Methods and compositions for treating inflammatory conditions

Abstract

The present invention provides novel compositions for inhibiting inflammation and for treating inflammatory diseases. These compositions comprise at least one anti-inflammatory peptide or polypeptide derived from PARS and / or at least one anti-inflammatory peptide or polypeptide derived from PAR1. PARS-derived and / or PARI-derived peptides generally comprise PARS or PARI that mimics activated protein C cleavage (e.g., activated protein C cleavage at residue Arg41 in human PARS and residue Arg51 in human PARI). 46 The present invention also provides methods of using the anti-inflammatory compositions described herein to inhibit unwanted inflammation and treat inflammatory diseases. The present invention additionally provides methods of screening candidate compounds to identify new anti-inflammatory agents.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 862,977, filed on June 18, 2019. The entire disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.

[0003] Government Support Statement

[0004] This invention was made with government support under Grant Nos. HL052246, HL104165, and HL142975 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0005] Inflammation is fundamentally a protective response, by which the body responds to a variety of injuries, infections and stresses. Its ultimate goal is to eliminate damage inducing agents (which can be microorganisms, physical irritants, chemicals, etc.), prevent tissue damage and / or start a repair process. Inflammation can be local or systemic, and can be acute or chronic. Although inflammatory responses are essential for stress response, resistance to infection and wound healing, inflammation can also be destructive. In fact, inflammation is an important component of the pathogenic process of many diseases and illnesses. In addition, the presence of inflammation indicates a less good prognosis in many diseases (such as cancer). Finally, in extreme cases, if improperly treated, inflammation can lead to a life-threatening systemic response.

[0006] In addition to its traditional anticoagulant function, activated protein C (APC) also exerts protective effects on multiple cell types and organs. APC can be anti-inflammatory by inhibiting the NLRP3 inflammasome. Inflammasomes play a central role in inflammation driven by the innate immune system by promoting the maturation and release of proinflammatory cytokines interleukin (IL)-1β and IL-18. Excessive inflammation can lead to many diseases, including cardiovascular disease, suggesting that limiting the elaboration of inflammasomes in the context of the innate immune system may be beneficial for human disease. For example, in the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS study), anti-IL-1β monoclonal antibodies reduced overall non-fatal myocardial infarction, stroke, and cardiovascular death.

[0007] Current methods for treating inflammation are inadequate, and inflammation often remains untreated or ineffectively treated. As a result, subjects suffering from abnormal inflammation may be subject to considerable harm, and may even endanger the subject's life. Therefore, there is an unmet need in the art for better or alternative means for combating inflammation and inflammatory diseases. The present invention addresses this and other unmet needs in the art. Summary of the invention

[0008] In one aspect, the present invention provides methods for inhibiting undesirable inflammation and / or treating inflammatory conditions in a subject. These methods include administering to the subject a pharmaceutical composition comprising an anti-inflammatory peptide derived from protease activated receptor-3 (PAR3). In various embodiments, the anti-inflammatory peptide derived from PAR3 comprises (1) Met 1 -Arg 41 A fragment of a human PAR3 extracellular domain deleted (SEQ ID NO: 3) or a conservatively modified variant thereof, the fragment consisting of at least the first 4 N-terminal residues of SEQ ID NO: 3; or (2) at least 4 consecutive amino acid residues of a human PAR3-derived peptide P3R (SEQ ID NO: 4) or a conservatively modified variant thereof, the at least 4 consecutive amino acid residues comprising Phe of SEQ ID NO: 4. 10 -Phe 12 .

[0009] In some embodiments, the anti-inflammatory peptide derived from PAR3 used also has APC-like cell protection activity. In some embodiments, the peptide derived from PAR3 used comprises at least the first 6, 7, 8, 9, 10, 11, 12, 13 or more N-terminal residues of SEQ ID NO: 3. In some embodiments, the peptide derived from PAR3 used comprises GAPPNSFEEFPFS (SEQ ID NO: 8) or GAPPNSFEEFPFSALEGWTGATIT (SEQ ID NO: 4). In some embodiments, the peptide derived from PAR3 used comprises SEQ ID NO: 4 comprising Phe 10 -Phe 12 In some of these embodiments, the PAR3-derived peptide comprises FPFSALEGW (SEQ ID NO: 10) or FPFSALEGWT GATIT (SEQ ID NO: 9). In some embodiments, the PAR3-derived peptide used is conjugated to a carrier moiety. For example, the carrier moiety can be a carrier protein, an immunoglobulin, an Fc domain, or a PEG molecule.

[0010] Some methods of the present invention further comprise administering to the subject an anti-inflammatory peptide derived from protease activated receptor-1 (PAR1). In various embodiments, the administered PAR1 derived peptide comprises (1) Met 1 -Arg 46 (2) a variant of the human PAR1-derived peptide TR47 (SEQ ID NO: 7) having at least the first 4 N-terminal residues of a deleted human PAR1 extracellular domain (SEQ ID NO: 6) or a conservatively modified variant thereof, or (3) a variant of the human PAR1-derived peptide TR47 (SEQ ID NO: 7) having at least one residue deleted or substituted at its N-terminus. In some of these embodiments, the administered PAR1-derived peptide comprises the first 4, 5, 6, 7, 8, 9, 10 or more N-terminal residues of the human PAR1-derived peptide TR47 (SEQ ID NO: 7). In some embodiments, the administered PAR1-derived peptide comprises a TR47 variant having a replaced N-terminal residue. In some of these embodiments, the TR47 variant used comprises SEQ ID NO: 49 (TR47ΔQ) or SEQ ID NO: 50 (TR47ΔA). In some embodiments, the administered PAR1-derived peptide comprises a TR47 variant having one or more N-terminal residues deleted. In some of these embodiments, the TR47 variant used comprises SEQ ID NO:47 (TR47(N-1)) or SEQ ID NO:48 (TR47(N-5)).

[0011] In some methods including administering a peptide from PAR3 and a peptide from PAR3, the two peptides are sequentially administered to an object in any order. In some other methods, the two peptides are administered to an object simultaneously. In some of these embodiments, a pharmaceutical composition comprising a peptide from PAR3 and a peptide from PAR1 is administered to an object in need of treatment. In some of these embodiments, a peptide from PAR3 is conjugated to a peptide from PAR1. For example, a peptide from PAR3 can be covalently bonded to a peptide from PAR1 (e.g., SEQ ID NO:61). In some of these embodiments, a linker portion can be used to covalently connect a peptide from PAR3 (e.g., SEQ ID NO:9) to a peptide from PAR1 (e.g., SEQ ID NO:36), as exemplified herein. In some embodiments, a peptide from PAR3 and / or a peptide from PAR1 is conjugated to a carrier portion. In some of these embodiments, the ratio of a peptide from PAR3 to a peptide from PAR1 is at least about 2:1, 4:1, 6:1, 8:1, or 10:1.

[0012] In some methods comprising administering both a PAR3-derived peptide and a PAR3-derived peptide, each of the PAR3-derived peptide and the PAR1-derived peptide is administered to the subject in a daily amount of up to about 0.25 mg / kg, 0.1 mg / kg, 0.05 mg / kg, 0.025 mg / kg, 0.01 mg / kg, 0.005 mg / kg, 0.0025 mg / kg, 0.001 mg / kg, 0.0005 mg / kg, 0.00025 mg / kg or less of the subject's body weight. In some other methods, a daily amount of up to about 0.25 mg / kg, 0.1 mg / kg, 0.05 mg / kg, 0.025 mg / kg, 0.01 mg / kg, 0.005 mg / kg, 0.0025 mg / kg, 0.001 mg / kg, 0.0005 mg / kg, 0.00025 mg / kg or less of one peptide is administered to a subject and a daily amount of at least about 0.01 mg / kg, 0.025 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 25 mg / kg or more of the subject's body weight of another peptide is administered to the subject.

[0013] In a number of embodiments, the methods of the present invention are intended to treat inflammatory diseases. The inflammatory disease to be treated can be any one selected from the following: asthma, autoimmune disease, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivity, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sterile inflammation, transplant rejection, viral-related inflammation and vasculitis. In some embodiments, the methods of the present invention are intended to treat the disorder associated with undesirable immune activation or undesirable immune response. For example, the disorder to be treated associated with undesirable immune activation or undesirable immune response can be a neuropathological condition, a viral infection or malaria. In some methods, the subject to be treated with the methods of the present invention suffers from or is suspected of suffering from acute neuroinflammation, chronic neuroinflammation or malarial inflammation.

[0014] In another aspect, the present invention provides a composition comprising an anti-inflammatory peptide derived from PAR3 and an anti-inflammatory peptide derived from PAR1. In some of these embodiments, the anti-inflammatory peptide derived from PAR3 comprises (1) Met 1 -Arg 41(1) a human PAR3 extracellular domain deleted (SEQ ID NO: 3) or an N-terminal fragment thereof with conservative modifications, the fragment consisting of at least the first 4 N-terminal residues of SEQ ID NO: 3, or (2) at least 4 consecutive amino acid residues of a PAR3-derived peptide P3R (SEQ ID NO: 4) or a conservatively modified variant thereof, the at least 4 consecutive amino acid residues comprising Phe of SEQ ID NO: 4. 10 -Phe 12 ; and the PAR1-derived anti-inflammatory peptide comprises (1) Met 1 -Arg 46 A deleted N-terminal fragment of the extracellular domain of human PAR1 (SEQ ID NO: 6) or a conservatively modified variant thereof, wherein the fragment consists of at least the first 4 N-terminal residues of SEQ ID NO: 6, or (2) a variant of the human PAR1-derived peptide TR47 (SEQ ID NO: 7) having at least one residue deleted or substituted at its N-terminus.

[0015] In some embodiments, the PAR3-derived peptide used comprises at least the first 13 N-terminal residues of SEQ ID NO:3 or a conservatively modified variant thereof, and the PAR1-derived peptide comprises at least the first 8 N-terminal residues of SEQ ID NO:6 or a conservatively modified variant thereof. In various embodiments, the PAR3-derived peptide used comprises SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or a conservatively modified variant thereof, and the PAR1-derived peptide comprises SEQ ID NO:7, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, or a conservatively modified variant thereof. In some compositions of the present invention, the amount of the PAR3-derived peptide is in a ratio of at least about 2:1, 4:1, 6:1, 8:1, or 10:1 to the amount of the PAR1-derived peptide. In some compositions, the PAR3-derived peptide is conjugated to the PAR1-derived peptide. In some compositions, the peptide derived from PAR3 is conjugated to the peptide derived from PAR1 through a linker portion or a carrier portion. For example, a peptide derived from PAR1 (e.g., SEQ ID NO: 36) can be covalently bonded to a peptide derived from PAR3 (e.g., SEQ ID NO: 9) through a glycine linker, as exemplified by SEQ ID NO: 61 herein. Some compositions of the invention are formulated for oral, intravenous, subcutaneous, intramuscular, intranasal, intraocular, topical or intraperitoneal administration to a subject.

[0016] Some compositions of the invention comprise two peptides at a daily dose of each of up to about 0.25 mg / kg, 0.1 mg / kg, 0.05 mg / kg, 0.025 mg / kg, 0.01 mg / kg, 0.005 mg / kg, 0.0025 mg / kg, 0.001 mg / kg, 0.0005 mg / kg, 0.00025 mg / kg or less of the average body weight of the subject group for which the composition is intended. Some other compositions of the invention comprise (1) one of the two peptides at a daily dose of each at up to about 0.25 mg / kg, 0.1 mg / kg, 0.05 mg / kg, 0.025 mg / kg, 0.01 mg / kg, 0.005 mg / kg, 0.0025 mg / kg, 0.001 mg / kg, 0.0005 mg / kg, 0.00025 mg / kg, or less of the average body weight of the group for which the composition is intended, and (2) a daily dose of the other peptide at least about 0.01 mg / kg, 0.025 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 25 mg / kg, or more of the average body weight of the group for which the composition is intended.

[0017] In another aspect, the present invention provides methods for inhibiting inflammation and treating inflammatory conditions in a subject. These methods require administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an anti-inflammatory peptide derived from protease activated receptor 1 (PAR1). In some methods, the administered PAR1-derived anti-inflammatory peptide comprises (1) Met 1 -Arg 46 (2) a variant of a human PAR1-derived peptide TR47 (SEQ ID NO: 7) having at least the first 4 N-terminal residues of a human PAR1 extracellular domain (SEQ ID NO: 6) or a conservatively modified variant thereof, or (3) a variant of a human PAR1-derived peptide TR47 (SEQ ID NO: 7) having at least one residue deleted or replaced at its N-terminus. In some embodiments, the PAR1-derived peptide used comprises the first 4, 5, 6, 7, 8, 9, 10 or more N-terminal residues of a human PAR1-derived peptide TR47 (SEQ ID NO: 7). In some methods, the PAR1-derived peptide used comprises a TR47 variant containing a replaced N-terminal residue. In some of these methods, the TR47 variant used comprises SEQ ID NO: 49 (TR47ΔQ) or SEQ ID NO: 50 (TR47ΔA). In some methods, the PAR1-derived peptide used comprises a TR47 variant containing one or more N-terminal residues deleted. In some of these methods, the TR47 variant used comprises SEQ ID NO:47 (TR47(N-1)) or SEQ ID NO:48 (TR47(N-5)).

[0018] In yet another aspect, the present invention provides a method for identifying an anti-inflammatory agent. The method requires (1) culturing a human THP-1 cell population, (2) contacting the cultured cells with an inflammatory inducing agent, (3) contacting the cultured cells with a candidate agent and an anti-inflammatory peptide derived from PAR3 or PAR1, respectively, and (4) measuring inflammation-related activity in cells contacted with the candidate agent and in cells contacted with the anti-inflammatory peptide derived from PAR3 or PAR1, respectively. If the inflammatory activity value measured in the cells contacted with the candidate agent is equal to or less than the inflammatory activity value measured in the cells contacted with the anti-inflammatory peptide derived from PAR3 or PAR1, the candidate agent is identified as an anti-inflammatory agent. In some methods, the anti-inflammatory peptide derived from PAR3 or PAR1 used is P3R (SEQ ID NO: 4) or TR47 (SEQ ID NO: 7), or a conservatively modified variant thereof. In multiple methods, the candidate agent used is a Met-mimicking peptide. 1 -Arg 41 Deletion of the extracellular domain of human PAR3 (SEQ ID NO: 3) or Met 1 -Arg 46 A peptide or polypeptide of the N-terminal sequence of the deleted human PAR1 extracellular domain (SEQ ID NO: 6), a variant, derivative or mimetic compound of the peptide or polypeptide. In some methods, the inflammation-related activity to be measured is caspase 1 enzyme activity. In some other methods, the inflammation-related activity to be measured is IL-1β release. In some methods, the inflammatory inducing agent used is lipopolysaccharide (LPS).

[0019] A further understanding of the nature and advantages of the present invention may be realized by reference to the remainder of the specification and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The anti-inflammatory activities of the PAR1-derived peptide TR47 and the PAR3-derived peptide P3R are shown. THP1-null (THP1) cells were cultured at 1×10 6The cells were plated in 96-well plates at a final concentration of 1 μg / mL and incubated with PMA (0.5 μM) in supplemented RPMI at 37° C. for 3 h. The medium was then replaced every 24 h for 3 days. Multiple subsets of wells were selected and cells were treated with the following in serum-free RPMI at 37°C for 60 min: (A) APC (4 μg / mL), TR47 (50 μM to 2 nM), scrTR47 (50 μM), SFLLRN (50 μM), TFLLRN (50 μM); (B) P3R (50 μM to 500 nM), P3K (50 μM); (C) APC (4 or 1 μg / mL), TR47 (8 nM), P3R (500 nM) or TR47+P3R (8 nM and 500 nM, respectively); (D) TR47 (0 to 128 nM) (●) ± P3R (500 nM) (□); (E) P3R (0 to 500 nM) (●) ± TR47 (1 nM) (□). After washing with DPBS, cells were incubated with LPS (1 μg / mL) in serum-free RPMI at 37°C for 3 hours. After washing with DPBS, cells were incubated with ATP (5 mM) at 37°C for 45 minutes in the presence or absence of 10 μM YVAD (caspase 1 inhibitor) or 2.5 μM ZVAD (pan-caspase inhibitor), and then caspase 1 activity was measured according to the manufacturer's instructions (relative luminescent unit, RLU). (D and E) Experimental caspase 1 activity was normalized to YVAD (caspase 1 inhibitor) luminescent units. (A to E) Data points represent mean ± SD of at least 3 independent experiments. *P<0.05 (compared with LPS+ATP); #P<0.05 (compared with LPS+ATP and APC, TR47 or P3R); **P<0.005 (compared with LPS+ATP and TR47+500 nM P3R); ns not significant.

[0021] Figure 2 APC and PAR1- and PAR3-derived peptides partially reduced NLRP3-driven inflammasome caspase 1 activity via EPCR, PAR1, and PAR3. THP1 or (A) NLRP3-deficient cells (THP1-defNLRP3) were incubated at 1×10 6 / mL final concentration plate was inoculated in a 96-well plate and incubated with PMA (0.5 μM) in supplemented RPMI at 37 ° C for 3 hours. The culture medium was subsequently replaced every 24 hours for 3 days. In brief, the selected wells were treated with APC (4 μg / mL), TR47 (50 μM) or P3R (50 μM) for 1 hour, washed with DPBS, and incubated with LPS (1 μg / mL) in serum-free RPMI at 37 ° C for 3 hours. After washing with DPBS, cells were incubated with ATP (5 mM) at 37 ° C for 45 minutes in the presence or absence of 10 μM YVAD (caspase 1 inhibitor) or 2.5 μM ZVAD (pan-caspase inhibitor). In (B), after PMA differentiation, selected wells were treated with APC (4 μg / mL), TR47 (50 μM) or P3R (50 μM) for 1 hour, washed with DPBS, and incubated with LPS (1 μg / mL) in serum-free RPMI at 37°C for 3 hours. The cells were washed with DPBS and then treated with NLRP3 inhibitor MCC950 (10 μM) or DMSO for 30 minutes. The cells were washed with DPBS again and incubated with ATP (5 mM) for 45 minutes in the presence or absence of 10 μM YVAD (caspase 1 inhibitor) or 2.5 μM ZVAD (pan-caspase inhibitor), and then luminescence was measured according to the manufacturer's instructions for caspase 1 activity. In (C to F), cells were incubated with antibodies or inhibitors before APC, TR47 or P3R treatment. Among them, cells were incubated with (C) blocking EPCR antibody RCR-252 (50 μg / mL); (D) mouse monoclonal antibody clone 19b against PAR3 (clone 19b; 25 μg / mL) for 30 minutes. In (E), cells were treated with mouse monoclonal antibodies against PAR1, WEDE15 (20 μg / mL), or ATAP2 (10 μg / mL) for 15 minutes. In (F), cells were treated with small molecule PAR1 inhibitor SCH79797 (SCH; 20 μM) for 20 minutes. After antibody or small molecule treatment (C to F), selected wells were treated with APC (4 μg / mL), TR47 (50 μM), or P3R (50 μM) for 1 hour, washed with DPBS, and incubated with LPS (1 μg / mL) in serum-free RPMI at 37°C for 3 hours. After washing with DPBS, cells were incubated with ATP (5 mM) at 37°C for 45 min in the presence or absence of 10 μM YVAD (caspase 1 inhibitor) or 2.5 μM ZVAD (pan-caspase inhibitor) and then luminescence (relative luminescence units, RLU) was measured according to the manufacturer's instructions for caspase 1 activity assay. (A to F) Data points represent the mean ± SD of at least 3 independent experiments.*P<0.05 (compared with LPS+ATP); #P<0.05 (compared with LPS+ATP and APC, TR47 or P3R); ##P<0.005 (compared with LPS+ATP and APC, TR47 or P3R); ns not significant.

[0022] Figure 3 It was shown that APC and PAR1- and PAR3-derived peptides reduced IL-1β release. THP1 cells were cultured at 1×10 6 The final concentration of / mL was plated in a 96-well plate and incubated with PMA (0.5μM) in supplemented RPMI at 37°C for 3 hours. The culture medium was subsequently replaced every 24 hours for 3 days. The selected wells were treated with APC (4μg / mL), TR47 (50μM), P3R (50μM) or TR47 / P3R (8nM and 500nM, respectively) for 1 hour, washed with DPBS, and incubated with LPS (1μg / mL) in serum-free RPMI at 37°C for 3 hours. The cells were washed with DPBS and incubated with ATP (5mM) for 45 minutes in the presence or absence of 10μM YVAD (caspase 1 inhibitor), and the resulting cell supernatant was measured for IL-1βQuantikine ELISA according to the instructions of the manufacturer (R&D), and the concentration of IL-1β was calculated after measuring the standard curve. The data points represent the mean ± SEM of at least 3 independent experiments. *P<0.05 (compared with LPS+ATP).

[0023] Figure 4 The anti-inflammatory activity of various fragments of PAR3-derived peptide P3R was shown. THP1 cells were cultured at 1×10 6 / mL final concentration plate was inoculated in a 96-well plate and incubated with PMA (0.5μM) in supplemented RPMI at 37°C for 3 hours. The medium was then replaced every 24 hours for 3 days. The selected wells were treated with APC (4μg / mL) (A) 50μM or (B) 2μM of P3R, P3Rm, P3R 51-65, P3R 51-59, P3R 54-59 or P3R 59-65 for 1 hour, washed with DPBS, and incubated with LPS (1μg / mL) in serum-free RPMI at 37°C for 3 hours. Cells were washed with DPBS and incubated with ATP (5 mM) at 37°C for 45 min in the presence or absence of 10 μM YVAD (caspase 1 inhibitor; C1i) or 2.5 μM ZVAD (pan-caspase inhibitor; pCi) and then the caspase 1 activity assay was performed by measuring luminescence (relative luminescence units, RLU) according to the manufacturer's instructions. (C) A graph showing PAR3 sequences, peptide sequences, and relative (anti-inflammatory) activity. These include sequences of P3R (SEQ ID NO: 4), P3Rm (SEQ ID NO: 8), P3R 51-65 (SEQ ID NO: 9), P3R 51-59 (SEQ ID NO: 10), P3R 54-59 (SEQ ID NO: 54), and P3R 59-65 (SEQ ID NO: 55).

[0024] Figure 5 The anti-inflammatory activity of variants of the PAR1-derived peptide TR47 is shown. THP1 cells were cultured at 1×10 6 / mL final concentration plated in 96-well plates and incubated with PMA (0.5μM) in supplemented RPMI at 37°C for 3 hours. The medium was then replaced every 24 hours for 3 days. Selected wells were treated with APC (4μg / mL), 50μM or 10nM TR47, 9-mer (TR479-mer; NPNDKYEPF (SEQ ID NO: 36)) or Ac-TR47 (acylated TR47) in (A), or (B) 50μM 16-mer-TR47, 8-mer-TR47, 6-mer-TR47, TR47 N-1, TR47 N-2, TR47 N-5, TR47ΔQ, TR47ΔD, TR47ΔA or TR48-54 for 1 hour, washed with DPBS, and incubated with LPS (1μg / mL) in serum-free RPMI at 37°C for 3 hours. Cells were washed with DPBS and incubated with ATP (5 mM) for 45 min at 37°C in the presence or absence of 10 μM YVAD (caspase 1 inhibitor; C1i) or 2.5 μM ZVAD (pan-caspase inhibitor; pCi) and then the caspase 1 activity assay was performed by measuring luminescence (relative luminescence units, RLU) according to the manufacturer's instructions. #P<0.001 (compared with LPS+ATP), **P<0.005 (compared with LPS+ATP), *P<0.05 (compared with LPS+ATP). (C) A diagram showing PAR1 sequence, peptide sequence and relative (anti-inflammatory) activity. These include the sequences of TR47 (SEQ ID NO:7), Ac-TR47 (SEQ ID NO:56), 16-mer-TR47 (SEQ ID NO:43), 9-mer-TR47 (SEQ ID NO:36), 8-mer-TR47 (SEQ ID NO:35), 6-mer-TR47 (SEQ ID NO:33), TR47 N-1 (SEQ ID NO:47), TR47 N-2 (SEQ ID NO:51), TR47 N-5 (SEQ ID NO:48), TR47ΔQ (SEQ ID NO:49), TR47ΔD (SEQ ID NO:52), TR47ΔA (SEQ ID NO:50) and TR48-54 (SEQ ID NO:53).

[0025] Figure 6 It was shown that PAR3-derived and PAR1-derived peptide variants exhibited synergy in reducing caspase 1 activity. THP1 cells were cultured at 1×10 6 / mL final concentration plate was inoculated in 96-well plates and incubated with PMA (0.5μM) in supplemented RPMI at 37°C for 3 hours. The culture medium was subsequently replaced every 24 hours for 3 days. In (A), the selected wells were treated with P3 (42-65) or P3 (42-54) (0 or 16nM) at 37°C for 60 minutes in the presence or absence of TR47 (1nM). In (B), the selected wells were treated with TR47 (0 or 4nM) or TR47 (N-1) (0 or 4nM) at 37°C for 60 minutes in the presence or absence of 500nM P3R. The cells were washed with DPBS and incubated with LPS (1μg / mL) in serum-free RPMI at 37°C for 3 hours. The cells were washed with DPBS and incubated with ATP (5 mM) in the presence or absence of 10 μM YVAD (caspase 1 inhibitor; C1i) or 2.5 μM ZVAD (pan-caspase inhibitor; pCi) at 37°C for 45 min, and then the luminescence (relative luminescence units, RLU) was measured for caspase 1 activity assay according to the manufacturer's instructions.

[0026] Figure 7 The results show that variants of PAR1-derived peptides or PAR3-derived peptides synergistically reduce caspase 1 activity. THP-1-null (THP-1 from Invivogen) cells were cultured at 1×10 6 The cells were plated in 96-well plates at a final concentration of 1 μg / mL and incubated with PMA (0.5 μM) in supplemented RPMI at 37° C. for 3 h. The medium was then replaced every 24 h for 3 days. A subset of wells were selected and cells were treated in serum-free RPMI for 60 min at 37°C with: (A) APC (4 μg / mL) or 50 μM of a peptide including: "TR47" (PAR1(47-66); SEQ ID NO:7), "TR47ΔQ" (PAR1(N47Q)-66; SEQ ID NO:49), "P3R" (PAR3(42-65); SEQ ID NO:4), or "P3Rm" (PAR3(42-54); SEQ ID NO:8); (B) TR47 (0 to 16 nM) alone (■) or with P3R (500 nM) (□) or TR47ΔQ (0 to 16 nM) alone (▲) or with P3R (500 nM) (▲) (C) P3R (0 to 16 nM) alone (■) or with TR47 (1 nM) (□) or P3Rm (0 to 16 nM) alone (▲) or with TR47 (1 nM) Alternatively (D) a subset of wells was selected and cells were treated with P3R (0 to 16 nM) (●) ± TR47 (1 nM) (○) or P3R51-65 (SEQ ID NO: 9) (0 to 16 nM) (▲; dotted line) ± TR47 (1 nM) (△; dotted line) in serum-free RPMI for 60 minutes at 37°C. After washing with DPBS, cells were incubated with LPS (1 μg / mL) in serum-free RPMI for 3 hours at 37°C. After washing with DPBS, cells were incubated with ATP (5 mM) at 37°C for 45 minutes in the presence or absence of 10 μM YVAD (caspase 1 inhibitor) or 2.5 μM ZVAD (pan-caspase inhibitor), and then caspase 1 activity was measured according to the manufacturer's instructions (relative luminescence units, RLU). Experimental caspase 1 activity was normalized to the value of YVAD (caspase 1 inhibitor) luminescence units. (B), (C) and (D): Experimental caspase 1 activity was normalized to the value of YVAD (caspase 1 inhibitor) luminescence units. For (A to C): Data points represent the mean ± SD of at least 3 independent experiments. *P < 0.005 (compared to LPS + ATP or compared to LPS + ATP and TR47 or P3R alone or variants); #P < 0.05 (compared to LPS + ATP and P3R alone or P3 (42-54) alone); ns not significant. For (D), *P < 0.05 (compared to LPS + ATP and P3 peptide alone); **P < 0.005 (LPS + ATP and P3 peptide alone); ns not significant.

[0027] Figure 8 It is shown that the PAR1:PAR3 covalently linked peptide reduces caspase 1 activity. THP-1 cells were cultured at 1×10 6 / mL final concentration was plated in a 96-well plate and incubated with PMA (0.5 μM) in supplemented RPMI at 37°C for 3 hours. The medium was then changed every 24 hours for 3 days. Selected wells were inoculated with PAR1 9-mer peptide P1 (47-55) (SEQ ID NO: 36) (0 to 500 nM) (○), PAR3 peptide P3 (51-65) (SEQ ID NO: 9) (0 to 500 nM) Or Gly10-linked PAR1 / PAR3 fusion peptide "G10" (0 to 500nM) (□) was treated at 37°C for 60 minutes. The cells were washed with DPBS and incubated with LPS (1 μg / mL) in serum-free RPMI at 37°C for 3 hours. The cells were washed with DPBS and incubated with ATP (5mM) at 37°C for 45 minutes in the presence or absence of 10 μM YVAD (caspase 1 inhibitor), and then the luminescence (relative luminescence unit, RLU) was measured according to the manufacturer's instructions for caspase 1 activity assay, and then the experimental caspase 1 activity was adjusted for caspase 1 inhibitor luminescence units and expressed as caspase 1 activity. The data points represent the mean ± SD of at least 3 independent experiments. *P<0.05 (compared with LPS+ATP).

[0028] Fig. 9 PAR1-derived, PAR3-derived and PAR1 / PAR3 fusion peptides are shown to inhibit thrombin-induced endothelial barrier destruction. The bottom curve of each figure in the figure is marked as IIa for a single thrombin, and represents the effect of thrombin (IIa) observed in the absence of peptides. In each figure, for each peptide added, IIa is always present. The numbers in each figure represent the concentration of each peptide of the corresponding curve. The peptide concentration of 0 is represented by the bottom curve marked as "IIa". (A) TER dose response inhibition over time, represented by the normalized cell index (Normalized Cell Index, NCI) of peptide P1 (47-66) or TR47 (using 5 to 50nM concentration) in PAR1 source. (B) NCI dose response inhibition over time of peptide P3 (42-65) or P3R (using 5 to 50nM concentration range) in PAR3 source. (C) NCI dose response inhibition over time of the short sequence PAR1-derived peptide P1(47-55) (using a concentration range of 50 nM to 100 μM peptide). (D) NCI dose response inhibition over time of the short sequence PAR3-derived peptide P3(42-54) (using a concentration range of 1 μM to 200 μM peptide). (E) NCI dose response inhibition over time of the hybrid peptide G10 (in which the P1(47-55) peptide described in Figure C is linked to the PAR3 peptide P3(51-65) using 10 Gly residues). In this case, the concentration range for studying dose response inhibition was 3 to 50 nM peptide. An estimate of the percentage activity of each peptide dilution in the presence of thrombin can be quantified by obtaining the area under the curve (AUC) for each peptide dilution and comparing it to the AUC value of thrombin alone in the absence of any peptide. These values ​​are used to calculate the potency of each PAR peptide. DETAILED DESCRIPTION

[0029] I. Overview

[0030] The present invention relates to the anti-inflammatory activity of various peptides or polypeptides derived from PAR1 and PAR3. The present invention is based in part on the discovery of the inventors that some peptides that simulate the N-terminal sequences of PAR3 and PAR1 (which are cut by APC at respective atypical sites) have anti-inflammatory activity. Unexpectedly, it is also found that there is a strong synergistic effect in the anti-inflammatory activity between the peptides in the observed PAR3 source and the peptides in the PAR1 source.

[0031] Activated protein C (APC) is a 41 and Arg 46 APC cleaves PAR1 at both positions. 41 APC cleaves PAR3. It is known that APC cleavage has cytoprotective and anti-apoptotic effects. The core of the present invention is the following discovery. Based on the action of APC on Arg 46 and Asn 47 The cleavage of the peptide bond between 47 Peptides containing a partial PAR1 sequence starting with 47-NPNDKYEPFWEDEEKNESGL-66 ("TR47" or "P1(47-66)") induce anti-inflammatory cell signaling, which can generally involve inhibition of inflammasomes. 41 and Gly 42 APC cleavage of the peptide bond between 42 Peptides that begin with a partial PAR3 sequence, such as residue 42-GAPPNSFEEFPFSALEGWTGATIT-65 ("P3R"), also induce anti-inflammatory cell signaling.

[0032] As described in detail herein, the inventors describe the role of EPCR, PAR1 and PAR3 in enhancing the anti-inflammatory activity of APC using induced human macrophage-like cell lines. The inventors found that TR47 (a peptide derived from PAR1) and P3R (a peptide derived from PAR3) each have an anti-inflammatory effect alone, and the combination of these two peptides can synergistically exert an anti-inflammatory effect. Specifically, the inventors evaluated the relevance of APC to the inhibition of inflammasomes in the human immune system by using the THP1 model cell line that has been used historically to study the cytoprotective activity of inflammasomes and APCs. Caspase 1 is a cysteine ​​protease in the NLRP3 inflammasome for the diagnosis of inflammasome activation. The inventors monitored the caspase 1 activity of activated THP1 cells to evaluate the anti-inflammatory effect of APC on human cells. The inventors also evaluated whether protease-activated receptors (PAR1-derived and PAR3-derived peptides, which mimic the atypical cleavage of APC in these two G-protein-coupled receptors (GPCRs), TR47 and P3R, respectively) exert anti-inflammatory activity in human THP1 cells. It was found that in the human THP1 cell line, APC, TR47 and P3R can each limit NLRP3 inflammasomes, as measured by caspase 1 activity and IL-1β release. Unexpectedly, it was observed that the two peptides can synergistically inhibit inflammasome activity. The anti-inflammatory effect of PAR peptides points to their utility as new anti-inflammatory pharmacological agents.

[0033] In order to further prove the observed synergy, the inventors also produced PAR1 / PAR3 fusion peptides by covalently linking the peptides in PAR1 source and PAR3 source, and detected its anti-inflammatory efficacy. Not surprisingly, it was found that the covalently linked PAR1 / PAR3 peptides significantly reduced caspase 1 activity at a certain concentration, while the anti-inflammatory effect of each component peptide alone was less. Importantly, it was also observed that the covalently linked PAR1 / PAR3 fusion peptides also inhibited the reduction of thrombin-induced transendothelial electrical resistance (transendothelial electrical resistance, TEER). These data further provide that the peptides in PAR1 source and PAR3 source can play the evidence of anti-inflammatory activity and cytoprotective activity (e.g., preventing thrombin-induced endothelial barrier destruction) in a synergistic manner.

[0034] Based on these discoveries, the present invention provides novel methods of inhibiting or suppressing inflammation, as well as methods for treating inflammatory diseases or disorders. Also provided herein are therapeutic compositions useful for practicing the therapeutic methods of the present invention.

[0035] Unless otherwise indicated, the present invention can be performed using standard procedures, such as described, for example, in Methods in Enzymology, Vol. 289: Solid-Phase Peptide Synthesis, JN Abelson, MI Simon, GB Fields (eds.), Academic Press; 1st edition (1997) (ISBN-13:978-0121821906); U.S. Pat. Nos. 4,965,343 and 5,849,954; Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques, Volume 152, edited by SLBerger and ARKimmerl, Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan et al., edited by John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (edited by Juan S. Bonifacino et al., John Wiley and Sons, Inc.) and Culture of Animal Cells: AManual of BasicTechnique by R.Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Volume 57, Jennie P. Mather and David Barnes, eds., Academic Press, 1st edition, 1998). The following sections provide additional guidance for practicing the compositions and methods of the present invention.

[0036] II. definition

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the invention belongs. The following references provide general definitions of many terms used in the present invention for technical personnel: Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (ed.), Oxford University Press (revised edition, 2000); Dictionary of Microbiology and Molecular Biology, Singleton et al. (ed.), John Wiley & Sons (3 PrdP ed., 2002); and Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (ed.), Oxford University Press (4 PthP ed., 2000). In addition, the following definitions are provided to help readers implement the present invention.

[0038] Unless the context clearly indicates otherwise, nouns modified by unquantified words mean one, one or more. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[0039] The term "amino acid" of a peptide as used herein refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, i.e., carbon, carboxyl, amino, and R groups bound to hydrogen, such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. The protective polypeptides derived from the PAR1 of the present invention include derivatives or analogs that have been modified with non-naturally encoded amino acids.

[0040] As used herein, the terms "comprising" or "including" are used to refer to compositions, methods and their respective components that are essential to the invention, but are open to the inclusion of unspecified elements, whether essential or not.

[0041] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0042] The term "consisting of" refers to the compositions, methods, and corresponding components described herein, which do not include any elements not recited in the description of the embodiment.

[0043] The term "conservatively modified variants" or "conservative substitution variants" applies to both amino acids and nucleic acid sequences. For a particular nucleic acid sequence, conservatively modified variants refer to those nucleic acids that encode the same or substantially the same amino acid sequence, or in the case where the nucleic acid does not encode an amino acid sequence, to substantially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at each position of alanine specified by a codon, the codon can be changed to any of the corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[0044] For polypeptide sequences, "conservatively modified variants" refer to variants with conservative amino acid substitutions, i.e., amino acid residues are replaced with other amino acid residues with similarly charged side chains. Families of amino acid residues with similarly charged side chains have been defined in the art. These families include amino acids with the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0045] As used herein, a "derivative" of a reference molecule (e.g., an anti-inflammatory peptide disclosed herein) is a molecule that has been chemically modified relative to the reference molecule while substantially retaining biological activity. The modification may be, for example, oligomerization or polymerization, modification of amino acid residues or peptide backbones, cross-linking, cyclization, conjugation, fusion to another heterologous amino acid sequence, or other modifications that significantly alter the stability, solubility, or other properties of the peptide.

[0046] The terms "decrease", "reduced", "reduction", "lowering" or "inhibiting" are generally all used herein to mean a reduction in a statistically significant amount. However, for the avoidance of doubt, "reduced", "reduced" or "lowering" or "inhibiting" means a reduction of at least 10% compared to a reference level, such as a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a reduction of 100% (e.g., a level that is not present compared to a reference sample), or any reduction of 10% to 100%.

[0047] The term "modified cell" or "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the specific subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be the same as the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0048] The term "fragment" refers to any peptide or polypeptide having an amino acid residue sequence shorter than the full-length polypeptide described herein. The isolated peptide of PAR1 is shortened or truncated compared to its parent full-length PAR1. Relative to the full-length PAR1 sequence, the polypeptide or peptide derived from PAR3 or PAR1 of the present invention is generally at the conserved Arg 41 and Arg 46 The residues are followed by an N-terminal truncation and may have additional N-terminal deletions or substitutions as described herein. These fragments may additionally comprise a C-terminal truncation (e.g., a truncation of up to 50, 100, 200, 300 or more C-terminal residues) and / or internal deletions.

[0049] "Inflammation" or "inflammatory response" refers to the innate immune response that occurs when tissue is damaged by bacteria, trauma, toxins, heat or any other cause. Damaged tissue releases compounds, including histamine, bradykinin and serotonin. Inflammation refers to both acute responses (i.e., responses in which the inflammatory process is active) and chronic responses (i.e., responses characterized by slow progression and formation of new connective tissue). Acute and chronic inflammation can be distinguished by the types of cells involved. Acute inflammation usually involves polymorphonuclear neutrophils; while chronic inflammation is usually characterized by lymphohistiocytic and / or granulomatous responses. Inflammation includes both specific and nonspecific defense system responses. Specific defense system responses are specific immune system response responses to antigens (which may include self-antigens). Nonspecific defense system responses are inflammatory responses mediated by white blood cells that do not have immune memory capabilities. Such cells include granulocytes, macrophages, neutrophils and eosinophils.

[0050] Inflammatory diseases are diseases caused by abnormally regulated inflammatory responses, such as rheumatoid arthritis, hay fever, and atherosclerosis. Inflammation or inflammatory response refers to the innate immune response that occurs when tissues are damaged by bacteria, trauma, toxins, heat, or any other cause. Damaged tissues release compounds, including histamine, bradykinin, and 5-hydroxytryptamine. Inflammation includes both acute responses (i.e., responses in which the inflammatory process is active) and chronic responses (i.e., responses characterized by slow progression and new connective tissue formation). Acute and chronic inflammation can be distinguished by the types of cells involved. Acute inflammation usually involves polymorphonuclear neutrophils; while chronic inflammation is usually characterized by lymphoid tissue cells and / or granulomatous responses. Inflammation includes both specific and nonspecific defense system responses. Specific defense system responses are specific immune system response responses to antigens (which may include self-antigens). Nonspecific defense system responses are inflammatory responses mediated by leukocytes that do not have immune memory capabilities. Such cells include granulocytes, macrophages, neutrophils, and eosinophils.

[0051] The term "isolated" means that the protein is removed from its natural environment. However, some components found with it may continue to be present with the "isolated" protein. Thus, an "isolated polypeptide" is not as it occurs in nature, but can be substantially less than 100% pure protein.

[0052] In the context of two or more nucleic acids or peptide sequences, the term "identical" or percentage "identity" refers to two or more identical sequences or subsequences. When two sequences are compared and aligned in a comparison window or a specified region to obtain maximum correspondence, if the two sequences have a specified percentage of identical amino acid residues or nucleotides (that is, 60% identity in a specified region or when not specified over the entire sequence, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity), as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, the two sequences are "substantially identical". Optionally, identity is present in a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably in a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0053] Sequence alignment methods for comparison are well known in the art. The best alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482c, 1970; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443, 1970; by retrieving the similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444, 1988; by computerized implementation of these algorithms (Wisconsin Genetics software package, Genetics Computer Group, Madison, GAP, BESTFIT, FASTA and TFASTA in WI); or by manual alignment and visual inspection (see, for example, Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003)). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.

[0054] In addition to the above sequence identity percentages, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies produced against the polypeptide encoded by the second nucleic acid, as described below. Thus, for example, where the two peptides differ only by conservative substitutions, a polypeptide is typically substantially identical to the second polypeptide. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0055] Unless otherwise indicated, the terms "polypeptide" and "peptide" are used interchangeably herein (e.g., "anti-inflammatory polypeptides derived from PAR3" and "anti-inflammatory peptides derived from PAR1") to refer to polymers of amino acid residues. It includes both short oligopeptides (e.g., peptides of less than about 25 residues) and longer polypeptide molecules (e.g., polymers of more than about 25 or 30 amino acid residues). Typically, the anti-inflammatory peptides (oligopeptides) or polypeptides of the present invention may include a length of about 4 amino acid residues to about 350 or more amino acid residues. In some embodiments, the peptide or polypeptide includes a length of about 8 amino acid residues to about 60 amino acid residues. The anti-inflammatory peptides or polypeptides of the present invention include naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids. Unless otherwise indicated, a specific polypeptide sequence also implicitly includes its conservatively modified variants.

[0056] The term "peptide mimetic" as used herein refers to a derivative compound that mimics the biological function of a reference peptide (e.g., an anti-inflammatory polypeptide disclosed herein). Generally speaking, the peptide mimetic derivative of an anti-inflammatory polypeptide derived from PAR1 of the present invention has at least 50%, at least 75%, or at least 90% of the biological activity of the reference polypeptide (e.g., inhibiting caspase 1 activity).

[0057] The term "operably linked" refers to the functional relationship between two or more polynucleotide (e.g., DNA) segments. Generally speaking, it refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, if a promoter or enhancer sequence stimulates or regulates the transcription of a coding sequence in an appropriate host cell or other expression system, it is operably linked to the coding sequence. Generally speaking, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically continuous with the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically continuous or need not be located near the coding sequence that enhances transcription.

[0058] The term "ortholog" or "homolog" used herein refers to polypeptides that have the same or similar functions and share basic sequence identity from different species or organisms. For example, PAR3 and PAR1 from humans, rabbits, rats, mice, and many other animal species, respectively, are orthologs because their sequences and functions are similar.

[0059] The phrase "signal transduction pathway" or "signaling activity" (e.g., APC, PAR3 or PAR1-mediated cytoprotective signaling) refers to at least one biochemical reaction, but more usually a series of biochemical reactions, which is produced by the interaction of cells with stimulatory compounds or agents. Thus, the interaction of stimulatory compounds (e.g., PAR3-derived or PAR1-derived peptides) with cells produces a "signal" transmitted through a signal transduction pathway, ultimately resulting in a cellular response.

[0060] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein.

[0061] The term "agent" includes any substance, molecule, element, compound, entity, or combination thereof. It includes, but is not limited to, for example, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, etc. It can be a natural product, a synthetic compound or a chemical compound, or a combination of two or more substances. Unless otherwise indicated, the terms "agent", "substance" and "compound" are used interchangeably herein.

[0062] Administration "in combination with" one or more other therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0063] The term "contacting" has its normal meaning and refers to combining two or more agents (e.g., polypeptides or small molecule compounds) or combining agents with cells. Contacting can occur in vitro, such as combining two or more agents or combining agents with cells or cell lysates in a test tube or other container. Contacting can also occur in cells or in situ, for example, by contacting the two polypeptides in cells by co-expressing a recombinant polynucleotide encoding the two polypeptides in cells or in cell lysates. Contacting can also occur in vivo in a subject, for example, by administering to a subject an agent that subsequently interacts with a desired target (e.g., tissue or cell).

[0064] Protease activated receptor (PAR) is a subfamily of related G protein coupled receptors activated by cutting a part of its extracellular domain. It is highly expressed in platelets and also in endothelial cells, myocytes and neurons. There are 4 known protease activated receptors, PAR1, PAR2, PAR3 and PAR4. They are members of the seven transmembrane G protein coupled receptor superfamily. PAR is activated by the action of serine proteases such as thrombin (acting on PAR1, 3 and 4), activated protein C (acting on PAR1, 2 and 3), trypsin (acting on PAR2) or other proteases at the N-terminus to display the bound ligand. The rate-limiting step of PAR signal transduction is determined by the proteolytic efficiency of the N-terminus, which is regulated by allosteric binding sites, cofactors, membrane localization and receptor dimerization. This ultimately controls the start of PAR signal transduction. In addition, these factors also control cell responses by directing signal transduction to G protein or β-detained protein pathways. PAR1 signal transduction on endothelial cells is controlled by activated proteases and heterodimerization with PAR2 or PAR3. The cellular actions of thrombin are mediated by protease-activated receptors (PARs). Thrombin signaling in platelets contributes to hemostasis and thrombosis.

[0065] PAR1 is expressed not only in all types of blood cells, but also in epithelial, neuronal, astrocyte and immune cells. PAR1 was originally identified as a thrombin receptor, but other agonists, such as activated protein C (APC), were later identified. These protease agonists proteolytically remove part of the N-terminal extracellular region of PAR1, generating new binding ligands that interact with the receptor body to induce transmembrane signaling, thereby triggering a wide range of signaling pathways. For example, PAR1 is involved in regulating vascular tension and the permeability of endothelial cells. In vascular smooth muscle, it mediates contraction, proliferation and hypertrophy. PAR1 contributes to the proinflammatory response observed in atherosclerosis and restenosis.

[0066] Protease-activated receptor 3 (PAR3), also known as coagulation factor II receptor-like 2 (F2RL2) and thrombin receptor-like 2, is a protein encoded by the F2RL2 gene in humans. PAR3 is activated by proteolytic cleavage of its extracellular amino terminus. Like PAR1, the new amino terminus of PAR3 acts as a binding ligand and activates the receptor. PAR3 is known to be a cofactor for thrombin-activated PAR4 in mouse platelets, and PAR3 can promote the activation of PAR1 in human cells. Like PAR1, PAR3 is also expressed on vascular endothelial cells. Similar low-level but detectable expression of PAR3 is also evident in human platelets.

[0067] As used herein, "treating" or "improvement" refers to (i) preventing a pathological condition (e.g., an inflammatory disease) from occurring (e.g., prevention); (ii) inhibiting a pathological condition or arresting its development; and (iii) alleviating symptoms associated with a pathological condition. Thus, "treatment" includes administering a therapeutic compound or composition described herein to prevent or delay the onset of symptoms, complications, or biochemical indicators of a disease described herein, to alleviate or improve symptoms of a disease, condition, or disorder, or to arrest or inhibit further development of a disease, condition, or disorder. "Treatment" also refers to any sign of successful treatment or improvement or prevention of a disease, condition, or disorder described herein, including any objective or subjective parameter, such as alleviation; relief; attenuation of symptoms or making the disease condition more tolerable to the patient; slowing the rate of degeneration or decline; or making the endpoint of degeneration less debilitating. The detailed procedures for treating or ameliorating a disease or its symptoms may be based on objective or subjective parameters, including the results of a physician's examination.

[0068] The term "variant" as used herein refers to a molecule (e.g., polypeptide or polynucleotide) comprising a sequence substantially identical to that of a reference molecule. For example, a reference molecule may be an N-terminally truncated PAR3 or PAR1 polypeptide (e.g., SEQ ID NO: 2 or SEQ ID NO: 5) or a polynucleotide encoding the polypeptide. A reference molecule may also be an anti-inflammatory polypeptide from a PAR3 or PAR1 source disclosed herein or a polynucleotide encoding the anti-inflammatory polypeptide (e.g., P3R or TR47). In some embodiments, a variant may have at least 50%, at least 70%, at least 80%, at least 90%, at least 95% or more sequence identity with a reference molecule. In some other embodiments, a variant differs from a reference molecule in that it has one or more conservative amino acid substitutions. In some other embodiments, a variant of a reference molecule (e.g., an anti-inflammatory polypeptide P3R or TR47) has an altered amino acid sequence (e.g., having one or more conservative amino acid substitutions) but substantially retains the biological activity of the reference molecule (e.g., activating PAR3 or PAR1 signaling). Conservative amino acid substitutions are well known to those skilled in the art.

[0069] The term "vector" is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cell in which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0070] III. PAR3-derived and PAR1-derived anti-inflammatory peptides and derivative compounds

[0071] The present invention provides methods and compositions related to the use of anti-inflammatory peptides or polypeptides from PAR3 and PAR1 sources to inhibit inflammation and treat inflammatory conditions or diseases. The anti-inflammatory peptides from PAR3 and PAR1 sources refer to peptides or polypeptides having anti-inflammatory activity and derived from the N-terminal sequence or its fragments after cutting at atypical cleavage sites by APC, respectively. In a plurality of embodiments, the anti-inflammatory peptides from PAR3 and PAR1 sources may also have cytoprotective activity. The cytoprotective activity of the peptide includes any non-anticoagulant protective cell activity mediated by the APC signal transduction pathway, particularly the activity related to inhibiting apoptosis and promoting cell survival. It includes activation of the PI3K-Akt survival pathway, prevention of thrombin-induced endothelial barrier destruction, inhibition of endothelial cell apoptosis (e.g., by blocking the pro-apoptotic activity of p53 or by other mechanisms), secretion of TNF-α by macrophages, reduction of cellular NFκB activation in endothelial cells, prevention of leukocyte adhesion to activated endothelial cells, induction of stabilization of endothelial cell barrier integrity by sphingosine 1 phosphate release or sphingosine 1 phosphate receptor 1 (S1P1) activation or by Tie2 activation. Such activity can be easily assessed using methods known in the art, such as endothelial barrier assays and in vivo vascular permeability assays. See, e.g., Mosnier et al., Blood. 120: 5237-5246, 2012; Burnier and Mosnier, Blood 122: 807-816, 2013; and Stavenuiter and Mosnier, Blood 124: 3480-3489, 2014.

[0072] As exemplified herein by peptides P3R and TR47 or their various fragments, respectively, PAR3-derived and PAR1-derived anti-inflammatory peptides activate PARI and PAR3 differently than peptides that mimic the PAR1 sequence starting at residue 42 (so-called TRAP agents) or peptides that mimic the PAR3 sequence starting at residue 39 (P3K agents), and induce different types of cell signaling. Any PAR3-derived and PAR1-derived anti-inflammatory peptides derived from the N-terminal sequence of PAR3 and PAR1 after cleavage by APC at the atypical cleavage site can be used in the present invention. Specifically, PAR1 at Arg 41 The typical site of cleavage by APC (and other proteases, especially thrombin) is the Arg 41 The new N-terminal peptide generated when PAR1 is cleaved at the N-terminal end is often referred to as the Thrombin Receptor Activating Peptide (TRAP). Thrombin cleavage of PAR1 or treatment of cells with TRAP is generally pro-inflammatory and may be harmful to cells or animals in general, depending on the animal's cells, organs, or environment. APC also expresses a thrombin receptor at the atypical site Arg 46 PAR1 is cleaved at the canonical site (Lys38) to generate a new N-terminus, as shown in the peptide TR47 (also referred to herein as "P1 (47-66)") (47-NPNDKYEPFWEDEEKNESGL-66; SEQ ID NO:7). TRAP and TR47 are agonists of PAR1 activation. However, TR47 has been shown to act as an agonist with a spectrum of activity that is significantly different from that of TRAP. PAR3 is cleaved at the canonical site (Lys38) by proteases (e.g., thrombin). Peptides that mimic the new N-terminus generated when cleavage occurs at Lys38 (e.g., sequences beginning with Thr39, such as the peptide "P3K") can act as agonists of PAR3 in activating a thrombin-mediated pathway. Similar to PAR1, PAR3 can also be activated at a non-canonical site (Arg 41 ) is cleaved at the end of the PAR3, generating a new N-terminus, as shown in the peptide P3R (also referred to herein as "P3 (42-65)") (42-GAPPNSFEEFPFSALEGWTGATIT-65; SEQ ID NO: 4). The peptides P3R and P3K are agonists of PAR3 activation. However, it has been shown that P3R exhibits an activity profile that is significantly different from that of P3K.

[0073] As exemplified herein, the simulations were performed with the N-terminal residue Asn 47 Anti-inflammatory peptides derived from PAR1 with a PAR1 partial sequence starting with PAR1 (e.g., peptide TR47) can induce anti-inflammatory cell signaling. Anti-inflammatory cell signaling can be demonstrated by, for example, inhibiting the activity of inflammasomes and / or inhibiting caspase 1 activation. Similarly, the simulation of the N-terminal residue Gly42 Anti-inflammatory peptides derived from PAR3 of the initial PAR3 partial sequence (e.g., peptide P3R) or some fragments thereof can also cause anti-inflammatory cell signaling. P3R and P3K are agonists of PAR3 activation. Examples show that P3R is shown to act as an agonist with a significantly different activity spectrum from P3K.

[0074] Generally speaking, the PAR3-derived anti-inflammatory peptide used in the practice of the present invention comprises (1) Met 1 -Arg 41 (1) at least the first 4 N-terminal residues of a human PAR3 sequence (SEQ ID NO: 2) or a conservatively modified variant thereof, or (2) at least 4 consecutive amino acid residues of a peptide P3R (SEQ ID NO: 4) or a conservatively modified variant thereof derived from human PAR3, wherein the at least 4 consecutive amino acid residues comprise Phe of SEQ ID NO: 3. 10 -Phe 12 In some embodiments, the peptide has a motif derived from a soluble, Met 1 -Arg 41 The sequence of the N-terminal residues of the deleted human PAR3 extracellular sequence (SEQ ID NO:3). In some of these embodiments, the polypeptide derived from the anti-inflammatory PAR3 comprises the first 25 or more N-terminal residues of SEQ ID NO:3 or a conservatively modified variant sequence. In some embodiments, the polypeptide derived from the anti-inflammatory PAR3 comprises the first 4, 5, 6, 7, 8, 9, 10, 11, 12 or more N-terminal residues of the peptide P3R (SEQ ID NO:4) or a conservatively modified variant sequence. In some embodiments, the polypeptide derived from the anti-inflammatory PAR3 comprises the first 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 N-terminal residues of the peptide P3R (SEQ ID NO:4) or a conservatively modified variant sequence. In some embodiments, the polypeptide derived from the anti-inflammatory PAR3 comprises at least 5, 6, 7, 8, 9, 10, 11, 12 or more consecutive amino acid residues of SEQ ID NO:4 or its conservatively modified variants, which comprises Phe 10 -Phe 12 In some embodiments, the anti-inflammatory PAR3-derived polypeptide comprises at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more consecutive amino acid residues of SEQ ID NO: 4 or a conservatively modified variant thereof, which comprises Phe 10 -Phe 12 Some of these PAR3-derived peptides contain Phe 10 -Trp 18Several specific examples of PAR3-derived anti-inflammatory polypeptides suitable for use in the present invention are P3R (SEQ ID NO: 4), P3Rm or P3 (42-54) (GAPPNSFEEFPFS; SEQ ID NO: 8), P3R51-65 or P3 (51-65) (FPFSALEGWTGATIT; SEQ ID NO: 9), and P3R51-59 or P3 (51-59) (FPFSALEGW; SEQ ID NO: 10), or conservatively modified variants thereof. Some other examples of anti-inflammatory polypeptides derived from PAR3 include GAPPNSFEEFPFSA (SEQ ID NO: 11), GAPPNSFEEFPFSAL (SEQ ID NO: 12), GAPPNSFEEFPFSALE (SEQ ID NO: 13), GAPPNSFEEFPFSALEG (SEQ ID NO: 14), GAPPNSFEEFPFSALEGW (SEQ ID NO: 15), GAPPNSFEEFPFSALEGWT (SEQ ID NO: 16), GAPPNSFEEFPFSALEGWTG (SEQ ID NO: 17), GAPPNSFEEFPFSALEGWTGA (SEQ ID NO: 18), GAPPNSFEEFPFSALEGWTGAT (SEQ ID NO: 19), GAPPNSFEEFPFSALEGWTGATI (SEQ ID NO: 20), FPFSALEGWT (SEQ ID NO: 21), FPFSALEGWTG (SEQ ID NO: 22), FPFSALEGWTGA (SEQ ID NO: 23). NO: 23), FPFSALEGWTGAT (SEQ ID NO: 24), FPFSALEGWTGATI (SEQ ID NO: 25), FPFSALEG (SEQ ID NO: 26), FPFSALE (SEQ ID NO: 27), FPFSAL (SEQ ID NO: 28), EFPFSAL (SEQ ID NO: 29), and EEFPFSAL (SEQ ID NO: 30), or conservatively modified variants thereof. In the method of the present invention for inhibiting inflammation and / or for treating inflammatory disorders, any of these PAR3-derived peptides may be used alone or in combination with the PAR1-derived peptides described herein.

[0075] Generally speaking, the PAR1-derived anti-inflammatory peptides used to practice the present invention have a similar affinity to Met 1 -Arg 46The human PAR1 sequence (SEQ ID NO: 5) with deletions, variants (e.g., Met 1 -Arg 46 The peptide may be a human PAR1 sequence with a deletion), a homolog (e.g., a non-human PAR1 sequence with a similar deletion), or a TR47 (also known as P1 (47-66)) (SEQ ID NO: 7) variant having a further N-terminal modification (amino acid substitution or deletion). Preferably, the peptide has a peptide derived from a soluble, Met 1 -Arg 46 The sequence of the N-terminal residues of the deleted human PAR1 extracellular sequence (SEQ ID NO: 6). In some embodiments, the anti-inflammatory PAR1-derived polypeptide has at least the first 4 N-terminal residues that are substantially identical to the corresponding N-terminal residues of SEQ ID NO: 6. In some of these embodiments, the anti-inflammatory PAR1-derived polypeptide comprises the first 21 or more N-terminal residues of SEQ ID NO: 6 or a conservatively modified variant sequence. In some preferred embodiments, the anti-inflammatory peptide derived from PAR1 comprises the first 6, 7, 8, 9, 10, 11, 12 or more N-terminal residues of peptide TR47 (SEQ ID NO: 7) or a conservatively modified variant sequence. As exemplified herein with TR47 8-mer, TR47 9-mer and TR47 16-mer, these peptides retain substantial anti-inflammatory activity relative to TR47. In some embodiments, the anti-inflammatory peptide derived from PAR1 comprises the first 13, 14, 15, 16, 17, 18, 19 or more N-terminal residues of SEQ ID NO: 7 or a conservatively modified variant sequence. In some other embodiments, the anti-inflammatory peptide derived from PAR1 is a TR47 variant with a mutation at the N-terminus. These include TR47 variants with a deletion of the first 1, 2, 3, 4, 5, 6, 7, 8 or more residues at the N-terminus. It also includes TR47 variants with one or more amino acid substitutions at the N-terminus. As exemplified herein with TR47 (N-1), TR47 (N-5), TR47ΔQ and TR47ΔA, these TR47 variant peptides also maintain significant anti-inflammatory activity relative to TR47.

[0076] Some specific examples of PAR1-derived anti-inflammatory polypeptides suitable for use in the present invention are shown in SEQ ID NO: 6 and SEQ ID NO: 7 (peptide TR47), and peptide

[0077] NPND (SEQ ID NO: 31), NPNDK (SEQ ID NO: 32), NPNDKY (SEQ ID NO: 33), NPNDKYE (SEQ ID NO: 34), NPNDKYEP (SEQ ID NO: 35), NPNDKYEPF (SEQ ID NO: 36), NPNDKYEPFW (SEQ ID NO: 37), NPNDKYEPFWE (SEQ ID NO: 37) NO: 38), NPNDKYEPFWED (SEQ ID NO: 39), NPNDKYEPFWEDE (SEQ ID NO: 40), NPNDKYEPFWEDEE (SEQ ID NO: 41), NPNDKYEPFWEDEEK (SEQ ID NO: 42), NPNDKYEPFWEDEEKN (SEQ ID NO: 43), NPNDKYEPFWEDEEKNE (SEQ ID NO: 44), NPNDKYEPFWEDEEKNES (SEQ ID NO: 45), NPNDKYEPFWEDEEKNESG (SEQ ID NO: 46), PNDKYEPFWEDEEKNESGL (SEQ ID NO: 47), NDKYEPFWEDEEKNESGL (SEQ ID NO: 51), DKYEPFWEDEEKNESGL (SEQ ID NO: 57), KYEPFWEDEEKNESGL (SEQ ID NO: 58), YEPFWEDEEKNESGL (SEQ ID NO: 48), EPFWEDEEKNESGL (SEQ ID NO:59), PFWEDEEKNESGL (SEQ ID NO:60), QPNDKYEPFWEDEEKNESGL (SEQ ID NO:49) and APNDKYEPFWEDEEKNESGL (SEQ ID NO:50).

[0078] In the method for inhibiting inflammation and / or for treating inflammatory disorders of the present invention, any of these PAR1-derived peptides may be used alone or in combination with the PAR3-derived peptides described herein.

[0079] In addition to the above-mentioned peptides, anti-inflammatory compounds suitable for the present invention also include related or derivative compounds derived from anti-inflammatory peptides or polypeptides derived from PAR3 sources and PAR1 sources exemplified herein. These related or derivative compounds include variants, analogs, mimetics, complexes or fusion molecules (comprising the peptide or polypeptide), and other compounds derived from anti-inflammatory peptides or polypeptides derived from PAR3 sources and PAR1 sources. In some preferred embodiments, derivative compounds have substantially the same chemical or biological activity as the anti-inflammatory peptides or polypeptides (e.g., P3R and TR47 peptides) in PAR3 sources and PAR1 sources exemplified herein. Related or derivative compounds may include, for example, peptides or polypeptides with substantially the same sequence, such as conservatively modified variants. It also includes peptides or polypeptides derived from non-human orthologs PAR3 and PAR1 sequences, respectively. Related or derivative compounds suitable for the present invention also include variants, analogs, polymers, peptide mimetics, fusions with other molecules (e.g., carrier moieties), or other derivatives that can be produced from anti-inflammatory polypeptides (e.g., peptides P3R and TR47) in PAR3 sources and PAR1 sources exemplified herein. Appropriate assays or screening methods can be implemented for these derivative compounds to identify anti-inflammatory compounds with optimized activity. In some embodiments, the derivative compounds are modified forms of exemplary peptides produced by conservative amino acid substitutions. In some other embodiments, the derivative compounds are variants produced by non-conservative substitutions (to the extent that they substantially retain the activity of those peptides). Anti-inflammatory peptides can be modified using standard techniques routinely implemented in the art (e.g., U.S. Patent Applications 20080090760 and 20060286636).

[0080] In some embodiments, the derivative compounds of the exemplary PAR-3 and PAR1-derived anti-inflammatory polypeptides (e.g., peptide P3R or TR47) of the present invention are analogs, or they contain one or more amino acid derivatives of the twenty naturally occurring standard amino acids, for example, 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine, ornithine or carboxyglutamic acid, and may contain amino acids that are not linked by polypeptide bonds. Similarly, it may also be cyclic polypeptides and other conformationally restricted structures. Methods for modifying polypeptides to produce analogs and derivatives are well known in the art, for example, Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meinhofer, eds., Vol. 5, p. 341, Academic Press, Inc., New York, NY (1983); and Burger's Medicinal Chemistry and Drug Discovery, Manfred E. Wolff, ed., Chapter 15, pp. 619-620, John Wiley & Sons Inc., New York, NY (1995).

[0081] Some other derivative compounds of the anti-inflammatory polypeptides in exemplary PAR3 source and PAR1 source are peptide mimics. The peptide mimics based on the anti-inflammatory peptides (e.g., peptide P3R or TR47) in PAR3 source and PAR1 source substantially retain the activity of the reference peptide. It includes chemically modified peptides or polypeptides substantially identical to the reference polypeptide or peptide derived from the structure and peptide mimics, polypeptide-like molecules comprising non-natural amino acids, peptoids, etc. (see, e.g., Burger's Medicinal Chemistry and Drug Discovery, 1995, the same). For example, peptide mimics may have one or more residues chemically derivatized by functional side group reactions. Except for side group derivatization, chemical derivatives may have one or more skeleton modifications, including α-amino substitutions, such as N-methyl, N-ethyl, N-propyl, etc., and α-carbonyl substitutions, such as thioesters, thioamides, guanidine, etc. Generally speaking, when compared with a reference polypeptide, a peptide mimic shows considerable structural identity, and exhibits a feature that can be identified or known to be derived from a reference polypeptide or is associated with a reference polypeptide. Peptide mimetics include, for example, organic structures that exhibit similar properties (e.g., charge and charge spacing characteristics) as a reference polypeptide. Peptide mimetics may also include constrained structures to maintain optimal spacing and charge interactions of amino acid functional groups.

[0082] In some embodiments, the derivative compounds of the PAR3-derived and PAR1-derived anti-inflammatory peptides or polypeptides described herein are molecules having a peptide or polypeptide covalently or non-covalently conjugated to a carrier moiety by any conventional method. A "carrier moiety" ("carrier" or "carrier molecule") is a conjugation partner capable of enhancing the immunogenicity of a polypeptide. Suitable carriers are typically large, slowly metabolized macromolecules, such as proteins; polysaccharides (e.g., latex-functionalized SEPHAROSE TM , agarose, cellulose, cellulose beads, etc.); polymeric amino acids (such as polyglutamic acid, polylysine, etc.); amino acid copolymers; and inactive viral particles or attenuated bacteria, such as Salmonella. In various embodiments, the carrier portion can be a carrier protein, an immunoglobulin, an Fc domain, a PEG molecule, or other polymer. In some embodiments, the carrier portion is a protein. Integral membrane proteins from, for example, E. coli and other bacteria are useful conjugation partners. Particularly useful carrier proteins are serum albumin, keyhole limpet hemocyanin (KLH), certain immunoglobulin molecules, thyroglobulin, ovalbumin, bovine serum albumin (BSA), tetanus toxoid (TT), and diphtheria toxoid (CRM). In some other embodiments, the carrier portion can be a polymer other than a protein or polypeptide. Examples of such polymers include, for example, carbohydrates, such as dextran, mannose, or mannan.

[0083] In some embodiments, the anti-inflammatory peptides, variants or derivatives of the PAR3 source and PAR1 source described herein are dimerized or multimerized molecules. In some of these embodiments, the dimerization or multimerization of the anti-inflammatory agent can be achieved by covalently linking to at least one linker portion. In some embodiments of combined therapy, a peptide or variant derived from PAR3 (e.g., P3R or P3R (51-59)) can be conjugated to a peptide or variant derived from PAR1 (e.g., TR47 or TR47 8-mer). Homologous multimerization or heterologous multimerization of peptides derived from PAR3 and / or PAR1 can be achieved in a variety of suitable ways. For example, the peptides can be covalently linked by recombinant technology. In some embodiments, the multimerized molecules of the present invention can utilize the above-mentioned carrier portion. In some of these embodiments, a carrier molecule or portion can be used to conjugate a peptide derived from PAR3 (e.g., P3R or P3R (51-59)) to a peptide derived from PAR1 (e.g., TR47 or TR47 8-mer). In some embodiments, the peptide or polypeptide can also be conjugated to C 1-12 The linking moiety is conjugated to the C 1-12The linking moiety is optionally terminated with one or two -NH- linkages and is optionally substituted at one or more available carbon atoms with a lower alkyl substituent.

[0084] The peptides in PAR3 and / or PAR1 sources described herein can be connected by other chemical bonds, such as by disulfide bonds or by chemical bridges. In some other embodiments, the anti-inflammatory peptides described herein can be physically connected in series to form polymers of peptides in PAR3 sources or PAR1 sources. The peptides constituting such polymers can be separated from each other by peptide joints. For example, the peptides in PAR3 sources can be covalently bonded to the peptides in PAR1 sources by glycine joints, as exemplified herein. In a number of embodiments, the glycine joints can include about 2 to about 20 glycine residues. In these embodiments, the peptides in PAR3 sources can be bonded to the N-terminal or C-terminal of the peptides in PAR1 sources by joints. In some embodiments, other molecular biology techniques known in the art can be used to produce peptide polymers. In some embodiments, polyethylene glycol (PEG) can be used as a joint to dimerize two peptide monomers. For example, a single PEG moiety comprising two reactive functional groups can be connected to the N-terminals of two peptide chains of a peptide dimer at the same time. These peptides are referred to herein as "PEGylated peptides". In some embodiments, peptide monomers of the invention can be oligomerized using a biotin / streptavidin system.

[0085] In any of these conjugation schemes, different copies of a PAR3-derived peptide (e.g., P3R or P3R(51-59)) and a PAR1-derived peptide (e.g., P3R or P3R(51-59)) can be linked in a heteromultimerized molecule. In some of these embodiments, for each copy of a linked PAR1-derived peptide, multiple copies of the PAR3-derived peptide are linked to a carrier portion. For example, a multimerized molecule may comprise a carrier portion, wherein the PAR3-derived peptide and the PAR1-derived peptide are linked in a ratio of at least about 20:1, 15:1, 12:1, 10:1, 8:1, 6:1, 4:1, or 2:1. In some other embodiments, for each copy of a linked PAR3-derived peptide, multiple copies of a PAR1-derived peptide are linked to a carrier portion. For example, the multimeric molecule may comprise a carrier portion in which a PAR1-derived peptide and a PAR3-derived peptide are linked in a ratio of at least about 20: 1, 15: 1, 12: 1, 10: 1, 8: 1, 6: 1, 4: 1 or 2: 1. Depending on the specific dosage to be achieved for the two peptides or their derivative compounds as described herein, any of these conjugation ratios may be used to construct a heteromultimeric molecule to implement the methods of the present invention.

[0086] Methods for stabilizing peptides known in the art can be used together with the methods and compositions described herein. For example, using D-amino acids, using reduced amide bonds as peptide backbones, and using non-peptide bonds to connect side chains, including but not limited to pyrrolidinone and sugar mimics, can provide stability. The design and synthesis of sugar scaffold peptide mimics are described in the art, for example, Hirschmann et al., J.Med.Chem.36,2441-2448,1996. In addition, pyrrolidinone-based peptide mimics present peptide pharmacophores on a stable background with improved bioavailability characteristics. See, for example, Smith et al., J.Am.Chem.Soc.122,11037-11038,2000.

[0087] In some embodiments, the derivative compounds of exemplary anti-inflammatory PAR3 and PAR1 peptides or polypeptides include modifications within the sequence, such as modifications by terminal-NH2 acylation (e.g., acetylation), or by thioglycolic acid amidation modification of terminal carboxyl amidation, such as terminal modifications with ammonia, methylamine, etc. The amino and / or carboxyl termini of the polypeptides described herein may also be modified. The terminal modification may be used to reduce the sensitivity of protease digestion, and thus may be used to extend the half-life of the polypeptide in solution, particularly in biological fluids where proteases may be present. Amino terminal modifications include methylation (e.g., -NHCH3 or -N(CH3)2), acetylation (e.g., with acetic acid or its halogenated derivatives, such as α-chloroacetic acid, α-bromoacetic acid or α-iodoacetic acid), addition of benzyloxycarbonyl (Cbz) groups, or blocking the amino termini with any blocking groups containing carboxylate functionality defined by RCOO- or sulfonyl functionality defined by R-SO2-, wherein R is selected from alkyl, aryl, heteroaryl, alkylaryl, etc., and similar groups. A desamino acid may also be incorporated at the N-terminus (so that there is no N-terminal amino group) to reduce sensitivity to proteases or to constrain the conformation of the peptide compound. In some embodiments, the N-terminus is acetylated with acetic acid or acetic anhydride.

[0088] The carboxyl terminal modification includes replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxyl terminal to introduce structural constraints. The peptides described herein can also be cyclized, or a deamino or decarboxylation residue can be incorporated at the end of the peptide so that there is no terminal amino or carboxyl group to reduce sensitivity to proteases or restrict the conformation of the peptide. Methods for the synthesis of cyclic peptides are known in the art, for example, in U.S. Patent Application No. 20090035814; and Muralidharan and Muir, Nat. Methods, 3: 429-38, 2006. The C-terminal functional groups of the peptides described herein include amides, amide lower alkyls, amide di(lower alkyls), lower alkoxys, hydroxyls and carboxyls, and lower ester derivatives thereof, and pharmaceutically acceptable salts thereof.

[0089] The anti-inflammatory peptides or polypeptide compounds in the PAR3 source and PAR1 source described herein are also used as structural models for non-peptide compounds with similar biological activity. There are multiple techniques that can be used to construct peptides with the same or similar desired biological activity as those in the PAR3 source and PAR1 source, but with more favorable active compounds in terms of solubility, stability and sensitivity to hydrolysis and proteolysis. See, for example, Morgan and Gainor, Ann.Rep.Med.Chem.24:243-252,1989. These techniques include but are not limited to replacing the peptide backbone with a backbone consisting of phosphonates, amidates, carbamates, sulfonamides, secondary amines and N-methyl amino acids.

[0090] IV. Synthesis of PAR3- and PAR1-derived anti-inflammatory peptides and related compounds

[0091] The anti-inflammatory polypeptides in the PAR3 source and PAR1 source described herein, including variants and derivatives thereof, can be chemically synthesized and purified by standard chemical or biochemical methods well known in the art. Some methods for producing analogs or derivative compounds of the anti-inflammatory polypeptides in the PAR3 source and PAR1 source are described above. Other methods that can be used to produce the anti-inflammatory polypeptides of the present invention and derivative compounds thereof include, for example, solid phase peptide synthesis. For example, peptides can be synthesized using t-Boc (tert-butyloxycarbonyl) or FMOC (9-fluorenylmethoxycarbonyl) protecting groups described in the art. See, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154, 1963; "Peptide synthesis and applications" in Methods in molecular biology, Vol. 298, John Howl, ed.; "Chemistry of Peptide Synthesis" by N. Leo Benoiton, 2005, CRC Press, (ISBN-13:978-1574444544); and "Chemical Approaches to the Synthesis of Peptides and Proteins" by P. Lloyd-Williams et al., 1997, CRC-Press, (ISBN-13:978-0849391422), Methods in Enzymology, Vol. 289: Solid-Phase Peptide Synthesis, JN Abelson, MI Simon, GB Fields (eds.), Academic Press; 1st edition (1997) (ISBN-13:978-0121821906); U.S. Patent Nos. 4,965,343 and 5,849,954.

[0092] In some embodiments, peptides can be obtained by solid phase synthesis using a peptide synthesis machine. Commercial peptide synthesis machines can be used for solid phase peptide synthesis. For example, Advanced Chemtech Model 396 multiple peptide synthesizers and Applied Biosystems Model 432A peptide synthesizers are suitable. There are commercial companies that can customize synthetic peptides, such as Abbiotec, Abgent, AnaSpec Global Peptide Services, LLC., Invitrogen and rPeptide, LLC.

[0093] In some embodiments, the anti-inflammatory polypeptides and derivatives thereof from PAR3 and PAR1 sources can also be synthesized and purified by molecular methods well known in the art. Recombinant polypeptides can be expressed in bacteria, mammals, insects, yeast or plant cells. For example, using full-length PAR3 or PAR1 cDNA sequences as templates for PCR cloning, conventional polymerase chain reaction (PCR) cloning techniques can be used to clone polynucleotides encoding peptides or polypeptides from PAR3 or PAR1 sources. Alternatively, the sense and antisense strands of the encoding nucleic acid can be prepared synthetically and then annealed together to form a double-stranded encoding nucleic acid. Ideally, restriction enzyme recognition sites should be designed at the ends of the sense and antisense strands to facilitate connection to a cloning vector or other vector. Alternatively, for the purpose of TA cloning well known in the art, 3'A-overhangs may be included. Such encoding nucleic acids with 3'A-overhangs can be easily connected to Invitrogen topoisomerase-assisted TA vectors, such as pCR-TOPO, pCR-Blunt II-TOPO, pENTR / D-TOPO and pENTR / SD / D-TOPO. The encoding nucleic acid can be cloned into a universal cloning vector, such as pUC19, pBR322, pBluescript vector (Stratagene Inc.) or pCR-TOPO from Invitrogen Inc. The resulting recombinant vector carrying the polynucleotide encoding PAR3 or PAR1 peptide can then be used for further molecular biology operations. These include, for example, site-directed mutagenesis of variant PAR1 peptides and / or reduce the immunogenic properties of the peptide or improve protein expression in a heterologous expression system. The coding sequence can also be subcloned into a protein expression vector or a viral vector for the synthesis of fusion proteins comprising the PAR1 peptide and for synthesizing proteins in a variety of protein expression systems. These include expression systems based on host cells selected from mammalian cell lines, insect cell lines, yeast, bacteria and plant cells.

[0094] In some related embodiments, the present invention provides isolated or substantially purified polynucleotides (DNA or RNA) encoding anti-inflammatory polypeptides in PAR3 source and PAR1 source as described herein, including heteromultimers (e.g., dimers) comprising both peptides in PAR3 source and peptides in PAR1 source. The present invention also provides expression vectors and modified host cells comprising vectors for expressing polynucleotides encoding polypeptides. The polynucleotides encoding anti-inflammatory polypeptides (e.g., multimers of P3R (51-59) / TR47 8-mers) are operably connected to the promoter in the expression vector. The expression construct may also include a secretory sequence that helps to purify the peptide from the cell culture medium. The host cell into which the vector has been introduced may be any of a variety of expression host cells known in the art, such as bacteria (e.g., Escherichia coli), yeast cells, or mammalian cells.

[0095] Recombinant protein expression in different host cells can be constitutive or inducible with inducers such as copper sulfate or sugars such as galactose, methanol, methylamine, thiamine, tetracycline or IPTG. After the protein is expressed in the host cells, the host cells are lysed to release the expressed protein for purification. A preferred purification method is affinity chromatography, such as ion-metal affinity chromatography using nickel, cobalt or zinc affinity resins for histidine-tagged peptides. Clontech uses its Cobalt resins and Novagen describe methods for purifying histidine-tagged recombinant proteins in their pET system manual, 10th edition. Another preferred purification strategy is by immunoaffinity chromatography, for example, anti-Myc antibody conjugated resins can be used to affinity purify Myc-tagged peptides. Enzymatic digestion with serine proteases (e.g., thrombin and enterokinase) cleaves and releases the peptide from the histidine or Myc tag, releasing the recombinant peptide from the affinity resin while the histidine tag and Myc tag remain attached to the affinity resin.

[0096] Cell-free expression systems can also be used to produce anti-inflammatory polypeptides of the present invention. Compared with traditional cell-based expression methods, cell-free expression systems have several advantages, including easy modification of reaction conditions to facilitate protein folding, reduced sensitivity to product toxicity, and suitability for high-throughput strategies, such as rapid expression screening or large-scale protein production, because the reaction volume and processing time are reduced. Cell-free expression systems can use plasmids or linear DNA. In addition, the increase in translation efficiency leads to a yield of more than one milligram of protein / ml reaction mixture. Spirin et al., Science 242:1162, 1988 describes an example of a cell-free translation system that can produce proteins in high yields. The method uses a continuous flow design of a feed buffer containing amino acids, adenosine triphosphate (ATP) and guanosine triphosphate (GTP) throughout the reaction mixture, and continuous removal of the translated polypeptide product. The system uses E. coli lysate to provide a cell-free continuous feed buffer. This continuous flow system is compatible with both prokaryotic and eukaryotic expression vectors. Chang et al., Science 310:1950-3, 2005 describes an example of large-scale cell-free protein production.

[0097] Other commercially available cell-free expression systems include Expressway TM The cell-free expression system (Invitrogen), which utilizes an E. coli-based in vitro system for efficient, coupled transcription and translation reactions to produce up to milligram quantities of active recombinant protein in a tube reaction format; the Rapid Translation System (RTS) (Roche Applied Science), which also uses an E. coli-based in vitro system; and the TNT-coupled reticulocyte lysis system (Promega), which uses a rabbit reticulocyte-based in vitro system.

[0098] V. Inhibit inflammation and treat inflammatory conditions

[0099] Anti-inflammatory peptides and derivative compounds in the anti-inflammatory PAR3 source and PAR1 source described herein, including variants, analogs and peptide mimetics, can be used in many treatments or preventive applications to inhibit or suppress undesirable inflammation, and to treat many inflammatory diseases or conditions. In some embodiments, the anti-inflammatory peptide or polypeptide or derivative compound in the PAR3 source described herein is administered to the subject in need of treatment in a therapeutically effective amount. In some embodiments, the anti-inflammatory peptide or polypeptide or derivative compound in the PAR1 source described herein is administered to the subject in need of treatment in a therapeutically effective amount. In some other embodiments, the anti-inflammatory peptide or polypeptide or derivative compound in the PAR3 source described herein and the anti-inflammatory peptide or polypeptide or derivative compound in the PAR3 source described herein are administered to the subject in need of treatment. As described in detail herein, the combination of the two compounds, even at very low doses, can also achieve synergy in suppressing or suppressing inflammation. As described in detail below, anti-inflammatory peptides (e.g., P3R or TR47) with or without homologous or heterologous multimerization (conjugation or fusion) can be formulated in pharmaceutical compositions for use in treatment or preventive applications disclosed herein.

[0100] In one aspect, the invention provides methods for inhibiting inflammation and treating an object suffering from an inflammatory disease. Anti-inflammatory peptides, variants and mimetics in PAR3 source and PAR1 source described herein can be used to inhibit or improve the symptom relevant to undesirable inflammation, and for treating various diseases or illnesses relevant to undesirable inflammatory response or mediated by undesirable inflammatory response. These include illnesses characterized by undesirable immune activation or undesirable immune response or related thereto, such as allograft rejection, autoimmune diseases (such as lupus and multiple sclerosis), allergic reactions (such as asthma), epidermal inflammatory diseases (eczema), rheumatoid arthritis, aseptic inflammation and hay fever. Specific diseases or conditions include, for example, graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, neuromyelitis optica (NMO), type I or type II diabetes and diseases associated therewith, vasculitis, pernicious anemia, Sjogren's syndrome, uveitis, psoriasis, Graves' ophthalmopathy, alopecia areata, etc.; allergic diseases, such as allergic asthma, atopic dermatitis, allergic rhinitis / conjunctivitis, allergic contact dermatitis; inflammatory diseases optionally with potential abnormal reactions, such as inflammatory bowel disease, Crohn's disease or ulcerative colitis, intrinsic asthma, inflammatory lung damage, inflammatory liver damage, inflammatory glomerular damage, atherosclerosis, osteoarthritis, irritant contact dermatitis and further eczematous dermatitis, seborrheic dermatitis, skin manifestations of immune-mediated diseases, inflammatory eye diseases, keratoconjunctivitis and acute respiratory distress syndrome. Conditions characterized by or associated with undesired immune activation or an undesired immune response also include viral infections, including, for example, infection with chikungunya virus, HIV-1, coronavirus, and influenza virus.

[0101] In various embodiments, the methods of the present invention are intended to treat inflammatory conditions and infectious diseases. Inflammatory diseases suitable for the prognostic methods of the present invention include any disease or condition associated with abnormal inflammation, and in particular, conditions mediated by or associated with type I IFN amplification pathways and cytokine storms. These include, for example, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivity, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, transplant rejection, and vasculitis.Examples of specific inflammatory disorders include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, acute respiratory distress syndrome (ARDS), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome immune deficiency syndrome, syndrome, CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, Crohn's disease, Dego's disease, dermatomyositis, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura thrombocytopenia purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus (type 1), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, sepsis, Sjögren's syndrome, stiff-man syndrome, systemic inflammatory response syndrome (SIRS), Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0102] In some embodiments, the undesirable inflammation or inflammatory condition to be treated is associated with, mediated by, or manifested by caspase 1 activation. In some embodiments, the undesirable inflammation or inflammatory condition to be treated is associated with, mediated by, or manifested by inflammasome formation. In some embodiments, the subject to be treated is a subject suffering from or suspected of suffering from a condition or disease that has been shown to be suitable for treatment with APCs. These include, for example, ischemia / reperfusion of the brain, heart, and kidneys; inflammation of the lungs, kidneys, and gastrointestinal tract; sepsis; Ebola virus; diabetes; and total lethal body radiation. See, e.g., Griffin et al., Blood 125:2898-2907, 2015.

[0103] In some embodiments, the method of treatment of the present invention includes administering a pharmaceutical composition to a subject, which comprises an anti-inflammatory peptide from a PAR3 source as described herein, such as P3R. In some embodiments, the method includes administering a pharmaceutical composition to a subject, which comprises an anti-inflammatory peptide from a PAR1 source as described herein, such as TR47. In some other embodiments, a combination of an anti-inflammatory peptide from a PAR3 source as described herein and an anti-inflammatory peptide from a PAR1 source as described herein is administered to a subject in need of treatment. In some of these embodiments, the anti-inflammatory peptide from a PAR3 source and the anti-inflammatory peptide from a PAR1 source are administered to the subject simultaneously. In some other embodiments, the two peptides may be administered to the subject sequentially. In a number of embodiments, the two peptides may each be independently conjugated to a carrier portion as described above. In some embodiments, the peptides from the PAR3 source and the PAR1 source administered simultaneously are conjugated to each other, such as by covalent peptide fusions. In these embodiments, the peptide from the PAR3 source may be fused at the N-terminus or C-terminus of the peptide from the PAR1 source. In some of these embodiments, the two peptides may be connected by a linker portion, such as a linker or a spacer peptide. In some embodiments, the two peptides in different ratios can be conjugated to a carrier moiety, such as a carrier protein, an Fc domain, or a PEG molecule as described above.

[0104] Preferably, the therapeutic methods of the present invention are intended to treat mammalian subjects. In some of these embodiments, the subject is a human patient. Some embodiments of the present invention relate to inhibiting or suppressing undesirable inflammation or inflammatory responses that underlie or are associated with any inflammatory disease or condition described herein. Some embodiments of the present invention relate to treating a human subject suffering from or suspected of suffering from an autoimmune disease. Examples of autoimmune diseases and related diseases suitable for use in the methods of the invention include, for example, acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, Behcet's disease, celiac disease, Chagas disease, chronic obstructive pulmonary disease, Crohn's disease, dermatomyositis, type 1 diabetes, diabetes-related inflammation, endometriosis, Goodpasture's syndrome, Graves' disease, graft versus host disease, and disease (GVHD), Guillain-Barré syndrome (GBS), Hashimoto's disease, hidradenitis suppurativa, Kawasaki disease, idiopathic thrombocytopenic purpura, interstitial cystitis, lupus erythematosus, mixed connective tissue disease, morphea, multiple sclerosis (MS), myasthenia gravis, narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, rheumatoid arthritis, schizophrenia, scleroderma, Sjögren's syndrome, stiff-man syndrome, temporal arteritis (also known as "giant cell arteritis"), ulcerative colitis, vasculitis, vitiligo, microscopic polyangiitis, glomerulonephritis, and Wegener's granulomatosis.

[0105] In some embodiments, the subject to be treated by the methods of the present invention is a subject suffering from or suspected of suffering from neuroinflammation. Subjects suffering from acute neuroinflammation or chronic neuroinflammation are suitable for treatment. Neuroinflammation is known to cause acute and chronic neuropathological conditions. These include stroke, post-traumatic brain injury, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, frontotemporal dementia, Lewy body dementia, cerebral malaria and prion disease. Other central nervous system pathologies with excessive inflammation include CNS infectious diseases, such as Zika virus, HIV and West Nile virus, coronavirus infection and bacterial infection causing meningitis. See, e.g., Voet et al., EMBO Mol. Med. 11: e10248, 2019; Heneka et al., Nat. Rev. Neurosci. 19: 610-621, 2018; Ismael et al., Sci. Rep. 8: 5971, 2018. Treatment with the methods of the present invention will be beneficial in curing or improving the symptoms of a variety of neuropathological conditions associated with neuroinflammation. By substantially suppressing or inhibiting the underlying inflammation in the listed conditions, subjects suffering from any of these neuropathological conditions can obtain significant benefits from treatment using the methods of the present invention. In some other embodiments, the subject to be treated with the methods of the present invention is a subject suffering from malarial inflammation or at risk of developing malarial inflammation. Malaria is caused by infection of malarial parasite erythrocytes and is a highly inflammatory disease with characteristic periodic fever caused by the synchronous rupture of infected erythrocytes to release daughter parasites. By substantially suppressing or inhibiting the underlying inflammation, subjects suffering from malaria can obtain great benefits from treatment using the methods of the present invention.

[0106] VI. Screening methods for identifying anti-inflammatory agents

[0107] The anti-inflammatory peptides and in vitro assays in PAR3 sources and / or PAR1 sources described herein can be used to identify new agents with anti-inflammatory activity. In some embodiments, the invention provides methods for screening candidate agents for anti-inflammatory activity using exemplary peptides (e.g., P3R and / or TR47) as positive controls. Any known screening method or platform for identifying anti-inflammatory agents can be applied to incorporate the anti-inflammatory peptides described herein as positive controls. These include any animal model or other in vivo platform, in vitro screening method, and in vitro screening method. Such methods are described in the art, for example, delPalacio et al., J. Biomole. Screening 21:567-578, 2016; Jiang et al., PLoS One. 9:e96214, 2014; Hall et al., Disease Models & Mechanisms 7:1069-1081, 2014; Phanse et al., J. App. Pharm. Sci. 2:19-33, 2012; Giddings and Maitra, J. Biomole. Screening 15:1204-1210, 2010; Singh et al., Clinical Chemistry 51:2252-2256, 2005; Maurer et al., J. Anal. Toxicol. 25:237-44, 2001; Alener & Bingoul, Intl. J. Crude Drug Res.26:197-207,1988; Famaey and Whitehouse, Biochem. Pharmacol. 24: 1609-1616, 1975; VanArman, Clin. Pharmacol. Ther. 16: 900-904, 1974; Carrano et al., J. Pharm. Sci. 61: 1450-1454, 1972; Riesterer et al., Agents and Action 2, 27-32, 1971; Winter et al., Fed. Proc. 23, 284, 1964; and Winter and Portar, J. Amer. Pharm. Sci. Ed. 46:515-519, 1957.

[0108] Some screening methods of the present invention involve using in vitro cultured cell lines to monitor inflammation-related activity. In some of these embodiments, the inflammation-related activity to be monitored involves the assembly and activation of inflammasomes and the inflammasome-dependent activation of caspase 1, such as the enzymatic activity of caspase 1 or the secretion of IL-1β. Inflammasomes are large intracellular multiprotein complexes that play an indispensable role in inflammation-driven disease states. Inflammasomes may be involved in the activation of caspase 1, which is known to catalyze the maturation and processing of interleukin (IL)-1β and pro-IL-1β. In the Cannabinoid for Atherosclerotic Disease (CANTOS) trial, neutralization of IL-1β by monoclonal antibodies (see, e.g., Ridker et al., N. Engl. J. Med. 377: 1119-1131, 2017) has been shown to reduce the risk of atherosclerotic thrombosis without changes in lipid levels, and is strong evidence pointing to a wide range of inflammation, especially the pathological role of IL-1β in cardiovascular disease. Inflammasomes play a protective role in host defense, but activation can also lead to ischemia-reperfusion injury (I / RI) and autoinflammation. In mouse models, APCs have been shown to limit inflammasome activation following myocardial I / RI, demonstrating that APCs have an anti-inflammatory role in the inflammasome process.

[0109] In some embodiments, cultured human THP-1 cell lines are used in the screening methods of the present invention.THP-1 cells are human monocytic cell lines that can differentiate into macrophages.As shown herein, the use of selected agonists in THP-1 cells is an effective in vitro means of inducing inflammasomes and / or caspase I activity and ultimately inducing inflammation.These screening methods of the present invention require inducing inflammasomes and / or caspase I-dependent inflammation in THP-1 cells by inflammatory inducing agents (e.g., LPS exemplified herein), and evaluating the potential anti-inflammatory activity of candidate agents for exemplary anti-inflammatory peptides (e.g., P3R or TR47).If there is similar or stronger activity in inhibiting inflammation, as demonstrated by inflammation-related activity or a phenotype (e.g., caspase 1 activity or IL-1β secretion) showing inflammasome activation and / or caspase 1 activity, it can be identified as a new anti-inflammatory agent.

[0110] Candidate agents that can be used for screening methods of the present invention can have any chemical properties. These include for example, peptides include polypeptides, beta-turn mimetics (beta-turn mimetic), polysaccharides, phospholipids, hormones, prostaglandins, steroids, purines, pyrimidines, oligomeric N-substituted glycine, oligocarbamates, carbohydrates, fatty acids, and derivatives, structural analogs or combinations thereof. In some embodiments, the candidate agent for screening methods of the present invention is a small molecule organic compound. In some of these embodiments, the combinatorial library of small molecule candidate agents can be used to screen therapeutic agents. Such compound libraries are well known in the art, for example, as described in Schultz et al., Bioorg. Med. Chem. Lett. 8:2409-2414, 1988; Weller et al., Mol Divers. 3:61-70, 1997; Fernandes et al., Curr. Opin. Chem. Biol. 2:597-603, 1998; and Sittampalam et al., Curr. Opin. Chem. Biol. 1:384-91, 1997. Candidate agents include acyclic and unbranched small organic molecules, as well as other organic compounds such as aromatic compounds, heterocyclic compounds, and benzodiazepines. kind.

[0111] In some embodiments, the candidate agent to be screened in the method of the present invention is a peptide or polypeptide, variant, analog, peptide mimetic or other related or derivative compound of the PAR3 source or PAR1 source described herein. In some of these embodiments, the candidate agent screened by the method of the present invention can be a peptide or polypeptide that simulates the Met 1 -Arg 41 Deletion of the extracellular domain of human PAR3 (SEQ ID NO: 3) or Met 1 -Arg 46 Variants, derivatives or mimetic compounds of peptides or polypeptides of the N-terminal sequence of the deleted human PAR1 extracellular domain (SEQ ID NO:6). For example, the candidate agent can be a polypeptide derived from SEQ ID NO:3 or SEQ ID NO:6 with multiple C-terminal or internal deletions. In some embodiments, the P3R peptide (SEQ ID NO:4) or the TR47 peptide (SEQ ID NO:7) can be used as a scaffold to produce a library of variant or analog peptides and peptide mimetics. Candidate agent libraries based on reference peptides or polypeptides (e.g., P3R peptides) can be easily produced using conventional practice methods as described herein. Relative to a reference peptide (e.g., P3R or TR47) or polypeptide (e.g., SEQ ID NO:3 or 6), the candidate agent can also be a library of variant peptides or polypeptides comprising one or more amino acid substitutions.

[0112] Methods for preparing libraries containing different populations of peptides, peptoids and peptidomimetics are well known in the art, and a variety of libraries are commercially available. See, for example, Ecker and Crooke, Biotechnology 13: 351-360, 1995; and Blondelle et al., Trends Anal. Chem. 14: 83-92, 1995; and references cited therein. See also Goodman and Ro, Peptidomimetics for Drug Design, in "Burger's Medicinal Chemistry and Drug Discovery" Vol. 1 (ed. ME Wolff; John Wiley & Sons 1995), pp. 803-861; and Gordon et al., J. Med. Chem. 37: 1385-1401 (1994). It is understood by those skilled in the art that peptides can be produced directly in vitro or can be expressed by nucleic acids that can be produced in vitro. Peptide molecule libraries can also be produced, for example, by constructing cDNA expression libraries from mRNA collected from target tissues. Methods for generating such libraries are well known in the art (see, e.g., Sambrook et al., Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989)).

[0113] Design peptide derivatives and mimetics and the method for screening functional peptide mimetics are well known to those skilled in the art.A basic method for designing a molecule simulating a known protein or peptide is to first identify the active region of a known protein (for example, in the case of antibody-antigen interaction, identify which or which regions of the antibody allow the combination with the antigen), and then look for a mimetics simulating the active region.Although the active region of a known polypeptide is relatively small, it is expected that mimetics will be smaller than protein (for example, in terms of molecular weight), and correspondingly easier and cheaper to synthesize and / or have benefits with respect to stability or other favorable pharmacokinetics.Such mimetics can be used as a convenient substitute for reference polypeptides (for example, SEQ ID NO:3) or peptides (for example, P3R), as a reagent interacting with a target molecule (for example, PAR3).For example, Reineke et al. (Nat.Biotech.17; 271-275,1999) designed a mimetic molecule, which uses a large short synthetic peptide library to simulate the binding site of interleukin 10 protein, wherein each peptide corresponds to a short segment of interleukin 10. Each of these peptides is then tested individually for binding to the target (in this case an antibody against interleukin 10) by assay techniques to identify potentially relevant peptides. Phage display libraries of peptides and alanine scanning methods can be used.

[0114] Other methods for designing peptide mimetics of specific peptides or proteins include those described in: European Patent EP1206494, Goede et al., BMC Bioinformatics, 7:11, 2006 SuperMimic program; and Campbell et al., Microbiol.and Immunol.46:211-215, 2002 MIMETIC program. The SuperMimic program is intended to identify compounds that mimic parts of proteins, or positions in proteins suitable for inserting mimics. The library provided by the application contains peptide mimic building blocks on the one hand, and protein structures on the other hand. Finding promising peptide mimetic joints for a given peptide is based on the superposition of several conformers of the peptide and the mimic. New synthetic elements or proteins can be introduced and used for retrieval. W. Campbell et al., 2002 taught the MIMETIC computer program, which generates a series of peptides that interact with the target peptide sequence. An in-depth discussion of this topic is reviewed by James R. Damewood Jr. in "Peptide Mimetic Design with the Aid of Computational Chemistry," in Reviews in Computational Chemistry, Vol. 9, January 2007, ed.: Kenny B. Lipkowitz, Donald B. Boyd (John Wiley & Sons, Inc.), and in Tselios et al., Amino Acids, 14:333-341, 1998.

[0115] Once a candidate agent library is prepared, these candidate agents can be easily screened for optimization or improved anti-inflammatory activity relative to the activity of a reference polypeptide. Candidate agents can be screened to improve any anti-inflammatory activity of a reference polypeptide disclosed herein, such as inhibiting or suppressing the secretion of caspase 1 enzyme activity or IL-1 β (see Examples below). Variants or analog compounds based on anti-inflammatory peptides (e.g., P3R or TR47) in reference PAR3 sources or PAR1 sources optimized with such screening methods can be used for a variety of therapeutic applications described herein.

[0116] VII. Therapeutic Composition and Dosage

[0117] The present invention provides treatments or pharmaceutical compositions for implementing the treatment or prevention methods described herein. Anti-inflammatory peptides from PAR3 sources and / or PAR1 sources described herein and related compounds with anti-inflammatory activity (e.g., variants, derivatives and mimetics) and other therapeutic agents disclosed herein can be directly applied to objects in need of treatment. However, these therapeutic compounds are preferably applied to objects in the form of pharmaceutical compositions, which contain peptides, variants or mimetics and / or other active agents and pharmaceutically acceptable carriers, diluents or excipients in unit dosage forms. Therefore, the present invention provides medicines or therapeutic compositions comprising one or more anti-inflammatory peptides or derivative compounds disclosed herein. The present invention also provides the use of these peptides or related compounds in the preparation of pharmaceutical compositions or medicines for treating or preventing the above-mentioned diseases or medical conditions mediated by or associated with undesirable inflammation.

[0118] Some compositions of the present invention include an anti-inflammatory peptide or related compound (e.g., variant, derivative and mimetic) in a PAR3 source as described herein. Some of these compositions are pharmaceutical compositions, which include an anti-inflammatory peptide (e.g., P3R) or a derivative compound in a PAR3 source as described herein, and a pharmaceutically acceptable carrier, for treatment of an effective amount or dosage. Some compositions of the present invention include an anti-inflammatory peptide or related compound in a PAR1 source as described herein, for treatment of an effective amount or dosage. Some of these compositions are pharmaceutical compositions, which include an anti-inflammatory peptide (e.g., TR47) or a derivative compound in a PAR1 source as described herein, and a pharmaceutically acceptable carrier, for treatment of an effective amount or dosage.

[0119] Other compositions of the present invention also include anti-inflammatory peptides or related compounds in PAR3 source and PAR1 source as described herein. Some of these compositions are pharmaceutical compositions, which include anti-inflammatory peptides in PAR3 source and PAR1 source as described herein or related or derived compounds as described herein, and pharmaceutically acceptable carriers. As described above, anti-inflammatory peptides in PAR3 source and PAR1 source can be provided as single peptides in the composition, in monomer or polymer form. Alternatively, peptides can be conjugated to each other as heterodimers or heteromultimers in the composition as described above. In a number of embodiments, monomers, heterologous or homodimers and heterologous or homomultimer peptides in the composition can be further conjugated to one or more carrier moieties described herein by covalent or non-covalent bonding. In some compositions, peptides or related compounds in PAR3 source and peptides or related compounds in PAR1 source can be conjugated to carrier moieties with different ratios as described herein.

[0120] Pharmaceutically acceptable carriers are not undesirable agents in biology or other aspects. These agents can be applied to a subject together with peptides from PAR3 sources and / or PAR1 sources without causing any undesirable biological effects or interacting with any component of the pharmaceutical composition in a harmful manner. The composition may additionally contain other therapeutic agents suitable for treating or preventing undesirable inflammation or inflammatory diseases. Pharmaceutical carriers enhance or stabilize the composition or promote the preparation of the composition. Pharmaceutically acceptable carriers include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, etc. The pharmaceutically acceptable carriers used should be suitable for the various routes of administration described herein. The art provides additional guidance for selecting suitable pharmaceutically acceptable carriers, such as Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th edition, 2000. In addition to anti-inflammatory peptides and pharmaceutically acceptable carriers, the pharmaceutical composition of the present invention may also include other active or inactive agents for suppressing information and known drugs for treating specific inflammatory diseases. For example, the pharmaceutical composition may include known anti-inflammatory agents and analgesics. Examples of such agents include glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs), acetaminophen, opioids, diproqualone, and topical lidocaine.

[0121] The pharmaceutical composition comprising the anti-inflammatory peptide or derivative compound and / or other therapeutic agent in the PAR3 source and / or PAR1 source described herein can be applied by various methods known in the art, such as oral administration. The route of administration and / or mode vary according to the desired result. According to the route of administration, the active therapeutic agent can be coated in the material to protect the compound from the effects of other natural conditions that can inactivate the agent, such as acid and medicament. Conventional pharmaceutical practice can be used to provide a suitable preparation so that such a composition is applied to the object. Any suitable route of administration can be used. These include but are not limited to oral, intravenous, parenteral, transdermal, subcutaneous, intraperitoneal, intramuscular, intracranial, intraorbital, intraventricular, intrapulmonary, intracapsular and intraspinal administration. According to the specific circumstances of the object to be treated, the systemic or local delivery of the therapeutic agent can be used for treatment.

[0122] When administered to a subject in vivo, a pharmaceutical composition typically comprises a therapeutically effective amount or dose of an active anti-inflammatory compound. A therapeutically effective amount is the total amount of the anti-inflammatory compound that achieves the desired anti-inflammatory effect. The effective dose or dose varies according to many different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other drugs administered, and whether the treatment is preventive or therapeutic. Typically, the patient is a human, but non-human mammals may also be treated. The therapeutic dose needs to be adjusted to optimize safety and efficacy. As a general guide, the dose is about 0.0001 to 100 mg / kg of the host's body weight, and more typically 0.01 to 5 mg / kg. For example, the dose can be 1 mg / kg body weight or 10 mg / kg body weight or in the range of 1 to 10 mg / kg.

[0123] In some embodiments, each peptide or derivative compound in the composition is provided at a concentration of at least 10nM, 25nM, 50nM, 100nM, 250nM, 500nM, 1μM, 2.5μM, 5μM, 10μM, 25μM or higher. Depending on the circumstances, each administration to the object may include a dose of about 0.5ml, 1ml, 2.5ml, 5ml, 10ml, 25ml, 50ml, 100ml or more of the composition. In some embodiments, the dose of each administered peptide or derivative compound can be about 10ng to 1g, 100ng to 100mg, 1μg to 10mg or 30 to 300μg for each patient. In some embodiments, the dose of each administered anti-inflammatory peptide or derivative is adjusted to reach a plasma concentration of 1 to 1000μg / ml, and in some embodiments 25 to 300μg / ml. In some embodiments, the dose of each anti-inflammatory peptide or derivative administered can be adjusted to achieve a plasma concentration of about 0.01 μg / ml to about 1.6 μg / ml, preferably about 0.01 μg / ml to about 0.5 μg / ml. It is also within the technical scope of the art to start the dose at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. It is also within the technical scope of the art to determine the optimal concentration of the variant to achieve the desired effect in the in vitro and ex vivo preparations of the present invention. Based on the initial assay results, the optimal concentration may be, for example, in the range of about 1 to 1,000 nM or about 1 to 200 μM, depending on the general properties of the compound.

[0124] Exemplary treatment regimens include administration once a day, once every other day, once a week, once every two weeks, once a month, or once every 3 to 6 months. In some methods, two or more anti-inflammatory peptides (e.g., P3R and TR47) or their corresponding derivatives are administered simultaneously, in which case the dosage of each therapeutic peptide administered falls within the range shown. Pharmaceutical compositions are typically administered multiple times. As described above, the interval between single doses can be weekly, monthly, or even annual. As indicated by the blood levels of anti-inflammatory peptides or derivative compounds comprising peptides (e.g., a complex comprising a peptide and a carrier portion) measured in patients, the intervals can also be irregular. Alternatively, peptides from PAR3 sources and / or PAR1 sources can be administered as sustained release formulations, in which case less frequency of administration is required. Dosage and frequency of administration can also vary according to the half-life of the administered compound in the patient. The dosage and frequency of administration can vary according to whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment throughout their lives. In therapeutic applications, it is sometimes necessary to administer a relatively high dose at relatively short intervals until the progression of the disease is reduced or terminated, and preferably until the patient exhibits partial or complete improvement in disease symptoms. Thereafter, a preventive regimen may be administered to the patient.

[0125] Some methods of the present invention relate to the combination of the anti-inflammatory peptide in the anti-inflammatory peptide in the PAR3 source and the PAR1 source or its corresponding derivative compound described herein. As exemplified by peptide P3R and TR47 herein, the extremely low dose of one of the two peptides is enough to achieve synergy. Therefore, in some embodiments, in the pharmaceutical composition, the two peptides are each provided with a concentration of at most about 500nM, 250nM, 50nM, 25nM, 10nM, 5nM, 2.5nM, 1nM or lower. In some embodiments, the use of the pharmaceutical composition is that the two peptides each provide a daily dose of at most about 0.25mg / kg, 0.1mg / kg, 0.05mg / kg, 0.025mg / kg, 0.01mg / kg, 0.005mg / kg, 0.0025mg / kg, 0.001mg / kg, 0.0005mg / kg, 0.00025mg / kg or lower of the body weight of the host. According to the average body weight of the object (human or non-human animal, adult or teenager), the composition of appropriate amount (volume) is applied to the object to reach the total daily dose of expectation described herein. Therefore, for example, the average body weight of 75 kg can be used to calculate the total amount of peptides used each day for adult objects or animals with similar body weight. Similarly, the average body weight of 60 kg, 50 kg, 40 kg, 30 kg, 20 kg, 15 kg, 10 kg, 7.5 kg, 5 kg or less can be used to calculate the total amount of peptides used each day for non-adults (e.g., 15 to 18, 11 to 14, 7 to 10, 4 to 6, 2 to 3, 1 to 2 or younger age groups) or animals of different age groups with similar body weight.

[0126] In some other embodiments, the pharmaceutical composition may include a peptide (e.g., a peptide derived from PAR3) or a derivative compound at a dose much higher than other peptides (e.g., peptides derived from PAR1) or derivative compounds. For example, the composition to be administered to a subject may include a peptide (e.g., P3R or P3R variants or derivatives) at a concentration of at least about 10 nM, 25 nM, 50 nM, 100 nM, 250 nM, 500 nM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM or higher. The concentration of other peptides (e.g., TR47 or TR47 variants or derivatives) in such a composition may be at most about 500 nM, 250 nM, 50 nM, 25 nM, 10 nM, 5 nM, 2.5 nM, 1 nM or lower. In some of these embodiments, the dosage of one of the two peptides to be administered to the subject can be at least about 0.01 mg / kg, 0.025 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 25 mg / kg or higher daily amount of the host body weight. The dosage of another peptide in the pharmaceutical composition of these embodiments can be at most about 0.25 mg / kg, 0.1 mg / kg, 0.05 mg / kg, 0.025 mg / kg, 0.01 mg / kg, 0.005 mg / kg, 0.0025 mg / kg, 0.001 mg / kg, 0.0005 mg / kg, 0.00025 mg / kg or lower daily amount of the host body weight. Similarly, the total amount of peptide in the composition for these embodiments can be determined according to the average body weight of the object. For example, an average body weight of 75 kg can be used to calculate the total amount of peptides used each day for adult subjects or animals with similar body weights. Similarly, an average body weight of 60 kg, 50 kg, 40 kg, 30 kg, 20 kg, 15 kg, 10 kg, 7.5 kg, 5 kg or less can be used to calculate the total amount of peptides used each day for non-adults (e.g., 15 to 18, 11 to 14, 7 to 10, 4 to 6, 2 to 3, 1 to 2 or younger age groups) or animals of different age groups with similar body weights.

[0127] The pharmaceutical compositions of the present invention can be prepared according to methods well known and routinely practiced in the art. See, for example, Goodman & Gilman's The Pharmacological Bases of Therapeutics, Hardman et al., eds., McGraw-Hill Professional (10 th·ed., 2001); Remington: The Science and Practice of Pharmacy, Gennaro, ed., Lippincott Williams&Wilkins (20 th ·ed., 2003); Pharmaceutical Dosage Forms and Drug Delivery Systems, Ansel et al (editor), Lippincott Williams&Wilkins (7 th · ed., 1999); and Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. The pharmaceutical composition is preferably prepared under GMP conditions.

[0128] Example

[0129] The following examples are provided to further illustrate the present invention but not to limit its scope.Other variations of the present invention will be apparent to those of ordinary skill in the art and are encompassed by the appended claims.

[0130] Example 1 Synergistic anti-inflammatory activity of PAR1-derived and PAR3-derived peptides

[0131] We first observed that APC treatment reduced caspase 1 activity in human PMA-differentiated THP1-null cells treated with LPS and ATP ( Figure 1 APC exhibits biased signaling by cleaving PAR1 at the atypical residue Arg46, and a 20-amino acid peptide derived from PAR1, TR47, that mimics the novel N-terminus derived from Arg46 cleavage, induces protective signaling in endothelial cells in vitro and reduces vascular leakage in vivo. 50 μM TR47 was initially tested based on previously published in vivo data, and we found that 50 μM TR47 and lower concentrations similarly reduced caspase 1 activity, whereas scrambled TR47 and the peptides SFLLRN or TFLLRN, which resemble the novel N-terminal amino acid sequence generated by PAR1 cleavage at Arg41 by thrombin, had no effect on caspase 1 activity ( Figure 1A). Protective signaling by APC can follow its cleavage of PAR3 at Arg41 on endothelial cells, and the PAR3-derived peptide P3R exerts protection in vivo and reduces neutrophil extracellular trap formation in vitro. Similar to APC and TR47, the P3R peptide reduces caspase 1 activity. The control peptide P3K, which represents a PAR3 peptide cleaved by thrombin at Lys38, did not significantly alter caspase 1 activity in THP1 cells ( Figure 1 B).

[0132] We hypothesized that although low concentrations of either TR47 or P3R were insufficient to exert anti-inflammatory activity by reducing caspase 1 activity, the combination of these two GPCR agonists could synergistically reduce caspase 1 activity. When <10 nM TR47 was incubated with THP1 cells in the presence of 500 nM P3R followed by induction of caspase 1 activity, a reduction in caspase 1 activity was observed, which was in contrast to the effect of TR47 in the absence of P3R after normalization to caspase 1 inhibition conditions ( Figure 1 C and 1D). In addition, when P3R at concentrations ≤ 500 nM was incubated with 1 nM TR47, a significant decrease in normalized caspase 1 activity was observed, in contrast to P3R in the absence of TR47 ( Figure 1 E). Whether intracellular signaling initiated by APC, TR47, P3R or a combination of peptides is similar or conserved in multiple human cell types remains to be resolved. The anti-inflammatory synergy of TR47 and P3R suggests that PAR1 and PAR3 may exist as a heterodimeric GPCR complex in an interactome that promotes anti-inflammatory intracellular signaling. It is easy to imagine that linking these two GPCR agonist peptides may have therapeutic prospects in selected inflammatory diseases.

[0133] Additional studies were performed to further confirm the observed synergistic activity. In these studies, additional variants of the PAR peptide that had been prepared and shown to retain their anti-inflammatory activity ( Figure 4 , 5 and 7A) were tested for synergy in maintaining anti-inflammatory activity ( Figure 7 ), as shown by the combination of PAR1-derived TR47 and PAR3-derived P3R ( Figure 1 D to E). For an example of such a study, consider the PAR3 peptide P3(51-65) (also referred to herein as peptide "P3R51-65"), which alone has anti-inflammatory effects at relatively high concentrations (e.g., 50 μM). When it was tested at concentrations of 0 to 16 nM in the presence or absence of 1 nM peptide P1(47-66) (referred to herein as peptide "TR47"), a significant reduction in caspase 1 activity was seen only in the presence of 1 nM P1(47-66) ( Figure 7 D), as observed for peptide P3(42-65) (referred to herein as peptide "P3R") ( Figure 1 E). These findings ( Figure 7 B to D) demonstrate that mutation of PAR3 peptide ligands from the N-terminus or altering sequence or length can result in a cooperative, synergistic anti-inflammatory response.

[0134] Figure 7 The data in B to D show that the combination of different PAR1-derived and PAR3-derived peptides provides enhanced anti-inflammatory activity compared to the anti-inflammatory activity of any single peptide alone for a given combination studied. This emphasizes the value of a combination of multiple different PAR1-derived and PAR3-derived peptides relative to each peptide alone for biological efficacy. These data indicate that changes can be made to the sequence or length of the PAR peptides and still produce a collaborative, synergistic anti-inflammatory response ( Figure 1 D to E and Figure 7 B to D).

[0135] Example 2 Mechanisms of anti-inflammatory activity of PAR1- and PAR3-derived peptides

[0136] NLRP3 is the major canonical sensor protein for inflammasome formation and, notably, mutations in NLRP3 lead to chronic autoinflammatory diseases in humans. 23 Inhibition of NLRP3 in NLRP3-deficient (defNLRP3) THP1 cells or in THP1-null cells using the NLRP3 inhibitor MCC950 resulted in a significant decrease in caspase 1 activity in all treatments tested ( Figure 2 A and 2B). These data indicate that NLRP3 is required for the generation of caspase 1 activity that is inhibited by APC, TR47, and P3R.

[0137] EPCR is an important APC-bound cellular receptor required for the atypical, biased signaling induced by APC. Blocking EPCR with the anti-EPCR monoclonal antibody RCR-252 prior to APC or TR47 treatment resulted in a significant loss of its inhibition of caspase 1 activity, whereas P3R studies showed a trend but did not reach significance ( Figure 2 C) To test whether PAR3 contributes to the anti-inflammatory effects of APC, TR47 and P3R, PAR3 was inhibited using a blocking antibody prior to treatment. 16,26 Blockade of PAR3 very significantly reduced the anti-inflammatory activity of APC and P3R and, to a lesser extent, the activity of TR47 ( Figure 2D). APC-initiated signaling through PAR1 is widely considered to be critical for beneficial effects in many cell types. Inhibition of PAR1 using blocking monoclonal antibodies WEDE15 and ATAP2 or the PAR1 small molecule antagonist SCH79797 (SCH) 17 Significantly inhibited the reduction of caspase 1 activity by APC ( Figure 2 E). The action of TR47 was inhibited by SCH79797 and WEDE15, but not by ATAP2; however, the WEDE15 epitope partially contains the sequence of TR47. None of the PAR1-targeted inhibitors statistically significantly reduced the anti-inflammatory activity of the PAR3 peptide agonist P3R ( Figure 2 E and 2F).

[0138] IL-1β is a key cytokine produced by the NLRP3 inflammasome. APC, TR47, P3R, and the combination of TR47 and P3R significantly inhibited the production and release of IL-1β by THP1 cells subjected to the above-mentioned protocol for caspase 1 activity ( Figure 3 ). This confirms that these agents are anti-inflammatory based on reductions in two widely recognized biomarkers of inflammation.

[0139] Example 3 Structural requirements for the anti-inflammatory activity of PAR1- and PAR3-derived peptides

[0140] To determine the relationship between peptide sequence and activity, we next sought to test peptides of varying lengths starting at either the N-terminus or the C-terminus. First, we tested P3R peptide variants starting with the previously described 13-mer P3Rmed (P3Rm). 16 At 50 μM, a significant reduction in caspase 1 activity was observed similar to the 24-mer P3R (residues 42 to 65; 24-mer), and this effect was lost at 2 μM ( Figure 4 A to C). Subsequently, the peptide was shortened from the N-terminus, and both P3R 51-65 or 51-59 showed similar reduction in caspase 1 activity at 50 μM that was lost at 2 μM ( Figure 4 Further shortening of the peptide by 3 or 8 amino acids (P3R 54-59 or 59-65, respectively) only partially reduced caspase-1 activity at 50 μM ( Figure 4 A), indicating that the Phe, Pro, Phe sequence may be crucial for anti-inflammatory activity ( Figure 4 C), and the cryptic N-terminus of PAR3 after APC-mediated cleavage may not be the critical signaling part, which potentially challenges the paradigm of PAR (at least for PAR3) activation and signaling.

[0141] Next, we sought to define sequences that are critical for mediating TR47-mediated reduction of caspase 1 activity. Our preliminary experiments showed that the previously published 9-mer of TR47 (NPNDKYEPF; SEQ ID NO: 36) reduced caspase 1 activity at 50 μM, similar to the 20-mer TR47 ( Figure 5 A). In contrast, when the N-terminus of TR47 was acylated, no reduction in caspase-1 activity was observed at equimolar concentrations, indicating that the free amine N-terminus may be critical for the anti-inflammatory activity mediated by TR47. Next, we tested additional C-terminally shortened TR47 peptides, including the previously published 8-mer TR47 (NPNDKYEP; SEQ ID NO: 35), which similarly to the 16-mer TR47 significantly reduced caspase-1 activity, while the 6-mer TR47 lost its inhibitory activity, indicating that peptides longer than 6 amino acids are required for the anti-inflammatory activity of PAR1 ( Figure 5 B). Next, we sought to test the paradigm that the N-terminal Asn(N) is required for the anti-inflammatory activity of TR47. Loss of 1 amino acid from the N-terminus (TR47 N-1) still reduced caspase 1 activity, whereas removal of 2 amino acids (TR47 N-2) resulted in loss of inhibition of caspase 1 activity. Surprisingly, reduction in caspase 1 activity was observed with removal of 5 amino acids from the N-terminus (TR47 N-5), suggesting that the cryptic N-terminus of PAR1 is not solely responsible for the beneficial signaling effects following APC-mediated cleavage ( Figure 5 B). Next, point mutations at the N-terminal residue 47 revealed that the charge at the N-terminus had different effects on TR47-mediated reduction of caspase 1 activity. Mutations of N→Q (Asn to Gln, one carbon-carbon bond longer on the side chain) or N→A in TR47 produced peptides with similar reduced caspase 1 activity. However, mutation of residue 47 to N→D (Asn to Asp) resulted in loss of caspase 1 activity reduction ( Figure 5 B). Finally, the truncated TR peptide TR48-54, which removed both an amino acid from the N-terminus and part of the C-terminus, did not reduce caspase-1 activity, indicating that the peptide has a minimum length requirement at both ends and that the full anti-inflammatory effect observed for TR47 may require different recognition internal sequences ( Figure 5 B to C).

[0142] The variant peptides were also tested for their synergistic anti-inflammatory activity. Some exemplary results are given in Figure 6 These include a combination of the PAR1-derived peptide TR47 and the PAR3-derived peptide P3R variant P3Rm ( Figure 6 A) and the synergy observed in the case of the combination of the PAR1-derived peptide TR47 variant TR47(N-1) and the PAR3-derived peptide P3R ( Figure 6 B).

[0143] Example 4 Anti-inflammatory activity of PAR1 peptide / PAR3 peptide conjugates

[0144] This example describes studies showing that covalent linkage of PAR1 and PAR3 peptides improves anti-inflammatory activity.

[0145] PAR19 polymer peptide P1 (47-55) (residues 47 to 55; SEQ ID NO: 36) and PAR3 peptide P3 (51-65) (SEQ ID NO: 9) were used, each of which alone has an anti-inflammatory effect at relatively high concentrations. In order to demonstrate the anti-inflammatory efficacy of covalently linked PAR1-derived peptides and PAR3-derived peptides, a PAR1 / PAR3 fusion peptide ("G10") NPNDKYEPFGGGGGGGGGGGFPFSALEGWTGATIT (SEQ ID NO: 61) was generated by covalently linking with a linker consisting of 10 glycine residues. The anti-inflammatory activity of the fusion peptide and its two component peptides was then tested.

[0146] At the tested concentrations (0 to 500 nM), in the presence of the "G10" peptide, a significant reduction in caspase 1 activity was observed at 2 nM to 20 nM, whereas neither the isolated PAR1-derived peptide nor the isolated PAR3-derived peptide had any anti-inflammatory effect at 200 nM ( Figure 8 As expected, at relatively high concentrations, the P1(47-55) peptide alone had an anti-inflammatory effect at 500 nM ( Figure 8 ).

[0147] The results further highlight the synergistic activity of the PAR1 and PAR3 agonist peptides. While each of the two peptides independently has anti-inflammatory effects, the combination is more effective than either peptide alone.

[0148] Example 5 PAR1 / PAR3 fusion peptide promotes endothelial barrier stability

[0149] This example describes studies showing that PAR1-derived, PAR3-derived, and covalently linked PAR1 / PAR3 fusion ("G10") peptides inhibit thrombin-induced endothelial barrier disruption.

[0150] The destruction of endothelial barrier integrity caused by thrombin was monitored using a determination based on transendothelial resistance (TER). The anti-inflammatory, vascular protective effects of different PAR peptides on cultured endothelial cells (EA.hy926) to prevent thrombin-induced vascular leakage were tracked in real time using iCELLigence machines (ACEA Biosciences Inc, SanDiego, CA). EA.hy926 cells were pipetted into the holes of gold-plated microelectrode sensors (L8, Acea Biosciences) at a density of 10,000 / well. Subsequently, as cells grow and spread on microelectrodes, the increase in resistance was monitored to obtain the convergence (reaching a platform) on the TER plate of the recording instrument connected to the real-time detection of TER changes in each hole. When each peptide dilution was added 30 minutes before adding thrombin (ultimately 0.25nM in all wells), the effect of the dose response inhibition of different PAR-derived peptides on thrombin action at a specified concentration was tested. The percentage of inhibition was directly proportional to the increase in TER signal over time. Cell index values ​​reflecting TER signals were normalized before adding thrombin according to the manufacturer's instructions.

[0151] The results of the study are Fig. 9 The results show that the heterobivalent PAR1:PAR3 agonist peptide G10 exhibits remarkable potency in its ability to stabilize endothelial cell barrier integrity, similar to its ability to inhibit endothelial cell barrier integrity by reducing caspase 1 activity ( Figure 8 ), wherein half-maximal activity was observed at about 6 nM G10 peptide (SEQ ID NO: 61). Fig. 9 In the experiment, the G10 peptide was about 8 times more potent than the long PAR1 peptide TR47 (e.g., comparing the effect of 6 nM G10 ( Fig. 9 E) Effect of 50 nM PAR1 peptide P1(47-56) alone ( Fig. 9 A), and G10 peptide ( Fig. 9 E) with the PAR3 peptide P3(42-54) alone ( Fig. 9 D) was >100-fold more effective in protecting endothelial barrier integrity. Fig. 9 E) showed a very significant effect, compared with another PAR3 peptide P3(42-65) containing the 47-55 sequence at 5 to 50 nM ( Fig. 9 B) No significant effect when tested.

[0152] Example 6 Some exemplary methods

[0153] Caspase 1 assay. THP1-null (wild-type cells) or NLRP3-deficient THP1-(defNLRP3) cells were cultured at 1×10 6The cells were plated with a final concentration of 1 μg / mL in a 96-well plate and incubated with PMA (0.5 μM) in supplemented RPMI at 37°C for 3 hours. The culture medium was subsequently replaced every 24 hours for 3 days. Multiple well subsets were selected and cells were treated with various concentrations of APC, TR47 or P3R in serum-free RPMI for 60 minutes at 37°C. After washing with DPBS, cells were incubated with LPS (lipopolysaccharide) (1 μg / mL) in serum-free RPMI at 37°C for 3 hours to induce inflammasomes. After washing with DPBS, to fully activate caspase 1 activity, cells were incubated with ATP (5 mM) at 37°C for 45 min in the presence or absence of 10 μM YVAD (caspase 1 inhibitor) and 2.5 μM ZVAD (pan-caspase inhibitor), and then caspase 1 activity assay was performed according to the manufacturer's instructions (Promega); caspase 1 activity was monitored as the luminescence change caused by substrate hydrolysis and reported as relative luminescence units (RLU). 15 In selection experiments, caspase 1 activity of experimental conditions was normalized to YVAD (caspase 1 inhibitor) conditions and expressed as normalized caspase 1 activity.

[0154] IL-1β ELISA. THP1 cells were cultured at 1×10 6 The final concentration of / mL was plated in a 96-well plate and incubated with PMA (0.5μM) in supplemented RPMI at 37°C for 3 hours. The culture medium was subsequently replaced every 24 hours for 3 days. The selected wells were treated with APC (4μg / mL), TR47 (50μM), P3R (50μM) or a combination of TR47 / P3R (8nM and 500nM, respectively) for 1 hour. The cells were then washed with DPBS and then incubated with LPS (1μg / mL) in serum-free RPMI at 37°C for 3 hours. The cells were washed with DPBS and incubated with ATP (5mM) for 45 minutes. The cell supernatant was carefully collected and frozen until the absorbance was measured using IL-1βQuantikine ELISA according to the manufacturer's instructions (R&D Systems). The concentration of IL-1β was calculated after measuring the standard curve according to the manufacturer's instructions.

[0155] ***

[0156] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art that certain changes and modifications may be made in accordance with the teachings of the invention without departing from the spirit or scope of the appended claims.

[0157] All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each were specifically and individually indicated to be incorporated by reference.

[0158] Some exemplary polypeptide sequences

[0159] SEQ ID NO: 1. Human PAR3

[0160]

[0161] SEQ ID NO: 2. Human PAR3 Met 1 -Arg 41 Missing fragments

[0162]

[0163] SEQ ID NO: 3. Human PAR3 Met 1 -Arg 41 Deletion of the extracellular domain

[0164]

[0165] SEQ ID NO:4. PAR3-derived peptide P3R (first 24 residues of SEQ ID NO:3)

[0166]

[0167] SEQ ID NO: 5. Human PAR1 Met 1 -Arg 46 Missing fragments

[0168]

[0169] SEQ ID NO: 6. Human PAR1 Asn 47 -Trp 100 (Met 1 -Arg 46 Deletion of the extracellular domain)

[0170]

[0171] SEQ ID NO: 7. Peptide TR47 derived from human PAR1 (first 20 residues of SEQ ID NO: 6)

[0172]

Claims

1. A composition comprising an anti-inflammatory peptide derived from PAR3 and an anti-inflammatory peptide derived from PAR1, wherein the peptide derived from PAR3 consists of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, and the peptide derived from PAR1 consists of SEQ ID NO:7, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:49 or SEQ ID NO:

50.

2. The composition of claim 1, wherein the ratio of the amount of the PAR3-derived peptide to the amount of the PAR1-derived peptide is at least 2:

1.

3. The composition of claim 1, wherein the ratio of the amount of the PAR3-derived peptide to the amount of the PAR1-derived peptide is at least 4:

1.

4. The composition of claim 1, wherein the ratio of the amount of the PAR3-derived peptide to the amount of the PAR1-derived peptide is at least 6:

1.

5. The composition of claim 1, wherein the ratio of the amount of the PAR3-derived peptide to the amount of the PAR1-derived peptide is at least 8:

1.

6. The composition of claim 1, wherein the ratio of the amount of the PAR3-derived peptide to the amount of the PAR1-derived peptide is at least 10:

1.

7. The composition of claim 1, wherein the PAR3-derived peptide is conjugated to the PAR1-derived peptide.

8. The composition of claim 7, wherein the PAR3-derived peptide is conjugated to the PAR1-derived peptide via a linker moiety or a carrier moiety.

9. The composition of claim 1, comprising (a) a PAR1-derived peptide consisting of the sequence shown in SEQ ID NO: 36, and (b) a PAR3-derived peptide consisting of the sequence shown in SEQ ID NO: 9, wherein (a) and (b) are linked via a peptide portion.

10. The composition of claim 9, comprising the sequence shown in SEQ ID NO:

61.

11. The composition of claim 1, formulated for oral, intravenous, subcutaneous, intramuscular, intranasal, intraocular, topical or intraperitoneal administration to a subject.

12. The composition of claim 1, comprising two peptides each at a daily dose of up to 0.25 mg / kg of the average body weight of the subject group for which the composition is intended.

13. The composition of claim 1, comprising two peptides each at a daily dose of up to 0.1 mg / kg of the average body weight of the group of subjects for which the composition is intended.

14. The composition of claim 1, comprising two peptides each at a daily dose of up to 0.05 mg / kg of the average body weight of the group of subjects for which the composition is intended.

15. The composition of claim 1, comprising two peptides each at a daily dose of up to 0.025 mg / kg of the average body weight of the group of subjects for which the composition is intended.

16. The composition of claim 1, comprising two peptides each at a daily dose of at most 0.01 mg / kg of the average body weight of the group of subjects for which the composition is intended.

17. The composition of claim 1, comprising two peptides each at a daily dose of up to 0.005 mg / kg of the average body weight of the group of subjects for which the composition is intended.

18. The composition of claim 1, comprising two peptides each at a daily dose of up to 0.0025 mg / kg of the average body weight of the group of subjects for which the composition is intended.

19. The composition of claim 1, comprising two peptides at a daily dose of each of up to 0.001 mg / kg of the average body weight of the group of subjects for which the composition is intended.

20. The composition of claim 1, comprising two peptides each at a daily dose of up to 0.0005 mg / kg of the average body weight of the group of subjects for which the composition is intended.

21. The composition of claim 1, comprising two peptides each at a daily dose of up to 0.00025 mg / kg of the average body weight of the group of subjects for which the composition is intended.

22. The composition of claim 1, comprising two peptides at a daily dose of each less than 0.00025 mg / kg of the average body weight of the group of subjects for which the composition is intended.

23. The composition of claim 1, comprising (1) one of the two peptides at a daily dose of up to 0.25 mg / kg of the average body weight of the group of subjects for which the composition is intended, and (2) the other peptide at a daily dose of at least 0.01 mg / kg of the average body weight of the group of subjects for which the composition is intended.

24. The composition of claim 23, comprising (1) one of the two peptides at a daily dose of up to 0.1 mg / kg of the average body weight of the subject group for which the composition is intended.

25. The composition of claim 23, comprising (1) one of the two peptides at a daily dose of up to 0.05 mg / kg of the average body weight of the group of subjects for which the composition is intended.

26. The composition of claim 23, comprising (1) one of the two peptides at a daily dose of up to 0.025 mg / kg of the average body weight of the group of subjects for which the composition is intended.

27. The composition of claim 23, comprising (1) one of the two peptides at a daily dose of up to 0.01 mg / kg of the average body weight of the group of subjects for which the composition is intended.

28. The composition of claim 23, comprising (1) one of the two peptides at a daily dose of up to 0.005 mg / kg of the average body weight of the group of subjects for which the composition is intended.

29. The composition of claim 23, comprising (1) one of the two peptides at a daily dose of up to 0.0025 mg / kg of the average body weight of the group of subjects for which the composition is intended.

30. The composition of claim 23, comprising (1) each of the two peptides at a daily dose of up to 0.001 mg / kg of the average body weight of the group of subjects for which the composition is intended.

31. The composition of claim 23, comprising (1) one of the two peptides at a daily dose of up to 0.0005 mg / kg of the average body weight of the group of subjects for which the composition is intended.

32. The composition of claim 23, comprising (1) one of the two peptides at a daily dose of up to 0.00025 mg / kg of the average body weight of the group of subjects for which the composition is intended.

33. The composition of claim 23, comprising (1) a daily dose of one of the two peptides, each of which is less than 0.00025 mg / kg of the average body weight of the subject group for which the composition is intended.

34. The composition of any one of claims 23 to 33, comprising (2) another peptide at a daily dose of at least 0.025 mg / kg of the average body weight of the group of subjects for which the composition is intended.

35. The composition of any one of claims 23 to 33, comprising (2) another peptide at a daily dose of at least 0.05 mg / kg of the average body weight of the group of subjects for which the composition is intended.

36. The composition of any one of claims 23 to 33, comprising (2) another peptide at a daily dose of at least 0.1 mg / kg of the average body weight of the subject group for which the composition is intended.

37. The composition of any one of claims 23 to 33, comprising (2) another peptide at a daily dose of at least 0.25 mg / kg of the average body weight of the subject group for which the composition is intended.

38. The composition of any one of claims 23 to 33, comprising (2) another peptide at a daily dose of at least 0.5 mg / kg of the average body weight of the subject group for which the composition is intended.

39. The composition of any one of claims 23 to 33, comprising (2) another peptide at a daily dose of at least 1 mg / kg of the average body weight of the group of subjects for which the composition is intended.

40. The composition of any one of claims 23 to 33, comprising (2) another peptide at a daily dose of at least 2.5 mg / kg of the average body weight of the subject group for which the composition is intended.

41. The composition of any one of claims 23 to 33, comprising (2) another peptide at a daily dose of at least 5 mg / kg of the average body weight of the subject group for which the composition is intended.

42. The composition of any one of claims 23 to 33, comprising (2) another peptide at a daily dose of at least 10 mg / kg of the average body weight of the subject group for which the composition is intended.

43. The composition of any one of claims 23 to 33, comprising (2) another peptide at a daily dose of at least 25 mg / kg of the average body weight of the subject group for which the composition is intended.

44. The composition of any one of claims 23 to 33, comprising (2) another peptide at a daily dose greater than 25 mg / kg of the average body weight of the subject group for which the composition is intended.

45. A method for identifying an anti-inflammatory agent, comprising (1) culturing a human THP-1 cell population, (2) contacting the cultured cells with an inflammation-inducing agent, (3) contacting the cultured cells with a candidate agent and an anti-inflammatory peptide derived from PAR3 or PAR1, respectively, and (4) measuring inflammation-related activity in the cells contacted with the candidate agent and in the cells contacted with the anti-inflammatory peptide derived from PAR3 or PAR1, respectively; wherein the inflammatory activity value measured in the cells contacted with the candidate agent is equal to or less than the inflammatory activity value measured in the cells contacted with the anti-inflammatory peptide derived from PAR3 or PAR1, then the candidate agent is identified as an anti-inflammatory agent, wherein the anti-inflammatory peptide derived from PAR3 or PAR1 is P3R (SEQ ID NO: 4) or TR47 (SEQ ID NO: 7), or a conservatively modified variant thereof, and wherein the candidate agent is a Met-mimicking peptide. 1 -Arg 41 Deletion of the extracellular domain of human PAR3 SEQ ID NO: 3 or Met 1 -Arg 46 A peptide or polypeptide having the N-terminal sequence of SEQ ID NO: 6 deleted from the extracellular domain of human PAR1, or a variant, derivative or mimetic compound of the peptide or polypeptide.

46. ​​The method of claim 45, wherein the inflammation-related activity is caspase 1 enzymatic activity.

47. The method of claim 45, wherein the inflammation-related activity is IL-1β release.

48. The method of claim 45, wherein the inflammation inducing agent is lipopolysaccharide (LPS).

Images