Small peptides for inhibiting SARS-COV-2 spike protein-and LPS-induced inflammation and cytokine production

Abstract

Small anti-inflammatory peptide for blocking NF-κB are disclosed herein. The peptides as well as compositions including the peptides can be administered to inhibit cytokine production and / or inhibit pulmonary inflammation in a subject in need. Particularly embodiments include administration to a subject suffering from coronavirus disease 2019 (COVID-19).

Description

SMALL PEPTIDES FOR INHIBITING SARS-COV-2 SPIKE PROTEIN-AND LPS-INDUCED INFLAMMATION AND CYTOKINE PRODUCTIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application Serial No. 63 / 601,771, filed on November 22, 2023, the disclosure of which is hereby incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing containing the file named "3513285.0388 Sequence Listing. xml", which is 9,477 bytes in size (as measured in MICROSOFT WINDOWS® EXPLORER) and was generated November 17, 2022, is provided herein and is herein incorporated by reference. This Sequence Listing consists of SEQ ID Nos: 1-10.BACKGROUND OF THE DISCLOSURE

[0003] The present disclosure relates generally to compositions of antiinflammatory peptides that reduce inflammation. Inflammation and inflammatory cytokines significantly contribute to the development and progression of a variety of diseases including infectious diseases such as hepatitis and coronavirus disease, autoimmune disorders, and cardiovascular diseases (CVDs) such as hypertension, coronary artery diseases (CAD) and atherosclerosis. Macrophages are a major source of inflammatory cytokines and nuclear factor-KB (NF-KB) is critically involved in the generation of inflammatory cytokines including TNF-a. TNF-a is considered the “master” pro-inflammatory cytokine and contributes to both the initiation and maintenance of inflammation and oxidative stress, an important cytokine for the initiation and progression of inflammation and immune responses. Lipopolysaccharide (LPS) is the major component of Gram-negative bacteria cell walls and a potent activator of monocytes / macrophages

[0004] The compositions of the present disclosure can be used to treat and control inflammation due to infectious diseases, autoimmune disorders, injury, radiation, ischemia, chemotherapy, and the like. The anti-inflammatory peptides include lysosomal regulator proteins, tripartite motif-containing proteins and zinc finger proteins.

[0005] One particular infectious disease is the coronavims disease 2019 (COVID- 19), which is a severe systemic inflammatory illness caused by the severe acute respiratory syndrome coronavims 2 (SARS-CoV-2) with high morbidity and mortality. Massive productions of inflammatory cytokines including interleukin-6 (IL-6), interleukin- ip (IL- 1 ), interleukin- 18 (IL- 18), and tumor necrosis factor-a (TNF-a), and subsequent inflammatory storm caused by COVID-19 are well recognized as the primary cause for sudden and rapidly progressing clinical symptoms including respiratory failure, thrombosis, and cardiac damage. Particularly, recognition of SARS-CoV-2 by various pattern recognition receptors (PRRs), such as c-type lectin receptors, leads to increased expressions and release of a variety of pro- inflammatory cytokines, including IL-6, IL- 1 p, IL- 18, and TNF-a, which further prime naive T-cell differentiation to Thl or cytotoxic T lymphocytes, ultimately promoting massive production and secretion of cytokines. The positive feedback loop of pro-inflammatory cytokines on innate and adaptive immune cells reinforces the cytokine storm, leading to excessive systemic inflammation and acute respiratory distress syndrome with poor clinical outcomes.

[0006] The spike protein is an important surface protein of SARS- CoV-2 and potently induces the production and release of various inflammatory cytokines and chemokines, including IL-6, IL-1 p, and TNFa in human and mouse macrophages. It is known that inflammation and oxidative stress are important pathophysiology for a variety of diseases including COVID- 19 related conditions like COVID pneumonia and cardiovascular complications. Thus, targeting inflammatory cytokines production could prevent the development and / or progression of severe COVID- 19 disease and other fatal complications.

[0007] Although a few SARS-CoV-2 vaccines, including mRNA vaccines, have been successfully rolled out in the world, COVID-19 pandemic has not been under effective control, and novel and effective treatments are urgently needed for severe COVID-19.BRIEF DESCRIPTION OF THE DISCLOSURE

[0008] In the present disclosure, a group of small peptides were synthesized and shown to effectively prevent spike protein-induced pulmonary inflammation and attenuate the productions of inflammatory cytokines in male and female subjects.

[0009] In one aspect, the present disclosure is directed to an antiinflammatory peptide having a sequence selected from the group consisting of SEQ ID NO: 1 (SWQLDPTEG), SEQ ID NO:2 (PNRERRRLQR), SEQ ID NO:3 (CYLTIPNK), SEQ ID NO:4 (TDEDEESLGD), SEQ ID NO:5 (EESLGDEEEE), SEQ ID NO:6 (TDEDEESLGDEEEE), SEQ ID NOG (LHIKLEKTHP), SEQ ID NO:8 (PWNMKRH), SEQ ID NO:9 (HERTHTGEKP) and SEQ ID NO: 10 (SWQLDPTEGPNRERRRLQRCYLTIPNK).

[0010] In one aspect, the present disclosure is directed to a composition including at least one anti-inflammatory peptide, the peptide being a lysosomal regulatory peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 (SWQLDPTEG), SEQ ID NO:2 (PNRERRRLQR), SEQ ID NOG (CYLTIPNK) and SEQ ID NO: 10 (SWQLDPTEGPNRERRRLQRCYLTIPNK).

[0011] In one aspect, the present disclosure is directed to a composition including at least one anti-inflammatory peptide, the peptide being a tripartite motif-containing peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4 (TDEDEESLGD), SEQ ID NOG (EESLGDEEEE), and SEQ ID NO:6 (TDEDEESLGDEEEE).

[0012] In one aspect, the present disclosure is directed to a composition including at least one anti-inflammatory peptide, the peptide being a zinc finger peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7 (LHIKLEKTHP), SEQ ID NO:8 (PWNMKRH), and SEQ ID NO:9 (HERTHTGEKP).

[0013] In one aspect, the present disclosure is directed to a method of inhibiting cytokine production in a subject in need thereof. The method includes administering an effective amount of a composition comprising an anti-inflammatory peptide selected from a lysosomal regulatory peptide, a tripartite motif-containing peptide, a zinc finger peptide, and combinations thereof.

[0014] In one aspect, the present disclosure is directed to a method of inhibiting pulmonary inflammation in a subject in need thereof. The method includes administering an effective amount of a composition comprising an anti-inflammatory peptide selected from a lysosomal regulatory peptide, a tripartite motif-containing peptide, a zinc finger peptide, and combinations thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The disclosure will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

[0016] FIG. 1 is a schematic illustration of the experimental design of Example 1.

[0017] FIG. 2 shows that SPS1 exposure induced a significant pulmonary inflammation. H / E staining showed that there were areas of consolidation in the lung of mice 48 hours after intratracheal instillation of SPS1. Treatment of mice with P3 effectively prevented SPS1 -induced pulmonary consolidation. Ctrl: mice exposed to PBS control; SPS1 : mice exposed to SPS1; SPS1+P3: mice exposed to SPS1 and treated with P3.

[0018] FIGS. 3A-3F show that P3 treatment effectively prevented SPS1 exposure-induced significant pulmonary inflammation in mice. Quantitative analysis with Fiji ImageJ (FIGS. 3A & 3B) showed that a significant increase in the percent solid lung tissue areas (FIGS. 3C) and a significant loss of the percent air apace in the lung (FIG. 3D) were observed in male mice 48 hours after intratracheal instillation of SPS1. Treatment of mice with P3 effectively prevented SPSl-induced pulmonary inflammation without increase in the lung solid tissue and preserved lung air space 48 hours after intratracheal instillation of SPS1. Ctrl: mice exposed to PBS and treated with saline; Media Ctrl: mice exposed to vehicle solution for SPS1 and treated with saline; SPS1: mice exposed to SPS1 and treated with saline; SPS1+P3: mice exposed to SPS1 and treated with P3 in saline. Data were presented as mean ± standard error, n=5-6.

[0019] FIG. 4 shows that P3 treatment effectively attenuated SPS1 exposure-induced significant accumulation of inflammatory cells in the lung tissue in mice. Microscopic examination showed that a significant accumulation of inflammatory cells was present in the lung tissue of mice (both male and female mice) 48 hours after intratracheal instillation of SPS1 with significant loss of air space. Treatment of mice with P3 effectively prevented SPSl-induced accumulations of inflammatory cells in the lung tissue with preserved lung air space. Sham: mice without exposure to PBS or SPS1 or any treatment; Ctrl: mice exposed to PBS; SPS1 (Female): female mice exposed to SPS1; SPS1 (Male): male mice exposed to SPS1; SPS1+P3 (Female): female mice exposed to SPS1 and treated with P3. SPS1+P3 (Male): male mice exposed to SPS1 and treated with P3.

[0020] FIG. 5 shows that P3 treatment effectively suppressed SPS 1 exposure-induced increases in the levels of protein and inflammatory cytokines in BALF in mice. Quantitative analysis showed that the levels of protein and inflammatory cytokines IL-6 and TNF-a were significantly increased in BALF from mice (both male and female) 48 hours after intratracheal instillation of SPS 1. Treatment of mice with P3 effectively prevented SPSl-induced increases in the levels of protein and inflammatory cytokines IL-6 and TNF-a in BALF. Ctrl: mice exposedto PBS; SPS1: mice exposed to SPS1; SPS1+P3: mice exposed to SPS1 and treated with P3. Data were presented as mean ± standard error, n=3-6.

[0021] FIG. 6 shows that P3 treatment effectively prevented SPS 1 exposure-induced increases in the levels of plasma inflammatory cytokines in BALF in mice. ELISA analysis showed that the levels of plasma inflammatory cytokines IL- 6 and TNF-a were significantly increased in mice (both male and female) 48 hours after intratracheal instillation of SPS 1. Treatment of mice with P3 effectively prevented SPS 1 -induced increases in the levels of plasma inflammatory cytokines IL- 6 and TNF-a. Ctrl: mice exposed to PBS; SPS1 : mice exposed to SPS1; SPS1+P3: mice exposed to SPS1 and treated with P3. Data were presented as mean ± standard error, n=5-7.

[0022] FIGS. 7A & 7B show that P2 treatment effectively prevented SPS1 exposure-induced pulmonary inflammation and cytokine production in mice. Histological study (FIG. 7A) showed that a significant increase in the solid lung tissue areas with a significant loss of the air apace in the lung were observed in male mice 48 hours after intratracheal instillation of SPS 1. Exposure to SPS1 also significantly increased the levels of BALF protein and IL-1 p, as well as well plasma IL-6 (FIG. 7B). Treatment of mice with P2 (PNRERRRLQR (SEQ ID NO:2), 10 mg / kg a day via i.p.) effectively prevented SPS l-induced pulmonary inflammation and cytokine productions in BALF and plasma. Ctrl: mice exposed to PBS and treated with saline; SPS1: mice exposed to SPS1 and treated with saline; SPS1+P2: mice exposed to SPS 1 and treated with P2 in saline. Data were presented as mean ± standard error, n=3-15.

[0023] FIGS. 8 A & 8B show that P12 treatment significantly attenuated SPS 1 exposure-induced pulmonary inflammation and cytokine production in mice. Histological study (FIG. 8A) showed that a significant increase in the solid lung tissue areas with a significant loss of the air apace in the lung were observed in male mice 48 hours after intratracheal instillation of SPS1. Exposure to SPS1 also significantly increased the levels of BALF protein and BALF TNF-a, IL-6, and IL-1 , as well as well plasma IL-6 (FIG. 8B). Treatment of mice with P12 (SWQLDPTEG(SEQ ID NO: 1), 10 mg / kg a day via i.p.) effectively prevented SPSl-induced pulmonary inflammation and cytokine productions in BALF and plasma. Ctrl: mice exposed to PBS and treated with saline; SPS1 : mice exposed to SPS1 and treated with saline; SPS 1+P12: mice exposed to SPS1 and treated with P12 in saline. Data were presented as mean ± standard error, n=4-15.

[0024] FIGS. 9A & 9B show that P13 treatment significantly attenuated SPS 1 exposure-induced pulmonary inflammation and cytokine production in mice. Histological study (FIG. 9A) showed that a significant increase in the solid lung tissue areas with a significant loss of the air apace in the lung were observed in male mice 48 hours after intratracheal instillation of SPS1. Exposure to SPS1 also significantly increased the levels of BALF protein and BALF TNF-a, IL-6, and IL-1 P, as well as well plasma IL-6 (FIG. 9B). Treatment of mice with Pl 3 (CYLTIPNK (SEQ ID NO:3), 10 mg / kg a day via i.p.) effectively prevented SPSl-induced pulmonary inflammation and cytokine productions in BALF and plasma. Ctrl: mice exposed to PBS and treated with saline; SPS1 : mice exposed to SPS1 and treated with saline; SPS 1+P13: mice exposed to SPS1 and treated with Pl 3 in saline. Data were presented as mean ± standard error, n=4-15.

[0025] FIGS. 10A & 10B show that P 14 treatment significantly attenuated SPS 1 exposure-induced pulmonary inflammation and cytokine production in mice. Histological study (FIG. 10A) showed that a significant increase in the solid lung tissue areas with a significant loss of the air apace in the lung were observed in male mice 48 hours after intratracheal instillation of SPS1. Exposure to SPS1 also significantly increased the levels of BALF protein and plasma IL-6 (FIG. 10B). Treatment of mice with P14 (SWQLDPTEGPNRERRRLQRCYLTIPNK (SEQ ID NO: 10), 10 mg / kg a day via i.p.) effectively prevented SPSl-induced pulmonary inflammation and cytokine productions in plasma. Ctrl: mice exposed to PBS and treated with saline; SPS1 : mice exposed to SPS1 and treated with saline; SPS1+P14: mice exposed to SPS1 and treated with P14 in saline. Data were presented as mean ± standard error, n=4-15.

[0026] FIGS. 11A & 1 IB show that P15 treatment significantly attenuated SPS 1 exposure-induced pulmonary inflammation and cytokine production in mice. Histological study (FIG. 11 A) showed that a significant increase in the solid lung tissue areas with a significant loss of the air apace in the lung were observed in male mice 48 hours after intratracheal instillation of SPS1. Exposure to SPS1 also significantly increased the levels of BALF protein and BALF TNF-a, IL-6, and IL-1 P, as well as well plasma IL-6 (FIG. 1 IB). Treatment of mice with P15 (EESLGDEEEE (SEQ ID NO:5), 10 mg / kg a day via i.p.) effectively prevented SPSl-induced pulmonary inflammation and cytokine productions in BALF and plasma. Ctrl: mice exposed to PBS and treated with saline; SPS1 : mice exposed to SPS1 and treated with saline; SPS1+P15: mice exposed to SPS1 and treated with P15 in saline. Data were presented as mean ± standard error, n=4-15.

[0027] FIGS. 12A & 12B show that P16 treatment significantly attenuated SPS 1 exposure-induced pulmonary inflammation and cytokine production in mice. Histological study (FIG. 12A) showed that a significant increase in the solid lung tissue areas with a significant loss of the air apace in the lung were observed in male mice 48 hours after intratracheal instillation of SPS1. Exposure to SPS1 also significantly increased the levels of BALF protein and plasma IL-6 (FIG. 12B). Treatment of mice with P16 (TDEDEESLGDEEEE (SEQ ID NO:6), 10 mg / kg a day via i.p.) effectively prevented SPSl-induced pulmonary inflammation and cytokine productions in BALF and plasma. Ctrl: mice exposed to PBS and treated with saline; SPS1: mice exposed to SPS1 and treated with saline; SPS1+P16: mice exposed to SPS1 and treated with P 16 in saline. Data were presented as mean ± standard error, n=4-15.

[0028] FIGS. 13A & 13B show that P17 treatment significantly attenuated SPS 1 exposure-induced pulmonary inflammation and cytokine production in mice. Histological study (FIG. 13 A) showed that a significant increase in the solid lung tissue areas with a significant loss of the air apace in the lung were observed in male mice 48 hours after intratracheal instillation of SPS1. Exposure to SPS1 also significantly increased the levels of BALF protein and BALF TNF-a, IL-6, and IL-1 P, as well as well plasma IL-6 (FIG. 13B). Treatment of mice with P17 (LHIKLEKTHP(SEQ ID N0:7), 10 mg / kg a day via i.p.) effectively prevented SPSl-induced pulmonary inflammation and cytokine productions in BALF and plasma. Ctrl: mice exposed to PBS and treated with saline; SPS1 : mice exposed to SPS1 and treated with saline; SPS 1+P 17: mice exposed to SPS1 and treated with Pl 7 in saline. Data were presented as mean ± standard error, n=4-15.

[0029] FIGS. 14A & 14B show that P18 treatment significantly attenuated SPS 1 exposure-induced pulmonary inflammation and cytokine production in mice. Histological study (FIG. 14A) showed that a significant increase in the solid lung tissue areas with a significant loss of the air apace in the lung were observed in male mice 48 hours after intratracheal instillation of SPS1. Exposure to SPS1 also significantly increased the level of BALF IL-1 P (FIG. 14B). Treatment of mice with P18 (PWNMKRH (SEQ ID NO:8), 10 mg / kg a day via i.p.) effectively prevented SPSl-induced pulmonary inflammation and cytokine productions in BALF. Ctrl: mice exposed to PBS and treated with saline; SPS1 : mice exposed to SPS1 and treated with saline; SPS1+P18: mice exposed to SPS1 and treated with P18 in saline. Data were presented as mean ± standard error, n=4-15.

[0030] FIGS. 15A & 15B show that P19 treatment significantly attenuated SPS 1 exposure-induced pulmonary inflammation and cytokine production in mice. Histological study (FIG. 15 A) showed that a significant increase in the solid lung tissue areas with a significant loss of the air apace in the lung were observed in male mice 48 hours after intratracheal instillation of SPS1. Exposure to SPS1 also significantly increased the levels of BALF protein and BALF IL-1 p, as well as well plasma IL-6 (FIG. 15B). Treatment of mice with P19 (HERTHTGEKP (SEQ ID NO:9), 10 mg / kg a day via i.p.) significantly decreased SPSl-induced pulmonary inflammation and cytokine productions in BALF and plasma. Ctrl: mice exposed to PBS and treated with saline; SPS1 : mice exposed to SPS1 and treated with saline; SPS1+P19: mice exposed to SPS1 and treated with Pl 9 in saline. Data were presented as mean ± standard error, n=4-15.

[0031] FIG. 16 shows that P3 treatment effectively prevented LPS- induced activation, NF-kB signaling and production of TNF-a in macrophages invitro. Western blotting analysis showed that exposure to LPS triggered a timedependent increase of NF-kB phosphorylation in macrophages and a significant increase in TNF-a level in the cells. Pretreatment of P3 for 30 min effectively prevented LPS-induced phosphorylation of NF-kB and the increase in TNF-a production in the cells. Random: random sequence; P3: peptide 3; TNF-a: tumor necrosis factor a; Phospho-NF-kB: phosphorylated NF-kB. Experiments were repeated at least 3 times.

[0032] FIG. 17 shows that P3 treatment effectively prevented LPS- induced activation NF-KB signaling in blood monocytes and productions of serum TNF-a and IL- 1 p in mice in vivo. Western blotting analysis showed that LPS treatment triggered a significant increase of NF-kB phosphorylation in blood monocytes and a significant increase in serum TNF-a and IL-1 levels in male mice. Pretreatment of P3 for 12 hours effectively attenuated LPS-induced phosphorylation of NF-kB in blood monocytes and the increases in serum TNF-a and IL- 1 P levels in mice. Random: random sequence; P3: peptide 3; TNF-a: tumor necrosis factor a; 1L- i p: interleukin- 1 beta; Phospho-NF-kB: phosphorylated NF-kB. Experiments were repeated at least 3 times.DETAILED DESCRIPTION

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0034] NF-KB acts as the central signaling hub that interacts with the imiate and adaptive immune systems to enhance cytokines production. Blocking NF- KB expression and / or activation has been shown to decrease the production of various inflammatory mediators, including innate cytokines and chemokines (TNF-a, IL-6, IL- 1 P, CXCL1 and CXCL2) and adaptive cytokines (IL-22, IL- 17 and IFN-y). NF-KB is abundantly expressed in immunoreactive cells especially macrophages. Increased macrophage infiltration has been observed in 18 cases from 21 consecutive COVID- 19 patients. A recent study showed that SARS-CoV-2 spike protein can trigger theproduction of pro-inflammatory cytokines and chemokines in epithelial cells and macrophages by binding with Toll-like receptors (one of the PRRs) and NF-KB activation. Thus, targeting NF-KB or NF-KB -mediated signaling may abolish inflammatory storm and effectively prevent and treat severe COVID-19 disease as well as cardiovascular diseases.

[0035] Accordingly, the present disclosure is directed to the use of small anti-inflammatory peptide for blocking NF-KB expression and / or activation. In one embodiment, the peptide reduces NF-KB signaling activity or function. In one embodiment, the peptide is a lysosomal regulatory peptide including an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (SWQLDPTEG), SEQ ID NO:2 (PNRERRRLQR) and SEQ ID NO:3 (CYLTIPNK). Particularly, the lysosomal regulator}7peptide is one of: SEQ ID NO: 1 (SWQLDPTEG), SEQ ID NO:2 (PNRERRRLQR) and SEQ ID NO:3 (CYLTIPNK).

[0036] In another embodiment, the peptide is a tripartite motifcontaining peptide including an amino acid sequence selected from the group consisting of SEQ ID NO:4 (TDEDEESLGD), SEQ ID NO:5 (EESLGDEEEE), and SEQ ID NO:6 (TDEDEESLGDEEEE). Particularly, the tripartite motif-containing peptide is one of: SEQ ID NO:4 (TDEDEESLGD), SEQ ID NO: 5 (EESLGDEEEE), and SEQ ID NO: (TDEDEESLGDEEEE)

[0037] In yet another embodiment, the peptide is a zinc finger peptide including an amino acid sequence selected from the group consisting of SEQ ID NO: 7 (LHIKLEKTHP), SEQ ID NO: 8 (PWNMKRH), and SEQ ID NO: 9 (HERTHTGEKP). Particularly, the zinc finger peptide is one of: SEQ ID NO: 7 (LHIKLEKTHP), SEQ ID NO: 8 (PWNMKRH), and SEQ ID NO: 9 (HERTHTGEKP).

[0038] Any one or more of the anti-inflammatory peptides described above can be included in a composition. This composition can further include pharmaceutically acceptable carriers. In some suitable embodiments, the pharmaceutically acceptable carriers are, for example, excipients, vehicles, diluents,and combinations thereof. For example, where the combination therapies are to be administered orally, they may be formulated as tablets, capsules, granules, powders, or syrups; or for parenteral administration, they may be formulated as injections (intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intravitreal), drop infusion preparations, or suppositories. In yet other embodiments, the compositions can be administered sublingually or even topically (via skin patch). These compositions can be prepared by conventional means, and, if desired, the active compounds (i.e., anti -infl ammatory peptide) may be mixed with any conventional additive, such as an excipient, a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent, a coating agent, or combinations thereof.

[0039] As noted above, the anti-inflammatory peptides target NF-KB to inhibit or reduce NF-KB-mediated signaling, which can decrease the production of various inflammatory mediators, including innate cytokines and chemokines. Further, this decrease in production prevents inflammation, particularly pulmonary inflammation associated with respirator}' and cardiovascular diseases.

[0040] Accordingly, the compositions of the present disclosure, including the anti-inflammatory peptides described above, alone or with a pharmaceutically acceptable carrier, can be administered in an effective amount to inhibit or reduce cytokine production in a subject in need thereof. As used herein, "effective amount" is that amount of active compound (i.e., anti-inflammatory peptide) that produces the desired effect. One skilled in the art (e.g., physician, medical professional and the like) capable of determining the effective amounts to provide in the composition.

[0041] In another embodiment, the compositions of the present disclosure, including the anti-inflammatory peptides described above, alone or with a pharmaceutically acceptable carrier, can be administered in an effective amount to inhibit or reduce pulmonary inflammation in a subject in need thereof.

[0042] In one embodiment, as used herein, a "subject in need" refers to an individual at risk for or having increased inflammation due to infectious disease, autoimmune disorders, injury, stroke, and the like. In particular embodiments, a "subject in need" refers to an individual suffering from coronavirus disease 2019 (COVID- 19). Additionally, a "subject in need" is also used herein to refer to an individual at risk for or diagnosed by a medical professional as having COVID- 19. As such, in some embodiments, the methods disclosed herein are directed to a subset of the general population such that, in these embodiments, not all of the general population may benefit from the methods. Based on the foregoing, because some of the method embodiments of the present disclosure are directed to specific subsets or subclasses of identified individuals (that is, the subset or subclass of subjects "in need" of assistance in addressing one or more specific conditions noted herein), not all individuals will fall within the subset or subclass of subjects as described herein. In particular, the subject in need is a human. The subject in need can also be, for example, a research animal such as, for example, a non-human primate, a mouse, a rat, a rabbit, a cow. a pig, and other types of research animals known to those skilled in the art.

[0043] Various functions and advantages of these and other embodiments of the present disclosure will be more fully understood from the examples shown below . The examples are intended to illustrate the benefits of the present disclosure, but do not exemplify the full scope of the disclosure.EXAMPLEEXAMPLE 1

[0044] In this Example, a pulmonary inflammation model in mice treated with different amounts of Intratracheal instillation (OP A) of spike protein S I subunit (SPS1) w as used to analyze the effects of small peptides as made herein. Particularly, it was found that small peptides such as those made herein effectively prevented SPS1 -induced pulmonary inflammation and productions of inflammatory cytokines in mice.Materials and MethodsReagents:

[0045] 1) Recombinant SARS-CoV-2 Spike Protein, subunit 1 (SPS1) and its elution solution for control were purchased from RayBiotech (No. 230- 011101-1000).

[0046] 2) Small peptides (P2, P3, P12, P13, P14, P15, P16, P17,Pl 8, and Pl 9) were all designed by the inventors and synthesized by GenScript USA. Inc.

[0047] The sequences of the small peptides:P2 PNRERRRLQR (SEQ ID NO:2)P3 TDEDEESLGD (SEQ ID NO: 4)P 12 S WQLDPTEG (SEQ ID NO : 1 )P 13 CYLTIPNK (SEQ ID NO : 3)P14SWQLDPTEGPNRERRRLQRCYLTIPNK (SEQ ID NO: 10)P15 EESLGDEEEE (SEQ ID NO:5)P16 TDEDEESLGDEEEE (SEQ ID NO:6)P17 LHIKLEKTHP (SEQ ID NO:7)P 18 P WNMKRH (SEQ ID NO : 8)P19 HERTHTGEKP (SEQ ID NO:9)

[0048] 3) Mouse TNF-a high sensitivity ELISA kits (BMS607HS) were purchased from Invitrogen. Mouse IL-6 (ELM-IL6) ELISA detection kits were from RayBiotech, and mouse IL-lb (SMLB00C) detection kits were from R&D Systems.Animal models, study design, and treatments:

[0049] All procedures conducted on mice were in accordance with the Guide for the Care and Use of Laboratory Animals of National Institutes of Health(NIH). All protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Missouri. MO, USA (Protocol Number 10118). Both male and female C57BL / 6 mice (8-12 weeks old) were purchased from Jackson Lab (Bar Harbor, Main, USA). Mice were fed a standard laboratory chow and maintained under controlled conditions (22±1 °C, 12-hour light / 12-hour dark cycle) with free access to water until euthanasia. Mice were randomly divided into experimental groups as shown in FIG. 1 with 5-10 mice in each group as detailed below:

[0050] Group 1: Control (Ctrl). The mice in this group received the same procedures as mice in the other two groups. However, the mice in this group were treated with the vehicle solution used for small peptides (saline, via i.p. injection in a total volume of 250 pl) once daily for 4 days, starting 48 hours prior to intratracheal instillation (OP A) of the vehicle solution for spike protein S 1 subunit (SPS 1, elution solution in PBS with the total volume of 50 pl). At 48 hours of vehicle solution OP A, mice were euthanized to collect lung tissue, BALF, and blood for analysis.

[0051] Group 2: SPS1. The mice in this group were treated with the vehicle solution used for small peptide (saline, via i.p. injection in a total volume of 250 pl) once daily for 4 days, starting 48 hours prior to intratracheal instillation (OP A) of SPS1 (400 ug / kg in PBS with the total volume of 50 pl). At 48 hours of SPS1 OP A, mice were euthanized to collect lung tissue, BALF, and blood for analysis.

[0052] Group 3: Peptides + SPS1. The mice in this group were treated with small peptide (10 mg / Kg for P2, P3, P12. P13, P14, P15, P15, P17, P19, or 5 mg / Kg for P 16, or 2 mg / Kg for P 18. via i.p. injection in a total volume of 250 pl) once daily for 4 days, starting 48 hours prior to intratracheal instillation (OP A) of SPS1 (400 ug / kg in PBS with the total volume of 50 pl). At 48 hours of SPS1 OP A, mice were euthanized to collect lung tissue, and blood for analysis.Lung histology:

[0053] The left lung was collected from each mouse and fixed in 4% formaldehyde for 48 hours followed by 70% EtOH. Lung tissue sections of 5-mm thickness were prepared from upper lobe, middle, and lower lobes, and stained with hematoxylin-eosin (H&E) by BioRepository, School of Medicine, University of Missouri, Columbia, MO.

[0054] Histologically, pulmonary inflammation was evaluated using: 1) percent area of solid tissue in the lung as calculated using the total solid area (ASolid) divided by the total lung area (Atotal) and timing 100 using Fiji ImageJ and Excel software; and 2) percent area of air space in the lung using Fiji ImageJ and Excel softw are. Infiltration of inflammatory cells in the lung was evaluated microscopically.Measurements of BALF total protein and cytokines in BALF and plasma:

[0055] Bronchoalveolar lavage fluid (BALF) was collected from the right lung of each mouse using 0.3 mL PBS. Both BALF and blood samples from each mouse w ere centrifuged at 2,500 g for 10 minutes, and the supernatants were collected for total protein and cytokine analysis. The total protein concentration in each sample was quantitatively determined using Gen5 Microplate Reader by Agilent BioTech as per manufacturer’s protocol. The levels of cytokines in BALF and plasma including IL-6, TNF-a, and IL-lb were quantitatively measured using ELISA kits.ResultsIntratracheal instillation (OP A) of spike protein SI subunit (SPS1) induced significant pulmonary inflammation in mice.

[0056] The condition to establish a pulmonary inflammation model in mice was optimized by treating the mice w ith different amount of SPS1 for different times. Mice developed significant pulmonary inflammation 24 hours after SPS 1 treatment and reached the maximum 48 hours after SPS1 treatment as reflected by an increase in pulmonary consolidation, loss of air space in the lung, increased infiltration of inflammatory cells in the lung tissue, increased BALF protein level, andincreased levels of inflammatory cytokines in BALF and plasma (FIGS. 2-6). SPS1 treatment at the concentration of 400 pg / kg or 500 pg / kg or 600 pg / kg for 48 hours triggered a similar level of pulmonary inflammation (data not shown). Thus, the mice were treated with 400 pg / kg for 48 hours to determine the effect of small peptides on the development of pulmonary' inflammation.Treatment with small peptides effectively prevented SPSl-induced pulmonary inflammation and cytokine production in mice.

[0057] Experiments were then conducted to determine if treatment with the small peptides could attenuate SPS l-induced pulmonary inflammation and the production of inflammatory cytokines in mice. One of the important features for pulmonary inflammation is consolidation of lung tissue with loss of air space in the lung. As shown in FIGS. 2 and 3, SPS1 exposure induced a significant pulmonary inflammation with patched areas of consolidation in the lung tissue (FIG. 2) with significant increase in the percent solid lung tissue areas and a significant loss of the percent air apace in the lung in mice 48 hours after SPS1 exposure (FIG. 3). Treatment of mice with P3 effectively prevented SPSl-induced pulmonary inflammation without visible consolidation of the lung tissue and preserved lung air space.

[0058] Another feature for pulmonary inflammation is an increase in the infiltration of inflammatory cells in the lung tissue. Indeed, microscopic examination of the lung tissue showed that there was a significant accumulation of inflammatory cells in the lung tissue of mice (both male and female mice) 48 hours after intratracheal instillation of SPS1 with significant loss of air space (FIG. 4). Treatment of mice with P3 effectively prevented SPSl-induced accumulations of inflammatory cells in the lung tissue with preserved lung air space (FIG. 4).

[0059] One of the hallmarks for tissue inflammation is an increase in capillary' permeability' with increased leakage of proteins to extracellular space, and increased production of inflammatory cytokines both locally and systematically. The protein level in BALF was significantly increased in mice (both male and female,FIG. 5). ELISA assay showed that the levels of inflammatory cytokines including IL- 6, TNF-a, and IL-ip were significantly increased in BALF and plasma in mice (both male and female. FIGS. 5 and 6) 48 hours after intratracheal instillation of SPS 1. Treatment of mice with P3 effectively attenuated SPS 1 -induced increases in BALF protein level and the levels of inflammatory cytokines in BALF and plasma (FIGS. 5 and 6). In addition to P3, other small peptides on the list were also tested in mice with similar results (data shown in FIGS. 7-15 for each peptide).

[0060] The data from this Example demonstrated that SPS1 exposure triggered a significant pulmonary' inflammation with significant increase in pulmonary consolidation, loss of air space in the lung, increased infiltration of inflammatory7cells in the lung tissue, increased BALF protein level, and increased levels of inflammatory' cytokines in BALF and plasma in both male and female mice 48 hours after SPS1 exposure. P3 treatment effectively prevented SPSl-induced pulmonary inflammation and production of inflammatory cytokines in mice.

[0061] The findings from the present Example are highly significant with tremendous translational and clinical implications. Although the mechanism(s) for the action of these small peptides on SPSl-induced inflammation and productions of inflammatory cytokines is not clear at this moment and needs further investigation, these small peptides can be used in the prevention and / or treatment of the following (but not limited to) conditions:1. COVID-19 pneumonia or myocarditis or other associated conditions.2. Viral myocarditis or other viral infections.3. Autoimmune disorders.4. Non-infectious inflammation (both acute and chronic) including but not limited to CAR-T therapy-related inflammation, arthritis and chronic pain.5. Prevention of rejection after organ transplantations.6. Arthrosclerosis.7. Acute and chronic heart failure.EXAMPLE 2

[0062] In this Example, the ability of small peptides of the present disclosure to inhibit LPS-induced activation of NF-kB and suppress the production of TNF-a in macrophages was analyzed.Methods and Results:A. In Vitro Study with Macrophages:

[0063] Mouse macrophages were cultured and expanded in Complete Macrophage Medium (from Cell Biologies) at a density of 1 x 105cells / mL in 6-well plates at 37 °C in a 5% v / v CO2 atmosphere. After 24 hours of culture, the cells were treated with P3 (10 pg / ml) for 30 minutes, and then exposed to LPS (final concentration of 0.2 pg / ml) for up to 24 hours as described in Bartosh TJ, et al. Macrophage Inflammatory Assay. Bio Protoc. 2014 Jul 20;4(14):el l80. The cells were collected at the baseline, and 3, 6, 12, and 24 hours after exposure to LPS to determine the levels of phosphorylation of NF-KB and intracellular TNF-a using western blotting analysis.

[0064] As shown in FIG. 16, LPS treatment triggered a significant and time-dependent activation of NF-KB signaling as reflected by a significant increase in the level of phosphorylated NF-KB in macrophages, along with a time-dependent production of TNF-a in the cells. As expected, pretreatment of P3 effectively prevented LPS-induced phosphorylation of NF-KB and the increase in TNF-a production in the cells.B. In Vivo Study in Mice:

[0065] To determine if P3 treatment could prevent LPS-induced activation of NF-KB and the productions of inflammatory cytokines, male mice (8-12 weeks of age) were pre-treated with P3 (10 mg / kg, once, via i.p injection) 12 hours prior to LPS injection (25 mg / kg, once, via i.p. injection). Random sequence of the peptide was used as control. After 24 hours of LPS injection, mice were sacrificed to collect the blood to measure the level of NF-KB in blood monocytes and serum levels of IL-1 [3 and TNF-a using Western blotting.

[0066] As shown in FIG. 17, LPS treatment significantly increased the level of NF-KB activation in blood monocytes as reflected by a significant increase in the level of phosphorylated NF-KB, along with a significant increase in the levels of serum TNF-a and IL- 1 p in mice. As expected, pretreatment of P3 effectively prevented LPS-induced phosphorylation of NF-KB and the increases in serum TNF-a and IL-1 p levels in mice.Conclusion:

[0067] P3 treatment effectively suppresses LPS-induced activation of NF-KB signaling in blood monocytes and production of inflammatory cytokines including TNF-a and IL- 1 both in vitro and in vivo (in mice).Significance and clinical implications:

[0068] It is known that inflammation and associated inflammatory cytokines especially TNF-a are critically involved in the development and progression of many diseases including CVDs, autoimmune diseases, diabetes, and COVID- 19 related conditions such as COVID pneumonia and cardiovascular complications. Thus, targeting inflammatory cytokines production could prevent the development and / or progression of diseases associated with inflammation.

[0069] In the present Example, it was demonstrated that LPS exposure activated the NF-KB signaling pathway and increased the production of an important inflammatory cytokine, TNF-a, in macrophages in a time-dependent manner. Treatment with P3 effectively prevented LPS-induced activation of NF-KB and increase in the production of TNF-a in macrophages in vitro.

[0070] The data from the present Example is highly significant with substantial translational and clinical implications. The small peptide can be used in the prevention and / or treatment of the conditions associated with inflammation including (but not limited to):1. COVID-19 pneumonia or myocarditis or other associated conditions.2. Viral myocarditis or other viral infections.

Claims

CLAIMSWhat is claimed is:

1. An anti-inflammatory peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (SWQLDPTEG), SEQ ID NO:2 (PNRERRRLQR), SEQ ID NO:3 (CYLTIPNK), SEQ ID NO:4 (TDEDEESLGD), SEQ ID NO:5 (EESLGDEEEE), SEQ ID NO:6 (TDEDEESLGDEEEE), SEQ ID NO:7 (LHIKLEKTHP), SEQ ID NO: 8 (PWNMKRH), SEQ ID NO: 9 (HERTHTGEKP) and SEQ ID NO: 10 (SWQLDPTEGPNRERRRLQRCYLTIPNK).

2. The anti-inflammatory peptide of claim 1, wherein the peptide reduces of NF-KB signaling activity.

3. A composition comprising at least one anti-inflammator ' peptide, wherein the at least one anti-inflammatory peptide is a lysosomal regulatory peptide and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (SWQLDPTEG), SEQ ID NO:2 (PNRERRRLQR), SEQ ID NO:3 (CYLTIPNK) and SEQ ID NOTO (SWQLDPTEGPNRERRRLQRCYLTIPNK).

4. The composition of claim 3, wherein the lysosomal regulatory peptide is selected from the group consisting of SEQ ID NO: 1 (SWQLDPTEG), SEQ ID NO:2 (PNRERRRLQR), SEQ ID NO:3 (CYLTIPNK) and SEQ ID NOTO (SWQLDPTEGPNRERRRLQRCYLTIPNK).

5. A composition comprising at least one anti-inflammatory peptide, wherein the at least one anti-inflammatory peptide is a tripartite motif-containing peptide and comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4 (TDEDEESLGD), SEQ ID NO:5 (EESLGDEEEE), and SEQ ID NO:6 (TDEDEESLGDEEEE).

6. The composition of claim 5, wherein the tripartite motif-containing peptide is selected from the group consisting of SEQ ID NO:4 (TDEDEESLGD). SEQ ID NO:5 (EESLGDEEEE), and SEQ ID NO:6 (TDEDEESLGDEEEE).

7. A composition comprising at least one anti-inflammatory peptide, wherein the at least one anti-inflammatory peptide is a zinc finger peptide and compnses an amino acid sequence selected from the group consisting of SEQ ID NOY (LHIKLEKTHP), SEQ ID NO: 8 (PWNMKRH), and SEQ ID NO: 9 (HERTHTGEKP).

8. The composition of claim 7, wherein the zinc finger peptide is selected from the group consisting of SEQ ID NO:7 (LHIKLEKTHP), SEQ ID NO:8 (PWNMKRH), and SEQ ID NO: 9 (HERTHTGEKP).

9. The composition of any one of claims 3, 5 or 7 further comprising a pharmaceutically acceptable carrier.

10. A method of inhibiting cytokine production in a subj ect in need thereof, the method comprising administering an effective amount of the composition of any one of claims 3, 5, or 7.

11. The method of claim 10, wherein the subject has or had coronavirus 2019 (COVID-19).

12. A method of inhibiting pulmonary' inflammation in a subject in need thereof, the method comprising administering an effective amount of the composition of any one of claims 3, 5, or 7.

13. The method of claim 12, wherein the subject has or had suffered from COVID 19.

14. Use of an anti-inflammatory’ peptide fortreating COVID19 by inhibiting cytokine production wherein the anti-inflammatory peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (SWQLDPTEG), SEQ ID NO:2 (PNRERRRLQR), SEQ ID NO:3 (CYLTIPNK), SEQ ID NO:4 (TDEDEESLGD), SEQ ID NO:5 (EESLGDEEEE), SEQ ID NO:6 (TDEDEESLGDEEEE). SEQ ID NO:7 (LHIKLEKTHP). SEQ ID NO:8 (PWNMKRH), SEQ ID NO:9 (HERTHTGEKP) and SEQ ID NO: 10 (SWQLDPTEGPNRERRRLQRCYLTIPNK).

15. Use of an anti-inflammatory peptide for treating COVID 19 by inhibiting pulmonary inflammation wherein the anti-inflammatory peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (SWQLDPTEG). SEQ ID NO:2 (PNRERRRLQR), SEQ ID NO:3 (CYLTIPNK), SEQ ID NO:4 (TDEDEESLGD), SEQ ID NO:5 (EESLGDEEEE), SEQ ID NO:6 (TDEDEESLGDEEEE), SEQ ID NO:7 (LHIKLEKTHP). SEQ ID NO:8 (PWNMKRH). SEQ ID NOV (HERTHTGEKP) and SEQ ID NO: 10 (SWQLDPTEGPNRERRRLQRCYLTIPNK).

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