WO2016038608A1 - Peptides derived from angiotensin receptor and use thereof - Google Patents

Abstract

Peptides derived from or corresponding to the carboxy-tail of the angiotensin II type receptor are provided. Further, pharmaceutical compositions comprising said peptides and use thereof in treating, ameliorating or preventing COX-2 associated disease including but not limited to inflammatory associated diseases and pain are provided.

Description

PEPTIDES DERIVED FROM ANGIOTENSIN RECEPTOR AND USE THEREOF

FIELD OF THE INVENTION

The invention relates to, inter alia, peptides derived from angiotensin II type 1 receptor and methods of treating cyclooxygenase-2 associated diseases and disorders using said peptides.

BACKGROUND OF THE INVENTION

Prostaglandins are bioactive lipids that function as major regulators of cardiovascular homeostasis. They are derived from a common H₂ prostaglandin endoperoxide (PGH₂), a metabolite of arachidonic acid (AA) that is formed by the rate-limiting enzyme cyclooxygenase (COX). COXs exist in two main isoforms, COX-1 and COX-2, both of which reside on the luminal surfaces of the endoplasmic reticulum and the inner and outer membranes of the nuclear envelope. Both enzymes display similar catalytic mechanisms but differ in their expression patterns. COX-1 is expressed almost ubiquitously and fulfills many housekeeping functions, while COX-2 is usually absent from most tissues but undergoes a rapid and transient increase of its expression by a broad range of pathological stimuli (Smith, W. L., and Langenbach, R., 2001, Clin Invest 107, 1491-1495). As such, inhibition of its activity by non-steroidal antiinflammatory drugs (NSAIDs) is one of the most common therapeutic targets for treatment of inflammation. However, COX-2 is also normally expressed in some tissues where it has some important physiological roles. In the kidney, the products of COX-2 catalysis increase the generation of the vasoconstricting hormone angiotensin II (Angll), which in turn downregulates the expression of COX-2 through activation of mainly the angiotensin II type 1 receptor (ATi).

The ATi receptor belongs to the super family of G protein coupled receptors (GPCRs) that relay signals by activating heterotrimeric G proteins, followed by second-messenger-mediated intracellular signaling. Studies in the last decade showed that ATi signals through two distinct signaling pathways, whereby binding of ligand initiates activation of G proteins, but quickly thereafter switches to β arrestin- mediated G protein-independent pathways. Coupling ATi to G proteins is mediated primarily through a DRY motif located in the third intracellular loop of the receptor. Mutation of this motif abrogates coupling to G proteins but β arrestin recruitment and activation of the ERK MAP kinase pathway remains intact. In contrast, the absence of certain phosphorylation sites in the CT of ATi prevents β arrestin-mediated signaling while preserving the G-protein pathway.

High levels of COX-2 are characteristic of many types of chronic ailments suggesting that tight regulation of its levels is critical for normal physiological function. Whereas the signaling cascades that lead to the induction of COX-2 are well-studied, there is much less information about the regulatory pathways that mediate its degradation. In the absence of its substrate AA, the mature COX-2 protein undergoes continuous turnover by shuttling from the endoplasmic reticulum to the cytosol via the ER-associated degradation (ERAD) pathway, where it is subsequently degraded by the Ubiquitin-Proteasome System. Degradation of COX-2 in the proteasome is preceded by its polyubiquitination, and was recently shown to be facilitated by caveolin-1 and also through its interaction with the G-protein coupled receptor (GPCR) prostaglandin El receptor (EP1) and βΐ adrenergic receptors.

There is a need for improved compositions and methods for treating COX-2 associated diseases and disorders including but not limited to inflammation and pain.

SUMMARY OF THE INVENTION

The present invention provides peptides derived from angiotensin II type 1 (ATi) receptor and particularly from the carboxy-tail (CT) of said ATi receptor, compositions comprising same and methods of use thereof in treatment of COX-2 associated diseases and disorders including but not limited to inflammation and pain.

The present invention is based, in part, on the surprising finding that short amino acid sequences of the carboxy-tail of ATi down-regulate COX-2 expression, thereby being useful for treating COX-2 associated diseases and disorders.

According to a first aspect, the present invention provides an isolated peptide of no more than 45 amino acids comprising an amino acid sequence as set forth in SEQ ID NO: 1 (KSHSXiLSTKMSTLSYRPSDNX₂SSSX₃KKPAX₄CFEVE), wherein Xi is Asn (N) or Ser (S), X₂ is Val (V) or Met (M), X₃ is Thr (T) or Ala (A) and X₄ is Pro (P) or Ser (S), or an analog, a derivative or fragment thereof.

According to one embodiment, said analog comprises the amino acid sequence as set forth in SEQ ID NO: 2 (KS HS NLS TKMS TLS YRPS DN VS S S TKKP APCFE VE) or an analog, a derivative or a fragment thereof. According to one embodiment, said analog comprises the amino acid sequence as set forth in SEQ ID NO: 3 (KS HS S LS TKMS TLS YRPS DNMS S S AKKP AS CFE VE) or an analog, a derivative or a fragment thereof. According to another embodiment, said analog comprises the amino acid sequence as set forth in SEQ ID NO: 4 (KS HSNLS TKMS PLS YRPS DN VS S S TKKP APCFE VE) or an analog, a derivative or a fragment thereof. According to another embodiment, said analog comprises the amino acid sequence as set forth in SEQ ID NO: 5 (KS HS NLS TKMS TLS YRHS DN VS S S TKKP APCFE VE) or an analog, a derivative or a fragment thereof.

According to another embodiment, said fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 6 (KS HS X i LS TKMS TLS YRPS ) wherein Xi is Asn (N) or Ser (S). According to another embodiment, said fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 7 (KSHSNLSTKMSTLSYRPS). According to another embodiment, said fragment comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 8 (KS HS S LS TKMS TLS YRPS ) . According to another embodiment, said fragment has a length of no more than 40 amino acids. According to another embodiment, said fragment has a length of no more than 35 amino acids. According to another embodiment, said fragment has a length of no more than 30 amino acids. According to another embodiment, said fragment has a length of no more than 25 amino acids. According to another embodiment, said fragment has a length of no more than 20 amino acids. According to another embodiment, said fragment has a length of no more than 15 amino acids.

According to another aspect, there is provided a pharmaceutical composition comprising as an active ingredient an isolated peptide of the present invention, and a pharmaceutically acceptable carrier. According to another aspect, there is provided a method of treating, preventing or alleviating a disease in a subject, particularly a disease associated with COX-2. According to some embodiments, the method comprises administering to the subject in need thereof an effective amount of the peptide of the invention or the pharmaceutical composition comprising same. According to another embodiment, the COX-2 associated disease or disorder is selected from the group consisting of an inflammatory associated disease or disorder, pain and stress - related pathologies. According to another embodiment, said inflammatory associated disease or disorder is selected from the group consisting of: multiple sclerosis, arthritis, asthma, inflammatory bowel disease (Crohn's disease), psoriasis, and systemic lupus erythematosus (SLE).

According to another embodiment, said pain is selected from the group consisting of acute pain, chronic pain, cancer pain, central pain, labor pain, myocardial infarction pain, pancreatic pain, colic pain, post-operative pain, headache pain, muscle pain, pain associated with intensive care, arthritic pain, neuropathic pain, and pain associated with an oral or periodontal disease, including gingivitis and periodontitis.

According to another embodiment, said stress-related pathologies is selected from posttraumatic stress disorder, acute stress disorder, adjustment disorder, bereavement related disorder, general anxiety disorder, social anxiety disorder and anxiety disorder due to a medical condition.

According to another aspect, there is provided a peptide of the invention or a pharmaceutical composition comprising same for use in treating, preventing or alleviating a disease in a subject, particularly a COX-2 associated disease or disorder.

According to another aspect, there is provided use of a peptide of the invention (or a pharmaceutical composition comprising same) for the preparation of a medicament for treating, preventing or alleviating a disease in a subject, particularly a COX-2 associated disease or disorder. This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-D. ATi receptor lowers COX-2 expression. (1A) HEK 293 cells were transfected with COX-2 and either empty vector (Mock) or HA-ATi at a ratio of 1:5. 36 h post transfection cells were stimulated with 50 μΜ AA for 30 min. The culture media was collected and probed for PGE₂ production by RIA. (n=4, in triplicates). (IB) HEK 293 were transfected with YFP-COX-2 and HA-ATi as in A and levels of YFP COX-2 were analyzed by flow cytometry. (1C) The expression of COX-2 (cy5) and GFP-ATi was analyzed by fluorescent microscopy. (ID) The effect of CFP-ATi on YFP-tagged COX-1 or COX-2 was measured in the presence of increasing levels of CFP-ATi at the indicated ratios. Total DNA levels were equal in all samples («=4).

Figures 2A-E. The effect of ATi on COX-2 does not require receptor activation. (2A) cells were transfected with COX-2 and empty vector (first two lanes), HA-ATi and empty vector (two middle lanes) and COX-2 with HA-ATi (last two lanes), and treated with Angll (1 μΜ, 10 min). Receptor activation was evaluated by measuring activation of ERK MAP kinase (pERK). Shown is a representative immunoblot of n=3. (2B) Quantification of ERK activation (ratio of phospho to total ERK) by 1 μΜ Angll at the indicated time points (n=3). (2C) Cells were transfected with either ATi alone or in the presence of wild type COX-2 or its catalytically inactive mutant G533A COX-2. ERK activation was measured after 10 min stimulation with 1 μΜ Angll (n=5). (2D) Cells were transfected as above and treated with the PKC inhibitors GFX at the indicated concentrations, throughout transfection. Levels of YFP-COX-2 and CFP-ATi were analyzed by flow cytometry (n=3). (2E) YFP-COX-2 was transfected with CFP-tagged wild type ATi, DRY/AAY ATi or TSTS/A ATi at ratios of 1 :5. Levels of YFP-COX-2 (black columns) and CFP-ATi (grey columns) were obtained by flow cytometry (n=5).

Figures 3A-D. Inhibition of the proteasome lowers ATi and rescues COX-2. Cells transfected with YFP- COX-2 and either empty vector or CFP-ATi were treated with or without 10 μΜ MG132 for 16 h. (3A) Summary graph of the effect of MG132 on YFP-COX-2 levels. (n=6). (3B) Expression of CFP-ATi from the same experiments. (3C) Representative immunoblot of cells transfected and treated as above that were analyzed for COX-2 and ATi using specific antibodies. (3D) Dose-dependent effect of MG132 treatment of YFP-COX-2 and CFP-ATi levels (n=3).

Figures 4A-C. ATi enhances proteasomal degradation of COX-2 by increasing its ubiquitination. ATi co-immunoprecipitates with COX-2. Cells were transfected with empty vector, COX-2, or HA-ATi and COX-2 with HA-ATi at a ratio of 1 : 1. Immunoprecipitation of (4A) HA or (4B) COX-2, was performed 16 h after transfection (representative blots of n=4). (4C) COX-2 ubiquitination is elevated in the presence ATi. COX-2 was immunoprecipitated from cells expressing COX-2 or HA-ATi alone or together. 16 h after transfection samples were collected and probed first for ubiquitin content followed by COX-2 and ATi antibodies (representative blot of n=4). Figures 5A-B. The carboxyl terminus of ATi is necessary for its effect on COX-2. (5A) HEK293 cells were transfected with COX-2 and either empty vector, wild type ATi (wt) or the Δ324 mutant at a 1 :5 ratio. Shown is a representative immunoblot of n=5 experiments. (5B) The effect of wild type and Δ324 mutant on YFP-COX-2 expression was measured by flow cytometry (n=5, in triplicates).

Figures 6A-D. The tail sequence of ATi (CT) is sufficient to downregulate COX-2 expression. (6A) HEK 293 cells were transiently transfected with YFP-COX-2, CFP-CT or both at a ratio of 1 :5. The effect on COX-2 expression was detected by fluorescent microscopy. CFP without the CT sequence was used as control and did not lower COX-2 expression (last panel). (6B) HEK 293 cells were transfected with wild type ATi or CT-Myc with or without COX-2. Representative immunoblot of n=5 experiments. (6C) NIH 3T3 fibroblasts were transfected with either empty vector or CT-Myc for 16 h in starvation media, followed by stimulation with 20% serum for 4 h (representative blots of n=4). (6D) The effect of wild type ATi or CT-Myc on YFP- COX-2 was measured using flow cytometry (n=4, in triplicates).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides peptides derived from or corresponding to the carboxy-tail of said ATi receptor, or analogs, derivatives or fragments thereof. The present invention further provides compositions comprising said peptides, and methods of use thereof in treatment of COX-2 associated diseases and disorders including but not limited to inflammation and pain.

As exemplified herein below, ATi receptor plays an important role in facilitating COX-2 degradation, thus constituting a feedback loop that does not depend on classical signaling pathways. Without wishing to be bound by any specific theory or mechanism of action, the demonstrated regulation of ATi constitutes physiological means of controlling normal COX-2 turnover, thereby treating pathological conditions associated by COX-2. Unexpectedly, short peptides derived from ATi CT downregulated COX-2 expression, thereby indicating use of said peptide in therapeutic approach for eliminating excess COX-2 protein.

In some embodiments, the peptides of the invention comprise an amino acid sequence derived from or correspond to residues 325-359 of an ATi receptor such as a human ATi receptor (Uniprot accession no. P30556). According to some embodiments, the present invention provides an isolated peptide comprising an amino acid sequence selected from the group consisting of: KSHSXiLSTKMSTLSYRPSDNX₂SSSX₃KKPAX₄CFEVE), wherein Xi is Asn (N) or Ser (S), X₂ is Val (V) or Met (M), X is Thr (T) or Ala (A) and X₄ is Pro (P) or Ser (S) or an analog, a derivative or fragment thereof.

According to some embodiments, the present invention provides an isolated peptide comprising the amino acid sequence as set forth in any one of: SEQ ID NO: 2

(KS HS NLS TKMS TLS YRPS DN VS S S TKKP APCFE VE) ; SEQ ID NO: 3

(KSHSSLSTKMSTLSYRPSDNMSSSAKKPASCFEVE); SEQ ID NO: 4 (KS HS NLS TKMS PLS YRPS DN VS S S TKKP APCFE VE) ; SEQ ID NO: 5

(KS HS NLS TKMS TLS YRHS DN VS S S TKKP APCFE VE) ; SEQ ID NO: 7

(KSHSNLSTKMSTLSYRPS); SEQ ID NO: 8 (KSHSSLSTKMSTLSYRPS); or an analog, a derivative or fragment thereof.

According to some embodiments of the invention, the peptides may be between about 8 and 45 amino acids in length. The peptides may comprise a sequence having between about 8 and about 45 amino acids of the carboxy-tail of an ATi receptor of a mammalian (e,g., human) variant. In some embodiments, the peptide comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 amino acids derived from, or corresponds to, the carboxy-tail of an ATi receptor and particularly to SEQ ID NO: 1. According to another embodiment, said fragment has a length of no more than 35 amino acids, no more than 34 amino acids, no more than 33 amino acids, no more than 32 amino acids, no more than 31 amino acids, no more than 30 amino acids, no more than 29 amino acids, no more than 28 amino acids, no more than 27 amino acids, no more than 26 amino acids, no more than 25 amino acids, no more than 24 amino acids, no more than 23 amino acids, no more than 22 amino acids, no more than 21 amino acids, no more than 20 amino acids, no more than 19 amino acids, no more than 18 amino acids, no more than 17 amino acids, no more than 16 amino acids, no more than 15 amino acids, no more than 14 amino acids, no more than 13 amino acids, no more than 12 amino acids, no more than 11 amino acids or no more than 10 amino acids, wherein each possibility represents a separate embodiment of the invention. The term "peptide" as used herein encompasses native peptides (degradation products, synthetic peptides or recombinant peptides), pep tidomime tics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids, and may have, for example, modifications rendering the peptides more stable while in the body or more capable of penetrating into cells.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

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 aspartic acid (D). According to an embodiment of the invention, the peptide of the invention comprises a sequence derived from residues 325-359 of an ATi receptor, with a conservative substitution. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has a similar property.

Exemplary conservative substitutions are set out in the Table 1 below:

According to an embodiment of the invention, the peptide disclosed herein comprises a sequence homologous to a sequence of residues 325-359 of an ATi receptor. According to an embodiment of the invention, the peptide comprises a sequence having greater than 75%, 80%, 85%, 90% or 95% homology to a sequence of residues 325-359 of an ATi receptor. According to another embodiment, the peptide comprises a sequence having greater than 75%, 80%, 85%, 90% or 95% homology to a sequence of SEQ ID NOs: 1-8. Each possibility represents a separate embodiment of the present invention.

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 of binding to COX-2, mediating COX-2 expression (e.g., degradation) and/or affecting inflammatory levels or other therapeutic use as specified herein.

Typically, the present invention encompasses derivatives of the peptides. 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-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted or serine; and ornithine may be substituted for lysine.

In addition, a peptide derivative can differ from the natural sequence of the peptides 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, which conformations can be achieved using methods well known in the art.

According to some embodiments of the methods and compositions of the invention, said peptide is coupled to a hydrophobic moiety. The term "hydrophobic" refers to the tendency of chemical moieties with nonpolar atoms to interact with each other rather than water or other polar atoms. Materials that are "hydrophobic" are, for the most part, insoluble in water. Non- limiting examples of natural products with hydrophobic properties include lipids, fatty acids, phospholipids, sphingolipids, acylglycerols, waxes, sterols, steroids, terpenes, prostaglandins, thromboxanes, leukotrienes, isoprenoids, retinoids, biotin, and hydrophobic amino acids such as tryptophan, phenylalanine, isoleucine, leucine, valine, methionine, alanine, proline, and tyrosine. A chemical moiety is also hydrophobic or has hydrophobic properties if its physical properties are determined by the presence of nonpolar atoms. The term includes lipophilic groups.

The term "lipophilic group", in the context of being attached to a peptide, refers to a group having high hydrocarbon content thereby giving the group high affinity to lipid phases. A lipophilic group can be, for example, a relatively long chain alkyl or cycloalkyl (preferably n- alkyl) group having approximately 6 to 30 carbons. The alkyl group may terminate with a hydroxyl, primary amine or any other reactive group. To further illustrate, lipophilic molecules include naturally-occurring and synthetic aromatic and non-aromatic moieties such as fatty acids, esters and alcohols, other lipid molecules, cage structures such as adamantane, and aromatic hydrocarbons such as benzene, perylene, phenanthrene, anthracene, naphthalene, pyrene, chrysene, and naphthacene.

According to some embodiments, the hydrophobic moiety comprises an aliphatic group and a reactive group through which the aliphatic group may be linked to the peptide. Non limiting examples of such reactive groups include: a carboxyl group, a carbonyl group, an amine group, a thiol group, a hydroxyl group, a maleimide, an imido ester, an N-hydroxysuccinimide, alkyl halide, and aryl azide.

The term "aliphatic", "aliphatic group" or "aliphatic chain, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more unsaturated bonds. Unless otherwise specified, aliphatic groups contain at least aliphatic carbon atoms. In some embodiments, aliphatic groups contain between 6 and 30 aliphatic carbon atoms. In other embodiments, aliphatic groups contain at least 8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain at least 10 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain at least 12 aliphatic carbon atoms, and in yet other embodiments aliphatic groups contain at least 16 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl, and heteroalkyl groups.

According to some embodiments, the hydrophobic moiety is selected from the group consisting of a fatty acid, a sterol or a fat soluble vitamin According to some embodiments, said hydrophobic moiety is a fatty acid. The fatty acid that can be coupled to the peptides of the invention is selected from saturated, unsaturated, monounsaturated, and polyunsaturated fatty acids. Typically, the fatty acid consists of at least six carbon atoms, preferably, at least eight carbon atoms. According to some embodiments, the fatty acid is an essential fatty acid. "Essential fatty acids" may refer to certain fatty acids, in particular polyunsaturated fatty acids that an organism must ingest in order to survive, being unable to synthesize the particular essential fatty acid de novo. Examples include the essential fatty acid C9, C12-linoleic acid and their structural variants. Essential fatty acids may be found in nature or produced synthetically. Non-limiting examples to fatty acids according to some embodiments of the invention include: decanoic acid (DA), undecanoic acid (UA), dodecanoic acid (lauric acid), myristic acid (MA), palmitic acid (PA), stearic acid, arachidic acid, lignoceric acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, trans - hexadecanoic acid, elaidic acid, lactobacillic acid, tuberculostearic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, docosapentaenoic acid and cerebronic acid, conjugated linolenic acid, omega 3 fatty acids (for example: docosahexaenoic acid (DHA), eicosapentaenoic acid, a-linolenic acid, stearidonic acid eicosatrienoic acid, eicosatetraenoic acid, docosapentaenoic acid and glycerol ester derivatives thereof), omega 6 fatty acids (for example: linoleic acid, gamma-linolenic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, docosadienoic acid, adrenic acid, docosapentaenoic acid and calendic acid), omega 9 fatty acids (for example: oleic acid, eicosenoic acid, mead acid, erucic acid and nervonic acid), polyunsaturated fatty acids, long- chained polyunsaturated fatty acids, arachidonic acid, monounsaturated fatty acids, precursors of fatty acids, and derivatives of fatty acids.

Vitamins: In certain embodiments, the present invention relates to vitamins selected from the group consisting of: vitamin A, vitamin D, vitamin E and vitamin K. According to other embodiments, the present invention relates to any other vitamin, salts and derivatives thereof known in the art. According to some embodiments, the hydrophobic moiety is conjugated to the N-terminus or C-terminus of said peptide. According to some embodiments, the hydrophobic moiety may be coupled to the peptide through any other free functional group along the peptide chain. For example, when the hydrophobic moiety is palmitic acid, the coupling is typically to a cysteine or otherwise to a serine or threonine. According to further embodiments, more than one hydrophobic moiety may be coupled to the peptide, through the N-terminus, C-terminus or through any other functional group along the peptide chain. Each possibility represents a separate embodiment of the present invention. It should be understood that the hydrophobic moiety is covalently coupled to the peptide. The terms "coupling" and "conjugation" are used herein interchangeably and refer to the chemical reaction, which results in covalent attachment of a hydrophobic moiety to a peptide to yield a lipophilic conjugate. Coupling of a hydrophobic moiety to a peptide is performed similarly to the coupling of an amino acid to a peptide during peptide synthesis. Alternatively, the coupling of a hydrophobic moiety to a peptide may be performed by any coupling method known in the art.

The peptide derivatives and analogs 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-, -CH2-0-, -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.

The present invention also encompasses peptide derivatives and analogs in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonylamino groups, carbobenzoxyamino groups, t-butyloxycarbonylamino groups, chloroacetylamino groups or formylamino groups. Free carboxyl groups may be derivatized to form, for example, salts, methyl and ethyl esters or other types of esters or hydrazides. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine. The peptide analogs can also contain non-natural amino acids. Examples of non-natural amino acids include, but are not limited to, sarcosine (Sar), norleucine, ornithine, citrulline, diaminobutyric acid, homoserine, isopropyl Lys, 3-(2'-naphtyl)-Ala, nicotinyl Lys, amino isobutyric acid, and 3-(3'-pyridyl-Ala).

Furthermore, the peptide analogs can contain other derivatized amino acid residues including, but not limited to, methylated amino acids, N-benzylated amino acids, O-benzylated amino acids, N-acetylated amino acids, O-acetylated amino acids, carbobenzoxy-substituted amino acids and the like. Specific examples include, but are not limited to, methyl- Ala (Me Ala), MeTyr, MeArg, MeGlu, MeVal, MeHis, N-acetyl-Lys, O-acetyl-Lys, carbobenzoxy-Lys, Tyr-O-Benzyl, Glu-O-Benzyl, Benzyl-His, Arg-Tosyl, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, and the like.

The invention further includes peptide analogs, which can contain one or more D-isomer forms of the 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 acid residues 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 requisite function of binding to COX-2, mediating COX-2 expression (e.g., degradation) and or affecting inflammatory levels or other therapeutic use as specified herein.

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.

The peptides of the invention may be synthesized or prepared by techniques well known in the art. The peptides can be synthesized by a solid phase peptide synthesis method of Merrifield (see J. Am. Chem. Soc, 85:2149, 1964). Alternatively, the peptides of the present invention can be synthesized using standard solution methods well known in the art (see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer- Verlag, 1984) or by any other method known in the art for peptide synthesis. In general, these 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.

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 suitable solid support. Suitable solid supports useful for the above synthesis are those materials, which are inert to the reagents and reaction conditions of the stepwise condensation-deprotection 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, Ν,Ν-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.

The peptides of the invention may alternatively be synthesized such that one or more of the bonds, which link the amino acid residues of the peptides are non-peptide bonds. These alternative 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 skilled in the art.

The peptides of the present invention, analogs or derivatives thereof produced by recombinant techniques can be purified so that the peptides will be substantially pure when administered to a subject. The term "substantially pure" refers to a compound, e.g., a peptide, which has been separated from components, which naturally accompany it.

Typically, a peptide is substantially pure when at least 50%, preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the peptide of interest. Purity can be measured by any appropriate method, e.g., in the case of peptides by HPLC analysis.

Included within the scope of the invention are peptide conjugates comprising the peptides of the present invention derivatives or analogs thereof joined at their amino or carboxy-terminus or at one of the side chains via a peptide bond to an amino acid sequence of a different protein. Conjugates comprising peptides of the invention and a protein can be made by protein synthesis, e. g., by use of a peptide synthesizer, or by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the conjugate by methods commonly known in the art. Addition of amino acid residues may be performed at either terminus of the peptides of the invention for the purpose of providing a "linker" by which the peptides of this invention can be conveniently bound to a carrier. Such linkers are usually of at least one amino acid residue and can be of 40 or more residues, more often of 1 to 10 residues. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.

According to an embodiment of the invention, the peptide of the invention has increased selectively to cyclooxygenase, particularly to COX-2. In some embodiments, the peptide of the invention has substantially greater selectively to COX-2 as compared to COX-1.

As used in connection with selective binding affinity, "substantially greater" means at least a two-fold, at least a three-fold, at least a four-fold, at least a five-fold, at least a six fold, at least a seven-fold, at least a eight-fold, at least a nine-fold, at least a ten-fold, at least a fifteen-fold, at least a twenty-fold, at least a thirty-fold, at least a forty-fold, at least a fifty-fold or at least a hundred-fold increase in the amount of peptide bound to a receptor. Pharmaceutical compositions of the invention

In some embodiments, there is provided pharmaceutical compositions comprising as an active ingredient a therapeutically effective amount of a peptide of the present invention, and a pharmaceutically acceptable carrier or diluents. The pharmaceutical compositions of the invention can be formulated in the form of a pharmaceutically acceptable salt of the peptides of the present invention or their analogs, or derivatives thereof. Pharmaceutically acceptable salts include those salts formed with free amino groups such as salts 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.

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.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic compound 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, glycerine, 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 compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, gels, creams, ointments, foams, pastes, sustained-release formulations and the like. The compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth 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 peptide 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. An embodiment of the invention relates to a peptide presented in unit dosage form and are 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.

Depending on the location of the tissue of interest, the peptides of the present invention can be supplied in any manner suitable for the provision of the peptide to cells within the tissue of interest. Thus, for example, a composition containing the peptides of the present invention can be introduced, for example, into the systemic circulation, which will distribute said 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.).

In an embodiment of the invention, peptides are 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 to be 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 peptides 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 compositions 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 compositions 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.

For topical application, a peptide of the present invention, derivative, analog or a fragment thereof can be combined with a pharmaceutically acceptable carrier so that an effective 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.

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 corn 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.

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.

The compositions of the present invention are generally administered in the form of a pharmaceutical composition comprising at least one of the active components of this invention together with a pharmaceutically acceptable carrier or diluent. Thus, the compositions of this invention can be administered either individually or together in any conventional oral, parenteral or transdermal dosage form.

Pharmaceutical compositions according to embodiments of the invention may contain 0.1%-95% of the active components(s) of this invention, preferably l%-70%. In any event, the composition or formulation to be administered may contain a quantity of active components according to embodiments of the invention in an amount effective to treat the condition or disease of the subject being treated.

The peptides of the present invention, derivatives, or analogs thereof can be delivered in a controlled release system. Thus, 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 one 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. Pharmaceutical use

According to another aspect, there is provided a method of treating, preventing or alleviating a disease in a subject, particularly a disease associated with COX-2. According to some embodiments, the method comprises administering to the subject in need thereof an effective amount of the peptide of the invention or the pharmaceutical composition comprising same.

According to another embodiment, the peptides and compositions described herein are useful in lowering or decreasing COX-2 expression levels. According to another embodiment, the peptides and compositions described herein are useful in enhancing COX-2 degradation such as by ubiquitination. According to another embodiment, the COX-2 associated disease or disorder is a disease or disorder having elevated or increase COX-2 levels. Methods for determining elevated or increase COX-2 levels are well known in the art. According to another embodiment, the COX-2 associated disease or disorder is selected from the group consisting of an inflammatory associated disease or disorder, pain and stress-related pathologies.

Inflammatory associated disease or disorder which may be treated by the peptides or compositions disclosed herein include but is not limited to inflammatory bowel disease, irritable bowel syndrome, colitis, ulcerative colitis, arthritis, neuroinflammation, Alzheimer's disease, Parkinson's disease, pain, fever, fibrotic diseases, cardiovascular diseases, post-ischemic reperfusion injury and congestive heart failure, cardiomyopathy, atherosclerosis, reperfusion injury, renal reperfusion injury, brain edema, neurotrauma and brain trauma, neurodegenerative disorders, central nervous system disorders, liver disease, hepatitis and nephritis, gastrointestinal conditions, ulcerative diseases, Crohn's disease, ophthalmic diseases, ophthalmological conditions, glaucoma, acute injury to the eye tissue and ocular traumas, diabetic nephropathy, skin-related conditions, myalgias due to infection, influenza, endotoxic shock, toxic shock syndrome, autoimmune disease, graft rejection, bone resorption diseases, multiple sclerosis, autoimmune encephalomyelitis, psoriasis, dermatitis, eczema, diverticulitis, coeliac disease, disorders of the female reproductive system, pathological (but non-malignant) conditions, such as hemaginomas, angiofibroma of the nasopharynx, and avascular necrosis of bone, benign and malignant tumors/neoplasia, obesity-related inflammation and infection.

According to another embodiment, said inflammatory associated disease or disorder is selected from the group consisting of: multiple sclerosis, arthritis, asthma, inflammatory bowel disease (Crohn's disease), psoriasis, and systemic lupus erythematosus (SLE).

According to another embodiment, said inflammatory associated disease or disorder is multiple sclerosis.

The effects of the peptides and compositions described herein for treating multiple sclerosis may be determined by methods known in the art, such as by the experimental autoimmune encephalomyelitis ("EAE") model (see, e.g., Miller, S. D. and Karpus, W. J. 2007. Experimental Autoimmune Encephalomyelitis in the Mouse. Current Protocols in Immunology. 78: 15.1: 15.1.1-15.1.18). EAE is a well-characterized and widely used model of MS. It is one of the few animal models in which drug therapeutic activity in the model often mimics and anticipates the drug's effects in human disease. In this model, mice are immunized with well- defined antigenic fragments of central nervous system myelin, such as fragments of the proteolipid protein (PLP). Over the course of 2 to 3 weeks the immunized animals develop a clinical and histopathologic syndrome of T-cell mediated cerebral autoimmunity that is highly reminiscent of relapsing/remitting MS (RRMS). The similarities between EAE and RRMS include inflammatory cerebral infiltrated and accompanying Thl/Thl7 responses to both the disease initiating myelin PLP peptide, and subsequent epitope spread, seen in disease relapse.

The term "pain" is used herein to represent all categories of physical pain. This includes traumatic pain resulting from injury, surgery or inflammation as well as pain associated with diseases such as cancer, AIDS, arthritis, and herpes. Pain can be associated with neuropathy such as diabetic neuropathy, causalgia, brachial plexus avulsion, occipital neuralgia, fibromyalgia, vulvodynia, prostadynia, pelvic pain, gout, and other forms of neuralgia, such as neuropathic and idiopathic pain syndromes. Specific organ- or site-localized pain, such as headache, ocular and corneal pain, bone pain, urogenital pain, heart pain, skin/burn pain, lung pain, visceral (kidney, gall bladder, etc.) pain, joint pain, dental pain and muscle pain are included in this invention. The general term "pain" also covers pain symptoms of varying severity, i.e. mild, moderate and severe pain, as well as those of acute and chronic pain.

Non limiting examples of pain includes inflammatory pain selected from the group consisting of organ transplant rejection; reoxygenation injury resulting from organ transplantation, chronic inflammatory diseases of the joints, arthritis, rheumatoid arthritis, osteoarthritis, bone diseases associated with increased bone resorption, inflammatory lung diseases, asthma, adult respiratory distress syndrome, chronic obstructive airway disease, inflammatory diseases of the eye, corneal dystrophy, trachoma, onchocerciasis, uveitis, sympathetic ophthalmitis endophthalmitis, chronic inflammatory diseases of the gum, gingivitis, periodontitis, tuberculosis, leprosy, inflammatory diseases of the kidney, uremic complications, glomerulonephritis, nephrosis, inflammatory diseases of the skin, sclerodermatitis, psoriasis and eczema, inflammatory diseases of the central nervous system, chronic demyelinating diseases of the nervous system, multiple sclerosis, AIDS -related neurodegeneration, Alzheimer s disease, infectious meningitis, encephalomyelitis, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, viral or autoimmune encephalitis, autoimmune diseases, Type I and Type II diabetes mellitus, diabetic complications, diabetic cataract, glaucoma, retinopathy, nephropathy, microaluminuria, progressive diabetic nephropathy, polyneuropathy, mononeuropathies, autonomic neuropathy, gangrene of the feet, atherosclerotic coronary arterial disease, peripheral arterial disease, nonketotic hyperglycemic -hyperosmolar coma, foot ulcers, joint problems, skin or mucous membrane complication, immune-complex vasculitis, systemic lupus erythematosus (SLE), inflammatory diseases of the heart, cardiomyopathy, ischemic heart disease hypercholesterolemia, atherosclerosis, preeclampsia, chronic liver failure, brain and spinal cord trauma, and inflammatory associated cancer.

According to another embodiment, the peptide disclosed herein is useful in treating epithelial cancer or carcinomas showing elevated COX-2 levels, including but not limited to breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, mouth cancer, esophageal cancer, small bowel cancer, stomach cancer, colon cancer, liver cancer, bladder cancer, pancreas cancer, ovary cancer, cervical cancer, lung cancer, skin cancer, prostate cancer, and renal cell carcinoma.

According to another embodiment, the peptide disclosed herein is useful in treating stress- related pathologies including but not limited to posttraumatic stress disorder, acute stress disorder, adjustment disorder, bereavement related disorder, general anxiety disorder, social anxiety disorder and anxiety disorder due to a medical condition.

According to another embodiment, the peptide disclosed herein is useful in treating affective disorders including but not limited to anxiety disorders (e.g., generalized anxiety disorder (GAD), social anxiety disorder (SAD; alternatively known as social phobia), panic disorder (with or without agoraphobia), posttraumatic stress disorder (PTSD), obsessive- compulsive disorder (OCD), separation anxiety disorder), mood disorders (e.g., depressive disorder, bipolar disorder) and psychotic disorders (e.g., schizophrenia, schizoaffective disorder, delusional disorder), substance-related disorders (e.g., substance abuse, substance-induced disorder, substance withdrawal).

A "therapeutically effective amount" of the peptide is that amount of peptide which is sufficient to provide a beneficial effect to the subject to which the peptide is administered. More specifically, a therapeutically effective amount means an amount of the peptide effective to prevent, alleviate or ameliorate tissue damage or symptoms of a disease of the subject being treated.

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" rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins. 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.

EXAMPLES

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. Materials- Goat polyclonal anti-COX-2 (C-20), mouse monoclonal ubiquitin (P4D1), goat polyclonal anti-actin (1-19) were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA), as was N-methylmaleimide (NEM). Mouse monoclonal HA.11 was from Covance (Emeryville, CA). Mouse monoclonal GFP was from MBL International Corporation (Woburn, MA). Horseradish peroxidase-conjugated bovine anti-goat IgG, goat anti-rabbit IgG, and goat anti- mouse IgG were obtained from Jackson ImmunoRe search Laboratories (West Grove, PA). Alexa Fluor 647 (Cy5) donkey anti-goat IgG for microscopy imaging experiments was obtained from Life Technologies (Carlsbad, CA). PGE2 Rabbit antisera for radioimmunoassays and 1-Oleoyl- 2-acetyl-sn-glycerol (OAG) were purchased from Sigma Aldrich (Rehovoth, Israel). Tritium- labeled PGE2 (190 Ci/mmol) was obtained from Perkin Elmer (Waltham, MA). (s)-MG132 was from Cayman Chemical (Ann Arbor, MI). GF109203X (GFX) was from Tocris Bioscience (Bristol, UK). All other reagents were standard laboratory grade. Cell culture and transfection- HEK-293 cells were grown in Eagle's MEM media, supplemented with 10% fetal bovine serum and 100 U/ml penicillin and streptomycin. Transient transfections were carried subconfluent (70-80%) monolayers using PolyJet (SignaGen Laboratories) at a ratio of 1:3 cDNA: PolyJet, according to the manufacturer's instructions. All samples contained the same amount of total cDNA. cDNA Constructs- Cloning of fluorescent versions of COX-2, wild type and mutant ATi was done as follows:

COX2-YFP: Forward 5'- ATTAAGCTTATGCTCGCCCGCGCCCTG-3' and reverse a 5'- ATTGGATCCTTCAGTTCAGTCGAACGTTC-3' and inserted into pEYFP-Nl vector (Clontech) between BamHI and Hindlll sites. The following constructs were cloned into pECFP- Nl between Xhol and BamHI sites: CFP- ATI: forward primer 5'- ATACTCGAGATGGCCCTTGACTCTTCT-3' and reverse primer 5'- ATAGGATCCCGCTCC ACCTC AAAAC-3 ' ; CFP- TSTS/A ATi: forward primer 5'- ATACTCGAGATGGCCCTTGACTCTTCT-3' and the reverse primer 5'- ATAGGATCCC GCTCCACCTCAAAAC-3'; CFP-DRY/AAY ATi: forward p5'- ATACTCGAGA TGGCCCTTGACTCTTCT-3' and the reverse primer 5'- ATAGGATCCCGCT CCACCTCAAAAC-3'. CFP- ATi carboxyl-tail (CT; amino acids 325-359) was cloned using the oligo overlap cloning method into pECFP-Nl vector (Clontech) between EcoRI and BamHI sites using the overlapping primers: 5'-

AATTCTAAGTCCCACTCAAGCCTGTCTACGAAAATGAGCACGCTTTCTTACCGGCC TTCGGATAACATGAGCTCATCGGCCAAAAAGCCTGCGTCTTGTTTTGAGGTGGAGT GAG-3' and 5'-GATCCTCACTCC ACCTC AAAACAAGACGCAGGCTT

TTTGGCCGATGAGCTCATGTTATCCGAAGGCCGGTAAGAAAGCGTGCTCATTTTCG TAGAC AGGCTTGAGTGGGACTTAG-3 '.

His-Myc tagged ATi- CT was cloned using the gBlocks Gene Fragment ACGGCGGATCCACCATGGCCAAGTCCCACTCAAGCCTGTCTACGAAAATGAGCAC GCTTTCTTACCGGCCTTCGGATAACATGAGCTCATCGGCCAAAAAGCCTGCGTCTT GTTTTGAGGTGGAGAAGCTTGGCCTT. The fragment was design to carry BamHI and Hindlll restriction sites (bold) and a Kozak translation initiation sequence (underline). Cloning was performed using the standard manufacture protocol.

All construct were confirmed by restriction digestion analysis and sequenced at the core sequencing facilities of the Technion Israel Institute of Technology and Hylabs (Rehovoth, Israel).

Immunoprecipitation and immunoblotting- monolayers in 100-mm culture dishes were washed twice with ice-cold PBS and lysed as described in Haddad et al. 2012, J Biol Chem 287, 17214-17223. Nitrocellulose membranes containing the immuno-complexes or total cell lysate proteins were incubated with primary antibodies at a dilution of 1:500 (COX-2 and HA), and 1:250 for Ub. Proteins were visualized by a WesternBright ECL (Advansta, CA) and quantified using a CCD camera and Quantity One software (XRS, Bio Rad).

Radioimmunoassay- 140,000 cells were plated in 12- well dishes and transfected with COX-2, with either pcDNA or ATi, as indicated above. Radioimmunoassays were performed as describe in Haddad 2012, ibid. Flow cytometry- Cells were washed twice with PBS, and resuspended in 150-200 μΐ PBS for cytometric analysis. All experiments were performed in triplicates. The samples were analyzed using BD FACSCanto II flow cytometer with DACSDiva software (BD Biosciences, San Jose, CA).

Microscopy: Cells were grown on 13 mm glass coverslips. Following transfection, cells were fixed with 4% paraformaldehyde, washed with PBS and blocked in PB buffer (1% BSA and 0.1% Triton X-100) in for 5 min. Samples were then incubated with anti COX-2 (1:200) for 1 h, washed three times with PBS and incubated with Alexa Fluor 647 donkey anti-goat IgG (1:200) for 1 h. Following additional three washes with PBS, samples were mounted onto glass slides using Mowiol (Sigma Aldrich, Israel) and visualized under an ApoTome.2 laser scanning confocal microscope (Zeiss) at a 63x magnification. All images were acquired using the same exposure conditions.

Statistical analysis- Experiments shown are mean +SEM for data averaged from at least three independent experiments. To determine statistical significance student's t test or one-way ANOVA were used. Post-hoc analysis was performed with Tukey multi-comparison test when appropriate. P values < 0.05 were considered significant. Analyses were done using version of the GraphPad Prism software. Example 1

Angiotensin II type 1 receptor downregulates the expression of COX-2

To test whether expression of the ATi receptor affects COX-2, HEK293 cells were co- transfected with COX-2 together with either empty plasmid or the receptor, and analyzed for the ability of COX-2 to generate PGE₂. Since HEK 293 cells do not express detectable amounts of either COX isoform, the data reflects only the activity of transfected COX-2 (Haddad 2012, ibid.). As depicted in Fig. 1A, co-transfection of COX-2 with ATi at a ratio of 1:5 reduced PGE₂ secretion by nearly half. To find out whether this decrease is due to diminished COX-2 levels, flow cytometry was used to analyze the levels of YFP-tagged COX-2 in the absence or presence of ATi. Co-expression of both proteins under the same conditions as the RIA experiment resulted in a marked 80% reduction in COX-2 expression (Fig. IB), suggesting that the most of the reduction in PGE₂ secretion may be attributed to a reduction in COX-2 levels. Consistent with this, immunofluorescence microscopy showed that compared to co-expression with an empty plasmid, the expression of YFP-COX-2 is severely downregulated in the presence of GFP-ATi (Fig. 1C).

To test whether ATi has a similar effect on the expression of COX-1, the inventors expressed either COX-1 or COX-2 together with increasing amounts of CFP-ATi. The total DNA levels were kept the same at all times. As shown in Fig. ID, while increasing the levels of ATi caused a marked drop in COX-2 levels, it did not have a significant effect on the levels of COX-1.

Example 2

Downregulation of COX-2 by ATi is not mediated via classical signaling pathways

The inventors next sought to determine whether the effect of ATi on COX-2 is mediated via its classical signaling pathways (Wei et al., 2003, Proc Natl Acad Sci U S A 100, 10782- 10787). First, the inventors expressed COX-2, ATi, or both in HEK 293 cells, stimulated them with the ATi ligand Angll and measured COX-2 levels and phosphorylation of the ERK MAP kinase as an indication for receptor activation. Cells transfected with COX-2 alone did not show a response to Angll, indicating that they do not express significant amount of endogenous ATi (Fig. 2A, first two lanes). Expression of ATi alone elicited a marked response to Angll stimulation, as detected by activation of ERK (Fig. 2A two middle lanes). Co-expression of COX-2 together with ATi significantly lowered COX-2 expression, and this phenomenon was not affected by the presence of Angll (Fig. 2A, last two lanes). Time response experiments in the presence of ATi alone or ATi and COX-2 showed that there were no differences in the kinetics of ERK activation by Angll, both showing a peak response at 5-10 minutes of stimulation (Fig. 2B). However, ERK activation by ATi was dampened in the presence of COX- 2 (Fig. 2C). The same reduction in the ability of ATi to stimulate COX-2 was observed using the catalytically inactive mutant G533A COX-2 suggesting that COX-2 protein interferes with signaling in this pathway in a manner that is independent of its catalytic activity.

To further exclude the involvement of possible downstream signaling by ATi in its effect on COX-2, PKC, the major downstream protein kinase activated by ATi, was inhibited. Cells were transfected with COX-2 in the presence or absence of ATi and treated with different concentrations of the PKC inhibitor GFX for the full duration of transfection. As depicted in Fig. 2D, this treatment did not reverse the reduction caused by ATi. A similar result was obtained using OAG, another potent inhibitor of PKC.

Coupling of the ATi receptor to G-proteins and β arrestin is mediated via specific motifs on the receptor. Thus, G protein coupling to ATi is abolished by mutating the highly conserved DRY sequence to AAY, and association with β arrestin is defective in ATi with a TSTS/A substitutions in the CT of the receptor. To test whether these motifs are involved in the effect of ATi on COX-2, the mutant receptors were tagged with cyan fluorescent protein (CFP) and co- expressed them together with YFP-COX-2 (Fig. 2E). Flow cytometry measurements showed that the wild type and mutant receptors expressed to similar extents (Fig. 2E right panel), and both significantly lowered COX-2 expression.

Example 3

ATi promotes degradation of COX-2 via the proteasome

Previously published data showed that the prostaglandin EP1 receptor downregulates COX-2 expression by enhancing its ubiquitination and subsequent proteasomal degradation (Haddad, 2012, ibid.). To test whether proteasomal degradation is involved in the effect of ATI on COX-2, cells expressing YFP-tagged COX-2, alone or with CFP-AT1, were treated overnight with or without the specific proteasome inhibitor MG132. Blockade of proteasomal activity abolished the effect of ATI, resulting in more than double the amount of COX-2 (Fig 3A). Treatment with MG132 also caused a parallel reduction in the levels of CFP-AT1 (Fig 3B). Similar results were observed using untagged proteins (Fig. 3C), thus excluding the possibility that the observed effect is an artifact of the fluorescent tags. A dose-response experiment with increasing concentrations of MG132 showed that COX-2 recovery was observed as soon as the levels of the receptor began falling at concentrations as low as 0.1 μΜ MG132 (Fig. 3D).

The inventors next tested whether COX-2 and ATI interact with each other. For this, cells were transfected with each protein alone or together, and samples were subject to immunoprecipitation. To enable detection of a possible interaction, a transfection ratio of 1: 1 COX-2: ATI that was found in dose-titration experiments to have a minimal effect on COX-2 expression (Fig. ID), was used. As shown in Fig. 4A and 4B, only cells that expressed both proteins showed the reciprocal protein in co- precipitates.

Next, the levels of ubiquitinated COX-2 were measured in the presence or absence of ATI. COX-2 was immunoprecipitated from all samples and membranes were probed first for ubiquitination levels and then for the presence of COX-2 and ATI. Under conditions of 1: 1 co- expression ATI did not cause a significant reduction in COX-2 but the levels of its ubiquitination were elevated compared to those of COX-2 alone (Fig. 4C).

Example 4

The effect of ATi on COX-2 is mediated via its carboxyl-tail

The CT of most GPCRs has a critical role in their interactions with intracellular proteins. To determine whether the effect of ATi on COX-2 can be localized to its CT, COX-2 was overexpressed with wild type receptor or a truncated receptor mutant that lacks its entire cytosolic tail (Δ324). As demonstrated by both western blot (Fig. 5 A) and flow cytometry (Fig. 5B) analyses, the effect of ATi on COX-2 was completely abolished in the absence of its CT.

The inventors next reasoned that if the CT is required for downregulation of COX-2 expression by ATi, then the inhibitory effect of ATi on COX-2 may be mimicked by the CT amino acid sequence itself. To test this, the inventors appended the amino-acid sequence (325-

359) of the CT to CFP or c-Myc and followed its effect on COX-2 expression. As depicted by fluorescent microscopy (Fig. 6A) and western blotting (Fig. 6B) co-expression of the CT of ATi together with COX-2 in HEK 293 cells, significantly downregulates the expression of the latter. To test whether wild type ATi or the CT lower the levels of endogenously expressing COX-2, we tested their effect on NIH3T3 fibroblasts that express endogenous COX-2 following serum- stimulation (15). Cells were transfected with empty vector, wild type ATi or CT-Myc and stimulated one day later with 20% serum. As shown in Fig. 6C, both wild type and CT lowered endogenous COX-2 expression in these cells, in a similar manner to the effect they had on the HEK 293 cells that were transfected with COX-2. Flow cytometry analysis showed both wild type ATi and CT cause a reduction of approximately 50% in COX-2 expression (Fig. 6D). The data presented herein show that ATi downregulates COX-2 expression in a mechanism that is not mediated by classical signaling pathways. Agonist stimulation of ATi promotes its coupling to Gaq, thereby initiating PLC-dependent activation of PKC. Immediately thereafter, the receptor is phosphorylated by G protein-coupled receptor kinases (GRKs) on distinct serine/threonine site located on its carboxyl terminus, thereby initiating a second wave of β arrestin-dependent signaling. However, the data presented herein show that Angll- mediated activation of the receptor, or inhibition of PKC activity, do not reverse its effect on COX-2 expression. Moreover, ATi mutants that are defective in their ability to engage with G proteins (DRY/AAY) or β arrestin (TSTS/A) do not cause a significant reversal of the receptor on COX- 2 expression, results that are in line with no significant role for classical signaling in this phenomenon. A role for β arrestin cannot completely be ruled out because although the Δ324 mutant that lacks the CT of the receptor has no effect on COX-2, it was shown to maintain a certain degree of phosphorylation-independent β arresting recruitment. Nonetheless, since ATi- mediated decrease in COX-2 is observed in the absence of any ligand, it is more likely that the CT of ATi interacts directly with COX-2 or with other currently unknown scaffold proteins that accelerate degradation of COX-2.

The CT of ATi harbors specific motifs that interact with many different molecules involved in downstream signaling such as G proteins and β arrestin, proteins of the JAK/STAT pathway and others. While not yet identified in ATi, other receptors such as rhodopsin contain a sorting signal that binds to the GTPase ARF4, a regulator of protein sorting. In terms of ubiquitination, the β₂ adrenergic receptor was shown to recruit the E3 ubiquitin ligase Nedd4 and to undergo ubiquitination prior to its degradation. The observed increase in the ubiquitination content of COX-2 in the presence of both EPi and ATi, may signify the involvement of a yet to be identified E3 ligase or other adaptors that direct COX-2 for degradation, the identification of which is under investigation.

As opposed to its highly inducible nature in most tissues, COX-2 is constitutively expressed in the cortex of the mammalian kidney (macula densa and the thick ascending limb of Henle), where it generates prostaglandins that raise the levels of renin. Elevated renin (e.g. due to salt depletion or inhibition of the angiotensin converting enzyme (ACE) cause a significant increase in COX-2 expression thus constituting positive feedback loop between renin and COX- 2. In contrast, the end product of renin, Angll, negatively regulates the expression of COX-2. This effect was shown to involve receptor signaling since administration of ACE inhibitors or angiotensin receptor blockers to rodents in vivo caused a marked elevation in COX-2 expression. Interestingly however, mice with a genetic depletion of ATi (Agtrla-/-, Agtrb-/-) also display significantly higher levels of COX-2 in their macula densa thus providing support that the actual presence of the ATi receptor is required to keep COX-2 expression at bay.

Descriptions of embodiments of the invention in the present application are provided by way of example and are not intended to limit the scope of the invention. The described embodiments comprise different features, not all of which are required in all embodiments of the invention. Some embodiments utilize only some of the features or possible combinations of the features. Variations of embodiments of the invention that are described, and embodiments of the invention comprising different combinations of features noted in the described embodiments, will occur to persons of the art. The scope of the invention is limited only by the claims.

Claims

1. An isolated peptide of no more than 45 amino acids comprising an amino acid sequence as set forth in SEQ ID NO: 1 (KSHSXiLSTKMSTLSYRPSDNX₂SSSX₃KKPAX₄CFEVE), wherein Xi is Asn (N) or Ser (S), X₂ is Val (V) or Met (M), X₃ is Thr (T) or Ala (A) and X₄ is Pro (P) or Ser (S), or an analog, a derivative or fragment thereof.

2. The peptide according to claim 1, comprising the amino acid sequence as set forth in SEQ ID NO: 2 (KS HS NLS TKMS TLS YRPS DN VS S S TKKPAPCFE VE) or an analog, a derivative or a fragment thereof.

3. The peptide according to claim 1, comprising the amino acid sequence as set forth in SEQ ID NO: 3 (KS HS S LS TKMS TLS YRPS DNMS S S AKKP AS CFE VE) or an analog, a derivative or a fragment thereof.

4. The peptide according to claim 1, comprising the amino acid sequence as set forth in SEQ ID NO: 4 (KSHSNLSTKMSPLSYRPSDNVSSSTKKPAPCFEVE) or an analog, a derivative or a fragment thereof.

5. The peptide according to claim 1, comprising the amino acid sequence as set forth in SEQ ID NO: 5 (KS HS NLS TKMS TLS YRHS DN VS S S TKKP APCFE VE ) or an analog, a derivative or a fragment thereof.

6. The peptide according to claim 1, wherein said fragment has a length of no more than 20 amino acids.

7. The peptide according to claim 1, wherein said fragment comprises an amino acid sequence as set forth in SEQ ID NO: 6 (KSHSXiLSTKMSTLSYRPS) wherein is Asn (N) or Ser (S).

8. The peptide according to claim 7, comprising the amino acid sequence as set forth in SEQ ID NO: 7 (KSHS NLS TKMS TLS YRPS).

9. The peptide according to claim 7, comprising the amino acid sequence as set forth in SEQ ID NO: 8 (KS HS S LS TKMS TLS YRPS ) .

10. A pharmaceutical composition comprising as an active ingredient a pharmaceutically acceptable amount of an isolated peptide according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10, for use in treating COX-2 associated diseases or disorders. A method of treating or alleviating a cyclooxygenase (COX)-2 associated disease or disorder in a subject in need thereof, the method comprises administering to the subject an effective amount of the pharmaceutical composition of claim 10.

The method according to claim 12, wherein the cyclooxygenase (COX)-2 associated disease or disorder is selected from the group consisting of an inflammatory associated disease or disorder, pain and stress-related pathologies.

The method according to claim 13, wherein said inflammatory associated disease or disorder is selected from the group consisting of: multiple sclerosis, arthritis, asthma, inflammatory bowel disease (Crohn's disease), psoriasis, and systemic lupus erythematosus (SLE).

The method according to claim 13, wherein said pain is selected from the group consisting of acute pain, chronic pain, cancer pain, central pain, labor pain, myocardial infarction pain, pancreatic pain, colic pain, post-operative pain, headache pain, muscle pain, pain associated with intensive care, arthritic pain, neuropathic pain, and pain associated with an oral or periodontal disease, including gingivitis and periodontitis.

The method according to claim 13, wherein said stress-related pathologies is selected from the group consisting of posttraumatic stress disorder, acute stress disorder, adjustment disorder, bereavement related disorder, general anxiety disorder, social anxiety disorder and anxiety disorder due to a medical condition.