WO2020175931A1 - 알부민이 결합된, slit3 단백질의 lrrd2를 포함하는 근육 질환 예방 또는 치료용 조성물 - Google Patents

알부민이 결합된, slit3 단백질의 lrrd2를 포함하는 근육 질환 예방 또는 치료용 조성물 Download PDF

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WO2020175931A1
WO2020175931A1 PCT/KR2020/002809 KR2020002809W WO2020175931A1 WO 2020175931 A1 WO2020175931 A1 WO 2020175931A1 KR 2020002809 W KR2020002809 W KR 2020002809W WO 2020175931 A1 WO2020175931 A1 WO 2020175931A1
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lrrd2
slit3
fusion protein
protein
albumin
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PCT/KR2020/002809
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English (en)
French (fr)
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고정민
김성섭
안경훈
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주식회사 대웅제약
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Priority to JP2021550267A priority Critical patent/JP7295261B2/ja
Priority to EP20763321.5A priority patent/EP3950707A4/en
Priority to CN202080017137.1A priority patent/CN113544142B/zh
Priority to US17/434,388 priority patent/US20220125889A1/en
Priority to BR112021016973A priority patent/BR112021016973A2/pt
Publication of WO2020175931A1 publication Critical patent/WO2020175931A1/ko

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to a composition for the prevention or treatment of muscle diseases including LRRD2 of Slit3 protein bound to albumin. More specifically, fusion protein including LRRD2 of Slit3 protein, the fusion protein to which albumin is bound Nucleic acid molecule encoding N, a recombinant vector containing the nucleic acid molecule, a transformant containing the recombinant vector, a method for producing a fusion protein using the transformant, a composition for preventing or treating muscle diseases containing the fusion protein , And it provides a composition for enhancing the in vivo half-life of LRRD2 of Slit3 protein comprising the fusion protein.
  • the Slit protein regulates the movement of neurons and axons during the development of the nervous system.
  • Slit protein is known to play a role as a factor that regulates various intracellular processes in various tissues such as heart, lung, kidney, breast tissue, and can regulate physiological activity by interacting with Robo receptors. Recently, cell growth and attachment As it has been reported that there is an important role in the regulation of function and mobility, it has been reported that the Slit protein may be involved in migration in the process of cell differentiation and in the development and metastasis of cancer. Specifically, the embryonic development stage of vertebrates Slit protein and Robo protein are expressed in, and the expression of Slit3, Robol and Robo2 proteins has been reported to increase in muscle tissue (Neil Vargesson et.al., Mechanisms of development 106.1 (2001): 175-180). According to this report, it is stated that the expression of Slit3 protein is increased in myoblasts of the muscle tissue of the embryonic hindlimb, but it is only involved in migration and not related to the differentiation of myoblasts.
  • HSA-Slit3 LRRD2 fusion protein HSA-Slit3 LRRD2 fusion protein
  • the purpose of the present invention is to improve the in vivo half-life of LRRD2 of Slit3 protein. 2020/175931 1»(:1 ⁇ 1 ⁇ 2020/002809 It is to provide a fusion protein.
  • Another object of the present invention is a nucleic acid molecule encoding the aforementioned fusion protein
  • Another object of the present invention is to provide a method for producing the fusion protein described above.
  • Another object of the present invention is the prevention of LRRD2 muscle diseases of Slit3 protein or
  • Another object of the present invention is to provide a composition that enhances the in vivo half-life of LRRD2 of Slit3 protein.
  • the present invention is a combination of albumin and Slit3 protein.
  • the albumin is human serum
  • the fusion protein may be that the human serum albumin is bound to the N-terminus of LRRD2 of the Slit3 protein.
  • the human serum albumin is N-(13] According to another preferred embodiment of the present invention.
  • LRRD2 of the Slit3 protein may include the amino acid sequence of SEQ ID NO: 3.
  • a linker may additionally be included between the albumin and LRRD2 of the Slit3 protein.
  • the linker is (GGGGS)n
  • n may be an integer of 1 to W.
  • the present invention also provides a nucleic acid molecule that encodes the aforementioned fusion protein.
  • the present invention also provides a recombinant vector comprising the aforementioned nucleic acid molecule and a transformant comprising the same.
  • the present invention also includes the step of culturing the above-described transformant, fusion
  • This invention also uses the aforementioned fusion protein to prevent or treat muscle diseases.
  • composition is provided.
  • the pharmaceutical composition may be administered as an injection.
  • the muscle disease [22] According to another preferred embodiment of the present invention, the muscle disease
  • the present invention also provides a composition for enhancing half-life of LRRD2 of Slit3 protein in vivo, including LRRD2 of Slit3 protein, to which albumin is bound.
  • Albumin-conjugated, LRRD2 of Slit3 protein of the present invention exhibits the same cytological efficacy as LRRD2 of Slit3 protein without albumin, and has a remarkably half-life in vivo compared to LRRD2 of Slit3 protein without albumin. It is increased so that bone-related diseases can be more effectively prevented or treated.
  • Figure 2 shows the results of SDS-PAGE after separation and purification of SP cystatin S-HSA-Slit3 LRRD2 fusion protein.
  • Figure 3 is a graph showing the receptor-binding ability of various types of HSA-Slit3 LRRD2 fusion protein (HSA-Slit3 LRRD2 fusion protein).
  • Figure 4 shows the plasma concentration time profile of Slit3 LRRD2 after IV administration of Slit3 LRRD2 ( ⁇ , “Slit3”) and HSA-Slit3 LRRD2 ( ⁇ , “HSA-Slit3”) in fasted male ICR mice.
  • LRRD2 of the Slit3 protein can be used for the prevention or treatment of sarcopenia by promoting the differentiation of muscle stem cells and inducing an increase in muscle mass.
  • it is administered in order to exert the efficacy of a child with a very short in vivo half-life. It was expected that the cycle would inevitably be shortened, resulting in a problem of decreasing efficacy due to excessive drug use.
  • the present inventors improved the efficacy of LRRD2 by enhancing its half-life in vivo.
  • LRRD2 of the Slit3 protein bound to albumin of the present invention is an unbound Slit3 protein of albumin.
  • the present invention provides a fusion protein of LRRD2 of Slit3 protein to which albumin is bound.
  • LRRD2 of Slit3 protein refers to the second leucine rich repeat domain (LRRD2) in the Slit3 protein.
  • LRRD2 second leucine rich repeat domain
  • the inventors of the present invention have shown that Slit3 protein or LRRD2 in this protein is Robol. Alternatively, it is combined with the Robo2 receptor and the Slit-Robo system
  • Slit3 LRRD2 means "LRRD2 of the Slit3 protein", and can be used interchangeably.
  • the albumin is human serum albumin, rhesus serum albumin (RhSA), cynomolgous monkey serum albumin (CySA), or murine.
  • (murine) may be serum albumin (MuSA), preferably human serum albumin.
  • the in vivo half-life of Slit3 LRRD2 in the presence of human serum albumin is at least less than the in vivo half-life of Slit3 LRRD2 in the absence of human serum albumin.
  • the serum half-life of Slit3 LRRD2 in the presence of human serum albumin is 14 times longer than that of Slit3 LRRD2 in the absence of human serum albumin.
  • the fusion protein according to the present invention is in the order of human serum albumin and Slit3 LRRD2.
  • the human serum albumin contains 609
  • human serum albumin can be used as a fragment containing the full length consisting of amino acids or a partial amino acid sequence thereof.
  • the full length amino acid sequence of human serum albumin is disclosed in NCBI GenBank: AAA98797.1, and in a specific embodiment of the present invention, it is composed of 609 amino acids.
  • human serum albumin consists of the amino acid sequence of SEQ ID NO: 2:
  • the Slit3 LRRD2 is human-derived, and can be used as a fragment containing the full length of LRRD2 or a partial amino acid sequence thereof in the Slit3 protein consisting of 1523 amino acids.
  • the full-length amino acid sequence of the Slit3 protein is disclosed in NCBI GenBank: AAQ89243.1.
  • Slit3 LRRD2 is the 278th to 486th amino acid (209 amino acids) in the full-length Slit3 protein consisting of 1523 amino acids.
  • Slit3 LRRD2 consists of the amino acid sequence of SEQ ID NO:3:
  • Slit3 LRRD2 is the amino acid of SEQ ID NO: 3
  • deletion having at least 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more sequence homology with the amino acid sequence of SEQ ID NO: 1 to 4 of the present invention, It refers to a protein or peptide that exhibits substantially the same physiological activity as a protein or peptide composed of the amino acid sequence of SEQ ID NO: 1 to 4.
  • the fusion protein may be included in the scope of the present invention as well as proteins or peptides having a wild-type amino acid sequence thereof, as well as amino acid sequence variants thereof.
  • the amino acid sequence variant refers to a wild-type amino acid sequence of Slit3 LRRD2 and at least one amino acid sequence.
  • Amino acid residues refer to proteins or peptides having different sequences by deletion, insertion, non-conservative or conservative substitution, or combinations thereof.
  • Amino acid exchange is known in 6 l : (H.Neurath, R ⁇ . Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchange is amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/ It is an exchange between Val, Ala/Glu, and Asp/Gly. In some cases, phosphorylation, sulMion,
  • Slit3 LRRD2 of the present invention is extracted from nature or
  • the Slit3 LRRD2 of the present invention can be subjected to IgG Fc protein fusion or PEGylation, in addition to albumin fusion, to increase its in vivo half-life.
  • a linker may be additionally included between albumin and LRRD2 of the Slit3 protein.
  • the preferred linker type may be (GGGGS) n (SEQ ID NO: 5), where n is 1 to It can be an integer of W, and preferably n can be an integer of 1 to 5.
  • the present invention also provides a nucleic acid molecule encoding a fusion protein in which human serum albumin is linked to the N-terminus of Slit3 LRRD2, and cystatin S may be linked to the N-terminus of the human serum albumin.
  • the base sequence encoding cystatin S as a signal peptide is linked to the 5'end of the base sequence encoding human serum albumin, and the human serum albumin is encoded.
  • the base sequence is Slit3
  • Cystatin S-HSA-Slit3 LRRD2 can be produced by linking to the 5'end of the base sequence encoding LRRD2.
  • the cystatin S is human-derived, and can be used as a nucleotide sequence encoding a full length consisting of 141 amino acids or a partial amino acid sequence thereof.
  • the full length amino acid sequence of cystatin S is NCBI GenBank: As disclosed in EAX10135.1, in a specific embodiment of the present invention, the first to 20th cystatin S of the full length consisting of 141 amino acids
  • cystatin S is a base sequence encoding the amino acid sequence of SEQ ID NO: 1.
  • the present invention also provides a recombinant vector comprising a base sequence encoding the fusion protein and a promoter functionally linked to the base sequence.
  • the term "functionally linked” refers to a functional link between a nucleic acid expression regulatory sequence (a promoter, a signal sequence, or an array of transcriptional regulatory factor binding sites) and a secondary base sequence, and the expression regulatory sequence corresponds to a secondary sequence. It affects the transcription and/or translation of nucleic acids.
  • vectors can be prepared for cloning or expression purposes.
  • vectors can be prepared for use in eukaryotic or prokaryotic host cells. For example, if the vector is prepared for expression in prokaryotic cells, it is transcribed.
  • Powerful promoters for initiating eg pU promoter, trp promoter, lac promoter, tac promoter and T7 2020/175931 1» (:1 ⁇ 1 ⁇ 2020/002809 promoter), ribosome binding site or translation start and transcription/translation stop sequence.
  • E. coli is used as a host cell, an operator in the promoter and operon for the biosynthesis of trimtophan in E. coli (Yanofsky, C., J. BacterioL,
  • phage X pLX promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980)
  • pLX promoter Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980)
  • a promoter for the gene encoding the toxin protein of Bacillus thuringensis Appl.Environ.Microbiol.
  • Typical vectors used in the present invention are pSClOl, pGVl 106, pACYC177, ColEl, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFRl, pHV14, pGEX series, pET series, pUC19,
  • a vector for a eukaryotic host cell, among others, promoters derived from the genome of animal cells, stray animal cells (eg metallothionein promoter) or mammalian viruses (eg adeno Viral rate promoters; promoters from vaccinia virus 7.5 K promoter, SV40 promoter, cytomegalovirus promoter and HSV's tk promoter) may be used.
  • stray animal cells eg metallothionein promoter
  • mammalian viruses eg adeno Viral rate promoters; promoters from vaccinia virus 7.5 K promoter, SV40 promoter, cytomegalovirus promoter and HSV's tk promoter
  • Vectors generally contain the polyadenylation site of the transcript.
  • virus-based vectors examples include pcDNA 3 (Invitrogen; including cytomegalovirus promoter and polyadenylation signal), pSI (Promega; including SV 40 promoter and polyadenylation signal), pCI (Promega; cytomegalovirus promoter, and Polyadenylation signal), and pREP7 (Invitrogen; including RSV promoter and SV 40 polyadenylation signal).
  • Promoters of genes for glyceraldehyde-3-phosphate dehydrogenase, lactase, enolase and alcohol dehydrolase can be used as regulatory sequences.
  • Plant-functional promoters may be used, cauliflower mosaic virus (CaMV) 35S promoter, pigwort mosaic virus 35S promoter, sugar cane basiliform virus promoter, comerina yellow motor virus promoter, ribulose-1,5 -bis- Light-inducible promoter from phosphate carboxylase subunit (ssRUBISCO), rice cytosolic triocell phosphate isomerase (TPI) promoter, adenine from Arabidopsis
  • CaMV cauliflower mosaic virus
  • pigwort mosaic virus 35S promoter pigwort mosaic virus 35S promoter
  • sugar cane basiliform virus promoter comerina yellow motor virus promoter
  • TPI rice cytosolic triocell phosphate isomerase
  • the recombinant vector of the present invention includes a nucleotide sequence that can easily separate the expressed fusion protein, but is not necessarily limited to glutathione
  • the fusion protein expressed by the sequence is friendly in a fast and easy way.
  • the protein contains the amino acid sequence of SEQ ID NO: 4.
  • the amino acid sequence of SEQ ID NO: 4 is as follows:
  • the fusion protein is separated by affinity chromatography.
  • affinity chromatography For example, in the case of glutathione transferase, an elution buffer containing glutathione is used, and the foreign protein can be separated quickly and easily. If you use 6 for,
  • the expression vector of the present invention preferably contains one or more markers capable of selection of the transformed host, for example, ampicillin, gentamicin, chloramphenicol, streptomycin, kanaicin, neomycin, geneticin. And
  • genes that are resistant to antibiotics such as tetracycline show 3 gene, and genes that are resistant to other toxic compounds such as metal ions.
  • the present invention also includes the recombinant vector of the present invention described above
  • Hosts useful for making transformants are well known in the art.
  • prokaryotic cell hosts 001/ JM109, ⁇ . Lee" 61G1, ⁇ . 001/ 1 ⁇ 1, ⁇ . ⁇ 1/ 1 ⁇ 392, ⁇ . ⁇ 1/ ⁇ .
  • Salmonella typhimurium, Serratia marcences, and various Pseudomonas, Corynebacterium and Streptomyces can be used.
  • yeast Sacharomyces cerevisiae
  • insect cells eg, ⁇ 0, ⁇ V138,:813 ⁇ 4, 008-7, 293, HepG2, 313, and MDCK cell lines
  • plant cells can be used.
  • Transformation of host cells can be carried out by a number of methods known in the art. For example, when prokaryotic cells are used as host cells, the method, the Hanson method
  • the present invention also provides a method of producing the fusion protein of Sho- ⁇ 011102.
  • the method includes a step of culturing under conditions for expressing the above-described transformants; and) obtaining the produced fusion protein.
  • the cultivation of the transformant for producing the fusion protein of the present invention can be carried out according to the appropriate medium and cultivation conditions known in the art. Such cultivation process is performed.
  • Cell culture is divided into suspension culture, adherent culture, and batch culture, fed-batch, and continuous culture methods according to the cell growth method.
  • the medium to be used for this method should adequately meet the requirements of the specific strain.
  • the medium used for this animal cell culture contains various carbon sources, nitrogen sources, and trace elements.
  • carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, and sunflower. Fats such as milk, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These carbon sources can be used alone or in combination.
  • nitrogen sources examples include organic nitrogen sources and urea, such as peptone, yeast extract, gravy, malt extract, corn steep liquor, and soybean meal, inorganic substances such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. These nitrogen sources may be used alone or in combination.
  • the medium may contain, as human sources, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodum-containing salt.
  • Metal salts such as magnesium sulfate or iron sulfate may be included.
  • amino acids, vitamins, and suitable precursors may be included.
  • the culture compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the ! ⁇ of the culture.
  • an antifoam such as fatty acid polyglycol ester can be used during the culture.
  • oxygen or oxygen-containing gas e.g. air
  • the temperature of the culture is usually 20°0 to 45°0. It is between 25°0 and 40°(:.
  • Non-stage acquisition can be performed to obtain a fusion protein in a separate form.
  • a fusion protein when expressed by a bacteria that has been transformed into a large amount, it is usually expressed after induction of a promoter, but is expressed. Is persistent, and the protein forms an insoluble precipitate (i.e., the inclusion body).
  • the fusion protein that forms the inclusion body uses a suitable buffer to use a suitable buffer. It can be reformed through dilution or dialysis. Then, the fusion protein is ammonium sulfate, size-differential filtration 2020/175931 1»(:1 ⁇ 1 ⁇ 2020/002809
  • the fusion protein of the present invention may be expressed in a plant body.
  • Transformation can be carried out according to conventional methods known in the art, and can be carried out according to electric shock, particle shock and Agrobacterium-induced transformation. Of these, Agrobacterium-induced transformation is most preferred. Do.
  • Agrobacterium-induced transformation can usually be performed using leaf slices and other tissues such as cotyledons and hypocotyls.
  • Selection of transformed cells can be performed by exposing the transformed culture to selection agents such as metabolic inhibitors, antibiotics, and herbicides.
  • selection agents such as metabolic inhibitors, antibiotics, and herbicides.
  • the cells are transformed and the marker gene resistant to the selection gene is stable.
  • markers are not necessarily limited to this, but the glycophosphate resistance gene and neomycin
  • phosphotransferase nptll
  • the development or regeneration of plants from plant prototyping agents or various foreign substances is well known in the art.
  • the transgenic rooted shoots produced are placed on a suitable plant growth medium.
  • the generation or regeneration of plants containing foreign genes induced by Agrobacterium can be performed by methods known in the art.
  • the present invention provides a pharmaceutical composition for preventing or treating muscle diseases comprising LRRD2 of albumin-conjugated Slit3 protein.
  • the pharmaceutical composition of the present invention may be a variety of oral or parenteral formulations.
  • one or more buffering agents eg saline or PBS
  • antioxidants e.g. saline or PBS
  • bacteriostatic agents e.g. EDTA
  • chelating agents e.g, EDTA
  • glutathione e.g., glutathione
  • fillers e.g., extenders, binders, adjuvants (for example, aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.
  • adjuvants for example, aluminum hydroxide
  • suspending agents for example, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc.
  • solids are included in one or more compounds, such as at least one excipient such as starch (including corn starch, wheat starch, rice starch, potato starch, etc.),
  • lubricants such as magnesium stearate and talc are also used.
  • liquid preparations for oral administration suspensions, liquid solutions, emulsions or syrups are used.
  • 2020/175931 1»(:1 ⁇ 1 ⁇ 2020/002809, etc., etc., which are commonly used simple diluents, liquid paraffin, and various excipients, such as humectants, sweeteners, fragrances or preservatives, etc. may be included.
  • cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant, and may additionally contain anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives.
  • Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations or suppositories.
  • Non-aqueous solutions and suspensions include
  • Vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and injectable esters such as ethyl oleate may be used.
  • injectable esters such as ethyl oleate
  • a base for suppositories witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. can be used.
  • composition of the present invention may be administered orally or parenterally, and for parenteral administration, for external use of the skin; intraperitoneal, rectal, intravenous, muscle, subcutaneous, intrauterine dura mater or intracerebral vascular injection; transdermal administration; Alternatively, it can be formulated according to methods known in the art in the form of a nasal inhalant.
  • suitable carriers for injections are, but not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol), mixtures thereof and/or vegetable oils. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, 10% ethanol, 40% propylene glycol, and 5%
  • PBS phosphate buffered saline
  • antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc. may additionally be used to protect the injections from microbial contamination.
  • the above injections may be used as an enteric solution such as dextrose. In most cases, it may contain a stabilizing agent such as sugar or sodium chloride at the stock price.
  • transdermal administration means that a pharmaceutical composition is topically administered to the skin to deliver an effective amount of the active ingredient contained in the pharmaceutical composition to the skin.
  • the fusion protein used according to the present invention is suitable propellant, for example, dichlorofluoromethane, trichlorofluoromethane,
  • the dosage unit can be determined by providing a valve that delivers a weighed amount, e.g. gelatin capsules and cartridges used in the inhaler or insufflator are a compound, and a powder of a suitable powder base such as lactose or starch. 2020/175931 1»(:1 ⁇ 1 ⁇ 2020/002809
  • Formulations for parenteral administration can be formulated to contain mixtures. Formulations for parenteral administration are generally known for all pharmaceutical chemistry (Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour).
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, activity of the drug, and Sensitivity, time of administration, route and rate of administration, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.
  • the pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or used in combination with other therapeutic agents. Can be administered by using
  • the therapeutic agent may be administered sequentially or simultaneously, and may be administered single or multiple, i.e., the total effective amount of the composition of the present invention can be administered to the patient as a single dose, and long-term administration as multiple doses. It can be administered by a fractionated treatment protocol. It is important to take into account all of the above factors and administer the amount that will achieve the maximum effect in the minimum amount without side effects, which can be easily determined by the person skilled in the art. .
  • the dosage of the pharmaceutical composition of the present invention varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and the severity of the disease. ,For parenteral administration, based on HSA-Slit3 LRRD2, preferably 0.01 to 50 mg per 1 kg of body weight per day, more preferably 0.1 to 30 mg
  • the dosage may increase or decrease depending on the route of administration, the severity of obesity, sex, weight, and age, so the above dosage does not limit the scope of the present invention in any way.
  • composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response modifiers.
  • the pharmaceutical composition of the present invention can also be provided in the form of an external preparation.
  • an external preparation for the skin additionally fatty substances, organic solvents, dissolving agents, thickening agents and gels Agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blockers , Wetting agents, essential oils, dyes, pigments, hydrophilic activators, lipophilic activators or any other ingredients commonly used in external skin preparations, such as lipid vesicles, etc., may contain adjuvants commonly used in the field of dermatology.
  • the ingredients can be introduced in amounts commonly used in the field of dermatology.
  • the pharmaceutical composition for preventing and treating muscle diseases of the present invention is provided for external use of the skin, 2020/175931 1»(:1 ⁇ 1 ⁇ 2020/002809 It may be a formulation such as an ointment, patch, gel, cream or spray, but is not limited thereto.
  • the muscle disease of the present invention is preferably a disease reported in the art as a muscle disease caused by muscle function decline, muscle wasting or muscle degeneration, and specifically
  • One or more selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle degeneration, myasthenia, cachexia, and sarcopenia is more preferable. Not limited.
  • This muscle wasting or deterioration is caused by congenital factors, acquired factors, aging, etc., and muscle consumption is characterized by gradual loss of muscle mass, weakness and degeneration of muscles, especially skeletal or voluntary muscles and heart muscles.
  • the above muscle refers to tendons, muscles, tendons comprehensively, and muscle function or muscle function refers to the ability to exert power by contraction of the muscle, and the maximum contraction force for the muscle to overcome resistance Muscle endurance, the ability to demonstrate how long or how many times a muscle can contract and relax at a given weight; and impetus, the ability to exert a strong force within a short period of time.
  • the above muscle function is proportional to muscle mass and is proportional to muscle mass.
  • improving muscle function means improving muscle function in a more positive direction.
  • the present invention also provides a health functional food composition for preventing or improving muscle diseases comprising LRRD2 of the Slit3 protein in which albumin is bound.
  • LRRD2 of the Slit3 protein in which albumin is bound.
  • the health functional food composition according to the present invention can be manufactured in various forms according to conventional methods known in the art.
  • General foods are not limited thereto, but beverages (including alcoholic beverages), fruits and processed foods thereof (Example: canned fruit, canned bottle, jam, marmalade, etc.), fish, meat and processed foods (eg, ham, sausage corn beef, etc.), bread and noodles (eg: udon, soba, ramen, spagate, etc.) Macaroni), fruit juice, various drinks, cookies, syrup, dairy products (eg butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort food, frozen food, various seasonings (eg miso, soy sauce, sauce, etc.) )Can be manufactured by adding the HSA-Slit3 LRRD2 fusion protein of the present invention to etc.
  • beverages including alcoholic beverages
  • fruits and processed foods thereof Example: canned fruit, canned bottle, jam, marmalade, etc.
  • fish, meat and processed foods eg,
  • the HSA-Slit3 LRRD2 fusion protein of the present invention itself is manufactured in the form of tea, juice and drinks.
  • the HSA-Slit3 LRRD2 fusion protein of the present invention can be prepared and used in the form of a powder or concentrate.
  • the HSA-Slit3 LRRD2 fusion protein of the present invention has the effect of preventing muscle disease and improving muscle function. 2020/175931 1»(:1 ⁇ 1 ⁇ 2020/002809 It is known that it can be prepared in the form of a composition by mixing with the active ingredients of vacuum paper.
  • the health drink composition can contain various flavors or natural carbohydrates as main components, like a regular drink.
  • the above-described natural carbohydrates are monosaccharides such as glucose and fructose; Disaccharides such as toss and sucrose; polysaccharides such as dextrin and cyclodextrin; may be sugar alcohols such as xylitol, sorbitol, and erythritol; sweeteners are natural sweeteners such as taumatin and stevia extract; saccharin, aspartame and The same synthetic sweetener can be used.
  • the ratio of the natural carbohydrate is generally about 0.01 to 0.04 per 100 11 ⁇ of the composition of the present invention, preferably about 0.02-0.03.
  • Protein can be contained as an active ingredient in a food composition for preventing muscle disease and improving muscle function, and the amount is not specifically limited to an amount effective to achieve the action for preventing muscle disease and improving muscle function, but 0.01 to 100 weight based on the total weight of the composition. It is desirable to be %. Health of the present invention
  • the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective properties. It may contain preservatives, glycerin, alcohol or carbon dioxide, etc.
  • the health food of the present invention may contain the flesh for the manufacture of natural fruit juice, fruit juice drink, or vegetable drink. These ingredients may be independently or mixed together. The ratio of these additives is not very important, but it is generally selected from 0.01 to 0.1 parts by weight per 0 parts by weight of the composition of the present invention.
  • the present invention contains 011102 fusion protein, for improving muscle function
  • a cosmetic composition is provided.
  • the composition of the active ingredients contained in the cosmetic composition of the present invention and its effects are the same as those for the aforementioned pharmaceutical composition, so the description is omitted.
  • the cosmetic composition is not particularly limited, but can be used externally to the skin.
  • Basic cosmetic composition face wash such as lotion, cream, essence, cleansing foam and cleansing water, pack, body oil
  • toned cosmetic composition foundation, lipstick, mascara, make-up base
  • hair with a dermatologically acceptable excipient It can be manufactured in the form of product composition (shampoo, rinse, hair conditioner, hair gel) and soap.
  • excipients are not limited thereto, for example, emollients, skin penetration 2020/175931 1»(:1 ⁇ 1 ⁇ 2020/002809 May contain enhancers, colorants, fragrances, emulsifiers, thickeners and solvents. In addition, flavors, colors, disinfectants, antioxidants, preservatives and moisturizers are additionally included. It can, and can contain thickeners, inorganic salts, synthetic polymer substances, etc. for the purpose of improving physical properties. For example, in the case of manufacturing face wash and soap with the cosmetic composition of the present invention, it can be easily manufactured by adding HSA-Slit3 LRRD2 fusion protein to a common face wash and soap base.
  • HSA-Slit3 LRRD2 fusion protein can be manufactured by adding HSA-Slit3 LRRD2 fusion protein to the oil-in-water (0/W) cream base.
  • flavorings, chelating agents, pigments, antioxidants, preservatives, and proteins, minerals, etc. for the purpose of improving physical properties.
  • Synthetic or natural materials such as vitamins can be added additionally.
  • the content of the HSA-Slit3 LRRD2 fusion protein contained in the cosmetic composition of the present invention is not limited thereto, but is preferably 0.001 to 10% by weight based on the total weight of the composition, and more preferably 0.01 to 5% by weight.
  • the content is less than 0.0% by weight, the desired effect cannot be expected, and when the content exceeds W% by weight, safety or formulation may be difficult to manufacture.
  • the present invention also provides a feed additive for improving muscle function containing albumin-conjugated, Slit3 protein of LRRD2.
  • the composition of the active ingredient and its effect in the feed additive of the present invention are described in the aforementioned pharmaceutical composition. As it is the same as for, the description is omitted.
  • the feed additive of the present invention is equivalent to the supplementary charge under the Feed Management Act.
  • the term "feed” may mean any natural or artificial diet, one meal meal, etc., or a component of the above one meal meal, for or suitable for eating, ingesting, and digesting animals.
  • feed can be used.
  • Non-limiting examples of the above feed include cereals, root fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, meals or grain by-products.
  • Vegetable feeds such as protein, inorganic logistics, oils and fats, minerals, unicellular proteins, zooplanktons, food, etc. These can be used alone or in combination of two or more.
  • the feed additive may additionally contain a carrier acceptable for the unit animal.
  • the feed additive may be used as it is or a known carrier, stabilizer, etc. may be added, and vitamins, amino acids, minerals, etc. may be added as necessary.
  • Various nutrients, such as, etc., antioxidants and other additives may be added, and the shape may be in an appropriate state such as powder, granules, pellets, suspensions, etc.
  • the feed additive of the present invention is supplied to the unit animal alone. Or it can be mixed with feed.
  • the present invention also provides a composition for enhancing the in vivo half-life of LRRD2 of Slit3 protein, including LRRD2 of Slit3 protein, to which albumin is bound.
  • PC DNA 3.1 vector SP cystatin S-HSA-Slit3 LRR D2-FLAG DNA 1.6 mg/ml was transfected into Expi293F suspension cells to carry out expression. After incubating the cells up to 4.5 ⁇ 5xl0 6 cells/ml in 125ml 293F cell suspension and replacing only the medium, Expifectamine 400 [ xl and Opti-mem 7.5ml (Samneul A) were reacted for 5 minutes at room temperature, 150ug of DNA and 7.5ml of Opti-mem (sample B) were reacted at room temperature for 5 minutes, then A and B samples were mixed and reacted at room temperature for 20 minutes to proceed with transfection. After 24 hours, enhancers 1 and 2 were mixed and treated, followed by incubation for 7 days.
  • FIG. 2 shows the results of performing SDS-PAGE after separating and purifying the fusion protein by the above process, and it was confirmed that the size of the fusion protein shown in FIG. 1 prepared in this example was 75 KDa.
  • HSA-S1U3 LRRD2 fusion protein HSA-S1U3 LRRD2 fusion protein
  • Slit3 LRRD2 binds to Robol or Robo2 receptor and is bound to M-cadherin in myoblasts through the Slit-Robo system (releases 3-catenin).
  • the receptor-binding ability of 12 types of HSA-Slit3 LRRD2 fusion proteins prepared in 2-1 was confirmed.
  • the binding capacity of 12 types of HSA-Slit3 LRRD2 fusion protein and Robol receptor was quantified using an ELISA system. The detailed conditions are as follows.
  • HSA-Slit3 LRRD2 fusion proteins are 96-well microtiter plates for 18 hours at 4°C to be 0, 1, 10, 100, 1000 nM per well considering molecular weight.
  • the coated material was PBS containing 0.05% Tween 20
  • PBST PBST
  • PBST PBST
  • lysis buffer 0.5% NP40, 50mM Tris pH 7.5, 150mM NaCl, ImM EDTA, 0.2mM NaF, ImM Na 3 V0 4 , ImM DTT, ImM PMSF, protease inhibitor Cocktail (Proteinase inhibitor cocktail)
  • Robol antibody diluted 1:1000 with 0.1% BSA after washing 3 times with PBST
  • the TMB solution was reacted for 30 minutes at 37 O C.
  • 100 M of 1N H 2 SO 4 was used, and the absorbance was measured at 450 nm.
  • Example 2 based on the results of Example 2, LRRD2-3 having the most excellent receptor binding ability was selected, and a drug kinetics study was conducted.
  • mice fasted for 4 hours before the experiment, and kept fasting for up to 4 hours after administration.
  • the farm gives 12 hours of light and dark, and the appropriate temperature (20 ⁇ 25 ⁇ 0 And humidity (40-60%) was maintained.
  • Slit3 LRRD2 was prepared by dissolving in PBS at a dose of 1 mg/mL.
  • HSA-Slit3 LRRD2 (LRRD2-3) was prepared by dissolving in PBS at a dose of 3.5 mg/mL (1 mg/mL as Slit3 LRRD2) considering the molecular weight.
  • the dose was W mL/kg for both groups, left It was administered through the coccygeal vein.
  • T 1/2 The half-life (T 1/2 ) was obtained by dividing LN2 by K e , and the area under the blood drug concentration versus time curve (AUC q- ) and the area under the blood drug moment versus time curve (AUMCV oo ) were linear trapezoidal method (linear trapezoidal rule) and standard area extrapolation method. Clearance (CL) and steady state volume of distribution (Vss) were calculated from [Equations 1] to [Equations 1].
  • V ss MRT X CL
  • Albumin-conjugated, LRRD2 of the Slit3 protein of the present invention exhibits the same cytological efficacy as LRRD2 of the Slit3 protein without albumin, and has a significant half-life in vivo compared to LRRD2 of the Slit3 protein without albumin. It can be increased to prevent or treat bone-related diseases more effectively.

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Abstract

본 발명은 알부민이 결합된, Slit3 단백질의 LRRD2를 포함하는 근육 질환 예방 또는 치료용 조성물에 관한 것으로, 보다 구체적으로 알부민이 결합된, Slit3 단백질의 LRRD2를 포함하는 융합 단백질, 상기 융합 단백질을 암호화하는 핵산분자, 상기 핵산분자를 포함하는 재조합 벡터, 상기 재조합 벡터를 포함하는 형질전환체, 상기 형질전환체를 이용한 융합 단백질의 제조방법, 상기 융합 단백질을 포함하는 근육 질환 예방 또는 치료용 조성물, 및 상기 융합 단백질을 포함하는 Slit3 단백질의 LRRD2의 생체 내 반감기 증진용 조성물을 제공한다.

Description

2020/175931 1»（：1/10公020/002809 명세서
발명의명칭:알부민이결합된， SLIT3단백질의 LRRD2를
포함하는근육질환예방또는치료용조성물 기술분야
[1] 본발명은알부민이결합된, Slit3단백질의 LRRD2를포함하는근육질환예방 또는치료용조성물에관한것으로,보다구체적으로알부민이결합된, Slit3 단백질의 LRRD2를포함하는융합단백질,상기융합단백질을암호화하는 핵산분자,상기핵산분자를포함하는재조합벡터,상기재조합벡터를포함하는 형질전환체,상기형질전환체를이용한융합단백질의제조방법,상기융합 단백질을포함하는근육질환예방또는치료용조성물,및상기융합단백질을 포함하는 Slit3단백질의 LRRD2의생체내반감기증진용조성물을제공한다. 배경기술
[2] Slit단백질은신경계의발생과정동안에뉴런과액손의이동을조절하는
것으로서잘알려져 있는단백질이다. Slit단백질은 Robo수용체와작용하여 생리학적활성을조절할수있으며 ,심장,폐 ,신장,유방조직등다양한조직에서 다양한세포내과정을조절하는인자로서역할을하는것으로알려졌으며,최근 세포의생장,부착능및이동능의조절에중요한역할이 있음이보고되면서 세포의분화과정에서의이동및암의발생과전이과정에 Slit단백질이관여할 수있음이보고된바있다.구체적으로,척추동물의배아발달단계에서 Slit 단백질및 Robo단백질이발현되며, Slit3, Robol및 Robo2단백질의발현이근육 조직에서증가하는것이보고된바있다 (Neil Vargesson et. al., Mechanisms of development 106.1 (2001): 175-180).이보고에따르면, Slit3단백질은배아뒷다리 근육조직의근육모세포에서발현이증가하나,이동능 (migration)에관여될뿐 근육모세포의분화에는관련하지않을것으로언급하고있다.
[3] 본발명자들은 Slit3단백질의 LRRD2가근육모세포의분화를촉진시켜
근육량의증가를유도함으로써근감소증의예방또는치료에사용할수있음을 밝힌바있다 (대한민국공개특허제 10-2017-0138920호). LRRD2는주사제로 환자가병원에내원하여투여받아야하지만,생체내반감기가매우짧아이의 약효가발휘되기위해서는투여주기가짧아질수밖에없고,이에따른과도한 약제사용으로효능이감소되는문제가발생할것으로예상된다.
[4] 이에본발명자들은 LRRD2의생체내반감기를증진함으로써이의효능을 개선시킨 HSA-Slit3 LRRD2융합단백질 (HSA-Slit3 LRRD2 fusion protein)을 개발하고,본발명을완성하였다.
발명의상세한설명
기술적과제
[5] 본발명의목적은 Slit3단백질의 LRRD2의생체내반감기를개선시기키위한 2020/175931 1»（：1^1{2020/002809 융합단백질을제공하는것이다.
[6] 본발명의다른목적은전술한융합단백질을암호화하는핵산분자,상기
핵산분자를포함하는재조합벡터 및상기 재조합벡터를포함하는
형질전환체를제공하는것이다.
[7] 본발명의또다른목적은전술한융합단백질의 제조방법을제공하는것이다.
[8] 본발명의다른목적은 Slit3단백질의 LRRD2의근육질환의 예방또는
치료효능을개선시킨조성물을제공하는것이다.
[9] 본발명의다른목적은 Slit3단백질의 LRRD2의 생체내반감기를증진시킨 조성물을제공하는것이다.
과제해결수단
[1이 상술한과제를해결하기위해,본발명은알부민이 결합된, Slit3단백질의
LRRD2의융합단백질을제공한다.
[11] 본발명의 바람직한일실시예에 따르면,상기 알부민은인간혈청
알부민 (human serum albumin)일수있다.
[12] 본발명의 바람직한또다른일실시예에 따르면,상기융합단백질은상기 인간 혈청 알부민은 Slit3단백질의 LRRD2의 N-말단에 결합된것일수있다.
[13] 본발명의 바람직한또다른일실시예에 따르면,상기 인간혈청 알부민은
서열번호 2의아미노산서열을포함할수있다.
[14] 본발명의 바람직한다른일실시예에따르면,상기 Slit3단백질의 LRRD2는 서열번호 3의아미노산서열을포함할수있다.
[15] 본발명의 바람직한또다른일실시예에 따르면,상기 알부민과 Slit3단백질의 LRRD2사이에 링커를추가로포함할수있다.
[16] 본발명의 바람직한다른일실시예에따르면,상기 링커는 (GGGGS)n
(서열번호 5)이고,여기서 n은 1내지 W의 정수일수있다.
[17] 본발명은또한,전술한융합단백질을암호화하는핵산분자를제공한다.
[18] 본발명은또한,전술한핵산분자를포함하는재조합벡터 및 이를포함하는 형질전환체를제공한다.
[19] 본발명은또한,전술한형질전환체를배양하는단계를포함하는,융합
단백질의 제조방법을제공한다.
[2이 본발명은또한,전술한융합단백질을근육질환예방또는치료용약학
조성물을제공한다.
[21] 본발명의 바람직한다른일실시예에따르면,상기 약학조성물은주사제로 투여될수있다.
[22] 본발명의 바람직한또다른일실시예에 따르면,상기근육질환은
긴장감퇴증 (atony),근위죽증 (muscular atrophy),근이영양증 (muscular dystrophy), 근무력증,악액질 (cachexia)및근감소증 (sarcopenia)으로이루어진군으로부터 선택되는어느하나이상일수있다. 2020/175931 1»（：1/10公020/002809 본발명은또한,알부민이결합된, Slit3단백질의 LRRD2를포함하는, Slit3 단백질의 LRRD2의 생체내반감기증진용조성물을제공한다.
발명의효과
24] 본발명의 알부민이결합된, Slit3단백질의 LRRD2는알부민이 결합되지 않은 Slit3단백질의 LRRD2와동일한세포학적효능을나타내고,알부민이결합되지 않은 Slit3단백질의 LRRD2와비교하여 생체내반감기가현저하게증가되어 골 관련질환을보다효과적으로예방또는치료할수있다.
도면의간단한설명
도 1은본발명의 알부민이 Slit3 LRRD2의 N-말단에 결합된융합단백질의 구성 및이의 아미노산서열을나타낸다.
도 2는 SP시스타틴 S-HSA-Slit3 LRRD2융합단백질을분리정제후 SDS-PAGE를시행한결과를나타낸것이다.
도 3은다양한형태의 HSA-Slit3 LRRD2융합단백질 (HSA-Slit3 LRRD2 fusion protein)의수용체결합능을그래프로나타낸것이다.
도 4는단식시킨수컷 ICR마우스에서 Slit3 LRRD2 (·,” Slit3”)와 HSA-Slit3 LRRD2 (■,”HSA-Slit3”)의 IV투여후 Slit3 LRRD2의 혈장농도시간프로파일을 나타낸것이다.
발명의실시를위한최선의형태
[29] 상술한바와같이, Slit3단백질의 LRRD2는근육모세포의분화를촉진시켜 근육량의증가를유도함으로써근감소증의 예방또는치료에사용할수있으나, 생체내반감기가매우짧아이의 약효가발휘되기위해서는투여주기가짧아질 수밖에 없고,이에 따른과도한약제사용으로효능이감소되는문제가발생할 것으로예상되었다.
] ] ] ] ] ] ]
231 이에,본발명자들은 LRRD2의 생체내반감기를증진함으로써 이의효능을
22222 33330735681
개선시킨 HSA-Slit3 LRRD2융합단백질 (HSA-Slit3 LRRD2 fusion protein)을 개발함으로써상술한문제의 해결방안을모색하였다.본발명의 알부민이 결합된, Slit3단백질의 LRRD2는알부민의 결합되지 않은 Slit3단백질의
LRRD2와동일한세포학적효능을나타내고,알부민이결합되지 않은 Slit3 단백질의 LRRD2와비교하여 생체내반감기가현저하게증가되어근육관련 질환을보다효과적으로예방또는치료할수있다.
이하,본발명을보다상세히 설명한다.
본발명은알부민이결합된, Slit3단백질의 LRRD2의융합단백질을제공한다. 본발명의융합단백질에 있어서, "Slit3단백질의 LRRD2”는 Slit3단백질내의 두번째류신풍부반복도메인 (leucine rich repeat domain, LRRD2)을말한다.본 발명자들은이전연구를통해 Slit3단백질또는이 단백질내의 LRRD2가 Robol 또는 Robo2수용체와결합하여 Slit-Robo시스템을통해근육모세포의
M-카데린 (M-caderin)에결합되어있던 (3-카테닌 ((3-catenin)을유리하여 2020/175931 1»（：1^1{2020/002809
(3-카테닌을활성화하고미오게닌 (my 미의발현을증가시켜,
근육모세포 (myoblast)의분화를유도함으로써근육형성을족진한다는것을 확인하였다.본발명에서,용어 "Slit3 LRRD2”는 "Slit3단백질의 LRRD2”를 의미하며 ,상호호환적으로사용될수있다.
[34] 본발명의융합단백질에있어서 ,상기알부민은인간혈청알부민 (human serum albumin),래서스 (rhesus)혈청알부민 (RhSA),시노몰구스원숭이 (cynomolgous monkey)혈청알부민 (CySA),또는뮤린 (murine)혈청알부민 (MuSA)일수있고, 바람직하게는인간혈청알부민일수있다.상기 Slit3 LRRD2는인간혈청 알부민존재하에 Slit3 LRRD2의생체내반감기는인간혈청알부민부재 하에서의 Slit3 LRRD2의생체내반감기보다적어도 W배이상더길다.본 발명의구체적인일실시예에서,인간혈청알부민존재하의 Slit3 LRRD2의혈청 반감기는인간혈청알부민부재하의 Slit3 LRRD2보다 14배더길다.
[35] 본발명에따른융합단백질은인간혈청알부민및 Slit3 LRRD2의순서로
결합되거나,그반대일수있다.바람직하게는인간혈청알부민및 Slit3
LRRD2의순서로결합된다.예를들어,인간혈청알부민이 Slit3 LRRD2의 N 말단에결합하는경우, Slit3 LRRD2의생체내반감기및이의근육질환관련 효능이가장우수하며,인간혈청알부민이 C말단에결합하는경우,정도의 차이가있을수있으나유효한효능을나타낼수있다.
[36] 본발명의융합단백질에있어서 ,상기인간혈청알부민은 609개의
아미노산으로이루어진전장또는이의일부아미노산서열을포함하는 단편으로사용될수있다.인간혈청알부민의전장아미노산서열은 NCBI GenBank: AAA98797.1에개시된것으로,본발명의구체적인일실시예에서는 609개의아미노산으로이루어진전장의인간혈청알부민에서 25번째내지 609번째의아미노산 (585개의아미노산)으로이루어진단편의형태를
사용하였다.본발명의융합단백질에서,인간혈청알부민은하기서열번호 2의 아미노산서열로이루어진다:
[37] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK TCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLF FAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGE RAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADL AKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKD VCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVS TPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSD RVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKK QTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL (서열번호 2). 2020/175931 1»（：1^1{2020/002809
[38] 본발명의융합단백질에 있어서 ,상기 Slit3 LRRD2는인간유래이며 , 1523개의 아미노산으로이루어진 Slit3단백질내의 LRRD2의전장또는이의일부 아미노산서열을포함하는단편으로사용될수있다. Slit3단백질의전장 아미노산서열은 NCBI GenBank: AAQ89243.1에개시된것으로,본발명의 구체적인일실시예에서 Slit3 LRRD2는 1523개의아미노산으로이루어진전장의 Slit3단백질에서 278번째내지 486번째의아미노산 (209개의아미노산)으로 이루어진단편의형태를사용하였다.본발명의융합단백질에서 Slit3 LRRD2는 하기서열번호 3의아미노산서열로이루어진다:
[39] ISCPSPCTCSNNIVDCRGKGLMEIPANLPEGIVEIRLEQNSIKAIPAGAFTQYKK LKRIDISKNQISDIAPDAFQGLKSLTSLVLYGNKITEIAKGLFDGLVSLQLLLLN ANKINCLRVNTFQDLQNLNLLSLYDNKLQTISKGLFAPLQSIQTLHLAQNPFVC DCHLKWLADYLQDNPIETSGARCSSPRRLANKRISQIKSKKFRCS (서열번호 3).
[4이 본발명의융합단백질에 있어서 , "Slit3 LRRD2”는서열번호 3의아미노산
서열과의기능적동등물을포함할수있다.
[41] 상기 "기능적동등물”은단백질또는펩티드의아미노산부가,치환또는
결실로인해,본발명의서열번호 1내지 4의아미노산서열과적어도 70%이상, 바람직하게는 80%이상,보다바람직하게는 90%이상,더욱바람직하게는 95% 이상의서열상동성을갖는것으로,서열번호 1내지 4의아미노산서열로 구성된단백질또는펩티드와실질적으로동질의생리활성을나타내는단백질 또는펩티드를말한다.
[42] 구체적으로,상기융합단백질은이의야생형아미노산서열을갖는단백질 또는펩티드뿐만아니라이의아미노산서열변이체또한본발명의범위에 포함될수있다.상기아미노산서열변이체란 Slit3 LRRD2의야생형아미노산 서열과하나이상의아미노산잔기가결실,삽입,비보존적또는보존적치환 또는이들의조합에의하여상이한서열을가지는단백질또는펩티드를 의미한다.
[43] 분자의활성을전체적으로변경시키지않는단백질및펩티드에서가능한
아미노산교환은당해분6ᅵ:에공지되어 있다 (H.Neurath, R丄. Hill, The Proteins, Academic Press, New York, 1979).가장통상적으로일어나는교환은아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly간의 교환이다.경우에따라서는인산화 (phosphorylation),황화 (sulMion),
아세틸화 (acetylation),당화 (glycosylation),메틸화 (methylation),
파네실화 (famesylation)등으로수식 (modification)될수도있다.
[44] 본발명의 Slit3 LRRD2,또는이의변이체는천연에서추출하거나
합성 (Merrifleld, J. Amer. chem. Soc. 85:2149-2156, 1963)또는 DNA서열을 기본으로하는유전자재조합방법에의해제조될수있다 (Sambrook et al, Molecular Cloning, Cold Spring Harbour Laboratory Press, New York, USA, 2판, 2020/175931 1»（：1/10公020/002809
1989）.
본발명의 Slit3 LRRD2는이의 생체내반감기를증진시키기 위해 ,알부민융합 이외에도 IgG의 Fc단백질융합또는페길레이션（PEGylation）등이수행될수 있다.
[46] 본발명의융합단백질에 있어서 ,알부민과 Slit3단백질의 LRRD2사이에는 링커가추가로포함될수있다.바람직한링커의종류로는（GGGGS）n（서열번호 5）일수있고,여기서 n은 1내지 W의 정수일수있으며,바람직하게는 n은 1내지 5의 정수일수있다.
본발명은또한, Slit3 LRRD2의 N말단에 인간혈청 알부민이 연결된형태의 융합단백질을암호화하는핵산분자를제공하며,추가로,상기 인간혈청 알부민의 N말단에는시스타틴 S가연결될수있다.
[48] 본발명의 핵산분자에 있어서,시스타틴 S는신호펩티드로서상기시스타틴 S를암호화하는염기서열은인간혈청 알부민을암호화하는염기서열의 5’ 말단에 연결되고,상기 인간혈청 알부민을암호화하는염기서열은 Slit3
LRRD2를암호화하는염기서열의 5’말단에 연결되어시스타틴 S-HSA-Slit3 LRRD2가생산될수있다.
본발명의 핵산분자에 있어서,상기시스타틴 S는인간유래이며, 141개의 아미노산으로이루어진전장또는이의 일부아미노산서열을암호화하는 염기서열로사용될수있다.시스타틴 S의 전장아미노산서열은 NCBI GenBank: EAX10135.1에 개시된것으로,본발명의구체적인일실시예에서는 141개의 아미노산으로이루어진전장의시스타틴 S에서 1번째내지 20번째의
아미노산（20개의 아미노산）으로이루어진단편을암호화하는염기서열을 사용하였다.본발명의 핵산분자에서,시스타틴 S는하기서열번호 1의 아미노산 서열을암호화하는염기서열이다.
347
55 50 4 495] MARPLCTLLLLMATLAGALA（서열번호 1）.
본발명은또한,상기융합단백질을암호화하는염기서열및상기 염기서열에 기능적으로연결된프로모터를포함하는재조합벡터를제공한다.
52] 용어 "기능적으로연결된”은핵산발현조절서열（프로모터,신호서열,또는 전사조절인자결합부위의 어레이）및이차염기서열사이가기능적으로연결된 것을의미하고,상기발현조절서열은이차서열에상응하는핵산의 전사 및/또는번역에 영향을준다.
본발명의 벡터시스템은문헌 [Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press（2001）]에기재 ¾
Figure imgf000008_0001
¾-o| 당업계에 잘알려진방법에 따라제조될수있다.
54] 일반적으로,벡터는클로닝또는발현의목적으로제조될수있다.또한,벡터는 진핵또는원핵숙주세포에서사용하기위해제조될수있다.예를들어,벡터가 원핵세포에서발현하기 위해제조된다면,전사를개시하기위한강력한 프로모터（예 , pU프로모터 , trp프로모터 , lac프로모터 , tac프로모터및 T7 2020/175931 1»（：1^1{2020/002809 프로모터 ),리보좀결합부위또는번역 개시 및전사/번역중지서열을포함한다. 특히, E. coli가숙주세포로서사용될경우, E. coli에 있는트림토판생합성을 위해프로모터 및오페론에 있는오퍼레이터 (Yanofsky, C., J. BacterioL,
158: 1018- 1024(1984))및파지 X의 왼쪽방향프로모터 (pLX프로모터 , Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445(1980))가조절서열로써사용될수 있다.숙주세포로서바실러스가사용될경우,바실러스츄린겐시스의톡신 단백질을코딩하는유전자에 대한프로모터 (Appl. Environ. Microbiol.
64:3932-3938(1998); and Mol. Gen. Genet. 250:734-741(1996))또는바실러스에 있는다른작동가능한프로모터가조절서열로서사용될수있다.
[55] 원핵세포에사용가능한수많은통상적인벡터들은당업자들에게잘알려져 있고,적당한벡터의선별은선별의문제이다.본발명에서사용된통상적인 벡터는 pSClOl, pGVl 106, pACYC177, ColEl, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFRl, pHV14, pGEX series, pET series, pUC19,
Xgt · 4XB, X-charon, XAzl및 M13을포함하며,반드시 이에 한정되는것은아니다.
[56] 예를들어,벡터가진핵숙주세포를위해제조될경우,그중에서도,동물세포, 표유동물세포의 게놈으로부터유래된프로모터 (예,메탈로티오네인프로모터) 또는포유동물의바이러스 (예,아데노바이러스레이트프로모터;백시니아 바이러스 7.5 K프로모터 , SV40프로모터 ,사이토메갈로바이러스프로모터 및 HSV의 tk프로모터)로부터의프로모터가사용될수있다.벡터는일반적으로 전사체의폴리아데닐화부위를포함한다.상업적으로이용가능한
바이러스-기초의 벡터의 예는 pcDNA 3 (Invitrogen;사이토메갈로바이러스 프로모터 및폴리아데닐화신호를포함), pSI (Promega; SV 40프로모터 및 폴리아데닐화신호포함), pCI (Promega;사이토메갈로바이러스프로모터 및 폴리아데닐화신호를포함),및 pREP7 (Invitrogen; RSV프로모터 및 SV 40 폴리아데닐화신호포함).
[57] 벡터가효모에 대해제조될경우,포스포글리세레이트키나아제 ,
글리세르알데히드- 3 -포스페이트디하이드로게나아제 ,락타아제 ,에놀라아제 및 알코올디하이드롤라아제에 대한유전자의프로모터가조절서열로서사용될 수있다.
[58] 발현벡터가식물세포에 대해제조될경우,당업계에 알려진다양한
식물체-기능적프로모터가사용될수있으며,콜리플라우어모자이크바이러스 (CaMV) 35S프로모터,피그워트모자이크바이러스 35S프로모터,슈가케인 바실리폼바이러스프로모터,코메리나옐로우모틀바이러스프로모터, 리불로스- 1,5 -비스-포스페이트카르복실라아제의소단위 (ssRUBISCO)로부터의 빛-유도성프로모터,쌀사이토졸릭트리오세포스페이트이소머라아제 (TPI) 프로모터 ,아라비돕시스 (Arabidopsis)로부터의 아데닌
포스포리보실트랜스퍼라아제 (APRT)프로모터 ,쌀액틴 1유전자프로모터 및 만노핀신타아제및옥토핀신타아제프로모터를포함한다. 2020/175931 1»（：1/10公020/002809 또한,본발명의 재조합벡터는발현된융합단백질을간편하게분리할수있는 염기서열을포함하며 ,반드시 이에 한정되는것은아니지만글루타티온
Figure imgf000010_0001
서열로인해발현된융합단백질은빠르고간편한방법으로친화
크로마토그래피로분리할수있다.
60] 본발명의구체적인일실시예에서는 서열을사용하였으며 ,
Figure imgf000010_0002
단백질은서열번호 4의 아미노산서열을포함한다.서열번호 4의 아미노산 서열은아래와같다:
(서열번호 4).
62] 본발명의다른일실예에 따르면,융합단백질은친화크로마토그래피에 의해 분리된다.예를들어,글루타치온 트랜스퍼라아제의경우,글루타티온을 포함하는용출완충액이사용되며,외래단백질을빠르고간편하게분리하기 위해 6 를사용하는경우,
Figure imgf000010_0003
사용된다.
본발명의 발현벡터는형질전환된숙주를선별가능하게할수있는하나또는 그이상의마커를포함하는것이 바람직하며,예를들어,암피실린,젠타마이신, 클로람페니콜,스트렙토마이신,카나 이신,네오마이신,제네티신및
테트라사이클린과같은항생제에 내성을갖는유전자, 1¾쇼3유전자,금속 이온과같은다른독성화합물에 저항성을가진유전자가있다.
[64] 본발명은또한,상기에기재된본발명의 재조합벡터를포함하는
형질전환체를제공한다.
[65] 형질전환체를제조하는데유용한숙주는당업계에 잘알려져 있다.예를들어, 원핵세포숙주,及. 001/ JM109,及.이" 61그1,及. 001/ 1^1,及. ^1/ 1止392,及. ^1/ 及.
91
5 6 6 6361 0011 X 1776, ^^3110,바실러스서브틸리스,바실러스츄린겐시스,
살모넬라타이피뮤리움,세라티아마르센세스및다양한슈도모나스, 코리네박테리움및스트렙토마이세스가사용될수있다.
진핵세포에서 ,효모 (사카로마이세스세레비시애 ),곤충세포,인간세포 (예 , ^0, \V138,：81¾, 008-7, 293, HepG2, 313,요 및 MDCK세포주)및식물체 세포가사용될수있다.
[671 숙주세포의 형질전환은당업계에 알려진수많은방법에 의해수행될수있다. 예를들어,숙주세포로서,원핵세포를사용하는경우, 방법,핸슨방법
!、!. 산., ¾0(：. ^1:1. . 801. 9:2110-2114(1973);크 모 I)., 1. Mol. 6101., 166:557-580(1983))및전기영동이 형질전환을위해사용될수있다. 또한,숙주세포로서,진핵세포를사용하는경우,미세주입,칼슘포스페이트 침전,전기충격,리포좀-매개의 형질감염, -덱스트란처리법,및입자 충격법이 형질전환을위해사용될수있다.또한,식물세포가숙주세포로서 사용될경우,아그로박테리움-매개의 형질전환이 가장바람직한방법이며,이는 2020/175931 1»（：1^1{2020/002809 원형질체로부터인접한식물체의재분화를위해필요한우회를가능하게하기 때문이다.
[68] 본발명은또한, 쇼- 此 011102의융합단백질을생산하는방법을제공한다. 상기방법은知）상기기재된형질전환체를발현시키기위한조건하에서 배양하는단계;및 ）생산된융합단백질을수득하는단계를포함한다.
[69] 본발명의융합단백질을제조하기위한형질전환체의배양은당업계에알려진 적당한배지와배양조건에따라이루어질수있다.이러한배양과정은
당업자라면선택되는균주에따라용이하게조정하여사용할수있다.세포 배양은,세포의성장방식에따라현탁배양과부착배양,배양방법에따라회분식 , 유가식및연속배양식의방법으로구분된다.배양에사용되는배지는특정한 균주의요구조건을적절하게만족시켜야하다.
이 동물세포배양에사용하는상기배지는다양한탄소원,질소원및미량원소 성분을포함한다.사용될수있는탄소원의 예는,포도당,자당,유당,과당, 말토오스,전분및셀룰로오스와같은탄수화물,대두유,해바라기유,피마자유 및코코넛유와같은지방,팔미트산,스테아린산및리놀레산과같은지방산, 글라이세롤및에탄올과같은알코올,그리고아세트산과같은유기산을 포함한다.이들탄소원은단독또는조합되어사용될수있다.사용될수있는 질소원의 예는,펩톤,효모추출물,육즙,맥아추출물,옥수수침지액 8니및 대두밀과같은유기질소원및요소,황산암모늄,염화암모늄,인산암모늄, 탄산암모늄및질산암모늄과같은무기질소원을포함한다.이들질소원은단독 또는조합되어사용될수있다.상기배지에는인원으로서,인산이수소칼륨, 인산수소이칼륨및대응되는소둠-함유염이포함될수있다.또한,
황산마그네슘또는황산철과같은금속염을포함할수있다.그외에,아미노산, 비타민,및적절한전구체등이포함될수있다.
1] 배양중에수산화암모늄,수산화칼륨,암모니아,인산및황산과같은화합물을 배양물에적절한방식으로첨가하여，배양물의 !^를조정할수있다.또한,배양 중에는지방산폴리글리콜에스테르와같은소포제를사용하여기포생성을 억제할수있다.또한,배양물의호기상태를유지하기위하여 ,배양물내로산소 또는산소-함유기체（예,공기）를주입한다.배양물의온도는보통 20°0내지 45°0 바람직하게는 25°0내지 40ᄋ（：이다.
[72] 본발명의 쇼- 此 011102의융합단백질을생산하는방법에 있어서,상기
（비단계의수득은분리된형태에있는융합단백질을수득하기위해수행될수 있다.예를들어,융합단백질이대용량으로형질전환된박테리아에의해발현될 경우,일반적으로는프로모터유도후에발현되며,그러나발현은지속되고, 단백질이불용성침전물을형성하게된다（즉,인클루젼바디）.인클루젼바디의 분리를위한적합한몇가지프로토콜이있다.인클루젼바디를형성한융합 단백질은적합한완충액을이용하여희석또는투석을통해재형성될수있다. 그리고난뒤,융합단백질은암모늄설페이트,크기차별여과 2020/175931 1»（：1^1{2020/002809
（울트라필트레이션）및컬럼크로마토그래피（크기,네트표면전하,소수성또는 친화도에의존）의사용에의해용해도분획화를포함하는당업계에알려진기본 방법으로수행될수있다.
3] 본발명의융합단백질은식물체에서발현될수도있다.식물세포의
형질전환은당업계에알려진통상적인방법에따라수행할수있으며,전기충격, 입자충격및아그로박테리움-유도된형질전환에따라수행될수있다.이들 중에서,아그로박테리움-유도된형질전환이가장바람직하다.
아그로박테리움-유도된형질전환은일반적으로잎절편및자엽및배축과같은 다른조직을이용하여수행할수있다.
4] 형질전환된세포의선별은형질전환된배양을대사저해제（metabolic inhibitor）, 항생제및제초제와같은선별제제에노출시킴으로써수행할수있다.세포가 형질전환되고선별유전자에저항성을갖는마커유전자를안정적으로
발현하면배양동안에성장및분화한다.예를들어마커는반드시이에한정되는 것은아니지만글리코포스페이트저항성유전자및네오마이신
포스포트랜스퍼라아제（nptll）시스템이있다.식물원형질제또는다양한외래 물질로부터의식물체의발생또는재분화는당업계에잘알려져있다.생산된 형질전환발근된신초는적합한식물체성장배지에치상된다.
아그로박테리움에의해유도된외래유전자를포함하는식물체의발생또는 재분화는당업계에알려진방법에의해수행될수있다.
5] 본발명은알부민이결합된 Slit3단백질의 LRRD2를포함하는근육질환예방 또는치료용약학조성물을제공한다.
6] 본발명의약학조성물은경구또는비경구의여러가지제형일수있다.상기 약학조성물을제형화할경우에는하나이상의완충제（예를들어,식염수또는 PBS）,항산화제,정균제,킬레이트화제（예를들어, EDTA또는글루타치온）, 충진제,증량제,결합제,아쥬반트（예를들어,알루미늄하이드록사이드）,현탁제, 농후제습윤제,붕해제또는계면활성제,희석제또는부형제를사용하여조제될 수있다.
刀 경구투여를위한고형제제에는정제,환제,산제,과립제,캡슐제등이
포함되며,이러한고형제는하나이상의화합물에적어도하나이상의부형제 예를들면,전분（옥수수전분,밀전분,쌀전분,감자전분등포함）,
칼슘카보네이트（calcium carbonate）,수크로스（sucrose）,락토오스（lactose）, 덱스트로오스,솔비톨,만니톨,자일리톨,에리스리톨말티톨,셀룰로즈,메틸 셀룰로즈,나트륨카르복시메틸셀룰로오즈및하이드록시프로필메틸-셀룰로즈 또는젤라틴등을섞어조제된다.예컨대,활성성분을고체부형제와배합한 다음이를분쇄하고적합한보조제를첨가한후과립혼합물로가공함으로써 정제또는당의정제를수득할수있다.
8] 또한,단순한부형제이외에스테아린산마그네슘,탈크등과같은윤활제들도 사용된다.경구투여를위한액상제제로는현탁제,내용액제,유제또는시럽제 2020/175931 1»（：1^1{2020/002809 등이 해당되는데,흔히사용되는단순희석제인물,리퀴드파라핀이외에 여러 가지부형제,예를들면습윤제,감미제,방향제또는보존제등이포함될수 있다.또한,경우에 따라가교결합폴리비닐피롤리돈,한천,알긴산또는나트륨 알기네이트등을붕해제로첨가할수있으며 ,항응집제 ,윤활제 ,습윤제 ,향료, 유화제 및방부제등을추가로포함할수있다.
P9] 비경구투여를위한제제에는멸균된수용액,비수성용제,현탁용제，유제, 동결건조제제또는좌제등이포함된다.비수성용제 및현탁용제로는
프로필렌글리콜 (propylene glycol),폴리에틸렌글리콜,올리브오일과같은 식물성 기름,에틸올레이트와같은주사가능한에스테르등이사용될수있다. 좌제의 기제로는위텝솔 (witepsol),마크로골,트윈 (tween) 61,카카오지 , 라우린지,글리세롤,젤라틴등이사용될수있다.
[8이 본발명의 약학조성물은경구또는비경구로투여될수있으며,비경구투여시 피부외용;복강내,직장,정맥,근육,피하,자궁내경막또는뇌혈관내주사하는 주사제 ;경피투여제 ;또는비강흡입제의 형태로당업계에공지된방법에따라 제형화할수있다.
[81] 상기주사제의 경우에는반드시 멸균되어야하며 박테리아및진균과같은
미생물의오염으로부터보호되어야한다.주사제의경우적합한담체의 예로는 이에 한정되지는않으나,물,에탄올,폴리올 (예를들어 ,글리세롤,프로필렌 글리콜및 액체폴리에틸렌글리콜등),이들의혼합물및/또는식물유를 포함하는용매또는분산매질일수있다.보다바람직하게는,적합한담체로는 행크스용액,링거용액,트리에탄올아민이함유된 PBS (phosphate buffered saline)또는주사용멸균수, 10%에탄올, 40%프로필렌글리콜및 5%
덱스트로즈와같은등장용액등을사용할수있다.상기주사제를미생물 오염으로부터보호하기위해서는파라벤,클로로부탄올,페놀,소르빈산, 티메로살등과같은다양한항균제 및항진균제를추가로포함할수있다.또한, 상기주사제는대부분의 경우당또는나트륨클로라이드와같은등장화제를 주가로포함할수있다.
[82] 경피투여제의 경우연고제,크림제,로션제,겔제,외용액제,파스타제,
리니멘트제 ,에어롤제등의 형태가포함된다.상기에서경피투여는약학 조성물을국소적으로피부에투여하여 약학조성물에함유된유효한양의 활성성분이 피부내로전달되는것을의미한다.
[83] 흡입투여제의 경우,본발명에 따라사용되는융합단백질은적합한추진제, 예를들면,디클로로플루오로메탄,트리클로로플루오로메탄,
디클로로테트라플루오로에탄,이산화탄소또는다른적합한기체를사용하여 , 가압팩또는연무기로부터 에어로졸스프레이 형태로편리하게전달할수있다. 가압에어로졸의경우,투약단위는계량된양을전달하는밸브를제공하여 결정할수있다.예를들면,흡입기또는취입기에사용되는젤라틴캡슐및 카트리지는화합물,및락토즈또는전분과같은적합한분말기제의분말 2020/175931 1»（：1^1{2020/002809 혼합물을함유하도록제형화할수있다.비경구투여용제형은모든제약화학에 일반적으로공지된처방서인문헌 (Remington’s Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour)에기재되어 있다.
[84] 본발명의 약학조성물은약제학적으로유효한양으로투여한다.본발명에
있어서 ,”약제학적으로유효한양”은의학적치료에 적용가능한합리적인 수혜/위험 비율로질환을치료하기에충분한양을의미하며,유효용량수준은 환자의 질환의종류,중증도,약물의 활성,약물에 대한민감도,투여시간,투여 경로및배출비율,치료기간,동시사용되는약물을포함한요소및기타의학 분야에 잘알려진요소에따라결정될수있다.본발명의 약학조성물은개별 치료제로투여하거나다른치료제와병용하여투여될수있고종래의
치료제와는순차적또는동시에투여될수있으며,단일또는다중투여될수 있다.즉,본발명의조성물의총유효량은단일투여량 (single dose)으로환자에게 투여될수있으며 ,다중투여량 (multiple dose)으로장기간투여되는분할치료 방법 (fractionated treatment protocol)에의해투여될수있다.상기한요소들을 모두고려하여부작용없이최소한의 양으로최대효과를얻을수있는양을 투여하는것이중요하며,이는당업자에의해용이하게결정될수있다.
[85] 본발명의 약학조성물의투여량은환자의 체중,연령,성별,건강상태，식이, 투여시간,투여방법，배설율및질환의중증도에따라그범위가다양하다.일일 투여량으로는,비경구투여시 HSA-Slit3 LRRD2를기준으로하루에 체중 1 kg당 바람직하게 0.01내지 50 mg,더바람직하게는 0.1내지 30 mg의 양으로
투여되도록,그리고경구투여시는본발명의 HSA-Slit3 LRRD2를기준으로 하루에 체중 1 kg당바람직하게 0.01내지 100 mg,더바람직하게는 0.01내지 10 mg의 양으로투여되도록 1내지수회에나누어투여할수있다.그러나투여 경로,비만의중증도,성별,체중,연령등에 따라서증감될수있으므로상기 투여량이 어떠한방법으로도본발명의 범위를한정하는것은아니다.
[86] 본발명의 약학조성물은단독으로,또는수술,방사선치료,호르몬치료,화학 치료및생물학적 반응조절제를사용하는방법들과병용하여사용할수있다.
[87] 본발명의 약학조성물은또한외용제의 제형으로제공할수있다.본발명의 근육질환예방및치료용약학조성물을피부외용제로사용하는경우,추가로 지방물질,유기용매,용해제,농축제 및겔화제,연화제,항산화제,현탁화제, 안정화제,발포제 (foaming agent),방향제,계면활성제,물,이온형유화제, 비이온형유화제,충전제,금속이온봉쇄제,킬레이트화제,보존제,비타민, 차단제,습윤화제,필수오일,염료,안료,친수성활성제,친유성 활성제또는 지질소낭등피부외용제에통상적으로사용되는임의의다른성분과같은피부 과학분야에서통상적으로사용되는보조제를함유할수있다.또한상기 성분들은피부과학분야에서 일반적으로사용되는양으로도입될수있다.
[88] 본발명의근육질환예방및치료용약학조성물이피부외용제로제공될경우, 2020/175931 1»（：1^1{2020/002809 이에 제한되는것은아니나,연고,패취,겔,크림또는분무제등의 제형일수 있다.
[89] 본발명의근육질환은근기능저하,근육소모또는근육퇴화로인한근육 질환으로당업계에보고된질병인것이 바람직하며,구체적으로
긴장감퇴증（atony）,근위죽증（muscular atrophy）,근이영양증（muscular dystrophy）, 근육퇴화,근무력증,악액질（cachexia）및근육감소증（sarcopenia）으로이루어진 군으로부터선택되는어느하나이상인것이보다바람직하나,이에 한정되지 않는다.
[9이 상기근육소모또는퇴화는선천적요인,후천적요인,노화등을원인으로 발생하며,근육소모는근육량의 점진적손실,근육,특히 골격근또는수의근및 심장근육의 약화및퇴행을특징으로한다.
[91] 보다구체적으로,상기근육은심줄,근육,건을포괄적으로지칭하고,근기능 또는근육기능은근육의수축에 의해힘을발휘할수있는능력을의미하며, 근육이 저항을이겨내기 위하여최대한의수축력을발휘할수있는근력 ;근육이 주어진중량에 얼마나오랫동안또는얼마나여러 번수축과이완을반복할수 있는지 나타내는능력인근지구력 ;및단시간내에강한힘을발휘하는능력인 순발력을포함한다.상기근기능은근육량에 비례하며,용어근기능개선은근 기능을보다긍정적인방향으로향상시키는것을의미한다.
[92] 본발명은또한,알부민이결합된, Slit3단백질의 LRRD2를포함하는근육질환 예방또는개선용건강기능식품조성물을제공한다.본발명의 건강기능식품 조성물에포함되는유효성분의구성 및 이의효과는전술한약학조성물에 대한 것과동일하므로,그기재를생략한다.
[93] 본발명에 따른건강기능식품조성물은당업계에 공지된통상적인방법에따라 다양한형태로제조할수있다.일반식품으로는이에 한정되지 않지만 음료（알콜성 음료포함）,과실및그의가공식품（예:과일통조림,병조림,잼, 마아말레이드등）,어류,육류및그가공식품（예:햄,소시지콘비이프등）,빵류 및면류（예:우동,메밀국수,라면,스파게이트,마카로니등）,과즙,각종드링크, 쿠키,엿,유제품（예:버터,치이즈등）,식용식물유지 ,마아가린,식물성단백질, 레토르트식품,냉동식품,각종조미료（예:된장,간장,소스등）등에본발명의 HSA-Slit3 LRRD2융합단백질을첨가하여 제조할수있다.또한,
영양보조제로는이에 한정되지 않지만캡슐,타블렛,환등에본발명의
HSA-Slit3 LRRD2융합단백질을첨가하여 제조할수있다.또한,
건강기능식품으로는이에 한정되지 않지만예를들면,본발명의 HSA-Slit3 LRRD2융합단백질자체를차,쥬스및드링크의 형태로제조하여
음용（건강음료）할수있도록액상화,과립화,캡슐화및분말화하여 섭취할수 있다.또한,본발명의 HSA-Slit3 LRRD2융합단백질을식품첨가제의 형태로 사용하기 위해서는분말또는농축액 형태로제조하여사용할수있다.또한,본 발명의 HSA-Slit3 LRRD2융합단백질과근육질환예방및근기능개선효과가 2020/175931 1»（：1^1{2020/002809 있다고알려진공지의활성성분과함께혼합하여조성물의형태로제조할수 있다.
[94]
Figure imgf000016_0001
011102융합단백질을건강음료로이용하는경우,상기 건강음료조성물은통상의음료와같이여러가지향미제또는천연탄수화물 등을주가성분으로함유할수있다.상술한천연탄수화물은포도당,과당과 같은모노사카라이드;말토스,슈크로스와같은디사카라이드;덱스트린, 사이클로덱스트린과같은폴리사카라이드;자일리톨,소르비톨,에리트리톨 등의당알콜일수있다.감미제는타우마틴,스테비아추출물과같은천연 감미제 ;사카린,아스파르탐과같은합성감미제등을사용할수있다.상기천연 탄수화물의비율은본발명의조성물 10011止당일반적으로약 0.01~0.04 바람직하게는약 0.02-0.03은이다.
[95] 또한,
Figure imgf000016_0002
단백질은근육질환예방및근기능 개선용식품조성물의유효성분으로함유될수있는데,그양은근육질환예방 및근기능개선용작용을달성하기에유효한양으로특별히한정되는것은 아니나,전체조성물총중량에대하여 0.01내지 100중량%인것이바람직하다. 본발명의건강기
Figure imgf000016_0003
질환예방및근기능개선용조성물에효과가있는것으로알려진다른활성 성분과함께혼합하여제조될수있다.
[96] 상기외에본발명의건강기능식품조성물은여러가지영양제，비타민,전해질, 풍미제,착색제,펙트산,펙트산의염,알긴산,알긴산의염,유기산,보호성
Figure imgf000016_0004
방부제 ,글리세린,알코올또는탄산화제 등을함유할수있다.그밖에본발명의건강식품은천연과일주스,과일주스 음료,또는야채음료의제조를위한과육을함유할수있다.이러한성분은 독립적으로또는혼합하여사용할수있다.이러한첨가제의비율은크게 중요하진않지만본발명의조성물 0중량부당 0.01 ~ 0.1중량부의범위에서 선택되는것이일반적이다.
[97] 또한,본발명은 011102융합단백질을포함하는,근기능개선용
화장료조성물을제공한다.본발명의화장료조성물에포함되는유효성분의 구성및이의효과는전술한약학조성물에대한것과동일하므로,그기재를 생략한다.
[98] 상기화장료조성물은특히제한되는것은아니나,피부외용으로사용할수 있다.
[99]
Figure imgf000016_0005
단백질을유효성분으로
함유하며피부학적으로허용가능한부형제와함께기초화장품조성물（화장수, 크림,에센스,클렌징폼및클렌징워터와같은세안제,팩,보디오일）,색조 화장품조성물（화운데이션,립스틱,마스카라,메이크업베이스）,두발제품 조성물（샴푸,린스,헤어컨디셔너,헤어젤）및비누등의형태로제조될수있다.
[100] 상기부형제로는이에한정되지는않으나예를들어,피부연화제,피부침투 2020/175931 1»（：1^1{2020/002809 증강제,착색제,방향제,유화제,농화제및용매를포함할수있다.또한,향료, 색소,살균제,산화방지제,방부제및보습제등을추가로포함할수있으며 , 물성개선을목적으로점증제 ,무기염류,합성고분자물질등을포함할수있다. 예를들면,본발명의화장료조성물로세안제및비누를제조하는경우에는 통상의세안제및비누베이스에 HSA-Slit3 LRRD2융합단백질을첨가하여 용이하게제조할수있다.크림을제조하는경우에는일반적인
수중유적형 (0/W)의크림베이스에 HSA-Slit3 LRRD2융합단백질을첨가하여 제조할수있다.여기에향료,킬레이트제,색소,산화방지제,방부제등과 물성개선을목적으로한단백질,미네랄,비타민등합성또는천연소재를 추가로첨가할수있다.
[101] 본발명의화장료조성물에함유되는 HSA-Slit3 LRRD2융합단백질의함량은 이에한정되지않지만전체조성물총중량에대하여 0.001내지 10중량%인것이 바람직하고, 0.01내지 5중량%인것이더욱바람직하다.상기함량이 0.0이중량% 미만에서는목적하는효과를기대할수없고, W중량%초과에서는안전성또는 제형상의제조에어려움이 있을수있다.
[102] 본발명은또한,알부민이결합된, Slit3단백질의 LRRD2를포함하는근기능 개선용사료첨가물을제공한다.본발명의사료첨가물에포함되는유효성분의 구성및이의효과는전술한약학조성물에대한것과동일하므로,그기재를 생략한다.
[103] 본발명의사료첨가물은사료관리법상의보조사료에해당한다.
[104] 본발명에서용어,”사료”는동물이먹고,섭취하며,소화시키기위한또는이에 적당한임의의천연또는인공규정식 ,한끼식등또는상기한끼식의성분을 의미할수있다.
[105] 상기사료의종류는특별히제한되지아니하며,당해기술분야에서
통상적으로사용되는사료를사용할수있다.상기사료의비제한적인예로는, 곡물류,근과류,식품가공부산물류,조류,섬유질류,제약부산물류,유지류, 전분류,박류또는곡물부산물류등과같은식물성사료;단백질류,무기물류, 유지류,광물성류,단세포단백질류,동물성플랑크톤류또는음식물등과같은 동물성사료를들수있다.이들은단독으로사용되거나 2종이상을혼합하여 사용될수있다.
[106] 또한,상기사료첨가물은추가적으로단위동물에허용되는담체를함유할수 있다.본발명에있어서는상기사료첨가물을그대로또는공지의담체,안정제 등을가할수있으며，필요에따라비타민,아미노산류,미네랄등의각종양분, 항산화제및기타의첨가제등을가할수도있으며,그형상으로서는분체,과립, 펠릿,현탁액등의적당한상태일수있다.본발명의사료첨가물을공급하는 경우는단위동물에대하여단독으로또는사료에혼합하여공급할수있다.
[107] 본발명은또한,알부민이결합된, Slit3단백질의 LRRD2를포함하는, Slit3 단백질의 LRRD2의생체내반감기증진용조성물을제공한다. 2020/175931 1»(：1^1{2020/002809
[108] 이하,실시예를통하여본발명을더욱상세히설명하고자한다.이들실시예는 오로지본발명을예시하기위한것으로서,본발명의범위가이들실시예에 의해제한되는것으로해석되지않는것은당업계에서통상의지식을가진자에 있어서자명할것이다.
[109] 하기실시예에사용된약어및이의의미는아래표 1에나타낸바와같다.
[11이 [표 1]
Figure imgf000018_0001
발명의실시를위한형태
[111] [실시예 1]
[112] HSA-S1U3 LRRD2융합단백질 (HSA-S1U3 LRRD2 fusion protein)의제조
[113] PC DNA 3.1벡터 SP시스타틴 S-HSA-Slit3 LRR D2-FLAG DNA 1.6mg/ml를 Expi293F현탁액세포에트랜스펙션시켜발현을진행하였다. 125ml 293F세포 현탁액에서세포를 4.5 ~ 5xl06세포/ ml까지배양하고배지만새로교체한후, 엑스피펙타민 (Expifectamine) 400[xl와 Opti-mem 7.5ml(A샘늘)을상온에서 5분 반응하고, DNA 150ug, Opti-mem 7.5ml(B샘플)을상온에서 5분반응후 A와 B 샘플을서로섞어 20분상온반응하여트랜스펙션을진행하였다. 24시간후, 인핸서 1과 2를섞어처리한후, 7일동안배양하였다.
[114] 7일된배양액을원심분리기 4°C 8000rpm에서 20분동안세포를침전시킨뒤에 상증액을코닝 (coming)사의 0.22[xm필터로여과하여사용하였다.레진은 시그마 (Sigma)사의항- FLAG레진을사용하여진행하였다.레진을각각 1.2ml씩 사용하였으며정제는 4OC 1ml/분속도에서진행하였다. TBS (Tris Glycine pH 2020/175931 1»(：1^1{2020/002809
7.4）를이용한워싱버퍼를레진의 20배로흘려넣어주었다.용출은시그마사의 FLAG펩티드 200나1와 TBS 9.8ml를섞어사용하였는데,분획（fraction）당 500 씩 8개를얻었고,단백질분획을모아버퍼를 DPBS로바꿔농축한다음,농도를 측정하였다.
[115] 도 2는상기과정으로융합단백질을분리정제후 SDS-PAGE를시행한결과를 나타낸것으로,본실시예에서제조된도 1에도시된융합단백질의크기가 75 KDa임을확인하였다.
[116] [실시예 2]
[117] 다양한형태의 HSA-S1U3 LRRD2융합단백질（HSA-S1U3 LRRD2 fusion
protein）의수용체결합능확인
[118] 2-1.다양한형태의 HSA-Slit3 LRRD2유함다백짐제조
[119] 실시예 1의제조방법을바탕으로,하기표 2와같이 12종의다양한 HSA-Slit3 LRRD2융합단백질을제조하였다.링커는（GGGGS）3（서열번호 6）을
사용하였다.
[12이 [표 2]
Figure imgf000019_0001
2020/175931 1»（：1/10公020/002809
[121]
Figure imgf000020_0001
단백질의아미노산서열은표 3에
나타내었다.
2020/175931 1»(：1/10公020/002809
[122] [표 3]
Figure imgf000021_0001
2020/175931 1»（：1^1{2020/002809
Figure imgf000029_0001
[123] *상기표 3에서밑줄로표시된서열은 HSA와 LRRD2를연결하는 GS링커이며, 볼드체로표시된서열은융합단백질을최종형태로발현시키기위해 C-말단에 부가되는서열을연결하는 GS링커이다.
[124] 2-2.다양하형태의 HSA-S1it3 LRRD2유함다백짐 rHSA-S1it3 LRRD2 fusion protein、의수용체 합능환이
[125] Slit3 LRRD2는 Robol또는 Robo2수용체와결합하여 Slit-Robo시스템을통해 근육모세포의 M-카데린에결합되어있던 (3-카테닌을방출시켜 (release)
(3-카테닌을활성화하고미오게닌의발현을증가시킴으로써,근육모세포의 분화를유도하여근육형성을족진한다.따라서,본실시예에서는실시예
2-1에서제조된 12종의 HSA-Slit3 LRRD2융합단백질의수용체결합능을 확인하였다. 12종의 HSA-Slit3 LRRD2융합단백질과 Robol수용체의결합능은 ELISA시스템을사용하여정량하였다.상세한조건은다음과같다.
[126] 12종의 HSA-Slit3 LRRD2융합단백질은분자량을고려하여웰당 0, 1, 10, 100, 1000 nM이되게 4°C에서 18시간동안 96 -웰미량정량판 (microtiter plates)
(NUNC사)에코팅하였다.코팅된물질은 0.05% Tween 20이포함된 PBS
(PBST)를사용하여 3번세척하였다.비특이적인바인딩을차단하기위해 1% BSA가첨가된 PBST로실온에서 2시간동안블러킹을하였다.블러킹버퍼를 제거하기위해 PBST로 3번세척하였다.세척후해당세포주로부터얻어진단백 30 ug을 (용해버퍼 (lysis buffer): 0.5% NP40, 50mM Tris pH 7.5, 150mM NaCl, ImM EDTA, 0.2mM NaF, ImM Na3V04, ImM DTT, ImM PMSF,단백분해효소 억제제칵테일 (Proteinase inhibitor cocktail))실온에서 2시간동안부착시켰다. PBST를사용하여 3번세척한후 0.1% BSA로 1:1000희석시킨 Robol항체
(abeam: ab7279)를실온에서 2시간동안부착시켰다. PBST를사용하여 3번 세척한후 0.1% BSA로 1:2000희석시킨 HRP결합항체 (cell signaling: 7074)를 실온에서 2시간동안부착시켰다. PBST를사용하여 5번세척한후
TMB용액으로 37OC에서 30분동안반응시켰다.상기반응을중지시키기위해 100M의 1N H2S04를사용하였고 450nm에서흡광도를측정하였다.
[127] 그결과,도 3에나타난바와같이 LRRD2-3과 LRRD2-6의수용체결합능이
가장우수한것으로확인되었다.
[128] [실시예 3]
[129] 마우스에서 Slit3 LRRD2및 HSA-S1U3 LRRD2융합단백질의약물동태연구
[130] 본실시예에서는실시예 2의결과를바탕으로,수용체결합능이가장우수한 LRRD2-3를선정하여,약물동태연구를수행하였다.
[131] 약동학연구는신약개발과정중의한부분으로,시간에따른체내의약물
농도의변화평가를통해시험약물의흡수,분포,대사및배설에대한정보를 2020/175931 1»（：1^1{2020/002809 얻는것을목표로한다.본실시예에서는 此 1요10)2-3및 1요10)2 융합단백질 (011102-3)의 1회정맥투여후마우스에서약물동태특성을 확인하였다.
[132] 3-1.화학물짐및용매
[133] 본실시예에서사용된카르바
구입하였고,아세토니트릴과메
Figure imgf000030_0001
구입하였다.
[134] 3-2.동물및투여조거
[135] 본실시예에서는체중 30~32.5은범위의 1이계웅성마우스 (6주령,
(주)오리엔트바이오,성남,대한민국)를사용하였다.마우스는실험전 4시간동안 절식하였고,투여후 4시간까지절식을유지하였다.사육장은 12시간씩명암을 주며적정온도 (20~25ᄋ 0및습도 (40~60%)를유지하였다.
[136] [표 4]
약물동태시험
Figure imgf000030_0002
[137] Slit3 LRRD2를 1 mg/mL의용량으로 PBS에녹여준비하였다. HSA-Slit3 LRRD2 (LRRD2-3)의경우,분자량을고려하여 3.5 mg/mL의용량 (Slit3 LRRD2로서 1 mg/mL)으로 PBS에녹여준비하였다.투여용량은두군모두 W mL/kg으로, 좌측미정맥을통해투여하였다.
[138] 3-3.약물동태시험
[139] 약물동태시험의경우,절식시킨마우스에 Slit3 LRRD2와 HSA-Slit3 LRRD2 (LRRD2-3)를각각 10 mg/kg및 35 mg/kg의용량으로미정맥을통해각각 투여하였다.투여후각각 0.05, 0.12, 0.33, 1, 3, 7, 10, 24, 48,및 72시간에 마우스를손으로고정한후헤파린코팅된모세관으로우측안와정맥총에서 혈액 70ᅡ 를채혈하였다.취해진혈액은 5분간원심분리한후혈장을분리하여 분석전까지 -20 에서냉동보관하였다.
[140] 3-4.분석 방법
[141] 혈장시료중 Slit3 LRRD2의농도는 HPLC/MS/MS시스템을이용하여
정량하였다.시료전처리전,혈장시료는 Ni-NTA마그네틱비드를이용하여 정제하였다.정제된 Slit3 LRRD2및 HSA-Slit3 LRRD2 (LRRD2-3)에 6M의 요소와 18 mM의디티오트레이톨 (DTT)을가해변성시킨후, 225 mM의 요오드아세트아미드를이용하여알킬화를유도하였다.이후,시그니처 펩타이드 (signature peptide)를얻기위해, 850 ng의재조합돼지트립신 (V5117, 2020/175931 1»(：1^1{2020/002809
Promega, Madison, WI, USA)을가해 24시간동안 37OC로설정된워터배스 (water bath)에서반응시켰다.반응후생성된트림신소화물 70 에 MeOH에용해한 3%의포름산 50 [ 를가한후,볼텍스믹서 (vortex mixer)를이용하여 W분간 혼합시료를현탁하고 13,500 rpm에서 W분간원심분리하여상징액 160ᅡ 을 취해분석용기에옮겨이중 5ᅡ 를 HPLC/MS/MS시스템에주입하여분석을 진행하였다
[142] 상세한분석조건은다음과같다.
[143] - HPLC시스템 : Agilent 1100 (Agilent Technologies, Santa Clara, CA)
[144] -컬럼 : ZORBAX® C8 3.5 [xm, 2.1*50 mm (Agilent)
[145] -이동상 (Mobile phase):
[146] A:증류수에용해한 0.1%의포름산
[147] R아세토니트릴
[148] (등용매용리)
[149] , ,
시간 0 ® Q . 1 ® 1 . 0 ® 1 . 5 ® 2. 5 ® 3 ® 5
B (% ) 一 5 ® 5 ® 5 ® 95 ® 95 ® 5 ® 5
[150] -유속: 300 [xL/min-온도:컬럼에서 20°C,및자동샘플기트레이 (autosampler tray)에서 1(TC
[151] -런타임: 5분
[152] -검출:탠덤사중극자질량분석기 (API 4000, QTRAP®, A抑 lied Biosystems/ MDS SCIEX, Foster City, CA, USA)
[153] -커튼가스 (curtain gas): 20 psi
[154] -이온소스가스 l(ion source gas 1): 50 psi
[155] -이온소스가스 2(ion source gas 2): 60 psi
[156] -이온스프레이전압 (ionspray voltage): 5500 V
[157] -온도: 600°C
[158] -다중반응모니터링 (multiple-reaction-monitoring, MRM)모드:양성
[159] Silt3 LRRD2의시그니처펩타이드 (P6)의분자이온은 23V의충돌
에너지 (collision energy)에의해조각화되었으며 ,중돌가스 (collision gas)는 장비에서 'medium (8 psi)’으로설정하였다.이온의검출은 ESI양성 MRM모드로 진행하였고, P6는 m/z 587.97 ® 491.50에서정량하였다.검출피크의적분은 Analyst software version 1.4.2 (Applied Biosystems/MDS SCIEX)을이용하여 진행하였다.혈장중 Silt3 LRRD2의정량가능범위는 1니 00 ^ig/mL이었고, HSA-Silt3 LRRD2 (LRRD2-3)의경우, 3 ~ 100 ^ig/mL이었다.해당분석에서 Slit3 LRRD2는 3.29분의피크머무름시간을나타내었다.
[16이 3-5.데이터분석
[161] 시간에따른혈장중 CNC00000의농도를상기실시예 3-4에기재된 LC-MS/MS 분석법을이용하여구하고, WinNonlin® 4.2 (Pharsight Corp., Cary, NC, USA) 2020/175931 1»（：1^1{2020/002809 소프트웨어의 비구획적분석법 (non-compartmental analysis)으로약동학적 매개변수 (PK parameters)를계산하였다.최고농도 (Cmax)와최고농도도달시간 (T max)은혈중약물농도대비시간에따른곡선에서경시적으로구하였고, 소실속도상수 (Ke)는로그스케일 (log scale)의최종단계 (terminal phase)에서 선형회귀분석을통해계산하였다.반감기 (T1/2)는 LN2를 Ke로나누어구하였고, 혈중약물농도대시간곡선하면적 (AUCq- )및혈중약물모멘트대시간 곡선하면적 (AUMCVoo)은선형사다리꼴법 (linear trapezoidal rule)과표준면적 외삽법 (standard area extrapolation method)으로계산하였다.클리어런스 (clearance, CL)와분포용적 (steady state volume of distribution, Vss)는다음의 ［식 1］내지 ［식
3］으로계산하였다:
[162] ［식 1］
[163]
Dose
Figure imgf000032_0001
AUC0 -¥
[164] [식 2]
[165]
Vss = MRT X CL
[166] [식 3]
[167]
Figure imgf000032_0002
농도는도
4및표 5와 6에 나타내었고,약동학파라미터는표 7에 나타내었다.관련된 파라미터와모든값들은개체별로산출한후평균으로나타내었다.시간에 따른 혈중농도패턴과동물실험 기록지를참고하여 이상이 있는개체군은데이터 분석에서 배제하였으며,데이터해석에사용된실험군은최소 11=3이상이 되도록하였다. 2020/175931 1»(：1^1{2020/002809
[17이 [표 5]
比3의정맥내투여후혈장농도
Figure imgf000033_0002
[171] =¾（ :정량한계미만은”0”으로처리함. ）2-3）의정맥투여후혈장농도
Figure imgf000033_0003
[173] =¾（ :정량한계미만
[174] 하기표 7에확인되는
Figure imgf000033_0001
011102대비약 14배개선된반감기를나타내었다. 2020/175931 1»(：1^1{2020/002809
［175］ ［표 7］
Figure imgf000034_0001
[176] [실시예 4]
[177] HSA-결합 Slit3 LRRD2의생체효능확인
[178] 9주령의 Balbc-nude마우스를 9주령에난소절제한후 11주령부터 4주간
알부민이결합되지않은 Slit3 LRRD2또는 HSA-Slit3 LRRD2융합단백질 (LRRD2-3)를처리하였다.각약물은하루한번,주당 5회정맥주사로 투여하였고, Slit3 LRRD2는매일 10 mg, HSA-결합 Slit3 LRRD2 (LRRD2-3)는 매일 37.13 mg (Slit3 LRRD2는매일 10 mg에해당)주사하였다.투여완료후 가자미근 (Soleus muscle)을채취하여근육무게를측정하였고,그결과를하기표 8에나타내었다.
[179] [표 8]
Figure imgf000034_0002
[180] * I) < 0.05, vs.너 1처리대조군
[181] 표 8에나타난바와같이,알부민이결합되지않은 81113 1^10)2와 쇼 -결합 比3 1^10)2모두가자미근의근육량을유의하게증가시켰다.그러나 -결합 81113 1^10)2가알부민이결합되지않은 81113 1^10)2보다더강한치료효능을 보였다.
산업상이용가능성 2020/175931 1»（：1^1{2020/002809
[182] 본발명의알부민이결합된, Slit3단백질의 LRRD2는알부민이결합되지않은 Slit3단백질의 LRRD2와동일한세포학적효능을나타내고,알부민이결합되지 않은 Slit3단백질의 LRRD2와비교하여생체내반감기가현저하게증가되어골 관련질환을보다효과적으로예방또는치료할수있다.
[183]
[184] 본발명을지원한국가연구개발사업은하기와같다.
[185] (1) [이발명을지원한국가연구개발사업]
[186] [과제고유번히 2017-1229 (HI15C0377010017)
[187] [부처명]보건복지부
[188] [연구관리전문기관]한국보건산업진흥원
[189] [연구사업명]질병중심중개중점연구
[19이 [연구과제명]골형성촉진작용을가지는거핵세포분비인자발굴
[191] [기여율] 75/100
[192] [주관기관]서울아산병원
[193] [연구기간] 2017.09.07 ~ 2018.09.06
[194]
[195] (2) [이발명을지원한국가연구개발사업]
[196] [과제고유번히 2013-2234 (HI 13C 1634060018)
[197] [부처명]보건복지부
[198] [연구관리전문기관]한국보건산업진흥원
[199] [연구사업명]질병중심중개중점연구
[20이 [연구과제명 ] Slit3 LRRD2의약동학연구및 Slit3 TG마우스를이용한 in vivo 독성검증
[201] [기여율] 25/100
[202] [주관기관]충남대학교산학협력단
[203] [연구기간] 2013.11.01 ~ 2019.06.30

Claims

2020/175931 1»（：1/10公020/002809 청구범위
[청구항 1] 알부민이결합된, Slit3단백질의 LRRD2를포함하는융합단백질.
[청구항 2] 제 1항에 있어서 ,상기알부민은인간혈청알부민 (human serum
albumin)인,융합단백질.
[청구항 3] 제 2항에 있어서 ,상기인간혈청알부민은 Slit3단백질의 LRRD2의
N-말단에결합되는,융합단백질.
[청구항 4] 제 3항에 있어서,상기인간혈청알부민은서열번호 2의아미노산서열을 포함하고,상기 Slit3단백질의 LRRD2는서열번호 3의아미노산서열을 포함하는,융합단백질.
[청구항 5] 제 1항에 있어서 ,상기알부민과 Slit3단백질의 LRRD2사이에링커를
추가로포함하는,융합단백질.
[청구항 6] 제 5항에 있어서 ,상기링커는 (GGGGS)n이고,여기서 n은 1내지 10의
정수인,융합단백질.
[청구항 7] 제 1항내지제 6항중어느한항의융합단백질을암호화하는핵산분자.
[청구항 8] 제 7항의핵산분자를포함하는재조합벡터 .
[청구항 9] 제 6항의재조합벡터를포함하는형질전환체 .
[청구항 10] 제 9항의형질전환체를배양하는단계를포함하는,알부민이결합된, Slit3 단백질의 LRRD2를포함하는융합단백질의제조방법 .
[청구항 11] 제 1항내지제 6항중어느한항의융합단백질을포함하는,근육질환
예방또는치료용약학조성물.
[청구항 12] 제 11항에 있어서 ,주사제로투여되는,근육질환예방또는치료용약학 조성물.
[청구항 13] 제 11항에 있어서,상기근육질환은긴장감퇴증 (atony),근위죽증 (muscular atrophy),근이영양증 (muscular dystrophy),근무력증,악액질 (cachexia)및 근감소증 (sarcopenia)으로이루어진군으로부터선택되는어느하나 이상인,근육질환예방또는치료용약학조성물.
[청구항 14] 제 1항내지제 6항중어느한항의융합단백질을포함하는, Slit3단백질의
LRRD2의생체내반감기증진용조성물.
PCT/KR2020/002809 2019-02-27 2020-02-27 알부민이 결합된, slit3 단백질의 lrrd2를 포함하는 근육 질환 예방 또는 치료용 조성물 WO2020175931A1 (ko)

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