Pharmaceutical composition for preventing or treating idiopathic pulmonary fibrosis containing sACE2 nanodiscs

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of idiopathic pulmonary fibrosis containing sACE2 nanodiscs, wherein the 'sACE2_MSP_Fc nanodiscs' or 'sACE2_Fc_MSP' of the present invention exhibited therapeutic and preventive effects against IPF.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of idiopathic pulmonary fibrosis, and more specifically, to a pharmaceutical composition for the prevention or treatment of idiopathic pulmonary fibrosis containing sACE2 nanodiscs. Background Technology

[0003] Fibrosis refers to a phenomenon in which parts of an organ harden due to various causes, rendering them unable to function normally; pulmonary fibrosis and hepatic fibrosis are typical examples. Currently, there are no drugs available to cure fibrosis, and treatment focuses on slowing its progression or alleviating symptoms.

[0004] Tissue transplantation is currently the only curative method for treating fibrosis. Pulmonary fibrosis is a chronic lung disease in which lung tissue thickens and hardens. This leads to a decline in lung function and difficulty breathing, making daily life impossible in severe cases.

[0005] Pulmonary fibrosis is defined as the process of excessive accumulation of connective tissue and collagen in the interstitium, the stromal tissue of the lungs. These pathological changes can be caused by various factors, and idiopathic pulmonary fibrosis, in which the clear cause is unknown, is the most common form.

[0006] The 5-year survival rate after diagnosis for idiopathic pulmonary fibrosis is poor, at 43%, and the 10-year survival rate is 15%. Although many studies are being conducted, there is currently no treatment method that has improved survival rates. The most common causes of death are respiratory failure (39%) and heart disease (27%), and others include lung cancer, pulmonary embolism, and pneumonia. Prior art literature

[0008] As a pharmaceutical composition for the treatment and prevention of pulmonary fibrosis, Korean Patent Publication No. 1020110119108 (2011.11.02) describes a pharmaceutical composition for the treatment and prevention of pulmonary fibrosis containing grape seed proanthocyanidin extract (GSPE) as an active ingredient. According to this invention, since pulmonary fibrosis is inhibited in a bleomycin-induced pulmonary fibrosis animal model, it is described as being useful as a treatment for idiopathic pulmonary fibrosis by expanding clinical indications. The problem to be solved

[0009] Treatment methods for idiopathic pulmonary fibrosis involve the use of steroids and cytotoxic drugs; recently, anti-fibrotic agents have become the dominant approach, with various attempts being made. Currently, pirfenidone and nintedanib are approved drugs for idiopathic pulmonary fibrosis. However, these drugs do not serve as a cure but rather delay pulmonary fibrosis and alleviate symptoms. Therefore, there is a need to develop more effective drugs to enable patients to lead normal daily lives. means of solving the problem

[0011] The present invention provides a pharmaceutical composition for the prevention or treatment of idiopathic pulmonary fibrosis containing a nanodisk, comprising: a lipid bilayer having a flat disc-shaped bilayer structure formed from phospholipids, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; and a membrane scaffold protein (MSP) surrounding the side of the lipid bilayer where the hydrophobic groups are exposed outward, wherein the membrane scaffold protein has an angiotensin-converting enzyme 2 (ACE2) bound to one end and an Fc fragment bound to the other end, and the bound angiotensin-converting enzyme 2 (ACE2) and the Fc fragment protrude outward from the nanodisk.

[0012] In addition, the present invention comprises a lipid bilayer having a flat disc-shaped bilayer structure formed from phospholipids, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; A pharmaceutical composition for the prevention or treatment of idiopathic pulmonary fibrosis containing a nanodisk comprising: a membrane scaffold protein (MSP) surrounding the side of the lipid bilayer where the hydrophobic group is exposed to the outside, wherein the membrane scaffold protein has an angiotensin-converting enzyme 2 (ACE2) bound to one end and an Fc fragment bound to the other end, and the membrane scaffold protein is a membrane scaffold protein with an elongated length formed by the interconnection of multiple membrane scaffold proteins through the mutual binding of the angiotensin-converting enzyme 2 of the membrane scaffold protein to form a dimer and the mutual binding of the Fc fragment of the membrane scaffold protein to form a dimer, and the bound angiotensin-converting enzyme 2 (ACE2) and the Fc fragment protrude to the outside of the nanodisk.

[0013] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of idiopathic pulmonary fibrosis containing a nanodisk, comprising: a lipid bilayer having a flat disc-shaped bilayer structure formed from phospholipids, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; and a membrane scaffold protein (MSP) surrounding the side of the lipid bilayer where the hydrophobic groups are exposed outward, wherein the membrane scaffold protein has angiotensin-converting enzyme 2 (ACE2) and an Fc fragment sequentially bound to the N-terminus in the direction from the N-terminus to the C-terminus, and said bound angiotensin-converting enzyme 2 (ACE2) and Fc fragment protruding outward from the nanodisk.

[0014] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of idiopathic pulmonary fibrosis containing a nanodisk, comprising: a lipid bilayer having a flat disc-shaped bilayer structure formed from phospholipids, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; and a membrane scaffold protein (MSP) surrounding the side of the lipid bilayer where the hydrophobic groups are exposed outward, wherein the membrane scaffold protein has an Fc fragment and angiotensin-converting enzyme 2 (ACE2) sequentially bound to the C-terminus from the N-terminus to the C-terminus, and the bound angiotensin-converting enzyme 2 (ACE2) and the Fc fragment protrude outward from the nanodisk.

[0015] In the pharmaceutical composition of the present invention, the phospholipid may preferably be one or more selected from phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol.

[0016] In the pharmaceutical composition of the present invention, the phospholipid may be one or more selected from DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine).

[0017] In the pharmaceutical composition of the present invention, the membrane scaffold protein may preferably be an amphiphilic protein having a helix structure. In this case, the membrane scaffold protein may be, for example, an apolipoprotein or a fragment of an apolipoprotein in which the 'helix structure and amphiphilic properties' of the apolipoprotein are maintained.

[0018] In the pharmaceutical composition of the present invention, the angiotensin-converting enzyme 2 may preferably be a water-soluble angiotensin-converting enzyme 2 from which the transmembrane domain has been removed.

[0019] In the pharmaceutical composition of the present invention, the nanodisk may preferably have one layer of the double layer surrounded by a membrane structuring protein, and the other layer of the double layer surrounded by another membrane structuring protein.

[0020] In the pharmaceutical composition of the present invention, the nanodisc may preferably be formed by mutually binding angiotensin-converting enzyme 2 of a membrane-structuring protein surrounding one layer of the double layer and angiotensin-converting enzyme 2 of a membrane-structuring protein surrounding the other layer of the double layer to form a dimer.

[0021] In the pharmaceutical composition of the present invention, the nanodisk may preferably be formed by mutually combining an Fc fragment of a membrane-structuring protein surrounding one layer of the double layer and an Fc fragment of a membrane-structuring protein surrounding the other layer of the double layer to form a dimer.

[0022] In the pharmaceutical composition of the present invention, the nanodisk may preferably be such that one layer of the double layer is surrounded by an extended membrane-structuring protein, and the other layer of the double layer is surrounded by another extended membrane-structuring protein. Effects of the invention

[0024] According to the present invention, 'sACE2_Fc' of Preparation Example 1 of the present invention inhibited the progression of IPF by about 40%. Nintedanib, used as a positive control, inhibited the progression of IPF by about 50%. However, the 'sACE2_MSP_Fc nanodisc' and 'sACE2_Fc_MSP nanodisc' of the present invention inhibited the progression of IPF by about 70%, and when sACE2_Fc, sACE2_MSP_Fc nanodisc, and Nintedanib were co-administered, sACE2_Fc had no effect, while sACE2_MSP_Fc inhibited the progression of IPF by about 85%. From the experimental results of the present invention, it was confirmed that the 'sACE2_MSP_Fc nanodisc' and 'sACE2_Fc_MSP nanodisc' of the present invention have potential as therapeutic and preventive substances for IPF. Brief explanation of the drawing

[0026] FIG. 1 is a schematic diagram of the fabrication of Preparation Example 2 'sACE2_Fc_MSP nanodisk' and Preparation Example 3 'sACE2_MSP_Fc nanodisk' of the present invention. The upper part of Fig. 2 shows the SEC-FPLC separation and purification results confirming that Preparation Example 2 'sACE2_Fc_MSP nanodisk' and Preparation Example 3 'sACE2_MSP_Fc nanodisk' were successfully fabricated by the lipid film manufacturing method, and the lower part of Fig. 2 shows the SEC-FPLC separation and purification and DLS analysis results confirming that Preparation Example 4 'sACE2_Fc_MSP nanodisk' and Preparation Example 5 'sACE2_MSP_Fc nanodisk' were successfully fabricated by the microfluidic system manufacturing method. FIG. 3 is an overview of a mouse model experiment according to Example 1 of the present invention. In FIG. 1, Sham: administration of PBS, BLM: administration of PBS after inducing pulmonary fibrosis with bleomycin, BLM+N: administration of Nintedanib after inducing pulmonary fibrosis with bleomycin, SF: administration of sACE2_Fc after inducing pulmonary fibrosis with bleomycin, SF+N: administration of sACE2_Fc and Nintedanib after inducing pulmonary fibrosis with bleomycin, SFM: administration of 'sACE2_Fc_MSP nanodisc' after inducing pulmonary fibrosis with bleomycin, SMF: administration of 'sACE2_MSP_Fc nanodisc' after inducing pulmonary fibrosis with bleomycin, SMF+N: administration of 'sACE2_MSP_Fc nanodisc and Nintedanib' after inducing pulmonary fibrosis with bleomycin. Figure 4 is an Ashcroft score graph. In Figure 2, Sham: PBS administration, BLM: PBS administration after inducing pulmonary fibrosis with bleomycin, BLM+N: Nintedanib administration after inducing pulmonary fibrosis with bleomycin, SF: sACE2_Fc administration after inducing pulmonary fibrosis with bleomycin, SF+N: sACE2_Fc and Nintedanib administration after inducing pulmonary fibrosis with bleomycin, SFM: 'sACE2_Fc_MSP nanodisc' administration after inducing pulmonary fibrosis with bleomycin, SMF: 'sACE2_MSP_Fc nanodisc' administration after inducing pulmonary fibrosis with bleomycin, SMF+N: 'sACE2_MSP_Fc nanodisc and Nintedanib' administration after inducing pulmonary fibrosis with bleomycin. Figure 5 shows the results of the comparison of lung tissue fibrosis. In Figure 3, Sham: administration of PBS, BLM: administration of PBS after inducing pulmonary fibrosis with bleomycin, BLM+N: administration of Nintedanib after inducing pulmonary fibrosis with bleomycin, SF: administration of sACE2_Fc after inducing pulmonary fibrosis with bleomycin, SF+N: administration of sACE2_Fc and Nintedanib after inducing pulmonary fibrosis with bleomycin, SFM: administration of 'sACE2_Fc_MSP nanodiscs' after inducing pulmonary fibrosis with bleomycin, SMF: administration of 'sACE2_MSP_Fc nanodiscs' after inducing pulmonary fibrosis with bleomycin, SMF+N: administration of 'sACE2_MSP_Fc nanodiscs and Nintedanib' after inducing pulmonary fibrosis with bleomycin. Specific details for implementing the invention

[0027] Angiotensin-converting enzyme 2 (ACE2) plays an important role in the renin-angiotensin-aldosterone system (RAAS), which regulates body fluids and blood pressure. In the renin-angiotensin-aldosterone system (RAAS), angiotensinogen is converted to angiotensin I by renin, and angiotensin-converting enzyme (ACE) converts angiotensin I into angiotensin II. Angiotensin II directly has an 'adverse effect' on the cardiovascular system, such as inflammatory responses within blood vessels and the promotion of atherosclerosis. However, angiotensin II in the body is converted into angiotensin (1-7) by ACE2, and as a result, homeostasis can be maintained. Therefore, ACE2 can be considered an important enzyme for maintaining homeostasis in the body.

[0028] Meanwhile, ACE2 / ANG(1-7) / MasR signaling is reported to have anti-inflammatory, antioxidant, vasodilating, anti-fibrotic, and anti-apoptotic effects, contrary to ACE / ANG2 / AT1R signaling. In particular, regarding liver fibrosis, ANG2 activates AT1R to induce liver fibrosis, and it has been reported that when ANG2 is converted to ANG(1-7) by ACE2, it inhibits downstream signaling of AT1R, thereby attenuating liver fibrosis. In addition, there are reports that idiopathic pulmonary fibrosis is reduced when ACE2 is overexpressed in mice, as well as reports that idiopathic pulmonary fibrosis is reduced when recombinant ACE2 (sACE2) is administered to mice.

[0029] Meanwhile, the inventors prepared nanodiscs using 'sACE2_MSP_Fc' or 'sACE2_Fc_MSP' fusion proteins, in which sACE2 and Fc are respectively bound to both ends of a membrane-structuring protein, and sought to elucidate their role in idiopathic pulmonary fibrosis. As a result of evaluating efficacy against idiopathic pulmonary fibrosis using a BLM-induced model, it was confirmed that fibrosis was reduced in the 'sACE2_MSP_Fc nanodiscs' or 'sACE2_Fc_MSP nanodiscs', and the present invention was completed.

[0030] In the present invention, angiotensin converting enzyme 2 (ACE2) is used, and the angiotensin converting enzyme 2 (ACE2) is used by binding it to a membrane scaffold protein (MSP). At this time, it is preferable to use sACE2, in which the hydrophobic transmembrane domain at the C-terminus of the ACE2 has been removed.

[0031] ACE2 is composed of a binding domain located at the N-terminus and a transmembrane domain located at the C-terminus; sACE2 is obtained by removing this C-terminal transmembrane domain. sACE2 with the C-terminal transmembrane domain removed is expressed in a soluble state, which is why sACE2 is also called 'soluble angiotensin-converting enzyme 2 (soluble ACE2)'. Alternatively, it is also called recombinant angiotensin-converting enzyme 2 (recombinant ACE2).

[0032] Meanwhile, the inventors have disclosed, through Korean Patent Registration No. 102438720 (August 26, 2022), a pharmaceutical composition and a food composition for the prevention or treatment of coronavirus infection having a spike protein capable of binding to ACE2, characterized by containing a nanodisc comprising: a lipid bilayer having a flat disc-shaped bilayer structure formed from phospholipids, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; a membrane scaffold protein (MSP) surrounding the side of the lipid bilayer where the hydrophobic groups are exposed outwardly with hydrophobic bonds; and an angiotensin converting enzyme 2 (ACE2) hydrophobically bonded to the inside of the lipid bilayer.

[0033] In addition, the inventors have disclosed a method for manufacturing a nanodisc containing angiotensin converting enzyme 2 through Korean Patent Registration No. 102610178 (November 30, 2023), wherein the membrane scaffold protein (MSP) and angiotensin converting enzyme 2 are mixed to produce a nanodisc containing angiotensin converting enzyme 2, wherein the membrane scaffold protein (MSP) and angiotensin converting enzyme 2 are fused.

[0034] In addition, the inventors of the present invention [apply] Korean Patent Application 10-2023-0133618 (filing date Oct. 2023.5) Through the word, “a lipid bilayer having a flat, disc-shaped bilayer structure formed from phospholipids, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; A nanodisk comprising a membrane scaffold protein (MSP) surrounding the 'side where hydrophobic groups are exposed to the outside' of the lipid bilayer, wherein the membrane scaffold protein has angiotensin-converting enzyme 2 (ACE2) bound to one end and an Fc fragment bound to the other end, and the bound angiotensin-converting enzyme 2 (ACE2) and the Fc fragment protruding to the outside of the nanodisk, and a lipid bilayer formed from a flat disc-shaped bilayer structure in which hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; A nanodisk comprising a membrane scaffold protein (MSP) surrounding the side of the lipid bilayer where the hydrophobic group is exposed to the outside, wherein the membrane scaffold protein has an angiotensin-converting enzyme 2 (ACE2) attached to one end and an Fc fragment attached to the other end, and the membrane scaffold protein is a membrane scaffold protein with an elongated length formed by multiple membrane scaffold proteins interconnected through the mutual binding of the angiotensin-converting enzyme 2 of the membrane scaffold protein to form a dimer and the mutual binding of the Fc fragment of the membrane scaffold protein to form a dimer, and the attached angiotensin-converting enzyme 2 (ACE2) and the Fc fragment protruding to the outside of the nanodisk.

[0035] Following the filing of the aforementioned patent, the inventors conducted follow-up research using the nanodiscs and confirmed that nanodiscs prepared using "sACE2_MSP_Fc" or "sACE2_Fc_MSP" reduced pulmonary fibrosis in "idiopathic pulmonary fibrosis." Furthermore, they confirmed that nanodiscs prepared using "sACE2_MSP_Fc" showed a higher fibrosis reduction effect compared to nanodiscs prepared using "sACE2_Fc_MSP".

[0036] The present invention uses nanodiscs prepared using membrane-structuring proteins in which angiotensin-converting enzyme 2 and an Fc fragment are respectively bound to both ends (LMW A-ND-Fc, MMW A-ND-Fc, HMW A-ND-Fc, see FIG. 1). Additionally, the present invention uses nanodiscs prepared using membrane-structuring proteins in which angiotensin-converting enzyme 2 and an Fc fragment are sequentially bound (see FIG. 1).

[0037] Meanwhile, in the present invention, the phospholipid may be one or more selected from the group consisting of, for example, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol.

[0038] 상기 포스파티딜콜린(phosphatidylcholine)은 일 예로 DOPC(1,2-Dioleoyl-sn-glycero-3-phosphocholine), DLPC(1,2-Dilauroyl-sn-glycero-3-phosphocholine), DMPC(1,2-Dimyristoyl-sn-glycero-3-phosphocholine), DPPC(1,2-Dipalmitoyl-sn-glycero-3-phosphocholine), POPC(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), C13PC, DDPC(1,2-Didecanoyl-sn-glycero-3-phosphocholine), DSPC(1,2-Distearoyl-sn-glycero-3-phosphocholine), DEPC(1,2-Dierucoyl-sn-glycero-3-phosphocholine), DLOPC(1,2-Dilinoleoyl-sn-glycero-3-phosphocholine), EPC(Egg phosphatidylcholine), MSPC( 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine), PMPC(1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine), PSPC(1-Palmitoyl-2- stearoyl-sn-glycero-3-phosphocholine), SMPC(1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine) 또는 SPPC(1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine)일 수 있다.

[0039] In addition, the above phosphatidylglycerol is, for example, DMPG(1,2-Dimyristoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), DPPG(1,2-Dipalmitoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), DSPG(1,2-Distearoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), POPG(1-Palmitoyl-2-oleoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), DEPG(1,2-Dierucoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), DLPG(1,2-Dilauroyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), It may be DOPG(1,2-Dioleoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]) or DSPG(1,2-Distearoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]).

[0040] In addition, the phosphatidylethanolamine includes DMPE (1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine), DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), It may be DEPE (1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine), or POPE (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine).

[0041] In addition, the phosphatidylserine may be DOPS (1,2-Dioleoyl-sn-glycero-3-phosphoserine), DLPS (1,2-Dilauroyl-sn-glycero-3-phosphoserine), DMPS (1,2-Dimyristoyl-sn-glycero-3-phosphoserine), DPPS (1,2-Dipalmitoyl-sn-glycero-3-phosphoserine), DSPS (1,2-Distearoyl-sn-glycero-3-phosphoserine) or POPS (POPS).

[0042] In addition, the above phosphatidylinositol may be phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5-bisphosphate, or phosphatidylinositol-3,4,5-bisphosphate.

[0043] Meanwhile, in the present invention, the membrane scaffold protein (MSP) has a helix structure and possesses amphiphilic characteristics, serving to surround the sides of the lipid bilayer.

[0044] Multiple membrane-structuring proteins may surround the sides of a nanodisk lipid bilayer. Each layer of the lipid bilayer may be surrounded by a membrane-structuring protein, or a single layer of the lipid bilayer may be surrounded by a membrane-structuring protein with an elongated length formed by combining multiple membrane-structuring proteins. In this case, when a single layer is surrounded by a membrane-structuring protein with an elongated length formed by combining multiple membrane-structuring proteins, a medium or large nanodisk with an increased diameter is formed.

[0045] An example of a membrane-structuring protein is apolipoprotein. Apolipoprotein is a protein that is specifically present in plasma lipoproteins and is known to stabilize the structure of lipoproteins, activate enzymes involved in lipoprotein metabolism, and function as a ligand for lipoprotein receptors present on the cell surface. The above apolipoproteins include, as examples, apolipoprotein A1 (ApoA-I), apolipoprotein A2 (ApoA-2), apolipoprotein B (ApoB), apolipoprotein C (ApoC), and apolipoprotein E (ApoE), MSP1 (Membrane scaffold protein 1), MSP1D1, MSP1D2, MSP1E1, MSP1E2, MSP1E3, MSP1E3D1, MSP2, MSP2N1, MSP2N2, MSP2N3, etc.

[0046] ApoA-I, mentioned above as an example, is known to be a component of high-density lipoprotein (HDL) that performs the direct role of removing cholesterol from surrounding tissues and transporting it to the liver or other lipoproteins. ApoA1 consists of a single polypeptide with 243 amino acids and a molecular weight of 28 kDa. It has eight repeating unit domains consisting of 11 or 22 amino acids, and is a protein in which the proportion of alpha-helix in the secondary structure constituting HDL is 60 to 75%. In addition, ApoE is known to be involved in cholesterol transport, similar to ApoA1, and is a protein composed of a single polypeptide with 299 amino acids and a molecular weight of 33 kDa.

[0047] In addition, as the membrane structuring protein in the present invention, a fragment of an apolipoprotein in which the 'helix structure and amphiphilic properties of the apolipoprotein' are maintained may be used. That is, within a range that does not lose the 'helix structure and amphiphilic properties' of the apolipoprotein, a part (fragment) of the apolipoprotein, rather than the whole apolipoprotein, may be used.

[0048] Meanwhile, in the present invention, angiotensin-converting enzyme 2 (ACE2) is an enzyme that is widely distributed on the surface of human cells, especially in the lungs, and exhibits substrate conversion ability to produce angiotensin (1-7) by cleaving angiotensin II (Ang II) or to produce angiotensin (1-9) by cleaving angiotensin I (Ang I).

[0049] Angiotensin-converting enzyme 2 has a hydrophobic transmembrane domain at the C-terminus, but in the present invention, it is preferable to use water-soluble angiotensin-converting enzyme 2 (sACE2) with the transmembrane domain removed.

[0050] In addition, in the case where angiotensin-converting enzyme 2, Fc is bound to a membrane-structuring protein, the membrane-structuring protein may be used by binding a gene encoding angiotensin-converting enzyme 2 or a gene encoding Fc to a gene encoding a membrane-structuring protein and then expressing the result.

[0051] Specifically, a gene encoding angiotensin-converting enzyme 2 (preferably a gene encoding sACE2), a gene encoding a membrane structure protein, and a gene encoding an Fc fragment are combined and expressed to produce a fusion protein (ACE2_MSP_Fc), and a nanodisk is manufactured using this. In addition, a gene encoding angiotensin-converting enzyme 2 (preferably a gene encoding sACE2), a gene encoding an Fc fragment, and a gene encoding a membrane structure protein are combined and expressed to produce a fusion protein (ACE2_Fc_MSP), and a nanodisk is manufactured using this.

[0052] Meanwhile, when nanodiscs are prepared using the ACE2_MSP_Fc binding protein, nanodiscs of the Low molecular weight (LMW) sA-ND-Fc, Middle molecular weight (MMW) sA-ND-Fc, and High molecular weight (HMW) sA-ND-Fc forms can be prepared (see Fig. 1).

[0053] Among the nanodiscs of the present invention, the nanodisc using the ACE2_MSP_Fc fusion protein contains different proteins at both ends of the membrane structuring protein (MSP), unlike the membrane structuring protein forms used in the manufacture of conventional nanodiscs. Nevertheless, the reason why the ACE2_MSP_Fc fusion protein can surround a phospholipid bilayer and form a nanodisc structure is that the angiotensin-converting enzyme 2 (ACE2) and the Fc fragment are combined in a homodimer form, allowing the membrane structuring protein (MSP) to fully form a disc shape. That is, multiple ACE2_MSP_Fc fusion proteins each combine their own angiotensin-converting enzyme 2 (ACE2) and Fc fragment to form a disc structure.

[0054] For example, when two ACE2_MSP_Fc fusion proteins are linked together, an angiotensin-converting enzyme 2 and Fc region surrounding one layer of the lipid bilayer of the nanodisk is coupled with an angiotensin-converting enzyme 2 and Fc region surrounding the other layer of the lipid bilayer to form a dimer, a nanodisk of normal size is formed (see LMW sA-ND-Fc in Fig. 1).

[0055] In addition, when four or eight ACE2-MSP-Fc fusion proteins are linked together to form an elongated membrane-structuring protein, a single large disc is formed, and medium- or large-sized nanodiscs with increased diameter are created (see MMW sA-ND-Fc and HMW sA-ND-Fc in Fig. 1). That is, when four or more ACE2-MSP-Fc fusion proteins are used, the 'Angiotensin-Converting Enzyme 2' of the membrane-structuring protein finds its partner and binds to form a dimer, and the 'Fc fragment' of the membrane-structuring protein also finds its partner and binds to form a dimer, thereby forming an elongated membrane-structuring protein and creating medium- or large-sized nanodiscs. At this time, the formation of dimers between different layers in the phospholipid bilayer is also possible.

[0057] Meanwhile, the pharmaceutical composition in the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using pharmaceutically acceptable carriers and / or excipients according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. At this time, the formulation may be prepared in various forms such as oral medications and injections, and may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0058] The pharmaceutical composition of the present invention may be administered orally or parenterally, and may be administered, for example, intrathecal administration, intravenous administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intrasternal administration, intratumoral administration, intranasal administration, intracerebral administration, intracranial administration, intrapulmonary administration, and rectal administration, but is not limited thereto.

[0059] Suitable dosages of the pharmaceutical composition of the present invention vary depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a physician of ordinary skill can easily determine and prescribe a dosage (pharmaceutical effective dose) effective for the desired treatment or prevention. As an example, the daily dosage of the pharmaceutical composition of the present invention may be 0.0001 to 100 mg / kg.

[0061] Hereinafter, the contents of the present invention will be explained in more detail through the following examples or experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes variations of equivalent technical concepts.

[0063] [Preparation Examples 1-3: Preparation of sACE2_Fc, 'sACE2_MSP_Fc nanodiscs' and 'sACE2_Fc_MSP nanodiscs']

[0064] (1) Manufacture of sACE2_Fc, 'sACE2_MSP_Fc nanodisk' and 'sACE2_Fc_MSP nanodisk'

[0065] A sACE2_Fc fusion protein (SEQ No. 4) was prepared by combining the soluble angiotensin-converting enzyme 2, Fc fragment gene (SEQ No. 1; SEQ No. 2) and expressing it (Preparation Example 1).

[0066] Meanwhile, the genes for water-soluble angiotensin-converting enzyme 2, membrane structuring protein, and Fc fragment were combined (SEQ No. 1, SEQ No. 3, SEQ No. 2), and the result was expressed to produce a sACE2-MSP-Fc fusion protein (SEQ No. 5).

[0067] Meanwhile, the genes for water-soluble angiotensin-converting enzyme 2, the Fc fragment, and the membrane structuring protein were combined (SEQ No. 1; SEQ No. 2, SEQ No. 3), and the sACE2-Fc-MSP fusion protein (SEQ No. 6) was prepared by expressing them.

[0068] LMW A-ND-Fc, MMW A-ND-Fc, and HMW A-ND-Fc of various sizes were prepared by mixing sACE2-MSP-Fc and lipids at a molar ratio of 1:300 and through the self-assembly process described below (Preparation Example 2, Fig. 1). In the case of nanodiscs prepared using sACE2-MSP-Fc, it was confirmed that nanodiscs were formed in the form of nanodiscs of normal diameter and medium and large nanodiscs with large diameters.

[0069] Meanwhile, A-Fc-ND was prepared by mixing sACE2-Fc-MSP and lipids so that the molar ratio was 1:300 and through the self-assembly process described below (Preparation Example 3, Fig. 1).

[0071] (2) Self-assembly (repid film manufacturing method)

[0072] As a lipid, DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) was dissolved in chloroform to prepare a lipid solution with a concentration of 25 mg / ml. Subsequently, 242 μL of the DMPC solution was transferred to a glass tube such that when dissolved in nanodisk (ND) buffer (40 mM Tris-Cl, 300 mM NaCl, 0.5 mM EDTA, 50 mM NaC, pH 7.4), the total lipid concentration was 10 mM and the volume was 1 mL. Then, nitrogen gas was added, and the solution was left under vacuum for at least 4 hours to remove the solvent, after which a lipid film was obtained. 1 mL of the ND buffer was mixed with the obtained lipid film to hydrate the lipid film, and the solution was subjected to ultrasound at 55°C for 30 minutes to obtain a lipid suspension with uniformly distributed lipids.

[0073] Subsequently, sACE2_MSP_Fc was mixed so that the molar ratio of sACE2_MSP_Fc to lipid was 1:300 (as in Preparation Example 2). (Meanwhile, in Preparation Example 3, sACE2_Fc_MSP was mixed so that the molar ratio of sACE2_Fc_MSP to lipid was 1:300). Then, a total of two times, once for 5 hours at room temperature and once for 16 hours at 4°C, were treated with an equal amount of bio-beads as the total mixture to produce nanodiscs containing sACE2 and Fc fragments through a self-assembly process.

[0074] The nanodiscs containing the prepared sACE2 and Fc fragments were separated and purified by size exclusion chromatography.

[0075] The upper figure of Fig. 2 is the SEC-FPLC separation and purification result confirming that Preparation Example 2 'sACE2_Fc_MSP nanodisk' and Preparation Example 3 'sACE2_MSP_Fc nanodisk' were successfully prepared by the lipid film manufacturing method.

[0077] (3) Self-assembly (microfluidic system manufacturing method)

[0078] As a lipid, DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) was dissolved in ethanol to prepare a lipid solution at a concentration of 5 mg / ml. A sACE2-MSP-Fc protein solution was prepared by diluting the lipid solution with PBS pH 7.4 buffer to a volume of 4 mL, with a molar ratio of 1:300 based on 1 mL of the lipid solution (Preparation Example 4). Additionally, a sACE2_Fc_MSP protein solution was prepared by diluting to the same concentration (Preparation Example 5).

[0079] Subsequently, the lipid solution was prepared by purging the lipid phase pump of the microfluidic system, and the sACE2_MSP_Fc protein solution or sACE2_Fc_MSP protein solution was prepared by purging the aquatic phase pump.

[0080] Nanodiscs containing sACE2 and Fc fragments were prepared through a self-assembly process by driving the pump so that the flow rate ratio of the lipid phase to the aquatic phase was 1:4 and the total system flow rate was 5 mL / min. The prepared nanodiscs containing sACE2 and Fc fragments were dialysised with PBS pH 7.4 buffer to remove residual ethanol, and then separated and purified by size exclusion chromatography.

[0081] The bottom figure of Fig. 2 shows the SEC-FPLC separation, purification, and DLS analysis results confirming that the 'sACE2_Fc_MSP nanodisk' of Preparation Example 4 and the 'sACE2_MSP_Fc nanodisk' of Preparation Example 5 were successfully fabricated by the microfluidic system manufacturing method.

[0083] [Example 1: Confirmation of therapeutic efficacy of Preparation Examples 1–3 in an IPF-induced model]

[0084] 1. Experimental Objective

[0085] In this embodiment, the therapeutic efficacy of the samples from Preparation Examples 1 to 3 was confirmed in an IPF-induced model.

[0087] 2. Experimental Method

[0088] (1) 6-week-old C57BL / 6 male mice were acclimatized for one week after arrival.

[0089] (2) Anesthetized mice by administering an anesthetic (Avertin) into the abdominal cavity.

[0090] (3) Induce idiopathic pulmonary fibrosis by administering 50 μl of bleomycin at a concentration of 0.4 mg / mL (working conc. 1 MPK) via intratracheal instillation or intranasal. The negative control group used PBS instead of bleomycin.

[0091] (4) Body weight was measured daily for 2 weeks starting from the day after administration of bleomycin, and three test substance samples {sACE2 three types of structures; sACE2_Fc (Preparation Example 1), 'sACE2_MSP_Fc nanodisc' (Preparation Example 2), 'sACE2_Fc_MSP nanodisc' (Preparation Example 3)} were administered via intranasal injection. 1 mpk of each of the three test substances was administered based on sACE2.

[0092] (5) After two weeks, lung tissue from mice was extracted and stained with H&E to measure and compare the Ashcroft score.

[0094] 3. Experimental Results

[0095] The efficacy of the above preparation examples 1 to 3 was evaluated by administering them to a mouse model in which pulmonary fibrosis was induced with bleomycin (BLM) (Fig. 3), and the results were as shown in Figs. 4 and 5.

[0096] sACE2_Fc of Preparation Example 1 inhibited the progression of IPF by about 40%. However, 'sACE2_MSP_Fc nanodiscs and sACE2_Fc_MSP' of Preparation Examples 2 and 3 inhibited the progression of IPF by about 70%. In addition, when co-administered with Nintedanib, a positive control, there was no additional inhibition of IPF progression with sACE2_Fc co-administration, but there was additional inhibition of IPF progression with sACE2_MSP_Fc.

[0097] The Ashcroft score described in Figure 4 is a pathological scoring system used to evaluate the degree of 'pulmonary fibrosis' occurring in lung tissue. It is primarily used to quantitatively assess the severity of pulmonary fibrosis through histological examination in diseases such as 'Idiopathic Pulmonary Fibrosis (IPF).' The Ashcroft score classifies the degree of fibrosis in lung tissue into a score ranging from 0 to 8. 0 points: Normal lung tissue; 1–3 points: Mild fibrosis with thickening of alveolar walls; 4–5 points: Moderate fibrosis with progressive damage or destruction of alveolar structures; 6–7 points: Severe fibrosis where alveolar structures are almost completely destroyed and replaced by fibrous tissue; 8 points: Complete fibrosis of alveolar structures, resulting in irreversible damage to the lung tissue.

[0098] As described above, the Ashcroft score is used by researchers to assign a score by observing lung biopsy samples under a microscope, and plays an important role in monitoring the progression of pulmonary fibrosis or evaluating the effectiveness of treatment in research or clinical practice.

[0099] Figure 4 is a graph showing the Ashcroft score, which evaluates the degree of fibrosis in lung tissues for each group according to the above criteria. Compared to the BLM group, the degree of fibrosis progression for the positive control group Nintedanib decreased by 50%, and the degree of fibrosis progression (Ashcroft score) for sACE2_Fc, sACE2_MSP_Fc, and sACE2_Fc_MSP decreased by 40%, 70%, and 70%, respectively. However, sACE2_Fc co-administered with Nintedanib did not show an additional decrease in the degree of fibrosis progression, while when co-administered with sACE2_MSP_Fc, the degree of fibrosis progression decreased further, confirming an 85% inhibition of IPF progression.

[0100] Meanwhile, Figure 5 is a representative image of the average score of the degree of fibrosis evaluated according to the above criteria for the lung tissue of each group. Sham: Representative image of normal lung tissue from the negative control group administered PBS instead of BLM. BLM: Group in which pulmonary fibrosis was induced by BLM administration; severe fibrosis progressed, with fibrosis occurring in most areas. BLM+N: Group in which pulmonary fibrosis was induced by BLM administration followed by oral administration of Nintedanib; the degree of fibrosis was significantly reduced compared to the BLM group. This serves as a positive control group for verification purposes in this study. SF: Group in which sACE2_Fc was administered intranasally after BLM induction; the degree of fibrosis was significantly reduced compared to the BLM group, indicating that SF effectively inhibits fibrosis. SF+N: A group administered intranasally with sACE2_Fc and orally with Nintedanib after BLM induction; the degree of fibrosis was significantly reduced compared to the BLM group, but there was no difference when compared to the group administered sACE2_Fc alone. This implies that there is no effect from co-administration. SMF: A group administered intranasally with sACE2_MSP_Fc after BLM induction; the degree of fibrosis was significantly reduced compared to the BLM group, indicating that SMF effectively inhibits fibrosis. SMF+N: A group administered intranasally with sACE2_MSP_Fc and orally with Nintedanib after BLM induction; the degree of fibrosis was significantly reduced compared to the BLM group, and it was confirmed that additional fibrosis was inhibited when compared to SMF alone. SFM: A group administered sACE2_Fc_MSP intranasally after BLM induction; compared to the BLM group, the degree of fibrosis was significantly reduced, and the degree of reduction was similar to that of SMF. This implies that SFM also effectively inhibits fibrosis.

[0101] The results of this experiment confirmed that 'sACE2_MSP_Fc nanodiscs' or 'sACE2_Fc_MSP nanodiscs' have potential as preventive substances for IPF.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of idiopathic pulmonary fibrosis containing a nanodisk comprising: a lipid bilayer having a flat, disc-shaped bilayer structure formed from phospholipids, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; and a membrane scaffold protein (MSP) surrounding the side of the lipid bilayer where the hydrophobic groups are exposed outward, wherein the membrane scaffold protein has an angiotensin-converting enzyme 2 (ACE2) bound to one end and an Fc fragment bound to the other end, and the bound angiotensin-converting enzyme 2 (ACE2) and the Fc fragment protrude outward from the nanodisk. Claim 2 A lipid bilayer having a flat, disc-shaped bilayer structure formed from phospholipids, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; A pharmaceutical composition for the prevention or treatment of idiopathic pulmonary fibrosis comprising a nanodisk including a membrane scaffold protein (MSP) surrounding the side of the lipid bilayer where the hydrophobic group is exposed to the outside, wherein the membrane scaffold protein is a membrane scaffold protein with an elongated length formed by the interconnection of multiple membrane scaffold proteins through the mutual binding of the 'Angiotensin-converting enzyme 2 (ACE2)' to one end and the mutual binding of the 'Fc fragment' to the other end to form a dimer, and the bound Angiotensin-converting enzyme 2 (ACE2) and the Fc fragment protrude to the outside of the nanodisk. Claim 3 A pharmaceutical composition for the prevention or treatment of idiopathic pulmonary fibrosis containing a nanodisk, comprising: a lipid bilayer having a flat, disc-shaped bilayer structure formed from phospholipids, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; and a membrane scaffold protein (MSP) surrounding the side of the lipid bilayer where the hydrophobic groups are exposed outward, wherein the membrane scaffold protein has an angiotensin-converting enzyme 2 (ACE2) Fc fragment sequentially bound to the N-terminus in the direction from the N-terminus to the C-terminus, and said bound angiotensin-converting enzyme 2 (ACE2) Fc fragment protruding outward from the nanodisk. Claim 4 A pharmaceutical composition for the prevention or treatment of idiopathic pulmonary fibrosis comprising a nanodisk having a flat, disc-shaped bilayer structure formed from phospholipids, wherein the lipid bilayer has hydrophilic groups oriented outward and hydrophobic groups oriented inward; and a membrane scaffold protein (MSP) surrounding the side of the lipid bilayer where the hydrophobic groups are exposed outward, wherein the membrane scaffold protein has an Fc fragment and angiotensin-converting enzyme 2 (ACE2) sequentially bound to the C-terminus from the N-terminus to the C-terminus, and the bound angiotensin-converting enzyme 2 (ACE2) and the Fc fragment protrude outward from the nanodisk. Claim 5 A pharmaceutical composition according to any one of claims 1 to 4, wherein the phospholipid is one or more selected from phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. Claim 6 A pharmaceutical composition according to any one of claims 1 to 4, wherein the phospholipid comprises one or more selected from DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). Claim 7 A pharmaceutical composition characterized in that, in any one of claims 1 to 4, the membrane scaffold protein is an amphiphilic protein having a helix structure. Claim 8 A pharmaceutical composition according to claim 7, wherein the membrane scaffold protein is an apolipoprotein or a fragment of an apolipoprotein in which the 'helix structure and amphiphilic properties' of the apolipoprotein are maintained. Claim 9 A pharmaceutical composition according to any one of claims 1 to 4, wherein the angiotensin-converting enzyme 2 is a water-soluble angiotensin-converting enzyme 2 with the transmembrane domain removed. Claim 10 A pharmaceutical composition according to any one of claims 1 to 4, wherein the nanodisk is characterized in that one layer of the double layer is surrounded by a membrane-structuring protein, and the other layer of the double layer is surrounded by another membrane-structuring protein. Claim 11 A pharmaceutical composition according to any one of claims 1 to 4, wherein the nanodisk is characterized in that angiotensin-converting enzyme 2 of a membrane-structuring protein surrounding one layer of the double layer and angiotensin-converting enzyme 2 of a membrane-structuring protein surrounding the other layer of the double layer mutually combine to form a dimer. Claim 12 A pharmaceutical composition according to any one of claims 1 to 4, wherein the nanodisk is characterized by the formation of a dimer by mutually combining an Fc fragment of a membrane-structuring protein surrounding one layer of the double layer and an Fc fragment of a membrane-structuring protein surrounding the other layer of the double layer. Claim 13 A pharmaceutical composition according to claim 2, wherein the nanodisk is characterized in that one layer of the double layer is surrounded by an extended membrane-structuring protein, and the other layer of the double layer is surrounded by another extended membrane-structuring protein.

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