effects of the parent peptide YGFGG (SEQ ID NO: 8) to synthetic derivatives thereof, in a well-known model of dextran sulphate sodium (DSS)-induced colitis. The colon length, which is the measure of colon inflammation in mice, was greatly reduced by DSS, indicating developed colitis. YGFGG (SEQ ID NO: 8), SEQ ID NO: 9, and SEQ ID NO: 10 were shown to reduce colon shortening (Fig. 5A). Specifically, the greatest inhibition of colon shortening was observed in mice treated with SEQ ID NO: 9 (Fig. 5A). Reduced weight is also a hallmark of colitis, as mice suffering from colitis cannot absorb food properly, as was evident in the DSS treatment. SEQ ID Nos: 8-10 were shown to inhibit weight reduction in DSS -treated mice (Fig. 5B). In that regard, the weight reduction inhibitory effect attributed to SEQ ID Nos: 9-10 was more pronounced compared to SEQ ID NO: 8 (YGFGG). Further, the inventors examined the biochemical inflammatory state of the treat mice, as reflected by blood TNFa levels. The results show that any one of SEQ ID Nos: 8-10 significantly reduced TNFa levels in the blood of DDS-treated mice. In that regard, the reduction of TNFa blood levels in mice treated with SEQ ID Nos: 9-10 was more pronounced compared to SEQ ID NO: 8 (YGFGG).

[0185] To this end, the inventors conclude that the peptidomimetics disclosed herein, are highly stable in blood, and have significant attributes related to treatment of pain, aswell as inflammatory diseases, including, but are not limited to inflammatory bowel disease.

[0186] Therefore, the results highlight that the pep tidomime tics of the invention are of high potential for pain therapy, being dual ligands / agonists of CB2-OR, and importantly having no opioid side or adverse effects.

[0187] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

CLAIMSWhat is claimed is:

1. A peptide being a dual agonist of cannabinoid receptor 2 (CB2) and of opioid receptor (OR).

2. The peptide of claim 1, being a precursor peptide comprising said dual agonist of CB2 and of OR.

3. The peptide of claim 1, being of 5 to 7 amino acids.

4. The peptide of any one of claims 1 to 3, being characterized by increased stability compared to a control.

5. The peptide of claim 4, wherein said stability is in-blood or in-plasma stability.

6. The peptide of claim 4 or 5, wherein said stability is determined as the half-life of said peptide in plasma and / or in blood at a temperature ranging between 30 °C and 40 °C.

7. The peptide of any one of claims 1 to 6, being a cyclic peptide.

8. The peptide of any one of claims 1 to 7, comprising an amino acid sequence selected from the group consisting of: XiGFGG (SEQ ID NO: 1), YX2FGG (SEQ ID NO: 2), YGYGG (SEQ ID NO: 3), YSFPG (SEQ ID NO: 4), X3AFGG (SEQ ID NO: 5), YAX4GG (SEQ ID NO: 6), YAX5GG (SEQ ID NO: 7), and any combination thereof, wherein: Xi is L-DOPA; X2 is D-2,4-diaminobutyric acid (DA2bu); X3 is 2,6-dimethyl- tyrosine (Dmt); X4 is D-l -naphthylalanine (D-l-Nal); and X5 is meta- aminobenzoic acid (mABA).

9. The peptide of claim 8, wherein the amino acid at position 2 of any one of SEQ ID Nos: 4-7 is a D amino acid.

10. The peptide of claim 8 or 9, wherein said SEQ ID NO: 2 is a cyclic peptide.

11. The peptide of claim 10, wherein said DA2bu of said SEQ ID NO: 2 being bound to a carboxyl group of the C-terminal glycine residue of said SEQ ID NO: 2, thereby forming said cyclic peptide.

12. The peptide of any one of claims 1 to 11, wherein said peptide excludes SEQ ID Nos: 8-10.

13. A composition comprising the peptide of any one of claims 1 to 12, and a pharmaceutically acceptable carrier.

14. The composition of claim 13, being formulated for injection or systemic administration.

15. The composition of claim 13 or 14, for use in the treatment of pain in a subject in need thereof.

16. The composition for use according to claim 15, wherein said pain is acute pain or chronic pain.

17. The composition for use according to claim 15, wherein said pain is a symptom of or induced by: inflammation, post-surgery, cancer, nerve injury or damage, or any combination thereof.

18. A composition comprising a therapeutically effective amount of a dual peptide agonist of CB2 and OR, or a precursor peptide comprising thereof, for use in the treatment of pain in a subject in need thereof.

19. The composition for use of claim 18, wherein said peptide is of 5 to 7 amino acids.

20. The composition for use of claim 18 or 19, wherein said peptide being characterized by increased in-blood or in-plasma stability, compared to a control.

21. The composition for use of any one of claims 18 to 20, being a cyclic peptide.

22. The composition for use of any one of claims 18 to 21, wherein said peptide comprises the amino acid sequence: X1X2X3GG (SEQ ID NO: 11) or a retro-verso isoform thereof, wherein: Xi is Tyrosine or an analog thereof, X2 is a naturally or non- naturally occurring aliphatic amino acid, X3 is a naturally or non-naturally occurring aromatic amino acid.

23. The composition for use of claim 22, wherein said Tyrosine analog is L-DOPA or Dmt.

24. The composition for use of claim 22 or 23, wherein X2 is Glycine, DA2bu, or Alanine.

25. The composition for use of any one of claims 22 to 24, wherein X3 is Phenylalanine, Tyrosine, or D-l-Nal.

26. The composition for use of any one of claims 22 to 25, wherein the amino acid at position 2 or 3 of said peptide is a D amino acid.

27. The composition for use of any one of claims 22 to 26, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 1-3, 5-6, and 8-10.

28. The composition for use of any one of claims 22 to 27, wherein said retro verso isoform of said peptide is SEQ ID NO: 9.

29. The composition for use of any one of claims 18 to 28, wherein pain is acute pain or chronic pain.

30. The composition for use of any one of claims to 18 to 29, wherein said pain is a symptom of or induced by: inflammation, post-surgery, cancer, nerve injury or damage, or any combination thereof.

31. The composition for use of any one of claims 18 to 30, wherein said composition further comprises a pharmaceutically acceptable carrier.

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