SERPIN peptide derivatives and methods of use thereof

Abstract

SERPIN-derived peptides, such as SP16 and SP163M, are useful in the treatment of a variety of conditions associated with LRP1 mediation. It is necessary to develop novel SERPIN peptide derivatives to further improve the stability, bioavailability and / or therapeutic effect of existing SERPIN-derived peptides. The present technology includes SERPIN peptide derivatives, methods for their preparation, and their use for the treatment of various conditions associated with LRP1 mediation.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 363,840, filed on April 29, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that complies with the ST.26 standard, which is simultaneously submitted through the Patent Center in .xml format and is incorporated herein by reference in its entirety. The .xml copy was created on April 26, 2023, named Serpin138536-8004WO00sequencelisting.xml, and is 78KB in size. Background Art

[0005] Serine protease inhibitors (SERPINs) are a large family of proteins involved in a variety of biological functions, such as fibrinolysis, blood coagulation, and inflammatory responses. When SERPINs bind to target serine proteases to inactivate enzyme activity, a conformational change occurs, exposing a unique short peptide motif (5-11 amino acids) 8,43 The protease inhibitor complex binds to low-density lipoprotein receptor-related protein (LRP1) at a newly exposed short peptide motif, a process that is conserved across the entire spectrum of serine protease inhibitors (SERPINs), such as alpha-1 antitrypsin (AAT) and antithrombin III (ATIII). 22,25,43 .

[0006] Previously disclosed is a family of SERPIN-derived peptides that were found to bind to LRP1 and to exert healing and homeostatic functions in addition to their anti-inflammatory functions. 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 variety of conditions associated with LRP1 mediation. It is necessary to develop novel SERPIN peptide derivatives to further improve the stability, bioavailability and / or efficacy of existing SERPIN-derived peptides. Summary of the invention

[0007] One aspect of the present technology is a SERPIN peptide derivative comprising, consisting essentially of, or consisting of a pentapeptide having an amino acid sequence of FVFLM (SEQ ID NO: 1), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQID 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, so that the peptide derivative can be cyclized; (iii) one or more amino acid residues in the pentapeptide are replaced by one or more amino acid residues of less hydrophobicity; (iv) one or more amino acid residues in the pentapeptide are replaced by one or more amino acid residues of greater hydrophobicity; and (v) one or more amino acid residues in the pentapeptide are deleted.

[0008] In certain embodiments, the SERPIN peptide derivatives of the present technology are linear peptides. In certain embodiments, the SERPIN peptide derivatives of the present technology are cyclized peptides. In certain embodiments, the SERPIN peptide derivatives are cyclized by the disulfide bond formed between two Cys residues. In certain embodiments, the SERPIN peptide derivatives are cyclized by the connector between two amino acid residues (such as two amino acid residues outside the pentapeptide sequence). In certain embodiments, the polar head or polar tail include two or more charged amino acids, such as positively charged amino acids selected from Arg, Lys and His. In certain embodiments, the SERPIN peptide derivatives are fused with one or more other peptides comprising an epitope tag, a half-life extender or an epitope tag and a half-life extender, to form a fusion protein or a fusion peptide. In certain embodiments, the SERPIN peptide derivatives are conjugated with a penetration enhancer.

[0009] Another aspect of the present technology is a composition comprising a SERPIN peptide derivative, fusion, or conjugate of the present technology and one or more pharmaceutically acceptable carriers. In certain embodiments, the composition is formulated into a dosage form suitable for oral, transdermal, or parenteral administration.

[0010] Another aspect of the present technology is a method for treating various conditions or diseases associated with LRP1 binding, such as respiratory viral or bacterial infections (such as COVID), and inflammatory diseases such as acute respiratory distress, asthma, atopic dermatitis, or eosinophilic esophagitis. Other diseases include central and peripheral nervous system diseases, such as peripheral nerve damage and neurodegenerative diseases. The method requires administering an effective amount of one or more SERPIN peptide derivatives, fusions thereof, or a composition comprising one or more SERPIN peptide derivatives or fusions thereof of the present technology to a subject suffering from a disease associated with LRP1 binding.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] This application contains at least one drawing executed in color. Copies of this application with color drawings will be provided upon request and payment of the necessary fee.

[0013] Figure 1 A peptide sequence (SEQ ID NO: 8) is shown. 1 -Crystal structure of antitrypsin.

[0014] Figure 2 The α-peptide sequence (SEQ ID NO: 8) was shown. 1 -Distances between amino acids in the antitrypsin crystal structure.

[0015] Figure 3 The ring-closing strategy is illustrated using the Lys-βAla-Glu 13C-C bond as an example.

[0016] Figures 4A-4B Shown are the structure-activity relationships of SERPIN-derived peptide derivatives SA1-SA8. Figure 4A The activity of peptide derivatives SA1-SA8 at reducing NFκB activation at different concentrations (0, 1, 10, 50 and 100 μg / mL) is shown. Figure 4B The activities of SA3 and SA7 were compared with those of SP163M and SP22. Compared to the previously published SERPIN peptide SP163M, the truncated peptides while retaining the LRP1 binding site and adding an arginine residue improved activity in the NFκB reporter gene assay.

[0017] Figure 5A-5BThe anti-inflammatory function of peptide derivatives A1-A15 compared to SP163M is shown. The peptide derivatives (50 or 100 μg / ml) were tested in response to LPS (5 ng / ml) in the NFκB reporter gene assay and screened for TNFα secretion in response to LPS (E. coli 0111:B4) stimulation (100 ng / ml, 24 hours) in IMG microglia. NFκB-induced secreted embryonic alkaline phosphatase (SEAP) was measured in the supernatant in NFκB reporter cells. TNFα secretion in the supernatant was detected by ELISA.

[0018] Figure 6A-6B The anti-inflammatory activities of peptide derivatives A2-1 to A2-9 comprising modifications to the cyclic structure and amino acid substitutions were shown. Fig. 6A Shown is the reduction of TNFα in the supernatant of IMG microglia after activation with LPS (100 ng / ml, 24 hours). Figure 6B The percentage reduction of NFKB activation after LPS stimulation (5 ng / ml, 24 hours) is shown. SP163M, SA7, A5 and A15 peptides were used for comparison.

[0019] Figure 7A-7B The dose response effect of peptide derivatives on NFκB and TNFα activation is shown. The peptide derivatives were tested at concentrations of 3.125, 6.25, 12.5, 25, and 50 μg / ml. SP163M, SP16, and SA7 were tested at concentrations up to 100 μg / ml. The dose response concentrations are shown in a logarithmic scale. The activity of peptide derivatives A2-1, A2-2, A2-3, A2-4, and A2-5 was superior to that of SA7, SP16, and SP163M, and was dose-dependent.

[0020] Figure 8 The results of the cytotoxicity test on IMG cells (microglia) are shown. IMG cells were treated for 24 hours with peptide derivatives at concentrations reaching or exceeding the test concentrations in the activity test (Figures 5-7). All peptides did not show any cytotoxic effect.

[0021] Fig. 9 The effect of peptide derivatives A3-1 to A3-16 (including further optimization of the ring structure) on NFκB activation under LPS stimulation (5ng / ml, 24 hours) is shown. The test concentrations of the peptide derivatives were 1.56μg / ml to 12.5μg / ml. SP163M and A2-5, for comparison. The test concentrations of SP163M were 12.5μg / ml to 100μg / ml. The dose response is shown in a logarithmic scale.

[0022] Fig.10Peptide derivatives A3-1, A3-2, A3-3, A3-5, A3-6, A3-7, A3-9, A3-14, A3-15, and A3-16 are shown to reduce the activity of IL-6 secretion in IMG after LPS stimulation (100ng / ml, 24 hours). The secretion of IL-6 was determined by ELISA. Peptide derivatives A2-5 and SP163M were used for comparison. The test concentration of SP163M was 12.5 to 100μg / ml. The test concentration of peptide derivatives including A2-5 was up to 12.5μg / ml. The dose response is shown on a logarithmic scale.

[0023] Figures 11A-11B NFκB reporter cells ( Fig.11A ) and IMG microglia ( Fig. 11B ) cytotoxicity test results. Peptide derivatives A3-1, A3-2, A3-5, A3-6, A3-7, A3-9, A3-14, A3-15, and A3-16 showed no cytotoxic effect at test concentrations (up to 12.5 μg / ml) that were all effective in reducing NFκB, cytokines IL-6 and TNFα. SP163M was used for comparison and had no cytotoxic effect at concentrations up to 100 μg / ml. Cells were treated with SP163M or the indicated concentrations of 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.

[0024] Fig.12 The ability of peptide derivatives A15 and A2-5 to block capsaicin-induced pain behavior in mice was tested. Mice were subcutaneously administered peptide derivatives 1 hour before injection of capsaicin (25 ng). SP163M was administered at a dose of 50 μg / mouse, while peptide derivatives A15 and A2-5 were administered at a dose of 5 μg / mouse. n = 4-9 mice / group. One-way ANOVA. *p<0.05, **p<0.01.

[0025] Fig.13 The cytokine profiles measured in microglia treated with the SERPIN peptide derivative SA7 or the SERPIN peptide SP163M were compared. SA7 showed significantly enhanced activity in reducing multiple cytokines including IL-6 and IL-1β.

[0026] Figures 14A-14B The results showed that the SERPIN peptide derivative SA7 had a significant effect on the neuroinflammation model induced by LPS compared with vehicle (LPS-induced) and SP163M ( Fig. 14B ) compared with the control group ( Fig.14A), and reduced the severity of weight loss. SP163M and SA7 were administered at a dose of 100 μg / mouse by subcutaneous injection, 1 hour after LPS (1 mg / kg) administration, for four consecutive days. Body weight and clinical scores were measured daily, and brain tissue was collected and processed for analysis at the end of the study. The scoring criteria are as follows: 0: bright, alert, responsive; 1-4: slightly slovenly / hunched, not very active; 5-10: hunched, lethargic, weight loss; ≥11 moribund.

[0027] Fig.15 It was shown that in the neuroinflammation model of Figure 14, animals treated with SA7 had significant differences in the levels of many cytokines measured in brain lysates, including IL-17A, IL-12, TNFα, and GM-CSF, compared to animals treated with vehicle and SP163M. IL-6 had the largest decrease, with no detectable levels measured.

[0028] Figures 16A-16B Western blot analysis of neurofilament light chain (NfL), a neuronal axon-specific marker, and anti-glial fibrillary acidic protein (GFAP), a marker specific for astrocyte activation, was shown in brain lysates extracted from a previously described neuroinflammation model. The peptide derivative SA7 significantly increased the expression of NfL compared to vehicle-treated and SP163M animals ( Fig.16A ), while the expression of GFAP was significantly decreased ( Fig. 16B ).

[0029] Figures 17A-17B The researchers showed that SA7, like the SERPIN peptide SP163M, regulates autophagy during inflammation. Microglia were treated with SP163M or SA7 and then exposed to LPS. Western blot analysis of the LC3 II to LC3 1 ratio (the starting point of autophagosome formation, a widely used biomarker for detecting autophagy) showed that SA7 increased autophagy compared to LPS alone. Fig.17A ) and LRP1 expression ( Fig. 17B ), which was comparable to SP163M. Compared with SP163M, SA7 also significantly increased the expression of LRP1 without LPS treatment.

[0030] Fig.18 Protease TMPRSS2 activity and IC50 of SP163M and peptide derivative SA7 are shown, and SA7 showed enhanced inhibition of TMPRSS2 activity in an overexpression cell system compared to SP163M.

[0031] Figures 19A-19BThe results showed that LRP1 expression was reduced in the esophagus of OVA-induced mice, while SP163M restored LRP1 protein expression. In addition, SA7 inhibited the production of the cytokine TSLP in SPINK7 knockout cells more effectively than SP163M compared with control cells.

[0032] Figures 20A-20C It was shown that in the OVA-induced allergic asthma model, the peptide derivative A2-5 reduced TSLP, total lung protein and 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.

[0033] Fig.21 Shown are conjugates comprising a peptide derivative of the present technology (eg, A2-5, SEQ ID NO: 42) and a penetration enhancer. DETAILED DESCRIPTION

[0034] The present technology includes SERPIN peptide analogs and 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 having an amino acid sequence or structural similarity to 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 containing one or more modifications (including insertions, deletions, or substitutions) to improve its stability, bioavailability, and / or biological activity or efficacy. In the present disclosure, the terms "derivative", "variant", and "analog" can be used interchangeably.

[0035] Promoting resolution of inflammation and mitigating tissue damage requires a precise orchestration of the immune response, and targeting a single cytokine or signaling pathway does not address all pathogenic factors in the pathology of certain diseases, such as those discussed in this article. A balanced inflammatory response plays a critical role in regeneration and repair, and anti-inflammatory drugs are associated with opposing effects on regeneration and tissue repair. 3 .

[0036] SERPIN peptides have previously been shown to: (1) have neurotrophic effects; (2) have regenerative and healing properties; (3) have analgesic effects; (4) have antiviral and antimicrobial properties; and / or (5) have anti-allergic effects. This combination of activities provides a unique mechanism for treating conditions associated with peripheral neuropathy, such as diabetic peripheral neuropathy, degenerative diseases, lung injury, allergic diseases, and infectious diseases. The SERPIN peptide analogs and variants and derivatives of the present technology have improved LRP1 binding activity, improved solubility, and / or improved pharmacokinetic properties and oral bioavailability. For example, as shown in the working examples, the SERPIN peptide derivatives of the present technology show improved anti-inflammatory effects and improved therapeutic effects in a neuroinflammation model.

[0037] Therefore, the present technology includes SERPIN peptide derivatives, pharmaceutical compositions comprising SERPIN peptide derivatives, and methods of using the same to treat a variety of conditions with dysregulated immune responses or impaired endocytic function or diseases whose pathologies are associated with LRP1 mediation, such as conditions associated with peripheral nerve damage and the pain caused by it, lung damage, infectious diseases, and allergic inflammation.

[0038] 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 replacements. In certain embodiments, the SERPIN peptide derivative comprises one or more hydrophobic amino acid replacements. 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.

[0039] SERPIN peptide derivatives and modifications

[0040] The present technology includes SERPIN peptide derivatives designed to target LRP1 with higher affinity, thereby exerting stronger anti-inflammatory and cell regenerative effects. These peptide derivatives are modified from original SERPIN-derived peptides based on structure-activity relationship studies and 3D 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 with previously disclosed SERPIN peptides such as SP16 (VKFNKPFVFLMIEQNTK) (SEQ ID NO: 4) and SP163M (Ac-VKFNKPFVFL[Nle]IEQNTK-NH 2)(SEQ IDNO: 5) (wherein Nle represents norleucine), the peptide derivatives of the present technology not only show improved LRP1 activity, but also show improved solubility, pharmacokinetic properties and bioavailability, especially oral and transdermal bioavailability.

[0041] Previous studies have demonstrated that small peptide fragments at the C-terminus of alpha-1 antitrypsin (the prototype of SERPINs) can bind to LRP1 and exert powerful cell regeneration, tissue protection and immunomodulatory functions. However, the tertiary structure of alpha-1 antitrypsin (AAT) hinders its direct binding to LRP1. Instead, AAT can only bind to LRP1 when interacting with the target protease, because AAT undergoes a conformational change that exposes a short 5-11 amino acid binding motif. Surprisingly, the entire highly conserved core sequence VKFNKPFVFLM (SEQ ID NO: 6) is not necessary for the anti-inflammatory effects of the peptide derivatives, as demonstrated by structure-activity relationship studies performed on these derivatives. These derivatives do not contain the FNKP (SEQ ID NO: 7) motif, which is highly conserved in SERPINS, while retaining LRP1 binding activity. However, the LRP1 binding motif is highly hydrophobic and unstable in solution, so the SERPIN peptide sequence needs to be modified.

[0042] Therefore, various modifications have been made to the SP16 / SP163M peptides, particularly to the peptides in or around the pentapeptide, to produce SERPIN peptide derivatives with improved performance. 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, in which the Met residue is replaced by the Nle residue. The pentapeptide is responsible for most of the interactions with LRP1. In the present technology, various modifications are made to the pentapeptide and / or the sequence around the pentapeptide to obtain novel SERPIN peptide derivatives with improved performance. 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 have been developed to obtain better oral bioavailability and blood-brain barrier permeability without affecting the anti-inflammatory activity or LRP1 binding activity. In certain embodiments, the size of the peptide derivatives of the present technology is 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, preferably 8, 9, or 10 amino acid residues. For example, the size of the peptide derivatives of the present technology is 8 amino acid residues, 9 amino acid residues or 10 amino acid residues.

[0043] In certain embodiments, a polar head comprising two or more amino acid residues with charged side chains is added to the N-terminus of the pentapeptide, and a polar tail comprising two or more amino acid residues with charged side chains is added to the C-terminus, or the polar head and the polar tail are added simultaneously to improve solubility. In some embodiments, the amino acid residues in the polar head or polar tail are positively charged and include Arg, His and Lys. The polar head or polar 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 polar 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 polar tail have a reverse structure. For example, reverse Lys refers to that the Lys residue is bound to the peptide backbone through the carboxylic acid group and the ε-amino group carried by the α-carbon in the side chain instead of the amino group and the carboxylic acid group carried by the α-carbon. Reverse Arg refers to the incorporation of an Arg residue into the peptide backbone via a guanidine group carried by the α-carbon rather than the δ-carbon. In some embodiments, two or three Arg residues are added to either or both ends of the pentapeptide. In some embodiments, two or three Arg residues are added to the N-terminus of the pentapeptide.

[0044] 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 connector between two amino acid residues. According to the prior art, two Cys residues can be added to the two ends of the pentapeptide, so that a cyclic peptide derivative can be obtained through a disulfide bond. Placing the Cys residues in a selected position in the peptide derivative to obtain the desired cyclic structure with an optimized ring size is within the purview of those of ordinary skill in the art. Alternatively, other natural, non-natural or, modified amino acid residues can also be added to either or both ends of the pentapeptide to form a connector between these amino acid residues. Specific amino acid residues can be selected and placed in a selected position to achieve a desired cyclic structure with an optimized ring size. According to cyclization strategies such as amides, disulfides, ring-closing metathesis (RCM), or olefin metathesis, amino acid residue replacement can be selected for cyclization without significant loss of activity. For example, amino acid residues with carboxylic acids on the side chain or C-terminus (including but not limited to Asp and Glu) or amino acid residues with amino groups on the side chain or N-terminus (including but not limited to Lys, Dab, and Dap) can be used for amide cyclization, while Cys or any non-natural amino acid with a sulfhydryl group on the side chain can be used for -SS-cyclization. Amino acids can be placed at any desired position of the peptide derivative so that a ring of the desired size can be formed without substantially comprising the activity of the peptide derivative. In some embodiments, head-to-tail cyclization can be formed. In some embodiments, the linker includes β-Ala. In some embodiments, the linker includes 2-[(2-amino)-ethoxy]-ethoxy-acetic acid (AEEA). In certain embodiments, the closed loop length between the amino acid residues is 5 to 15 CC bonds, for example, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 CC bonds.

[0045] In certain embodiments, the peptide derivatives of the present technology include one or more substitutions in the pentapeptide sequence to improve plasma stability and achieve improved binding affinity to the peptide cognate receptor. For example, one or more hydrophilic amino acid residues can be used to replace one or more amino acid residues in the pentapeptide that have no interaction or minimal interaction with LRP1. In addition, one or more amino acid residues in the pentapeptide that interacts with LRP1 can be replaced by one or more amino acid residues with similar but more significant physicochemical properties. For example, there is an aromatic ring on the side chain of the Phe residue. In peptide derivatives 1-5 and 1-6, the Phe residue can be replaced by Nal (naphthalene alanine) or Trp, which is displayed as a naphthalene ring or an indole ring instead of a benzene ring. These substitutions greatly improve the aromatic properties of the amino acid residues, making them more hydrophobic and more aromatic (π stacking) interactions. In some embodiments, one or more amino acid residues in the pentapeptide can be replaced by one or more natural or non-natural 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 substitution of Val to Thr to retain some hydrophobicity while introducing some hydrogen bonds, and a substitution of Phe to Nal to increase hydrophobic and aromatic interactions. In another embodiment, peptide derivative A3-8 includes a substitution of Nle to D-Ser. Based on crystal structure studies, Nle does not interact with LRP-1, but is in the aqueous phase. The side chain of Nle is a hydrophobic linear hydrocarbon chain that requires energy to dissolve. Replacing the Nle residue with D-Ser having a hydrophilic side chain can promote dissolution by reducing the enthalpic penalty and converting it into a stronger binding energy. Therefore, in some embodiments, the Nle residue of the pentapeptide is missing. In some embodiments, one or more 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 hydrophobic residues in the pentapeptide are replaced by one or more more hydrophobic residues such as Nal. In some embodiments, the Nle residue in the pentapeptide is replaced with an amino acid residue having a D-configuration, such as D-Dap, D-Lys, and D-Asp.

[0046] The Met or Nle residues in the pentapeptide have long linear hydrophobic side chains. Replacing Met or Nle with a hydrophilic amino acid in the D configuration greatly improved the binding activity of the peptide derivatives to LRP1. Substitution with amino acid residues containing carboxylic acids in the side chain can improve the binding activity to a certain extent, while substitution with amino acids with short side chains containing hydroxyl (Ser) or amino (Dap) can achieve the best effect.

[0047] In certain embodiments, the SERPIN peptide derivatives of the present technology can be further modified to extend shelf life and / or bioavailability by using one or more non-natural peptide bonds or amino acids, or by attachment to a peptide functional group, such as polyethylene glycol (PEG).

[0048] In certain embodiments, the peptide derivatives of the present technology include, consist essentially of, or consist of a pentapeptide having the amino acid sequence 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 polar tail contains 2-9 charged amino acid residues, such as Arg, Lys, and His. In some embodiments, the polar head or polar tail contains 2 or 3 charged amino acid residues. Some examples of peptide derivatives shown in Table 1 were used to study the role of positively charged amino acids in improving solubility and regulating NFκB activation, using SP163M and SP22 as positive controls. These peptide derivatives have different tripeptide sequences added to either or both ends of the LRP1 binding site.

[0049] In some embodiments, the peptide derivative comprises, consists essentially of, or consists of a peptide having the following amino acid sequence: HHHPFVFLMHHH (SEQ ID NO: 10), HHHPFVFL[Nle]HHH (SEQ ID NO: 11), RRRPFVFL[Nle]RRR (SEQ ID NO: 12), KKKPFVFL[Nle]KKK (SEQ ID NO: 13), EEEVKFNKPFVFL[Nle]EEE (SEQ ID NO: 14), RRRCPFVFL[Nle]CRRR (SEQID NO: 15), RRRCPFVFL[Nle]C (SEQ ID NO: 16), CPFVFL[Nle]CRRR (SEQ ID NO: 17), RRRVKFNKPFVFLMRRR (SEQ ID NO: 18), or VKFNKPFVFL[Nle]IEQNTK (SEQ ID NO: 5).

[0050]

[0051] *The underlined sequence indicates cyclization. Disulfide bonds are formed between C residues to cyclize the peptide.

[0052] 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, the activity of reducing NFκB increases. However, adding one or more other charged amino acids, such as positively charged amino acids (e.g., histidine (H) or lysine (K)) or negatively charged amino acids (e.g., glutamic acid (E)) on both sides of the LRP1 binding site does not confer activity, indicating the importance and uniqueness of arginine residues.

[0053] In certain embodiments, the peptide derivative of the present technology is a cyclic peptide comprising, consisting essentially of, or consisting 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. Cyclization can be between any two residues, for example, cyclization can be head-to-tail cyclization. Additional residues, such as Cys, can be inserted to promote the formation of SS bonds to connect two residues. In some embodiments, one or more of the hydrophobic residues F, M or Nle in the pentapeptide are replaced by neutral or hydrophilic residues. Therefore, peptide derivatives with various positively charged amino acid residue additions and / or amino acid substitution combinations are designed (some examples are shown in Table 2) to improve their solubility, stability and / or oral bioavailability without affecting their NFκB regulatory activity.

[0054] 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, the P amino acid residue is located at the C-terminus of the pentapeptide, so the peptide derivative includes the amino acid sequence PFVFLM (SEQ ID NO: 8) or PFVFL [Nle] (SEQ ID NO: 9). In some aspects, the peptide derivative comprising the amino acid sequence PFVFLM (SEQ ID NO: 8) or PFVFL [Nle] (SEQ ID NO: 9) can be a cyclized peptide. In some aspects, the peptide derivative comprising the amino acid sequence PFVFLM (SEQ ID NO: 8) or PFVFL [Nle] (SEQ ID NO: 9) can be cyclized by further comprising a Cys residue to promote the formation of an SS bond, such as a peptide derivative having the amino acid sequence CPFVFLMC (SEQ ID NO: 19) or CPFVFL [Nle] C (SEQ ID NO: 20). In some aspects, the peptide derivative having the sequence CPFVFLMC (SEQ ID NO: 19) or CPFVFL[Nle]C (SEQ ID NO: 20) may further comprise a polar head, such as three R amino acid residues. In some aspects, the peptide derivative may comprise the sequence RRRCPFVFLMC (SEQ ID NO: 21) or RRRCPFVFL[Nle]C (SEQ ID NO: 22). In some aspects, the peptide derivative having the sequence RRRCPFVFLMC (SEQ ID NO: 21) or RRRCPFVFL[Nle]C (SEQ ID NO: 22) may further be acetylated. In some aspects, the peptide derivative may be peptide SA7.

[0055] In certain embodiments, the peptide derivatives of the present technology comprise, consist essentially of, or consist of the following amino acid sequence: RCFVFL[Nle]C (SEQ ID NO: 23), RRCFVFL[Nle]C (SEQID NO: 24), RRCFVFL[Nle]C (SEQ ID NO: 25), RRRCFVFL[Nle]C (SEQ ID NO: 26), RRRCFVFT[Nle]C (SEQ ID NO: 27), RRRCFTFL[Nle]C (SEQ ID NO: 28), or RRRCTVFL[Nle]C (SEQ ID NO: 29).

[0056]

[0057]

[0058] *The underlined sequence indicates cyclization. Disulfide bonds are formed between C residues to cyclize the peptide.

[0059] like Figure 1 As shown, the F, F, and M residues of the pentapeptide FVFLM (SEQ ID NO: 1) point in one direction, while the V and L residues point in the opposite direction. The P residue at the N-terminus of the pentapeptide forms a turn in the structure but does not interact with LRP1. It is unlikely that all five residues of the pentapeptide interact directly with LRP1. In addition, peptide derivatives A7-A9 were designed to replace one of the hydrophobic amino acid residues (F, V, and L) with a more hydrophilic residue (such as T) to investigate whether solubility and / or activity could be improved.

[0060] Table 3 lists some examples of peptide derivatives having one F residue replaced by 1-naphthylalanine, a non-natural amino acid with an additional aromatic ring. The replacement of naphthylalanine (Nal) increases the hydrophobicity and may result in stronger hydrophobic interactions with LRP1.

[0061] In certain embodiments, the peptide derivatives of the present technology comprise, consist essentially of, or consist of the following amino acid sequence: RRRCFV[Nal]L[Nle]C (SEQ ID NO:30) or RRRC[Nal]VFL[Nle]C (SEQ ID NO:31).

[0062]

[0063]

[0064] *The underlined sequence indicates cyclization. Disulfide bonds are formed between C residues to cyclize the peptide.

[0065] Additional peptide derivatives were designed to determine the optimal ring size. For example, the Cys-Cys bridge could be replaced with a longer linker to improve cyclization. Based on the crystal structure of the LRP1 binding site, the distance between the alpha carbon of the N-terminal amino acid and the alpha carbon of the C-terminal amino acid of the pentapeptide FVFLM (SEQ ID NO: 1) was measured. The length of the CC bond was assumed to be Converting the distance between amino acids into the number of CC bonds can be used to design cyclization strategies. For example, the distance between the α carbons of K368 and I375 is (about 15 CC-bonds), the distance between the α-carbons of P369 and I375 is (about 13.5 CC-bonds). Figure 2In order to cyclize the derivative and mimic the distance between P369 and I375, a β-Ala linker with 13 to 14 CC bonds can be used to connect the side chain of the Lys residue to the side chain of the Glu residue, so that the peptide derivative is cyclized and the closed length of the ring is 13 CC bonds. Figure 3 .

[0066] In certain embodiments, the peptide derivatives of the present technology comprise, consist essentially of, or consist of the following amino acid sequence: RRRDFVFL[Nle][Dap] (SEQ ID NO: 32) 、RRRDFVFL[Nle][Dap] (SEQ ID NO:33) 、RRRDFVFL[Nle][Dap] (SEQ ID NO:34) 、RRREFVFL[Nle]K (SEQ ID NO:35) 、RRREFVFL[Nle]K (SEQ ID NO:36) ,RRRFVFL[Nle]D (SEQ ID NO:37).

[0067] Table 4 illustrates peptide derivatives with various ring closure designs, where ring-closing linkers were used to replace the Cys-Cys bridge.

[0068]

[0069]

[0070] *The underlined sequence indicates circularization.

[0071] Example 2 shows that peptide derivatives A5, A8, A10 and A15 exhibit improved activity in reducing TNFα activation. Table 5 lists some examples of other SERPIN peptide derivatives with similar modifications, as well as peptide derivatives with substitutions in the pentapeptide. For example, Nle in the pentapeptide was replaced or deleted by Ala to determine whether Nle is involved in the interaction with LRP1. In addition, each residue of the pentapeptide was replaced by D-Ser (dS) to improve solubility and resistance to proteases.

[0072] In certain embodiments, the peptide derivatives of the present technology comprise, consist essentially of, or consist of the following amino acid sequence: RRR[Dap]FT[Nal]L[Nle]D (SEQ ID NO:38), RRRFT[Nal]L[Nle]D (SEQ ID NO: 39) RRRFVFLAD (SEQ ID NO: 40) RRRFVFLD (SEQ ID NO: 41) ,RRRFVFL[dS]D (SEQ ID NO: 42 )、RRRFVF[dS][Nle]D (SEQ ID NO: 43) 、RRRFV[dS]L[Nle]D (SEQ ID NO: 44) 、RRRF[dS]FL[Nle]D (SEQ ID NO: 45) or RRR[dS]VFL[Nle]D (SEQ ID NO: 46)。

[0073]

[0074]

[0075] *The underlined sequence indicates circularization.

[0076] As shown in Figure 7, peptide derivatives A2-1, A2-2, A2-3, A2-4, and A2-5 were more active than SP163M and SA7 in reducing TNFα and NFκB activation. None of the tested peptide derivatives showed significant toxicity to the tested cell lines ( Figure 8 ).

[0077] In certain embodiments, the peptide derivatives of the present technology comprise, consist essentially of, or consist of the following amino acid sequence: RRRFT[Nal]LD (SEQ ID NO:47) , [rK]RRFT[Nal]LD (SEQ ID NO:48) , [rR]RRFT[Nal]LD (SEQ ID NO:49) 、[rR][rR]FT[Nal]LD (SEQ ID NO:50) 、RRR[Dap]FT[Nal]LD (SEQ ID NO:51) ,RR[Dap]FT[Nal]LD (SEQ ID NO:52 )RRRKFT[Nal]LD (SEQ ID NO:53 ),RR[Dap]FT[Nal]L[dS]D (SEQ ID NO:54 )、RRRFVFL[dDap]D (SEQ ID NO: 56 )、RRRFVFL[dK]D (SEQ ID NO:57) ,RRRFVFL[dD]D (SEQ ID NO:58) ,RRRFTF[Nal]A[Nle]D (SEQ ID NO:59) 、RRRAT[Nal]L[Nle]D (SEQ ID NO: 60) 、RRRFT[Nal]L[dS]D (SEQ ID NO: 61 )、RRRFTWL[dS]D (SEQ ID NO:62) .

[0078] Table 6 provides examples of further optimizing SERPIN peptide derivatives by shortening peptide length, optimizing cyclization and / or residue substitution to further improve solubility, activity and / or oral availability. In certain embodiments, one or more amino acid residues in the peptide derivative pentapeptide are deleted or replaced by one or more natural or non-natural amino acid residues. For example, the Nle residue can be replaced by less hydrophobic amino acids such as Ala or Ser (D-Ser) to improve the agonist activity bound to LRP1. In certain embodiments, D-configuration amino acids can be used to change the orientation of amino acids in the three-dimensional structure of the peptide and / or 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. As shown in the following example, the charged amino acids can have a "reverse" chemical structure to optimize the size of the ring:

[0079]

[0080]

[0081]

[0082]

[0083] *The underlined sequence indicates circularization.

[0084] The peptide derivatives of the present technology can be further modified at the amide bond to improve the stability and absorption of proteases. These modifications include but are not limited to peptide bond isostere, N-methylation and / or D-configuration amino acid substitution.

[0085] In certain embodiments, a peptide derivative is disclosed that comprises, consists essentially of, or consists of the following amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 (SEQ ID NO: 3), wherein:

[0086] X1 is a hydrophilic amino acid residue or none;

[0087] X2 is a hydrophilic amino acid residue or none;

[0088] X3 is a hydrophilic amino acid residue or none;

[0089] X4 is a Cys amino acid residue or none;

[0090] X5 is a Pro amino acid residue or none;

[0091] X6 is the first hydrophobic amino acid residue;

[0092] X7 is a short branched-chain amino acid residue;

[0093] X8 is the second hydrophobic amino acid residue;

[0094] X9 is a saturated hydrophobic amino acid residue;

[0095] X10 is a hydrophilic amino acid in the D configuration; and

[0096] X11 is any amino acid residue which allows cyclization of the peptide derivative.

[0097] 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.

[0098] 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 chain 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 D configuration hydrophilic amino acid of X10 is Asp, Glu, Lys, Dap or Cys residue of D configuration.

[0099] In some embodiments, X1 is a basic residue showing a positive charge or none; X2 is a basic residue showing a positive charge or none; X3 is a basic residue showing a positive charge or none. In some embodiments, X1 is an Arg, Lys, or His residue, or none; X2 is an Arg, Lys, or His residue, or none; X3 is an Arg, Lys, or His residue, or none.

[0100] In some embodiments, X6 is Phe or Nal. In some embodiments, X8 is Phe or Nal. In some embodiments, X6 is Ala, Phe or Nal, and X4 is Nal. In some embodiments, X7 is Asp, Glu, Lys, Dap, or Cys.

[0101] In some embodiments, the peptide derivative is linear or cyclized. In some embodiments, the size of the peptide derivative is less than 15 amino acid residues. In some embodiments, the size of the peptide derivative is 7, 8, 9, 10, 11 or 12 amino acid residues.

[0102] In certain embodiments, the peptide derivatives of the present technology are acetylated at the N-terminus, amidated at the C-terminus, or acetylated at the N-terminus and amidated at the C-terminus.

[0103] In certain embodiments, the peptide derivative is cyclized with 10 C-C bonds or equivalents to close the ring between the alpha carbon of X4 and X11 of SEQ ID NO:3. Cyclization can also occur between X11 and the additional amino acid residues added between X4 and X5 of SEQ ID NO:3. Cyclization can also occur between X11 and the additional amino acid residues added between X5 and X6 of SEQ ID NO:3. In this case, the optimal ring is closed using 5 C-C bonds (or equivalents). In certain embodiments, the cyclization is a head-to-tail cyclization.

[0104] In certain embodiments, the SERPIN peptide derivatives of the present technology have 5 to 30 amino acids, such as 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 size. In some embodiments, the size of the SERPIN peptide derivatives of the present technology is 20 amino acids or less. Longer peptides may have reduced solubility, while shorter peptides may have reduced stability. In certain embodiments, the size of the peptide derivative is 9 amino acid residues; however, the peptide derivatives with a size of 8 amino acid residues combined with an optimized cyclization strategy have minimal activity loss.

[0105] In certain embodiments, the SERPIN peptide derivatives of the present technology are fused with one or more other peptides or compounds to form a fusion peptide or fusion protein. For example, one or more other peptides include epitope tags, such as ALFA-tags, V5-tags, Myc-tags, HA-tags, Spot-tags, T7-tags, NE-tags, half-life extenders, such as poly (ethylene glycol) (PEG), lipidation, FC fusion or albumin fusion, or both epitope tags and half-life extenders.

[0106] In certain embodiments, such as Fig.21 As shown, the peptide derivatives of the present technology can be conjugated with permeation enhancers (such as oleic acid, bile acid, cationic detergents or lactams) to further improve transdermal, transepithelial, nasal, gastric or topical bioavailability.

[0107] Pharmaceutical compositions comprising SERPIN peptide derivatives

[0108] On the other hand, the present disclosure relates to a pharmaceutical composition, including one or more SERPIN peptide derivatives or fusion peptides of the present technology treatment effective amount, or consisting essentially of, or consisting of. 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, which are not peptide derivatives of the present technology. In some embodiments, the pharmaceutical composition further comprises one or more penetration enhancers in an amount of 0.1% to 5% (w / w amount) to promote the penetration of peptide derivatives or fusion peptides into the skin, mucosa, nasal mucosa, or epithelial layer. Non-limiting examples of penetration enhancers include fatty acids (such as oleic acid), bile acids (such as cholic acid), cationic detergents (such as hexadecyltrimethylammonium) and lactams (such as laurocapron). In some embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable carrier, excipient, additive, preservative, or a combination thereof. Examples of acceptable carriers include physiologically acceptable solutions, such as sterile saline and sterile buffered saline.

[0109] As used herein, the term "effective amount" refers to the amount of a composition that produces the desired effect. An effective amount of a composition can be used to produce a preventive or therapeutic effect on a subject, such as preventing or treating a target condition, alleviating symptoms associated with the condition, or producing a desired physiological effect. In this case, the effective amount of the composition is a "therapeutically effective amount," "therapeutically effective concentration," or "therapeutically effective dose." The precise effective amount or therapeutically effective amount refers to the amount of the composition that produces the most effective therapeutic effect on a given object or cell population. The amount varies depending on a variety of factors, including but not limited to the properties of the composition (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the cell or subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose, and drug type), the properties of one or more pharmaceutically acceptable carriers in the preparation, and the route of administration. In addition, the effective amount or therapeutically effective amount may vary depending on whether the composition is administered alone or in combination with another composition, drug, therapy, or other treatment method or approach. A person skilled in clinical and pharmacological techniques can determine the effective amount or therapeutically effective amount by routine experimentation, i.e., by monitoring the response of the cell or subject to the administration of the composition and adjusting the dose accordingly. For further guidance, see Remington: The Science and Practice of Pharmacy, 21st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0110] In some embodiments, the peptide derivatives or pharmaceutical compositions of the present technology can be formulated for oral administration, parenteral administration (such as intravenous administration), intramuscular administration, subcutaneous administration (injection or by devices such as infusion pumps), intradermal administration, transdermal administration, topical administration, and intranasal administration. In some embodiments, a subcutaneous injection pump can be used to deliver the peptides or pharmaceutical compositions of the present technology. The peptides or pharmaceutical compositions can be administered multiple times. More specifically, after the first administration, one or more doses can be given as a synergist.

[0111] The SERPIN peptide derivatives, fusion peptides or pharmaceutical compositions of the present technology have various functions. In certain embodiments, the present technology is a method for treating a subject in need 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 that causes pathology mediated by LRP1, such as a condition associated with peripheral nerve damage and pain caused thereby, lung injury, infectious diseases and allergic inflammation (such as eosinophilic esophagitis). In some embodiments, the subject suffers from a disease associated with an immune response disorder, the disease being selected from peripheral neuropathy, neuropathic pain, COVID-19 infection, acute respiratory distress syndrome (ARDS), sepsis, SARS-CoV-2, influenza, alpha virus infection, and cytokine storm.

[0112] As used herein, "treating" a condition may refer to preventing the condition, slowing the onset or development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, alleviating or ending symptoms associated with the condition, causing complete or partial regression of the condition, or a combination of the above. Treatment may also refer to prophylactic treatment of a condition.

[0113] The term "subject" as used herein is 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 been treated with any serine protease inhibitors, such as alpha-1-antitrypsin, prior to treatment with the peptides of the present technology.

[0114] In some embodiments, the peptides can be used to reduce the level of TNF-α in the serum of human individuals with elevated pathological TNF-α levels. When an effective amount of the peptide is administered to a human subject, the serum TNF-α level can be reduced by 75%. In certain embodiments, when an effective amount of the peptide is administered to a human subject, the peptide causes a 50% or 75% decrease in serum TNF-α levels compared to the level before administration of the peptide.

[0115] Diseases associated with LRP-1

[0116] LRP1 is an endocytic and cell signaling receptor with several ligands that induce specific cell signaling cascades that contribute to cell survival and anti-inflammatory mechanisms 5,18,22,25. LRP1 is ubiquitously expressed in many different organs and is present in large quantities in the brain, lungs, heart, and immune cells. Due to these unique functions and its widespread expression in tissues and immune cells, it plays a crucial role in regulating inflammation, cellular metabolism, and maintaining homeostasis. For example, LRP1 can regulate inflammatory signaling pathways such as the NFκB and JNK pathways, thereby inducing the transformation of pro-inflammatory macrophages (M1) to anti-inflammatory macrophages (M2) phenotypes, regulating cytokine output, and contributing to efficient migration and phagocytosis. 22,26,51 In neutrophils, LRP1-dependent mechanisms enhance the cell adhesion, chemotaxis, and antimicrobial properties of these cells, thereby protecting against immunosuppression 25 During acute infection or injury, LRP1 also promotes resolution of inflammation by clearing PAMPs and DAMPs from dying or damaged tissues, thereby preventing cycles of tissue damage. 25 LRP1 also mediates autophagy during infection, an important metabolic process that has recently been shown to play an important protective role in a variety of diseases. 4,10 Therefore, due to its multifunctional ability to modulate inflammation, targeting LRP1 has great potential to mitigate multiple aspects of the immune response that contribute to the pathology of diseases as diverse as neurological disorders, infectious diseases, and allergic inflammation.

[0117] Neurological disorders

[0118] In terms of nerve damage and associated pain, peripheral nervous system injury leads to increased expression of LRP1. Previous studies have shown that LRP1 agonists can promote axonal growth in the CNS and induce regeneration after spinal cord injury. 53 LRP1 is an endocytic receptor for multiple ligands, including tissue plasminogen activator (tPA), matrix metalloproteinase-9 (MMP-9), and activated α2-macroglobulin. 14 These ligands are able to induce anti-inflammatory activity 39 , activates the Schwann cell repair program 21 , and transcriptionally activates cell signaling pathways in neurons associated with axon regeneration 38 . LRP1 requires ligand binding to activate cell signaling, but different ligands elicit distinct and sometimes opposing cell signaling responses, reflecting the ability of different ligands to assemble unique co-receptor complexes. In addition, many LRP1 ligands are multidomain proteins with numerous effects on cell physiology that do not involve LRP1 binding. For example, tissue plasminogen activator (tPA) binding to LRP1 promotes the survival and migration of Schwann cells (SCs). 23 However, tPA induces pain through LRP1-independent activities 1EI-tPA promotes the survival of human iPSC-derived neural progenitor cells (iNPCs), and transplantation of EI-tPA-activated iNPCs into rodents with severe spinal cord injury improves recovery of motor function 40 Imbalanced microenvironment following nerve injury may have serious consequences, including the development of chronic neuropathic pain states. 12 In peripheral nervous system (PNS) injuries, inflammatory cytokines such as TNFα, IL-6, and IL-1β and anti-inflammatory cytokines such as IL-10 play a central role in axonal regeneration and repair. 6 .

[0119] The role of LRP1 has been extensively studied in neurodegenerative diseases such as Alzheimer's disease (AD). AD is characterized by a 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 patients. The spread of protein aggregates during disease progression is a common theme in 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 the transcytosis of Aβ from the brain to the periphery, targeting LRP1 with one or more peptide derivatives of the present technology can be used as a novel therapeutic. In addition, tau protein aggregates that form NTFs play a central role in the pathogenesis of Alzheimer's disease. The function of LRP1 is to regulate the endocytosis, aggregation and diffusion of tau protein associated with pathological deterioration. 32 Therefore, the SERPIN peptide derivatives of the present technology can be used as an intervention to repair plaque and tangle pathology by regulating LRP1-associated tau and amyloid Aβ protein aggregation.

[0120] LRP1 has been shown to be involved in the development of neurodegenerative diseases and the regulation of amyloid beta peptide (Aβs) metabolism in the brain and periphery. Neuroinflammation plays a crucial role in neurodegenerative diseases such as Alzheimer's disease (AD). LRP1 is highly expressed in CNS cells and has been shown to play a crucial role in the survival of primary neurons under stress conditions. 56,57 Microglia are resident immune cells of the brain that play a role in maintaining environmental homeostasis. Microglia are considered to be the prototypical tissue-resident macrophage-like innate immune cells of the CNS. 58 These cells are involved in chemotaxis, phagocytosis, antigen presentation, and cytokine production; however, damage or infection of microglia can lead to neuroinflammation and neurodegeneration. 59Reactive glial cells (e.g., microglia) and associated neuroinflammation play a key role in the development and progression of the disease and are activated through the dysregulated clearance of amyloid-β and other damage-associated molecular patterns (DAMPs). 13 AB deposition and tau hyperphosphorylation lead to microglial activation, activation of the NFκB inflammatory pathway, and associated proinflammatory cytokines such as TNFα, IL-6, and IL-1β, which can lead to neuronal damage and loss. Impaired autophagy, a homeostatic process that degrades and recycles proteins such as amyloid-β, has been implicated in AD. 7 LRP1 has been shown to mediate healthy lysosomal processing associated with autophagy. Therefore, through LRP1, the SERPIN peptide derivatives of the present disclosure can mediate multiple aspects of AD, including healthy cellular metabolism to reduce the spread of protein aggregation, reduce neuroinflammation, and improve neuronal dysfunction, thereby allowing these cells to survive and possibly regenerate.

[0121] Acute lung injury (acute respiratory distress syndrome)

[0122] Acute lung injury (ALI), leading to acute respiratory distress syndrome (ARDS), can be triggered by a variety of inflammatory insults, including pneumonia, trauma, and / or infection. A key initiating step in ALI is the dysregulated innate immune response to damage-associated or pathogen-associated molecular patterns (DAMPs or PAMPs, respectively). Alveolar macrophages are activated by infectious microorganisms in the Toll-like and Nod-like receptor signaling pathways, leading to further recruitment of macrophages and circulating neutrophils. Neutrophils accumulate in the lungs and release proinflammatory cytokines and other cytotoxic substances, leading to increased injury. The lung epithelium is damaged by these cells, and their secreted products lead to pulmonary edema and potential respiratory distress (ARDS). Increased production of proinflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, and IL-18 leads to a "cytokine storm," which portends morbidity and mortality in sepsis. LRP1 has been reported to play a crucial role in sepsis and ARDS as it regulates lung inflammation and lung tissue repair. 49 . The SERPIN peptide derivatives in the present technology target LRP1, thereby precisely coordinating the immune response and restoring homeostasis. Therefore, the peptide derivatives disclosed in the present invention can initiate multiple mechanisms by inducing specific signaling pathways mediated by LRP1, which have a significant impact on reducing lung injury and improving survival. These mechanisms include rebalancing cytokine output to promote inflammation resolution, mediating autophagy to restore proper cellular metabolic processes, restoring immune cell function, and clearing receptors to regulate tissue damage cycles. These mechanisms help clear infections and allow the host to better resist infections. Based on these mechanisms, the disclosed SERPIN peptide derivatives can be used as immunomodulatory therapeutics to prevent or treat ARDS by mediating LRP1 signaling.

[0123] Infectious Diseases

[0124] Acute respiratory distress syndrome (ARDS) is a major complication of severe COVID-19 patients. Clinical evaluation and retrospective studies in Wuhan, China showed that all admitted patients developed pneumonia, 29% of them developed ARDS, and among non-surviving severe patients, the vast majority (81%) developed ARDS. 17,52 . Therefore, patients who develop ARDS are at greatly increased risk of death. Virus-mediated innate immune responses through Toll-like receptors (TLRs) initiate an inflammatory response necessary to eliminate the infection. In some cases, the initial viral insult and triggers are highly amplified and uncontrolled, leading to overstimulation of immune cells and abnormal cytokine release (known as a cytokine storm), causing tissue damage that can lead to organ failure and death. Cytokine regulation is a key factor in preventing the harmful effects of an overactivated immune response, and controlling the cytokine storm can 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 factor of death for a subset of critically ill patients. 27 . Recently, clinical trials of the interleukin-6 inhibitor (IL-6) Kevzara (salilumab) have been initiated for the treatment of patients with pulmonary complications of coronavirus infection. IL-6 is a biomarker associated with higher mortality in patients with pneumonia. These trials will provide key insights into the effectiveness of single cytokine-targeted therapies. However, historically, targeting a single cytokine or pathway has not been an effective approach in patients with ARDS.

[0125] LRP1 signaling mediated by the SERPIN peptide derivatives of this technology may have therapeutic potential as an immunomodulatory strategy to improve the prognosis of COVID-19 patients by modulating multiple mechanisms of lung inflammation, including inhibition of cytokine storm, improvement of cell survival, regulation of autophagy and cell metabolism to clear infection, and control of tissue repair signaling pathways to promote normal healing and prevent fibrosis. LRP1 is known to regulate protease / antiprotease activity and mediate viral entry 43 The novel coronavirus utilizes a protease (TMPRSS2) to regulate cell entry and infection, and treatment with its corresponding protease inhibitors (such as SERPINs) blocked infection of lung cells 16,24,28. SARS-CoV-2 virus entry depends on the serine protease TMPRSS2 that directs the viral S protein involved in host cell entry, which is the first step in the viral replication cycle. Camostat mesylate is a natural SEPPIN that functions as a TMPRSS2 protease inhibitor and can effectively reduce the entry of SARS-CoV-2 virus, thereby limiting infection and spread of the disease. 16 . The alpha-1 antitrypsin derivatives in 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 can inhibit the entry of the virus through the mechanism of controlling the host protease / viral protein interaction through LRP1, thereby reducing the replication of SARS-CoV-2.

[0126] Arthropod-borne viruses are an important cause of acute encephalitis and an emerging global problem with an increasing risk of introduction into new areas 11,31 . Mosquito-borne encephalitis alphaviruses include Venezuelan (VEEV), Eastern (EEEV), and Western equine encephalitis viruses (WEEV), which are endemic in the Americas, causing outbreaks of encephalomyelitis and may have spread to the United States. VEEV causes a febrile illness in humans, with typical symptoms of fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system (CNS). Mortality rates for neurologic cases are as high as 35% in children and 10% in adults, and survivors typically suffer long-term neurologic deficits. 42 The overall estimate of VEEV survivors suffering from neurological sequelae is 4-14%, but this rate is likely higher due to misdiagnosis of arboviral encephalitis. 33 .

[0127] VEEV infection is known to inhibit cellular transcription and translation, thereby downregulating innate immune responses 45,48 In contrast, in the central nervous system (CNS), VEEV infection leads to the upregulation of many genes involved in inflammatory responses 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 VEEV pathogenesis. 9,20,30,34,35 Analysis of gene expression changes in brain tissue of mice infected with VEEV revealed alterations in immune pathways involved in antigen presentation, inflammation, apoptosis, and traditional antiviral responses 36In addition, viral regulation of the extracellular matrix and adhesion genes (such as 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 showed reduced brain inflammation and delayed onset of clinical disease 37 These studies suggest that alphavirus-induced inflammation contributes substantially to neuronal damage and that controlling inflammation is a viable therapeutic strategy.

[0128] The SERPIN peptide derivatives of this technology can bind to LRP1 and initiate the immune regulatory cascade. The expression of LRP1 increases during ischemia, tissue damage and viral infection. 2,22,25,43 Binding of one or more disclosed peptide derivatives to LRP1 can inhibit inflammatory responses and induce pro-survival signaling through phosphorylation of protein kinase Akt. Therefore, targeting LRP1 has the potential to become a broad-spectrum therapeutic strategy for infectious diseases.

[0129] Through LRP1-guided host-mediated mechanisms, SERPIN peptide derivatives can suppress the harmful cytokine storm associated with severe COVID-19 disease, activate protective pathways to prevent lung damage, and / or clear infection faster, and have a direct impact on inhibiting viral infection. Therefore, the dual anti-inflammatory and antiviral mechanisms of SERPIN peptide derivatives can effectively improve the survival rate of patients with ARDS caused by SARS-COV2.

[0130] Eosinophilic esophagitis

[0131] Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory response mediated primarily by type 2 immunity and associated with esophageal dysfunction and epithelial barrier dysfunction. Esophageal inflammation can lead to dysphagia, persistent heartburn, chest and abdominal pain, weight loss, and food impaction. It is characterized by high levels of eosinophils, proteases, cathelicidins, serine proteases including kallikrein (KLK5), and thymic stromal lymphopoietin (TSLP), a cytokine and master regulator of allergic type 2 inflammatory responses in the local environment. 41In esophageal epithelial cells, loss of function of the serine peptidase inhibitor Kazal type 7-SPINK7 results in uncontrolled protease activity, release of proinflammatory cytokines such as TNFα, CCL2, GM-CSF, IL-8, and CXCL10, and induction of inflammation. Recently, the serine protease KLK-5, an important mediator of epithelial barrier function, was found to be a direct target of SPINK7, and loss of SPINK7 mediates EoE pathogenesis primarily through uncontrolled KLK-5 protease activity. Interestingly, SERPIN α-1 antitrypsin was able to inhibit KLK5 activity in vitro and suppress allergen-induced esophageal eosinophilia in vivo. However, the mechanism of this activity is unclear and may involve LRP1 rather than or in addition to direct proteolytic inhibition.

[0132] LRP1 levels are low in eosinophilic asthma patients. Furthermore, in a model of allergic airway disease, LRP1-specific deletion of mouse CD11b and CD11c dendritic cells leads to enhanced allergic inflammatory responses. 29 LRP1-deficient mice have increased antigen uptake, eosinophilic inflammation, allergic sensitization, increased Th2-mediated cytokine production, and decreased T regulatory cells 29 Therefore, LRP1 can help maintain the homeostasis of proteases / inhibitors in the esophageal environment, mediate TH2 responses and inhibit inflammatory signaling pathways (NFκB, JNK), thereby repairing esophageal dysfunction.

[0133] 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 has been shown to be significantly increased in nasal and airway epithelial cells in type 2 asthma and allergic rhinitis. Studies have also shown that the expression of TMPRSS2 is positively correlated with TH2-mediated immune responses important in allergic reactions. The disclosed SERPIN peptide derivatives may be of great significance in alleviating eosinophilic esophagitis by mediating the control of LRP1.

[0134] The following examples are intended to illustrate various embodiments of the present technology. Therefore, the specific embodiments discussed do not constitute a limitation on the scope of the present technology. It is obvious that those skilled in the art can make various equivalents, changes, and modifications without departing from the scope of the present technology, and it is understood that these equivalent embodiments will be included herein. In addition, all references cited in this disclosure are incorporated herein by reference in their entirety, as if fully listed here.

[0135] Various embodiments of the present technology are described in paragraphs 0096 to 0175 below.

[0136] A SERPIN peptide derivative comprising the core sequences FVFLM (SEQ ID NO: 1), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQ ID NO: 8), PFVFL[Nle] (SEQ ID NO: 9), and one or more of the following modifications:

[0137] (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 a polar head added to the N-terminus of the core sequence and a polar tail added to the C-terminus of the core sequence;

[0138] (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 the N-terminus and the C-terminus of the core sequence, so that the peptide derivative can be cyclized;

[0139] (iii) one or more amino acid residues in the core sequence are replaced by one or more alternative amino acid residues that are less hydrophobic than the one or more amino acid residues in the core sequence;

[0140] (iv) one or more amino acid residues in the core sequence are replaced by one or more alternative amino acid residues that are more hydrophobic than the one or more amino acid residues in the core sequence; and

[0141] (v) One or more amino acid residues in the core sequence are deleted.

[0142] The SERPIN peptide derivative according to paragraph 0096, wherein the SERPIN peptide derivative is a linear peptide.

[0143] The SERPIN peptide derivative according to paragraph 0096, wherein the SERPIN peptide derivative is a cyclized peptide.

[0144] The SERPIN peptide derivative according to paragraph 0098, wherein the SERPIN peptide derivative is cyclized via a disulfide bond formed between two Cys residues.

[0145] The SERPIN peptide derivative according to paragraph 0098, wherein the SERPIN peptide derivative is cyclized via a linker between two amino acid residues.

[0146] The SERPIN peptide derivative according to any 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 Arg, Lys, and His.

[0147] The SERPIN peptide derivative according to any of paragraphs 0096-0101, wherein the size of the SERPIN peptide derivative is less than 15 amino acid residues.

[0148] The SERPIN peptide derivative according to any of paragraphs 0096-0102, wherein the size of the SERPIN peptide derivative is 7, 8, 9, 10, 11, or 12 amino acid residues.

[0149] A SERPIN peptide derivative according to any of paragraphs 0096-0103, wherein one or more amino acid residues are D-amino acids.

[0150] A SERPIN peptide derivative comprising an amino acid sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 (SEQ ID NO: 3), wherein:

[0151] X1 is a hydrophilic amino acid residue or none;

[0152] X2 is a hydrophilic amino acid residue or none;

[0153] X3 is a hydrophilic amino acid residue or none;

[0154] X4 is a Cys amino acid residue or none;

[0155] X5 is a Pro amino acid residue or none;

[0156] X6 is the first hydrophobic amino acid residue;

[0157] X7 is a short branched-chain amino acid residue;

[0158] X8 is the second hydrophobic amino acid residue;

[0159] X9 is a saturated hydrophobic amino acid residue;

[0160] X10 is a hydrophilic amino acid in the D configuration; and

[0161] X11 is any amino acid residue that allows cyclization of the SERPIN peptide derivative.

[0162] The SERPIN peptide according to paragraph 0105, wherein the first and second hydrophobic amino acid residues are aromatic amino acid residues.

[0163] A SERPIN peptide according to any of paragraphs 0105-0106, wherein the short branched amino acid residue is Val or Thr.

[0164] A SERPIN peptide according to any of paragraphs 0105-0107, wherein the saturated hydrophobic amino acid is Leu.

[0165] A SERPIN peptide according to any of paragraphs 0105-0108, wherein the hydrophilic amino acid in the D configuration is an Asp, Glu, Lys, Dap, or Cys residue in the D configuration.

[0166] A SERPIN peptide according to paragraphs 0105-0109, wherein

[0167] X1 is a basic residue showing a positive charge or none;

[0168] X2 is a basic residue showing a positive charge or none; and

[0169] X3 is a basic residue showing a positive charge or none.

[0170] A SERPIN peptide derivative according to paragraphs 0105-0110, wherein

[0171] X1 is an Arg, Lys, or His residue, or none;

[0172] X2 is an Arg, Lys, or His residue, or none; and

[0173] X3 is an Arg, Lys, or His residue, or none.

[0174] A SERPIN peptide derivative according to any of paragraphs 0105-0111, wherein X6 is Phe or Nal.

[0175] A SERPIN peptide derivative according to any of paragraphs 0105-0112, wherein X8 is Phe or Nal.

[0176] A SERPIN peptide derivative according to any of paragraphs 0105-0113, wherein X6 is Ala, Phe, or Nal, and X4 is Nal.

[0177] A SERPIN peptide derivative according to any of paragraphs 0105-0114, wherein X7 is Asp, Glu, Lys, Dap, or Cys.

[0178] The SERPIN peptide derivative according to any of paragraphs 0105-0115, wherein the SERPIN peptide derivative is linear or cyclized.

[0179] The SERPIN peptide derivative according to any of paragraphs 0105-0116, wherein the size of the SERPIN peptide derivative is less than 15 amino acid residues.

[0180] The SERPIN peptide derivative according to any of paragraphs 0105-0117, wherein the size of the SERPIN peptide derivative is 7, 8, 9, 10, 11, or 12 amino acid residues.

[0181] A SERPIN peptide derivative comprising the core sequences FVFLM (SEQ ID NO: 1), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQ ID NO: 8), PFVFL[Nle] (SEQ ID NO: 9), and one or more of the following modifications:

[0182] (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 one or more Lys, Glu, or His residues added to the N-terminus of the core sequence and a polar tail added to the C-terminus of the core sequence;

[0183] (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 the N-terminus and the C-terminus of the core sequence, so that the SERPIN peptide derivative can be cyclized by forming a disulfide bond between two Cys residues;

[0184] (iii) one or more amino acid residues in the core sequence are replaced by one or more Thr amino acid residues; and

[0185] (iv) one or more amino acid residues in the core sequence are replaced by one or more alternative amino acid residues that are more hydrophobic than the one or more amino acid residues in the core sequence.

[0186] The SERPIN peptide derivative according to paragraph 0119, wherein the SERPIN peptide derivative is a linear peptide.

[0187] The SERPIN peptide derivative according to paragraph 0119 or 0120, wherein the size of the SERPIN peptide derivative is less than 15 amino acid residues.

[0188] The SERPIN peptide derivative according to any of paragraphs 0119-0121, wherein the size of the SERPIN peptide derivative is 7, 8, 9, 10, 11, or 12 amino acid residues.

[0189] A SERPIN peptide derivative comprising an amino acid sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 (SEQ ID NO: 3), wherein:

[0190] X1 is a hydrophilic amino acid residue or none;

[0191] X2 is a hydrophilic amino acid residue or none;

[0192] X3 is a hydrophilic amino acid residue or none;

[0193] X4 is a Cys amino acid residue or none;

[0194] X5 is a Pro amino acid residue or none;

[0195] X6 is the first hydrophobic amino acid residue;

[0196] X7 is a short branched-chain amino acid residue;

[0197] X8 is the second hydrophobic amino acid residue;

[0198] X9 is a saturated hydrophobic amino acid residue;

[0199] X10 is a hydrophilic amino acid in the D configuration; and

[0200] X11 is any amino acid residue that allows cyclization of the SERPIN peptide derivative;

[0201] wherein X4 is Cys, X5 is Pro, X7 is Thr, and / or X10 is a Lys residue in the D-configuration.

[0202] The SERPIN peptide according to paragraph 0123, wherein the first and second hydrophobic amino acid residues are aromatic amino acid residues.

[0203] A SERPIN peptide according to paragraph 0123 or 0124, wherein the short branched-chain amino acid residue is Val or Thr.

[0204] A SERPIN peptide according to any of paragraphs 0123-0125, wherein the saturated hydrophobic amino acid is Leu.

[0205] A SERPIN peptide according to any of paragraphs 0123-0126, wherein the hydrophilic amino acid in the D configuration is an Asp, Glu, Lys, Dap, or Cys residue in the D configuration.

[0206] A SERPIN peptide according to any of paragraphs 0123-0127, wherein

[0207] X1 is a basic residue showing a positive charge or none;

[0208] X2 is a basic residue showing a positive charge or none; and

[0209] X3 is a basic residue showing a positive charge or none.

[0210] A SERPIN peptide derivative according to any of paragraphs 0123-0128, wherein

[0211] X1 is an Arg, Lys or His residue, or none;

[0212] X2 is an Arg, Lys or His residue, or none; and

[0213] X3 is an Arg, Lys or His residue, or none.

[0214] A SERPIN peptide derivative according to any of paragraphs 0123-0129, wherein X6 is Phe or Nal.

[0215] A SERPIN peptide derivative according to any of paragraphs 0123-0130, wherein X8 is Phe or Nal.

[0216] A SERPIN peptide derivative according to any of paragraphs 0123-0131, wherein X6 is Ala, Phe or Nal, and X4 is Nal.

[0217] The SERPIN peptide derivative according to any of paragraphs 0123-0132, wherein X7 is Asp, Glu, Lys, Dap, or Cys.

[0218] The SERPIN peptide derivative according to any of paragraphs 0123-0133, wherein the SERPIN peptide derivative is linear or cyclized.

[0219] The SERPIN peptide derivative according to any of paragraphs 0123-0134, wherein the size of the SERPIN peptide derivative is less than 15 amino acid residues.

[0220] The SERPIN peptide derivative according to any of paragraphs 0123-0135, wherein the size of the SERPIN peptide derivative is 7, 8, 9, 10, 11, or 12 amino acid residues.

[0221] A SERPIN peptide derivative comprises a sequence of SEQ ID NO: 10-20 or 23-62, wherein the SERPIN peptide derivative comprises 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 a polar head added to the N-terminus of the core sequence and a polar tail added to the C-terminus of the core sequence.

[0222] A SERPIN peptide derivative comprises a 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 the N-terminus and C-terminus of the core sequence, so that the peptide derivative can be cyclized.

[0223] A SERPIN peptide derivative comprises a sequence of SEQ ID NO: 27-29, 38, 39 or 42-62, wherein one or more amino acid residues in the core sequence are replaced by one or more alternative amino acid residues having less hydrophobicity than the one or more amino acid residues in the core sequence.

[0224] A SERPIN peptide derivative comprises the sequence of SEQ ID NO: 30 or 31, wherein one or more amino acid residues in the core sequence are replaced by one or more alternative amino acid residues having greater hydrophobicity than the one or more amino acid residues in the core sequence.

[0225] A SERPIN peptide derivative comprises the sequence of SEQ ID NO: 41 or 47-53, wherein one or more amino acid residues in the core sequence are deleted.

[0226] A SERPIN peptide derivative comprises 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 and a polar tail is added to the C-terminus of the core sequence, or one or more Lys, Glu or His residues are added to the N-terminus of the core sequence and a polar tail is added to the C-terminus of the core sequence.

[0227] A SERPIN peptide derivative comprises 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 the N-terminus and the C-terminus of the core sequence, so that the SERPIN peptide derivative can be cyclized through a disulfide bond formed between two Cys residues.

[0228] A SERPIN peptide derivative comprising the 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.

[0229] A fusion protein comprising the SERPIN peptide derivative of any one of paragraphs 0096-0144, and an epitope tag, a half-life extender, or an epitope tag and a half-life extender.

[0230] A conjugate comprising the SERPIN peptide derivative of any one of paragraphs 0096-0144, and a permeability enhancer.

[0231] A pharmaceutical composition comprising the SERPIN peptide derivative of any one of paragraphs 0096-0144, the fusion protein of paragraph 0145, or the conjugate of paragraph 0146.

[0232] The pharmaceutical composition according to paragraph 0147, further comprising one or more additional therapeutic agents.

[0233] The pharmaceutical composition according to paragraph 0147 or 0148 further comprises a pharmaceutically acceptable carrier, excipient, additive, preservative, or a combination thereof.

[0234] The pharmaceutical composition according to any of paragraphs 0147-0149, further comprising a penetration enhancer.

[0235] The pharmaceutical composition according to any one of paragraphs 0147-0150, wherein the pharmaceutical composition is formulated for oral administration, transdermal administration, or parenteral administration.

[0236] A fusion protein comprising the SERPIN peptide derivative of any one of paragraphs 0119-0136, 0140, or 0142-0144, and an epitope tag, a half-life extender, or both the epitope tag and the half-life extender.

[0237] A conjugate comprising the SERPIN peptide derivative of any one of paragraphs 0119-0136, 0140, or 0142-0144, and a penetration enhancer.

[0238] A pharmaceutical composition comprising the SERPIN peptide derivative of any one of paragraphs 0119-0136, 0140, or 0142-0144, the fusion protein of paragraph 0152, or the conjugate of paragraph 0153.

[0239] The pharmaceutical composition according to paragraph 0154, further comprising one or more additional therapeutic agents.

[0240] The pharmaceutical composition according to paragraph 0154 or 0155, further comprising a pharmaceutically acceptable carrier, excipient, additive, preservative, or a combination thereof.

[0241] The pharmaceutical composition according to any of paragraphs 0154-0156, further comprising a penetration enhancer.

[0242] The pharmaceutical composition according to any one of paragraphs 0154-0157, wherein the pharmaceutical composition is formulated for oral administration, transdermal administration, or parenteral administration.

[0243] A method for treating a subject suffering from a disease or condition associated with LRP1, comprising administering to the subject an effective amount of the SERPIN peptide derivative of paragraphs 0096-0144, the fusion protein of paragraph 0145, the conjugate of paragraph 0146, or the pharmaceutical composition of any of paragraphs 0147-0151 to treat the disease or condition associated with LRP1.

[0244] The method according to paragraph 0159, wherein administration of the SERPIN peptide reduces NFkB activation in the subject.

[0245] The method of paragraph 0159 or 0160, wherein administration of the SERPIN peptide reduces TNFa in the subject.

[0246] The method according to any of paragraphs 0159-0161, wherein administration of the SERPIN peptide reduces IL-6.

[0247] The method according to any of paragraphs 0159-0162, wherein the disease or condition associated with LRP1 is acute or neuropathic pain, nociceptive pain, or inflammatory pain.

[0248] The method according to any of paragraphs 0159-0163, wherein the disease or disorder associated with LRP1 is a disease or condition caused by a viral infection.

[0249] The method according to paragraph 0164, wherein administration of the SERPIN peptide reduces viral replication.

[0250] The method according to any of paragraphs 0159-0162, wherein the disease or condition associated with LRP1 is eosinophilic esophagitis.

[0251] The method according to any of paragraphs 0159-0162, wherein the disease or condition associated with LRP1 is acute lung injury.

[0252] A method of treating a subject having a disease or condition associated with LRP1, comprising administering to the subject an effective amount of a SERPIN peptide derivative of any of paragraphs 0119-0136, 0140, or 0142-0144, a fusion protein of paragraph 0152, a conjugate of paragraph 0153, or a pharmaceutical composition of any of paragraphs 0154-0158 to treat the disease or condition associated with LRP1.

[0253] The method according to paragraph 0168, wherein administration of the SERPIN peptide reduces NFkB activation in the subject.

[0254] The method according to paragraph 0168 or 0169, wherein administration of the SERPIN peptide reduces TNFa in the subject.

[0255] The method according to any of paragraphs 0168-0170, wherein administration of the SERPIN peptide reduces IL-6.

[0256] The method according to any of paragraphs 0168-0170, wherein the disease or condition associated with LRP1 is a disease or condition caused by a viral infection.

[0257] The method according to paragraph 0172, wherein administration of the SERPIN peptide reduces viral replication.

[0258] The method according to any of paragraphs 0168-0170, wherein the disease or condition associated with LRP1 is eosinophilic esophagitis.

[0259] The method according to any of paragraphs 0168-0170, wherein the disease or condition associated with LRP1 is acute lung injury.

[0260] Example

[0261] Example 1: Anti-inflammatory effects of SERPIN derivatives

[0262] The anti-inflammatory effects of the SERPIN peptide derivatives SA1-SA8 listed in Table 1 were tested with SP163M and SP22 as positive controls. 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 stimulated with LPS (5 ng / ml) and then incubated overnight. NFκB-induced secreted embryonic alkaline phosphatase (SEAP) was measured in the supernatant and the absorbance was read. Figure 1 As shown, SERPIN peptide derivatives (such as SA3, SA6, and SA7) that retained the shortest LRP1 binding sequence FVFL[Nle] and the tripeptide RRR as flanks to improve solubility showed higher activity in inhibiting NFκB compared with SP163M. However, SERPIN peptide derivatives (such as SA1, SA2, SA4, and SA5) with the tripeptide HHH or KKK or the negatively charged tripeptide EEE as flanks showed very low NFκB inhibitory activity.

[0263] This data suggests that shortened peptides such as SA3 can be prepared and are more effective in 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).

[0264] Example 2: Evaluation of the biological activity of SERPIN peptide derivatives

[0265] 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 varying concentrations of each peptide derivative (up to 100 μg / ml) before being stimulated with LPS (5 ng / ml) and cultured overnight. NFκB-induced secreted embryonic alkaline phosphatase (SEAP) was measured in the supernatant and the absorbance was read. The percentage reduction in NFκB activation compared to vehicle (LPS-stimulated) cells is shown. IMG microglia were treated with varying concentrations of each peptide derivative (up to 100 μg / ml) before being stimulated with LPS (100 ng / ml) for 24 hours. TNFα (pg / ml) in the supernatant was measured by ELISA.

[0266] The SERPIN peptide derivatives A1-A15 listed in Tables 2-4 were tested for TNFα activity as shown in Figure 5. Peptide derivatives A5 and A8 with amino acid substitutions and peptide derivatives A10 and A15 with ring closure modifications showed improved activity in reducing TNFα activation.

[0267] FIG6 shows that peptide derivatives A2-1, A2-2, A2-3, A2-4, and A2-5 have a significant effect on reducing TNFα ( Fig. 6A ) and NFκB( Figure 6B ) activation. 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 unmodified SP16, SP163M and SA7 were tested at concentrations up to 100 μg / mL. All peptide derivatives showed no cytotoxicity ( Figure 8 In the cytotoxicity assay, cells (NFκB reporter cells or IMG microglia) were treated with SP163M or 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 h and then used according to the manufacturer's instructions. The cell viability was determined by luminescent cell viability assay.

[0268] Fig. 9 The peptide derivatives A3-10 and A3-14 showed excellent activity in reducing NFκB activation. Fig.10It was shown that peptide derivative A3-14 exhibited excellent activity in reducing the secretion of IL-6 in IMG after LPS stimulation. Due to synthesis-related problems, peptide derivative A3-10 was not included in the IL-6 assay. None of the tested peptide derivatives showed significant cytotoxicity (Figure 11).

[0269] Table 7 below summarizes the EC50 of some peptide derivatives of the present technology tested in vitro.

[0270]

[0271]

[0272] Example 3: In vivo effects of SERPIN peptide derivatives

[0273] The ability of SERPIN peptide derivatives A15 and A2-5 to reduce capsaicin-induced pain-related behaviors in mice was tested 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 minutes. Fig.12 As shown, the pain blocking effects of both peptide derivatives at a low dose of 5 μg / mouse were similar to the effects of SP163M at a higher dose.

[0274] Example 4: Effects of SERPIN peptide derivatives on neuroinflammation

[0275] like Fig.13 As shown, the cytokine profile was explored in LPS-induced microglia treated with the SERPIN peptide derivatives SA7, SP163M or vehicle control. Cytokines were induced by LPS and measured in the supernatant. In microglia, SA7 was more effective than peptide SP163M in reducing LPS-mediated multiple cytokines, including IL-6, TNFα, IL-1β, IL-12, and CXCL1. IMG microglia were treated with SA7 (100μg / mL) and SP163M (100μg / mL) for 24 hours with or without the addition of LPS (100ng / mL). Cytokine levels (pg / ml) in the supernatant were determined by multiplex assay.

[0276] Next, the efficacy of the peptide derivative SA7 was explored in an in vivo neuroinflammation model. The LPS model of neuroinflammation has been extensively studied to understand neurodegenerative diseases. 60-63These studies confirmed that LPS induces cognitive impairment in C57BL / 6 mice. Furthermore, acute systemic LPS resulted in microglial activation in the brain, impaired clearance of amyloid-β, and increased blood and brain proinflammatory cytokines. Therefore, the LPS-induced neuroinflammation model was used to test the activity of the peptide derivative SA7.

[0277] Female C57BL / 6 mice (n=3-4) were injected intraperitoneally with 1 mg / kg LPS (E. coli 0111: B4) for 4 consecutive days, followed by subcutaneous injection of SP163M (100 μg) or SA7 (100 μg) 1 hour after LPS injection every day. Body weight and clinical assessment were recorded daily. At the end of the study (day 5) (24 hours after the last LPS injection), mice were weighed, scored and sacrificed. Figure 3 The clinical scores of SA7-treated mice were significantly improved compared with those of vehicle- and SP163M-treated mice ( Fig.14A ). In addition, the SA7-treated mice also experienced a slight benefit in terms of weight loss, whereas the SP163M-treated mice did not ( Fig. 14B ).

[0278] Brain homogenate using LEGENDplex TM The Mouse Inflammation Panel (flow cytometric multiplex assay using fluorescently encoded beads) was evaluated for cytokine profiling. This panel allows for the simultaneous quantitative detection 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. Fig.15 The results showed that the SA7-treated group did show differences in the levels of many LPS-induced cytokines compared to the vehicle-treated and SP163M-treated animals. For example, moderate levels of IL-6 were measured in the brain homogenates of the vehicle and SP163M, 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 lower levels. This corresponds to the improvement of clinical symptoms after SA7 treatment.

[0279] Next, brain homogenates were analyzed for two biomarkers of CNS injury: neurofilament light chain (NfL), expressed in neuronal axons, and glial fibrillary acidic protein (GFAP), expressed in astrocytes, a marker of astrocyte activation. It has been reported that the level of inflammation in the cerebral cortical region is negatively correlated with plasma NfL levels. The study found that LPS selectively reduced the expression of neurofilament light chain in neurons. NfL is required for neuronal regeneration, synaptic connectivity, and balanced signaling operations of neurons, suggesting that an increase in NfL may indicate that neurons are protected from LPS-induced injury. Fig.16A The peptide derivative SA7 significantly increased NfL protein expression in brain homogenates after LPS-activated neuroinflammation as determined by Western blot analysis compared to vehicle and SP163M. The abnormal expression of GFAP indicated the presence of reactive astrocytes and neuroinflammation. Fig. 16B The peptide derivative SA7 was shown to significantly reduce the expression of GFAP in brain lysates as determined by Western blot analysis compared to SP163M and vehicle treated animals. These results suggest that the peptide derivative has a strong neuroprotective effect.

[0280] Example 5: Effects of SERPIN peptide derivatives on autophagy

[0281] In addition to neuroinflammation, impaired autophagy, a homeostatic process that degrades and recycles proteins such as amyloid-β, has also been implicated in AD. 7 . LRP1 has been shown to mediate healthy lysosomal processing associated with autophagy. Therefore, it is possible that multiple aspects of AD could be mediated by LRP1 agonists, such as derivatives derived from SERPIN peptides, including healthy cellular metabolism to reduce the spread of protein aggregates, reduce neuroinflammation, and improve neuronal dysfunction, allowing these cells to survive and potentially regenerate. The effects of SP163M and the peptide derivative SA7 on autophagy markers were tested in microglia. IMG microglia were treated with SP163M (100 μg / ml) or SA7 (100 μg / ml) prior to the addition of LPS (100 ng / ml) for 24 h. Lysates were collected and subjected to western blot analysis for LRP1 and microtubule-associated protein light chain 3 (LC3I and II). The LC3II / I ratio, a common marker of autophagy, was determined to indicate autophagic flux. Fig.17A showed that in LPS-activated microglia, autophagic flux was reduced, indicating that normal lysosomal processing was impaired. In the presence of LPS, treatment with SP163M or the peptide derivative SA7 restored autophagic function to near baseline levels. LRP1 protein expression was measured. Fig. 17Bshowed that LPS caused a decrease in protein expression levels (possibly indicating increased LRP1 shedding). SP163M and the peptide derivative SA7 increased LRP1 protein expression in LPS-activated microglia. Interestingly, treatment with SA7 significantly increased LRP1 expression in the absence of LPS compared to SP163M. These results suggest that regulation of LRP1 by SP163M contributes to an alternative mechanism by which homeostatic balance of dysregulated cellular processes is regulated.

[0282] Example 6: Effects of SERPIN peptide derivatives on viral infection

[0283] The SERPIN-derived peptide SP163M exhibited antiviral effects. SP163M was able to significantly reduce viral replication of neuroinflammatory alphaviruses such as Eastern Equine Encephalitis (EEEV). SP163M also inhibited viral replication of the novel coronavirus SARS-CoV-2. It is well known that SARS-CoV-2 and other viruses such as influenza utilize host cell proteases for viral entry. TMPRSS2 processes the S protein on the SARS-CoV2 envelope, a process known as priming. Priming of the S protein is necessary for the binding of the S protein to the host receptor ACE2. It is well known that LRP1 regulates protease / antiprotease activity. Protease inhibitors such as alpha-1 antitrypsin can reduce the proteolytic activity of TMPRSS2 and prevent priming of the S protein, thereby preventing viral entry. In addition, inhibition of TMPRSS2 prevents processing by ACE2, thereby reducing the infectivity of the coronavirus. Fig.18 The inhibitory effects of different concentrations of SP163M and the peptide derivative SA7 on TMPRSS2 are shown. The IC50 value of SP163M is 1125 ng / mL, while the IC50 value of the SA7 derivative is 83 ng / mL, which is more potent.

[0284] Example 7: Effects of SERPIN peptide derivatives on eosinophilic esophagitis

[0285] In the case of eosinophilic esophagitis (EoE), A1AT has been shown to attenuate experimental EoE in mouse models and in vitro. It is unclear whether the A1AT effect is mediated by inhibition of proteolytic activity or by activation of LRP1 signaling. LRP1 is lower in patients with eosinophilic asthma, and in animal models, loss of LRP1 is associated with exacerbated allergic reactions. SP163M does not contain any antiprotease activity sequences and may act through the mediation of LRP1 rather than proteolytic activity. In a mouse model of eosinophilic esophagitis, mice were intraperitoneally sensitized with ovalbumin (OVA) and then intranasally challenged with OVA on day 4. Esophageal lysates from EoE-induced mice showed lower levels of LRP1 protein expression as determined by Western blot analysis. Treatment of mice with the SERPIN-derived peptide SP163M increased LRP1 levels ( Fig.19A ). Evidence suggests that loss of serine peptidase inhibitor kazolin-type 7 (SPINK7) expression in the esophagus is an upstream event in the pathogenesis of EoE. Therefore, 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 the generation of dendritic cells polarizing type 2 T cells (Th2), key cells involved in the pathogenesis of EoE, following loss of SPINK7. Fig.19B SP163M was shown to inhibit poly-inosinic-cytidylic acid (polyI:C)-induced TSLP production by SPINK7 knockout EPC2 cells (human esophageal epithelial cells). SPINK7 KO cells and control cells were plated under high calcium and high density for 48 h and then treated with SP163M or the derivative SA7 (200 μg / ml) and poly-inosinic-cytidylic acid (5 μg / ml, or left untreated) for 8 h. TSLP production in the supernatant was measured by ELISA.

[0286] Furthermore, in an allergic asthma mouse model, mice were sensitized to ovalbumin by two intraperitoneal injections of ovalbumin containing the adjuvant alum (a strong inducer of innate immune responses and TH2-mediated immune responses) and then challenged by intranasal instillation of ovalbumin on four days, mice treated with the peptide derivative A2-5 showed a reduction in inflammatory mediators. Lung homogenates from OVA-induced mice treated with vehicle (vehicle / OVA) showed a significant increase in TSLP levels compared to non-OVA-induced mice (saline / saline) (p=0.001). OVA-induced mice treated with the peptide derivative A2-5 (A2-5 / OVA) showed a significant decrease in TSLP levels compared to OVA-induced vehicle-treated mice (p=0.026) ( Fig. 20A). Exposure to allergens also resulted in an increase in total protein in lung tissue compared to animals that were not exposed to the allergens. Infiltration of immune cells into the lungs may be responsible for the increased levels of total protein in the lungs. Fig. 20B The results showed that the protein levels in the lungs of mice treated with SP163M (SP163M / OVA), the steroid dexamethasone (Dex / OVA), and the peptide derivative A2-5 were significantly reduced compared with vehicle-treated animals (p<0.005). In A2-5-treated mice, this was associated with a reduction in the TH-2-mediated cytokines IL-5, IL-4, and IL-13 in lung tissue compared with vehicle-treated mice ( Fig. 20C ).

[0287] References

[0288] The references, patents and published patent applications listed below, as well as all references cited in the above specification, are hereby incorporated by reference in their entirety as if fully set forth herein.

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Claims

1. A SERPIN peptide derivative comprising the core sequences FVFLM (SEQ ID NO: 1), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQ ID NO: 8), 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 core sequence, a polar tail added to the C-terminus of the core sequence, or a polar head added to the N-terminus of the core sequence and a polar tail added to the C-terminus of the core sequence; (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 the N-terminus and the C-terminus of the core sequence, so that the peptide derivative can cyclize; (iii) one or more amino acid residues in the core sequence are replaced by one or more alternative amino acid residues that are less hydrophobic than the core sequence; (iv) one or more amino acid residues in the core sequence are replaced by one or more alternative amino acid residues having greater hydrophobicity than the core sequence; and (v) One or more amino acid residues in the core sequence are deleted. 2 . The SERPIN peptide derivative according to claim 1 , wherein the SERPIN peptide derivative is a linear peptide.

3. The SERPIN peptide derivative according to claim 1, wherein the SERPIN peptide derivative is a cyclized peptide. The SERPIN peptide derivative according to claim 3 , wherein the SERPIN peptide derivative is cyclized via a disulfide bond formed between two Cys residues. The SERPIN peptide derivative according to claim 3 , wherein the SERPIN peptide derivative is cyclized via a linker between two amino acid residues.

6. The SERPIN peptide derivative according to any one of claims 1 to 5, wherein the polar head or the polar tail comprises two or more charged amino acids, such as positively charged amino acids selected from Arg, Lys, and His.

7. The SERPIN peptide derivative according to any one of claims 1 to 6, wherein the size of the SERPIN peptide derivative is less than 15 amino acid residues.

8. The SERPIN peptide derivative according to any one of claims 1 to 7, wherein the size of the SERPIN peptide derivative is 7, 8, 9, 10, 11, or 12 amino acid residues.

9. The SERPIN peptide derivative according to any one of claims 1 to 8, wherein one or more amino acid residues are D-amino acids.

10. 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 none; X2 is a hydrophilic amino acid residue or none; X3 is a hydrophilic amino acid residue or none; X4 is a Cys amino acid residue or none; X5 is a Pro amino acid residue or none; X6 is the first hydrophobic amino acid residue; X7 is a short branched-chain amino acid residue; X8 is the 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 according to claim 10 , wherein the first hydrophobic amino acid residue and the second hydrophobic amino acid residue are aromatic amino acid residues.

12. The SERPIN peptide according to claim 10 or 11, wherein the short branched-chain amino acid residue is Val or Thr.

13. The SERPIN peptide according to any one of claims 10 to 12, wherein the saturated hydrophobic amino acid is Leu.

14. The SERPIN peptide according to any one of claims 10 to 13, wherein the hydrophilic amino acid in D configuration is an Asp, Glu, Lys, Dap, or Cys residue in D configuration.

15. The SERPIN peptide according to claims 10-14, wherein: X1 is a basic residue showing a positive charge or none; X2 is a basic residue showing a positive charge or none; and X3 is a basic residue showing a positive charge or none.

16. The SERPIN peptide derivative according to claims 10-15, wherein: X1 is an Arg, Lys, or His residue, or none; X2 is an Arg, Lys, or His residue, or none; and X3 is an Arg, Lys, or His residue, or none.

17. The SERPIN peptide derivative according to any one of claims 10 to 16, wherein X6 is Phe or Nal.

18. The SERPIN peptide derivative according to any one of claims 10 to 17, wherein X8 is Phe or Nal.

19. The SERPIN peptide derivative according to any one of claims 10 to 18, wherein X6 is Ala, Phe, or Nal, and X4 is Nal.

20. The SERPIN peptide derivative according to any one of claims 10 to 19, wherein X7 is Asp, Glu, Lys, Dap, or Cys.

21. The SERPIN peptide derivative according to any one of claims 10 to 20, wherein the SERPIN peptide derivative is linear or cyclized.

22. The SERPIN peptide derivative according to any one of claims 10 to 21, wherein the size of the SERPIN peptide derivative is less than 15 amino acid residues.

23. The SERPIN peptide derivative according to any one of claims 10 to 22, wherein the SERPIN peptide derivative has a size of 7, 8, 9, 10, 11, or 12 amino acid residues.

24. A SERPIN peptide derivative comprising the core sequences FVFLM (SEQ ID NO: 1), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQ ID NO: 8), PFVFL[Nle] (SEQ ID NO: 9), and one or more of 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 one or more Lys, Glu, or His residues added to the N-terminus of the core sequence and a polar tail added to the C-terminus of the core sequence; (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 the N-terminus and the C-terminus of the core sequence, so that the SERPIN peptide derivative can be cyclized via a disulfide bond formed between two Cys residues; (iii) one or more amino acid residues in the core sequence are replaced by one or more Thr amino acid residues; and (iv) one or more amino acid residues in the core sequence are replaced by one or more alternative amino acid residues that are more hydrophobic than the core sequence.

25. The SERPIN peptide derivative according to claim 24, wherein the SERPIN peptide derivative is a linear peptide.

26. The SERPIN peptide derivative according to claim 24 or 25, wherein the size of the SERPIN peptide derivative is less than 15 amino acid residues.

27. The SERPIN peptide derivative according to any one of claims 24 to 26, wherein the SERPIN peptide derivative is 7, 8, 9, 10, 11, or 12 amino acid residues in size.

28. 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 none; X2 is a hydrophilic amino acid residue or none; X3 is a hydrophilic amino acid residue or none; X4 is a Cys amino acid residue or none; X5 is a Pro amino acid residue or none; X6 is the first hydrophobic amino acid residue; X7 is a short branched-chain amino acid residue; X8 is the 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; wherein X4 is Cys, X5 is Pro, X7 is Thr, and / or X10 is a Lys residue in the D-configuration.

29. The SERPIN peptide according to claim 28, wherein the first hydrophobic amino acid residue and the second hydrophobic amino acid residue are aromatic amino acid residues.

30. The SERPIN peptide according to claim 28 or 29, wherein the short branched-chain amino acid residue is Val or Thr.

31. The SERPIN peptide according to any one of claims 28-30, wherein the saturated hydrophobic amino acid is Leu.

32. The SERPIN peptide according to any one of claims 28 to 31, wherein the hydrophilic amino acid in D configuration is an Asp, Glu, Lys, Dap, or Cys residue in D configuration.

33. The SERPIN peptide according to any one of claims 28 to 32, wherein: X1 is a basic residue showing a positive charge or none; X2 is a basic residue showing a positive charge or none; and X3 is a basic residue showing a positive charge or none.

34. The SERPIN peptide derivative according to any one of claims 28 to 33, wherein: X1 is an Arg, Lys, or His residue, or none; X2 is an Arg, Lys, or His residue, or none; and X3 is an Arg, Lys, or His residue, or none.

35. The SERPIN peptide derivative according to any one of claims 28 to 34, wherein X6 is Phe or Nal.

36. The SERPIN peptide derivative according to any one of claims 28 to 35, wherein X8 is Phe or Nal.

37. The SERPIN peptide derivative according to any one of claims 28 to 36, wherein X6 is Ala, Phe, or Nal, and X4 is Nal.

38. The SERPIN peptide derivative according to any one of claims 28 to 37, wherein X7 is Asp, Glu, Lys, Dap, or Cys.

39. The SERPIN peptide derivative according to any one of claims 28 to 38, wherein the SERPIN peptide derivative is linear or cyclized.

40. The SERPIN peptide derivative according to any one of claims 28-39, wherein the size of the SERPIN peptide derivative is less than 15 amino acid residues.

41. The SERPIN peptide derivative according to any one of claims 28-40, wherein the size of the SERPIN peptide derivative is 7, 8, 9, 10, 11 or 12 amino acid residues.

42. A SERPIN peptide derivative comprising the sequence of SEQ ID NO: 10-20 or 23-62, wherein the SERPIN peptide derivative comprises 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 a polar head added to the N-terminus of the core sequence and a polar tail added to the C-terminus of the core sequence.

43. 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 the N-terminus and the C-terminus of the core sequence to allow the peptide derivative to cyclize.

44. A SERPIN peptide derivative comprising the sequence of SEQ ID NO: 27-29, 38, 39, or 42-62, wherein one or more amino acid residues in the core sequence are replaced by one or more alternative amino acid residues that are less hydrophobic than the core sequence.

45. A SERPIN peptide derivative comprising the sequence of SEQ ID NO: 30 or 31, wherein one or more amino acid residues in the core sequence are replaced by one or more alternative amino acid residues that are more hydrophobic than the core sequence.

46. ​​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.

47. A SERPIN peptide derivative comprising 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 and a polar tail is added to the C-terminus of the core sequence, or one or more Lys, Glu or His residues are added to the N-terminus of the core sequence and a polar tail is added to the C-terminus of the core sequence.

48. A SERPIN peptide derivative comprising the 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 the N-terminus and C-terminus of the core sequence, so that the SERPIN peptide derivative can be cyclized through a disulfide bond formed between two Cys residues.

49. A SERPIN peptide derivative comprising the 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.

50. A fusion protein comprising the SERPIN peptide derivative according to any one of claims 1 to 49, and an epitope tag, a half-life extender, or an epitope tag and a half-life extender.

51. A conjugate comprising the SERPIN peptide derivative according to any one of claims 1 to 49, and a penetration enhancer.

52. A pharmaceutical composition comprising the SERPIN peptide derivative according to any one of claims 1-49, the fusion protein according to claim 50, or the conjugate according to claim 51.

53. The pharmaceutical composition of claim 52, further comprising one or more additional therapeutic agents.

54. The pharmaceutical composition according to claim 52 or 53, further comprising a pharmaceutically acceptable carrier, excipient, additive, preservative, or a combination thereof.

55. The pharmaceutical composition of any one of claims 52-53, further comprising a penetration enhancer.

56. A pharmaceutical composition according to any one of claims 52-55, wherein the pharmaceutical composition is formulated for oral administration, transdermal administration, or parenteral administration.

57. A fusion protein comprising the SERPIN peptide derivative according to any one of claims 24-41, 45, or 47-49, and an epitope tag, a half-life extender, or an epitope tag and a half-life extender.

58. A conjugate comprising the SERPIN peptide derivative according to any one of claims 24-41, 45, or 47-49, and a penetration enhancer.

59. A pharmaceutical composition comprising the SERPIN peptide derivative according to any one of claims 24-41, 45, or 47-49, the fusion protein according to claim 57, or the conjugate according to claim 58.

60. The pharmaceutical composition of claim 59, further comprising one or more additional therapeutic agents.

61. The pharmaceutical composition according to claim 59 or 60, further comprising a pharmaceutically acceptable carrier, excipient, additive, preservative, or a combination thereof.

62. The pharmaceutical composition of any one of claims 59-61, further comprising a penetration enhancer.

63. A pharmaceutical composition according to any one of claims 59-62, wherein the pharmaceutical composition is formulated for oral administration, transdermal administration, or parenteral administration.

64. A method of 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 according to claims 1-49, a fusion protein according to claim 50, a conjugate according to claim 51, or a pharmaceutical composition according to any one of claims 52-56 to treat the disease or condition associated with LRP1.

65. The method of claim 64, wherein administration of the SERPIN peptide reduces NFkB activation in the subject.

66. The method of claim 64 or 65, wherein administration of the SERPIN peptide reduces TNFa in the subject.

67. The method of any one of claims 64-66, wherein administration of the SERPIN peptide reduces IL-6.

68. The method of any one of claims 64-67, wherein the disease or condition associated with LRP1 is acute or neuropathic pain, nociceptive pain, or inflammatory pain.

69. The method of any one of claims 64-68, wherein the disease or condition associated with LRP1 is a disease or condition caused by a viral infection.

70. The method of claim 69, wherein administration of the SERPIN peptide reduces viral replication.

71. The method of any one of claims 64-67, wherein the disease or condition associated with LRP1 is eosinophilic esophagitis.

72. The method of any one of claims 64-67, wherein the disease or condition associated with LRP1 is acute lung injury.

73. A method of 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 according to any one of claims 24-41, 45, or 47-49, a fusion protein according to claim 57, a conjugate according to claim 58, or a pharmaceutical composition according to any one of claims 59-63 to treat the disease or condition associated with LRP1.

74. The method of claim 73, wherein administration of the SERPIN peptide reduces NFkB activation in the subject.

75. The method of claim 73 or 74, wherein administration of the SERPIN peptide reduces TNFa in the subject.

76. The method of any one of claims 73-75, wherein administration of the SERPIN peptide reduces IL-6.

77. The method of any one of claims 73-75, wherein the disease or condition associated with LRP1 is a disease or condition caused by a viral infection.

78. The method of claim 77, wherein administration of the SERPIN peptide reduces viral replication.

79. The method of any one of claims 73-75, wherein the disease or condition associated with LRP1 is eosinophilic esophagitis.

80. The method of any one of claims 73-75, wherein the disease or condition associated with LRP1 is acute lung injury.

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