WO2019184993A1 - 聚乙二醇与雷帕霉素的结合物及其用途 - Google Patents

聚乙二醇与雷帕霉素的结合物及其用途 Download PDF

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WO2019184993A1
WO2019184993A1 PCT/CN2019/080194 CN2019080194W WO2019184993A1 WO 2019184993 A1 WO2019184993 A1 WO 2019184993A1 CN 2019080194 W CN2019080194 W CN 2019080194W WO 2019184993 A1 WO2019184993 A1 WO 2019184993A1
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factor
group
disease
cancer
use according
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French (fr)
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汪进良
冯泽旺
王雷敏
赵宣
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天津键凯科技有限公司
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Priority to US17/042,887 priority Critical patent/US20210023232A1/en
Publication of WO2019184993A1 publication Critical patent/WO2019184993A1/zh
Priority to US17/901,108 priority patent/US20230036997A1/en

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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • 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
    • C08G65/34—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from hydroxy compounds or their metallic derivatives
    • C08G65/48—Polymers modified by chemical after-treatment

Definitions

  • the present invention relates to the field of medical technology, and in particular to a combination of polyethylene glycol and rapamycin and use thereof, particularly in the preparation of a medicament for reducing an immune response, more specifically polyethylene glycol and The use of a conjugate of rapamycin in the manufacture of a medicament for reducing an excessive immune response elicited by a recipient to an exogenous immunogen such as a therapeutic, a transplant or the like.
  • Immunogenicity refers to the ability to stimulate the body to form specific antibodies or sensitized lymphocytes, that is, the antigen can stimulate specific immune cells, activate, proliferate and differentiate immune cells, and finally produce immune effector antibodies and sensitized lymphocytes. Characteristics.
  • the immune response refers to the physiological process by which the immune system of the body stimulates the immunogen to exclude antigens.
  • Some therapeutic agents are also based on their own chemical properties, as well as immunogenicity, such as certain macromolecular proteins and polysaccharides, small molecular peptides and nucleic acids, and grafts such as cells, tissues, organs, artificial materials, etc., may also induce Immune response.
  • the cells in the immune system recognize the therapeutic agent, the graft, and initiate an immunological reaction of attack, destruction, and elimination, which is not conducive to the treatment of the patient, causes more pain to the patient, and may even aggravate the condition. life threatening.
  • Rapamycin also known as sirolimus
  • Rapamycin is a macrolide antibiotic produced by Streptomyces hygroscopicus found in the soil of Easter Island in Chile in 1975. It was used as an immunosuppressant in 1989.
  • the US Home Products Company developed a new drug for anti-transplant rejection reaction into the clinic, and was approved by the US FDA in September 1999.
  • rapamycin has a good clinical prospect, there are still defects such as low bioavailability and poor water solubility.
  • some researchers have used rapamycin in combination with nanocarriers to reduce the above-mentioned excessive immune response, as disclosed in patent application CN201480031937.3 for therapeutic macromolecules and synthetic nanocarriers that are not linked to synthetic nanocarriers.
  • the present invention provides a use of a combination of polyethylene glycol and rapamycin for the preparation of a medicament for reducing an immune response, said polyethylene glycol and rapamycin
  • the conjugate has the structure shown in Formula I:
  • PEG is a polyethylene glycol residue
  • D is a residue of rapamycin or a derivative thereof
  • X is a linking group between PEG and D.
  • the X is selected from the group consisting of: -(CR 1 R 2 ) a -, -(CH 2 ) a NH-, -(CH 2 ) a NHCO-, -(CH 2 ) a CONH -, -(CH 2 ) a CO-, -(CH 2 ) a COO-, -(CH 2 ) a O-, a cycloalkyl group, an aryl group, a heterocyclic group, one of -A- and -Op- Combination of species or multiple,
  • a is an integer from 0 to 10
  • R 1 and R 2 are independently selected from: -H, C1-6 alkyl, -OR', -NHR', -N(R') 2 , -CN, -F, -Cl, -Br, -I One or a combination of two or more of -COR', -COOR', -OCOR', -CONHR', and -CON(R') 2 ,
  • R' is selected from the group consisting of: -H, C1-6 alkyl, -F, -Cl, -Br and -I,
  • -A- is an amino acid residue
  • -Op- is an oligopeptide residue (e.g., a polypeptide comprising 2-10 (specifically 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues).
  • the X has the structure: -X 1 -X 2 -X 3 -, wherein X 1 and X 3 are independently selected from: -(CR 1 R 2 ) a -, - (CH 2 ) a NH-, -(CH 2 ) a NHCO-, -(CH 2 ) a CONH-, -(CH 2 ) a CO-, -(CH 2 ) a COO- and -(CH 2 ) a
  • a combination of one or more of O-, X 2 is selected from the group consisting of cycloalkyl, aryl, heterocyclic, -A-, and -Op-.
  • amino acid residue -A- is selected from the structures shown below: One of them,
  • G is selected from the group consisting of: -H, -CH 3 , -CH 2 -S-CH 3 , -CH 2 OH, -CH 2 SH, -CH 2 CONH 2 , -CH 2 CH 2 CONH 2 , Preferably from: -H, -CH 3 , More preferably, G is -H or -CH 3 .
  • the oligopeptide residue -Op- contains 2-10 amino acid residues, which are the same or different, and are selected from the above defined scheme of the above amino acid residue -A-;
  • the oligopeptide residue keto-Op- may be a linear oligopeptide residue, such as a dipeptide of glycine Tripeptide Etc., at this time, the oligopeptide residue -Op- has two connectable sites;
  • the oligopeptide residue -Op- may also be a branched oligopeptide residue, such as (m is 1, 2, 3 or 4), wherein A 1 is selected from the group consisting of residues of aspartic acid, glutamic acid, and lysine, and A 2 and A 3 are the same or different, and are selected from the above-mentioned amino acid residues of the present invention.
  • a definition of the basal-A- specifically as described in the Chinese patent application CN201410715522.X, particularly as
  • R 1 and R 2 are independently selected from: -H, C1-6 alkyl, -OR', -NHR', -N(R') 2 , -CN, -F, -Cl, -Br, -I a combination of one or more of -COR', -COOR', -OCOR', -CONHR', and -CON(R') 2 , and R' is selected from the group consisting of: -H, C1-6 alkyl, -F, -Cl, -Br, and -I,
  • a is an integer from 0-10.
  • said R' is selected from the group consisting of: -H and C1-3 alkyl groups (such as methyl, ethyl, n-propyl or isopropyl).
  • said R 1 and R 2 are independently selected from the group consisting of: H, C1-3 alkyl (such as methyl, ethyl, n-propyl or isopropyl), -OH, C1-3 alkoxy (i.e., -OR ', R' is C1-3 alkyl), - NH 2, -F, -Cl, -Br , and -I or a combination of one of more of; more preferably selected from: H, -CH 3 , -OH, -OCH 3 and -OCH 2 CH 3 .
  • the a is an integer from 0 to 5, such as 0, 1, 2, 3, 4 or 5.
  • the X 1 and X 3 are independently selected from the group consisting of: a single bond, -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CO-, -CH 2 CH 2 CO -, -CH 2 NH-, -CH 2 NHCH 2 -, -CH 2 CONH-, -CH 2 CONHCH 2 -, -CH 2 NHCO-, -CH 2 NHCOCH 2 -, -CH 2 COO-, -CH 2 A combination of one or more of COOCH 2 -, -CH 2 O-, -CH 2 OCH 2 -.
  • said X 1 is -CH 2 CO-.
  • said X 1 is -CH 2 OCH 2 -.
  • said X 1 is -CH 2 CONHCH 2 -.
  • said X 3 is a single bond.
  • the X 3 is -CH 2 CO-.
  • the -Op- has the following structure:
  • n 1, 2, 3 or 4
  • a 1 is selected from the group consisting of residues of aspartic acid, glutamic acid, and lysine.
  • a 2 and A 3 are the same or different and are independently selected from the following structures: Wherein G has the above definition of the invention.
  • the A 1 is a glutamic acid residue
  • the A 2 and A 3 are the same, both are glycine residues
  • the -Op- has the following structure:
  • m is 2 or 3.
  • the linking group X may contain only two linking sites, respectively linking PEG and D; the X may also contain more than three linking sites, and one polyethylene-2 is attached.
  • An alcohol residue and two or more residues of rapamycin or a derivative thereof for the purpose of increasing the drug loading, etc., or a residue of rapamycin or a derivative thereof and two or more polyethylene glycol residues To achieve the purpose of improving the water solubility or steric hindrance of the combination.
  • said X is -X 1 -Op-X 3 -, -Op- has the above definition of the invention.
  • the X is Preferably, m is 2 or 3.
  • the PEG is a linear, Y-type, multi-branched polyethylene glycol residue, including, for example, monomethoxypolyethylene glycol (mPEG), linear double-ended PEG, Y Type PEG, 4-arm branched PEG, 6-arm branched PEG or 8-arm branched PEG, and the like.
  • mPEG monomethoxypolyethylene glycol
  • Y Type PEG 4-arm branched PEG
  • 6-arm branched PEG or 8-arm branched PEG and the like.
  • the PEG may have a molecular weight of 1-100 KDa, such as 1-10 KDa (specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 KDa), 10-50 KDa (specifically 10, 15, 20, 25, 30, 35, 40, 45 or 50 KDa) or 50-100 KDa (specifically 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 KDa), etc. Further preferably, it is 10-50 KDa.
  • 1-10 KDa specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 KDa
  • 10-50 KDa specifically 10, 15, 20, 25, 30, 35, 40, 45 or 50 KDa
  • 50-100 KDa specifically 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 KDa
  • it is 10-50 KDa.
  • the PEG is a linear polyethylene glycol residue having the structure shown in Formula II or III:
  • p and q are independently selected from an integer from 1 to 280, preferably an integer from 220 to 1140,
  • Y is a terminal group and has a -L-T structure.
  • L is a linking group between oxygen (O) and a terminal group T, and is selected from the group consisting of: -(CH 2 ) b -, -(CR 3 R 4 ) b -, -(CH 2 ) b NH-, One or more of -(CH 2 ) b NHCO-, -(CH 2 ) b CONH-, -(CH 2 ) b CO-, -(CH 2 ) b COO- and -(CH 2 ) b O- Combination of species, where b is an integer from 0 to 10,
  • R 3 and R 4 are independently selected from: -H, C1-6 alkyl, -OR", -NHR", -N(R") 2 , -CN, -F, -Cl, -Br, -I a combination of one or more of -COR", -COOR", -OCOR", -CONHR", and -CON(R") 2 ,
  • R" is selected from the group consisting of: -H, C1-6 alkyl, -F, -Cl, -Br and -I,
  • T is a terminal group selected from the group consisting of: H, a C1-6 alkyl group, a C3-6 cycloalkyl group, a C4-10 cycloalkylalkyl group, a C6-10 aryl group, a C7-14 aralkyl group. , monosaccharides and oligosaccharide residues.
  • the L is selected from the group consisting of: a single bond, -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CONH-, -NH-, -CO-, -CONHCH 2 -, a combination of one or more of -CH 2 NH-, -CH 2 CONH-, and -COCH 2 -.
  • the T is selected from the group consisting of methyl, ethyl, isopropyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclohexyl, benzyl,
  • the Y is selected from the group consisting of methyl, ethyl, isopropyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclohexyl, benzyl,
  • said Y is a methyl group.
  • the Y is
  • the PEG is a Y-type polyethylene glycol residue having a structure represented by Formula IV or V:
  • i and h are independently selected from an integer of from 1 to 140, preferably an integer from 110 to 570,
  • Y has the structure defined in the above formula II of the present invention, preferably from: methyl, ethyl, isopropyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclohexyl, benzyl, More preferably from: methyl,
  • the PEG is a multi-branched polyethylene glycol residue having the structure shown in Formula VI:
  • k is an integer from 1 to 760, preferably an integer from 70 to 380,
  • j is an integer of 3-8
  • R is a core molecule of a multi-branched polyethylene glycol, and R is selected from the group consisting of: pentaerythritol, oligo-pentaerythritol, methyl glucoside, sucrose, diethylene glycol, propylene glycol, glycerin, and polyglycerol residues.
  • R is selected from the group consisting of: residues of pentaerythritol, oligo-pentaerythritol, glycerol, and polyglycerol; more preferably: from pentaerythritol, dipentaerythritol, tripent pentaerythritol, glycerol, and hexaglycerol residues.
  • the multi-branched polyethylene glycol residue contains only one connectable site having the following structure:
  • Y has the structure defined in the above formula II of the present invention, preferably from: methyl, ethyl, isopropyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclohexyl, benzyl, More preferably from: methyl,
  • the multi-branched polyethylene glycol residue has the structure:
  • k is an integer from 1 to 570, preferably an integer from 55 to 285,
  • x is an integer of 1-10 (specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably an integer of 1-6,
  • Y has the structure defined in the above formula II of the present invention, preferably from: methyl, ethyl, isopropyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclohexyl, benzyl, More preferably from: methyl,
  • the multi-branched polyethylene glycol residue has the structure:
  • k is an integer from 1 to 280, preferably an integer from 28 to 140,
  • Y is an integer of 1-10 (specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably an integer of 1-5, more preferably an integer of 1-3,
  • Y has the structure defined in the above formula II of the present invention, preferably from: methyl, ethyl, isopropyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclohexyl, benzyl, More preferably from: methyl,
  • a PEG molecule when the PEG has the structure of the above formula II, IV or V, a PEG molecule can bind to the drug D through the linking group X through its linking site; when the PEG has the above formula III Or the structure of VI, one PEG molecule has more than two connectable sites, and PEG can bind to drug D through one of the linking sites through linker X, in which case the conjugate contains only one drug residue PEG can also bind to drug D through a linking group X through a plurality of linking sites, in which case there are more than two drug residues in the conjugate.
  • the PEG when it is a PEG having two or more connectable sites represented by Formula III or VI, it binds to Drug D through a linking group through a linking group X, and other connectable sites can be linked.
  • the terminal group Y e.g., methyl group
  • the terminal group Y is as shown in the formula VII.
  • the D has the following structure:
  • R 5 is selected from the group consisting of: H, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C6-C12 aryl group, and a C7-C14 aralkyl group;
  • R 6 is selected from the group consisting of: H, a hydroxyl group, and a C1-C6 alkoxy group;
  • R 7 is selected from the group consisting of: H, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C6-C12 aryl group, a C7-C14 aralkyl group, and -C(O) R71 , wherein R71 is selected from the group consisting of: H, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C6-C12 aryl group, and a C7-C14 aralkyl group;
  • R 8 is selected from the group consisting of: H, a hydroxyl group, and a C1-C6 alkoxy group;
  • R 9 is selected from the group consisting of: -O-, -OR 51 -O-, Wherein R 91 is a C1-C6 alkylene group;
  • Z is H or a hydroxy protecting group.
  • said R 5 is H or a C1-C6 alkyl group, more preferably H, methyl or ethyl; in one embodiment of the invention, said R 5 is methyl.
  • the R 6 is selected from the group consisting of H, a hydroxyl group and a C1-C3 alkoxy group, more preferably -OCH 3 , -OCH 2 CH 3 , -O(CH 2 ) 2 CH 3 ; In one embodiment of the invention, the R 6 is methoxy.
  • the R 7 is selected from the group consisting of: H, a C 1 -C 6 linear alkyl group, a C 2 -C 10 linear alkenyl group, a phenyl group, a halogenated phenyl group, a benzyl group, Phenylethyl, -C(O)R 71 , wherein R 71 is selected from: H, C 1 -C 6 linear alkyl, more preferably H, -OCH 3 , -OCH 2 CH 3 , -O(CH 2 ) 2 CH 3 ; In one embodiment of the invention, said R 7 is H.
  • the R 8 is selected from the group consisting of: H, a hydroxyl group and a C1-C3 alkoxy group, more preferably -OCH 3 , -OCH 2 CH 3 , -O(CH 2 ) 2 CH 3 ; in one embodiment of the present invention, R 8 is a methoxy group.
  • R 91 is a C1-C3 alkylene group such as -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -.
  • R 5 is a methyl group
  • R 6 is a methoxy group
  • R 7 is H
  • R 8 is a methoxy group
  • the D is selected from the group consisting of: rapamycin, everolimus, temsirolimus, pilomos A9, ABT-578, and one of the phosphatisole base.
  • the D is selected from the group consisting of: One of them,
  • Z is H or a hydroxy protecting group.
  • the D has the following structure:
  • the combination has the following structure:
  • the PEG has the structure of the above Formula II of the present invention; more preferably, the PEG has the following structure: p has the above definition of the invention.
  • the PEG has a molecular weight of from 10 to 50 KDa, more preferably 20 KDa.
  • the m is 2 or 3.
  • the D has the following structure:
  • the immune response is caused by an exogenous immunogen.
  • the use is a combination of polyethylene glycol and rapamycin in the preparation for reducing exogenous immunity of the recipient.
  • the recipient is a mammal, preferably a primate, more preferably a human.
  • the exogenous immunogen is selected from the group consisting of a therapeutic agent, a graft, an artificial antigen carrier, a toxin, and the like.
  • the exogenous immunogen is a therapeutic agent; preferably, the therapeutic agent is a therapeutic macromolecule, more preferably a therapeutic protein such as a cytokine, human hemoglobin, blood factor or coagulation A factor, a vascular endothelial growth factor antibody antagonist, a hormone, an antibody, an enzyme, and a coenzyme; more preferably, the therapeutic protein is an antibody or an enzyme and a coenzyme.
  • the therapeutic agent is a therapeutic macromolecule, more preferably a therapeutic protein such as a cytokine, human hemoglobin, blood factor or coagulation A factor, a vascular endothelial growth factor antibody antagonist, a hormone, an antibody, an enzyme, and a coenzyme; more preferably, the therapeutic protein is an antibody or an enzyme and a coenzyme.
  • examples of the cytokine include, but are not limited to, interleukin, colony stimulating factor, interferon, growth factor, tumor necrosis factor, transforming growth factor-beta family or chemokine family, and the like.
  • examples of the growth factor include, but are not limited to, adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FFGF), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF) , granulocyte-macrophage colony-stimulating factor (GM-CSF), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), liver cancer-derived growth factor (HDGF), insulin-like growth factor (IGF), Promoting migration factor, myostatin (GDF-8), nerve growth factor (NGF) and other neurotrophic factors, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF
  • AM a
  • examples of the blood factor or coagulation factor include, but are not limited to, Factor I, Factor II, Tissue Factor, Factor V, Factor VII (eg, Long-acting Factor VII), Factor VIII, Factor IX, Factor X, Factor Xa, Factor XII, Factor XIII, Recombinant Coagulation Factor (eg, Recombinant Factor VIII), von Willebrand Factor, Prekallikrein, High Molecular Weight Kininogen, Fibronectin, Antithrombin III , heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitor (ZPI), plasminogen, ⁇ 2-antiplasmin, tissue plasminogen activator (tPA), urokinase , plasminogen activator inhibitor-1 (PAI1), plasminogen activator inhibitor-2 (PAI2), cancer procoagulant or afaripin.
  • Factor I Factor II
  • Tissue Factor Factor V
  • the blood factor or coagulation factor is Factor VIII.
  • the hormone includes, but is not limited to, melatonin (N-acetyl-5-methoxytryptamine), serotonin, thyroxine (or tetraiodothyronine) (thyroid hormone), three Iodine (thyroid hormone), adrenaline (or adrenal hormone), norepinephrine (or norepinephrine), dopamine (or prolactin inhibitor), anti-Miller tube hormone (or Miller tube inhibitor) Factor or hormone), adiponectin, adrenocorticotropic hormone (or cortisol), angiotensinogen and angiotensin, vasopressin (or vasopressin, arginine vasopressin), atrial natriuretic peptide (or atrial natriuretic peptide), calcitonin, cholecystokinin, corticotropin releasing hormone, erythropoietin, follicle stimulating hormone
  • the antibody comprises: a monoclonal antibody, a polyclonal antibody, a dimer, a multimer, a bispecific antibody, a multispecific antibody, and an antibody fragment (eg, Fab, Fab', F(ab) 2 , F(ab') 2 and Fv, etc.) and the like; examples of the monoclonal antibody are specifically adalimumab (HUMIRA), tocilizumab, orelizumab, Bailey Mulimumab, Infliximab, Rituximab, Trastuzumab, Bevacizumab, Alemtuzumab , Cetuximab, Panitumumab, Nimotuzumab, Pertuzumab, Ibritumomab, Tosimizumab (Tositumomab), Omalizumab, Abagovomab, Abciximab, Adecatumumab, Afelimoma
  • the monoclonal antibody is adalimumab.
  • the enzyme and the coenzyme include, but are not limited to, an immunozyme, a microbial enzyme, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase or a ligase; and the like, specifically, imiglucerase, ⁇ -galactosidase A ( ⁇ -galA), agalsidase beta, acid alpha-glucosidase (GAA), recombinant aglucosidase alpha (LUMIZYME), alpha-glucosidase, arylsulfatase, pull Ranizyme, ALDURAZYME, idursulfase, ELAPRASE, sulfurase (NAGLAZYME), PEGylated recombinant uricase (KRYSTEXXA), PEGylated recombinant Candida urate, asparaginase, and the like.
  • the enzyme is ⁇ -galactosidase A ( ⁇ -galA), acid ⁇ -glucosidase (GAA), PEGylated recombinant uricase (KRYSTEXXA), polyethylene Alcoholized recombinant Candida uricase or asparaginase.
  • ⁇ -galA ⁇ -galactosidase A
  • GAA acid ⁇ -glucosidase
  • KRYSTEXXA PEGylated recombinant uricase
  • Candida uricase polyethylene Alcoholized recombinant Candida uricase or asparaginase.
  • the exogenous immunogen is an artificial antigen vector
  • the artificial antigen carrier refers to a carrier (eg, coupled) to a hapten or other antigen to enhance its immunogenicity.
  • Immunogenic molecules for the preparation of artificial antigens including, for example, proteins, polypeptide polymers, macromolecular polymers, and certain particles.
  • the protein carrier also known as carrier protein
  • the protein carrier includes, but is not limited to, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), human serum albumin (HSA), ovalbumin (OVA), and the like.
  • the polypeptide polymer carrier is, for example, polylysine (PLL) or the like.
  • the particulate carrier is such as certain bacterial particles, viruses, and the like.
  • the artificial antigen vector is a protein carrier.
  • the protein carrier is keyhole limpet hemocyanin (KLH).
  • the exogenous immunogen is a graft; preferably, the graft is a cell, tissue, organ or other biocompatible graft for transplantation, the graft may It is a cell, tissue, or organ of autologous or allogeneic (like a species but different genotypes), and may also be an artificial material (such as artificial bone, artificial trachea, etc.).
  • the graft is a cell, preferably a stem cell, such as a hematopoietic stem cell, a neural stem cell, or the like.
  • the graft is tissue, preferably from: skin, bone and cartilage, dermis and fat, mucosa, fascia, cornea, muscle, nerve, artificial biomaterial, and the like.
  • the graft is an organ, preferably from the heart, pancreas, kidney, lung and liver, and the like.
  • the conjugate is administered prior to, concurrently with, or after administration of the exogenous immunogen.
  • exemplary therapeutic agents include, but are not limited to, antimetabolites, platinum-based agents, alkylating agents, tyrosine kinase inhibitors, anthracyclines, vinca alkaloids, proteasomes Inhibitors and topoisomerase inhibitors, and the like.
  • chemotherapeutic agents include, but are not limited to, antimetabolites, platinum-based agents, alkylating agents, tyrosine kinase inhibitors, anthracyclines, vinca alkaloids, proteasomes Inhibitors and topoisomerase inhibitors, and the like.
  • chemotherapeutic agents include, but are not limited to, antimetabolites, platinum-based agents, alkylating agents, tyrosine kinase inhibitors, anthracyclines, vinca alkaloids, proteasomes Inhibitors and topoisomerase inhibitors, and the like.
  • the above therapeutic agents are known to those skilled in the art, and the invention is not limited in this regard.
  • the invention also provides a use of a combination of PEG and rapamycin for preparing a medicament for enhancing the therapeutic effect of a first medicament, wherein the first medicament is selected from the group consisting of a cytokine, a human hemoglobin, a blood factor or a blood coagulation factor, At least one of a vascular endothelial growth factor antibody antagonist, a hormone, an antibody, an enzyme, and a coenzyme.
  • the first medicament is for treating a tumor, a blood disease, an infectious disease, an immune system disease, a hereditary disease, a metabolic disease.
  • the tumor is selected from the group consisting of lung cancer, kidney cancer, melanoma, liver cancer head and neck cancer, skin cancer, squamous cell carcinoma, ovarian cancer, bone cancer, colorectal cancer, bladder cancer, stomach cancer, pancreatic cancer, prostate cancer.
  • the infectious disease is selected from the group consisting of HIV virus infection, hepatitis virus (type A, type B and type C) infection, herpes virus infection, influenza virus infection;
  • the immune system disease is selected from the group consisting of lupus erythematosus, rheumatoid arthritis, ankylosing spine Inflammation, myasthenia gravis, multiple sclerosis, autoimmune hemolytic anemia, autoimmune hepatitis, scleroderma, nodular polyarteritis, Wegener granulomatosis;
  • the blood disease is selected from hemophilia;
  • the hereditary disease is selected from the group consisting of Fabry disease, Pompe disease, lysozyme volume disease,
  • the invention also provides a use of a combination of PEG and rapamycin for the preparation of a medicament for enhancing the effect of gene therapy.
  • the gene therapy is a non-immunogenic gene therapy, such as a non-immunogenic gene therapy using a viral vector.
  • the gene therapy is for the treatment of cancer, blood diseases, infectious diseases, immune system diseases, hereditary diseases, metabolic diseases.
  • the tumor is selected from the group consisting of lung cancer, kidney cancer, melanoma, liver cancer head and neck cancer, skin cancer, squamous cell carcinoma, ovarian cancer, bone cancer, colorectal cancer, bladder cancer, stomach cancer, pancreatic cancer, prostate cancer.
  • Hodgkin's lymphoma Hodgkin's lymphoma, follicular lymphoma, chronic or acute leukemia, mesothelioma, pancreatic cancer, breast cancer, multiple myeloma and other solid tumors;
  • the blood disease is selected from hemophilia;
  • the infectious disease is selected from the group consisting of HIV virus infection, hepatitis virus (type A, type B and type C) infection, herpes virus infection, influenza virus infection;
  • the immune system disease is selected from the group consisting of lupus erythematosus, rheumatoid arthritis, and tonicity.
  • the blood disease is selected from hemophilia
  • the genetic disease is selected from the group consisting of Fabry disease, Pompe's disease, lysozyme volume disease, hereditary muscle disease, genetic metabolic disease; the metabolic disease is selected from amino acid metabolism disorders (such as branch chain diseases, High amino acidemia, high amino aciduria), urea metabolism disorder, hyperamineemia, mucopolysaccharidosis, storage diseases (such as glycogen storage disease and lipid storage disease), glycogen storage disease I (such as Cory's disease), malabsorption diseases (such as intestinal carbohydrate malabsorption), oligosaccharidase deficiency (such as maltase deficiency, lactase deficiency, insulase deficiency), fructose metabolism disorder, galacto
  • the present invention provides a use of a combination of polyethylene glycol and rapamycin for reducing an immune response.
  • the invention also provides a use of a combination of PEG and rapamycin for enhancing the therapeutic effect of a second medicament.
  • the invention also provides a use of a combination of PEG and rapamycin for enhancing the effect of gene therapy.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a first drug and a conjugate of PEG and rapamycin according to the present invention, the first drug being selected from the group consisting of a cytokine, a human At least one of hemoglobin, blood factor or coagulation factor, vascular endothelial growth factor antibody antagonist, hormone, antibody, enzyme, and coenzyme.
  • the pharmaceutical or pharmaceutical composition of the present invention is selected from the group consisting of a tablet, a pill, a powder, a lozenge, a powder, an elixir, a suspension, an emulsion, a solution, a syrup, an aerosol, an ointment, a transdermal absorbent, and a soft Capsules, hard capsules, suppositories, sterile injectable solutions, and lyophilized powders.
  • the pharmaceutical or pharmaceutical composition of the present invention is a sterile injectable solution or a lyophilized powder.
  • the pharmaceutical or pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable adjuvant.
  • the adjuvant is selected from the group consisting of a carrier and a diluent (lactose, dextrose, sucrose, sorbitol, mannitol, starch) , gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate and hydroxyl
  • a carrier lactose, dextrose, sucrose, sorbitol, mannitol, starch
  • gum arabic calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate and hydroxyl
  • propyl benzoate talc, magnesium stearate
  • the method for administering the medicament or the pharmaceutical composition of the present invention is selected from the group consisting of oral, subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous, intranasal, epidural, sublingual, intranasal, intracerebral, intrathecal, Transdermal, rectal, etc.
  • the present invention provides a conjugate of PEG and rapamycin and its use, particularly in the preparation for reducing the immune response (especially reducing the recipient's exogenous immunogens such as therapeutic agents, grafts, etc.)
  • New use in drugs for immune response wherein the combination of PEG and rapamycin can significantly reduce antibody productivity against exogenous immunogens such as therapeutic agents, grafts, and reduce the excessive immune response caused by it, It has the beneficial effects of ensuring or even improving the therapeutic effect of the therapeutic agent, improving the immune status of the recipient, reducing or even eliminating the rejection of the transplant, and the preparation process is relatively simple, the cost is low, and the industrial production is easy to realize, and the application value is very good. .
  • Alkyl means a straight or branched hydrocarbon chain radical which does not contain an unsaturated bond
  • a C1-C6 alkyl group means an alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, N-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, etc.; if the alkyl group is substituted by a cycloalkyl group, which is correspondingly a "cycloalkylalkyl” radical,
  • the cycloalkylalkyl group of C4-10 means a cycloalkylalkyl group having 4 to 10 carbon atoms, such as cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexyl.
  • aralkyl group of C7-14 means an aralkyl group having 7-14 carbon atoms, such as a benzyl group, Benzyl or phenethyl.
  • alkoxy means a substituent formed by substituting a hydrogen in a hydroxy group with an alkyl group
  • alkoxy group of C1-C6 means an alkoxy group having 1 to 6 carbon atoms, such as a methoxy group or an ethoxy group. , propoxy, butoxy, and the like.
  • Cycloalkyl means an alicyclic hydrocarbon such as containing from 1 to 4 monocyclic and/or fused rings containing from 3 to 18 carbon atoms, preferably from 3 to 10 carbon atoms, such as cyclopropyl, cyclohexyl or Adamantyl and the like
  • cycloalkyl group of C3-6 as used in the present invention means a cycloalkyl group having 3 to 6 carbon atoms such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group and a cyclohexyl group.
  • Aryl means a monocyclic or polycyclic radical, including polycyclic radicals containing a monoaryl group and/or a fused aryl group, such as containing 1-3 monocyclic or fused rings and 6-
  • the 18 carbon atom, the aryl group of C6-10 in the present invention means an aryl group having 6 to 10 carbon ring atoms such as a phenyl group or a naphthyl group.
  • Heterocyclyl includes heteroaromatic groups and heteroalicyclic groups containing from 1 to 3 monocyclic and/or fused rings and from 3 to about 18 ring atoms. Preferred heteroaromatic groups and heteroalicyclic groups contain from 5 to about 10 ring atoms. Suitable heteroaryl groups in the compounds of the invention contain 1, 2 or 3 heteroatoms selected from N, O or S atoms, said nitrogen-containing heterocycles being referred to as N atoms in the heteroatoms thereof. .
  • the "monosaccharide” is generally a polyhydroxy aldehyde or a polyhydroxy ketone having 3 to 6 carbon atoms.
  • the monosaccharide may be classified into triose, butyrate, pentose, hexose or the like according to the number of carbon atoms.
  • Common monosaccharides such as glucose, fructose, galactose, ribose, deoxyribose and the like.
  • “Oligosaccharide” refers to a compound formed by polymerizing 2-10 glycosidic bonds, which are formed by dehydration condensation of a monosaccharide hydroxyl group with a hydroxyl group of another monosaccharide, common oligosaccharides.
  • disaccharide refers to a compound formed by linking two monosaccharide molecules together by a glycosidic bond, such as sucrose, lactose, maltose and the like.
  • the "trisaccharide” trisole monosaccharide is a compound formed by glycosidic linkage, such as gentiotriose, raffinose and the like.
  • rapamycin also known as sirolimus, in which rapamycin derivatives include, but are not limited to, compounds having a structure and function similar to rapamycin, such as ivimo Division (everolimus, RAD 001 (Novartis)), temsirolimus (also known as temsirolimus, CC1-779 (Wyeth)), Biomus A9 (biolimus A9), ABT-578 (zotarolimus) ), deforolimus (ridaforolimus, AP23573, MK-8669) and the like.
  • ivimo Division everolimus, RAD 001 (Novartis)
  • temsirolimus also known as temsirolimus, CC1-779 (Wyeth)
  • Biomus A9 biolimus A9
  • ABT-578 zotarolimus
  • deforolimus ridaforolimus, AP23573, MK-8669
  • the "therapeutic agent” as used in the present invention means a substance which can be administered to a recipient and has a therapeutic effect, such as a macromolecular substance such as a protein, a carbohydrate, a lipid, and a nucleic acid.
  • the therapeutic agent is a therapeutic macromolecule.
  • Administration of therapeutic macromolecules to a recipient may result in an overreactive immune response, such as the production of anti-therapeutic macromolecular specific antibodies.
  • the therapeutic macromolecule is a therapeutic protein.
  • excessive immune response is meant to promote or aggravate a disease, disorder or condition (or symptom thereof) as described herein, or to be a disease or disorder as described herein. Or illness (or its symptoms). Such an immune response generally has an adverse effect on the health of the subject or is a symptom of adverse effects on the health of the subject. Excessive immune responses include antigen-specific antibody production, antigen-specific B cell proliferation and/or activity, or antigen-specific CD4+ T cell proliferation and/or activity. In general, these excessively strong immune responses are therapeutic macromolecular specific and counteract the desired beneficial effects of administering the therapeutic macromolecule.
  • the “antibody” as used in the present invention is used in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (eg, bispecific antibodies) and antibody fragments, As long as they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology, 170: 4854-4861).
  • the antibody can be murine, human, humanized, chimeric, or derived from other species.
  • Antibodies can be of any type (eg, IgG, IgE, IgM, IgD, and IgA) and classes (eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2).
  • the "recipient” as used in the present invention is a mammal, including but not limited to primates (such as humans, monkeys, baboons, etc.), cloven-hoofed animals (such as pigs, sheep, cattle, etc.), and hoofed animals (such as horses). , rodents (such as mice, capybaras, etc.), carnivores (including canines (such as wolves, dogs), cats (such as tigers, cats), bears (such as pandas), etc., preferably human.
  • primates such as humans, monkeys, baboons, etc.
  • cloven-hoofed animals such as pigs, sheep, cattle, etc.
  • hoofed animals such as horses.
  • rodents such as mice, capybaras, etc.
  • carnivores including canines (such as wolves, dogs), cats (such as tigers, cats), bears (such as pandas), etc., preferably human.
  • transplantation refers to a process in which a certain cell, tissue or organ of the living body is irreparably damaged due to damage, and the process of implanting a corresponding healthy cell, tissue or organ into the body is called a cell. Transplantation of tissues or organs. Common are autologous transplantation, allogeneic transplantation and isogenic transplantation.
  • pharmaceutically acceptable excipient means that the ingredient has no biological activity or other undesirable activity impurities, for example, the ingredient can be incorporated into a disclosed pharmaceutical preparation and administered to a patient without causing significant adverse biological effects or Interaction occurs in a deleterious manner with other ingredients contained in the formulation.
  • Treatment includes inhibiting, delaying, alleviating, attenuating, limiting, alleviating or ameliorating a disease, disorder, condition or condition, its occurrence and/or progression, and/or its symptoms.
  • the rapamycin used in the examples was purchased from Wuhan Yuancheng Co-creation Technology Co., Ltd., and monomethoxy polyethylene glycol-glutamic acid dipeptide and glycine rapamycin ester were provided by Tianjin Keykai Technology Co., Ltd. The rest are commercially available reagents.
  • the experimental animals were Balb/c female mice, purchased from Beijing Huakangkang Biotechnology Co., Ltd.; the immunogen was keyhole limpet hemocyanin (KLH), purchased from Sigma, USA.
  • reaction solution was concentrated the next day, and the residue was crystallized from isopropanol to obtain monomethoxypolyethylene glycol (20KDa)-glutamic acid dipeptide-rapamycin conjugate (JK1208R) 4.7 g (n was about 450 ).
  • Animals were housed in an IVC (independent air supply system) cage at the SPF level animal house (4 per cage).
  • the animal information card for each cage indicates the number of animals in the cage, gender, strain, date of receipt, dosing schedule, experiment number, group, and start date of the experiment. All cages, litter, feed and drinking water are sterilized prior to use. Cage, feed and drinking water are changed twice a week.
  • the feeding environment and lighting conditions are as follows:
  • Light cycle 12 hours light, 12 hours without light (lights at 8 am - 8 pm)
  • Group G1 (model group):
  • Tail blood test On the 28th to 29th day, about 20ul of tail blood was collected from the tail vein, RT30min, centrifugation at 5000rpm for 5min, blood cell sediment was discarded, and serum was taken for Elisa detection titer;
  • Group G2 (Group IV): Control immunization was performed with the G1 group, and 620 mg of JK1208R (prepared in Example 1) was intravenously administered on Days 0 and 7, respectively.
  • G3 group (PO group): Control immunization was performed with the G1 group, and 52 ⁇ g of rapamycin was orally administered on the 0th day and the 7th day, respectively.
  • Tail blood test On the 40th to 41st day, about 20ul of tail blood was collected from the tail vein, RT30min, centrifugation at 5000rpm for 5min, blood cell sediment was discarded, and serum was taken for ELISA to check the titer.
  • secondary antibody goat anti-mouse IgG-HRP 1:10000 1

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Abstract

一种PEG与雷帕霉素的结合物及其用途，特别是在制备用于减少免疫应答的药物中的用途，其中所述PEG与雷帕霉素的结合物可显著降低针对外源免疫原的抗体生产率，减少因其施用而引起的过强的免疫应答，具有可保证甚至改善治疗剂的治疗效果、改善接受者自身免疫状况、降低甚至消除移植排斥反应等有益效果，且其制备工艺相对简单，成本较低，易实现工业化生产，有非常好的应用价值。

Description

聚乙二醇与雷帕霉素的结合物及其用途 技术领域
本发明涉及医药技术领域，具体涉及一种聚乙二醇与雷帕霉素的结合物及其用途，特别是在制备用于减少免疫应答的药物中的用途，更具体为聚乙二醇与雷帕霉素的结合物在制备用于减少接受者对外源免疫原如治疗剂、移植物等引起的过强的免疫应答的药物中的用途。
背景技术
免疫原性，是指能够刺激机体形成特异抗体或致敏淋巴细胞的能力，即指抗原能刺激特定的免疫细胞，使免疫细胞活化、增殖、分化，最终产生免疫效应物质抗体和致敏淋巴细胞的特性。免疫应答是指机体免疫系统对免疫原刺激所产生的以排除抗原为目的的生理过程。
一些治疗剂基于其自身的化学特性，也具有免疫原性，如某些大分子蛋白质和多糖、小分子多肽和核酸等，及移植物如细胞、组织、器官、人工材料等，也可能诱导产生免疫应答。在这种情况下，免疫系统中的细胞识别该治疗剂、移植物，并发起攻击、破坏和清除的免疫学反应，不利于患者的治疗，给患者带来更多的痛苦，甚至可能加重病情危及生命。
雷帕霉素(又名西罗莫司)是1975年在智利复活节岛的土壤中发现的由吸水链霉菌产生的一种大环内酯类抗生素，其作为免疫抑制剂，在1989年被美国Home Products公司研制开发作为抗移植排异反应的新药进入临床，并于1999年9月通过美国FDA批准上市。虽然雷帕霉素在临床上表现出良好的前景，但仍存在生物利用度低、水溶性差等缺陷。近年来，一些研究人员通过雷帕霉素与纳米载体结合用于降低上述过强的免疫应答，如专利申请CN201480031937.3中公开了未与合成纳米载体连接的治疗性大分子和与合成纳米载体连接的免疫抑制剂的给药组合，用于降低不期望的体液免疫应答。
但上述雷帕霉素与纳米载体的结合物制备工艺复杂，成本较高，纳米载体粒径分布较宽，产品批次稳定性较差，治疗效果不理想，不利于工业化放大生产和实际应用。
发明内容
为克服现有技术的不足，本发明提供一种聚乙二醇与雷帕霉素的结合物在制备用于减少免疫应答的药物中的用途，所述聚乙二醇与雷帕霉素的结合物具有通式Ⅰ所示结构：
PEG-X-D
(Ⅰ)
其中，
PEG为聚乙二醇残基，
D为雷帕霉素或其衍生物残基，
X为PEG和D之间的连接基团。
优选地，所述结合物中，所述X选自：-(CR 1R 2) a-、-(CH 2) aNH-、-(CH 2) aNHCO-、-(CH 2) aCONH-、-(CH 2) aCO-、-(CH 2) aCOO-、-(CH 2) aO-、环烷基、芳基、杂环基、-A-和-Op-中的一种或多种的组合，
其中，a为0-10的整数，
R 1和R 2独立地选自：-H、C1-6的烷基、-OR'、-NHR'、-N(R') 2、-CN、-F、-Cl、-Br、-I、-COR'、-COOR'、-OCOR'、-CONHR'和-CON(R') 2中的一种或两种以上的组合，
R'选自：-H、C1-6的烷基、-F、-Cl、-Br和-I，
-A-为氨基酸残基，
-Op-为寡肽残基(如含2-10(具体如2、3、4、5、6、7、8、9或10)个氨基酸残基的多肽)。
在本发明的一个实施方式中，所述X具有如下结构：-X 1-X 2-X 3-，其中，X 1和X 3独立地选自：-(CR 1R 2) a-、-(CH 2) aNH-、-(CH 2) aNHCO-、-(CH 2) aCONH-、-(CH 2) aCO-、-(CH 2) aCOO-和-(CH 2) aO-中的一种或多种的组合，X 2选自：环烷基、芳基、杂环基、-A-和-Op-。
优选地，所述氨基酸残基-A-选自如下所示结构：
Figure PCTCN2019080194-appb-000001
Figure PCTCN2019080194-appb-000002
中的一种，
其中，G选自：-H、-CH 3、
Figure PCTCN2019080194-appb-000003
Figure PCTCN2019080194-appb-000004
-CH 2-S-CH 3、
Figure PCTCN2019080194-appb-000005
-CH 2OH、-CH 2SH、-CH 2CONH 2、-CH 2CH 2CONH 2、
Figure PCTCN2019080194-appb-000006
Figure PCTCN2019080194-appb-000007
优选自：-H、-CH 3、
Figure PCTCN2019080194-appb-000008
Figure PCTCN2019080194-appb-000009
更优选地，G为-H或-CH 3。
优选地，所述寡肽残基-Op-含有2-10个氨基酸残基，所述氨基酸残基相同或不同，选自本发明上述氨基酸残基-A-的限定方案；所述寡肽残基-Op-可为直链状寡肽残基，如甘氨酸的二肽
Figure PCTCN2019080194-appb-000010
三肽
Figure PCTCN2019080194-appb-000011
等，此时所述寡肽残基-Op-有两个可连接位点；所述寡肽残基-Op-也可为分支状寡肽残基，如
Figure PCTCN2019080194-appb-000012
(m为1、2、3或4)，其中A 1选自：天冬氨酸、谷氨酸、赖氨酸的残基，A 2和A 3相同或不同，选自本发明上述氨基酸残基-A-的限定方案，具体如中国专利申请 CN201410715522.X中所述的多爪寡肽，特别如
Figure PCTCN2019080194-appb-000013
Figure PCTCN2019080194-appb-000014
R 1和R 2独立地选自：-H、C1-6的烷基、-OR'、-NHR'、-N(R') 2、-CN、-F、-Cl、-Br、-I、-COR'、-COOR'、-OCOR'、-CONHR'和-CON(R') 2中的一种或两种以上的组合，R'选自：-H、C1-6的烷基、-F、-Cl、-Br和-I，
a为0-10的整数。
优选地，所述R'选自：-H和C1-3的烷基(如甲基、乙基、正丙基或异丙基)。
优选地，所述R 1和R 2独立地选自：H、C1-3的烷基(如甲基、乙基、正丙基或异丙基)、-OH、C1-3的烷氧基(即-OR'，R'为C1-3的烷基)、-NH 2、-F、-Cl、-Br和-I中的一种或多种的组合；更优选自：H、-CH 3、-OH、-OCH 3和-OCH 2CH 3。
优选地，所述a为0-5的整数，如0、1、2、3、4或5。
优选地，所述X 1和X 3独立选自：单键、-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-、-CH 2CO-、-CH 2CH 2CO-、-CH 2NH-、-CH 2NHCH 2-、-CH 2CONH-、-CH 2CONHCH 2-、-CH 2NHCO-、--CH 2NHCOCH 2-、-CH 2COO-、-CH 2COOCH 2-、-CH 2O-、-CH 2OCH 2-中的一种或多种的组合。
在本发明的一个实施例中，所述X 1为-CH 2CO-。
在本发明的另一个实施例中，所述X 1为-CH 2OCH 2-。
在本发明的另一个实施例中，所述X 1为-CH 2CONHCH 2-。
在本发明的一个实施例中，所述X 3为单键。
在本发明的一个实施例中，所述X 3为-CH 2CO-。
在本发明的一个实施方式中，所述-Op-具有如下结构：
Figure PCTCN2019080194-appb-000015
其中，m为1、2、3或4，
A 1选自：天冬氨酸、谷氨酸、赖氨酸的残基，
A 2和A 3相同或不同，独立地选自如下结构：
Figure PCTCN2019080194-appb-000016
其中G具有本发明上述定义。
在本发明的一个实施例中，所述A 1为谷氨酸残基
Figure PCTCN2019080194-appb-000017
在本发明的一个实施例中，所述A 2和A 3相同，均为甘氨酸残基
Figure PCTCN2019080194-appb-000018
在本发明的一个具体实施方式中，所述-Op-具有如下结构：
Figure PCTCN2019080194-appb-000019
优选地，m为2或3。本发明通式Ⅰ所示结合物中，所述连接基团X可以仅含有两个连接位点，分别连接PEG和D；所述X也可含有三个以上连接位点，连接一个聚乙二醇残基和两个以上雷帕霉素或其衍生物残基，达到提高药物负载量等目的，也可连接一个雷帕霉素或其衍生物残基和两个以上聚乙二醇残基，达到提高结合物水溶性或空间位阻等目的。
在本发明另一个实施方式中，所述X为-X 1-Op-X 3-，-Op-具有本发明上述定义。
在本发明的一个实施例中，所述X为
Figure PCTCN2019080194-appb-000020
优选地，m为2或3。
优选地，所述结合物中，所述PEG为直链、Y型、多分支的聚乙二醇残基，例如包括单甲氧基聚乙二醇(mPEG)、直链双端PEG、Y型PEG、4臂支链PEG、6臂支链PEG或8臂支链PEG等。
优选地，所述的PEG的分子量可为1-100KDa，例如1-10KDa(具体可为1、2、3、4、5、6、7、8、9或10KDa)、10-50KDa(具体可为10、15、20、25、30、35、40、45或50KDa)或50-100KDa(具体可为50、55、60、65、70、75、80、85、90、95或100KDa)等；进一步优选为10-50KDa。
在本发明一具体实施方式中，所述PEG为直链聚乙二醇残基，具有通式Ⅱ或Ⅲ所示的结构：
Figure PCTCN2019080194-appb-000021
其中，p和q独立地选自1-2280的整数，优选为220-1140的整数，
Y为端基，具有-L-T结构，
其中，L为氧(O)和封端基T之间的连接基团，选自：-(CH 2) b-、-(CR 3R 4) b-、-(CH 2) bNH-、-(CH 2) bNHCO-、-(CH 2) bCONH-、-(CH 2) bCO-、-(CH 2) bCOO-和-(CH 2) bO-中的一种或多种 的组合，其中b为0-10的整数，
R 3和R 4独立地选自：-H、C1-6的烷基、-OR”、-NHR”、-N(R”) 2、-CN、-F、-Cl、-Br、-I、-COR”、-COOR”、-OCOR”、-CONHR”和-CON(R”) 2中的一种或多种的组合，
R”选自：-H、C1-6的烷基、-F、-Cl、-Br和-I，
T为封端基，选自：H、C1-6的烷基、C3-6的环烷基、C4-10的环烷基烷基、C6-10的芳基、C7-14的芳烷基、单糖和低聚糖残基。
优选地，所述L选自：单键、-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-、-CONH-、-NH-、-CO-、-CONHCH 2-、-CH 2NH-、-CH 2CONH-和-COCH 2-中的一种或多种的组合。
优选地，所述T选自：甲基、乙基、异丙基、环丙基、环丙基甲基、环丁基、环己基、苄基、
Figure PCTCN2019080194-appb-000022
优选地，所述Y选自：甲基、乙基、异丙基、环丙基、环丙基甲基、环丁基、环己基、苄基、
Figure PCTCN2019080194-appb-000023
在本发明的一个优选实施例中，所述Y为甲基。
在本发明的另一个优选实施例中，所述Y为
Figure PCTCN2019080194-appb-000024
在本发明一具体实施方式中，所述PEG为Y型聚乙二醇残基，具有通式Ⅳ或Ⅴ所示的结构：
Figure PCTCN2019080194-appb-000025
Figure PCTCN2019080194-appb-000026
其中，i和h独立地选自1-1140的整数，优选为110-570的整数，
Y具有本发明上述通式Ⅱ中定义的结构，优选自：甲基、乙基、异丙基、环丙基、环丙基甲基、环丁基、环己基、苄基、
Figure PCTCN2019080194-appb-000027
更优选自：甲基、
Figure PCTCN2019080194-appb-000028
在本发明一具体实施方式中，所述PEG为多分支聚乙二醇残基，具有通式Ⅵ所示的结构：
Figure PCTCN2019080194-appb-000029
其中，k是1-760的整数，优选为70-380的整数，
j是3-8的整数，
R是多分支聚乙二醇的核心分子，R选自：季戊四醇、寡聚季戊四醇、甲基葡萄糖苷、蔗糖、二甘醇、丙二醇、甘油和聚甘油的残基。
优选地，R选自：季戊四醇、寡聚季戊四醇、甘油和聚甘油的残基；更优选自：季戊四醇、二聚季戊四醇、三聚季戊四醇、甘油和六聚甘油残基。
在本发明的一个实施例中，所述多分支聚乙二醇残基仅含有一个可连接位点，其具有如下结构：
Figure PCTCN2019080194-appb-000030
其中，Y具有本发明上述通式Ⅱ中定义的结构，优选自：甲基、乙基、异丙基、环丙基、环丙基甲基、环丁基、环己基、苄基、
Figure PCTCN2019080194-appb-000031
更优选自：甲基、
Figure PCTCN2019080194-appb-000032
在本发明一个优选实施例中，所述多分支聚乙二醇残基具有如下结构：
Figure PCTCN2019080194-appb-000033
其中，k是1-570的整数，优选为55-285的整数，
x为1-10的整数(具体如1、2、3、4、5、6、7、8、9或10)，优选为1-6的整数，
其中，Y具有本发明上述通式Ⅱ中定义的结构，优选自：甲基、乙基、异丙基、环丙基、环丙基甲基、环丁基、环己基、苄基、
Figure PCTCN2019080194-appb-000034
更优选自：甲基、
Figure PCTCN2019080194-appb-000035
在本发明另一个优选实施例中，所述多分支聚乙二醇残基具有如下结构：
Figure PCTCN2019080194-appb-000036
其中，k是1-280的整数，优选为28-140的整数，
y为1-10的整数(具体如1、2、3、4、5、6、7、8、9或10)，优选为1-5的整数，更优选为1-3的整数，
Y具有本发明上述通式Ⅱ中定义的结构，优选自：甲基、乙基、异丙基、环丙基、环丙基甲基、环丁基、环己基、苄基、
Figure PCTCN2019080194-appb-000037
更优选自：甲基、
Figure PCTCN2019080194-appb-000038
在本发明中，当所述PEG具有上述通式Ⅱ、Ⅳ或Ⅴ的结构时，一个PEG分子可通过其连接位点通过连接基团X与药物D结合；当所述PEG具有上述通式Ⅲ或Ⅵ的结构时，一个PEG分子具有两个以上可连接位点，PEG可通过其中一个连接位点通过连接基团X与药物D结合，此时，所述结合物中只含有一个药物残基，PEG也可通过多个连接位点通过连接基团X与药物D结合，此时，结合物中有两个以上药物残基。优选地，所述PEG为通式Ⅲ或Ⅵ所示的具有两个以上可连接位点的PEG时，其通过一个连接位点通过连接基团X与药物D结合，其他可连接位点可连接封端基Y(如甲基)，如通式Ⅶ所示结构。
优选地，所述结合物中，所述D具有如下结构：
Figure PCTCN2019080194-appb-000039
其中，
R 5选自：H、C1-C6的烷基、C2-C6的烯基、C6-C12的芳基和C7-C14的芳烷基；
R 6选自：H、羟基和C1-C6的烷氧基；
R 7选自：H、C1-C6的烷基、C2-C6的烯基、C6-C12的芳基、C7-C14的芳烷基和-C(O)R 71，其中R 71选自：H、C1-C6的烷基、C2-C6的烯基、C6-C12的芳基和C7-C14的芳烷基；
R 8选自：H、羟基和C1-C6的烷氧基；
R 9选自：-O-、-O-R 51-O-、
Figure PCTCN2019080194-appb-000040
其中R 91为C1-C6的亚烷基；
Z为H或羟基保护基团。
优选地，式Ⅷ中，所述R 5为H或C1-C6的烷基，更优选为H、甲基或乙基；在本发明的一个实施例中，所述R 5为甲基。
优选地，式Ⅷ中，所述R 6选自H、羟基和C1-C3的烷氧基，更优选为-OCH 3、-OCH 2CH 3、-O(CH 2) 2CH 3；在本发明的一个实施例中，所述R 6为甲氧基。
优选地，式Ⅷ中，所述R 7选自：H、C 1-C 6的直链烷基、C 2-C 10的直链烯基、苯基、 卤代苯基、苯甲基、苯乙基、-C(O)R 71，其中R 71选自：H、C 1-C 6的直链烷基，更优选为H、-OCH 3、-OCH 2CH 3、-O(CH 2) 2CH 3；在本发明的一个实施例中，所述R 7为H。
优选地，式Ⅷ中，所述R 8选自：H、羟基和和C1-C3的烷氧基，更优选为-OCH 3、-OCH 2CH 3、-O(CH 2) 2CH 3；在本发明的一个实施例中，所述R 8为甲氧基。
优选地，所述R 91为C1-C3的亚烷基，如-CH 2-、-CH 2CH 2-、-CH 2CH 2CH 2-。
在本发明的一个优选实施例中，所述式Ⅷ中，所述R 5为甲基，R 6为甲氧基，R 7为H，R 8为甲氧基。
优选地，所述结合物中，所述D选自：雷帕霉素、依维莫司、坦西莫司、比奥莫司A9、ABT-578和地磷莫司中的一种的残基。
更优选地，所述结合物中，所述D选自：
Figure PCTCN2019080194-appb-000041
Figure PCTCN2019080194-appb-000042
Figure PCTCN2019080194-appb-000043
中的一种，
其中，Z为H或羟基保护基团。
此处所述羟基保护基团，本领域技术人员在本发明的基础上可根据实际情况选择本领域常用的羟基保护基团，如-CH 3、-C(CH 3) 3、-CH 2OCH 3、-COCH 3、-COC(CH 3) 3、-CH 2CH＝CH 2、-Si(CH 3) 3、
Figure PCTCN2019080194-appb-000044
等，本发明对此不做具体限定。
在本发明的一个优选实施例中，所述结合物中，所述D具有如下结构：
Figure PCTCN2019080194-appb-000045
在本发明的一个具体实施方式中，所述结合物具有如下结构：
Figure PCTCN2019080194-appb-000046
Figure PCTCN2019080194-appb-000047
上式中，PEG、m、D具有本发明上述定义。
优选地，式Ⅸ中，所述PEG具有本发明上述通式Ⅱ的结构；更优选地，所述PEG具有如下结构：
Figure PCTCN2019080194-appb-000048
p具有本发明上述定义。
优选地，式Ⅸ中，所述PEG的分子量为10-50KDa，更优选为20KDa。
优选地，式Ⅸ中，所述m为2或3。
优选地，式Ⅸ中，所述D具有如下结构：
Figure PCTCN2019080194-appb-000049
在本发明的一方面，所述的免疫应答是由外源免疫原引起的，优选地，所述用途为聚乙二醇与雷帕霉素的结合物在制备用于减少接受者对外源免疫原的过强的免疫应答的药物中的用途。
优选地，所述接受者为哺乳动物，优选为灵长目动物，更优选为人。
优选地，所述的外源免疫原选自：治疗剂、移植物、人工抗原载体、毒素等。
在本发明的一个实施方式中，所述外源免疫原为治疗剂；优选地，所述治疗剂为治疗性大分子，更优选为治疗性蛋白质，如细胞因子、人血红蛋白、血液因子或凝血因子、血管内皮生长因子抗体拮抗剂、激素、抗体、酶及辅酶；更优选地，所述治疗性蛋白质为抗体或酶及辅酶。
本发明中，所述细胞因子的实例包括但不限于：白细胞介素、集落刺激因子、干扰素、生长因子、肿瘤坏死因子、转化生长因子-β家族或趋化因子家族等。
本发明中，所述生长因子的实例包括但不限于：肾上腺髓质素(AM)、血管生成素(Ang)、 自分泌运动因子、骨形态生成蛋白(BMP)、脑源性神经营养因子(BDNF)、表皮生长因子(EGF)、红细胞生成素(EPO)、成纤维细胞生长因子(FFGF)、神经胶质细胞系源性神经营养因子(GDNF)、粒细胞集落刺激因子(G-CSF)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、生长分化因子-9(GDF9)、肝细胞生长因子(HGF)、肝癌源性生长因子(HDGF)、胰岛素样生长因子(IGF)、促移行因子、肌生长抑制素(GDF-8)、神经生长因子(NGF)及其他神经营养因子、血小板源性生长因子(PDGF)、血小板生成素(TPO)、转化生长因子α(TGF-α)、转化生长因子β(TGF-β)、肿瘤坏死因子α(TNF-α)、血管内皮生长因子(VEGF)、Wnt信号传导途径、胎盘生长因子P1GF)、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6和IL-7。
本发明中，所述血液因子或凝血因子的实例包括但不限于：因子I、因子II、组织因子、因子V、因子VII(例如长效凝血因子VII)、因子VIII、因子IX、因子X、因子Xa、因子XII、因子XIII、重组凝血因子(例如重组凝血因子VIII)、冯·维勒布兰德因子、前激肽释放酶、高分子量激肽原、纤连蛋白、抗凝血酶III、肝素辅因子II、蛋白C、蛋白S、蛋白Z、蛋白Z相关蛋白酶抑制剂(ZPI)、纤溶酶原、α2-抗纤溶酶、组织纤溶酶原激活物(tPA)、尿激酶、纤溶酶原激活物抑制剂-1(PAI1)、纤溶酶原激活物抑制剂-2(PAI2)、癌促凝物质或阿法依伯汀等。
在本发明的一个实施例中，所述血液因子或凝血因子为因子VIII。
本发明中，所述激素包括但不限于：褪黑激素(N-乙酰基-5-甲氧基色胺)、血清素、甲状腺素(或四碘甲腺原氨酸)(甲状腺激素)、三碘甲腺氨酸(甲状腺激素)、肾上腺素(或肾上腺激素)、去甲肾上腺素(或去甲肾上腺激素)、多巴胺(或催乳素抑制激素)、抗米勒管激素(或米勒管抑制因子或激素)、脂连蛋白、促肾上腺皮质激素(或促皮质素)、血管紧张素原和血管紧张素、抗利尿激素(或加压素、精氨酸加压素)、心房钠尿肽(或心钠素)、降钙素、胆囊收缩素、促皮质素释放激素、红细胞生成素、促卵泡激素、胃泌素、生长素释放肽、胰高血糖素、胰高血糖素样肽(GLP-1)、GIP、促性腺素释放激素、生长激素释放激素、人绒毛膜促性腺素、人胎盘催乳素、生长激素、抑制素、胰岛素、胰岛素样生长因子(或生长调节素)、瘦素、黄体化激素、黑素细胞刺激激素、食欲素、催产素、甲状旁腺激素、催乳素、 松弛素、分泌素、生长激素抑制素、血小板生成素、甲状腺刺激激素(或促甲状腺素)、促甲状腺素释放激素、皮质醇、醛固酮、睾酮、去氢表雄酮、雄烯二酮、二氢睾酮、雌二醇、雌酮、雌三醇、黄体酮、骨化三醇(1，25-二羟基维生素D3)、骨化二醇(25-羟基维生素D3)、前列腺素类、白三烯类、前列环素、血栓烷类、催乳素释放激素、促脂解素、脑钠尿肽、神经肽Y、组胺、内皮素、胰多肽、肾素和脑啡肽等。
本发明中，所述抗体包括：单克隆抗体、多克隆抗体、二聚体、多聚体、双特异性抗体、多特异性抗体和抗体片段(例如Fab、Fab'、F(ab) 2、F(ab') 2和Fv等)等；所述单克隆抗体的实例具体如阿达木单抗(Adalimumab，HUMIRA)、托珠单抗(Tocilizumab)、奥瑞珠单抗(Ocrelizumab)、贝利木单抗(Belimumab)、英利昔单抗(Infliximab)、利妥昔单抗(Rituximab)、曲妥珠单抗(Trastuzumab)、贝伐珠单抗(Bevacizumab)、阿伦珠单抗(Alemtuzumab)、西妥昔单抗(Cetuximab)、帕尼单抗(Panitumumab)、尼妥珠单抗(Nimotuzumab)、帕妥珠单抗(Pertuzumab)、替依莫单抗(Ibritumomab)、托西莫单抗(Tositumomab)、奥马珠单抗(Omalizumab)、阿巴伏单抗(Abagovomab)、阿昔单抗(Abciximab)、阿德木单抗(Adecatumumab)、阿非莫单抗(Afelimomab)、阿夫土珠单抗(Afutuzumab)、培化阿珠单抗(Alacizumab pegol)、喷替酸阿妥莫单抗(Altumomabpentetate)、麻安莫单抗(Anatumomabmafenatox)、安芦珠单抗(Anrukinzumab)(IMA-638)、阿泊珠单抗(Apolizumab)、阿西莫单抗(Arcitumomab)、阿塞珠单抗(Aselizumab)、阿托木单抗(Atorolimumab)、巴匹珠单抗(Bapineuzumab)、巴利昔单抗(Basiliximab)、巴土昔单抗(Bavituximab)、贝妥莫单抗(Bectumomab)、贝利木单抗(Belimumab)、柏替莫单抗(Bertilimumab)、贝索单抗(Besilesomab)、贝伐单抗(Bevacizumab)、比西单抗(Biciromab)、比伐单抗-DM1(Bivatuzumabmertansine)、兰妥莫单抗(Blinatumomab)、本妥昔单抗(Brentuximabvedotin)、ABT-874(Briakinumab)、卡那单抗(Canakinumab)、美坎珠单抗(Cantuzumabmertansine)、卡罗单抗喷地肽(Capromabpendetide)、卡妥索单抗(Catumaxomab)、西利珠单抗(Cedelizumab)、培舍珠单抗(Certolizumabpegol)、泊西他珠单抗(Citatuzumabbogatox)、西妥木单抗(Cixutumumab)、克立昔单抗(Clenoliximab)、 Clivatuzumabtetraxetan、CNTO148(戈利木单抗(golimumab))、CNTO1275(优特克单抗(ustekinumab))、可那木单抗(Conatumumab)、达西珠单抗(Dacetuzumab)、达克珠单抗(Daclizumab)、地舒单抗(Denosumab)、地莫单抗(Detumomab)、阿托度单抗(Dorlimomabaritox)、Dorlixizumab、依美昔单抗(Ecromeximab)、依库珠单抗(Eculizumab)、埃巴单抗(Edobacomab)、依决洛单抗(Edrecolomab)、依法珠单抗(Efalizumab)、依夫单抗(Efungumab)、艾西莫单抗(Elsilimomab)、培戈赖莫单抗(Enlimomabpegol)、西依匹莫单抗(Epitumomabcituxetan)、依帕珠单抗(Epratuzumab)、厄利珠单抗(Erlizumab)、厄马索单抗(Ertumaxomab)、埃达珠单抗(Etaracizumab)、艾韦单抗(Exbivirumab)、法索单抗(Fanolesomab)、法拉莫单抗(Faralimomab)、非维珠单抗(Felvizumab)、非扎奴单抗(Fezakinumab)、芬妥木单抗(Figitumumab)、芳妥珠单抗(Fontolizumab)、福拉韦单抗(Foravirumab)、夫苏木单抗(Fresolimumab)、加利昔单抗(Galiximab)、Gantenerumab、加维莫单抗(Gavilimomab)、吉妥珠单抗奥佐米星(Gemtuzumabozogamicin)、戈利木单抗(Golimumab)、戈利昔单抗(Gomiliximab)、Ibalizumab、替伊莫单抗(Ibritumomabtiuxetan)、伊戈伏单抗(Igovomab)、英西单抗(Imciromab)、英利昔单抗(Infliximab)、英妥木单抗(Intetumumab)、伊诺莫单抗(Inolimomab)、伊珠单抗奥佐米星(Inotuzumabozogamicin)、伊匹木单抗(Ipilimumab)、伊妥木单抗(Iratumumab)、凯利昔单抗(Keliximab)、拉贝珠单抗(Labetuzumab)、来金珠单抗(Lebrikizumab)、来马索单抗(Lemalesomab)、乐地单抗(Lerdelimumab)、来沙木单抗(Lexatumumab)、利韦单抗(Libivirumab)、林妥珠单抗(Lintuzumab)、鲁卡木单抗(Lucatumumab)、鲁昔单抗(Lumiliximab)、马帕木单抗(Mapatumumab)、马司莫单抗(Maslimomab)、马妥珠单抗(Matuzumab)、美泊利单抗(Mepolizumab)、美替木单抗(Metelimumab)、米拉珠单抗(Milatuzumab)、明瑞莫单抗(Minretumomab)、米妥莫单抗(Mitumomab)、莫罗木单抗(Morolimumab)、莫他珠单抗(Motavizumab)、莫罗单抗-CD3(Muromonab-CD3)、MYO-029(司他莫单抗(stamulumab))、他那可单抗(Nacolomabtafenatox)、他那莫单抗(Naptumomabestafenatox)、那他珠单抗(Natalizumab)、奈巴库单抗(Nebacumab)、奈昔木单抗(Necitumumab)、奈瑞莫单抗 (Nerelimomab)、尼妥珠单抗(Nimotuzumab)、巯诺莫单抗(Nofetumomabmerpentan)、奥瑞珠单抗(Ocrelizumab)、奥度莫单抗(Odulimomab)、奥法木单抗(Ofatumumab)、莫奥珠单抗(Oportuzumabmonatox)、奥戈伏单抗(Oregovomab)、奥昔珠单抗(Otelixizumab)、帕昔单抗(Pagibaximab)、帕利珠单抗(Palivizumab)、帕木单抗(Panitumumab)、帕诺库单抗(Panobacumab)、帕考珠单抗(Pascolizumab)、帕尼单抗(Pemtumomab)、培妥珠单抗(Pertuzumab)、培克珠单抗(Pexelizumab)、平妥莫单抗(Pintumomab)、普立昔单抗(Priliximab)、普立木单抗(Pritumumab)、PRO140、雷韦单抗(Rafivirumab)、雷莫芦单抗(Ramucirumab)、雷珠单抗(Ranibizumab)、雷昔库单抗(Raxibacumab)、瑞加韦单抗(Regavirumab)、瑞利珠单抗(Reslizumab)、利妥木单抗(Rilotumumab)、罗妥木单抗(Robatumumab)、罗利珠单抗(Rontalizumab)、罗维珠单抗(Rovelizumab)、鲁利珠单抗(Ruplizumab)、沙妥莫单抗(Satumomab)、司韦单抗(Sevirumab)、西罗珠单抗(Sibrotuzumab)、西法木单抗(Sifalimumab)、Siltuximab、西利珠单抗(Siplizumab)、苏兰珠单抗(Solanezumab)、ASONEP(Sonepcizumab)、松妥珠单抗(Sontuzumab)、司他芦单抗(Stamulumab)、硫索单抗(Sulesomab)、他珠单抗(Tacatuzumabtetraxetan)、他度珠单抗(Tadocizumab)、他利珠单抗(Talizumab)、他尼珠单抗(Tanezumab)、帕他莫单抗(Taplitumomabpaptox)、替非珠单抗(Tefibazumab)、阿替莫单抗(Telimomabaritox)、替妥莫单抗(Tenatumomab)、替奈昔单抗(Teneliximab)、替利珠单抗(Teplizumab)、TGN1412、替西木单抗(Ticilimumab)、曲美木单抗(tremelimumab)、替加珠单抗(Tigatuzumab)、TNX-355(伊巴珠单抗(ibalizumab))、TNX-650、TNX-901(他利珠单抗(talizumab))、托利珠单抗(Toralizumab)、托西莫单抗(Tositumomab)、曲美木单抗(Tremelimumab)、西莫白介素单抗(Tucotuzumabcelmoleukin)、妥韦单抗(Tuvirumab)、乌珠单抗(Urtoxazumab)、优特克单抗(Ustekinumab)、伐利昔单抗(Vapaliximab)、维多珠单抗(Vedolizumab)、维妥珠单抗(Veltuzumab)、维帕莫单抗(Vepalimomab)、维西珠单抗(Visilizumab)、伏洛昔单抗(Volociximab)、伏妥昔单抗(Votumumab)、扎芦木单抗(Zalutumumab)、扎木单抗(Zanolimumab)、齐拉木单抗(Ziralimumab)、阿佐莫单抗(Zolimomabaritox)等。
在本发明的一个实施例中，所述单克隆抗体为阿达木单抗。
本发明中，所述酶及辅酶包括但不限于：免疫酶、微生物酶、氧化还原酶、转移酶、水解酶、裂合酶、异构酶或连接酶等；具体如伊米苷酶、α-半乳糖苷酶A(α-galA)、阿加糖酶β、酸性α-葡糖苷酶(GAA)、重组阿葡糖苷酶α(LUMIZYME)、α-葡糖苷酶、芳基硫酸酯酶、拉罗尼酶、ALDURAZYME、艾杜硫酶、ELAPRASE、加硫酶(NAGLAZYME)、聚乙二醇化重组尿酸酶(KRYSTEXXA)、聚乙二醇化重组假丝酵母尿酸酶、门冬酰胺酶等。
在本发明的一个实施例中，所述酶为α-半乳糖苷酶A(α-galA)、酸性α-葡糖苷酶(GAA)、聚乙二醇化重组尿酸酶(KRYSTEXXA)、聚乙二醇化重组假丝酵母尿酸酶或门冬酰胺酶。
在本发明的另一个实施方式中，所述外源免疫原为人工抗原载体，所述的人工抗原载体是指作为载体与半抗原或其他抗原结合(如偶联)以增强其免疫原性，用于制备成人工抗原的具有免疫原性的分子，其包括如蛋白质、多肽聚合物、大分子聚合物和某些颗粒等。所述蛋白质载体(又称为载体蛋白)，包括但不限于，钥孔血蓝蛋白(KLH)、牛血清蛋白(BSA)、人血清白蛋白(HSA)、卵清蛋白(OVA)等。所述的多肽聚合物载体如多聚赖氨酸(PLL)等。所述的颗粒载体如某些细菌颗粒、病毒等。
在本发明的一个具体实施方式中，所述人工抗原载体为蛋白质载体。
在本发明的一个实施例中，所述的蛋白质载体为钥孔血蓝蛋白(KLH)。
在本发明的另一个实施方式中，所述外源免疫原为移植物；优选地，所述移植物为用于移植的细胞、组织、器官或其他生物适合的移植物，所述移植物可以是自体或异体(如同物种但不同基因型个体)的细胞、组织、器官，也可为人工材料(如人工骨、人工气管等)等。
在本发明的一个具体实施方式中，所述移植物为细胞，优选为干细胞，如造血干细胞、神经干细胞等。
在本发明的一个具体实施方式中，所述移植物为组织，优选自：皮肤、骨及软骨、真皮及脂肪、黏膜、筋膜、角膜、肌肉、神经、人工生物材料等。
在本发明的一个具体实施方式中，所述移植物为器官，优选自：心脏、胰腺、肾脏、 肺和肝脏等。
优选地，本发明所述用途中，所述结合物在施用外源免疫原之前、同时或之后施用。
其他示例性的治疗剂如化学治疗剂，其包括但不限于：抗代谢药物、基于铂的药剂、烷化剂、酪氨酸激酶抑制剂、蒽环类抗生物、长春花生物碱、蛋白酶体抑制剂和拓扑异构酶抑制剂等。上述治疗剂是本领域技术人员已知的，本发明在此方面不受限制。
本发明还提供了一种PEG与雷帕霉素的结合物在制备增强第一药物治疗效果的药物中的应用，所述的第一药物选自细胞因子、人血红蛋白、血液因子或凝血因子、血管内皮生长因子抗体拮抗剂、激素、抗体、酶及辅酶类药物中的至少一种。
优选地，所述的第一药物用于治疗肿瘤、血液疾病、感染性疾病、免疫系统疾病、遗传性疾病、代谢病。
更优选地，所述的肿瘤选自肺癌、肾癌、黑色素瘤、肝癌头颈癌、皮肤癌、鳞状细胞癌、卵巢癌、骨癌、结直肠癌、膀胱癌、胃癌、胰腺癌、前列腺癌、霍奇金淋巴瘤、滤泡性淋巴瘤、慢性或急性白血病、间皮瘤、胰腺癌、乳腺癌、多发性骨髓瘤和其他实体瘤；所述的血液疾病选自血友病；所述的感染性疾病选自HIV病毒感染、肝炎病毒(A型、B型和C型)感染、疱疹病毒感染、流感病毒感染；所述免疫系统疾病选自红斑狼疮、类风湿关节炎、强直性脊柱炎、重症肌无力、多发性硬化症、自身免疫性溶血性贫血、自身免疫性肝炎、硬皮病、结节性多动脉炎、Wegener肉芽肿病；所述的血液疾病选自血友病；所述的遗传性疾病选自法布里病、庞培氏病、溶酶体积存疾病、遗传性肌肉疾病、遗传代谢疾病；所述的代谢病选自氨基酸代谢紊乱(如分支链疾病、高氨基酸血症、高氨基酸尿症)、尿素代谢紊乱、高胺血症、粘多醣病、贮积病(如肝醣贮积病及脂质贮积病)、糖原贮积病I(如柯里氏病)、吸收不良疾病(如肠碳水化合物吸收不良)、寡醣酶缺乏症(如麦芽糖酶不足、乳糖酶不足、蔗糖酶不足)、果糖代谢障碍、半乳糖代谢障碍、半乳糖血症、碳水化合物利用紊乱(如糖尿病)、低血糖症、丙酮酸盐代谢紊乱、低脂血症、低脂蛋白血症、高脂血症、高脂蛋白血症、肉毒碱或肉毒碱酰基转移酶缺乏症、卟啉代谢紊乱、卟啉、嘌呤代谢紊乱、溶酶体病、神经及神经系统代谢疾病(如神经节苷脂沉积症、神经类脂增多症)、硫酸脑苷 脂病、脑白质病、莱施-奈恩综合征。
本发明还提供了一种PEG与雷帕霉素的结合物在制备增强基因疗法效果的药物中的应用。
优选地，所述的基因疗法为非免疫原性的基因疗法，例如是采用病毒载体的非免疫原性的基因疗法。
更优选地，所述的基因疗法用于治疗癌症、血液疾病、感染性疾病、免疫系统疾病、遗传性疾病、代谢病。
进一步优选地，所述的肿瘤选自肺癌、肾癌、黑色素瘤、肝癌头颈癌、皮肤癌、鳞状细胞癌、卵巢癌、骨癌、结直肠癌、膀胱癌、胃癌、胰腺癌、前列腺癌、霍奇金淋巴瘤、滤泡性淋巴瘤、慢性或急性白血病、间皮瘤、胰腺癌、，乳腺癌、多发性骨髓瘤和其他实体瘤；所述的血液疾病选自血友病；所述的感染性疾病选自HIV病毒感染、肝炎病毒(A型、B型和C型)感染、疱疹病毒感染、流感病毒感染；所述免疫系统疾病选自红斑狼疮、类风湿关节炎、强直性脊柱炎、重症肌无力、多发性硬化症、自身免疫性溶血性贫血、自身免疫性肝炎、硬皮病、结节性多动脉炎、Wegener肉芽肿病；所述的血液疾病选自血友病；所述的遗传性疾病选自法布里病、庞培氏病、溶酶体积存疾病、遗传性肌肉疾病、遗传代谢疾病；所述的代谢病选自氨基酸代谢紊乱(如分支链疾病、高氨基酸血症、高氨基酸尿症)、尿素代谢紊乱、高胺血症、粘多醣病、贮积病(如肝醣贮积病及脂质贮积病)、糖原贮积病I(如柯里氏病)、吸收不良疾病(如肠碳水化合物吸收不良)、寡醣酶缺乏症(如麦芽糖酶不足、乳糖酶不足、蔗糖酶不足)、果糖代谢障碍、半乳糖代谢障碍、半乳糖血症、碳水化合物利用紊乱(如糖尿病)、低血糖症、丙酮酸盐代谢紊乱、低脂血症、低脂蛋白血症、高脂血症、高脂蛋白血症、肉毒碱或肉毒碱酰基转移酶缺乏症、卟啉代谢紊乱、卟啉、嘌呤代谢紊乱、溶酶体病、神经及神经系统代谢疾病(如神经节苷脂沉积症、神经类脂增多症)、硫酸脑苷脂病、脑白质病、莱施-奈恩综合征。
本发明提供一种聚乙二醇与雷帕霉素的结合物在用于减少免疫应答中的应用。
本发明还提供了一种PEG与雷帕霉素的结合物在增强第二药物治疗效果中的应用。
本发明还提供了一种PEG与雷帕霉素的结合物在增强基因疗法效果中的应用。
本发明还提供了一种药物组合物，所述的药物组合物中包括第一药物和本发明所述的PEG与雷帕霉素的结合物，所述的第一药物选自细胞因子、人血红蛋白、血液因子或凝血因子、血管内皮生长因子抗体拮抗剂、激素、抗体、酶及辅酶类药物中的至少一种。
本发明所述的药物或药物组合物选自片剂、丸剂、粉剂、锭剂、散剂、酏剂、悬浮液、乳液、溶液、糖浆剂、气雾剂、软膏剂、透皮吸收剂、软胶囊、硬胶囊、栓剂、无菌注射溶液以及冻干粉剂等形式。优选的，本发明所述的药物或药物组合物为无菌注射溶液或冻干粉剂。
本发明所述的药物或药物组合物中还含有药学上可接受的辅料，优选的，所述的辅料选自载体和稀释剂(乳糖、右旋糖、蔗糖、山梨醇、甘露醇、淀粉类、阿拉伯树胶、磷酸钙、藻酸盐、黄蓍胶、明胶、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲基酯和羟基苯甲酸丙基酯、滑石、硬脂酸镁和矿物油中的一种或两种以上)、润滑剂、增溶剂、湿润剂、乳化剂和悬浮剂、防腐剂、甜味剂或矫味剂。
本发明所述的药物或药物组合物的施用方法选自口服、皮下、皮内、肌内、腹膜内、静脉内、鼻内、硬膜外、舌下、鼻内、脑内、叶鞘内、透皮、直肠等。
本发明提供了一种PEG与雷帕霉素的结合物及其用途，特别是在制备用于减少免疫应答(特别是减少接受者对外源免疫原如治疗剂、移植物等引起的过强的免疫应答)的药物中的新用途，其中所述PEG与雷帕霉素的结合物可显著降低针对外源免疫原如治疗剂、移植物的抗体生产率，减少其引起的过强的免疫应答，具有可保证甚至改善治疗剂的治疗效果、改善接受者自身免疫状况、降低甚至消除移植排斥反应等有益效果，且其制备工艺相对简单，成本较低，易实现工业化生产，有非常好的应用价值。
具体实施方式
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义，如：
“烷基”指的是直链或支链的且不含不饱和键的烃链自由基，C1-C6的烷基指含有1-6 个碳原子的烷基，如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、正己基等；如烷基被环烷基取代，其相应为“环烷基烷基”自由基，C4-10的环烷基烷基是指含有4-10个碳原子的环烷基烷基，如环丙基甲基、环丙基乙基、环丁基甲基、环戊基甲基、环己基甲基等；如烷基被芳基取代，那么其相应为“芳烷基”自由基，C7-14的芳烷基是指含7-14个碳原子的芳烷基，如苄基、二苯甲基或苯乙基等。
“烷氧基”指的是羟基中的氢被烷基取代后形成的取代基，C1-C6的烷氧基指含有1-6个碳原子的烷氧基，如甲氧基、乙氧基、丙氧基、丁氧基等。
“环烷基”指的是脂环烃，如含1至4个单环和/或稠环、含3-18个碳原子，优选3-10个碳原子，如环丙基、环己基或金刚烷基等，本发明中所述“C3-6的环烷基”指含有3-6个碳原子的环烷基，如环丙基、环丁基、环戊基和环己基。
“芳基”指的是单环或多环自由基，包括含单芳基基团和/或稠芳基基团的多环自由基，如包含1-3个单环或稠环及6-18个碳环原子，本发明中所述C6-10的芳基是指含有6-10个碳环原子的芳基，如苯基、萘基。
“杂环基”包括含1至3个单环和/或稠环及3至约18个环原子的杂芳香族基团和杂脂环基团。优选的杂芳香族基团和杂脂环基团含5至约10个环原子。本发明的化合物中的合适的杂芳基含1、2或3种杂原子，所述杂原子选自N、O或S原子，所述的含氮杂环是指其杂原子中包括N原子。
“单糖”一般是含有3-6个碳原子的多羟基醛或多羟基酮，按碳原子数目，单糖可分为丙糖、丁糖、戊糖、己糖等。常见的单糖如葡萄糖、果糖、半乳糖、核糖、脱氧核糖等。“低聚糖”是指含有2-10个糖苷键聚合而成的化合物，所述糖苷键是一个单糖的苷羟基和另一单糖的某一羟基脱水缩合形成的，常见的低聚糖如二糖、三糖等。所述“二糖”是指由两个单糖分子通过糖苷键连接在一起形成的化合物，如蔗糖、乳糖、麦芽糖等。所述“三糖”三分子单糖以糖苷键连结形成的化合物，如龙胆三糖、棉子糖等。
“雷帕霉素(rapamycin)”，也称为西罗莫司(sirolimus)，本发明中雷帕霉素衍生物包括但不限于结构和功能类似于雷帕霉素的化合物，如依维莫司(everolimus，RAD 001 (Novartis))、坦西莫司(temsirolimus，又称为替西罗莫司，CC1-779(Wyeth))、比奥莫司A9(biolimus A9)、ABT-578(zotarolimus)、地磷莫司(deforolimus，ridaforolimus，AP23573，MK-8669)等。上述雷帕霉素及其衍生物的化学结构如下所示：
Figure PCTCN2019080194-appb-000050
本发明中所述“治疗剂”是指可向接受者施用并且具有治疗效果的物质，如蛋白质、碳水化合物、脂质和核酸等大分子物质等。在本发明的一个实施方式中，所述治疗剂为治疗性大分子。向接受者施用治疗性大分子时可能会引起过强的免疫应答，如抗治疗性大分子特异性抗体的产生。在本发明的一个具体实施方式中，所述治疗性大分子为治疗性蛋白质。
本发明中所述“过强的免疫应答”是指由由暴露于抗原引起的、促进或加重本文中所述疾病、紊乱或病症(或其症状)，或者其为本文中所述疾病、紊乱或病症(或其症状)。这样的免疫应答一般对对象的健康具有不良影响或者为对对象之健康的不良影响的症状。过强的免疫应答包括抗原特异性抗体产生、抗原特异性B细胞增殖和/或活性或抗原特异性CD4+T细胞增殖和/或活性。一般来说，这些过强的免疫应答是治疗性大分子特异性的并且抵消施用所述治疗性大分子所期望的有益效果。
本发明中所述“抗体”以其最广泛的含义使用并且特别覆盖单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体(例如：双特异性抗体)和抗体片段，只要它们表现出所需的生物活性(Miller等(2003)Jour.of Immunology,170:4854-4861)。抗体可以为鼠、人、人源化、嵌合抗体或来源于其它物种。抗体可具有任何类型(例如IgG、IgE、IgM、IgD及IgA)及类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)。
本发明中所述“接受者”为哺乳动物，包括但不限于灵长目动物(如人、猴、猿等)、偶蹄目动物(如猪、羊、牛等)、奇蹄目动物(如马)、啮齿类动物(如老鼠、水豚等)、食肉目动物(包括犬科(如狼、狗)、猫科(如虎、猫)、熊科(如熊猫)等)，优选为人。
本发明中所述“移植”是指机体的某种细胞、组织或器官因损伤而导致不可复性的结构及功能损害时，将相应健康细胞、组织或器官植入机体的过程称为细胞、组织或器官的移植(transplantation)。常见的有自体移植、同种异体移植及同基因型移植等。
本发明中所述“药学上可接受的辅料”含义是该成分没有生物活性或其他不良活性的杂质，例如该成分可以纳入公开的药物制剂并给与患者，但不引起显著的不良生物效果或以有害的方式和该制剂中包含的其他成分产生相互作用。
本发明中所述“治疗”包括抑制、延迟、缓和、减弱、限制、减轻或消退疾病、障碍、病症或状态，其发生和/或进程，和/或其症状。
本发明中所述“包含”表示"开放"或"包含性"用语，使得它们包括列举的要素，而且还允许包括额外的、未提及的要素。
本发明中所述“约”通常意指所述值的+/-5％，更通常指所述值的+/-4％，更通常指所述值的+/-3％，更通常指所述值的+/-2％，更通常指所述值的+/-1％，更通常指所述值的+/-0.5％。
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例
实施例中所用的雷帕霉素从武汉远成共创科技有限公司购得，单甲氧基聚乙二醇－谷氨酸二肽、甘氨酸雷帕霉素酯由天津键凯科技有限公司提供，其余为市售试剂。实验动物为Balb/c雌鼠，购自北京华阜康生物科技股份有限公司；免疫原为钥孔血蓝蛋白(KLH)，购自美国Sigma公司。
实施例1单甲氧基聚乙二醇(数均分子量20000)－谷氨酸二肽－雷帕霉素结合物(JK1208R)(结构式如下)的制备
Figure PCTCN2019080194-appb-000051
将单甲氧基聚乙二醇－谷氨酸二肽(20KDa，5g，0.25mmol)、甘氨酸雷帕霉素酯486mg(0.5mmol)、HOBt(34mg，0.25mmol)和DMAP(61mg，0.5mmol)加到反应瓶中，用二氯甲烷溶解，冰浴冷却，再滴入DCC(155mg，0.75mmol)溶于二氯甲烷的溶液，滴完后自然升到室温，反应过夜。次日将反应液浓缩，残分用异丙醇结晶，得单甲氧基聚乙二醇(20KDa)－谷氨酸二肽－雷帕霉素结合物(JK1208R)4.7g(n约为450)。
实施例2评价针对钥孔血蓝蛋白(KLH)抗体水平的影响
2.1实验动物
种属：小鼠
品系：Balb/c
等级：SPF
繁育单位：北京华阜康生物科技股份有限公司
购入时周龄：5周
购入时体重范围：14-16g
2.2实验动物饲养条件
动物到达后在实验环境饲养3天后方开始实验。动物在SPF级动物房以IVC(独立送风系统)笼具饲养(每笼4只)。每笼动物信息卡注明笼内动物数目，性别，品系，接收 日期，给药方案，实验编号，组别以及实验开始日期。所有笼具、垫料、饲料及饮水在使用前均灭菌。笼具、饲料及饮水每周更换两次。饲养环境及光照情况如下：
温度：20-26℃
湿度：40-70％
光照周期：12小时光照，12小时无光照(上午8点开灯～下午8点关灯)
2.3仪器与耗材
2.3.1主要试剂和耗材
所用试剂耗材 生产厂家
弗氏完全佐剂 Sigma公司Cat.No F5881
弗氏不完全佐剂 Sigma公司Cat.No F5506
2.3.2主要仪器
所用仪器 生产厂家
DEM-3型自动洗板机 北京拓普分析仪器有限责任公司
酶标仪 BIO-RAD 680
2.4实验方法及步骤
2.4.1.抗原信息：免疫原：KLH；检测原：KLH
2.4.2试验过程：
G1组(模型组)：
动物免疫：
初免：第1天，100ug抗原(免疫原)/只×18只老鼠+同体积弗式完全佐剂，用注射器相互注射的方式形成油包水化合物，皮下多点注射；
二免：第14天，100ug抗原/只×18只老鼠+同体积弗式不完全佐剂，用注射器相互注射的方式形成油包水化合物，皮下多点注射；
三免：第24天，100ug抗原/只×18只老鼠+同体积弗式不完全佐剂，用注射器相互注射的方式形成油包水化合物，皮下多点注射；
尾血检测：第28-29天，尾静脉采集约20ul尾血，RT30min，5000转离心5min，弃血细胞沉淀，吸取血清，用于Elisa检测效价；
四免：第34天，100ug抗原/只×18只老鼠+同体积弗式不完全佐剂，用注射器相互注射的方式形成油包水化合物，皮下多点注射；
G2组(IV组)：同G1组进行对照免疫，同时，在第0天和第7天分别静脉注射JK1208R(实施例1制备)620mg。
G3组(PO组)：同G1组进行对照免疫，同时在第0天和第7天分别口服雷帕霉素52μg。
尾血检测：第40-41天，尾静脉采集约20ul尾血，RT30min，5000转离心5min，弃血细胞沉淀，吸取血清，用于ELISA检测效价。
2.4.3效价检测：(酶联免疫吸附法(ELISA))
包被：10ug抗原(检测原)+10ml包被液(1.6gNaCO 3+2.92gNaHCO 3+100ml去离子水)，混匀，100ul/孔铺Elisa板，37℃孵育2h或者4℃过夜；
封闭：包被好的板子吸弃抗原包被液，直接加入封闭液(5％脱脂奶粉+包被液)150ul/孔，37℃下处理2h后洗板3次，去离子水洗即可，4℃暂存；
检测：血清1:1000 1×PBS，混匀，吸150ul稀释液加入第一个孔，第2-8孔均加入100ul1×PBS，从第一个孔吸50ul液体加入第二孔，吸吹10次后吸取50ul到第三孔，吸吹10次后吸50ul到第四孔，依次吸吹到第7孔后吸取50ul丢弃，第八孔作为阴性对照；37℃孵育1h后去离子水洗3次，加二抗(羊抗鼠IgG-HRP 1:10000 1×PBS)100ul/孔，37℃孵育30min后去离子水洗3次，加入显色液(依照试剂盒说明书使用)，100ul/孔，37℃10min后终止(2M硫酸30ul/孔)，酶标仪检测OD值。
2.5实验结果：
G1、G2和G3三组动物四免效价数据统计结果如表1和附图1所示。
表1 PO组和IV组对KLH抗体产生的影响
Figure PCTCN2019080194-appb-000052
Figure PCTCN2019080194-appb-000053
注：与模型组比较*P<0.05，**P<0.01，***P<0.001
由结果可知，三免后PO组和IV组与免疫模型组相比较，在1:3K时数据经过统计分析，IV组和免疫模型组有显著性差异，而PO组数据无显著差异。四免后PO组和IV组与免疫模型组相比较，在1:1K和1:3K时数据经过统计分析，IV组和免疫模型组均有显著性差异，而PO组数据无显著差异。说明IV组四免后能显著降低KLH抗体的产生。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。

Claims (18)

  1. 一种聚乙二醇与雷帕霉素的结合物在制备用于减少免疫应答的药物中的用途。
  2. 如权利要求1所述的用途，其特征在于，所述聚乙二醇与雷帕霉素的结合物具有通式Ⅰ所示结构：
    PEG-X-D
    (Ⅰ)
    其中，
    PEG为聚乙二醇残基，
    D为雷帕霉素或其衍生物残基，
    X为PEG和D之间的连接基团。
  3. 如权利要求2所述的用途，其特征在于，所述结合物中，所述X具有如下结构：-X 1-X 2-X 3-，其中，X 1和X 3独立地选自：-(CR 1R 2) a-、-(CH 2) aNH-、-(CH 2) aNHCO-、-(CH 2) aCONH-、-(CH 2) aCO-、-(CH 2) aCOO-和-(CH 2) aO-中的一种或多种的组合，X 2选自：环烷基、芳基、杂环基、-A-和-Op-，R 1和R 2独立地选自：-H、C1-6的烷基、-OR'、-NHR'、-N(R') 2、-CN、-F、-Cl、-Br、-I、-COR'、-COOR'、-OCOR'、-CONHR'和-CON(R') 2中的一种或两种以上的组合，R'选自：-H、C1-6的烷基、-F、-Cl、-Br和-I，
    a为0-10的整数，
    -A-为氨基酸残基，
    -Op-为寡肽残基，具有如下结构：
    Figure PCTCN2019080194-appb-100001
    其中，m为1、2、3或4，
    A 1选自：天冬氨酸、谷氨酸和赖氨酸的残基，
    A 2和A 3为相同或不同的氨基酸残基。
  4. 如权利要求3所述的用途，其特征在于，所述结合物具有如下结构：
    Figure PCTCN2019080194-appb-100002
  5. 如权利要求1-4任一项所述的用途，其特征在于，所述的免疫应答是由外源免疫原引起的过强的免疫应答，所述的外源免疫原选自：治疗剂、移植物、人工抗原载体和毒素。
  6. 如权利要求5所述的用途，其特征在于，所述治疗剂为治疗性蛋白质，选自：细胞因子、人血红蛋白、血液因子或凝血因子、血管内皮生长因子抗体拮抗剂、激素、抗体、酶及辅酶中的一种或多种。
  7. 如权利要求6所述的用途，其特征在于，所述血液因子或凝血因子选自：因子I、因子II、组织因子、因子V、因子VII、因子VIII、因子IX、因子X、因子Xa、因子XII、因子XIII、重组凝血因子、冯·维勒布兰德因子、前激肽释放酶、高分子量激肽原、纤连蛋白、抗凝血酶III、肝素辅因子II、蛋白C、蛋白S、蛋白Z、蛋白Z相关蛋白酶抑制剂(ZPI)、纤溶酶原、α2-抗纤溶酶、组织纤溶酶原激活物(tPA)、尿激酶、纤溶酶原激活物抑制剂-1(PAI1)、纤溶酶原激活物抑制剂-2(PAI2)、癌促凝物质和阿法依伯汀中的一种或多种。
  8. 如权利要求6所述的用途，其特征在于，所述抗体包括：单克隆抗体、多克隆抗体、二聚体、多聚体、双特异性抗体、多特异性抗体和抗体片段。
  9. 如权利要求6所述的用途，其特征在于，所述酶及辅酶选自：伊米苷酶、α-半乳糖苷酶A、阿加糖酶β、酸性α-葡糖苷酶、重组阿葡糖苷酶α、α-葡糖苷酶、芳基硫酸酯酶、拉罗尼酶、ALDURAZYME、艾杜硫酶、ELAPRASE、加硫酶、聚乙二醇化重组尿酸酶、聚乙二醇化重组假丝酵母尿酸酶和门冬酰胺酶中的一种或多种。
  10. 如权利要求5所述的用途，其特征在于，所述人工抗原载体为蛋白质载体，选自： 钥孔血蓝蛋白、牛血清蛋白、人血清白蛋白和卵清蛋白。
  11. 如权利要求10所述的用途，其特征在于，所述人工抗原载体为钥孔血蓝蛋白。
  12. 一种PEG与雷帕霉素的结合物在制备增强第一药物治疗效果的药物中的用途，所述的第一药物选自细胞因子、人血红蛋白、血液因子或凝血因子、血管内皮生长因子抗体拮抗剂、激素、抗体、酶及辅酶类药物中的至少一种。
  13. 如权利要求12所述的用途，其特征在于，所述PEG与雷帕霉素的结合物具有通式Ⅰ所示结构：
    PEG-X-D
    (Ⅰ)
    其中，
    PEG为聚乙二醇残基，
    D为雷帕霉素或其衍生物残基，
    X为PEG和D之间的连接基团。
  14. 如权利要求12-13任一项所述的用途，其特征在于，所述血液因子或凝血因子选自：因子I、因子II、组织因子、因子V、因子VII、因子VIII、因子IX、因子X、因子Xa、因子XII、因子XIII、重组凝血因子、冯·维勒布兰德因子、前激肽释放酶、高分子量激肽原、纤连蛋白、抗凝血酶III、肝素辅因子II、蛋白C、蛋白S、蛋白Z、蛋白Z相关蛋白酶抑制剂(ZPI)、纤溶酶原、α2-抗纤溶酶、组织纤溶酶原激活物(tPA)、尿激酶、纤溶酶原激活物抑制剂-1(PAI1)、纤溶酶原激活物抑制剂-2(PAI2)、癌促凝物质和阿法依伯汀中的一种或多种；优选为因子VIII。
  15. 如权利要求12-13任一项所述的用途，其特征在于，所述抗体包括：单克隆抗体、多克隆抗体、二聚体、多聚体、双特异性抗体、多特异性抗体和抗体片段；优选地，所述单克隆抗体为阿达木单抗。
  16. 如权利要求12-13任一项所述的用途，其特征在于，所述酶及辅酶选自：伊米苷酶、α-半乳糖苷酶A、阿加糖酶β、酸性α-葡糖苷酶、重组阿葡糖苷酶α、α-葡糖苷酶、芳 基硫酸酯酶、拉罗尼酶、ALDURAZYME、艾杜硫酶、ELAPRASE、加硫酶、聚乙二醇化重组尿酸酶、聚乙二醇化重组假丝酵母尿酸酶和门冬酰胺酶中的一种或多种；优选为α-半乳糖苷酶A、酸性α-葡糖苷酶、聚乙二醇化重组尿酸酶、聚乙二醇化重组假丝酵母尿酸酶或门冬酰胺酶。
  17. 如权利要求12-16任一项所述的用途，其特征在于，所述的第一药物用于治疗肿瘤、血液疾病、感染性疾病、免疫系统疾病、遗传性疾病或代谢病。
  18. 如权利要求17所述的用途，其特征在于，所述的肿瘤选自：肺癌、肾癌、黑色素瘤、肝癌头颈癌、皮肤癌、鳞状细胞癌、卵巢癌、骨癌、结直肠癌、膀胱癌、胃癌、胰腺癌、前列腺癌、霍奇金淋巴瘤、滤泡性淋巴瘤、慢性或急性白血病、间皮瘤、胰腺癌、乳腺癌、多发性骨髓瘤和其他实体瘤；
    所述的血液疾病选自血友病；
    所述的感染性疾病选自：HIV病毒感染、肝炎病毒感染、疱疹病毒感染和流感病毒感染；
    所述免疫系统疾病选自：红斑狼疮、类风湿关节炎、强直性脊柱炎、重症肌无力、多发性硬化症、自身免疫性溶血性贫血、自身免疫性肝炎、硬皮病、结节性多动脉炎和Wegener肉芽肿病；
    所述的遗传性疾病选自：法布里病、庞培氏病、溶酶体积存疾病、遗传性肌肉疾病和遗传代谢疾病；
    所述的代谢病选自：氨基酸代谢紊乱、糖原贮积病I、吸收不良疾病、寡醣酶缺乏症、果糖代谢障碍、半乳糖代谢障碍、半乳糖血症、碳水化合物利用紊乱、低血糖症、丙酮酸盐代谢紊乱、低脂血症、低脂蛋白血症、高脂血症、高脂蛋白血症、肉毒碱或肉毒碱酰基转移酶缺乏症、卟啉代谢紊乱、卟啉、嘌呤代谢紊乱、溶酶体病、神经及神经系统代谢疾病、硫酸脑苷脂病、脑白质病和莱施-奈恩综合征。
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