PAR2 mimetic peptides and uses thereof

Abstract

This invention is in the field of medicinal pharmacology. In particular, the invention relates to protease activated receptor type 2 (PAR2) modulating compounds (e.g., mimetic peptides), compositions comprising such modulating compounds, and their use as therapeutics for the treatment of conditions involving PAR2 activity.

Claims

1. A composition comprising a protease activator receptor type-2 (PAR2) mimetic peptide having Formula I: [heterocycle moiety]-[peptide sequence]-[linker moiety]-[cell membrane anchoring moiety], including pharmaceutically acceptable salts, lipidated analogs, pegylated analogs, and/or prodrugs thereof; wherein the heterocycle is selected from the group consisting of an aminothiazoyl moiety, a penicillamine moiety, a homoserine moiety, a thiazolamine moiety, and a hydroxyproline moiety; wherein the linker moiety is one or more polyethylene glycol (PEG) moieties; wherein the cell membrane anchoring moiety is hexadecyl; wherein the PAR2 mimetic peptide is configured to activate or inhibit PAR2 biological activity, and wherein the peptide sequence is the amino acid sequence selected from the group consisting of Ile-Gly, Ile-Gly-Arg, Ser-Leu-Ile-Gly (SEQ ID NO:2), Thr-Ile-Gly, Thr-Ile-Gly-Arg (SEQ ID NO:4), Ser-Lys-Gly-Arg-Ser (SEQ ID NO:5), Ser-Lys-Gly-Arg (SEQ ID NO:6), His-Ile-Gly-Arg (SEQ ID NO:7), Val-Ile-Gly-Arg (SEQ ID NO:8), Ser-Lys-Gly-Ser (SEQ ID NO: 22), Gly-Arg-Ser, Ser-Lys-Arg-Ser (SEQ ID NO: 23), and Leu-Lys-Gly-Arg (SEQ ID NO: 14).

2. The composition of claim 1, wherein the PAR.sub.2 mimetic peptide is selected from [2-aminothiazoyl]-[His-Ile-Gly-Arg (SEQ ID NO: 7)]-[PEG.sub.3]-[hexadecyl], [2-aminothiazoyl]-[Val-Ile-Gly-Arg (SEQ ID NO: 8)]-[PEG.sub.3]-[hexadecyl], [2-aminothiazoyl]-[(homoserine)IGR]-[PEG.sub.3]-[hexadecyl], [2-aminothiazoyl]-[Thr-Ile-Gly-Arg (SEQ ID NO:4)]-[PEG.sub.3]-[hexadecyl], [2-aminothiazoyl]-[Ser-Lys-Gly-Arg-Ser (SEQ ID NO:5)]-[PEG.sub.3]-[hexadecyl], and [2-aminothiazoyl]-[Ser-Lys-Gly-Arg (SEQ ID NO:6)]-[PEG.sub.3]-[hexadecyl].

3. A method for activating or inhibiting the activity of PAR.sub.2 in a subject, comprising administering to the subject in need thereof a composition as recited in claim 1.

4. The method of claim 3, wherein the subject has or is experiencing aberrant PAR.sub.2 activity.

5. The method of claim 3, wherein the subject is a human subject.

6. The method of claim 3, wherein the subject has or is at risk for developing an inflammatory condition involving aberrant PAR.sub.2 activity.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the primary PAR.sub.2 signalling pathways.

(2) FIG. 2A shows a schematic for PAR.sub.2 signalling, and the measuring of PAR.sub.2 signalling.

(3) FIG. 2B shows raw traces over time illustrating increases in impedance (Cell Index) over 25 hrs.

(4) FIG. 2C shows transformed traces of agonist addition only, normalized for comparison.

(5) FIG. 2D shows averaged responses from four experiments with appropriate concentration noted at right.

(6) FIG. 2E shows a concentration response curve.

(7) FIG. 3 shows PAR.sub.2 tethered ligand probe development—trypsin site.

(8) FIGS. 4A-C show PAR.sub.2 tethered ligand probe development—kallikrein site.

(9) FIGS. 5A-B show in vitro physiological PAR.sub.2 agonist screening using xCELLigence.

(10) FIGS. 6A-C show 2-at-TIGR (SEQ ID NO:4)-PEG.sub.3-Hdc signalling assays.

DETAILED DESCRIPTION OF THE INVENTION

(11) The protease-activated receptor-2 (PAR.sub.2) is one of the four members of the family of GPCRs that are activated after proteolytic cleavage of their extracellular, amino terminus (Adams et al., Pharmacol. Ther. 130, 248-282; Ramachandran, R., et al., (2012) Nat. Rev. Drug Discov. 11, 69-86). The resulting ‘tethered-peptide’ sequence (ending with SLIGRL (SEQ ID NO: 19) in the rodent receptor and SLIGKV (SEQ ID NO: 20) in the human receptor) exposed after proteolytic cleavage activates PAR.sub.2. A variety of potent and effective peptidomimetic agonists based upon the exposed tethered sequences have been developed to PAR.sub.2 (Adams et al., Pharmacol. Ther. 130, 248-282; Boitano S, et al. (2014) PLoS ONE 9: e99140). These compounds have been very useful in understanding the consequences of PAR.sub.2 activation across experimental models. However, the natural tethered agonist presentation for PAR.sub.2, and its corresponding access to the PAR.sub.2 binding pocket, has proved a difficult target for development of antagonists. Despite this difficulty, a number of PAR.sub.2 antagonists have been proposed (Suen J Y, et al., (2012) Br J Pharmacol 165:1413-1423; Yau M K, et al., (2013) J Med Chem 56: 7477-7497). Further complicating the issue of drug development is the growing evidence for ‘biased signalling’ that can follow PAR.sub.2 agonism (Hollenberg, M. D., et al., 2014. 171(5): p. 1180-94) or antagonism (Goh, F. G., et al., British journal of pharmacology, 2009. 158(7): p. 1695-704; Suen, J. Y., et al., 2014. 171(17): p. 4112-24).

(12) PAR.sub.2 plays an important role in a variety of diseases linked to proteinase release from endogenous sources or exposure to exogenous proteinases (Ramachandran R, et al., (2012) Nat Rev Drug Discov 11: 69-86, Hollenberg, M. D., et al., 2014. 171(5): p. 1180-94). One consequence of PAR.sub.2 activation in the peripheral nervous system is sensitization of neurons responsible for transmitting noxious information to the CNS. These nociceptive neurons express PAR.sub.2, and PAR.sub.2 activation on these neurons leads to enhanced signalling via a variety of channels including the capsaicin and noxious heat receptor, TRPV1 (Dai Y, et al., (2004) J Neurosci 24: 4293-4299). PAR.sub.2 is responsible for proteinase sensitization of TRPV1 in vivo, leading to thermal hyperalgesia. PAR.sub.2 null animals have deficits in pain sensitization in a variety of inflammatory pain models, and PAR.sub.2 activation is sufficient to induce a transition to a chronic pain state, making this receptor an important target for drug development for pathological pain (Vergnolle N (2009) Pharmacol Ther 123: 292-309; Bao Y, et al., 2014 Expert Opin Ther Targets 18: 15-27; Tillu D V, et al. (2015) Pain 156: 859-867). Additionally, a broad variety of preclinical and clinical findings link exogenous proteinases, and more specifically PAR.sub.2, to asthma (Reed C E, Kita H (2004) J Allergy Clin Immunol 114: 997-1008, quiz 1009; Vergnolle N (2009) Pharmacol Ther 123: 292-309; Jacquet A (2011) Clin Exp Allergy 41: 305-311; Snelgrove R J, et al., (2014) J Allergy Clin Immunol 134: 583-592). While there is a strong rationale for PAR.sub.2 antagonist drug discovery for these indications, few PAR.sub.2 antagonists have been described and even fewer have been demonstrated to exhibit efficacy in preclinical disease models (Yau, M. K., et al., J Med Chem, 2013. 56(19): p. 7477-97).

(13) The present invention provides PAR.sub.2 mimetic peptides that utilize this ligand chemistry combined with alternative PAR.sub.2 cleavage sites. Indeed, experiments conducted during the course of developing embodiments for the present invention identified highly potent PAR.sub.2 peptides and mimetic activators and agonists.

(14) Accordingly, the present invention relates to modulating compounds which function as activators and inhibitors of PAR.sub.2 proteins. The invention further relates to methods of treating, ameliorating, or preventing disorders in a patient, such as those that are responsive to either PAR.sub.2 activation or PAR.sub.2 inhibition, comprising administering to a subject (e.g., a human patient) a composition comprising one or more of the PAR.sub.2 mimetic peptides off the invention and, potentially, additional agent(s). Such disorders include those characterized by aberrant PAR.sub.2 activity (e.g., inflammatory disorders).

(15) In a particular embodiment, modulating compounds include mimetic peptides which function as activators and inhibitors of PAR.sub.2 proteins. Indeed, in some embodiments, the present invention provides PAR.sub.2 mimetic peptides having Formula I: [heterocycle moiety]-[peptide sequence]-[linker moiety]-[cell membrane anchoring moiety],
including pharmaceutically acceptable salts, lipidated analogs, pegylated analogs, and/or prodrugs thereof.

(16) Formula I is not limited to particular chemical moieties for the heterocyle moiety, the peptide sequence, the linker moiety, and/or the cell membrane anchoring moiety.

(17) In some embodiments, the heterocycle moiety is any aromatic heterocycle moiety that comprises at least one atom selected from Nitrogen, Oxygen and Sulfur. Examples of such heterocyle moieties include, but are not limited to, a thiazole moiety, a pyridine moiety, an azabicycloalkane moiety, an aminothiazoyl moiety, and/or an aminonicotinyl moiety.

(18) The heterocyle moiety is not limited to a particular positioning within the PAR.sub.2 mimetic peptide. In some embodiments, the aromatic heterocycle moiety is positioned at the N-terminus of the PAR.sub.2 mimetic peptide.

(19) In some embodiments, the peptide sequence is any peptide sequence that comprises two or more contiguous amino acid residues. In some embodiments, the peptide sequence is any combination of two or more contiguous amino acid residues that confers PAR.sub.2 activating properties or PAR.sub.2 antagonizing properties onto the PAR.sub.2 mimetic peptide. Examples of the two or more contiguous amino acid residues include, but are not limited to Ile-Gly, Ile-Gly-Arg, Leu-Ile-Gly, Leu-Ile-Gly, Leu-Ile-Gly-Arg (SEQ ID NO:1), Ser-Leu-Ile-Gly (SEQ ID NO:2), Ser-Leu-Ile-Gly-Arg (SEQ ID NO:3), Thr-Ile-Gly, Thr-Ile-Gly-Arg (SEQ ID NO:4), Ser-Lys-Gly-Arg-Ser (SEQ ID NO:5), SKGR (SEQ ID NO:6), HIGR (SEQ ID NO:7), VIGR (SEQ ID NO:8), any of the peptide sequences described in Example 1, and any of the peptide sequences described in Tables 1, 2, 3, 4 and 5.

(20) In some embodiments, the linker moiety is a chemical moiety configured to bridge the peptide sequence and cell membrane anchoring moiety. Examples of such linker moieties include, but are not limited to, a substituted aliphatic chain, an unsubstituted aliphatic chain, substituted aromatic chain, an unsubstituted aromatic chain, a linear polymer, one or more polyethylene glycol (PEG) moieties, one or more 3,19-dioxo-2,8,11,14,21-pentaoxa-4,18-diazatricosan-23-oic acid residue derivative moieties, and/or any combination thereof. In some embodiments, the linker moiety comprises a polyethylene glycol (PEG) moiety. In some embodiments, the linker moiety comprises multimers of 3,19-dioxo-2,8,11,14,21-pentaoxa-4,18-diazatricosan-23-oic acid.

(21) In some embodiments, the linker moiety is a linear polymer that comprises monomeric subunits. For example, in some embodiments, the linear polymer comprises saccharide moieties, peptide moieties, lactone moieties, acrylate moieties, and/or synthetic polymer moieties. In some embodiments, the linear polymer comprises collagen-like polypeptides and/or synthetic surrogates of spider silk.

(22) In some embodiments, the cell membrane anchoring moiety is any chemical moiety that comprises a hydrophobic chemical moiety or a synthetic structure that forms a non-covalent binding interaction with a cell membrane. The PAR.sub.2 mimetic peptides are not limited to a particular type or kind of a cell membrane anchoring moiety. In some embodiments, cell membrane moiety comprises a lipid moiety. In some embodiments, the cell membrane anchoring moiety comprises a saturated or unsaturated hydrocarbon moiety. In some embodiments, the cell membrane anchoring moiety is hexadecyl. In some embodiments, the cell membrane anchoring moiety is a saturated C.sub.12-C.sub.20 alkyl residue. In some embodiments, the cell membrane anchoring moiety is a cell-penetrating moiety. In some embodiments, the cell membrane anchoring moiety is a transmembrane domain.

(23) The cell membrane anchoring moiety is not limited to a particular positioning within the PAR.sub.2 mimetic peptide. In some embodiments, the cell membrane anchoring moiety is positioned at the C-terminus of the PAR.sub.2 mimetic peptide.

(24) In some embodiments, the length of the [linker moiety]-[cell membrane anchoring moiety] is approximately 30-50 Angstroms.

(25) In some embodiments, the PAR.sub.2 mimetic peptide is configured to modulate PAR.sub.2 biological activity. For example, in some embodiments, the PAR.sub.2 mimetic peptide is configured to activate PAR.sub.2 biological activity. In some such embodiments, the mimetic peptide is: [2-aminothiazoyl]-[LIGR (SEQ ID NO:1)]-[PEG.sub.3]-[hexadecyl]. In some such embodiments, the mimetic peptide is: [2-aminothiazoyl]-[HIGR (SEQ ID NO:7)]-[PEG.sub.3]-[hexadecyl]. In some such embodiments, the mimetic peptide is: [2-aminothiazoyl]-[VIGR (SEQ ID NO:8)]-[PEG.sub.3]-[hexadecyl]. In some such embodiments, the mimetic peptide is: [2-aminothiazoyl]-[(homoserine)IGR]-[PEG.sub.3]-[hexadecyl].

(26) In some embodiments, the PAR.sub.2 mimetic peptide is configured to antagonize PAR.sub.2 biological activity.

(27) In some such embodiments, the mimetic peptide is configured to antagonize PAR.sub.2 activity resulting from interaction between trypsin and PAR.sub.2. In some such embodiments, the mimetic peptide is: [2-aminothiazoyl]-[Thr-Ile-Gly-Arg (SEQ ID NO:4)]-[PEG.sub.3]-[hexadecyl].

(28) In some embodiments, the mimetic peptide is configured to antagonize PAR.sub.2 activity resulting from interaction between kallikrein 5 and PAR.sub.2. In some such embodiments, the mimetic peptide is: [2-aminothiazoyl]-[Ser-Lys-Gly-Arg-Ser (SEQ ID NO:5)]-[PEG.sub.3]-[hexadecyl]. In some such embodiments, the mimetic peptide is: [2-aminothiazoyl]-[Ser-Lys-Gly-Arg (SEQ ID NO:6)]-[PEG.sub.3]-[hexadecyl].

(29) In some such embodiments, the mimetic peptide is: [2-aminothiazol-4yl]-[SKGRS (SEQ ID NO:10)]-[PEG.sub.3]-[Hdc].

(30) In some such embodiments, the mimetic peptide is: [2-aminothiazol-4yl]-[SKGR (SEQ ID NO:6)]-[PEG.sub.3]-[Hdc].

(31) In some such embodiments, the mimetic peptide is: [2-aminothiazol-4yl]-[LIGR (SEQ ID NO:1)]-[PEG.sub.3]-[Hdc].

(32) In some such embodiments, the mimetic peptide is: [2-aminothiazol-4yl]-[TIGR (SEQ ID NO: 4)]-[PEG.sub.3]-[Hdc].

(33) In some such embodiments, the mimetic peptide is shown in Tables 1, 2, 3, 4, and/or 5.

(34) In some embodiments, the mimetic peptide is 2-at-LIGRL (SEQ ID NO:18)-PEG.sub.3-Hdc

(35) ##STR00011##

(36) In certain embodiments, the modulating compounds which function as activators and inhibitors of PAR.sub.2 proteins are small molecules. For example, in some embodiments the present invention provides small molecule compounds encompassed within Formula I:

(37) ##STR00012##
including pharmaceutically acceptable salts, solvates, and/or prodrugs thereof.

(38) Formula I is not limited to a particular chemical moiety for R.sub.1, R.sub.2, R.sub.3, and R.sub.4. In some embodiments, the particular chemical moiety for R.sub.1, R.sub.2, R.sub.3, and R.sub.4 independently include any chemical moiety that permits the resulting compound to function as an inhibitor of PAR.sub.2 protein activity. In some embodiments, the particular chemical moiety for R.sub.1, R.sub.2, R.sub.3, and R.sub.4 independently include any chemical moiety that permits the resulting compound to function as an activator of PAR.sub.2 protein activity.

(39) Such compounds are not limited to a particular chemical moiety for R.sub.1. In some embodiments, R.sub.1 is selected from

(40) ##STR00013##

(41) In some embodiments, R.sub.2 is an amino acid selected from a Leu, Ile, Val, Cha, Arg, Orn, Lys, Dap, Thr, Ser, and Tyr.

(42) In some embodiments, R.sub.3 is selected from 2-furoyl

(43) ##STR00014##
acetyl

(44) ##STR00015##
3-methylbutyryl

(45) ##STR00016##

(46) In some embodiments, R.sub.4 is selected from 2-furoyl

(47) ##STR00017##
acetyl

(48) ##STR00018##
3-methylbutyryl

(49) ##STR00019##

(50) In certain embodiments, the modulating compound is the PAR.sub.2 antagonist C391

(51) ##STR00020##
In some embodiments, the C391 is lipidated.

(52) Indeed, an important aspect of the present invention is that the compositions of the present invention are useful in treating conditions characterized with aberrant PAR.sub.2 activity. For example, in some embodiments, compositions comprising PAR.sub.2 modulating compounds (e.g., mimetic peptides, small molecules) are used to treat inflammatory conditions through antagonizing PAR.sub.2 activity. Such conditions include, but are not limited to, asthma, chronic pain, cancer and/or vascular disorders. In some embodiments, the compositions and methods of the present invention are used to treat cells, tissues, organs, or pathological conditions and/or disease states in an animal (e.g., a mammalian patient including, but not limited to, humans and veterinary animals) having aberrant PAR.sub.2 activity. In this regard, various diseases and pathologies are amenable to treatment or prophylaxis using the present methods and compositions. A non-limiting exemplary list of these diseases and conditions includes, but is not limited to, cancers having aberrant activity, inflammatory conditions having aberrant PAR.sub.2 activity, asthma, chronic pain, and/or vascular disorders having aberrant PAR.sub.2 activity.

(53) Some embodiments of the present invention provide methods for administering an effective amount of a PAR.sub.2 modulating compound (e.g., mimetic peptide, small molecule) of the invention and at least one additional therapeutic agent (including, but not limited to, pain relieving agents, chemotherapeutic antineoplastics, apoptosis-modulating agents, antimicrobials, antivirals, antifungals, and anti-inflammatory agents) and/or therapeutic technique (e.g., surgical intervention, and/or radiotherapies).

(54) A number of suitable anti-inflammatory agents are contemplated for use in the methods of the present invention. Examples include steroidal anti-inflammatory agents (e.g., albuterol), and non-steroidal anti-inflammatory agents.

(55) A number of suitable anticancer agents are contemplated for use in the methods of the present invention. Indeed, the present invention contemplates, but is not limited to, administration of numerous anticancer agents such as: agents that induce apoptosis; polynucleotides (e.g., antisense, ribozymes, siRNA); polypeptides (e.g., enzymes and antibodies); biological mimetics; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., antibodies conjugated with anticancer drugs, toxins, defensins), toxins; radionuclides; biological response modifiers (e.g., interferons (e.g., IFN-α) and interleukins (e.g., IL-2)); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all-trans-retinoic acid); gene therapy reagents (e.g., antisense therapy reagents and nucleotides); tumor vaccines; angiogenesis inhibitors; proteosome inhibitors: NF-KB modulators; anti-CDK compounds; HDAC inhibitors; and the like. Numerous other examples of chemotherapeutic compounds and anticancer therapies suitable for co-administration with the disclosed compounds are known to those skilled in the art.

(56) In some embodiments, the pain relieving agents include, but are not limited to, analgesic drugs and respective antagonists. Examples of analgesic drugs include, but are not limited to, paracetamol and Non-steroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, opiates and morphonimimetics, and specific analgesic agents.

(57) Examples of NSAIDs include, but are not limited to, salicylates (e.g., Acetylsalicylic acid (Aspirin), Aloxiprin, Benorylate/Benorilate, Choline magnesium salicylate, Diflunisal, Ethenzamide, Faislamine, Methyl salicylate, Magnesium salicylate, Salicyl salicylate, Salicylamide), arylalkanoic acids (e.g., Diclofenac, Aceclofenac, Acemethacin, Alclofenac, Bromfenac, Etodolac, Indometacin, Nabumetone, Oxametacin, Proglumetacin, Sulindac, Tolmetin), 2-arylpropionic acids (profens) (e.g., Ibuprofen, Alminoprofen, Benoxaprofen, Carprofen, Dexibuprofen, Dexketoprofen, Fenbufen, Fenoprofen, Flunoxaprofen, Flurbiprofen, Ibuproxam, Indoprofen, Ketoprofen, Ketorolac, Loxoprofen, Naproxen, Oxaprozin, Pirprofen, Suprofen, Tiaprofenic acid), N-arylanthranilic acids (fenamic acids) (e.g., Mefenamic acid, Flufenamic acid, Meclofenamic acid, Tolfenamic acid), pyrazolidine derivatives (e.g., Phenylbutazone, Ampyrone, Azapropazone, Clofezone, Kebuzone, Metamizole, Mofebutazone, Oxyphenbutazone, Phenazone, Sulfinpyrazone), oxicams (e.g., Piroxicam, Droxicam, Lornoxicam, Meloxicam, Tenoxicam), sulphonanilides (e.g., nimesulide), licofelone, and omega-3 fatty acids.

(58) Examples of COX-2 inhibitors include, but are not limited to Celecoxib, Etoricoxib, Lumiracoxib, Parecoxib, Rofecoxib, Valdecoxib.

(59) Examples of opiates include, but are not limited to, natural opiates (e.g., alkaloids contained in the resin of the opium poppy including morphine, codeine and thebaine), semi-synthetic opiates (e.g., created from the natural opioids, such as hydromorphone, hydrocodone, oxycodone, oxymorphone, desomorphine, diacetylmorphine (Heroin), nicomorphine, dipropanoylmorphine, diamorphine, benzylmorphine, Buprenorphine, Nalbuphine, Pentazocine, meperidine, diamorphine, and ethylmorphine), fully synthetic opioids (e.g., such as fentanyl, pethidine, Oxycodone, Oxymorphone, methadone, tramadol, Butorphanol, Levorphanol, and propoxyphene), and endogenous opioid peptides (e.g., produced naturally in the body, such as endorphins, enkephalins, dynorphins, and endomorphins).

(60) Examples of analgesics include, but are not limited to, tricyclic antidepressants (e.g., amitriptyline, carbamazepine, gabapentin, and pregabalin), Tetrahydrocannabinol, ketamine, clonidine, α.sub.2-adrenoreceptor agonists, mexiletine, Orphenadrine, cyclobenzaprine, scopolamine, atropine, gabapentin, first-generation antidepressants and other drugs possessing anticholinergic and/or antispasmodic.

(61) In some embodiments, pain-relieving agents include anesthetic drugs. Examples of anesthetic drugs include, but are not limited to, local anesthetics (e.g., procaine, amethocaine, cocaine, lidocaine, prilocaine, bupivacaine, levobupivacaine, ropivacaine, dibucaine), inhaled anesthetics (e.g., Desflurane, Enflurane, Halothane, Isoflurane, Nitrous oxide, Sevoflurane, Xenon), intravenous anesthetics (e.g., Barbiturates (e.g., amobarbital (AMYTAL), pentobarbital (Nembutal), secobarbital (Seconal), Phenobarbital, Methohexital, Thiopental, Methylphenobarbital, Metharbital, Barbexaclone)), Benzodiazepines (e.g., alprazolam, bromazepam (Lexotan), chlordiazepoxide (Librium), Clobazam, Clonazepam, Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam, temazepam, nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax), temazepam (Restoril, Normison, Planum, Tenox, and Temaze, Triazolam, Etomidate, Ketamine, Propofol).

(62) In some embodiments, pain-relieving agents include anticonvulsant drugs. Examples of anticonvulsant drugs include, but are not limited to, aldehydes (e.g., paraldehyde), aromatic allylic alcohols (e.g., stiripentol), barbiturates (e.g., amobarbital (AMYTAL), pentobarbital (Nembutal), secobarbital (Seconal), Phenobarbital, Methohexital, Thiopental, Methylphenobarbital, Metharbital, Barbexaclone), benzodiazepines (e.g., alprazolam, bromazepam (Lexotan), chlordiazepoxide (Librium), Clobazam, Clonazepam, Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam, temazepam, nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax), temazepam (Restoril, Normison, Planum, Tenox, and Temaze), Triazolam), bromides (e.g., potassium bromide), carbamates (e.g., felbamate), carboxamides (e.g., carbamazepine, oxcarbazepine), fatty acids (e.g., valproates (e.g., valproic acid, sodium valproate, and divalproex sodium), Vigabatrin, Progabide, Tiagabine), fructose derivatives (e.g., topiramate), gaba analogs (e.g., gabapentin, pregabalin), hydantoins (e.g., Ethotoin, Phenytoin, Mephenytoin, Fosphenytoin), Oxazolidinediones (e.g., paramethadione, trimethadione, ethadione), priopionates (e.g., primidone), pyrrolidines (e.g., brivaracetam, levetiracetam, seletracetam), succinimides (e.g., Ethosuximide, Phensuximide, Mesuximide), sulfonamides (e.g., Acetazolamide, Sulthiame, Methazolamide, Zonisamide), triazines (e.g., lamotrigine), ureas (e.g., pheneturide, phenacemide), and valproylamdies (amide derivatives of valproate) (e.g., valpromide, valnoctamide).

(63) In some embodiments, pain-relieving agents include muscle relaxant drugs. Examples of muscle relaxant drugs include, but are not limited to, depolarizing muscle relaxants (e.g., Succinylcholine), short acting non-depolarizing muscle relaxants (e.g., Mivacurium, Rapacuronium), intermediate acting non-depolarizing muscle relaxants (e.g., Atracurium, Cisatracurium, Rocuronium, Vecuronium), and long acting non-depolarizing muscle relaxants (e.g., Alcuronium, Doxacurium, Gallamine, Metocurine, Pancuronium, Pipecuronium, d-Tubocurarine).

(64) In some embodiments, a PAR.sub.2 modulating compound (e.g., mimetic peptide, small molecule) of the invention and one or more additional agents (e.g., anti-inflammatory agents, anti-cancer agents, pain-relieving agents) are administered to an animal (e.g., a human patient) under one or more of the following conditions: at different periodicities, at different durations, at different concentrations, by different administration routes, etc. In some embodiments, the PAR.sub.2 modulating compound (e.g., mimetic peptide, small molecule) is administered prior to the one or more additional agents (e.g., anti-inflammatory agents, anti-cancer agents, pain-relieving agents), e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks prior to the one or more additional agents (e.g., anti-inflammatory agents, anti-cancer agents, pain-relieving agents). In some embodiments, the PAR.sub.2 modulating compound (e.g., mimetic peptide, small molecule) is administered after the one or more additional agents (e.g., anti-inflammatory agents, anti-cancer agents, pain-relieving agents), e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks after the administration of the additional agent. In some embodiments, the PAR.sub.2 modulating compound (e.g., mimetic peptide, small molecule) and the additional agent are administered concurrently but on different schedules, e.g., the PAR.sub.2 modulating compound (e.g., mimetic peptide, small molecule) is administered daily while the additional agent is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In other embodiments, the PAR.sub.2 modulating compound (e.g., mimetic peptide, small molecule) is administered once a week while the additional agent is administered daily, once a week, once every two weeks, once every three weeks, or once every four weeks.

(65) Compositions within the scope of this invention include all compositions wherein the PAR.sub.2 modulating compounds (e.g., mimetic peptides, small molecules) of the present invention are contained in an amount that is effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typically, the PAR.sub.2 modulating compounds (e.g., mimetic peptides, small molecules) may be administered to mammals, e.g. humans, orally at a dose of 0.0025 to 50 mg/kg, or an equivalent amount of the pharmaceutically acceptable salt thereof, per day of the body weight of the mammal being treated for disorders responsive to activation or inhibition of PAR.sub.2 activity. In one embodiment, about 0.01 to about 25 mg/kg is orally administered to treat, ameliorate, or prevent such disorders. For intramuscular injection, the dose is generally about one-half of the oral dose. For example, a suitable intramuscular dose would be about 0.0025 to about 25 mg/kg, or from about 0.01 to about 5 mg/kg.

(66) The unit oral dose may comprise from about 0.01 to about 1000 mg, for example, about 0.1 to about 100 mg of the PAR.sub.2 modulating compound (e.g., mimetic peptide, small molecule). The unit dose may be administered one or more times daily as one or more tablets or capsules each containing from about 0.1 to about 10 mg, conveniently about 0.25 to 50 mg of the PAR.sub.2 modulating compound (e.g., mimetic peptide, small molecule) or its solvates.

(67) In a topical formulation, the PAR.sub.2 modulating compound (e.g., mimetic peptide, small molecule) may be present at a concentration of about 0.01 to 100 mg per gram of carrier. In a one embodiment, the PAR.sub.2 modulating compound (e.g., mimetic peptide, small molecule) is present at a concentration of about 0.07-1.0 mg/ml, for example, about 0.1-0.5 mg/ml, and in one embodiment, about 0.4 mg/ml.

(68) In addition to administering the PAR.sub.2 modulating compound (e.g., mimetic peptide, small molecule) as a raw chemical, the PAR.sub.2 modulating compounds (e.g., mimetic peptides, small molecule) of the invention may be administered as part of a pharmaceutical preparation containing suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the PAR.sub.2 modulatign compounds into preparations which can be used pharmaceutically. The preparations, particularly those preparations which can be administered orally or topically and which can be used for one type of administration, such as tablets, dragees, slow release lozenges and capsules, mouth rinses and mouth washes, gels, liquid suspensions, hair rinses, hair gels, shampoos and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by intravenous infusion, injection, topically or orally, contain from about 0.01 to 99 percent, in one embodiment from about 0.25 to 75 percent of active mimetic peptide(s), together with the excipient.

(69) The pharmaceutical compositions of the invention may be administered to any patient that may experience the beneficial effects of the PAR.sub.2 modulating compounds (e.g., mimetic peptides, small molecules) of the invention. Foremost among such patients are mammals, e.g., humans, although the invention is not intended to be so limited. Other patients include veterinary animals (cows, sheep, pigs, horses, dogs, cats and the like).

(70) The PAR.sub.2 modulating compounds (e.g., mimetic peptides, small molecules) and pharmaceutical compositions thereof may be administered by any means that achieve their intended purpose. For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal or topical routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

(71) The pharmaceutical preparations of the present invention are manufactured in a manner that is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active mimetic peptides with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.

(72) Suitable excipients are, in particular, fillers such as saccharides, for example lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices. For this purpose, concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate, are used. Dye-stuffs or pigments may be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active mimetic peptide doses.

(73) Other pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the active mimetic peptides in the form of granules that may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active mimetic peptides are in one embodiment dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin. In addition, stabilizers may be added.

(74) Possible pharmaceutical preparations that can be used rectally include, for example, suppositories, which consist of a combination of one or more of the active mimetic peptides with a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules that consist of a combination of the active mimetic peptides with a base. Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

(75) Suitable formulations for parenteral administration include aqueous solutions of the active mimetic peptides in water-soluble form, for example, water-soluble salts and alkaline solutions. In addition, suspensions of the active mimetic peptides as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides or polyethylene glycol-400. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.

(76) The topical compositions of this invention are formulated in one embodiment as oils, creams, lotions, ointments and the like by choice of appropriate carriers. Suitable carriers include vegetable or mineral oils, white petrolatum (white soft paraffin), branched chain fats or oils, animal fats and high molecular weight alcohol (greater than C.sub.12). The carriers may be those in which the active ingredient is soluble. Emulsifiers, stabilizers, humectants and antioxidants may also be included as well as agents imparting color or fragrance, if desired. Additionally, transdermal penetration enhancers can be employed in these topical formulations. Examples of such enhancers can be found in U.S. Pat. Nos. 3,989,816 and 4,444,762.

(77) Ointments may be formulated by mixing a solution of the active ingredient in a vegetable oil such as almond oil with warm soft paraffin and allowing the mixture to cool. A typical example of such an ointment is one that includes about 30% almond oil and about 70% white soft paraffin by weight. Lotions may be conveniently prepared by dissolving the active ingredient, in a suitable high molecular weight alcohol such as propylene glycol or polyethylene glycol.

(78) One of ordinary skill in the art will readily recognize that the foregoing represents merely a detailed description of certain preferred embodiments of the present invention. Various modifications and alterations of the compositions and methods described above can readily be achieved using expertise available in the art and are within the scope of the invention.

(79) Having now fully described the invention, it will be understood by those of skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any embodiment thereof. All patents, patent applications and publications cited herein are fully incorporated by reference herein in their entirety.

EXPERIMENTAL

Example I

(80) Protease-activated receptor-2 (PAR.sub.2) belongs to a four-member family of G-Protein coupled receptors (GPCRs) that contain internal ligands exposed following exogenous or endogenous protease cleavage of the extracellular amino terminus. PAR.sub.2 is associated with a variety of inflammatory conditions, including asthma and pain. The contributions of PAR.sub.2 signalling to disease has been hindered by the lack of potent, efficacious antagonists, and their potential for biased-ligand signalling. It was recently demonstrated that lipid tethering of known PAR.sub.2 peptidomimetic agonists based on the primary trypsin cleavage sequence (SLIGRL (SEQ ID NO: 19)) increased their potency >200 fold.

(81) Here, lipid tethering (hexadecyl (Hdc) group with polyethylene glycol (PEG) spacers) and heterocycle (2-aminothiazoyl; 2-at) substitution of hexapeptide sequence derived from the primary cleavage site of kallikreins 4/16 (SSKGRS (SEQ ID NO:9)) was used to elucidate novel PAR.sub.2 antagonists. Compound 562 (C562), 2-aminothiazol-4yl-SKGRS (SEQ ID NO:10)-PEG.sub.3-Hdc blocks PAR.sub.2 Ca.sup.2+ signalling elicited via peptidomimetics (2-at-LIGRL (SEQ ID NO:18)-NH.sub.2) or via asthma associated protease activation (Alternaria alternata filtrates) in cultured human bronchial epithelial cells (16HBE14o−). This compound was a biased-signalling antagonist in that it had no effect on mitogen activated protein kinase (MAPK) signalling, the other major signalling pathway activated via PAR.sub.2. A shortened version of C562, 2-at-SKGR (SEQ ID NO: 6)-PEG.sub.3-Hdc (C595), maintained antagonistic activity against peptidomimetic activation in an in vitro physiological signalling assay (xCELLigence). C595 is closely related to the previously described potent and specific PAR.sub.2 agonist, 2-at-LIGR (SEQ ID NO: 1)-PEG.sub.3-Hdc. Thus, experiments screened a series of potential PAR.sub.2 ligands with a heterocycle serine substitute followed by four amino acids (XXGR) and the PEG.sub.3-Hdc lipid tether. Several potent agonists are described, and one partial agonist (C608, 2-at-TIGR (SEQ ID NO:4)-PEG.sub.3-Hdc) that also acts as a potent, specific and biased signalling antagonist of PAR.sub.2. When used in nanomolar concentrations, C608 blocked PAR.sub.2-dependent Ca.sup.2+ signalling via protease or peptidomimetics without effects on MAPK signalling. C562, C595 and C608 are novel pharmacological tools that can be used to evaluate the physiological consequences of PAR.sub.2 full and biased ligand signalling.

(82) FIG. 2 shows a schematic for PAR.sub.2 signalling, and the measuring of PAR.sub.2 signalling.

(83) FIG. 3 shows PAR.sub.2 tethered ligand probe development—trypsin site.

(84) FIG. 4 shows PAR.sub.2 tethered ligand probe development—kallikrein site.

(85) FIG. 5 shows in vitro physiological PAR.sub.2 agonist screening using xCELLigence.

(86) FIG. 6 shows 2-at-TIGR (SEQ ID NO:4)-PEG.sub.3-Hdc signalling assays.

(87) Tables 1, 2, 3, 4 and 5 provide additional PAR.sub.2 mimetic peptide is configured to modulate PAR.sub.2 biological activity.

(88) It was concluded that synthetic tethering allows for screening and development of novel PAR.sub.2 ligands.

(89) It was concluded that kallikrein site directed tethered ligands are biased antagonists: a) RTCA signalling antagonists; b) Ca.sup.2+ signalling anatagonist; c) no effect on MAPK pathways.

(90) It was concluded that C608 is a partial agonist and biased antagonist a) Partial RTCA agonist and potent anatagonist (low concentrations); b) Partial Ca.sup.2+ signalling agonist and potent antagonist (low concentrations); c) Full MAPK agonist.

(91) It was concluded that a partial and biased agonist/antagonist can provide novel in vivo applications.

(92) TABLE-US-00001 TABLE 1 Kallikrein Peptide Sequence with Truncations Compound # in Order Compound Summary as of appearance Compound Name Determined by RTCA Screen 1 SSKGRSO-NH2 Not Included 2 2at-SKGRSO-NH2 Not Included 3 SSKGRSO-PEG.sub.3-Pam Not Included 4 2at-SKGRSO-PEG.sub.3-Pam Not Included 5 2at-SKGRS-PEG.sub.3-Hdc PAR2 Antagonist 6 2at-SKGR-PEG.sub.3-Hdc Reduces a PAR2 physiological response

(93) TABLE-US-00002 TABLE 2 amino acid substitutions based on truncated of 2at-SKGR- PEG.sub.3-Hdc from above to try to improve truncation Compound # in Order Compound Summary as of appearance Compound Name Determined by RTCA Screen 7 2at-SRGR-PEG.sub.3-Hdc No Activity at PAR2 Detected 8 2at-SHGR-PEG.sub.3-Hdc No Activity at PAR2 Detected 9 2at-S-Dap-GR-PEG.sub.3- Reduces a PAR2 physiological Hdc response 10 2at-TKGR-PEG.sub.3-Hdc No Activity at PAR2 Detected 11 2at-LKGR-PEG.sub.3-Hdc Full PAR2 Agonist

(94) TABLE-US-00003 TABLE 3 amino acid L2 substitutions of 2at-LIGR-PEG.sub.3- Hdc to assess block based 2at-X-IGR (Note premise thought better binding from this sequence) Compound # in Order Compound Summary as of appearance Compound Name Determined by RTCA Screen 12 2at-LIGR-PEG.sub.3-Hdc Full PAR2 Agonist 13 2at-SIGR-PEG.sub.3-Hdc No Activity at PAR2 Detected 14 2at-TIGR-PEG.sub.3-Hdc PAR2 Agonist and Antagonist 15 2at-DIGR-PEG.sub.3-Hdc No Activity at PAR2 Detected 16 2at-HIGR-PEG.sub.3-Hdc PAR2 Agonist 17 2at-VIGR-PEG.sub.3-Hdc PAR2 Agonist

(95) TABLE-US-00004 TABLE 4 non-lipidated TIGR Compound # in Order Compound Summary as of appearance Compound Name Determined by RTCA Screen 18 2at-TIGR-NH2 No Classical Activation or Block at PAR2 19 2at-TIGRL-NH2 No Classical Activation or Block at PAR2

(96) TABLE-US-00005 TABLE 5 alternative amino acid L2 substitutions of 2at-LIGRL-PEG.sub.3-Hdc Compound # in Order Compound Summary as of appearance Compound Name Determined by RTCA Screen 20 2at-hydroxyproline- No Activity at PAR2 Detected IGR-PEG.sub.3-Hdc 21 2at-homoserine-IGR- PAR2 Agonist PEG.sub.3-Hdc 22 2at-penicillamine-IGR- No Activity at PAR2 Detected PEG.sub.3-Hdc 23 2at-4thiazolamine-IGR- No Activity at PAR2 Detected PEG.sub.3-Hdc 24 2at-(dL)IGR-PEG.sub.3-Hdc No Activity at PAR2 Detected 25 2at-(dl)IGR-PEG.sub.3-Hdc No Activity at PAR2 Detected 26 2at-(dT)IGR-PEG.sub.3-Hdc No Activity at PAR2 Detected

EQUIVALENTS

(97) The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

INCORPORATION BY REFERENCE

(98) The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.