Novel peptide having binding affinity to angiotensin-converting enzyme 2(ACE2) and uses thereof

Abstract

The present invention relates to a novel peptide having binding affinity to ACE2 and uses thereof and, more specifically, to: a peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4, and having binding affinity to ACE2; a nanocarrier and a drug complex, each comprising the peptide; and a pharmaceutical composition for preventing or treating COVID-19, comprising the peptide, the nanocarrier, or the drug complex. The novel peptide according to the present invention can be used to increase the COVID-19 therapeutic effect through specific binding to ACE2-expressing cells.

Description

Novel peptides having binding ability to angiotensin-converting enzyme 2 (ACE2) and uses thereof

[0001] The present invention relates to a novel peptide having binding ability to angiotensin-converting enzyme 2 (ACE2), known as a receptor on a cell membrane to which the spike protein of SARS-CoV-2 binds, and a use thereof, and more particularly, to a peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 and having binding ability to ACE2, a nanocarrier comprising the peptide, a drug complex, and a pharmaceutical composition for preventing or treating COVID-19 comprising the peptide, the nanocarrier, or the drug complex.

[0002]

[0003] Angiotensin-converting enzyme (ACE) is an 805-amino acid metalloproteinase located on the X chromosome (Xp22.2). It is a transmembrane glycoprotein with a single extracellular catalytic domain. ACE plays a key regulatory role in the renin-angiotensin system (RAS), converting inactive angiotensin I (Ang I) to angiotensin II (Ang II), which regulates vasoconstriction.

[0004] Angiotensin-converting enzyme 2 (ACE2) is a homolog of ACE that acts on the pathway that reduces the action of angiotensin II (Ang II) to produce angiotensin 1-7 (Ang 1-7). ACE2 is mostly distributed in blood vessels and myocardial tissues and has been reported to have effects opposite to those of angiotensin II, such as reducing blood pressure and inhibiting vascular damage (M Donoghue, et al., Circ Res. 2000 Sep 1;87(5):E1-9).

[0005] It is known that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding of the coronavirus' spike protein (a protrusion-shaped protein on the outer surface of the viral membrane) to a receptor on the cell membrane, allowing the virus to enter the cell. SARS-CoV-1 utilizes ACE2 as a receptor (Jie Cui, et al., Nat Rev Microbiol. 2019 Mar;17(3):181-192). It was confirmed that the spike proteins of SARS-CoV-2 and SARS-CoV-1 have quite similar shapes, and because of this morphological similarity, it was revealed that SARS-Cov-2 also strongly attaches to the surface of host cells through the ACE2 receptor (Alexandra C Walls, et al. Cell. 2020 Apr 16;181(2):281-292.e6; Daniel Wrapp, et al., Science. 2020 Mar 13;367(6483):1260-1263; Peng Zhou, et al., Nature. 2020 Mar;579(7798):270-273). After the coronavirus's spike protein binds to ACE2, the protein scissors present in the host cell cut off a part of the spike protein, allowing the virus to penetrate the cell. In the case of SARS-COV-2, it was found that 'TMPRSS2' in the respiratory cell membrane was used as a protein scissors (Markus Hoffmann, et al., Cell. 2020 Apr 16;181(2):271-280.e8).

[0006] ACE2 has been reported to be distributed in alveolar and small intestinal epithelial cells (I Hamming, et al., J Pathol. 2004 Jun;203(2):631-7). In addition, ACE2 expression was reported to increase after the onset of infection during the course of infection in 624 lung tissues, bronchoalveolar lavage samples, and epithelial cell samples collected from six independent studies (Guoping Li, et al., J Autoimmun. 2020 Aug:112:102463). ACE2 activation is thought to be related to innate or adaptive immune responses, and is suggested to be involved in the regulation of various immune cells, including B cells, and the secretion of cytokines such as IL-1, IL-10, IL-6, and IL-8. That is, the hypothesis is that after the spike protein of the virus binds to ACE2 and enters the alveolar cells, an immune response is triggered and ACE2 expression is enhanced, creating conditions favorable for the entry and proliferation of the virus through genetic changes in epithelial cells and stimulating T cells.

[0007] Therefore, ACE2 is predicted to play a crucial role in SARS-CoV-2 infection and immune responses. Since the binding of SARS-CoV-2 to host cells is the first step in viral infection, strategies to block this pathway are being utilized in the development of vaccines and early therapeutics. It is necessary to develop targeting moieties that preemptively bind to ACE2 to prevent SARS-CoV-2 from binding to ACE2, or that facilitate the delivery of COVID-19 therapeutic drugs to ACE2, thereby targeting SARS-CoV-2 distributed around ACE2.

[0008] Accordingly, the inventors of the present invention have made diligent efforts to solve the problems of the above-mentioned prior art, and as a result, have developed a peptide capable of binding to ACE2, and confirmed that it can be applied as a delivery vehicle containing a COVID-19 treatment agent, thereby completing the present invention.

[0009]

[0010] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0011]

[0012] Summary of the invention

[0013] The purpose of the present invention is to provide a novel peptide capable of binding to ACE2 and a use of the peptide as a delivery vehicle or therapeutic agent for delivering a drug specifically to ACE2-expressing cells.

[0014]

[0015] To achieve the above purpose, the present invention provides a peptide having binding ability to ACE2, represented by any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4.

[0016] The present invention also provides a nanocarrier comprising the peptide, lipid and stabilizer.

[0017] The present invention also provides a drug complex comprising the nanocarrier and a drug.

[0018] The present invention also provides a pharmaceutical composition for preventing or treating COVID-19 comprising the peptide, the nanocarrier, or the drug complex.

[0019] The present invention also provides a method for preventing or treating COVID-19, comprising administering the peptide, the nanocarrier, or the drug complex to a subject in need thereof.

[0020] The present invention also provides a use of the peptide, the nanocarrier, or the drug complex for preventing or treating COVID-19.

[0021] The present invention also provides the use of the peptide, the nanocarrier or the drug complex for the manufacture of a medicament for the prevention or treatment of COVID-19.

[0022]

[0023] Figure 1 shows the results confirming that peptides of sequence numbers 1 to 4 bind to ACE2 protein in a concentration-dependent manner.

[0024] Figure 2 is a confocal microscopic photograph confirming that the peptides of sequence numbers 1 to 4 bind to ACE2 on the cell membrane.

[0025] Figure 3 is a photograph of the properties of solid lipid nanocarriers containing diphenoxylate manufactured by homogenization and sonication (from left, composition 1, composition 2, composition 3, and composition 4).

[0026] Figure 4 shows the results of observing solid lipid nanocarriers (from left, composition 1, composition 2, and composition 4) manufactured by homogenization and ultrasonic treatment using a transmission electron microscope.

[0027] Figure 5 is a confocal micrograph confirming that the solid lipid nanocarriers of compositions 2, 3, and 4 bind to ACE2 in the cell membrane.

[0028] Figure 6 is a graph showing changes in mouse body weight (A) and survival rate (B) according to the viral infection dose.

[0029] Figure 7 is a graph showing the change in body weight (A) and survival rate (B) of mice infected with a virus by diphenoxylate contained in a solid lipid nanocarrier administered intranasally.

[0030] Figure 8 is a graph showing the change in body weight (A) and survival rate (B) of mice infected with a virus by diphenoxylate contained in a solid lipid nanocarrier administered intraperitoneally.

[0031]

[0032] Detailed description of the invention and preferred embodiments

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0034]

[0035] In the present invention, peptide sequences having ACE2 binding ability were discovered using a phage display method, and it was confirmed that they exhibited ACE2 binding ability in a concentration-dependent manner.

[0036]

[0037] Accordingly, the present invention relates to a peptide having binding ability to ACE2, which is represented by any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4.

[0038] Sequence number 1: FDLPKYVTNSDV

[0039] Sequence number 2: AYSHSHASTSAS

[0040] Sequence number 3: GDLTNMHHRHSE

[0041] Sequence number 4: HDASFIGHLPKH

[0042] In this specification, the terms "polypeptide", "peptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues, such as those commonly found in proteins in their natural state.

[0043] Although the peptide of the present invention has been described with respect to a specific amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 4, it is obvious to those skilled in the art that not only the sequence of the peptide of the present invention described in the present specification but also an equivalent range of amino acid sequences with some modifications thereof are interpreted as falling within the scope of the rights of the present invention within the scope of the core technical idea of ​​the present invention having binding ability to ACE2. For example, additional changes may be made to the amino acid sequence in order to further improve the physiological activity properties of the peptide, and such modifications include, for example, deletion, insertion and / or substitution of amino acid sequence residues.

[0044] The peptides of the present invention also include peptides in which a portion of the amino acid sequence is replaced through conservative substitutions. As used herein, the term "conservative substitution" refers to a modification of a polypeptide that involves replacing one or more amino acids with amino acids having similar biochemical properties without causing a loss of the biological or biochemical function of the polypeptide. A "conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains are well known and defined in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It is anticipated that the peptides of the present invention may have conservative amino acid substitutions and still retain activity.

[0045] In the present invention, the peptide may be characterized by having at least 60%, preferably 70%, more preferably 80%, and most preferably 90% sequence homology (or identity) with the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 4, but is not limited thereto, and may be characterized by being a peptide that exhibits substantially the same physiological activity as the peptide of the present invention. Here, the term "substantially" means a state of exhibiting all or nearly the same degree of a specific property. Therefore, in the present specification, "substantially the same physiological activity" means exhibiting a COVID-19 preventive or therapeutic effect by specifically binding to ACE2.

[0046] In one embodiment of the present invention, it was confirmed that the peptides of sequence numbers 1 to 4 actually bind to ACE2 present in the cell membrane.

[0047] In the present invention, a targeting moiety is provided that increases the distribution of the peptide-containing COVID-19 therapeutic agent to ACE2.

[0048]

[0049] In another aspect, the present invention relates to a nucleic acid encoding the peptide.

[0050] In the present invention, the nucleic acid may be characterized by being represented by a base sequence selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 14, but is not limited thereto.

[0051]

[0052] In one embodiment of the present invention, solid lipid nanoparticles were prepared using a peptide having ACE2 binding ability conjugated to palmitic acid, a lipid, and a stabilizer as a delivery vehicle to deliver diphenoxylate, a COVID-19 treatment agent.

[0053] Accordingly, the present invention, from another aspect, relates to a nanocarrier comprising the peptide, lipid and stabilizer.

[0054] In the present invention, the nanocarrier may be characterized by being selected from the group consisting of cationic polymers, liposomes, micelles, emulsions, and solid lipid nanoparticles, but is not limited thereto.

[0055] In the present invention, the lipid may be characterized by being a saturated fatty acid, an unsaturated fatty acid, or a cationic lipid having 12 to 20 carbon atoms, but is not limited thereto. For example, it may be a monoglyceride including 1-glycerol monostearate and 2-glycerol monostearate, glyceryl monooleate, or trimyristin.

[0056] In the present invention, the stabilizer may be, but is not limited to, poloxamer 188, Tween 80, polyvinyl alcohol (PVA), polysorbate (Span) 80, glycerin fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, lecithin, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 (DSPE-PEG2000), D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS), dimyristoylphosphatidylethanolamine-PEG2000 (DMPE-PEG2000), or dipalmitoylphosphatidylethanolamine-PEG2000 (DPPE-PEG2000).

[0057] In the present invention, the peptide may be characterized in that it is coupled through a covalent bond to a fatty acid having 6 to 22 carbon atoms.

[0058] In the present invention, the fatty acid may be selected from the group consisting of oleic acid, linoleic acid, lauric acid, sapienic acid, stearic acid, and palmitic acid, but is not limited thereto.

[0059] In one embodiment of the present invention, the peptide may be characterized as being a peptide in which palmitic acid is linked to the N-terminus of a peptide having sequence numbers 1 to 4.

[0060] In the present invention, the nanocarrier may be characterized in that it is manufactured by a homogenization and ultrasonic treatment method.

[0061] In the present invention, the homogenization and ultrasonic treatment method may be characterized by including, but is not limited to, a first step of dissolving lipids in a solvent; a second step of dissolving a stabilizer in water; a third step of mixing the second step with the solution prepared in the first step; a fourth step of concentrating the mixture formed in the third step to remove the solvent; a fifth step of homogenizing the concentrated solution in the fourth step; and a sixth step of ultrasonicating the mixture formed in the fifth step.

[0062] In the present invention, the fifth step of homogenization utilizes a homogenizer such as a high-pressure homogenizer to enhance the stability of the composition, and may also utilize an ultrasonic emulsifier or homomixer. To further stabilize the manufactured nanoparticles, the homogenization process may be performed more than once.

[0063] Another manufacturing method may be characterized by comprising: a first step of dissolving a lipid in a solvent; a second step of dissolving a stabilizer in water; a third step of mixing the second step with the solution prepared in the first step; a fourth step of spray-drying the mixture formed in the third step to remove the solvent; and a fifth step of obtaining solid lipid nanoparticles produced in the fourth step.

[0064]

[0065] In another aspect, the present invention relates to a drug conjugate comprising the nanocarrier and a drug.

[0066] In the present invention, the drug may be characterized as a SARS-CoV-2 infection treatment agent, and the SARS-CoV-2 infection treatment agent may be characterized as being diphenoxylate, but is not limited thereto.

[0067] In the present invention, the peptide in the drug delivery system may be characterized as being in the range of about 5 to about 10 weight (w / w) percent.

[0068] In the present invention, the lipid in the drug delivery system may be characterized as being in the range of about 15 to about 40 weight (w / w) percent.

[0069] In the present invention, the stabilizer in the drug delivery system may be characterized as being in the range of about 45 to about 80 weight (w / w) percent.

[0070]

[0071] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating coronavirus disease-19 (COVID-19) comprising the peptide, the nanocarrier, or the drug delivery system.

[0072] In another aspect, the present invention relates to a method for preventing or treating COVID-19, comprising a step of administering the peptide, the nanocarrier, or the drug delivery system to a subject in need thereof.

[0073] In another aspect, the present invention relates to the use of the peptide, nanocarrier or drug delivery system for the prevention or treatment of COVID-19.

[0074] In another aspect, the present invention relates to the use of the peptide, the nanocarrier or the drug complex for the manufacture of a medicament for the prevention or treatment of COVID-19.

[0075] COVID-19 (Coronavirus disease 2019, COVID-19) originated in China in December 2019 and spread worldwide. The World Health Organization (WHO) declared a pandemic, the highest level of infectious disease response (March 11, 2020). The International Committee on Taxonomy of Viruses (ICTV) reported that the pathogen of COVID-19 is a variant of SARS-CoV and named it SARS-CoV-2 on February 11, 2020.

[0076] In the present invention, the pharmaceutical composition may be characterized in that it is formulated into any one dosage form selected from the group consisting of injections, oral administration preparations, patches, liquids, capsules, granules, tablets, powders, sprays, nasal administration preparations, inhalation sprays, ointments, gels, mucosal administration preparations, and suppositories, but is not limited thereto. These preparations may be prepared by a conventional method used for formulation in the art or by a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA, and may be formulated into various preparations depending on each disease or ingredient. However, the above description is exemplary, and the preparations to which the present invention is applicable are not limited to the above description.

[0077] In the present invention, the pharmaceutical composition may be characterized by further containing an acceptable adjuvant, and the adjuvant may be, for example, a carrier. Pharmaceutically acceptable carriers include saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, or emulsion, a formulation for nasal administration, an inhalation spray, a pill, a capsule, a granule, or a tablet. However, the above description is exemplary, and the adjuvants or carriers usable in the present invention are not limited to the above description.

[0078] The composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, nasally, bronchially, or topically) depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The daily dosage of the peptide of the present invention is about 1 μg / kg to 100 mg / kg, preferably 5 μg / kg to 50 mg / kg, and can be administered daily or divided into 1 to 3 times a week, but the dosage and administration interval are not limited thereto.

[0079] In this case, the dosage and route of administration will vary depending on the individual patient's age, weight, and response. Appropriate dosage and route of administration can be readily selected by those skilled in the art, taking these factors into account.

[0080]

[0081] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0082]

[0083] Example 1: Identification of peptides with binding ability to ACE2

[0084] Example 1-1: Identification of peptide sequences with binding ability to ACE2 using phage display.

[0085] Phage display was used to discover specific peptide sequences that bind to ACE2. Among them, subtractive panning, a new phage display technique that can be used when trying to discover sequences that bind to a specific substance while not binding to others, was used. M13 phages labeled with a random peptide library of high diversity and complexity were bound to the wells of a polystyrene plate coated with the ACE2 protein. Unbound phages were washed away and only bound phages were recovered. This process was repeated several times, and the final phages obtained were recovered by applying subtractive panning. The DNA sequences encoded by the recovered phages were analyzed to discover peptide sequences that have the ability to bind to the ACE2 protein.

[0086] The phage display kit used was the Ph.D.-12 Phage Display Kit from New England Biolabs (USA), and the final 19 clones discovered using the phages labeled with the random peptide library consisting of 12 amino acids provided in the kit were subjected to sequence analysis by Cosmogene Tech Co., Ltd. in the form of an E. coli culture medium.

[0087] As a result, as shown in Table 1, four sequences showing the same DNA sequence in two or more clones were confirmed, and by translating these, the final peptide sequences consisting of 12 amino acids were obtained as SEQ ID NO. 1 (FDLPKYVTNSDV), SEQ ID NO. 2 (AYSHSHASTSAS), SEQ ID NO. 3 (GDLTNMHHRHSE), and SEQ ID NO. 4 (HDASFIGHLPKH). Since the above peptide sequences are likely to be specific sequences binding to the ACE2 protein, the above peptide sequences were selected as candidates for specific peptide sequences binding to the ACE2 protein.

[0088]

[0089]

[0090]

[0091]

[0092] Example 1-2: Synthesis of a peptide having binding ability to ACE2

[0093] Peptides of SEQ ID NOs. 1 to 4 were synthesized sequentially from the N-terminus using F-moc solid phase peptide synthesis. Palmitic acid and the peptide of SEQ ID NO. 3 were synthesized by removing the F-moc protecting group of the last amino acid of the N-terminus of SEQ ID NO. 3, and then adding DIPEA (10 equivalents), HBTU (5 equivalents), and palmitic acid (5 equivalents) in the presence of DMF. The synthesized peptide sequences were cleaved from the resin, washed, lyophilized, and then separated and purified by liquid chromatography. The molecular weight of the purified peptides was confirmed using MALDI-TOF analysis.

[0094]

[0095] Example 1-3: Confirmation of binding affinity for ACE2

[0096] To confirm the binding affinity with ACE2 protein, the peptides of Example 1-2 were labeled with biotin. Each peptide was biotinylated using EZ-Link Sulfo-NHS-Biotin (Pierce Biotechnology, USA) according to the manufacturer's experimental method, and unbound byproducts were removed through ultrafiltration, a membrane separation method that uses pressure difference as a driving force. Afterwards, the molecular weight of the synthesized product was confirmed by measuring the molecular weight using mass spectrometry. Analysis and purification were performed using analytical reverse phase liquid chromatography. C with a diameter of 4.6 mm 18 The analysis was performed by flowing 0.1% TFA / H2O and 0.092% TFA / acetonitrile at a flow rate of 1 ml / min for 30 minutes with a change of 0 to 60% using a column, and the wavelength of the UV detector was 220 nm. Purification was performed using a 2.2 cm diameter column at a flow rate of 20 ml / min under the same solvent and detection wavelength conditions. Only a portion of the biotin-bound peptide was collected, and the solvent was removed using a rotary evaporator, and then lyophilized.

[0097] The binding affinity for ACE2 protein was measured using an avidin-biotin complex binding assay. ACE2 protein was coated onto each well of a 96-well polystyrene plate at a concentration of 0.02 μg / μl for 16 hours, and then blocked with blocking solution (0.1 M NaHCO3 (pH 8.6), 5 mg / ml BSA, 0.02% NaN3 (optional), filter sterilized) for at least 1 hour. The blocking solution was removed, and the plates were washed vigorously at least six times with washing solution (TBS + 0.1% [v / v] Tween-20). Then, 100 μl of biotin-labeled peptides of SEQ ID NOs: 1 to 4 were dispensed onto the surface of the ACE2 protein-coated wells at concentrations ranging from 0 μM to 500 μM. After reacting for 1 hour at room temperature, each well was washed vigorously more than 6 times with an appropriate washing solution, and 100 μl of ExtrAvidin-Peroxidase (Cat. #E2886, Sigma-Aldrich, USA) was diluted 1:5000 in blocking solution and dispensed into each well. After reacting for 1 hour at room temperature, the wells were washed vigorously more than 6 times with the washing solution, and 100 μl of substrate solution (2,2'-AZINO-BIS, Cat. #A3219, Sigma-Aldrich, USA) was dispensed into each well and the color reaction was performed for 20 minutes at room temperature. After 20 minutes, 100 μl of 1% SDS solution was treated to stop the reaction, and the absorbance was measured at 405 nm.

[0098] As a result, as shown in Fig. 1, when peptides having ACE2 binding ability of SEQ ID NOs: 1 to 4 were treated, the tendency of the peptides to bind to the ACE2 protein in a concentration-dependent manner was confirmed. Among them, the peptide of SEQ ID NO: 3 was measured to have the highest binding ability.

[0099]

[0100] Example 1-4: Confirmation of binding of peptides of sequence numbers 1 to 4 to ACE2 on cell membrane

[0101] To confirm the binding between the ACE2 protein expressed on the cell membrane and the peptides of SEQ ID NO: 1 to SEQ ID NO: 4, a fluorescent material (Alexa Fluor™680 NHS, Cat. # A20008, Thermo Fisher, USA) was attached to the peptides of Example 1-2. Caco-2 cells were seeded at 1x10 in a 4-well chamber slide (Cat. # 154526, Thermo Fisher, USA). 5 Cells were cultured, and treated with fluorescently labeled peptides at 100 μM for 30 minutes, and then washed three times with DPBS. The washed cells were fixed with 4% paraformaldehyde solution for 20 minutes at room temperature. The fixed cells were washed three times with DPBS. ACE2 antibody (Cat. #ab272500, Abcam, UK) was diluted 1:100 in DPBS, treated with the cells, and reacted for 16 hours at 4°C. After washing three times with DPBS, the cells were treated with fluorescently labeled secondary antibody (Mouse anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, FITC, Cat. #31584, Thermo Fisher, USA) at a ratio of 1:200 for 1 hour at room temperature. After washing three times with DPBS, 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Cat. #D1306, Thermo Fisher, USA) was dissolved in PBS at a concentration of 300 nM and treated with the cells for 15 minutes. After washing three times with DPBS, the cells were sealed with Fluoroshield™ (Cat. #F6182-20ML, Merck, Germany). The sealed slides were observed for peptides, ACE2 protein, and cell nuclei using a confocal fluorescence microscope.

[0102] As a result, as shown in Fig. 2, when peptides having ACE2 binding ability of sequence numbers 1 to 4 were treated, it was confirmed that the peptide of sequence number 3 most effectively bound to the ACE2 protein expressed by the cell.

[0103]

[0104] Example 2: Development of a delivery vehicle comprising an ACE2 binding peptide

[0105] Example 2-1: Preparation of solid lipid nanocarriers containing ACE2 binding peptides

[0106] Nanocarriers containing ACE2 binding peptides and drug conjugates containing diphenoxylate (DHL) were prepared by homogenization followed by sonication (Composition 1). Diphenoxylate was dissolved in ethanol at a concentration of 10% (w / v) by stirring or sonication. Monoglyceride (1-glycerol monostearate (GMS)) was dissolved in a 1:1 mixture of ethanol and chloroform at a concentration of 6% (w / v), and the palmitic acid-SEQ ID NO: 3 peptide was dissolved at a concentration of 1% (w / v). 1 ml of ethanol containing dissolved DHL was added to 1 ml of the solution containing dissolved GMS, and the mixture was homogenized using a Mini Homogenizer for 5 minutes. After mixing, the solvent was completely removed using a rotary evaporator. Poloxamer 188 was dissolved in DW at a concentration of 1.5% (w / v), and 5 ml was added to the solid lipid from which the solvent had been completely removed. The mixture was homogeneously mixed for 5 minutes using a Mini Homogenizer (NT-30K, Hangzhou Miu Instruments Co., Ltd.) and then sonicated for 30 minutes (ultrasonic cleaner NXPC-B5020S, KODO Technical Research Co., Ltd.). The sonicated sample was separated using a centrifuge at 4000 rpm for 4 minutes, and the supernatant was filtered through a 0.2 μm filter. The filtered sample was stored in a refrigerator (4°C) or rapidly frozen in liquid nitrogen and lyophilized.

[0107]

[0108] Drug complex composition 2 was prepared using the same homogenization and sonication method as above. Diphenoxylate was dissolved in ethanol at a concentration of 10% (w / v) by stirring or sonication. Monoglyceride (1-glycerol monostearate (GMS)) was dissolved in a 1:1 mixture of ethanol and chloroform at a concentration of 6% (w / v), and the palmitic acid-SEQ ID NO: 3 peptide was dissolved at a concentration of 1% (w / v). Then, 1 ml of ethanol containing DHL was added to 1 ml of the solution containing GMS, and the mixture was homogeneously mixed for 5 minutes using a Mini Homogenizer. After mixing, the solvent was completely removed using a rotary evaporator. Poloxamer 188 was dissolved in DW at a concentration of 2% (w / v), and 5 ml was added to the solid lipid from which the solvent had been completely removed. After homogeneous mixing for 5 minutes using a mini homogenizer, the sample was sonicated for 30 minutes. After sonication, the sample was separated using a centrifuge at 4000 rpm for 4 minutes, and the supernatant was filtered through a 0.2 μm filter. The filtered sample was stored in a refrigerator (4°C) or rapidly frozen in liquid nitrogen and lyophilized.

[0109]

[0110] Drug complex composition 3 was prepared using the same homogenization and sonication method as above. Diphenoxylate was dissolved in ethanol at a concentration of 10% (w / v) by stirring or sonication. Monoglyceride (1-glycerol monostearate (GMS)) was dissolved in a 1:1 mixture of ethanol and chloroform at a concentration of 6% (w / v), and palmitic acid-SEQ ID NO: 3 peptide was dissolved at a concentration of 2% (w / v). Then, 1 ml of ethanol in which DHL was dissolved was added to 1 ml of the solution containing GMS and palmitic acid-SEQ ID NO: 3 peptide, and the mixture was sonicated for 5 minutes to mix. After mixing, the solvent was completely removed using a rotary evaporator. Poloxamer 188 was dissolved in DW at a concentration of 6% (w / v), and 30 ml was added to the solid lipid from which the solvent had been completely removed. After homogeneous mixing for 15 minutes using a homogenizer (HG-15A, DAIHAN Scientific Co., Ltd.), the samples were sonicated for 30 minutes. The sonicated samples were centrifuged at 15,000 rpm at 4°C for 30 minutes, and the supernatant was filtered through a 0.2 μm filter. The filtered samples were stored in a refrigerator (4°C) or rapidly frozen in liquid nitrogen and lyophilized.

[0111]

[0112] Drug complex composition 4 was prepared using the same homogenization and sonication method as above. Diphenoxylate was dissolved in ethanol at a concentration of 10% (w / v) by stirring or sonication. Monoglyceride (1-glycerol monostearate (GMS)) was dissolved in a 1:1 mixture of ethanol and chloroform at a concentration of 6% (w / v), and palmitic acid-SEQ ID NO: 3 peptide was dissolved at a concentration of 2% (w / v). Then, 1 ml of ethanol in which DHL was dissolved was added to 1 ml of the solution containing GMS and palmitic acid-SEQ ID NO: 3 peptide, and the mixture was sonicated for 5 minutes to mix. After mixing, the solvent was completely removed using a rotary evaporator. Tween 80 was dissolved in DW at a concentration of 6% (w / v), and 30 ml was added to the solid lipid from which the solvent had been completely removed. After homogenizing for 15 minutes with a homogenizer, the samples were sonicated for 30 minutes. After sonication, the samples were separated using a centrifuge at 15,000 rpm at 4°C for 30 minutes, and the supernatant was filtered through a 0.2 μm filter. The filtered samples were stored in a refrigerator (4°C) or rapidly frozen in liquid nitrogen and lyophilized.

[0113]

[0114] The composition of each component of Compositions 1, 2, 3, and 4 is as shown in Table 2, and the properties of the drug complexes of Compositions 1, 2, 3, and 4 manufactured are as shown in Figure 3.

[0115]

[0116] The entrapment efficiency of each composition was measured as follows. Solid lipid nanocarriers loaded with diphenoxylate were dissolved in water. Centrifuged at 13,000 rpm, 4℃, for 30 minutes, the supernatant was filtered, and then analyzed by HPLC. The column used was Shim-pack GIS C18, 5㎛, 4.6x150㎜, and the mobile phase A was purified water with 0.1% formic acid added, and the mobile phase B was acetonitrile. The column temperature was set to 30℃, the wavelength was 220nm, and 20㎕ of the solution was injected and analyzed for 25 minutes at an isocratic flow of 30%. The entrapment efficiency was evaluated by centrifuging the aqueous phase and analyzing it by HPLC, and the concentration of the drug not captured in the aqueous phase was calculated as the entrapment efficiency (% entrapment efficiency) using the following calculation formula.

[0117]

[0118] The capture efficiency of the solid lipid nanocarrier of each composition was measured to be over 60%, and compositions 3 and 4 were measured to be over 90% (Table 3).

[0119]

[0120] Example 2-2: Particle size, potential, and properties of solid lipid nanocarriers containing ACE2 binding peptides

[0121] The average diameter and zeta potential of the solid lipid nanocarrier and drug complex containing diphenoxylate were analyzed using particle size analysis equipment and zeta potential measurement equipment. After preparing according to Example 2, the nanoparticles were passed through a 0.2 μm hydrophilic filter and analyzed using a particle size analyzer (ZETASIZER, ZETASIZER Ultra, Malvern Panalytical / UK). In addition, the formed nanoparticles were observed using a transmission electron microscope (TEM, JEM-1230, JEOL).

[0122] Depending on the composition, ratio, and manufacturing method, the average diameter of 13 to 856 nm and the zeta potential of 3 to 54 mV were measured, and compositions 3 and 4 were measured to have an average diameter of less than 100 nm (Table 4).

[0123]

[0124] The characteristics of the drug complex prepared by homogenization and sonication were analyzed using transmission electron microscopy (Fig. 4). The particle morphology confirmed the formation of spherical solid lipid nanoparticles. A single layer was formed on the outside, and the lipid layer encapsulated the diphenoxylate inside.

[0125]

[0126] Example 2-3: Confirmation of binding of solid lipid nanocarriers to cell membrane ACE2

[0127] The binding of the ACE2 protein expressed on the cell membrane and the drug complexes of compositions 2, 3, and 4 of Example 2-1 was confirmed. A fluorescent material (Alexa Fluor™ 680 NHS, Cat. # A20008, Thermo Fisher, USA) was attached to the peptide of palmitic acid-SEQ ID NO: 3, and compositions 1-4 were prepared. Caco-2 cells were seeded at 1x10 in a 4-well chamber slide (Cat. # 154526, Thermo Fisher, USA). 5After culturing the cells, the drug conjugates of compositions 2, 3, and 4 were treated at a concentration of 1 mg / ml for 30 minutes and then washed three times with DPBS. The washed cells were fixed with 4% paraformaldehyde solution for 20 minutes at room temperature. The fixed cells were washed three times with DPBS. ACE2 antibody (Cat. #ab272500, Abcam, UK) was diluted 1:100 in DPBS and treated with the cells, and reacted at 4°C for 16 hours. After washing three times with DPBS, the cells were treated with a fluorescently labeled secondary antibody (Mouse anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, FITC, Cat. #31584, Thermo Fisher, USA) at a ratio of 1:200 for 1 hour at room temperature. After washing three times with DPBS, 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Cat. #D1306, Thermo Fisher, USA) was dissolved in PBS at a concentration of 300 nM and treated with the cells for 15 minutes. After washing three times with DPBS, the cells were sealed with Fluoroshield™ (Cat. #F6182-20ML, Merck, Germany). The sealed slides were observed for solid lipid nanocarriers, ACE2 protein, and cell nuclei using a confocal fluorescence microscope.

[0128] As a result, as shown in Fig. 5, it was confirmed that the drug conjugate of composition 2-4 was bound to the ACE2 protein when treated.

[0129]

[0130] Example 3: Results of an efficacy test of a drug complex containing diphenoxylate against SARS-CoV-2.

[0131] Example 3-1: Experimental Method

[0132] The experiment was conducted with reference to the guidelines for in vivo efficacy testing of COVID-19 therapeutics issued by the National Institute of Food and Drug Safety Evaluation. The efficacy testing was conducted at the BL3 Animal Laboratory of the Korea Research Institute of Chemical Technology.

[0133] SARS-CoV-2 delta (B.1.617.2, NCCP#43390) was used as the virus. Experimental animals were purchased from B6.Cg-Tg(K18-ACE2)2Prlmn / J mice (The Jackson Laboratory, USA). SARS-CoV-2 was injected at 10 pfu to 10 pfu per mouse. 4 Infection was performed by intranasal administration at a concentration of 10 pfu. Body weight changes and survival rates according to the viral infection concentration were observed for 14 days.

[0134] As a result, as shown in Fig. 6, 10 4 When infected with PFU, body weight decreased by less than 80% after infection, and all died within 6 days. 10 3 When infected with PFU, body weight decreased by less than 80% after infection, and all died within 8 days. 10 2 When infected with 10 pfu and 10 pfu of the virus, body weight decreased to its maximum for 8 days and then recovered. The survival rate was 10 2 pfu was about 60%, and 10 pfu was about 70%. When converted to LD50, 10 4 pfu is about 100 LD50, 10 3 pfu was calculated to be approximately 10 LD50. To test the antiviral therapeutic efficacy of diphenoxylate, 10 3 It was decided to inoculate the virus with pfu.

[0135]

[0136] Example 3-2: Confirmation of the therapeutic efficacy of a drug complex containing diphenoxylate against COVID-19

[0137] The therapeutic efficacy of a drug complex containing diphenoxylate against SARS-CoV-2 infection was evaluated. Solid lipid nanocarrier particles (composition 3) were dispersed in 200 μL of water for injection at doses of 0.1, 1, and 10 mg / kg of diphenoxylate and administered intranasally twice (9:00 and 16:00) one day before virus infection. On the day of infection, SARS-CoV-2 was administered at a dose of 10 3 Mice were infected with pfu at 1:00 PM, and the drug was administered intranasally at 9:00 AM and 4:00 PM on the same day. Solid lipid nanocarriers were then administered intranasally twice daily at the same time for four days. Weight changes and survival rates were observed for 14 days after viral infection.

[0138] As a result, as shown in Fig. 7, mice infected with only the virus continued to lose weight and all died on the 8th day. The group administered 0.1 mg / kg of diphenoxylate also lost weight until the 8th day and all died on the 8th day. In the group administered 1 mg / kg of diphenoxylate, body weight decreased to the lowest level until the 11th day. The survival rate was low, with animals dying on days 8, 9, and 10, and all died on the 11th day. In the group administered 10 mg / kg of diphenoxylate, body weight decreased to the lowest level until the 11th day. The survival rate was low, with animals dying on days 7, 8, 9, and 10, and all died on the 11th day. The survival period was 3 days longer than that of the group administered only the virus.

[0139]

[0140] Solid lipid nanocarriers (Formulation 4) were dispersed in 200 μL of water for injection to administer diphenoxylate at doses of 0.1, 1, and 10 mg / kg, and administered intraperitoneally twice (at 9:00 and 16:00) on the day of virus infection. On the same day, SARS-CoV-2 was infected at 13:00 with 103 pfu. Thereafter, solid lipid nanocarriers were administered intraperitoneally at 9:00 and 17:00 for 4 days. Body weight changes and survival rates were observed for 14 days after virus infection.

[0141] As a result, as shown in Fig. 8, mice infected only with the virus showed continuous weight loss and all died on the 6th day. The group administered 0.1 mg / kg of diphenoxylate also showed weight loss until the 6th day and all died on the 6th day. In the group administered 1 mg / kg of diphenoxylate, body weight decreased to the lowest level until the 7th day and then increased again, recovering to the body weight at the time of virus infection on the 14th day. The survival rate was approximately 40% on the 9th day, with deaths starting on the 7th day, but the survival rate was maintained until the 14th day. In the group administered 10 mg / kg of diphenoxylate, body weight decreased to the lowest level until the 7th day, similar to the 1 mg / kg group, but then increased again, recovering to the body weight at the time of virus infection on the 14th day. The survival rate died from the 7th day, was approximately 40% on the 9th day, and was maintained until the 14th day. The results above demonstrate that intraperitoneal administration of solid lipid nanocarriers containing 1 mg / kg and 10 mg / kg of diphenoxylate demonstrated therapeutic efficacy against SARS-CoV-2 infection. Furthermore, intraperitoneal administration, which allows for systemic delivery, was more effective than intranasal administration.

[0142]

[0143] According to the present invention, a novel peptide having binding ability to ACE2, a receptor to which the spike protein of SARS-CoV-2 binds, can be used for targeting a COVID-19 therapeutic agent or targeting a delivery vehicle loaded with a COVID-19 therapeutic agent, thereby increasing the COVID-19 therapeutic effect through specific binding to ACE2-expressing cells.

[0144]

[0145] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0146]

[0147] Electronic file attached.

Claims

1. A peptide having binding ability to ACE2, represented by any one amino acid sequence selected from the group consisting of sequence numbers 1 to 4.

2. A nucleic acid encoding the peptide of paragraph 1.

3. A nanocarrier comprising the peptide, lipid and stabilizer of paragraph 1.

4. A nanocarrier according to claim 3, characterized in that the nanocarrier is selected from the group consisting of cationic polymers, liposomes, micelles, emulsions, and solid lipid nanoparticles.

5. A nanocarrier according to claim 3, characterized in that the peptide is coupled through a covalent bond to a fatty acid having 6 to 22 carbon atoms.

6. A nanocarrier according to claim 5, characterized in that the fatty acid is selected from the group consisting of oleic acid, linoleic acid, lauric acid, sapienic acid, stearic acid, and palmitic acid.

7. A nanocarrier according to claim 3, characterized in that the nanocarrier is manufactured by a homogenization and ultrasonic treatment method.

8. Drug conjugate comprising the nanocarrier of paragraph 3 and a drug.

9. A drug complex according to claim 8, characterized in that the drug is diphenoxylate.

10. A pharmaceutical composition for preventing or treating coronavirus disease-19 (COVID-19), comprising the peptide of paragraph 1, the nanocarrier of paragraph 3, or the drug complex of paragraph 8.

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