WO2017048018A1 - Pharmaceutical composition containing anionic drug, and preparation method therefor - Google Patents

Pharmaceutical composition containing anionic drug, and preparation method therefor Download PDF

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Publication number
WO2017048018A1
WO2017048018A1 PCT/KR2016/010269 KR2016010269W WO2017048018A1 WO 2017048018 A1 WO2017048018 A1 WO 2017048018A1 KR 2016010269 W KR2016010269 W KR 2016010269W WO 2017048018 A1 WO2017048018 A1 WO 2017048018A1
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Prior art keywords
acid
formula
group
integer
copolymer
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PCT/KR2016/010269
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French (fr)
Korean (ko)
Inventor
남혜영
김봉오
서민효
손지연
최지혜
김상훈
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주식회사 삼양바이오팜
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Priority to EP16846839.5A priority Critical patent/EP3357491B1/en
Priority to JP2018533597A priority patent/JP6638072B2/en
Application filed by 주식회사 삼양바이오팜 filed Critical 주식회사 삼양바이오팜
Priority to ES16846839T priority patent/ES2883290T3/en
Priority to DK16846839.5T priority patent/DK3357491T3/en
Priority to RU2018113459A priority patent/RU2721558C2/en
Priority to AU2016324450A priority patent/AU2016324450B2/en
Priority to CN201680053810.0A priority patent/CN108024960B/en
Priority to US15/759,943 priority patent/US11253598B2/en
Priority to SG11201802073YA priority patent/SG11201802073YA/en
Priority to MX2018003096A priority patent/MX2018003096A/en
Priority to CA2998092A priority patent/CA2998092C/en
Priority claimed from KR1020160117053A external-priority patent/KR101828877B1/en
Publication of WO2017048018A1 publication Critical patent/WO2017048018A1/en
Priority to IL257937A priority patent/IL257937B/en
Priority to ZA2018/02303A priority patent/ZA201802303B/en
Priority to US17/572,195 priority patent/US20220125929A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/107Emulsions ; Emulsion preconcentrates; Micelles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a pharmaceutical composition containing an anionic drug and to delivering the same, and to a method of preparing the same.
  • nucleic acid materials Numerous studies have been conducted on non-viral carriers used for the delivery of nucleic acid materials. The most representative ones are cationic lipids using cationic lipids and lipoplexes and polycation polymers and nucleic acids.
  • the cationic lipids or polycationic polymers studied so far have less results than viral carriers, but have severe toxicity when used in amounts necessary to achieve a striking effect, resulting in inadequate use in medicine.
  • the lipid-nucleic acid complexes which combine the cationic lipids with the nucleic acids and deliver the nucleic acids into the cells, are very widely used in cell line experiments, but do not exhibit a structure that can have stability in blood. It is possible to use it in vivo (see US Pat. No. 6,458,382).
  • anionic drug delivery compositions that form a complex by electrostatic interaction of a nucleic acid with a cationic lipid such that the complex is encapsulated within the micellar structure of the amphiphilic block copolymer.
  • this also provides room for improvement in blood stability of the nucleic acid and specific targeting to cancer tissues.
  • Korean Patent No. 1296326 discloses an anionic drug as an active ingredient; Cationic Lipids; An amphiphilic block copolymer and a polylactic acid, wherein the anionic drug forms a complex with the cationic lipid, and the complex has a structure encapsulated inside a micellar structure formed by the amphiphilic block copolymer and the polylactic acid.
  • An anionic drug delivery composition is disclosed.
  • polylactic acid used in this patent is a general polylactic acid polymer having a carboxyl group at its terminal, and has a problem of insufficient drug delivery effect.
  • siRNA short interfering RNA
  • siRNAs are short, double-stranded RNA strands that inhibit the expression of glycolysis by cleaving mRNAs of genes complementary to these (McManus and Sharp, Nature Rev. Genet. 3: 737 (2002); Elbashir, et. al., Genes Dev. 15: 188 (2001).
  • siRNAs are known to be rapidly degraded by nucleases in blood and rapidly excreted in vitro through the kidneys.
  • siRNA is known to have a strong negative charge does not easily cross the cell membrane. Therefore, in order to use siRNA as a therapeutic agent, siRNA needs to be stabilized in the blood, efficiently delivered into a target tissue or cell, and development of a non-toxic carrier.
  • an object of the present invention is to provide a composition for anionic drug delivery comprising a micellar structure containing polylactic acid salt to effectively deliver the anionic drug in the body.
  • the present invention is an anionic comprising administering the composition It is an object to provide a method of delivery of a drug.
  • an object of the present invention is to provide a method for preparing a pharmaceutical composition that can effectively deliver such a negative drug.
  • the present invention comprises an anionic drug delivery composition comprising a micellar structure containing polylactic acid salt to effectively deliver an anionic drug to the body, uses for the anionic drug delivery of the composition and administering the composition. It relates to a method of delivery of anionic drugs.
  • the anionic drug delivery composition comprising the micelle structure according to the present invention is a structure containing a complex of a drug and a divalent compound in the micelle structure of the amphiphilic block co-polymer and the polylactic acid salt.
  • the anionic drug forms a complex by electrostatic interaction with the cationic compound, and the complex thus formed is enclosed in a micelle structure formed by an amphiphilic block copolymer and a polylactic acid salt. It is done.
  • the composition is soluble in water and comprises polylactic acid as a component to form micelle structures, thereby increasing the stability of blood and avoiding reticulum endothelial system (RES) during infusion into the target site, specifically cancer tissue. It is also useful for evasion and / or targeting of reticulum endothelial system (RES) because of its high efficiency in delivery.
  • RES reticulum endothelial system
  • step (a) Dissolving a polylactate in a water-soluble organic solvent or a mixed solvent of an aqueous solution and an organic solvent; (b) removing the organic solvent layer of step (a);
  • step (c) process of micellizing by adding an aqueous solution to the mixture from which the organic solvent of step (b) is removed
  • the anionic drug is isolated from the outside by using a micellar structure formed of a cationic compound and an amphiphilic block polymer and a polylactic acid salt. You can increase the stability. Therefore, the pharmaceutical composition can increase the stability of the anionic drug in the blood or body fluid when administered in the body, and particularly has the advantage that the anionic drug can be efficiently delivered into the cell avoiding the reticuloendothelial system.
  • FIG. 1 is a diagram showing a schematic structure of a polymer micelle delivery vehicle in which a complex of an anionic drug and a bipolar compound according to the present invention is encapsulated.
  • Figure 2 is a view showing the NMR results of the polylactic acid sodium salt according to Preparation Example 8.
  • composition of the composition according to the present invention the anionic drug and the cationic compound is enclosed in the micellar structure formed by the amphiphilic block polymer and polylactic acid salt, the complex of such anionic drug and cationic compound is enclosed
  • the approximate structure of the polymerized micelle transporter is shown in FIG. 1.
  • anionic drugs bind to each other through an electrostatic interaction with a cationic compound to form a complex of a negative drug and a cationic compound.
  • the anionic drug and cationic compound complex formed are encapsulated in a micellar structure formed by an amphiphilic block copolymer and a polylactic acid salt.
  • the micellar structure formed by the amphiphilic block copolymer and the polylactic acid salt is a hydrophilic portion of the amphiphilic block copolymer in an aqueous environment.
  • the polylactic acid salt that forms the outer wall of the micelle and contains the hydrophobic portion of the amphiphilic block copolymer and the amphiphilic block co-polymer as a separate component forms the inner wall of the micelle, and the anionic drug and the cation inside the formed micelle. It is a structure encapsulated with a compound of a sex compound.
  • the anionic drug and the cationic compound complex is maintained in the micelle structure formed by the amphiphilic block copolymer and the polylactic acid salt to improve stability in blood or body fluids.
  • the particle size of the micelles 10 to 200 m, more preferably 10 to 150nm.
  • the standard charge of the micelle particles is -20 to 20 mV, more preferably -10 to 10 mV. The particle size and standard charge are most preferred in terms of stability of the micellar structure and the content of constituents, the absorption of anionic drugs in the body, and the convenience of sterilization as a pharmaceutical composition.
  • the anionic drug included as an active ingredient in the composition according to the present invention is a concept including all substances having a negatively charged pharmacological activity in the molecule in aqueous solution.
  • the anionicity may be imparted from one or more functional groups selected from the group consisting of carboxyl groups, phosphate groups and sulfate groups.
  • the anionic drug may be a polyanionic drug or nucleic acid such as peptide, protein or heparin.
  • the nucleic acid material may be a deoxyribonucleic acid, a ribo nucleic acid or a backbone, a nucleic acid drug such as a polynucleotide derivative whose sugar or base is chemically modified or modified at the end, and more specifically, RNA, DNA, siRNA (short interfering RNA), aptamer, antisense oligodeoxynucleotide (ODN), antisense RNA (antisense RNA), ribozyme (ribozyme) and DNAzyme (DNAzyme) may be one or more selected from the group consisting of. .
  • the nucleic acid may be chemically modified or terminally modified with a backbone, sugar or base for the purpose of increasing blood stability or weakening the immune response. Specifically, some of the phosphodiester bonds of the nucleic acid
  • a phosphorothioate or boranophosphate bond or a methyl, methoxyethyl group, at the 2'-OH position of some ribose base, It may include one or more modified nucleotides in which various functional groups such as fluorine are introduced.
  • At least one terminus of the nucleic acid is cholesterol, tocopherol and carbon number
  • siRNA can be modified at the 5 'end, or 3' end, or both ends of the sense and / or antisense strand, and preferably at the end of the sense strand.
  • the cholester, tocopherol and fatty acids having 10 to 24 carbon atoms having 10 to 24 carbon atoms
  • the siRNA mediates the degradation of mRNA complementary to the siRNA sequence when present in the same cell as the target gene, thereby exerting the expression of the target gene.
  • duplex RNA or single-stranded RNA that forms a double strand inside single-stranded RNA that can be reduced or inhibited. Bonding between the double strands is via hydrogen bonds between the nucleotides, and not all nucleotides within the double strands must complementarily bind to each other, and both strands may or may not be separated.
  • the length of the siRNA is about 15 to 60 (the number of single nucleotides of the double-stranded RNA, that is, the number of base pairs, and in the case of a single-stranded RNA, the length of the double stranded RNA within the single-stranded RNA). Nucleotides, specifically about 15 to 30 nucleotides, and more specifically about 19 to 25 nucleotides
  • SiRNA which is a nucleotide.
  • the double-stranded siRNA may have an overhang of 1-5 nucleotides at either the 3 'or 5' end, at one end, or at both ends.
  • both ends may be in the form of blunts without protrusions.
  • siRNA disclosed in US Patent Publication No. 2002-0086356, US Patent No. 7,056,704, which is incorporated herein by reference.
  • the siRNA may have a symmetrical structure in which the two strands are the same length, or an asymmetric double stranded structure in which one strand is shorter than the other strand.
  • antisense of 19 to 21 nucleotides nucleotides, nt
  • siRNA can be an asymmetric siRNA with nucleotide overhangs. Specifically, it may be siRNA disclosed in International Patent Publication No. 09/078685.
  • the anionic drugs by weight of the total composition, from 0.001 to 10 parts by weight 0/0, specifically, is preferably contained in an amount of 0.01 to 5% by weight.
  • the anionic When the content of the drug 0.001 0/0 is less than an amount of the delivery system to be used as compared to the drug may cause side effects due to too many delivery system, when it exceeds 10% by weight, the size of the micelle becomes too large stability misael There is a risk of lowering and increasing the loss rate during filter sterilization.
  • the cationic compound is bonded by an electrostatic interaction with the anionic drug to form a complex, the complex is encapsulated inside the micelle structure of the amphiphilic block copolymer.
  • the cationic compound includes all types of compounds capable of forming a complex by electrostatic interaction with the anionic drug, and may be, for example, a lipid and a polymer type.
  • Cationic lipids include ⁇ , ⁇ -dioleyl- ⁇ , ⁇ -dimethylammonium chloride (DODAC), ⁇ , ⁇ -distearyl- ⁇ , ⁇ -dimethylammonium bromide (DDAB), N- (l- (2, 3-dioleoyloxy) propyl- ⁇ , ⁇ , ⁇ -trimethylammonium chloride (DOTAP), N, N-dimethyl- (2,3-dioleoyloxy) propylamine (DODMA), ⁇ , ⁇ , ⁇ - Trimethyl- (2,3-dioleoyloxy) propylamine (DOTMA), 1,2-diacyl-3-trimethylammonium-propane (TAP), 1,2-diacyl-3-dimethylammonium-propane (DAP ), 3beta- [ ⁇ - ( ⁇ ', ⁇ ', ⁇ '-trimethylaminoethane) carbamoyl] cholester (
  • (Aminoethane) carbamoyl] cholesterol AC-cholesterol
  • cholesteryloxypropane-1-amine COPA
  • N- (N'-aminoethane) carbamoylpropanoic tocopherol AC-tocopherol
  • N- It may be one or a combination of two or more selected from the group consisting of (N′_methylaminoethane) carbamoylpropanoic tocopherol (MC-tocopherol).
  • Such cationic When using lipids, it is desirable to use less polycationic lipids with high cation density in the molecule in order to reduce the toxicity caused by cationic lipids, and more specifically, the functional groups capable of exhibiting cations in aqueous solution per molecule. It can be one.
  • the cationic lipid is 3beta- [ ⁇ - ( ⁇ ', ⁇ ', ⁇ '-trimethylaminoethane) carbamoyl] cholester (TC-cholester) : 3beta [ ⁇ - ( ⁇ ', ⁇ '-dimethylaminoethane) carbamoyl] cholesterol (DC-cholesterol), 3beta [N- ( ⁇ '-monomethylaminoethane) carbamoyl] cholester (MC -Cholester), 3-beta [ ⁇ - (aminoethane) carbamoyl] cholesterol (AC-cholesterol), N- (l- (2,3-dileoyloxy) propyl- ⁇ , ⁇ , ⁇ -trimethylammonium chloride (DOTAP), ⁇ , ⁇ -dimethyl- (2,3-dileoyloxy) propylamine (DODMA), and ⁇ , ⁇ , ⁇ -trimethyl-
  • DC-cholesterol 3
  • polyarginine polyamidoa
  • PAMAM polyethylenimine
  • dextran dextran
  • hyaluronic acid albumin
  • albumin albumin
  • PEI polymer 3 ⁇ 4riethyleneimine
  • PVAm polyamine and polyvinylamine
  • the cationic lipid may be a cationic lipid of Formula 7:
  • n and m are each 0 to 12
  • a and b are each 1 to 6
  • R1 and R2 are each independently selected from the group consisting of saturated and unsaturated hydrocarbons of 1 to 25 carbon atoms will be.
  • n and m are independently 1 to 9, and may be 2 ⁇ n + m ⁇ 10.
  • a and b may be 2 to 4.
  • R 1 and R 2 are each independently lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, capita Lignoceryl, cerotyl, myristoleyl, palmitoleyl, sapienyl, oleyl, linoleyl, arachidyl It may be selected from the group consisting of (arachidonyl), eicosapentaenyl, ethucyl, erucyl, docososahexaenyl, and serrotyl.
  • cationic lipids include 1,6-dileoyltriethylenetetramide, 1,8-dilinoleylyltetraethylenementamide, 1,4-dimyristoleoyldiethylenetriamide, 1,10- At least one selected from the group consisting of distearoylpentaethylenenucleamide and 1,10-dioleoylpentaethylenenucleamide.
  • Cationic compounds used in the present invention based on the weight of the total composition may be included 0.01 to 50% by weight, specifically 1 to 10% by weight.
  • the cationic content of the lipids not being a sufficient amount to form an anionic drug and the complex is less than 0.01%, 50 weight exceeds 0/0, the size of the micelle so increases the stability of the misael decreases filter sterilization The loss rate may increase.
  • the ratio of the amount of charge of the anionic drug ( ⁇ ) and the cationic compound ( ⁇ ) ( ⁇ / ⁇ ; cationic charge ratio of the cationic compound to the anionic charge of the anionic drug) is 0.1 to 128. Specifically, it is 5 to 64, more specifically 1 to 32, and even more specifically 1 to 24, most preferably 6 to 24. If the ratio ( ⁇ / ⁇ ) is less than 0.1, since it is difficult to form a complex containing a sufficient amount of anionic drugs, it is advantageous to form a complex containing a sufficient amount of anionic drugs at a ⁇ or more. On the other hand, when the ratio ( ⁇ / ⁇ ) exceeds 128, it may cause toxicity, so it is preferable to set it to 128 or less.
  • amphiphilic block copolymer is a hydrophilic A block and
  • the AB type block copolymer may be a hydrophobic B block copolymer comprising a hydrophobic B block forming a core (inner wall) and a hydrophilic A block forming a shell (outer wall). Forms shell-type polymer micelles.
  • the hydrophilic A block may be at least one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyridone, polyacrylamide and derivatives thereof. More specifically, the hydrophilic A block
  • hydrophilic A block It may be at least one selected from the group consisting of monomethoxy polyethylene glycol, monoacetoxy polyethylene glycol, polyethylene glycol, copolymers of polyethylene and propylene glycol and polyvinylpyridone.
  • the hydrophilic A block It may be at least one selected from the group consisting of monomethoxy polyethylene glycol, monoacetoxy polyethylene glycol, polyethylene glycol, copolymers of polyethylene and propylene glycol and polyvinylpyridone.
  • the number average molecular weight may be 200 to 50,000 Daltons, more specifically 1,000 to 20,000 Daltons, and more specifically 1,000 to 5,000 Daltons.
  • a functional group capable of reaching a specific tissue or cell, a ligand, or a functional group capable of promoting intracellular delivery at the end of the hydrophilic A block is necessary.
  • the functional group or ligand may be one or more selected from the group consisting of monosaccharides, polysaccharides, vitamins, peptides, proteins and antibodies to cell surface receptors.
  • the functional group or ligand is an anamide (anisamide), vitamin B9 (folic acid), vitamin B12, vitamin A, galactose, lactose, mannose, hyaluronic acid, RGD peptide, NGR peptide, transferrin, transferrin receptor It may be one or more selected from the group consisting of.
  • the hydrophobic B block is a biocompatible biodegradable polymer, and in one embodiment, may be at least one selected from the group consisting of polyester, polyanhydride, polyamino acid, polyorthoester and polyphosphazine. More specifically, the hydrophobic B specific polylactide, polyglycolide,
  • Polycaprolactone polydioxan-2-one, copolymer of polylactide and glycolide, copolymer of polylactide and polydioxan-2-one, copolymer of polylactide and polycaprolacron and polyglycol Consisting of a copolymer of a lide and a polycaprolactone
  • the hydrophobic B Blocks have a number average molecular weight of 50 to 50,000 Daltons, more specifically 200 to
  • tocopherols tocopherols, cholesterol, or fatty acids having 10 to 24 carbon atoms
  • Amphiphilic block comprising said hydrophilic block (A) and hydrophobic block (B)
  • the content of the copolymer composition is, based on the total dry weight, 40 to 99.98% by weight, in particular from 85 to 99.8 parts by weight 0/0, more specifically, is preferably 90 to 99.8 wt. 0/0. If the amphiphilic block the amount of the copolymer 40 parts by weight 0 / under 0 reduced is too large the stability of the micelle size of the micelles and may cause greater the city filter sterilized loss rate, which when to embedded content exceeds 99.98 increase% The content of anionic drugs becomes too small.
  • the composition ratio of the hydrophilic block (A) and the hydrophobic block (B) is 40 to 70 weight 0 based on the weight of the copolymer .
  • specifically, may range from 50 to 60% by weight.
  • hydrophilic beulteuk (A) ratio is 40 parts by weight 0/0 if less difficult to form the misael solubility in water of the polymer is low
  • the copolymer is a hydrophilic block in order to have the solubility in a cheungbun water to form micelles ol because of the difficulty of use as a solubilizing composition of the drug / cationic lipid complex
  • - is (a) not less than the ratio of 40% by weight of the good on the other hand, 70 parts by weight when it is more than 0 / lowers the hydrophilicity is so high that the stability of the polymeric micelles anionic.
  • the ratio of the hydrophilic block (A) is preferably 70% by weight or less.
  • the amphiphilic block copolymer encapsulates the anionic drug and the cationic lipid complex inside the micellar structure in an aqueous solution, wherein the anionic drug and the cationic lipid are compared to the weight (b) of the amphiphilic block copolymer.
  • Weight (a) ratio of the composite [a / b X 100; (Anionic drug weight + amount Lee Eun-sung lipid weight) / amphiphilic block copolymer weight X 100] is from 0.001 to 100 parts by weight 0/0, and specifically from 0.01 to 50 parts by weight 0 /., More specifically from 0.1 to 10 parts by weight 0/0 can be.
  • the weight ratio is less than 0.001 weight 0 /., The content of the anionic drug and the cationic lipid complex is too low to stratify the effective content that the negatively effective drug can act effectively. This is difficult, on the contrary, when it exceeds 100% by weight, it does not form a micelle structure of the appropriate size in consideration of the molecular weight of the amphiphilic block-heap sieve and the amount of the anionic drug and lipid complex.
  • the micellar structure of the composition according to the invention is characterized in that it comprises polylactic acid salt (PLANa).
  • the polylactic acid salt is distributed in the core (inner wall) of micelles to enhance the hydrophobicity of the core to stabilize the micelles and to effectively avoid the reticulum endothelial system (RES) in the body. That is, the carboxylic acid anion of polylactic acid is more effectively combined with the cationic complex than polylactic acid to reduce the surface potential of the polymer micelles, thereby reducing the positive charge of the surface potentials compared to the polymer micelles that do not contain polylactic acid. Is less trapped by and has the advantage of excellent delivery efficiency to the desired site (eg cancer cells, inflammatory cells, etc.).
  • the desired site eg cancer cells, inflammatory cells, etc.
  • the polylactic acid salt included may have a number average molecular weight of 500 to 50,000 Daltons, specifically 1,000 to 10,000 Daltons. If the molecular weight is less than 500 Daltons, the hydrophobicity is too low to be present in the core (inner wall) of the micelle, and if the molecular weight exceeds 50,000 Daltons, there is a problem that the particles of the polymer micelle become large.
  • the polylactic acid salt may be used in an amount of 1 to 200 parts by weight, specifically 10 to 100 parts by weight, more specifically 30 to 60 parts by weight, based on 100 parts by weight of the amphipathic block polymer.
  • the content of the polylactic acid salt exceeds 200 parts by weight relative to 100 parts by weight of the amphiphilic block polymer, the size of the micelle increases, making it difficult to filter using a sterile membrane, and when the content is less than 1 part by weight, the desired effect cannot be sufficiently obtained.
  • it can contain 10 to 1,000 parts by weight of the amphipathic block copolymer relative to 1 part by weight of anionic drug, and 5 to 500 parts by weight of polylactic acid salt.
  • the amphiphilic block copolymer may be contained in an amount of 50 to 800 parts by weight, more preferably 100 to 500 parts by weight. Preferably, it may contain 10 to 300 parts by weight of polylactic acid salt, more preferably 50 to 100 parts by weight.
  • the terminal opposite of sodium carboxylate in the terminal of the polylactic acid salt is hydroxy, aceoxy, benzoyloxy, decanoyloxy, palmitoyloxy and It may be substituted with one selected from the group consisting of alkoxy having 1 to 2 carbon atoms.
  • the polylactic acid salt of the present invention is characterized in that at least one selected from the group consisting of compounds of the formula (1) to (6).
  • A is -COO-CHZ-; ⁇ is -COO-CHY-, -COO-CH 2 CH 2 CH 2 CH 2 CH 2 -or -COO-CH 2 CH 2 OCH 2 ; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl, or ethyl group; Z and Y are each a hydrogen atom or a methyl or phenyl group; M is Na, K, or Li; n is an integer from 1 to 30; m is an integer of 0-20.
  • X is a methyl group
  • Y ' is a hydrogen atom or a phenyl group
  • p is an integer from 0 to 25
  • q is an integer from 0 to 25, provided that p + q is an integer from 5 to 25
  • R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group
  • M is Na, K, or Li
  • Z is a hydrogen atom, a methyl or a phenyl group.
  • D, L-lactic acid, D-polylactic acid, polymandelic acid, copolymer of D, L-lactic acid and glycolic acid, copolymer of D, L-lactic acid and mandelic acid, D, L-lactic acid and caprolactone It is selected from the group consisting of a co-polymer of and a copolymer of D, L- lactic acid and 1,4-dioxane-2-one;
  • R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group;
  • M is independently Na, K, or Li.
  • S-0-PAD-C00-Q In Equation 4, S is High; L is -NR,-or -0-, where
  • Ri is a hydrogen atom or C 1-10 alkyl
  • Q is CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 , CH 2 CH 2 CH 2 C3 ⁇ 4, or CH 2 C 6 H 5
  • a is an integer from 0 to 4
  • b is an integer from 1 to 10
  • M is Na,, or Li
  • PAD is composed of D, L-polylactic acid, D-polylactic acid, polymandelic acid, co-polymer of D, L-lactic acid and glycolic acid, co-polymer of D, L-lactic acid and mandelic acid, D, L-lactic acid and
  • R ' is -PAD-0-C (0) -CH 2 CH 2 -C (0) -OM, wherein PAD is
  • X and X ' are independently hydrogen, alkyl having 1 to 10 carbon atoms or aryl having 6 to 20 carbon atoms; Y and Z are independently Na, K, or Li; m and n are independently integers from 0 to 95, with 5 ⁇ m + n ⁇ 100; a and b are independently an integer from 1 to 6; R is-(CH 2 ) k- , divalent alkenyl having 2 to 10 carbon atoms, divalent aryl having 6 to 20 carbon atoms, or a combination thereof, wherein k is 0 to 10 Is an integer.
  • the polylactic acid salt is a compound of Formula 1 or Formula 2
  • compositions of the present invention improve the intracellular delivery efficiency of anionic drugs.
  • it can further include a fusible lipid of from 0.1 to 10% increase.
  • the fusion lipid When the fusion lipid is combined with the complex of the anionic drug and the cationic lipid, the hydrophobic interaction is combined to form a complex of the anionic drug, the cationic lipid and the fusion lipid, and the complex including the fusion lipid is Encapsulated Inside the Micellar Structure of the Amphiphilic Block Copolymer
  • the fusion lipid may be one or a combination of two or more selected from the group consisting of phospholipids, cholesterol, and tocopheres.
  • the phospholipid may be at least one selected from the group consisting of phosphatidyl ethanolamine (PE), phosphatidylcholine (PC), and phosphatidic acid. remind
  • PE Phosphatidylethanolamine
  • Phosphatidylcholine (PC) and phosphatidic acid can be in the form combined with one or two C10-24 fatty acids.
  • the cholesterol and tocope include ' analogues, derivatives, and metabolites of cholesterol and tocope.
  • the fusion lipids include dilauuroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, and dipalmitoyl phosphatidylethanolamine.
  • phosphatidylethanolamine dilinoleoyl phosphatidylethanolamine, 1-palmitoyl-2-leleoyl phosphatidylethanolamine (1-palmitoyl-2-oleoyl phosphatidylethanolamine), 1,2-dipitanoyl-3-sn -Phosphatidylethanolamine (I, 2 -diphytanoyl-3-sn-phosphatidylethanolamine), dilauuroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dipalmitoylline Work
  • Dioleoyl phosphatidic acid Dioleoyl phosphatidic acid, dilinoleoyl phosphatidic acid, 1-palmitoyl-2-oleoyl phosphatidic acid (l-pahnitoyl-2-oleoyl
  • phosphatidic acid 1,2-dipitanoyl-3-sn-phosphatidic acid (l, 2-diphytanoyl-3-sn-phosphatidic acid), one or more selected from the group consisting of cholesterol and tocophere May be a combination.
  • the fusion lipid is dioleoyl
  • Dioleoyl phosphatidylethanolamine DOPE
  • dipalmitoleoylphosphocholine (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, DPPC
  • dioleoylphosphocholine l, 2 -dioleoyl- sn-glycero-3-phosphocholine, DOPC
  • Dipalmitooleoylphosphoethanolamine (l, 2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, DPPE) may be one or more selected from the group consisting of ⁇ amphipathic block according to one embodiment Copolymer and
  • compositions containing anionic drug-cationic compound complexes encapsulated in a polylactic acid micellar construct can be administered via routes of administration such as blood vessels, muscles, subcutaneous, oral, bone, transdermal or topical tissue, and are suitable for such routes of administration. And may be formulated into a variety of oral or parenteral formulations.
  • the oral dosage formulation may include various preparations, such as eye drops and injections, such as tablets, capsules, powder formulations, liquids, and the like, and parenteral dosage formulations.
  • the composition may be an injection formulation. For example, when lyophilizing the composition according to the invention, it is distilled water for injection, 0.9%
  • the present invention also provides a method of preparing a pharmaceutical composition comprising an amphiphilic block copolymer micelle containing the anionic drug.
  • the anionic drug, bi-lipid lipid, amphiphilic blotting Method for producing a composition for anionic drug delivery comprising a copolymer and a polylactic acid salt
  • step (b) removing the organic solvent layer from the mixture prepared in step (a);
  • step (c) adding the aqueous solution to the mixture from which the organic solvent of step (b) has been removed, and then micellizing.
  • an anionic drug, a cationic compound, an amphiphilic block copolymer and a polylactic acid salt are mixed to form a complex in a water-soluble organic solvent or a mixed solvent of an aqueous solution and an organic solvent.
  • the water-miscible organic solvent may be at least one selected from the group consisting of acetone, ethanol, methanol and acetic acid
  • the organic solvent of the mixed solvent is a group consisting of ethyl acetate, acetonitrile, methylene chloride, chloroform and dioxane It may be one or more selected from.
  • the aqueous solution may be distilled water, water for injection, or a complete solution.
  • the mixing ratio of the organic solvent and the aqueous solution in the mixed solvent is not particularly limited, and may be, for example, 1: 0.1 to 50, more specifically 1: 0.5 to 10 (organic solvent volume: aqueous solution volume), but is not limited thereto. It is not.
  • step (b) the organic solvent is removed by evaporation from the mixture prepared in step (a).
  • step (c) the remaining mixture after the organic solvent is evaporated is dissolved in an aqueous solution, thereby encapsulating the complex of the anionic drug and the cationic compound in the micelle structure formed by the amphiphilic block copolymer and the polylactic acid salt.
  • the aqueous solution and the amount of use thereof are as described above.
  • step (c) it may further comprise a step of lyophilizing by adding a (d) lyophilization aid.
  • the preparation method sterilizing the aqueous solution of the polymer micelle obtained in step (c) before lyophilization of the step (d) with a sterile filter
  • the process may further include.
  • the lyophilization aid used in the present invention may be used to help the lyophilized composition to maintain a cake form or to uniformly dissolve quickly in the process of reconstitution after lyophilizing the amphiphilic block copolymer composition.
  • By adding, specifically, may be at least one selected from the group consisting of lactose, manny, sorbbi and sucrose ⁇
  • the content of the lyophilization aid based on the total dry weight of the lyophilized composition, 1 to 90 weight 0/0, and more specifically 10 to 60 wt. 0/0.
  • a composition in which an anionic drug and a cationic compound complex is encapsulated in an amphiphilic block copolymer and a polylactic acid salt structure is prepared.
  • the micelle particles in the prepared composition are stable in bloodemia, and have a size of 10 to 200 nm, more specifically 10 to
  • MPEG-PLA of 4,000 Daltons was synthesized [Preparation Example 2].
  • MPEG-PLA block having a number average molecular weight of 2,000-1,750 Daltons using monomethoxypolyethylene glycol (molecular weight 2,000 Daltons or less, NOF corporation) in the same manner Co-polymer was synthesized [Preparation Example 3].
  • MPEG-PLA-tocopherol (number average molecular weight 5,000-4,000-530 Daltons) was obtained according to the process described in Preparation Example 2 of WO2012-091523 [Preparation Example 4].
  • aqueous sodium hydrogen carbonate solution (0.1 ⁇ 1) 150 1 was slowly added and stirred at 60 rpm for 60 to 2 hours. After adding 15 g of sodium chloride at room temperature and dissolving with stirring, the aqueous layer was removed using a separatory funnel.
  • 1,6 dioTETA (/ P ratio 18) was dissolved in 94.63 ⁇ of chloroform and siRNA 100 was dissolved in 80 ⁇ l of distilled water.
  • 505.37 ⁇ of chloroform was added so that the volume ratio of the organic layer to the aqueous layer was 10 times as a whole.
  • An emulsion was prepared using an ultrasonic grinder while siRNA was added to the solution mixture of 1,6 dioTETA and mPEG-PLA-tocopherol in chloroform.
  • the emulsion was added to 2320 ⁇ of distilled water to prepare a composite emulsion using an ultrasonic mill.
  • the prepared complex emulsion was placed in a 1-neck equilateral polar flask and distilled under reduced pressure in a rotary evaporator to selectively remove chloroform, thereby siRNA / 1,6-dioleoyl.
  • Polymeric micelles containing triethylenetetraamide (dioTETA) / mPEG-PLA-tocopherol (2k-1.7k) were prepared (see Table 1).
  • siRNA lipid N / P ratio mg of polymer. The same applies in the following table.
  • the dioTETA solution, PLA solution, and 24 mg solution of mPEG-PLA-tocopherol were mixed together, and an emulsion was prepared using an ultrasonic mill while adding dropwise siRNA solution.
  • the solvent was removed by distillation under reduced pressure in a 1-neck equiaxed polar flask coated with 6 mg of oil emulsion mPEG-PLA-tocopherol and a rotary evaporator. 3 ml of distilled water was added to the flask, and the mixture was shaken gently to prepare siRNA / dioTETA / mPEG-PLA-tocopherol (2k-1.7k) / PLA-containing polymer micelle (see Table 2).
  • Example 3-6 Preparation of a Composition Containing siRNA / dioTETA / mPEG-PLA-Tocope (2k-1.7k) / PLANa (1.7k)
  • composition Composition siRNA lipid Polymer 1 Polymer 2 Example 3 siR A / dioTETA / mPEG-5-8-1-0.3 150 ⁇ ⁇ 1.26 mg 30 mg 9 mg
  • Example 4 siRNA / dioTETA / mPEG- 5-24-1-0.3 150 ⁇ 8 3.79 mg 30 mg 9 mg PLA-Tocope (2k- 1.7k) / PLANa (1.7k)
  • Example 1 2 1 Preparation of a composition containing 1 siRNA-cholester / dioTETA / mPEG-PLA-tocope ( 2 k-1.7k) PLANa (1.7k)
  • siRNA-cholester was used to prepare a composition containing siRNA-cholesterol / dioTETA / mPEG-PLA-tocopherol (2k-1.7k) / PLANa (1.7k) using the same procedure as in Example 1.
  • the mixture was distilled under reduced pressure in a rotary distillation concentrator to remove the solvent. Add 3 ml of distilled water to the flask, shake gently to dissolve the composition
  • siRNA-PEG / dioTETA / mPEG-PLA-tocopherol (2k-1.7k) / PLANa (1.7k) containing composition was subjected to the same procedure as in Example 1 using siRNA-PEG.
  • bPEI Dissolve 0.3 mg in distilled water was dissolved 15 ⁇ siRNA 150 ⁇ ⁇ in distilled water to 120 ⁇ .
  • the bPEI aqueous solution and the siRNA aqueous solution are mixed with 105 ⁇ l of HBS complete aqueous solution (10 mM HEPES, 1 mM NaCl).
  • 9 mg PLANa (1.7k) was dissolved in 180 ⁇ l of chloroform and 30 mg of mPEG-PLA-tocopherol (2k-1.7k) was dissolved in 100 ⁇ l of chloroform. 2140 ⁇ of chloroform was added so that the volume ratio of the organic layer to the aqueous layer was 10 times as a whole.
  • the PLANa solution was mixed with a 24 mg solution of mPEG-PLA-tocopherol, and an emulsion was prepared by using an ultrasonic mill while adding dropwise HBS complete aqueous solution mixed with bPEI and siRNA.
  • the emulsion was placed in a 1-neck equilateral flask coated with 6 mg of mPEG-PLA-tocopherol and distilled under reduced pressure in a rotary evaporator to remove the solvent. 6 ml of distilled water was added to the flask, and the mixture was shaken gently to prepare a siRNA / bPEI / mPEG-PLA-tocopherol (2k-1.7k) / PLANa (1.7k) -containing composition (Table 12).
  • Table 12 Table 12
  • lipids were prepared in the same manner as in Example 1 to contain siRNA / 1,10-dioleoyl pentaethylenenucleamide (dioPEHA) / mPEG-PLA-tocofe (2k-1.7k) / PLANa (1.7k) SiRNA / 1,8-dilinoleoyl tetraethylenepentamide with composition 9
  • composition 10 (dilTEPA) / mPEG-PLA-tocophere (2k-1.7k) / PLANa (L7k) containing composition 10
  • siRNA-PEG was used to prepare siRNA-PEG / dioTETA / mPEG-PLA-tocopherol (2k-1.7k) / PLANa (1.7k) / DOPE containing composition.
  • the amount of DOPE was added 1 and 4 times the amount of dioTETA, respectively. This mixture
  • the solvent was removed by distillation under reduced pressure in a rotary distillation thickener. 3 ml of distilled water was added to the polar flask, and the composition was prepared by gently shaking to dissolve it (Table 14).
  • Example 1 siR A / dioTETA / mPEG-PLA-5-16-1-0.3 28.07 nm -1.29 mV tocopherol (2k-1.7k) / PLANa (1.7k)
  • Example 12 siRNA-cholester /dioTETA/mPEG-5-16-1-0.3 27.73 nm -4.38 mV
  • the formulations were injected intravenously into Balb / c mice at lmg / kg and blood was drawn 5 hours and 6 hours later.
  • the blood is collected in a new tube by centrifugation at I3000 rpm for 4 minutes at 4 minutes, and the standard formulation is prepared by diluting in PBS at a total of 11 concentrations ranging from 4 ⁇ to 0.00256 ⁇ .
  • ⁇ ⁇ of the diluted standard formulation was added to the 96 well plate for PCR, and 9 ⁇ l of Balb / c mouse serum and 90 ⁇ of 0.25% triton X-100 were added.
  • Triton X-100 90 ⁇ l of 0.25% Triton X-100 was added to the blood sample ⁇ ⁇ of the experimental group, followed by a pretreatment step to release the carrier.
  • the siRNA exposed as the formulation was released was synthesized into cDNA via reverse transcription (RT) step and qRT-PCR (Bio-Rad CFX96 Real-Time System) was performed using the synthesized cDNA. Analysis was performed using the Bio-Rad CFX Manager program.
  • Example 2 1 1650.82 5362.87 As can be seen in Table 16, the formulations prepared in Examples 1 and 2 of the present invention have a concentration of 5 to 8 times the concentration of the formulations present in blood compared to Comparative Examples 1 and 2 at 0.5 hours. The higher the degree, the more stable the blood.
  • siRNA / dioTETA / mPEG-PLA-tocope / PLANa polymer micelles distributed in liver, liver and cancer tissues was confirmed.
  • mice were prepared by injecting Balb / c nude mice subcutaneously with an A2780cis human ovarian cancer cell line.
  • the formulation was administered intravenously with a total of four doses, one at a dose of l mg / kg. Liver and cancer tissues were removed and weighed 200 mg 24 hours after the last dose, and then placed in 1.8 mL of 0.25% Triton X-100 and ground with a tissue grinder. Threadard tissue sample is to use the tissue administered with saline and change the tissue in the same way. Concentrations of standard formulations are prepared by dilution in PBS at 11 concentrations ranging from 4 ⁇ to 0.00256 ⁇ .
  • tissue ground for the standard is added to a 96-well plate for PCR, and the formulation ⁇ ⁇ was added to the standard.
  • Tissue samples to be analyzed in the experimental group are subjected to a pretreatment step in which 100 ⁇ is added and the formulation is released.
  • the exposed siRNA was synthesized into cDNA by reverse transcription (RT) step and qRT-PCR (Bio-Rad CFX96 Real-Time System) was performed using the synthesized cDNA. The analysis was performed using the Bio- Rad CFX Manager program.
  • Example 2 according to the present invention was shown to reduce the liver tissue distribution, cancer tissue distribution increased compared to Comparative Example 1.
  • PLANa-containing polymer micelle carrier according to the present invention can be specifically targeted to cancer tissues.
  • siRNA / dioTETA / mPEG-PLA-tocope / PLANa polymer micelles was confirmed by analysis of gene suppression ability.
  • Cancer-induced mice were prepared by feeding A549 human lung cancer tumor isolates to Balb / c nude mice.
  • the formulation was administered intravenously three times, once per dose, at a dose of 0.5 mg / kg.
  • Physiological saline was administered as a control group, 5 dogs for each formulation.
  • the cancer tissue was extracted, and the first milling was performed using a mortar and pestle under liquid nitrogen, and the tissue was once again changed using a QIAGEN tissue crusher (TissueLyser).
  • HPRT mRNA is removed from the cells using 600 ⁇ 1 of working homogenizing solution, Homogenizing solution 600 ⁇ 1 + Proteinase K (23 mg / mL) 6 ⁇ 1, which is prepared in 10 mg of twice-broken cancer tissue.
  • Samples obtained by the above method were analyzed using a bDNA assay kit. The assay was followed by the manufacturer of the kit (Panomics bDNA assay).
  • GAPDH mRNA a gene that is not affected by HPRT siRNA, was also analyzed in the same manner, and the average amount of HPRT mRNA expression in cancer tissues was calculated by correcting the measured HPRT mRNA amount.
  • Table 18 The analysis results are shown in Table 18 below.
  • Example 2 according to the present invention can be seen that inhibits the mRNA 54% of the target gene HPRT in cancer tissues in vivo.

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Abstract

Disclosed are a pharmaceutical composition for anionic drug delivery, and a preparation method therefor, the pharmaceutical composition for anionic drug delivery containing: an anionic drug as an active ingredient; a cationic compound; an amphiphilic block copolymer; and a polylactate, wherein the anionic drug formed a complex with the cationic lipid, and the complex is encapsulated within a micelle structure formed by the amphiphilic block copolymer and the polylactate.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
음이온성 약물 함유 약제학적 조성물 및 그 제조방법  Anionic Drug-Containing Pharmaceutical Compositions and Methods of Making the Same
【기술분야】  Technical Field
본 발명은 음이온성 약물을 함유하고, 이를 전달하기 위한 약제학적 조성물 및 그 제조방법에 관한 것이다.  The present invention relates to a pharmaceutical composition containing an anionic drug and to delivering the same, and to a method of preparing the same.
【배경기술】  Background Art
음이온성 약물, 특히 핵산 물질을 이용한 치료에 있어서, 안전하고 효율적인 약물 전달기술은 오랫동안 연구되어 왔으며, 다양한 전달체 및 전달기술이 개발되어 왔다. 특히, 아데노바이러스나 레트로바이러스 등을 이용한 바이러스성 전달체, 및 양이온성 지질, 양이은성 고분자 등을 이용한  In the treatment with anionic drugs, especially nucleic acid materials, safe and efficient drug delivery techniques have been studied for a long time and various carriers and delivery techniques have been developed. In particular, viral carriers using adenoviruses or retroviruses, and cationic lipids, cationic polymers, etc.
비바이러스성 전달체를 이용한 전달기술들이 개발되어 왔다. Delivery techniques using nonviral carriers have been developed.
그러나, 바이러스를 이용한 전달체를 이용하는 기술은, 비특이적 면역 반응 등의 위험성에 노출되어 있으며 생산 공정이 복잡하여 상용화하는 데 많은 문제점이 있는 것으로 알려져 있다. 따라서, 최근 연구 방향은 양이온성 지질이나 양이온성 고분자를 이용하는 비바이러스성 전달체를 이용하여 그 단점을 개선하는 방향으로 진행되고 있다. 이러한 비바이러스성 전달체는, 바이러스성 전달체에 비하여 효율성에서 뒤떨어지지만, 생체 내 안전성의 측면에서 부작용이 적고, 경제성 측면에 '서 생산 가격이 저렴하다는 장점을 가지고 있다. However, a technique using a delivery vehicle using a virus is exposed to risks such as a nonspecific immune response, and it is known that there are many problems in commercialization due to the complicated production process. Therefore, recent research directions have been made to improve the disadvantages by using a non-viral carrier using a cationic lipid or a cationic polymer. These non-viral delivery system is, but poor in efficiency compared to the viral delivery system, has the advantage that fewer side effects in terms of in vivo stability, low price "books produced in an economic point of view.
핵산 물질의 전달에 이용되는 비바이러스성 전달체에 대해서 많은 연구가 이루어졌는데, 가장 대표적인 것은 양이온성 지질을 이용한 양이온성 지질과 핵산의 복합체 (lipoplex) 및 풀리양이온성 (polycation) 고분자와 핵산의  Numerous studies have been conducted on non-viral carriers used for the delivery of nucleic acid materials. The most representative ones are cationic lipids using cationic lipids and lipoplexes and polycation polymers and nucleic acids.
복합체 (polyplex)이다. 이러한 양이온성 지질 혹은 폴리양이온성 고분자는, 음이온성 약물과 정전기적 상호 작용을 통해 복합체를 형성함으로써 음이은성 약물을 안정화시키고, 세포 내 전달을 증가시킨다는 점에서 많은 연구가 진행되어 왔다 (De Paula D, Bentley MV, Mahato RI, Hydrophobization and bioconj ligation for enhanced siRNA delivery and targeting, R A 13 (2007) 431 -56; Gary DJ, Puri N, Won YY, Polymer-based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery, J Control release 121 (2007) 64-73). It is a polyplex. These cationic lipids or polycationic polymers have been studied in that they form complexes through electrostatic interactions with anionic drugs to stabilize negatively drug and increase intracellular delivery (De Paula D). , Bentley MV, Mahato RI, Hydrophobization and bioconj ligation for enhanced siRNA delivery and targeting, RA 13 (2007) 431 -56; Gary DJ, Puri N, Won YY, Polymer-based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery, J Control release 121 (2007) 64-73).
그러나, 이제까지 연구된 양이온성 지질 또는 폴리양이온성 고분자들은, 층분한 효과를 얻기 위해 필요한 양을 사용할 경우에 , 바이러스성 전달체보다는 덜하지만, 심각한 독성을 유발하여 의약품으로 사용이 부적당하다는 결과를 나타내었다. 또한, 양이온성 지질과 핵산의 결합을 통해 착화합물을 이루어 세포 내로 핵산을 전달시키는 지질 -핵산 복합체의 경우는 세포주 실험에서는 매우 광범위하게 이용되지만, 혈중에서의 안정성을 가질 수 있는 구조를 나타내지 못하기 때문에 생체 내에서 이용하기에는 블가능하다 (미국특허 제 6,458,382호 참고).  However, the cationic lipids or polycationic polymers studied so far have less results than viral carriers, but have severe toxicity when used in amounts necessary to achieve a striking effect, resulting in inadequate use in medicine. . In addition, the lipid-nucleic acid complexes, which combine the cationic lipids with the nucleic acids and deliver the nucleic acids into the cells, are very widely used in cell line experiments, but do not exhibit a structure that can have stability in blood. It is possible to use it in vivo (see US Pat. No. 6,458,382).
또한, 핵산 물질 자체에 지질 혹은 고분자를 직접 접합시킨 후, 미셀이나 다른 고분자와 복합체를 이루게 하여 나노 입자를 형성시키는 연구도 진행되고 있는데, 핵산 물질에 직접 지질 혹은 고분자를 접합시키는 경우, 접합 효율이나 품질 관리의 측면에서 어려움을 가지고 있으며 아직까지 명확하게 핵산 전달의 효율에 있어서는 검증이 되어 있지 않다.  In addition, research has been conducted to form nanoparticles by directly conjugating lipids or polymers to nucleic acid materials themselves and complexing them with micelles or other polymers. There are difficulties in terms of quality control and there is no clear verification of the efficiency of nucleic acid delivery.
따라서, 독성을 유발할 수 있는 양이온성 고분자 또는 양이온성 지질의 사용량을 최소화하여 독성을 감소시키면서, 혈중 및 체액 내에서 안정하고, 세포 내 전달이 가능하여 충분한 효과를 얻을 수 있는 음이온성 약물 전달 기술의 개발이 필요하다. 한편, 양친성 블록 공중합체를 이용하여 고분자 미셀의 형태로 난용성 약물을 가용화하고 수용액상에서 안정하게 함으로써 약물 전달체로서 이용하려는 노력이 다양하게 진행되었다 (WO 1997-010849). 그러나, 이러한 양친성 블록 공중합체는 내부에 소수성을 띄는 고분자 미셀을 형성함으로써 소수성을 띄는 난용성 약물을 가용화할 수는 있지만, 음이은을 띄는 핵산 등의 친수성 약물은 고분자 미셀 내부에 봉입할 수 없으므로, 이들 핵산을 포함하는 음이온성 약물의 전달에는 적당하지 않다. 이에, 핵산과 양이온성 지질의 정전기적 상호작용에 의한 복합체를 형성하여 상기 복합체가 양친성 블록 공중합체의 미샐 구조 내부에 봉입되도록 하는 음이온성 약물 전달 조성물을 개시한 바 있다. 그러나, 이 역시 핵산의 혈중 안정성 및 암조직에 대한 특이적 표적화에 있어서 개선의 여지를 안고 있다.  Therefore, it is possible to reduce the toxicity by minimizing the amount of cationic polymer or cationic lipid that can cause toxicity, while maintaining stability in the blood and body fluids and enabling intracellular delivery to obtain sufficient effects. Need development On the other hand, various efforts have been made to use them as drug carriers by solubilizing poorly soluble drugs in the form of polymer micelles using an amphiphilic block copolymer and making them stable in aqueous solution (WO 1997-010849). However, these amphiphilic block copolymers can solubilize hydrophobic poorly soluble drugs by forming hydrophobic polymer micelles, but hydrophilic drugs such as nucleic acids having negative hydrophobicity cannot be encapsulated inside polymer micelles. Not suitable for delivery of anionic drugs comprising these nucleic acids. Thus, anionic drug delivery compositions have been disclosed that form a complex by electrostatic interaction of a nucleic acid with a cationic lipid such that the complex is encapsulated within the micellar structure of the amphiphilic block copolymer. However, this also provides room for improvement in blood stability of the nucleic acid and specific targeting to cancer tissues.
한국등록특허 계 1296326호에는 유효성분으로서 음이온성 약물; 양이온성 지질; 양친성 블록 공중합체 및 폴리락트산을 포함하며, 상기 음이온성 약물은 상기 양이온성 지질과 복합체를 형성하고, 상기 복합체가 양친성 블록 공증합체 및 폴리락트산이 형성하는 미셀 구조 내부에 봉입된 구조를 갖는 것을 특징으로 하는 음이온성 약물 전달용 조성물이 개시되어 있다. 그러나 이 특허에서 사용된 폴리락트산은 말단에 카르복시기를 갖는 일반적인 폴리락트산 고분자로서, 약물 전달 효과가 미흡한 문제점올 가지고 있다. Korean Patent No. 1296326 discloses an anionic drug as an active ingredient; Cationic Lipids; An amphiphilic block copolymer and a polylactic acid, wherein the anionic drug forms a complex with the cationic lipid, and the complex has a structure encapsulated inside a micellar structure formed by the amphiphilic block copolymer and the polylactic acid. An anionic drug delivery composition is disclosed. However, polylactic acid used in this patent is a general polylactic acid polymer having a carboxyl group at its terminal, and has a problem of insufficient drug delivery effect.
한편, 많은 질병은 여러 요인으로 인하여 질병 유전자의 발현이 , 증가하거나 돌연변이에 의해 비정상적인 활성이 나타남으로써 발생하게 된다. siRNA(short interfering RNA)는, 전사후 공정에서 서열 특이적으로 특정 유전자의 발현을 억제하므로, 유전자 치료제로서 많은 관심이 집중되고 있다. 특히, siRNA의 높은 활성과 정밀한 유전자 선택성으로 인해, 기존의 안티센스 뉴클레오티드나 리보자임 등의 문제점을 해결할수 있는 핵산 치료제로 기대되고 있다. siRNA는 짧은 이중 나선의 RNA 가닥으로, 이들과 염기 서열이 상보적인 유전자의 mRNA를 절단함으로써 해당유전자의 발현을 억제시킨다 (McManus and Sharp, Nature Rev. Genet. 3:737 (2002); Elbashir, et al., Genes Dev. 15: 188 (2001). On the other hand, many diseases are caused by the expression of genes due to several factors, disease, or increased by mutation appears unusual activity. Since siRNA (short interfering RNA) suppresses the expression of a specific gene in a sequence-specific manner in a post-transcription process, much attention has been focused on gene therapy agents. In particular, due to the high activity and precise gene selectivity of siRNA, it is expected to be a nucleic acid therapeutic agent that can solve problems such as antisense nucleotides and ribozymes. siRNAs are short, double-stranded RNA strands that inhibit the expression of glycolysis by cleaving mRNAs of genes complementary to these (McManus and Sharp, Nature Rev. Genet. 3: 737 (2002); Elbashir, et. al., Genes Dev. 15: 188 (2001).
그러나, 이러한 장점에도 불구하고, siRNA는 혈증에서 핵산분해 효소에 의해 빠르게 분해되고, 신장을 통하여 빠르게 체외로 배설되는 것으로 알려져 있다. 또한 siRNA는 강한 음전하를 띄어 세포막을 쉽게 통과하지 못하는 것으로 알려져 있다. 따라서 siRNA를 치료제로 사용하기 위해서는 siRNA를 혈액에서 안정화시키고, 목표로 삼은 조직이나 세포 안으로 효율적으로 전달할 수 있으며, 독성을 나타내지 않는 전달체의 개발이 필요하다.  However, despite these advantages, siRNAs are known to be rapidly degraded by nucleases in blood and rapidly excreted in vitro through the kidneys. In addition, siRNA is known to have a strong negative charge does not easily cross the cell membrane. Therefore, in order to use siRNA as a therapeutic agent, siRNA needs to be stabilized in the blood, efficiently delivered into a target tissue or cell, and development of a non-toxic carrier.
【발명의 상세한 설명】  [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
상기와 같은 문제점을 해소하고자, 본 발명은 폴리락트산염을 함유하여 음이온성 약물을 체내에 효과적으로 전달할 수 있는 미셀 구조체를 포함하는 음이온성 약물 전달용 조성물을 제공하는 것을 목적으로 한다.  In order to solve the above problems, an object of the present invention is to provide a composition for anionic drug delivery comprising a micellar structure containing polylactic acid salt to effectively deliver the anionic drug in the body.
또한, 본 발명은 상기 조성물의 음이온성 약물 전달을 위한 용도를 제공하는 것을 목적으로 한다.  It is also an object of the present invention to provide a use for the anionic drug delivery of the composition.
또한, 본 발명은 상기 조성물을 투여하는 단계를 포함하는 음이온성 약물의 전달 방법을 제공하는 것을 목적으로 한다. In addition, the present invention is an anionic comprising administering the composition It is an object to provide a method of delivery of a drug.
또한, 본 발명은 상기와 같은 음이은성 약물을 체내에 효과적으로 전달할 수 있는 약학적 조성물의 제조방법을 제공하는 것을 목적으로 한다.  In addition, an object of the present invention is to provide a method for preparing a pharmaceutical composition that can effectively deliver such a negative drug.
【기술적 해결방법】  Technical Solution
본 발명은 폴리락트산염올 함유하여 음이온성 약물을 체내에 효과적으로 전달할 수 있는 미셀 구조체를 포함하는 음이온성 약물 전달용 조성물, 상기 조성물의 음이온성 약물 전달을 위한 용도 및 상기 조성물을 투여하는 단계를 포함하는 음이온성 약물의 전달 방법에 관한 것이다.  The present invention comprises an anionic drug delivery composition comprising a micellar structure containing polylactic acid salt to effectively deliver an anionic drug to the body, uses for the anionic drug delivery of the composition and administering the composition. It relates to a method of delivery of anionic drugs.
본 발명에 따른 미셀 구조체를 포함하는 음이온성 약물 전달용 조성물은, 양친성 블록 공증합체와 폴리락트산염의 미셀 구조체에 약물 및 양이은성 화합물의 복합체가포함된 구조로서 , 구체적으로는  The anionic drug delivery composition comprising the micelle structure according to the present invention is a structure containing a complex of a drug and a divalent compound in the micelle structure of the amphiphilic block co-polymer and the polylactic acid salt.
유효성분으로서 음이온성 약물;  Anionic drugs as active ingredients;
양이온성 화합물;  Cationic compounds;
양친성 블록 공증합체; 및  Amphiphilic block co-polymers; And
폴리락트산염  Polylactic acid salt
을 포함하며 , 상기 음이온성 약물은 상기 양이온성 화합물과 정전기적 상호작용에 의해 복합체를 형성하고, 이와 같이 형성된 복합체가 양친성 블록 공중합체 및 폴리락트산염에 의하여 형성된 미샐 구조 내부에 봉입되는 것을 특징으로 한다.  Wherein the anionic drug forms a complex by electrostatic interaction with the cationic compound, and the complex thus formed is enclosed in a micelle structure formed by an amphiphilic block copolymer and a polylactic acid salt. It is done.
상기 조성물은 물에 용해가 가능하고, 미셀 구조체를 형성하는 한 성분으로서 폴리락트산염을 포함함으로써, 체내 주입시 혈증 안정성 증가 및 세망내피계 (RES)를 회피하는 것을 통하여 표적 부위, 구체적으로 암조직으로의 전달 효율이 우수하므로, 세망내피계 (RES) 회피 및 /또는 표적화증진 용도로도 유용하다.  The composition is soluble in water and comprises polylactic acid as a component to form micelle structures, thereby increasing the stability of blood and avoiding reticulum endothelial system (RES) during infusion into the target site, specifically cancer tissue. It is also useful for evasion and / or targeting of reticulum endothelial system (RES) because of its high efficiency in delivery.
또 다른 본 발명의 일 양태로서, 상기 음이온성 약물 전달용 조성물의 제조방법은,  In another aspect of the present invention, the method for preparing the composition for anionic drug delivery,
(a) 음이은성 약물, 양이은성 화합물, 양친성 블록 공중합체 및  (a) negative silver drugs, positive silver compounds, amphiphilic block copolymers and
플리락트산염을 수흔화성 유기용매 또는 수용액과 유기용매의 흔합 용매에 용해시키는 공정; (b) 상기 (a) 공정의 유기용매층를 제거하는 공정; Dissolving a polylactate in a water-soluble organic solvent or a mixed solvent of an aqueous solution and an organic solvent; (b) removing the organic solvent layer of step (a);
(c)상기 (b) 공정의 유기용매가 제거된 혼합물에 수용액을 첨가하여 미셀화하는 공정  (c) process of micellizing by adding an aqueous solution to the mixture from which the organic solvent of step (b) is removed
을 포함할 수 있다.  It may include.
【발명의 효과】  【Effects of the Invention】
본 발명에 따른 음이온성 약물 전달용 약제학적 조성물은, 양이온성 화합물, 그리고 양친성 블록 고분자 및 폴리락트산염으로 형성된 미셀 구조체을 사용하여 음이온성 약물을 외부로부터 격리시킴에 따라 음이온성 약물의 혈중 혹은 체액 내 안정성을 높일 수 있다. 따라서, 본 약제학적 조성물은 체내에 투여하였을 경우 음이온성 약물의 혈중 혹은 체액 내 안정성을 높일 수 있으며, 특히 세망내피계를 회피하여 음이온성 약물이 세포 내로 효율적으로 전달될 수 있다는 장점이 있다.  In the pharmaceutical composition for anionic drug delivery according to the present invention, the anionic drug is isolated from the outside by using a micellar structure formed of a cationic compound and an amphiphilic block polymer and a polylactic acid salt. You can increase the stability. Therefore, the pharmaceutical composition can increase the stability of the anionic drug in the blood or body fluid when administered in the body, and particularly has the advantage that the anionic drug can be efficiently delivered into the cell avoiding the reticuloendothelial system.
【도면의 간단한 설명】  [Brief Description of Drawings]
도 1은 본 발명에 따른 음이온성 약물 및 양이은성 화합물의 복합체가 봉입된 고분자 미셀 전달체의 개략적인 구조를 나타낸 도면이다.  1 is a diagram showing a schematic structure of a polymer micelle delivery vehicle in which a complex of an anionic drug and a bipolar compound according to the present invention is encapsulated.
도 2는 제조예 8에 따른 폴리락트산 나트륨염의 NMR 결과를 나타낸 도면이다.  Figure 2 is a view showing the NMR results of the polylactic acid sodium salt according to Preparation Example 8.
【발명의 실시를 위한 최선의 형태】  [Best form for implementation of the invention]
이하, 본 발명을 보다상세히 설명한다.  Hereinafter, the present invention will be described in more detail.
본 발명에 따른 조성물의 구성성분 증, 상기 음이온성 약물과 양이온성 화합물은, 양친성 블록 고분자와 폴리락트산염에 의하여 형성된 미셀 구조 내부에 봉입되고, 이러한 음이온성 약물 및 양이온성 화합물의 복합체가 봉입된 고분자 미셀 전달체의 대략적인 구조를 도 1에 나타내었다. 도 1을 참조하면, 음이온성 약물은 양이온성 화합물과 정전기적 상호작용을 통해 서로 결합하여, 음이은성 약물과 양이온성 화합물 복합체를 형성한다. 형성된 음이온성 약물과 양이온성 화합물 복합체는, 양친성 블록 공중합체 및 폴리락트산염에 의해 형성되는 미샐 구조 내에 봉입된다.  Component composition of the composition according to the present invention, the anionic drug and the cationic compound is enclosed in the micellar structure formed by the amphiphilic block polymer and polylactic acid salt, the complex of such anionic drug and cationic compound is enclosed The approximate structure of the polymerized micelle transporter is shown in FIG. 1. Referring to FIG. 1, anionic drugs bind to each other through an electrostatic interaction with a cationic compound to form a complex of a negative drug and a cationic compound. The anionic drug and cationic compound complex formed are encapsulated in a micellar structure formed by an amphiphilic block copolymer and a polylactic acid salt.
도 1에 나타낸 바와 같이, 양친성 블록 공중합체 및 폴리락트산염에 의해 형성되는 미셀 구조체는 수성 환경에서 양친성 블록 공증합체의 친수성 부분이 미셀의 외벽을 형성하고 양친성 블록 공중합체의 소수성 부분과 상기 양친성 블록 공증합체와 별도의 성분으로 함유된 폴리락트산염이 미셀의 내벽을 형성하고, 그 형성된 미씰의 내부에 음이온성 약물과 양이온성 화합물 복합체가 봉입된 구조이다. As shown in FIG. 1, the micellar structure formed by the amphiphilic block copolymer and the polylactic acid salt is a hydrophilic portion of the amphiphilic block copolymer in an aqueous environment. The polylactic acid salt that forms the outer wall of the micelle and contains the hydrophobic portion of the amphiphilic block copolymer and the amphiphilic block co-polymer as a separate component forms the inner wall of the micelle, and the anionic drug and the cation inside the formed micelle. It is a structure encapsulated with a compound of a sex compound.
상기 음이온성 약물과 양이온성 화합물 복합체는 상기 양친성 블록 공증합체 및 폴리락트산염에 의해 형성되는 미셀 구조체 내에 봉입된 상태를 유지하여 혈중 또는 체액 내에서의 안정성이 향상된다. 일실시예에서, 상기 미셀의 입자 크기는 , 10 내지 200 m이며, 더욱 구체적으로는 10 내지 150nm인 것이 좋다. 또한, 상기 미셀 입자의 표준 전하는 -20 내지 20 mV 이며, 더욱 구체적으로 -10 내지 10 mV인 것이 좋다. 상기 입자 크기 및 표준 전하는, 미샐 구조의 안정성 및 구성성분들의 함량과 체내에서 음이온성 약물의 흡수도 및 약제학적 조성물로서 멸균의 편의성면에서 가장 바람직하다. 본 발명에 따른 조성물에 유효성분으로 포함되는 음이온성 약물은 수용액 증에서 분자 내에 음전하를 띄는 약리학적 활성을 가진 모든 물질을 포함하는 개념이다. 구체적인 일 양태로서, 상기 음이온성은, 카르복시기, 포스페이트기 및 설페이트기로 구성된 군으로부터 선택되는 하나 이상의 작용기로부터 부여될 수 있다. 또한, 본 발명의 일 양태로서 음이온성 약물은 펩타이드, 단백질 또는 헤파린 등의 다중 음이온성 약물 또는 핵산일 수 있다.  The anionic drug and the cationic compound complex is maintained in the micelle structure formed by the amphiphilic block copolymer and the polylactic acid salt to improve stability in blood or body fluids. In one embodiment, the particle size of the micelles, 10 to 200 m, more preferably 10 to 150nm. In addition, the standard charge of the micelle particles is -20 to 20 mV, more preferably -10 to 10 mV. The particle size and standard charge are most preferred in terms of stability of the micellar structure and the content of constituents, the absorption of anionic drugs in the body, and the convenience of sterilization as a pharmaceutical composition. The anionic drug included as an active ingredient in the composition according to the present invention is a concept including all substances having a negatively charged pharmacological activity in the molecule in aqueous solution. In a specific embodiment, the anionicity may be imparted from one or more functional groups selected from the group consisting of carboxyl groups, phosphate groups and sulfate groups. In addition, as an aspect of the present invention, the anionic drug may be a polyanionic drug or nucleic acid such as peptide, protein or heparin.
또한, 상기 핵산 물질은, 디옥시리보 핵산, 리보 핵산또는 백본 (backbone), 당 또는 염기가 화학적으로 변형되거나 말단이 수식된 폴리뉴클레오타이드 유도체 등의 핵산 약물일 수 있으며, 보다 구체적으로는 RNA, DNA, siRNA(short interfering RNA), 압타머 (aptamer), 안티센스 ODN(antisense oligodeoxynucleotide), 안티센스 RNA(antisense RNA), 리보자임 (ribozyme) 및 디엔에이자임 (DNAzyme) 등으로 구성된 군으로부터 선택되는 하나 이상의 핵산일 수 있다. 나아가, 상기 핵산은 혈중 안정성을 증가시키거나 면역 반웅올 약화시키는 등의 목적을 위해 백본 (backbone), 당또는 염기가 화학적으로 변형되거나 말단이 수식될 수 있다. 구체적으로, 핵산의 포스포다이에스테르 (phosphodiester) 결합의 일부를  In addition, the nucleic acid material may be a deoxyribonucleic acid, a ribo nucleic acid or a backbone, a nucleic acid drug such as a polynucleotide derivative whose sugar or base is chemically modified or modified at the end, and more specifically, RNA, DNA, siRNA (short interfering RNA), aptamer, antisense oligodeoxynucleotide (ODN), antisense RNA (antisense RNA), ribozyme (ribozyme) and DNAzyme (DNAzyme) may be one or more selected from the group consisting of. . Furthermore, the nucleic acid may be chemically modified or terminally modified with a backbone, sugar or base for the purpose of increasing blood stability or weakening the immune response. Specifically, some of the phosphodiester bonds of the nucleic acid
포스포로티오에이트 (phosphorothioate) 또는 보라노포스페이트 (boranophosphate) 결합으로 대체하거나, 일부 리보오스 염기의 2'-OH 위치에 메틸기, 메록시에틸기, 불소 등의 다양한 작용기가도입된 수식된 뉴클레오티드를 1종 이상 포함할 수 있다. Replace with a phosphorothioate or boranophosphate bond, or a methyl, methoxyethyl group, at the 2'-OH position of some ribose base, It may include one or more modified nucleotides in which various functional groups such as fluorine are introduced.
또한, 상기 핵산의 하나 이상의 말단은 콜레스테롤, 토코페롤 및 탄소수 In addition, at least one terminus of the nucleic acid is cholesterol, tocopherol and carbon number
10 내지 24개의 지방산으로 구성된 군으로부터 선택되는 하나 이상으로 수식될 수 있다. 예를 들어, siRNA의 경우 센스 및 /또는 안티센스 가닥의 5' 말단, 또는 3' 말단, 또는 양 말단에 수식될 수 있으며, 바람직하게 센스 가닥의 말단에 수식될 수 있다. It may be modified to one or more selected from the group consisting of 10 to 24 fatty acids. For example, siRNA can be modified at the 5 'end, or 3' end, or both ends of the sense and / or antisense strand, and preferably at the end of the sense strand.
상기 콜레스테를, 토코페롤 및 탄소수 10 내지 24개의 지방산에는  The cholester, tocopherol and fatty acids having 10 to 24 carbon atoms
콜레스테롤, 토코페롤 및 지방산의 각 유사체, 유도체, 및 대사체가 포함된다. Each analog, derivative, and metabolite of cholesterol, tocopherol and fatty acids is included.
상기 siRNA는, 표적 유전자와 동일한 세포에 존재하는 경우에 siRNA의 서열에 상보적인 mRNA의 분해를 매개함으로써, 표적 유전자의 발현을  The siRNA mediates the degradation of mRNA complementary to the siRNA sequence when present in the same cell as the target gene, thereby exerting the expression of the target gene.
감소시키거나 억제할 수 있는 이증가닥 RNA(duplex RNA),또는 단일가닥 RNA 내부에서 이중가닥의 형태를 띄는 단일가닥 RNA를 지칭한다. 이중가닥사이의 결합은, 뉴클레오티드 간의 수소 결합을 통해 이루어지며, 이중가닥 내부의 모든 뉴클레오티드가상보적으로 서로 결합해야 하는 것은 아니며, 상기 양 가닥은 분리되어 있거나 (separate) 분리되어 있지 않을 수 있다. 일 양태로서, 상기 siRNA의 길이는 약 15 내지 60 개의 (이중 가닥 RNA의 한쪽 뉴클레오티드의 갯수, 즉, 염기쌍의 갯수를 의미하며, 단일 가닥 RNA인 경우에는 단일 가닥 RNA 내부의 이증 가닥의 길이를 의미한다) 뉴클레오티드이며, 구체적으로는, 약 15 내지 30 개의 뉴클레오티드이고, 보다구체적으로는 약 19 내지 25 개의 It refers to duplex RNA, or single-stranded RNA that forms a double strand inside single-stranded RNA that can be reduced or inhibited. Bonding between the double strands is via hydrogen bonds between the nucleotides, and not all nucleotides within the double strands must complementarily bind to each other, and both strands may or may not be separated. In one embodiment, the length of the siRNA is about 15 to 60 (the number of single nucleotides of the double-stranded RNA, that is, the number of base pairs, and in the case of a single-stranded RNA, the length of the double stranded RNA within the single-stranded RNA). Nucleotides, specifically about 15 to 30 nucleotides, and more specifically about 19 to 25 nucleotides
뉴클레오티드인 siRNA를 포함한다. SiRNA which is a nucleotide.
일 양태로서, 이중가닥 siRNA는 3' 또는 5' 말단에 1-5 뉴클레오티드의 돌출부 (overhang)를 한쪽 말단에, 또는 양쪽 말단에 가질 수 있다. 또 다른 예에서는 양 말단이 돌출부를 갖지 않는 블런트 (bhmt) 형태일 수 있다.  In one aspect, the double-stranded siRNA may have an overhang of 1-5 nucleotides at either the 3 'or 5' end, at one end, or at both ends. In another example, both ends may be in the form of blunts without protrusions.
구체적으로는 미국특허공개 제 2002-0086356호, 미국특허 제 7,056,704에 개시된 siRNA일 수 있다 (상기 문헌은 본 명세서에 참조로서 포함된다). Specifically, it may be an siRNA disclosed in US Patent Publication No. 2002-0086356, US Patent No. 7,056,704, which is incorporated herein by reference.
또한, 상기 siRNA는 두 가닥의 길이가 동일한 대칭적인 구조를 갖거나, 한 가닥이 다른 가닥보다 짧은 비대칭적인 이증가닥 구조일 수 있다. 구체적으로, 19 내지 21 뉴클레오티드 (nucleotide, nt)의 안티센스 (antisense); 및 상기 안티센스에 상보적인 서열을 갖는 15 내지 19nt의 센스 (sense);로 구성되는 이중가닥 (double strand)의 siRNA분자 (small interfering RNA molecule)로서, 상기 siRNA는 안티센스의 5' 방향의 말단이 블런트 말단 (blunt end)이고 안티센스의 3' 말단에 1-5 In addition, the siRNA may have a symmetrical structure in which the two strands are the same length, or an asymmetric double stranded structure in which one strand is shorter than the other strand. Specifically, antisense of 19 to 21 nucleotides (nucleotides, nt); And the antisense A double stranded siRNA molecule consisting of 15 to 19nt of sense having a complementary sequence; a small interfering RNA molecule, wherein the siRNA has a blunt end at the 5 'end of the antisense. ) And 1-5 at the 3 'end of the antisense
뉴클레오타이드 돌출부 (overhang)를 갖는 비대칭 siRNA일 수 있다. 구체적으로 국제특허공개 제 09/078685호에 개시된 siRNA일 수 있다. It can be an asymmetric siRNA with nucleotide overhangs. Specifically, it may be siRNA disclosed in International Patent Publication No. 09/078685.
본 발명에서, 음이온성 약물은, 전체 조성물의 중량을 기준으로, 0.001 내지 10 중량0 /0, 구체적으로는 0.01 내지 5중량 %로 포함되는 것이 좋다. 상기 음이온성 약물의 함량이 0.001 중량0 /0 미만이면 약물에 비하여 사용되는 전달체의 양이 너무 많아서 전달체에 의한부작용이 있을 수 있고, 10 중량%을 초과하면, 미셀의 크기가 너무 커져 미샐의 안정성이 저하되고 필터 멸균시 손실율이 커질 우려가 있다. In the present invention, the anionic drugs, by weight of the total composition, from 0.001 to 10 parts by weight 0/0, specifically, is preferably contained in an amount of 0.01 to 5% by weight. The anionic When the content of the drug 0.001 0/0 is less than an amount of the delivery system to be used as compared to the drug may cause side effects due to too many delivery system, when it exceeds 10% by weight, the size of the micelle becomes too large stability misael There is a risk of lowering and increasing the loss rate during filter sterilization.
구체적인 일 양태에서, 상기 양이온성 화합물은, 음이온성 약물과 정전기적 상호작용에 의해 결합되어 복합체를 형성하고, 상기 복합체는 양친성 블록 공중합체의 미샐 구조 내부에 봉입된다. 따라서, 상기 양이온성 화합물은, 음이온성 약물과 정전기적 상호작용에 의해 복합체를 형성할 수 있는 모든 형태의 화합물을 포함하며, 예를 들어, 지질과 고분자 종류일 수 있다. 양이온성 지질은, Ν,Ν-디올레일 -Ν,Ν-디메틸암모늄클로라이드 (DODAC), Ν,Ν-디스테아릴 -Ν,Ν- 디메틸암모늄브로마이드 (DDAB), N-(l-(2,3-디올레오일옥시)프로필 -Ν,Ν,Ν- 트리메틸암모늄클로라이드 (DOTAP), N,N-디메틸 -(2,3- 디올레오일옥시 )프로필아민 (DODMA), Ν,Ν,Ν-트리메틸 -(2,3- 디올레오일옥시 )프로필아민 (DOTMA), 1 ,2-디아실 -3-트리메틸암모늄-프로판 (TAP), 1,2-디아실 -3-디메틸암모늄-프로판 (DAP), 3베타 -[Ν-(Ν',Ν',Ν'- 트리메틸아미노에탄)카바모일]콜레스테를 (TC-콜레스테를), 3베타 -[Ν-(Ν',Ν'- 디메틸아미노에탄)카바모일]콜레스테롤 (DC-콜레스테롤), 3베타 -[Ν-(Ν'- 모노메틸아미노에탄)카바모일]콜레스테를 (MC-콜레스테롤), 3베타- [Ν- In a specific embodiment, the cationic compound is bonded by an electrostatic interaction with the anionic drug to form a complex, the complex is encapsulated inside the micelle structure of the amphiphilic block copolymer. Thus, the cationic compound includes all types of compounds capable of forming a complex by electrostatic interaction with the anionic drug, and may be, for example, a lipid and a polymer type. Cationic lipids include Ν, Ν-dioleyl-Ν, Ν-dimethylammonium chloride (DODAC), Ν, Ν-distearyl-Ν, Ν-dimethylammonium bromide (DDAB), N- (l- (2, 3-dioleoyloxy) propyl-Ν, Ν, Ν-trimethylammonium chloride (DOTAP), N, N-dimethyl- (2,3-dioleoyloxy) propylamine (DODMA), Ν, Ν, Ν- Trimethyl- (2,3-dioleoyloxy) propylamine (DOTMA), 1,2-diacyl-3-trimethylammonium-propane (TAP), 1,2-diacyl-3-dimethylammonium-propane (DAP ), 3beta- [Ν- (Ν ', Ν', Ν'-trimethylaminoethane) carbamoyl] cholester (TC-cholester), 3beta- [Ν- (Ν ', Ν'-dimethyl) Aminoethane) carbamoyl] cholesterol (DC-cholesterol), 3beta- [Ν- (Ν'-monomethylaminoethane) carbamoyl] cholesterol (MC-cholesterol), 3beta- [Ν-
(아미노에탄)카바모일]콜레스테롤 (AC-콜레스테롤), 콜레스테릴옥시프로판 -1- 아민 (COPA), N-(N'-아미노에탄)카바모일프로파노익 토코페롤 (AC-토코페롤) 및 N- (N'_메틸아미노에탄)카바모일프로파노익 토코페롤 (MC-토코페롤)로 구성된 군으로부터 선택된 하나또는 둘 이상의 조합일 수 있다. 이러한 양이온성 지질을 사용하는 경우, 양이온성 지질로부터 유발되는 독성을 감소시키기 위하여 분자 내의 양이온 밀도가 높은 폴리양이온성 지질을 적게 사용하는 것이 바람직하고, 보다구체적으로는 분자당 수용액 상에서 양이온을 나타낼 수 있는 작용기가 하나일 수 있다. 이에 따라, 보다 바람직한 일 양태에서, 상기 양이온성 지질은 3베타 -[Ν-(Ν',Ν',Ν'-트리메틸아미노에탄)카바모일]콜레스테를 (TC-콜레스테를) : 3베타 [Ν-(Ν',Ν'- 디메틸아미노에탄)카바모일]콜레스테를 (DC-콜레스테를), 3베타 [N- (Ν'- 모노메틸아미노에탄)카바모일]콜레스테를 (MC-콜레스테를), 3베타 [Ν- (아미노에탄)카바모일]콜레스테를 (AC-콜레스테를), N-(l-(2,3-디을레오일옥시) 프로필 -Ν,Ν,Ν-트리메틸암모늄클로라이드 (DOTAP), Ν,Ν-디메틸 -(2,3- 디을레오일옥시)프로필아민 (DODMA), 및 Ν,Ν,Ν-트리메틸 -(2,3- 디올레오일옥시)프로필아민 (DOTMA)으로 이루어진 군에서 선택된 1종 이상의 것일 수 있다. 한편, 양이온성 고분자는 키토산 (chitosan), 글라이콜 키토산 (glycol chitosan), 프로타민 (protamine), 폴리라이신 (polylysine), 폴리아르기닌 (polyarginine), 폴리아미도아민 (PAMAM), 폴리에틸렌이민 (polyethylenimine), 덱스트란 (dextran), 히알루론산 (hyaluronic acid), 알부민 (albumin), 고분자 ¾리에틸렌이민 (PEI), 플리아민 및 폴리비닐아민 (PVAm)으로 구성된 군으로부터 선택되는 것을 특징으로 하며, 바람직하게는 고분자폴리에틸렌이민 (PEI), 폴리아민 및 폴리비닐아민 (PVA)으로 이루어진 군으로부터 선택되는 1종 이상의 것일 수 있다. (Aminoethane) carbamoyl] cholesterol (AC-cholesterol), cholesteryloxypropane-1-amine (COPA), N- (N'-aminoethane) carbamoylpropanoic tocopherol (AC-tocopherol) and N- It may be one or a combination of two or more selected from the group consisting of (N′_methylaminoethane) carbamoylpropanoic tocopherol (MC-tocopherol). Such cationic When using lipids, it is desirable to use less polycationic lipids with high cation density in the molecule in order to reduce the toxicity caused by cationic lipids, and more specifically, the functional groups capable of exhibiting cations in aqueous solution per molecule. It can be one. Thus, in a more preferred embodiment, the cationic lipid is 3beta- [Ν- (Ν ', Ν', Ν'-trimethylaminoethane) carbamoyl] cholester (TC-cholester) : 3beta [Ν- (Ν ', Ν'-dimethylaminoethane) carbamoyl] cholesterol (DC-cholesterol), 3beta [N- (Ν'-monomethylaminoethane) carbamoyl] cholester (MC -Cholester), 3-beta [Ν- (aminoethane) carbamoyl] cholesterol (AC-cholesterol), N- (l- (2,3-dileoyloxy) propyl-Ν, Ν, Ν-trimethylammonium chloride (DOTAP), Ν, Ν-dimethyl- (2,3-dileoyloxy) propylamine (DODMA), and Ν, Ν, Ν-trimethyl- (2,3-dioleoyloxy) The cationic polymer may be at least one selected from the group consisting of propylamine (DOTMA), while the cationic polymer may be chitosan, glycol chitosan, protamine, polylysine, or polyarginine. (polyarginine), polyamidoa (PAMAM), polyethylenimine, dextran, hyaluronic acid, albumin, albumin, polymer ¾riethyleneimine (PEI), polyamine and polyvinylamine (PVAm) It is characterized in that it is selected, preferably at least one selected from the group consisting of polymer polyethyleneimine (PEI), polyamine and polyvinylamine (PVA).
구체적인 일 양태에서, 양이온성 지질은 하기 화학식 7의 양이온성 지질일 수 있다:  In a specific embodiment, the cationic lipid may be a cationic lipid of Formula 7:
[화학식 7]  [Formula 7]
Figure imgf000011_0001
Figure imgf000011_0001
상기 식에서  In the above formula
n과 m은 각각 0 내지 12이되, 2≤n + m 12이며, a와 b는 각각 1 내지 6이며, R1과 R2는 각각 독립적으로 탄소수 1 1 내지 25개의 포화 및 불포화 탄화수소로 이루어진 군에서 선택된 것이다.  n and m are each 0 to 12, 2≤n + m 12, a and b are each 1 to 6, R1 and R2 are each independently selected from the group consisting of saturated and unsaturated hydrocarbons of 1 to 25 carbon atoms will be.
바람직하게는, n과 m은 독립적으로 1 내지 9이며, 2≤n+m≤ 10일 수 있다. 바람직하게는, a와 b가 2 내지 4일 수 있다. Preferably, n and m are independently 1 to 9, and may be 2≤n + m≤10. Preferably, a and b may be 2 to 4.
바람직하게는, R1과 R2는, 각각 독립적으로, 라우릴 (lauryl), 미리스틸 (myristyl), 팔미틸 (palmityl), 스테아릴 (stearyl), 아라키딜 (arachidyl), 베헨닐 (behenyl), 리그노세릴 (lignoceryl), 세로될 (cerotyl), 미리스트올레일 (myristoleyl), 팔미트올레일 (palmitoleyl), 사피에닐 (sapienyl), 올레일 (oleyl), 리놀레일 (linoleyl), 아라키도닐 (arachidonyl), 에이코사펜타에닐 (eicosapentaenyl), 에투실 (erucyl), 도코사핵사에닐 (docosahexaenyl), 및 세로틸 (cerotyl)로 이루어진 군에서 선택된 것일 수 있다.  Preferably, R 1 and R 2 are each independently lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, ligne Lignoceryl, cerotyl, myristoleyl, palmitoleyl, sapienyl, oleyl, linoleyl, arachidyl It may be selected from the group consisting of (arachidonyl), eicosapentaenyl, ethucyl, erucyl, docososahexaenyl, and serrotyl.
양이온성 지질의 구체적인 예는 1,6-디을레오일트리에틸렌테트라마이드, 1,8-디리놀레오일테트라에틸렌멘타마이드, 1,4- 디미리스틀레오일디에틸렌트리아마이드, 1,10-디스테아로일펜타에틸렌핵사마이드 및 1 ,10-디올레오일펜타에틸렌핵사마이드로 구성된 그룹으로부터 선택되는 하나 이상일 수 있다.  Specific examples of the cationic lipids include 1,6-dileoyltriethylenetetramide, 1,8-dilinoleylyltetraethylenementamide, 1,4-dimyristoleoyldiethylenetriamide, 1,10- At least one selected from the group consisting of distearoylpentaethylenenucleamide and 1,10-dioleoylpentaethylenenucleamide.
본 발명에서 사용되는 양이온성 화합물은, 전체 조성물의 중량을 기준으로 0.01 내지 50중량%, 구체적으로는으1 내지 10중량 %포함될 수 있다. 상기 양이온성 지질의 함량이 0.01% 미만이면 음이온성 약물과 복합체를 형성할 수 있는 충분한 양이 되지 못하고 , 50중량0 /0을 초과하면, 미셀의 크기가 너무 커져 미샐의 안정성이 저하되고 필터 멸균시 손실율이 커질 우려가 있다. Cationic compounds used in the present invention, based on the weight of the total composition may be included 0.01 to 50% by weight, specifically 1 to 10% by weight. The cationic content of the lipids not being a sufficient amount to form an anionic drug and the complex is less than 0.01%, 50 weight exceeds 0/0, the size of the micelle so increases the stability of the misael decreases filter sterilization The loss rate may increase.
상기 양이온성 화합물과 음이온성 약물은, 정전기적 상호작용을 통해 결합하여, 복합체를 형성한다. 구체적인 일 양태로서, 상기 음이온성 약물 (Ρ)과 양이온성 화합물 (Ν)의 전하량의 비율 (Ν/Ρ; 음이온성 약물의 음이온 전하에 대한 양이온성 화합물의 양이온 전하 비율)은, 0.1 내지 128이며, 구체적으로는으 5 내지 64, 더 구체적으로는 1 내지 32이고, 보다 더 구체적으로는 1 내지 24, 가장 구체적으로는 6 내지 24인 것이 좋다. 상기 비율 (Ν/Ρ)이 0.1 미만인 경우에는 충분한 양의 음이온성 약물을 포함하는 복합체를 형성하기 어렵기 때문에 , ι 이상이어야 충분한 양의 음이온 약물을 포함하는 복합체를 형성할 수 있어서 유리하다. 반면, 비율 (Ν/Ρ)이 128 초과시에는 독성을 유발할 우려가 있으므로, 128 이하로 하는 것이 좋다.  The cationic compound and the anionic drug combine through an electrostatic interaction to form a complex. As a specific aspect, the ratio of the amount of charge of the anionic drug (Ρ) and the cationic compound (Ν) (Ν / Ρ; cationic charge ratio of the cationic compound to the anionic charge of the anionic drug) is 0.1 to 128. Specifically, it is 5 to 64, more specifically 1 to 32, and even more specifically 1 to 24, most preferably 6 to 24. If the ratio (Ν / Ρ) is less than 0.1, since it is difficult to form a complex containing a sufficient amount of anionic drugs, it is advantageous to form a complex containing a sufficient amount of anionic drugs at a π or more. On the other hand, when the ratio (Ν / Ρ) exceeds 128, it may cause toxicity, so it is preferable to set it to 128 or less.
구체적인 일 양태에서, 상기 양친성 블톡 공중합체는, 친수성 Α블록 및 소수성 B 블록을 포함하는 A-B 형 블록 공중합체일 수 있다ᅳ 상기 A-B 형 블록 공증합체는, 수용액 상에서, 소수성 B 블록이 코어 (내벽)를 형성하고 친수성 A 블록이 쉘 (외벽)을 형성하는 코어-쉘 타입의 고분자 미셀을 형성한다. In a specific embodiment, the amphiphilic block copolymer is a hydrophilic A block and The AB type block copolymer may be a hydrophobic B block copolymer comprising a hydrophobic B block forming a core (inner wall) and a hydrophilic A block forming a shell (outer wall). Forms shell-type polymer micelles.
이와 관련하여, 상기 친수성 A 블록은 폴리알킬렌글리콜, 폴리비닐알콜, 폴리비닐피를리돈, 폴리아크릴아미드 및 그 유도체로 구성된 군으로부터 선택되는 하나 이상일 수 있다. 보다 구체적으로, 친수성 A 블록은  In this regard, the hydrophilic A block may be at least one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyridone, polyacrylamide and derivatives thereof. More specifically, the hydrophilic A block
모노메특시폴리에틸렌클리콜, 모노아세톡시폴리에틸렌글리콜, 폴리에틸렌글리콜, 폴리에틸렌과 프로필렌글리콜의 공중합체 및 폴리비닐피를리돈으로 구성된 군으로부터 선택되는 하나 이상일 수 있다. 상기 친수성 A블록은 It may be at least one selected from the group consisting of monomethoxy polyethylene glycol, monoacetoxy polyethylene glycol, polyethylene glycol, copolymers of polyethylene and propylene glycol and polyvinylpyridone. The hydrophilic A block
수평균분자량이 200 내지 50,000달톤, 보다구체적으로는 1,000 내지 20,000달톤, 보다 더 구체적으로는 1,000 내지 5,000달톤인 것일 수 있다. The number average molecular weight may be 200 to 50,000 Daltons, more specifically 1,000 to 20,000 Daltons, and more specifically 1,000 to 5,000 Daltons.
또한, 필요에 따라 친수성 A블록의 말단에 특정 조직이나 세포에 도달할 수 있는 작용기, 리간드, 또는 세포내 전달올 촉진할 수 있는 작용기를  In addition, if necessary, a functional group capable of reaching a specific tissue or cell, a ligand, or a functional group capable of promoting intracellular delivery at the end of the hydrophilic A block.
화학적으로 결합시켜 양친성 블록 공중합체와 폴리락트산염으로 형성된 고분자 미셀 전달체의 체내 분포를 조절하거나상기 미셀 전달체가 세포 내로 전달되는 효율을 높일 수 있다. 상기 작용기나 리간드는 단당류, 다당류, 비타민, 펩타이드, 단백질 및 세포 표면 수용체에 대한 항체로 이루어진 군에서 선택된 1종 이상일 수 있다. 보다구체적으로 > 상기 작용기나 리간드는 아니사마이드 (anisamide), 비타민 B9(엽산), 비타민 B12, 비타민 A, 갈락토오스, 락토오스, 만노오스, 히알루론산, RGD 펩타이드, NGR 펩타이드, 트랜스페린, 트랜스페린 수용체에 대한 항체 등으로 이루어진 군에서 선택된 1종 이상일 수 있다. By chemically binding, it is possible to control the distribution in the body of the polymer micelle transporter formed of the amphiphilic block copolymer and the polylactic acid salt or increase the efficiency of the micelle transporter to be delivered into the cell. The functional group or ligand may be one or more selected from the group consisting of monosaccharides, polysaccharides, vitamins, peptides, proteins and antibodies to cell surface receptors. More specifically > The functional group or ligand is an anamide (anisamide), vitamin B9 (folic acid), vitamin B12, vitamin A, galactose, lactose, mannose, hyaluronic acid, RGD peptide, NGR peptide, transferrin, transferrin receptor It may be one or more selected from the group consisting of.
상기 소수성 B 블록은, 생체적합성 생분해성 고분자로서, 일실시예에서, 폴리에스테르, 폴리언하이드라이드, 폴리아미노산, 폴리오르소에스테르 및 폴리포스파진으로 구성된 군으로부터 선택되는 하나 이상일 수 있다. 보다 구체적으로는, 상기 소수성 B 블특은 폴리락타이드, 폴리글리콜라이드,  The hydrophobic B block is a biocompatible biodegradable polymer, and in one embodiment, may be at least one selected from the group consisting of polyester, polyanhydride, polyamino acid, polyorthoester and polyphosphazine. More specifically, the hydrophobic B specific polylactide, polyglycolide,
폴리카프로락톤, 폴리디옥산 -2-온, 플리락타이드와 글리콜라이드의 공중합체, 폴리락타이드와폴리디옥산 -2-온의 공중합체, 폴리락타이드와폴리카프로락론의 공중합체 및 폴리글리콜라이드와폴리카프로락톤의 공중합체로 구성된 Polycaprolactone, polydioxan-2-one, copolymer of polylactide and glycolide, copolymer of polylactide and polydioxan-2-one, copolymer of polylactide and polycaprolacron and polyglycol Consisting of a copolymer of a lide and a polycaprolactone
군으로부터 선택되는 하나 이상일 수 있다. 또 다른 일실시예에서, 상기 소수성 B 블록은 수평균분자량이 50 내지 50,000달톤, 보다 구체적으로는 200 내지 It may be one or more selected from the group. In another embodiment, the hydrophobic B Blocks have a number average molecular weight of 50 to 50,000 Daltons, more specifically 200 to
20,000달톤, 보다 더 구체적으로는 1,000 내지 5,000달톤인 것일 수 있다. 또한, 소수성 블록의 소수성을 증가시켜 미셀의 안정성을 향상시키기 위하여 토코페롤, 콜레스테롤, 또는 탄소수 10 내지 24개의 지방산을 소수성 블록 말단의 It may be 20,000 daltons, more specifically 1,000 to 5,000 daltons. In addition, to increase the hydrophobicity of the hydrophobic block to improve the stability of the micelles, tocopherols, cholesterol, or fatty acids having 10 to 24 carbon atoms
히드록시기에 화학적으로 결합시킬 수 있다. It can be chemically bonded to a hydroxyl group.
상기 친수성 블록 (A)과 소수성 블록 (B)을 포함하는 양친성 블록  Amphiphilic block comprising said hydrophilic block (A) and hydrophobic block (B)
공중합체의 함량은, 조성물 전체 건조중량을 기준으로, 40 내지 99.98중량%이며, 구체적으로는 85 내지 99.8중량0 /0, 더 구체적으로는 90 내지 99.8중량0 /0인 것이 좋다. 상기 양친성 블록 공중합체의 함량이 40 중량0 /0 미만이면 미셀의 크기가 너무 커져 미셀의 안정성이 저하되고 필터 멸균시 손실율이 커질 우려가 있고, 함량이 99.98 증량%를 초과하면 함입할 수 있는 음이온성 약물의 함량이 너무 적어지게 된다. The content of the copolymer composition is, based on the total dry weight, 40 to 99.98% by weight, in particular from 85 to 99.8 parts by weight 0/0, more specifically, is preferably 90 to 99.8 wt. 0/0. If the amphiphilic block the amount of the copolymer 40 parts by weight 0 / under 0 reduced is too large the stability of the micelle size of the micelles and may cause greater the city filter sterilized loss rate, which when to embedded content exceeds 99.98 increase% The content of anionic drugs becomes too small.
또 다른 일실시예에서, 상기 양친성 블록 공중합체에 있어서, 친수성 블록 (A)과 소수성 블록 (B)의 /조성비는, 공증합체 중량을 기준으로, 친수성 블록 (A)이 40 내지 70중량0 Λ, 구체적으로는 50 내지 60중량 % 범위일 수 있다. 친수성 블특 (A)의 비율이 40중량0 /0 미만이면 고분자의 물에 대한 용해도가 낮아서 미샐을 형성하기 어렵기 때문에, 공중합체가 미셀올 형성하기에 층분한 물에 대한 용해도를 갖기 위하여 친수성 블록 (A)의 비율이 40중량 % 이상인 것이 좋은 한편, 70중량0 /。를 초과하면 친수성이 너무 높아 고분자 미셀의 안정성이 낮아져서 음이온성-약물 /양이온성 지질 복합체의 가용화 조성물로 사용하기 어려우므로, 미샐 안정성을 고려하여 친수성 블록 (A)의 비율이 70중량 % 이하인 것이 좋다. 구체적인 일 양태에서, 상기 양친성 블록 공중합체는 수용액 상에서 음이온성 약물과 양이온성 지질 복합체를 미셀 구조 내부에 봉입시키는데, 이 때 양친성 블록 공중합체의 중량 (b) 대비 음이온성 약물 및 양이온성 지질 복합체의 중량 (a) 비율 [a/b X 100; (음이온성 약물 중량+양이은성 지질 중량) /양친성 블록 공중합체 중량 X 100]은, 0.001 내지 100중량0 /0, 구체적으로는 0.01 내지 50중량0 /。, 보다구체적으로는 0.1 내지 10중량0 /0일 수 있다. 상기 중량 비율이, 0.001중량0 /。 미만인 경우에는 음이온성 약물 및 양이온성 지질 복합체의 함량이 지나치게 낮아져서 음이은성 약물이 효과적으로 작용할 수 있는 유효 함량을 층족시키기 어려우며, 반대로 100중량% 초과시에는 양친성 블록 공중힙 "체의 분자량과 음이온성 약물 및 지질 복합체의 양을 고려할 때 적절한 크기의 미셀 구조를 형성하지 못하기 때문이다. In another embodiment, in the amphiphilic block copolymer, the composition ratio of the hydrophilic block (A) and the hydrophobic block (B) is 40 to 70 weight 0 based on the weight of the copolymer . Λ, specifically, may range from 50 to 60% by weight. Since hydrophilic beulteuk (A) ratio is 40 parts by weight 0/0 if less difficult to form the misael solubility in water of the polymer is low, the copolymer is a hydrophilic block in order to have the solubility in a cheungbun water to form micelles ol because of the difficulty of use as a solubilizing composition of the drug / cationic lipid complex, - is (a) not less than the ratio of 40% by weight of the good on the other hand, 70 parts by weight when it is more than 0 / lowers the hydrophilicity is so high that the stability of the polymeric micelles anionic. In consideration of the microscopic stability, the ratio of the hydrophilic block (A) is preferably 70% by weight or less. In one specific embodiment, the amphiphilic block copolymer encapsulates the anionic drug and the cationic lipid complex inside the micellar structure in an aqueous solution, wherein the anionic drug and the cationic lipid are compared to the weight (b) of the amphiphilic block copolymer. Weight (a) ratio of the composite [a / b X 100; (Anionic drug weight + amount Lee Eun-sung lipid weight) / amphiphilic block copolymer weight X 100] is from 0.001 to 100 parts by weight 0/0, and specifically from 0.01 to 50 parts by weight 0 /., More specifically from 0.1 to 10 parts by weight 0/0 can be. When the weight ratio is less than 0.001 weight 0 /., The content of the anionic drug and the cationic lipid complex is too low to stratify the effective content that the negatively effective drug can act effectively. This is difficult, on the contrary, when it exceeds 100% by weight, it does not form a micelle structure of the appropriate size in consideration of the molecular weight of the amphiphilic block-heap sieve and the amount of the anionic drug and lipid complex.
본 발명에 따른 조성물 중 미셀 구조체는 폴리락트산염 (PLANa)를 포함하는 것을 특징으로 한다. 상기 폴리락트산염은 미샐의 코어 (내벽)에 분포하여 코어의 소수성을 강화시켜 미셀을 안정시킴과 동시에 체내에서 세망내피계 (RES)를 효과적으로 회피하는 역할을 한다. 즉, 폴리락트산염의 카르복실산 음이온이 폴리락트산보다 효과적으로 양이온성 복합체와 결합하여 고분자 미셀의 표면전위를 감소시켜 폴리락트산염을 포함하지 않는 고분자 미셀에 비해 표면전위의 양성 전하가 감소하여 세망내피계에 의해 덜 포획되고, 이로 인하여 목적하는 부위 (예컨대, 암세포, 염증세포 등)로의 전달 효율이 우수하다는 장점이 있다ᅳ  The micellar structure of the composition according to the invention is characterized in that it comprises polylactic acid salt (PLANa). The polylactic acid salt is distributed in the core (inner wall) of micelles to enhance the hydrophobicity of the core to stabilize the micelles and to effectively avoid the reticulum endothelial system (RES) in the body. That is, the carboxylic acid anion of polylactic acid is more effectively combined with the cationic complex than polylactic acid to reduce the surface potential of the polymer micelles, thereby reducing the positive charge of the surface potentials compared to the polymer micelles that do not contain polylactic acid. Is less trapped by and has the advantage of excellent delivery efficiency to the desired site (eg cancer cells, inflammatory cells, etc.).
상기 ᅵ양친성 블록 공증합체와 별도의 성분으로 미셀 내벽 성분으로 . 포함되는 폴리락트산염은 수평균분자량이 500 내지 50,000달톤, 구체적으로 1 ,000 내지 10,000달톤인 것이 좋다. 분자량이 500달톤 미만이면 소수성이 너무 낮아 미셀의 코어 (내벽)에 존재하기 어렵고, 분자량이 50,000달톤을 초과하면 고분자 미샐의 입자가 커지는 문제가 있다. Said ᅵ as a separate component from the amphiphilic block co-polymer as a micelle inner wall component . The polylactic acid salt included may have a number average molecular weight of 500 to 50,000 Daltons, specifically 1,000 to 10,000 Daltons. If the molecular weight is less than 500 Daltons, the hydrophobicity is too low to be present in the core (inner wall) of the micelle, and if the molecular weight exceeds 50,000 Daltons, there is a problem that the particles of the polymer micelle become large.
상기 폴리락트산염은 양친성 블록 고분자 100 증량부에 대하여 1 내지 200중량부, 구체적으로 10 내지 100증량부, 더 구체적으로 30 내지 60중량부로 사용될 수 있다. 폴리락트산염의 함량이 양친성 블록 고분자 100 증량부 대비 200증량부를 초과하면 미셀의 크기가 증가하여, 멸균막을사용한 여과가 어렵게 되고, 1중량부 미만이면 목적하는 효과를 충분히 얻을 수 없다.  The polylactic acid salt may be used in an amount of 1 to 200 parts by weight, specifically 10 to 100 parts by weight, more specifically 30 to 60 parts by weight, based on 100 parts by weight of the amphipathic block polymer. When the content of the polylactic acid salt exceeds 200 parts by weight relative to 100 parts by weight of the amphiphilic block polymer, the size of the micelle increases, making it difficult to filter using a sterile membrane, and when the content is less than 1 part by weight, the desired effect cannot be sufficiently obtained.
일 구체예에서, 음이온성 약물 1 증량부 대비 양친성 블록 공중합체를 10 내지 1 ,000 중량부, 폴리락트산염을 5 내지 500 중량부로 함유할 수 있다.  In one embodiment, it can contain 10 to 1,000 parts by weight of the amphipathic block copolymer relative to 1 part by weight of anionic drug, and 5 to 500 parts by weight of polylactic acid salt.
바람직하게는 양친성 블록 공중합체를 50 내지 800 중량부, 보다 바람직하게는 100 내지 500 중량부로 함유할 수 있다. 바람직하게는, 폴리락트산염올 10 내지 300 중량부, 보다 바람직하게는 50 내지 100 중량부로 함유할 수 있다. Preferably, the amphiphilic block copolymer may be contained in an amount of 50 to 800 parts by weight, more preferably 100 to 500 parts by weight. Preferably, it may contain 10 to 300 parts by weight of polylactic acid salt, more preferably 50 to 100 parts by weight.
한 구체예에서, 상기 폴리락트산염의 말단 중 카르복실산나트륨의 반대편의 말단은, 히드록시, 아세록시, 벤조일옥시, 데카노일옥시, 팔미토일옥시 및 탄소수 1 내지 2개의 알콕시로 이루어진 그룹 중에서 선택된 하나로 치환될 수 있다. In one embodiment, the terminal opposite of sodium carboxylate in the terminal of the polylactic acid salt is hydroxy, aceoxy, benzoyloxy, decanoyloxy, palmitoyloxy and It may be substituted with one selected from the group consisting of alkoxy having 1 to 2 carbon atoms.
바람직한 하나의 양태로서, 본 발명의 폴리락트산염은 하기 화학식 1 내지 6의 화합물로 이루어진 군으로부터 선택되는 하나 이상인 것을 특징으로 한다.  As a preferred embodiment, the polylactic acid salt of the present invention is characterized in that at least one selected from the group consisting of compounds of the formula (1) to (6).
[화학식 1]  [Formula 1]
RO-GHZ- [A]n- [B ] m-COOM RO-GHZ- [A] n- [B] m -COOM
상기 식 1에서 , Α는 -COO-CHZ-이고; Β는 -COO-CHY-, -COO- CH2CH2CH2CH2CH2-또는 -COO-CH2CH2OCH2이며; R은 수소원자, 또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸, 또는 에틸기이고; Z와 Y는 각각 수소원자, 또는 메틸 또는 페닐기이며; M은 Na, K, 또는 Li이고; n은 1 내지 30의 정수이며; m은 0 내지 20의 정수이다. In formula 1, A is -COO-CHZ-; Β is -COO-CHY-, -COO-CH 2 CH 2 CH 2 CH 2 CH 2 -or -COO-CH 2 CH 2 OCH 2 ; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl, or ethyl group; Z and Y are each a hydrogen atom or a methyl or phenyl group; M is Na, K, or Li; n is an integer from 1 to 30; m is an integer of 0-20.
[화학식 2]  [Formula 2]
R0-CHZ- [ C0O-CHX ] p- [ COO-CHY ' ]q-C00-CHZ-C00M R0-CHZ- [C0O-CHX] p- [COO-CHY '] q -C00-CHZ-C00M
상기 식 2에서, X는 메틸기이고; Y'는 수소원자 또는 페닐기이며; p는 0 내지 25의 정수이고, q는 0 내지 25의 정수이되, 단 p+q는 5 내지 25의 정수이고; R은 수소원자, 또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸 또는 에틸기이며; M은 Na, K, 또는 Li이고; Z는 수소 원자, 메틸 또는 페닐기이다.  In formula 2, X is a methyl group; Y 'is a hydrogen atom or a phenyl group; p is an integer from 0 to 25, q is an integer from 0 to 25, provided that p + q is an integer from 5 to 25; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; M is Na, K, or Li; Z is a hydrogen atom, a methyl or a phenyl group.
[화학식 3]  [Formula 3]
R0-PAD-C00— W-M'  R0-PAD-C00— W-M '
상기 식 3에서, W-M'는
Figure imgf000016_0001
C C00M이고; PAD는
In Formula 3, W-M 'is
Figure imgf000016_0001
C C00M; PAD
D,L-폴리락트산, D-폴리락트산, 폴리만델릭산, D,L-락트산과 글리콜산의 공중합체, D,L-락트산과 만델릭산의 공중합체, D,L-락트산과 카프로락톤의 공증합체 및 D,L- 락트산과 1 ,4-디옥산 -2-온의 공중합체로 구성된 그룹으로부터 선택되는 것이며; R은 수소원자, 또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸 또는 에틸기이고; M은 독립적으로 Na, K, 또는 Li이다. D, L-lactic acid, D-polylactic acid, polymandelic acid, copolymer of D, L-lactic acid and glycolic acid, copolymer of D, L-lactic acid and mandelic acid, D, L-lactic acid and caprolactone It is selected from the group consisting of a co-polymer of and a copolymer of D, L- lactic acid and 1,4-dioxane-2-one; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; M is independently Na, K, or Li.
[화학식 4]  [Formula 4]
S-0-PAD-C00-Q 상기 식 4에서, S는
Figure imgf000017_0001
고; L은 -NR,- 또는 -0-이며, 여기서
S-0-PAD-C00-Q In Equation 4, S is
Figure imgf000017_0001
High; L is -NR,-or -0-, where
Ri은 수소원자 또는 C1-10알킬이고; Q는 CH3, CH2CH3, CH2CH2CH3, CH2CH2CH2C¾, 또는 CH2C6H5이고; a는 0 내지 4의 정수이며; b는 1 내지 10의 정수이고; M은 Na, , 또는 Li이며; PAD는 D,L-폴리락트산, D-폴리락트산, 폴리만델릭산, D,L-락트산과 글리콜산의 공증합체, D,L-락트산과 만델릭산의 공증합체, D,L-락트산과 Ri is a hydrogen atom or C 1-10 alkyl; Q is CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 , CH 2 CH 2 CH 2 C¾, or CH 2 C 6 H 5 ; a is an integer from 0 to 4; b is an integer from 1 to 10; M is Na,, or Li; PAD is composed of D, L-polylactic acid, D-polylactic acid, polymandelic acid, co-polymer of D, L-lactic acid and glycolic acid, co-polymer of D, L-lactic acid and mandelic acid, D, L-lactic acid and
카프로락톤의 공중합체, 및 D,L-락트산과 1,4-디옥산 -2-온의 공증합체로 이루어진 군에서 선택되는 하나 이상이다. At least one selected from the group consisting of a copolymer of caprolactone and a co-polymer of D, L-lactic acid and 1,4-dioxan-2-one.
[화학식 5]  [Formula 5]
Figure imgf000017_0002
Figure imgf000017_0002
상기 식 5에서, R'는 -PAD-0-C(0)-CH2CH2-C(0)-OM이고, 여기서 PAD는In Formula 5, R 'is -PAD-0-C (0) -CH 2 CH 2 -C (0) -OM, wherein PAD is
D,L-폴리락트산, D—폴리락트산, 폴리만델릭산, D,L-락트산과 글리콜산의 공증합체, D,L-락트산과 만델릭산의 공증합체, D,L-락트산과 카프로락톤의 공중합체, D,L- 락트산과 1,4-디옥산 -2-온의 공중합체로 구성된 그룹으로부터 선택되는 것이고, M은 Na, K, 또는 Li이며; a는 1 내지 4의 정수이다. D, L-polylactic acid, D—polylactic acid, polymandelic acid, co-polymer of D, L-lactic acid and glycolic acid, co-polymer of D, L-lactic acid and mandelic acid, D, L-lactic acid and caprolactone Is selected from the group consisting of copolymers of D, L-lactic acid and copolymers of 1,4-dioxane-2-one, and M is Na, K, or Li; a is an integer of 1-4.
[화학식 6]  [Formula 6]
YO-[-C(O)-(CHX)a^-]te-C:(.0)-R-C(Q)- r-O-(CHX' )b-C(0)-]il-OZ YO-[-C (O)-(CHX) a ^-] te -C :(. 0) -RC (Q)-rO- (CHX ') b -C (0)-] il -OZ
상기 식 6에서, X 및 X'은 독립적으로 수소, 탄소수가 1~10인 알킬 또는 탄소수가 6~20인 아릴이고; Y 및 Z는 독립적으로 Na, K, 또는 Li이며; m 및 n은 독립적으로 0 내지 95의 정수이되, 5 < m + n < 100이고; a 및 b는 독립적으로 1 내지 6의 정수이며; R은 -(CH2)k-, 탄소수가 2~10인 2가 알케닐 (divalent alkenyl), 탄소수가 6~20인 2가 아릴 (divalent aryl) 또는 이들의 조합이고, 여기서 k는 0 내지 10의 정수이다. In Formula 6, X and X 'are independently hydrogen, alkyl having 1 to 10 carbon atoms or aryl having 6 to 20 carbon atoms; Y and Z are independently Na, K, or Li; m and n are independently integers from 0 to 95, with 5 <m + n <100; a and b are independently an integer from 1 to 6; R is-(CH 2 ) k- , divalent alkenyl having 2 to 10 carbon atoms, divalent aryl having 6 to 20 carbon atoms, or a combination thereof, wherein k is 0 to 10 Is an integer.
상기 폴리락트산염은 화학식 1 또는 화학식 2의 화합물인 것인  The polylactic acid salt is a compound of Formula 1 or Formula 2
바람직하다. desirable.
일 구체예에서, 본 발명의 조성물은 음이온성 약물의 세포 내 전달 효율을 증가시키기 위하여 전체 조성물의 증량을 기준으로 0.01 내지 50증량0 /0, 구체적으로는 0.1 내지 10증량 %의 융합성 지질을 추가로 포함할수 있다. In one embodiment, the compositions of the present invention improve the intracellular delivery efficiency of anionic drugs. To increase relative to the increase of the total composition, 0.01 to 50 increased 0/0, and to be more specific, it can further include a fusible lipid of from 0.1 to 10% increase.
상기 융합성 지질은 음이온성 약물과 양이온성 지질의 복합체에 흔합시, 소수성 상호작용으로 결합하여 음이온성 약물, 양이온성 지질 및 융합성 지질의 복합체를 형성하고, 상기 융합성 지질을 포함하는 복합체는 양친성 블록 공중합체의 미셀 구조 내부에 봉입된다ᅳ 일 구체예에서, 상기 융합성 지질은 인지질, 콜레스테를, 및 토코페를로 구성된 군으로부터 선택된 하나 또는 둘 이상의 조합일 수 있다.  When the fusion lipid is combined with the complex of the anionic drug and the cationic lipid, the hydrophobic interaction is combined to form a complex of the anionic drug, the cationic lipid and the fusion lipid, and the complex including the fusion lipid is Encapsulated Inside the Micellar Structure of the Amphiphilic Block Copolymer In one embodiment, the fusion lipid may be one or a combination of two or more selected from the group consisting of phospholipids, cholesterol, and tocopheres.
구체적으로, 상기 인지질은 포스파티딜에탄을아민 (phosphatidylethanolamin, PE), 포스파티딜콜린 (phosphatidylcholine, PC) 및 포스파티딘산 (phosphatidic acid)으로 이루어진 군에서 선택된 1종 이상일 수 있다. 상기  Specifically, the phospholipid may be at least one selected from the group consisting of phosphatidyl ethanolamine (PE), phosphatidylcholine (PC), and phosphatidic acid. remind
포스파티딜에탄올아민 (phosphatidylethanolamin, PE), Phosphatidylethanolamine (PE),
포스파티딜콜린 (phosphatidylcholine, PC) 및 포스파티딘산은 하나또는 2 개의 C10- 24 지방산과 결합된 형태일 수 있다. 상기 콜레스테롤 및 토코페를에는 ' 콜레스테를 및 토코페를의 각 유사체, 유도체, 및 대사체가포함된다. Phosphatidylcholine (PC) and phosphatidic acid can be in the form combined with one or two C10-24 fatty acids. The cholesterol and tocope include ' analogues, derivatives, and metabolites of cholesterol and tocope.
구체적으로는 융합성 지질은 디라우로일 포스파티딜에탄을아민 (dilauroyl phosphatidylethanolamine), 디미리스토일 포스파티딜에탄을아민 (dimyristoyl phosphatidylethanolamine), 디팔미토일 포스파티딜에탄올아민 (dipalmitoyl  Specifically, the fusion lipids include dilauuroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, and dipalmitoyl phosphatidylethanolamine.
phosphatidylethanolamine), 디스테아로일 포스파티딜에탄을아민 (distearoyl phosphatidylethanolamine), distearoyl phosphatidylethane
phosphatidylethanolamine), 디올레오일 포스파티딜에탄올아민 (dioleoyl phosphatidylethanolamine), dioleoyl phosphatidylethanolamine
phosphatidylethanolamine), 디리놀레오일 포스파티딜에탄을아민 (dilinoleoyl phosphatidylethanolamine), 1-팔미토일 -2-을레오일 포스파티딜에탄올아민 (1-palmitoyl- 2-oleoyl phosphatidylethanolamine), 1,2-디피타노일 -3-sn-포스파티딜에탄올아민 (I ,2- diphytanoyl-3-sn-phosphatidylethanolamine), 디라우로일 포스파티딜콜린 (dilauroyl phosphatidylcholine), 디 )리스토일 포스파티딜콜린 (dimyristoyl phosphatidylcholine), 디팔미토일 포스파티딜콜린 (dipalmitoyl phosphatidylcholine), 디스테아로일 phosphatidylethanolamine), dilinoleoyl phosphatidylethanolamine, 1-palmitoyl-2-leleoyl phosphatidylethanolamine (1-palmitoyl-2-oleoyl phosphatidylethanolamine), 1,2-dipitanoyl-3-sn -Phosphatidylethanolamine (I, 2 -diphytanoyl-3-sn-phosphatidylethanolamine), dilauuroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dipalmitoylline Work
포스파티딜콜린 (distearoyl phosphatidylcholine), 디을레오일 포스파티딜콜린 (dioleoyl phosphatidylcholine), 디리놀레오일 포스파티딜콜린 (dilinoleoyl phosphatidylcholine), 1 - 팔미토일 -2-올레오일 포스파티딜콜린 (l-pahnitoyl-2-oleoyl phosphatidylcholine), 1,2- 디피타노일 -3-sn-포스파티딜콜린 (l ,2-diphytanoyl-3-sn-phosphatidyk;holine), 디라우로일 포스파티딘산 (dilauroyl phosphatidic acid), 디미리스토일 포스파티딘산 (dimyristoyl phosphatidic acid), 디팔미토일 포스파티딘산 (dipalmitoyl phosphatidic acid), Distatiyl phosphatidylcholine, dioleoyl phosphatidylcholine, dilinoleoyl phosphatidylcholine, 1- palmitoyl-2-oleoyl phosphatidylcholine, l-pahnitoyl-2-oleylyl phospho Diphytanoyl-3-sn-phosphatidylcholine (l, 2-diphytanoyl-3-sn-phosphatidyk; holine), dilauuroyl phosphatidic acid, dimyristoyl phosphatidic acid , Dipalmitoyl phosphatidic acid,
디스테아로일 포스파티딘산 (distearoyl phosphatidic acid), 디올레오일 Distearoyl phosphatidic acid, dioleoyl
포스파티딘산 (dioleoyl phosphatidic acid), 디리놀레오일 포스파티딘산 (dilinoleoyl phosphatidic acid), 1-팔미토일 -2-올레오일 포스파티딘산 (l-pahnitoyl-2-oleoyl Dioleoyl phosphatidic acid, dilinoleoyl phosphatidic acid, 1-palmitoyl-2-oleoyl phosphatidic acid (l-pahnitoyl-2-oleoyl
phosphatidic acid), 1,2-디피타노일 -3-sn-포스파티딘산 (l,2-diphytanoyl-3-sn-phosphatidic acid), 콜레스테를 및 토코페를로 구성된 군으로부터 선택된 하나또는 둘 이상의 조합일 수 있다. phosphatidic acid), 1,2-dipitanoyl-3-sn-phosphatidic acid (l, 2-diphytanoyl-3-sn-phosphatidic acid), one or more selected from the group consisting of cholesterol and tocophere May be a combination.
바람직한 구체예에서, 상기 융합성 지질은 디올레오일  In a preferred embodiment, the fusion lipid is dioleoyl
포스파티딜에탄올아민 (dioleoyl phosphatidylethanolamine, DOPE), 、 디팔미토올레오일포스포콜린 (1 ,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, DPPC), 디올레오일포스포콜린 (l,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), Dioleoyl phosphatidylethanolamine (DOPE), dipalmitoleoylphosphocholine (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, DPPC), dioleoylphosphocholine (l, 2 -dioleoyl- sn-glycero-3-phosphocholine, DOPC),
디팔미토올레오일포스포에탄올아민 (l,2-dipalmitoleoyl-sn-glycero-3- phosphoethanolamine, DPPE) 등으로 이루어진 군에서 선택된 1종 이상일 수 있다ᅳ 구체적인 일 양태로서, 본 발명에 따른 양친성 블록 공중합체 및 Dipalmitooleoylphosphoethanolamine (l, 2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, DPPE) may be one or more selected from the group consisting of ᅳ amphipathic block according to one embodiment Copolymer and
폴리락트산염 미셀 구조체에 봉입된 음이온 약물-양이온성 화합물 복합체 함유 조성물은, 혈관, 근육, 피하, 경구, 뼈, 경피 또는 국소 조직 등의 투여 경로를 통하여 투여될 수 있고, 이러한 투여 경로에 적합하게, 다양한 경구 또는 비경구 투여 제제로 제형화될 수 있다. 상기 경구 투여 제제로는 정제, 캡슐, 분체 제제, 액제 등, 비경구 투여 제제로는 점안제, 주사제 등 다양한 제제를 예시할 수 있는데, 바람직한 일 양태로서,상기 조성물은 주사용 제제일 수 있다. 예를 들어, 본 발명에 따른 조성물을 동결건조하는 경우, 이를 주사용 증류수 , 0.9% Compositions containing anionic drug-cationic compound complexes encapsulated in a polylactic acid micellar construct can be administered via routes of administration such as blood vessels, muscles, subcutaneous, oral, bone, transdermal or topical tissue, and are suitable for such routes of administration. And may be formulated into a variety of oral or parenteral formulations. The oral dosage formulation may include various preparations, such as eye drops and injections, such as tablets, capsules, powder formulations, liquids, and the like, and parenteral dosage formulations. In one preferred embodiment, the composition may be an injection formulation. For example, when lyophilizing the composition according to the invention, it is distilled water for injection, 0.9%
생리식염수 및 5% 덱스트로스 수용액 등으로 재건하여 주사용 제제 형태로 제조할 수 있다. 본 발명은 또한, 상기 음이온성 약물을 함유하는 양친성 블록 공중합체 미셀을 포함하는 약제학적 조성물을 제조하는 방법을 제공한다. It may be prepared in the form of an injectable preparation by reconstitution with physiological saline, 5% dextrose aqueous solution and the like. The present invention also provides a method of preparing a pharmaceutical composition comprising an amphiphilic block copolymer micelle containing the anionic drug.
구체적인 일 양태로서, 상기 음이온성 약물, 양이은성 지질, 양친성 블톡 공중합체 및 폴리락트산염을 포함하는 음이온성 약물 전달용 조성물을 제조하는 방법은, In one specific embodiment, the anionic drug, bi-lipid lipid, amphiphilic blotting Method for producing a composition for anionic drug delivery comprising a copolymer and a polylactic acid salt,
(a) 음이온성 약물, 양이온성 화합물, 양친성 블록 공중합체 및  (a) anionic drugs, cationic compounds, amphiphilic block copolymers, and
폴리락트산염을 수흔화성 유기용매, 또는 수용액 및 유기용매의 혼합 용매에 용해시키는 공정; -Dissolving the polylactic acid salt in a water-soluble organic solvent or a mixed solvent of an aqueous solution and an organic solvent; -
(b) 상기 (a)공정에서 제조된 흔합물에서 유기용매층를 제거하는 공정;(b) removing the organic solvent layer from the mixture prepared in step (a);
(c) 상기 (b)공정의 유기용매가 제거된 흔합물에 수용액을 첨가하여 미셀화하는 공정을 포함한다. (c) adding the aqueous solution to the mixture from which the organic solvent of step (b) has been removed, and then micellizing.
보다 구체적으로, 상기 제조방법은,  More specifically, the manufacturing method,
상기 (a) 공정에서는, 수흔화성 유기용매, 또는 수용액 및 유기용매의 흔합 용매 내에서 음이온성 약물, 양이온성 화합물, 양친성 블록 공증합체 및 폴리락트산염을 흔합하여 복합체를 형성한다. 구체적으로는, 상기 수혼화성 유기용매는 아세톤, 에탄올, 메탄올 및 아세트산으로 이루어진 그룹 중에서 선택되는 하나 이상일 수 있고, 상기 혼합 용매의 유기용매는 아세트산 에틸, 아세토니트릴, 메틸렌클로라이드, 클로로포름 및 다이옥산으로 이루어진 그룹 중에서 선택되는 하나 이상일 수 있다. 상기 수용액은 증류수, 주사용수,또는 완층액일 수 있다. 상기 흔합 용매 중의 유기용매와 수용액의 흔합비는 특별한 한정은 없으며, 예컨대 부피 기준으로 1 :0.1 내지 50, 보다 구체적으로 1 :0.5 내지 10(유기용매 부피:수용액 부피)일 수 있으나, 이에 제한되는 것은 아니다.  In the step (a), an anionic drug, a cationic compound, an amphiphilic block copolymer and a polylactic acid salt are mixed to form a complex in a water-soluble organic solvent or a mixed solvent of an aqueous solution and an organic solvent. Specifically, the water-miscible organic solvent may be at least one selected from the group consisting of acetone, ethanol, methanol and acetic acid, and the organic solvent of the mixed solvent is a group consisting of ethyl acetate, acetonitrile, methylene chloride, chloroform and dioxane It may be one or more selected from. The aqueous solution may be distilled water, water for injection, or a complete solution. The mixing ratio of the organic solvent and the aqueous solution in the mixed solvent is not particularly limited, and may be, for example, 1: 0.1 to 50, more specifically 1: 0.5 to 10 (organic solvent volume: aqueous solution volume), but is not limited thereto. It is not.
상기 (b)공정에서, 상기 (a) 단계에서 제조된 흔합물에서 유기용매를 증발시킴으로써 제거한다.  In the step (b), the organic solvent is removed by evaporation from the mixture prepared in step (a).
상기 (c) 공정은 유기용매가 증발되고 남은 흔합물을 수용액 증에 용해시킴으로써, 음이온성 약물과 양이온성 화합물과의 복합체를 양친성 블록 공중합체와 폴리락트산염이 형성하는 미셀 구조체 내부에 봉입시킨다. 상기 수용액 및 그 사용량은 상기에서 기재한 바와 같다.  In the step (c), the remaining mixture after the organic solvent is evaporated is dissolved in an aqueous solution, thereby encapsulating the complex of the anionic drug and the cationic compound in the micelle structure formed by the amphiphilic block copolymer and the polylactic acid salt. . The aqueous solution and the amount of use thereof are as described above.
또 다론 하나의 추가적인 양태로서, 상기 (c) 공정 이후에, (d) 동결건조 보조제를 가하여 동결건조 하는 공정을 더 포함할 수 있다.  In addition, as an additional aspect, after the step (c), it may further comprise a step of lyophilizing by adding a (d) lyophilization aid.
또 다른 하나의 추가적인 양태로서, 상기 제조방법은, 상기 (d) 공정의 동결 건조 전에 (c) 공정에서 얻은 고분자 미샐 수용액을 멸균 필터로 멸균하는 공정을 추가로 포함할 수 있다. As yet another additional aspect, the preparation method, sterilizing the aqueous solution of the polymer micelle obtained in step (c) before lyophilization of the step (d) with a sterile filter The process may further include.
본 발명에서 사용되는 동결건조 보조제는 동결건조된 조성물이 케이크 형태를 유지할 수 있도록 하거나, 양친성 블록 공중합체 조성물을 동결건조 후, 재건 (reconstitution)하는 과정에서 빠른 시간 내에 균일하게 녹는 것을 도와주기 위해 첨가하는 것으로, 구체적으로, 락토스, 만니를, 솔비를 및 슈크로스로 이루어진 군으로부터 선택되는 하나 이상일 수 있다ᅳ 상기 동결건조 보조제의 함량은, 동결건조 조성물 전체 건조중량을 기준으로, 1 내지 90 중량0 /0, 더 구체적으로는 10 내지 60 중량0 /0 이다. The lyophilization aid used in the present invention may be used to help the lyophilized composition to maintain a cake form or to uniformly dissolve quickly in the process of reconstitution after lyophilizing the amphiphilic block copolymer composition. By adding, specifically, may be at least one selected from the group consisting of lactose, manny, sorbbi and sucrose 함량 The content of the lyophilization aid, based on the total dry weight of the lyophilized composition, 1 to 90 weight 0/0, and more specifically 10 to 60 wt. 0/0.
이러한 본 발명에 따른 제조방법을 통해 음이온성 약물과 양이온성 화합물 복합체가 양친성 블록 공중합체 및 폴리락트산염 미샐 구조체에 봉입된 형태의 조성물이 제조된다. 구체적으로 제조된 조성물 내의 미샐 입자는 혈증에서 안정하며, 그 크기는 10 내지 200 nm이며, 더욱 구체적으로는 10 내지 Through the preparation method according to the present invention, a composition in which an anionic drug and a cationic compound complex is encapsulated in an amphiphilic block copolymer and a polylactic acid salt structure is prepared. Specifically, the micelle particles in the prepared composition are stable in bloodemia, and have a size of 10 to 200 nm, more specifically 10 to
150 nm이다. 150 nm.
【발명의 실시를 위한 형태】  [Form for implementation of invention]
이하, 본 발명을 하기 실시예에 의거하여 보다자세하게 설명하나, 이들은 본 발명을 설명하기 위한 것일 뿐 이들에 의하여 본 발명의 범위가 어떤 식으로든 제한되는 것은 아니다.  Hereinafter, the present invention will be described in more detail with reference to the following examples, but these are merely for illustrating the present invention, and the scope of the present invention is not limited by them in any way.
[제조예 1] 1,6-디올레오일 트리에틸렌테트라마이드 (1,6-dioleoyl  Preparation Example 1 1,6-dioleoyl triethylenetetramide (1,6-dioleoyl
triethylenetetramide)의 합성 synthesis of triethylenetetramide)
WO2012-091523의 실시예 1에 기재된 공정에 따라 표제 화합물을 합성 및 확인하였다. . . The title compound was synthesized and identified according to the process described in Example 1 of WO2012-091523. . .
1제조예 2 및 3] 모노메록시폴리에틸렌글리콜-폴리락타이드 (mPEG-PLA) 블록 공증합체 (A-B)의 중합 1 Preparation Examples 2 and 3] Polymerization of Monomethoxypolyethyleneglycol-polylactide (mPEG-PLA) block co-polymer (A-B)
WO2012-091523의 제조예 1에 기재된 공정에 따라수평균 분자량은 5,000- According to the process described in Preparation Example 1 of WO2012-091523, the number average molecular weight was 5,000-
4,000 달톤의 mPEG-PLA를 합성하였다 [제조예 2]. 동일한 방법으로 모노메록시폴리에틸렌글리콜 (분자량 2,000 달톤 이하, NOF corporation)을 사용하여 수평균 분자량이 2,000-1 ,750 달톤인 mPEG-PLA블록 공증합체를 합성하였다 [제조예 3]. MPEG-PLA of 4,000 Daltons was synthesized [Preparation Example 2]. MPEG-PLA block having a number average molecular weight of 2,000-1,750 Daltons using monomethoxypolyethylene glycol (molecular weight 2,000 Daltons or less, NOF corporation) in the same manner Co-polymer was synthesized [Preparation Example 3].
[제조예 4 및 5] mPEG-PLA-토코페를의 중합 Production Examples 4 and 5 Polymerization of mPEG-PLA-Tocope
WO2012-091523의 제조예 2에 기재된 공정에 따라 mPEG-PLA-토코페롤 (수평균 분자량 5,000-4,000-530 달톤) 을 얻었다 [제조예 4].  MPEG-PLA-tocopherol (number average molecular weight 5,000-4,000-530 Daltons) was obtained according to the process described in Preparation Example 2 of WO2012-091523 [Preparation Example 4].
동일한 방법으로 수평균 분자량이 2,000-1,75-530 달론인 mPEG-PLA- 토코페롤을 얻었다 [제조예 5].  In the same manner, mPEG-PLA-tocopherol having a number average molecular weight of 2,000-1,75-530 Dalons was obtained [Production Example 5].
[제조예 6 및 7] 폴리락트산 (PLA) 합성 Preparation Examples 6 and 7 Synthesis of Polylactic Acid (PLA)
한국등록특허 제 1296326호의 제조예 8에 기재된 공정에 따라 PLA (수평균 분자량 1,700 달톤) 이며, 수율은 87% 였다 [제조예 6], According to the process described in Preparation Example 8 of Korean Patent No. 1296326, it was PLA (number average molecular weight 1, 7 Da Dalton), and the yield was 87% [Preparation Example 6],
동일한 방법으로 24 시간 반웅하여 수평균 분자량이 4,000 달톤인 폴리락트산을 중합하였다. 정제된 폴리락트산은 1H-NMR을 통해 확인하였고, 수율은 85% 였다 [제조예 7].  The reaction was carried out in the same manner for 24 hours to polymerize polylactic acid having a number average molecular weight of 4,000 Daltons. Purified polylactic acid was confirmed by 1 H-NMR, the yield was 85% [Preparation Example 7].
[제조예 8 및 9] D,L-폴리락틱산나트륨염 (PLANa) 합성 Preparation Examples 8 and 9 Synthesis of D, L-polylactic acid sodium salt (PLANa)
제조예 6에서 얻어진 폴리락틱산 (수평균 분자량 1 ,700) 100 g에  To 100 g of polylactic acid (number average molecular weight 1,700) obtained in Production Example 6
아세토니트릴 150 ml를 첨가하여 용해시켰다. 그런 다음, 탄산수소나트륨 수용액 (0.1 ^1 ) 150 1 를 서서히 첨가하고, 60에서 2 시간 동안 lOO rpm에서 교반하였다. 상온에서 염화나트륨 15 g을 첨가하고 교반하면서 용해시킨 후, 분별 깔대기를 이용하여 수용액층을 제거하였다. 150 ml of acetonitrile were added and dissolved. Then, aqueous sodium hydrogen carbonate solution (0.1 ^ 1) 150 1 was slowly added and stirred at 60 rpm for 60 to 2 hours. After adding 15 g of sodium chloride at room temperature and dissolving with stirring, the aqueous layer was removed using a separatory funnel.
남아있는 유기용매충에 증류수 100 ml와 염화나트륨 10 g을 첨가하여 교반하면서 용해시켰다. 다시 분별 깔대기를 이용하여 유기용매층만 회수한후, 얻어진 유기용매층을 80에서 진공조건으로 2시간 동안 분별 증류하여, 유기용매와 증류수를 완전히 제거하였다.  To the remaining organic solvent, 100 ml of distilled water and 10 g of sodium chloride were added and dissolved with stirring. After recovering only the organic solvent layer using a separatory funnel again, the organic solvent layer was fractionally distilled under vacuum conditions at 80 for 2 hours to completely remove the organic solvent and distilled water.
그런 다음, 무수 아세톤 150 ml를 첨가하여 고분자를 용해시키고, 용해되지 않은 침전물은 여과 분리하여 제거하였다. 80에서 진공조건으로 2 시간 동안 분별 증류하여, 아세톤을 제거하였다. 그 결과, 정제된 풀리락틱산 나트륨염 69 g을 얻었다. 정제된 폴리락틱산 나트륨염은 NMR올 통하여 확인되었다 [제조예 8]. 제조예 7에서 얻어진 폴리락트산 (수평균 분자량 4,000)을 이용하여 폴리락틱산 나트륨염을 얻었다 [제조예 9]. Then, 150 ml of anhydrous acetone was added to dissolve the polymer, and the undissolved precipitate was removed by filtration. Fractional distillation under vacuum at 80 for 2 hours to remove acetone. As a result, 69 g of purified sodium saline salt was obtained. Purified polylactic acid sodium salt was confirmed through NMRol [Preparation Example 8]. The polylactic acid sodium salt was obtained using the polylactic acid (number average molecular weight 4,000) obtained by the manufacture example 7. [Manufacture example 9].
[비교예 1] siRNA/1,6-디올레오일 트리에틸렌테트라미드 (dioTETA)/mPEG- PLA-토코페를 (2k-l 7k) 함유 고분자 미셀 제조 Comparative Example 1 Preparation of a polymer micelle containing siRNA / 1,6-dioleoyl triethylenetetramide (dioTETA) / mPEG-PLA-tocope (2k-1 7k)
1 ,6 dioTETA 1.89 mg ( /P 비율 18)을 클로로포름 94.63 μΐ에 녹이고 siRNA 100 을 증류수 80 μ1에 녹였다 . mPEG-PLA-토코페롤 (2k- 1.7k) 60 mg을 클로로포름 200 μΐ에 녹였다. 전체적으로 수층에 대한 유기층의 부피비율이 10배가 되도록 클로로포름 505.37 μΐ을 추가하였다. 1 ,6 dioTETA 와 mPEG-PLA-토코페를올 클로로포름에 녹인 용액 흔합물에 siRNA를 한 방을씩 가하면서 초음파분쇄기를 이용하여 유상액을 제조하였다. 유상액을 증류수 2320 μΐ에 더하면서 초음파 분쇄기를 이용하여 복합유상액을 제조하였다. 제조한 복합 유상액을 1 -구 등근 폴라스크에 넣고 증류농축장치 (rotary evaporator)에서 감압 증류함으로써 클로로포름을 선택적으로 제거하여 siRNA/1,6-디올레오일  1.89 mg of 1,6 dioTETA (/ P ratio 18) was dissolved in 94.63 μΐ of chloroform and siRNA 100 was dissolved in 80 μl of distilled water. 60 mg of mPEG-PLA-tocopherol (2k-1.7k) was dissolved in 200 μΐ of chloroform. 505.37 μΐ of chloroform was added so that the volume ratio of the organic layer to the aqueous layer was 10 times as a whole. An emulsion was prepared using an ultrasonic grinder while siRNA was added to the solution mixture of 1,6 dioTETA and mPEG-PLA-tocopherol in chloroform. The emulsion was added to 2320 μΐ of distilled water to prepare a composite emulsion using an ultrasonic mill. The prepared complex emulsion was placed in a 1-neck equilateral polar flask and distilled under reduced pressure in a rotary evaporator to selectively remove chloroform, thereby siRNA / 1,6-dioleoyl.
트리에틸렌테트라아마이드 (dioTETA)/mPEG-PLA-토코페롤 (2k-1.7k) 함유 고분자 미샐을 제조하였다 (표 1 참조).  Polymeric micelles containing triethylenetetraamide (dioTETA) / mPEG-PLA-tocopherol (2k-1.7k) were prepared (see Table 1).
【표 11  Table 11
Figure imgf000023_0001
Figure imgf000023_0001
(조성비에서 각성분의 단위는 siRNA 지질 N/P 비율, 고분자 mg이다. 이하 표에서 동일하게 적용된다.)  (The unit of each component in composition ratio is siRNA lipid N / P ratio, mg of polymer. The same applies in the following table.)
【비교예 2】 siRNA/1,6-디을레오일 트리에틸렌테트라미드 (dioTETA)/mPEG- PLA-토코페를 (2k-1.7k) /PLA (1.7k) 함유 고분자 미샐 제조 [Comparative Example 2] Preparation of polymer micelle containing siRNA / 1,6-dileoyl triethylenetetramide (dioTETA) / mPEG-PLA-tocope (2k-1.7k) / PLA (1.7k)
1 ,6 dioTETA 2.52 mg (N/P 비율 16)을 클로로포름 126.18 μΐ에 녹이고 siRNA 150 을 증류수 120 μΐ에 녹였다 . PLA-COOH (1.7k) 9 mg을 클로로포름 180 μΐ에 녹이고 mPEG-PLA-토코페를 (2k- 1.7k) 30 mg을 클로로포름 100 μΐ에 녹였다.  2.52 mg (16 N / P ratio) of 1,6 dioTETA was dissolved in 126.18 μΐ of chloroform and siRNA 150 was dissolved in 120 μΐ of distilled water. 9 mg PLA-COOH (1.7k) was dissolved in 180 μl of chloroform and mPEG-PLA-tocope (2k-1.7k) 30 mg was dissolved in 100 μl of chloroform.
전체적으로 수층에 대한유기층의 부피비율이 10배가 되도록 클로로포름 813.82 μΐ을 추가하였다. 상기 mPEG-PLA-토코페를 30 mg을 클로로포름에 녹인 용액 중 mPEG-PLA-토코페롤 6 1 (20중량%)에 해당하는 양인 20 μ 클로로포름 1564 μΐ을 첨가하여 1-구 등근 플라스크에 넣고 증류농축장치 (rotary evaporator)에서 감압 증류하여 용매를 제거하였다. Chloroform 813.82 so that the volume ratio of organic layer to water layer is 10 times as a whole. μΐ was added. 30 ml of the mPEG-PLA-tocophere in a solution of 30 mg of chloroform was added to 1564 μΐ of 20 μ chloroform, an amount corresponding to mPEG-PLA-tocopherol 6 1 (20% by weight), and placed in a 1-neck back flask. The solvent was removed by distillation under reduced pressure in a rotary evaporator.
상기 dioTETA 용액, PLA용액 그리고 mPEG-PLA-토코페롤 24 mg 용액올 섞어주고, siRNA수용액을 한 방울씩 가하면서 초음파 분쇄기를 이용하여 유상액을 제조하였다. 유상액올 mPEG-PLA-토코페롤 6 mg이 도포된 1 -구 등근 폴라스크에 넣고 증류농축장치 (rotary evaporator)에서 감압 증류하여 용매를 제거하였다. 플라스크에 증류수 3 ml을 가하고, 부드럽게 흔들어 녹임으로써 siRNA/dioTETA/mPEG-PLA-토코페롤 (2k-1.7k)/PLA함유 고분자 미셀올 제조하였다 (표 2 참조).  The dioTETA solution, PLA solution, and 24 mg solution of mPEG-PLA-tocopherol were mixed together, and an emulsion was prepared using an ultrasonic mill while adding dropwise siRNA solution. The solvent was removed by distillation under reduced pressure in a 1-neck equiaxed polar flask coated with 6 mg of oil emulsion mPEG-PLA-tocopherol and a rotary evaporator. 3 ml of distilled water was added to the flask, and the mixture was shaken gently to prepare siRNA / dioTETA / mPEG-PLA-tocopherol (2k-1.7k) / PLA-containing polymer micelle (see Table 2).
【표 2】  Table 2
Figure imgf000024_0001
Figure imgf000024_0001
(고분자 1 : mPEG-PLA-토코페롤, 고분자 2: PLA) 【실시예 1-2】 siRNA/1,6-디올레오일 트리에틸렌테트라미드  (Polymer 1: mPEG-PLA-Tocopherol, Polymer 2: PLA) [Example 1-2] siRNA / 1,6-dioleoyl triethylenetetramide
(dioTETA)/mPEG-PLA-토코페를 (2k-1.7k) /PLANa (1.7k) 함유 조성물 제조  Preparation of a composition containing (dioTETA) / mPEG-PLA-tocope (2k-1.7k) / PLANa (1.7k)
l,6 dioTETA 2.52 mg을 클로로포름 126.18 μΐ에 녹이고 siRNA 150 을 증류수 120 μΐ에 녹였다 . PLANa (1.7k) 9 mg을 클로로포름 180 μ 녹이고 mPEG- PLA-토코페를 (2k-1.7k) 30 mg을 클로로포름 100 μΐ에 녹였다. 전체적으로 수층에 대한 유기층의 부피비율이 10배가 되도록 클로로포름 813.82 μΐ을 추가하였다. 상기 mPEG-PLA-토코페를 30 mg을 클로로포름에 녹인 용액 중 mPEG-PLA- 토코페롤 6 1 (20중량%)에 해당하는 양인 20 μ1에 클로로포름 1564 μΐ을 첨가하여 1-구 등근 플라스크에 넣고 증류농축장치 (rotary evaporator)에서 감압 증류하여 용매를 제거하였다. 2.52 mg of l, 6 dioTETA was dissolved in 126.18 μΐ of chloroform and siRNA 150 was dissolved in 120 μΐ of distilled water. 9 mg of PLANa (1.7k) was dissolved in 180 μl of chloroform and 30 mg of mPEG-PLA-tocope (2k-1.7k) was dissolved in 100 μl of chloroform. In total, 813.82 μΐ of chloroform was added so that the volume ratio of the organic layer to the aqueous layer was 10 times. 30 ml of mPEG-PLA-tocophere was dissolved in chloroform and chloroform 1564 μΐ was added to 20 μ1, which is equivalent to mPEG-PLA-tocopherol 6 1 (20% by weight), and placed in a 1-neck back flask. The solvent was removed by distillation under reduced pressure in a rotary evaporator.
상기 dioTETA 용액, PLANa용액 그리고 mPEG-PLA-토코페를 24 mg 용액을 섞어주고, siRNA 수용액을 한 방울씩 가하면서 초음파 분쇄기를 이용하여 유상액을 제조하였다. 유상액을 mPEG-PLA-토코페롤 6 mg이 도포된 1-구 등근 플라스크에 넣고 증류농축장치 (rotary evaporator) 에서 감압 증류하여 용매를 제거하였다. 플라스크에 증류수 3 ml을 가하고, 부드럽게 흔들어 녹임으로써 siRNA/dioTETA7mPEG-PLA-토코페를 (2k-1.7k)/PLANa 함유 조성물을 제조하였다. 위와 유사한 방법으로 dioTETA와 PLANa 양을 달리하여 고분자 미셀 2을 제조하였다. 24 mg of the dioTETA solution, PLANa solution and mPEG-PLA-tocope The solution was mixed, and an emulsion was prepared by using an ultrasonic mill while dropwise adding an siRNA aqueous solution. The emulsion was placed in a 1-neck equipotential flask coated with 6 mg of mPEG-PLA-tocopherol and distilled under reduced pressure in a rotary evaporator to remove the solvent. 3 ml of distilled water was added to the flask, and the mixture was shaken gently to prepare a siRNA / dioTETA 7mPEG-PLA-tocofe (2k-1.7k) / PLANa-containing composition. In a similar manner to the above, polymer micelles 2 were prepared by varying amounts of dioTETA and PLANa.
【표 3】  Table 3
Figure imgf000025_0001
Figure imgf000025_0001
(고분자 1 : mPEG-PLA-토코페를, 고분자 2: PLANa)  (Polymer 1: mPEG-PLA-tocofe, polymer 2: PLANa)
[실시예 3-6] siRNA/dioTETA/mPEG-PLA-토코페를 (2k-1.7k) /PLANa (1.7k) 함유 조성물 제조 Example 3-6 Preparation of a Composition Containing siRNA / dioTETA / mPEG-PLA-Tocope (2k-1.7k) / PLANa (1.7k)
실시예 1과 동일한 방법으로 dioTETA또는 mPEG-PLA-토코페를 (2k-1.7k) 양을 달리 하여 siRNA/dioTETA/mPEG-PLA-토코페롤 (2k-l .7k)/PLANa (l.7k) 함유 조성물 3-6을 제조하였다. SiRNA / dioTETA / mPEG-PLA-tocopherol (2k-1 .7 k) / PLANa (l. 7 k) by varying the amount of dioTETA or mPEG-PLA-tocophere (2k-1.7k) in the same manner as in Example 1. ) Containing composition 3-6 was prepared.
실시예 3 내지 6에서 얻어진 조성물은 아래의 표 4에 정리하였다:  The compositions obtained in Examples 3-6 are summarized in Table 4 below:
【표 4】  Table 4
조성물 조성비 siRNA lipid 고분자 1 고분자 2 실시예 3 siR A/dioTETA/mPEG- 5-8-1-0.3 150 μ§ 1.26 mg 30 mg 9 mg Composition Composition siRNA lipid Polymer 1 Polymer 2 Example 3 siR A / dioTETA / mPEG-5-8-1-0.3 150 μ § 1.26 mg 30 mg 9 mg
PLA-토코페롤 (2k- 1.7k)/PLANa (1.7k)  PLA-tocopherol (2k- 1.7k) / PLANa (1.7k)
실시예 4 siRNA/dioTETA/mPEG- 5-24-1-0.3 150 μ8 3.79 mg 30 mg 9 mg PLA-토코페를 (2k- 1.7k)/PLANa (1.7k) Example 4 siRNA / dioTETA / mPEG- 5-24-1-0.3 150 μ 8 3.79 mg 30 mg 9 mg PLA-Tocope (2k- 1.7k) / PLANa (1.7k)
실시예 5 siRNA/dioTETA/mPEG- 5-16-0.5- 150 μ§ 2.52 mg 15 mg 9 mg Example 5 siRNA / dioTETA / mPEG-5-16-0.5- 150 μ § 2.52 mg 15 mg 9 mg
PLA-토코페롤 (2k- 0.3  PLA-tocopherol (2k- 0.3
1.7k)/PLANa (1.7k)  1.7k) / PLANa (1.7k)
실시예 6 siRNA/dioTETA/mPEG- 5-16-3-0.3 150 μ§ 2.52 mg 90 mg 9 mg Example 6 siRNA / dioTETA / mPEG-5-16-3-0.3 150 μ § 2.52 mg 90 mg 9 mg
PLA-토코페를 (2k- 1.7k)/PLANa (1.7k)  PLA-Tocope (2k- 1.7k) / PLANa (1.7k)
(고분자 1 : mPEG-PLA-토코퍼 1롤, 고분7 - 2: PLANa) (Polymer 1: mPEG-PLA- Sat copper roll 1, mounds 7 - 2: PLANa)
[실시예 7 및 8】 siRNA/dioTETA/mPEG-PLA-토코페를 (2k-1.7k) /PLANa (1.7k) 함유 조성물 제조 Examples 7 and 8 Preparation of a composition containing siRNA / dioTETA / mPEG-PLA-tocope (2k-1.7k) / PLANa (1.7k)
실시예 _ 1과동일한 방법을 사용하되, PLANa (1.7k)의 양을 달리하여 siRNA Example _ siRNA using the same method, but varying the amount of PLANa (1.7k)
/dioTETA/mPEG-PLA-토코페롤 (2k-1.7k)/PLANa (1.7k) 함유 고분자 미샐을 제조하였다 (표 5). Polymeric micelles containing / dioTETA / mPEG-PLA-tocopherol (2k-1.7k) / PLANa (1.7k) were prepared (Table 5).
【표 5】  Table 5
Figure imgf000026_0001
Figure imgf000026_0001
(고분자 l : mPEG-PLA-토코페롤, 고분ᄌ' 2: PLANa) (Polymer l: mPEG-PLA-tocopherol, polymer ' 2: PLANa)
[실험예 1】 siRNA/dioTETA/mPEG-PLA-토코페를 (2k-1.7k) /PLANa (1.7k) 미셀의 조성비 변화에 따른 입자크기 및 표면 전하 비교 Experimental Example 1 Comparison of particle size and surface charge of siRNA / dioTETA / mPEG-PLA-tocope with varying composition ratio of (2k-1.7k) / PLANa (1.7k) micelles
siR A/dioTETA 의 비율 (N/P 비율), 양친성 블록 공증합체 (2k- 1.7k) 양 및 PLANa (1.7k) 양에 따른 나노 입자 형성 여부를 확인하기 위하여 미셀의 크기와 표면 전하를 확인하였다. 동적 광산란 (DLS; dynamic light scattering) 방법을 이용하여 입자의 크기를 측정하였다. 구체적으로, He-Ne 레이져를 광원으로 사용하였으며, MALVERN사의 Zetasizer Nano ZS90 기기를 매뉴얼에 따라 작동하였다. Determine the size and surface charge of micelles to determine whether nanoparticles were formed according to the ratio of siR A / dioTETA (N / P ratio), amphiphilic block copolymer (2k-1.7k) and PLANa (1.7k). It was. Dynamic light scattering (DLS) Particle size was measured. Specifically, He-Ne laser was used as a light source, and MALVERN's Zetasizer Nano ZS90 instrument was operated according to the manual.
N/P 비율에 따른 실시예 1 내지 4 미셀의 크기와 표면 전하는 아래의 표 6에 나타내었다:  The size and surface charge of the micelles 1 to 4 according to the N / P ratio are shown in Table 6 below:
【표 6]  [Table 6]
Figure imgf000027_0001
양친성 블특 공중합체 (2k-1.7k) 양을 달리한 실시예 1, 5 및 6의 크기와 표면 전하는 아래의 표 7에 나타내었다.
Figure imgf000027_0001
The magnitudes and surface charges of Examples 1, 5 and 6 with varying amounts of amphiphilic unspecific copolymers (2k-1.7k) are shown in Table 7 below.
【표 7】  Table 7
mPEG-PLA-토코페롤 (2k-1.7k) 양의 변화 Changes in the amount of mPEG-PLA-tocopherol (2k-1.7k)
Figure imgf000027_0002
Figure imgf000027_0002
PLANa (1.7k) 양을 달리한 실시예 1, 7 및 8의 크기와 표면 전하는 아래의 표 8에 나타내었다.  The sizes and surface charges of Examples 1, 7 and 8 with varying amounts of PLANa (1.7k) are shown in Table 8 below.
【표 8] PLA-COONa (1.7k) 양의 변화 [Table 8] Change in PLA-COONa (1.7k) Amount
Figure imgf000028_0001
Figure imgf000028_0001
[실시예 9-11] siRNA/dioTETA/mPEG-PLA-토코페를 PLANa함유 조성물 제조 Example 9-11 Preparation of PLANa-containing Composition of siRNA / dioTETA / mPEG-PLA-Tocope
실시예 1과 동일한 방법을 사용하되, mPEG-PLA-토코폐를 (2k-1.7k) 대신 mPEG-PLA-토코페를 (5k-4k) 를사용하거나 PLANa (1.7k) 대신 PLANa (4k) 를 사용하여 고분자 미샐 9, 10, 1 1을 제조하였다.  Use the same method as in Example 1, except mPEG-PLA-tocophere (5k-4k) instead of (2k-1.7k) or PLANa (4k) instead of PLANa (1.7k) Polymeric micelles 9, 10, and 1 1 were prepared.
【표 9】Table 9
Figure imgf000028_0002
Figure imgf000028_0002
(고분자 1 : mPEG-PLA-토코페를, 고분자 2: PLANa)  (Polymer 1: mPEG-PLA-Tocope, Polymer 2: PLANa)
【실험예 2] siRNA/dioTETA/mPEG-PLA-토코페를/ PLANa 미셀의 조성물과 조성비 변화에 따른 입자크기 및 표면 전하 비교 양친성 블록 공중합체 분자량과 PLA-COONa분자량에 따른 나노 입자 형성 여부를 확인하기 위하여 실시예 1, 9, 10 및 11 미셀의 크기와 표면 전하를 실험예 1과 동일한 방법으로 확인하였다. 얻어진 결과를 아래의 표 10에 Experimental Example 2 Comparison of SiRNA / dioTETA / mPEG-PLA-Tocope / PLANa micelles with Particle Size and Surface Charge According to Composition Ratio In order to confirm the formation of nanoparticles according to the amphiphilic block copolymer molecular weight and PLA-COONa molecular weight, the size and surface charge of Example 1, 9, 10 and 11 micelles were confirmed in the same manner as in Experimental Example 1. The results obtained are shown in Table 10 below.
나타내었다:  Indicated:
【표 10】  Table 10
Figure imgf000029_0001
Figure imgf000029_0001
【실시예 121 siRNA-콜레스테를 /dioTETA/mPEG-PLA-토코페를 (2k- 1.7k) PLANa (1.7k) 함유 조성물 제조 Example 1 2 1 Preparation of a composition containing 1 siRNA-cholester / dioTETA / mPEG-PLA-tocope ( 2 k-1.7k) PLANa (1.7k)
siRNA-콜레스테를을 사용하여 실시예 1와 동일한 과정을 거쳐 siRNA- 콜레스테롤 / dioTETA /mPEG-PLA-토코페롤 (2k-1.7k)/PLANa (1.7k) 함유 조성물을 제조하였다. 이 흔합물을 회전증류농축장치에서 감압 증류하여 용매를 제거하였다. 플라스크에 증류수 3 ml을 가하고, 부드럽게 흔들어 녹임으로써 조성물을  siRNA-cholester was used to prepare a composition containing siRNA-cholesterol / dioTETA / mPEG-PLA-tocopherol (2k-1.7k) / PLANa (1.7k) using the same procedure as in Example 1. The mixture was distilled under reduced pressure in a rotary distillation concentrator to remove the solvent. Add 3 ml of distilled water to the flask, shake gently to dissolve the composition
제조하였다. ' [실시예 13] siRNA-PEG/dioTETA/mPEG-PLA-토코페롤 (2k-1.7k) PLANaPrepared. "[Example 13] siRNA-PEG / dioTETA / mPEG-PLA- tocopherol (2k-1.7k) PLANa
(1.7k) 함유 조성물 제조 (1.7k) Containing Composition Preparation
siRNA-PEG를 사용하여 실시예 1와동일한 과정을 거쳐 siRNA- PEG/dioTETA/mPEG-PLA-토코페롤 (2k-1.7k)/PLANa (1.7k) 함유 조성물을  A siRNA-PEG / dioTETA / mPEG-PLA-tocopherol (2k-1.7k) / PLANa (1.7k) containing composition was subjected to the same procedure as in Example 1 using siRNA-PEG.
제조하였다. 이 혼합물을 회전증류농축장치에서 감압 증류하여 용매를 제거하였다. 플라스크에 증류수 3 ml을 가하고, 부드럽게 흔들어 녹임으로써 조성물을 Prepared. The mixture was distilled under reduced pressure in a rotary distillation concentrator to remove the solvent. Add 3 ml of distilled water to the flask and shake gently to dissolve the composition.
제조하였다. 실시예 12 내지 13에서 얻어진 조성물을 아래의 표 11에 정리하였다:Prepared. The compositions obtained in Examples 12-13 are summarized in Table 11 below:
【표 11】 Table 11
Figure imgf000030_0001
Figure imgf000030_0001
(고분자 1 : mPEG-PLA-토코페를, 고분자 2: PLANa) [실시예 14] siRNA/bPEI/mPEG-PLA-토코페를 (2k-1.7k)/PLANa (1.7k) 함유한조성물 제조  (Polymer 1: mPEG-PLA-Tocope, Polymer 2: PLANa) [Example 14] Preparation of a composition containing siRNA / bPEI / mPEG-PLA-Tocope (2k-1.7k) / PLANa (1.7k)
bPEI 0.3 mg을 증류수 15 μΐ에 녹이고 siRNA 150 μβ을 증류수 120 μΐ에 녹였다. 상기 bPEI수용액과 siRNA수용액을 HBS 완층수용액 (lO mM HEPES, 1 mM NaCl) 105 μΐ에 흔합한다. PLANa (1.7k) 9 mg을 클로로포름 180 μΐ에 녹이고 mPEG-PLA-토코페롤 (2k-1.7k) 30 mg을 클로로포름 100 μΐ에 녹였다. 전체적으로 수층에 대한유기층의 부피비율이 10배가 되도록 클로로포름 2140 μΐ을 추가하였다. 상기 tnPEG-PLA-토코페를 30 mg을 클로로포름에 녹인 용액 중 mPEG-PLA-토코페를 6 1 (20중량%)에 해당하는 양인 20 μΐ에 클로로포름 1564 μΐ을 첨가하여 1-구 등근 플라스크에 넣고 증류농축장치 (rotary evaporator)에서 감압 증류하여 용매를 제거하였다. bPEI Dissolve 0.3 mg in distilled water was dissolved 15 μΐ siRNA 150 μ β in distilled water to 120 μΐ. The bPEI aqueous solution and the siRNA aqueous solution are mixed with 105 µl of HBS complete aqueous solution (10 mM HEPES, 1 mM NaCl). 9 mg PLANa (1.7k) was dissolved in 180 μl of chloroform and 30 mg of mPEG-PLA-tocopherol (2k-1.7k) was dissolved in 100 μl of chloroform. 2140 μΐ of chloroform was added so that the volume ratio of the organic layer to the aqueous layer was 10 times as a whole. In a solution of 30 mg of tnPEG-PLA-tocope dissolved in chloroform, mPEG-PLA-tocope was added to 20 μΐ, which is equivalent to 6 1 (20% by weight), and chloroform 1564 μΐ was added to a 1-neck back flask. The solvent was removed by distillation under reduced pressure in a rotary evaporator.
상기 PLANa용액 그리고 mPEG-PLA-토코페롤 24 mg 용액을 섞어주고, bPEI와 siRNA가 흔합된 HBS 완층수용액을 한 방울씩 가하면서 초음파 분쇄기를 이용하여 유상액을 제조하였다. 유상액을 mPEG-PLA-토코페롤 6 mg이 도포된 1- 구 등근 플라스크에 넣고 증류농축장치 (rotary evaporator)에서 감압 증류하여 용매를 제거하였다. 플라스크에 증류수 6 ml올 가하고, 부드럽게 흔들어 녹임으로써 siRNA/bPEI/mPEG-PLA-토코페롤 (2k-1.7k)/PLANa (1.7k) 함유 조성물을 제조하였다 (표 12). 【표 12] The PLANa solution was mixed with a 24 mg solution of mPEG-PLA-tocopherol, and an emulsion was prepared by using an ultrasonic mill while adding dropwise HBS complete aqueous solution mixed with bPEI and siRNA. The emulsion was placed in a 1-neck equilateral flask coated with 6 mg of mPEG-PLA-tocopherol and distilled under reduced pressure in a rotary evaporator to remove the solvent. 6 ml of distilled water was added to the flask, and the mixture was shaken gently to prepare a siRNA / bPEI / mPEG-PLA-tocopherol (2k-1.7k) / PLANa (1.7k) -containing composition (Table 12). Table 12
Figure imgf000031_0001
Figure imgf000031_0001
(고분자 l : mPEG-PLA-토코페롤, 고분자 2: PLANa)  (Polymer l: mPEG-PLA-tocopherol, polymer 2: PLANa)
[실시예 15 및 16] siRNA/양이온성 지질 /mPEG-PLA-토코페를 (2k-1.7k) /PLANa (1.7k) 함유 조성물 제조 Examples 15 and 16 Preparation of a composition containing siRNA / cationic lipids / mPEG-PLA-tocope (2k-1.7k) / PLANa (1.7k)
실시예 1과 동일한 방법으로 양이온성 지질을 달리하여 siRNA/ 1,10- 디올레오일 펜타에틸렌핵사마이드 (dioPEHA)/mPEG-PLA-토코페를 (2k-1.7k)/PLANa (1.7k) 함유 조성물 9와 siRNA/ 1,8-디리놀레오일 테트라에틸렌펜타마이드  Different cationic lipids were prepared in the same manner as in Example 1 to contain siRNA / 1,10-dioleoyl pentaethylenenucleamide (dioPEHA) / mPEG-PLA-tocofe (2k-1.7k) / PLANa (1.7k) SiRNA / 1,8-dilinoleoyl tetraethylenepentamide with composition 9
(dilTEPA)/mPEG-PLA-토코페를 (2k-1.7k)/PLANa (L7k) 함유 조성물 10을 (dilTEPA) / mPEG-PLA-tocophere (2k-1.7k) / PLANa (L7k) containing composition 10
제조하였다 (표 13).  Prepared (Table 13).
【표 13】  Table 13
Figure imgf000031_0002
Figure imgf000031_0002
(고분자 l : mPEG-PLA-토코페롤, 고분자 2: PLANa) [실시예 17 및 18] siRNA-PEG/dioTETA/mPEG-PLA-토코페롤 (2k- (Polymer l: mPEG-PLA-tocopherol, polymer 2: PLANa) [Examples 17 and 18] siRNA-PEG / dioTETA / mPEG-PLA-tocopherol (2k-
1.7k)/PLANa (1.7k) DOPE(dioleoylphosphatidylethanolamine) 함유 조성물 제조 Preparation of 1.7k) / PLANa (1.7k) DOPE (dioleoylphosphatidylethanolamine) containing composition
siRNA-PEG를 사용하여 실시예 1와 동일한 과정을 거쳐 siRNA- PEG/dioTETA/mPEG-PLA-토코페롤 (2k-1.7k)/PLANa (1.7k)/DOPE 함유 조성물을 제조하였다. DOPE 양은 dioTETA 양의 1배, 4배로 각각 넣어주었다. 이 혼합물을 회전증류농축장치에서 감압 증류하여 용매를 제거하였다. 폴라스크에 증류수 3 ml을 가하고, 부드럽게 흔들어 녹임으로써 조성물을 제조하였다 (표 14). siRNA-PEG was used to prepare siRNA-PEG / dioTETA / mPEG-PLA-tocopherol (2k-1.7k) / PLANa (1.7k) / DOPE containing composition. The amount of DOPE was added 1 and 4 times the amount of dioTETA, respectively. This mixture The solvent was removed by distillation under reduced pressure in a rotary distillation thickener. 3 ml of distilled water was added to the polar flask, and the composition was prepared by gently shaking to dissolve it (Table 14).
【표 14]  Table 14
Figure imgf000032_0001
Figure imgf000032_0001
(고분자 l : mPEG-PLA-토코페롤, 고분자 2: PLANa)  (Polymer l: mPEG-PLA-tocopherol, polymer 2: PLANa)
【실험예 3] siRNA/양이온성 물질 /양친성 블록 공중합체 PLANa(/DOPE) 미셀의 크기 및 표면 전하비교 Experimental Example 3 Comparison of Size and Surface Charge of siRNA / Cation Material / Amphiphilic Block Copolymer PLANa (/ DOPE) Micelles
siRNA 종류, 양이온성 물질, PLANa 및 DOPE 여부에 따른 나노 입자 형성 여부를 확인하기 위하여 미샐의 크기와표면 전하를 실험예 1과 동일한 방법으로 확인하였다. 얻어진 결과를 아래의 표 15에 나타내었다:  In order to confirm the formation of nanoparticles according to siRNA type, cationic material, PLANa and DOPE, the size and surface charge of micelles were confirmed in the same manner as in Experimental Example 1. The results obtained are shown in Table 15 below:
【표 15】  Table 15
조성물 종류 조성비 입자크기 표면 전하 비교예 1 siR A/dioTETA/mPEG-PLA- 5-18-3 25.32 nm 12.37 mV 토코페를 (2k- 1.7k)  Composition Type Composition Ratio Particle Size Surface Charge Comparative Example 1 siR A / dioTETA / mPEG-PLA-5-18-3 25.32 nm 12.37 mV Tocope (2k-1.7k)
비교예 2 siR A/dioTETA/mPEG-PLA- 5-16-1-0.3 25.71 nm 5.48 mV 토코페를 (2k-1.7k)/PLA (1.7k) Comparative Example 2 siR A / dioTETA / mPEG-PLA-5-16-1-0.3 25.71 nm 5.48 mV tocope (2k-1.7k) / PLA (1.7k)
실시예 1 siR A/dioTETA/mPEG-PLA- 5-16-1-0.3 28.07 nm -1.29 mV 토코페롤 (2k-1.7k)/PLANa (1.7k) Example 1 siR A / dioTETA / mPEG-PLA-5-16-1-0.3 28.07 nm -1.29 mV tocopherol (2k-1.7k) / PLANa (1.7k)
실시예 12 siRNA-콜레스테를 /dioTETA/mPEG- 5-16-1-0.3 27.73 nm -4.38 mV Example 12 siRNA-cholester /dioTETA/mPEG-5-16-1-0.3 27.73 nm -4.38 mV
PLA-토코페를 (2k-1.7k)/PLANa PLA-Tocope (2k-1.7k) / PLANa
Figure imgf000033_0001
Figure imgf000033_0001
[실험예 4] siRNA/양이온성 물질 /양친성 블특 공중합체/ PLANa 미셀의 혈중 농도 분석 Experimental Example 4 Analysis of Blood Concentration of siRNA / Cationous / Amphiphilic Specific Copolymer / PLANa Micelle
실험예 1에서 제조된 제형을 동물에 투여하고 투여 0.5시간, 6시간후에 채혈을 하여 RT (Reverse Transcription)과 qRT-PCR (quantitative Reverse Transcription- Polymerase Chain Reaction) 방법을 이용해 아래와 같은 방법으로 미셀의 혈증 농도를 분석한다.  0.5 mg, 6 hours after the administration of the formulation prepared in Experiment 1 to the animal, blood was collected by RT (Reverse Transcription) and qRT-PCR (quantitative Reverse Transcription- Polymerase Chain Reaction) method Analyze the concentration.
제형을 lmg/kg로 Balb/c 마우스에 정맥주사하여 으5시간, 6시간후에 각각 채혈한다. 혈액을 I3000 rpm, 4에서 15분간 원심분리기를 돌려 상충 부분만 새 튜브에 모으고, 스탠다드용 제형의 농도는 4 μΜ에서 0.00256 μΜ까지 총 11개의 농도로 PBS에 희석하여 준비한다. PCR용 96웰 플레이트에 스탠다드용으로 희석한 제형을 Ι μΐ 넣고 9 μ1의 Balb/c마우스 혈청과 90 μΐ의 0.25% triton X-100을 넣어준다. 실험군 혈액 샘플 ΙΟ μΙ에 90 μ1의 0.25% Triton X-100 를 넣어준 후 전달체를 풀어주는 전처리 단계를 거친다. 제형이 풀어짐으로써 노출된 siRNA를 역전사 (RT) 단계를 거쳐 cDNA로 합성하고 합성된 cDNA를 이용하여 qRT-PCR (Bio-Rad CFX96 Real-Time System) 을 수행하였다. 분석은 Bio-Rad CFX Manager 프로그램을 이용하여 분석하였다.  The formulations were injected intravenously into Balb / c mice at lmg / kg and blood was drawn 5 hours and 6 hours later. The blood is collected in a new tube by centrifugation at I3000 rpm for 4 minutes at 4 minutes, and the standard formulation is prepared by diluting in PBS at a total of 11 concentrations ranging from 4 μΜ to 0.00256 μΜ. Ι μΐ of the diluted standard formulation was added to the 96 well plate for PCR, and 9 μl of Balb / c mouse serum and 90 μΐ of 0.25% triton X-100 were added. 90 μl of 0.25% Triton X-100 was added to the blood sample ΙΟ μΙ of the experimental group, followed by a pretreatment step to release the carrier. The siRNA exposed as the formulation was released was synthesized into cDNA via reverse transcription (RT) step and qRT-PCR (Bio-Rad CFX96 Real-Time System) was performed using the synthesized cDNA. Analysis was performed using the Bio-Rad CFX Manager program.
【표 16】 혈중 농도 (ng/mL) Table 16 Blood concentration (ng / mL)
0.5시간 6시간 0.5 hours 6 hours
비교예 1 1565.6 856.82 Comparative Example 1 1565.6 856.82
비교예 2 808.43 158.75 Comparative Example 2 808.43 158.75
실시예 1 7483.83 3449.33 Example 1 7483.83 3449.33
실시예 2 1 1650.82 5362.87 표 16에서 알 수 있는 바와 같이, 본 발명의 실시예 1 및 2 에서 제조된 제형은 비교예 1 및 2 에 비해 혈중에서 존재하는 제형의 농도가 0.5시간에서 5 내지 8배 정도높은 편으로 혈중 안정성이 뛰어남을 알 수 있다ᅳ Example 2 1 1650.82 5362.87 As can be seen in Table 16, the formulations prepared in Examples 1 and 2 of the present invention have a concentration of 5 to 8 times the concentration of the formulations present in blood compared to Comparative Examples 1 and 2 at 0.5 hours. The higher the degree, the more stable the blood.
[실험예 5] siRNA/양이은성 물질 /양친성 블록 공중합체/ PLANa 미셀의 생체 내 조직 분포' Experimental Example 5 In vivo Tissue Distribution of siRNA / Cationic Compounds / Amphiphilic Block Copolymers / PLANa micelles ''
siRNA/dioTETA/mPEG-PLA-토코페를 /PLANa고분자 미샐이 생체 내에서, 간과 암조직에 분포된 양을 확인하였다.  The amount of siRNA / dioTETA / mPEG-PLA-tocope / PLANa polymer micelles distributed in liver, liver and cancer tissues was confirmed.
Balb/c 누드 마우스에 A2780cis 인간 난소암 세포주를 피하에 주입하여 암이 유발된 생쥐를 제조하였다. 제형을 l mg/kg의 용량으로 이를에 한번씩 총 4희 정맥투여 하였다. 마지막투여 후 24시간에 간과 암조직을 적출하여 200 mg씩 무게를 잰 후 1.8 mL의 0.25% Triton X- 100 에 넣고 조직분쇄기로 조직을 갈아준다. 스랜다드용조직샘플은 생리식염수를 투여한 조직을 이용하고 같은 방법으로 조직올 갈아준다. 스탠다드용 제형의 농도는 4 μΜ에서 0.00256 μΜ까지 총 11개 농도로 PBS에 회석하여 준비한다. PCR용 96웰 플레이트에 스탠다드용으로 갈아준 조직을 99 μΐ 넣고 스탠다드용으로 회석한 제형 Ι μΐ를 넣어준다. 실험군으로 분석할 조직 샘플은 100 μΐ씩 넣고 제형을 풀어주는 전처리 단계를 거친다.  Cancer-induced mice were prepared by injecting Balb / c nude mice subcutaneously with an A2780cis human ovarian cancer cell line. The formulation was administered intravenously with a total of four doses, one at a dose of l mg / kg. Liver and cancer tissues were removed and weighed 200 mg 24 hours after the last dose, and then placed in 1.8 mL of 0.25% Triton X-100 and ground with a tissue grinder. Threadard tissue sample is to use the tissue administered with saline and change the tissue in the same way. Concentrations of standard formulations are prepared by dilution in PBS at 11 concentrations ranging from 4 μΜ to 0.00256 μΜ. 99 μl of the tissue ground for the standard is added to a 96-well plate for PCR, and the formulation Ι μΐ was added to the standard. Tissue samples to be analyzed in the experimental group are subjected to a pretreatment step in which 100 μΐ is added and the formulation is released.
노출된 siRNA를 역전사 (RT) 단계를 거쳐 cDNA로 합성하고 합성된 cDNA를 이용하여 qRT-PCR (Bio-Rad CFX96 Real-Time System)을 수행하였다. 분석은 Bio- Rad CFX Manager 프로그램을 이용하여 분석하였다. The exposed siRNA was synthesized into cDNA by reverse transcription (RT) step and qRT-PCR (Bio-Rad CFX96 Real-Time System) was performed using the synthesized cDNA. The analysis was performed using the Bio- Rad CFX Manager program.
분석 결과는 아래의 표 17에 나타내었다. 【표 17] The analysis results are shown in Table 17 below. Table 17
Figure imgf000035_0001
표 17에 나타난 바와 같이, 본 발명에 따른 실시예 2는 비교예 1 에 비하여 간조직 분포는 감소하고, 암조직 분포량이 증가한 것으로 나타났다.
Figure imgf000035_0001
As shown in Table 17, Example 2 according to the present invention was shown to reduce the liver tissue distribution, cancer tissue distribution increased compared to Comparative Example 1.
이러한 결과는 본 발명에 따른 PLANa포함 고분자 미셀 전달체가 암조직에 특이적으로 표적화할 수 있는 가능성을 제시해 주는 것이라 할수 있다. These results suggest that the PLANa-containing polymer micelle carrier according to the present invention can be specifically targeted to cancer tissues.
【실험예 6] siRNA/양이온성 물질 /양친성 블록 공중합체/ PLANa 미셀의 생체 내 활성 (유전자 억제능력 분석) Experimental Example 6 In vivo activity of siRNA / cationic substance / amphiphilic block copolymer / PLANa micelle
siRNA/dioTETA/mPEG-PLA-토코페를 /PLANa고분자 미셀의 생체 내 활성을 유전자 억제 능력 분석을 통해 확인 하였다ᅳ  In vivo activity of siRNA / dioTETA / mPEG-PLA-tocope / PLANa polymer micelles was confirmed by analysis of gene suppression ability.
Balb/c 누드 마우스에 A549 인간 폐암 종양편올 이,식하셔 암이 유발된 생쥐를 제조하였다. 제형을 0.5 mg/kg의 용량으로 이를에 한번씩 총 3회 정맥투여 하였다. 대조군으로는 생리식염수를 투여하였고, 각 제형당 5 마리씩 실시하였다. 마지막 투여 후 24시간에 암조직을 적출하여 액체 질소 하에서 막자사발을 이용하여 일차 분쇄 후, QIAGEN조직 분쇄기 (TissueLyser)를 이용하여 다시 한번 조직을 갈아준다. 두 번에 걸쳐 파쇄된 암조직 10 mg 에 미리 준비해 놓은 작업용 용액 (working homogenizing solution, Homogenizing solution 600μ1 + Proteinase K (23mg/mL) 6μ1) 600μ1을 이용하여 HPRT mRNA를 세포 밖으로 꺼낸다. 상기의 방법으로 확보된 샘플을 bDNA assay kit을 이용하여 분석하였다. 분석방법은 kit 제조사 (Panomics bDNA분석법) 의 지시에 따랐다.  Cancer-induced mice were prepared by feeding A549 human lung cancer tumor isolates to Balb / c nude mice. The formulation was administered intravenously three times, once per dose, at a dose of 0.5 mg / kg. Physiological saline was administered as a control group, 5 dogs for each formulation. After 24 hours after the last administration, the cancer tissue was extracted, and the first milling was performed using a mortar and pestle under liquid nitrogen, and the tissue was once again changed using a QIAGEN tissue crusher (TissueLyser). HPRT mRNA is removed from the cells using 600 μ1 of working homogenizing solution, Homogenizing solution 600 μ1 + Proteinase K (23 mg / mL) 6 μ1, which is prepared in 10 mg of twice-broken cancer tissue. Samples obtained by the above method were analyzed using a bDNA assay kit. The assay was followed by the manufacturer of the kit (Panomics bDNA assay).
HPRT siRNA 의 영향을 받지 않는 유전자인 GAPDH mRNA 도 동일한 방법으로 분석하여, 측정된 HPRT mRNA 양올 보정하여 암조직에서의 HPRT mRNA 의 상대적 발현양을 평균값을 계산하였다. 분석 결과는 아래 표 18에 나타내었다. 【표 18] GAPDH mRNA, a gene that is not affected by HPRT siRNA, was also analyzed in the same manner, and the average amount of HPRT mRNA expression in cancer tissues was calculated by correcting the measured HPRT mRNA amount. The analysis results are shown in Table 18 below. Table 18
Figure imgf000036_0001
표 18에 나타난 바와 같이, 본 발명에 따른 실시예 2는 생체 내 암조직에서 타겟 유전자 HPRT의 mRNA 54% 억제함을 알 수 있다.
Figure imgf000036_0001
As shown in Table 18, Example 2 according to the present invention can be seen that inhibits the mRNA 54% of the target gene HPRT in cancer tissues in vivo.

Claims

【청구의 범위】 【청구항 1】 유효성분으로서 음이온성 약물; 양이온성 화합물; 양친성 블록 공중합체; 및 하기 화학식 1 내지 6의 화합물로 이루어진 군으로부터 선택되는 하나 이상인 폴리락트산염을 포함하며, 상기 음이온성 약물은 상기 양이온성 화합물과 정전기적 상호작용에 의해 복합체를 형성하고, 상기 복합체는 양친성 블록 공중합체 및 폴리락트산염이 형성하는 미셀 구조 내부에 봉입되어 있는 것을 특징으로 하는, 음이온성 약물 전달용 조성물: Claims Claim 1 Anionic drugs as active ingredients; Cationic compounds; Amphiphilic block copolymers; And at least one polylactic acid salt selected from the group consisting of compounds of Formulas 1 to 6, wherein the anionic drug forms a complex by electrostatic interaction with the cationic compound, and the complex is an amphiphilic block An anionic drug delivery composition, characterized in that encapsulated inside a micellar structure formed by the copolymer and polylactic acid salt:
[화학식 1]  [Formula 1]
RO-CHZ- [A] rt- [B]m-COOM RO-CHZ- [A] rt - [B] m -COOM
상기 식 1에서 , Α는 -COO-CHZ-이고; Β는 -COO-CHY-, -COO- CH2CH2CH2CH2CH2- 또는 -COO-C¾CH2OCH2이며; R은 수소원자, 또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸, 또는 에틸기이고; Z와 Y는 각각 수소원자, 또는 메틸 또는 페닐기이며; M은 Na, K, 또는 Li이고; n은 1 내지 30의 정수이며; m은 0 내지 20의 정수이다. In formula 1, A is -COO-CHZ-; Β is -COO-CHY-, -COO-CH 2 CH 2 CH 2 CH 2 CH 2 -or -COO-C¾CH 2 OCH 2 ; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl, or ethyl group; Z and Y are each a hydrogen atom or a methyl or phenyl group; M is Na, K, or Li; n is an integer from 1 to 30; m is an integer of 0-20.
[화학식 2]  [Formula 2]
RO-CHZ- [C0< GHX]p- [00Q-CHY1 ],-CO0-CHZ-CM RO-CHZ- [C0 <GHX] p- [00Q-CHY 1 ],-CO0-CHZ-CM
상기 식 2에서, X는 메틸기이고; Y'는 수소원자 또는 페닐기이며; p는 0 내지 25의 정수이고, q는 0 내지 25의 정수이되, 단 p+q는 5 내지 25의 정수이고; R은 수소원자, 또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸 또는 에틸기이며; M은 Na, K,또는 Li이고; Z 는 수소 원자, 메틸 또는 페닐기이다.  In formula 2, X is a methyl group; Y 'is a hydrogen atom or a phenyl group, p is an integer from 0 to 25, q is an integer from 0 to 25, provided that p + q is an integer from 5 to 25; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; M is Na, K, or Li; Z is a hydrogen atom, methyl or phenyl group.
[화학식 3]  [Formula 3]
R0-PAD-C00-W-M'  R0-PAD-C00-W-M '
C0O  C0O
C― CH2CO0M C00  C― CH2CO0M C00
상기 식 3에서, W-M'는 ¾CO0M 또는 ᅳ CHᅳ CHaCOCH이고; PAD는 D,L-폴리락트산, D-폴리락트산, 폴리만델릭산 , D,L-락트산과 글리콜산의 공중합체, D,L-락트산과 만델릭산의 공중합체, D,L-락트산과 카프로락톤의 공중합체 및 D,L- 락트산과 1,4-디옥산 -2-온의 공중합체로 구성된 그룹으로부터 선택되는 것이며; R은 수소원자, 또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸 또는 에틸기이고; M은 독립적으로 Na, K, 또는 Li이다. In Formula 3, W-M 'is ¾CO0M or ᅳ CH ᅳ CHaCOCH; PAD D, L-lactic acid, D-polylactic acid, polymandelic acid, copolymer of D, L-lactic acid and glycolic acid, copolymer of D, L-lactic acid and mandelic acid, D, L-lactic acid and caprolactone And a copolymer of D, L-lactic acid and 1,4-dioxane-2-one; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; M is independently Na, K, or Li.
[화학식 4]  [Formula 4]
S-0-PAD-C00-Q  S-0-PAD-C00-Q
r  r
Η·十 L—CH~ C+  Η · 十 L—CH ~ C +
상기 식 4에서, S는 0 ) a-cooM이고; L은 -Ν -또는 -0-이며, 여기서In formula 4, S is 0 ) a -cooM; L is -Ν -or -0-, where
Ri은 수소원자 또는 C1-10알킬이고; Q는 CH3, CH2CH3, CH2CH2CH3, CH2CH2CH2CH3, 또는 CH2C6H5이고; a는 0 내지 4의 정수이며; b는 1 내지 10의 정수이고; M은 Na, K, 또는 Li이며; PAD는 D,L-폴리락트산, D-폴리락트산, 폴리만델릭산, D,L-락트산과 글리콜산의 공중합체, D,L-락트산과 만델릭산의 공증합체, D,L-락트산과 Ri is a hydrogen atom or C 1-10 alkyl; Q is CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 , CH 2 CH 2 CH 2 CH 3 , or CH 2 C 6 H 5 ; a is an integer from 0 to 4; b is an integer from 1 to 10; M is Na, K, or Li; PAD is composed of D, L-polylactic acid, D-polylactic acid, polymandelic acid, copolymers of D, L-lactic acid and glycolic acid, co-polymers of D, L-lactic acid and mandelic acid, D, L-lactic acid and
카프로락톤의 공중합체, 및 D,L-락트산과 1,4-디옥산 -2-온의 공중합체로 이루어진 군에서 선택되는 하나 이상이다. At least one selected from the group consisting of a copolymer of caprolactone and a copolymer of D, L-lactic acid and 1,4-dioxane-2-one.
[화학식 5]  [Formula 5]
Figure imgf000038_0001
Figure imgf000038_0001
상기 식 5에서, R'는 -PAD-0-C(0)-CH2CH2-C(0)-OM이고, 여기서 PAD는 D,L-폴리락트산, D-폴리락트산, 폴리만델릭산, D,L—락트산과 글리콜산의 공증합체, D,L-락트산과 만델릭산의 공중합체, D,L-락트산과 카프로락톤의 공중합체, D,L- 락트산과 1,4-디옥산 -2-온의 공중합체로 구성된 그룹으로부터 선택되는 것이고, M은 Na, K, 또는 Li이며; a는 1 내지 4의 정수이다. In Formula 5, R 'is -PAD-0-C (0) -CH 2 CH 2 -C (0) -OM, wherein PAD is D , L-polylactic acid, D-polylactic acid, polymandelic acid , D, L—Lactic acid and glycolic acid co-polymer, D, L-lactic acid and mandelic acid copolymer, D, L-lactic acid and caprolactone copolymer, D, L-lactic acid and 1,4-dioxane Is selected from the group consisting of copolymers of 2-ones, M is Na, K, or Li; a is an integer of 1-4.
[화학식 6]  [Formula 6]
Y0- [-C(0)-(CHX)a-0-]m-C(0) - -C(O) - [— CM: CHX' )b-C(Q)- J tt—OZ 상기 식 6에서, X 및 X'은 독립적으로 수소, 탄소수가 1~10인 알킬 또는 탄소수가 6~20인 아릴이고; Y 및 Z는 독립적으로 Na, K, 또는 Li이며; m 및 n은 독립적으로 0 내지 95의 정수이되, 5 < m + n < 100이고; a 및 b는 독립적으로 1 내지 6의 정수이며; R은 -(CH2)k-, 탄소수가 2~10인 2가 알케닐 (divalent alkenyl), 탄소수가 6~20인 2가 아릴 (divalent aryl) 또는 이들의 조합이고, 여기서 k는 0 내지 10의 정수이다. Y0- [-C (0)-(CHX) a -0-] m -C (0)--C (O)-[— CM: CHX ') b -C (Q)-J tt —OZ At 6, X and X 'are independently hydrogen, alkyl having 1 to 10 carbon atoms or aryl having 6 to 20 carbon atoms; Y and Z are independently Na, K, or Li; m and n are Independently an integer from 0 to 95, with 5 <m + n <100; a and b are independently an integer from 1 to 6; R is-(CH 2 ) k- , divalent alkenyl having 2 to 10 carbon atoms, divalent aryl having 6 to 20 carbon atoms, or a combination thereof, wherein k is 0 to 10 Is an integer.
【청구항 2】  [Claim 2]
제 1항에 있어서,  The method of claim 1,
상기 음이온성 약물은 핵산인 것을 특징으로 하는, 음이온성 약물 전달용 조성물. 【청구항 3】 The anionic drug is characterized in that the nucleic acid, anionic drug delivery composition. [Claim 3]
제 2항에 있어서,  The method of claim 2,
상기 핵산은 RNA, DNA, siRNA(short interfering RNA), 압타머 (aptamer), 안티센스 ODN(antisense oligodeoxynucleotide), 안티센스 R A(antisense RNA), The nucleic acid may be RNA, DNA, siRNA (short interfering RNA), aptamer, antisense oligodeoxynucleotide (ODN), antisense RNA (A),
리보자임 (ribozyme) 및 디엔에이자임 (DNAzyme)로 이루어진 군으로부터 선택되는 하나 이상인 것을 특징으로 하는, 음이온성 약물 전달용 조성물. Ribozyme (ribozyme) and dienezyme (DNAzyme), characterized in that at least one selected from the group consisting of, anionic drug delivery composition.
【청구항 4】  [Claim 4]
제 3항에 있어서,  The method of claim 3, wherein
상기 핵산은 하나 이상의 말단이 콜레스테를, 토코페를, 및 탄소수 10 내지 The nucleic acid has at least one end of cholester, tocope, and 10 to 10 carbon atoms.
24개의 지방산으로 구성된 군으로부터 선택되는 하나 이상으로 수식된 것을 특징으로 하는, 음이온성 약물 전달용 조성물. Characterized in the one or more selected from the group consisting of 24 fatty acids, anionic drug delivery composition.
【청구항 5】  [Claim 5]
제 1항에 있어서,  The method of claim 1,
상기 양이온성 화합물은 양이온성 지질 및 양이온성 고분자로 구성된 군으로부터 선택되는 하나 이상인 것을 특징으로 하는, 음이온성 약물 전달용 조성물. The cationic compound is at least one selected from the group consisting of cationic lipids and cationic polymers, anionic drug delivery composition.
【청구항 6】  [Claim 6]
제 5항에 있어서,  The method of claim 5,
상기 양이은성 지질은 Ν,Ν-디을레일 -Ν,Ν-디메틸암모늄클로라이드 (DODAC), N,N- 디스테아릴 -Ν,Ν-디메틸암모늄브로마이드 (DDAB), N-(l -(2,3-디올레오일옥시 )프로필- Ν,Ν,Ν-트리메틸암모늄클로라이드 (DOTAP), Ν,Ν-디메틸 -(2,3- 디을레오일옥시)프로필아민 (DODMA), 1,2-디아실 -3-트리메틸암모늄-프로판 (TAP), 1,2-디아실 -3-디메틸암모늄-프로판 (DAP), 3베타 -[Ν-(Ν',Ν',Ν'- 트리메틸아미노에탄)카바모일]콜레스테롤 (TC 콜레스테롤), 3베타 [Ν-(Ν',Ν'- 디메틸아미노에탄)카바모일 ]콜레스테롤 (DC-콜레스테를), 3베타 [Ν-(Ν'- 모노메틸아미노에탄)카바모일]콜레스테롤 (MC-콜레스테롤), 3베타 [Ν- (아미노에탄)카바모일]콜레스테롤 (AC-콜레스테롤), 콜레스테릴옥시프로판 - 1 - 아민 (COPA), N-(N'-아미노에탄)카바모일프로파노익 토코페롤 (AC-토코페롤) 및 N- (Ν'-메틸아미노에탄)카바모일프로파노익 토코페롤 (MC-토코페를)로 이루어진 군에서 선택된 1종 이상인, 음이온성 약물 전달용 조성물. The cationic lipids are Ν, Ν-dialeyl -Ν, Ν-dimethylammonium chloride (DODAC), N, N- distearyl -Ν, Ν-dimethylammonium bromide (DDAB), N- (l-(2, 3-dioleoyloxy) propyl- Ν, Ν, Ν-trimethylammonium chloride (DOTAP), Ν, Ν-dimethyl- (2,3-dileoyloxy) propylamine (DODMA), 1,2-diacyl 3-trimethylammonium-propane (TAP), 1,2-diacyl-3-dimethylammonium-propane (DAP), 3beta- [N- (Ν ', Ν', Ν'-) Trimethylaminoethane) carbamoyl] cholesterol (TC cholesterol), 3beta [Ν- (Ν ', Ν'-dimethylaminoethane) carbamoyl] cholesterol (DC-cholesterol), 3beta [Ν- (Ν'- Monomethylaminoethane) carbamoyl] cholesterol (MC-cholesterol), 3beta [Ν- (aminoethane) carbamoyl] cholesterol (AC-cholesterol), cholesteryloxypropane-1-amine (COPA), N- ( N'-aminoethane) carbamoylpropanoic tocopherol (AC-tocopherol) and N- (Ν'-methylaminoethane) carbamoylpropanoic tocopherol (MC-tocopher); Composition for anionic drug delivery.
【청구항 7】  [Claim 7]
제 5항에 있어서,  The method of claim 5,
상기 양이온성 지질은 하기 화학식 7의 양이온성 지질인, 음이온성 약물 전달용 조성물: The cationic lipid is a cationic lipid of Formula 7, wherein the composition for anionic drug delivery:
[화학식  Formula
Figure imgf000040_0001
Figure imgf000040_0001
상기 식에서,  In the above formula,
η과 m은 각각 0 내지 12이되, 2≤n + m≤12이며, a와 b는 각각 1 내지 6이며, R1과 R2는 각각 독립적으로 탄소수 1 1 내지 25개의 포화 및 불포화 탄화수소로 이루어진 군에서 선택된 것이다. η and m are each 0 to 12, 2≤n + m≤12, a and b are each 1 to 6, and R1 and R2 are each independently a group consisting of saturated and unsaturated hydrocarbons having 1 to 25 carbon atoms It is selected.
【청구항 8】  [Claim 8]
제 7항에 있어서 , η과 m은 독립적으로 1 내지 9이며, 2≤n+m < 10인, 음이온성 약물 전달용 조성물.  8. The composition for anionic drug delivery according to claim 7, wherein η and m are independently 1 to 9 and 2 ≦ n + m <10.
【청구항 9]  [Claim 9]
제 7항에 있어서, a와 b가 2 내지 4인, 음이은성 약물 전달용 조성물.  8. The composition of claim 7, wherein a and b are 2-4.
【청구항 10】  [Claim 10]
거 17항에 있어서, R1과 R2는, 각각 독립적으로, 라우릴 (lauryl), 미리스틸 (myristyl), 팔미틸 (palmityl), 스테아릴 (stearyl), 아라키딜 (arachidyl), 베헨닐 (behenyl), 리그노세릴 (lignoceryl), 세로틸 (cerotyl), 미리스트올레일 (myristoleyl), 팔미트올레일 (palmitoleyl),사피에닐 (sapienyl), 올레일 (oleyl), 리놀레일 (linoleyl), 아라키도닐 (a chidonyl), 에이코사펜타에닐 (eicosapentaenyl), 에루실 (erucyl), 도코사핵사에닐 (docosahexaenyl), 및 세로틸 (cerotyl)로 이루어진 군에서 선택된 것인, 음이온성 약물 전달용 조성물. According to claim 17, R1 and R2 are each independently lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl , Lignoceryl, cerotyl, myristoleyl, palmitoleyl, sapienyl, oleyl, linoleyl, Anionic drug delivery, selected from the group consisting of a chidonyl, eicosapentaenyl, erucyl, docosahexaenyl, and cerotyl Composition.
【청구항 1 1 ]  [Claim 1 1]
제 1항에 있어서,  The method of claim 1,
상기 양이온성 고분자는 키토산 (chitosan), 글라이콜 키토산 (glycol chitosan), 프로타민 (protamine), 폴리 라이신 (polylysine), 폴리 아르기닌 (polyarginine), 폴리아미도아민 (PAMAM), 폴리에틸렌이민 (polyethylenimine), 덱스트란 (dextran), 히알루론산 (hyaluronic acid), 알부민 (albumin), 고분자폴리에틸렌이민 (PEI),폴리아민 및 폴리비닐아민으로 구성된 군으로부터 선택되는 것을 특징으로 하는, 음이은성 약물 전달용 조성물ᅳ The cationic polymer is chitosan, glycol chitosan, protamine, polylysine, polyarginine, polyamidoamine (PAMAM), polyethylenimine, dex. A composition for the delivery of a negative drug which is selected from the group consisting of dextran, hyaluronic acid, albumin, albumin, high molecular polyethyleneimine (PEI), polyamine and polyvinylamine.
【청구항 12]  [Claim 12]
제 5항에 있어서,  The method of claim 5,
상기 음이온성 약물 (P)과 양이온성 지질 (N)의 전하량의 비율 (N/P)은 0.1 내지 128인 것을 특징으로 하는 음이온성 약물 전달용 조성물. The ratio (N / P) of the charge amount of the anionic drug (P) and the cationic lipid (N) is 0.1 to 128, the composition for delivery of anionic drugs.
【청구항 13】  [Claim 13]
제 1항에 있어서,  The method of claim 1,
상기 양친성 블록 공중합체는 친수성 A 블록과 소수성 B 블록으로 구성되는 A-B 형 이증 블록 공중합체이며, 상기 친수성 A블록은 폴리알킬렌글리콜, 폴리비닐알콜, 폴리비닐피를리돈, 폴리아크릴아미드 및 그 유도체로 이루어진 군에서 선택되는 1종 이상이며, 상기 소수성 B 블록은 폴리에스테르, The amphiphilic block copolymer is an AB type diblock block copolymer composed of a hydrophilic A block and a hydrophobic B block, and the hydrophilic A block is polyalkylene glycol, polyvinyl alcohol, polyvinylpyridone, polyacrylamide and At least one selected from the group consisting of derivatives, the hydrophobic B block is polyester,
폴리언하이드라이드, 폴리아미노산, 풀리오르소에스테르 및 폴리포스파진으로 이루어진 군에서 선택되는 1종 이상인 것을 특징으로 하는, 음이온성 약물 전달용 조성물. A composition for anionic drug delivery, characterized in that at least one selected from the group consisting of polyanhydrides, polyamino acids, pulliolysoesters and polyphosphazines.
【청구항 14】  [Claim 14]
제 13항에 있어서,  The method of claim 13,
상기 소수성 B 블록의 말단 히드록시기는 콜레스테롤, 토코페롤, 및 탄소수 10 내지 24개의 지방산으로 구성된 군으로부터 선택되는 하나 이상으로 수식된 것을 특징으로 하는, 음이온성 약물 전달용 조성물. The terminal hydroxyl group of the hydrophobic B block is cholesterol, tocopherol, and the number of carbon atoms from 10 to 24 fatty acids in the formula, it characterized in that the at least one selected from the group consisting of the anionic drug delivery composition.
【청구항 15】 [Claim 15]
제 M항에 있어서,  The method of claim M,
상기 친수성 A 블특의 수평균 분자량은 200 내지 '50,000달톤이고, 친수성 B 블록의 수평균 분자량은 50 내지 50,000달톤인 것을 특징으로 하는, 음이온성 약물 전달용 조성물. The hydrophilic A beulteuk a number average molecular weight of 200 to '50,000 Daltons, the number of the hydrophilic B block has an average molecular weight, characterized in that 50 to 50,000 Daltons, the anionic drug delivery composition.
【청구항 16】  [Claim 16]
제 5항에 있어서,  The method of claim 5,
상기 양친성 블록 공중합체의 중량 (b) 대비 음이온성 약물 및 양이온성 지질 복합체의 중량 (a) 비율 (a/b X I 00)은, 0.001 내지 100 중량 %인 것을 특징으로 하는, 음이온성 약물 전달용 조성물. The anionic drug delivery, characterized in that the weight (a) ratio (a / b XI 00) of the anionic drug and the cationic lipid complex to the weight (b) of the amphiphilic block copolymer is 0.001 to 100% by weight. Composition.
【청구항 17】  [Claim 17]
제 1항에 있어서,  The method of claim 1,
상기 폴리락트산염은 화학식 1 또는 화학식 2로 나타내어지는 것을 특징으로 하는, 음이온성 약물 전달용 조성물. The polylactic acid salt is an anionic drug delivery composition, characterized in that represented by the formula (1) or (2).
【청구항 18]  [Claim 18]
제 1항에 있어서, 상기 폴리락트산염의 수평균 분자량이 500 내지  According to claim 1, wherein the number average molecular weight of the polylactic acid salt is 500 to
50,000달톤인 것을 특징으로 하는, 음이온성 약물 전달용 조성물. It is 50,000 Daltons, anionic drug delivery composition.
【청구항 19]  [Claim 19]
제 13항에 있어서,  The method of claim 13,
음이온성 약물 1 중량부 대비 양친성 블록 공중합체를 10 내지 1 ,000 증량부 및 폴리락트산염을 5 내지 500 중량부로 함유하는 것을 특징으로 하는, 음이온성 약물 전달용 조성물. An anionic drug delivery composition comprising 10 to 1,000 parts by weight of an amphiphilic block copolymer relative to 1 part by weight of anionic drug and 5 to 500 parts by weight of polylactic acid salt.
【청구항 20]  [Claim 20]
거 U항에 있어서, 미샐의 표면 전하가 -20 내지 20 mV인 것을 특징으로 하는, 음이은성 약물 전달용 조성물.  The composition according to claim U, wherein the surface charge of the micelle is -20 to 20 mV.
【청구항 21】  [Claim 21]
제 1항에 있어서, 입자 크기가 10 내지 200 nm인 것을 특징으로 하는, 음이온성 약물 전달용 조성물.  The composition for anionic drug delivery according to claim 1, wherein the particle size is 10 to 200 nm.
【청구항 22] 제 1항에 있어서, 동결건조 보조제를 추가로 포함하는 것을 특징으로 하는, 음이온성 약물 전달용 조성물. [Claim 22] The composition of claim 1, further comprising a lyophilization aid.
【청구항 23】  [Claim 23]
제 1항에 있어서, 융합성 지질 (ftisogenic lipid)을 추가로 포함하는, 음이온성 약물 전달용 조성물.  The composition for anionic drug delivery according to claim 1, further comprising a fusogenic lipid.
【청구항 24]  [Claim 24]
제 23항에 있어서, 상기 융합성 지질은 디라우로일  The method of claim 23, wherein the fusion lipid is dilauroyl
포스파티딜에탄올아민 (dUauroyl phosphatidylethanolamine), 디미리스토일 Phosphatidylethanolamine (dUauroyl phosphatidylethanolamine), dimyristoyl
포스파티딜에탄을아민 (dimyristoyl phosphatidylethanolamine), 디팔미토일 Dimyristoyl phosphatidylethanolamine, dipalmitoyl
포스파티딜에탄올아민 (dipalmitoyl phosphatidylethanolamine), 디스테아로일 Phosphatidylethanolamine (dipalmitoyl phosphatidylethanolamine), distearoyl
포스파티딜에탄올아민 (distearoyl phosphatidylethanolamine), 디올레오일 Distatiyl phosphatidylethanolamine, dioleoyl
포스파티딜에탄올아민 (dioleoyl phosphatidylethanolamine), 디리놀레오일 Dioleoyl phosphatidylethanolamine, dilinoleyl
포스파티딜에탄올아민 (dilinoleoyl phosphatidylethanolamine), 1-팔미토일 -2-올레오일 포스파티딜에탄올아민 (l-palmitoyl-2-oleoyl phosphatidylethanolamine), 1 ,2-디피타노일- 3-sn-포스파티딜에탄올아민 (l,2-diphytanoyl-3-sn-phosphatidyletl nolainine), 디라우로일 포스파티딜콜린 (dilauroyl phosphatidylcholine), 디미리스토일 Dilinoleoyl phosphatidylethanolamine, 1-palmitoyl-2-oleoyl phosphatidylethanolamine, 1,2-dipitanoyl 3-sn-phosphatidylethanolamine (l, 2 -diphytanoyl-3-sn-phosphatidyletl nolainine, dilauuroyl phosphatidylcholine, dimyristoyl
포스파티딜콜린 (dimyristoyl phosphatidylcholine), 디팔미토일 Phosphatidylcholine (dimyristoyl phosphatidylcholine), dipalmitoyl
포스파티딜콜린 (dipalmitoyl phosphatidylcholine), 디스테아로일 Phosphatidylcholine (dipalmitoyl phosphatidylcholine), distearoyl
포스파티딜콜린 (distearoyl phosphatidylcholine), 디올레오일 포스파티딜콜린 (dioleoyl phosphatidylcholine), 디리놀레오일 포스파티딜콜린 (dilinoleoyl phosphatidylcholine), 1- 팔미토일 -2-올레오일 포스파티딜콜린 (l-palmitoyl-2-oleoyl phosphatidylcholine), 1 ,2- 디피타노일 -3-sn-포스파티딜콜린 (l,2-diphytanoyl-3-sn-phosphatidylcholine), 디라우로일 포스파티딘산 (dilauroyl phosphatidic acid), 디미리스토일 포스파티딘산 (dimyristoyl phosphatidic acid), 디팔미토일 포스파티딘산 (dipalmitoyl phosphatidic acid), Distatiyl phosphatidylcholine, dioleoyl phosphatidylcholine, dilinoleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine (l-palmitoyl-2-oleylyl, 2-dyl2-ylline) Diphytanoyl-3-sn-phosphatidylcholine (l, 2-diphytanoyl-3-sn-phosphatidylcholine), dilauuroyl phosphatidic acid, dimyristoyl phosphatidic acid, dimyristoyl phosphatidic acid Palmitoyl phosphatidic acid,
디스테아로일 포스파티딘산 (distearoyl phosphatidic acid), 디을레오일 Distearoyl phosphatidic acid, dioleoyl
포스파티딘산 (dioleoyl phosphatidic acid), 디리놀레오일 포스파티딘산 (dilinoleoyl phosphatidic acid), 1-팔미토일 -2-을레오일 포스파티딘산 (l-palmitoyl-2-oleoyl phosphatidic acid), 1,2-디피타노일 -3-sn-포스파티딘산 (l,2-diphytanoyl-3-sn-phosphatidic acid), 콜레스테롤, 및 토코페롤로 이루어진 군에서 선택된 1종 이상인, 음이온성 약물 전달용 조성물. Dioleoyl phosphatidic acid, dilinoleoyl phosphatidic acid, 1-palmitoyl-2-oleoyl phosphatidic acid, 1,2- Anionic, at least one selected from the group consisting of diphytanoyl-3-sn-phosphatidic acid, cholesterol, and tocopherols Composition for drug delivery.
【청구항 25]  [Claim 25]
(a) 음이은성 약물, 양이온성 화합물, 양친성 블록 공증합체 및  (a) negative silver drugs, cationic compounds, amphiphilic block co-polymers and
폴리락트산염을 수흔화성 유기용매, 또는 수용액 및 유기용매의 혼합 용매에 용해시키는 공정 ; Dissolving the polylactic acid salt in a water-soluble organic solvent or a mixed solvent of an aqueous solution and an organic solvent;
(b) 상기 (a)공정의 유기용매층를 제거하는 공정;  (b) removing the organic solvent layer of step (a);
(c) 상기 (b) 공정의 유기용매가 제거된 흔합물에 수용액을 첨가하여 미셀화하는 공정을 포함하는,  (c) adding an aqueous solution to the mixture from which the organic solvent of step (b) has been removed, and including the step of micellizing;
저 U항 내지 제 22항 증 어느 한 항에 따른 음이온성 약물 전달용조성물의 제조방법.  A method for preparing an anionic drug delivery composition according to any one of claims U to 22.
【청구항 26]  [Claim 26]
유효성분으로서 음이온성 약물;  Anionic drugs as active ingredients;
양이온성 화합물; Cationic compounds;
양친성 블록 공중합체; 및 Amphiphilic block copolymers; And
하가화학식 1 내지 6의 화합물로 이루어진 군으로부터 선택되는 하나 이상인 폴리락트산염을 포함하며, At least one polylactic acid salt selected from the group consisting of compounds of Formulas 1 to 6,
상기 음이온성 약물은 상기 양이온성 화합물과 정전기적 상호작용에 의해 복합체를 형성하고, 상기 복합체는 양친성 블록 공중합체 및 폴리락트산염이 형성하는 미셀 구조 내부에 봉입되어 있는 것을 특징으로 하는 조성물의 음이온성 약물 전달을 위한 용도: The anionic drug forms a complex by electrostatic interaction with the cationic compound, and the complex is encapsulated inside a micellar structure formed by an amphiphilic block copolymer and a polylactic acid salt. Uses for sex drug delivery:
[화학식 1]  [Formula 1]
RO-CHZ- [A] n- [B ]m-COOM RO-CHZ- [A] n- [B] m -COOM
상기 식 1에서 , Α는 -COO-CHZ-이고; Β는 -COO-CHY-, -COO- CH2CH2CH2CH2CH2-또는 -COO-CH2CH2OCH2이며; R은 수소원자, 또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸, 또는 에틸기이고; Z와 Y는 각각 수소원자, 또는 메틸 또는 페닐기이며; M은 Na, K, 또는 Li이고; n은 1 내지 30의 정수이며; m은 0 내지 20의 정수이다. In formula 1, A is -COO-CHZ-; Β is -COO-CHY-, -COO-CH 2 CH 2 CH 2 CH 2 CH 2 -or -COO-CH 2 CH 2 OCH 2 ; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl, or ethyl group; Z and Y are each a hydrogen atom or a methyl or phenyl group; M is Na, K, or Li; n is an integer from 1 to 30; m is an integer of 0-20.
[화학식 2]  [Formula 2]
RO-CHZ- [COCKHX] p-[C00-CHY' ] q-C00-CHI-C00M 상기 식 2에서, X는 메틸기이고; Y'는 수소원자또는 페닐기이며; p는 0 내지 25의 정수이고, q는 0 내지 25의 정수이되, 단 p+q는 5 내지 25의 정수이고; R은 수소원자,또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸 또는 에틸기이며; M은 Na, K, 또는 Li이고; Z 는수소 원자, 메틸 또는 페닐기이다. RO-CHZ- [COCKHX] p- [C00-CHY '] q -C00-CHI-C00M In formula 2, X is a methyl group; Y 'is a hydrogen atom or a phenyl group; p is an integer from 0 to 25, q is an integer from 0 to 25, provided that p + q is an integer from 5 to 25; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; M is Na, K, or Li; Z is a hydrogen atom, a methyl or a phenyl group.
[화학식 3] [Formula 3]
0-PAD-C00-W-M'  0-PAD-C00-W-M '
C0OM  C0OM
—— C— CH2COOM COOM  —— C— CH2COOM COOM
상기 식 3에서, W-M'는 CH2GO0M 또는 ᅳ CH— C C00M이고; PAD는In Formula 3, W-M 'is CH2 GO0M or ᅳ CH— C C00M; PAD
D,L-폴리락트산, D-폴리락트산, 폴리만델릭산, D,L-락트산과 글리콜산의 공증합체, D,L-락트산과 만델릭산의 공중합체, D,L-락트산과 카프로락톤의 공중합체 및 D,L- 락트산과 1,4-디옥산 -2-온의 공중합체로 구성된 그룹으로부터 선택되는 것이며 ; R은 수소원자,또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸 또는 에틸기이고; M은 독립적으로 Na, K, 또는 Li이다. D, L-polylactic acid, D-polylactic acid, polymandelic acid, co-polymer of D, L-lactic acid and glycolic acid, copolymer of D, L-lactic acid and mandelic acid, D, L-lactic acid and caprolactone It is selected from the group consisting of a copolymer of and a copolymer of D, L-lactic acid and 1,4-dioxane-2-one; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; M is independently Na, K, or Li.
[화학식 4] [Formula 4 ]
S-0-PAD-C00-Q 상기 식 4에서, S는
Figure imgf000045_0001
고; L은 -Ν -또는 -0-이며, 여기서
S-0-PAD-C00-Q In Formula 4, S is
Figure imgf000045_0001
High; L is -Ν -or -0-, where
¾은 수소원자또는 C1-10알킬이고; Q는 CH3, CH2CH3, C¾CH2CH3, CH2CH2CH2CH3, 또는 C¾C6H5이고; a는 0 내지 4의 정수이며; b는 1 내지 10의 정수이고; M은 Na, K, 또는 Li이며; PAD는 D,L-폴리락트산, D-폴리락트산, 폴리만델릭산 , D,L-락트산과 글리콜산의 공증합체, D,L-락트산과 만델릭산의 공증합체, D,L-락트산과 ¾ is a hydrogen atom or C 1-10 alkyl; Q is CH 3 , CH 2 CH 3 , C¾CH 2 CH 3 , CH 2 CH 2 CH 2 CH 3 , or C¾C 6 H 5 ; a is an integer from 0 to 4; b is an integer from 1 to 10; M is Na, K, or Li; PAD is composed of D, L-polylactic acid, D-polylactic acid, polymandelic acid, co-polymer of D, L-lactic acid and glycolic acid, co-polymer of D, L-lactic acid and mandelic acid, D, L-lactic acid and
카프로락톤의 공증합체, 및 D,L-락트산과 1,4-디옥산 -2-온의 공중합체로 이루어진 군에서 선택되는 하나 이상이다. At least one selected from the group consisting of a co-polymer of caprolactone and a copolymer of D, L-lactic acid and 1,4-dioxane-2-one.
[화학식 5]  [Formula 5]
Figure imgf000045_0002
상기 식 5에서, R'는 -PAD-0-C(0)-CH2CH2-C(0)-OM이고, 여기서 PAD는 D,L-폴리락트산, D-폴리락트산, 폴리만델릭산, D,L-락트산과 글리콜산의 공증합체, D,L-락트산과 만델릭산의 공중합체, D,L-락트산과 카프로락톤의 공증합체, D,L— 락트산과 1,4-디옥산 -2-온의 공중합체로 구성된 그룹으로부터 선택되는 것이고, M은 Na, K, 또는 Li이며; a는 1 내지 4의 정수이다.
Figure imgf000045_0002
In Formula 5, R 'is -PAD-0-C (0) -CH 2 CH 2 -C (0) -OM, wherein PAD is D, L-polylactic acid, D-polylactic acid, polymandelic acid , Copolymer of D, L-lactic acid and glycolic acid, Copolymer of D, L-lactic acid and mandelic acid, Copolymer of D, L-lactic acid and caprolactone, D, L—Lactic acid and 1,4-dioxane Is selected from the group consisting of copolymers of 2-ones, M is Na, K, or Li; a is an integer of 1-4.
[화학식 6]  [Formula 6]
Y0-[ -C(O) -(CI^ )a^- ]BrC(0)-R-C(Ό -[-0-(CHX, )b-c(o)-]d-oz 상기 식 6에서, X 및 X'은 독립적으로 수소, 탄소수가 1~10인 알킬 또는 탄소수가 6~20인 아릴이고; Y 및 Z는 독립적으로 Na, K, 또는 Li이며; m 및 n은 독립적으로 0 내지 95의 정수이되, 5 < m + n < 100이고; a 및 b는 독립적으로 1 내지 6의 정수이며; R은 -(CH2)k-, 탄소수가 2~10인 2가 알케닐 (divalent alkenyl), 탄소수가 6~20인 2가 아릴 (divalent aryl)또는 이들의 조합이고, 여기서 k는 0 내지 10의 정수이다. Y0- [-C (O)-(CI ^) a ^-] B rC (0) -RC (Ό-[-0- (CHX , ) b -c (o)-] d -oz X and X 'are independently hydrogen, alkyl having 1 to 10 carbon atoms or aryl having 6 to 20 carbon atoms; Y and Z are independently Na, K, or Li; m and n are independently 0 to 95 An integer of 5 <m + n <100; a and b are independently integers of 1 to 6; R is-(CH 2 ) k- , divalent alkenyl having 2 to 10 carbon atoms And divalent aryl having 6 to 20 carbon atoms or a combination thereof, wherein k is an integer of 0 to 10.
【청구항 27】  [Claim 27]
유효성분으로서 음이온성 약물;  Anionic drugs as active ingredients;
양이온성 화합물; Cationic compounds;
양친성 블록 공증합체; 및 Amphiphilic block co-polymers; And
하기 화학식 1 내지 6의 화합물로 이루어진 군으로부터 선택되는 하나 이상인 폴리락트산염을 포함하며, To include at least one polylactic acid salt selected from the group consisting of compounds of the formula 1 to 6,
상기 음이온성 약물은 상기 양이온성 화합물과 정전기적 상호작용에 의해 복합체를 형성하고, 상기 복합체는 양친성 블록 공중합체 및 폴리락트산염이 형성하는 미셀 구조 내부에 봉입되어 있는 것을 특징으로 하는 조성물을, 음이온성 약물의 전달을 필요로 하는 환자에게 투여하는 단계를 포함하는 음이온성 약물의 전달 방법: The anionic drug forms a complex by electrostatic interaction with the cationic compound, wherein the complex is encapsulated inside a micelle structure formed by the amphiphilic block copolymer and polylactic acid salt, A method of delivering an anionic drug, comprising administering to a patient in need thereof.
[화학식 1]  [Formula 1]
RO-CHZ- [A]n- [B]m-COOM RO-CHZ- [A] n- [B] m -COOM
상기 식 1에서 , Α는 -COO-CHZ-이고; Β는 -COO-CHY-, -COO- C¾CH2CH2CH2CH2- 또는 -COO-CH2CH2OCH2이며; R은 수소원자,또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸, 또는 에틸기이고; Z와 Y는 각각수소원자,또는 메틸 또는 페닐기이며; M은 Na, K, 또는 Li이고; n은 1 내지 30의 정수이며; m은 0 내지 20의 정수이다. In formula 1, A is -COO-CHZ-; Β is -COO-CHY-, -COO-C¾CH 2 CH 2 CH 2 CH 2 -or -COO-CH 2 CH 2 OCH 2 ; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl, or ethyl group; Z and Y are each hydrogen atom, or Methyl or phenyl group; M is Na, K, or Li; n is an integer from 1 to 30; m is an integer of 0-20.
[화학식 2]  [Formula 2]
RD-CHZ- [C0O-CHX] p- [COO-GHY' ]g-C00-CHZ-C0OM RD-CHZ- [C0O-CHX] p- [COO-GHY '] g -C00-CHZ-C0OM
상기 식 2에서, X는 메틸기이고; Y'는 수소원자또는 페닐기이며; p는 0 내지 25의 정수이고, q는 0 내지 25의 정수이되, 단 p+q는 5 내지 25의 정수이고; R은 수소원자,또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸 또는 에틸기이며; M은 Na, K, 또는 Li이고; Z 는 수소 원자, 메틸 또는 페닐기이다.  In formula 2, X is a methyl group; Y 'is a hydrogen atom or a phenyl group; p is an integer from 0 to 25, q is an integer from 0 to 25, provided that p + q is an integer from 5 to 25; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; M is Na, K, or Li; Z is a hydrogen atom, a methyl or a phenyl group.
[화학식 3]  [Formula 3]
R으 PAD— COO-W-tf  R PAD— COO-W-tf
C00M  C00M
—— C— CI½C00M COO  —— C— CI½C00M COO
상기 식 3에서, W-M'는 CH2CO0M 또는 ᅳ CHᅳ C C00M이고; PAD는 D,L-폴리락트산 , D-폴리락트산, 폴리만델릭산, D,L-락트산과 글리콜산의 공중합체, D,L-락트산과 만델릭산의 공중합체, D,L-락트산과 카프로락톤의 공중합체 및 D,L- 락트산과 1,4-디옥산 -2-온의 공중합체로 구성된 그룹으로부터 선택되는 것이며; R은 수소원자,또는 아세틸, 벤조일, 데카노일, 팔미토일, 메틸 또는 에틸기이고; M은 독립적으로 Na, K, 또는 Li이다. In Formula 3, W-M 'is CH2CO0M or ᅳ CH ᅳ C C00M; PAD is D, L-polylactic acid, D-polylactic acid, polymandelic acid, copolymer of D, L-lactic acid and glycolic acid, copolymer of D, L-lactic acid and mandelic acid, D, L-lactic acid and A copolymer of caprolactone and a copolymer of D, L-lactic acid and 1,4-dioxane-2-one; R is a hydrogen atom or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; M is independently Na, K, or Li.
[화학식 4]  [Formula 4]
S-0-PAD-C00-Q 상기 식 4에서, S는
Figure imgf000047_0001
고; L은 또는 -0-이며, 여기서
S-0-PAD-C00-Q In Formula 4, S is
Figure imgf000047_0001
High; L is or -0-, where
Ri은 수소원자또는 CM0알킬이고; Q는 CH3, CH2CH3, CH2CH2CH3, CH2CH2CH2CH3, 또는 CH2C6H5이고; a는 0 내지 4의 정수이며; b는 1 내지 10의 정수이고; M은 Na, K, 또는 Li이며; PAD는 D,L-폴리락트산, D-폴리락트산, 폴리만델릭산 , D,L-락트산과 글리콜산의 공중합체, D,L-락트산과 만델릭산의 공중합체, D,L-락트산과 Ri is a hydrogen atom or C M0 alkyl; Q is CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 , CH 2 CH 2 CH 2 CH 3 , or CH 2 C 6 H 5 ; a is an integer from 0 to 4; b is an integer from 1 to 10; M is Na, K, or Li; PAD is D, L-polylactic acid, D-polylactic acid, polymandelic acid, copolymer of D, L-lactic acid and glycolic acid, copolymer of D, L-lactic acid and mandelic acid, D, L-lactic acid and
카프로락톤의 공중합체, 및 D,L-락트산과 1,4-디옥산 -2-온의 공중합체로 이루어진 군에서 선택되는 하나 이상이다. At least one selected from the group consisting of a copolymer of caprolactone and a copolymer of D, L-lactic acid and 1,4-dioxane-2-one.
[화학식 5]
Figure imgf000048_0001
[Formula 5]
Figure imgf000048_0001
상기 식 5에서, R'는 -PAD-0-C(0)-CH2CH2-C(0)-OM이고, 여기서 PAD는 D,L-폴리락트산, D-폴리락트산, 폴리만델릭산, D,L-락트산과 글리콜산의 공증합체, D,L-락트산과 만델릭산의 공증합체, D,L-락트산과 카프로락톤의 공중합체, D,L- 락트산과 1,4-디옥산 -2-온의 공증합체로 구성된 그룹으로부터 선택되는 것이고, M은 Na,K, 또는 Li이며; a는 1 내지 4의 정수이다. In Formula 5, R 'is -PAD-0-C (0) -CH 2 CH 2 -C (0) -OM, wherein PAD is D, L-polylactic acid, D-polylactic acid, polymandelic acid , D, L-lactic acid and glycolic acid co-polymer, D, L-lactic acid and mandelic acid co-polymer, D, L-lactic acid and caprolactone copolymer, D, L-lactic acid and 1,4-dioxane 2-one is selected from the group consisting of co-polymers, M is Na, K, or Li; a is an integer of 1-4.
[화학식 6]  [Formula 6]
YO-L-C(O') - eHX)a-o- ]M-C(0)- -C O)- L-O-(CHX' )b—( 0)— ]n— oz 상기 식 6에서, X 및 X'은 독립적으로 수소, 탄소수가 1~10인 알킬 또는 탄소수가 6~20인 아릴이고; Y 및 Z는 독립적으로 Na,K, 또는 Li이며; m 및 n은 독립적으로 0 내지 95의 정수이되, 5<m + n<100이고; a및 b는 독립적으로 1 내지 6의 정수이며; R은 -(CH2)k-, 탄소수가 2~10인 2가 알케닐 (divalent alkenyl), 탄소수가 6 20인 2가 아릴 (divalent aryl) 또는 이들의 조합이고, 여기서 k는 0 내지 10의 정수이다. YO-LC (O ' )-eHX) a -o-] M -C (0)--CO)-LO- (CHX') b — (0) —] n — oz In Equation 6 above, X and X Is independently hydrogen, alkyl having 1 to 10 carbon atoms or aryl having 6 to 20 carbon atoms; Y and Z are independently Na, K, or Li; m and n are independently integers from 0 to 95, with 5 <m + n <100; a and b are independently an integer of 1 to 6; R is-(CH 2 ) k- , divalent alkenyl having 2 to 10 carbon atoms, divalent aryl having 6 20 carbon atoms, or a combination thereof, wherein k is 0 to 10 Is an integer.
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