Classifications

• • 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
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • 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
  • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
  • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
  • C08G65/32—Polymers modified by chemical after-treatment
• • 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
  • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
  • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
  • C08G65/32—Polymers modified by chemical after-treatment
  • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
  • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
  • C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
• • 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
  • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
  • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
  • C08G65/32—Polymers modified by chemical after-treatment
  • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
  • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
  • C08G65/33331—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing imide group
  • C08G65/33337—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing imide group cyclic
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L2203/00—Applications
  • C08L2203/02—Applications for biomedical use

Definitions

• the present invention relates to a medical four-branched water-soluble polymer that can improve the solubility of a conjugate with a biologically relevant substance and suppress the aggregation of the conjugate.
• bio-related substances such as antibodies, hormones, cytokines, and enzymes are usually administered into the body and are rapidly excreted from the body through glomerular filtration in the kidney and uptake by macrophages in the liver and spleen. put away. Therefore, it has a short half-life in blood, and it is often difficult to obtain sufficient pharmacological effects.
• bio-related substances with water-soluble polymers such as polyethylene glycol (PEG), albumin, and the like.
• PEG polyethylene glycol
• albumin albumin
• Antibodies which are one of the bio-related substances, have high binding affinity, binding specificity, and high stability in blood, so they are currently applied to many diagnostic agents and pharmaceuticals.
• Escherichia coli which is widely used as a means of producing general bio-related substances, does not have a glycosylation function
• most antibody drugs are currently produced from mammalian cells (CHO cells, NSO cells, etc.) that add human-type sugar chains. ) is manufactured by Since the sugar chains of antibodies produced by CHO cells, etc. are biosynthesized by glycosyltransferases, the sugar chain structure and the amount of glycosylation change with different passages even in the same cell line.
• Non-Patent Document 1 Antibodies also have the problem of high production costs due to the use of animal cells compared to inexpensive E. coli, and the tendency to aggregate (Non-Patent Document 2).
• antibodies are huge molecules with a molecular weight of approximately 160,000, and although they have a long half-life in the blood, they have the problem of extremely slow migration from blood to tissues. Therefore, studies are being conducted on antibody fragments with reduced molecular weights and enhanced biotransportability. Since antibody fragments have a small molecular weight and do not have sugar chains, unlike antibodies, they can be produced in E. coli, which is advantageous in terms of production cost.
• antibody fragments having antibody variable regions include Fv, Fab, Fab', F(ab') 2 , single chain antibodies (scFv), bispecific antibodies (diabodies), and various animal-derived nanobodies.
• scFv single chain antibodies
• scFv bispecific antibodies
• various animal-derived nanobodies include Fv, Fab, Fab', F(ab') 2 , single chain antibodies (scFv), bispecific antibodies (diabodies), and various animal-derived nanobodies.
• PEG water-soluble polymer
• Non-Patent Document 3 Non-Patent Document 3
• these PEGylated antibody fragments also cause aggregation in the same way as antibodies.
• Non-Patent Document 4 Non-Patent Document 4
• Cyclic peptides are less soluble than general peptides and are known to cause aggregation due to insolubilization and self-association, so efforts are being made to add solubilizers and modify them with water-soluble polymers (patented Document 3, Non-Patent Document 5). Furthermore, in recent years, enhancement of pharmacological activity has been demanded so that the best therapeutic effect can be obtained. efforts are being made to
• the problem to be solved by the present invention is to provide a medical tetra-branched water-soluble polymer that can suppress aggregation and insolubilization that occur when two molecules of bio-related substances are combined.
• X 1 and X 2 are each independently a functional group capable of reacting with a bio-related substance
• P 1 to P 4 are each independently a divalent group of a water-soluble polymer
• Q 1 has the formula (q1):
• Q 2 is a trivalent group represented by Q 2 is represented by formulas (q1) to (q3):
• X 1 is a functional group capable of reacting with a bio-related substance
• P 1 and P 3 are each independently a divalent group of a water-soluble polymer
• Q 1 has the formula (q1):
• Q 2 is a trivalent group represented by Q 2 is represented by formulas (q1) to (q3):
• the functional group capable of reacting with a biomaterial is an active ester group, an active carbonate group, a formyl group, an isocyanate group, an isothiocyanate group, an epoxy group, a maleimidyl group, a vinylsulfonyl group, an acryloyl group, an alkylsulfonyloxy group; any one of the above [1] to [4], which is a carboxy group, a mercapto group, a pyridyldithio group, an ⁇ -haloacetyl group, an alkynyl group, an allyl group, a vinyl group, an amino group, an aminooxy group, a hydrazide group, or an azide group; 1. Medical tetra-branched water-soluble polymer according to one.
• the four-branched water-soluble polymer for medical use of the present invention can improve aggregation and solubility, which have been problems with antibodies and cyclic peptides (bio-related substances). Furthermore, since the four-branched water-soluble polymer for medical use of the present invention has functional groups capable of reacting with two bio-related substances in one molecule, it is possible to combine a conventional water-soluble polymer with one bio-related substance. Compared with the body, the conjugate of the medical tetra-branched water-soluble polymer of the present invention and two bio-related substances may have improved pharmacological activity.
• the present invention will be described in detail below.
• the four-branched water-soluble polymer for medical use of the present invention (hereinafter sometimes abbreviated as "the water-soluble polymer of the present invention") may be used alone or in combination of two or more.
• the water-soluble polymer of the present invention is represented by the following formula (1) or (2).
• the end of the straight line "-" in the chemical formula representing the group in this specification indicates a bonding position, not a carbon atom.
• X 1 and X 2 are each independently a functional group capable of reacting with a bio-related substance
• P 1 to P 4 are each independently a divalent group of a water-soluble polymer
• Q 1 has the formula (q1):
• Q 2 is a trivalent group represented by Q 2 is represented by formulas (q1) to (q3):
• L 1 to L 8 are each independently a single bond or a divalent spacer
• L9 is a divalent spacer
• R1 and R2 are each independently a hydrocarbon group.
• water-soluble polymer of the present invention represented by formula (1) may be abbreviated as “water-soluble polymer (1)”.
• Water-soluble polymers of the present invention represented by other formulas may be similarly abbreviated.
• the water-soluble polymer of the present invention corresponds to both water-soluble polymer (1) and water-soluble polymer (2), the water-soluble polymer is classified as water-soluble polymer (1) in the present invention.
• the water-soluble polymer of the present invention is preferably represented by formula (3) or formula (4) below.
• the water-soluble polymer of the present invention represented by formula (3) or formula (4) is the "X 2 -L 3 -P 2 -L 4 -" and " -L 7 -P 4 -L 8 -R 2 '' are respectively "X 1 -L 1 -P 1 -L 2 -" and "-L 5 -P 3 -L 6 -R 1 '' is.
• the definitions of symbols in formulas (3) and (4) are as described above. Further, unless otherwise specified, the explanations (preferred embodiments, etc.) of the symbols in formula (3) or (4) are the same as the explanations of the symbols in formula (1) or (2), respectively, which will be described later. is.
• the water-soluble polymer of the present invention corresponds to both water-soluble polymer (3) and water-soluble polymer (4), the water-soluble polymer is classified as water-soluble polymer (3) in the present invention.
• the groups in formula (1) or (2) are described below in order.
• the divalent groups of the water-soluble polymers of P 1 to P 4 are preferably each independently divalent groups of polyethylene glycol (PEG), polyoxazoline or polysialic acid.
• the divalent group of polyethylene glycol refers to a divalent group having a structure in which hydrogen atoms are removed from hydroxy groups at both ends of polyethylene glycol (that is, —O—(C 2 H 4 O) n - (wherein n is the number of repeating units).
• divalent group of polyoxazoline is represented by the following formula:
• R is an alkyl group and n is the number of repeating units.
• R is an alkyl group and n is the number of repeating units.
• the divalent group of polysialic acid means a divalent group having a structure in which hydrogen atoms are removed from the hydroxy groups at both ends of polysialic acid.
• the divalent groups of the water-soluble polymer P 1 to P 4 are each independently divalent groups of polyethylene glycol or polysialic acid, more preferably divalent groups of polyethylene glycol. is the base of the valence.
• P 1 to P 4 are divalent groups of polyethylene glycol, the number average molecular weights of the divalent groups of polyethylene glycol may be the same or different.
• the total number average molecular weight of the divalent groups of the water-soluble polymers P 1 to P 4 is preferably 4,000 or more and 160,000 or less, and more It is preferably 10,000 or more and 120,000 or less, more preferably 20,000 or more and 80,000 or less.
• the total number average molecular weight of the water-soluble polymers P 1 to P 4 is 20,000 or more and 160,000. It is below.
• the number average molecular weight of water-soluble polymers can be measured by gel permeation chromatography (GPC).
• the sum of the number average molecular weights of the divalent groups of the two P1 and two P3 water-soluble polymers is preferably 4,000 or more and 160,000 or less. , more preferably 10,000 or more and 120,000 or less, and still more preferably 20,000 or more and 80,000 or less. In one preferred embodiment of the present invention, the sum of the number average molecular weights of the two P1 and the two P3 water-soluble polymers in both Formula (3) and Formula (4) of the present invention is 20,000. Above 160,000 and below.
• the number average molecular weights of the divalent groups of the water-soluble polymers P 1 to P 4 are each independently preferably 1,000 to 80,000, more preferably 2,000 to 60,000, More preferably 2,500 to 40,000.
• Q 1 is a trivalent group represented by formula (q1)
• Q 2 is a trivalent group represented by any one of formulas (q1) to (q3).
• a trivalent group represented by formula (q1) can be formed, for example, from glycerin or a derivative thereof.
• a trivalent group represented by formula (q2) can be formed, for example, from glutamic acid or a derivative thereof.
• a trivalent group represented by formula (q3) can be formed, for example, from lysine or a derivative thereof.
• Q2 is preferably a trivalent group represented by formula (q1) or formula (q2), more preferably a trivalent group represented by formula (q1).
• X 1 and X 2 are each independently a functional group capable of reacting with a bio-related substance.
• the functional group is not particularly limited as long as it forms a covalent bond by reacting with a functional group present in biologically relevant substances such as physiologically active proteins, peptides, antibodies, nucleic acids, and anticancer agents to be chemically modified.
• Examples of the functional group include "Harris, J. M. Poly(Ethylene Glycol) Chemistry; Plenum Press: New York, 1992", “Hermanson, G. T. Bioconjugate Techniques, 2nd ed.; Academic Press: San Diego, CA, 2008", and "PEGylated Protein Drugs: Basic Science and Clinical Applications; Veronese, F. M., Ed.; Birkhauser: Basel, Switzerland, 2009”.
• a functional group capable of reacting with a bio-related substance is particularly a functional group capable of forming a chemical bond with a functional group possessed by the bio-related substance, such as an amino group, a mercapto group, a formyl group, a carboxy group, an unsaturated bond, or an azide group. Not restricted.
• Examples of functional groups that can react with biological substances include active ester groups, active carbonate groups, formyl groups, isocyanate groups (also known as “isocyanato groups”), isothiocyanate groups (also known as “isothiocyanato groups”), epoxy groups, carboxy group, mercapto group, maleimidyl group, substituted maleimidyl group, hydrazide group, pyridyldithio group, substituted sulfonate group, vinylsulfonyl group, amino group, aminooxy group (H 2 N—O—), iodoacetamide group, alkylcarbonyl group, Examples include alkenyl groups (eg, allyl groups, vinyl groups), alkynyl groups, azide groups, acryloyl groups, ⁇ -haloacetyl groups and the like.
• isocyanate groups also known as “isocyanato groups”
• isothiocyanate groups also known as “isothiocyanato
• an active ester group means an ester group having an alkoxy group with high leaving ability (that is, an alkoxycarbonyl group).
• alkoxy groups with high leaving ability include alkoxy groups derived from nitrophenol, N-hydroxysuccinimide, pentafluorophenol, etc. a group having a structure).
• the active ester group is preferably an ester group having an alkoxy group derived from N-hydroxysuccinimide (ie, an N-succinimidyloxycarbonyl group).
• an active carbonate group means a carbonate group having an alkoxy group with high leaving ability (that is, an alkoxycarbonyloxy group).
• alkoxy groups with high leaving ability include alkoxy groups derived from nitrophenol, N-hydroxysuccinimide, pentafluorophenol, etc. a group having a structure).
• the activated carbonate group is preferably a carbonate group having an alkoxy group derived from nitrophenol or N-hydroxysuccinimide (ie, a nitrophenyloxycarbonyloxy group or a succinimidyloxycarbonyloxy group), more preferably 4 - a carbonate group having an alkoxy group derived from nitrophenol or N-hydroxysuccinimide (ie, a 4-nitrophenyloxycarbonyloxy group or an N-succinimidyloxycarbonyloxy group).
• a maleimidyl group means an N-maleimidyl group (that is, a 1-maleimidyl group), and a substituted maleimidyl group is a maleimidyl group in which a hydrocarbon group is bonded to one carbon atom of the double bond of the maleimidyl group.
• a maleimidyl group with The hydrocarbon group is preferably a hydrocarbon group having 1 to 5 carbon atoms, more preferably an alkyl group having 1 to 5 carbon atoms. Examples of alkyl having 1 to 5 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, butyl group and tert-butyl group.
• the hydrocarbon group is more preferably a methyl group or an ethyl group.
• the substituted sulfonate group is a group represented by —O—(SO 2 )—R (wherein R is a hydrocarbon group which may contain a fluorine atom).
• Hydrocarbon groups which may contain a fluorine atom include, for example, methyl group, ethyl group, propyl group, isopropyl group, butyl group, tert-butyl group, hexyl group, nonyl group, vinyl group, phenyl group and benzyl group.
• Substituted sulfonate groups are preferably alkylsulfonyloxy groups.
• the functional group capable of reacting with a biological substance is preferably an active ester group, an active carbonate group, a formyl group, an isocyanate group, an isothiocyanate group, an epoxy group, a maleimidyl group, a substituted maleimidyl group, a vinylsulfonyl group, an acryloyl group, a substituted sulfonate group, carboxyl group, mercapto group, pyridyldithio group, ⁇ -haloacetyl group, alkynyl group, allyl group, vinyl group, amino group, aminooxy group, hydrazide group, or azide group.
• the functional group capable of reacting with a biological substance is more preferably an active ester group, an active carbonate group, a formyl group, a maleimidyl group, a substituted maleimidyl group, a carboxy group, an amino group, or an aminooxy group, and more preferably an active ester. group, activated carbonate group, maleimidyl group, substituted maleimidyl group, carboxy group, amino group or aminooxy group, particularly preferably activated ester group, maleimidyl group, substituted maleimidyl group, carboxy group or amino group, most preferably is an active ester, maleimidyl, carboxy or amino group.
• the functional group capable of reacting with a bio-related substance is the following group (I), group (II), group (III), group (IV), group (V) and group (VI)
• Group (I) a functional group capable of reacting with an amino group possessed by a bio-related substance, the following formula (a), formula (b), formula (c), formula (d), formula (e), formula (f), formula (g), a functional group represented by formula (j), or formula (k).
• Group (II) a functional group capable of reacting with a mercapto group possessed by a biologically relevant substance, the following formula (a), formula (b), formula (c), formula (d), formula (e), formula (f), formula (g), formula (h), formula (i), formula (j), formula (k), or a functional group represented by formula (l).
• Group (IV) Functional Group Reactive with Carboxy Group of Bio-related Substance
• Functional groups represented by the following formula (h), formula (m), formula (n), or formula (p) can be mentioned.
• Group (V) Functional groups capable of reacting with unsaturated bonds possessed by bio-related substances
• Functional groups represented by the following formula (h), formula (m), or formula (o) can be mentioned.
• U 1 in formula (j) represents a halogen atom, preferably a chlorine atom (Cl), a bromine atom (Br) or an iodine atom (I), more preferably Br or I, still more preferably I.
• Y 1 in formula (e) is a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms, preferably a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.
• alkyl groups having 1 to 5 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, butyl group, tert-butyl group and the like.
• Y 1 is preferably a hydrogen atom, a methyl group or an ethyl group, more preferably a hydrogen atom.
• Y 3 in formula (l) represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, preferably an alkyl group having 1 to 5 carbon atoms.
• the alkyl group having 1 to 5 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, butyl group, tert-butyl group and the like, preferably methyl group or ethyl group.
• Y 2 in formula (k) represents a hydrocarbon group having 1 to 10 carbon atoms which may contain a heteroatom.
• Heteroatoms include, for example, fluorine atoms and oxygen atoms.
• Hydrocarbon groups having 1 to 10 carbon atoms which may contain a heteroatom include, for example, methyl group, ethyl group, propyl group, isopropyl group, butyl group, tert-butyl group, hexyl group, nonyl group, vinyl group, phenyl group, benzyl group, 4-methylphenyl group, trifluoromethyl group, 2,2,2-trifluoroethyl group, 4-(trifluoromethoxy)phenyl group and the like, preferably methyl group, vinyl group, 4-methylphenyl group, 2,2,2-trifluoroethyl group and the like.
• the functional group capable of reacting with a biologically relevant substance is preferably a functional group represented by formula (a) (that is, an N-succinimidyloxycarbonyl group), formula (b) functional group represented by formula (i.e., N-succinimidyloxycarbonyloxy group), functional group represented by formula (c) (i.e., 4-nitrophenyloxycarbonyloxy group), represented by formula (e) (i.e., a maleimidyl group or a substituted maleimidyl group), a functional group represented by formula (g) (i.e., a carboxy group), a functional group represented by formula (m) (i.e., an amino group), or a functional group represented by (n) (that is, an aminooxy group), more preferably an N-succinimidyloxycarbonyl group, a functional group represented by formula (e), a carboxy group, or an amino group; and more preferably an N-succinimidyloxycarbony
• L 1 to L 8 are each independently a single bond or a divalent spacer, and L 9 is a divalent spacer.
• the bivalent spacers of L 2 , L 4 , L 5 and L 7 are each independently preferably (i) a degradable oligopeptide chain, (ii) an alkylene group, (iii) an amide bond (-NH-CO- or -CO-NH-), (iv) an ether bond (-O-), (v) a thioether bond (-S-), (vi) a urethane bond (-NH-CO-O- or -O-CO-NH-), (vii) an imino group (-NH-), (viii) a carbonyl group (-CO-), (ix) a urea bond (--NH--CO--NH--), or (x) a combination of (i)-(ix) above.
• the degradable oligopeptide chain is not particularly limited as long as it is stable in blood in vivo and has the ability to be degraded by intracellular enzymes.
• the degradable oligopeptide chain is preferably an oligopeptide chain composed of 2 to 5 residues of neutral amino acids excluding cysteine.
• Degradable oligopeptide chains include, for example, glycine-phenylalanine-leucine-glycine chains, glycine-glycine-phenylalanine-glycine chains, glycine-phenylalanine-glycine chains, glycine-leucine-glycine chains, valine-citrulline-glycine chains, valine- Examples include alanine-glycine chains, phenylalanine-glycine chains, and the like.
• the degradable oligopeptide chain is preferably a glycine-phenylalanine-leucine-glycine chain, glycine-glycine-phenylalanine-glycine chain, glycine-phenylalanine-glycine chain, valine-citrulline-glycine chain, valine-alanine-glycine chain, or phenylalanine -glycine chains, more preferably glycine-phenylalanine-leucine-glycine chains, glycine-phenylalanine-glycine chains, valine-citrulline-glycine chains, or phenylalanine-glycine chains, more preferably glycine-phenylalanine-leucine-glycine chains, or phenylalanine-glycine chains.
• a phenylalanine (Phe)-glycine (Gly) chain can be either -Gly-Phe- or -Phe-Gly-.
• the divalent spacers of L 1 , L 3 , L 6 , L 8 and L 9 are each independently preferably (i) an alkylene group, (ii) an amide bond, (iii) an ether bond, (iv) a thioether bond, (v) a urethane bond; (vi) an imino group, (vii) a carbonyl group, (viii) a urea bond, (ix) A combination of (i) to (viii) above.
• L 1 to L 4 , L 6 and L 8 are each independently a single bond selected from the following group (VII) (the following formula (z1) where s is 0) or a bivalent spacer, or a bivalent spacer consisting of a combination of 2 to 4 selected from group (VII), wherein L 5 and L 7 are each independently a single bond (s is 0 a formula (z1) below), or a combination of a divalent spacer and a degradable oligopeptide chain selected from group (VII) below, and L9 is 2 selected from group (VII) below is a valent spacer (provided that s in formula (z1) is an integer of 1 or more).
• a divalent spacer containing an ester bond and/or a carbonate bond is not preferable because it is gradually degraded in blood in vivo.
• s of formula (z1) to s of formula (z11) are each independently an integer of 0 to 10, preferably an integer of 0 to 6, more preferably an integer of 0 to 3.
• s when s is 0, it indicates that —(CH 2 ) s — in the above formula is a single bond. Therefore, when s is 0, formula (z1) is a single bond.
• formulas (z2) to (z11) multiple s in the same formula may be the same or different.
• the divalent spacer represented by formula (z3) is bound to P 1 on its left side and X 1 or It may be bound to D1 , or may be bound to X1 or D1 on its left side and bound to P1 on its right side.
• L 1 and L 3 are each independently preferably represented by formulas (z12) to (z14):
• s1 to s5 are each independently an integer of 0 to 10.
• s1 to s5 are each independently an integer of 0 to 10.
• the case where s2 to s5 are 0 is the same as the case where s1 is 0.
• * in formulas (z12) to (z14) indicates the bonding position with X1
• ** indicates the bonding position with P1 .
• * in formulas (z12) to (z14) indicates the bonding position with X2
• s1 and s5 are each independently preferably an integer of 0-6, more preferably an integer of 0-3.
• s2-s4 are each independently preferably an integer of 0-6, more preferably an integer of 1-3.
• L 2 , L 4 , L 6 and L 8 are preferably single bonds.
• L 9 preferably has formula (z15) or formula (z16):
• s6 is an integer from 1 to 10.
• s6 is a bivalent spacer represented by
• * in formulas (z15) and (z16) indicates the bonding position with Q1
• s6 is preferably an integer of 1-6, more preferably an integer of 1-5.
• L 5 and L 7 are each independently preferably a single bond or formula (z17):
• s7 is preferably an integer of 1-6, more preferably an integer of 1-3.
• L 10 is preferably a phenylalanine-glycine chain
• L 10 is a phenylalanine-glycine chain
• R 1 and R 2 are each independently a hydrocarbon group having 1 to 5 carbon atoms, preferably an alkyl group having 1 to 5 carbon atoms. Examples of alkyl groups having 1 to 5 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, butyl group, tert-butyl group and the like. R 1 and R 2 are each independently more preferably a methyl group or an ethyl group.
• Suitable examples of water-soluble polymer (1) or water-soluble polymer (2) are as follows.
• X 1 and X 2 are each independently an active ester group, a maleimidyl group, a substituted maleimidyl group, a carboxy group or an amino group; P 1 to P 4 are each independently a divalent group of polyethylene glycol or polysialic acid; Q 1 is a trivalent group represented by formula (q1), Q 2 is a trivalent group represented by formula (q1) or formula (q2), L 1 and L 3 are each independently represented by any of formulas (z12) to (z14) (wherein s1 to s5 are each independently an integer of 0 to 10) is a single bond or a divalent spacer, L 2 , L 4 , L 6 and L 8 are single bonds, L 9 is a divalent spacer represented by formula (z15) or formula (z16) (wherein s6 is an integer of 1 to 10), L 5 and L 7 are each independently a single bond or represented by the formula (z
• R 1 and R 2 are each independently a hydrocarbon group having 1 to 5 carbon atoms, Water-soluble polymer (1) or water-soluble polymer (2).
• L 10 is a phenylalanine-glycine chain
• L 10 is a phenylalanine-glycine chain
• the glycine of L 10 More preferably, the carbonyl group (--CO--) of the residue is bound and the imino group (--NH--) of the phenylalanine residue of L 10 is bound to Q 1 or Q 2 .
• the number average molecular weight of the divalent groups of P 1 to P 4 of polyethylene glycol or polysialic acid and the total number (preferred range, etc.) of the above water-soluble polymer of P 1 to P 4 It is the same as the number average molecular weight of the divalent group and the sum thereof.
• X 1 and X 2 are each independently an active ester group, a functional group represented by formula (e) (wherein Y 1 is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms); a carboxy group or an amino group, P 1 to P 4 are each independently a divalent group of polyethylene glycol, Q 1 is a trivalent group represented by formula (q1), Q 2 is a trivalent group represented by formula (q1) or formula (q2), L 1 and L 3 are each independently represented by any of formulas (z12) to (z14) (wherein s1 to s5 are each independently integers of 0 to 6) is a single bond or a divalent spacer, L 2 , L 4 , L 6 and L 8 are single bonds, L 9 is represented by formula (z15) or formula (z16) (wherein s6 is an integer of 1 to 6) is a bivalent spacer represented by L 5 and L 7 are
• L 10 is a phenylalanine-glycine chain
• L 10 is a phenylalanine-glycine chain
• the imino group (—NH—) of —(CH 2 ) s7 —NH— and the glycine of L 10 More preferably, the carbonyl group (--CO--) of the residue is bound and the imino group (--NH--) of the phenylalanine residue of L 10 is bound to Q 1 or Q 2 .
• the number average molecular weight of the divalent groups of polyethylene glycol of P 1 to P 4 and the total number thereof are the divalent Same as the number average molecular weight of the groups and their sum.
• X 1 and X 2 are each independently a functional group represented by formula (a) (that is, an N-succinimidyloxycarbonyl group), a maleimidyl group, a carboxy group or an amino group; P 1 to P 4 are each independently a divalent group of polyethylene glycol, Q 1 is a trivalent group represented by formula (q1), Q 2 is a trivalent group represented by formula (q1) or formula (q2), L 1 and L 3 are each independently represented by formulas (z12) to (z14) (wherein s1 and s5 are each independently an integer of 0 to 3, and s2 to s4 are each independently is independently an integer of 1 to 3.) is a single bond or a divalent spacer represented by L 2 , L 4 , L 6 and L 8 are single bonds, L 9 is a divalent spacer represented by formula (z15) or formula (z16) (wherein s6 is an integer
• R 1 and R 2 are each independently a methyl group or an ethyl group, Water-soluble polymer (1) or water-soluble polymer (2).
• L 10 is a phenylalanine-glycine chain
• L 10 is a phenylalanine-glycine chain
• the carbonyl group (--CO--) of the residue is bound and the imino group (--NH--) of the phenylalanine residue of L 10 is bound to Q 1 or Q 2 .
• the number average molecular weight of the divalent groups of polyethylene glycol of P 1 to P 4 and the total number thereof are the divalent Same as the number average molecular weight of the groups and their sum.
• X 1 and X 2 are each independently a functional group represented by formula (a) (that is, an N-succinimidyloxycarbonyl group), a maleimidyl group, a carboxy group or an amino group; P 1 to P 4 are each independently a divalent group of polyethylene glycol, Q 1 is a trivalent group represented by formula (q1), Q 2 is a trivalent group represented by formula (q1) or formula (q2), L 1 and L 3 are each independently a single bond or a divalent spacer represented by the formula (z13) (wherein s3 is an integer of 1 to 3), L 2 , L 4 , L 6 and L 8 are single bonds, L 9 is a divalent spacer represented by formula (z15) or formula (z16) (wherein s6 is an integer of 1 to 5), L 5 and L 7 are each independently a single bond or represented by the formula (z17), where s7 is an integer from
• R 1 and R 2 are each independently a methyl group or an ethyl group, Water-soluble polymer (1) or water-soluble polymer (2).
• L 10 is a phenylalanine-glycine chain
• L 10 is a phenylalanine-glycine chain
• the carbonyl group (--CO--) of the residue is bound and the imino group (--NH--) of the phenylalanine residue of L 10 is bound to Q 1 or Q 2 .
• the number average molecular weight of the divalent groups of polyethylene glycol of P 1 to P 4 and the total number thereof are the divalent Same as the number average molecular weight of the groups and their sum.
• Preferred examples of the water-soluble polymer (3) or the water-soluble polymer (4) include the above [water-soluble polymer (1-1) or water-soluble polymer (2-1)] to [water-soluble polymer (1-4) or water-soluble polymer (2-4)], “X 1 and X 2 ”, “P 1 to P 4 ”, “L 1 and L 3 ”, “L 2 , L 4 , L 6 and L 8 ”, “L 5 and L 7 ” and “Water-soluble polymer (1) or Water-soluble polymer (2)” are replaced with “X 1 ”, “P 1 and P 3 ”, “L 1 ”, “L 2 and L 6 ", "L 5 " and those changed to "water-soluble polymer (3) or water-soluble polymer (4)".
• the water-soluble polymer of the present invention is particularly preferably the compound (p3), the compound (p4), the compound (p4-a), the compound (p6), the compound (p7), the compound (p7-a), and the compound (p7-a) of the synthesis examples described later.
• water-soluble polymer of the present invention can be produced as shown in Synthesis Examples below.
• water-soluble polymer (1) has the following formula:
• X 3 and X 4 are a functional group or a divalent spacer to which a functional group is attached, the functional group of X 3 and the functional group of X 4 are capable of reacting with each other, and the definitions of other symbols are as described above.
• polymer (1a) and polymer (1b) corresponding to the left and right structures of water-soluble polymer (1), as shown in FIG.
• polymer (1a) and polymer (1b) are separated via the compound (i.e. spacer) may be combined.
• Polymer (1a) and polymer (1b) may be commercially available products or may be produced using known reactions, as shown in Synthesis Examples below.
• Polymer (1a) and polymer (1b) are, for example, glycerin or derivatives thereof, glutamic acid or derivatives thereof, or lysine or derivatives thereof, and water-soluble polymers corresponding to P 1 to P 4 are bound directly or via spacers. to form an intermediate, and to the resulting intermediate, a compound having X 1 or X 2 can be bound directly or via a spacer.
• Synthesis of Compound (p1) Compound (p1-b) (18 g, 1.8 mmol) obtained in Synthesis Example 1-2 was mixed with 2,6-di-tert-butyl-p-cresol ( hereinafter referred to as "BHT") (18 mg) was dissolved in toluene (144 g) at 40°C, heated to 110°C, and dehydrated under reflux for 30 minutes.
• BHT 2,6-di-tert-butyl-p-cresol
• ethylenediamine monohydrate (14.1 g, 180 mmol) was added to the resulting solution and reacted at 40° C. under a nitrogen atmosphere for 5 hours. After completion of the reaction, the solution was cooled to room temperature, 20% saline (112.5 g) was added, and the mixture was stirred for 10 minutes. Dichloromethane (180 g) was then added and the solution was stirred at room temperature under a nitrogen atmosphere for 30 minutes. After stirring, the solution was allowed to stand for 30 minutes to separate the layers, and then the organic layer was recovered. After the collected organic layer was concentrated to dryness, the obtained concentrate was dissolved in ethyl acetate (180 g).
• the compound (p1) (3.0 g, 0.3 mmol) obtained in Synthesis Example 1-3 and SUNBRIGHT GL2-200TS (6.0 g, 0.3 mmol) manufactured by NOF Corporation were dissolved in toluene (36 g), The reaction was carried out at 40° C. under nitrogen atmosphere for 3 hours. After completion of the reaction, the solution was diluted with toluene (108 g) and stirred at room temperature for 5 minutes. Hexane (60 g) was then added and the mixture was stirred at room temperature under a nitrogen atmosphere for 15 minutes to precipitate the product.
• the precipitate was collected by suction filtration using 5A filter paper, washed with hexane (36 g), suction filtered using 5A filter paper, and dried in vacuo to obtain the compound (p2) (yield: 7.8 g). ).
• the precipitate was collected by suction filtration using 5A filter paper, dissolved again in ethyl acetate (100 g), and hexane (50 g) was added to precipitate the product.
• the precipitate was collected by suction filtration using 5A filter paper, washed with hexane (50 g), suction filtered using 5A filter paper, and dried in vacuo to obtain the above compound (p3) (yield: 2.3 g). , number average molecular weight (Mn) 34,510).
• the solution was allowed to stand for 30 minutes to separate the layers, and then the organic layer was recovered. After the collected organic layer was concentrated to dryness, the obtained concentrate was dissolved in ethyl acetate (100 g).
• Sodium sulfate (3 g) was added to the resulting solution, and the mixture was stirred at room temperature under a nitrogen atmosphere for 30 minutes for dehydration. After dehydration, sodium sulfate was removed by suction filtration using 5A filter paper. Hexane (50 g) was added to the resulting filtrate and the mixture was stirred at room temperature for 15 minutes to precipitate the product.
• the precipitate was collected by suction filtration using 5A filter paper, dissolved again in ethyl acetate (100 g), and hexane (50 g) was added to precipitate the product. Suction filtration was performed using 5A filter paper, and the precipitate was collected, washed with hexane (50 g), suction filtered using 5A filter paper, and dried in vacuo to obtain the compound (p6) (yield: 2.4 g). , number average molecular weight (Mn) 35,780).
• the solution was allowed to stand for 30 minutes to separate the layers, and then the organic layer was recovered. After the collected organic layer was concentrated to dryness, the obtained concentrate was dissolved in ethyl acetate (100 g).
• Sodium sulfate (3 g) was added to the resulting solution, and the mixture was stirred at room temperature under a nitrogen atmosphere for 30 minutes for dehydration. After dehydration, sodium sulfate was removed by suction filtration using 5A filter paper. Hexane (50 g) was added to the resulting filtrate and the mixture was stirred at room temperature for 15 minutes to precipitate the product.
• the resulting concentrate was diluted with 100 mM phosphate buffer (20 mL) of pH 6.0 and concentrated to 1 mL again by ultrafiltration. . After repeating the same operation twice, the absorbance at 280 nm was measured using Nanodrop (Thermo Scientific) to confirm the protein concentration. After confirmation, ultrafiltration was performed, and a 4 mg/mL Trast F(ab') 2 solution (0.8 mL) was prepared by concentration.
• the resulting concentrate is diluted with 100 mM phosphate buffer (20 mL) containing 20 mM EDTA at pH 6.0, and concentrated to 1 mL again by ultrafiltration. bottom.
• the absorbance at 280 nm was measured using Nanodrop (Thermo Scientific) to confirm the protein concentration.
• a 4 mg/mL Trast Fab' solution (0.2 mL) was prepared by concentration by ultrafiltration.
• magnesium sulfate was removed by suction filtration using 5A filter paper.
• the resulting filtrate was diluted with toluene (100 mL) and stirred until the mixture was homogeneous.
• hexane 100 mL was added to the solution and stirred for 15 minutes at room temperature under a nitrogen atmosphere to precipitate the product.
• Suction filtration was performed using 5A filter paper, the precipitate was collected, washed with hexane (50 mL), suction filtration was performed using 5A filter paper, and the compound (p10-a) was obtained by vacuum drying (yield: 110 mg). ).
• SUNBRIGHT DE-200HC 100 mg, 0.005 mmol
• cyclosporine A 60 mg, 0.05 mmol
• FUJIFILM Wako Pure Chemical Industries, Ltd. were dissolved in dichloromethane (0.5 mL).
• Dicyclohexylcarbodiimide (10 mg, 0.05 mmol) was added to the solution and reacted at room temperature under nitrogen atmosphere for 6 hours. After completion of the reaction, the reaction mixture was diluted with toluene (100 mL), and suction filtered using 5A filter paper to remove insoluble matter by filtration.
• SUNBRIGHT DE-200HS (30 mg, 0.0015 mmol) manufactured by NOF Corporation and linaclotide (11.4 mg, 0.0075 mmol) manufactured by Medchemexpress were dissolved in dimethyl sulfoxide (0.5 mL) and stirred at room temperature for 6 hours under a nitrogen atmosphere. reacted. After completion of the reaction, the solution was diluted with toluene (100 mL) and stirred until the mixture became homogeneous. After stirring, suction filtration was performed using 5A filter paper, hexane (100 mL) was added to the resulting filtrate, and the mixture was stirred at room temperature for 15 minutes under a nitrogen atmosphere to precipitate the product. The precipitate was collected by suction filtration using 5A filter paper, washed with hexane (50 mL), suction filtered using 5A filter paper, and dried in vacuo to obtain the compound (p12) (yield: 23 mg).
• Synthesis example 2 except that SUNBRIGHT GL2-100TS (3.0 g, 0.3 mmol) manufactured by NOF Corporation was used instead of SUNBRIGHT GL2-200TS (6.0 g, 0.3 mmol) manufactured by NOF Corporation.
• the above compound (p17) was obtained in the same manner (yield: 4.1 g).
• the compound (p8) was dissolved in ultrapure water, and 1M hydrochloric acid was added to adjust the pH to 1.0. Thereafter, 1M sodium hydroxide aqueous solution was added to adjust the pH to 10.0, and then 1M hydrochloric acid was added again to adjust the pH to 6.0 to obtain an aqueous solution of compound (p8).
• trastuzumab was dissolved in PBS containing 5% sucrose, and the same operation as above was performed to obtain an aqueous trastuzumab solution with a pH of 6.0.
• the four-branched water-soluble polymer for medical use of the present invention can improve the solubility of the conjugate between it and a biologically relevant substance, and can suppress aggregation of the conjugate. Furthermore, since the four-branched water-soluble polymer for medical use of the present invention has functional groups capable of reacting with two bio-related substances in one molecule, it is possible to combine a conventional water-soluble polymer with one bio-related substance. Compared with the body, the conjugate of the medical tetra-branched water-soluble polymer and two bio-related substances may have improved pharmacological activity.

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