WO2009136572A1 - 葉酸若しくは葉酸誘導体の高分子結合体 - Google Patents
葉酸若しくは葉酸誘導体の高分子結合体 Download PDFInfo
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- WO2009136572A1 WO2009136572A1 PCT/JP2009/058325 JP2009058325W WO2009136572A1 WO 2009136572 A1 WO2009136572 A1 WO 2009136572A1 JP 2009058325 W JP2009058325 W JP 2009058325W WO 2009136572 A1 WO2009136572 A1 WO 2009136572A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular 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
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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
- A61K31/74—Synthetic polymeric materials
- A61K31/785—Polymers containing nitrogen
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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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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
- C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
- C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
- C08G69/10—Alpha-amino-carboxylic acids
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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
- C08G69/48—Polymers modified by chemical after-treatment
Definitions
- the present invention relates to a polymer conjugate of folic acid or a folic acid derivative, a production method thereof and use thereof.
- Folic acid is a kind of water-soluble vitamin and is a coenzyme required for the synthesis of amino acids and nucleic acids. Folic acid is converted into tetrahydrofolic acid by a reductase in the body, and further converted to be used for dTMP (deoxythymidine monophosphate) and purine synthesis. Some folic acid derivatives have the action of inhibiting the conversion of folic acid in vivo and stopping cell division. These group of compounds are called antifolate inhibitors, and the representative compound methotrexate has long been used for the treatment of leukemia, sarcoma, gastric cancer and the like.
- methotrexate also has an immunomodulatory action and has become an essential drug for pharmacotherapy of rheumatoid arthritis.
- folic acid itself and the folic acid derivative leucovorin are also used as drugs that neutralize folate deficiency and the toxicity of methotrexate.
- folic acid and folic acid derivatives are useful compounds as pharmaceuticals, and various derivative studies aiming at improving their effects.
- These derivatives include, for example, aminopterin, plalatrexate, previtrexed, edatrexate, pemetrexed, raltitrexed, raltitrexed, and the like shown below.
- the mechanism of action of these antifolates is mainly an inhibitory action on dihydrofolate reductase, but some newly developed drugs have an inhibitory action on thymidylate synthetase and are more effective. Development is underway for new drugs.
- pemetrexed is characterized by inhibiting a plurality of enzymes involved in the folate metabolic system and is approved as a therapeutic agent for malignant pleural mesothelioma.
- DDS drug delivery system
- Patent Document 1 and Patent Document 3 describe a water-soluble polymerized pharmaceutical preparation using a micelle formed by an amphiphilic polymer as a carrier.
- Patent Document 1 binds methotrexate as an example of a hydrophobic segment. Although there is a description of a polymer carrier, methotrexate and an amphiphilic polymer are amide-bonded.
- Patent Document 3 does not describe a polymer conjugate to which folic acid or a folic acid derivative is bound.
- Patent Document 2 reports that chemical stability and in vivo drug release efficiency were simultaneously achieved by bonding a polymer compound and camptothecins with a phenyl ester bond. However, this method cannot be used as it is for the carboxy group of folic acid derivatives represented by methotrexate (including folic acid itself).
- Non-Patent Document 1 and Non-Patent Document 2 describe a polymer conjugate in which an amphiphilic polymer and an alkyl ester bond are used using a carboxy group of methotrexate, but the release rate of the drug is slow, There are doubts about the effectiveness of the drug.
- the release of the drug from the polymer conjugate depends on the enzyme in the living body, the enzyme activity varies among individuals, so that the drug release rate varies, and as a result, the drug efficacy may vary.
- the bond between the drug and the amphiphilic polymer in Patent Document 1 is an amide bond
- the amide bond is chemically stable, and when the polymer conjugate is administered in vivo, the release rate of the drug is considerably high. Slow and impractical.
- the drug needs to be released from the polymer conjugate at an appropriate rate, and it is not sufficient to simply bind to the polymer compound.
- the bond between the drug and the polymer compound is weak, not only is the polymer conjugate unstable, making it extremely difficult to formulate, but the release of the drug is fast and the pharmacokinetics can be improved by binding to the polymer compound. I can't expect it. If the binding is too strong, even if the pharmacokinetics are improved, the drug is not released from the polymer conjugate, making it difficult to exert a pharmacological effect.
- a block copolymer comprising a polymer having polyethylene glycol and a carboxylic acid in the side chain, and folic acid or a folic acid derivative are bound via a specific linker molecule.
- the present inventors have reached a present invention.
- the present invention relates to the following (1) to (11).
- A represents a monocyclic or condensed aromatic group
- G represents an optionally substituted (C1-C6) alkylene group
- Y represents a hydrogen atom or a substituent
- E represents Shows residues of folic acid or folic acid derivatives
- D is an unsubstituted (C1-C6) alkyl group, the average value of n is 50-1000, the average value of c + d + e is 5-100, and B is an (C1-C6) acyl group A polymer conjugate of folic acid or a folic acid derivative or a pharmacologically acceptable salt thereof according to (4) above.
- the substituent represented by the formula (I) is represented by the following formula (III) [Wherein E represents a residue of folic acid or a folic acid derivative]
- An anticancer agent comprising as an active ingredient a polymer conjugate of folic acid or a folic acid derivative according to any one of (1) to (8), or a pharmacologically acceptable salt.
- a therapeutic agent for inflammatory diseases comprising as an active ingredient a polymer conjugate of folic acid or a folic acid derivative according to any one of (1) to (8) above, or a pharmacologically acceptable salt.
- high molecular weight compounds have dispersion in molecular weight and composition and are not a single molecule, so it is difficult to perform rigorous chemical analysis, and folic acid or folic acid derivatives have multiple amino groups and carboxy groups. There are multiple modes of binding.
- separation and purification can be performed after condensing folic acid or a folic acid derivative and a linker molecule, and the condensation reaction between the obtained compound and the block copolymer proceeds relatively easily.
- the quality of the polymer conjugate of the obtained folic acid or folic acid derivative can be made constant.
- the polymer conjugate obtained in the present invention has a phenyl ester bond between the drug and the linker molecule, it is possible to simultaneously achieve practical chemical stability and excellent in vivo drug release efficiency, It is possible to enhance the pharmacological effect and reduce side effects.
- the release rate of the drug from the polymer conjugate of folic acid or folic acid derivative of the present invention can be controlled by changing the substituent of the linker moiety or selecting the carboxy group of the drug to be bound.
- the release rate of the drug can be adjusted by mixing these polymer conjugates having different release rates, a wider application is possible.
- the block copolymer comprising a polyethylene glycol and a polymer having a carboxy group in the side chain has the above formula (I) [wherein A represents a monocyclic or condensed aromatic group; Represents an optionally substituted (C1-C6) alkylene group, Y represents a hydrogen atom or a substituent, and E represents a residue of folic acid or a folic acid derivative]. Or a pharmacologically acceptable salt thereof.
- Polyethylene glycols include polyethylene glycol modified at both ends or one end, and the modifying groups at both ends may be the same or different.
- the terminal modifying group include an optionally substituted (C1 to C6) alkyl group, preferably a methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, s -Butyl group, t-butyl group, benzyl group, dimethoxyethyl group, dietoxyethyl group, aminomethyl group, aminoethyl group, 3-aminopropyl group, 4-aminobutyl group and the like.
- the molecular weight of the polyethylene glycol moiety is usually about 300 to 500,000, preferably about 500 to 100,000, and more preferably about 1,000 to 50,000.
- the molecular weight in the present invention is a peak top molecular weight measured by gel permeation chromatography (GPC method).
- the polymer having a carboxy group in the side chain may be a polymer containing a component having a carboxy group in the side chain.
- the component having a carboxy group in the side chain include acrylic acid, methacrylic acid, and malic acid. Aspartic acid, glutamic acid, and the like.
- the polymer having a carboxy group in the side chain include polyacrylic acid, polymethacrylic acid, polymalic acid, polyaspartic acid, polyglutamic acid, and the like.
- polyaspartic acid and polyglutamic acid are preferable, and polyaspartic acid is particularly preferable.
- the number of carboxy groups per molecule of the block copolymer is preferably about 3 to 200, and more preferably about 5 to 100.
- Examples of the block copolymer in the present invention include compounds in which polyethylene glycols having a functional group at a terminal and polycarboxylic acids having a functional group at a terminal are bonded, and terminals described in Patent Document 1, Patent Document 3, and the like. And compounds obtained by a polymerization reaction of an amino acid activated product that starts polymerization with polyethylene glycol having an amino group.
- a in the above formula (I) is a monocyclic or condensed aromatic group, and is not particularly limited, but is preferably an aromatic group containing no hetero atoms (C6-C18), such as a phenyl group, a naphthyl group, an anthryl group.
- a phenyl group is particularly preferable.
- the substitution position of the phenolic hydroxyl group and aminoalkyl group is not particularly limited.
- Y in A in the above formula (I) is a hydrogen atom or a substituent bonded to a monocyclic or condensed aromatic group.
- substituent include a methyl group, an ethyl group, and an isopropyl group.
- the binding strength between folic acid or a folic acid derivative and a linker molecule is greatly influenced by the acidity of the phenolic hydroxyl group when the linker molecule is cleaved.
- the acidity is changed by changing Y
- the folic acid or the folic acid derivative and the linker molecule The strength of molecular bonds also changes.
- A is a phenyl group
- an electron-withdrawing group such as a cyano group, a nitro group, or a halogen atom is preferable to accelerate release of folic acid or a folic acid derivative, and an electron donating group is used to delay release of folic acid or a folic acid derivative.
- the alkyl group etc. which are are preferable.
- the number of Y substitutions is not particularly limited as long as it is a substitutable number. When Y is a substituent, the substitution position is not particularly limited, although it is necessary to consider the influence on the release rate.
- G in the above formula (I) is an optionally substituted (C1-C6) alkylene group such as methylene group, ethylene group, trimethylene group, tetramethylene group, methylethylene group, dimethylethylene.
- alkylene group such as methylene group, ethylene group, trimethylene group, tetramethylene group, methylethylene group, dimethylethylene.
- a linear alkylene group such as a methylene group, an ethylene group, a trimethylene group and a tetramethylene group is preferable, and an ethylene group is particularly preferable.
- E in the above formula (I) is not particularly limited as long as it is a residue of folic acid or a folic acid derivative that means folic acid or a folic acid derivative in E-CO 2 H, and is a folic acid derivative having a folic acid or a carboxy group.
- E-CO 2 H includes folic acid, methotrexate, aminopterin, plalatrexate, previtrexed, edatrexate, pemetrexed, raltitrexed, lometrexol, etc. Among them, methotrexate, pemetrexed and the like are preferable.
- Examples of the polymer conjugate of the present invention include compounds represented by the above formula (II).
- Examples of the (C1-C6) alkyl group optionally having a substituent in D of the formula (II) include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, an s -Butyl group, t-butyl group, n-pentyl group, n-hexyl group, benzyl group, dimethoxyethyl group, diethoxyethyl group, aminomethyl group, aminoethyl group, 3-aminopropyl group, 4-aminobutyl group, etc. Can be mentioned.
- D is preferably an unsubstituted (C1 to C6) alkyl group, and particularly preferably a methyl group.
- n is about 5 to 11,500, preferably about 50 to 1,000, and more preferably about 100 to 300.
- Examples of the (C2 to C6) alkylene group in J of the above formula (II) include linear alkylene groups such as ethylene group, trimethylene group, tetramethylene group, hexamethylene group, and among them, trimethylene group is preferable.
- c + d + e represents the total number of aspartic acid in one molecule of the polymer conjugate, and the average value is about 3 to 200, preferably about 5 to 100, more preferably 6 to 60. Degree.
- Each constituent unit of polyaspartic acid may be bonded at random or may be bonded by forming a block, but c + d is a positive number and e may be 0.
- the ratio of the ⁇ -amino acid structural unit (c) to the total number of aspartic acids (c + d + e) is preferably 10 to 100%, particularly preferably 20 to 100%. This ratio can be appropriately changed by, for example, selecting the deprotection conditions for the protecting group of polyaspartic acid during the process of the production method according to Patent Document 1 and the like.
- Examples of the (C1 to C6) acyl group in B of the above formula (II) include formyl group, acetyl group, propionyl group, butyryl group, valeryl group, isovaleryl group, pivaloyl group, hexanoyl group and the like.
- B is preferably a (C2 to C4) acyl group, such as an acetyl group or a propionyl group, and an acetyl group is particularly preferred.
- R in the above formula (II) examples include a hydroxyl group or a substituent represented by the above formula (I), and at least one R in one molecule is a substituent represented by the formula (I).
- the substituent represented by formula (I) is as described above, and is preferably a substituent represented by formula (III).
- E in formula (III) has the same meaning as E in formula (I) above, and preferred compounds are also the same.
- the polymer conjugate of folic acid or a folic acid derivative of the present invention will be described in the case where the folic acid derivative is methotrexate and the linker molecule is tyramine (4-aminoethylphenol).
- the present invention is limited to this compound. None happen. Since methotrexate has two carboxy groups, there are two positional isomers in the compound in which tyramine and methotrexate are ester-linked.
- a compound in which the ⁇ -carboxy group of glutamic acid, which is a partial structure of methotrexate, is bonded to tyramine is called an ⁇ -substituted product
- a compound in which a ⁇ -carboxy group is bonded to tyramine is called a ⁇ -substituted product.
- the manufacturing method of these compounds is demonstrated.
- the amino group of tyramine is protected with a protecting group that can be removed after formation of an ester bond.
- the protecting group is not particularly limited as long as it is a general amino protecting group. However, there is a protecting group that can be deprotected under neutral or acidic conditions in which the ester bond between tyramine and methotrexate is stable. Desirable examples include benzyloxycarbonyl group, t-butoxycarbonyl (Boc) group, and allyloxycarbonyl group.
- the tyramine whose amino group is protected is subjected to dehydration condensation using methotrexate and a dehydration condensing agent in an organic solvent.
- the reaction temperature of the dehydration condensation reaction is usually 4 to 60 ° C., preferably 15 to 50 ° C., and the reaction time is 1 hour to several days, preferably 4 to 48 hours.
- the organic solvent is not particularly limited as long as the reaction proceeds.
- aromatic hydrocarbons such as toluene and xylene
- halogenated hydrocarbons such as methylene chloride, chloroform and 1,2-dichloroethane, tetrahydrofuran, dioxane
- Ethers such as dimethoxyethane and diethylene glycol dimethyl ether
- nitriles such as acetonitrile and propionitrile
- amides such as dimethylformamide, dimethylacetamide and N-methylpyrrolidone
- ureas such as 1,3-dimethylimidazolidinone or the above solvents
- amides or ureas preferably dimethylformamide or 1,3-dimethylimidazolidinone.
- the dehydrating condensing agent is not particularly limited as long as the condensation reaction of amines and carboxyl groups proceeds, but preferably dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-dimethylaminopropyl-3-ethylcarbodiimide, carbonyldiimidazole, isobutyl chloroformate, Pivalic acid chloride, DMT-MM (4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride), TFFH (tetramethylfluoroformamidinium hexafluorophosphate) ), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinolinone (EEDQ) or BOP (benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphine It is over door).
- a reaction aid may be used in the dehydration condensation reaction.
- the reaction aid include N-hydroxysuccinimide, 1-hydroxybenzotriazole, 4-dimethylaminopyridine, 2,6-di-t-butyl-4-methylpyridine, and the like.
- an appropriate purification treatment is performed as necessary to obtain a mixture of the above ⁇ - and ⁇ -substituted products in which the amino group of tyramine is protected or each separated isomer. Further, the amino-protecting group is deprotected by appropriate treatment to obtain a condensate ( ⁇ -substituted product, ⁇ -substituted product, or mixture thereof) of methotrexate and tyramine.
- the above folic acid or folic acid derivative and the phenolic hydroxyl group of the compound represented by the above formula (IV) are ester-bonded to remove the amino protecting group
- Production of a polymer conjugate of folic acid or a folic acid derivative, characterized by dehydrating condensation of the carboxy group of a block copolymer comprising a polyethylene glycol and a polymer having a carboxy group in the side chain to form an amide bond A method is also included. Next, the manufacturing method will be described.
- the reaction temperature of the dehydration condensation reaction is usually 4 to 60 ° C., preferably 15 to 50 ° C., and the reaction time is 1 hour to several days, preferably 4 to 48 hours.
- Examples of the organic solvent include the same organic solvents as those used in the condensation reaction of methotrexate and amino-protected tyramine, and preferred organic solvents are also the same.
- Examples of the dehydrating condensing agent include the same dehydrating condensing agent as the dehydrating condensing agent in the condensation reaction of methotrexate and aminoamine-protected tyramine.
- a reaction aid may be used, and examples of the reaction aid include the same reaction aids as those in the condensation reaction of methotrexate and tyramine protected with an amino group.
- the side chain carboxy group of the block copolymer is first activated and then reacted with the amino group of the substituted product.
- the activation method a method usually used in the production of peptide bonds can be applied, and examples thereof include a method using the above-described reagent. That is, a method of condensing a carboxylic acid and a reaction aid such as N-hydroxysuccinimide with the above-mentioned dehydrating condensing agent, isolating it as an active ester, and then adding an amine to obtain an amide.
- the amount of folic acid and folic acid derivative bound to the block copolymer composed of polyethylene glycol and a polymer having a carboxy group in the side chain via a linker is:
- the amount is not particularly limited as long as it has a medicinal effect, but it is usually 1 to 100%, preferably 10 to 90% of the total number of carboxy groups of the polymer.
- the amount of folic acid and folic acid derivative bound can be determined, for example, from the intensity of the ultraviolet absorption spectrum.
- the folic acid or folic acid derivative liberated by alkaline hydrolysis of the polymer conjugate of folic acid or folic acid derivative of the present invention can also be determined by quantifying it using, for example, high performance liquid chromatography (HPLC). it can.
- HPLC high performance liquid chromatography
- the side chain carboxy group to which the linker molecule is not bound or the carboxy group of folic acid and folic acid derivative that are not an ester may be in a free form or a salt form.
- the free form it can be converted into the target salt by a method known per se or a method analogous thereto, and when obtained in the salt, the free form or the purpose can be obtained by a method known per se or a method analogous thereto.
- the salt include lithium salt, sodium salt, potassium salt, magnesium salt, ammonium salt or triethylammonium salt.
- the polymer conjugate of folic acid or folic acid derivative of the present invention may form micelles having polyethylene glycol as an outer shell in water. By forming micelles, good water solubility, stability in aqueous solution, and enhancement of drug efficacy are expected.
- the present invention also includes an anticancer agent, an inflammatory disease therapeutic agent, or an antirheumatic agent comprising the above-mentioned folic acid or folic acid derivative polymer conjugate as a medicinal ingredient.
- the polymer conjugate of folic acid or folic acid derivative can be administered as it is, or as a pharmaceutical composition mixed with a pharmaceutically acceptable substance.
- the pharmaceutical composition may have any dosage form such as injection, powder, granule, tablet, suppository and the like.
- These preparations may contain various adjuvants used for pharmaceuticals, that is, additives such as carriers and other auxiliary agents, such as stabilizers, preservatives, soothing agents and emulsifiers.
- the content of the polymer conjugate of folic acid and folic acid derivative in the preparation varies depending on the preparation, but is usually 0.1 to 100% by weight, preferably 1 to 98% by weight.
- the obtained precipitate was dissolved in a methylene chloride-methanol mixed solvent (1: 1), dehydrated with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure.
- the residue was purified with a silica gel column to obtain two types of MTX monoesters (1.711 g of ⁇ -monoester, 1.332 g of ⁇ -monoester).
- the retention times for the ⁇ -monoester and ⁇ -monoester were 13.3 minutes and 13.7 minutes, respectively, under HPLC condition 1.
- HPLC condition 2 Column: Inertsil ODS-3 (5 ⁇ m) 20 ⁇ 250 mm Column temperature: Room temperature Eluent: Liquid A 0.1% trifluoroacetic acid aqueous solution, liquid B Acetonitrile gradient time (min) 0 4.9 5 13 13.1 20 20.1 30 B liquid (%) 10 10 20 20 40 40 10 10 Flow rate: 20 mL / min Detector: UV (254 nm)
- the produced urea was filtered with a Kiriyama funnel, and the resulting filtrate was diluted with ethyl acetate (39 mL), and then hexane (58 mL) was added to precipitate crystals. After stirring for 15 minutes, the stirring was stopped, the supernatant was removed, and a mixed solvent of hexane-ethyl acetate (3: 2) was further added and stirred. The precipitate was filtered with a Kiriyama funnel and dried under vacuum to obtain PEG-Asp33 (OSu) -Ac (454 mg).
- the precipitate was filtered with a Kiriyama funnel and dried under vacuum to obtain crude crystals (261 mg) of the target compound.
- the crude crystals were dissolved in 52 mL of acetonitrile-water mixed solvent (1: 1), and the ion exchange resins Muromac® C1002 (2.61 g) and Muromac® A203T (2.61 g) (both Muromachi Technos) were used. (Made by Co., Ltd.) was added, and impurities were purified by adsorption. After filtration of the resin, the filtrate was concentrated under reduced pressure and lyophilized to obtain PEG-Asp33 ( ⁇ -Tyramine-MTX) -Ac (172 mg).
- the MTX content of this polymer conjugate was 19.6% (w / w).
- the drug (MTX) content was calculated by hydrolyzing the obtained polymer conjugate with an aqueous sodium hydroxide solution and analyzing the released drug (MTX) by HPLC. The same applies to the following polymer conjugates.
- the precipitate was filtered with a Kiriyama funnel and dried under vacuum to obtain crude crystals (270 mg) of the target compound.
- This crude crystal is dissolved in 54 mL of acetonitrile-water mixed solvent (1: 1), ion exchange resins Muromac® C1002 (2.70 g) and Muromac® A203T (2.70 g) are added, and impurities are added. Adsorption purification. After filtration of the resin, the filtrate was concentrated under reduced pressure and lyophilized to obtain PEG-Asp33 ( ⁇ -Tyramine-MTX) -Ac (170 mg). The MTX content of this polymer conjugate was 25.0% (w / w).
- the produced urea was filtered with a Kiriyama funnel, and the resulting filtrate was diluted with ethyl acetate (34 mL), and then hexane (50 mL) was added to precipitate crystals. After stirring for 40 minutes, the stirring is stopped, the supernatant is removed, and a mixed solvent of hexane-ethyl acetate (4: 1) is further added and stirred. The precipitate was filtered with a Kiriyama funnel and dried under vacuum to obtain PEG-Asp40 (OSu) -Ac (424 mg).
- the precipitate was filtered with a Kiriyama funnel and dried under vacuum to obtain crude crystals (413 mg) of the target compound.
- the crude crystals are dissolved in 83 mL of acetonitrile-water mixed solvent (1: 1), ion exchange resins Muromac® C1002 (4.13 g) and Muromac® A203T (4.13 g) are added, and impurities are added. It was purified by adsorption. After filtration of the resin, the filtrate was concentrated under reduced pressure and lyophilized to obtain PEG-Asp40 ( ⁇ -Tyramine-MTX) -Ac (263 mg). The MTX content of this polymer conjugate was 23.6% (w / w).
- the precipitate was filtered with a Kiriyama funnel and dried under vacuum to obtain crude crystals (189 mg) of the target compound.
- the crude crystals are dissolved in 38 mL of acetonitrile-water mixed solvent (1: 1), ion exchange resins Muromac® C1002 (1.89 g) and Muromac® A203T (1.89 g) are added, and impurities are added. It was purified by adsorption.
- the resin was filtered, concentrated under reduced pressure, and lyophilized to obtain PEG-Asp40 ( ⁇ -tyramine-MTX) -Ac (126 mg).
- the MTX content of this polymer conjugate was 28.6% (w / w).
- the precipitate was filtered with a Kiriyama funnel and dried under vacuum to obtain the target compound crude crystals (158 mg).
- This crude crystal is dissolved in 32 mL of acetonitrile-water mixed solvent (1: 1), ion exchange resins Muromac® C1002 (1.58 g) and Muromac® A203T (1.58 g) are added, and impurities are added. It was purified by adsorption. After filtration of the resin, the filtrate was concentrated under reduced pressure and lyophilized to obtain PEG-Asp44 ( ⁇ -Tyramine-MTX) -Ac (109 mg). The MTX content of this polymer conjugate was 22.7% (w / w).
- Test Example 1 Measurement of drug release rate (drug release in the absence of hydrolase) About 5 mg of the sample was weighed and dissolved in acetonitrile (250 ⁇ L) and water (250 ⁇ L) (solution A). 0.1N sodium hydroxide solution (900 ⁇ L) was added to solution A (100 ⁇ L) and allowed to stand at room temperature for 1 hour. 50 ⁇ L of this solution was sampled, neutralized with 50 ⁇ L of 0.1N hydrochloric acid, and diluted with PBS solution (400 ⁇ L). This solution was analyzed by HPLC and used as a reference for the total amount of drug contained in the sample.
- solution A 200 ⁇ L was diluted with a PBS solution (1800 ⁇ L), placed in a 37 ° C. thermostat, sampled over time, and analyzed by HPLC.
- the drug release rate at each time point is calculated by comparing the peak area of the drug with the reference peak area of the total drug amount, and the results obtained are shown in FIG.
- MTX_A-33 is PEG-Asp33 ( ⁇ -tyramine-MTX) -Ac obtained in Example 1
- MTX_G-33 is PEG-Asp33 ( ⁇ -tyramine-MTX) obtained in Example 2.
- MTX_G-44 is PEG-Asp44 ( ⁇ -tyramine-MTX) -Ac obtained in Example 6
- MTX_G-40 is PEG-Asp40 ( ⁇ -tyramine-MTX) obtained in Example 4.
- MTX_A-40 is PEG-Asp40 ( ⁇ -tyramine-MTX) -Ac obtained in Example 3
- PEM_G-40 is PEG-Asp40 ( ⁇ , ⁇ -tyramine- PEM) -Ac.
- the polymer conjugate of folic acid or folic acid derivative of the present invention can release a drug in the absence of an enzyme, and the drug release rate depends on the acidity of the carboxylic acid of the drug molecule.
- the release rate of the ⁇ -substituted product is faster than the release rate of the ⁇ -substituted product.
- PEM_G-40 since a mixture of ⁇ and ⁇ is bound, the release rate is intermediate between the ⁇ -substituted product and the ⁇ -substituted product. This indicates that the drug release rate can be controlled by mixing the ⁇ -substituted product and the ⁇ -substituted product at an appropriate ratio.
- Test Example 2 Anti-tumor effect on mouse colon cancer Colon 26 transplanted mouse BALB / c mouse subcultured mouse colon cancer Colon 26 was made into a block of about 2 mm square and transplanted subcutaneously on the dorsal side of CDF1 mice using a trocar. did.
- the polymer conjugate MTX_A-40 PEG-Asp40 ( ⁇ -tyramine-MTX) -Ac of the present invention obtained in Example 3
- MTX_G-44 PEG
- the relative tumor volume on the day of determination for the untreated group was 12.3.
- 20 mg / kg of MTX was administered daily for 5 days, all animals died from toxicity.
- the relative tumor volume of 15 mg / kg was almost the same as that of the untreated group, which was 12.2, and no antitumor effect was observed.
- the polymer conjugate MTX_A-40 of the present invention died of toxicity at 50 mg / kg as MTX, but the relative tumor volume was 4.8.
- the relative tumor volume of 25 mg / kg is 6.8, and tumor growth is suppressed depending on the dose.
- MTX_G-44 at 25 mg / kg as MTX died of toxicity, but the relative tumor volume was 2.3.
- the relative tumor volume of 12.5 mg / kg is 8.2, which suppresses tumor growth depending on the dose.
- the high molecular weight conjugates of MTX (MTX_A-40, MTX_G-44) of the present invention strongly suppress the growth of tumors after a single administration, and their effects greatly exceed MTX that has been continuously administered, and are useful as anticancer agents. is there.
- the MTX_A-40 2.5 mg / kg group (MTX equivalent; high dose group) and the MTX_A-40 1.25 mg / kg group (MTX equivalent; low dose group)
- the 2.5 mg / kg administration group was administered into the tail vein on the 1st and 8th days.
- Leflonomide (10 mg / kg) was orally administered by gavage for 28 consecutive days from the sensitization date.
- the left footpad of the rat was observed to score the arthritis that developed, and the results are shown in FIG.
- the arthritis score criteria in Table 2 were used for scoring.
- the MTX administration group developed inflammation almost at the same time as the control group, and no anti-inflammatory effect was observed.
- the MTX_A-40 administration group delayed the onset of inflammation despite the total dose being 1 ⁇ 2 compared to MTX, and the polymer conjugate of MTX enhanced and sustained the anti-inflammatory action of MTX. It has been confirmed that the polymer conjugate of folic acid or folic acid derivative of the present invention is useful as a therapeutic agent for inflammatory diseases.
- Test Example 4 Anti-inflammatory evaluation 2 using rat collagen arthritis model 2 A 5 mg / mL aqueous solution of MTX_G-40 (PEG-Asp40 ( ⁇ -Tyramine-MTX) -Ac) obtained in Example 4 and a comparative control substance MTX was prepared and used for administration, and the same procedure as in Test Example 3 was performed. The positive target substance leflonomide was suspended in an aqueous solution of sodium carboxymethylcellulose (CMC) and used for administration.
- MTX_G-40 1.00 mg / kg (converted to MTX), 1.25 mg / kg (converted to MTX) and MTX 2.5 mg / kg were administered on the 1st, 8th and 15th days after sensitization.
- Leflonomide (10 mg / kg) was orally administered by gavage for 28 consecutive days from the sensitization date. After sensitization, the rat left footpad was observed, and the onset arthritis was scored using Table 2 above, and the results are shown in FIG.
- the MTX administration group delayed the onset of inflammation compared to the control group.
- MTX_G-40 was administered at a lower dose of 1.00 mg / kg than MTX and delayed the onset of inflammation further than MTX, and the 28-day onset of inflammation observed in the 1.25 mg / kg administration group It was confirmed that the polymer conjugate of MTX enhanced and sustained the anti-inflammatory action of MTX.
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Abstract
Description
また、葉酸自身や葉酸誘導体のロイコボリン等は葉酸欠乏症やメトトレキサートの毒性を中和する薬剤としても使用されている。
特許文献2には高分子化合物とカンプトテシン類をフェニルエステル結合で結合させることによって、化学的な安定性と生体内での薬剤遊離効率を同時に達成したことが報告されている。しかしながら、この方法をそのままメトトレキサートに代表される葉酸誘導体(葉酸自身も含む)のカルボキシ基に用いることはできない。
薬剤を高分子結合体とすることで薬理効果を向上させるためには薬剤が適度な速度で高分子結合体から遊離する必要があり、単純に高分子化合物に結合させるだけでは不十分である。薬剤と高分子化合物との結合が弱い場合、高分子結合体が不安定となり製剤化が極めて難しくなるばかりではなく、薬剤の遊離が速くて高分子化合物と結合させることによる薬物動態等の改良が期待できない。また、結合が強すぎる場合、薬物動態は改良されたとしても薬剤が高分子結合体から遊離されず薬理効果の発揮が困難となる。
(1)ポリエチレングリコール類と側鎖にカルボキシ基を有するポリマーからなるブロック共重合体の該カルボキシ基に下記式(I)
で表される置換基が結合している葉酸若しくは葉酸誘導体の高分子結合体、またはその薬理学的に許容される塩。
(3)ポリ酸性アミノ酸がポリアスパラギン酸またはポリグルタミン酸である上記(2)に記載の葉酸若しくは葉酸誘導体の高分子結合体、またはその薬理学的に許容される塩。
で表される上記(1)~(3)のいずれか一項に記載の葉酸若しくは葉酸誘導体の高分子結合体、または薬理学的に許容される塩。
(6)式(I)で表される置換基が下記式(III)
で表される置換基である上記(1)~(5)のいずれか一項に記載の葉酸若しくは葉酸誘導体の高分子結合体、または薬理学的に許容される塩。
(8)葉酸の誘導体がペメトレキセドである上記(1)~(6)のいずれか一項に記載の葉酸若しくは葉酸誘導体の高分子結合体、または薬理学的に許容される塩。
(10)上記(1)~(8)のいずれか一項に記載の葉酸若しくは葉酸誘導体の高分子結合体、または薬理学的に許容される塩を有効成分とする炎症疾患治療薬。
また、本発明で得られる高分子結合体は薬剤とリンカー分子がフェニルエステル結合しているので、実用的な化学的安定性と優れた生体内薬剤遊離効率を同時に達成することが可能であり、薬理効果を増強し、副作用を軽減することが可能となる。
更に、本発明の葉酸若しくは葉酸誘導体の高分子結合体から薬剤の遊離速度をリンカー部分の置換基を変えたり、結合する薬剤のカルボキシ基を選ぶことにより制御することができる。加えて、これらの遊離速度の異なる高分子結合体を混合することでも薬剤の放出速度を調整することができるため、より広い応用が可能である。
ポリエチレングリコール類部分の分子量は、通常300~500000程度であり、好ましくは500~100000程度、更に好ましくは1000~50000程度である。なお、本発明における分子量とはゲル浸透クロマトグラフィー(GPC法)で測定したピークトップ分子量である。
本発明におけるブロック共重合体1分子当たりのカルボキシ基の数は、3~200個程度が好ましく、5~100個程度がより好ましい。
葉酸若しくは葉酸誘導体とリンカー分子の結合の強さは、リンカー分子が開裂した際のフェノール性水酸基の酸性度に大きく影響され、Yを変えることによりその酸性度を変化させると葉酸若しくは葉酸誘導体とリンカー分子の結合の強さも変化する。例えば、Aがフェニル基の場合、葉酸若しくは葉酸誘導体の遊離を早めるためには電子吸引基であるシアノ基、ニトロ基、ハロゲン原子等が好ましく、葉酸若しくは葉酸誘導体の遊離を遅らせるには電子供与基であるアルキル基等が好ましい。Yの置換数は置換可能な数であれば特に限定されない。
Yが置換基である場合、その置換位置は、遊離速度に対する影響を考慮する必要はあるが、特に限定されない。
上記式(I)のEとしては、E-CO2Hで葉酸若しくは葉酸誘導体を意味する葉酸若しくは葉酸誘導体の残基であり、葉酸若しくはカルボキシ基を有する葉酸誘導体であれば特に限定されず、例えば、E-CO2Hとして上記の葉酸、メトトレキサート、アミノプテリン、プララトレキセート、プレビトレキセド、エダトレキセート、ペメトレキセド、ラルチトレキセド、ロメトレキソール等が挙げられ、中でもメトトレキサート、ペメトレキセド等が好ましい。
式(II)のDにおける置換基を有していてもよい(C1~C6)アルキル基としては、例えば、メチル基、エチル基、n-プロピル基、i-プロピル基、n-ブチル基、s-ブチル基、t-ブチル基、n-ペンチル基、n-ヘキシル基、ベンジル基、ジメトキシエチル基、ジエトキエチル基、アミノメチル基、アミノエチル基、3-アミノプロピル基、4-アミノブチル基等が挙げられる。Dとしては無置換(C1~C6)アルキル基が好ましく、中でもメチル基が特に好ましい。
上記式(II)中、nの平均値は5から11500程度であり、好ましくは50~1000程度であり、より好ましくは100~300程度である。
上記式(II)中、c+d+eは高分子結合体1分子中の全アスパラギン酸数を表し、その平均値は3~200程度であり、好ましくは5~100程度であり、より好ましくは6~60程度である。ポリアスパラギン酸の各構成単位はランダムに結合していてもブロックを形成して結合していてもよいが、c+dは正数であり、eは0でもよい。
また、全アスパラギン酸数(c+d+e)に対するα-アミノ酸型構成単位(c)の割合は好ましくは10~100%であり、特に好ましくは20~100%である。この割合は、例えば、特許文献1等に準じた製造方法の工程中、ポリアスパラギン酸の保護基の脱保護条件等を選ぶことにより適宜変えることが可能である。
メトトレキサートはカルボキシ基を2個有していることから、チラミンとメトトレキサートがエステル結合している化合物にも2つの位置異性体が存在する。以下に示すように、メトトレキサートの部分構造であるグルタミン酸のα-カルボキシ基がチラミンと結合したものをα置換体、γ-カルボキシ基がチラミンと結合したものをγ置換体と呼ぶ。
まず、チラミンのアミノ基をエステル結合生成後に除去可能な保護基で保護する。該保護基としては、一般的なアミノ基の保護基であれば特に問わないが、チラミンとメトトレキサートのエステル結合が安定である脱保護条件、即ち、中性若しくは酸性条件で脱保護できる保護基が望ましく、例えば、ベンジルオキシカルボニル基、t-ブトキシカルボニル(Boc)基、アリルオキシカルボニル基等が挙げられる。
脱水縮合反応の反応温度は、通常4~60℃、好ましくは15~50℃であり、反応時間は1時間~数日、好ましくは4~48時間である。
該有機溶媒としては反応が進行する限り特に限定されないが、例えば、トルエン、キシレン等の芳香族炭化水素類、塩化メチレン、クロロホルム、1,2-ジクロロエタン等のハロゲン化炭化水素類、テトラヒドロフラン、ジオキサン、ジメトキシエタン、ジエチレングリコールジメチルエーテル等のエーテル類、アセトニトリル、プロピオニトリル等のニトリル類、ジメチルホルムアミド、ジメチルアセトアミド、N-メチルピロリドン等のアミド類、1,3-ジメチルイミダゾリジノン等のウレア類または前記溶媒の混合溶媒等が挙げられ、好ましくはアミド類またはウレア類であり、より好ましくはジメチルホルムアミドまたは1,3-ジメチルイミダゾリジノンである。
脱水縮合反応を行った後、必要に応じて適当な精製処理を行い、チラミンのアミノ基が保護された上記のα置換体とγ置換体の混合物若しくは分離された各異性体を得る。更に、アミノ基の保護基を適当な処理を行い脱保護し、上記のメトトレキサートとチラミンの縮合体(α置換体、γ置換体、若しくはその混合体)を得る。
続いて該製造方法について説明する。
脱水縮合反応の反応温度は、通常4~60℃、好ましくは15~50℃であり、反応時間は1時間~数日、好ましくは4~48時間である。
該脱水縮合剤としては、上記のメトトレキサートとアミノ基を保護したチラミンの縮合反応における脱水縮合剤と同様な脱水縮合剤が挙げられる。
脱水縮合反応の際には反応助剤を用いてもよく、該反応助剤としては、上記のメトトレキサートとアミノ基を保護したチラミンの縮合反応における反応助剤と同様な反応助剤が挙げられる。
活性化の方法としては、通常ペプチド結合製造の際に用いられる方法が適用可能であり、例えば、上記の試薬を使用した方法が挙げられる。即ち、カルボン酸類とN-ヒドロキシスクシンイミド等の反応助剤を上記の脱水縮合剤で縮合し、活性エステル体として単離した後にアミン類を加えてアミドを得る方法等である。
葉酸及び葉酸誘導体の結合量は、例えば、紫外線吸収スペクトルの強度から求めることができる。また、本発明の葉酸若しくは葉酸誘導体の高分子結合体をアルカリ加水分解することにより遊離する葉酸または葉酸誘導体を、例えば、高速液体クロマトグラフィー(HPLC)等を用いて定量することによっても求めることができる。
該塩としては、例えば、リチウム塩、ナトリウム塩、カリウム塩、マグネシウム塩、アンモニウム塩またはトリエチルアンモニウム塩等が挙げられる。
該製剤中における葉酸及び葉酸誘導体の高分子結合体の含量は製剤により種々異なるが、通常0.1~100重量%、好ましくは1~98重量%である。
Tyramine(5.49g)をジオキサン110mL、水110mLに溶解し、(Boc)2O(ジ-t-ブチルジカーボネート:9.60g)を加えた。室温で4時間攪拌した後、酢酸エチルで抽出した。有機層を無水硫酸ナトリウムで脱水した後、減圧濃縮した。残渣をシリカゲルカラム(ヘキサン-酢酸エチル)で精製し、目的物の画分を減圧濃縮することでBoc-Tyramine(9.05g)を得た。
MTX(メトトレキサート:8.18g)、Boc-Tyramine(8.54g)、DMAP(ジメチルアミノピリジン:4.40g)をDMF(ジメチルホルムアミド:164mL)に溶解した後、ジイソプロピルカルボジイミド(5.64mL)を加えた。室温で4時間攪拌した後、酢酸エチル(1.64L)、水(1.64L)で抽出した。水層に20mMクエン酸緩衝液(pH4.6)2.9Lを加え、生成した沈殿を桐山ロートでろ過した。得られた沈殿を塩化メチレン-メタノール混合溶媒(1:1)に溶解させ、無水硫酸ナトリウムで脱水した後、減圧下溶媒を留去した。残渣をシリカゲルカラムで精製し、2種のMTXモノエステル体(α-モノエステル体 1.711g、γ-モノエステル体 1.332g)を得た。α-モノエステル体とγ-モノエステル体はHPLC条件1において保持時間がそれぞれ13.3分、13.7分であった。
カラム:Inertsil ODS-3(5μm) 4.6x150mm
カラム温度:40℃
溶離液:A液 0.1%リン酸水溶液、B液 アセトニトリル
グラジエント
時間(分) 0 30 35 35.1 45
B液(%) 10 90 90 10 10
流速:1.0mL/分
検出器:UV(254nm)
J.Med.Chem.,1992,35,p4450-4454に記載されている方法で得られたペメトレキセド(PEM:624mg)、Boc-Tyramine(693mg)、DMAP(357mg)をDMF(12.5mL)に溶解した後、ジイソプロピルカルボジイミド(457μL)を加えた。室温で45分攪拌した後、酢酸(334μL)を加えた。HPLC条件2で精製を行い、Boc-Tyramine-PEM(α、γ-モノエステルの混合体:約1:1)277mgを得た。α-エステルとγ-エステルはHPLC条件1において保持時間がそれぞれ18.0分、18.3分であった。
HPLC条件2
カラム:Inertsil ODS-3(5μm) 20x250mm
カラム温度:室温
溶離液:A液 0.1%トリフルオロ酢酸水溶液、B液 アセトニトリル
グラジエント
時間(分) 0 4.9 5 13 13.1 20 20.1 30
B液(%) 10 10 20 20 40 40 10 10
流速:20mL/分
検出器:UV(254nm)
参考例2で得られたα-Boc-Tyramine-MTX(100mg)に酢酸エチル(1.5mL)を加え懸濁させたのち、4N HCl/EtOAc(0.5mL)を加え、室温で1時間攪拌した。沈殿を桐山ロートでろ過し、真空乾燥してα-Tyramine-MTX(111mg)を得た。回収率から3塩酸塩であると考えられる。
参考例2で得られたγ-Boc-Tyramine-MTX(100mg)に酢酸エチル(1.5mL)を加え懸濁させたのち、4N HCl/EtOAc(0.5mL)を加え、室温で1時間攪拌した。沈殿を桐山ロートでろ過し、真空乾燥してγ-Tyramine-MTX(108mg)を得た。回収率から3塩酸塩であると考えられる。
参考例3で得られたBoc-Tyramine-PEM(α、γ混合体)100mgに酢酸エチル(1.5mL)を加え懸濁させたのち、4N HCl/ジオキサン(0.5mL)を加え、室温で1時間攪拌した。沈殿を桐山ロートでろ過し、真空乾燥してTyramine-PEM(α、γ混合体)93mgを得た。回収率から2塩酸塩であると考えられる。
特許文献3に記載の方法に準じて合成したPEG-Asp33-Ac(片末端メチル片末端アミノプロピルポリエチレングリコールポリアスパラギン酸(33ユニット)のブロック共重合体N-アセチル体:485mg)、HOSu(N-ヒドロキシスクシンイミド:115mg)をDMF(9.7mL)に溶解し33℃のオイルバスで加温した。DCC(ジシクロヘキシルカルボジイミド:206mg)を添加して1時間攪拌した。生成したウレアを桐山ロートでろ過し、得られたろ液に酢酸エチル(39mL)を加え希釈した後に、ヘキサン(58mL)を添加し結晶を析出させた。15分攪拌した後に攪拌をとめ上澄みを除去し、更にヘキサン-酢酸エチル混合溶媒(3:2)を追加し攪拌した。桐山ロートで沈殿をろ過し、真空乾燥してPEG-Asp33(OSu)-Ac(454mg)を得た。
参考例7で得られたPEG-Asp33(OSu)-Ac(150mg)と参考例4で得られたα-Tyramine-MTX(113mg)をDMF(1.5mL)に溶解し、トリエチルアミン(69μL)を添加した。室温で4時間攪拌したのち、ヘキサン-酢酸エチル混合溶媒(4:1)100mL中に滴下した。10分間攪拌した後に攪拌をとめ上澄みを除去し、更にヘキサン-酢酸エチル混合溶媒(4:1)を追加し攪拌した。桐山ロートで沈殿をろ過し、真空乾燥して目的化合物の粗結晶(261mg)を得た。
この粗結晶をアセトニトリル-水混合溶媒(1:1)52mLに溶解し、イオン交換樹脂 Muromac(登録商標)C1002(2.61g)、Muromac(登録商標)A203T(2.61g)(共に、ムロマチテクノス(株)製)を加え、不純物を吸着精製した。樹脂をろ過した後、ろ液を減圧濃縮、凍結乾燥してPEG-Asp33(α-Tyramine-MTX)-Ac(172mg)を得た。この高分子結合体のMTX含有率は19.6%(w/w)であった。薬剤(MTX)含有率は、得られた高分子結合体を水酸化ナトリウム水溶液で加水分解させ、遊離してきた薬剤(MTX)をHPLCで分析することで算出した。以下の高分子結合体でも同様である。
参考例7で得られたPEG-Asp33(OSu)-Ac(146mg)と参考例5で得られたγ-Tyramine-MTX(110mg)をDMF(1.5mL)に溶解し、トリエチルアミン(67μL)を添加した。室温で4時間攪拌したのち、ヘキサン-酢酸エチル混合溶媒(4:1)100mL中に滴下した。10分間攪拌した後に攪拌をとめ上澄みを除去し、更にヘキサン-酢酸エチル混合溶媒(4:1)を追加し攪拌した。桐山ロートで沈殿をろ過し、真空乾燥して目的化合物の粗結晶(270mg)を得た。
この粗結晶をアセトニトリル-水混合溶媒(1:1)54mLに溶解し、イオン交換樹脂 Muromac(登録商標)C1002(2.70g)、Muromac(登録商標)A203T(2.70g)を加え、不純物を吸着精製する。樹脂をろ過した後、ろ液を減圧濃縮、凍結乾燥してPEG-Asp33(γ-Tyramine-MTX)-Ac(170mg)を得た。この高分子結合体のMTX含有率は25.0%(w/w)であった。
特許文献3に記載の方法に準じて合成したPEG-Asp40-Ac(片末端メチル片末端アミノプロピルポリエチレングリコールポリアスパラギン酸(40ユニット)のブロック共重合体N-アセチル体:420mg)、HOSu(115mg)をDMF(8.4mL)に溶解し37℃のオイルバスで加温した。DCC(206mg)を添加して1時間攪拌した。生成したウレアを桐山ロートでろ過し、得られたろ液に酢酸エチル(34mL)を加え希釈した後に、ヘキサン(50mL)を添加し結晶を析出させた。40分攪拌した後に攪拌をとめ上澄みを除去し、更にヘキサン-酢酸エチル混合溶媒(4:1)を追加し攪拌する。桐山ロートで沈殿をろ過し、真空乾燥してPEG-Asp40(OSu)-Ac(424mg)を得た。
参考例8で得られたPEG-Asp40(OSu)-Ac(220mg)と参考例4で得られたα-Tyramine-MTX(177mg)をDMF(2.2mL)に溶解し、トリエチルアミン(109μL)を添加した。室温で4時間攪拌したのち、ヘキサン-酢酸エチル混合溶媒(4:1)100mL中に滴下した。70分間攪拌した後に攪拌をとめ上澄みを除去し、更にヘキサン-酢酸エチル混合溶媒(4:1)を追加し攪拌した。桐山ロートで沈殿をろ過し、真空乾燥して目的化合物の粗結晶(413mg)を得た。
この粗結晶をアセトニトリル-水混合溶媒(1:1)83mLに溶解し、イオン交換樹脂 Muromac(登録商標)C1002(4.13g)、Muromac(登録商標)A203T(4.13g)を加え、不純物を吸着精製した。樹脂をろ過した後、ろ液を減圧濃縮、凍結乾燥してPEG-Asp40(α-Tyramine-MTX)-Ac(263mg)を得た。この高分子結合体のMTX含有率は23.6%(w/w)であった。
参考例8で得られたPEG-Asp40(OSu)-Ac(100mg)と参考例5で得られたγ-Tyramine-MTX(81mg)をDMF(1.0mL)に溶解し、トリエチルアミン(49μL)を添加した。室温で4時間攪拌したのち、ヘキサン-酢酸エチル混合溶媒(4:1)100mL中に滴下した。25分間攪拌した後に攪拌をとめ上澄みを除去し、更にヘキサン-酢酸エチル混合溶媒(4:1)を追加し攪拌した。桐山ロートで沈殿をろ過し、真空乾燥して目的化合物の粗結晶(189mg)を得た。
この粗結晶をアセトニトリル-水混合溶媒(1:1)38mLに溶解し、イオン交換樹脂 Muromac(登録商標)C1002(1.89g)、Muromac(登録商標)A203T(1.89g)を加え、不純物を吸着精製した。樹脂をろ過した後、減圧濃縮、凍結乾燥してPEG-Asp40(γ-Tyramine-MTX)-Ac(126mg)を得た。この高分子結合体のMTX含有率は28.6%(w/w)であった。
参考例8で得られたPEG-Asp40(OSu)-Ac(129mg)と参考例6で得られたTyramine-PEM(α、γ混合体:93mg)をDMF(1.3mL)に溶解し、トリエチルアミン(42μL)を添加した。室温で2.5時間攪拌したのち、ヘキサン-酢酸エチル混合溶媒(4:1)130mL中に滴下した。10分間攪拌した後に攪拌をとめ上澄みを除去し、更にヘキサン-酢酸エチル混合溶媒(4:1)を追加し攪拌した。桐山ロートで沈殿をろ過し、真空乾燥して目的化合物の粗結晶(246mg)を得た。
この粗結晶をアセトニトリル-水混合溶媒(1:1)50mLに溶解し、イオン交換樹脂 Muromac(登録商標)C1002(2.5g)、Muromac(登録商標)A203T(2.5g)を加え、不純物を吸着精製した。樹脂をろ過した後、ろ液を減圧濃縮、凍結乾燥してPEG-Asp40(α、γ-Tyramine-PEM)-Ac(150mg)を得た。この高分子結合体のPEM含有率は22.1%(w/w)であった。
特許文献3に記載の方法に準じて合成したPEG-Asp44-Ac(片末端メチル片末端アミノプロピルポリエチレングリコールポリアスパラギン酸(44ユニット)のブロック共重合体N-アセチル体:582mg)、HOSu(173mg)をDMF(11.6mL)に溶解し37℃オイルバスで加温した。DCC(309mg)を添加して1時間攪拌した。生成したウレアを桐山ロートでろ過した。得られたろ液に酢酸エチル(46mL)を加え希釈した後に、ヘキサン(70mL)を添加し結晶を析出させた。30分攪拌した後に攪拌をとめて上澄みを除去し、更にヘキサン-酢酸エチル混合溶媒(4:1)を追加し攪拌した。桐山ロートで沈殿をろ過し、真空乾燥してPEG-Asp44(OSu)-Ac(543mg)を得た。
参考例9で得られたPEG-Asp44(OSu)-Ac(100mg)と参考例5で得られたγ-Tyramine-MTX(57mg)をDMF(2mL)に溶解し、トリエチルアミン(35μL)を添加した。室温で4時間攪拌したのち、ヘキサン-酢酸エチル混合溶媒(4:1)100mL中に滴下した。15分間攪拌した後に攪拌をとめ上澄みを除去し、更にヘキサン-酢酸エチル混合溶媒(4:1)を追加し攪拌した。桐山ロートで沈殿をろ過し、真空乾燥して目的化合物の粗結晶(158mg)を得た。
この粗結晶をアセトニトリル-水混合溶媒(1:1)32mLに溶解し、イオン交換樹脂 Muromac(登録商標)C1002(1.58g)、Muromac(登録商標)A203T(1.58g)を加え、不純物を吸着精製した。樹脂をろ過した後、ろ液を減圧濃縮、凍結乾燥してPEG-Asp44(γ-Tyramine-MTX)-Ac(109mg)を得た。この高分子結合体のMTX含有率は22.7%(w/w)であった。
サンプル約5mgを秤量し、アセトニトリル(250μL)、水(250μL)に溶解させた(溶液A)。溶液A(100μL)に0.1N水酸化ナトリウム溶液(900μL)を加えて1時間室温で静置した。この溶液50μLをサンプリングし、0.1N塩酸50μLで中和後、PBS溶液(400μL)で希釈した。この溶液をHPLCで分析しサンプルに含まれる総薬剤量の基準とした。
一方、溶液A(200μL)をPBS溶液(1800μL)で希釈後、37℃の恒温槽中に靜置し、経時的にサンプリングしてHPLCで分析を行った。薬剤のピーク面積を上記総薬剤量の基準ピーク面積と比較して各経時点における薬剤放出率を計算し、得られた結果を図1に示す。
BALB/cマウスの皮下で継代したマウス結腸癌Colon26を約2mm角のブロックにし、套管針を用いてCDF1マウスの背側部皮下に移植した。腫瘍移植後7日目に実施例3で得られた本発明の高分子結合体MTX_A-40(PEG-Asp40(α-Tyramine-MTX)-Ac、実施例6で得られたMTX_G-44(PEG-Asp44(γ-Tyramine-MTX)-Acと比較対照物質としてのMTXを尾静脈に投与した。本発明の高分子結合体は注射用水に溶解し単回投与した。対照としたMTXは蒸留水で溶解・稀釈して、1日1回、5日間連日投与した。投与後、腫瘍の長径(Lmm)及び短経(Wmm)を定期的に測定し、腫瘍体積を(L×W2)/2により算出した。投与開始日の腫瘍体積を1.0とし、投与開始後12日目を判定日として各投与群の相対腫瘍体積を求めた。その結果を表1に示す。本発明の化合物の用量はMTX換算である。
MTXの20mg/kgを5日間連日投与すると動物は全匹毒性により死亡した。15mg/kgの相対腫瘍体積は12.2と無処置群の相対腫瘍体積とほとんど変わらず、抗腫瘍効果は見られなかった。
一方、本発明の高分子結合体MTX_A-40はMTXとして50mg/kgでは1匹が毒性によって死亡したが、相対腫瘍体積は4.8であった。同じく25mg/kgの相対腫瘍体積は6.8であり、投与量に依存して腫瘍増殖を抑制している。MTX_G-44はMTXとして25mg/kgでは1匹が毒性によって死亡したが、相対腫瘍体積は2.3であった。同じく12.5mg/kgの相対腫瘍体積は8.2であり投与量に依存して腫瘍増殖を抑制している。
以上、本発明のMTXの高分子結合体(MTX_A-40、MTX_G-44)は単回投与において腫瘍の増殖を強く抑制し、その効果は連投したMTXを大きく上回るものであり、抗癌剤として有用である。
DA/Slcラットの背部にウシII型コラーゲンIIとFreund’s incomplete adjuvantとの乳濁液を背部皮内4箇所に合計0.4mL皮内投与(感作)し、関節炎を誘発した。実施例3で得られたMTX_A-40(PEG-Asp40(α-Tyramine-MTX)-Ac)と比較対照物質MTXは5mg/mL水溶液を調製し投与に用いた。陽性対象物質のレフロノミドはカルボキシメチルセルロースナトリウム(CMC)水溶液で懸濁させて投与に用いた。MTX_A-40の2.5mg/kg投与群(MTX換算;高用量群)は感作後1日目に、MTX_A-40の1.25mg/kg投与群(MTX換算;低用量群)とMTXの2.5mg/kg投与群は1日目と8日目に尾静脈内に投与した。また、レフロノミド(10mg/kg)は感作日から28日間連続して強制経口投与した。感作後、ラット左足蹠を観察し発症した関節炎をスコア化し、結果を図2に示す。スコア化には表2の関節炎スコア基準を用いた。
一方、MTX_A-40投与群はMTXと比べ総投与量が1/2であるにも関わらず炎症の発症時期を遅延させており、MTXの高分子結合体はMTXの抗炎症作用を増強、持続させていることが確認され、本発明の葉酸若しくは葉酸誘導体の高分子結合体は炎症疾患治療薬として有用であることを示している。
実施例4で得られたMTX_G-40(PEG-Asp40(γ-Tyramine-MTX)-Ac)と比較対照物質MTXの5mg/mL水溶液を調製し投与に用い、試験例3と同様に行った。陽性対象物質のレフロノミドはカルボキシメチルセルロースナトリウム(CMC)水溶液で懸濁させて投与に用いた。MTX_G-40の1.00mg/kg(MTX換算)、1.25mg/kg(MTX換算)投与群とMTXの2.5mg/kg投与群は感作後1日目、8日目、15日目、22日目に尾静脈内に投与した。また、レフロノミド(10mg/kg)は感作日から28日間連続して強制経口投与した。感作後、ラット左足蹠を観察し、発症した関節炎を上記表2を用いてスコア化し、結果を図3に示す。
Claims (11)
- 側鎖にカルボキシ基を有するポリマーがポリ酸性アミノ酸である請求項1に記載の葉酸若しくは葉酸誘導体の高分子結合体、またはその薬理学的に許容される塩。
- ポリ酸性アミノ酸がポリアスパラギン酸またはポリグルタミン酸である請求項2に記載の葉酸若しくは葉酸誘導体の高分子結合体、またはその薬理学的に許容される塩。
- Dが無置換(C1~C6)アルキル基であり、nの平均値が50~1000であり、c+d+eの平均値が5~100であり、Bが(C1~C6)アシル基である請求項4記載の葉酸若しくは葉酸誘導体の高分子結合体、または薬理学的に許容される塩。
- 葉酸の誘導体がメトトレキサートである請求項1~6のいずれか一項に記載の葉酸若しくは葉酸誘導体の高分子結合体、または薬理学的に許容される塩。
- 葉酸の誘導体がペメトレキセドである請求項1~6のいずれか一項に記載の葉酸若しくは葉酸誘導体の高分子結合体、または薬理学的に許容される塩。
- 請求項1~8のいずれか一項に記載の葉酸若しくは葉酸誘導体の高分子結合体、または薬理学的に許容される塩を有効成分とする抗癌剤。
- 請求項1~8のいずれか一項に記載の葉酸若しくは葉酸誘導体の高分子結合体、または薬理学的に許容される塩を有効成分とする炎症疾患治療薬。
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KR101203475B1 (ko) * | 2004-09-22 | 2012-11-21 | 니폰 가야꾸 가부시끼가이샤 | 신규 블록 공중합체, 미셀 제제물 및 이를 유효성분으로함유하는 항암제 |
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Also Published As
Publication number | Publication date |
---|---|
US20110294980A1 (en) | 2011-12-01 |
EP2284209B1 (en) | 2016-08-31 |
JPWO2009136572A1 (ja) | 2011-09-08 |
EP2284209A4 (en) | 2014-11-26 |
EP2284209A1 (en) | 2011-02-16 |
JP5366940B2 (ja) | 2013-12-11 |
US9149540B2 (en) | 2015-10-06 |
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