WO2013073697A1 - フェニルボロン酸基が導入されたブロックコポリマーおよびその使用 - Google Patents
フェニルボロン酸基が導入されたブロックコポリマーおよびその使用 Download PDFInfo
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- WO2013073697A1 WO2013073697A1 PCT/JP2012/079897 JP2012079897W WO2013073697A1 WO 2013073697 A1 WO2013073697 A1 WO 2013073697A1 JP 2012079897 W JP2012079897 W JP 2012079897W WO 2013073697 A1 WO2013073697 A1 WO 2013073697A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
- A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/40—Polyamides containing oxygen in the form of ether groups
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/42—Polyamides containing atoms other than carbon, hydrogen, oxygen, and nitrogen
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G2650/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G2650/28—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule characterised by the polymer type
Definitions
- the present invention relates to a block copolymer having a phenylboronic acid group introduced therein and a complex containing the block copolymer and a drug.
- Biopharmaceuticals that use biopolymers such as proteins and nucleic acids are more likely to be degraded by enzymes or eliminated by the immune system than conventional drugs that use low molecular weight compounds.
- the present inventor has developed a drug delivery system (DDS) using a polyamino acid-based block copolymer from the viewpoint of improving the reachability of such bio-based drugs to the affected area.
- DDS drug delivery system
- One of the goals of the development is to provide a carrier that achieves both stability in the blood (retention of bio-based drugs) and drug release at the affected area.
- a phenylboronic acid compound in which a phenyl ring is fluorinated has been produced as a biocompatible material (Patent Document 1).
- Patent Document 2 is a related art relating to a reagent that is applied to the behavior and structural analysis of nucleic acids and nucleic acid-related substances by supporting a nucleic acid on a polyamino acid derivative using a phenylboronic acid group.
- JP 2011-140537 A Japanese Patent Laid-Open No. 2002-179683
- the main object of the present invention is to provide a carrier in which the stability of the bio-based drug in blood and the release at the affected area are compatible.
- Patent Document 2 describes that a reagent carrying a nucleic acid-related substance is applied and developed as a DDS. However, the DDS developed in this way is only a prodrug such as a pseudo-RNA and is in a state where precipitation is likely to occur.
- the technique described in Patent Document 2 is directed to a DDS that is different from the DDS that the inventor has developed in the first place, and cannot be easily applied as a carrier having excellent stability in blood.
- a biocompatible material produced for a purpose different from the development of DDS greatly improves the stability of bio-based drugs in blood by polyamino acid block copolymers, and completed the present invention.
- a block copolymer comprising a polyamino acid chain segment and a hydrophilic polymer chain segment, wherein the polyamino acid chain segment comprises an amino acid residue having a cationic group in the side chain and a side chain.
- a block copolymer comprising an amino acid residue having a phenylboronic acid group in which at least one hydrogen of the phenyl ring has been substituted to have a pKa near physiological pH.
- a complex is provided. The complex is a complex of the block copolymer and the biopolymer.
- a carrier that can achieve both the stability of a bio-based drug in blood and the release in a target cell.
- the block copolymer of the present invention comprises a polyamino acid chain segment and a hydrophilic polymer chain segment.
- the polyamino acid chain segment includes an amino acid residue having a cationic group in the side chain (hereinafter sometimes referred to as “cationic amino acid residue”) and a phenyl group having a pKa in the vicinity of physiological pH in the side chain.
- An amino acid residue having a phenylboronic acid group substituted with at least one hydrogen of the ring hereinafter sometimes referred to as “substituted PBA group” (hereinafter referred to as “substituted PBA group-containing amino acid residue”). ).
- the cationic amino acid residue and the substituted PBA group-containing amino acid residue may be different amino acid residues or the same amino acid residue.
- the polyamino acid chain segment may include a cationic amino acid residue having no substituted PBA group in the side chain and a substituted PBA group-containing amino acid residue having no cationic group in the side chain.
- an amino acid residue having both a cationic group and a substituted PBA group in the side chain may be included.
- the substituted PBA group has at least one hydrogen of the phenyl ring constituting the phenylboronic acid group so as to have a pKa around physiological pH. Is substituted by any substituent.
- the pKa of the substituted PBA group is preferably less than 8, and more preferably less than 7.5.
- the number of hydrogens to be substituted is 1, 2, 3, or 4, and when only one hydrogen is substituted, the substituent and the introduction site of B (OH) 2 can be any of ortho, meta, and para. Good.
- the substituent include halogens such as fluorine, chlorine and bromine, nitro groups, and the like.
- the substituted PBA group is a fluorinated phenylboronic acid group (hereinafter referred to as “FPBA group”) represented by the following formula (I). It may be called The pKa of the substituted PBA group is specified from the substituted PBA group-containing amino acid synthesized as a monomer.
- the lower limit value of the pKa of the substituted PBA group is not particularly limited, but may be 2 or 3 for example.
- F is independently present, n is 1, 2, 3 or 4, and when n is 1, the introduction site of F and B (OH) 2 is ortho, meta Or any of Para.
- the block copolymer of the present invention has a cationic group in the side chain portion of the polyamino acid chain segment, and can associate with a biopolymer to form a complex, for example, a polyion complex (PIC).
- PIC polyion complex
- the block copolymer of the present invention has a substituted PBA group in the side chain portion of the polyamino acid chain segment, the following effects can be obtained.
- a hydrophobic interaction preferably works in addition to the electrostatic interaction between a plurality of the block copolymers of the present invention in an aqueous medium.
- the association force between the polymers is enhanced so that the block copolymers of the present invention can form very stable polymer micelles in an aqueous medium.
- the block copolymer of the present invention is presumed to be arranged radially with the polyamino acid chain segment on the inside and the hydrophilic polymer chain segment on the outside.
- aqueous medium include aqueous buffers such as water, physiological saline, phosphate buffer, carbonate buffer, borate buffer, and acetate buffer.
- the phenylboronic acid compound has a reversible covalent bonding ability with a molecule having 1,2-diol (cis-diol) or 1,3-diol in an aqueous medium. Therefore, the block copolymer of the present invention includes a substituted PBA group in addition to an electrostatic bond between a biopolymer having 1,2-diol or the like (for example, siRNA) and a cationic group in an aqueous medium. As a result, a stable complex (for example, a polymer micelle encapsulating the biopolymer) can be formed. In particular, since a siRNA having a cis-diol at each 3 'terminal ribose of a double strand can be bound at the two ends, a very stable complex can be formed.
- a biopolymer having 1,2-diol or the like for example, siRNA
- a cationic group in an aqueous medium.
- the pKa of phenylboronic acid is usually about 8 to 9, and the covalent binding ability with the molecule having the 1,2-diol etc. is maximized in the vicinity of the pKa.
- the use of phenylboronic acid in the environment has been considered difficult in principle.
- the substituted PBA groups introduced into the block copolymers of the present invention have a pKa near physiological pH (preferably exhibiting a pKa of less than 8, more preferably less than 7.5).
- the above binding ability can be suitably exerted in a living environment.
- the substituted PBA group is highly hydrophobized under a pH environment of pKa or lower, and therefore, by appropriately controlling pKa, it is possible to react with a molecule having the 1,2-diol or the like in a biological environment. Both bonding and hydrophobic interactions between block copolymers can be enhanced. As a result, extremely stable complexes with these molecules can be obtained.
- the polyamino acid chain segment includes a cationic amino acid residue having a cationic group in the side chain and a substituted PBA group-containing amino acid residue having a substituted PBA group in the side chain.
- the cationic amino acid residue is preferably a cationic amino acid residue having an amino group in the side chain.
- the amino group can be coordinated to boron of the substituted PBA group in an aqueous medium.
- the hydrophobicity of the block copolymer due to the introduction of the substituted PBA group is avoided, and high hydrophilicity can be maintained.
- the amino group is coordinated, the binding ability of the substituted PBA group to the molecule having the cis-diol can be maintained.
- amino acid from which the cationic amino acid residue having an amino group in the side chain is derived for example, any appropriate amine compound is introduced into basic amino acids such as lysine, ornithine, arginine, homoarginine, histidine and acidic amino acids. And amino acid derivatives.
- amino acid derivatives in which the carboxyl group (—C ( ⁇ O) OH) of lysine and acidic amino acid is substituted with any group of the following formulas (i) to (iv) are preferable, and lysine and asparagine
- the —OH part of the carboxyl group at the ⁇ -position or ⁇ -position of the acid or the ⁇ -position or ⁇ -position of the glutamic acid (—C ( ⁇ O) OH) is substituted with any group of the following formulas (i) to (iv)
- Amino acid derivatives are more preferred, and the —OH part of the carboxyl group (—C ( ⁇ O) OH) at the ⁇ -position or ⁇ -position of lysine and aspartic acid or the ⁇ -position or ⁇ -position of glutamic acid is substituted with a group of the following formula (i)
- the amino acid derivatives obtained are more preferred.
- p1 to p4 and q1 to q6 are independently of each other, preferably 2 or 3, more preferably 2.
- R1 and r2 are each independently an integer of 1 to 3, preferably.
- the cationic amino acid residue is an amino acid residue in which the —OH part of the carboxyl group (—C ( ⁇ O) OH) of the acidic amino acid is substituted with any group of the above formulas (i) to (iv).
- these residues have different amine functional groups, so pKa exhibits multiple steps, and at physiological conditions pH 7.4, the multiple amine functional groups are partially protonated, It has been shown that damage to cells is low.
- the substituted PBA group is typically introduced into the side chain via a divalent linking group.
- the divalent linking group include an amide bond, a carbamoyl bond, an alkyl bond, an ether bond, an ester bond, a thioester bond, a thioether bond, a sulfonamide bond, a urethane bond, a sulfonyl bond, a thymine bond, a urea bond, and a thiourea bond.
- any appropriate amino acid residue can be selected as long as the substituted PBA group can be introduced through the linking group.
- the substituted PBA group is preferably introduced into a cationic amino acid residue having an amino group in the side chain.
- a substituted PBA group is a reaction between a cationic amino acid residue having an amino group in a side chain and carboxyphenylboronic acid or an ester thereof in which at least one hydrogen of the phenyl ring is substituted with the amino group.
- a cationic amino acid residue having a plurality of amino groups in the side chain only one substituted PBA group or a plurality of substituted PBA groups may be introduced.
- the substituted PBA group-containing amino acid residue after introduction is also a cationic amino acid residue because it has both an amino group and a substituted PBA group in the side chain. Therefore, in the present invention, it is understood that a polyamino acid chain segment containing only such amino acid residues also contains both cationic amino acid residues and substituted PBA group-containing amino acid residues.
- a polyamino acid chain segment containing only such amino acid residues also contains both cationic amino acid residues and substituted PBA group-containing amino acid residues.
- the polyamino acid chain segment is an amino acid residue having a hydrophobic group in the side chain (hereinafter sometimes referred to as “hydrophobic amino acid residue”).
- hydrophobic amino acid residue By including the hydrophobic amino acid residue, the hydrophobic interaction acting between the block copolymers of the present invention is increased in an aqueous medium, and as a result, more stable polymer micelles can be formed. Furthermore, a hydrophobic amino acid residue can pierce the hydrophobic part of the cell membrane and function as an anchor for fixing the polymer micelle to the cell membrane. Therefore, when a biopolymer such as nucleic acid is encapsulated in the polymer micelle, the introduction rate of the biopolymer into cells can be improved.
- the amino acid from which the hydrophobic amino acid residue is derived is preferably an amino acid having a solubility in 100 g of water at 25 ° C. of 5 g or less, more preferably 4 g or less.
- amino acids include nonpolar natural amino acids such as leucine, isoleucine, phenylalanine, methionine, and tryptophan, and hydrophobic derivatives of amino acids having a hydrophobic group introduced in the side chain.
- the hydrophobic derivative of the amino acid preferably include a derivative in which a hydrophobic group is introduced into the side chain of an acidic amino acid such as aspartic acid or glutamic acid.
- the hydrophobic group to be introduced is preferably a saturated or unsaturated linear or branched aliphatic hydrocarbon group having 6 to 27 carbon atoms, an aromatic hydrocarbon group having 6 to 27 carbon atoms or a steryl group. Can be exemplified.
- saturated linear or branched aliphatic hydrocarbon group having 6 to 27 carbon atoms examples include a pentacosyl group, a hexacosyl group, a heptacosyl group and the like in addition to an alkyl group having 6 to 27 carbon atoms and the alkyl group. Illustrated.
- the unsaturated straight chain or branched aliphatic hydrocarbon group having 6 to 27 carbon atoms includes 1 to 5 carbon-carbon single bonds in the chain of the alkyl group having 6 to 27 carbon atoms. Examples thereof include a carbon double bond group.
- unsaturated aliphatic hydrocarbons from which such groups are derived include lauric acid (or dodecanoic acid), myristic acid (or tetradecanoic acid), palmitic acid (or hexadecanoic acid), palmitooleic acid (or 9 -Hexadecenoic acid), stearic acid (or octadecanoic acid), oleic acid, linoleic acid, linolenic acid, eleostearic acid (or 9,11,13-octadecatrienoic acid), arachidic acid, arachidonic acid, behenic acid, lignoserine Examples include acids, nervonic acid, serotic acid, and montanic acid.
- aromatic hydrocarbon group having 6 to 27 carbon atoms examples include aryl groups and aralkyl groups. Specific examples of these include phenyl group, naphthyl group, tolyl group, xylyl group, benzyl group, phenethyl group and the like.
- the sterol from which the steryl group is derived means a natural, semi-synthetic or synthetic compound based on a cyclopentanone hydrophenanthrene ring (C 17 H 28 ), or a derivative thereof.
- a natural sterol Includes, but is not limited to, cholesterol, cholestanol, dihydrocholesterol, cholic acid, campesterol, cystosterol and the like
- semi-synthetic or synthetic compounds include, for example, synthetic precursors of these natural products (If necessary, if present, certain functional groups, some or all of the hydroxy groups are protected by hydroxy protecting groups known in the art, or the carboxyl group is protected by a carboxyl protecting group. Compound).
- the sterol derivative means that a C 1-12 alkyl group and a halogen atom such as chlorine, bromine, and fluorine are introduced into the cyclopentanone hydrophenanthrene ring within a range that does not adversely affect the object of the present invention. It means that the ring system can be saturated, partially unsaturated, and the like.
- the sterol from which the steryl group is derived is preferably a sterol derived from animal or vegetable oils such as cholesterol, cholestanol, dihydrocholesterol, cholic acid, campesterol, cystosterol, and more preferably cholesterol, cholestanol, or dihydroxycholesterol. Particularly preferred is cholesterol.
- the polyamino acid chain segment may contain only one type of amino acid residue or two or more types of amino acid residues as a cationic amino acid residue, a substituted PBA group-containing amino acid residue, and a hydrophobic amino acid residue.
- a cationic amino acid residue a substituted PBA group-containing amino acid residue
- a hydrophobic amino acid residue a hydrophobic amino acid residue.
- the order of binding of the cationic amino acid residue, the substituted PBA group-containing amino acid residue, and the hydrophobic amino acid residue in the polyamino acid chain segment is arbitrary, and may be a random structure or a block structure.
- the number of cationic amino acid residues, substituted PBA group-containing amino acid residues and hydrophobic amino acid residues contained in the polyamino acid chain segment can be appropriately adjusted depending on the type of each amino acid residue, the use of the block copolymer, and the like. From the viewpoint of more reliably improving biopolymer retention (stability in blood), it is preferable to adjust the number of each amino acid residue so as to satisfy the following relational expression.
- a plurality of cationic groups are present in the repeating unit (amino acid residue) constituting the polyamino acid chain segment, a plurality of substituted PBA groups can be introduced into one repeating unit.
- the total number of substituted PBA groups in the polyamino acid chain segment is the number of cationic amino acid residues into which the substituted PBA groups are not introduced and the amino acid residues into which the substituted PBA groups are introduced (substituted amino acid residues containing substituted PBA groups). May exceed the sum of Moreover, in the analysis by a general-purpose structural analysis device, it is not easy to distinguish whether a plurality of substituted PBA groups are introduced into one repeating unit or dispersedly introduced into a plurality of repeating units. There may not be.
- the number of cationic amino acid residues not having a substituted PBA group is calculated from the sum (theoretical value) of the number of cationic amino acid residues and the substituted PBA group-containing amino acid residue. It is allowed to be set to a value obtained by subtracting the number of the substituted PBA groups (actual value) (however, 0 is the lower limit value).
- the upper limit of the following relational expression is not particularly limited, it may be 40, 38, 35, or 25, for example. When the value of the following relational expression is 14 or more, further 15 or more, the stability in blood is more reliably improved.
- the hydrophilic polymer chain segment can be constituted by any suitable hydrophilic polymer.
- the hydrophilic polymer include poly (ethylene glycol), polysaccharide, poly (vinyl pyrrolidone), poly (vinyl alcohol), poly (acrylamide), poly (acrylic acid), poly (methacrylamide), and poly (methacrylic). Acid), poly (methacrylic acid ester), poly (acrylic acid ester), polyamino acid, poly (malic acid), or derivatives thereof.
- Specific examples of polysaccharides include starch, dextran, fructan, galactan and the like.
- poly (ethylene glycol) is commercially available as terminal-reactive polyethylene glycol having various functional groups at its terminals, and various molecular weights are commercially available and can be easily obtained. It can be preferably used.
- [A-3. Specific example of block copolymer] Specific examples of the block copolymer in the particularly preferred embodiment of the present invention are shown in the formulas (1) to (4).
- the substituted PBA group is introduced into the side chain of the cationic amino acid residue.
- R 1 is a hydrogen atom or an unsubstituted or substituted linear or branched alkyl group having 1 to 12 carbon atoms
- R 2 is a hydrogen atom, an unsubstituted or substituted linear or branched alkyl group having 1 to 12 carbon atoms, or an unsubstituted or substituted linear or branched group having 1 to 24 carbon atoms.
- An alkylcarbonyl group, R 3 is a hydroxyl group, an unsubstituted or substituted linear or branched alkyloxy group having 1 to 12 carbon atoms, an unsubstituted or substituted linear or branched group having 2 to 12 carbon atoms.
- R 4a , R 4b , R 5a and R 5b are, independently of one another, a methylene group or an ethylene group;
- the groups R 6a and R 6b are independently selected from the groups (i) to (iv) above;
- the groups of R 7a and R 7b are, independently of one another, —O— or —NH—;
- R 8a and R 8b are each independently a saturated or unsaturated linear or branched aliphatic hydrocarbon group having 6 to 27 carbon atoms, or an aromatic hydrocarbon group having 6 to 27 carbon atoms.
- L 1 and L 3 are, independently of each other, —SS— or a valence bond;
- L 2 is —NH—, —O—, —O (CH 2 ) p1 —NH—, or —L 2a — (CH 2 ) q1 —L 2b —, wherein p1 and q1 are Independently, an integer from 1 to 5,
- L 2a is OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO
- L 2b is NH or O
- L 4 is —OCO— (CH 2 ) p2 —CO—, —NHCO— (CH 2 ) p3 —CO—, or —L 4a — (CH 2 ) q2 —CO—, where p2, p3 , And q2 are
- k representing the repeating number of ethylene glycol (or oxyethylene) is an integer of 30 to 20,000, preferably 40 to 2,000, more preferably 50 to 1,500. It is.
- Examples of the linear or branched alkyloxy group having 1 to 12 carbon atoms, the alkyl-substituted imino group, and the alkyl moiety of the alkyl group defined by the above R 1 to R 3 groups include, for example, methyl group, ethyl group N-propyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group, n-hexyl group, decyl group, and undecyl group.
- alkenyl or alkynyl moiety in the straight chain or branched alkenyloxy group having 2 to 12 carbon atoms or the straight chain or branched alkynyloxy group having 2 to 12 carbon atoms is exemplified in the above examples having 2 or more carbon atoms And those containing a double bond or a triple bond in the alkyl group.
- the substituents when “substituted” include, but are not limited to, C 1-6 alkoxy groups, aryloxy groups, aryl C 1-3 oxy groups, cyano groups, carboxyls A group, an amino group, a C 1-6 alkoxycarbonyl group, a C 2-7 acylamide group, a tri-C 1-6 alkylsiloxy group, a siloxy group, a silylamino group, or an acetalized formyl group, formyl group, chlorine or Mention may be made of halogen atoms such as fluorine.
- a display such as C 1-6 means 1 to 6 carbon atoms, and the same meaning is used hereinafter.
- the unsubstituted or substituted linear or branched alkylcarbonyl group having 1 to 12 carbon atoms in the unsubstituted or substituted linear or branched alkylcarbonyl group having 1 to 24 carbon atoms is as described above.
- Examples of the alkyl moiety having 13 or more carbon atoms include a tridecyl group, a tetradecyl group, a pentadecyl group, a nonadecyl group, a docosanyl group, and a tetracosyl group.
- the group R 1 may be substituted with a group that includes a target binding site.
- the group containing the target binding site may be any suitable group as long as it has directivity or functionality for the target tissue, for example, an antibody or a fragment thereof, or other functionality or targeting property It may be a group derived from a physiologically active substance such as a protein, a peptide, an aptamer, a sugar such as lactose, a folic acid, or a derivative thereof.
- D is preferably a peptide having a weight average molecular weight of 50 to 20,000, more preferably a peptide having a weight average molecular weight of 100 to 10,000, and still more preferably a peptide having a weight average molecular weight of 150 to 3,000. It is.
- D is preferably a peptide having 1 to 200 amino acid residues, more preferably a peptide having 1 to 100 amino acid residues, and having 1 to 30 amino acid residues. More preferably, it is a peptide.
- the peptides include peptides that can specifically bind to integrins involved in angiogenesis, intimal thickening, and malignant tumor growth, and specifically include RGD peptides.
- RGD peptide refers to a peptide containing an arginine-glycine-aspartic acid (RGD) sequence.
- RGD peptide is a cyclic RGD (cRGD) peptide.
- D can be represented by the following formula (IV).
- the groups of Q, R 6a , R 6b , R 8a and R 8b the same group may be selected for all the repeating units to which they belong, or different groups may be selected.
- s is 1, 3, or 4, for example.
- R 4a and R 4b groups represent ethylene groups
- n is the integer 0 or mn is the integer 0.
- the former represents, for example, poly- ⁇ -glutamic acid obtained by polymerization of N-carboxylic acid anhydride of glutamic acid ⁇ -benzyl ester, and the latter is, for example, a strain belonging to the genus Bacillus including Bacillus natto. Represents poly- ⁇ -glutamic acid produced.
- both groups of R 4a and R 4b represent methylene groups, it is understood that the respective repeating units having these groups can coexist.
- the groups R 5a and R 5b are preferably methylene groups, and the groups R 5a and R 5b are ethylene groups.
- the total number z of the cationic amino acid residues and substituted PBA group-containing amino acid residues contained in the block copolymer of the formula (1) or (2) is 1 to 300, preferably from the viewpoint of the stability of the polymer micelle formed. It is an integer of 20 to 300, more preferably 30 to 200, still more preferably 40 to 150.
- the total number m of the cationic amino acid residues and substituted PBA group-containing amino acid residues contained in the block copolymer of the formula (3) or (4) is 1 to 300, preferably from the viewpoint of the stability of the polymer micelle formed. It is an integer of 1 to 200, more preferably 1 to 150.
- the block copolymer contains hydrophobic amino acid residues (that is, when x is not 0)
- the number of cationic amino acid residues is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, More preferably, it can be an integer of 1 to 5. According to such a block copolymer, the release of a biopolymer represented by a nucleic acid can be performed more suitably.
- the number x of hydrophobic amino acid residues that can be contained in the block copolymer of the formula (3) or (4) can be appropriately adjusted depending on the type or number of cationic amino acid residues, the use of the block copolymer, and the like.
- the number x of the hydrophobic amino acid residues is preferably an integer of 1 to 80, more preferably 5 to 70, and still more preferably 10 to 60.
- the number of substituted PBA group-containing amino acid residues can be appropriately adjusted depending on the type or number of cationic amino acid residues, the use of the block copolymer, and the like.
- the substituted PBA group is preferably introduced into the side chain of the cationic amino acid residue so as to satisfy the following relationship. When such a relationship is satisfied, the retention of the biopolymer by the block copolymer (stability in blood) is more reliably improved.
- the upper limit of the following relational expression is not particularly limited, it may be 40, 38, 35, or 25, for example. When the value of the following relational expression is 14 or more, further 15 or more, the stability in blood is more reliably improved.
- the block copolymers of the present invention can be made by any suitable synthetic method.
- An example of a method for synthesizing a block copolymer in a preferred embodiment of the present invention is as follows.
- an anion living polymerization is performed using an initiator capable of imparting R 1 to form a polyethylene glycol chain, and an amino group is introduced to the growth terminal side of the polyethylene glycol chain; NCA-Lys from the amino terminal Polymerizing a protected amino acid derivative such as (TFA) to form a polyamino acid chain segment; deprotecting the side chain of the polyamino acid to expose an amino group; and the exposed amino group and fluorine By reacting with the carboxyl group of the activated carboxyphenylboronic acid and introducing the desired number of FPBA groups into the side chain via an amide bond.
- an initiator capable of imparting R 1 to form a polyethylene glycol chain
- an amino group is introduced to the growth terminal side of the polyethylene glycol chain
- NCA-Lys from the amino terminal Polymerizing a protected amino acid derivative such as (TFA) to form a polyamino acid chain segment
- the block copolymer in another preferred embodiment of the present invention can be prepared, for example, as follows. That is, an anion living polymerization is performed using an initiator capable of imparting R 1 to form a polyethylene glycol chain, and an amino group is introduced to the growth terminal side of the polyethylene glycol chain; Polymerizing a protected amino acid N-carboxylic acid anhydride such as L-aspartate or ⁇ -benzyl-L-glutamate to form a polyamino acid chain segment; then, the polyamino acid and diethylenetriamine (DET), etc.
- an anion living polymerization is performed using an initiator capable of imparting R 1 to form a polyethylene glycol chain, and an amino group is introduced to the growth terminal side of the polyethylene glycol chain; Polymerizing a protected amino acid N-carboxylic acid anhydride such as L-aspartate or ⁇ -benzyl-L-glutamate to form a polyamino acid chain segment; then, the polyamino acid
- An amine residue such as a DET group is introduced into the amino acid side chain by an ester-amide exchange reaction; then the amino group of the amine residue and the carboxyl group of fluorinated carboxyphenylboronic acid And reacting with the desired number of FPBA by amide bonds It may be prepared by: introducing into the side chain.
- ⁇ -benzyl-L-aspartate and ⁇ -benzyl-L-glutamate are used in combination to form a polyamino acid chain segment
- the subsequent transesterification reaction is performed on ⁇ -benzyl-L-aspartate. Preferentially occurs.
- a block copolymer containing an amino acid residue derived from ⁇ -benzyl-L-glutamate as a hydrophobic amino acid residue can be obtained.
- a structural change (for example, formation of an imide ring by dealcoholization of an amino acid ester residue) may occur in a part of the amino acid ester residue due to nucleophilic attack of an amine.
- the polyamino acid chain segment may further include residues that have undergone such a structural change.
- the residue that has undergone the above structural change is not included in any of the cationic amino acid residue, the substituted PBA group-containing amino acid residue, and the hydrophobic amino acid residue.
- some NH groups and NH 2 groups in the cationic amino acid residue may be converted into a salt (mainly hydrochloride) due to the use of an acid (mainly hydrochloric acid) in the synthesis process.
- the polyamino acid chain segment may contain such a structure. That is, some NH groups and NH 2 groups in the groups of Q, R 6a and R 6b may be salts (for example, hydrochlorides).
- a block copolymer having a target binding site at the end of a hydrophilic polymer can be synthesized by introducing a group containing a target binding site after synthesizing a block copolymer as described above using .
- Various methods can be used as a method for introducing a group containing a target binding site.
- a block copolymer having a polyethylene glycol chain acetalized at the ⁇ -terminus and a compound having a desired target binding site having a cysteine terminus are used.
- the target binding site can be imparted to the end of the polyethylene glycol chain.
- the complex of the present invention is a complex of the block copolymer described in the above section A and a biopolymer.
- the complex may be a PIC of a plurality of the block copolymers and a biopolymer.
- the PIC may be in the form of a polymer micelle in which a biopolymer is encapsulated by a plurality of the block copolymers.
- the conventional carrier has a problem that the complex is dissociated in a medium having an ionic concentration similar to physiological conditions, but the complex of the present invention is excellent in stability. Even in a medium containing, the form of the complex can be maintained.
- biopolymer examples include proteins, lipids, and nucleic acids. As used herein, protein also includes peptides.
- the block copolymer can suitably form a complex with a nucleic acid.
- An example of a biopolymer suitable for forming a complex is an anionically chargeable compound having a negative charge in a pH range of pKa or less of the block copolymer.
- the block copolymer has a cationic group in the amino acid side chain of the polyamino acid chain segment, so that the block copolymer is a complex that is stable under physiological conditions even with a small molecular weight nucleic acid (for example, Vesicles or aggregates).
- Nucleic acid that can provide a complex with a block copolymer means a poly or oligonucleotide having a nucleotide unit consisting of a purine or pyrimidine base, pentose, and phosphate, as an oligo or poly double-stranded RNA, oligo or poly dinucleotide.
- nucleic acid may be a natural type or a non-natural type that has been chemically modified, and may have a molecule such as an amino group, a thiol group, or a fluorescent compound added thereto. Also good.
- the nucleic acid is RNA
- the RNA since the RNA has a 1,2-cis-diol at the 3 ′ end ribose, in addition to the electrostatic interaction with the cationic group of the cationic amino acid residue, the substituted PBA It can be reversibly covalently bonded to the block copolymer via a group.
- a composite having excellent stability can be obtained.
- replacement of the RNA molecule with blood usually about 4 to 7 mM
- glucose having a cis-diol structure in the molecule
- RNA since a cross-linked structure having the 3′-terminal ribose of each strand as a binding site can be formed inside the micelle, a complex with extremely excellent stability can be obtained.
- the complex with the RNA is incorporated into an endosome that is in a low pH environment, the hydrophobic interaction is increased by hydrophobizing the substituted PBA group, so that the stability can be further improved.
- RNA is rapidly released by replacement with other molecules having 1,2-cis-diol present at a relatively high concentration in the cytoplasm, such as ATP and ribonucleic acid. It is possible.
- the chain length of the nucleic acid can be, for example, 4 to 20,000 bases, preferably 10 to 10,000 bases, more preferably 18 to 30 bases.
- nucleic acid examples include plasmid DNA, siRNA, micro RNA, shRNA, antisense nucleic acid, decoy nucleic acid, aptamer, and ribozyme in view of its function or action.
- the content ratio of the block copolymer and the biopolymer in the composite of the present invention can be appropriately set according to the types of the block copolymer and the biopolymer, the use of the composite, and the like.
- the complex of the present invention preferably has an N / P ratio of 2 or more from the viewpoint of improving the stability under physiological conditions, and the viewpoint of suppressing toxicity due to the polymer.
- the N / P ratio is preferably 200 or less.
- the N / P ratio means [the amino group concentration of the polyamino acid side chain in the block copolymer contained in the complex] / [the phosphate group concentration in the nucleic acid].
- the complex of the present invention when the biopolymer is siRNA has a [positive number of polyamino acid chain segments] / [polyamino acid] at pH 7.4 from the viewpoint of improving stability under physiological conditions.
- the total number of negative charges of the strand segments and the negative charges of the siRNA] can be, for example, 1/2 to 20/1, preferably 1/1 to 10/1.
- the complex of the present invention can be used, for example, in an aqueous solution buffered as necessary. And can be prepared only by mixing biopolymers. That is, the complex of the present invention does not need to be separately modified at the terminal of the block copolymer, and may have an advantage that it can be prepared by a simple method.
- the present invention will be specifically described by way of examples, but the present invention is not limited to these examples.
- the polymer structure is described in the order of “molecular weight of PEG (kDa) ⁇ number of amino acid residues containing FPBA group / total number of amino acid residues constituting the polyamino acid chain segment”.
- the molecular weight of PEG is 10,000 and the polyamino acid chain segment is a polylysine having a polymerization degree of 40 and 5 FPBA groups are introduced, it is expressed as “PEG-PLL 10-5 / 40”.
- the molecular weight of PEG is 10,000 and the polyamino acid chain segment is polylysine having a polymerization degree of 40 into which no FPBA group is introduced, it is expressed as “PEG-PLL 10-0 / 40”.
- each number in the notation is an average value of the entire block copolymer.
- MeO-PEG-PLys (TFA).
- MeO-PEG-PLys and 5 equivalents of D-Mannitol with respect to the amino group of PLys are dissolved in 50 mM aqueous NaHCO 3 solution.
- GPC gel permeation chromatograph
- the molecular weight of the obtained block copolymer was measured by gel permeation chromatograph (GPC). At that time, a solution in which 50 mg / mL D-Sorbitol was dissolved in 50 mM phosphate buffer (pH 7.4) was used as a mobile phase. The amount of FPBA group introduced was estimated from the integral ratio of the side chain to the phenyl ring in the 1 H-NMR spectrum. At that time, a solution in which the polymer was dissolved in D 2 O containing a small amount of NaOD was used as a measurement sample.
- siRNA The sequence of siRNA used in the following experimental examples is as follows. All labels such as Cy3 were introduced at the 5 ′ end of the sense strand.
- hVEGF-siRNA siRNA against human vascular endothelial growth factor
- Sense strand 5′-GAUCUCAUCAGGGUACUCCdTdT-3 ′
- Antisense strand 5'-GAGAGUACCCUGAUGAAUGdTdT-3 '(SEQ ID NO: 2)
- scramble-siRNA non-therapeutic sequence siRNA
- Sense strand 5′-UUCCUCCGAACGUGUCACGGUUU-3 ′
- Antisense strand 5′-ACGUGACACGUUCGGGAGAAUU-3 ′
- GL3-siRNA siRNA against firefly luciferase
- Sense strand 5′-CUUACGCUGACUACUCUCAUU
- the obtained diluted solution was allowed to stand at room temperature for about 1 hour, and the diffusion time of siRNA was measured (10 sec ⁇ 10 times) with a confocal laser scanning microscope (manufactured by Carl Zeiss, product name “LSM510”). The results are shown in Table 1 and FIG.
- the diffusion time (diffusion time of siRNA alone) when measured in the same manner except that it is not mixed with the block copolymer is 157.5 ⁇ sec. Note that GL3-siRNA was used as the siRNA.
- the complex was added to and mixed with each block copolymer and GL3-siRNA in 10 mM HEPES buffer (pH 7.3) so that the N / P ratio was 4 and the siRNA concentration was 80 nM. After standing at room temperature for about an hour, it was diluted to various concentrations before use.
- the block copolymer treatment group into which FPBA groups were introduced showed higher cell viability than the block copolymer treatment group into which FPBA groups were not introduced.
- the composite treatment group formed using the block copolymer introduced with the FPBA group has higher cell survival than the complex treatment group formed using the block copolymer into which the FPBA group has not been introduced. Showed the rate. From the above results, cytotoxicity due to the introduction of FPBA groups is not observed, and it can be seen that the block copolymer having FPBA groups introduced has sufficient biocompatibility.
- Block copolymer (PEG-PLL 12-23 / 43) and GL3-siRNA labeled with Cy3 were dissolved in various pH buffers (phosphate buffer or phosphate-citrate buffer), and N / P ratio was 4 and the siRNA concentration was 50 nM.
- the obtained mixture was allowed to stand at room temperature for about 1 hour, and the diffusion time of siRNA was measured (10 sec ⁇ 10 times) with a confocal laser scanning microscope (manufactured by Carl Zeiss, product name “LSM510”). The results are shown in FIG.
- siRNA-encapsulating polymer micelle As shown in FIG. 4, since the diffusion time of siRNA is longer than 800 ⁇ sec in a wide pH range from the in vivo environment (about pH 7.4) to the acidic environment (about pH 5.5) in the endosome, the present invention. It can be seen that this block copolymer can form a stable complex (siRNA-encapsulating polymer micelle) with siRNA in the pH range.
- the block copolymer of the present invention may It can be seen that a stable complex (siRNA-encapsulating polymer micelle) can be formed.
- a stable complex siRNA-encapsulating polymer micelle
- the diffusion time gradually decreases at higher glucose concentrations than in blood, it can be seen that at these concentrations, micelles collapse and siRNA is released quickly.
- micelles collapse occurs when an RNA molecule bound to the FPBA group via a 1,2-diol structure in the micelle is replaced with glucose having the same 1,2-diol structure.
- the micelle shape can be maintained when the concentration of UMP is low, but when the concentration of UMP exceeds 1 mM, the micelle is replaced by an RNA molecule and UMP. It can be seen that siRNA is rapidly released.
- dTMP having a structure similar to UMP but not having a 1,2-diol structure
- the micelle shape is stably maintained even at a concentration exceeding 10 mM. This is presumed to be because no replacement with RNA molecules occurs.
- A549-Luc cells were seeded in a 48-well dish at 10,000 cells / well and cultured in an incubator for 24 hours using 10% fetal bovine serum-containing DMEM medium. The medium was replaced with a fresh DMEM medium containing 10% fetal bovine serum, and a si3-labeled siRNA or a complex of the siRNA and a block copolymer was added to the medium so that the siRNA was 100 nM / well. After culturing at 37 ° C. for 5 or 9 hours in an incubator, the cells were washed 3 times with 1 mL of PBS buffer, and the cells were detached from the dish with trypsin-EDTA solution.
- a block copolymer (PEG-PLL 12-23 / 43) and Cy5-GL3-siRNA were added to 10 mM HEPES buffer (pH 7.3) so that the N / P ratio was 8 and the siRNA concentration was 7.5 ⁇ M.
- the complex solution was prepared by mixing and allowing to stand at room temperature for about 1 to 2 hours.
- FIG. 8 shows the relationship between the relative value of the tumor volume relative to the tumor volume on the administration start date and the number of days elapsed after administration.
- the block copolymer of the present invention can be suitably applied in the field of DDS.
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Abstract
Description
本発明の別の局面によれば、複合体が提供される。当該複合体は、上記ブロックコポリマーと生体高分子との複合体である。
本発明のブロックコポリマーは、ポリアミノ酸鎖セグメントと親水性ポリマー鎖セグメントとを含む。当該ポリアミノ酸鎖セグメントは、側鎖にカチオン性基を有するアミノ酸残基(以下、「カチオン性アミノ酸残基」と称する場合がある)と、側鎖に生理的pH付近にpKaを有するようにフェニル環の少なくとも1つの水素が置換されたフェニルボロン酸基(以下、「置換PBA基」と称する場合がある)を有するアミノ酸残基(以下、「置換PBA基含有アミノ酸残基」と称する場合がある)とを含む。ここで、カチオン性アミノ酸残基と置換PBA基含有アミノ酸残基とは、異なるアミノ酸残基であってもよく、同一のアミノ酸残基であってもよい。具体的には、当該ポリアミノ酸鎖セグメントは、側鎖に置換PBA基を有さないカチオン性アミノ酸残基と側鎖にカチオン性基を有さない置換PBA基含有アミノ酸残基とを含んでもよいし、これらの一方または両方に代えて、あるいは、これらに加えて、側鎖にカチオン性基と置換PBA基との両方を有するアミノ酸残基を含んでもよい。
上記ポリアミノ酸鎖セグメントは、側鎖にカチオン性基を有するカチオン性アミノ酸残基と側鎖に置換PBA基を有する置換PBA基含有アミノ酸残基とを含む。
-NH-(CH2)p1-〔NH-(CH2)q1-〕r1NH2 (i);
-NH-(CH2)p2-N〔-(CH2)q2-NH2〕2 (ii);
-NH-(CH2)p3-N{〔-(CH2)q3-NH2〕〔-(CH2)q4-NH-〕r2H} (iii);および
-NH-(CH2)p4-N{-(CH2)q5-N〔-(CH2)q6-NH2〕2}2 (iv)
(式(i)~(iv)において、p1~p4、q1~q6、およびr1~r2は、それぞれ相互に独立して、1~5の整数である。)
上記親水性ポリマー鎖セグメントは、任意の適切な親水性ポリマーによって構成され得る。該親水性ポリマーとしては、例えば、ポリ(エチレングリコール)、ポリサッカライド、ポリ(ビニルピロリドン)、ポリ(ビニルアルコール)、ポリ(アクリルアミド)、ポリ(アクリル酸)、ポリ(メタクリルアミド)、ポリ(メタクリル酸)、ポリ(メタクリル酸エステル)、ポリ(アクリル酸エステル)、ポリアミノ酸、ポリ(リンゴ酸)、またはこれらの誘導体が挙げられる。ポリサッカライドの具体例としては、デンプン、デキストラン、フルクタン、ガラクタン等が挙げられる。これらのなかでも、ポリ(エチレングリコール)は、末端に種々の官能基を有する末端反応性ポリエチレングリコールが市販されており、また、種々の分子量のものが市販されており、容易に入手できることから、好ましく用いられ得る。
本発明の特に好ましい実施形態におけるブロックコポリマーの具体例を式(1)~(4)に示す。当該式(1)~(4)のブロックコポリマーにおいては、置換PBA基は、カチオン性アミノ酸残基の側鎖に導入される。
R1の基は、水素原子あるいは未置換もしくは置換された炭素数1~12の直鎖または分枝状のアルキル基であり、
R2の基は、水素原子、炭素数1~12の未置換もしくは置換された直鎖または分枝状のアルキル基あるいは炭素数1~24の未置換もしくは置換された直鎖または分枝状のアルキルカルボニル基であり、
R3の基は、ヒドロキシル基、炭素数1~12の未置換もしくは置換された直鎖または分枝状のアルキルオキシ基、炭素数2~12の未置換もしくは置換された直鎖または分枝状のアルケニルオキシ基、炭素数2~12の未置換もしくは置換された直鎖または分枝状のアルキニルオキシ基あるいは炭素数1~12の未置換もしくは置換された直鎖または分枝状のアルキル置換イミノ基であり、
R4a、R4b、R5aおよびR5bの基は、相互に独立して、メチレン基またはエチレン基であり、
R6aおよびR6bの基は、相互に独立して、上記(i)~(iv)の基から選択される基であり、
R7aおよびR7bの基は、相互に独立して、-O-または-NH-であり、
R8aおよびR8bの基は、相互に独立して、炭素数6~27の飽和もしくは不飽和の直鎖または分枝状の脂肪族炭化水素基、炭素数6~27の芳香族炭化水素基あるいはステリル基であり、
Qの基は、-NH2、-NHC(=NH)NH2、または以下の式(II)で表される基であり、
L2は、-NH-、-O-、-O(CH2)p1-NH-、または-L2a-(CH2)q1-L2b-であり、ここで、p1およびq1は、相互に独立して、1~5の整数であり、L2aはOCO、OCONH、NHCO、NHCOO、NHCONH、CONHまたはCOOであり、L2bはNHまたはOであり、
L4は、-OCO-(CH2)p2-CO-、-NHCO-(CH2)p3-CO-、または-L4a-(CH2)q2-CO-であり、ここで、p2、p3、およびq2は、相互に独立して、1~5の整数であり、L4aは、OCONH、-CH2NHCO-、NHCOO、NHCONH、CONHまたはCOOであり、
kは、30~20,000の整数であり、
sは、1~6の整数であり、
mは、1~300の整数であり、
nは、0~mの整数であり、
xは、0~80の整数であり
yは、0~xの整数であり
zは、2~300の整数であり、
ただし、z個のQの基に含有される1級アミノ基および2級アミノ基の総数または(m-n)個のR6aの基とn個のR6bの基とに含有される1級アミノ基および2級アミノ基の総数をwとしたとき、1以上w未満の当該アミノ基の水素原子が置換PBA基(例えば、上記式(I)で示されるFPBA基)を有するアシル基で置換されている。)
本発明のブロックコポリマーは、任意の適切な合成方法によって作製され得る。本発明の好ましい実施形態におけるブロックコポリマーの合成方法の一例は次の通りである。すなわち、R1を付与できる開始剤を用いてアニオンリビング重合を行うことによりポリエチレングリコール鎖を形成すること、当該ポリエチレングリコール鎖の成長末端側にアミノ基を導入すること;当該アミノ末端からNCA-Lys(TFA)等の保護されたアミノ酸誘導体を重合させてポリアミノ酸鎖セグメントを形成すること;当該ポリアミノ酸の側鎖を脱保護してアミノ基を露出させること;および、当該露出したアミノ基とフッ素化カルボキシフェニルボロン酸のカルボキシル基とを反応させて、アミド結合により所望の数のFPBA基を当該側鎖に導入すること;によって作製され得る。
本発明の複合体は、上記A項に記載のブロックコポリマーと生体高分子との複合体である。当該複合体は、複数の当該ブロックコポリマーと生体高分子とのPICであり得る。また、当該PICは、複数の当該ブロックコポリマーによって生体高分子が内包されたポリマーミセルの形態であり得る。従来型のキャリアでは、生理条件と同程度のイオン濃度の媒体中で複合体が解離してしまうという問題があるが、本発明の複合体は安定性に優れるので、当該媒体中、さらにはタンパク質を含む媒体中であっても、複合体の形態を維持し得る。
上記生体高分子としては、例えば、タンパク質、脂質、核酸等が挙げられる。本明細書において、タンパク質はペプチドも含む。生体高分子のなかでも、上記ブロックコポリマーは核酸と好適に複合体を形成し得る。また、複合体の形成に適した生体高分子としては、上記ブロックコポリマーのpKa以下のpHの範囲において負の電荷を有する、アニオン荷電性の化合物が例示できる。
以下の実験例で用いたポリリシン系のブロックコポリマーの合成スキームAを以下に示す。具体的には、(1-i)~(1-iii)に記載の通りにしてブロックコポリマーを作製した。
(1-ii) MeO-PEG-PLys(TFA)を1NのNaOHaq./メタノール=1/10中、35℃で12時間撹拌し、透析(外液は水)および凍結乾燥を経て、MeO-PEG-PLysを得る。
(1-iii) MeO-PEG-PLysとPLysのアミノ基に対して5当量のD-Mannitolを50mMのNaHCO3水溶液中に溶解させる。得られた溶液にメタノールに溶解した3-フルオロ-4-カルボキシフェニルボロン酸を添加し、次いで、縮合剤DMT-MMを加える。得られた溶液を室温にて終夜撹拌し、透析(外液は水)および凍結乾燥を経て、MeO-PEG-[PLys(FPBA)/PLys]を得る。
以下の実験例で用いたPAsp(DET)系のブロックコポリマーの合成スキームBを以下に示す。具体的には、(2-i)~(2-iii)に記載の通りにしてブロックコポリマーを作製した。
(2-ii) ベンゼン凍結乾燥したMeO-PEG-PBLAをN-メチル-2-ピロリドン(NMP)中に溶解する。得られた溶液をジエチレントリアミンのNMP溶液中に滴下し、5~10℃で1時間撹拌する。氷冷下で塩酸にて中和し、透析(外液は0.01Nの塩酸)および凍結乾燥を経て、MeO-PEG-PAsp(DET)を得る。
(2-iii) MeO-PEG-PAsp(DET)およびPAsp(DET)の一級アミンに対して5当量のD-Mannitolを氷冷下(0℃)において50mMのNaHCO3水溶液中に溶解する。得られた溶液に、メタノールに溶解した3-フルオロ-4-カルボキシフェニルボロン酸を添加し、次いで、縮合剤DMT-MMを加える。得られた溶液を6時間撹拌し、5℃で透析(外液は、任意にソルビトールを含む、0.01Nの塩酸)し、凍結乾燥を経て、MeO-PEG-P[Asp(DET-FPBA/DET)]を得る。
以下の実験例で使用したsiRNAの配列は次の通りである。Cy3等の標識は、全てセンス鎖の5’末端に導入した。
(1)hVEGF-siRNA(ヒト血管内皮成長因子に対するsiRNA):
センス鎖:5’-GAUCUCAUCAGGGUACUCCdTdT-3’(配列番号1)
アンチセンス鎖:5’-GGAGUACCCUGAUGAGAUCdTdT-3’(配列番号2)
(2)scramble-siRNA(非治療用配列siRNA):
センス鎖:5’-UUCUCCGAACGUGUCACGUUU-3’(配列番号3)
アンチセンス鎖:5’-ACGUGACACGUUCGGAGAAUU-3’(配列番号4)
(3)GL3-siRNA(ホタルルシフェラーゼに対するsiRNA)
センス鎖:5’-CUUACGCUGACUACUUCGAUU-3’(配列番号5)
アンチセンス鎖:5’-UCGAAGUACUCAGCGUAAGUU-3’(配列番号6)
上記(1-i)~(1-iii)および(2-i)~(2-iii)の記載の通りにして、アミノ酸重合度およびFPBA基含有量が異なる種々のブロックコポリマーを作製した。なお、Methoxy-PEG-NH2としては、PEGの分子量(Mw)が12,000であるものを用いた。当該ブロックコポリマーとCy3で蛍光標識したsiRNAとをN/P比が8となるように混合した。得られた混合液を冷蔵庫内で1~2時間静置後、血清溶液(150mM NaCl, 10mM Hepes, 10% FBS)でsiRNA濃度が50nMとなるように希釈した。得られた希釈液を1時間程度室温で静置し、共焦点レーザスキャン顕微鏡(Carl Zeiss社製、製品名「LSM510」)にてsiRNAの拡散時間を測定した(10sec×10times)。結果を表1および図1に示す。なお、ブロックコポリマーと混合しないこと以外は同様に測定した場合における拡散時間(siRNA単独の拡散時間)は、157.5μsecである。なお、siRNAとしては、GL3‐siRNAを用いた。
A549-Luc細胞を96ウェルの培養ディッシュに2,500cells/wellとなるように播種し、10%牛胎児血清含有DMEM培地を用いて、インキュベーターで24時間培養した。培地を新しい10%牛胎児血清含有DMEM培地に交換するとともに、生理食塩水に溶解した各ブロックコポリマーを、種々のアミン濃度となるように添加した。次いで、48時間培養した後、製品名「Cell Counting Kit8」(同仁化学研究所社製)によって生細胞数を測定し、細胞生存率を算出した(N=4)。結果を図2に示す。
A549-Luc細胞を96ウェルの培養ディッシュに2,500cells/wellとなるように播種し、10%牛胎児血清含有DMEM培地を用いて、インキュベーターで24時間培養した。培地を新しい10%牛胎児血清含有DMEM培地に交換するとともに、siRNAとブロックコポリマーとの複合体を、種々のsiRNA濃度となるように添加した。次いで、48時間培養した後、製品名「Cell Counting Kit8」(同仁化学研究所社製)によって生細胞数を測定し、細胞生存率を算出した(N=4)。結果を図3に示す。なお、上記複合体は、各ブロックコポリマーとGL3-siRNAとを、N/P比が4でsiRNA濃度が80nMとなるように10mM HEPES緩衝液(pH7.3)に添加および混合し、1~2時間程度室温で静置後、種々の濃度に希釈して用いた。
ブロックコポリマー(PEG-PLL 12-23/43)とCy3で標識したGL3-siRNAとを種々のpHの緩衝液(リン酸緩衝液またはリン酸-クエン酸緩衝液)に溶解し、N/P比が4でsiRNA濃度が50nMとなるように混合した。得られた混合液を1時間程度室温で静置し、共焦点レーザスキャン顕微鏡(Carl Zeiss社製、製品名「LSM510」)にてsiRNAの拡散時間を測定した(10sec×10times)。結果を図4に示す。
ブロックコポリマー(PEG-PLL 12-23/43)とCy3で標識したGL3‐siRNAとを10mM HEPES緩衝液(pH7.3)にそれぞれ溶解し、N/P比が4となるように混合した。得られた混合液を1~2時間程度冷蔵庫内で静置した後、種々の濃度のグルコース、dTMPまたはUMPを含む10mM HEPES緩衝液(pH7.3)でsiRNA濃度が50nMとなるように希釈した。得られた希釈液を室温で1時間程度静置した。次いで、共焦点レーザスキャン顕微鏡(Carl Zeiss社製、製品名「LSM510」)にてsiRNAの拡散時間を測定した(10sec×10times)。結果を図5に示す。
48wellディッシュに10,000cells/wellとなるようにA549-Luc細胞をまき、10%牛胎児血清含有DMEM培地を用いて、インキュベーターで24時間培養した。培地を新しい10%牛胎児血清含有DMEM培地に交換し、siRNAが100nM/wellとなるよう、Cy3で標識したsiRNAまたは該siRNAとブロックコポリマーとの複合体を培地に添加した。インキュベーターで37℃で5時間または9時間培養した後、細胞を1mLのPBS緩衝液で3回洗浄し、トリプシン-EDTA溶液で細胞をディッシュからはがした。はがした細胞を、Cy3フィルターをセットしたフローサイトメーター(BD社製、LSRII)を用いてヒストグラム解析を行い、細胞内へのsiRNAの取り込み量を評価した(N=4)。なお、siRNAとしては、GL3-siRNAを用いた。細胞内へのsiRNA取り込み量を示すグラフを図6に示す。なお、上記複合体は、各ブロックコポリマーとsiRNAとを、N/P比が8でsiRNA濃度が4μMとなるように10mM HEPES緩衝液(pH7.3)に添加および混合し、1~2時間程度室温で静置することによって調製した。
ブロックコポリマー(PEG-PLL 12-23/43)とCy5-GL3-siRNAとを、N/P比が8でsiRNA濃度が7.5μMとなるように10mM HEPES緩衝液(pH7.3)に添加および混合し、1~2時間程度室温で静置することによって複合体溶液を調製した。該複合体溶液またはsiRNA溶液(10mM HEPES緩衝液)をマウス(メスのBalb/C nude、6週齢、N=4)にsiRNAの投与量が20μgとなるように尾静脈投与し、siRNAの血中残存量を経時的に調べた。結果を図7に示す。
(1)FPBA基含有ブロックコポリマーの調製
スキームBと類似のスキームによって、Ace-PEG-PAsp(DET) 12-62/75のブロックコポリマーを得た。具体的には、PEG側末端にアセタール基を有するAce-PEG-PBLA(12-110)をDETでアミノリシスし、Ace-PEG-PAsp(DET)(12-90)を得た。次いで、NaCl溶液で透析して対イオンを酢酸からクロライドとした後に、縮合剤(DMT-MM)を用いて4-カルボキシ-3-フルオロフェニルボロン酸を縮合させて、Ace-PEG-PAsp(DET) 12-62/75のブロックコポリマーを得た。
次いで、アセタール基に対して5当量のcRGDペプチド(Cyclic(Arg-Gly-Asp-D-Phe-Cys)をpH7.2でDTTと1時間混合し、得られた混合液と上記ブロックコポリマーとを混合し、pHを2に調整した。1時間撹拌後、2Mの酢酸ナトリウムおよびNaOHを用いてpHを5とした。その後、ポリマー濃度が20mg/mLとなるように水を加えて一晩撹拌した。純水に対して透析し、未反応物及び不要物を除去後、凍結乾燥によってPEG鎖の末端にcRGDを導入されたブロックコポリマー cRGD-PEG-PAsp(DET) 12-62/75を得た。なお、反応、透析は4℃(冷蔵庫)にて行った。
ブロックコポリマーを10mg/mLとなるように10mM HEPES緩衝液に溶解した。12.18μLの該ポリマー溶液と、75μLのsiRNA(15μM 10mM HEPES緩衝液)と、7.81μLの10mM HEPES緩衝液とを混合し、一晩静置した。該混合液に投与数時間前に3M NaCl 10mM HEPES緩衝液を5μL添加し、NaCl濃度が150mMとなるように調整した(全量 100μL)。これにより、複合体(siRNA内包ポリマーミセル)を得た。調製した複合体におけるsiRNAとブロックコポリマーとの組み合わせおよび混合比を表2に示す。
6週齢のマウス(メスのBalb/C nude、N=4)を購入後、1週間飼育し、5.0×107cells/mLとしたHela-luc細胞を各マウスに100μLずつ皮下に注射した。その後さらに4日間飼育し、治療(すなわち、複合体の投与)を開始した。具体的には、上記(3)で調製した複合体を一日おきに6日目まで尾静脈より投与した(すなわち、投与開始日を0日目として、0、2、4、6日目の計4回投与)。1回の投与につき、15μg/100μLのsiRNAを投与した。対照群に対しては、1回の投与につき100μLのHEPES溶液を投与した。投与開始日における腫瘍体積に対する腫瘍体積の相対値と投与後の経過日数との関係を図8に示す。
Claims (9)
- ポリアミノ酸鎖セグメントと親水性ポリマー鎖セグメントとを含むブロックコポリマーであって、
該ポリアミノ酸鎖セグメントが、側鎖にカチオン性基を有するアミノ酸残基と、側鎖に生理的pH付近にpKaを有するようにフェニル環の少なくとも1つの水素が置換されたフェニルボロン酸基を有するアミノ酸残基とを含む、ブロックコポリマー。 - 前記置換されたフェニルボロン酸基のpKaが8未満である、請求項1に記載のブロックコポリマー。
- 前記カチオン性基が、アミノ基である、請求項1から3のいずれか1項に記載のブロックコポリマー。
- 前記側鎖にカチオン性基を有するアミノ酸残基が、リシン残基、または、酸性アミノ酸のカルボキシル基(-C(=O)OH)の-OH部が下記式(i)~(iv)のいずれかの基で置換されたアミノ酸残基である、請求項1から4のいずれか1項に記載のブロックコポリマー。
-NH-(CH2)p1-〔NH-(CH2)q1-〕r1NH2 (i);
-NH-(CH2)p2-N〔-(CH2)q2-NH2〕2 (ii);
-NH-(CH2)p3-N{〔-(CH2)q3-NH2〕〔-(CH2)q4-NH-〕r2H} (iii);および
-NH-(CH2)p4-N{-(CH2)q5-N〔-(CH2)q6-NH2〕2}2 (iv)
(式(i)~(iv)において、p1~p4、q1~q6、およびr1~r2は、それぞれ相互に独立して、1~5の整数である。) - 前記ポリアミノ酸鎖セグメントが、側鎖に疎水性基を有するアミノ酸残基をさらに含む、請求項1から6のいずれか1項に記載のブロックコポリマー。
- 請求項1から7のいずれか1項に記載のブロックコポリマーと生体高分子との複合体。
- 前記生体高分子が核酸である、請求項8に記載の複合体。
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- 2012-11-19 WO PCT/JP2012/079897 patent/WO2013073697A1/ja active Application Filing
- 2012-11-19 EP EP12850688.8A patent/EP2781536B1/en not_active Not-in-force
- 2012-11-19 CN CN201280056639.0A patent/CN104093768B/zh not_active Expired - Fee Related
- 2012-11-19 US US14/358,816 patent/US9114177B2/en not_active Expired - Fee Related
- 2012-11-19 CA CA2853902A patent/CA2853902A1/en not_active Abandoned
- 2012-11-19 AU AU2012337721A patent/AU2012337721B2/en not_active Ceased
- 2012-11-19 RU RU2014124333/04A patent/RU2014124333A/ru not_active Application Discontinuation
- 2012-11-19 IN IN3580CHN2014 patent/IN2014CN03580A/en unknown
- 2012-11-19 BR BR112014011831A patent/BR112014011831A2/pt not_active IP Right Cessation
- 2012-11-19 KR KR1020147013186A patent/KR101912978B1/ko active IP Right Grant
- 2012-11-19 JP JP2013529476A patent/JP5481614B2/ja not_active Expired - Fee Related
- 2012-11-19 MX MX2014005540A patent/MX2014005540A/es unknown
- 2012-11-19 ES ES12850688.8T patent/ES2603632T3/es active Active
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US9561284B2 (en) | 2011-03-31 | 2017-02-07 | Nanocarrier Co., Ltd. | Pharmaceutical composition containing a block copolymer bound to a boronic acid compound |
EP3106177A4 (en) * | 2014-02-12 | 2018-01-03 | AccuRna, Inc. | COMPOSITION FOR mRNA DELIVERY |
US10232054B2 (en) | 2014-02-12 | 2019-03-19 | Accurna, Inc. | Composition for mRNA delivery |
JPWO2015170757A1 (ja) * | 2014-05-08 | 2017-04-20 | 国立大学法人 東京大学 | 医薬組成物 |
US10993960B1 (en) * | 2014-05-08 | 2021-05-04 | Kawasaki Institute Of Industrial Promotion | Pharmaceutical composition |
US20210187005A1 (en) * | 2014-05-08 | 2021-06-24 | Kawasaki Institute Of Industrial Promotion | Pharmaceutical composition |
WO2018025699A1 (ja) * | 2016-08-02 | 2018-02-08 | 日本化薬株式会社 | アクティブターゲティング型高分子誘導体、その高分子誘導体を含む組成物、及びそれらの用途 |
JPWO2018025699A1 (ja) * | 2016-08-02 | 2019-05-30 | 日本化薬株式会社 | アクティブターゲティング型高分子誘導体、その高分子誘導体を含む組成物、及びそれらの用途 |
US10973762B2 (en) | 2016-08-02 | 2021-04-13 | Nippon Kayaku Kabushiki Kaisha | Active-targeting-type polymer derivative, composition containing said polymer derivative, and uses of said polymer derivative and said composition |
WO2020241819A1 (ja) | 2019-05-29 | 2020-12-03 | 国立大学法人東京工業大学 | 複合体、医薬、癌治療剤、キット及び結合体 |
Also Published As
Publication number | Publication date |
---|---|
MX2014005540A (es) | 2014-09-22 |
CN104093768B (zh) | 2016-09-21 |
EP2781536A4 (en) | 2015-06-10 |
BR112014011831A2 (pt) | 2017-05-09 |
US9114177B2 (en) | 2015-08-25 |
ES2603632T3 (es) | 2017-02-28 |
JP5481614B2 (ja) | 2014-04-23 |
AU2012337721B2 (en) | 2015-12-10 |
RU2014124333A (ru) | 2015-12-27 |
CN104093768A (zh) | 2014-10-08 |
US20150051347A1 (en) | 2015-02-19 |
EP2781536A1 (en) | 2014-09-24 |
KR101912978B1 (ko) | 2018-10-29 |
KR20140106511A (ko) | 2014-09-03 |
AU2012337721A1 (en) | 2014-05-22 |
CA2853902A1 (en) | 2013-05-23 |
EP2781536B1 (en) | 2016-08-17 |
JPWO2013073697A1 (ja) | 2015-04-02 |
IN2014CN03580A (ja) | 2015-10-09 |
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