SERPIN PEPTIDE DERIVATIVES AND METHODS OF USE THEREOF

Abstract

SERPIN-derived peptides such as SP16 and SP163M can be used to treat a number of conditions related to the mediation of LRP1.In order to further improve the stability, bioavailability and / or efficacy of existing SERPIN-derived peptides, it is necessary to develop new SERPIN peptide derivatives.SERPIN peptide derivatives, their preparation methods, and their use for treating various conditions related to the mediation of LRP1 are related to the present technology. TIFF2025515349000031.tif95170

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 363,840, filed April 29, 2022, the disclosure of which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing in accordance with ST.26, which has been submitted contemporaneously herewith via the Patent Center in .xml format and is hereby incorporated by reference in its entirety. The .xml copy created on April 26, 2023 is named Serpin 138536-8004WO00 Sequence Listing.xml and is 78KB in size. [Background technology]

[0003] background Serine protease inhibitors (SERPINs) are a large family of proteins involved in diverse biological functions such as fibrinolysis, blood coagulation, and inflammation. When SERPINs bind to their target serine proteases and inactivate their enzymatic activity, a conformational change occurs that exposes unique short peptide motifs (5–11 amino acids) 8、43 The protease-inhibitor complex binds to the low-density lipoprotein receptor-related protein (LRP1) at a newly exposed short peptide motif, a process that is conserved across a range of serine protease inhibitors (SERPINs) such as alpha-1 antitrypsin (AAT) and antithrombin III (ATIII). 22、25、43 .

[0004] Previously disclosed is a family of SERPIN-derived peptides that bind to LRP1 and have been found to exert healing and homeostatic functions beyond its anti-inflammatory function.See, for example, U.S. Patent Nos. 8,975,224; 9,951,104; 11,020,462; and U.S. Patent Application Publication Nos. 2021 / 0188912 and 2021 / 0369822, the contents of which are incorporated herein by reference.SERPIN-derived peptides such as SP16 and SP163M can be used to treat a number of conditions associated with the mediation of LRP1.There is a need to develop novel SERPIN peptide derivatives to further improve the stability, bioavailability, and / or efficacy of existing SERPIN-derived peptides. Summary of the Invention

[0005] overview In one aspect, of the present technology, FVFLM (SEQ ID NO: 1), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQ ID NO: 8), or PFVFL[Nle] (SEQ ID NO: 9) and one or more of the following modifications: (i) a polar head added to the N-terminus of the pentapeptide, a polar tail added to the C-terminus of the pentapeptide, or both; (ii) one or more amino acid residues added to the N-terminus of the pentapeptide, the C-terminus of the pentapeptide, or both, such that the peptide derivative may be cyclized; (iii) one or more amino acid residues in the pentapeptide being replaced by one or more amino acid residues having a lower hydrophobicity; (iv) one or more amino acid residues in the pentapeptide being replaced by one or more amino acid residues having a higher hydrophobicity; and (v) one or more amino acid residues in the pentapeptide being deleted.

[0006] In certain embodiments, the SERPIN peptide derivative of the present technology is a linear peptide. In certain embodiments, the SERPIN peptide derivative of the present technology is a cyclized peptide. In certain embodiments, the SERPIN peptide derivative is cyclized by forming a disulfide bond between two Cys residues. In certain embodiments, the SERPIN peptide derivative is cyclized by a linker between two amino acid residues, for example between two amino acid residues outside the pentapeptide sequence. In certain embodiments, the polar head or polar tail comprises two or more charged amino acids, such as positively charged amino acids selected from the group consisting of Arg, Lys, and His. In certain embodiments, the SERPIN peptide derivative is fused with one or more other peptides, including an epitope tag, a half-life extender, or both an epitope tag and a half-life extender, to form a fusion protein or fusion peptide. In certain embodiments, the SERPIN peptide derivative is conjugated to a permeability enhancer.

[0007] In another aspect, the present technology relates to a composition comprising the SERPIN peptide derivative, fusion or conjugate of the present technology and one or more pharma- ceutically acceptable carriers.In some embodiments, the composition is formulated into a dosage form suitable for oral, transdermal or parenteral administration.

[0008] In another aspect, the present technology relates to a method for treating various conditions or diseases associated with LRP1 binding, such as respiratory viral or bacterial infections (e.g., COVID), and inflammatory diseases such as acute respiratory distress, asthma, atopic dermatitis, or eosinophilic esophagitis.Other conditions include conditions of the central and peripheral nervous system, such as peripheral nerve injury and neurodegenerative diseases.The present method involves administering an effective amount of one or more SERPIN peptide derivatives of the present technology, their fusions, or a composition comprising one or more SERPIN peptide derivatives or their fusions to a subject suffering from a condition associated with LRP1 binding. [Brief description of the drawings]

[0009] This application contains at least one drawing executed in color. Copies of this application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1] 1 shows the crystal structure of α1-antitrypsin containing the pentapeptide sequence (SEQ ID NO:8). [Diagram 2] 1 shows the distances between amino acids based on the crystal structure of α1-antitrypsin containing the pentapeptide sequence (SEQ ID NO:8). [Diagram 3] The ring-closing strategy is illustrated using the 13 CC bonds of Lys-βAla-Glu as an example. [Figure 4] Figures 4A-4B demonstrate the structure-activity relationship of SERPIN-derived peptide derivatives SA1-SA8. Figure 4A shows the activity of peptide derivatives SA1-SA8 at various concentrations of 0, 1, 10, 50, and 100 μg / mL in reducing NFκB activation. Figure 4B compares the activity of SA3 and SA7 with that of SP163M and SP22. Compared to previously disclosed SERPIN peptide SP163M, truncating the peptide while retaining the LRP1 binding site and adding an arginine residue conferred improved activity in the NFκB reporter assay. [Diagram 5] Figures 5A-5B demonstrate the anti-inflammatory function of peptide derivatives A1-A15 compared to SP163M. Peptide derivatives (50 or 100 μg / ml) were tested in NFκB reporter assays in response to LPS (5 ng / ml) and screened for TNFα secretion in IMG microglial cells in response to LPS (E. coli 0111:B4) stimulation (100 ng / ml, 24 h). NFκB-induced secreted embryonic alkaline phosphatase (SEAP) was measured in the supernatant in NFκB reporter cells. TNFα secretion in the supernatant was measured via ELISA. [Figure 6]Figures 6A-6B demonstrate the anti-inflammatory activity of peptide derivatives A2-1 to A2-9, which contain modifications to the ring structure and amino acid substitutions. Figure 6A shows the reduction of TNFα in the supernatant of IMG microglial cells after activation with LPS (100 ng / ml, 24 h). Figure 6B shows the percentage reduction of NFκB activation after LPS stimulation (5 ng / ml, 24 h). SP163M, SA7, A5, and A15 peptides were included for comparison. [Figure 7] 7A-7B show the dose-response effect of peptide derivatives on NFκB and TNFα activation. Peptide derivatives were tested at concentrations of 3.125, 6.25, 12.5, 25, and 50 μg / ml. SP163M, SP16, and SA7 were included at concentrations up to 100 μg / ml. Dose-response concentrations are shown on a log scale. Peptide derivatives A2-1, A2-2, A2-3, A2-4, and A2-5 exhibited superior activity to SA7, SP16, and SP163M in a dose-dependent manner. [Figure 8] The results of the cytotoxicity assay on IMG cells (microglia) are shown. IMG cells were treated for 24 hours with peptide derivatives at or above the concentrations tested for the activity assay (Figures 5-7). None of the peptides exhibited any cytotoxic effects. [Figure 9] Figure 1 shows the effect of peptide derivatives A3-1 to A3-16, including further optimization of the ring structure, on NFκB activation in response to LPS stimulation (5ng / ml, 24 hours). Peptide derivatives were tested at concentrations between 1.56μg / ml and 12.5μg / ml. SP163M and A2-5 were included for comparison. SP163M was tested at concentrations between 12.5μg / ml and 100μg / ml. Dose response is shown on a log scale. [Figure 10]Figure 1 shows the activity of peptide derivatives A3-1, A3-2, A3-3, A3-5, A3-6, A3-7, A3-9, A3-14, A3-15, and A3-16 in reducing IL-6 secretion in IMG following LPS stimulation (100 ng / ml, 24 h). IL-6 secretion was measured by ELISA. Peptide derivatives A2-5 and SP163M were included for comparison. SP163M was tested at concentrations between 12.5 and 100 μg / ml. Peptide derivatives including A2-5 were tested at concentrations up to 12.5 μg / ml. Dose response is shown on a log scale. [Figure 11] 11A-11B show the results of cytotoxicity assays on NFκB reporter cells (FIG. 11A) and IMG microglial cells (FIG. 11B). Peptide derivatives A3-1, A3-2, A3-5, A3-6, A3-7, A3-9, A3-14, A3-15, and A3-16 did not exhibit cytotoxic effects when tested at concentrations effective to reduce both NFκB and cytokines IL-6 and TNFα (up to 12.5 μg / ml). SP163M was included for comparison and did not demonstrate cytotoxic effects at concentrations up to 100 μg / ml. Cells were treated with the indicated concentrations of SP163M or peptide derivatives A3-1, A3-2, A3-5, A3-6, A3-7, A3-9, A3-14, A3-15, A3-16, or A2-5 for 24 hours. [Figure 12] Peptide derivatives A15 and A2-5 were tested for their ability to block capsaicin-induced pain behavior in mice. Mice were treated with peptide derivatives by subcutaneous administration 1 h prior to capsaicin injection (25 ng). SP163M was administered at a dose of 50 μg / mouse, whereas peptide derivatives A15 and A2-5 were administered at a dose of 5 μg / mouse. n=4-9 mice / cohort. One-way ANOVA. *P<0.05, **P<0.01. [Figure 13]The cytokine profiles examined in microglial cells treated with either the SERPIN peptide derivative SA7 or the SERPIN peptide SP163M are compared. SA7 demonstrated significantly increased activity in reducing several cytokines, including IL-6 and IL-1β. [Figure 14] Figures 14A-14B show that the SERPIN peptide derivative SA7 reduced the clinical scores (measured daily with a scoring system) (Figure 14A) and the severity of weight loss (Figure 14B) in an LPS-induced neuroinflammation model compared to both vehicle (induced with LPS) and SP163M. SP163M and SA7 were administered by subcutaneous injection at a dose of 100 μg / mouse 1 hour after LPS administration (1 mg / kg) for 4 consecutive days. Body weight and clinical scores were measured daily, and brain tissue was collected and processed at the end of the study for analysis. The score legend is as follows: 0: lively, alert, and responsive; 1-4: slightly disheveled / hunched, less active; 5-10: hunched, inactive, and weight loss; >= 11 moribund. [Figure 15] In the neuroinflammation model in Figure 14, SA7-treated animals showed marked differences compared to both vehicle- and SP163M-treated animals in the levels of many cytokines measured in brain lysates, including IL-17A, IL-12, TNFα, and GM-CSF, with IL-6 being most substantially reduced, with no detectable levels measured. [Figure 16] 16A-16B show Western blot analysis of neurofilament light chain (NfL), a marker specific for axons, and anti-glial fibrillary acidic protein (GFAP), a marker specific for astrocyte activation, in brain lysates from previously described neuroinflammation models. The peptide derivative SA7 significantly increased the expression of NfL (FIG. 16A), whereas GFAP expression was significantly decreased (FIG. 16B), compared to vehicle-treated and SP163M animals. [Figure 17]Figures 17A-17B show that SA7 regulated autophagy during inflammation similarly to the SERPIN peptide SP163M. Microglial cells were treated with SP163M or SA7 and then exposed to LPS. Western blot analysis of the LC3 II to LC3 1 ratio (the initiation of autophagosome formation - a biomarker widely used to detect autophagy) shows that SA7 increased autophagy (Figure 17A) and LRP1 expression (Figure 17B) compared to LPS alone and to a similar extent as SP163M. LRP1 expression was also significantly higher with SA7 without LPS treatment compared to SP163M. [Figure 18] The protease TMPRSS2 activity and IC50 of SP163M and peptide derivative SA7 are shown, with SA7 showing increased inhibition of TMPRSS2 activity in overexpressing cell lines compared to SP163M. [Figure 19] 19A-19B demonstrate that LRP1 was reduced in the esophagus of OVA-induced mice and that SP163M restored LRP1 protein expression. Furthermore, SA7 more potently inhibited the production of the cytokine TSLP in SPINK7 knockout cells than SP163M and compared to control cells. [Figure 20] Figures 20A-20C demonstrate that in a model of OVA-induced allergic asthma, peptide derivative A2-5 reduces TSLP, total lung protein, and the TH-2-mediated cytokines IL-5, IL-4, and IL-13 in lung homogenates to a greater extent than both OVA-induced vehicle-treated and SP163M mice. [Figure 21] Illustrated is a conjugate comprising a peptide derivative of the present technology (e.g., A2-5, SEQ ID NO: 42) and a permeability enhancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description The present technology relates to SERPIN peptide analogs, as well as variants and derivatives thereof, and their use in preventing or treating various conditions by targeting low density lipoprotein receptor-related protein-1 (LRP-1). As used herein, the term "derivative" refers to a peptide that shares similarity in amino acid sequence or structure with the pentapeptide FVFLM (SEQ ID NO: 1), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQ ID NO: 8), or PFVFL[Nle] (SEQ ID NO: 9), and contains one or more modifications, including insertions, deletions, or substitutions, to improve stability, bioavailability, and / or biological activity or biological efficacy compared to the pentapeptide. In this disclosure, the terms "derivative", "variant", and "analog" may be used interchangeably.

[0011] Promoting resolution of inflammation and reducing tissue injury requires precise coordination of immune responses, and targeting a single cytokine or signaling pathway does not address all contributing factors in the pathology of a particular disease, as discussed here. A balanced inflammatory response plays an essential role in regeneration and repair, and anti-inflammatory drugs are associated with opposing effects on regeneration and tissue repair. 3 .

[0012] SERPIN peptides have been previously shown to (1) exert neurotrophic effects, (2) have regenerative and healing properties, (3) exhibit analgesic effects, (4) have antiviral and antibacterial properties, and / or (5) exert antiallergic effects. This combination of activities provides a unique mechanism in the treatment of conditions related to peripheral neuropathy, such as diabetic peripheral neuropathies, degenerative disorders, lung injury, allergic diseases, and infectious diseases. The SERPIN peptide analogs of the present technology, as well as their variants and derivatives, have improved LRP1 binding activity, improved solubility, and / or improved pharmacokinetic properties, oral bioavailability. For example, as demonstrated in the data-supported examples, the SERPIN peptide derivatives of the present technology show improved anti-inflammatory effects and improved efficacy in models of neuroinflammation.

[0013] Thus, the present technology relates to SERPIN peptide derivatives, pharmaceutical compositions comprising SERPIN peptide derivatives, and methods of using them to treat a number of conditions in which the immune response is dysregulated or endocytic function is impaired, such as in conditions associated with peripheral nerve injury and the resulting pain, lung injury, infectious diseases, and allergic inflammation, or diseases in which LRP1 mediation contributes to the pathology.

[0014] In certain embodiments, the SERPIN peptide derivative is a synthetic peptide. In certain embodiments, the SERPIN peptide derivative is cyclized. In certain embodiments, the SERPIN peptide derivative comprises one or more hydrophilic amino acid substitutions. In certain embodiments, the SERPIN peptide derivative comprises one or more hydrophobic amino acid substitutions. In certain embodiments, the SERPIN peptide derivative comprises one or more positively charged amino acids at the N-terminus, the C-terminus, or both the N-terminus and the C-terminus.

[0015] SERPIN peptide derivatives and modifications The technology concerns SERPIN peptide derivatives designed to target LRP1 with higher affinity to exert stronger anti-inflammatory and cell regenerative effects. These peptide derivatives are modified from the original SERPIN-derived peptides based on structure-activity relationship studies and 3-D modeling of peptide / LRP1 interactions. These derivatives overcome some of the challenges associated with peptide therapeutics, such as solubility, plasma stability, and oral bioavailability. Compared to previously disclosed SERPIN peptides such as TIFF2025515349000002.tif18153 (where Nle stands for norleucine), the peptide derivatives of the present technology exhibit not only improved LRP1 activity, but also improved solubility, pharmacokinetic properties, and bioavailability, particularly oral and transdermal bioavailability.

[0016] It has previously been demonstrated that a small peptide fragment from the C-terminal part of alpha-1 antitrypsin (the prototypical SERPIN) can bind to LRP1 and exert potent cell regenerative, tissue protective, and immunomodulatory functions. However, the tertiary structure of alpha-1 antitrypsin (AAT) prevents its direct binding to LRP1. Rather, AAT can bind to LRP1 only when it is interacting with its target protease due to a conformational change that occurs in AAT that exposes a short 5-11 amino acid binding motif. Surprisingly, as demonstrated by structure-activity relationship studies performed on these derivatives, the highly conserved core sequence The entire TIFF2025515349000003.tif4128 is not required for the anti-inflammatory effect of the peptide derivative. Although the derivative retains LRP1-binding activity, it does not contain the FNKP (SEQ ID NO: 7) motif, which is highly conserved among SERPINs. However, the LRP1-binding motif is highly hydrophobic and unstable in solution, necessitating modifications to the SERPIN peptide sequence.

[0017] Therefore, various modifications are made to the SP16 / SP163M peptide, particularly to the pentapeptide or its surroundings, to produce SERPIN peptide derivatives with improved properties. As used herein, "pentapeptide" refers to the FVFLM (SEQ ID NO: 1) sequence in SP16 or the FVFL[Nle] (SEQ ID NO: 2) sequence in SPM163, where the Met residue is replaced with an Nle residue. The pentapeptide is responsible for most of the interaction with LRP1. At the time of the present technology, various modifications are made to the pentapeptide and / or the sequence surrounding the pentapeptide to obtain novel SERPIN peptide derivatives with improved properties. For example, the sequence of the SP163M peptide is further modified by deletion, substitution, and / or cyclization to further improve anti-inflammatory activity, solubility, LRP1 binding activity, and / or oral bioavailability. Compared with the sequence of SP16 or SP163M, shorter peptide derivatives are developed to achieve better oral bioavailability and brood brain barrier permeability without compromising anti-inflammatory activity or LRP1 binding activity.In certain embodiments, the peptide derivative of the present technology has a size of 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, preferably 8, 9, or 10 amino acid residues.For example, the peptide derivative of the present technology has a size of 8 amino acid residues, 9 amino acid residues, or 10 amino acid residues.

[0018] In certain embodiments, to improve solubility, an N-terminal polar head containing two or more amino acid residues with charged side chains, a C-terminal polar tail containing two or more amino acid residues with charged side chains, or both a polar head and a polar tail are added to the pentapeptide. In some embodiments, the amino acid residues in the polar head or tail are positively charged and include Arg, His, and Lys. The polar head or tail can use a combination of the same charged amino acid residues or a combination of different amino acid residues. For example, the polar head or tail includes the amino acid sequence RR, RRR, KK, KKK, HH, HHH, KRR, KR, or RRK. In some embodiments, one or more positively charged amino acid residues in the polar head or tail have an inverted structure. For example, an inverted Lys means that the Lys residue is incorporated into the peptide backbone using the carboxylic acid group carried by the alpha carbon and the epsilon amino group in the side chain, rather than both the amino group and the carboxylic acid group carried by the alpha carbon. Inverted Arg means that the Arg residue is incorporated into the peptide backbone using the guanidinium group carried by the alpha carbon instead of the delta carbon. In some embodiments, two or three Arg residues are added to either or both termini of the pentapeptide. In some embodiments, two or three Arg residues are added to the N-terminus of the pentapeptide.

[0019] In certain embodiments, the peptide derivatives of the present technology are cyclized, for example, by forming a disulfide bond between two Cys residues or by a linker between two amino acid residues. At the time of the present technology, two Cys residues can be added to both ends of the pentapeptide so that a cyclic peptide derivative can be obtained through a disulfide bond. It is within the understanding of a person skilled in the art to place Cys residues at selected positions in the peptide derivative to achieve a desired cyclic structure with optimized ring size. Alternatively, other natural, non-natural, or modified amino acid residues can be added to either or both ends of the pentapeptide so that a linker can be formed between these amino acid residues. Specific amino acid residues can be selected and placed at selected positions to achieve a desired cyclic structure with optimized ring size. Depending on cyclization strategies such as amide, disulfide, and ring-closing metathesis (RCM) or olefin metathesis, substitutions of amino acid residues for cyclization can be selected without significant loss of activity. For example, amino acid residues having a carboxylic acid at their side chain or at their C-terminus, including but not limited to Asp and Glu, or an amino group at their side chain or at their N-terminus, including but not limited to Lys, Dab, and Dap, can be used for amide cyclization, and Cys or any unnatural amino acid carrying a sulfhydryl group at its side chain can be used for -SS-cyclization. The amino acid can be placed at any desired position of the peptide derivative such that a ring of the desired size can be formed without substantially comprising the activity of the peptide derivative. In some embodiments, a head-to-tail cyclization is formed. In some embodiments, the linker comprises β-Ala. In some embodiments, the linker comprises 2-[(2-amino)-ethoxy]-ethoxy-acetic acid (AEEA). In some embodiments, the ring closing length between amino acid residues is 5 to 15 C-C bonds, e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 C-C bonds.

[0020] In certain embodiments, the peptide derivatives of the present technology contain one or more substitutions in the sequence of the pentapeptide to enhance plasma stability and to achieve increased binding affinity to the peptide's cognate receptor. For example, one or more amino acid residues in the pentapeptide that have no or minimal interaction with LRP1 can be replaced by one or more hydrophilic amino acid residues. Moreover, one or more amino acid residues in the pentapeptide that interact with LRP1 can be replaced by one or more amino acid residues with similar but more pronounced physicochemical characteristics. For example, the Phe residue has an aromatic ring in its side chain. The Phe residue can be replaced by Nal (naphthylalanine) in peptide derivatives 1-5 and 1-6, or by Trp, which presents a naphthyl or indole ring instead of a phenyl ring. These substitutions greatly improve the aromaticity of the amino acid residues, allowing for more hydrophobic and more aromatic (pi-stacking) interactions. In some embodiments, one or more amino acid residues in the pentapeptide can be replaced by one or more natural or unnatural amino acid residues. In some embodiments, one or more amino acid residues in the pentapeptide have a D-configuration. In some embodiments, the side chains of one or more amino acid residues in the pentapeptide are modified. For example, peptide derivative A3-1 includes a Val to Thr substitution to retain some hydrophobicity while introducing a few hydrogen bonds, and a Phe to Nal substitution to increase hydrophobic and aromatic interactions. In another example, peptide derivative A3-8 includes a Nle to D-Ser substitution. Based on crystal structure studies, Nle does not interact with LRP-1, but rather is in the aqueous phase. The side chain of Nle is a hydrophobic linear hydrocarbon chain, which requires energy to solvate. Replacing the Nle residue with D-Ser, which has a hydrophilic side chain, promotes solvation by reducing the enthalpic penalty, which translates into stronger binding energy.Thus, in some embodiments, the Nle residue of the pentapeptide is deleted. In some embodiments, one or more of the hydrophobic residues in the pentapeptide are replaced by one or more less hydrophobic residues, such as Ala, or by one or more neutral or hydrophilic residues, such as Thr and Ser. In some embodiments, one or more of the hydrophobic residues in the pentapeptide are replaced by one or more residues with higher hydrophobicity, such as Nal. In some embodiments, the Nle residue of the pentapeptide is replaced by an amino acid residue with a D configuration, such as D-Dap, D-Lys, and D-Asp.

[0021] The Met or Nle residues in the pentapeptide have long, linear, hydrophobic side chains. Substitution of Met or Nle with hydrophilic amino acids of the D configuration greatly improves the binding activity of the peptide derivatives to LRP1. Substitution with amino acid residues bearing carboxylic acids in the side chains results in some improvement, whereas substitution with amino acids bearing short side chains presenting hydroxyl groups (Ser) or amino groups (Dap) achieves the best results.

[0022] In certain embodiments, the SERPIN peptide derivatives of the present technology can be further modified using one or more non-natural peptide bonds or amino acids, or by attachment to peptide functional groups such as polyethylene glycol (PEG), to extend shelf life and / or bioavailability.

[0023] In certain embodiments, the peptide derivatives of the present technology comprise, consist essentially of, or consist of a pentapeptide having an amino acid sequence of FVFLM (SEQ ID NO: 1) or FVFL[Nle] (SEQ ID NO: 2) and a polar head added to the N-terminus of the pentapeptide, a polar tail added to the C-terminus of the pentapeptide, or both. The polar head or tail comprises 2-9 charged amino acid residues, such as Arg, Lys, and His. In some embodiments, the polar head or tail comprises 2 or 3 charged amino acid residues. Some examples of peptide derivatives shown in Table 1 were used to investigate the role of positively charged amino acids in improving solubility and modulating NFκB activation, using SP163M and SP22 as positive controls. The peptide derivatives have various tripeptide sequences added to either or both ends of the LRP1 binding site.

[0024] In some aspects, the peptide derivative is The peptide comprises, consists essentially of, or consists of a peptide having the amino acid sequence of TIFF2025515349000004.tif37156.

[0025] Table 1. Peptide derivatives with various tripeptide modifications TIFF2025515349000005.tif102147*The underlined sequences represent cyclization. Disulfide bonds are formed between the C residues to cyclize the peptide.

[0026] As demonstrated in Example 1, when one or more positively charged amino acids, such as arginine (R) residues, are added to the LRP1 binding motif, activity in reducing NFκB is increased. However, addition of one or more other charged amino acids, such as positively charged amino acids, such as histidine (H) or lysine (K), or negatively charged amino acids, such as glutamic acid (E), flanking either side of the LRP1 binding site does not confer activity, indicating the importance and uniqueness of the arginine residues.

[0027] In certain embodiments, the peptide derivatives of the present technology are cyclic peptides and comprise, consist essentially of, or consist of a pentapeptide having an amino acid sequence of FVFLM (SEQ ID NO: 1) or FVFL[Nle] (SEQ ID NO: 2) and a polar head added to the N-terminus of the pentapeptide, a polar tail added to the C-terminus of the pentapeptide, or both. The cyclization can be between any two residues, e.g., the cyclization can be a head-to-tail cyclization. An additional residue, such as Cys, can be inserted to facilitate the formation of an S-S bond to link the two residues. In some embodiments, one or more hydrophobic residues of F, M, or Nle in the pentapeptide are replaced with a neutral or hydrophilic residue. Thus, peptide derivatives with various combinations of additions of positively charged amino acid residues and / or amino acid substitutions, some examples of which are shown in Table 2, were designed to improve solubility, stability, and / or oral bioavailability without compromising their activity of modulating NFκB.

[0028] In certain embodiments, the peptide derivatives of the present technology include a pentapeptide having an amino acid sequence of FVFLM (SEQ ID NO: 1) or FVFL[Nle] (SEQ ID NO: 2) and an additional proline ("P") amino acid residue. In some aspects, since the P amino acid residue is at the C-terminal end of the pentapeptide, the peptide derivative includes an amino acid sequence of PFVFLM (SEQ ID NO: 8) or PFVFL[Nle] (SEQ ID NO: 9). In some aspects, a peptide derivative including an amino acid sequence of PFVFLM (SEQ ID NO: 8) or PFVFL[Nle] (SEQ ID NO: 9) can be a cyclized peptide. In some aspects, peptide derivatives comprising the amino acid sequence of PFVFLM (SEQ ID NO:8) or PFVFL[Nle] (SEQ ID NO:9) can be cyclized by further including a Cys residue to facilitate the formation of an S-S bond, such as a peptide derivative having an amino acid sequence of CPFVFLMC (SEQ ID NO:19) or CPFVFL[Nle]C (SEQ ID NO:20). In some aspects, peptide derivatives having the sequence of CPFVFLMC (SEQ ID NO:19) or CPFVFL[Nle]C (SEQ ID NO:20) can further include a polar head, such as three R amino acid residues. In some aspects, peptide derivatives can include In some aspects, the sequence may include: The peptide derivative having the sequence of TIFF2025515349000007.tif4140 may be further acetylated. In some aspects, the peptide derivative may be peptide SA7.

[0029] In certain aspects, the peptide derivatives of the present technology include It comprises, consists essentially of, or consists of the amino acid sequence of TIFF2025515349000008.tif17148.

[0030] Table 2. Peptide derivatives for improving solubility TIFF2025515349000009.tif160149*The underlined sequences represent cyclization. Disulfide bonds are formed between the C residues to cyclize the peptide.

[0031] As shown in Figure 1, the F, F, and M residues of the pentapeptide FVFLM (SEQ ID NO: 1) point in one direction, whereas the V and L residues point in the opposite direction. The P residue at the N-terminus of the pentapeptide creates a turn in the structure but has no interaction with LRP1. It is unlikely that all five residues of the pentapeptide directly interact with LRP1. Moreover, peptide derivatives A7-A9 have been designed to replace one of the hydrophobic amino acid residues (F, V, and L) with a more hydrophilic residue, such as T, to examine whether solubility and / or activity could be improved.

[0032] Table 3 shows some examples of peptide derivatives in which one of the F residues is replaced by 1-naphylalanine, an unnatural amino acid with an additional aromatic ring. The naphylalanine (Nal) substitution increases hydrophobicity and may have stronger hydrophobic interactions with LRP1.

[0033] In certain aspects, the peptide derivatives of the present technology include It comprises, consists essentially of, or consists of the amino acid sequence of TIFF2025515349000010.tif4148.

[0034] Table 3. Peptide derivatives with substitutions to improve activity TIFF2025515349000011.tif48149*The underlined sequences represent cyclization. Disulfide bonds are formed between the C residues to cyclize the peptide.

[0035] Additional peptide derivatives were designed to determine the optimal ring size. For example, the Cys-Cys bridge can be replaced by a longer linker to improve cyclization. Based on the crystal structure of the LRP1 binding site, the distance between the α-carbon of the amino acid on the N-terminus and the α-carbon of the amino acid on the C-terminus of the pentapeptide FVFLM (SEQ ID NO: 1) was measured. Assuming a CC bond length of 1.5 Å, the distance between amino acids is converted to the number of CC bonds, which can be used to design cyclization strategies. For example, the distance between the α-carbons of K368 and I375 is 22.4 Å (approximately 15 CC bonds), and the distance between the α-carbons of P369 and I375 is 20.2 Å (approximately 13.5 CC bonds). See Figure 2. To cyclize the derivative and mimic the distance between P369 and I375, a linker of 13-14 C-C bonds using β-Ala can be used to link the side chain of the Lys residue to the side chain of the Glu residue, resulting in a peptide derivative with a ring closure length of 13 C-C bonds. See Figure 3.

[0036] In certain aspects, the peptide derivatives of the present technology include It comprises, consists essentially of, or consists of the amino acid sequence of TIFF2025515349000012.tif17148.

[0037] Table 4 illustrates some examples of peptide derivatives with various ring-closing designs, in which ring-closing linkers are used to replace the Cys-Cys bridge.

[0038] Table 4: Peptide derivatives with various cyclizations TIFF2025515349000013.tif155153TIFF2025515349000014.tif162153*Underlined sequences represent cyclization.

[0039] Example 2 demonstrates that peptide derivatives A5, A8, A10, and A15 exhibit improved activity in reducing TNFα activation. Table 5 lists additional SERPIN peptide derivatives with similar modifications, and some examples of peptide derivatives with substitutions in the pentapeptide. For example, Nle in the pentapeptide is replaced with Ala or deleted to determine whether Nle is involved in the interaction with LRP1. Moreover, each residue in the pentapeptide is replaced with D-Ser (dS) to improve solubility and resistance to proteases.

[0040] In certain aspects, the peptide derivatives of the present technology include TIFF2025515349000015.tif24149

[0041] Table 5. Peptide derivatives with various ring closures and AA substitutions TIFF2025515349000016.tif152149*Underlined sequences represent cyclization.

[0042] As shown in Figure 7, peptide derivatives A2-1, A2-2, A2-3, A2-4, and A2-5 demonstrated superior activity to SP163M and SA7 in reducing TNFα and NFκB activation. None of the tested peptide derivatives exhibited any significant toxicity to the tested cell lines (Figure 8).

[0043] In certain aspects, the peptide derivatives of the present technology include TIFF2025515349000017.tif44149

[0044] Table 6 provides examples of SERPIN peptide derivatives that are further optimized by shortening peptide length via deletion, optimizing cyclization, and / or substituting residues to further improve solubility, activity, and / or oral availability. In certain embodiments, one or more amino acid residues in the pentapeptide of the peptide derivative are deleted or substituted with one or more natural or non-natural amino acid residues. For example, the Nle residue can be substituted by a less hydrophobic amino acid such as Ala or Ser (D-Ser) to improve agonist activity binding to LRP1. In certain embodiments, amino acids in the D configuration can be used to change the orientation of the amino acids in the 3-D structure of the peptide and / or to confer protease stability. In certain embodiments, two charged amino acid residues, such as Arg and / or Lys, are added to the N-terminus of the pentapeptide. In certain embodiments, three charged amino acid residues, such as Arg and / or Lys, are added to the N-terminus of the pentapeptide. Charged amino acids can have a "flipped" chemical structure to optimize ring size, as illustrated in the following example: TIFF2025515349000018.tif83128

[0045] Table 6. Peptide derivatives with additional modifications TIFF2025515349000019.tif109153TIFF2025515349000020.tif209153TIFF2025515349000021.tif175153*Underlined sequences represent cyclization.

[0046] The peptide derivatives of the present technology can be further modified at the amide bonds to improve protease stability and absorbency, including, but not limited to, peptide bond isosteres, N-methylation, and / or D-configuration amino acid substitutions.

[0047] In certain aspects, disclosed are peptide derivatives comprising, consisting essentially of, or consisting of the amino acid sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 (SEQ ID NO:3), wherein: X1 is a hydrophilic amino acid residue or is absent; X2 is a hydrophilic amino acid residue or is absent; X3 is a hydrophilic amino acid residue or is absent; X4 is a Cys amino acid residue or is absent; X5 is a Pro amino acid residue or is absent; X6 is the first hydrophobic amino acid residue; X7 is a short branched amino acid residue; X8 is a second hydrophobic amino acid residue; X9 is a saturated hydrophobic amino acid residue; X10 is a hydrophilic amino acid in the D configuration; and X11 is any amino acid residue that allows for cyclization of the peptide derivative.

[0048] In some embodiments, SEQ ID NO:3 further comprises one or more of the following: X4 is Cys, X5 is Pro, X7 is Thr, and X10 is a Lys residue in the D configuration.

[0049] In some embodiments, the first and second hydrophobic amino acid residues of X6 and X8 are aromatic amino acid residues. In some embodiments, the short branched amino acid residue of X7 is Val or Thr. In some embodiments, the saturated hydrophobic amino acid of X9 is Leu. In some embodiments, the hydrophilic amino acid of D configuration of X10 is Asp, Glu, Lys, Dap, or Cys residue in D configuration.

[0050] In some embodiments, X1 is a basic residue exhibiting a positive charge or is absent; X2 is a basic residue exhibiting a positive charge or is absent; and X3 is a basic residue exhibiting a positive charge or is absent. In some embodiments, X1 is an Arg, Lys, or His residue or is absent; X2 is an Arg, Lys, or His residue or is absent; and X3 is an Arg, Lys, or His residue or is absent.

[0051] In some embodiments, X6 is Phe or NaI. In some embodiments, X8 is Phe or NaI. In some embodiments, X6 is Ala, Phe, or NaI, and X4 is NaI. In some embodiments, X7 is Asp, Glu, Lys, Dap, or Cys.

[0052] In some embodiments, the peptide derivative is linear or cyclized. In some embodiments, the peptide derivative has a size of less than 15 amino acid residues. In some embodiments, the peptide derivative has a size of 7, 8, 9, 10, 11, or 12 amino acid residues.

[0053] In certain embodiments, the peptide derivatives of the present technology are acetylated at the N-terminus, amidated at the C-terminus, or both.

[0054] In certain embodiments, the peptide derivative is cyclized with 10 CC bonds or equivalent to close the ring between the alpha carbon of X4 and X11 of SEQ ID NO: 3. Cyclization can also occur between X11 of SEQ ID NO: 3 and an additional amino acid residue added between X4 and X5. Cyclization can also occur between X11 of SEQ ID NO: 3 and an additional amino acid residue added between X5 and X6. In this situation, the optimal ring is closed using five CC bonds (or equivalent). In certain embodiments, the cyclization is a head-to-tail cyclization.

[0055] In certain embodiments, the SERPIN peptide derivative of the present technology has a size of 5 to 30 amino acids, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the SERPIN peptide derivative of the present technology has a size of 20 amino acids or less. Longer peptides may have reduced solubility, while shorter peptides may have reduced stability. In certain embodiments, the peptide derivative has a size of 9 amino acid residues; however, peptide derivatives with a size of 8 amino acid residues in conjunction with an optimized cyclization strategy suffer only minimal loss of activity.

[0056] In certain embodiments, the SERPIN peptide derivative of the present technology is fused with one or more other peptides or compounds to form a fusion peptide or fusion protein.For example, the one or more other peptides include epitope tags such as ALFA tag, V5 tag, Myc tag, HA tag, Spot tag, T7 tag, NE tag, poly(ethylene glycol) (PEG), lipidation, FC fusion, or albumin fusion, or both epitope tags and half-life extenders.

[0057] In certain embodiments, the peptide derivatives of the present technology can be conjugated with permeability enhancers such as oleic acid, cholic acid, cationic surfactants, or lactams to further improve transdermal, transepithelial, nasal, gastric, or topical bioavailability, as illustrated in FIG. 21.

[0058] Pharmaceutical Compositions Comprising SERPIN Peptide Derivatives In another aspect, the present disclosure relates to a pharmaceutical composition comprising, consisting essentially of, or consisting of an effective amount of one or more SERPIN peptide derivatives or fusion peptides of the present technology. The pharmaceutical composition of the present technology can be formulated into any suitable dosage form for transdermal, sublingual, nasal, oral, inhalation, rectal, or ocular administration. In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents that are not SERPIN peptide derivatives of the present technology. In some embodiments, the pharmaceutical composition further comprises one or more permeability enhancers in an amount of 0.1-5% (w / w) to facilitate the penetration of the peptide derivatives or fusion peptides into the skin, mucosa, nasal mucosa, or epithelial layers. Non-limiting examples of permeability enhancers include fatty acids such as oleic acid, bile acids such as cholic acid, cationic surfactants such as cetyltrimethylammonium, and lactams such as laurocapram. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier, excipient, additive, preservative, or combination thereof. Examples of acceptable carriers include physiologically acceptable solutions, such as sterile saline and sterile buffered saline.

[0059] The term "effective amount" as used herein refers to the amount of a composition that produces a desired effect. An effective amount of a composition can be used to produce a prophylactic or therapeutic effect in a subject, such as preventing or treating a target condition, alleviating symptoms associated with the condition, or producing a desired physiological effect. In such cases, the effective amount of a composition is a "therapeutically effective amount", "therapeutically effective concentration", or "therapeutically effective dose". The exact effective amount or therapeutically effective amount is the amount of the composition that produces the most effective results in terms of efficacy of treatment in a given subject or cell population. This amount will vary depending on a wide variety of factors, including but not limited to the characteristics of the composition (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological state of the subject (including age, sex, type and stage of disease, general physical condition, responsiveness to a given dosage, and type of medication) or cell, the nature of the pharmacologic acceptable carrier in the formulation, and the route of administration. Furthermore, the effective amount or therapeutically effective amount can vary depending on whether the composition is administered alone or in combination with another composition, drug, therapy, or other treatment method or modality. Those skilled in the clinical and pharmacological arts will be able to determine effective or therapeutically effective amounts through routine experimentation, i.e., by monitoring the response of a cell or subject to administration of the composition and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21 st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0060] In certain embodiments, the peptide derivative or pharmaceutical composition of the present technology can be formulated for oral administration, parenteral administration, for example, intravenous administration, intramuscular administration, subcutaneous administration (through a device such as a bolus injection or an infusion pump), intradermal administration, transdermal administration, topical administration, and intranasal administration.In certain embodiments, a subcutaneous infusion pump can be used to deliver the peptide or pharmaceutical composition of the present technology.The peptide or pharmaceutical composition can be administered more than once.More specifically, after the initial administration, one or more additional doses can be given as a booster.

[0061] The SERPIN peptide derivative, fusion peptide, or pharmaceutical composition of the present technology has various functions. In certain embodiments, the present technology relates to a method of treating a subject in need of treatment with an effective amount of one or more SERPIN peptide derivatives, fusion peptides, or pharmaceutical compositions disclosed herein. In some embodiments, the subject suffers from a disease or condition in which LRP1 mediation contributes to the pathology, such as in peripheral nerve injury and the resulting pain, lung injury, infectious disease, and conditions associated with allergic inflammation, such as eosinophilic esophagitis. In some embodiments, the subject suffers from a disease associated with a dysregulated immune response, selected from the group consisting of peripheral neuropathy, neuropathic pain, COVID-19 infection, acute respiratory distress syndrome (ARDS), sepsis, SARS-CoV-2, influenza, alphavirus infection, and cytokine storm.

[0062] As used herein, "treating" a condition or "treatment" of a condition can refer to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, or some combination thereof. Treatment can also refer to prophylactic or preventative treatment of the condition.

[0063] As used herein, the term "subject" refers to an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has not undergone any pretreatment with a serine protease inhibitor, such as alpha-1-antitrypsin treatment, before treatment with the peptide of the present technology.

[0064] In some embodiments, the peptide can be used to reduce serum TNF-α levels in human individuals with pathologically elevated TNF-α levels. When administered to a human subject in an effective amount, the peptide causes a 75% reduction in serum TNF-α levels. In certain embodiments, when administered to a human subject in an effective amount, the peptide causes a 50% or 75% reduction in serum TNF-α levels compared to the levels before administration of the peptide.

[0065] LRP-1 Related Conditions LRP-1 functions as an endocytosis receptor and cell signaling receptor and has several ligands that induce specific cell signaling cascades that can contribute to cell survival and anti-inflammatory mechanisms. 5、18、22、25 LRP1 is ubiquitously expressed in many different organs, with abundant expression in the brain, lung, heart, and immune cells. Due to these unique capabilities and its wide expression in both tissues and immune cells, LRP1 plays an essential role in regulating inflammation, cellular metabolism, and maintaining homeostasis. As an illustration, LRP1 regulates inflammatory signaling pathways, such as the NFκB and JNK pathways, which induce the conversion of proinflammatory (M1) macrophages to anti-inflammatory (M2) macrophage phenotypes, modulates cytokine output, and contributes to effective migration and phagocytosis. 22、26、51 In neutrophils, LRP1-dependent mechanisms result in enhanced cell adhesion, chemotaxis, and antibacterial effects of these cells, thereby resisting immune suppression. 25During acute infection or injury, LRP1 also promotes resolution of inflammation through scavenging PAMPS and DAMPS from dying or damaged tissues, preventing the tissue injury cycle. 25 During infection, LRP1 has also been shown to mediate autophagy, a key metabolic process that has recently been shown to play an important protective role in a wide variety of diseases. 4、10 Thus, targeting LRP1, due to its multifunctional ability to regulate inflammation, has substantial potential to alleviate several aspects of the immune response that contribute to the pathology of several diseases, including neurological disorders, infectious diseases, and allergic inflammatory diseases.

[0066] Neurological disorders With regard to nerve injury and the associated pain, damage to the peripheral nervous system induces increased LRP1 expression. It has previously been demonstrated that LRP1 agonists can promote axonal growth in the CNS and induce regeneration after spinal cord injury. 53 LRP1 is an endocytic receptor for a diverse number of ligands, including tissue-type plasminogen activator (tPA), matrix metalloproteinase-9 (MMP-9), and activated α2-macroglobulin. 14 These ligands induce anti-inflammatory activity. 39 , activating the Schwann cell repair program 21 and transactivates cell signaling pathways in neurons involved in axon regeneration 38 Although LRP1 requires ligand binding to activate cell signaling, different ligands elicit distinct and sometimes opposing cell signaling responses, reflecting the ability of different ligands to assemble unique co-receptor complexes. Furthermore, many LRP1 ligands are multi-domain proteins with numerous effects on cell physiology that do not involve LRP1 binding. For example, tissue-type plasminogen activator (tPA) binds to LRP1 to promote Schwann cell (SC) survival and migration. 23However, tPA induces pain through LRP1-independent activity. 1 EI-tPA promotes survival of human iPSC-derived neural progenitor cells (iNPCs), and iNPCs activated with EI-tPA and transplanted into rodents with severe spinal cord injury demonstrate improved recovery of motor function. 40 Imbalances in the microenvironment following nerve injury can have serious consequences, including the development of chronic neuropathic pain conditions. 12 In peripheral nervous system (PNS) injury, both proinflammatory cytokines such as TNFα, IL-6, and IL-1β, and anti-inflammatory cytokines such as IL-10 have been shown to play a central role in axonal regeneration and repair. 6 .

[0067] The role of LRP1 has been extensively studied with respect to neurodegenerative diseases such as Alzheimer's disease (AD). AD is characterized by progressive loss of cognitive abilities and the formation of senile plaques composed primarily of amyloid-β (Aβ) and tau protein aggregates called neurofibrillary tangles (NFTs) in the hippocampus and cortex of affected humans. The spread of protein aggregates during disease progression is a common theme underlying the pathology of neurodegenerative diseases. As an endocytic receptor, LRP1 has been shown to regulate the clearance, degradation, and production of amyloid-β peptide in the brain and throughout the body. 54 Since LRP1-mediated clearance of Aβ across the blood-brain barrier (BBB) ​​is a key event in regulating Aβ transcytosis from the brain to the periphery, targeting LRP1 with one or more peptide derivatives of the present technology may serve as a novel treatment. In addition, tau protein aggregates forming NTFs play a central role in the pathogenesis of Alzheimer's disease. LRP1 functions to regulate the endocytosis, accumulation, and diffusion of tau protein, which are associated with the worsening of the pathology. 32Thus, the SERPIN peptide derivatives of the present technology may serve as an intervention to correct both plaque and tangle pathologies through mediating LRP1-associated tau and amyloid aβ protein aggregation. The SERPIN peptide derivatives of the present technology can also act against neuroinflammation, which plays an essential role in neurodegenerative diseases.

[0068] LRP1 has been shown to be involved in the development of neurodegenerative diseases and in regulating the metabolism of amyloid beta peptide (Aβ) in the brain and periphery. Neuroinflammation plays an essential role in neurodegenerative diseases such as Alzheimer's disease (AD). LRP1 is highly expressed in cells of the CNS and has been shown to play an essential role in the survival of primary neurons under stressful conditions. 56、57 Microglial cells act as resident immune cells in the brain and help maintain homeostasis in the environment. Microglia are thought to be the prototype of tissue-resident macrophage-like innate immune cells in the CNS. 58 These cells are involved in chemotaxis, phagocytosis, antigen presentation, and cytokine production, whereas impaired or infected microglia may contribute to neuroinflammation and neurodegeneration. 59 Reactive glial cells (such as microglial cells) and associated neuroinflammation play a key role in both disease initiation and progression, becoming active through the dysregulated clearance of beta-amyloid and other injury-associated molecular patterns (DAMPs). 13 AB deposition and tau hyperphosphorylation contribute to microglial activation, activation of the NFκB inflammatory pathway, and associated proinflammatory cytokines such as TNFα, IL-6, and IL-1β, which contribute to neuronal injury and loss. Impaired autophagy, the homeostatic process that breaks down and recycles proteins such as beta-amyloid, has been associated with AD. 7LRP1 has been shown to mediate normal lysosomal processing associated with autophagy. Thus, through LRP1, the disclosed SERPIN peptide derivatives can mediate several aspects of AD, including normal cellular metabolism, reducing the spread of protein aggregates, mitigating neuroinflammation, and improving neuronal dysfunction, resulting in the survival and potentially regeneration of these cells.

[0069] Acute lung injury (acute respiratory distress syndrome) Acute lung injury (ALI) leading to acute respiratory distress syndrome (ARDS) can be initiated by a wide variety of inflammatory insults, such as pneumonia, trauma, and / or infection. The primary initiating step in ALI is a dysregulated innate immune response to injury-associated molecular patterns or pathogen-associated molecular patterns (DAMPs or PAMPs, respectively). Alveolar macrophages are activated by infectious microorganisms in Toll-like and Nod-like receptor signaling pathways that result in the recruitment of additional macrophages and circulating neutrophils. Neutrophils accumulate in the lung and release proinflammatory cytokines and other cytotoxic substances, causing exacerbation of injury. The lung epithelium is injured by these cells, and their secretory products cause pulmonary edema and potential respiratory distress (ARDS). An increase in the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, IL-8, and IL-18 occurs, resulting in a "cytokine storm" that is predictive of morbidity and mortality in sepsis. It has been reported that LRP1 regulates lung inflammation and lung tissue repair, and thus plays an essential role in sepsis and ARDS. 49The SERPIN peptide derivatives of the present technology target LRP1, resulting in precise coordination of immune responses to restore homeostasis. Thus, the disclosed peptide derivatives can have a significant impact on reducing lung injury and improving survival through multiple mechanisms initiated by inducing specific LRP1-mediated signaling pathways. These mechanisms include rebalancing cytokine output to promote resolution of inflammation, mediating autophagy to restore proper cellular metabolic processes, restoring immune cell function, and receptor scavenging to regulate the tissue damage cycle. These mechanisms aid in the clearance of infection and allow the host to better fight infection. Based on these mechanisms, the disclosed SERPIN peptide derivatives can be used as immunomodulatory therapeutic agents to prevent or treat ARDS by mediating LRP1 signaling.

[0070] infectious disease Acute respiratory distress syndrome (ARDS) is a major complication in patients with severe COVID-19 disease. Clinical evaluation and retrospective studies from Wuhan, China, showed that 100% of hospitalized patients had pneumonia, 29% of whom developed ARDS, and the majority (81%) of critically ill non-survivors developed ARDS. 17、52Thus, patients who develop ARDS are at substantially higher risk of mortality. Virus-mediated activation of the innate immune response through Toll-like receptors (TLRs) initiates the inflammatory response necessary to clear the infection. In some cases, the initiated viral invasion and triggers are highly amplified and uncontrolled, resulting in immune cell overstimulation and abnormal cytokine release (known as cytokine storm), resulting in tissue injury that may lead to organ failure and death. Cytokine regulation is a major factor in preventing the deleterious effects of a hyperactive immune response, and controlling the cytokine storm may have a significant impact on the progression of viral pneumonia to ARDS. In the case of SARS-CoV-2, emerging evidence suggests that cytokine storm is a contributing factor to mortality for a subpopulation of patients with severe disease. 27 Recently, clinical trials have begun for Kevzara (sarilumab), an interleukin-6 inhibitor (IL-6), for the treatment of coronavirus-infected patients with pulmonary complications. IL-6 is a biomarker associated with higher mortality in individuals with pneumonia. These trials will provide essential insight into the efficacy of treatments targeting single cytokines. However, historically, targeting a single cytokine or pathway in ARDS patients has not proven to be an effective approach.

[0071] LRP1 signaling mediated by the SERPIN peptide derivatives of the present technology may have therapeutic potential as an immunomodulatory strategy to improve outcomes in COVID-19 patients through regulating several mechanisms of lung inflammation, including suppressing cytokine storm, improving cell survival, regulating autophagy and cell metabolism to clear infection, and controlling tissue repair signaling pathways to promote proper healing and prevent fibrosis. LRP1 is widely known to regulate protease / antiprotease activity and mediate viral entry. 43SARS-CoV-2 utilizes a protease (TMPRSS2) to regulate cell entry and infection, and treatment with the corresponding protease inhibitors, such as SERPIN, blocked infection of lung cells. 16、24、28 SARS-CoV-2 viral entry is dependent on TMPRSS2, a serine protease that primes the viral S protein, which is involved in host cell entry, the first step of the viral replication cycle. Camostat mesylate, a natural SERPIN with TMPRSS2 protease inhibitory function, was effective in reducing SARS-CoV-2 viral entry, thereby limiting both infection and spread of the disease. 16 The alpha-1 antitrypsin derivatives of the present technology, such as SP163M, can also inhibit TMPRSS2, possibly through LRP1, and may have the effect of reducing SARS-CoV-2 viral replication. The disclosed SERPIN peptide derivatives may reduce SARS-CoV-2 replication and inhibit viral entry through the mechanism of LRP1 control of host protease / viral protein interactions.

[0072] Arthropod-borne viruses are an important cause of acute encephalitis and an emerging global problem with an increasing risk of introduction into new regions. 11、31 Mosquito-borne encephalitic alphaviruses, including Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), and Western equine encephalitis virus (WEEV), are endemic to the Americas and have caused outbreaks of encephalomyelitis with potential for spread to the United States. In humans, VEEV causes a febrile illness typically characterized by fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system (CNS). Neurologic cases have a mortality rate as high as 35% in children and 10% in adults, and survivors often have long-term neurologic deficits. 42 Overall estimates of VEEV survivors with neurological sequelae range from 4% to 14%, but may be higher due to misdiagnosis of arboviral encephalitis. 33 .

[0073] VEEV infection is known to inhibit cellular transcription and translation to downregulate innate immune responses. 45、48 In contrast, in the central nervous system (CNS), VEEV infection results in the upregulation of numerous genes in the inflammatory response and apoptotic pathways. 45、48 Specifically, proinflammatory cytokines, including interleukin-1β (IL-1β), IL-6, IL-12, and tumor necrosis factor-α (TNF-α), play a role in the pathogenesis of VEEV. 9、20、30、34、35 Changes in gene expression were analyzed in brain tissue from VEEV-infected mice, revealing modifications in immune pathways involved in antigen presentation, inflammation, apoptosis, and conventional antiviral responses. 36 In addition, viral modulation of extracellular matrix and adhesion genes, including integrin, cadherin-1, cadherin-2, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1 (ICAM-1), was observed in the brains of VEEV-infected mice. 37 ICAM-1 knockout mice demonstrate reduced inflammation in the brain and subsequent delayed onset of clinical disease. 37 These studies suggest that alphavirus-induced inflammation contributes substantially to neurological injury and that control of inflammation is a viable therapeutic strategy.

[0074] The SERPIN peptide derivatives of the present technology can bind to LRP1 and initiate an immunoregulatory cascade. LRP1 expression is increased during ischemia, tissue injury, and viral infection. 2、22、25、43 Binding of one or more of the disclosed peptide derivatives to LRP1 can inhibit inflammatory responses and induce pro-survival signaling through phosphorylation of the protein kinase Akt. Thus, targeting LRP1 has potential as a broad-spectrum therapeutic strategy against infectious diseases.

[0075] SERPIN peptide derivatives, through host-mediated mechanisms directed at LRP1, can suppress the harmful cytokine storm associated with severe COVID-19 disease, activate protective pathways to prevent lung injury, and / or clear the infection faster, and have a direct impact on suppressing viral infection. Thus, the dual anti-inflammatory and antiviral mechanisms of SERPIN peptide derivatives may have the effect of improving survival in patients with SARS-COV2-induced ARDS.

[0076] Eosinophilic esophagitis Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory response, primarily mediated by type 2 immunity, associated with esophageal dysfunction and disturbed epithelial barrier function. Esophageal inflammation results in dysphagia, persistent heartburn, chest and abdominal pain, weight loss, and food impaction. It is characterized by high numbers of eosinophils, proteases, serine proteases including cathelicidin, kallikrein (KLK5), and thymic stromal lymphopoietin (TSLP), a cytokine and master regulator of allergic type 2 inflammatory responses in the local environment. 41 In esophageal epithelial cells, loss of function of the serine peptidase inhibitor Kazal7-SPINK7 leads to uncontrolled protease activity, release of proinflammatory cytokines such as TNFα, CCL2, GM-CSF, IL-8, and CXCL10, and inflammation. Recently, it has been found that the serine protease KLK-5, a key mediator of epithelial barrier function, is a direct target of SPINK7, and loss of SPINK7 mediates EoE pathogenesis mainly through uncontrolled KLK-5 protease activity. Interestingly, the SERPIN alpha-1 antitrypsin is able to inhibit KLK5 activity in vitro and inhibit allergen-induced esophageal eosinophilia in vivo. However, the mechanism of the activity is still unclear and may involve LRP1 instead of or in addition to direct proteolytic inhibition.

[0077] Patients with eosinophilic asthma have lower levels of LRP1. In addition, specific deletion of LRP1 in CD11b and CD11c dendritic cells in mice results in increased allergic inflammatory responses in a model of allergic airway disease. 29 Mice lacking LRP1 had increased antigen uptake and suffered from increased eosinophilic inflammation, increased allergic sensitization, increased Th2-mediated cytokine production, and reduced regulatory T cells. 29 Thus, LRP1 may help maintain protease / inhibitor homeostasis in the esophageal environment, mediate TH2 responses, and inhibit inflammatory signaling pathways (NFκB, JNK) to result in repair of esophageal dysfunction.

[0078] As demonstrated herein, the disclosed SERPIN peptide derivatives, such as SA7, a potent LRP1 agonist without protease inhibitor function, can reduce the expression of TMPRSS2. TMPRSS2 expression has been shown to be significantly increased in nasal and airway epithelial cells of type 2 asthma and allergic rhinitis. Research has also shown that TMPRSS2 expression is positively associated with TH2-mediated immune response, which is important for allergic response. The disclosed SERPIN peptide derivatives may have significant significance in alleviating eosinophilic esophagitis by mediating control over LRP1.

[0079] The following examples are intended to illustrate various aspects of the present technology. Therefore, the specific aspects discussed should not be constructed as limitations on the scope of the present technology. It is clear to those skilled in the art that various equivalents, changes and modifications can be made without departing from the scope of the present technology, and it is understood that such equivalent aspects are included herein. Furthermore, all references cited in this disclosure are incorporated herein by reference in their entirety as if fully set forth herein.

[0080] Various aspects of the present technology are described in the following paragraphs 0096-0175 of this specification.

[0081] SERPIN peptide derivatives comprising the core sequence of FVFLM (SEQ ID NO: 1), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQ ID NO: 8), PFVFL[Nle] (SEQ ID NO: 9) and the following modifications: (i) a polar head added to the N-terminus of the core sequence, a polar tail added to the C-terminus of the core sequence, or both; (ii) one or more amino acid residues added to the N-terminus of the core sequence, the C-terminus of the core sequence, or both, such that the peptide derivative may be cyclized; (iii) one or more amino acid residues in the core sequence that are replaced by one or more substituting amino acid residues that have a lower hydrophobicity compared to the one or more amino acid residues in the core sequence that are replaced; (iv) one or more amino acid residues in the core sequence that are replaced by one or more substituting amino acid residues that have a higher hydrophobicity compared to the one or more amino acid residues in the core sequence that are replaced; and (v) one or more amino acid residues in the core sequence are deleted and one or more of the following:

[0082] 0097. A SERPIN peptide derivative of paragraph 0096 which is a linear peptide.

[0083] 0098. A SERPIN peptide derivative of paragraph 0096 which is a cyclized peptide.

[0084] 0099. A SERPIN peptide derivative of paragraph 0098, which is cyclized by forming a disulfide bond between two Cys residues.

[0085] 0100. A SERPIN peptide derivative of paragraph 0098, which is cyclized by a linker between two amino acid residues.

[0086] 0101. The SERPIN peptide derivative of paragraphs 0096-0100, wherein the polar head or polar tail comprises two or more charged amino acids, such as positively charged amino acids selected from the group consisting of Arg, Lys, and His.

[0087] 0102. A SERPIN peptide derivative of paragraphs 0096 to 0101 having a size of less than 15 amino acid residues.

[0088] 0103. A SERPIN peptide derivative of paragraphs 0096 to 0102 having a size of 7, 8, 9, 10, 11, or 12 amino acid residues.

[0089] 0104. The SERPIN peptide derivative of paragraphs 0096 to 0103, wherein one or more amino acid residues are D-amino acids.

[0090] A SERPIN peptide derivative comprising the amino acid sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 (SEQ ID NO: 3), wherein: X1 is a hydrophilic amino acid residue or is absent; X2 is a hydrophilic amino acid residue or is absent; X3 is a hydrophilic amino acid residue or is absent; X4 is a Cys amino acid residue or is absent; X5 is a Pro amino acid residue or is absent; X6 is the first hydrophobic amino acid residue; X7 is a short branched amino acid residue; X8 is a second hydrophobic amino acid residue; X9 is a saturated hydrophobic amino acid residue; X10 is a hydrophilic amino acid in the D configuration; and X11 is any amino acid residue that allows cyclization of the SERPIN peptide derivative; The SERPIN peptide derivative.

[0091] 0106. The SERPIN peptide of paragraph 0105, wherein the first and second hydrophobic amino acid residues are aromatic amino acid residues.

[0092] 0107. The SERPIN peptide of paragraphs 0105 to 0106, wherein the short branched amino acid residue is Val or Thr.

[0093] 0108. The SERPIN peptide of paragraphs 0105 to 0107, wherein the saturated hydrophobic amino acid is Leu.

[0094] 0109. The SERPIN peptide of paragraphs 0105 to 0108, wherein the hydrophilic amino acid in the D configuration is an Asp, Glu, Lys, Dap, or Cys residue in the D configuration.

[0095] X1 is a basic residue exhibiting a positive charge or is absent; X2 is a basic residue exhibiting a positive charge or is absent; and X3 is a basic residue exhibiting a positive charge or is absent; SERPIN peptides of paragraphs 0105 to 0109.

[0096] X1 is an Arg, Lys, or His residue or is absent; X2 is an Arg, Lys, or His residue or is absent; and X3 is an Arg, Lys, or His residue or is absent; SERPIN peptide derivatives of items 0105 to 0110.

[0097] 0112. The SERPIN peptide derivative of paragraphs 0105 to 0111, wherein X6 is Phe or Nal.

[0098] 0113. The SERPIN peptide derivative of paragraphs 0105 to 0112, wherein X8 is Phe or Nal.

[0099] 0114. The SERPIN peptide derivative of paragraphs 0105 to 0113, wherein X6 is Ala, Phe, or Nal and X4 is Nal.

[0100] 0115. The SERPIN peptide derivative of paragraphs 0105 to 0114, wherein X7 is Asp, Glu, Lys, Dap, or Cys.

[0101] A SERPIN peptide derivative of any one of paragraphs 0105 to 0115, which is linear or cyclized.

[0102] A SERPIN peptide derivative of any one of paragraphs 0105 to 0116, having a size of less than 15 amino acid residues.

[0103] 0118. A SERPIN peptide derivative of paragraphs 0105 to 0117 having a size of 7, 8, 9, 10, 11, or 12 amino acid residues.

[0104] SERPIN peptide derivatives comprising the core sequence of FVFLM (SEQ ID NO: 1), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQ ID NO: 8), PFVFL[Nle] (SEQ ID NO: 9) and the following modifications: (i) one or more Lys, Glu, or His residues added to the N-terminus of the core sequence, a polar tail added to the C-terminus of the core sequence, or both; (ii) one or more amino acid residues added to the N-terminus of the core sequence, the C-terminus of the core sequence, or both, such that the SERPIN peptide derivative may be cyclized by forming a disulfide bond between two Cys residues; (iii) one or more amino acid residues in the core sequence that are substituted by one or more Thr amino acid residues; and (iv) one or more amino acid residues in the core sequence that are replaced by one or more substituting amino acid residues that have a higher hydrophobicity than the one or more amino acid residues being replaced. and one or more of the following:

[0105] 0120. A SERPIN peptide derivative of paragraph 0119, which is a linear peptide.

[0106] A SERPIN peptide derivative of paragraph 0119 or 0120 having a size of less than 15 amino acid residues.

[0107] 0122. A SERPIN peptide derivative of paragraphs 0119 to 0121 having a size of 7, 8, 9, 10, 11, or 12 amino acid residues.

[0108] A SERPIN peptide derivative comprising the amino acid sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 (SEQ ID NO: 3), wherein: X1 is a hydrophilic amino acid residue or is absent; X2 is a hydrophilic amino acid residue or is absent; X3 is a hydrophilic amino acid residue or is absent; X4 is a Cys amino acid residue or is absent; X5 is a Pro amino acid residue or is absent; X6 is the first hydrophobic amino acid residue; X7 is a short branched amino acid residue; X8 is a second hydrophobic amino acid residue; X9 is a saturated hydrophobic amino acid residue; X10 is a hydrophilic amino acid in the D configuration; and X11 is any amino acid residue that allows for cyclization of the SERPIN peptide derivative; wherein X4 is Cys, X5 is Pro, X7 is Thr, and / or X10 is a Lys residue in the D configuration; The SERPIN peptide derivative.

[0109] The SERPIN peptide of paragraph 0123, wherein the first and second hydrophobic amino acid residues are aromatic amino acid residues.

[0110] 0125. The SERPIN peptide of paragraph 0123 or 0124, wherein the short branched amino acid residue is Val or Thr.

[0111] 0126. The SERPIN peptide of paragraphs 0123 to 0125, wherein the saturated hydrophobic amino acid is Leu.

[0112] 0127. The SERPIN peptide of paragraphs 0123 to 0126, wherein the hydrophilic amino acid in the D configuration is an Asp, Glu, Lys, Dap, or Cys residue in the D configuration.

[0113] X1 is a basic residue exhibiting a positive charge or is absent; X2 is a basic residue exhibiting a positive charge or is absent; and X3 is a basic residue exhibiting a positive charge or is absent; SERPIN peptides of paragraphs 0123 to 0127.

[0114] X1 is an Arg, Lys, or His residue or is absent; X2 is an Arg, Lys, or His residue or is absent; and X3 is an Arg, Lys, or His residue or is absent; SERPIN peptide derivatives according to items 0123 to 0128.

[0115] 0130. The SERPIN peptide derivative of paragraphs 0123 to 0129, wherein X6 is Phe or Nal.

[0116] 0131. The SERPIN peptide derivative of paragraphs 0123 to 0130, wherein X8 is Phe or Nal.

[0117] 0132. The SERPIN peptide derivative of paragraphs 0123 to 0131, wherein X6 is Ala, Phe, or Nal and X4 is Nal.

[0118] 0133. The SERPIN peptide derivative of paragraphs 0123 to 0132, wherein X7 is Asp, Glu, Lys, Dap, or Cys.

[0119]

[0134] A SERPIN peptide derivative of paragraphs 0123 to 0133, which is linear or cyclized.

[0120] A SERPIN peptide derivative of paragraphs 0123 to 0134, having a size of less than 15 amino acid residues.

[0121] 0136. A SERPIN peptide derivative of paragraphs 0123 to 0135 having a size of 7, 8, 9, 10, 11, or 12 amino acid residues.

[0122] A SERPIN peptide derivative comprising a sequence of SEQ ID NO: 10-20 or 23-62, comprising a polar head appended to the N-terminus of the core sequence, a polar tail appended to the C-terminus of the core sequence, or both.

[0123] A SERPIN peptide derivative comprising the sequence of SEQ ID NO: 15-17 or 23-62, wherein one or more amino acid residues are added to the N-terminus of the core sequence, the C-terminus of the core sequence, or both, such that the peptide derivative can be cyclized.

[0124] 139. A SERPIN peptide derivative comprising a sequence of SEQ ID NO: 27-29, 38, 39, or 42-62, wherein one or more amino acid residues in a core sequence are replaced by one or more replacement amino acid residues having a lower hydrophobicity compared to the one or more amino acid residues in the core sequence that are replaced.

[0125] 140. A SERPIN peptide derivative comprising the sequence of SEQ ID NO: 30 or 31, wherein one or more amino acid residues in a core sequence are replaced by one or more substituted amino acid residues having a higher hydrophobicity compared to the one or more amino acid residues in the core sequence that are replaced.

[0126] A SERPIN peptide derivative comprising the sequence of SEQ ID NO: 41 or 47-53, wherein one or more amino acid residues in the core sequence are deleted.

[0127] A SERPIN peptide derivative comprising the sequence of SEQ ID NO: 10, 11, 13, or 14, 35, or 36, wherein one or more Lys, Glu, or His residues are added to the N-terminus of the core sequence, a polar tail is added to the C-terminus of the core sequence, or both.

[0128] A SERPIN peptide derivative comprising a sequence of SEQ ID NO: 15-17 or 23-31, wherein one or more amino acid residues are added to the N-terminus of the core sequence, the C-terminus of the core sequence, or both, such that the SERPIN peptide derivative can be cyclized by forming a disulfide bond between two Cys residues.

[0129] A SERPIN peptide derivative comprising a sequence of SEQ ID NO: 27-29, 38, 39, 47-55, or 59-62, wherein one or more amino acid residues in the core sequence are replaced by one or more Thr amino acid residues.

[0130] A fusion protein comprising the SERPIN peptide derivative of any one of paragraphs 0096 to 0144 and an epitope tag, a half-life extender, or both.

[0131] A conjugate comprising a SERPIN peptide derivative according to any one of items 0096 to 0144 and a permeability enhancer.

[0132] A pharmaceutical composition comprising the SERPIN peptide derivative of any one of claims 0096 to 0144, the fusion protein of any one of claims 0145, or the conjugate of any one of claims 0146.

[0133]

[0148] The pharmaceutical composition of paragraph 0147, further comprising one or more additional therapeutic agents.

[0134]

[0149] The pharmaceutical composition of paragraph 0147 or paragraph 0148, further comprising a pharma- ceutically acceptable carrier, excipient, additive, preservative, or a combination thereof.

[0135]

[0150] The pharmaceutical composition of paragraphs 0147 to 0149, further comprising a permeability enhancer.

[0136]

[0151] A pharmaceutical composition of paragraphs 0147 to 0150, formulated for oral, transdermal, or parenteral administration.

[0137] A fusion protein comprising a SERPIN peptide derivative of paragraphs 0119-0136, 0140, or 0142-0144 and an epitope tag, a half-life extender, or both.

[0138] A conjugate comprising a SERPIN peptide derivative of any one of items 0119 to 0136, 0140, or 0142 to 0144 and a permeability enhancer.

[0139] A pharmaceutical composition comprising a SERPIN peptide derivative of paragraph 0119 to 0136, 0140, or 0142 to 0144, a fusion protein of paragraph 0152, or a conjugate of paragraph 0153.

[0140]

[0155] The pharmaceutical composition of paragraph 0154, further comprising one or more additional therapeutic agents.

[0141]

[0156] The pharmaceutical composition of paragraph 0154 or paragraph 0155, further comprising a pharma- ceutically acceptable carrier, excipient, additive, preservative, or a combination thereof.

[0142]

[0157] The pharmaceutical composition of paragraphs 0154 to 0156, further comprising a permeability enhancer.

[0143]

[0158] A pharmaceutical composition of paragraphs 0154 to 0157, which is formulated for oral, transdermal, or parenteral administration.

[0144] A method for treating a subject suffering from a disease or condition associated with LRP1, comprising administering to the subject an effective amount of a SERPIN peptide derivative of any one of paragraphs 0096 to 0144, a fusion protein of paragraph 0145, a conjugate of paragraph 0146, or a pharmaceutical composition of paragraphs 0147 to 0151, for treating the disease or condition associated with LRP1.

[0145]

[0160] The method of paragraph 0159, wherein administering the SERPIN peptide reduces activation of NFkB in the subject.

[0146]

[0161] The method of paragraph 0159 or 0160, wherein administering the SERPIN peptide reduces TNFa in the subject.

[0147]

[0162] The method of paragraphs 0159 to 0161, wherein administering the SERPIN peptide reduces IL-6.

[0148]

[0163] The method of paragraphs 0159 to 0162, wherein the disease or condition associated with LRP1 is acute or neuropathic pain, nociceptive pain, or inflammatory pain.

[0149]

[0164] The method of paragraphs 0159 to 0163, wherein the disease or condition associated with LRP1 is a disease or condition caused by a viral infection.

[0150]

[0165] The method of paragraph 0164, wherein administering the SERPIN peptide reduces viral replication.

[0151]

[0166] The method of paragraphs 0159 to 0162, wherein the disease or condition associated with LRP1 is eosinophilic esophagitis.

[0152] The method of paragraphs 0159 to 0162, wherein the disease or condition associated with LRP1 is acute lung injury.

[0153] A method for treating a subject suffering from a disease or condition associated with LRP1, comprising administering to the subject an effective amount of a SERPIN peptide derivative of paragraph 0119 to 0136, 0140, or 0142 to 0144, a fusion protein of paragraph 0152, a conjugate of paragraph 0153, or a pharmaceutical composition of paragraphs 0154 to 0158, for treating the disease or condition associated with LRP1.

[0154]

[0169] The method of paragraph 0168, wherein administering the SERPIN peptide reduces activation of NFkB in the subject.

[0155]

[0170] The method of paragraph 0168 or 0169, wherein administering the SERPIN peptide reduces TNFa in the subject.

[0156]

[0171] The method of paragraphs 0168 to 0170, wherein the step of administering a SERPIN peptide reduces IL-6.

[0157]

[0172] The method of paragraphs 0168 to 0170, wherein the disease or condition associated with LRP1 is a disease or condition caused by a viral infection.

[0158]

[0173] The method of paragraph 0172, wherein administering the SERPIN peptide reduces viral replication.

[0159]

[0174] The method of paragraphs 0168 to 0170, wherein the disease or condition associated with LRP1 is eosinophilic esophagitis.

[0160]

[0175] The method of paragraphs 0168 to 0170, wherein the disease or condition associated with LRP1 is acute lung injury. EXAMPLES

[0161] Example 1: Anti-inflammatory effects of SERPIN-derived derivatives Using SP163M and SP22 as positive controls, the SERPIN peptide derivatives SA1-SA8 listed in Table 1 were tested for their anti-inflammatory effects. Reporter cells (THP1-XBlue-MD2-CD14 cells) were treated with each peptide derivative SA1-SA8, as well as SP22 and SP163M (50 μg / ml) before being invaded with LPS (5 ng / ml) and incubated overnight. NFκB-inducible secreted embryonic alkaline phosphatase (SEAP) was measured in the supernatant and absorbance was read. As shown in Figure 1, SERPIN peptide derivatives (such as SA3, SA6, and SA7) that retained the shortest LRP1-binding sequence FVFL[Nle] and the RRR tripeptide as a flank to improve solubility demonstrated improved activity in NFκB inhibition when compared to SP163M. However, SERPIN peptide derivatives flanked by either the HHH or KKK tripeptides, or the negatively charged EEE tripeptides (SA1, SA2, SA4, SA5, etc.), demonstrated minimal NFκB inhibitory activity.

[0162] This data shows that truncated peptides (e.g., SA3) can be made and are more effective at reducing NFκB activation than the SERPIN peptides SP163M and SP22, which contain a highly conserved core sequence including the FNKP and LRP1 binding motifs (FVFLM / Nle).

[0163] Example 2: Evaluation of the biological activity of SERPIN peptide derivatives Various SERPIN peptide derivatives were screened in vitro for their ability to reduce TNFα and NFκB secretion via LRP-1 activation. NFκB reporter cells (THP1-XBlue-MD2-CD14 cells) were treated with various concentrations of each peptide derivative up to 100 μg / ml, then invaded with LPS (5 ng / ml) and incubated overnight. NFκB-induced secreted embryonic alkaline phosphatase (SEAP) was measured in the supernatant and absorbance was read. The percentage reduction in NFκB activation relative to vehicle (LPS-stimulated) cells is shown in the figure. IMG microglial cells were treated with various concentrations of each peptide derivative up to 100 μg / ml, then stimulated with LPS (100 ng / ml) for 24 h. TNFα (pg / ml) was measured in the supernatant using ELISA.

[0164] The SERPIN peptide derivatives A1-A15 listed in Tables 2-4 were tested for their TNFα activity, as shown in Figure 5. Peptide derivatives A5 and A8, which have amino acid substitutions, and peptide derivatives A10 and A15, which have modified ring closures, demonstrated improved activity in reducing TNFα activation.

[0165] Figure 6 demonstrates that peptide derivatives A2-1, A2-2, A2-3, A2-4, and A2-5 exhibited superior activity in reducing activation of TNFα (Figure 6A) and NFκB (Figure 6B). The activity of these peptide derivatives was dose-dependent and showed improved potency (Figure 7). The peptide derivatives were tested at concentrations up to 50 μg / mL, while SP16 unmodified, SP163M, and SA7 were tested at concentrations up to 100 μg / mL. None of the peptide derivatives exhibited cytotoxicity (Figure 8). For cytotoxicity assays, cells (NFκB reporter cells or IMG microglial cells) were treated with SP163M or either peptide derivatives SA7, A5, A15, A2-1, A2-2, A2-3, A2-4, A2-5, A2-6, A2-7, A2-8, and A2-9 at concentrations up to 100 μg / mL for 24 hours, and then cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay according to the manufacturer's instructions.

[0166] Figure 9 demonstrates that peptide derivatives A3-10 and A3-14 exhibited excellent activity in reducing NFκB activation, and Figure 10 demonstrates that peptide derivative A3-14 exhibited excellent activity in reducing IL-6 secretion in IMG after LPS stimulation. Peptide derivative A3-10 was not included in the IL-6 assay due to synthesis issues. None of these tested peptide derivatives showed any significant cytotoxicity (Figure 11).

[0167] Table 7 below summarizes the EC50 of in vitro tests for some of the peptide derivatives of the present technology.

[0168] Table 7: EC50 (μg / mL) of various peptide derivatives TIFF2025515349000022.tif83128

[0169] Example 3: In vivo effects of SERPIN peptide derivatives The SERPIN peptide derivatives A15 and A2-5 were tested for their ability to reduce pain-related behaviors induced by capsaicin in mice compared to SP163M. SP163M (50 μg), A15 (5 μg), or A2.5 (5 μg), or capsaicin (25 ng) were injected into the hind paws of male mice, and pain-related behaviors such as flinching or licking were observed for 10 min. As shown in Figure 12, both peptide derivatives at a low dose of 5 μg / mouse exhibited a level of pain-blocking effect similar to that of SP163M at much higher doses.

[0170] Example 4: Effects of SERPIN peptide derivatives on neuroinflammation As shown in Figure 13, cytokine profiles were investigated in LPS-induced microglial cells treated with SERPIN peptide derivatives SA7, SP163M, or vehicle control. Cytokines were induced with LPS and measured in the supernatant. In microglial cells, SA7 reduced several LPS-mediated cytokines, including IL-6, TNFα, IL-1β, IL-12, and CXCL1, more potently than peptide SP163M. IMG microglial cells were treated with SA7 (100 μg / ml), SP163M (100 μg / ml), with or without LPS (100 ng / ml) for 24 hours. Cytokine levels (pg / ml) in the supernatant were measured by multiplex assay.

[0171] Next, the efficacy of the peptide derivative SA7 was investigated in an in vivo model of neuroinflammation. The LPS model of neuroinflammation has been used in numerous studies to understand neurodegenerative diseases. 60~63 These studies have confirmed that LPS induces cognitive impairment in C57BL / 6 mice. Furthermore, acute systemic LPS induces activation of microglial cells in the brain, impaired amyloid beta clearance, and increased proinflammatory cytokines in the blood and brain. Therefore, we used an LPS-induced neuroinflammation model to test the activity of the peptide derivative SA7.

[0172] Female C57BL / 6 mice (n=3-4) were administered 1 mg / kg LPS (E. coli 0111:B4) by intraperitoneal injection for 4 consecutive days, followed by daily subcutaneous administration of either SP163M (100 μg) or SA7 (100 μg) 1 h after LPS injection. Body weight and clinical assessments were recorded daily. At the end of the study (day 5) (24 h after the last LPS injection), mice were weighed, scored, and sacrificed. Figure 3 shows that the clinical scores of SA7-treated mice were significantly improved compared to both vehicle- and SP163M-treated mice (Figure 14A). SA7-treated mice also had a slight advantage in weight loss that was not seen in SP163M-treated mice (Figure 14B).

[0173] Brain homogenates were assessed for cytokine analysis using the LEGENDplex™ Mouse Inflammation Panel, a flow-based multiplex assay using fluorescently coded beads. This panel allows for simultaneous quantification of 13 mouse cytokines, including IL-1α, IL-1β, IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, MCP-1, IFN-β, IFN-γ, TNF-α, and GM-CSF. Figure 15 shows that the SA7-treated group showed differences in the levels of many LPS-induced cytokines compared to vehicle- and SP163M-treated animals. As an illustration, moderate levels of IL-6 were measured in vehicle and SP163M brain homogenates, but no detectable levels of IL-6 were measured in any of the SA7-treated animals, while IL-17A, IL-12, TNFα, and GM-CSF also showed significantly lower levels. This is consistent with the improvement in clinical signs seen with SA7 treatment.

[0174] Brain homogenates were then analyzed for two biomarkers of CNS injury: neurofilament light chain (NfL), expressed in nerve axons, and glial fibrillary acidic protein (GFAP), a marker of astrocyte activation and expressed in astrocytes. There are reports of an inverse correlation between inflammation levels in cortical regions and plasma NfL levels. LPS was found to selectively reduce the expression of neurofilament light chain in neurons. NfL is required for neuronal regeneration, synaptic connectivity, and neuronal homeostatic signaling functions, indicating that increased NfL may point to protection of neurons from LPS-induced injury. Figure 16A shows that the peptide derivative SA7 significantly increases NfL protein expression in brain homogenates following LPS-activated neuroinflammation compared to both vehicle and SP163M, as measured by Western blot. Aberrant expression of GFAP points to reactive astrocytes and neuroinflammation. Figure 16B shows that peptide derivative SA7 significantly reduces GFAP expression in brain lysates as measured by Western blot analysis compared to both SP163M- and vehicle-treated animals. These results suggest a highly neuroprotective effect of the peptide derivative.

[0175] Example 5: Effects of SERPIN peptide derivatives on autophagy In addition to neuroinflammation, impaired autophagy, a homeostatic process that breaks down and recycles proteins such as beta-amyloid, has been linked to AD. 7LRP1 has been shown to mediate normal lysosomal processing associated with autophagy. Thus, through LRP1 agonists such as SERPIN peptide-derived derivatives, it has the potential to mediate several aspects of AD including normal cellular metabolism, reducing the spread of protein aggregates, mitigating neuroinflammation, and improving neuronal dysfunction, resulting in the survival and possibly regeneration of these cells. The effect of SP163M and the peptide derivative SA7 on autophagy markers was tested in microglial cells. IMG microglial cells were treated with SP163M (100 μg / ml) or SA7 (100 μg / ml) before the addition of LPS (100 ng / ml) for 24 h. Lysates were collected and western blot analysis of both LRP1 and microtubule-associated protein light chain 3 (LC3 I and II) was performed. The LC3II / I ratio, a commonly used marker for autophagy that indicates autophagic flux, was determined. Figure 17A shows that autophagy flux is reduced in microglial cells activated by LPS, indicating that normal lysosomal processing is impaired. Treatment with either SP163M or the peptide derivative SA7 in the presence of LPS restored autophagy function to near baseline levels. LRP1 protein expression was measured, and Figure 17B shows that LPS caused a decrease in levels, likely indicative of increased LRP1 shedding. SP163M and the peptide derivative SA7 increased LRP1 protein expression in microglial cells activated by LPS. Interestingly, in the absence of LPS, treatment with SA7 significantly increased LRP1 expression compared to SP163M. These results point to another mechanism by which regulation of LRP1 by SP163M contributes to the homeostatic balance of dysregulated cellular processes.

[0176] Example 6: Effects of SERPIN peptide derivatives on viral infections The SERPIN-derived peptide SP163M demonstrated antiviral effects. SP163M was able to significantly reduce the viral replication of neuroinflammatory alphaviruses such as Eastern Equine Encephalitis (EEEV). SP163M also inhibited the viral replication of the novel coronavirus SARS-CoV-2. SARS-CoV-2 and other viruses, such as influenza, are known to use host cell proteases for viral entry. TMPRSS2 processes the S protein on the SARS-CoV2 envelope in a process called priming. Priming of the S protein is required for binding between the S protein and the host receptor ACE2. LRP1 is widely known to regulate protease / antiprotease activity. Protease inhibitors such as alpha-1 antitrypsin block viral entry by reducing the proteolytic activity of TMPRSS2 and preventing the priming of the S protein. In addition, inhibition of TMPRSS2 prevents the processing of ACE2, which reduces the infectivity of coronaviruses. Figure 18 shows the inhibitory effect of SP163M and the peptide derivative SA7 on TMPRSS2 at various concentrations. The IC50 of SP163M was 1125 ng / mL, whereas the SA7 derivative was much more potent with an IC50 of 83 ng / mL.

[0177] Example 7: Effect of SERPIN peptide derivatives on eosinophilic esophagitis With regard to eosinophilic esophagitis (EoE), A1AT has been shown to attenuate experimental EoE in murine models and in vitro. It is unclear whether the effect of A1AT is mediated by inhibition of proteolytic activity or through activation of LRP1 signaling. Patients with eosinophilic asthma have lower LRP1, and in animal models, loss of LRP1 is associated with exacerbated allergic responses. SP163M does not contain any sequence for antiprotease activity and likely acts through mediation of LRP1 rather than through proteolytic activity. In a mouse model of eosinophilic esophagitis, in which mice were sensitized intraperitoneally to ovalbumin (OVA) and then challenged intranasally with OVA on four separate days, esophageal lysates from EoE-induced mice show lower levels of LRP1 protein expression as measured by Western blot analysis. Treatment of mice with the SERPIN-derived peptide SP163M shows increased levels of LRP1 (Figure 19A). Evidence indicates that loss of esophageal expression of serine peptidase inhibitor kazal type 7 (SPINK7) is an upstream event in the pathogenesis of EoE. Thus, after loss of SPINK7, we tested the in vitro efficacy of SP163M and the peptide derivative SA7 in reducing thymic stromal lymphopoietin (TSLP), a key immune checkpoint cytokine involved in stimulating dendritic cells to polarize the generation of type 2 T cells (Th2), the key cells involved in the pathogenesis of EoE. Figure 19B shows that SP163M inhibited poly I:C-induced TSLP production by SPINK7 knockout EPC2 cells (human esophageal epithelial cells). SPINK7 KO and control cells were plated at high calcium and high density for 48 h and then treated with either SP163M or derivative SA7 (200 μg / ml) and poly I:C (5 μg / ml, or untreated) for 8 h. TSLP production in the supernatants was measured by ELISA.

[0178] Furthermore, in an allergic asthma mouse model in which mice were sensitized to ovalbumin by two intraperitoneal injections containing the adjuvant alum, a potent inducer of both innate and TH2-mediated immune responses, and then challenged with ovalbumin given by intranasal instillation on four separate days, mice treated with peptide derivative A2-5 showed reduced inflammatory mediators. Lung homogenates of vehicle-treated ova-induced mice (vehicle / OVA) demonstrated significantly increased levels of TSLP compared to non-ova-induced mice (saline / saline) (p=0.001). ova-induced mice treated with peptide derivative A2-5 (A2-5 / OVA) demonstrated significantly lower levels of TSLP compared to ova-induced vehicle-treated mice (p=0.026) (Figure 20A). Allergen exposure also resulted in an increase in total protein in lung tissue compared to non-exposed animals. Infiltration of immune cells into the lungs is likely to explain the increase in total protein levels in the lungs. Figure 20B shows that protein levels in the lungs of mice treated with SP163M (SP163M / OVA), mice treated with the steroid dexamethasone (Dex / OVA), and mice treated with peptide derivative A2-5 were significantly reduced compared to vehicle-treated animals (p<0.005). In mice treated with A2-5, this was associated with a reduction in the TH-2-mediated cytokines IL-5, IL-4, and IL-13 in lung tissue compared to vehicle-treated mice (Figure 20C).

[0179] References The references, patents, and published patent applications listed below, as well as all references cited in the specification above, are hereby incorporated by reference in their entireties as if fully set forth herein. TIFF2025515349000023.tif191143TIFF2025515349000024.tif206142TIFF2025515349000025.tif205143TIFF2025515349000026.tif220143TIFF2025515349000027.tif222143TIFF2025515349000028.tif222143TIFF2025515349000029.tif220143TIFF2025515349000030.tif22142

Claims

1. A SERPIN peptide derivative comprising the core sequences of FVFLM (SEQ ID NO: 1), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQ ID NO: 8), PFVFL[Nle] (SEQ ID NO: 9), and the following modifications: (i) one or more Lys, Glu, or His residues added to the N-terminus of the core sequence, a polar tail added to the C-terminus of the core sequence, or both; (ii) one or more amino acid residues added to the N-terminus of the core sequence, the C-terminus of the core sequence, or both, such that the SERPIN peptide derivative can be cyclized by forming a disulfide bond between two Cys residues; (iii) one or more amino acid residues in the core sequence substituted by one or more Thr amino acid residues; and (iv) one or more amino acid residues in the core sequence substituted by one or more substituted amino acid residues having a higher hydrophobicity than the one or more amino acid residues to be substituted one or more of which and comprising the said SERPIN peptide derivative.

2. The SERPIN peptide derivative according to claim 1, which is a linear peptide.

3. The SERPIN peptide derivative according to claim 1 or 2, having a size of less than 15 amino acid residues.

4. The SERPIN peptide derivative according to claim 1, having a size of 7, 8, 9, 10, 11, or 12 amino acid residues.

5. A SERPIN peptide derivative comprising the amino acid sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 (SEQ ID NO: 3), wherein X1 is a hydrophilic amino acid residue or does not exist; X2 is a hydrophilic amino acid residue or does not exist; X3 is a hydrophilic amino acid residue or does not exist; X4 is a Cys amino acid residue or does not exist; X5 is a Pro amino acid residue or does not exist; X6 is a first hydrophobic amino acid residue; X7 is a short branched amino acid residue; X8 is a second hydrophobic amino acid residue; X9 is a saturated hydrophobic amino acid residue; X10 is a hydrophilic amino acid in the D configuration or Nle; and X11 is any amino acid residue that enables cyclization of the SERPIN peptide derivative; Here, X4 is Cys, X5 is Pro, X7 is Thr, and / or X10 is a D-configuration Lys residue. The SERPIN peptide derivative.

6. The SERPIN peptide derivative according to claim 5, wherein the first and second hydrophobic amino acid residues are aromatic amino acid residues; the short branched amino acid residue is Val or Thr; and / or the saturated hydrophobic amino acid is Leu.

7. The SERPIN peptide derivative according to claim 5 or 6, wherein the hydrophilic amino acid in the D-configuration is a D-configuration Asp, Glu, Lys, Dap, or Cys residue.

8. X1 is a basic residue having a positive charge or does not exist; X2 is a basic residue having a positive charge or does not exist; and X3 is a basic residue having a positive charge or does not exist. The SERPIN peptide derivative according to claim 5.

9. X1 is an Arg, Lys, or His residue or does not exist; X2 is an Arg, Lys, or His residue or does not exist; and X3 is an Arg, Lys, or His residue or does not exist. The SERPIN peptide derivative according to claim 5.

10. The SERPIN peptide derivative according to claim 5, wherein X6 is Phe or Nal; X8 is Phe or Nal; X6 is Ala, Phe, or Nal and X4 is Nal; and / or X7 is Asp, Glu, Lys, Dap, or Cys.

11. The SERPIN peptide derivative according to claim 5, which is linear or cyclized.

12. The SERPIN peptide derivative according to claim 5, which has a size of less than 15 amino acid residues.

13. The SERPIN peptide derivative according to claim 5, which has a size of 7, 8, 9, 10, 11, or 12 amino acid residues.

14. A fusion protein comprising the SERPIN peptide derivative according to claim 1 or 5, and an epitope tag, a half-life extender, or both.

15. The SERPIN peptide derivative according to any one of claims 1 or 5, or A fusion protein comprising the SERPIN peptide derivative and an epitope tag, a half-life extender, or both A pharmaceutical composition comprising the same

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