Peptides and methods of treating sepsis, atherosclerosis, thrombosis, stroke, heart attack, and inflammation

Abstract

The present disclosure provides peptides that inhibit the binding interaction between beta integrins and G protein subunits and compositions, e.g., pharmaceutical compositions, particularly nanoparticle compositions, comprising the peptides, and methods of using the peptides to treat atherosclerosis, thrombosis, stroke, heart attack, inflammation, acute respiratory distress syndrome (ARDS), autoimmune disease, hemodialysis AV fistula, or organ transplantation.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 932,024, filed November 7, 2019, the entire contents of which are incorporated herein by reference.

[0003] Statement Regarding Federally Sponsored Research

[0004] This disclosure was made with government support under license numbers HL080264, HL062350, HL125356, HL150797, HL142396 and contract numbers HHSN268201400007C and HHSN268201700002C granted by the National Institutes of Health. The government holds certain rights to this invention.

[0005] By referencing materials incorporated into electronic submissions

[0006] The sequence list and specification, which are part of this disclosure, are submitted together as a text file. The text file containing the sequence list is named "56205_Seqlisting.txt", was created on November 9, 2020, and has a size of 12,040 bytes. The subject of the sequence list is incorporated herein by reference in its entirety. Technical Field

[0007] This disclosure relates to peptides that inhibit the binding interaction between β-integrin and G protein subunits, and compositions comprising said peptides, such as pharmaceutical compositions, specifically nanoparticle compositions. This disclosure also relates to methods of using peptides to treat atherosclerosis, thrombosis, stroke, heart attack, inflammation, acute respiratory distress syndrome (ARDS), autoimmune diseases, hemodialysis AV fistulas, or organ transplantation. This disclosure further relates to nanoparticle compositions for delivering peptides in vitro into cells and for in vivo therapeutic and diagnostic purposes. Background Technology

[0008] In vivo delivery of peptides into cells remains a major obstacle to the development of peptide-based drugs that target intracellular compartments. Therefore, despite the generally low toxicity and high specificity of natural peptides (1, 2), success in developing this type of drug is rare. Developing an efficient method for delivering peptides into cells in vivo will have significant implications for advancing peptide-based drug development.

[0009] Thrombotic cardiovascular disease causes more deaths than any other disease in the world (3). Platelets physiologically mediate hemostasis and thrombus formation to prevent bleeding, but they are also crucial in the development of occlusive thrombosis (4, 5). Therefore, antiplatelet therapy is essential in the treatment of thrombotic diseases and in the prevention of thrombosis in patients undergoing invasive vascular surgery (6, 7). The important role of platelets in hemostasis and thrombosis requires the adhesion receptor integrin αIIbβ3 (also known as glycoprotein (GP)IIb-IIIa) (8). Currently available antiplatelet drugs inhibit thrombus formation by inhibiting the activation of integrin αIIbβ3 (e.g., cyclooxygenase (COX) inhibitors aspirin, adenosine diphosphate (ADP) P2Y12 receptor inhibitors, clopidogrel, ticagrelor, and cangrelor) or by directly blocking the ligand binding function of integrin αIIbβ3 (integrin antagonists abciximab, eptifibatide, and tirofiban) (9, 10). P2Y12 inhibitors, with or without aspirin (such as clopidogrel), are currently the standard of care (9, 10). However, due to the importance of integrin-mediated primary platelet adhesion and aggregation in hemostasis, these drugs have a serious side effect of excessive bleeding (11-14). Bleeding is closely associated with increased adverse outcomes and mortality (11, 15-17). Therefore, there is a need for a new generation of antiplatelet drugs with minimal impact on hemostasis (18). Recently, thrombin receptor inhibitors, namely the protease-activated receptor (PAR)1 inhibitor vorapaxar (19) and the PAR4 inhibitor BMS-986120 (20), have been reported to partially reduce bleeding effects compared to P2Y12 inhibitors in animal studies. However, clinical trials have revealed significant adverse bleeding effects associated with vorapaxar (19). Although the mechanisms by which these inhibitors reduce bleeding in animal models remain unclear, the residual but significant bleeding effects of both PAR1 and PAR4 inhibitors are consistent with the understanding that these thrombin receptors are important for activating the ligand-binding function of integrin αIIbβ3.

[0010] Currently, three classes of integrin inhibitors are in clinical use or under development: monoclonal antibodies targeting the extracellular ligand-binding domain of heterodimers (e.g., Reopro, Eli Lilly, Indianapolis; Vitaxin, MedImmune, Gaithersburg, MD); synthetic peptides containing RGD or KGD sequences (e.g., Integrillin, Millennium Pharmaceuticals; cilengitide, Merck KGaA, Darmstadt, Germany); and peptide mimics (e.g., aggrestat (tirofiban), Merck, White House Station, NJ; S247, Pfizer, St. Louis, MO).

[0011] The first integrin-specific drug targets integrin αIIbβ3, which is central to hemostasis and plays a crucial role in platelet adhesion and thrombosis. αIIbβ3 also plays a role in the inflammatory response. The first FDA-approved αIIbβ3 antagonist has been shown to be beneficial for indications including acute coronary syndrome and myocardial infarction prevention. However, the use of some drugs is limited due to the pharmacokinetic properties of some of these drugs—some have demonstrated rapid plasma clearance, rapid metabolism, poor oral bioavailability, and / or large variations in plasma levels. Furthermore, some antagonists of αIIbβ3 integrins induce thrombocytopenia. See, for example, *Advances in Immunology*, Vol. 91, Elsevier Academic Press (San Diego, CA), 2006. A common and potentially life-threatening side effect of integrin inhibitors is bleeding (because integrins are important in hemostasis).

[0012] A new type of Gα has recently been discovered. 13 A novel strategy for selectively targeting integrin αIIbβ3 ligand binding without affecting the ligand binding function of integrin β3 has been developed, involving Gα-ligands derived from the cytoplasmic domain of integrin β3 (21, 22). 13 Synthetic peptides bound to the ExE motif (mP6) have shown inhibition of thrombus formation without affecting hemostasis (21). However, the in vivo delivery efficiency of this peptide with liposomes or lipid micelles is low. Summary of the Invention

[0013] To enhance the in vivo delivery of the disclosed peptides, high-load peptide nanoparticles (HLPNs) (by total nanoparticle mass) comprising more than 30 molar percentages of one or more amphiphilic peptides, peptide-lipid conjugates, or peptide-hydrophobic molecular conjugates, or combinations thereof. For certain peptides, such as peptide mP6 (derived from the Gα of the β3 cytoplasmic domain), high-load peptide nanoparticles (HLPNs) have been developed. 13 The synthesis of a 6-amino acid peptide bound to an ExE motif facilitates the preparation of high-load peptide nanoparticles by exchanging the C-terminal amino acid residues for amino acids with longer hydrophobic side chains (e.g., replacing alanine with leucine, isoleucine, or methionine). This change allows for higher concentrations of peptides to form stable nanoparticles together with phospholipids and PEG-attached phospholipids. The HLPN can be used to efficiently deliver peptides to their intracellular targets in vitro and in vivo. The peptide concentration incorporated into the stable nanoparticle suspension is significantly increased to greater than 10 mM, enabling bolus injection in human subjects. Compared to current encapsulated peptides using lipid micelles or liposomes, this novel lipid-stabilized HLPN is primarily formed by lipid conjugation or amphiphilic peptides (such as M3mP6-Myr-FEEERL (SEQ ID NO:25)) linked with a low percentage of phospholipid phosphatidylcholine (PC) (2-10%) and polyethylene glycol (DSPE-PEG) (10-60%) linked with 1,2-distearate-sn-glycerol-3-phosphate ethanolamine.

[0014] Using this in vivo delivery technology, the data presented in this paper demonstrate that the HLPN effectively inhibits occlusive thrombosis. For example, the data presented in this paper demonstrate the superior properties of M3mP6 in inhibiting occlusive thrombosis without causing excessive bleeding. M3mP6 HLPN also synergistically enhances the antithrombotic effect of current standard antiplatelet therapy while minimizing the side effect of bleeding. Furthermore, post-ischemic injection of M3mP6 HLPN significantly improved the outcome of myocardial ischemia / reperfusion injury in mice.

[0015] Therefore, in one aspect, this disclosure provides lipid-stabilized, high-load peptide nanoparticles comprising: i) a peptide of greater than about 10 mol% based on the total nanoparticle mass; ii) one or more lipids free of water-soluble polymers, of about 2 to about 20 mol%; and iii) one or more lipids covalently linked to a water-soluble polymer of about 10 to about 60 mol%, wherein the peptide is an esterified peptide, an amphiphilic peptide, or a peptide-hydrophobic conjugate, and the peptide consists of about 2 to about 50 amino acid residues. In some exemplary aspects, the total nanoparticle mass is 30 mol%.

[0016] In all aspects, the peptides in the lipid-stabilized, high-load peptide nanoparticles are lipotropic peptides conjugated with fatty acids. In all aspects, the fatty acids are covalently linked to the peptides. In all aspects, the lipotropic peptides are myristoylated.

[0017] In all respects, the lipids of the lipid-stabilized, high-load peptide nanoparticles that are free of water-soluble polymers are phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylethanolamine (PE), or mixtures thereof.

[0018] In all respects, the lipid covalently linked to the water-soluble polymer of the lipid-stabilized high-load peptide nanoparticles is peg-phosphatidylethanolamine (PEG-PE), PEG-phosphatidylcholine (PEG-PC), PEG-phosphatidylglycerol (PEG-PG), PEG-phosphatidylinositol (PEG-PI), PEG-phosphatidylserine (PEG-PS), PEG-1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[amino(polyethylene glycol)]] (PEG-DSPE) or a mixture thereof. In various aspects, the PEG-DSPE may comprise PEG with a molecular weight of about 200 to about 100,000 or other PEGs known in the art, such as PEG200, PEG300, PEG400, PEG500, PEG600, PEG700, PEG800, PEG900, PEG1000, PEG2000, PEG3000, PEG4000, PEG5000, PEG6000, PEG7000, PEG8000, PEG9000, PEG10,000, PEG20,000, PEG30,000, PEG40,000, PEG50,000, PEG60,000, PEG70,000, PEG80,000, PEG90,000 or PEG100,000. In one exemplary aspect, the lipid covalently linked to the water-soluble polymer is PEG-1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)-2000] (PEG2000-DSPE). In another aspect, the lipid covalently linked to the water-soluble polymer is PEG-1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)-2000] (PEG2000-DSPE), and the lipid without the water-soluble polymer is phosphatidylcholine.

[0019] In each aspect, the peptide in the lipid-stabilized, high-load peptide nanoparticles comprises the amino acid sequence FEX1ERX2 (SEQ ID NO:1), wherein X1 and X2 are selected from natural and non-natural amino acids, wherein the peptide is a hexamer, heptamer, octamer, or decimer, and wherein the peptide inhibits both integrin-dependent and integrin-independent platelet-granule secretion, or wherein the peptide inhibits leukocyte integrin outward-inward signaling. In each aspect, X1 is glutamic acid or lysine. In each aspect, X2 is isoleucine, alanine, methionine, or lysine. In each aspect, X1 is glutamic acid or lysine, and X2 isoleucine, alanine, methionine, or lysine. In a related aspect, the peptide is myristylated.

[0020] In all respects, the peptide in any of the disclosed lipid-stabilized high-load peptide nanoparticles is FEEERI (SEQ ID NO:2), FEKEKI (SEQ ID NO:3), FEKERI (SEQ ID NO:4), RGT, EEERA (SEQ ID NO:5), FEEERA (SEQ ID NO:6), FEEERM (SEQ ID NO:7), FEEERL (SEQ ID NO:8), FEKEKM (SEQ ID NO:9), FEKEKL (SEQ ID NO:10), FEKERM (SEQ ID NO:11), FEKERL (SEQ ID NO:12), CFEEERAC (SEQ ID NO:13), FEEERAR (SEQ ID NO:14), FEEERARA (SEQ ID NO:15), SIRYSGHpSL (SEQ ID NO:16), KFEEERARAKWDT (SEQ ID NO:17), or LLARRPTKGIHEY (SEQ ID NO:18).

[0021] In other respects, the peptides in any of the disclosed lipid-stabilized high-load peptide nanoparticles are myr-FEEERI (SEQ ID NO:19), myr-FEKEKI (SEQ ID NO:20), myr-FEKERI (SEQ ID NO:21), myr-RGT, myr-EEERA (SEQ ID NO:22), myr-FEEERA (SEQ ID NO:23), myr-FEEERM (SEQ ID NO:24), myr-FEEERL (SEQ ID NO:25), myr-FEKEKM (SEQ ID NO:26), myr-FEKEKL (SEQ ID NO:27), myr-FEKERM (SEQ ID NO:28), myr-FEKERL (SEQ ID NO:29), myr-CFEEERAC (SEQ ID NO:30), myr-FEEERAR (SEQ ID NO:31), myr-FEEERARA (SEQ ID NO:29), myr-CFEEERAC (SEQ ID NO:30), myr-FEEERAR (SEQ ID NO:31), myr-FEEERARA (SEQ ID NO:29), myr-FEEEERAC (SEQ ID NO:30), myr-FEEERAR (SEQ ID NO:31), myr-FEEERARA (SEQ ID NO:29), myr-FEEEERAC (SEQ ID NO:20), myr-FEEEERAR (SEQ ID NO:31), myr-FEEERARA (SEQ ID NO:29), myr-FEEEERAR (SEQ ID NO:20 ... ID NO:32), myr-SIRYSGHpSL (SEQ ID NO:33), myr-KFEEERARAKWDT (SEQ ID NO:34) or myr-LLARRPTKGIHEY (SEQ ID NO:35).

[0022] In all respects, the lipid-stabilized high-load peptide nanoparticles comprise about 10 to about 80 mol% peptides based on the total nanoparticle mass. In related respects, the peptides in the lipid-stabilized high-load peptide nanoparticles are myr-FEEERL (SEQ ID NO:25) or myr-FEKEKL (SEQ ID NO:27).

[0023] In an exemplary aspect, the lipid-stabilized high-load peptide nanoparticles comprise 2-10% phosphatidylcholine and 10-60% PEG-DSPE.

[0024] This disclosure also provides pharmaceutical compositions comprising any of the lipid-stabilized, high-load nanoparticles of this disclosure, and pharmaceutically acceptable carriers, diluents, or excipients.

[0025] On the other hand, this disclosure provides a method for preparing lipid-stabilized, high-load peptide nanoparticles. The method includes contacting peptide nanoparticles comprising one or more lipotropic peptides consisting of about 2 to about 50 amino acid residues with 2-20% by weight of a lipid free of water-soluble polymers and 20-50% by weight of a lipid covalently linked to a water-soluble polymer. The mixture is dissolved in methanol and chloroform, then evaporated using a rotary evaporator to form a thin film. The film is then rehydrated with isotonic brine (0.9% NaCl) or pH-buffered isotonic brine to prepare the lipid-stabilized, high-load nanoparticles.

[0026] Furthermore, this disclosure provides a method for treating thrombotic or inflammatory symptoms in a subject in need, the method comprising administering to the subject an effective amount of any of the disclosed lipid-stabilized, high-load nanoparticles. In related aspects, the inflammatory symptoms are sepsis, acute respiratory distress syndrome (ARDS), autoimmune diseases, hemodialysis AV fistulas, organ transplantation, atherosclerosis, vasculitis, deep vein thrombosis, or ischemia-reperfusion injury. In related aspects, the thrombotic symptoms are arterial thrombosis, venous thrombosis and microvascular thrombosis, prosthetic valve thrombosis, AV fistula thrombosis, heart attack, or ischemic stroke.

[0027] On the other hand, this disclosure provides a method for treating a subject in need of stroke or heart attack, the method comprising administering to the subject an effective amount of any of the disclosed lipid-stabilized high-load nanoparticles.

[0028] In another aspect, this disclosure provides the use of an effective amount of any of the disclosed lipid-stabilized high-load nanoparticles or any of the disclosed pharmaceutical compositions in the preparation of a medicament for treating thrombotic or inflammatory symptoms in a subject of need. In a related aspect, the inflammatory symptoms are sepsis, acute respiratory distress syndrome (ARDS), autoimmune diseases, hemodialysis AV fistulas, organ transplantation, atherosclerosis, vasculitis, deep vein thrombosis, or ischemia-reperfusion injury. In a related aspect, the thrombotic symptoms are arterial thrombosis, venous thrombosis and microvascular thrombosis, prosthetic valve thrombosis, AV fistula thrombosis, heart attack, or ischemic stroke.

[0029] On the other hand, this disclosure provides the use of an effective amount of any of the disclosed lipid-stabilized high-load nanoparticles or any of the disclosed pharmaceutical compositions in the preparation of a medicament for treating a subject in need of stroke or heart attack.

[0030] This disclosure also provides compositions for treating thrombotic or inflammatory symptoms in subjects of need, wherein the compositions comprise an effective amount of any of the disclosed lipid-stabilized high-load nanoparticles or any of the disclosed pharmaceutical compositions. In related aspects, the inflammatory symptoms are sepsis, acute respiratory distress syndrome (ARDS), autoimmune diseases, hemodialysis AV fistulas, organ transplantation, atherosclerosis, vasculitis, deep vein thrombosis, or ischemia-reperfusion injury. In related aspects, the thrombotic symptoms are arterial thrombosis, venous thrombosis and microvascular thrombosis, prosthetic valve thrombosis, AV fistula thrombosis, heart attack, or ischemic stroke.

[0031] In another aspect, this disclosure provides compositions for treating stroke or heart attack in a subject of need, said compositions comprising an effective amount of any of the disclosed lipid-stabilized high-load nanoparticles or any of the disclosed pharmaceutical compositions.

[0032] In any of the disclosed methods, uses, and compositions, the peptide is an esterified peptide comprising the amino acid sequence of FEEERM (SEQ ID NO:7), FEEERL (SEQ ID NO:8), FEEERI (SEQ ID NO:2), FEKEKM (SEQ ID NO:9), FEKEKL (SEQ ID NO:10), FEKEKI (SEQ ID NO:3), FEKERM (SEQ ID NO:11), FEKERL (SEQ ID NO:12), FEKERI (SEQ ID NO:4), or CFEEERAC (SEQ ID NO:13).

[0033] In any of the disclosed methods, uses, and compositions, the peptide is myristylated and comprises the amino acid sequence of myr-FEEERM (SEQ ID NO:24), myr-FEEERL (SEQ ID NO:25), myr-FEEERI (SEQ ID NO:19), myr-FEKEKM (SEQ ID NO:26), myr-FEKEKL (SEQ ID NO:27), myr-FEKEKI (SEQ ID NO:20), myr-FEKERM (SEQ ID NO:28), myr-FEKERL (SEQ ID NO:29), myr-FEKERI (SEQ ID NO:21), or myr-CFEEERAC (SEQ ID NO:30).

[0034] On the other hand, this disclosure provides a peptide comprising the amino acid sequence FEEERI (SEQ ID NO:2), FEKEKI (SEQ ID NO:3), or FEKERI (SEQ ID NO:4). In a related aspect, this disclosure provides a peptide composed of the amino acid sequence FEEERI (SEQ ID NO:2), FEKEKI (SEQ ID NO:3), or FEKERI (SEQ ID NO:4). In a related aspect, the peptide further comprises a membrane-permeable portion. For example, the membrane-permeable portion is a membrane-permeable peptide, a membrane-permeable synthetic compound, or a fatty acid. In some aspects, the membrane-permeable portion is a fatty acid. In a related aspect, the fatty acid is covalently linked to the peptide. In a related aspect, the peptide is myristicated. In a related aspect, the peptide is myr-FEEERI (SEQ ID NO:19), myr-FEKEKI (SEQ ID NO:20), or myr-FEKERI (SEQ ID NO:21).

[0035] In various aspects, this disclosure provides pharmaceutical compositions comprising the peptides disclosed herein, as well as pharmaceutically acceptable carriers, diluents, or excipients. In various aspects, the pharmaceutical compositions comprise the amino acid sequence FEEERI (SEQ ID NO:2), FEKEKI (SEQ ID NO:3), or FEKERI (SEQ ID NO:4), as well as pharmaceutically acceptable carriers, diluents, or excipients. In various aspects, the pharmaceutical compositions comprise peptides, as well as pharmaceutically acceptable carriers, diluents, or excipients, wherein the peptide is myr-FEEERI (SEQ ID NO:19), myr-FEKEKI (SEQ ID NO:20), or myr-FEKERI (SEQ ID NO:21).

[0036] On the other hand, this disclosure provides a method for treating thrombotic or inflammatory symptoms in a subject in need, the method comprising administering to the subject an effective amount of the disclosed peptide or pharmaceutical composition. In related aspects, the inflammatory symptoms are sepsis, acute respiratory distress syndrome (ARDS), autoimmune diseases, hemodialysis AV fistulas, organ transplantation, atherosclerosis, vasculitis, deep vein thrombosis, or ischemia-reperfusion injury. In related aspects, the thrombotic symptoms are arterial thrombosis, venous thrombosis and microvascular thrombosis, prosthetic valve thrombosis, AV fistula thrombosis, heart attack, or ischemic stroke.

[0037] On the other hand, this disclosure provides a method for treating ischemic events, such as stroke, heart attack, or AV fistula thrombosis, in subjects in need, the method comprising administering to the subject an effective amount of the peptide or pharmaceutical composition disclosed herein.

[0038] On the other hand, this disclosure provides the use of an effective amount of the disclosed peptide or pharmaceutical composition in the preparation of a medicament for treating thrombotic or inflammatory symptoms in a subject of need. In related aspects, the inflammatory symptoms are sepsis, acute respiratory distress syndrome (ARDS), autoimmune diseases, hemodialysis AV fistulas, organ transplantation, atherosclerosis, vasculitis, deep vein thrombosis, or ischemia-reperfusion injury. In related aspects, the thrombotic symptoms are arterial thrombosis, venous thrombosis and microvascular thrombosis, prosthetic valve thrombosis, AV fistula thrombosis, heart attack, or ischemic stroke.

[0039] On the other hand, this disclosure provides the use of an effective amount of the disclosed peptide or pharmaceutical composition in the preparation of a medicament for treating stroke and heart attack or AV fistula thrombosis in subjects of need.

[0040] On the other hand, this disclosure provides compositions for treating thrombotic or inflammatory symptoms in subjects of need, said compositions comprising an effective amount of the peptides or pharmaceutical compositions disclosed herein. In related aspects, said inflammatory symptoms are sepsis, acute respiratory distress syndrome (ARDS), autoimmune diseases, hemodialysis AV fistulas, organ transplantation, atherosclerosis, vasculitis, deep vein thrombosis, or ischemia-reperfusion injury. In related aspects, said thrombotic symptoms are arterial thrombosis, venous thrombosis and microvascular thrombosis, prosthetic valve thrombosis, AV fistula thrombosis, heart attack, or ischemic stroke.

[0041] On the other hand, this disclosure provides compositions for treating stroke and heart attack in subjects in need, said compositions comprising an effective amount of the peptide or pharmaceutical composition disclosed herein.

[0042] There are two types of granule secretion: integrin-dependent and integrin-independent. The platelet adhesion receptor integrin αIIbβ3 is known to induce integrin-dependent platelet granule secretion. However, it has been found that blocking the ligand binding function of integrin αIIbβ3 with currently available integrin inhibitors (such as integrilin) ​​or mutating the Gα13 binding site in the cytoplasmic domain of integrin β3 actually enhances integrin-independent granule secretion, even if it also inhibits integrin-dependent granule secretion.

[0043] Therefore, on the other hand, this disclosure provides a method for treating a subject with a disease or symptom regulated by platelet granule secretion, Gα13 function, or leukocyte integrin outward-inward signaling, the method comprising administering to the subject an effective amount of a peptide comprising the amino acid sequence FEX1ERX2 (SEQ ID NO:1), wherein X1 and X2 are selected from natural and non-natural amino acids, wherein the peptide is a hexamer, heptamer, octamer, or decimer, and wherein the peptide inhibits both integrin-dependent and integrin-independent platelet granule secretion, or wherein the peptide inhibits leukocyte integrin outward-inward signaling.

[0044] On the other hand, this disclosure provides a method for inhibiting integrin-inward signaling in a subject without inhibiting integrin-independent platelet granule secretion, the method comprising administering to the subject an effective amount of a peptide consisting of the amino acid sequence of EEERA (SEQ ID NO:5), EEERM (SEQ ID NO:36), EEERL (SEQ ID NO:37), EKEKM (SEQ ID NO:38), EKEKL (SEQ ID NO:39), EKERM (SEQ ID NO:40), EKERL (SEQ ID NO:41), or CEEERAC (SEQ ID NO:42). Such a peptide may be useful in conditions requiring platelet granule secretion during the treatment of thrombosis (e.g., for promoting wound healing). Attached Figure Description

[0045] Figure 1A-1E :( Figure 1A A schematic diagram of lipid-stabilized high-load peptide nanoparticles (HLPN). Figure 1B The structure of the M3mP6(Myr-FEEERL(SEQ ID NO:25)) peptide. Figure 1C Compared to disordered peptides, M3mP6 has a greater effect on integrin β3 and Gα in human platelets stimulated by α-thrombin (0.025 U / mL). 13 The dose-dependent inhibition of co-immunoprecipitation. Figure 1D ) Figure 1C Quantitative analysis of Western blots obtained from COVID-19 (n=3). OD, optical density. Figure 1E For example, the size distribution of a single formulation of M3mP6 HLPN (6.6±0.8nm) by dynamic light scattering analysis.

[0046] Figure 2A-2I The functional analysis of M3mP6 HLPN is shown. Figure 2A M3mP6 HLPN inhibited thrombin-induced human platelet secretion in an in vitro dose-dependent manner. Data were obtained from 3-4 experiments. Figure 2B M3mP6 HLPN showed dose-dependent inhibition of thrombin (0.03 U / mL)-induced platelet aggregation. Figure 2C The inhibitory effect of M3mP6 HLPN on thrombin-induced human platelet aggregation is limited to low-dose thrombin (Scra: disordered control peptide). Figure 2D M3mP6 HLPN inhibits both low-dose-induced and high-dose-induced platelet secretion in human platelets. Figure 2E Compared with the disordered peptide control, M3mP6 HLPN partially inhibited collagen (1 μg / mL)-induced platelet aggregation in mice. Figure 2F Compared to the disordered peptide HLPN, M3mP6 HLPN partially inhibited U46619 (0.5 μM)-induced platelet aggregation in mice. Figure 2G M3mP6 HLPN had no effect on ADP (5 μM)-induced human platelet aggregation. Figure 2H M3mP6 HLPN had no effect on the binding of the PE-conjugated antibody JON / A against activated αIIbβ3 induced by PAR4 agonist (PAR4-AP) to mouse platelets. Figure 2I M3mP6HLPN had no effect on the binding of Oregon Green-labeled fibrinogen to mouse platelets induced by the PAR4 agonist (PAR4-AP). *P<0.05, ****P<0.0001, ns, not significant.

[0047] Figure 3A-3G The pharmacokinetics of M3mP6 HLPN are shown. Figure 3A Flow cytometry was used to compare the uptake of fluorescently labeled M3mP6 dissolved in DMSO with that dissolved in HLPN into mouse platelets. Figure 3B The effects of 40 μM M3mP6 dissolved in HLPN and 160 μM DMSO on human platelet aggregation are comparable. Figure 3C M3mP6 HLPN (n=15) inhibited FeCl3-induced carotid artery thrombosis, while DMSO-dissolved M3mP6 (n=6) had no effect compared to the saline control (n=14). Figure 3D The dose-response of M3mP6 HLPN injected retroorbitally 15 minutes before surgery in inhibiting FeCl3-induced carotid artery occlusive thrombosis (n=3). Figure 3E Pharmacokinetic study of M3mP6 HLPN plasma levels after retroorbital injection (5 μmol / kg). Figure 3FPharmacokinetic study of whole blood levels of M3mP6 HLPN after retroorbital injection (5 μmol / kg). Figure 3G Kinetics of the antithrombotic effect of 5 μmol / kg M3mP6 HLPN (retroorbital injection) in inhibiting FeCl3-induced carotid artery occlusive thrombosis (n=3). **P<0.01, ****P<0.0001, ns, not significant.

[0048] Figures 4A-4D This study compares the effects of M3mP6 HLPN and aspirin on thrombosis and hemostasis. Figure 4A and 4B The effects of M3mP6 HLPN (10 μmol / kg, 15 minutes before surgery), aspirin (4.3 mg / kg, orally 2 hours before surgery), and M3mP6 HLPN plus aspirin on FeCl3-induced thrombosis were compared. Figure 4A ) and tail bleeding time ( Figure 4B ). ( Figure 4A For the normal control, n=17; for M3mP6 HLPN, n=18; for aspirin, n=11; for aspirin + M3mP6 HLPN, n=11. Figure 4B For the normal control group, n=16; for M3mP6 HLPN, n=18; for aspirin, n=11; for aspirin + M3mP6 HLPN, n=11. Figure 4C and 4D The effects of M3mP6 HLPN (5 μmol / kg), high-dose aspirin (36 mg / kg, intraperitoneal, 1 hour before surgery), and M3mP6 HLPN plus high-dose aspirin on FeCl3-induced carotid artery thrombosis were compared. Figure 4C ) and tail bleeding time ( Figure 4D ). ( Figure 4C D: For normal control, n=8; for M3mP6 HLPN, n=8; for aspirin, n=10; for aspirin + M3mP6 HLPN, n=6; **P<0.01, ***P<0.001, ****P<0.0001.

[0049] Figures 5A-5D This study compares the effects of M3mP6 HLPN and oral P2Y12 inhibitors on thrombosis and hemostasis, as well as their synergistic antithrombotic effects. Figure 5A and 5BThe effects of M3mP6 HLPN (10 μmol / kg, 15 minutes before surgery) on FeCl3-induced carotid artery thrombosis were compared with those of clopidogrel (4 mg / kg, 2 hours before surgery), M3mP6 HLPN plus clopidogrel, ticagrelor (3 mg / kg, 2 hours before surgery), and M3mP6 HLPN plus ticagrelor. Figure 5A ) and tail bleeding time ( Figure 5B ). ( Figure 5A and 5B For normal controls, n=30; for M3mP6 HLPN, n=18; for clopidogrel, n=8; for clopidogrel + M3mP6 HLPN, n=8; for ticagrelor, n=13; for ticagrelor + M3mP6 HLPN, n=14. Figure 5C and 5D Compare the effects of M3mP6 HLPN, clopidogrel plus aspirin, M3mP6 HLPN, and clopidogrel plus aspirin on FeCl3-induced carotid artery thrombosis. Figure 5C ) and tail bleeding time ( Figure 5D ). ( Figure 5C and 5D (For the normal control group, n=27; for M3mP6 HLPN, n=18; for aspirin, n=11; for clopidogrel, n=8; for aspirin + clopidogrel, n=9; for aspirin + clopidogrel + M3mP6 HLPN, n=10). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0050] Figures 6A-6F This study compares the effects of M3mP6 HLPN and cangrarol on thrombosis, hemostasis, platelet thrombosis, and coagulation, as well as their synergistic antithrombotic effects. Figure 6A The effects of M3mP6 HLPN (10 μmol / kg) and canagrelor (30 μg / kg) on ​​occlusive thrombosis when injected 5 minutes after FeCl3 (3.75%)-induced carotid artery injury. Figure 6B )like( Figure 6A Analysis of mouse tail hemorrhage time as described in [reference needed]. A and B: n=7 for normal controls, n=9 for M3mP6 HLPN, and n=6 for cangrelox. Figure 6C and 6DThe effects of pre-injury injection of M3mP6 HLPN (10 μmol / kg) on ​​FeCl3-induced carotid artery thrombosis were compared with those of high-dose canagrelor (30 μg / kg), high-dose canagrelor (30 μg / kg) plus M3mP6 HLPN, low-dose canagrelor (10 μg / kg), and low-dose canagrelor (10 μg / kg) plus M3mP6 HLPN, as well as the normal control. Figure 6C ) and tail bleeding time ( Figure 6D ). ( Figure 6C For the normal control, n=29; for M3mP6 HLPN, n=26; for high-dose canagrelor, n=20; for high-dose canagrelor + M3mP6 HLPN, n=15; for low-dose canagrelor, n=17; for low-dose canagrelor + M3mP6 HLPN, n=14. Figure 6D For the normal control, n=29; for M3mP6 HLPN, n=25; for canagrelor 30 μg / kg, n=17; for canagrelor 30 μg / kg + M3mP6 HLPN, n=17; for canagrelor 10 μg / kg, n=17; for canagrelor 10 μg / kg + M3mP6 HLPN, n=14. Figure 6E The median integrated platelet fluorescence intensity (DyLight 649-labeled anti-GPIbβ) at the laser-induced cremasteric arteriole injury sites was compared between control (black), M3mP6 HLPN (10 μmol / kg) treated (red), and cangrelor (30 μg / kg) treated (blue) mice. (The control group, M3mP6 treated group, and cangrelor treated group each had 27 injury sites. (The difference between control and M3mP6 or cangrelor was not statistically significant; there was no significant difference between M3mP6 and cangrelor.) Figure 6F The median integrated fibrin fluorescence intensity (Alexa Fluor 488 labeled antifibrin) at the laser-induced cremasteric artery lesion sites was compared between control (black), M3mP6 HLPN (10 μmol / kg) treated (red), and cangrelo (30 μg / kg) treated (blue) mice. (27 lesion sites were found in each of the control, M3mP6 treated, and cangrelo treated groups. (Control vs. M3mP6 or cangrelo, P < 0.0001; M3mP6 vs. cangrelo, P < 0.01)

[0051] Figures 7A-7B This demonstrates that M3mP6 does not induce bleeding in a mouse model of surgical bleeding with arterial perforation and in a canine buccal mucosal bleeding time (BMBT) test. Figure 7A Illustration of a novel mouse surgical hemorrhage model. Figure 7BA comparison was made between M3mP6 HLPN (10 μmol / kg) and high-dose canagrelor (30 μg / kg), high-dose canagrelor plus M3mP6 HLPN, low-dose canagrelor (10 μg / kg), and low-dose canagrelor plus M3mP6 HLPN in a surgical bleeding arterial perforation model (n=27 for normal control, n=20 for M3mP6 HLPN, n=16 for canagrelor 30 μg / kg, n=17 for canagrelor 30 μg / kg + M3mP6 HLPN, n=11 for canagrelor 10 μg / kg, and n=12 for canagrelor 10 μg / kg + M3mP6 HLPN). Figure 7B BMBT test of M3mP6HLPN for hemostasis in dogs. (n=3). ****P<0.0001, ns, not significant.

[0052] Figure 8A-8L This study demonstrates the effect of post-ischemic injection of M3mP6 HLPN on myocardial ischemia and reperfusion (MI / R) injury in mice. Figure 8A A schematic protocol for a MI / R study. The left anterior descending artery (LAD) of mice was completely ligated for 45 minutes before reopening (reperfusion). The mouse chest was then closed. Thirty-five minutes after LAD ligation, M3mP6 HLPN or a disordered peptide HLPN control (Scra) was injected via jugular vein at a bolus of 5 μmol / kg, followed by continuous infusion at a rate of 2.5 μmol / kg / hour for 24 hours. Echocardiography and / or histological examination were then performed on the mice. Figure 8B Representative images of heart slices from mice treated with M3mP6 HLPN or out-of-order peptide HLPN 24 hours after reperfusion. Figure 8C The infarct area (white) is quantified as a percentage of the at-risk area (non-blue), as shown in Figure A. Figure 8D The risk area was quantified as a percentage of the entire heart slice. Vital tissue within the risk area was stained red. (For each group, n = 4, *p < 0.05) Figure 8E For the following representative M-mode long-axis echo images: (i) sham-operated control; (ii) MI / R processed with scrambled control HLPN; and (iii) MI / R processed with M3mP6HLPN. Figure 8F Left ventricular ejection fraction (LVEF) in mice was measured by echocardiography and calculated using Vevo 2100 software. Data are presented as mean ± SEM, and statistical data were analyzed using one-way ANOVA with Graphpad PRISM 5.0. Figure 8GTwenty-four hours after MI / R, platelets in mouse heart sections were immunohistochemically stained using rat anti-integrin αIIb antibody. Representative sections were from: (i) sham-operated controls; (ii) MI / R treated with out-of-order peptide HLPN; and (iii) MI / R treated with M3mP6 HLPN. (iv) αIIb positive staining was quantified using ImageJ software and expressed as a percentage of total tissue area. Statistical analysis was examined using the Mann-Whitney nonparametric t-test. Figure 8H Twenty-four hours after MI / R, neutrophils in mouse heart sections were immunohistochemically stained using rat anti-Ly6G antibody. Representative sections were from: (i) sham-operated controls; (ii) MI / R treated with out-of-order peptide HLPN; and (iii) MI / R treated with M3mP6 HLPN. (iv) Ly6G-positive cells / mm² were counted using imageJ software. Statistical analysis was performed using the Mann-Whitney nonparametric t-test. **p<0.01. Figure 8I Plasma MPO levels in mice 24 hours after MI / R. (Sham surgery: n=6, disordered HLPN treatment: n=11, M3mP6 HLPN treatment: n=9). Figure 8J Kaplan-Meier survival curves of mice 7 days after MI / R surgery treated with M3mP6HLPN or out-of-order peptide HLPN. The sham surgery did not cause death in any of the 6 tested mice. Figure 8K The percentage of left ventricular ejection fraction (LVEF) in mice treated with M3mP6 HLPN, cangrelox, saline, or sham surgery. Figure 8L The Kaplan-Meyer survival curves for mice show the percentage of survival and reperfusion time after MI in mice treated with M3mP6 HLPN, cangrelo, saline, or sham surgery.

[0053] Figures 9A-9C The EXE motif peptide MB2mP6, derived from the cytoplasmic domain of integrin β2, does not affect neutrophil adhesion but inhibits neutrophil migration. Figure 9A Compared with the control peptide (n=3), the adhesion of mouse neutrophils to the β2-integrin ligand ICAM1 was not affected after MB2mP6 (50 μM). Figure 9B Neutrophil transendothelial migration was dose-dependently inhibited by treatment with MB2mP6 peptide. Figure 9C ) in control neutrophils (Gα13) treated with MB2mP6 peptide fl / flIn this study, neutrophil transendothelial migration was significantly reduced, similar to that observed in Gα13 knockout neutrophils (Gα13...). fl / fl The inhibitory effect of LysM cre) was observed. It should be noted that MB2mP6 treatment did not further reduce Gα13. fl / flLysMCre Neutrophil transmembrane migration within neutrophils suggests that inhibition via MB2mP6 is Gα13-specific.

[0054] Figure 10A-10D MB2mP6 was shown to inhibit cytokine expression in CLP-induced septic mice. Figures 10A-10B The expression of selected cytokines (IL-6 and TNFα) in mouse serum at protein levels, measured by ELISA, is shown 24 hours after CLP. Sham operation, n=6; randomized, n=8; MB2mP6, n=10. Figure 10C-10D The expression of selected cytokines at the mRNA level in mouse lung tissue 24 hours after CLP is shown. Sham operation, n=4; randomized, n=6; MB2mP6, n=6. All data are shown as mean ± sem. Data were analyzed by two-tailed unpaired t-test with Welch's correction; *P<0.05, **P<0.01, ***P<0.001.

[0055] Figure 11 This study demonstrates that MB2mP6 protects mice from death and prevents sepsis-induced organ damage in a CLP sepsis model. a) Immediate treatment with MB2mP6 after CLP surgery enhances the survival rate of sepsis-affected C57BL / 6 mice in the CLP model compared to the sham-operated group or the saline treatment group. All mice in the sham-operated group survived. Significance analysis was determined using GraphPad Prism software via the Mantel-Cox test. Sham-operated, n=6; sham-operated, n=23; MB2mP6, n=24; saline, n=18.

[0056] Figures 12A-12E This study demonstrates that thrombin-induced platelet secretion is enhanced in integrin β3- / - platelets and is further enhanced by the integrin antagonist integrilin. Figure 12A Washed wild-type (WT) and β3- / - mouse platelets were dissolved and immunoblotted using rabbit antibodies recognizing integrin β3 and anti-α13 antibodies. Figure 12BThrombin was used to stimulate washed wild-type (WT) and β3- / - platelets. Platelet aggregation and ATP secretion were simultaneously recorded using a Chronolog platelet aggregator at 37°C and 1000 rpm in the presence of a luciferin-luciferase reagent. Figure 12C Quantification of secretion. *p<0.05 (n=3, Stourden t-test). Figure 12D Washed human platelets pre-incubated with thrombin-stimulated buffer or 10 μg / ml Integrilin were used. Platelet aggregation and ATP secretion were simultaneously recorded using a Chronolog platelet aggregator in the presence of luciferin-luciferase reagent. Figure 12E Quantification of secretion. *p<0.05, Student t test (n=3).

[0057] Figures 13A-13E This demonstrates the dual role of integrin αIIbβ3 in regulating platelet granule secretion. Figure 13A Washed wild-type (WT) and β3- / - platelets were stimulated with U46619. Platelet aggregation and ATP secretion were monitored using a Chronolog Lumi aggregator at 37°C and 1000 rpm. Note the two ATP secretion waves in wild-type platelets and the higher single (first) secretion wave in β3 knockout platelets. Figure 13B and 13C Using the Stourden t-test (n=3) for ( Figure 13A Quantification is performed on any secretion wave in the sequence. Figure 13D Washed human platelets pre-incubated with U46619 stimulation buffer or 10 μg / ml Integrilin. (As in...) Figure 13A Similar to [previous study], it monitors platelet aggregation and ATP secretion. Figure 13E Using the Stourden t-test (n=3) for ( Figure 13D Quantitative analysis was performed on the secretion data of ) .

[0058] Figures 14A-14E This demonstrates the dual role of Gα13-β3 interaction in platelet granule secretion. Figure 14A Flow cytometry analysis of platelet expression of wild-type (EEE) and AAA mutant integrin β3 in β3- / - mice transplanted with bone marrow stem cells transfected with wild-type or AAA mutant human β3 cDNA. Platelets were stained with anti-β3 monoclonal antibody mAb15. Figure 14B Thrombin was used to stimulate washed wild-type (EEE) and AAA-expressing mouse platelets. Platelet aggregation / ATP secretion was monitored as shown in Figure 1. Figure 14CUsing the Stourden t-test (n=3) from ( Figure 14B Quantitative data. Figure 14D Stimulation of washed EEE and AAA platelets with U46619. (As in...) Figure 14B Similar to the previous method, it monitors platelet aggregation. Figure 14E Using the Stourden t-test (n=3) from ( Figure 14D Quantitative data.

[0059] Figures 15A-15E The role of Gα13 in integrin-independent and integrin-dependent platelet granule secretion is illustrated. Figure 15A Equal volumes of washed wild-type (WT) and Gα13- / - mouse platelets were dissolved and immunoblotting was performed using a rabbit antibody that specifically recognizes Gα13 and integrin β3. Figure 15B Washed wild-type (WT) and Gα13- / - platelets were stimulated with thrombin. Platelet aggregation and ATP secretion were simultaneously recorded in the presence of a luciferin-luciferase agent, as shown in Figure 1. Figure 15C )right( Figure 15B Quantification of secretion in ) . Studen's t-test (n=3). Figure 15D U46619 was used to stimulate washed wild-type (WT) and Gα13- / - platelets. Platelet aggregation and ATP secretion were simultaneously recorded in the presence of a luciferin-luciferase agent. Figure 15E )right( Figure 15D Quantification of secretion in ) . Studen's t-test (n=3).

[0060] Figures 16A-16E This study compares the effects of mP6 and mP5 on platelet granule secretion. Figure 16A Thrombin was used to stimulate washed human platelets. As described above, platelet aggregation and ATP secretion were simultaneously recorded in the presence of a luciferin-luciferase agent. Figure 16B )right( Figure 16A Quantification of secretion in ) . Studen's t-test (n=3). Figure 16C Stimulate washed human platelets with U46619. Simultaneously record platelet aggregation and ATP secretion in the presence of a luciferin-luciferase agent. Note the two granule secretion waves and the opposing effects of mP5 and mP6 on the first wave. Figure 16D and 16E )right( Figure 16C Quantification of secretion in ) . Studen's t-test (n=3).

[0061] Figure 17 This is a schematic diagram of the dual regulation of platelet granule secretion by integrin and Gα13.

[0062] Figures 18A-18F The stability of M3mP6 HLPN during storage is shown. Figure 18A And B)M3mP6 HLPN should be stored at -20°C for 18 months before use. Figure 18A ) and afterwards ( Figure 18B Dynamic light scattering (DLS) analysis of particle size. Figure 18C Antithrombotic effects of M3mP6 HLPN in a FeCl3-induced carotid artery thrombosis model before and after 18 months of storage at -20°C. n=4, *p<0.05. (D and E) Antithrombotic effects of M3mP6 HLPN before 2 weeks of storage at room temperature (approximately 22°C). Figure 18D ) and afterwards ( Figure 18E DLS analysis of particle size. Figure 18F Antithrombotic effect of M3mP6 HLPN in a FeCl3-induced carotid artery thrombosis model before and after storage at room temperature for 2 weeks. *p<0.05.

[0063] Figure 19 This study illustrates the ingestion of fluorescently labeled M3mP6 HLPN into mouse platelets. Fluorescently labeled M3mP6 HLPN was incubated with mouse platelets at 37°C for 30 minutes, and then plates were seeded onto coverslips. Platelets were then stained with PE-conjugated rat anti-GPIbβ antibody at room temperature for 30 minutes. The ingestion of M3mP6 into platelets was observed using a confocal microscope (Zeiss LSM 710META).

[0064] Figures 20A-20B The effects of M3mP6 HLPN, the disordered control peptide (disordered) HLPN, the AAA mutant peptide (Myr-FAAARL (SEQ ID NO:43) HLPN), and physiological saline solution on 7.5% FeCl3-induced carotid artery thrombosis were demonstrated. Figure 20A ) and tail bleeding time ( Figure 20B Comparison of (saltwater: n=15, AAA: n=9, random order: n=6, M3mP6: n=15). ****p<0.0001.

[0065] Figure 21This study presents a dose-response analysis of tail vein injection of M3mP6 HLPN in inhibiting occlusive thrombosis using a 7.5% FeCl3-induced carotid artery thrombosis model. It should be noted that the effective dose is 1.25 μmol peptide / kg or higher. (For M3mP6 HLPN, 1.25 and 2.5 μmol / kg, n = 3; for M3mP6 HLPN, 5 μmol / kg, n = 5; for M3mP6 HLPN, 10 μmol / kg, n = 6).

[0066] Figure 22 The effects of 50% vs. 36.8% M3mP6 HLPN on FeCl3-induced thrombosis are shown. Using a FeCl3-induced carotid artery thrombosis model, the antithrombotic effect of HLPN composed of 50% (mass / mass) M3mP6 peptide, 40% DSPE-PEG, and 10% PC (5 μmol / kg peptide injection) was comparable to that of HLPN composed of 36.8% M3mP6, 51.7% DSPE-PEG, and 11.5% PC (5 μmol / kg peptide injection) (n=6).

[0067] Figures 23A-23B This paper presents a comparison of platelet thrombosis and fibrin production in mice treated with 10 μmol / kg M3mP6 HLPN and 30 μg / kg cangrarolol in an in vivo laser-induced cremasteric arteriolar thrombosis model. Platelet counts over time were measured in a total of 27 injuries. Figure 23A ) or fibrin ( Figure 23B The median of total fluorescence. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0068] Figure 24 The table of average pharmacokinetic parameters for the M3mP6 peptide is shown. * Standard error; NA - Standard error that cannot be calculated due to sample size <3, IVB - Intravenous bolus, IVI - Intravenous infusion, Cmax - Maximum plasma concentration, Tmax - Cmax time, Css - Steady-state plasma concentration during continuous intravenous infusion, AUC0-last - Area under the plasma concentration-time curve from time zero (administration of intravenous bolus) to the last plasma concentration (48 hours after cessation of infusion), AUC0-∞ - AUC from time zero to infinity, λz - Terminal elimination rate constant, t1 / 2-λz - Terminal elimination half-life, CLAUC - Clearance estimated by AUC0-∞, CLcss - Clearance estimated by Css, Vλz - Volume of distribution.

[0069] Figure 25A-25IMB2mP6 was shown to block the interaction of Gα13 with β2 and β3 integrins and inhibit macrophage and platelet function without causing bleeding. Figure 25A Representative immunoblotting was performed on THP-1 cells differentiated from PMA cells pretreated with MB2mP6 or control peptide micelles (50 μM) for 20 min and then stimulated with LPS (100 ng / mL) to obtain co-immunoprecipitates of β2 integrin and Gα13 with anti-β2 antibody. Immunoprecipitates and total cell lysates were then immunoblotted with anti-β2 integrin and anti-Gα13 antibodies. Figure 25B Representative immunoblots were generated from the co-immunoprecipitation of β3 integrin and Gα13 with anti-β3 antibody in human platelets pretreated with MB2mP6 or control peptide micelles (50 μM) for 5 min and then stimulated with thrombin (0.025 U / mL) at different time points. Immunoprecipitates and total cell lysates were immunoblotted separately with both anti-β3 integrin and anti-Gα13 antibodies. Figure 25C and 25D MB2mP6 micelles inhibited LPS (100 ng / mL)-stimulated expression of Il-1β (c) and Il-6 (d) mRNA in mouse BMDM. Changes in mRNA expression levels were detected by qRT-PCR. All groups, n = 4. A representative thrombin (0.03 U / mL)-induced human platelet aggregation tracer showed dose-dependent inhibition of aggregation by MB2mP6 micelles. Figure 25F Representative studies have shown that ADP secretion induced by thrombin (0.03 U / mL) in washed human platelets was inhibited in a dose-dependent manner by MB2mP6 micelles. Figure 25G and 25H The effects of MB2mP6 and M3mP6 (Myr-FEEERL (SEQ ID NO:25)) on FeCl3-induced carotid artery thrombosis ( Figure 25G ) and tail bleeding time ( Figure 25H Comparison of the following: Control, n=29; MB2mP6, n=14; M3mP6, n=29. In the reverse passive Arthus (rpA) assay, MB2mP6 did not enhance inflammation-induced bleeding compared to the disordered peptide. All data are shown as mean ± sem. Data in c and d were analyzed by two-way ANOVA and post-hoc Tukey multiple comparisons test; **p<0.01, ***p<0.001. Analysis was performed by two-tailed unpaired t-test. Figure 25G , 25H Data from 25I. ns, not significant, **p<0.01, ***p<0.001, ****p<0.0001.

[0070] Figures 26A-26FMB2mP6 was shown to enhance the survival rate of a CLP sepsis model and prevent sepsis-induced organ damage. Figure 26A Compared with the disordered peptide or saline treatment groups, immediate MB2mP6 treatment after CLP surgery enhanced the survival rate of sepsis-affected C57BL / 6 mice in the CLP model. All mice in the sham-operated group survived. Significance was determined using the Mantel-Cox test with GraphPad Prism software. Sham surgery, n=6; disordered peptide, n=23; MB2mP6, n=24; saline, n=18. Figure 26B Six hours after the onset of CLP, MB2mP6 treatment enhanced the survival rate of septic C57BL / 6 mice in the CLP model. All mice in the sham-operated group survived. Significance was determined using the Mantel-Cox test with GraphPad Prism software. Sham-operated, n=6; saline, n=24; MB2mP6, n=19. Figure 26C-26D MB2mP6 inhibited the selected cytokines IL-6 and [the protein level in mouse serum measured by ELISA 24 hours after CLP]. The expression. Sham surgery, n=6; random order, n=8; MB2mP6, n=10. Figure 26E Representative images of fibrin deposition detected in mouse glomeruli by PTAH staining 24 hours after CLP. Bar charts represent the percentage of PTAH-positive stained area in each glomerulus (30 random glomeruli from 6 mice / group). Sham operation, n=6; randomized, n=30; MB2mP6, n=30. Scale bar. Figure 26F Compared with mice treated with disordered peptides, plasma BUN levels in septic mice were suppressed 24 hours after CLP in MB2mP6-treated mice. Sham-operated, n=6; disordered, n=9; MB2mP6, n=9. All data are shown as mean ± sem. Analysis was performed using a two-tailed unpaired t-test and Welch correction. Figure 26C , 26D Data from 26F; analyzed using a two-tailed nonparametric Mann-Whitney test. Figure 26E The data in the table are: *p<0.05, **p<0.01, ***p<0.001.

[0071] Figure 27A-27J The study showed that leukocyte or platelet-specific Gα13 knockout reduced mortality in CLP-induced septic mice. Figure 27A , with Gα13 fl / fl Compared with control mice, Gα13 fl / fl-LysMCre CLP-induced mouse survival rate increased. All mice in the sham-operated group survived. Gα13fl / fl Sham surgery, n=6; Gα13 fl / fl CLP, n = 20; Gα13 fl / fl-LysMCre Sham surgery, n=6; Gα13 fl / fl-LysMCre CLP, n=25. Significance was determined using the Mantel-Cox test with GraphPad Prism software. Figure 27B and 27C Gα13 fl / fl-LysMCre and Gα13 fl / fl Serum levels of selected cytokines IL-6 and TNFα in septic mice 24 hours after CLP in control mice. All sham-operated groups, n=6; all CLP groups, n=10. Figure 27D Representative images of immunohistochemical staining of fibrin deposition in mouse kidneys 24 hours after CLP. Bar graphs represent the percentage of fibrin-positive stained area in each glomerulus (30 random glomeruli from 6 mice / group). All groups, n=30. Scale bar, 100 μm. Figure 27E Plasma levels of BUN in septic mice 24 hours after CLP showed Gα13 levels. fl / fl-LysMCre Mice and Gα13 fl / fl There were no differences among mice. All sham-operated groups, n=5; all CLP groups, n=10. Figure 27F , with Gα13 fl / fl Compared with control mice, Gα13 fl / fl-PF4Cre CLP-induced sepsis increased the survival rate of mice. All mice in the sham-operated group survived. Gα13 fl / fl Sham surgery, n=6; Gα13 fl / fl CLP, n = 26; Gα13 fl / fl-PF4Cre Sham surgery, n=7; Gα13 fl / fl-PF4Cre CLP, n=17. Significance was determined using the Mantel-Cox test with GraphPad Prism software. Figure 27G Representative images of immunohistochemical staining of fibrin deposition in mouse kidneys 24 hours after CLP. Bar graphs represent the percentage of fibrin-positive stained area in each glomerulus (30 random glomeruli from 6 mice / group). All groups, n=30. Scale bar, 100 μm. Figure 27H , with Gα13 fl / fl Compared to mice, in Gα13 fl / fl-PF4Cre In mice, plasma BUN levels were decreased in septic mice 24 hours after CLP. All groups, n=9. Figure 27I and 27J Gα13 fl / fl-PF4Cre and Gα13fl / fl Serum levels of selected cytokines TNFα and IL-6 in septic mice 24 hours after CLP in control mice. All groups, n=8. All data are shown as mean ± sem. Analysis was performed using two-way ANOVA and multiple comparisons. Figure 27B , 27C Data in 27H-J were analyzed using a two-tailed nonparametric Mann-Whitney test for d and g. The results were not significant (ns), *p<0.05, **p<0.01, ***p<0.001.

[0072] Figures 28A-28D This study demonstrated that platelet and leukocyte dual-specific Gα13 knockout protects mice from death in a CLP sepsis model. Figure 28A , with Gα13 fl / fl Compared with control mice, Gα13 fl / fl-PF4 / LysMCre CLP-induced sepsis increased the survival rate of mice. All mice in the sham-operated group survived. Gα13 fl / fl Sham surgery, n=6; Gα13 fl / fl CLP, n = 17; Gα13 fl / fl-PF4 / LysMCre Sham surgery, n=7; Gα13fl / fl-LysMCre CLP, n=13. Significance was determined using the Mantel-Cox test with GraphPadPrism software. Figure 28B , with Gα13 fl / fl Compared to mice, in Gα13 fl / fl-PF4 / LysMCre In mice, plasma BUN levels were decreased in septic mice 24 hours after CLP. All groups, n=9. Figure 28C and 28D , with Gα13 fl / fl Compared with control mice, in Gα13 fl / fl-PF4 / LysMCre In mice, serum levels of the selected cytokines IL-6 and TNFα were decreased in septic mice 24 hours after CLP. All groups, n=9. Figure 28E Representative images of immunohistochemical staining of fibrin deposition in mouse kidneys 24 hours after CLP. Bar charts represent the percentage of fibrin-positive stained area per glomerulus (30 random glomeruli from 6 mice / group). All groups, n=30. Scale bar, 100 μm. All data are shown as mean ± sem. Data in bd were analyzed by two-way ANOVA and multiple comparisons; data in e were analyzed by two-tailed nonparametric Mann-Whitney test; *p<0.05, **p<0.01, ***p<0.001.

[0073] Figures 29A-29DThe study demonstrated that MB2mP6 inhibited glomerular microvascular thrombosis and renal function impairment in CLP-infected mice. Figure 29A Representative images of immunohistochemical staining of fibrin in the lungs of mice after CLP (n=24). Bar charts represent the percentage of fibrin-positive stained area in each glomerulus (30 random glomeruli from 6 mice / group). Sham operation, n=6; randomized, n=30; MB2mP6, n=30. Scale bar, 100 mm. Figure 29B Representative images of immunohistochemical staining of platelet αIIb in lung 24 cells of mice after CLP. Bar graphs represent the percentage of αIIb-positive stained area in each glomerulus (30 random glomeruli from 6 mice / group). Sham operation, n=6; randomized, n=30; MB2mP6, n=30. Scale bar, 100 mm. Figure 29C Compared with mice treated with disordered peptides, serum creatinine levels in septic mice 24 hours after CLP were suppressed in MB2mP6-treated mice. (Sham operation, n=6; disordered peptide, n=8; MB2mP6, n=10) Figure 29D Compared with mice treated with disordered peptides, serum cystatin C levels were suppressed in MB2mP6-treated septic mice 24 hours after CLP. Sham operation, n=6; disordered peptide, n=8; MB2mP6, n=10. All data are shown as mean ± sem. Analysis was performed using a two-tailed nonparametric Mann-Whitney test. Figure 29A and 29B Data in c and d were analyzed by two-tailed unpaired t-test and Welch correction; *p<0.05, **p<0.01, ***p<0.001.

[0074] Figures 30A-30B The generation of platelet-specific, leukocyte-specific, and platelet- and leukocyte-specific Gα13 knockout mice. Figure 30A A schematic diagram of the reproductive strategy of Gα13 knockout mice. Figure 30B Representative protein blots for Gα13 protein analysis.

[0075] Figures 31A-31B The importance of Gα13 in transendothelial migration of mouse neutrophils. Figure 31A Gα13 in Gα13 fl / fl (Gα 13 + / + ) and Gα13 fl / fl LysM cre (Gα 13 - / - Comparison of Western blots of proteins expressed in mouse neutrophils (mPMNs) (n=3). Figure 31B and 31CThe total number of neutrophils isolated from bone marrow (B) (n=6) and blood (C) (n=6). Figure 31D Gα13 - / - Neutrophils and Gα13 + / + Comparison of in vitro transendothelial migration of formyl peptide among neutrophils (n=3). t-tests were used for statistical analysis. *P<0.05, **P<0.01, ***P<0.001, ns, not significant.

[0076] Figures 32A-32B The importance of ICAM1 and β2 integrins in Gα13-dependent transendothelial migration. Figure 32A In the absence or presence of 10 nM fMIVIL, Gα was compared using transporous filters pre-coated with 5% BSA, fibrinogen (60 μg / ml), and ICAM1 (10 μg / ml). 13 + / + and Gα 13 - / - Neutrophil migration. Figure 32B In the presence of 10 nM fMIVIL, the anti-β2 antibody effectively blocks the action of Gα13. fl / fl Neutrophils and Gα13 fl / fl LysM cre The effect of two types of neutrophils (n=3) on transendothelial migration. Two-way ANOVA and Tukey's multiple comparison test were used in Figures A and B. ****P<0.0001; ns, not significant.

[0077] Figures 33A-33B :Gα 13 Its role in neutrophil migration on the ICAM1 surface. Figure 33A Gα 13 fl / fl (Gα 13 + / + 78 cells), Gα 13 fl / fl LysM cre (Gα 13 - / - Comparison of the migration rates of neutrophils (47 cells) on a slide coated with ICAM1 (10 μg / ml). Figure 33B Gα 13 + / + (78 cells) and Gα 13 - / - Comparison of total distance traveled by neutrophils (47 cells) during migration. Figure 33C Gα 13 + / + (78 cells) and Gα 13 - / -Comparison of Euclidean distance (a straight line between the start and end points) between neutrophil migrations (47 cells). Figure 33D Rose diagram represents Gα 13 + / + (cells) and Gα 13 - / - Orientation of neutrophil migration trajectories (cells). Dark areas indicate the frequency of trajectory orientation towards 12 different segments over 30 minutes. Cell percentage in each segment is shown in black. Figure 33E Cell position at the 30-minute time point relative to the 0-minute time point was quantified in three categories: towards, away from, or neutral, relative to the fMIVIL loading site. (Black text indicates the percentage of cells in each region). Studen's t-test was used. Figure 33A and 33B The Mann-Whitney test is used for Figure 33C Furthermore, the chi-square test is used for... Figure 33E ns, not significant, *P<0.05, ***P<0.001, ****P<0.0001.

[0078] Figures 34A-34B MB2mP6 blocks Gα in human neutrophils 13 Interaction with β2 integrin. Figure 34A Representative immunoblots of β2 integrin and Gα13 in human neutrophils co-precipitated with anti-β2 antibody. PMNs were pretreated with MB2mP6 or control peptide micelles (50 mM) for 10 min and then stimulated with fMLP (1 μM) in 6-well plates pre-coated with ICAM1 (10 μg / mL). PMN samples were collected at specified time points. The samples were then treated with anti-β2 integrin and anti-Gα13 antibody. 13 Both antibodies were used to perform immunoblotting on immunoprecipitates and total cell lysates. Figure 34B The co-immunoprecipitation blots in Figure A (n=4) were quantitatively analyzed using ImageJ software. OD (optical density) was used.

[0079] Figures 35A-35B β2EXE motif peptide MB2mP6 inhibits transendothelial neutrophil migration. Figure 35A Comparison of mouse neutrophil adhesion to ICAM1 after treatment with MB2mP6 (50 μM) or control (Ctrl) peptide (50 μM) (n=3). Figure 35B The dose-dependent inhibitory effect of MB2mP6 on neutrophil transendothelial migration. Figure 35C MB2mP6 (50 μM) for Gα 13 + / + and Gα 13- / - The influence of neutrophil transendothelial migration. Figure 35D The effect of MB2mP6 (50 μM) pretreatment on neutrophil migration was investigated using a transporous filter coated with endothelial cells, ICAM1, or fibrinogen (60 μg / ml). One-way ANOVA was used... Figure 35B , 35C And 35D. *P<0.05, **P<0.01, ***P<0.001, ns, not significant.

[0080] Figures 36A-36B Ga 13 Its role in neutrophil adhesion and diffusion. Figure 36A , comparison and Ga 13 Adhesion of knockout neutrophils on ICAM1 was compared (t-test, ns, not significant, 20 frames randomly selected). Figure 36B DIC images of mouse neutrophils adhering to ICAM1. (t = 0 min, Gα13) + / + 3 cells, Gα13 - / - 2 cells, t=5 minutes, Gα13 + / + 23 cells, Gα13 - / - (26 cells). Scale bar, 5 μm. Figure 36C , such as in ( Figure 36B Similar to the previous method, the size of neutrophils plated on an ICAM-coated surface was quantified using real-time DIC images at the indicated time points. The size of non-adhesive neutrophils (t=0) was estimated by measuring cells (n=5) on the BSA surface. One-way ANOVA, ***P<0.001, ns, not significant. Figure 36D ,exist Figure 36B and 36E The percentage of cells with membrane folds in the fixed control (Gα) 13 + / + ) and Gα 13 Knockout (Gα) 13 - / - Neutrophil size was quantified by adhering to ICAM1 at 37°C for 15 minutes (n=36, p<0.0005, Mann-Whitney U test). Figure 36F Human neutrophils treated with fixed AAA or MB2mP6 peptides adhered to ICAM1 at 37°C for 15 minutes and were quantified by size (n=58, p<0.0001, Mann-Whitney U test).

[0081] Figures 37A-37B :Gα 13 Its role in neutrophil migration in vivo. Figure 37A Ga 13+ / + With Gα 13 - / - Comparison of peritoneal neutrophil infiltration induced by thioglycolate (n=4). Figure 37B , compared with control Ga 13 + / + Compared to mice (n=5), by using Gα13 - / - Lung neutrophil migration in mice stimulated by LPS implanted in the trachea. One-way ANOVA was used for all figures. *P<0.05, ***P<0.001.

[0082] Figures 38A-38C MB2mP6 inhibits CLP-induced microvascular thrombosis and vascular leakage in the lungs of septic mice. Figure 38A C57BL mice were immediately infused with MB2mP6 or out-of-order peptides after a CLP attack. Twenty-four hours later, the lungs were removed and sections were taken, and fibrin was stained with phosphotungstic acid hematoxylin (PTAH) (blue stain). Bar graph: mean (±SD) percentage of stained area per mm² (**P<0.01; t-test and Welch correction). Figure 38B and 38C C57BL mice immediately after CLP ( Figure 38B ) or 6 hours ( Figure 38C Mice were infused with MB2mP6 or saline. Twenty-three hours later, Evans blue albumin (EBA; 1%, 25 mg / kg body weight) was injected intravenously into the mice. One hour later, mouse lungs were harvested, and Evans blue was extracted with formamide at 60°C for 18 hours, and quantified by absorbance at 620 nm. Bar graph: mean (±SD), (*P<0.05, **P<0.01; one-way ANOVA). Detailed Implementation

[0083] This article discloses three significant advances: (1) the development of novel lipid-stabilized high-load peptide nanoparticles (HLPNs) for the efficient delivery of a novel ExE motif peptide (M3mP6) into intracellular compartments for therapeutic purposes in vivo; (2) data demonstrating that M3mP6 HLPN effectively inhibits occlusive thrombosis without observable side effects of bleeding; and (3) post-ischemic injection of M3mP6 HLPN also inhibits microvascular thrombosis / inflammation and improves cardiac function and survival in a mouse model of MI / R injury. Importantly, this novel drug not only demonstrates antithrombotic synergy with current standard-of-care antiplatelet drugs (P2Y12 inhibitors) while minimizing the risk of bleeding, but also shows significant therapeutic effects on MI / R injury when injected post-ischemic. Therefore, the drug M3mP6 HLPN has the potential to greatly advance current antiplatelet therapies for heart attacks and improve drug safety.

[0084] Despite their advantages of high specificity due to their unique amino acid sequences and low risk of nonspecific toxic effects, the development of peptide-based drugs targeting intracellular compartments remains lagging (1, 2). One of the main reasons is the lack of technology to efficiently deliver sufficient amounts of peptides into cells in vivo. Therefore, a breakthrough in the efficient delivery of peptides into cells should greatly facilitate the development and therapeutic use of peptide-based therapeutics. Many tools have been developed for delivering peptides into cells. Among them, lipolysis makes the peptide membrane permeable. However, myristylated peptide M3mP6, while membrane permeable in vitro and inhibiting platelet function, is ineffective in vivo. Therefore, membrane permeability is insufficient to deliver peptides into platelets in vivo. Liposomes and lipid micelles are relatively more efficient and are frequently used for in vivo drug delivery (2). However, these methods typically require large amounts of lipids to encapsulate limited amounts of peptides, thus requiring exceptionally high peptide affinity or affinity for their targets. For example, lipid micelle formulations of the synthetic peptide mP6 contain up to 4% (mol / mol) of peptide, with a maximum concentration of mP6 peptide <1 mM, which is insufficient for clinical use (21). In this study, the lipid-stabilized M3mP6 HLPN contained up to 70% (mol / mol) of peptide, achieving a high peptide concentration of >10 mM suitable for human bolus IV injection. Importantly, the lipid-stabilized HLPN also significantly facilitated the entry of the peptide drug into cells, resulting in a much smaller peptide concentration required to achieve comparable antiplatelet efficacy. These superior properties make M3mP6 HLPN a potent in vivo antithrombotic agent. Therefore, these data suggest that the lipid-stabilized HLPN significantly improves the intracellular delivery efficiency of peptide-based drugs in vivo and enables the efficient use of peptide drugs with relatively moderate affinity for their intracellular targets. Thus, this new technique may have general significance in promoting the development of peptide-based drugs targeting intracellular compartments for therapeutic purposes.

[0085] Platelets circulating in the bloodstream are typically in a resting state and are only activated upon exposure to platelet agonists. Platelet agonists induce platelet activation through various receptor-mediated intracellular signaling pathways (28, 29). These intracellular signals converge into an "inside-out" signal to transition αIIbβ3 from a "resting" state to an "activated" state (8, 30). This makes integrin ligand binding a necessary condition for platelet adhesion and aggregation (12). Current antiplatelet drugs either inhibit the platelet activation signaling pathways that lead to integrin activation or directly inhibit the ligand binding function of integrin αIIbβ3 (10, 12). Although these drugs have become cornerstones in the treatment of patients with cardiovascular disease (6, 12), they also inhibit hemostasis, leading to excessive bleeding, which can be life-threatening (11–14). The bleeding effects of these drugs limit the optimal use of effective doses and thus limit their antithrombotic efficacy. Therefore, bleeding in thrombotic patients undergoing peripheral coronary intervention is associated with poor prognosis and increased mortality (11, 15-17). Thus, balancing optimal platelet inhibition with minimal bleeding complications remains a significant clinical challenge, particularly for patients requiring invasive procedures or suffering from thrombotic stroke, where bleeding is life-threatening. Recently, various thrombin receptor inhibitors have been shown to reduce bleeding as a side effect in animal models (19, 20). However, the reduction in bleeding side effects is partial and associated with clopidogrel (19, 20). Bleeding was still significantly increased in animals administered these drugs compared to normal controls (19, 20). In contrast, no difference in bleeding was observed between normal controls and animals administered M3mP6 HLPN. Importantly, clinical trials of the PAR1 inhibitor vorapazal showed a significant increase in bleeding in human patients (19), indicating that the reduced bleeding side effects of thrombin receptor inhibitors still pose a clinically relevant risk. The hemostatic effect of these drugs is consistent with their inhibition of the activation of integrin αIIbβ3 ligand binding function, and therefore the hemostatic effect of these drugs is not fundamentally different from that of current antiplatelet drugs (29). The novel peptides disclosed herein are conceptually based on a completely different concept: selectively targeting integrin outward-inward signaling without affecting integrin ligand binding to platelets. Based on this concept, the disclosed peptides allow primary platelet adhesion and aggregation to occur, but prevent pathological thrombus expansion (21, 22). Using the HLPN delivery system of this peptide-based novel drug as described above, HLPNs including the peptides have been shown to effectively inhibit occlusive thrombus formation without any observable effect on hemostasis in mice and dogs, and this was demonstrated in mice using tail bleeding time and carotid perforation bleeding analysis.Therefore, by designing a lipid-stable HLPN delivery system, this paper discloses a potential clinically available drug that may be superior to currently used or under development antiplatelet drugs.

[0086] This disclosure provides a lipid-stabilized, high-load peptide nanoparticle comprising: i) a lipotropic peptide, amphiphilic peptide, or peptide-hydrophobic conjugate comprising more than about 10 mole percent of the total nanoparticle mass; ii) one or more lipids free of water-soluble polymers comprising about 2 to about 20 mole percent of the total nanoparticle mass; and iii) one or more lipids covalently linked to water-soluble polymers comprising about 10 to about 60 mole percent of the total nanoparticle mass. For example, lipoactive peptides consist of about 2 to about 50 amino acid residues, or about 2 to about 45 amino acids, or about 2 to about 40 amino acids, or about 2 to about 35 amino acids, or about 2 to about 30 amino acids, or about 2 to about 25 amino acids, or about 2 to about 20 amino acids, or about 2 to about 15 amino acids, or about 2 to about 10 amino acids, or about 2 to about 5 amino acids, or about 3 to about 40 amino acids, or about 3 to about 25 amino acids, or about 4 to about 35 amino acids, or about 4 to about 25 amino acids, or about 5 to about 50 amino acids, or about 5 to about 25 amino acids, or about 6 to about 50 amino acids, or about 6 to about 25 amino acids, or about 10 to about 25 amino acids.

[0087] In some embodiments, the lipid-stabilized high-load peptide nanoparticles comprise about 10 to about 80 mol%, or about 10 to about 75 mol%, or about 10 to about 70 mol%, or about 10 to about 65 mol%, or about 10 to about 60 mol%, or about 10 to about 55 mol%, or about 10 to about 50 mol%, or about 10 to about 45 mol%, or about 10 to about 40 mol%, or about 40 to about 55 mol%, or about 40 to about 75 mol%, or about 50 to about 80 mol%, or about 60 to about 80 mol%, or about 70 to about 80 mol%, based on the total nanoparticle mass.

[0088] In some embodiments, the lipid-stabilized high-load peptide nanoparticles comprise one or more lipids that do not contain water-soluble polymers, ranging from about 2 to about 20 mol percent, or about 2 to about 15 mol percent, or about 2 to about 10 mol percent, or about 2 to about 5 mol percent, or about 5 to about 20 mol percent, or about 5 to about 10 mol percent.

[0089] In some embodiments, the lipid-stabilized high-load peptide nanoparticles comprise one or more lipids covalently linked to a water-soluble polymer, in a molar percentage of about 10 to about 60, or about 10 to about 55, or about 10 to about 50, or about 10 to about 45, or about 10 to about 40, or about 10 to about 35, or about 10 to about 30, or about 10 to about 25, or about 10 to about 20, or about 10 to about 15.

[0090] The foregoing arguments of this disclosure have been presented for purposes of illustration and description. The foregoing is not intended to limit this disclosure to one or more of the forms disclosed herein. While the description of this disclosure includes one or more embodiments and descriptions of certain variations and modifications, other variations and modifications are also within the scope of this disclosure, for example, as may be within the skill and knowledge of one skilled in the art upon understanding this disclosure. It is intended to provide for the inclusion of alternative embodiments within the permitted scope, including structures, functions, scopes, or steps that are alternative, interchangeable, and / or equivalent to the claimed structure, function, scope, or steps, whether or not such alternative, interchangeable, and / or equivalent structures, functions, scopes, or steps are disclosed herein, and is not intended to disclose any patentable subject matter.

[0091] Nanoparticles

[0092] Suitable lipids for preparing micelles and liposomes for encapsulating compounds and peptides for drug delivery are known in the art. See, for example, Banerjee and Onyuksel, Peptide Delivery Using Phospholipid Micelles, WIREs Nanomed Nanobiotechnol 4:562-574 (2012). Representative lipids include phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), etc. Representative lipids linked to water-soluble polymers include fatty acids or mixtures of fatty acids conjugated to PEG (poly(ethylene glycol))-PE, PEG-PC, PEG-PG, PEG-PI, PEG-PS, PEG-DSPE, etc. In a particular embodiment, the lipid covalently linked to the water-soluble polymer is 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], and the lipid not containing the water-soluble polymer is phosphatidylcholine. In an exemplary embodiment, the lipid-stabilized, high-load peptide nanoparticles comprise 2-10% phosphatidylcholine and 10-60% PEG-DSPE. In various aspects, the PEG-DSPE may comprise PEG with a molecular weight of 200-100,000 or other PEGs known in the art.

[0093] In some embodiments, peptide modification allows the peptide to form the bulk of the micelle nanoparticle composition, thereby significantly increasing the peptide concentration incorporated into the micelle nanoparticles and thus increasing in vivo delivery efficiency. In an exemplary aspect, the peptide of the nanoparticle is covalently linked to a fatty acid or other lipid moiety. Lipidified peptides, amphiphilic peptides, or peptide-hydrophobic conjugates are capable of forming micelle nanoparticles, which can be stabilized by the presence of certain lipids. In some embodiments, HLPN-stabilized lipids can be covalently linked to a water-soluble polymer and a lipid without the water-soluble polymer. Such lipid-stabilized high-load peptide nanoparticles (HLPNs) differ from lipid micelles encapsulating peptides, where a high concentration of peptide forms the bulk of the micelle nanoparticles, thereby improving delivery efficiency. Lipids used for micelle synthesis are known in the art. See, for example, Banerjee and Onyuksel, Peptide Delivery Using Phospholipid Micelles, WIRE Nanomedicine & Nanobiotechnology 4:562-574 (2012). In an exemplary aspect, the lipid covalently linked to the water-soluble polymer is 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], and the lipid not containing the water-soluble polymer is phosphatidylcholine.

[0094] In other embodiments, the lipids free of the water-soluble polymer are selected from phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine. In a particular embodiment, the lipid covalently linked to the water-soluble polymer is PEG-DSPE, and the lipid free of the water-soluble polymer is phosphatidylcholine.

[0095] In an exemplary aspect, modifying esterified FEEERA (SEQ ID NO:6) into esterified FEEERL (SEQ ID NO:8), FEEERI (SEQ ID NO:2), FEKEKL (SEQ ID NO:10), FKEKEI (SEQ ID NO:44), or FEEERM (SEQ ID NO:7) promotes the formation of stable micelle nanoparticles, wherein the micelle nanoparticles contain a peptide concentration greater than about 4-10 mM compared to the original 1 mM peptide concentration in FEEERA (SEQ ID NO:6) in the micelle formulation. In some embodiments, the peptide concentration is approximately 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6... 9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.6, 9.6, 9.7, 9.8, 9.9 or approximately 10mM.

[0096] Representative peptides suitable for formulation into lipid-stabilized high-load peptide nanoparticles, consisting of approximately 2 to approximately 50 amino acid residues, include FEEERI (SEQ ID NO:2), FEKEKI (SEQ ID NO:3), FEKERI (SEQ ID NO:4), RGT, EEERA (SEQ ID NO:5), FEEERA (SEQ ID NO:6), FEEERM (SEQ ID NO:7), FEEERL (SEQ ID NO:8), FEKEKM (SEQ ID NO:9), FEKEKL (SEQ ID NO:10), FEKERM (SEQ ID NO:11), FEKERL (SEQ ID NO:12), CFEEERAC (SEQ ID NO:13), FEEERAR (SEQ ID NO:14), FEEERARA (SEQ ID NO:15), SIRYSGHpSL (SEQ ID NO:16), and KFEEERARAKWDT (SEQ ID NO:17). Other peptides can also be formulated into HLPNs for delivery into cells, such as RCLLPA (SEQ ID NO:45) (Rusu et al., Blood 123(3):442-50, 2014) and LLARRPTKGIHEY (SEQ ID NO:18) (Huang JS et al., JBC 282,10210-10222, 2007).

[0097] In some embodiments, the esterified peptide comprises a peptide consisting of about 2 to about 50 amino acid residues conjugated to a fatty acid. In other embodiments, the fatty acid is covalently linked to the peptide. In still other embodiments, the peptide is myristylated.

[0098] In other embodiments, the lipid-stabilized peptides are selected from myr-FEEERI (SEQ ID NO:19), myr-FEKEKI (SEQ ID NO:20), myr-FEKERI (SEQ ID NO:21), myr-RGT, myr-EEERA (SEQ ID NO:22), myr-FEEERA (SEQ ID NO:23), myr-FEEERM (SEQ ID NO:24), myr-FEEERL (SEQ ID NO:25), myr-FEKEKM (SEQ ID NO:26), myr-FEKEKL (SEQ ID NO:27), myr-FEKERM (SEQ ID NO:28), myr-FEKERL (SEQ ID NO:29), myr-CFEEERAC (SEQ ID NO:30), myr-FEEERAR (SEQ ID NO:31), myr-FEEERARA (SEQ ID NO:32), myr-SIRYSGHpSL (SEQ ID NO:19), myr-FEEERI (SEQ ID NO:20), myr-FEKEKI (SEQ ID NO:20), myr-FEKERI (SEQ ID NO:21), myr-FEEERARA (SEQ ID NO:32), myr-SIRYSGHpSL (SEQ ID NO:20), myr-FEEERI (SEQ ID NO:20), myr-FEKEKI ... (SEQ ID NO:33), myr-KFEEERARAKWDT (SEQ ID NO:34), or myr-LLARRPTKGIHEY (SEQ ID NO:35). In other embodiments, the lipid-stabilized peptides are myr-FEEERL (SEQ ID NO:25) or myr-FEKEKL (SEQ ID NO:27).

[0099] In some embodiments, the nanoparticles comprise about 10 to about 80 mol% of peptides based on the total mass of the nanoparticles, said peptides being composed of the amino acid sequences FEEERM (SEQ ID NO:7), FEEERL (SEQ ID NO:8), FEEERI (SEQ ID NO:2), FEKEKM (SEQ ID NO:9), FEKEKL (SEQ ID NO:10), FEKEKI (SEQ ID NO:3), FEKERM (SEQ ID NO:11), FEKERL (SEQ ID NO:12), FEKERI (SEQ ID NO:4), or CFEEERAC (SEQ ID NO:13).

[0100] In some embodiments, the nanoparticles comprise about 10 to about 80 mol% of peptides based on the total mass of the nanoparticles, said peptides being myr-FEEERL (SEQ ID NO:25) or myr-FEKEKL (SEQ ID NO:27).

[0101] In some embodiments, the nanoparticles include peptides disclosed in International Patent Application Publication No. WO / 2011 / 116026, U.S. Patent Nos. 8,685,921, 9,156,884, 10,011,634 and / or 10,738,080, all of which are incorporated herein by reference in their entirety.

[0102] This article also discloses a method for preparing lipid-stabilized high-load peptide nanoparticles, the method comprising stabilizing peptide nanoparticles comprising one or more lipotropic peptides consisting of about 2 to about 50 amino acid residues with 2-20% by weight of a water-insoluble polymer-free lipid and 10-60% by weight of a lipid covalently linked to a water-soluble polymer.

[0103] This article also discloses a method for treating thrombotic or inflammatory symptoms in a subject in need, the method comprising administering an effective amount of the nanoparticles or peptides disclosed herein to the subject. For example, this article discloses a method for treating thrombotic or inflammatory symptoms in a subject in need, the method comprising administering to the subject an effective amount of nanoparticles, the nanoparticles comprising: a lipotropic peptide of greater than about 10 mol percent based on the total nanoparticle mass; about 2 to about 20 mol percent of one or more lipids free of water-soluble polymers; and about 10 to about 60 mol percent of one or more lipids covalently linked to a water-soluble polymer, wherein the lipotropic peptide is myr-FEEERM (SEQ ID NO:24), myr-FEEERL (SEQ ID NO:25), myr-FEEERI (SEQ ID NO:19), myr-FEKEKM (SEQ ID NO:26), myr-FEKEKL (SEQ ID NO:27), myr-FEKEKI (SEQ ID NO:20), myr-FEKERM (SEQ ID NO:28), myr-FEKERL (SEQ ID NO:29), myr-FEKERI (SEQ ID NO:21), or myr-CFEEERAC (SEQ ID NO:28). IDNO:30).

[0104] In some embodiments, the inflammatory symptoms are sepsis, acute respiratory distress syndrome (ARDS), autoimmune diseases, AV fistulas in hemodialysis, organ transplantation, atherosclerosis, vasculitis, deep vein thrombosis, or ischemia-reperfusion injury.

[0105] In various exemplary embodiments, this disclosure provides a method for treating acute respiratory distress syndrome (ARDS), the method comprising administering to a subject an effective amount of the nanoparticles or peptides disclosed herein. ARDS can be caused by severe viral infections (such as COVID-19) and bacterial infections (such as sepsis) or aseptic inflammation (such as severe trauma and hypoxia). ARDS is often caused by excessive inflammation and blood clots obstructing small blood vessels in response to severe infection, trauma, etc., resulting in leakage of blood vessels in the lungs, causing a large amount of fluid to flow into the alveoli (small inflatable sacs lining the blood vessels used to exchange oxygen / CO2 with the blood). The fluid in the alveoli obstructs air exchange, thereby leading to hypoxia.

[0106] This article also discloses a method for treating a subject for a disease or symptom regulated by platelet granule secretion, Gα13 function, or leukocyte integrin outward-inward signaling, the method comprising administering to the subject an effective amount of a peptide comprising the amino acid sequence FEX1ERX2 (SEQ ID NO:1), wherein X1 and X2 are selected from natural and non-natural amino acids, wherein the peptide is a hexamer, heptamer, octamer, or decimer, and wherein the peptide inhibits integrin-dependent and integrin-independent platelet granule secretion, or wherein the peptide inhibits leukocyte integrin outward-inward signaling.

[0107] In some embodiments, X1 is glutamic acid (E), and X2 is alanine (A). In other embodiments, the peptide further includes a membrane-permeable portion. In other embodiments, the membrane-permeable portion is a membrane-permeable peptide, a membrane-permeable synthetic compound, and a fatty acid. In other embodiments, the membrane-permeable portion is a fatty acid. In other embodiments, the fatty acid is covalently linked to the peptide.

[0108] In a specific embodiment, the peptide is myr-FEEERA (SEQ ID NO:23), myr-FEEERM (SEQ ID NO:24), myr-FEEERL (SEQ ID NO:25), myr-FEKEKM (SEQ ID NO:26), myr-FEKEKL (SEQ ID NO:27), myr-FEKERM (SEQ ID NO:28), myr-FEKERL (SEQ ID NO:29), or myr-CFEEERAC (SEQ ID NO:30). In another specific embodiment, the peptide is incorporated into nanoparticles, wherein the nanoparticles comprise about 36 to about 80 mol% of the peptide.

[0109] In some embodiments, the disease or condition is regulated by platelet granule secretion. In specific embodiments, the disease or condition regulated by platelet granule secretion is selected from inflammatory conditions. More specifically, inflammatory conditions are selected from atherosclerosis, autoimmune diseases, and inflammation.

[0110] In other embodiments, the disease or symptom is regulated by leukocyte integrin outward-inward signaling. In a specific embodiment, the disease or symptom regulated by leukocyte integrin outward-inward signaling is selected from sepsis, atherosclerosis, vasculitis, deep vein thrombosis, and ischemia-reperfusion injury.

[0111] In various embodiments, the peptides disclosed herein are anti-inflammatory and inhibit leukocyte migration. In related embodiments, the inhibition of migration is achieved without completely eliminating the important cell adhesion function of leukocytes mediated by the adhesion receptor integrin family and without eliminating the function of neutrophils in sensing sites of infection or inflammation.

[0112] The methods disclosed herein for treating diseases or symptoms regulated by platelet granule secretion, Gα13 function, or leukocyte integrin outward-inward signaling may further include administering an effective amount of a P2Y12 inhibitor. In some embodiments, the P2Y12 inhibitor is selected from clopidogrel, ticagrelor, presugrel, and canagrelor. In other embodiments, the method may further include administering an effective amount of aspirin.

[0113] This article also discloses a method for inhibiting integrin-inward signaling in a subject without inhibiting integrin-independent platelet granule secretion, the method comprising administering to the subject an effective amount of a peptide comprising the amino acid sequence of EEERA (SEQ ID NO:5), EEERM (SEQ ID NO:36), EEERL (SEQ ID NO:37), EKEKM (SEQ ID NO:38), EKEKL (SEQ ID NO:39), EKERM (SEQ ID NO:40), EKERL (SEQ ID NO:41), or CEEERAC (SEQ ID NO:42). Such a peptide may be useful in conditions requiring platelet granule secretion during the treatment of thrombosis (e.g., for promoting wound healing).

[0114] In one embodiment, the peptide that inhibits both integrin-dependent and integrin-independent granule secretion and leukocyte function is FEEERA (SEQ ID NO:6).

[0115] This article also discloses peptides composed of the amino acid sequences FEEERI (SEQ ID NO:2), FEKEKI (SEQ ID NO:3), or FEKERI (SEQ ID NO:4).

[0116] In some embodiments, the peptide further includes a membrane-permeable portion. In other embodiments, the membrane-permeable portion is selected from membrane-permeable peptides, membrane-permeable synthetic compounds, and fatty acids. In other embodiments, the membrane-permeable portion is a fatty acid. In other embodiments, the fatty acid is covalently linked to the peptide. In a particular embodiment, the peptide is myristylated. In yet another particular embodiment, the peptide is selected from myr-FEEERI (SEQ ID NO:19), myr-FEKEKI (SEQ ID NO:20), and myr-FEKERI (SEQ ID NO:21).

[0117] This disclosure also provides a method for treating thrombotic or inflammatory symptoms in a subject in need, the method comprising administering to the subject an effective amount of a peptide consisting of the amino acid sequences FEEERI (SEQ ID NO:2), FEKEKI (SEQ ID NO:3), or FEKERI (SEQ ID NO:4).

[0118] The disclosed peptides can be obtained by methods known in the art. Suitable methods for de novo synthesis of peptides are described, for example, in Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, edited by Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, edited by Westwood et al., Oxford University Press, 2000; and U.S. Patent No. 5,449,752. Further exemplary methods for preparing the peptides of this disclosure are described herein.

[0119] In some embodiments, the peptides described herein are commercially synthesized by companies such as Synpep (Dublin, California), Peptide Technologies Corp. (Gaithersburg, Maryland), Multiple Peptide Systems (San Diego, California), Peptide2.0 Inc. (Chantilly, Virginia), and American Peptide Co. (Sunnyvale, California). In this respect, the peptides may be synthetic, recombinant, isolated, and / or purified.

[0120] In addition, in some respects, peptides are generated by recombination of nucleic acids encoding the amino acid sequence of a peptide using standard recombination methods. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd ed., Cold Spring Harbor Press, NY, 2001; and Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing Associates and John Wiley & Sons, NY, 1994.

[0121] The peptides disclosed herein can be isolated and / or purified. As used herein, the term "isolated" means removed from its natural environment. As used herein, the term "purified" means with improved purity, wherein "purity" is a relative term and is not necessarily interpreted as absolute purity. In exemplary aspects, the purity of the compound (e.g., in the composition) is at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 95%, or at least or about 98% or about 100%.

[0122] Other peptide modifications

[0123] In some embodiments, the peptides of this disclosure include one or more modifications, including, but not limited to, phosphorylation, glycosylation, hydroxylation, esterification, cyclization, sulfonation, amidation, acetylation, carboxylation, esterification (e.g., myristylation, palmitoylation), introduction of non-hydrolyzable bonds, disulfide formation and conjugation, or linkage with a target peptide or carrier peptide, or conversion to an acid addition salt and / or optionally dimerization or polymerization or conjugation, as further described herein. Modifications can improve the stability and / or activity of the peptide.

[0124] For example, the C-terminus can be modified by amidation, addition of peptide alcohols and aldehydes, addition of esters, addition of p-nitroaniline and thioesters, and multiple antigenic peptides. The N-terminus and side chains can be modified by polyethylene glycolation, acetylation, formylation, addition of fatty acids, addition of benzoyl groups, addition of bromoacetyl groups, addition of pyroglutamyl groups, succinylation, addition of tetrabutoxycarbonyl groups and addition of 3-mercaptopropyl groups, acylation (e.g., lipopeptides), biotinylation, phosphorylation, sulfation, glycosylation, introduction of maleimide groups, chelation moieties, chromophores, and fluorophores.

[0125] Conjugate

[0126] In some embodiments, the compounds of this disclosure are attached or conjugated to a second portion (e.g., a heterologous portion, a conjugated portion). As used herein, the term “heterologous portion” is synonymous with “conjugated portion” and refers to any molecule (chemical or biochemical, naturally occurring or uncoded) that is different from the disclosed compounds. Exemplary heterologous portions include, but are not limited to, polymers, carbohydrates, lipids, nucleic acids, oligonucleotides, DNA or RNA, amino acids, peptides, polypeptides, proteins, therapeutic agents (e.g., cytotoxic agents, cytokines), or diagnostic agents. Representative peptides of this disclosure include peptides conjugated to lipids, peptides conjugated to a second peptide, such as peptides including transmembrane domains (e.g., the α-transmembrane domain of the IL2 receptor), referred to herein as “amphiphilic peptides,” and peptides conjugated to hydrophobic portions (e.g., DSPE).

[0127] In some embodiments, the peptide incorporated into the HLPN is chemically modified with various substituents. In some embodiments, the chemical modification imparts additional desired properties as discussed herein. In some aspects, the chemical modification takes many different forms, such as heteropeptides, polysaccharides, lipids, radioisotopes, non-standard amino acid residues and nucleic acids, metal chelates, and various cytotoxic agents. In some embodiments, the compound may be used to mimic the structure of the amino acid residues in the peptide described herein.

[0128] In some embodiments, the compound is fused with a heteropeptide to impart various properties, such as increased solubility and / or stability and / or half-life, resistance to proteolytic cleavage, clearance regulation, and targeting of specific cell or tissue types. In some embodiments, the compound is linked to the Fc domain of IgG or other immunoglobulins. In some embodiments, the compound is fused with alkaline phosphatase (AP). Methods for preparing Fc or AP fusion constructs are found in WO 02 / 060950. The compounds of this disclosure can be endowed with properties such as half-life and bioavailability by fusing the compound with a protein domain having specific properties.

[0129] When the compound is a peptide, it can be modified, for example by glycosylation, amidation, carboxylation, or phosphorylation, or by generating acid addition salts, amides, esters, specifically C-terminal esters and N-acyl derivatives as described above. Peptides can also be modified to produce peptide derivatives by forming covalent or non-covalent complexes with other moieties. Covalently bound complexes can be prepared by linking the chemical moieties to functional groups on the side chains of amino acids comprising the peptide or at the N or C terminus.

[0130] Peptides can be conjugated to reporter groups and include, but are not limited to, radiolabeling, fluorescent labeling, enzymes (e.g., catalytic calorimetric or fluorescent reactions), substrates, solid matrices, or carriers (e.g., biotin or avidin). Examples of similar substances are described in WO 98 / 28621 and Olofsson et al., Proceedings of the National Academy of Sciences (Proc. Nat'l. Acad. Sci. USA), 95:11709-11714 (1998), U.S. Patent Nos. 5,512,545 and 5,474,982; and U.S. Patent Application Nos. 20020164687 and 20020164710.

[0131] Cysteine ​​residues most commonly react with haloacetic esters (and their corresponding amines), such as chloroacetic acid or chloroacetamide, to produce carboxymethyl or carboxyamidomethyl derivatives. Cysteine ​​residues are also derivatized by reactions with: bromotrifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0132] Histidine residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5–7.0, as this reagent has relative specificity for histidine side chains. Bromophenylacetyl bromide is also useful; this reaction is preferably carried out at pH 6.0 in 0.1 M sodium dimethylarsinate.

[0133] Lysyl acyl and amino-terminal residues react with succinic acid or carboxylic anhydrides. Derivatization with these reagents reverses the charge of the lysyl acyl residue. Other suitable reagents for derivatizing α-amino residues include imine esters such as methylpyridinium imide; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed glyoxylate reactions.

[0134] Arginine residues are modified by reaction with one or more conventional reagents, such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Due to the high pK of the guanidine functional group, the derivatization of arginine residues requires the reaction to be carried out under basic conditions. Furthermore, these reagents can react with lysine groups and arginine ε-amino groups.

[0135] Specific modifications to tyrosine residues themselves have been extensively studied, with particular interest in introducing spectral labeling into tyrosine residues through reactions with aromatic diazo compounds or tetranitromethane. Most commonly, N-acetylimidazolium and tetranitromethane are used to form O-acetyltyrosine acyl compounds and 3-nitro derivatives, respectively. 125 I or 131 I iodinate tyrosine residues to prepare labeled proteins for radioimmunoassay.

[0136] The carboxyl side group (aspartic or glutamic) can be selectively modified by reacting with carbodiimides (R1) such as 1-cyclohexyl-3-(2-morpholino-4-ethyl)carbodiimide or 1-ethyl-3-(4-azainium-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartic and glutamic residues can be converted to asparagyl and glutamicyl residues by reacting with ammonium ions.

[0137] Glutamine acyl and asparagine acyl residues are frequently deamidated to their corresponding glutamine and asparagine residues. Alternatively, these residues are deamidated under weakly acidic conditions. Any of these residues in either form falls within the scope of this disclosure.

[0138] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonyl residues, methylation of the α-amino groups of the lysine, arginine, and histidine side chains (TECreighton, *Proteins: Structure and Molecule Properties*, WH Freeman & Co., San Francisco, pp. 79-86, 1983), acetylation of the N-terminal amine, and, in some cases, amidation of the C-terminal carboxyl group. These derivatives are chemically modified peptide compositions in which the binding construct peptide is linked to a polymer.

[0139] Typically, chemical derivatization can be carried out under any suitable conditions for reacting a protein with an activated polymer molecule. Methods for preparing peptide chemical derivatives generally include the following steps: (a) reacting the peptide with an activated polymer molecule (such as a reactive ester or aldehyde derivative of the polymer molecule) under conditions where the binding construct is linked to one or more polymer molecules; and (b) obtaining the reaction product. Optimal reaction conditions will be determined based on known parameters and desired results. For example, a higher polymer molecule:protein ratio results in a greater amount of linked polymer molecules. In some embodiments, the compound may have a single polymer molecule moiety at the amino terminus (see, for example, U.S. Patent No. 5,234,784).

[0140] Compared to underivative molecules, the derivatized binding constructs disclosed herein may possess additional activities (i.e. enhanced or weakened biological activity) or other properties, such as increased or decreased half-life.

[0141] In some embodiments, the compound is directly connected to the conjugate moiety in the absence of a linker. Alternatively, the compound is indirectly connected to the conjugate moiety via one or more linkers. Whether directly or indirectly connected via a linker, the compounds can be linked by covalent bonds (e.g., peptide, ester, amide, or thiol-hydrogen bonds) or non-covalent bonds (e.g., via hydrophobic interactions, hydrogen bonds, van der Waals bonds, electrostatic or ionic interactions) or combinations thereof. The compounds and conjugate moiety disclosed herein can be linked in any manner known in the art, including but not limited to any disclosed linker. See, for example, the section entitled “Linker”.

[0142] Heterogeneous components: polymers, carbohydrates, and lipids

[0143] In some embodiments, the heterosource portion is a polymer. The polymer may be branched or unbranched. The polymer may have any molecular weight. In some embodiments, the average molecular weight of the polymer is between about 2 kDa and about 100 kDa (the term "about" indicates that in the preparation of the water-soluble polymer, some molecules will have a molecular weight heavier than the specified molecular weight, while some molecules will have a molecular weight lighter than the specified molecular weight). In some aspects, the average molecular weight of the polymer is between about 5 kDa and about 50 kDa, between about 12 kDa and about 40 kDa, or between about 20 kDa and about 35 kDa.

[0144] In some embodiments, the polymer is modified to have a single reactive group, such as an acylated active ester or an alkylated aldehyde, such that the degree of polymerization can be controlled. In some embodiments, the polymer is water-soluble, such that the proteins to which it is linked do not precipitate in an aqueous environment (such as a physiological environment). In some embodiments, for example, when the composition is used for therapeutic purposes, the polymer is pharmaceutically acceptable. Additionally, in some aspects, the polymer is a mixture of polymers (e.g., copolymers, block copolymers).

[0145] In some embodiments, the polymer is selected from the group consisting of: polyamides, polycarbonates, polyalkylene compounds and their derivatives, including polyalkylene glycols, polyepoxides, and polyalkylene terephthalates; polymers of acrylic acids and methacrylates, including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate); polyethylene Polymers including polyvinyl alcohol, polyvinyl ether, polyvinyl ester, polyvinyl halide, poly(vinyl acetate) and polyvinylpyrrolidone; polyglycolic acid, polysiloxane, polyurethane and copolymers thereof, including cellulose of alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, methylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxy-propyl methylcellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate and sodium cellulose sulfate; polypropylene, polyethylene and polystyrene including poly(ethylene glycol), poly(ethylene oxide) and poly(ethylene terephthalate).

[0146] In some respects, the polymer is a biodegradable polymer, including synthetic biodegradable polymers (e.g., polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide-caprolactone)) and natural biodegradable polymers (e.g., alginates and other polysaccharides, including dextran and cellulose, collagen, their chemical derivatives (substitution, addition, such as alkyl, alkylene, hydroxylation, oxidation, and other modifications conventionally performed by those skilled in the art), albumins and other hydrophilic proteins (e.g., corn gluten and other glutenin and hydrophobic proteins), and any copolymers or mixtures thereof. Typically, these materials degrade by enzymatic hydrolysis or in vivo exposure to water, by surface erosion, or by bulk erosion.

[0147] In some respects, the polymers are bioadhesive polymers, such as biodegradable hydrogels described by H.S. Sawhney, C.P. Pathak, and J.A. Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein by reference, polyhyaluronic acid, casein, gelatin, polyanhydride, polyacrylic acid, alginate, chitosan, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(laurate methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[0148] In some embodiments, the polymer is a water-soluble polymer or a hydrophilic polymer. Suitable water-soluble polymers are known in the art and include, for example, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; Klucel), hydroxypropyl methylcellulose (HPMC; Methocel), nitrocellulose, hydroxypropyl ethyl cellulose, hydroxypropyl butyl cellulose, hydroxypropyl pentyl cellulose, methyl cellulose, ethyl cellulose (Ethocel), hydroxyethyl cellulose, various alkyl celluloses and hydroxyalkyl celluloses, various cellulose ethers, cellulose acetate, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, vinyl acetate / crotonic acid copolymer, polyalkyl polymethacrylate, hydroxymethyl methacrylate, methacrylic acid copolymer, polymethacrylic acid, polymethyl methacrylate, maleic anhydride / methyl vinyl ether copolymer, polyvinyl alcohol, sodium and calcium polyacrylate, polyacrylic acid, acidic carboxyl polymers, carboxylated polymethylene, carboxylated vinyl polymers, polyoxyethylene polyoxypropylene copolymers, polymethyl vinyl ether-conmaleic anhydride, carboxymethylamide, potassium methacrylate divinylbenzene copolymer, polyethylene glycol, polyethylene oxide and its derivatives, salts and combinations thereof. In some aspects, the water-soluble polymer or mixture thereof comprises, but is not limited to, N-linked or O-linked carbohydrates, sugars, phosphates, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylethanolamine (PE); carbohydrates; sugars; phosphates; polyethylene glycol (PEG) (including PEG forms already used for protein derivatization, comprising mono-(C1-C10)alkoxy- or aryloxy-polyethylene glycol); monomethoxy polyethylene glycol; dextran (such as, for example, low molecular weight dextran of about 6 kD); cellulose; cellulose; other carbohydrate-based polymers, poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol or mixtures thereof. This disclosure also covers bifunctional crosslinked molecules that can be used to prepare covalently linked polymers.

[0149] An exemplary water-soluble polymer used herein is polyethylene glycol (PEG). As used herein, polyethylene glycol means any of the PEG forms that can be used to derive other proteins, such as mono(C1-C10)alkoxy- or aryloxy-PEG. PEG is a linear or branched neutral polyether obtained in a wide range of molecular weights and is soluble in water and most organic solvents. PEG effectively excludes other polymers or peptides in the presence of water, primarily through its high dynamic chain mobility and hydrophilicity, thus forming a water shell or hydrated sphere when attached to the surface of other proteins or polymers. PEG is non-toxic, non-immunogenic, and approved by the Food and Drug Administration for internal consumption.

[0150] Proteins or enzymes, when conjugated with PEG, exhibit proven biological activity, non-antigenic properties, and reduced clearance when administered in animals. (See F.M. Veronese et al., "Preparation and Properties of Monomethoxypoly(ethyleneglycol)-modified Enzymes for Therapeutic Applications," J.M. Harris, ed., *Poly(Ethylene Glycol) Chemistry—Biotechnical and Biomedical Applications*, 127-36, 1992, which are incorporated herein by reference.) These phenomena are due to the repulsive properties of PEG in preventing recognition by the immune system. Furthermore, PEG has been widely used in surface modification procedures to reduce protein adsorption and improve blood compatibility. SW Kim et al., *Annals of the New York Academy of Sciences* 516:116-30, 1987; Jacobs et al., *Artificial Organs* 12:500-501, 1988; Park et al., *Journal of Polymer Science*, Part A 29:1725-31, 1991, these references are incorporated herein by reference. Hydrophobic polymer surfaces, such as polyurethane and polystyrene, can be modified by grafting PEG (MW3,400) and used as non-thrombotic surfaces. Due to the hydration of PEG, surface properties (contact angle) can be more consistent with hydrophilic surfaces. More importantly, protein adsorption (albumin and other plasma proteins) caused by the high chain mobility, hydrated spheres, and protein repulsion properties of PEG can be significantly reduced.

[0151] PEG (MW 3,400) was identified as the optimal size for surface immobilization studies (Park et al., Journal of Biomedical Materials Research, 26:739-45, 1992), while PEG (MW 5,000) was most beneficial in reducing protein antigenicity (FM Veronese et al., JM Harris et al., Poly(Ethylene Glycol) Chemistry—Biotechnical and Biomedical Applications, 127-36).

[0152] A method for preparing a PEGylated compound may include the steps of: (a) reacting the compound with a polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions in which the compound is linked to one or more PEG groups; and (b) obtaining a reaction product. Generally, the optimal reaction conditions for the acylation reaction will be determined based on known parameters and the desired results. For example, a higher PEG:compound ratio will result in a higher percentage of PEGylated product. In some embodiments, the compound will have a single PEG moiety at the N-terminus. See U.S. Patent No. 8,234,784, which is incorporated herein by reference.

[0153] In some embodiments, the heterologous portion is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), or a polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose, kelp polysaccharide, xylan, mannan, fucoidan, galactomannan).

[0154] In some embodiments, the heterologous portion is a lipid. In some embodiments, lipids are fatty acids, eicosanoic acid, prostaglandins, leukotrienes, thromboxanes, N-acylethanolamine, glycerides (e.g., mono-, di-, and tri-substituted glycerides), glycerophospholipids (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), sphingolipids (e.g., sphingosine, ceramides), sterol lipids (e.g., steroids, cholesterol), enol esters, glycolipids or polyketides, oils, waxes, cholesterol, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids.

[0155] Lipidification

[0156] In an exemplary aspect, the peptide is esterified or otherwise linked to lipids. In some embodiments, the lipids are fatty acids, eicosanoic acid, prostaglandins, leukotrienes, thromboxanes, N-acylethanolamine, glycerides (e.g., mono-, di-, and tri-substituted glycerides), glycerophospholipids (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), sphingolipids (e.g., sphingosine, ceramides), sterol lipids (e.g., steroids, cholesterol), enol esters, glycolipids or polyketides, oils, waxes, cholesterol, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. In some embodiments, esterification is performed as described in Kowalczyk et al., Peptides and Peptide-based Biomaterials and their Biomedical Applications (2017; 1030:185-227, incorporated herein by reference in its entirety).

[0157] In one embodiment, the peptide is conjugated with a fatty acid, for example, the peptide is myristylated. For example, the fatty acid can be conjugated to the N-terminus of the peptide; such fatty acids include caprylic acid (C8), capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), and stearic acid (C18), etc. Furthermore, cysteine ​​residues in the peptide can be palmitoylated. In one embodiment, the fatty acid is covalently linked to the peptide. In one embodiment, the peptide is myristylated, stearylated, or palmitoylated at the N-terminal amino acid. In one embodiment, the peptide is myristylated at the N-terminal amino acid.

[0158] In an exemplary aspect, the peptide is covalently linked to a fatty acid. In some specific embodiments, the fatty acid is a C4 to C30 fatty acid. In an exemplary aspect, the fatty acid is any one of C4, C6, C8, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28, or C30 fatty acids. In some embodiments, the fatty acid is a C8 to C20 fatty acid, a C12 to C29 fatty acid, or a C14 to C18 fatty acid, such as a C14 or C16 fatty acid.

[0159] In one exemplary embodiment, the peptide is covalently linked to a fatty acid, and the fatty acid is linked to an N-terminal or C-terminal amino acid. In an alternative embodiment, the peptide is covalently linked to a fatty acid, and the fatty acid is linked to an internal amino acid of the peptide, for example, via a functional group on the side chain of the internal amino acid. For example, the fatty acid may be linked to an amine, hydroxyl, or thiol on the side chain of the internal amino acid. In another exemplary embodiment, the peptide is covalently linked to a fatty acid, and the fatty acid is linked to a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or twelfth amino acid.

[0160] In other embodiments, the peptide may be cyclized. For example, the peptide may include two Cys residues, the sulfur atoms of which participate in the formation of a disulfide bond. In an exemplary aspect, the peptide includes a Cys residue as a terminal residue. In a particular embodiment, the peptide is CFEEERAC (SEQ ID NO: 13). Suitable methods for modifying peptides with disulfide bonds or sulfur-based cyclization are described, for example, in Jackson et al., *Journal of the American Chemical Society* 113:9391-9392 (1991) and Rudinger and Jost, *Experientia* 20:570-571 (1964).

[0161] Other peptide cyclization methods are reviewed in Davies, *Journal of Peptide Science*, 9:471-501 (2003). These methods include the formation of amide bridges, thioether bridges, thioester bridges, urea bridges, carbamate bridges, sulfonamide bridges, etc. For example, a thioester bridge can be formed between the C-terminus of a Cys residue and its side chain. Alternatively, a thioester can be formed via a side chain of an amino acid (Cys) containing a thiol and a side chain containing a carboxylic acid (e.g., Asp, Glu). In another approach, a crosslinking agent (such as a dicarboxylic acid, e.g., octanoic acid (corticocyanate)) can introduce a link between two functional groups on the amino acid side chain, such as free amino, hydroxyl, thiol groups, and combinations thereof.

[0162] The peptides, peptide compositions, and nanoparticles disclosed herein are considered for therapeutic purposes. For example, certain peptides of this disclosure can be used to treat atherosclerosis, thrombosis, stroke or heart attack, acute respiratory distress syndrome (ARDS), autoimmune diseases, hemodialysis AV fistulas, organ transplantation, and / or inflammation in subjects in need. The method includes the step of administering to a subject an amount of the peptides or peptide compositions of this disclosure that is effective in treating atherosclerosis, thrombosis, stroke or heart attack, acute respiratory distress syndrome (ARDS), autoimmune diseases, hemodialysis AV fistulas, organ transplantation, and / or inflammation.

[0163] In some embodiments, the peptides, peptide compositions, and nanoparticles of this disclosure are administered in combination with effective amounts of P2Y12 inhibitors and / or aspirin to enhance antithrombotic effects while minimizing bleeding.

[0164] In some embodiments, the P2Y12 inhibitor is selected from clopidogrel, ticagrelor, pregrel, and canagrelor.

[0165] Because blood clotting and thrombosis play a role in stroke and heart attack, this disclosure further provides a method for treating or preventing stroke or heart attack in a subject in need. The method includes the step of administering to the subject an effective amount of the disclosed peptides, peptide compositions, and nanoparticles for treating or preventing stroke or heart attack.

[0166] Certain peptides provided herein can also be used to influence leukocyte function. Therefore, this disclosure provides a method for inhibiting leukocyte adhesion, diffusion, migration, or chemotaxis. The method includes the step of contacting leukocytes with an amount of the disclosed peptides, peptide compositions, and nanoparticles that effectively inhibit leukocyte adhesion, diffusion, migration, or chemotaxis. Since these leukocyte functions are related to inflammation, this disclosure further provides a method for inhibiting or treating inflammation in a subject in need. The method includes the step of administering to the subject an amount of the disclosed peptides, peptide compositions, and nanoparticles that effectively inhibit or treat inflammation. In exemplary embodiments, the disclosed peptides, peptide compositions, or nanoparticles are administered systemically to the subject, for example, parenterally (e.g., via intravenous injection).

[0167] In other embodiments, administering an effective dose of certain peptides, peptide compositions or nanoparticles provided herein can be used to treat sepsis, a systemic inflammatory state caused by microorganisms or their toxins entering the circulation.

[0168] As used herein, the term "treatment" and related terms do not necessarily imply 100% or complete treatment. Rather, different degrees of treatment exist, one of which is considered by those skilled in the art to have potential benefit or therapeutic effect. In this regard, the methods of this disclosure for treating atherosclerosis, thrombosis, stroke or heart attack, acute respiratory distress syndrome (ARDS), autoimmune diseases, hemodialysis AV fistulas, organ transplantation, and / or inflammation can provide any amount or level of treatment. Furthermore, treatment provided by the methods of this disclosure can comprise treatment of one or more symptoms or signs of atherosclerosis, stroke, heart attack, acute respiratory distress syndrome (ARDS), autoimmune diseases, hemodialysis AV fistulas, organ transplantation, or inflammation that is currently being treated. Additionally, treatment provided by the methods of this disclosure can cover slowing the progression of atherosclerosis, stroke, heart attack, acute respiratory distress syndrome (ARDS), autoimmune diseases, hemodialysis AV fistulas, organ transplantation, or inflammation. As used herein, "treatment" also encompasses "inhibition" and "prevention."

[0169] As used herein, the term "prevention" and words derived therefrom encompass delaying the onset of a prevented medical condition. As used herein, the term "prevention" and words derived therefrom encompass reducing the risk of a prevented medical condition.

[0170] As used herein, the term "inhibition" and words derived therefrom may not be 100% or complete inhibition or negation. Rather, varying degrees of inhibition exist, and those skilled in the art recognize the potential benefit or therapeutic effect therein. In this regard, compounds can inhibit the binding interaction between β-integrin and the G protein α subunit to any amount or level. In exemplary embodiments, the inhibition provided by the methods of this disclosure is at least or about 10% inhibition (e.g., at least or about 20% inhibition, at least or about 30% inhibition, at least or about 40% inhibition, at least or about 50% inhibition, at least or about 60% inhibition, at least or about 70% inhibition, at least or about 80% inhibition, at least or about 90% inhibition, at least or about 95% inhibition, at least or about 98% inhibition). In some embodiments, compounds completely eliminate the binding interaction between β-integrin and the G protein α subunit, such that the β-integrin-G protein α subunit binding complex is undetectable in samples obtained from subjects, as measured by, for example, immunoprecipitation, Western blotting, immunohistochemistry, etc.

[0171] As used herein, the term "amount effective" or "therapeutic effective amount" refers to the amount of the disclosed peptide or pharmaceutical composition comprising the peptide of the invention sufficient to achieve the desired outcome (e.g., treatment or limitation of the development of a symptom or disease characterized by hyperangiogenesis). The amount of peptide constituting an "effective" or "therapeutic effective amount" can vary depending on the severity of the disease, the condition of the patient being treated, weight or age, frequency of administration, or route of administration, but can be conventionally determined by those skilled in the art. Clinicians may adjust the dosage or route of administration to obtain optimal therapeutic effect. Based on the factors described above, typical dosages range from about 0.1 μg / kg to at most about 100 mg / kg or more. In some embodiments, the dosage may range from 0.1 μg / kg to about 100 mg / kg, or from 1 μg / kg to about 100 mg / kg, or from 5 μg / kg to about 100 mg / kg.

[0172] The pharmaceutical compositions, nanoparticles, or peptides disclosed herein can be specifically formulated for oral administration in solid or liquid form or for intravenous injection. The optimal pharmaceutical composition will be determined by those skilled in the art based on, for example, the intended route of administration, delivery format, and desired dosage. See, for example, *Remington's Pharmaceutical Sciences*, 18th edition (edited by ARGenrmo), 1990, Mack Publishing Company.

[0173] The peptides disclosed herein can be incorporated into conventional systemic dosage forms, such as tablets, capsules, soft gelatin capsules, elixirs, or injectable formulations. The dosage forms may also include necessary physiologically acceptable carrier materials, excipients, lubricants, buffers, surfactants, antibacterial agents, fillers (such as mannitol), antioxidants (ascorbic acid or sodium bisulfite), etc.

[0174] Acceptable formulation materials are preferably non-toxic to receptors at the dosage and concentration used. Pharmaceutical compositions may contain formulation materials used to modify, maintain, or preserve, for example, the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability. Suitable formulation materials include, but are not limited to: amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); swelling agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as sucrose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight peptides; and other components. Salt counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronics, PEG, dehydrated sorbitol esters, polysorbates such as polysorbate 20 and polysorbate 80, Triton, tromethamine, lecithin, cholesterol, or tyloxapal); stability enhancers (e.g., sucrose or sorbitol); tension enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, or mannitol or sorbitol); delivery mediators; diluents; excipients and / or pharmaceutical adjuvants. See, for example, Remington Pharmaceutical Sciences, Id.

[0175] The primary medium or carrier in the pharmaceutical compositions, nanoparticles, or peptides of this disclosure can be aqueous or non-aqueous. For example, suitable carriers or carriers may be water for injection, physiological saline, or artificial cerebrospinal fluid, possibly supplemented with other materials commonly found in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary carriers. The pharmaceutical composition may include a Tris buffer with a pH of about 7.0 to 8.5 or an acetate buffer with a pH of about 4.0 to 5.5, said buffer may further contain sorbitol or a suitable alternative thereof. Pharmaceutical compositions of this disclosure for storage can be prepared by mixing the selected composition having the desired purity with an optional formulation pharmaceutical agent (Remington Pharmaceutical Sciences, Id.) in the form of a lyophilized cake or aqueous solution. Furthermore, the peptides of this disclosure can be formulated into lyophilized products using suitable excipients (such as sucrose).

[0176] The drug compositions, nanoparticles, or peptides disclosed herein can be administered orally, intravenously, intraperitoneally, intracerebrally (intraparenchymal), intraventricularly, intramuscularly, intraocularly, intraarterially, intraportally, or intralesionally; via sustained-release systems or implantable devices. The drug compositions can be administered by bolus injection, continuous infusion, or via implantable devices. The drug compositions can be administered topically via implantable membranes, sponges, or other suitable materials that have absorbed or encapsulated the desired molecules. When using implantable devices, the devices can be implanted into any suitable tissue or organ, and the delivery of the desired molecules can be achieved through diffusion, timed-release bolus injection, or continuous administration.

[0177] The pharmaceutical compositions, nanoparticles, or peptides of this disclosure can be delivered parenterally. When considering parenteral administration, the therapeutic compositions of this disclosure can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired compound identified in the screening methods of this disclosure in a pharmaceutically acceptable medium. A particularly suitable carrier for parenteral injection is sterile distilled water, wherein the compound identified in the screening methods of this disclosure is formulated into a sterile isotonic solution and appropriately stored. Preparation may involve formulating the desired molecule with an agent (such as injectable microspheres, bio-erectible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes) that can provide controlled or sustained release of the product, which can then be delivered via reservoir injection. Formulas containing hyaluronic acid have the effect of promoting circulation duration. Implantable drug delivery devices can be used to introduce the desired molecule.

[0178] The compositions, nanoparticles, or peptides of this disclosure can also be formulated for inhalation. In these embodiments, the nanoparticles or peptides of this disclosure are formulated as dry powders for inhalation, or the inhalation solution may also be formulated with a propellant for aerosol delivery, such as by nebulization. Lung administration is further described in International Application PCT / US94 / 001875, which describes the lung delivery of chemically modified proteins and is incorporated herein by reference.

[0179] The pharmaceutical compositions, nanoparticles, or peptides of this disclosure can be delivered via the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the scope of the art. The nanoparticles or peptides of this disclosure administered in this manner may or may not be formulated with those carriers conventionally used in solid dosage forms such as tablets and capsules. Capsules can be designed to release the active portion of the formulation at a point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional pharmaceutical agents may be included to promote the absorption of the peptides of this disclosure. Diluents, flavoring agents, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be employed.

[0180] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include exfoliating agents such as sugars and sodium chloride. Prolonged absorption of injectable drug formulations can be achieved by including agents that delay absorption (such as aluminum monostearate and gelatin).

[0181] Pharmaceutically acceptable salts

[0182] Regarding this disclosure, the compositions, nanoparticles, or peptides disclosed herein are in some respects in the form of salts (e.g., pharmaceutically acceptable salts). Such salts can be prepared in situ or separately during the final separation and purification of the active agent by reacting a free base functional group with a suitable acid. Examples of acids that can be used to form pharmaceutically acceptable acid addition salts include, for example, inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid; and organic acids, such as oxalic acid, maleic acid, succinic acid, and citric acid.

[0183] Representative acid addition salts include, but are not limited to, acetates, adipates, alginates, citrates, aspartates, benzoates, benzenesulfonates, hydrogen sulfates, butates, camphorates, camphor sulfonates, diglucose, glyceryl phosphates, hemisulfates, heptanates, hexanoates, fumarates, hydrochlorides, hydrobromides, hydroiodates, 2-hydroxyethanesulfonate (hydroxyethyl sulfonate), lactates, maleates, methanesulfonates, nicotinates, 2-naphthalenesulfonate, oxalates, palmitates, pectinates, persulfates, 3-phenylpropionates, picrates, pentanoates, propionates, succinates, tartrates, thiocyanates, phosphates, glutamates, bicarbonates, p-toluenesulfonates, and undecanoates.

[0184] Base addition salts can also be prepared in situ during the final separation and purification of the compositions or peptides disclosed herein, or in situ by reacting the carboxylic acid moiety with a suitable base (such as a pharmaceutically acceptable metal cation hydroxide, carbonate, or bicarbonate) or with ammonia or an organic primary, secondary, or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, alkali metal or alkaline earth metal-based cations, such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts; and non-toxic quaternary ammonium and amine cations, including ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium, and ethylammonium. Other representative organic amines that can be used to form base addition salts include, for example, ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine.

[0185] Furthermore, the basic nitrogen-containing group can be quaternized with the compositions or peptides disclosed herein as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; long-chain halides, such as decyl, lauryl, myristyl, and stearoyl chlorides, bromides, and iodides; and aralkyl halides, such as benzyl bromide and phenethyl bromide, etc. This yields water-soluble, oil-soluble, or dispersible products.

[0186] Application time

[0187] The disclosed pharmaceutical compositions, nanoparticles, peptides, or formulations can be administered according to any regimen, including, for example, daily (once a day, twice a day, three times a day, four times a day, five times a day, six times a day), every two days, every three days, every four days, every five days, every six days, weekly, every two weeks, every three weeks, monthly, or every two months. Similar to dosing, the timing can be fine-tuned based on dose-response studies, efficacy and toxicity data, and is initially estimated based on the timing used for other therapeutic agents.

[0188] combination

[0189] In some embodiments, the compositions, nanoparticles, or peptides described herein are administered alone, and in alternative embodiments, the compositions or peptides described herein are administered in combination with another therapeutic agent, such as another active agent (e.g., structure) of a different type of this disclosure or another therapeutic agent that does not inhibit the binding interaction between β-integrin and the α subunit of the G protein. In some aspects, other therapeutic agents are intended to treat or prevent the diseases disclosed herein. In a specific aspect, another therapeutic agent is the one listed in the section entitled “Heterogeneous Section: Therapeutic Agents”.

[0190] In exemplary embodiments, the compositions, nanoparticles, or peptides described herein are administered or packaged in combination with an antithrombotic agent. In exemplary embodiments, the antithrombotic agent is an anticoagulant, such as fondaparinux and bivalirudin. In exemplary embodiments, the antithrombotic agent is an antiplatelet agent, such as aspirin, clopidogrel, dipyridamole, and abciximab.

[0191] In an exemplary aspect, the compositions, nanoparticles, or peptides described herein are administered or packaged in combination with an antiplatelet drug. In an exemplary aspect, the antiplatelet drug is an irreversible cyclooxygenase inhibitor (e.g., aspirin), an adenosine diphosphate (ADP) receptor inhibitor (e.g., clopidogrel, prasugrel, ticagrelor, ticlopidine), a phosphodiesterase inhibitor (e.g., cilostazol), a glycoprotein IIb / IIIa inhibitor (e.g., abciximab, eptifibatide), tirofiban, an adenosine reuptake inhibitor (e.g., dipyridamole), or a thromboxane inhibitor (e.g., a thromboxane synthase inhibitor, a thromboxane receptor antagonist (e.g., terutroban)). In an exemplary aspect, the antiplatelet drug is aspirin, thienopyridine, a cyclooxygenase inhibitor, or a P2Y12 inhibitor.

[0192] In an exemplary aspect, the compositions, nanoparticles, or peptides described herein are administered or packaged in combination with an integrin antagonist or integrin inhibitor. In an exemplary aspect, the integrin inhibitor is eptifibatide.

[0193] In exemplary embodiments, the composition, nanoparticles, or peptides are administered simultaneously with other therapeutic agents. In alternative embodiments, the composition or peptides are administered before or after other therapeutic agents.

[0194] The foregoing can be better understood by referring to the following examples, which are presented for illustrative purposes and are not intended to limit the scope of this disclosure.

[0195] Example

[0196] General Method

[0197] animal

[0198] The mice used in this study were 8- to 10-week-old C57BL / 6 mice. Animal use and protocols were approved by the Institutional Animal Care Committee of the University of Illinois at Chicago. Mice of similar age, weight, and sex ratio (1:1) were used for control and treatment.

[0199] In some experiments, 14- to 16-week-old mice with equal sex ratios were used. Gα13 fl / fl The mice were a gift from Dr. Stefan Offermanns' laboratory (Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany). PF4-Cre mice, LysM-Cre (Lyz2-Cre) and C57BL / 6 mice were obtained from the Jackson Laboratory. They were bred using Gα13... fl / fl Platelet-specific or leukocyte-specific Gα13 knockout mice were generated from PF4-Cre or LysM-Cre mice, and the results were confirmed by genotyping and Western blot analysis. Figures 30A-30B Control mice were negative for Cre recombinase with matched genetic background, age, and sex. Animal use and protocol were approved by the Institutional Animal Care Committee of the University of Illinois at Chicago. A randomized mouse selection method was used throughout the study, employing all mice with the correct genotype and without bias.

[0200] reagents

[0201] Peptides were synthesized and purified at the Research Resource Center at the University of Illinois at Chicago, or custom-made by the New England Biolabs, MA. Peptides purchased from China Peptide Company (CPC) were also tested in some experiments, but this supplier's peptides were abandoned due to unreliable quality. Clopidogrel bisulfate was purchased from Sigma-Aldrich Co. (St. Louis, Missouri). Cangrelor was purchased from Adooq Bioscience Co. (York, UK), and Brilinta (ticagrelor) was purchased from AstraZeneca pharmaceutical company (London, UK). The mouse anti-integrin β3 monoclonal antibody M15 was a gift from Dr. Mark Ginsberg (University of California, San Diego). Rabbit anti-integrin β3 antibody (catalog 18309-1-AP) was obtained from Proteintech Group Inc. (Chicago, Illinois). Rabbit anti-Gα13 antibody (GTX32613) was purchased from GeneTex Inc. (Irvine, California). PE-conjugated rat anti-mouse integrin αIIbβ3 (active) clone JON / A was obtained from Emfret Analytics (Germany). Rat anti-mouse Ly-6G (clone 1A8) antibody was purchased from BD Biosciences (San Jose, California).

[0202] Mouse anti-integrin β2 antibody (1.BB.246, sc-71397) and rat anti-integrin αIIb antibody (MWReg30, sc-19963) were purchased from Santa Cruz Biotechnology (Dallas, Texas). Rabbit anti-integrin β2 monoclonal antibody (D4N5Z, #73663) was purchased from CellSignaling Technology, Inc. (Denver, Massachusetts). Mouse anti-integrin β3 monoclonal antibody M15 was a gift from Dr. Mark Ginsberg (University of California, San Diego). Rabbit anti-integrin β3 antibody (18309-1-AP) was obtained from Protein Technologies Group, Inc. (Chicago, Illinois). Rabbit anti-Gα13 antibody (GTX32613) was purchased from GeneTex, Inc. (Irvine, California). Rabbit anti-fibrin / fibrinogen polyclonal antibody (A0080) was obtained from Dako / Agilent Technologies (Santa Clara, California). Mouse cytokine IL-6, TNFα, and IL-10 ELISA kits were obtained from R&D Systems, Inc. (Minneapolis, Minnesota). The blood urea nitrogen (BUN) assay kit was purchased from Arbor Assays (Ann Arbor, Michigan). The creatinine and alanine aminotransferase colorimetric assay kit was purchased from Cayman Chemical (Ann Arbor, Michigan). The mouse cystatin C ELISA kit was purchased from Thermo Fisher Scientific (Waltham, Massachusetts).

[0203] Anti-Gα 13 Antibody (GTX32613) was purchased from GeneTex, Inc. (Irvine, California). Integrin β2 antibodies (sc-19624, sc-8420, and sc-71397) were from Santa Cruz Biotechnology, Inc. (Dallas, Texas). Rabbit anti-integrin β2 monoclonal antibody (D4N5Z, #73663) was purchased from Cell Signaling Technologies, Inc. (Denver, Massachusetts). N-formyl-Met-Ile-Val-Ile-Leu (fMIVIL (SEQ ID NO:46)) was obtained from Dr. Richard Ye (Southgate et al., 2008). Anti-GAPDH (MAB5718) was purchased from R&D Systems, Inc. Hank's balanced salt solution (HBSS; with and without Ca) was used. 2+ and Mg 2+Phenol red and RPMI 1640 medium were obtained from Gibco / Life Technologies. Bovine serum albumin (BSA) and bacterial LPS (Escherichia coli O55: B5) were purchased from Sigma-Aldrich (St. Louis, Missouri).

[0204] Lipid-stabilized high-load M3mP6 peptide nanoparticles

[0205] Using film rehydration (21, 31), the peptide myr-FEEERL (SEQ ID NO: 25) was modified from the original β3 cytoplasmic domain sequence to make it more suitable for forming peptide-based nanoparticles, and formulated with PEG2000-DSPE (Avanti Polar Lipids Inc., Alabaster, AL) to form lipid-stabilized, high-load peptide nanoparticles, L-α-phosphatidylcholine (egg PC, type XI-E, Sigma-Aldrich, St. Louis, Missouri), wherein the molar ratio of peptide to PEG-DSPE to PC was 36.8:51.7:11.5 or 50:40:10 (a further improved formulation with the same in vivo effects). As previously described, dynamic light scattering (DLS) (Zetasizer Nano ZS90, Malvern, Worcestershire, UK) was used to determine the size distribution of nanoparticles (32). This method uses light scattering to monitor the Brownian motion of the particles. Particle size is expressed as a volume-weighted diameter distribution. In some studies, Dupage Medical Technology, Inc. has produced nanoparticles containing 60% (mol / mol) in large-scale production. Figure 3C M3mP6 HLPN (Figure 8) and 70-80% (mol / mol, not shown) M3mP6 also have similar antithrombotic effects.

[0206] Platelet preparation, aggregation and granule secretion

[0207] For human subjects, the University of Illinois at Chicago obtained approval from the Institutional Review Board and obtained informed consent from volunteers in accordance with the Declaration of Helsinki. Platelets were prepared as previously described (33). Platelet aggregation and adenosine triphosphate (ATP) secretion were simultaneously measured at 37°C with stirring (1000 rpm) in a luminescence aggregater (Chronolog) as previously described (34).

[0208] Immunoprecipitation

[0209] Immunoprecipitation of integrin β3 and Gα13 was performed in a manner similar to that described previously (21, 22, 35). Briefly, differentiated THP-1 cells (2 × 10^6) or human platelets (5 × 10^8 / mL, 300 μL) were treated with different concentrations of M3mP6 or out-of-order peptides. Ten minutes after treatment, THP-1 cells or platelets were stimulated at different time points with LPS (100 ng / mL) or thrombin (0.025 U / mL) or continuously in an aggregator for 5 minutes, and then lysed with NP40 lysis buffer (50 mM Tris, pH 7.4, 10 mM MgCl2, 150 mM NaCl, 1% NP-40, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM NaF) and a complete protease inhibitor mixture tablet (Roche). After centrifugation at 14,000 g for 10 minutes at 4°C, the lysate was collected and immunoprecipitated overnight at 4°C with mouse anti-integrin β2 antibody (1.BB.246) or mouse anti-integrin β3 IgG (M15) or an equivalent amount of mouse IgG. Immunoprecipitation was then performed for 1 hour at 4°C using Protein A / G and agarose beads (SC-2003, Raddas Santa Cruz Biotechnology, Texas). After washing three times with NP40 lysis buffer, the immunoprecipitate was analyzed by Western blotting.

[0210] Immunoprecipitation of integrin β2 and Gα13 was performed similarly to the previously described procedure (Gong et al., 2010; Shen et al., 2013). Briefly, human neutrophils (5 × 10^6) were pretreated with MB2mP6 (50 μM) or a scrambled peptide (50 μM). Ten minutes after treatment, the human neutrophils were stimulated with fMLF (1 μM) and loaded into ICAM1 pre-coated 6-well plates. At the specified time points, the neutrophils in each well were dissolved with NP40 lysis buffer (50 mM HEPES, pH 7.4, 10 mM MgCl2, 150 mM NaCl, 1% NP-40, 1 mM MEGTA, 1 mM sodium orthovanadate, 1 mM NaF) mixed with a complete protease inhibitor mixture tablet (Roche). After centrifugation at 14,000g for 10 minutes at 4°C, the lysate was collected and immunoprecipitated overnight at 4°C with mouse anti-integrin β2 antibody (1.BB.246, sc71937) or an equivalent amount of mouse IgG. Immunoprecipitation was then performed for 1 hour at 4°C using Protein A / G and agarose beads (sc-2003, Raddas Santa Cruz Biotechnology, Texas). After washing three times with NP40 lysis buffer, the immunoprecipitate was analyzed by Western blotting.

[0211] fibrinogen and JON / A binding assay

[0212] As previously described, washed mouse platelets in modified Tyrode buffer (5 × 10^7 / mL) were incubated at room temperature for 30 minutes with 10 μg / mL Oregon Green conjugated fibrinogen (Molecular Probes) and PAR4AP, or at room temperature with PE conjugated rat anti-mouse integrin αIIβ3 (active) antibody JON / A (Emfret) and PAR4AP. The reaction was diluted with PBS containing 1% BSA and analyzed by flow cytometry using Accuri C6 (BD Biosciences).

[0213] Drug administration

[0214] Two hours prior to the start of carotid artery injury surgery, C57BL / 6 mice were administered aspirin, clopidogrel, or ticagrelor via tube feeding using a feeding needle (20-gauge bent metal, Fine Science Tools, Heidelberg, Germany) and a syringe. In some experiments, high-dose aspirin was also administered intraperitoneally. M3mP6 was administered via retroorbital injection 15 minutes before surgery. Canagrelor was injected retroorbitally before the start of carotid artery injury. In some experiments determining the dose-response relationship of M3mP6, M3mP6 was also administered intravenously via the tail vein.

[0215] Maximum tolerated dose (MTD) and pharmacokinetics

[0216] CD-1 mice (6-8 weeks old and weighing 20-30g) were isolated for at least 3 days upon arrival and housed in groups under standard conditions (tap water, Harlan irradiated diet 7912, and Anderson bed-o'cob bedding). The first mouse was intravenously injected with the maximum feasible dose (60 mg / kg (60 μmol / kg)) of M3mP6 via the tail vein (day 0). No signs of toxicity were detected on the following day (day 1). The same dose was then administered to four more animals. Animals were observed twice daily for 8 days for clinical signs of toxicity. Since no clinical signs of toxicity were observed, this maximum feasible dose was considered the MTD.

[0217] For pharmacokinetic studies, CD-1 mice were intravenously injected with 5 mg / kg (5 μmol / kg) M3mP6 (potential therapeutic dose). Three treated mice were sacrificed at the following time points: 0 (before administration) min, 5 min, 10 min, 30 min, 1 hour, 2 hours, 4 hours, 6 hours, and 24 hours, and their blood was collected into K2EDTA tubes via the vena cava. Whole blood and plasma M3mP6 concentrations were analyzed using an Agilent 1290 Infinity LC and a 6430 triple quadrupole mass spectrometer and MassHunter software.

[0218] Toxicological studies of bolus and continuous intravenous infusion of M3mP6 HLPN were conducted using jugular cannulated (JVC) Sprague-Dawley (SD) rats (6–8 weeks old and weighing 250–350 kg) that were isolated for at least 3 days upon arrival. Four SD rats (n=2 of each sex) were administered an intravenous bolus dose of M3mP6 (100 mg / kg, dose volume 10 mL / kg), followed by an intravenous infusion of M3mP6 for 24 hours (25 mg / kg / hour, total dose 600 mg / kg / day). This dose level was considered a single-dose MTD because no toxicity was observed during days 3–4 post-administration, as it was the maximum feasible dose based on the maximum permissible dosing volume. A preliminary 7-day continuous infusion study was similarly conducted in rats at lower drug concentrations. Observed parameters included mortality, clinical signs, body weight, hematology, clinical chemistry, and organ weight. For pharmacokinetic studies, plasma samples for drug analysis were obtained from blood samples collected from 6 males and 6 females (3 rats / sex per time point) in dose groups 3 and 4 (phase 2) at the following time points on day 1 after the start of the bolus dose and infusion: 5 minutes, 10 minutes, and 30 minutes, and 1 hour, 2 hours, 6 hours, and 24 hours. Additional blood samples were collected before the end of the 5-day infusion, immediately after the cessation of infusion, and 30 minutes, 1 hour, 4 hours, 6 hours, 24 hours, and 48 hours after the end of the infusion.

[0219] Laser-induced thrombosis of small arteries in mouse cremaster muscle and in vivo microscopic examination

[0220] As previously described, laser-induced thrombosis and in vivo microscopy were performed in male C57BL / 6 mice (6–8 weeks old) anesthetized by intraperitoneal injection of ketamine and toluidine (36). Platelet thrombosis was indicated by infusion of anti-mouse GPIbβ labeled with DyLight 649 from Emfrey, Germany. Fibrin deposition was tracked using a monoclonal anti-fibrin antibody (clone 59D8) labeled with Alexa Fluor (AF) 488 (25), as previously described. A suspension of M3mP6 HLPN (10 μmol / kg) or the control peptide (Myr-ERAFEE (SEQ ID NO:47)) HLPN in 0.15 M NaCl or canagrelor was injected 15 minutes before or immediately before induced injury. Fluorescence and bright-field images of 27–30 lesion sites in each group of 7 mice were recorded via an intensifier using an Olympus BX61W microscope with a 603 / 1.0NA water immersion objective and a high-speed camera (5 frames / second). The images shown were compressed to 100 frames / second.

[0221] FeCl3-induced mouse carotid artery thrombosis model

[0222] Eight to ten-week-old C57BL / 6 mice were anesthetized with isoflurane inhalation. The right carotid artery was dissected (34, 37). As previously described, an MA-0.5SB nanoprobe (Transonic Systems, Ithaca, NY) was attached to the carotid artery to monitor blood flow using a TS420 flow meter (Transconic Systems) (38). After stabilization, carotid thrombosis was induced with 7.5% ferric chloride (FeCl3) (Sigma-Aldrich, St. Louis, Missouri, USA). A filter paper disc (2 mm in diameter) soaked in 1.2 μL of 7.5% FeCl3 was placed on top of the artery for 3 minutes and then removed. M3mP6 HLPN, HLPN with a disordered control peptide, HLPN with a second control peptide carrying the AAA mutation, or saline solution were injected retroorbitally 15 minutes prior to surgery. In preliminary studies, both control peptides showed no significant difference compared to saline solution. Figure 19 To investigate the effect of post-injury drug injection on thrombus development, a filter paper disc soaked in 1.2 μl of 3.75% FeCl3 was placed on the artery for 2 minutes, and the drug was injected retroorbitally 3 minutes after the filter paper was removed. Blood flow was continuously monitored until 5 minutes after occlusion, or 30 minutes if occlusion did not occur. Occlusion time was defined as the time from filter paper removal to stable occlusion (2 minutes without blood flow). The Mann-Whitney test was used to analyze the data to compare the median between treatments.

[0223] Tail bleeding time

[0224] Tail hemorrhage time was determined as previously described (21, 39). Eight to ten-week-old C57BL / 6 mice were anesthetized with isoflurane. After cutting a 0.5 cm segment from the distal tip of the tail, the mouse tail was immediately immersed in 0.15 M NaCl at 37°C. Tail hemorrhage time was defined as the time from tail cutting to stable cessation of bleeding (no rebleeding within 60 seconds). Bleeding was observed for up to 15 minutes. If bleeding persisted at the 15-minute timepoint, hemostasis was achieved by applying pressure. The Mann-Whitney test was used to analyze the data to compare medians between groups.

[0225] Analysis of arterial perforation and hemorrhage

[0226] To estimate bleeding during vascular surgery, 8- to 10-week-old C57BL / 6 mice were anesthetized with isoflurane inhalation. The right carotid artery was dissected as previously described, and a puncture was made in the middle of the carotid artery using a 27 1 / 2G needle (34, 37). The puncture wound was immediately covered with a 4×4 mm hemostatic pad (CERTI-GAUZE™, Kansas City, MO, Missouri), which serves to absorb extravascular blood and also aids in hemostasis, similar to hemostasis during vascular surgery. The pad was removed after 15 minutes, regardless of whether bleeding continued. The amount of blood absorbed on the pad during the 15-minute time period was quantified by estimating the amount of hemoglobin. For this purpose, the blood absorbed on the pad was dissolved in 30 mL of double-distilled water. 1 mL of 2×Drabkin solution was added to 1 mL of dissolved blood. After thorough mixing and incubation at room temperature for 20 minutes, the absorbance of the sample was analyzed at 540 nm using a spectrophotometer. Similarly, known volumes of blood were analyzed to establish a standard curve for calculating blood volume on the pad. The statistical significance of the data was analyzed using the Mann-Whitney test.

[0227] Bleeding time (BMBT) test for dogs

[0228] Following administration of M3mP6 HLPN (10 mg M3mP6 peptide / kg), BMBT (40) was performed in dogs using a Jorvet Surgicutt bleeding time device (J522J, Jorgensen Laboratories, Inc. CO) as previously described and according to the manufacturer's instructions. The normal bleeding time range for dogs was less than 4 minutes.

[0229] Mouse model of myocardial ischemia and reperfusion

[0230] The MI / R model was established as previously described (41, 42). C57BL / 6 mice were randomly assigned to the surgical and sham-operated groups. Mice were anesthetized with 1.5–3.0% isoflurane inhalation and cannulated orally with an 18G angiocath sleeve. Mice were given artificial ventilation using a rodent ventilator. A left thoracotomy was performed by carefully making a 1 cm incision along the sternum and layering the incision 1 mm to the left from the midline between the 2nd and 4th ribs. The left anterior descending artery (LAD) was located and ligated below the incision with a 1–2 mm 8-0 prolene suture. A 4-0 prolene suture was placed below the suture to aid in its removal. 35 minutes after ischemia induction, M3mP6 or control HLPN was injected at a bolus dose of 5 μmol / kg, followed by infusion via jugular vein cannulation at 2.5 μmol / kg / hour. The sutures were cut and removed 45 minutes after the ischemia period. Successful reperfusion is confirmed by observing the change in color of the anterior wall from pale to pink after 15-20 seconds. The chest wound is closed after surgical procedures.

[0231] Determination of infarct area in MI / R mice

[0232] Twenty-four hours after I / R, the LAD was reoccluded at the previous location, and 1% Evans blue dye (Sigma, Darmstadt, Germany) was injected into the cardiac chambers via the ascending aorta (42). Mice were then euthanized, and their hearts were harvested and rinsed in saline. The hearts were then frozen at -20°C for 15 minutes and transversely sliced ​​into 6-7 pieces. The slices were incubated in a dark room at 37°C with 1.5% 2,3,5-triphenyltetrasodium chloride (TTC, Sigma, America) for 20 minutes and then imaged (Nikon SMZ800N). The ischemic area, risk area, and total left ventricular area were measured and calculated using ImageJ.

[0233] Echocardiography

[0234] Echocardiography of mice was performed at the Center for Cardiovascular Research Physiology Core (CCVRPC) using a VisualSonics Vevo 770 echocardiography system. Sedation was maintained with 2% isoflurane throughout the examination. Both two-dimensional and M-mode images were obtained in long-axis and short-axis views. Ejection fraction (EF) was calculated using Vevo 2100 software. Measurements were performed on three images independently acquired for each animal by researchers unaware of the experimental group's status.

[0235] Immunohistochemistry

[0236] Mouse hearts were collected after perfusion and fixed overnight in 10% formalin. Twenty hours after fixation, the mouse hearts were dehydrated in 70% ethanol and embedded in paraffin. Four-micron sections were cut and stained with hematoxylin and eosin. Sections were dewaxed, rehydrated, peroxidase-blocked, and antigen-retrieval was performed by heating at 95°C in citrate buffer (pH 6.0) for 20 minutes. Platelet-rich thrombi were stained using rat anti-mouse integrin αIIb antibody (1:250, Clone MWReg 30, Santa Cruz Biotechnology) and an anti-rat IgG-avidin-biotin complex kit (Vector Laboratories). Neutrophils infiltrated into the mouse heart were stained with rat anti-mouse Ly6G (clone 1A8, BD Biosciences). Positive staining in each section was quantified using ImageJ software.

[0237] Detection of MPO in mouse plasma

[0238] Mouse blood samples were drawn from the retroorbital sinus into 1.5 ml Eppendorf tubes containing 7.5 μl heparin (1000 u / ml) and centrifuged at 600 g for 15 minutes at 4°C to obtain plasma. Plasma MPO was detected using the Mouse Myeloperoxidase DuoSet ELISA Kit (R&D Systems) according to the manufacturer's instructions.

[0239] Preparation of micelle peptides

[0240] MB2mP6 (Myr-FEKEKL (SEQ ID NO:27)) and the disordered control (Myr-EFKKLE (SEQ ID NO:48)) peptides were synthesized and purified by the Research Resource Center at the University of Illinois at Chicago. PEG2000-DSPE (Avanti Polar Lipids, Alabama, Alabama), L-α-phosphatidylcholine (Egg PC, Type XI-E, Sigma-Aldrich, St. Louis, Missouri) and the peptides were mixed at a molar ratio of 55.6:11.9:40. The membrane rehydration method was used as described previously. 16 Prepare micelles.

[0241] Detection of cytokine expression in mouse bone marrow-derived macrophages (BMDM)

[0242] Mouse BMDM 26 was isolated as described above. After differentiation into macrophages by incubation in DMEM medium containing 15% L929 conditioned medium and 10% FBS for 7 days, BMDM (2×10⁶ / mL) was seeded into 6-well plates for 14 hours and serum starved for 4 hours. Eight hours after LPS stimulation, mouse BMDM was collected, and total RNA was extracted and analyzed by quantitative RT-PCR (qRT-PCR) using SYBR Green (Roche) and various mouse cytokine-specific primers.

[0243] mIl1b forward: 5'-CGACAAAATACCTGTGGCCT-3'(SEQ ID NO:51)

[0244] mIl1b reverse: 5'-TTCTTTGGGTATTGCTTGGG-3'(SEQ ID NO:52)

[0245] mIl6 forward: 5'-TCCAGTTGCCTTCTTGGGAC-3' (SEQ ID NO:53)

[0246] mIl6 reverse: 5'-GTGTAATTAAGCCTCCGACTTG-3'(SEQ ID NO:54)

[0247] mTnfa forward: 5'-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC-3' (SEQ ID NO:55)

[0248] mTnfa reverse: 5'-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3' (SEQ ID NO:56)

[0249] mIl10 forward: 5'-GGTTGCCAAGCCTTATCGGAAATG-3'(SEQ ID NO:57)

[0250] mIl10 reverse: 5'-CACTCTTCACCTGCTCCACTGC-3'(SEQ ID NO:58)

[0251] mGapdh forward: 5'-TGCGACTTCAACAGCAACTC-3' (SEQ ID NO:59)

[0252] mGapdh reverse: 5'-CTTGCTCAGTGTCCTTGCTG-3'(SEQ ID NO:60)

[0253] Platelet preparation, aggregation and granule secretion

[0254] Blood was drawn from healthy volunteers via intravenous puncture. The University of Illinois at Chicago received approval from its institutional review board and obtained informed consent from the volunteers in accordance with the Declaration of Helsinki.

[0255] To prepare platelet-rich plasma (PRP), whole blood was anticoagulated with 3.8% trisodium citrate. To prepare human platelets, one-seventh volume of ACD was used as the anticoagulant. Platelets were washed twice and resuspended in modified Tyrode buffer as previously described (27). Platelet aggregation and adenosine triphosphate (ATP) secretion were simultaneously measured at 37°C with stirring (1000 rpm) in a Chronolog apparatus as previously described (28).

[0256] FeCl3-induced mouse carotid artery thrombosis model

[0257] Eight to ten-week-old C57BL / 6 mice (weighing 20–25 g) were anesthetized by isoflurane inhalation. The right carotid artery was dissected. As previously described, an MA-0.5SB nanoprobe (Transconic Systems, Ithaca, NY) was attached to the carotid artery to monitor blood flow using a TS420 flowmeter (Transconic Systems) (29). After stabilization, a filter paper disc (2 mm in diameter) soaked in 1.2 μL of 7.5% FeCl3 (Sigma-Aldrich, St. Louis, Missouri) was placed on top of the carotid artery for 3 minutes to induce thrombus formation and then removed. MB2mP6 or a disordered control peptide was injected retroorbitally 15 minutes prior to the procedure. Blood flow was continuously monitored until 5 minutes after occlusion, or 15 minutes if no occlusion occurred. Occlusion time was defined as the time from removal of the filter paper to stable occlusion (5 minutes without blood flow). The Mann-Whitney test was used to analyze the data to compare the median between treatments.

[0258] Tail bleeding time

[0259] Tail hemorrhage time was determined as previously described (16, 29). Eight- to ten-week-old C57BL / 6 mice weighing 20–25 g were anesthetized with isoflurane. After cutting a 0.5 cm segment from the distal tip of the tail, the mouse tail was immediately immersed in 0.15 M NaCl at 37 °C. Tail hemorrhage time was defined as the time from tail cutting to stable cessation of bleeding (no rebleeding within 60 seconds). Bleeding was observed for up to 15 minutes. If bleeding persisted after 15 minutes, hemostasis was achieved by applying pressure. The Mann-Whitney test was used to analyze the data to compare the medians between groups.

[0260] Reverse passive Arthus (rpA) reaction

[0261] C57 / BL6 mice were anesthetized by intraperitoneal injection of a mixture of ketamine and toluidine (100 mg / kg and 16 mg / kg mouse body weight), and the inner surface of the mice's backs was shaved. The shaved mice received an intradermal injection of rabbit anti-bovine serum albumin (BSA) antibody (6 μg / μL, MP Biomedicals, OH, Ohio) in 25 μL of 0.9% NaCl, followed by intravenous injection of BSA (75 μg / g mouse body weight) and 100 μL of sterile 0.9% NaCl containing a peptide inhibitor (5 μmol / kg mouse body weight). Additionally, a control site received 25 μL of PBS concurrently. Four hours post-injection, the mice were either euthanized and inflamed, or control skin samples were collected from each injection site. Hemoglobin (Hb) levels in the skin samples were quantified using a hemoglobin colorimetric assay kit (Cayman Chemicals, Ann Arbor, Michigan).

[0262] Cecal ligation and puncture (CLP) sepsis model

[0263] CLP sepsis was induced as previously described (32, 33). Briefly, mice (14 to 16 weeks old, equal numbers for each sex) were anesthetized by intraperitoneal administration of ketamine (100 mg / kg body weight) and toluidine (8 mg / kg). Following a midline laparotomy, the cecum was ligated approximately 1 cm from the distal cecum and then double-penetrated with an 18-gauge needle. Sham-operated mice underwent the same procedure except for ligation and cecal puncture. Immediately postoperatively, analgesia (buprenorphine, 0.1 mg / kg, subcutaneously) was administered and repeated every 12 hours thereafter for 3 days. At these time points, fluid resuscitation (pre-warmed 0.9% NaCl, 0.05 ml / g body weight) was administered subcutaneously to prevent fluid loss and aid in body temperature recovery. One hour postoperatively, antibiotics were administered subcutaneously (…). Solution; 10 mg / kg body weight (to simulate clinical conditions) for 5 days. Drug administration continued until the pre-arranged sacrifice, death, or completion of the study in surviving mice. Mice were observed every 6 hours for 8 days and analyzed using a logarithmic-rank test (using Prism GraphPad software (ver. 5.0), San Diego, CA, USA). For CLP-induced organ damage in mice, blood samples and kidney or lung tissue were collected 24 hours after CLP for cytokine and immunohistochemical analysis.

[0264] Mouse jugular vein cannulation and continuous peptide infusion

[0265] Mice were weighed, anesthetized with ketamine / toluidine (100 / 5 mg / kg), and transferred to a heated platform under a dissecting microscope. A 5 mm incision was made between the ear and scapula, penetrating the skin of the upper back of the mouse. The mouse was then placed in a supine position, and the skin over the right jugular vein was shaved and cleaned with hexachlorophenol and 70% ethanol. After making a vertical incision at the jugular vein site, the vein was dissected and exposed. A catheter was tunneled from the upper back incision to the jugular vein incision using a trocar kit (DSI, St. Paul, MN). The jugular vein was carefully dissected, and two sutures were loosely tied around the vessel. A small incision was then made in the vein, and a beveled catheter was inserted and tied in place with sutures without closing the catheter. The wound was closed with 6-0 sutures and cleaned with hexachlorophenol. For peptide treatment, MB2mP6 or control peptide micelles were injected immediately or 6 hours after CLP surgery at a bolus dose of 2.5 μmol / kg, followed by continuous infusion at a rate of 1.25 μmol / kg / hour for 5 days.

[0266] Measurement of organ damage

[0267] Twenty-four hours after CLP, mouse blood was collected and added to EDTA-washed microcentrifuge tubes or citrate buffer. Plasma was obtained after centrifugation at 1,000g for 5 minutes at 4°C. In some cases, mouse serum was collected from the blood without the addition of any anticoagulant. Renal function markers BUN, creatinine, cystatin C, and mouse liver function marker ALT were detected using the corresponding kits according to the manufacturer's instructions. Kidney, lung, and liver tissues from septic mice were collected from euthanized mice and sham-operated mice 24 hours after CLP and fixed in 10% formalin.

[0268] Immunohistochemistry

[0269] Twenty-four hours after CLP, mouse kidneys and lungs were collected with phosphate-buffered saline and then fixed in 10% formalin solution. Twenty hours after fixation, mouse tissues were dehydrated in 70% ethanol and embedded in paraffin. Four- to five-micron sections were cut and stained with hematoxylin and eosin for quality control. Sections were dewaxed, rehydrated, peroxidase-blocked, and subjected to antigen retrieval by heating at 95°C in citrate buffer at pH 6.0 for 20 minutes. Platelet-rich or fibrin-rich thrombi were stained using rat anti-mouse integrin αIIb antibody (1:250, clone MWReg 30), rabbit anti-fibrin / fibrinogen antibody (1:2,000), and an anti-rat or anti-rabbit IgG-avidin-biotin complex kit (Carrier Labs). Positive staining in each section was quantified using ImageJ software. Fibrin deposition was also identified by staining slides with Mallory phosphotungstic acid hematoxylin (PTAH) (PTAH staining kit, American MasterTech, McKinney, TX) and observed using a Leica DMI RB microscope with a 40× / 0.55NA objective. The total thrombus area / glomerulus ratio was quantified by analyzing 20–30 glomeruli from each group using ImageJ software.

[0270] Detection of cytokine expression

[0271] Twenty-four hours after CLP, blood was collected from mice with sham surgery and CLP-induced sepsis. Serum was separated and cytokine levels were analyzed using a specific mouse cytokine ELISA kit. Lungs were also collected from those euthanized mice after PBS perfusion. Cytokine transcripts expressed in lung tissue were detected by real-time PCR using SYBR green.

[0272] Table 1: Key Resources

[0273]

[0274]

[0275] Gα 13 Production of defective mice

[0276] Using Gα as described in *Natural Medicine* 9, 1418-1422 (2003) 13 flox / flox Mice. Using Gα mice hybridized with LysM-Cre mice. 13 flox / flox To produce tissue-specific Gα 13fl / fl LysM-Cre Mice. Age- and sex-matched mice were used in the study. Blood was collected and white blood cell and neutrophil counts were determined using the Hemavet System (Drew Scientific, Oxford, CT).

[0277] Cell culture

[0278] Human umbilical vein endothelial cells (HUVECs) and human lung microvascular endothelial cells (HLMVECs) were purchased from Lonza (Vauxville, Maryland) and cultured on a 0.2% gelatin-coated surface in EGM2 medium (Lonza, Morrisville, North Carolina).

[0279] Cells were washed with phosphate-buffered saline (PBS) (0.137M NaCl, 0.01M Na₂HPO₄, 0.0027M KCl, 0.9mM CaCl₂, 0.5mM MgCl₂, pH 7.4, Corning). Growing cells were collected by trypsinization (Hank's balanced salt solution with phenol red containing 0.05% porcine trypsin and 0.02% EDTA·4Na, Thermo Fisher Scientific). Cells were maintained in Endothelial Growth Medium-2 (EGM-2, Lonza) and a complement bullet kit (0.1% epidermal growth factor (hEGF), 0.1% hydrocortisone, 0.1% GA-1000, 0.4% bovine brain extract (BBE), 0.1% ascorbic acid, 2% FBS, Lonza, Morristown, NJ) and an additional 8% fetal bovine serum (FBS) (vol / vol, Lonza). The cells were cultured in T75 flasks and separated at a 1:3 ratio. These cells were used in the first four passages of the experiments.

[0280] All cells were cultured on cell culture dishes in a humidified incubator at 37°C and 5% CO2 until the confluence reached 80-90%.

[0281] In vivo mouse neutrophil migration model

[0282] For the peritonitis model, mice were challenged by intraperitoneal injection of 1 mL of 3% thioglycolate broth (Sigma-Aldrich, St. Louis, Missouri) or 1 mL of PBS (control). Four hours later, 10 mL of saline was injected into the peritoneum of the mice and massaged for 2 minutes. Peritoneal lavage fluid was collected with a syringe, and neutrophil counts were determined using HEMAVET (Call et al., 2001; Ray and Dittel, 2010).

[0283] Neutrophils were recruited to the lungs via intratracheal administration of LPS (2 mg / kg). Six hours later, mice were sacrificed, and cold saline was injected into the lungs via the trachea, followed by aspiration to collect bronchoalveolar lavage fluid. This collection procedure was performed twice. Neutrophils were counted using HEMAVET (Reutershan, 2005).

[0284] Mouse neutrophil isolation

[0285] Femurs and tibias from 8- to 12-week-old mice were isolated and washed on ice with cold HBSS containing 0.5% BSA. All remaining tissue on the bones was removed, and the cells were rinsed with HBSS containing 0.5% BSA using a 27-G needle. The combined bone marrow eluate was gently resuspended and filtered through a 70 μm nylon cell filter to remove cell clumps and bone particles. The filtrate was centrifuged at 1500 rpm for 5 min at 4 °C, and the cell pellet was resuspended with 3 mL of HBSS containing 0.5% BSA. Cells were layered through a discontinuous gradient consisting of a lower layer of 3 mL Nycoprep 1.077 and 3 mL 72% Percoll, and then centrifuged at 2500 rpm for 20 min at room temperature with slow acceleration and no braking. Neutrophils were isolated from the Nycoprep and 72% Percoll interface and washed with HBSS. After centrifugation, the cell trays were incubated on ice for 2 minutes with 2 mL of ACK (potassium ammonium chloride) lysis buffer (Lonza Bioscience, Morrisville, NC) to remove red blood cells. After a final wash with HBSS, the cells were resuspended in 2 mL of 0.1% BSA-RPMI 1640 and counted. Neutrophils were incubated in a CO2 incubator at 37°C for 1 hour prior to the experiment. (Luscinskas, 2008; Southgate et al., 2008).

[0286] Human neutrophil isolation

[0287] Blood was collected from healthy donors using acidic citrate-dextran (citrate buffer containing 2% dextran) as an anticoagulant. Human neutrophils were isolated from the blood using a density gradient based on a Percoll separation method, employing 55% and 74% Percoll. Red blood cells were lysed using RBC lysis buffer. Cell viability in routine preparations was greater than 98%, as determined by trypan blue exclusion.

[0288] Production of mouse ICAM-1

[0289] 293-T cells were transfected with the pVITRO-hygro vector (InvivoGen) containing the expression construct, which consists of the extracellular domain of mouse ICAM1 (Gln28-Asn485) and a 6×His tag. Transfected cells were selected using hygromycin (100 μg / ml) and maintained in serum-free environment. TM 293 (Thermo Fisher Scientific). The ICAM1-6×His fusion protein was purified from cell culture supernatant by Ni-NTA (nickel-nitrotriacetic acid) chromatography and dialyzed against PBS (pH 7.4). The ICAM1 protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific).

[0290] Real-time imaging of neutrophil migration

[0291] Neutrophils were isolated using a mouse neutrophil enrichment kit from Stemcell Technologies Inc., Cambridge, MA. Neutrophil plates were seeded onto Ibidi chemotaxis chambers pre-coated with appropriate ligands (Ibidi, Fitchburg, WI). Experiments were performed according to the manufacturer’s instructions (Weckmann et al., 2017). A chemical attractant (1 mM fMIVIL (SEQ ID NO: 46)) was loaded onto the left inlet to induce chemotaxis. Neutrophil migration was observed using a Meta 710 BIG microscope at 37°C and 5% CO2. Data were collected according to Ibidi’s instructions. Velocity, distance, and Euclidean distance were analyzed using ImageJ and R scripts (Kroon et al., 2018; RStudio team, 2020).

[0292] Transpore migration measurement

[0293] Confluent endothelial cells grown on 3 μm pore size transporous inserts (Lowell Corning, MA) were treated with 10 ng / mL TNF-α and 10 ng / mL IFN-γ for 4 h, and then used for neutrophil transendothelial cell migration assays. For non-endothelial-mediated migration assays, the transporous inserts were coated with ICAM1 or fibrinogen overnight at 4 °C. The inserts were then washed twice with PBS. Neutrophils in 0.1% BSA-RPMI 1640 medium were seeded in the upper chamber of the insert and allowed to migrate towards the lower chamber containing a specified concentration of formyl peptide (fMIVIL (SEQ ID NO:46)) in a CO2 incubator at 37 °C for 4 h. (Nuzzi, Paul; Lokuta, Mary; Huttenlocher, 2007; Southgate et al., 2008).

[0294] peptide inhibitors

[0295] Myristic acylated peptide MB2mP6 (Myr-FEKEKL (SEQ ID NO:27)) and control peptide (Myr-FKKEKL (SEQ ID NO:49)) were synthesized and purified at the Research Resource Center of the University of Illinois at Chicago. Micellar peptide formulations were prepared as previously described (Shen et al., 2013). The micellar peptides were resuspended in RPMI 1640 medium or HBSS.

[0296] Quantitative analysis of neutrophil diffusion area

[0297] Cell diffusion area was measured using ImageJ. Images were adjusted for brightness and contrast to enhance cell edge visibility. All cells were observed individually to determine if they exhibited membrane folds. Figure 5B The top right image shows wrinkled cells, while the bottom right image shows non-wrinkled cells.

[0298] Neutrophils migrate toward the chemical attractant in the migration chamber.

[0299] Live cell imaging

[0300] The central channel of an Ebid. μ-Slide chemotaxis chamber (Ebid., Fitchburg, Wisconsin, USA) was pre-coated with 10 μL of PBS containing 10 μg / mL purified ICAM1 (expressed in 293T cells) for 1 hour at 37°C. Mouse neutrophils were isolated using HBSS (5.33 mM KCl, 0.44 mM KH2PO4, 4.16 mM NaHCO3, 137.93 mM NaCl, 0.34 mM Na2HPO4, 5.55 mM D-glucose, pH 7.4, Gibco) and diluted to 10 × 10⁻⁶. 6Cells / mL. The side chamber of the Ibid. μ-Slide chemotactic cell was filled with 70 μL RPMI (2 g / L sodium bicarbonate, 2 g / L glucose, 5 mg / L phenol red, 6 g / L NaCl, 2 g / L NaHCO3, 1.512 g / L Na2HPO4, 400 mg / L KCl, 100 mg / L MgSO4, 100 mg / L Ca(NO3)2, Sigma-Aldrich) and 10 μM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, Thermo Fisher Scientific) and non-essential amino acids (0.1 mM glycine, 0.1 mM L-alanine, 0.1 mM L-asparagine, 0.1 mM L-aspartic acid, 0.1 mM L-glutamine, 0.1 mM L-proline and 0.1 mM serine, Thermo Fisher Scientific) and 10% FBS. With all other inlets blocked, 10 mL of neutrophils were loaded into the central channel through the central bottom inlet (inlet A) to prevent cell leakage from the observation chamber between A and B (Supplementary Figure I). The Ebids chamber (Weckmann et al., 2017) was then maintained in an incubator at 37°C for 15 minutes to allow cell adhesion. Following Ebids's recommendations, N-formyl-Met-Ile-Val-Ile-Leu (fMIVIL (SEQ ID NO:46)) from RPMI medium (Southgate et al., 2008) (1 μM) of Listeria monocytogenes (L. monocytogenes) was added to the lower left side chamber (inlet C), and the imaging position was set using Zen software (Carl Zeiss Microscopy GmbH, Germany). Video was recorded every 30 seconds and converted to TIFF images for image quantification.

[0301] Cell Tracking

[0302] Use Zen software to set the pixel size to 0.55μm. 2 / pixel. ImageJ was used for manual tracking of the TIFF image. The migration distance per cell was calculated by multiplying the pixels by 0.55 μm. Trace cell counts are shown in the supplementary video.

[0303] Cell migration patterns (cumulative distance, Euclidean distance, and velocity)

[0304] Cell positions (X and Y) were manually determined using ImageJ, and cell migration distances were calibrated using pixel / μm values. Cumulative distances were determined based on the accumulated individual distances over 30 seconds. Euclidean distances were calculated using the initial and final positions of each cell. Velocity was calculated by dividing the Euclidean distance by the migration time.

[0305] Direction of cell migration

[0306] Cell locations were determined manually using ImageJ. The location data was then adjusted to the actual distance (0.55 μm). 2 / pixel). Cell positions are adjusted to initial positions, where X = 0 and Y = 0. Movement and direction of movement are determined by comparing the final position with the initial position. Initial X position, Xf: final X position, Yi: initial Y position, Yf: final Y position. Cells moving toward the chemical attractant are defined by Xf-Xi < 0 (cells toward) and Xf-Xi > 0 (cells away). The percentage of cells moving toward and away from the chemical attractant for each type is divided by the total number of moving cells (combined cell toward and cell away). The direction of cell migration is also presented using a roseplot, where the number of cells migrating to the 12 segments around position 0 is quantified. The roseplot is calculated and plotted using Rstudio (RStudio Team, 2020).

[0307] statistics

[0308] For parametric data, Stourden's t-test or ANOVA was used to analyze statistical significance. For nonparametric data, the Mann-Whitney test was used to determine statistical significance. Fisher's exact test was performed on sample size estimates. Survival analysis was performed using Graphpad software with the Kaplan-Meier method. A p-value < 0.05 was considered significant.

[0309] Example 1: High-load ExE peptide nanoparticles as a novel antiplatelet drug

[0310] Gα of the β3 cytoplasmic domain (FEEERA (SEQ ID NO:6)) 13 Binding ExE motifs selectively mediate outward-to-inward signaling and occlusive thrombosis (21). However, efficiently delivering the synthetic ExE motif peptide mP6 into cells in vivo for therapeutic purposes remains a challenge. While liposomes or lipid micelles can be used for in vivo peptide delivery, the low concentrations of peptides incorporated into liposomes / lipid micelles rarely achieve effective doses for clinical use, except for a few very high-affinity drugs. In these studies, only 1 mM mP6 concentrations (<4% of total molar content) were achieved in lipid micelle suspensions. This concentration is impractical for clinical use.

[0311] To efficiently deliver ExE motif peptides into cells in vivo and thus translate novel concepts into clinically available drugs, novel lipid-stabilized high-load peptide nanoparticles (HLPNs) have been developed. The main component of HLPN is an amphiphilic peptide (or a peptide conjugated to a lipid or hydrophobic molecule) capable of self-assembling to form micellar nanoparticle structures (even if unstable). The peptide micellar nanoparticles are stabilized by a low percentage of phosphatidylcholine and protected by polyethylene glycol (PEG) linked to 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE), thereby forming a hydrophilic outer layer. Figure 1A A redesigned mP6 was developed to enhance its incorporation into HLPN. The novel peptide M3mP6 (Myr-FEEERL (SEQ ID NO:25)) contains a key ExE motif, and its N-terminal phenolic alanine is linked to a myristoyl group. The C-terminal alanine is replaced with a leucine with a long hydrophobic side chain, which facilitates the packaging of higher concentrations of peptides with lipids into nanoparticles. Figure 1B As shown by immunoprecipitation, this peptide retains mP6's ability to inhibit Gα. 13 The function of -β3 interaction ( Figure 1C and 1D M3mP6 HLPN achieves a high M3mP6 peptide loading of >80% (mol / mol) of total nanoparticles and a high peptide concentration of >10 mM in injectable suspension (>10 times higher than the original lipid micelle formulation of mP6 (21)).

[0312] As analyzed by dynamic light scattering (DLS), the vast majority (99.8–100%) of these lipid-stabilized PEG-coated M3mP6 HLPNs have an average size ranging from 6 nm to 20 nm (in different formulations), with occasional very small groups (0–0.2%) exhibiting larger diameters (approximately 50–500 nm). Figure 1E The lyophilized powder of M3mP6 is readily soluble in physiological saline for intravenous injection and is stable for more than 18 months when stored at -20°C, exhibiting similar DLS curves and pharmacological effects. It is also stable for at least 2 weeks when stored at room temperature (22°C). Figures 18A-18F ).

[0313] Acute MTD (maximum tolerated dose) studies in mice showed no signs of toxicity following a single bolus injection of up to 60 mg peptide / kg (approximately 60 μmol / kg). Further toxicity studies in rats showed no observable toxicity to M3mP6 HLPN after MTD exceeding 100 mg peptide bolus injection (80 × conversion efficacy dose) and after continuous infusion at 150 mg / kg / day for 5 days. M3mP6 HLPN dose-dependently inhibited low-dose thrombin-induced human platelet granule secretion and secretion-dependent platelet aggregation in vitro. Figure 2A and 2B However, it had no effect on high-dose thrombin-induced platelet aggregation. Figure 2C and 2D Even at high thrombin concentrations, platelet granule secretion is still partially inhibited by M3mP6. Figure 2D ).

[0314] M3mP6 HLPN also partially inhibits collagen production ( Figure 2E Platelet aggregation induced by U46619 (thromboxane A2 analogue) and U46619 (thromboxane A2 analogue) Figure 2F However, it does not affect ADP-induced platelet aggregation. Figure 2G It does not affect JonA ( ), nor does it affect JonA ( Figure 2H ) or fibrinogen ( Figure 2I M3mP6 binds to platelets induced by PAR4 agonist peptides. These data confirm that M3mP6 does not affect inward-outward signal transduction or the ligand binding function of αIIbβ3, but it does inhibit the secondary platelet response to integrin inward signal transduction.

[0315] Fluorescence microscopy of fluorescently labeled M3mP6 showed that M3mP6 HLPN entered platelets after incubation. Figure 19 Compared to the same concentration of M3mP6 dissolved in DMSO, as analyzed by flow cytometry, the amount of fluorescent M3mP6 HLPN entering platelets was significantly increased. Figure 3A Consistently, only 1 / 4 concentration of M3mP6 in HLPN is required to achieve comparable inhibition of platelet aggregation compared to the same peptide dissolved in DMSO. Figure 3B This demonstrates superior intracellular delivery. Importantly, the M3mP6 peptide dissolved in DMSO had no effect on arterial thrombosis in vivo. Figure 3C In contrast, M3mP6HLPN inhibited occlusive thrombus formation in a dose-dependent manner using a robust FeCl3-induced carotid artery thrombosis model in mice. Figure 3C , 3D See Figures 20A-20B(Comparison with negative control peptides) This demonstrates the effect of HLPN on peptide activity in vivo. When administered via retroorbital injection ( Figure 3D ) and administered via tail vein injection at a concentration of 1.25 μmol / kg ( Figure 21 At doses above 2.5 μmol / kg, the antithrombotic effect of M3mP6 HLPN was observed. When the same amount of peptide (5 μmol / kg) was injected, HLPN containing 50% (mass / mass) M3mP6 peptide had similar antithrombotic effects to HLPN containing 36% M3mP6, confirming that peptide concentration, rather than the formulation, determined the antithrombotic effect. Figure 22 Pharmacokinetic studies have shown that at t 1 / 2-λz =2.46 hours after intravenous injection of 5 mg / kg M3mP6 (5 μmol / kg) Figure 24 The blood and plasma levels of M3mP6 reached their maximum within 10 minutes, with a plasma level of 59.63 ng / ml. Figure 3E And the concentration in whole blood was 33.36 ng / ml. Figure 3F Consistently, the antithrombotic effect of M3mP6 was confirmed 5 minutes after injection and persisted until approximately 45 minutes after injection. Figure 3G This efficacy window is longer than that of current intravenous antiplatelet drugs canagrelor and eptifibatide. A 5-day infusion study in rats showed a t0.05 of 3.1 (male) and 3.7 (female) hours. 1 / 2-λz (Half-life after infusion cessation) (see also) Figure 24 (PK characteristics). Therefore, M3mP6 HLPN is a rapid-acting and reversible antiplatelet drug suitable for intravenous injection, and its therapeutic effect can be prolonged by continuous infusion if needed.

[0316] Example 2: Compared with high-dose aspirin, M3mP6 HLPN has superior antithrombotic effects but does not cause bleeding.

[0317] A robust FeCl3-induced mouse carotid artery thrombosis model was used to compare the effects of M3mP6 HLPN and aspirin in inhibiting occlusive thrombus formation. A mouse model was used because the β3 cytoplasmic domain is identical between humans and mice. In this model, wild-type C57BL / 6 mice had a median time of 199 seconds to form a stable occlusive thrombus after injury was induced by contact with 2 mm diameter filter paper soaked in 1.2 μl of 7.5% FeCl3 for 3 minutes, as measured by Doppler ultrasound. Compared with normal controls, a single bolus injection of M3mP6 HLPN (5 or 10 μmol / kg, 15 minutes before surgery, retroorbital) significantly delayed the formation of a stable occlusive thrombus. Figure 4AIn contrast, the tail hemorrhage time (10 μmol / kg) in mice treated with M3mP6 HLPN was not significantly different from that in the control group. Figure 4B In this effective thrombosis model, the occlusion time (4.3 mg / kg, 2 hours before surgery, orally) and tail hemorrhage time in aspirin-treated mice were not significantly different from those in normal controls. Figure 4A and 4B To rule out the possibility of insufficient aspirin administration, the effects of very high doses of aspirin (36 mg / kg, intraperitoneal, 1 hour before the experiment) on thrombosis and bleeding were also tested. Under these robust thrombotic conditions ( Figure 4C This dose of aspirin still had no significant effect on occlusive thrombosis, but significantly increased tail bleeding time. Figure 4D These effects are in contrast to those of M3mP6 HLPN (5 μmol / kg), which significantly inhibits occlusive thrombus formation but does not affect tail bleeding time. Figure 4C and 4D The combination of 5 μmol / kg M3mP6 HLPN and 36 mg / kg aspirin showed similar efficacy in significantly inhibiting thrombus formation as M3mP6 HLPN alone. Figure 4C Furthermore, the duration of tail bleeding was similar to that of aspirin alone. Figure 4D Therefore, M3mP6HLPN is significantly superior to aspirin in inhibiting FeCl3-induced arterial thrombosis without causing bleeding.

[0318] Example 3: Comparison and synergistic effect of M3mP6 HLPN and oral P2Y12 inhibitors on thrombosis and hemostasis

[0319] The antithrombotic effect of M3mP6 HLPN was compared with that of the P2Y12 antagonist clopidogrel, which is currently the standard of care for antiplatelet therapy that is more effective than aspirin. A single oral loading dose of clopidogrel (4 mg / kg, 2 hours before surgery) resulted in a moderate but significantly delayed FeCl3-induced post-injury occlusive thrombus formation. However, this dose of clopidogrel was significantly less effective than M3mP6 HLPN in inhibiting FeCl3-induced occlusive thrombus formation. Figure 5A The antithrombotic effect of M3mP6 HLPN was compared with that of ticagrelor, a more effective direct-oral P2Y12 antagonist that does not require hepatic transformation. A single injection of M3mP6 HLPN had a similar antithrombotic effect to a single high-dose oral administration of ticagrelor (3 mg / kg, 2 hours). Figure 5ATherefore, M3mP6 HLPN is similar to the potent P2Y12 inhibitor ticagrelor in its ability to inhibit occlusive thrombosis in vivo. Importantly, it was further demonstrated that mice treated with M3mP6 HLPN in combination with clopidogrel or ticagrelor had significantly longer median occlusion times than mice treated with each of the drugs alone, and their additive median occlusion times were longer than those of M3mP6 HLPN plus clopidogrel or M3mP6 HLPN plus ticagrelor. Figure 5A These data suggest that M3mP6 HLPN has a synergistic effect with clopidogrel and ticagrelor in inhibiting thrombus formation.

[0320] P2Y12 receptor inhibitors have been shown to cause excessive bleeding. In fact, clopidogrel and ticagrelor significantly prolonged tail bleeding time in mice compared to controls. Figure 5B In contrast, mice treated with M3mP6 HLPN had normal bleeding time. Figure 5B Additionally, the bleeding time in mice treated with a combination of M3mP6 HLPN and clopidogrel or ticagrelor was similar to that in mice treated with one of the P2Y12 receptor inhibitors alone. Figure 5B However, since the tail bleeding time analysis terminated at 900 seconds, and the high concentrations of clopidogrel or ticagrelor used already resulted in a significant increase in bleeding, approaching the maximum limit of this assay, the potential for exacerbation of bleeding by combining M3mP6 HLPN with P2Y12 inhibitors will be discussed further in the following sections. In summary, these data suggest that intravenous M3mP6 HLPN is superior to oral P2Y12 inhibitors in the acute treatment of arterial thrombosis and reduction of bleeding risk. Importantly, M3mP6 HLPN exhibits a synergistic antithrombotic effect when used in combination with P2Y12 inhibitors.

[0321] Example 4: Comparison and synergistic effects of M3mP6 HLPN with clopidogrel plus aspirin on thrombosis and hemostasis.

[0322] The combination of clopidogrel and aspirin is recommended for antiplatelet therapy in patients with coronary artery disease (CHD). Therefore, this study investigated the comparison and synergistic effects of combining M3mP6 with clopidogrel and aspirin. A single oral loading dose of clopidogrel combined with aspirin had similar antithrombotic effects to clopidogrel alone, and was significantly less effective than a single injection of M3mP6 HLPN alone in inhibiting occlusive thrombus formation. Figure 5C Compared with the combination of clopidogrel and aspirin, the combination of M3mP6 HLPN with aspirin and clopidogrel has a significantly enhanced antithrombotic effect. Figure 5CHowever, its antithrombotic effect is similar to that of the combination of M3mP6 HLPN and clopidogrel in the absence of aspirin. Figure 5A In the analysis of tail bleeding time, compared with normal controls or M3mP6HLPN alone, clopidogrel plus aspirin significantly increased bleeding time. Figure 5D This further underscores the benefits of M3mP6 HLPN in reducing the risk of bleeding.

[0323] Example 5: Comparison and synergistic effect between M3mP6 HLPN and intravenous P2Y12 inhibitor cangrarel

[0324] Recently, canagrelor, a novel intravenous direct P2Y12 inhibitor, was approved by the FDA for the treatment of acute thrombotic events. Compared with oral P2Y12 inhibitors, canagrelor has a faster and more effective antithrombotic effect, but also significantly leads to more severe bleeding (23, 24). The acute antithrombotic effect of canagrelor was compared with that of M3mP6 HLPN under experimental conditions simulating a thrombotic emergency when the drug was injected retroorbitally after the onset of carotid artery thrombosis. Under these conditions, there was no statistically significant difference in the antithrombotic effect between M3mP6 (10 μmol / kg) and a loading dose of canagrelor (30 μg / kg). Figure 6A However, using tail hemorrhage analysis, this concentration of cangrelor, rather than M3mP6, resulted in significant hemorrhage. Figure 6B Furthermore, when used in a laser-induced mouse model of cremasteric artery thrombosis, intravenous pre-injection of M3mP6 HLPN (10 μmol / kg) showed no significant difference in complete inhibition of thrombus formation compared to a loading dose of cangrarel (30 μg / kg). Figure 6E and Figure 23A The effects of M3mP6 HLPN injection versus a loading dose of cangrarel in preventing occlusive carotid artery thrombosis induced by high concentrations of FeCl3 were compared. Under these conditions, the effect of a single loading dose of cangrarel (30 g / kg, retroorbital) on occlusive thrombosis varied considerably among individual mice or in experiments. In most mice tested, the effect on vascular occlusion was similar to that of M3mP6 HLPN. However, a small group of mice treated with cangrarel showed a longer occlusion time (…). Figure 6C Therefore, Kangreilo's overall effect is moderate but significantly better than M3mP6 HLPN. Figure 6C However, cangrarol treatment significantly increased tail hemorrhage time, while M3mP6 HLPN had no effect on hemorrhage. Figure 6D The combination of M3mP6HLPN and Cangrelo showed a significantly longer median occlusion time than either M3mP6 HLPN or Cangrelo alone. Figure 6C Furthermore, its additive effect is longer than that of M3mP6 HLPN and cangrarel, indicating a synergistic effect between M3mP6 HLPN and cangrarel, which significantly enhances the antithrombotic efficacy.

[0325] Example 6: Comparative effect of M3mP6 HLPN and Cangrelo on intravascular coagulation using a mouse laser-induced cremasteric artery thrombosis model.

[0326] Recent studies have shown that outward-to-inward signal transduction plays a crucial role not only in platelet thrombosis but also in intravascular coagulation under flow shear, a significant aspect of thrombosis (25). This result contrasts with previous reports, suggesting that current antiplatelet drugs have little effect on intravascular coagulation (26, 27). Therefore, the effects of M3mP6 HLPN versus canagrelor on platelet thrombosis and intravascular coagulation were evaluated using a laser-induced cremasteric artery thrombosis model. Although both drugs have similar efficacy in inhibiting platelet thrombosis ( Figure 6E However, M3mP6 HLPN almost completely inhibited the formation of intravascular fibrin clots at the site of vascular injury, while cangrelo only had a moderate effect. Figure 6F and Figure 23B Therefore, M3mP6 HLPN not only effectively inhibits thrombus formation but also inhibits intravascular coagulation in vivo, and this effect is significantly superior to that of the most effective P2Y12 inhibitor, cangrarerol.

[0327] Example 7: Synergistic antithrombotic effect of M3mP6 HLPN combined with low-dose canagrelor in the absence of excessive bleeding.

[0328] These data indicate that the maximum clinical dose of canagrelor has effective antithrombotic efficacy. Figure 6A However, it also significantly increased bleeding, which contrasts with the selective effect of M3mP6 HLPN on thrombus formation. Figure 6B These data also indicate that the combination of M3mP6 HLPN with this high-dose canagrelor significantly enhanced its antithrombotic efficacy compared to high-dose canagrelor alone. Figure 6C However, it appears to have similar bleeding side effects as cangrelo alone. Figure 6D Therefore, it was hypothesized that the combination of M3mP6 HLPN and low-dose canagrelor could synergistically enhance antithrombotic efficacy while reducing bleeding side effects caused by high-dose canagrelor. In fact, when M3mP6 HLPN was used in combination with low-dose canagrelor (10 g / kg), the antithrombotic effect was even significantly greater than the maximum clinical dose of canagrelor (…). Figure 6CHowever, bleeding was significantly reduced. Figure 6D Therefore, the use of M3mP6 HLPN in combination with low-dose canagrelor would be the best option for acute antithrombotic therapy, as it has a strong antithrombotic effect and reduces the risk of bleeding.

[0329] Example 8: Comparison of M3mP6 HLPN and Cangrelo in an arterial perforation model of surgical bleeding

[0330] Clinically, the risk of bleeding during antiplatelet therapy is often associated with invasive interventions, particularly endovascular interventions where vessel wall perforation is a necessary consequence. To more closely simulate bleeding during endovascular / surgical procedures, a surgical bleeding model of carotid artery perforation was designed. In this model, the common carotid artery is surgically exposed and punctured with a needle. A hemostatic pad (CERTI-GAUZE) is immediately applied. TM The pad is used to cover the perforation site to achieve hemostasis in a manner similar to that performed during vascular intervention. Blood adsorbed on the pad is washed off and quantified to indicate the level of bleeding. In the normal control, bleeding at the perforation site is rapidly stopped by the hemostatic pad, with only a small amount of extravasation. Figure 3F Even with the use of a hemostatic pad, a loading dose of canagrelor resulted in excessive bleeding from the perforating artery. In contrast, in this surgical bleeding model, mice treated with M3mP6 HLPN showed no difference from control mice. Figure 7A This further demonstrates that M3mP6 HLPN is a safe antiplatelet drug that does not cause excessive bleeding under simulated vascular interventional conditions. To verify that the combination of M3mP6 HLPN with a P2Y12 antagonist does not exacerbate the side effects of the P2Y12 antagonist, M3mP6 was used in combination with high-dose and low-dose canagrelor. There was no difference in bleeding, as quantified by the amount of bleeding between canagrelor alone and canagrelor plus M3mP6 HLPN. Importantly, the combination of M3mP6 HLPN and low-dose canagrelor did not show a significant difference in bleeding compared to the normal control group. Figure 7B These data clearly demonstrate that M3mP6 HLPN is an effective antiplatelet drug that does not cause bleeding, and that the combination of M3mP6 HLPN with low-dose canagrelor minimizes the risk of bleeding caused by canagrelor, while exhibiting superior antithrombotic effects compared to even the highest clinical doses of canagrelor.

[0331] Example 9: M3mP6 HLPN did not cause prolonged bleeding in the canine buccal mucosal bleeding time (BMBT) test.

[0332] To determine whether M3mP6 might affect hemostasis in large animals, the BMBT, a routine bleeding time test, was used to measure bleeding time in three dogs before and during M3mP6 HLPN infusion. No significant difference in bleeding time was observed before and after M3mP6 HLPN administration; both were within the normal bleeding time range (dogs <4 minutes). Figure 7C These data indicate that M3mP6 HLPN does not cause excessive bleeding, not only in rodents but also in dogs.

[0333] Example 10: Treatment of myocardial infarction-reperfusion (MI / R) injury with M3mP6 HLPN

[0334] The current popular treatment for myocardial infarction / ischemia (MI) involves surgical or percutaneous coronary intervention to physically reopen the occluded artery. However, reperfusion of ischemic tissue can lead to myocardial ischemia / reperfusion (MI / R) injury, in which ischemic tissue undergoes an acute thromboinflammatory response upon re-exposure to oxygenated blood, resulting in impaired cardiac function and death. To evaluate the therapeutic effect of M3mP6 HLPN on MI and MI / R injury under conditions simulating a clinical MI process, severe MI was induced in mice by ligating the left anterior descending coronary artery (LAD) for 45 minutes, followed by reopening to allow reperfusion. To simulate clinical treatment, M3mP6 or control HLPN was administered post-ischemic injection 35 minutes after MI induction. Figure 8A Compared with the control group, such as by triphenyltetrazol chloride (TTC) / Evans blue staining ( Figure 8B-8D As shown, the M3mP6 HLPN treatment group exhibited a significantly lower infarct area / danger area ratio and prevented impairment of cardiac function, as indicated by echocardiography performed 24 hours post-procedure. Figure 8E And F). Histoimmunochemical studies showed that M3mP6 HLPN treatment significantly reduced MI / R-induced microvascular thrombosis in reperfused cardiac tissue. Figure 8G ) and interesting neutrophil infiltration ( Figure 8H M3mP6 also reduced plasma MPO levels in MI / R mice. Figure 8I This is an indicator of neutrophil activation. Importantly, M3mP6 significantly reduced mortality during the 7-day postoperative monitoring period. Figure 8J It was also found that M3mP6 can improve MI / R survival and is more effective than canagrelor in treating myocardial ischemia / reperfusion injury. Figure 8K-8L These data indicate that M3mP6 is effective in treating MI / R-induced thrombosis / inflammation and cardiac injury in mouse models.

[0335] Example 11: Effects of ExE motif peptide on leukocyte function and systemic inflammation.

[0336] The Gα13-binding ExE motif is conserved in different integrin β subunits with sequence variations, including the leukocyte-specific β2 integrin subunit. Therefore, to investigate the effects of MB2mP6 on leukocyte function and inflammation, a myristylated peptide of the Gα13-binding motif Myr-FEKEKL (SEQ ID NO:27) (MB2mP6) derived from the β2 cytoplasmic domain and lipid-stabilized high-MB2mP6-loaded nanoparticles were prepared. Pre-incubation of the MB2mP6 nanoparticles significantly attenuated the transendothelial migration of peripheral blood neutrophils induced by the bacterial chemokine (fMIVIL (SEQ ID NO:46)). Figures 9A-9C This indicates that MB2mP6 indeed inhibits leukocyte function. To determine the effect of MB2mP6 on systemic inflammation, sepsis was induced in mice using a standard cecal ligation-puncture (CLP) model, and sepsis-induced mortality was compared between the control group and the MB2mP6-treated group. MB2mP6 significantly reduced CLP-induced inflammation, as indicated by pro-inflammatory cytokine levels (…). Figure 10A-10D It also reduced sepsis-induced mortality. Figure 11 Consistent with this observation, M3mP6 was also observed to inhibit neutrophil infiltration into reperfused cardiac tissue following myocardial ischemia. Figure 8H These data provide evidence that the ExE peptide, as described in this patent, is an effective anti-inflammatory drug, which was previously unknown.

[0337] Example 12: Defective ligands enhance the binding function of integrin αIIbβ3 to thrombin-induced platelet granule secretion.

[0338] To investigate the mechanism by which integrin αIIbβ3 outward-to-inward signal transduction regulates platelet granule secretion, granule secretion was compared between wild-type and β3- / - platelets. Figure 1A Despite defective aggregation, β3- / - platelets stimulated by low concentrations of thrombin showed an enhanced ATP secretion response. Figure 12B and 12C However, at higher thrombin concentrations, no difference in ATP release was observed between wild-type and β3- / - platelets. Figure 12B and 12C To determine whether the effect of β3 deficiency on platelet secretion was caused by platelet developmental defects or ligand defects in integrin binding, wild-type mouse or human platelets were treated with the integrin antagonist integrilin and then stimulated with a low dose of thrombin. Integrilin enhanced platelet granule secretion in a similar manner to β3 deficiency. Figure 12D and 12E Therefore, the ligands that bind to integrin αIIbβ3 appear to negatively regulate granule secretion, which clearly contradicts the understanding that the ligands that bind to integrin αIIbβ3 promote platelet granule secretion.

[0339] Example 13: The dual role of integrin αIIbβ3 in regulating platelet granule secretion.

[0340] It is well known that platelet granule secretion can be mediated through two distinct pathways: integrin-dependent granule secretion induced by agonist receptor signaling and integrin-independent granule secretion induced by outward-to-inward signaling. When platelets are stimulated with thrombin, the secretions induced by these two pathways are usually not dissociated. However, platelet granule secretion induced by the stable thromboxane A2 analog U46619 showed a distinct dissociation between the integrin-independent and integrin-dependent waves. Therefore, U46619 was used to stimulate platelets and examine the effect of αIIbβ3 deficiency on integrin-independent first-wave and integrin-dependent second-wave granule secretion. First-wave granule secretion in β3- / - mouse platelets ( Figure 13A and 13B ) and Integrilin-treated mouse and human platelets ( Figure 13D and 13E Significant enhancement in both. In contrast, in β3- / -( Figure 13A and 13C ) and platelets treated with Integrilin ( Figure 13D and 13E In this study, integrin-dependent second-wave secretion was reduced. These data suggest that ligand binding to integrin αIIbβ3 negatively regulates GPCR-induced integrin-independent first-wave granule secretion, but stimulates integrin-dependent granule secretion.

[0341] Example 14: The binding of Gα13 to the cytoplasmic domain of integrin β3 mediates the dual role of integrins in platelet granule secretion.

[0342] Integrin-dependent platelet granule secretion requires out-to-in signaling. Previous studies have shown that early integrin out-to-in signaling is mediated by the direct binding of Gα13 to the cytoplasmic domain of the integrin β3 subunit. It has also been shown that the EEE motif in the cytoplasmic domain of Gα13 and β3 interacts, and mutating these glutamate residues to alanine (AAA mutation) eliminates the Gα13-β3 interaction and selectively eliminates integrin out-to-in signaling without affecting in-to-out signaling and ligand binding to integrin. To investigate whether the Gα13-integrin interaction is important for regulating platelet granule secretion, β3- / - mice were lethally irradiated and transplanted with bone marrow stem cells expressing wild-type or AAA mutant β3, respectively. Flow cytometry data showed that wild-type and AAA mutant platelets expressed similar levels of integrin β3 (… Figure 14A As expected, when stimulated with low doses of thrombin, platelets expressing this outward-to-inward signaling defective β3 mutant (AAA) showed reduced aggregation compared to wild-type platelets. Figure 14B However, ATP secretion is enhanced rather than reduced in AAA platelets. Figure 14B and 14C This is similar to β3- / - mouse platelets and integrilin-treated human platelets. Furthermore, in U46619-stimulated platelets, integrin-dependent second-wave granule secretion is selectively eliminated in AAA mutant platelets. Figure 14D and 14E In contrast, compared to platelets expressing wild-type β3, AAA mutant platelets showed enhanced integrin-independent first-wave platelet granule secretion. Figure 14D and 14E This indicates that Gα13-integrin interaction selectively inhibits integrin-independent platelet granule secretion, but stimulates integrin-dependent granule secretion.

[0343] Example 15: Effects of Gα13 inhibitory peptides mP6 and mP5 on platelet granule secretion.

[0344] Gα13 plays an important role in both integrin-independent and integrin-dependent granule secretion. Figures 15A-15EIn these studies on Gα13-integrin interactions, two integrin-based peptide inhibitors, mP5 (myr-EEERA (SEQ ID NO:22)) and mP6 (myr-FEEERA (SEQ ID NO:23)), were prepared. These peptide inhibitors inhibit Gα13-integrin interactions and outward-inward signaling without affecting ankle protein-integrin interactions and ligand-integrin binding. Therefore, the effects of these two inhibitors on platelet granule secretion were evaluated. As expected, platelets pretreated with mP5 partially inhibited thrombin-induced platelet aggregation but enhanced platelet ATP secretion. Figures 16A-16E Consistent with results obtained using platelets from AAA mutants expressing β3, mP5 significantly enhanced the first wave of ATP secretion induced by U46619, but reduced the integrin-dependent second wave of ATP secretion. Figure 16A and 16B These results, along with data obtained using Gα13- / - platelets, indicate that myr-EEERA (SEQ ID NO:22) selectively inhibits integrin-Gα13 interactions without eliminating the overall function of Gα13. These data also support the conclusion that Gα13-integrin interactions negatively regulate integrin-independent granule secretion but stimulate integrin-dependent granule secretion in mouse and human platelets. Figure 17 ).

[0345] Compared with mP5, pretreatment of platelets with mP6 effectively inhibited thrombin-induced platelet aggregation and also significantly inhibited thrombin-induced ATP secretion. Figure 5A and 5B This indicates that both the integrin-dependent and integrin-independent components of platelet granule secretion are inhibited. In fact, in mP6-treated platelets, both U46619-induced integrin-independent first-wave granule secretion and integrin-dependent second-wave secretion are reduced. Figures 5A-5D This contrasts with platelet formation after mP5 treatment. These results indicate that mP6 inhibits the function of Gα13 to stimulate both integrin-independent and integrin-dependent granule secretion.

[0346] The data presented in this article demonstrate that the exemplary M3mP6 HLPN is not only a potent antithrombotic agent on its own, but also exhibits a significant synergistic effect when used in combination with current standard-of-care P2Y12 inhibitors (with or without aspirin). This synergistic effect is observed even at the highest clinical doses with clopidogrel and the more potent novel direct P2Y12 inhibitors ticagrelor and canagrelor. These synergistic effects suggest that the integrin-dependent secondary amplification of thrombus formation requires the release of known agonists such as ADP and thromboxane A2 (TXA2), and also requires the activation of additional signaling pathways that are not inhibited by ADP receptor antagonists or TXA2 synthesis pathway inhibitors. Since M3mP6 HLPN does not increase the bleeding risk associated with aspirin and P2Y12 inhibitors, this synergistic effect also suggests that the combination of M3mP6 HLPN with P2Y12 inhibitors has the potential to enhance antithrombotic efficacy to a level currently unattainable with antiplatelet therapy, without the risk of devastating bleeding. Consistent with this view, the combination of M3mP6 and low-dose canagrelor has been demonstrated to not only enhance antithrombotic efficacy more effectively than the highest clinical dose of canagrelor, but also to significantly reduce bleeding associated with high-dose canagrelor. Therefore, the combination of M3mP6 HLPN with a lower dose of a P2Y12 inhibitor represents a novel antithrombotic strategy with significant clinical implications, as it enhances antithrombotic efficacy and mitigates the life-threatening bleeding risk associated with currently used antiplatelet drugs. These data further suggest that this rapidly acting new drug can be used as an effective post-ischemic treatment for acute myocardial ischemia / reperfusion (I / R) injury. Post-ischemic injection of M3mP6 HLPN inhibits both microvascular thrombosis and inflammation in reperfused cardiac tissue and improves cardiac function and survival in a mouse model of myocardial ischemia / reperfusion.

[0347] Example 16: MB2mP6 blocks the interaction between Gα13 and β2 and β3 integrins and inhibits macrophage and platelet function without causing bleeding.

[0348] Integrins play a crucial role in platelet and leukocyte function. Current antiplatelet drugs either inhibit or block integrin-activated “inside-out” signaling or block ligand binding to integrins. Because integrin αIIbβ3-mediated primary platelet adhesion / aggregation is important for hemostasis, these drugs can exacerbate bleeding. Ligand binding to integrins stimulates “outside-in” signaling, which is important for platelet-mediated thrombus expansion, leukocyte transport, and inflammation. Ligand binding to integrin αIIbβ3 induces the binding of the G protein subunit Gα13 to β3, thereby transmitting outside-in signaling (14, 15). Because outside-in signaling is a post-adhesion amplification mechanism, inhibition of this process has minimal impact on primary integrin-mediated platelet aggregation and hemostasis (16). Gα13 binds to the cytoplasmic ExE motif homologous in β2 and β3 integrins (16). Therefore, an inhibitory peptide, MB2mP6 (Myr-FEKEKL (SEQ ID NO:27)), was designed based on the ExE sequence of integrin β2. MB2mP6 effectively inhibited the co-immunoprecipitation of Gα13 and β2 integrin in LPS-stimulated macrophages differentiated from the human monocytic leukemia cell line (THP-1) (17). Figure 25A Furthermore, it cross-inhibits the co-immunoprecipitation of Gα13 and β3 in thrombin-stimulated human platelets. Figure 25B MB2mP6 effectively inhibits mouse bone marrow-derived macrophages (BMDM). Figure 25C and 25D The expression of LPS-induced pro-inflammatory cytokines IL-1β and IL-6 in macrophages suggests that Gα13-integrin interaction is important in the pro-inflammatory function of macrophages. Furthermore, MB2mP6 inhibits human platelet aggregation and secretion in vitro. Figure 25E and 25F It also inhibited FeCl3-induced carotid artery thrombosis in mice in vivo, although its antithrombotic effect appeared to be less effective than that of β3-derived M3mP6 peptide. Figure 25G Importantly, MB2mP6 did not affect the injury-induced tail hemorrhage time. Figure 25H It also does not affect inflammation-induced hemorrhage in the mouse reverse passive Arthus (rpA) response. Figure 25I These data indicate that MB2mP6 has anti-inflammatory and antithrombotic effects, but does not exacerbate bleeding.

[0349] Example 17: MB2mP enhances the survival rate of a CLP sepsis model and prevents sepsis-induced organ damage.

[0350] A mouse model of severe multimicrobial sepsis with cecal ligation and puncture (CLP) was used to test the potential therapeutic effect of MB2mP6 on systemic inflammation. Immediately after CLP, MB2mP6 or a disordered control peptide in a micelle nanoparticle formulation was intravenously infused via a pre-placed jugular vein cannula at a rate of 1.25 μmol / kg / h. The MB2mP6 group showed a significantly higher survival rate at 192 hours (8 days) (71% survival in the MB2mP6 group compared to 26% in the control peptide group); p = 0.002 ( Figure 26A The control peptide group was similar to the saline treatment group. Figure 26A To more closely mimic the clinical presentation of sepsis treatment, MB2mP6 infusion was initiated 6 hours after CLP onset. Under these conditions, MB2mP6 still significantly improved the 8-day survival rate (42% survival in the MB2mP6 group compared to 12.5% ​​in the control group, p = 0.034). Figure 26B Therefore, even with a 6-hour delay in injection, MB2mP6 effectively improved the survival rate of septic mice. To assess the effect of MB2mP6 on in vivo inflammation during sepsis, the levels of pro-inflammatory cytokines in mouse serum collected 24 hours after CLP were tested. Control mice showed a significant increase in IL-6 and TNFα secretion, which were significantly reduced by MB2mP6 treatment. Figure 26C and 26D Similarly, the expression of IL-6 and TNFα transcripts in the lungs of septic mice was also induced by CLP and significantly inhibited by MB2mP6 infusion. These data suggest that MB2mP6 has a potent anti-inflammatory effect in vivo during severe sepsis.

[0351] Severe sepsis can lead to glomerular microvascular thrombosis, thereby impairing renal function (18, 19). Indeed, microvascular thrombosis has been observed in the glomeruli of CLP-secreting mice, such as by Mallory phosphotungstic acid hematoxylin (PTAH) (…). Figure 26E Antifibrin staining for fibrin deposition and anti-αIIb staining for platelets are shown in the following: Figure 29A and 29B CLP sepsis also impairs kidney function, as evidenced by elevated levels of BUN, creatinine, and cystatin C in the control group 24 hours after CLP. Figure 26F and Figure 29C and 29D The elevation shown is indicated by MB2mP6 treatment. MB2mP6 treatment significantly inhibited glomerular thrombosis and reduced BUN, creatinine, and cystatin C. Figure 26E-26F and Figures 29A-29D Therefore, MB2mP6 inhibits both inflammation and renal thrombosis, and increases the survival rate of septic mice.

[0352] Example 18: Leukocyte or platelet-specific Gα13 knockout reduced mortality in CLP-induced sepsis mice.

[0353] To determine the role of leukocyte Gα13 in systemic inflammation and to evaluate the contribution of leukocyte Gα13 to the efficacy of MB2mP6 treatment, Gα13 was analyzed. fl / fl Mice were mated with LysM-Cre mice to generate leukocyte-specific Gα13 knockout mice, as described in *Nature Medicine* 9, 1418-1422 (2003) (20). Western blot analysis confirmed the presence of Gα13. fl / fl-LysMCre Gα13 protein is absent in both mouse macrophages and neutrophils, but is present in platelets. Figure 30A and 30B Compared with the control group, leukocyte-specific Gα13 knockout (Gα13) fl / fl-LysMCre The 8-day survival rate of mice in CLP-induced sepsis was moderately but significantly improved (44% in the MB2mP6 group, compared to 19% in the control group, p = 0.0475). Figure 27A However, the survival rate of leukocyte-specific Gα13 knockout mice was significantly lower than that of MB2mP6-treated mice. Figure 26A right Figure 27A Importantly, the increase in serum cytokine IL-6 and TNFα levels 24 hours after CLP occurred at Gα13. fl / fl-LysMCre Significantly inhibited in mice ( Figure 27B and 27C This indicates that leukocyte Gα13 plays a major role in the sepsis-induced inflammatory state. In contrast, compared to control Gα13... fl / fl Compared to mice, Gα13 fl / fl-LysMCre In mice, glomerular microvascular thrombosis indicated by fibrin deposition was mild but not significantly reduced. Figure 27D Furthermore, the elevation of the kidney injury marker BUN was not observed in Gα13. fl / fl-LysM-Cre The blood of septic mice showed a decrease in ( Figure 27E These data indicate that leukocyte Gα13 is not a key factor in renal microvascular thrombosis and injury, and that the protective effect of leukocyte-specific Gα13 knockout against systemic inflammation in sepsis is mainly due to the inhibition of leukocyte-mediated cytokine secretion and inflammation.

[0354] To evaluate the contribution of platelet Gα13 to the therapeutic effect of MB2mP6 and its role in systemic inflammation, platelet-specific knockout mice were generated by mating Gα13flox / flox mice with PF4-Cre mice. Figure 30A and 30B ). Compared with control Gα13fl / fl Compared to mice, CLP-induced platelet-specific Gα13 knockout (Gα13) after sepsis fl / fl-PF4Cre The 8-day (192-hour) survival rate of mice was moderately but significantly improved (39.2% vs. 9.6% in the control group; p = 0.0475). Figure 27F ), but significantly lower than MB2mP6 treated mice ( Figure 26A right Figure 27F Compared to Gα13 knockout in white blood cells, compared to Gα13... fl / fl Mice, Gα13 fl / fl-PF4Cre In mice, CLP-induced glomerular microvascular thrombosis and renal function impairment (indicated by blood BUN levels 24 hours after CLP) were significantly reduced. Figure 27G and 27H Therefore, platelet Gα13 plays an important role in glomerular microvascular thrombosis and kidney injury during sepsis, and may contribute to the therapeutic effects of MB2mP6 on glomerular thrombosis and kidney injury. However, in Gα13... fl / fl-PF4Cre In mice, serum levels of the inflammatory cytokine TNFα were not significantly reduced. Figure 27I Even though the level of the cytokine IL-6 in mouse serum was partially (and significantly) reduced ( Figure 27J Therefore, it appears that platelet-specific Gα13 knockout moderately protects mice from sepsis death primarily by inhibiting microvascular thrombosis, even though platelet Gα13 may exacerbate the inflammatory state.

[0355] Example 19: Platelet and leukocyte dual-specific Gα13 knockout protects mice from death in a CLP sepsis model.

[0356] To further determine whether Gα13 in platelets and leukocytes plays a non-dependent role in sepsis-induced mortality and organ damage, platelet / leukocyte double Gα13 knockout mice were developed. fl / fl-LysM / PF4 double Cre mice, Figure 30A and 30B The 8-day survival rate of CLP in double Gα13 knockout mice was 66%. Figure 28A The survival rate was similar to that of wild-type septic mice treated with MB2mP6 (71%). Figure 28A ), and not only significantly higher than control mice, but also higher than platelet-specific Gα13 knockout mice (39%) ( Figure 27F right Figure 28A ) and leukocyte-specific Gα13 knockout mice (44%) Figure 27A right Figure 28A Consistent with these results, 24 hours after CLP, the renal injury marker BUN (…) was found in the blood of control mice. Figure 26F) and cytokine IL-6 ( Figure 26C ) or TNFα ( Figure 26D Elevated levels of ) in double Gα13 knockout mice ( Figure 28B , 28C CLP-induced glomerular microvascular thrombosis was significantly inhibited in double Gα13 knockout mice, as evidenced by a substantial reduction in fibrin deposition. Figure 28E These data not only further support the unique role of Gα13 in platelets and leukocytes in systemic inflammation of sepsis, but also demonstrate that its role in sepsis is additive. Furthermore, these data suggest that the efficacy of MB2mP6 in treating systemic inflammation of sepsis in a CLP model may be due to its ability to inhibit Gα13-integrin interactions in both platelets and leukocytes.

[0357] In summary, these experimental results clearly demonstrate that thrombosis and inflammation play an additive role in exacerbating systemic inflammation, explaining the failure of past treatments for sepsis with anti-inflammatory drugs alone, and importantly, providing strong support for new approaches to treating systemic inflammation (including sepsis) with simultaneous antithrombotic and anti-inflammatory therapies. However, all current antithrombotic approaches carry a significant risk of excessive bleeding, which has been shown to outweigh the beneficial effects of Xigris (3, 6). In contrast, MB2mP6 has been shown to target the outward-inward signaling of integrins in both leukocytes and platelets without exacerbating bleeding. Importantly, MB2mP6 has been shown to effectively treat systemic inflammation of sepsis in a CLP mouse model, reducing both inflammation and thrombosis to improve survival. Notably, the effectiveness of MB2mP6 is not limited to preventing the onset of CLP sepsis, but also shows significant therapeutic effects when administered 6 hours after CLP in mice. In mice, sepsis-induced increases in most cytokines, as well as pulmonary neutrophil recruitment, alveolar leakage, endothelial injury, hepatic neutrophil and platelet recruitment, impaired sinusoidal perfusion, and acute kidney injury, were well-established 4–6 hours after CLP (21–25). Therefore, MB2mP6 is not purely prophylactic but disrupts the natural progression of systemic inflammation. If a patient / experimental animal has already succumbed to the consequences of inflammation and thrombosis (e.g., multiple organ dysfunction), it is unlikely to recover with these (or possibly any other) drugs. In fact, immediate infusion of MB2mP6 after sepsis onset resulted in higher survival rates compared to infusion 6 hours after CLP. Therefore, early use of this class of drugs is likely to effectively block the progression of systemic inflammation to an irreversible phase. Overall, these data reveal the independent and interdependent roles of inflammation, hemorrhage, and thrombosis in systemic inflammation and provide new concepts and drugs for the simultaneous administration of anti-inflammatory and antithrombotic therapies without inducing vascular leakage and hemorrhage. This new treatment concept should help defend against systemic inflammation, which poses an increasing challenge to human health, such as the recent SARS-CoV-2 infection.

[0358] Example 20: The Importance of Gα13 in Neutrophil Transendothelial Migration

[0359] Myeloid selective knockout mouse model used to study Gα 13 The importance of Cre in neutrophil migration (Moers et al., Nature Medicine 9, 1418-1422 (2003)). C57BL / 6 mice expressing Cre under the control of the mouse lysozyme 2 promoter (LysMcre) were compared with Gα mice with a side-linked loxP. 13 Alleles (Gα) 13 fl / fl Hybridization of homozygous mice to produce myeloid-specific conditional Gα 13Knockout mice (Gα) 13 fl / fl-LysMcre In these mice, Gα 13 Expression is defective in neutrophils, but normally expressed in platelets. Figure 31A ). In Gα 13 fl / fl-LysMcre Mice and Gα 13 fl / fl In both mice, the number of neutrophils in peripheral blood and bone marrow was similar. Figures 31B-31C Transporous chambers with an endothelial monolayer grown on gelatin-coated filter membranes (Lowell Corning, MA) were used to analyze neutrophil transendothelial migration. Endothelial cells were treated with both 10 ng / mL TNF-α and INF-γ to induce the expression of β2-integrin ligand intercellular adhesion molecule 1 (ICAM1), which is important for neutrophil transmembrane migration. Cells from control Gα13 were also analyzed. fl / fl (Gα 13 + / + ) or Gα 13 fl / fl-LysMcre (Gα 13 - / - Neutrophils from mice were sampled into the upper chamber, and different concentrations of a chemical inducer (formyl peptide derived from Listeria monocytogenes, fMIVIL (SEQ ID NO:46)(N-formyl-Met-Ile-Val-Ile-Leu) (Southgate et al., 2008)) were added to the lower chamber to induce neutrophil migration across the endothelial monolayer. A small amount of control Gα was also included. 13 + / + Neutrophils migrated across the endothelial monolayer without fMIVIL (SEQ ID NO:46) stimulation, but migrating Gα... 13 - / - The number of neutrophils is even less ( Figure 31D ). Control Gα for migration 13 + / + The number of neutrophils increased significantly with increasing fMIVIL (SEQ ID NO:46) concentration, peaking at 10 nM fMIVIL (SEQ ID NO:46). Figure 31D ), and then at a higher fMIVIL (SEQ ID NO:46) concentration, the concentration was reduced ( Figure 31D This is a typical chemotactic response. However, compared to the control Gα... 13 + / + Compared to neutrophils, Gα cells that migrated to the lower compartment using 10 nM fMIVIL (SEQ ID NO:46) 13The number of defective neutrophils was significantly lower, and a peak response was not achieved until a higher fMIVIL (SEQ ID NO:46) concentration (100 nM), and the peak response was still significantly reduced compared to the peak reached by control neutrophils at 10 nM fMIVIL (SEQ ID NO:46). Figure 31D Therefore, Gα 13 It is important in the transendothelial migration of neutrophils.

[0360] Example 21: ICAM1 and β2 integrins in Gα 13 Importance of dependence on transendothelial migration.

[0361] To determine Gα 13 To investigate whether endothelial-dependent neutrophil migration is mediated by the interaction of β2-integrin with its integrin ligand ICAM1 on the endothelial cell surface, transmembrane migration of neutrophils across a transporous filter coated with purified ICAM1 was tested. Compared to a BSA-coated filter, a significantly smaller number of neutrophils migrated across the ICAM1-coated filter in the absence of the chemical inducer fMIVIL (SEQ ID NO:46), which was observed in controls or Gα. 13 The defective mice are similar to each other. However, when using fMIVIL (SEQ ID NO:46) ( Figure 32A Upon stimulation, compared to surfaces coated with BSA or fibrinogen, control neutrophils showed significantly increased transpore migration through ICAM1-coated filters, even in the presence of fMIVIL (SEQ ID NO:46), migration through pores coated with BSA and fibrinogen was also increased. In contrast, in the presence of 10 nM fMIVIL (SEQ ID NO:46), Gα... 13 Defective neutrophils migrated significantly less through ICAM1-coated filters than control Gα. 13 + / + Neutrophil migration ( Figure 32A The migration of neutrophils stimulated by fMIVIL (SEQ ID NO:46) across a control BSA-coated surface was similar to or possibly slightly increased. β2-integrin also binds fibrinogen, and this binding has been shown to be important in bacterial clearance (Flick et al., 2004). Gα was also investigated. 13 Is it also important for leukocyte migration to fibrinogen? Interestingly, the migration of control neutrophils induced by fMIVIL (SEQ ID NO:46) through a fibrinogen-coated filter was similar to that induced by fMIVIL (SEQ ID NO:46) on a BSA surface, and control neutrophils showed similar migration to Gα.13 There was no difference in response among neutrophils knocked out, suggesting that leukocyte migration to fibrinogen occurs via a different mechanism than ICAM1 and does not require Gα. 13 Dependent on outward-to-inward signal transduction. In summary, these data indicate that Gα... 13 The selection importance of ICAM1-dependent neutrophil transendothelial migration was observed, but not in neutrophil migration to another β2-integrin ligand, fibrinogen, or control BSA. To further validate the dependence of neutrophil transendothelial migration on β2-integrin, transendothelial migration assays were performed in the presence of a functionally blocking anti-β2 monoclonal antibody, compared to the IgG control. Gα13-dependent neutrophil transendothelial migration was inhibited by the anti-β2 antibody. Figure 32B These data are consistent with the previously established finding that leukocyte transendothelial migration requires β2 integrin (Ding et al., 1999), and further suggest that Gα 13 It is important in β2 integrin-dependent transendothelial migration of neutrophils.

[0362] Example 22: The role of Gα13 in the migration velocity and directionality of neutrophils on ICAM1.

[0363] Cell migration toward chemotactic attractors involves both the sensing of directional chemotactic attractor signals (which is typically GPCR-dependent) and the coordination of migration mechanisms driving cell movement, integrins playing a crucial role. To determine whether Gα13 is important in orientation sensing and / or driving cell movement, a microfluidic chamber was used to measure the velocity and directionality of neutrophil migration along a chemotactic attractor gradient. Compared to control neutrophils, Gα13 knockout neutrophils migrated at a significantly reduced velocity toward the chemotactic attractor fMIVIL (SEQ ID NO:46). Figure 33A Furthermore, the total travel distance and Euclidean distance (distance between the start and end points) of Gα13-deficient neutrophils were significantly reduced. Figures 33B-33C However, the percentage of neutrophils migrating to the chemical inducer did not differ significantly between control and knockout neutrophils. Figure 33D , 33E These data indicate that Gα13 knockout is primarily associated with chemically induced, integrin-dependent neutrophil motility defects, but not with directionality.

[0364] Example 23: Gα derived from the integrin β2 subunit 13 Inhibitory peptides binding to ExE motifs inhibit Gα 13 Integrin-dependent interactions and neutrophil transendothelial migration.

[0365] It has been previously proven that Gα13 The ExE motif, found in several integrin β subunits, interacts directly with the β subunit (Haixia Gong, Bo Shen, Panagiotis Flevaris, Christina Chow, Stephen C.-T. Lam, Tatyana A. Voyno-Yasenetskaya, Tohru Kozasa, 2010; Shen et al., 2015, 2013). Further exploration of Gα... 13 The role of β2-β2 interaction in neutrophil transendothelial migration was investigated, examining the effects of the synthetic peptide myr-FEKEKL (SEQ ID NO:27) (MB2mP6) derived from the β2ExE motif or the control peptide (myr-FKKEKL (SEQ ID NO:47)) on neutrophil migration. Compared to the control peptide, MB2mP6 inhibited Gα-β2 interaction in human neutrophils stimulated by the formal peptide. 13 Interaction with β2 integrin ( Figures 34A-34B ).

[0366] However, compared with the control peptide, MB2mP6 (50 μM) had no significant effect on the adhesion of neutrophils to ICAM1. Figure 35A This indicates that Gα 13 - Integrin interactions are not important in inside-out signal transduction or ligand binding function of β2-integrin. In contrast, neutrophil transendothelial migration is dose-dependently inhibited by MB2mP6 ( Figure 35B To determine whether the peptide's function is Gα... 13 Dependence was also tested, and MB2mP6 was also tested for its effect on Gα. 13 - / - The role of neutrophils in transendothelial migration. As previously described, compared with control Gα... 13 + / + Compared to neutrophils, Gα 13 - / - Neutrophil transpore migration was significantly reduced, but MB2mP6 treatment did not further inhibit Gα. 13 - / - Transendothelial migration of neutrophils Figure 35C This suggests that the inhibitory effect on control neutrophils may be due to Gα. 13 It is not dependent on [the specific substance]. Furthermore, MB2mP6 inhibits the transmembrane migration of wild-type neutrophils on endothelial cells or ICAM1-coated transpore filters, but neutrophil migration on fibrinogen-coated transpores is not affected by MB2mP6. Figure 35D The results are consistent with Gα. 13 - / - The results for neutrophils were consistent. Figures 32A-32BThese data support Gα 13 The view that β2 interaction selectively mediates integrin / ICAM1-dependent neutrophil migration.

[0367] Example 24: Gα 13 The absence of ICAM1 does not affect neutrophil adhesion to ICAM1, but it inhibits the spread of adherent neutrophils.

[0368] The above data indicates that knocking out Gα 13 It inhibited integrin-dependent transendothelial migration of neutrophils. To further distinguish whether this effect is due to Gα... 13 (1) Due to its importance in regulating integrin-dependent cell adhesion or (2) in mediating ICAM1-induced integrin outward-to-inward signaling, Gα was tested. 13 + / + Neutrophils and Gα 13 - / - Neutrophils' adhesion to ICAM1-coated surfaces was examined, and Gα was found to be... 13 + / + Neutrophils and Gα 13 - / - There was no significant difference in the adhesion of neutrophils to ICAM1. Figure 36A Therefore, the ICAM1 binding function of β2 integrin does not require Gα. 13 To evaluate Gα 13 Whether Gα plays an important role in integrin outward signal transduction was investigated. 13 Does knockout affect neutrophil diffusion on ICAM1-coated surfaces? It has been established that cell diffusion at the cell front (plate-like pseudopodia formation) is a cellular response to integrin outward-inward signaling and is important in integrin-dependent cell migration. Gα 13 It has been shown to mediate outward-to-inward signal transduction, leading to cell proliferation within platelets (Shen et al., 2015, 2013). Data show that Gα... prior to cell adhesion to ICAM1... 13 The size of the defective neutrophils was similar to that of the control neutrophils. However, although the control neutrophils spread fairly quickly on the ICAM1-coated surface, Gα... 13 The spread of knockout neutrophils is reduced. Figure 36B , 36C Therefore, with Gα 13 Compared to neutrophils knocked out or control neutrophils treated with MB2mP6, control neutrophils that adhered to ICAM1 were significantly larger. Figures 36B-36F These data are consistent with knowledge regarding neutrophil migration and coordinated diffusion and contraction, and Gα 13It plays a crucial role in the outward-to-inward signaling that leads to integrin-dependent cell proliferation. These data further support the theory of Gα. 13 The idea that integrins play an important role in integrin-dependent cell migration by mediating integrin-out signal transduction.

[0369] Example 25: The role of Gα13 in neutrophil migration in vivo.

[0370] To determine the in vivo importance of Gα13-dependent transendothelial migration, two mouse models were used to further investigate the role of Gα13 in neutrophil migration to extravascular inflammatory sites. In a mercaptoglycol-induced peritonitis model, compared with control Gα13... + / + Compared to mice, Gα13- / - mice showed a significant reduction in neutrophil migration into the peritoneal cavity. Figure 37A In an LPS-induced lung inflammation model, compared with Gα13... + / + Compared to mice, neutrophils infiltrating the lungs 6 hours after intratracheal LPS infusion showed higher levels of Gα13. - / - Significantly reduced in mice ( Figure 37B These data suggest that Gα13 plays an important role in the migration of neutrophils to extravascular inflammatory sites in the body.

[0371] Example 26: MB2mP6 inhibits CLP-induced microvascular thrombosis and vascular leakage in the lungs of septic mice.

[0372] Acute respiratory distress syndrome (ARDS) can be caused by severe viral infections, COVID-19 infection, and bacterial infections such as sepsis.

[0373] The effects of ExE motif peptides in high-load peptide nanoparticle (HLPN) formulations on pulmonary vascular leakage and thrombosis were evaluated in a mouse model of systemic inflammation (sepsis).

[0374] In short, C57BL mice were infused with MB2mP6 or a disordered peptide immediately after CLP onset. Twenty-four hours later, the lungs were removed and sections were taken, and fibrin was stained with phosphotungstic acid hematoxylin (PTAH) (blue staining agent). Figure 38A C57BL mice immediately after CLP ( Figure 38B ) or 6 hours ( Figure 38C Mice were infused with MB2mP6 or saline. 23 hours later, Evans blue albumin (EBA; 1%, 25 mg / kg body weight) was injected intravenously into the mice. One hour later, mouse lungs were harvested, and Evans blue was extracted with formamide at 60°C for 18 hours, and quantified by absorbance at 620 nm.

[0375] like Figures 38A-38CAs shown, in addition to the anti-inflammatory effect of the ExE motif peptide in the high-load peptide nanoparticle (HLPN) formulation, the ExE motif peptide significantly reduced pulmonary vascular leakage and thrombosis in a mouse model of sepsis. The ExE peptide is an effective drug for treating ARDS.

[0376] This disclosure provides Gα 13 Examples of the role of Gα in integrin β2-mediated transendothelial neutrophil migration, and this role is selectively important in neutrophil migration to ICAM1 (an endothelial β2 ligand), but not in migration to fibrinogen (another β2 integrin ligand). These data further suggest that Gα 13 Its main function is to enhance integrin-dependent neutrophil motility without affecting the directionality of neutrophil migration. This integrin-dependent neutrophil motility is known to be mediated through GPCRs and integrin-mediated integrin-mediated outward signaling. Furthermore, Gα... 13 This effect is derived from β2-integrin Gα 13 Gα blocking sequence 13 - Peptide inhibition of integrin interactions suggests that it may be mediated through Gα 13 Mediated by interaction with β2 integrin. Importantly, Gα was also observed in vivo using two different in vivo models. 13 Role in neutrophil migration. Together with previous studies, these data suggest that while GPCR-induced integrin inward-outward signaling and subsequent cell adhesion are important for the directionality of neutrophil migration and the induction of outward-inward signaling, Gα... 13 Outward-to-inward signaling is selectively important for cell motility and is essential for neutrophil migration via β2-integrin ligands. Therefore, targeting Gα... 13 It is a novel anti-inflammatory therapy that does not affect the adhesion function of β2 integrin.

[0377] According to reports, integrin β2 and Gα 13 It plays a role in cell migration (Kolaczkowska and Kubes, 2013; Kourtzelis et al., 2017; Nourshargh and Alon, 2014; Baldassarre et al., 2004; Baldassarre et al., 2006; Tan et al., 2006). However, the relationship between β2 and Gα has not been investigated. 13 Possible links in leukocyte migration. Currently, Gα... 13Its importance in leukocyte function is attributed to its role in transmitting typical GPCR signaling, which is crucial in cytokine-mediated leukocyte activation and chemotaxis (Chen et al., 2012; Dorward et al., 2015; Francis et al., 2006; Goulimari et al., 2005; Surve et al., 2016). However, the data in this paper suggest that Gα in neutrophils... 13 Knockout does not affect the directionality of neutrophil migration or cell adhesion, but selectively inhibits integrin-dependent cell motility and cell diffusion. Therefore, the study described in this paper demonstrates that Gα 13 It plays an important role in the transendothelial migration of neutrophils through the integrin-outward signaling pathway.

[0378] Neutrophils migrate through two distinct mechanisms: slow integrin-dependent migration and rapid integrin-independent amoeba movement. (Sixt, 2009; Shen et al., 2013) The transendothelial migration of circulating blood neutrophils from the vascular system requires a tightly regulated β2-integrin-dependent process (Huttenlocher and Horwitz, 2011). Data show that Gα... 13 Gα plays a crucial role in neutrophil migration and ICAM1-dependent transendothelial migration via the β2-integrin ligand ICAM1. Neutrophil transendothelial migration is blocked by a specific monoclonal anti-β2 antibody, further supporting the role of Gα. 13 The idea that β2-dependent neutrophil migration is also β2-dependent is supported. Data indicate that on surfaces coated with BSA (not integrin ligands), Gα... 13 It is not important for neutrophil migration, and interestingly, Gα was also found to be... 13 It does not participate in the migration of neutrophils to fibrinogen, which is a β2 ligand neutralized in plasma and at vascular wound sites. This finding is interesting, suggesting that Gα... 13 ICAM1 plays a selective role not only in integrin-dependent cell migration but also in the selection of certain β2 ligands. This selectivity appears significant because neutrophils migrate faster on ICAM1 compared to fibrinogen-coated surfaces. An earlier study reported that fibrinogen-mediated leukocyte adhesion to vascular endothelium is ICAM1-dependent (Languino et al., 1993), suggesting that ICAM1 not only directly interacts with β2 integrins to mediate Gα... 13Fibrinogen plays a crucial role in leukocyte-dependent migration and is also important in fibrinogen (another β2-ligand)-mediated leukocyte-endothelial interactions. Therefore, fibrinogen can enhance leukocyte adhesion to the endothelium and leukocyte migration by modulating integrin-ICAM1 interactions and subsequent outward-to-inward signaling. In this regard, α-... L Conformational changes in the I domain can significantly enhance the affinity between ICAM1 and the I domain (McDowall et al., 1998), suggesting a potential mechanism for fibrinogen regulation. Although fibrinogen itself may also operate independently of Gα... 13 Mediating slower neutrophil migration, similar to BSA. Integrins (including β2-integrin) bidirectionally transmit signals: chemical attractants and cytokines induce or enhance “inside-out” signaling for ligand-integrin binding. Ligand binding not only mediates cell adhesion but also induces “outside-in” signaling into the cell, resulting in additional cellular responses, including cell diffusion and cell contraction. Coordinated cell diffusion (primarily at the leading edge) and contraction (primarily at the trailing edge) are thought to drive integrin-dependent cell migration. Consistent with this view, proteins mediating integrin inside-out signaling, such as ankle protein-1, focal adhesion protein-3, and RIAM, have been reported to play important roles in the directionality of neutrophil-like cell migration (Lagarrigue et al., 2016b; Yamahashi et al., 2015). Therefore, the directionality of neutrophil migration requires GPCR-induced integrin activation and adhesion. This paper shows that Gα… 13 Gα plays a crucial role in enhancing the directionality of motility rather than integrin-dependent neutrophil migration, suggesting that directionality is determined by the location of integrin activation and connection, while motility is determined by integrin-mediated outward-inward signaling. 13 Data that knockout does not affect leukocyte adhesion to ICAM1 but significantly affects cell diffusion on ICAM1 during migration support this. Importantly, this data suggests that the knockout is derived from the β2-integrin subunit (and Gα). 13 The peptide of the ExE motif (binding site) significantly inhibits β2-integrin-dependent transendothelial migration of neutrophils, similar to Gα. 13 The effect of knockout. As mentioned earlier, the ExE motif is in Gα. 13 - The importance of integrin interactions and outward-to-inward signaling in other cell types (Shen et al., 2015, 2013), these results indicate that Gα 13 Gα plays a crucial role in the outward-to-inward signal transduction-dependent neutrophil motility mechanism and is therefore essential for β2-integrin-dependent transendothelial neutrophil migration. Since transendothelial neutrophil migration plays a key role in inflammation, targeting Gα... 13This represents a novel approach to developing novel anti-inflammatory drugs that do not completely eliminate integrin and leukocyte functions in the immune system.

[0379] In summary, this paper discloses an engineered, lipid-stabilized HLPN for the efficient in vivo delivery of an inhibitory peptide of integrin outward-inward signaling. This HLPN is a rapidly acting, reversible, and potent antithrombotic agent without the side effect of bleeding. Importantly, post-ischemic injection of this novel drug effectively treats MI / R injury and significantly reduces MI / R mortality. Furthermore, an antiplatelet strategy was developed in which the combination of M3mP6 HLPN with current antiplatelet drugs significantly enhances antithrombotic efficacy and reduces the risk of bleeding. The studies described herein were conducted in a mouse model where the relevant β3 cytoplasmic domain sequence is identical to that in humans.

[0380] Although preferred embodiments, uses, and modifications of this disclosure have been depicted and disclosed, such descriptions are to be considered illustrative rather than restrictive, especially since those skilled in the art will understand that various modifications and changes can be made without departing from the spirit and scope of the appended claims.

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Claims

1. A lipid-stabilized, high-load peptide nanoparticle, comprising: i) Peptides comprising more than approximately 10 molar percentages based on the total mass of nanoparticles; ii) 2 to 20 mol percent of one or more lipids that do not contain water-soluble polymers; and iii) 10 to 60 mol% of one or more lipids covalently linked to a water-soluble polymer, The peptide is a lipotropic peptide, an amphiphilic peptide, or a peptide-hydrophobic conjugate, and the peptide consists of about 2 to about 50 amino acid residues; and The peptides mentioned therein are myr-FEEERL (SEQ ID NO:25) or myr-FEKEKL (SEQ ID NO:27).

2. The lipid-stabilized high-load peptide nanoparticles according to claim 1, wherein the one or more lipids free of water-soluble polymers are phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), or mixtures thereof.

3. The lipid-stabilized high-load peptide nanoparticles according to claim 1 or 2, wherein the one or more lipids covalently linked to the water-soluble polymer are peg-phosphatidylethanolamine (PEG-PE), PEG-phosphatidylcholine (PEG-PC), PEG-phosphatidylglycerol (PEG-PG), PEG-phosphatidylinositol (PEG-PI), PEG-phosphatidylserine (PEG-PS), PEG-1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[amino(polyethylene glycol)]] (PEG-DSPE) or mixtures thereof.

4. The lipid-stabilized high-load peptide nanoparticles according to any one of claims 1 to 3, wherein the lipid-stabilized high-load peptide nanoparticles comprise about 10 to about 80 mol% peptides based on the total mass of the nanoparticles.

5. The lipid-stabilized high-load peptide nanoparticles according to any one of claims 1 to 3, comprising 2-10% phosphatidylcholine and 10-60% PEG-DSPE.

6. A pharmaceutical composition comprising lipid-stabilized, high-load peptide nanoparticles according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier, diluent, or excipient.

7. Use of an effective amount of lipid-stabilized high-load peptide nanoparticles according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating acute respiratory distress syndrome (ARDS), prosthetic valve thrombosis, atherosclerosis, arterial thrombosis, AV fistula thrombosis, deep vein thrombosis, ischemia-reperfusion injury, ischemic stroke, microvascular thrombosis, sepsis, vasculitis or venous thrombosis in subjects of need.

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