Surfactant protein b analog and synthetic pulmonary surfactant comprising same

Abstract

The present invention relates to a surfactant protein B analog and a synthetic pulmonary surfactant comprising same. A pulmonary surfactant according to one aspect has excellent surface physical properties and thus is useful for treating breathing disorders such as infant respiratory distress syndrome.

Description

Surfactant protein B analogues and synthetic lung surfactants containing the same

[0001] It relates to surfactant protein B analogs and synthetic lung surfactants.

[0002] Respiratory distress syndrome (RDS) is a disease that causes respiratory failure in premature infants due to insufficient production of pulmonary surfactant (PS). It is the most common disease among newborns, especially premature infants, and has a high mortality rate, making it one of the most important diseases in the neonatal field. Intratracheal administration of artificial PS preparations has been proven effective in clinical trials for RDS in premature infants and newborns, and is currently established as the most reliable and universal treatment for this disease. It is contributing to improving the survival rate and even infant mortality rate of premature infants worldwide and in Korea.

[0003] The main components of lung surfactant are phospholipids such as dipalmitoyl phosphatidyl choline (DPPC), phosphatidyl glycerol (PG), other phospholipids, neutral lipids, etc., and surfactant proteins (SP) SP-A, B, C, and D. In addition, commercially used artificial PS products are largely divided into two groups: (i) PS preparations isolated from the lungs of animals (cows or pigs), and (ii) artificial synthetic PS preparations using synthetic peptides of SP-B or C of surfactant proteins (SP). Most of the artificial PS preparations currently in use are artificial PS preparations isolated from the lungs of cows or pigs. However, animal-derived PS preparations contain animal proteins, so they have the following disadvantages: 1) animal antigenicity, 2) potential risk of infectious diseases, 3) ethical issues of having to slaughter large numbers of animals such as cows and pigs, and 4) expensive manufacturing costs. Therefore, the need for the development of next-generation artificial synthetic PS preparations has been raised.

[0004] These researchers designed surfactant protein B analogs (SP-B analogs) with a new sequence to develop next-generation artificial PS similar to human PS rather than preparations derived from animal lungs, and completed the present invention by confirming their efficacy as new drug candidates.

[0005] One aspect is to provide a surfactant protein B (SP-B) analogue comprising at least one substitution of an amino acid corresponding to position 2 of the amino acid sequence of SEQ ID NO: 1 with norleucine and a substitution of an amino acid corresponding to position 16 with norleucine.

[0006] Another aspect is to provide a pulmonary surfactant comprising the surfactant protein B analog and the surfactant protein C analog.

[0007] Another aspect is to provide a composition for preventing, improving or treating a pulmonary disease comprising the above-described pulmonary surfactant.

[0008] Another aspect is to provide a composition for improving lung health comprising the above-described lung surfactant.

[0009] Another aspect provides a method for preventing, ameliorating or treating a lung disease comprising administering to a subject in need thereof an effective amount of the surfactant protein B analog or pulmonary surfactant.

[0010] Another aspect provides a use of the surfactant protein B analog or pulmonary surfactant for preventing, ameliorating or treating lung diseases.

[0011] Another aspect provides the use of the surfactant protein B analog or pulmonary surfactant for use in the manufacture of a pharmaceutical preparation for preventing, ameliorating or treating a lung disease.

[0012] One aspect provides a surfactant protein B (SP-B) analogue comprising at least one substitution of an amino acid corresponding to position 2 of the amino acid sequence of SEQ ID NO: 1 with norleucine and a substitution of an amino acid corresponding to position 16 with norleucine.

[0013] In the present invention, “pulmonary surfactant” is a mixture of proteins and lipids that coat the inner surface of the alveoli to enable normal breathing. Pulmonary surfactant plays an important role in maintaining respiratory function by contributing to the stabilization and elasticity of the lung surface by reducing the surface tension at the air-liquid surface of the alveoli, thereby reducing the pressure required for alveolar expansion and preventing alveolar collapse. Pulmonary surfactant mainly contains lipids and a small amount of proteins, and is composed of phospholipids such as dipalmitoyl phosphatidyl choline (DPPC), phosphatidyl glycerol (PG), other phospholipids, neutral lipids, etc., and surfactant proteins.

[0014] In the present invention, “surfactant proteins (SPs)” are classified into SP-A, B, C, and D. SP-A and SP-D are water-soluble proteins that participate in the production and secretion of lung surfactant as collectors, and SP-B and SP-C are hydrophobic membrane proteins that increase the rate at which surfactant spreads over the surface. The term “surfactant protein” in this specification may be used interchangeably with “pulmonary surfactant protein,” or “pulmonary surfactant protein.”

[0015] In the present invention, the amino acid sequence of SEQ ID NO: 1 may be a polypeptide having SP-B activity that is modified to produce the SP-B analog of the present invention. Specifically, it may be a naturally occurring polypeptide or a wild-type polypeptide, and may include a variant or functional fragment thereof, etc., as long as it can be a parent of the SP-B of the present invention, without limitation. The polypeptide may be a natural polypeptide of animal or human origin, or may be an artificially synthesized polypeptide.

[0016] In the present invention, “SP-B analogue” refers to a variant in which the amino acid corresponding to the 2nd position and / or the 16th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1, which is a polypeptide having SP-B activity, is substituted with norleucine. Specifically, it may be a variant in which the methionine at the 2nd position and / or the 16th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with norleucine. The substitution with norleucine can alleviate the risk of oxidation, thereby preventing the stability and functional damage of the peptide and helping to increase the resilience of the peptide.

[0017] In the present invention, the “corresponding amino acid” refers to an amino acid residue at a corresponding position in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the amino acid residue at that position. Identifying the amino acid at the corresponding position may determine a specific amino acid of a sequence that references a specific sequence. In the present invention, the “corresponding position” generally refers to a similar or corresponding position in the amino acid sequence of a related protein or a reference sequence. For example, any amino acid sequence may be aligned with SEQ ID NO: 1, and based on this, each amino acid residue of the amino acid sequence may be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the position of the corresponding amino acid, or the position where a modification such as a substitution, insertion, or deletion occurs, may be identified by comparing it with a query sequence (also referred to as a “reference sequence”) using a sequence alignment algorithm known in the art.

[0018] In one specific example, the SP-B analog may further comprise an addition of a serine (S), leucine-serine (LS), tryptophan-leucine-serine (WLS), or cysteine-tryptophan-leucine-serine (CWLS) sequence at the N-terminus.

[0019] In one specific example, the peptide may have an amino acid modified at the N-terminus, C-terminus, or both termini. Specifically, the modification of the amino acid at the N-terminus, C-terminus, or both termini may be palmitoylation, myristoylation, acetylation, acylation, esterification, sulfonylation, alkylation, formylation, glycosylation, amidation, prenylation, or PEGylation of the amino acid. More specifically, the SP-B analog may include acetylation of the N-terminal amino acid, amidation of the C-terminal amino acid, or a combination thereof.

[0020] In one specific example, the SP-B analog may be one in which a disulfide bond is formed between at least one pair of amino acids within the molecule and / or between molecules. Specifically, the disulfide bond may be formed between cysteine ​​residues within the SP-B analog (Cys-Cys intra disulfide bond). More specifically, the SP-B analog may be one in which a disulfide bond is formed between an amino acid corresponding to the first position of the added cysteine-tryptophan-leucine-serine (CWLS) sequence and an amino acid corresponding to position 14 of the amino acid sequence of SEQ ID NO: 1. The formation of the disulfide bond may provide structural stability of the protein and is important for maintaining a functional conformation, and thus may help maintain lung surface activity.

[0021] In one specific example, the SP-B analogue may be one in which the head and tail of the amino acid are cyclized.

[0022] In one specific embodiment, the cyclization may be by modification of amino acids at the N-terminus, C-terminus, or both termini. For example, head-to-tail cyclization may be promoted by an acetylated N-terminal amino acid and an amidated C-terminal amino acid.

[0023] In one specific example, the cyclization may be due to a disulfide bond within the SP-B analog.

[0024] In one specific example, the SP-B analogue may include an amino acid sequence having 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the amino acid sequence of SEQ ID NO: 2 or 3, but is not limited thereto, and is included within the scope of the present invention without limitation if it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or 3.

[0025] In the present invention, "homology" or "identity" refers to the degree of relationship between two given amino acid sequences or base sequences, and may be expressed as a percentage. In the present invention, "homology" and "identity" may be used interchangeably.

[0026] It is obvious that a polypeptide or protein having an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added is also included within the scope of the present application, provided that the amino acid sequence has the homology or identity mentioned in the present invention and exhibits an activity corresponding to the polypeptide or protein. That is, even if the present application describes “a polypeptide or protein having or including an amino acid sequence described by a specific sequence number,” it is obvious that a polypeptide or protein having (or including) an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added can also be used in the present application, provided that it has the same or corresponding activity as a polypeptide or protein consisting of the amino acid sequence of the corresponding sequence number.

[0027] As used herein, “amino acid” and “amino acid residue” refer to a natural amino acid, an unnatural amino acid, or a modified amino acid. Unless otherwise stated, all references to amino acids, either generically or by name, specifically include references to both the D and L stereoisomers (where the structure permits such stereoisomeric forms). Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Non-natural amino acids include modified amino acid residues that are chemically modified, or reversibly or irreversibly chemically blocked, at the N-terminal amino group or side chain, such as N-methylated D and L amino acids or residues in which a side chain functional group is chemically modified with another functional group.

[0028] It is clear that a polypeptide or protein having an amino acid sequence having a deletion, modification, substitution (e.g., conservative substitution) or addition of a portion of the sequence is also included within the scope of the present invention, provided that the amino acid sequence has homology or identity as described herein and exhibits an activity corresponding to the polypeptide or protein.

[0029] As used herein, "conservative substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain, without causing a loss of the biological or biochemical function of the polypeptide or protein. Classes of amino acid residues having similar side chains are well-defined and known 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 nonpolar 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).

[0030] Additionally, the variants of the present invention may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the polypeptide, and for example, the N-terminus of the variant may be conjugated with a signal (or leader) sequence that is involved in translocation of the protein co-translationally or post-translationally.

[0031] Additionally, the variants of the present invention may be conjugated to other sequences or linkers so that they can be identified, purified, or synthesized.

[0032] In addition, if it has the same or corresponding activity as the variant of the present invention, it does not exclude meaningless sequence additions before and after the amino acid sequence of the corresponding sequence number, mutations that can occur naturally, or silent mutations thereof, in addition to the variant of the present invention, and it is clear that even if it has such sequence additions or mutations, it falls within the scope of the present invention.

[0033]

[0034] Another aspect provides a lung surfactant comprising the surfactant B (SP-B) analogue and the surfactant C (SP-C) analogue.

[0035] In one specific example, the SP-C analogue may include, but is not limited to, an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity to the amino acid sequence of SEQ ID NO: 4 (CPVHLKRLLLLLLLLLLLLLL).

[0036] In one specific example, the surfactant may further comprise a phospholipid.

[0037] As used herein, a “phospholipid” is a lipid in which one fatty acid is replaced by a phosphate group and a simple organic molecule. Examples of the most common phospholipids that can be found in lung surfactants include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS). The glycerol moiety of the phospholipid is primarily esterified with long-chain fatty acids, which may in turn be saturated (e.g., myristic acid, palmitic acid, and stearic acid), monounsaturated (e.g., oleic acid), or polyunsaturated (linoleic acid and arachidonic acid). Specific examples include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), and palmatoyl-oleoyl-phosphatidylglycerol (POPG).

[0038] Specifically, the surfactant may further include, but is not limited to, dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), and / or palmitic acid (PA).

[0039] In one specific example, the SP-B analog and the SP-C analog in the surfactant may be included in a weight ratio of 0.1 to 10:1, 0.1 to 5:1, 0.1 to 2:1, 0.3 to 10:1, 0.3 to 5:1, 0.3 to 2:1, 0.5 to 10:1, 0.5 to 5:1, or 0.5 to 2:1. Specifically, the weight ratio may be 2:3, 3:3, 4:3, or 5:3.

[0040] In one specific example, the SP-B analogue in the lung surfactant may be included in an amount of at least 0.01 wt%, at least 0.05 wt%, at least 0.1 wt%, at least 0.5 wt%, at least 0.7 wt%, at least 0.8 wt%, at least 0.9 wt%, or at least 1 wt% of the total lung surfactant.

[0041] In one embodiment, the SP-B analogue in the surfactant may be comprised in an amount of 1 to 10 wt%, 1 to 7 wt%, 1 to 5 wt%, 1 to 3 wt%, or about 2 wt% of the total surfactant.

[0042] The description of the above SP-B analogues also applies to the above lung surfactants.

[0043]

[0044] Another aspect provides a composition for preventing, improving or treating a pulmonary disease comprising the above-described surfactant.

[0045] Another aspect provides a composition for improving lung health comprising the above-described lung surfactant.

[0046] As used herein, "prevention" refers to any action that inhibits or delays the onset of a disease by administering the composition of the present invention to a subject. For preventive purposes, the composition may be administered to a subject at risk of developing a specific disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0047] As used herein, "treatment" refers to any action that improves the symptoms of a disease or provides benefit by administering the composition of the present invention to a subject. As used herein, the terms "treatment," "palliation," and "improvement" may be used interchangeably. A therapeutic benefit refers to any therapeutically significant improvement or effect on one or more diseases, conditions, or symptoms under treatment.

[0048] The lung disease of the present specification may be at least one selected from the group consisting of pneumonia, respiratory distress syndrome (RDS), acute respiratory distress syndrome (ARDS), meconium aspiration syndrome, pulmonary hemorrhage, submersion, and acute lung injury (ALI).

[0049] The above composition may be a pharmaceutical composition.

[0050] The pharmaceutical composition of the present invention may be in any form suitable for the intended method of administration. In the pharmaceutical composition of the present invention, “administration” means introducing a predetermined substance into a patient by any suitable method, and the route of administration of the pharmaceutical composition may be administered by any common route as long as the drug can reach the target tissue. Administration may be done by any method known in the art, for example, continuous infusion by a conventional method, or intratracheally injection into a patient (e.g., a premature infant) under continuous or intermittent positive airway pressure (IPPV). In addition, administration may be done using a thin catheter, mask, prongs, etc. inserted into the trachea, or according to continuous positive airway pressure (nCPAP), but is not limited thereto.

[0051] The above administration is 0.00001 mg to 1,000 mg of the composition according to one specific example per subject per day, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, Or it may be administered in doses of 10 mg to 25 mg.

[0052] However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage by considering these factors. The frequency of administration may be once a day or twice or more within the range of clinically acceptable side effects, and the administration may be done in one or more sites, and the total number of administration days may be from 1 to 30 days per treatment, daily or at intervals of 2 to 5 days. If necessary, the same treatment may be repeated after an appropriate period. For animals other than humans, the same dosage as for humans per kg may be used, or the above dosage may be converted into an amount based on the volume ratio (e.g., average value) of the organs (e.g., heart) of the target animal and the human.

[0053] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., formulated according to conventional methods, or parenteral formulations such as suspensions, emulsions, lyophilized preparations, external preparations, suppositories, sterile injection solutions, and implantable preparations. The pharmaceutical composition may further comprise, in addition to the active ingredient, a pharmaceutically acceptable excipient that can be used in formulation.

[0054] The above excipients include carriers, vehicles, diluents, solvents, for example, monohydric alcohols, for example, ethanol, isopropanol, and polyhydric alcohols, for example, glycerol, and edible oils, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, oily esters, for example, ethyl oleate, isopropyl myristate; It may include at least one selected from the group consisting of binders, adjuvants, solubilizers, thickeners, stabilizers, disintegrants, glidants, lubricants, buffers, emulsifiers, wetting agents, suspending agents, sweeteners, coloring agents, flavoring agents, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starches, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low melting point waxes, ion exchange resins, etc., but is not limited thereto.

[0055] The carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0056] In addition, the pharmaceutical composition may be sterilized or may further contain auxiliary agents such as preservatives, stabilizers, wetting agents or emulsifying agents, salts for osmotic pressure control and / or buffers, and may further contain other therapeutically useful substances, and may be formulated according to conventional methods of mixing, granulating or coating.

[0057] The content of the lung surfactant in the pharmaceutical composition of the present invention can be appropriately adjusted depending on the purpose of use of the pharmaceutical composition, the form of the formulation, etc., and may be, for example, 0.001 to 99 wt%, 0.001 to 90 wt%, 0.001 to 50 wt%, 0.01 to 50 wt%, 0.1 to 50 wt%, or 1 to 50 wt% based on the total weight of the pharmaceutical composition.

[0058] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and may be adjusted according to factors including the type of the patient’s disease, the severity of the disease, the type of active ingredient administered, the type of formulation, the patient’s age, sex, weight, health condition, diet, sensitivity, the time and method of drug administration, the combination of the composition or concurrently used drugs, and other factors well known in the medical field.

[0059] The pharmaceutical composition of the present invention can prevent, improve or treat the condition of an individual, including a step of treating or administering it to an individual in need thereof in an amount effective to prevent or treat a disease.

[0060] The condition of the above entity may be related to lung disease or lung health.

[0061] The dosage of the pharmaceutical composition for the prevention or treatment of diseases according to the present invention may range from 0.01 ug / kg to 10 g / kg per day, specifically from 0.01 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once daily or divided into several doses. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0062] The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual in need of an effect for improving lung disease or lung health.

[0063] The above composition may be a health functional food.

[0064] The term "health functional food" as used herein refers to a food manufactured or processed for the purpose of health supplementation using a specific ingredient as a raw material or by extracting, concentrating, refining, mixing, or other methods of a specific ingredient contained in a food raw material. It refers to a food designed and processed so that the above-mentioned ingredients can sufficiently exert bioregulatory functions on the body, such as biodefense, regulation of biological rhythms, and disease prevention and recovery. In the present invention, it may be used for the purpose of improving lung health.

[0065] There are no specific restrictions on the types of the above foods. Examples of the above foods include formulations selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, gums, candies, and health drinks, and include all health foods in the conventional sense.

[0066] The above health functional food may include food additives that are food-related and acceptable, and may include an appropriate carrier commonly used in the manufacture of health functional foods.

[0067] The above composition may be a feed composition.

[0068] The feed composition can be prepared by adding the compound or its salt in an appropriate effective concentration range according to various feed preparation methods known in the art, and can be used for the purpose of preventing or improving lung disease or lung health.

[0069] The above feed may refer to any natural or artificial diet, meal, etc., or ingredients of the meal, which are suitable for or intended for animals to eat, ingest, and digest. The type of feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of the feed include plant feed such as grains, roots, fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils, starches, meal, or grain by-products; animal feed such as proteins, fat-free substances, oils, minerals, oils, single-cell proteins, zooplankton, or food.

[0070] The terms and methods described for the above surfactant protein B analog, lung surfactant and disease also apply to the above composition.

[0071]

[0072] Another aspect provides a method of preventing, ameliorating or treating a lung disease comprising administering to a subject in need thereof an effective amount of the surfactant protein B analog or pulmonary surfactant.

[0073] Another aspect provides the use of the surfactant protein B analog or pulmonary surfactant for preventing, ameliorating or treating lung diseases.

[0074] Another aspect provides the use of the surfactant protein B analog or pulmonary surfactant for use in the manufacture of a pharmaceutical preparation for preventing, ameliorating or treating a lung disease.

[0075] The terms and methods described for the above surfactant protein B analogue, lung surfactant and disease also apply to the above methods and uses.

[0076]

[0077] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0078] Surfactants, depending on their nature, exhibit excellent physical properties, including rapid surface diffusion, high surface adsorption efficiency, and low surface tension. These surfactants also exhibit excellent properties in increasing lung volume relative to pressure in animal lungs, making them useful for the treatment of lung-related diseases, including respiratory therapy.

[0079] Figure 1 is a graph comparing the surface diffusion rates of different types of SP-B analogs.

[0080] Figure 2 is a graph comparing the surface adsorption rates of different types of SP-B analogs.

[0081] Figure 3 is a graph comparing the surface tension of different types of SP-B analogs.

[0082] Figure 4 is a graph comparing the surface diffusion rate according to the content of SP-B analogues in the lung surfactant.

[0083] Figure 5 is a graph comparing the surface adsorption rate according to the content of SP-B analogue in the lung surfactant.

[0084] Figure 6 is a graph comparing surface tension according to the content of SP-B analogue in the lung surfactant.

[0085] Figure 7 is a graph showing a pressure-volume curve of the lungs after injection of a surfactant according to one specific example.

[0086] Figure 8 is an image showing the results of histological analysis of lung tissue after injection of a surfactant according to one specific example.

[0087] Figure 9 is a graph measuring the ratio of the aerated area within the alveoli after injection of a surfactant according to one specific example.

[0088] The following examples are provided for more detailed description. However, these examples are provided solely to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0089]

[0090] Example 1. Preparation of surfactant protein B (SP-B) analogues

[0091] A surfactant protein B (SP-B) analogue according to one specific example was prepared as follows.

[0092] Candidate SP-B was designed using the SP-B peptide sequence (RMLPQLVCRLVLRCSMD) (SEQ ID NO: 1) from a previous study as a basic structure (A Combination of Short and Simple Surfactant Protein B and C Analogues as a New Synthetic Surfactant: In Vitro and Animal Experiments. Yonsei Med J. 2017 Jul;58(4):823-828. doi: 10.3349 / ymj.2017.58.4.823.). Structural variations of the peptide were selected to improve its functional properties.

[0093] The peptide was produced using solid-phase peptide synthesis (SPPS). Specifically, after confirming the amino acid sequence of the SP-B peptide to be synthesized, each amino acid residue was prepared, along with the reagents and solvents required for peptide synthesis. The first amino acid residue was bound to an immobilized resin, and protected amino acids were sequentially added to extend the sequence. Deprotection and coupling processes were repeated at each binding step. Upon completion of synthesis, the peptide was cleaved from the resin and the protecting group was removed. The synthesized peptide was then purified using high-performance liquid chromatography (HPLC) to obtain a high-purity peptide. The purified peptide was analyzed using mass spectrometry and other methods to confirm that the expected sequence had been accurately synthesized. Finally, the peptide was lyophilized into a stable powder and packaged in a glass container. Sequence number 7 corresponds to the sequence in which the head and tail are cyclized in sequence number 5.

[0094] The candidate sequences are shown in Table 1 below.

[0095] SP-B #Sequence number SP-B sequence SP-B4Sequence number 2 CWLSR(Nle)LPQLVCRLVLRCS(Nle)DSP-B5Sequence number 3 R(Nle)LPQLVCRLVLRCS(Nle)DSP-B1Sequence number 5 R(Nle)LPQLVCRLVLRSS(Nle)DSP-B2Sequence number 6 AcR(Nle)LPQLVCRLVLRSS(Nle)DNH2SP-B3Sequence number 7 R(Nle)LPQLVCRLVLRSS(Nle)D

[0096]

[0097] Example 2. Preparation of an artificial synthetic lung surfactant formulation

[0098] A lung surfactant according to one specific example, including the SP-B analog of Example 1, was prepared as follows.

[0099] Specifically, SP-B and SP-C analog peptides were dissolved in a small amount of trifluoroacetic acid (TFA) together with DPPC, POPG, and PA. This dissolved mixture was then added to a mixture of CHCl3 (chloroform) and CH3OH (methanol) (2:1, v / v) to produce a peptide-lipid mixture containing 2% SP-C peptide in a 75:25:10 (w / w) DPPC / PG / PA ternary mixture. This SP-C peptide-lipid mixture was suspended in 10% ethanol, incubated at 40-45°C for 15 min, and then lyophilized to obtain a white powder. This powder was dispersed in saline, homogenized by sonication in ice-cold water for 1 min, and then stored at -20°C under nitrogen. The final phospholipid concentration was 10 mg / ml.

[0100] The types of SP-B variants used in each manufacturing example and the preparation ratio of the lung surfactant are summarized in Table 2 below.

[0101]

[0102] Manufacturing Example CHAsurf #SP-B #Preparation ratio (w / w) (DPPC:PG:PA:SP-B:SP-C) Manufacturing Example 1 CHAsurf-1SP-B1 75:25:10:3:3 Manufacturing Example 2 CHAsurf-2SP-B2 75:25:10:3:3 Manufacturing Example 3 CHAsurf-3SP-B3 75:25:10:3:3 Manufacturing Example 4 CHAsurf-4SP-B4 75:25:10:3:3 Manufacturing Example 5 CHAsurf-5SP-B5 75:25:10:3:3 Manufacturing Example 6 CHAsurf-4ASP-B4 75:25:10:2:3 Manufacturing Example 7 CHAsurf-4BSP-B4 75:25:10:3:3 Manufacturing Example 8CHAsurf-4CSP-B475:25:10:4:3 Manufacturing example 9CHAsurf-4DSP-B475:25:10:5:3

[0103]

[0104] Experimental Example 1. Surface Physical Properties of Lung Surfactant Preparations

[0105] 1.1 Surface physical properties examination according to SP-B analog type

[0106] To verify the surface physical properties of the surfactant according to one specific example, a modified Wilhelmy balance test was performed. The modified Wilhelmy balance test is an experimental technique that measures surface tension and is frequently used to evaluate the properties of biological surfactants. This test measures surface tension by contacting a solid surface (typically a platinum plate) with a liquid, and is a modified version that can evaluate how quickly and effectively the surfactant spreads across the liquid surface. In this experiment, a modified Wilhelmy balance machine was constructed, consisting of a Teflon water tank (15.0 × 6.0 × 2.2 cm) equipped with a motor-driven plunger connected to a K20 tensiometer (Krüss, Germany). The surface tension was measured with this device, and the temperature under the plunger was maintained constant with a hot plate. By observing and recording changes in surface tension over time, the performance and properties of the surfactant can be analyzed.

[0107] Specifically, a platinum plate was thoroughly cleaned and dried, then positioned so that it contacted the liquid surface. The length and angle of the plate in contact with the liquid were maintained constant. The surface tension acting on the plate upon contact with the liquid is determined by factors such as the liquid's density, gravitational acceleration, and contact angle, and this was transmitted to a measuring device to calculate the surface tension.

[0108] To examine surface diffusion, 1.3 μl of a 25 mg / mL surfactant solution was added to 80 mL of saline water in a tank maintained at 37°C, and the diffusion behavior of the surfactant on the saline water surface was closely observed over a 3.5-min observation period. To evaluate the interaction between the surfactant and the saline water, 66% compression-decompression cycles were performed, and for stability, ST-area diagrams were obtained, focusing on data from the seventh cycle.

[0109] To test the surface adsorption rate, 7 mL of saline solution and 1 mL (0.5 mg / mL) of surfactant were placed in a circular Teflon tank, stirred at 120 rpm with a Magnetic Striders MIX 1 eco rotator, and observed for 40 minutes.

[0110] Surface spreading test, ST-area diagram (hysteresis curve), and surface adsorption test were performed on the lung surfactants of Manufacturing Examples 1 to 5 and the commercial product Poractant alfa (Curosurf®, Chiesi Pharmaceutical, Italy), as described above, and the results are shown in Figures 1 to 3. The criteria for surface area characteristics of an ideal artificial lung surfactant formulation proposed by Fujiwara et al. are as follows:

[0111] 1) Fast surface spreading: less than 10 seconds to reach equilibrium surface tension of 24-27 mN / n;

[0112] 2) Rapid surface adsorption: less than 1 minute to reach surface tension of 27-30 mN / m (surface pressure 42-45 mN / m);

[0113] 3) Minimum surface tension less than 10 mN / m at 20-30% surface compression;

[0114] 4) Surface tensions up to 27-30 mN / m achieved without 100% surface compression area;

[0115] 5) Very low surface compressibility (less than 0.03 m / mm at a surface tension of 10 mN / m).

[0116] As shown in FIGS. 1 to 3, the lung surfactants according to Manufacturing Examples 4 and 5 showed rapid diffusion rates, excellent surface adsorption efficiency, and highly efficient reductions in surface tension, demonstrating results equivalent to or superior to those of the control drug Curosurf, thereby satisfying the criteria for an ideal artificial lung surfactant. That is, it can be confirmed that the lung surfactant comprising an SP-B analog according to one specific example has an excellent surface activity effect.

[0117]

[0118] 1.2 Surface physical properties test according to SP-B content

[0119] In order to examine the surface physical properties according to the content of the SP-B candidate in the surfactant according to one specific example, experiments were performed under the same conditions as in Experimental Example 1.1 for the surfactants of Preparation Examples 6 to 9 containing 2 mg, 3 mg, 4 mg, and 5 mg of SP-B, respectively, and the results are shown in FIGS. 4 to 6.

[0120] As shown in Figures 4 to 6, as the SP-B concentration increased, faster diffusion and higher adsorption rates were observed, and surface tension reduction was significantly improved. However, all of the lung surfactants of Preparation Examples 6 to 9 showed fast diffusion rates, excellent surface adsorption efficiency, and high efficiency reduction in surface tension, showing results equivalent to or superior to those of the control drug Curosurf, and meeting the standards of an ideal artificial lung surfactant.

[0121]

[0122] Experimental Example 2. In vivo efficacy test of lung surfactant preparations

[0123] To verify the in vivo efficacy of a lung surfactant according to one specific example, the following experiments were performed.

[0124] First, the New Zealand white rabbit model was used for the experiment, and all animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Kyung Hee University Medical Center (KHMC-IACUC 22-042). Eight anesthetized mother rabbits underwent cesarean section, and 15 mg / kg of Zoletil® (tiletamine + zolazepam) was administered via the ear vein. Premature infants were extracted at G27 and full-term infants at G31. Thirty-one premature infants and 10 full-term infants were used, and the average birth weight of premature infants was 36.1±3.1 g, and the average birth weight of full-term infants was 64.3±7.6 g. After tracheostomy was performed after delivery, gentle forced ventilation was performed using a syringe containing 0.3-0.5 cc of air. The fetus was placed in the supine position, a lateral hole was made in the trachea, an 18-gauge vascular catheter was inserted into the trachea, and the tip of the vascular catheter was positioned on the birth canal using an elastic thread. The control and experimental groups were set as (i) a control group of full-term infants who were not administered anything (term control, Term control, 10 animals), (ii) a control group of premature infants who were not administered anything (premature control, Preterm control, 11 animals), (iii) a group of premature infants administered Curosurf (control group, Preterm Curosurf, 10 animals), and (iv) a group of premature infants administered the CHAsurf preparation according to one specific example (preparation example 7 administration group, Preterm CHAsurf-4B, 10 animals). The dose of the administered surfactant was 100 mg / kg for all treatment groups. The experimental animals in each animal group were connected to a small animal ventilator (VentElite, Harvard Apparatus; Cambridge, MA, USA) through a tracheostomy tube, and a pressure-volume curve was measured to observe the change in lung volume increase according to the increase in pressure.Among these, the expiratory volume (ml / kg) at each expiratory pressure was measured and compared, and the results are shown in Figure 7.

[0125] As shown in Fig. 7, the tidal volume (Tv) measured at the pressure (Peak inspiration pressure, PIP) set in the small animal ventilator showed better results in the premature infant experimental group administered a surfactant according to one specific example than in the premature infant experimental group administered Curosurf.

[0126]

[0127] Additionally, the aerated area within the alveoli was analyzed histopathologically. Specifically, the tracheostomy was blocked with 10 cm H2O, and lung tissue samples were fixed in formalin solution. HE staining was performed on both left and right lung tissue samples to observe histological findings, and the ratio (%) of the aerated area to the total alveolar area was measured at a magnification of ×100 using the ImageJ program (V 1.48 for Windows, NIH, USA). The results are shown in Figures 8 and 9.

[0128] As shown in FIGS. 8 and 9 , the surfactant-administered group according to one embodiment exhibited significantly reduced alveolar collapse, improved lung compliance, and improved aeration characteristics similar to those of the Curosurf-administered group. In other words, it was observed that the surfactant-administered group according to one embodiment effectively restored alveolar structural integrity and improved respiratory dynamics, such as by increasing lung elasticity.

[0129]

[0130] In summary, surfactants for lungs according to their daily properties have excellent physical properties such as fast surface diffusion rate, high surface adsorption efficiency, and low surface tension, and also show excellent properties in increasing lung volume against pressure in animal lungs, so it can be seen that they are useful for treating lung-related diseases such as respiratory therapy.

[0131]

[0132] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

A surfactant protein B (SP-B) analogue comprising at least one substitution of the amino acid corresponding to position 2 of the amino acid sequence of SEQ ID NO: 1 with norleucine and at least one substitution of the amino acid corresponding to position 16 with norleucine. An SP-B analogue according to claim 1, further comprising an addition of a serine (S), leucine-serine (LS), tryptophan-leucine-serine (WLS), or cysteine-tryptophan-leucine-serine (CWLS) sequence at the N-terminus. An SP-B analogue according to claim 1, comprising acetylation of an N-terminal amino acid, amidation of a C-terminal amino acid, or a combination thereof. An SP-B analogue according to claim 1, wherein a disulfide bond is formed between at least one pair of amino acids within the SP-B analogue. An SP-B analogue according to claim 1, wherein the SP-B analogue comprises an amino acid sequence of SEQ ID NO: 2 or 3. A pulmonary surfactant comprising an SP-B analogue according to any one of claims 1 to 5; and a surfactant protein C (SP-C) analogue. A lung surfactant according to claim 6, wherein the SP-C analogue comprises an amino acid sequence of SEQ ID NO:

4. A pulmonary surfactant according to claim 6, wherein the SP-B analogue and the SP-C analogue are contained in a weight ratio of 0.1 to 10:

1. A pulmonary surfactant according to claim 6, wherein the SP-B analogue in the pulmonary surfactant is contained in an amount of 0.01 wt% or more of the total pulmonary surfactant. A surfactant according to claim 6, further comprising a phospholipid, a fatty acid, or a combination thereof. A pharmaceutical composition for preventing or treating lung diseases, comprising the surfactant of claim 6. A pharmaceutical composition according to claim 11, wherein the lung disease is at least one selected from the group consisting of pneumonia, respiratory distress syndrome (RDS), acute respiratory distress syndrome (ARDS), meconium aspiration syndrome, pulmonary hemorrhage, submersion, and acute lung injury (ALI). A health functional food for improving lung health, comprising the lung surfactant of claim 6.

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