WO2023282296A1 - ｐＨ応答性脂質誘導体 - Google Patents

ｐＨ応答性脂質誘導体 Download PDF

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WO2023282296A1
WO2023282296A1 PCT/JP2022/026863 JP2022026863W WO2023282296A1 WO 2023282296 A1 WO2023282296 A1 WO 2023282296A1 JP 2022026863 W JP2022026863 W JP 2022026863W WO 2023282296 A1 WO2023282296 A1 WO 2023282296A1
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French (fr)
Japanese (ja)
Inventor
公一 尾木
准也 道西
知之 大嶽
伸宏 西山
宏泰 武元
裕 三浦
貴大 野本
誠 松井
イーロン ソン
真広 豊田
アリア ガセミザデ
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NOF Corp
Tokyo Institute of Technology NUC
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NOF Corp
Tokyo Institute of Technology NUC
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Priority to JP2023533170A priority Critical patent/JPWO2023282296A1/ja
Priority to US18/577,158 priority patent/US20240374733A1/en
Priority to EP22837710.7A priority patent/EP4368657A4/en
Publication of WO2023282296A1 publication Critical patent/WO2023282296A1/ja
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    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12Y207/11021—Polo kinase (2.7.11.21)

Definitions

  • the present invention relates to a lipid derivative consisting of a pH-responsive polymer and a lipid. Further, the present invention relates to a transport carrier for drug delivery having a high tumor tissue accumulation property by capturing minute changes in pH around the tumor tissue and changing its physical properties.
  • Nucleic acid drugs can target with high specificity the causes of diseases that could not be targeted by conventional small molecule or antibody drugs, and are expected to be used in drug discovery in many disease areas.
  • a drug delivery system has been studied for the purpose of delivering nucleic acid drugs to target cells more efficiently.
  • studies are being conducted to reduce side effects by more efficiently delivering drugs only to target cells.
  • lipid nanoparticles (Lipid Nano Particles: LNP) are generally used, whose basic constituents are phospholipids or derivatives thereof, lipids other than sterols and phospholipids, PEG lipids, and the like.
  • LNP Lipid Nano Particles
  • PEG lipids play an important role in suppressing aggregation between carriers and improving blood retention in the body.
  • This PEG lipid improves blood retention by covering the carrier surface with a hydrated layer of PEG, which suppresses opsonization such as serum protein adsorption, resulting in phagocytosis by macrophages and uptake by reticuloendothelial tissue. can be avoided (hereinafter referred to as stealth).
  • stealth opsonization such as serum protein adsorption, resulting in phagocytosis by macrophages and uptake by reticuloendothelial tissue.
  • Patent Document 2 discloses the use of liposomes for intracellular nucleic acid delivery using a temperature-responsive polymer. It has been reported that the temperature change of the liposome changes from hydrophilic to hydrophobic, thereby increasing the affinity with the cell membrane and improving the accumulation and uptake efficiency. However, since temperature changes are required, there are cases where incidental equipment is required, and since the temperature-responsive polymer is a vinyl polymer, it cannot be expected to decompose in vivo, making it safe as a drug. concerns also remain. Furthermore, in the disclosed technology, carriers are limited to liposomes only, and drugs are also limited to nucleic acid drugs.
  • Non-Patent Document 1 discloses a liposome containing a phospholipid derivative in which a succinic acid skeleton that decomposes under acidic conditions is arranged between a PEG residue and a phospholipid residue. It has been reported that the liposome changes from hydrophilic to hydrophobic under acidic conditions of pH 5-6, thereby increasing affinity with cell membranes and improving accumulation and uptake efficiency. However, it is known that the pH around the tumor tissue is weakly acidic (about pH 6.5), and there is a possibility that the desired effect cannot be obtained around the tumor tissue. Furthermore, in the case of pH-responsive liposomes, there may be problems in terms of storage stability, such as decomposition during drug preparation and storage.
  • the transport carrier for drug delivery to tumor tissue can change its physical properties by capturing minute changes in pH around the tumor tissue, can contain low-molecular-weight drugs and nucleic acid drugs, and can be degraded in vivo. technology was desired.
  • a pH-responsive transport carrier effective as a transport carrier capable of achieving an introduction system capable of safely and efficiently delivering low-molecular-weight drugs and the like to tumor tissue and transfecting nucleic acid drugs and the like into tumor cells;
  • a further object is to provide a pH-responsive lipid derivative useful for forming such a transport carrier.
  • pH-responsive lipid derivative [1] structure represented by the following formula (i):
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms, an acyl group having 8 to 24 carbon atoms, or a sterol residue;
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms,
  • B is a hydrogen atom, an alkali metal, or an ammonium group;
  • X is a linking group comprising a linking group formed by a nucleophilic substitution reaction, or a Michael addition reaction, or a thiol-ene reaction, or a cycloaddition reaction;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, d, and e are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g and n are each independently 1 or 2
  • m is
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms, an acyl group having 8 to 24 carbon atoms, or a sterol residue;
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms,
  • B is a hydrogen atom, an alkali metal, or an ammonium group;
  • X is a linking group comprising a linking group formed by a nucleophilic substitution reaction, or a Michael addition reaction, or a thiol-ene reaction, or a cycloaddition reaction;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, d, and e are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g and n are each independently 1 or 2
  • m is
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms, an acyl group having 8 to 24 carbon atoms, or a sterol residue;
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms,
  • B is a hydrogen atom, an alkali metal, or an ammonium group;
  • X is a linking group comprising a linking group formed by a nucleophilic substitution reaction, or a Michael addition reaction, or a thiol-ene reaction, or a cycloaddition reaction;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, d, and e are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g and n are each independently 1 or 2
  • m is
  • X 1 and X 2 are each independently a group containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms, and Z is a Michael addition reaction, a thiol-ene reaction, or a ring is a group formed by addition reaction, p1, p2 and p3 are each independently 0 or 1, and q1 and q2 are each independently from 0 to 50.
  • m is from 5 to 150; The pH-responsive lipid derivative according to any one of [1] to [4] above.
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms
  • M is an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms
  • A is a carboxy group
  • a1, a2, b, and n are each 1
  • c, d, e, and f are each 0, g and h are each 2
  • the pH-responsive lipid derivative according to any one of [1] to [3] and [5] to [7] above.
  • R 1 is a sterol residue and a1, b, c, d are each 0, The pH-responsive lipid derivative according to any one of [1] to [8] above.
  • R 1 and R 2 are each independently an acyl group having 8 to 24 carbon atoms
  • M is an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms
  • B is a hydrogen atom, an alkali metal, or an ammonium group
  • a1, a2, b, c, d, e, and n are each 1
  • f is an integer from 2 to 5
  • X is the above formula (2)
  • X 1 and X 2 are groups containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms
  • p1, p2, and p3 are each 1, The pH-responsive lipid derivative according to any one of [1] to [7] and [9] above.
  • R 1 and R 2 are each independently an acyl group having 8 to 24 carbon atoms
  • M is an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms
  • B is a hydrogen atom, an alkali metal, or an ammonium group
  • a1, a2, b, c, d, e, and n are each 1
  • f is an integer from 2 to 5
  • X is the above formula (2)
  • X 1 and X 2 are groups containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms
  • Z is a group formed by a cycloaddition reaction
  • p1, p2, and p3 are each 1
  • q2 is from 0 to 5
  • the pH-responsive lipid derivative according to any one of [1] to [7] and [9] to [10] above.
  • R 1 and R 2 are each independently an acyl group having 8 to 24 carbon atoms
  • M is an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms
  • B is a hydrogen atom, an alkali metal, or an ammonium group
  • a1, a2, b, c, d, e, and n are each 1
  • f is an integer from 2 to 5
  • X is the above formula (2)
  • X 1 and X 2 are groups containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms
  • Z is a group formed by a cycloaddition reaction
  • p1, p2, and p3 are each 1
  • q is from 0 to 5
  • the pH-responsive lipid derivative according to any one of [1] to [7] and [9] to [11] above.
  • R 1 and R 2 are each independently an acyl group having 8 to 24 carbon atoms
  • M is an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms
  • B is a hydrogen atom, an alkali metal, or an ammonium group
  • A is a carboxy group, a1, a2, b, c, d, e, and n are each 1
  • f is an integer from 2 to 5
  • g and h are each 2
  • X is the above formula (2)
  • X 1 and X 2 are groups containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms
  • Z is a group formed by a cycloaddition reaction
  • p1, p2, and p3 are each 1
  • q1 and q2 are each independently 0 to 5
  • the pH-responsive lipid derivative according to any one of [1] to [7] and [9] to [12] above.
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms or an acyl group having 8 to 24 carbon atoms, b, e, and n are each 1; d is 0; f is an integer from 2 to 5, and X is the above formula (2), X 1 and X 2 are groups containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms, p1, p2 and p3 are each 1;
  • the pH-responsive lipid derivative according to any one of [1] to [7] and [9] to [13] above.
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms or an acyl group having 8 to 24 carbon atoms, A is a carboxy group, b, e, and n are each 1; d is 0; f is an integer from 2 to 5; g and h are each 2, and X is the above formula (2), X 1 and X 2 are groups containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms, Z is a group formed by a cycloaddition reaction, p1, p2, and p3 are each 1; q1 and q2 are each independently 0 to 5;
  • the pH-responsive lipid derivative according to any one of [1] to [7] and [9] to [14] above.
  • R 1a and R 2a are each independently an alkyl group having 8 to 24 carbon atoms or a sterol residue;
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms,
  • B is a hydrogen atom, an alkali metal, or an ammonium group;
  • X is a linking group comprising a linking group formed by a nucleophilic substitution reaction, or a Michael addition reaction, or a thiol-ene reaction, or a cycloaddition reaction;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, d, and e are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g and n are each independently 1 or 2
  • m is from 5 to 300;
  • R a is H,
  • P 1 represents a carboxy-protecting group, and g is 1 or 2.
  • Step (A1) Represented by the following formula (5) by ring-opening polymerization using a polymerization initiator represented by the following formula (3) and an ⁇ -amino acid-N-carboxylic acid anhydride represented by the following formula (4) obtain a polymer that Further, —COCH 3 or —CO—(CH 2 ) b1 —COOH (wherein b1 is 2 or 3) is introduced into the terminal amino group of the obtained polymer to obtain the following formula (5-1). Obtaining the indicated polymer.
  • P 1 represents a carboxy-protecting group, and g is 1 or 2.
  • Step (B) The polymer represented by the following formula (5-i) obtained in the step (A) or the step (A1) (the polymer represented by the formula (5) and the polymer represented by the formula (5-1) a step of obtaining a polymer represented by the following formula (7-i) by reacting a diethylenetriamine derivative represented by the following formula (6) with a diethylenetriamine derivative represented by the following formula (6).
  • P2 represents a protected carboxy group or a protected sulfo group, and h is as defined above.
  • Step (C) A step of obtaining the pH-responsive lipid derivative represented by the formula (47-i) by deprotecting the polymer represented by the formula (7-i) obtained in the step (B). [17] A structure represented by the following formula (36-i), characterized by performing the following step (D):
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms, an acyl group having 8 to 24 carbon atoms, or a sterol residue;
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms,
  • B is a hydrogen atom, an alkali metal, or an ammonium group;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, and d are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g is 1 or 2;
  • m is from 5 to 300;
  • X 1 and X 2 are each independently a group having 1 to 5 carbon atoms and containing a bonding group through a nucleophilic substitution reaction,
  • Z is a group formed by a Michael addition reaction, or
  • Y1 is an atomic group containing a functional group capable of forming a covalent bond with the functional group included in Y2 below, and other symbols are as described above.
  • Y 2 is an atomic group containing a functional group capable of forming a covalent bond with the functional group contained in Y 1 , and other symbols are as described above.
  • R 1a and R 2a are each independently an alkyl group having 8 to 24 carbon atoms or a sterol residue;
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms,
  • B is a hydrogen atom, an alkali metal, or an ammonium group;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, and d are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g is 1 or 2;
  • m is from 5 to 300;
  • X 1 and X 2 are each independently a group having 1 to 5 carbon atoms and containing a bonding group through a nucleophilic substitution reaction,
  • Z is a group formed by a Michael addition reaction, or a thiol-ene reaction, or a
  • Y1 is an atomic group containing a functional group capable of forming a covalent bond with the functional group included in Y2 below, and other symbols are as described above.
  • P 1 represents a carboxy-protecting group
  • Y 2 is an atomic group containing a functional group capable of forming a covalent bond with the functional group contained in Y 1 , and other symbols are as described above.
  • Step (F) Represented by the following formula (13-i) by reacting the polymer represented by the formula (12-i) obtained in the step (E) with the diethylenetriamine derivative represented by the formula (6) to obtain a polymer that
  • Step (G) A step of deprotecting the polymer represented by the formula (13-i) obtained in the step (F) to obtain the pH-responsive lipid derivative represented by the formula (46-i).
  • R 1a and R 2a are each independently an alkyl group having 8 to 24 carbon atoms or a sterol residue;
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms,
  • B is a hydrogen atom, an alkali metal, or an ammonium group;
  • X is a linking group comprising a linking group formed by a nucleophilic substitution reaction, or a Michael addition reaction, or a thiol-ene reaction, or a cycloaddition reaction;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, d, and e are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g and n are each independently 1 or 2
  • m is from 5 to 300;
  • P 1 represents a carboxy-protecting group, and g is 1 or 2.
  • Step (B1) A polymer represented by the following formula (7) is obtained by reacting the polymer represented by the formula (5) obtained in the step (A2) with a diethylenetriamine derivative represented by the following formula (6). process.
  • P2 represents a protected carboxy group or a protected sulfo group, and h is as defined above.
  • Step (C1) A step of obtaining the pH-responsive lipid derivative represented by the formula (47) by deprotecting the polymer represented by the formula (7) obtained in the step (B1).
  • Step (D1) A structure represented by the following formula (36), characterized by performing the following step (D1):
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms, an acyl group having 8 to 24 carbon atoms, or a sterol residue;
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms,
  • B is a hydrogen atom, an alkali metal, or an ammonium group;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, and d are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g is 1 or 2;
  • m is from 5 to 300;
  • X 1 and X 2 are each independently a group having 1 to 5 carbon atoms and containing a bonding group through a nucleophilic substitution reaction,
  • Z is a group formed by a Michael addition reaction, or
  • Y1 is an atomic group containing a functional group capable of forming a covalent bond with the functional group included in Y2 below, and other symbols are as described above.
  • Y2 is an atomic group containing a functional group capable of forming a covalent bond with the functional group contained in Y1, and other symbols are as described above.
  • R 1a and R 2a are each independently an alkyl group having 8 to 24 carbon atoms or a sterol residue;
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms,
  • B is a hydrogen atom, an alkali metal, or an ammonium group;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, and d are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g is 1 or 2;
  • m is from 5 to 300;
  • X 1 and X 2 are each independently a group having 1 to 5 carbon atoms and containing a bonding group through a nucleophilic substitution reaction,
  • Z is a group formed by a Michael addition reaction, or a thiol-ene reaction, or a
  • Y1 is an atomic group containing a functional group capable of forming a covalent bond with the functional group included in Y2 below, and other symbols are as described above.
  • P 1 represents a carboxyl-protecting group
  • Y 2 is an atomic group containing a functional group capable of forming a covalent bond with the functional group contained in Y 1 , and other symbols are as described above. as follows.
  • Step (F1) A polymer represented by the following formula (13) is obtained by reacting the polymer represented by the formula (12) obtained in the step (E1) with the diethylenetriamine derivative represented by the formula (6). process.
  • Step (G1) A step of obtaining a pH-responsive lipid derivative represented by the formula (46) by deprotecting the polymer represented by the formula (13) obtained in the step (F1).
  • R 1a and R 2a are each independently an alkyl group having 8 to 24 carbon atoms or a sterol residue;
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms,
  • B is a hydrogen atom, an alkali metal, or an ammonium group;
  • X is a linking group comprising a linking group formed by a nucleophilic substitution reaction, or a Michael addition reaction, or a thiol-ene reaction, or a cycloaddition reaction;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, d, and e are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g and n are each independently 1 or 2
  • m is from 5 to 300;
  • R b is —
  • P 1 represents a carboxy-protecting group, and g is 1 or 2.
  • Step (B2) Represented by the following formula (7-1) by reacting the polymer represented by the formula (5-1) obtained in the step (A3) with a diethylenetriamine derivative represented by the following formula (6). to obtain a polymer that
  • P2 represents a protected carboxy group or a protected sulfo group, and h is as defined above.
  • Step (C2) A step of obtaining the pH-responsive lipid derivative represented by the formula (47-1) by deprotecting the polymer represented by the formula (7-1) obtained in the step (B2).
  • Step (D2) A structure represented by the following formula (36-1), characterized by performing the following step (D2):
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms, an acyl group having 8 to 24 carbon atoms, or a sterol residue;
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms,
  • B is a hydrogen atom, an alkali metal, or an ammonium group;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, and d are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g is 1 or 2;
  • m is from 5 to 300;
  • R b is COCH 3 , or —CO—(CH 2 ) b1 —COOH, where b1 is 2 or 3;
  • X 1 and X 2 are each independently a group having 1 to 5 carbon
  • Y1 is an atomic group containing a functional group capable of forming a covalent bond with the functional group included in Y2 below, and other symbols are as described above.
  • Y 2 is an atomic group containing a functional group capable of forming a covalent bond with the functional group contained in Y 1 above, and other symbols are as described above.
  • R 1a and R 2a are each independently an alkyl group having 8 to 24 carbon atoms or a sterol residue;
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms,
  • B is a hydrogen atom, an alkali metal, or an ammonium group;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, and d are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g is 1 or 2;
  • m is from 5 to 300;
  • R b is —COCH 3 or —CO—(CH 2 ) b1 —COOH, where b1 is 2 or 3;
  • X 1 and X 2 are each independently a group having 1 to 5 carbon atoms and containing a bonding group through
  • Y1 is an atomic group containing a functional group capable of forming a covalent bond with the functional group included in Y2 below, and other symbols are as described above.
  • P 1 represents a carboxy-protecting group
  • Y 2 is an atomic group containing a functional group capable of forming a covalent bond with the functional group contained in Y 1 , and other symbols are as described above.
  • Step (F2) Represented by the following formula (13-1) by reacting the polymer represented by the formula (12-1) obtained in the step (E2) with the diethylenetriamine derivative represented by the formula (6). to obtain a polymer that
  • Step (G2) A step of deprotecting the polymer represented by the formula (13-1) obtained in the step (F2) to obtain the pH-responsive lipid derivative represented by the formula (46-1).
  • a transport carrier for drug delivery containing the pH-responsive lipid derivative (i) [25] A transport for drug delivery containing 0.1 to 30 mol% of the pH-responsive lipid derivative according to any one of [1] to [15] above. carrier. [26] The transport carrier for drug delivery according to [25] above, which is a solid lipid nanoparticle. [27] The transport carrier for drug delivery according to [25] above, characterized in that the zeta potential difference (Q) represented by the following formula (F1) is 2.5 mV or more.
  • Formula (F1): Q (zeta potential at pH 6.5) - (zeta potential at pH 7.4) [28] The drug according to [25] above, wherein the zeta potential difference (Q) represented by formula (F1) is 2.5 mV or more and the zeta potential at pH 6.5 is greater than 0. Vehicle for delivery. [29] The transport carrier for drug delivery according to [25] above, which has a particle size of 10 to 200 nm. [30] The drug delivery carrier according to [25] above, which further contains a phospholipid. [31] The drug delivery carrier according to [25] above, which further contains a cationic lipid.
  • cationic lipid is DOTAP (1,2-dioleoyloxy-3-trimethylammonium propane) or DODAP (dioleoyl-3-dimethylammonium propane).
  • transport carrier [33] The drug delivery carrier according to [25] above, which further contains a sterol.
  • pH-responsive lipid derivative of the present invention include, for example, the following.
  • [1A] A structure represented by the following formula (1):
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms, an acyl group having 8 to 24 carbon atoms, or a sterol residue;
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms,
  • B is a hydrogen atom, an alkali metal, or an ammonium group;
  • X is a linking group comprising a linking group formed by a nucleophilic substitution reaction, or a Michael addition reaction, or a thiol-ene reaction, or a cycloaddition reaction;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, d, and e are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g and n are each independently 1 or 2
  • m is
  • X 1 and X 2 are each independently a group containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms, and Z is a Michael addition reaction, a thiol-ene reaction, or a ring is a group formed by addition reaction, p1, p2 and p3 are each independently 0 or 1, and q1 and q2 are each independently from 0 to 50.
  • m is 5 to 150
  • g and h are each 2, and A is a carboxy group.
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms, b and n are each 1; d is 0; The pH-responsive lipid derivative according to any one of [1A] to [4A] above.
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms
  • M is an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms
  • A is a carboxy group, a1, a2, b, and n are each 1
  • c, d, e, and f are each 0, g and h are each 2
  • R1 is a sterol residue, and a1, b, c, and d are each 0, The pH-responsive lipid derivative according to any one of [1A] to [4A] above.
  • R 1 and R 2 are each independently an acyl group having 8 to 24 carbon atoms
  • M is an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms
  • B is a hydrogen atom, an alkali metal, or an ammonium group
  • a1, a2, b, c, d, e, and n are each 1
  • f is an integer from 2 to 5
  • X is the above formula (2)
  • X 1 and X 2 are groups containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms
  • p1, p2, and p3 are each 1, The pH-responsive lipid derivative according to any one of [1A] to [4A] above.
  • R 1 and R 2 are each independently an acyl group having 8 to 24 carbon atoms
  • M is an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms
  • B is a hydrogen atom, an alkali metal, or an ammonium group
  • a1, a2, b, c, d, e, and n are each 1
  • f is an integer from 2 to 5
  • X is the above formula (2)
  • X 1 and X 2 are groups containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms
  • Z is a group formed by a cycloaddition reaction
  • p1, p2, and p3 are each 1
  • q2 is from 0 to 5
  • the pH-responsive lipid derivative according to any one of [1A] to [4A] or [8A] above.
  • R 1 and R 2 are each independently an acyl group having 8 to 24 carbon atoms
  • M is an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms
  • B is a hydrogen atom, an alkali metal, or an ammonium group
  • a1, a2, b, c, d, e, and n are each 1
  • f is an integer from 2 to 5
  • X is the above formula (2)
  • X 1 and X 2 are groups containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms
  • Z is a group formed by a cycloaddition reaction
  • p1, p2, and p3 are each 1
  • q is from 0 to 5
  • the pH-responsive lipid derivative according to any one of [1A] to [4A], [8A] or [9A] above.
  • R 1 and R 2 are each independently an acyl group having 8 to 24 carbon atoms
  • M is an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms
  • B is a hydrogen atom, an alkali metal, or an ammonium group
  • A is a carboxy group, a1, a2, b, c, d, e, and n are each 1
  • f is an integer from 2 to 5
  • g and h are each 2
  • X is the above formula (2)
  • X 1 and X 2 are groups containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms
  • Z is a group formed by a cycloaddition reaction
  • p1, p2, and p3 are each 1
  • q1 and q2 are each independently 0 to 5
  • the pH-responsive lipid derivative according to any one of [1A] to [4A] or [8A] to [10A] above.
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms or an acyl group having 8 to 24 carbon atoms, b, e, and n are each 1; d is 0; f is an integer from 2 to 5, and X is the above formula (2), X 1 and X 2 are groups containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms, p1, p2 and p3 are each 1; The pH-responsive lipid derivative according to any one of [1A] to [4A] above.
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms or an acyl group having 8 to 24 carbon atoms, A is a carboxy group, b, e, and n are each 1; d is 0; f is an integer from 2 to 5; g and h are each 2, and X is the above formula (2), X 1 and X 2 are groups containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms, Z is a group formed by a cycloaddition reaction, p1, p2, and p3 are each 1; q1 and q2 are each independently 0 to 5;
  • the pH-responsive lipid derivative according to any one of [1A] to [4A] or [12A] above.
  • the pH-responsive lipid derivative of the present invention has the property of becoming electrically neutral under neutral conditions (pH 7.4) and becoming cationic under weakly acidic conditions (pH 6.5) around tumor tissue. are doing.
  • Transport carriers for drug delivery represented by spherical micelles and liposomes containing the pH-responsive lipid derivative of the present invention, show stealth properties against blood components and normal tissues, which are in a neutral environment in vivo, and around tumor tissues. Increased cationicity in response to minute pH changes improves the efficiency of accumulation and uptake into tumor tissue.
  • the low-molecular-weight drug or nucleic acid drug encapsulated in the transport carrier can be released around the tumor tissue or efficiently introduced into the tumor cells.
  • the pH-responsive lipid derivative of the present invention is composed of a polymer of amino acids contained in vivo. Therefore, after delivery of low-molecular-weight drugs and nucleic acid drugs, they are degraded in vivo and do not accumulate in vivo.
  • the effective pH-responsive transport carrier can achieve an introduction system capable of safely and efficiently delivering low-molecular-weight drugs, nucleic acid drugs, etc. to the vicinity of tumor tissue and transfecting them into tumor cells. It becomes possible to provide a transport vehicle, as well as a pH-responsive lipid derivative useful for forming such a transport vehicle.
  • FIG. 1 shows the evaluation results of the pH responsiveness of the tested polymers in the presence of heparin in Experimental Example 1 described later.
  • FIG. 2 shows the degree of cellular uptake of siRNA from the particles tested in Experimental Example 3 described later.
  • FIG. 3 shows the evaluation results of the tumor accumulation of siRNA from the tested particles in Experimental Example 4 described later.
  • FIG. 4 shows the evaluation results of the antitumor effect of siPLK1 using the tested particles in Experimental Example 5 below.
  • pH-responsive lipid derivative (i) of the present invention is represented by the structure below.
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms, or an alkyl group having 8 to 24 carbon atoms is an acyl group or a sterol residue of M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms, B is a hydrogen atom, an alkali metal, or an ammonium group;
  • X is a linking group comprising a linking group formed by a nucleophilic substitution reaction, or a Michael addition reaction, or a thiol-ene reaction, or a cycloaddition reaction;
  • A is a carboxy group or a sulfo group, a1, a2, b, c, d, and e are each independently 0 or 1;
  • f is an integer from 0 to 5;
  • h is an integer from 1 to 3;
  • g and n are each independently 1 or 2
  • the pH-responsive lipid derivative (i) includes two preferred embodiments, (A) pH-responsive lipid derivative (1) and (B) pH-responsive lipid derivative (1-1). These three derivatives structurally differ only at R a . That is, in (A) the pH-responsive lipid derivative (1), R a is H as one preferred embodiment, and (B) in the pH-responsive lipid derivative (1-1), another preferred embodiment.
  • R a is —COCH 3 or —CO—(CH 2 ) b1 —COOH (where b1 is 2 or 3)
  • the pH-responsive lipid derivative (i) encompasses both as R a is H, —COCH 3 , or —CO—(CH 2 ) b1 —COOH (where b1 is 2 or 3) and other structural features (R 1 , R 2 , M, B, X, A, a1, a2, b, c, d, e, f, h, g, n and m) are common in the three derivatives.
  • the pH-responsive lipid derivative (i) of the present invention will be described by taking (A) the pH-responsive lipid derivative (1) as a representative example.
  • a person skilled in the art can similarly understand the pH-responsive lipid derivative (i) and the pH-responsive lipid derivative (1-1) based on the description.
  • pH-responsive lipid derivative of the present invention is represented by the following formula (1).
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms, an acyl group having 8 to 24 carbon atoms, or a sterol residue.
  • the alkyl groups having 8 to 24 carbon atoms represented by R 1 and R 2 may be linear or branched, and may contain unsaturated bonds.
  • decyl group dodecyl group, tetradecyl group, hexadecyl group, octadecyl group, icosyl group, decenyl group, dodecenyl group, tetradecenyl group, hexadecenyl group, octadecenyl group, icosenyl group, decadienyl group, dodecadienyl group, tetradecadienyl group, preferably aliphatic hydrocarbon groups having 10 to 20 carbon atoms such as enyl group, hexadecadienyl group, octadecadienyl group and icosadienyl group, more preferably carbon atoms such as tetradecyl group, hexadecyl group, octadecyl group and icosyl group; It is an aliphatic hydrocarbon group of number 14-20.
  • the acyl groups having 8 to 24 carbon atoms represented by R 1 and R 2 may be linear or branched, and may contain an unsaturated bond. Specifically, octanoyl group, nonanoyl group, decanoyl group, undecanoyl group, dodecanoyl group, tridecanoyl group, tetradecanoyl group, pentadecanoyl group, hexadecanoyl group, heptadecanoyl group, octadecanoyl group, nonadecanoyl group and icosanoyl group.
  • Examples of sterol residues represented by R 1 and R 2 in formula (1) include cholesteryl group (cholesterol residue), cholesteryl group (cholestanol residue), stigmasteryl group (stigmasterol residue), ⁇ -sitosteryl group ( ⁇ -sitosterol residue), lanosteryl group (lanosterol residue), ergosteryl group (ergosterol residue) and the like.
  • the sterol residue is preferably cholesteryl or cholesteryl.
  • a1 and a2 are each independently 0 or 1.
  • b is 0 or 1.
  • the resulting lipid derivative represents a single-chain lipid derivative
  • M is a trivalent group represented by >N—CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms (linear or branched hydrocarbon group having 3 to 7 carbon atoms It is not particularly limited as long as it is trivalent, and examples thereof include groups represented by the following formulas (14) to (16). In addition, in order to clarify the bonding relationship between the group M and other groups, the following formula includes other groups.
  • r1 represents an integer from 1 to 5
  • r2 represents an integer from 0 to 4
  • R 1 and R 2 are each as described above and a1 and a2 are each as described above
  • c is as described below, and
  • c is 0 or 1.
  • d is 0 or 1.
  • formula (1) represents a phospholipid.
  • B is a hydrogen atom, an alkali metal, or an ammonium group.
  • Alkali metals include lithium, sodium, potassium and the like.
  • e is 0 or 1.
  • n 1 or 2.
  • X is a linking group including a linking group formed by a nucleophilic substitution reaction, a Michael addition reaction, a thiol-ene reaction, or a cycloaddition reaction.
  • X is not particularly limited as long as it is a divalent organic group, but preferably includes a linking group represented by the following formula (2).
  • p1 and p2 are each independently 0 or 1.
  • X 1 and X 2 are each independently a group having 1 to 5 carbon atoms and containing a bonding group formed by a nucleophilic substitution reaction, and are composed of a bond formed by the nucleophilic substitution reaction and a spacer. .
  • Bonds formed by nucleophilic substitution reactions include ester bonds, urethane bonds, amide bonds, ether bonds, carbonate bonds, divalent hydrocarbon groups containing secondary amino groups, single bonds, and divalent hydrocarbon groups. is mentioned.
  • the spacer is not particularly limited as long as it is a site formed by a covalent bond, and examples thereof include structures represented by formula (17) or formula (18).
  • q1 and q2 represent the degree of polymerization of the ethylene glycol structure, each independently from 0 to 50.
  • p3 is 0 or 1.
  • p3 When p3 is 0, it can be produced using the polymerization initiator described below as a starting material.
  • Z is a group formed by a Michael addition reaction, a thiol-ene reaction, or a cycloaddition reaction, and examples thereof include the following formulas (19) to (27).
  • the following formula includes other groups.
  • E represents a hydrogen atom or a methyl group
  • X2 and p2 are each as described above, and
  • X is a trivalent organic group, and is not particularly limited as long as it is trivalent. Examples thereof include groups represented by the following formulas (28) to (30). .
  • g is 1 or 2.
  • h 1 to 3.
  • A is a carboxy group or a sulfo group.
  • the functional group represented by the following formula (31) including A and h in formula (1) exhibits neutrality at pH 7.2 to 7.6 and changes to cationic at pH 6.0 to 6.6 have characteristics. That is, a polymer containing the pH-responsive functional group as a repeating unit also has similar pH-responsiveness. In addition, the lipid derivative represented by formula (1) having the pH-responsive polymer also has similar pH-responsiveness.
  • m is 5 to 300, and although the range of m depends on the lipid to be bound, it is preferably 5 to 200, most preferably 5 to 150. Furthermore, it is particularly preferably 5 to 110.
  • m When m is less than 5, the content of pH-responsive functional groups in the pH-responsive lipid derivative is reduced, which may make it difficult to form pH-responsive lipid nanoparticles.
  • m When m is greater than 300, the proportion of hydrophilic moieties in the pH-responsive lipid derivative increases, and the pH-responsive lipid derivative is easily detached from the produced lipid nanoparticles.
  • the degree of polymerization of the pH-responsive lipid derivative according to the present embodiment that is, m can be adjusted according to the required performance, for example, by the polymerization conditions of the intermediate polymer.
  • the N-terminus of the peptide structure in formula (1) is described as a hydrogen atom, the terminal amino group may be replaced by a
  • a change in pH may give an onium salt structure, or an intramolecular nucleophilic substitution reaction may give a ⁇ -lactam ring.
  • the N-terminal amino group is protected or converted to a functional group such as an acetyl group or carboxylic acid, represented by the following formula.
  • B pH-responsive polymer (1-1).
  • the structure represented by formula (1) is preferably a structure represented by formulas (32) to (34) below.
  • R3 is an alkyl group having 14 to 20 carbon atoms, A is a carboxy group or a sulfo group, and m is 5 to 150.
  • R 4 is an alkyl group having 14 to 20 carbon atoms or an acyl group having 14 to 20 carbon atoms, A is a carboxy group or a sulfo group, and m is 5 to 150.
  • R 4 is an alkyl group having 14 to 20 carbon atoms or an acyl group having 14 to 20 carbon atoms, A is a carboxy group or a sulfo group, and m is 5 to 150.
  • the pH-responsive lipid derivative (i) of the present invention can be obtained by performing step (A), step (B) and step (C) in that order when R a is H in formula (47-i).
  • step (A1), step (B) and step (C) are performed in that order to obtain the pH-responsive lipid derivative represented by the formula (47-i)
  • step (D) the pH-responsive lipid derivative represented by the formula (36-i) is obtained
  • step (E) step (F) and step (G) are performed in that order.
  • the pH-responsive lipid derivative represented by the above formula (46-i) can be obtained and can be produced.
  • the pH-responsive lipid derivative (i) of the present invention is a preferred embodiment of (A) pH-responsive lipid derivative (1) and (B) pH-responsive lipid derivative (1-1). It can be manufactured by obtaining Therefore, the method for producing (A) pH-responsive lipid derivative (1) and (B) pH-responsive lipid derivative (1-1) will be described below.
  • the pH-responsive lipid derivative represented by formula (1) according to the present invention can be produced by, for example, polymerization method, coupling method (1), or coupling method (2).
  • the polymerization method will be described below.
  • the following is a representative method for producing the pH-responsive lipid derivative represented by the above formula (1).
  • a pH-responsive lipid derivative represented by the structure of formula (47) below can be produced by a production method in which the following steps (A2), (B1), and (C1) are performed in this order.
  • R 1a and R 2a are each independently an alkyl group having 8 to 24 carbon atoms or a sterol residue, and M is 3 represented by >N—CH 2 —CH 2 —.
  • B is a hydrogen atom, an alkali metal or an ammonium group
  • X is a nucleophilic substitution reaction, or a Michael addition reaction
  • A is a carboxy group or a sulfo group
  • a1, a2, b, c, d, and e are each independently , 0 or 1
  • f is an integer of 0 to 5
  • h is an integer of 1 to 3
  • g and n are each independently 1 or 2
  • m is 5 to 300.
  • Step (A2) In the step (A2), a polymerization initiator represented by the following formula (3) and an ⁇ -amino acid-N-carboxylic acid anhydride represented by the following formula (4) are used to carry out ring-opening polymerization to give the following formula: This is the step of obtaining the polymer represented by (5).
  • R 1a and R 2a are each independently an alkyl group having 8 to 24 carbon atoms or a sterol residue, and the alkyl group may be linear or branched, It may contain an unsaturated bond. Other symbols are as described above.
  • p1 and p2 are each independently 0 or 1.
  • X 1 and X 2 are each independently a group having 1 to 5 carbon atoms containing a bonding group formed by a nucleophilic substitution reaction, and composed of a bond and a spacer formed by a nucleophilic substitution reaction. .
  • Bonds formed by nucleophilic substitution include urethane bonds, amide bonds, ether bonds, divalent hydrocarbon groups containing secondary amino groups, single bonds, and divalent hydrocarbon groups.
  • the spacer is not particularly limited as long as it is a site formed by a covalent bond, and examples thereof include structures represented by the above formula (17) or the above formula (18).
  • q1 and q2 represent the degree of polymerization of the ethylene glycol structure, each independently from 0 to 50.
  • X is a trivalent organic group, and is not particularly limited as long as it is trivalent. Examples include groups represented by formulas (28) to (30). be done.
  • g 1 or 2.
  • Acid anhydrides derived from naturally occurring L-amino acids are preferred from the viewpoint of use in pharmaceutical applications.
  • P 1 represents a carboxy-protecting group, preferably a carboxy-protecting group having 1 to 8 carbon atoms. It is not particularly limited as long as it is a general protecting group for a carboxy group, but specific examples include methyl group, ethyl group, t-butyl group, allyl group, benzyl group and the like. A benzyl group is preferred from the viewpoint of availability and ease of reaction in the subsequent step (B).
  • a known method can be used as a ring-opening polymerization method. Such methods are described, for example, in Peptide-Based Materials, 2011, 1-26, Angew. Chem. , 2018, 5151-5155, Proceedings of the National Academy of Sciences of the United States of America, 2019, 10658-10663.
  • Examples of the solvent used for the ring-opening polymerization reaction include various organic solvents such as tetrahydrofuran, acetonitrile, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dichloromethane and chloroform, and their mixtures. N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, dichloromethane and chloroform are preferred.
  • the total amount of the solvent used in the ring-opening polymerization reaction is 3 to 100 times, preferably 5 to 50 times by volume, the ⁇ -amino acid-N-carboxylic acid anhydride represented by formula (4). Most preferably 7 to 25 times the amount.
  • the ratio is not particularly limited as long as the various compounds represented by the formulas (3) and (4) are respectively dissolved.
  • the reaction temperature during the ring-opening polymerization reaction is usually 20 to 60°C, preferably 25 to 40°C.
  • the reaction time at this time varies depending on conditions such as the reaction temperature, but is usually preferably 3 to 24 hours.
  • m can be defined in the same way as m in formula (1) above. That is, in formula (5), m is 5 to 300, the range of m is preferably 5 to 200, and most preferably 5 to 150. Furthermore, it is particularly preferably 5 to 110.
  • the obtained polymer may be used as it is, or may be isolated and purified by treatments such as reprecipitation, gel filtration chromatography, and membrane purification.
  • the terminal amino group can be protected or converted to obtain a pH-responsive lipid derivative in which the terminal amino group is protected or converted.
  • Step (B1) the polymer represented by the formula (5) obtained in the step (A2) is subjected to an addition reaction with a diethylenetriamine derivative represented by the following formula (6) to obtain the polymer represented by the following formula (7). It is the process of obtaining the polymer shown.
  • P2 represents a protected carboxy group or a protected sulfo group.
  • the protected carboxyl group is not particularly limited as long as it is a commonly used derivative. group, carboxylic acid benzyl ester group, and the like. A carboxylic acid t-butyl ester group is preferred from the viewpoint of ease of deprotection.
  • the protected sulfo group is not particularly limited as long as it is a commonly used derivative. and the like.
  • a sulfonic acid 2,2,2-trifluoromethyl ester group is preferred from the viewpoint of ease of deprotection.
  • the amount of the diethylenetriamine derivative represented by the formula (6) is usually 2 to 10 times, preferably 3 to 8 times, most preferably 3 to 8 times the molar ratio of m of the polymer represented by the formula (5). It is preferably 4 to 6 times the amount.
  • the diethylenetriamine derivative may form a crosslinked structure between polymers or the addition rate of the diethylenetriamine derivative to the polymer may decrease. The undigested diethylenetriamine derivative is wasted.
  • a catalyst can also be used in this reaction, for example, a catalyst such as 2-hydroxypyridine, pyridine, triethylamine, or the like can be used.
  • the addition reaction can be carried out in various solvents.
  • the solvent is not particularly limited as long as it has no reactivity with the polymer represented by formula (5) and the diethylenetriamine derivative represented by formula (6).
  • Examples include tetrahydrofuran, toluene, acetonitrile, and chloroform. , N-methyl-2-pyrrolidone, N,N-dimethylformamide, and mixtures thereof. From the viewpoint of polymer solubility, N-methyl-2-pyrrolidone or tetrahydrofuran is preferred.
  • the total amount of the solvent used in the addition reaction is 1.5 to 70 times, preferably 1.8 to 50 times, most preferably 2 to 30 times the volume of the polymer represented by formula (5). quantity.
  • the reaction temperature during the addition reaction varies depending on the solvent used, but is usually 0-100°C.
  • the reaction time at this time varies depending on conditions such as the reaction temperature, but is usually preferably 3 to 72 hours.
  • the obtained polymer may be used as it is without being purified, or may be isolated and purified by treatments such as reprecipitation, gel filtration chromatography, and membrane purification.
  • Step (C1) is a step of deprotecting the polymer represented by formula (7) obtained in step (B1) to obtain the pH-responsive polymer represented by formula (47).
  • a known method can be used according to P2 of the diethylenetriamine derivative introduced in step ( B1).
  • P2 is a carboxylic acid t -butyl ester group
  • it is deprotected by hydrolysis under acidic conditions to form a carboxy group.
  • P2 is a sulfonic acid 2,2,2-trifluoromethyl ester group
  • it becomes a sulfo group by hydrolysis under basic conditions.
  • the acid used for the acid hydrolysis reaction is not particularly limited as long as it is an acid, but an inorganic acid is preferred, an aqueous hydrochloric acid solution, an aqueous nitric acid solution, or an aqueous phosphoric acid solution is more preferred, and an aqueous hydrochloric acid solution is most preferred.
  • the concentration of the acid varies depending on the type of acid.
  • concentration of the acid in the case of an aqueous solution of hydrochloric acid, it is usually 1 to 11.2N, preferably 1.5 to 11.2N, most preferably 2 to 11.2N.
  • the acid hydrolysis reaction can be carried out in various solvents, but solvents that are miscible with water are preferred.
  • Mixed solvents used in the acid hydrolysis reaction include, for example, water-THF and water-1,4-dioxane.
  • the total amount of the solvent used in the acid hydrolysis reaction is 3 to 100 times by volume, preferably 4 to 75 times by volume, most preferably 5 to 50 times by volume the polymer represented by formula (7). be.
  • the ratio is not particularly limited, but it is preferable that the various compounds represented by the formula (7) are dissolved in the amount of the organic solvent used.
  • the reaction temperature during the acid hydrolysis reaction is usually 20-80°C, preferably 25-60°C.
  • the reaction time at this time varies depending on conditions such as the reaction temperature, but is usually preferably 3 to 48 hours.
  • the pH-responsive lipid derivative represented by formula (1) can also be synthesized by covalently bonding a lipid derivative and a pH-responsive polymer.
  • the pH-responsive polymer of the present invention represented by formula (1) is as described above, but here, as a specific example, the lipid derivative and the pH-responsive polymer are covalently bonded at a molar ratio of 1:1.
  • the following formula (36) will be explained.
  • the pH-responsive lipid derivative represented by the structure of the following formula (36) is produced in the step (D1 ) can be manufactured through the manufacturing method shown in .
  • R 1 and R 2 are each independently an alkyl group having 8 to 24 carbon atoms, an acyl group having 8 to 24 carbon atoms, or a sterol residue, and M is >N- a trivalent group represented by CH 2 —CH 2 — or an optionally branched hydrocarbon trivalent group having 3 to 7 carbon atoms
  • B is a hydrogen atom, an alkali metal or an ammonium group
  • A is a carboxy or a sulfo group
  • a1, a2, b, c, and d are each independently 0 or 1
  • f is an integer of 0 to 5
  • h is an integer of 1 to 3
  • g is 1 or 2
  • m is 5 to 300
  • X 1 and X 2 are each independently a group containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms
  • Z is a Michael addition reaction, thiol-ene reaction, or cycloaddition reaction
  • Step (D1) is represented by the above formula (36) by reacting a functional group-containing lipid represented by the following formula (8) with a functional group-containing pH-responsive polymer represented by the following formula (9). It is a step of obtaining a pH-responsive lipid derivative.
  • p1 and p2 are each independently 0 or 1.
  • X 1 and X 2 are each independently a group having 1 to 5 carbon atoms containing a bonding group formed by a nucleophilic substitution reaction, and composed of a bond and a spacer formed by the nucleophilic substitution reaction. .
  • Bonds formed by nucleophilic substitution reactions include ester bonds, urethane bonds, amide bonds, ether bonds, carbonate bonds, divalent hydrocarbon groups containing secondary amino groups, single bonds, and divalent hydrocarbon groups. is mentioned.
  • the spacer is not particularly limited as long as it is a site formed by a covalent bond, and examples thereof include structures represented by the above formula (17) or the above formula (18).
  • Y1 represents an atomic group containing a functional group capable of forming a covalent bond with a functional group included in Y2 below.
  • the atomic group Y 1 is an atomic group contained in the lipid represented by the formula (8), and a functional group (Y 1 ') obtained by forming a covalent bond with a functional group contained in Y 2 below.
  • the binding site W is a linker responsible for binding to the lipid, and there is no particular limitation as long as it is a site formed by covalent bonding.
  • the binding site W is preferably an ester bond, a urethane bond, an amide bond, an ether bond, a carbonate bond, a divalent hydrocarbon group containing a secondary amino group, a single bond, a divalent hydrocarbon group, and a divalent valent oxyalkylene groups and repeating structures thereof.
  • the hydrocarbon group preferably has 12 or less carbon atoms, and includes methylene, ethylene, trimethylene, propylene, isopropylene, tetramethylene, butylene, isobutylene, pentamethylene, and hexamethylene. and the like.
  • the oxyalkylene group includes an oxyethylene group or an oxypropylene group. It is preferable that the number of repeating units of the oxylene alkylene skeleton is 12 or less.
  • Y2 represents an atomic group containing a functional group capable of forming a covalent bond with the functional group contained in the Y1, and the functional group contained in the atomic group Y1 in the formula ( 8 ). and the functional groups contained in atomic group Y 2 are different from each other.
  • the atomic group Y 2 is an atomic group contained in the functional group-containing pH-responsive polymer represented by the formula (9), and is a functional group capable of forming a covalent bond with the functional group contained in the Y 1 ( Y 2 ′).
  • Y 2 may be the functional group (Y 2 ') alone, or may consist of the binding site (W') between the functional group (Y 2 ') and the pH-responsive polymer.
  • the binding site W' in formula (9) is a linker responsible for binding to the pH-responsive polymer, and is not particularly limited as long as it is a site formed by covalent bonding.
  • the binding site W′ is preferably a urethane bond, an amide bond, an ether bond, a divalent hydrocarbon group containing a secondary amino group, a single bond, a divalent hydrocarbon group, and a divalent oxyalkylene. groups and repeating structures thereof.
  • the hydrocarbon group preferably has 12 or less carbon atoms, and includes methylene, ethylene, trimethylene, propylene, isopropylene, tetramethylene, butylene, isobutylene, pentamethylene, and hexamethylene. and the like.
  • the oxyalkylene group includes an oxyethylene group or an oxypropylene group. It is preferable that the number of repeating units of the oxylene alkylene skeleton is 12 or less.
  • the functional group Y 1 ' in the formula (8) and Y 2 ' in the formula (9) are different functional groups and are not particularly limited as long as they are functional groups capable of reacting with each other to form a covalent bond.
  • the pH-responsive lipid derivative represented by the formula (36) is obtained by reacting the functional group Y 1 ′ and the functional group Y 2 ′.
  • Preferred functional groups suitable for the functional group Y 1 ' include the following formulas (37) to (45).
  • E represents a hydrogen atom or a methyl group.
  • reaction conditions for covalently bonding the functional group Y 1 ' and the functional group Y 2 ' known methods can be used.
  • a responsive lipid derivative is obtained.
  • Suitable examples of the functional group Y 2 ' include formulas (37) to (41) and (43) to (45) among the functional groups exemplified for the functional group Y 1 '.
  • the functional group Y 1 ' is an internal alkyne compound represented by the formula (37)
  • the functional group Y 2 ' reacts with the formula (43) and is represented by the formula (19). to form
  • the functional group Y 1 ' is an internal alkyne compound represented by the formula (38)
  • the functional group Y 2 ' reacts with the formula (43) and is represented by the formula (20). to form Z.
  • the functional group Y 1 ' is an alkyne compound represented by the formula (39)
  • the functional group Y 2 ' reacts with the formula (43) and is represented by the formula (21). form Z.
  • the functional group Y 1 ' is a transcyclooctyne compound represented by the above formula (40)
  • the functional group Y 2 ' reacts with a tetrazine group or the like and is represented by the above formula (22). form Z.
  • the functional group Y 1 ' is an alkene compound represented by the formula (41)
  • the functional group Y 2 ' reacts with the formula (45) and is represented by the formula (23). form Z.
  • the functional group Y 1 ' is a maleimide compound represented by the above formula (42)
  • the functional group Y 2 ' reacts with the above formula (45) and is represented by the above formula (24) form Z.
  • the functional group Y 1 ' is the formula (43)
  • the functional group Y 2 ' reacts with the formula (39) to form Z represented by the formula (25).
  • the functional group Y 1 ' is a tetrazine compound represented by the formula (44)
  • the functional group Y 2 ' reacts with the formula (39) and is represented by the formula (26). form Z.
  • the functional group Y 1 ' is the formula (45)
  • the functional group Y 2 ' reacts with the formula (41) to form Z represented by the formula (27).
  • Y 1 ' in the formula (8) is a functional group represented by the formula (37), and Y 2 ' in the formula (9) is a functional group represented by the formula (43). A case will be described.
  • the amount of the functional group-containing lipid represented by formula (8) used in the cycloaddition reaction is usually 0.00 in molar ratio to the functional group-containing pH-responsive polymer represented by formula (9). 8 to 3.0 times the amount, preferably 1.0 to 2.5 times the amount, most preferably 1.2 to 2.0 times the amount.
  • the proportion of the pH-responsive polymer in the product increases, making purification difficult. If the amount is more than 3.0 times, the proportion of the functional group-containing lipid in the product will increase, making purification difficult, and the functional group-containing lipid that was not used in the reaction will be wasted.
  • the cycloaddition reaction can be carried out in various solvents. Any solvent may be used for the cycloaddition reaction as long as it can dissolve the functional group-containing lipid represented by formula (8) and the functional group-containing pH-responsive polymer represented by formula (9).
  • the solvent may be capable of dissolving the functional group-containing lipid represented by formula (8) and the functional group-containing pH-responsive polymer represented by formula (9), or two or more solvents capable of dissolving each of them. They may be mixed and used.
  • solvents used in the cycloaddition reaction include various organic solvents such as water, buffers, methanol, toluene, THF, dichloromethane, chloroform, and mixtures thereof.
  • Mixed solvents used in the cycloaddition reaction include combinations such as water-toluene, water-THF, water-dichloromethane, and water-chloroform.
  • the cycloaddition reaction may be in a homogeneous solvent system or in a two-phase separated solvent system.
  • water- A mixed solvent of dichloromethane is preferred.
  • the total amount of the solvent used in the cycloaddition reaction is 50 to 150 times, preferably 65 to 135 times, most preferably 50 to 150 times the volume of the functional group-containing pH-responsive polymer represented by formula (8). 80 to 120 times the amount.
  • the ratio is not particularly limited as long as the various compounds represented by the formulas (8) and (9) are respectively dissolved.
  • the reaction temperature during the cycloaddition reaction is usually 20 to 60°C, preferably 25 to 45°C.
  • the reaction time at this time varies depending on conditions such as the reaction temperature, but is usually preferably 3 to 72 hours.
  • a pH-responsive lipid derivative in which Z is the formula (19) is obtained.
  • the obtained pH-responsive lipid derivative can be purified by, for example, reprecipitation, gel filtration chromatography, membrane purification, or the like.
  • Y 1 ' in the formula (8) is a functional group represented by the formula (41), and Y 2 ' in the formula (9) is a functional group represented by the formula (45). A case will be described.
  • the amount of the functional group-containing lipid represented by formula (8) used in the thiol-ene reaction is usually 0.00 in molar ratio to the functional group-containing pH-responsive polymer represented by formula (9). 8 to 3.0 times the amount, preferably 1.0 to 2.5 times the amount, most preferably 1.2 to 2.0 times the amount.
  • the proportion of the pH-responsive polymer in the product increases, making purification difficult. If the amount is more than 3.0 times, the proportion of the functional group-containing lipid in the product will increase, making purification difficult, and the functional group-containing lipid that was not used in the reaction will be wasted.
  • the thiol-ene reaction can be performed in various solvents. Any solvent can be used for the thiol-ene reaction as long as it can dissolve the functional group-containing lipid represented by formula (8) and the functional group-containing pH-responsive polymer represented by formula (9).
  • the solvent may be capable of dissolving the functional group-containing lipid represented by formula (8) and the functional group-containing pH-responsive polymer represented by formula (9), or two or more solvents capable of dissolving each of them. They may be mixed and used.
  • solvents used for the thiol-ene reaction include various organic solvents such as water, buffers, methanol, toluene, THF, dichloromethane, chloroform, and mixtures thereof.
  • Mixed solvents used in the thiol-ene reaction include, for example, combinations of water-toluene, water-THF, water-dichloromethane, water-chloroform, and the like.
  • the cycloaddition reaction may be in a homogeneous solvent system or in a two-phase separated solvent system.
  • water- A mixed solvent of dichloromethane is preferred.
  • the total amount of the solvent used in the thiol-ene reaction is 50 to 150 times, preferably 65 to 135 times, most preferably 80 to 120 times the volume of the pH-responsive polymer represented by formula (9). Double the amount.
  • the ratio is not particularly limited as long as the various compounds represented by the formulas (8) and (9) are respectively dissolved.
  • a thermal radical generator or photoradical generator is used during the thiol-ene reaction between the functional group-containing lipid represented by formula (8) and the functional group-containing pH-responsive polymer represented by formula (9). .
  • Peroxides or azo compounds are preferred as thermal radical generators.
  • peroxides examples include tert-butyl hydroperoxide, dibenzoyl peroxide, ammonium persulfate, hydrogen peroxide, tert-butyl-2-ethylhequinoate, dilauroyl peroxide, preferably tert-butyl hydroperoxide.
  • Peroxide ammonium persulfate, hydrogen peroxide.
  • azo compounds include azobis(iso-butyronitrile), 2,2′-azobis(2-methylbutanenitrile), 2,2′-azobis(2-amidinopropane) hydrochloride, 4′4-azobis(4 -cyanopentanoic acid), 2'2-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2'2-azobis[N-(2-hydroxyethyl)-2-methylpropionamidine] 4 hydrate, 2'2-azobis(2-methylpropionamidine) dihydrochloride, 2'2-azobis[2-(2-imidazolin-2-yl)propane], 2'2-azobis[2-(2 -imidazolin-2-yl)propane]dihydrochloride.
  • Photoradical generators include, for example, lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate, diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide, 2-hydroxy-4'-(2-hydroxyethoxy) -2-methylpropiophenone, FOM-03011 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), Cyracure UVI-6970, UVI-6974, and UV-6990 (all manufactured by Union Carbide, USA), Irgacure 264 (BASF) (manufactured by Nippon Soda Co., Ltd.) and CIT-1682 (manufactured by Nippon Soda Co., Ltd.).
  • BASF Irgacure 264
  • CIT-1682 manufactured by Nippon Soda Co., Ltd.
  • the amount of the thermal radical generator or photoradical generator used in the thiol-ene reaction is usually 0.05 to 10 times the molar ratio of the functional group-containing pH-responsive polymer represented by formula (9). , preferably 0.10 to 8 times the amount, most preferably 0.15 to 5 times the amount.
  • the reaction temperature during the thiol-ene reaction between the functional group-containing lipid represented by formula (8) and the functional group-containing pH-responsive polymer represented by formula (9) is
  • the 10-hour half-life temperature of the thermal radical generator used is preferably ⁇ 20°C.
  • the reaction time at this time varies depending on conditions such as the reaction temperature, but is usually preferably 3 to 72 hours.
  • the reaction temperature during the thiol-ene reaction between the functional group-containing lipid represented by formula (8) and the functional group-containing pH-responsive polymer represented by formula (9) is The temperature is usually 0 to 60° C., and radicals are generated using a device that emits ultraviolet light, such as a high-pressure mercury lamp or an LED laser, according to the absorption wavelength of the photoradical generator used.
  • a device that emits ultraviolet light such as a high-pressure mercury lamp or an LED laser
  • the ultraviolet irradiation time varies depending on the type of the photoradical generator and the conditions of the device that emits ultraviolet rays, it is preferably 3 hours or less.
  • a pH-responsive lipid derivative in which Z is the formula (23) is obtained.
  • the obtained pH-responsive lipid derivative can be purified by, for example, reprecipitation, gel filtration chromatography, membrane purification, or the like.
  • a pH-responsive lipid derivative having two pH-responsive polymers can be obtained by reacting a lipid having two atomic groups Y1 and a pH-responsive polymer having one atomic group Y2.
  • a pH-responsive lipid derivative whose terminal group is protected or converted by reacting a pH-responsive polymer whose terminal amino group is protected or converted instead of the formula (9) having a terminal amino group. can be obtained.
  • the pH-responsive lipid derivative represented by the formula (1) is synthesized by covalently bonding the lipid derivative and the pH-responsive polymer precursor to synthesize the pH-responsive lipid derivative precursor, and then forming the pH-responsive lipid derivative. Induction is also possible.
  • the pH-responsive lipid derivative of the present invention represented by the formula (1) is as described above.
  • the combined formula (46) below will be described.
  • a pH-responsive lipid derivative represented by the structure of formula (46) below can be produced by a production method in which the following steps (E1), (F1), and (G1) are performed in this order.
  • R 1a and R 2a are each independently an alkyl group having 8 to 24 carbon atoms or a sterol residue, and M is 3 represented by >N—CH 2 —CH 2 —.
  • B is a hydrogen atom, an alkali metal or an ammonium group
  • A is a carboxy group or a sulfo group
  • a1, a2, b, c, and d are each independently 0 or 1
  • f is an integer of 0 to 5
  • h is an integer of 1 to 3
  • g is 1 or 2
  • m is 5 to 300
  • X 1 and X 2 are each independently a group containing a bonding group by a nucleophilic substitution reaction having 1 to 5 carbon atoms
  • Z is a Michael addition reaction, a thiol-ene reaction, or a ring p1 and p2 are each independently 0
  • the pH-responsive lipid derivative represented by the above formula (46), in which the terminal amino group is protected or converted is replaced with a pH-responsive polymer precursor having a terminal amino group.
  • a pH-responsive polymer precursor with protected or converted amino groups it can be produced by a production method in which the following step (E1), step (F1), and step (G1) are performed in this order.
  • Step (E1) a pH-responsive lipid derivative represented by the following formula (12) is obtained by reacting a functional group-containing lipid represented by the following formula (10) with a polymer represented by the following formula (11). This is the step of obtaining a precursor.
  • R 1a and R 2a are each independently an alkyl group having 8 to 24 carbon atoms or a sterol residue, and the alkyl group may be linear or branched, It may contain an unsaturated bond. Other symbols are as described above.
  • Each of X 1 and X 2 is independently a group having 1 to 5 carbon atoms containing a bonding group formed by a nucleophilic substitution reaction, and is composed of a bond formed by the nucleophilic substitution reaction and a spacer.
  • Bonds formed by nucleophilic substitution include urethane bonds, amide bonds, ether bonds, divalent hydrocarbon groups containing secondary amino groups, single bonds, and divalent hydrocarbon groups.
  • the spacer is not particularly limited as long as it is a site formed by a covalent bond, and examples thereof include structures represented by the above formula (17) or the above formula (18).
  • Y1 represents an atomic group containing a functional group capable of forming a covalent bond with a functional group included in Y2 below.
  • the atomic group Y 1 is an atomic group contained in the lipid represented by the formula (10), and a functional group (Y 1 ') obtained by forming a covalent bond with a functional group contained in Y 2 below.
  • the binding site W is a linker responsible for binding to the lipid, and there is no particular limitation as long as it is a site formed by covalent bonding.
  • the binding site W is preferably a urethane bond, an amide bond, an ether bond, a divalent hydrocarbon group containing a secondary amino group, a single bond, a divalent hydrocarbon group, a divalent oxyalkylene group and A repeating structure of this is mentioned.
  • the hydrocarbon group preferably has 12 or less carbon atoms, and includes methylene, ethylene, trimethylene, propylene, isopropylene, tetramethylene, butylene, isobutylene, pentamethylene, and hexamethylene. and the like.
  • the oxyalkylene group includes an oxyethylene group or an oxypropylene group. It is preferable that the number of repeating units of the oxylene alkylene skeleton is 12 or less.
  • Functional groups suitable as the functional group Y 1 ' preferably include the above formulas (37) to (45).
  • P 1 is a carboxy-protecting group having 1 to 8 carbon atoms. It is not particularly limited as long as it is a general protecting group for a carboxy group, but specific examples include methyl group, ethyl group, t-butyl group, allyl group, benzyl group and the like. A benzyl group is preferred from the viewpoint of availability and ease of reaction in the subsequent step (F).
  • Y2 represents an atomic group containing a functional group capable of forming a covalent bond with the functional group contained in the Y1, and the functional group contained in the atomic group Y1 in the formula ( 10 ). and the functional groups contained in atomic group Y 2 are different from each other.
  • the atomic group Y2 is an atomic group contained in the polymer represented by the formula (11) and includes a functional group ( Y2 ') capable of forming a covalent bond with the functional group contained in Y1.
  • Y 2 may be the functional group (Y 2 ') alone, or may consist of the binding site (W') between the functional group (Y 2 ') and the pH-responsive polymer.
  • the binding site W′ in formula (11) is a linker responsible for binding to the pH-responsive polymer precursor, and is not particularly limited as long as it is a site formed by covalent bonding. ) is similar to the binding site contained in Y2 in
  • the functional group Y 1 ' in the formula (10) and Y 2 ' in the formula (11) are different functional groups and are not particularly limited as long as they are functional groups capable of reacting with each other to form a covalent bond. .
  • As the reaction conditions for covalently bonding the functional group Y 1 ' and the functional group Y 2 ' a known method can be used. A responsive lipid derivative precursor is obtained.
  • the obtained pH-responsive lipid derivative precursor may be used as it is without purification, or may be isolated and purified by treatments such as reprecipitation, gel filtration chromatography, and membrane purification.
  • the cycloaddition reaction can be carried out in various solvents. Any solvent can be used for the cycloaddition reaction as long as it can dissolve the functional group-containing lipid represented by formula (10) and the polymer represented by formula (11).
  • the solvent may be capable of dissolving the functional group-containing lipid represented by formula (10) and the polymer represented by formula (11), or a mixture of two or more solvents capable of dissolving each may be used. good.
  • solvent used for the cycloaddition reaction examples include various organic solvents such as THF, toluene, N-methyl-2-pyrrolidone, N,N-dimethylformamide, dichloromethane and chloroform, and mixtures thereof.
  • pH-responsive lipid derivative precursor in which Z is the formula (19) is obtained.
  • the obtained pH-responsive lipid derivative precursor may be used as it is without purification, or may be isolated and purified by treatments such as reprecipitation, gel filtration chromatography, and membrane purification.
  • Step (F1) the polymer represented by the formula (12) obtained in the step (E) is subjected to an addition reaction with the diethylenetriamine derivative represented by the formula (6) to obtain the polymer represented by the formula (13) below. It is the process of obtaining the polymer shown.
  • the amount of the diethylenetriamine derivative represented by the formula (6) is usually 2 to 10 times, preferably 3 to 8 times, the molar ratio of m of the polymer represented by the formula (12). Most preferably, it is 4 to 6 times the amount.
  • the diethylenetriamine derivative may form a crosslinked structure between polymers or the addition rate of the diethylenetriamine derivative to the polymer may decrease. The undigested diethylenetriamine derivative is wasted.
  • a catalyst can also be used in this reaction, for example, a catalyst such as 2-hydroxypyridine, pyridine, triethylamine, or the like can be used.
  • the addition reaction can be carried out in various solvents.
  • the solvent is not particularly limited as long as it has no reactivity with the polymer represented by formula (12) and the diethylenetriamine derivative represented by formula (6).
  • Examples include tetrahydrofuran, toluene, acetonitrile,
  • Examples include various organic solvents such as chloroform, N-methyl-2-pyrrolidone, N,N-dimethylformamide, and mixtures thereof. From the viewpoint of polymer solubility, N-methyl-2-pyrrolidone or tetrahydrofuran is preferred.
  • the total amount of the solvent used in the addition reaction is 1.5 to 70 times, preferably 1.8 to 50 times, most preferably 2 to 30 times the volume of the polymer represented by formula (12). Double the amount.
  • the reaction temperature during the addition reaction varies depending on the solvent used, but is usually 0-100°C.
  • the reaction time at this time varies depending on conditions such as the reaction temperature, but is usually preferably 3 to 72 hours.
  • the obtained polymer may be used as it is without being purified, or may be isolated and purified by treatments such as reprecipitation, gel filtration chromatography, and membrane purification.
  • Step (G1) The step (G1) is a step of deprotecting the polymer represented by the formula (13) obtained in the step (F1) to obtain the pH-responsive lipid derivative represented by the formula (46). .
  • a known method can be used according to P2 of the diethylenetriamine derivative introduced in step ( F1).
  • P 2 in formula (13) is a carboxylic acid t-butyl ester group
  • the above step (C1) is repeated except that the polymer represented by formula (7) is replaced by formula (13).
  • lipid having two atomic groups Y1 and a pH - responsive polymer precursor having one atomic group Y2 are reacted, and the steps (E1) to (G1) are performed to obtain two pH-responsive polymers.
  • the present pH-responsive lipid derivative can be obtained.
  • the pH-responsive lipid derivative represented by formula (1-1) according to the present invention can be produced by, for example, polymerization method, coupling method (1), or coupling method (2).
  • the following is a representative method for producing the pH-responsive lipid derivative represented by the above formula (1-1). It is also possible to produce the pH-responsive lipid derivative represented by the formula (1-1) by using other production methods known in the field or by appropriately modifying them.
  • a pH-responsive lipid derivative represented by the structure of formula (47-1) below can be produced by carrying out the following steps (A3), (B2) and (C2) in that order.
  • Step (A3) is (i) ring-opening polymerization using the polymerization initiator represented by the formula (3) and the ⁇ -amino acid-N-carboxylic anhydride represented by the formula (4) to obtain the formula (5); to obtain a polymer represented by (step (i)), (ii) introducing COCH 3 or CO—(CH 2 ) b1 —COOH (wherein b1 is 2 or 3) to the terminal amino group of the obtained polymer to obtain the above formula (5-1) to obtain a polymer represented by (step (ii)), It is a process.
  • step (i) A person skilled in the art will refer to the explanation of the step (A2) described in detail above for the production of the pH-responsive lipid derivative (1), appropriately determine the reaction conditions, etc., and carry out the step (i). be able to.
  • step (ii) A person skilled in the art can refer to the specific description in the examples below, polymers and methods known in the art, and common general technical knowledge to appropriately determine reaction conditions and the like, and perform step (ii). can be implemented. Specifically, polymer (5-1) can be synthesized by protecting or converting the terminal amino group of the polymer represented by formula (5) (also referred to as polymer (5)). A known reaction can be used for protecting or converting the terminal amino group.
  • polymer (5-1) in which R b is —CO—CH 3 can be obtained by reacting acetic anhydride as an electrophile with the terminal amino group of polymer (5).
  • a polymer (5-1) in which R b is —CO—(CH 2 ) 2 —COOH is obtained by reacting succinic anhydride as an electrophilic agent with the terminal amino group of the polymer (5).
  • the amount of the electrophilic agent to be used is usually 2 to 10 times, preferably 3 to 8 times, and most preferably 4 to 6 times the molar ratio of the terminal amino group of the polymer (5). .
  • a catalyst can be used for this reaction, for example, a catalyst such as DMAP can be used.
  • the reaction can be performed in various solvents.
  • the solvent is not particularly limited as long as it can dissolve the polymer (5) and has no reactivity with the electrophilic agent. Examples include tetrahydrofuran, 1,4-dioxane, toluene, acetonitrile, dichloromethane, and chloroform. , N-methyl-2-pyrrolidone, N,N-dimethylformamide, and mixtures thereof.
  • the total amount of the solvent used in the reaction is 1.5 to 70 times, preferably 1.8 to 50 times, most preferably 2 to 30 times by volume the polymer (5).
  • the reaction temperature during the reaction varies depending on the solvent used, but is usually 0 to 100°C.
  • the reaction time at this time varies depending on conditions such as the reaction temperature, but is usually preferably 3 to 72 hours.
  • Step (B2) The step (B2) is Represented by the formula (7-1) by reacting the polymer represented by the formula (5-1) obtained in the step (A3) with the diethylenetriamine derivative represented by the formula (6). It is a process to obtain a polymer that
  • step (B1) a pH-responsive lipid derivative represented by formula (47-1) is obtained by deprotecting the polymer represented by formula (7-1) obtained in step (B2). is the process of obtaining
  • step (C2) a pH-responsive lipid derivative represented by formula (47-1) is obtained by deprotecting the polymer represented by formula (7-1) obtained in step (B2). is the process of obtaining
  • step (D2) A person skilled in the art can appropriately synthesize the functional group-containing pH-responsive polymer (9-1) with reference to polymers and methods known in the art, as well as common technical knowledge and step (ii) in step (A3). , can be used to carry out step (D2).
  • the pH-responsive lipid derivative represented by the formula (1-1) is synthesized by covalently bonding the lipid derivative and the pH-responsive polymer precursor to synthesize the pH-responsive lipid derivative precursor, and then the pH-responsive lipid derivative. It is also possible to guide to More specifically, in the pH-responsive lipid derivative (1-1), the pH-responsive lipid derivative represented by the structure of the following formula (46-1) is a pH-responsive polymer precursor having a terminal amino group.
  • step (E1), step (F1), and step (G1) detailed above for the production of the pH-responsive lipid derivative (1) using the pH-responsive polymer precursor substituted with can be produced by performing the corresponding step (E2), step (F2), and step (G2) in this order.
  • the hydrogen atom of the terminal amino group is R b (where R b is —COCH 3 or —CO—(CH 2 ) b1 —COOH (where b1 is 2 or 3)
  • the pH-responsive polymer precursor substituted with ) can be appropriately synthesized with reference to polymers and methods known in the art, as well as common technical knowledge and step (ii) in step (A3).
  • the methods for producing the pH-responsive lipid derivatives (1) and (1-1) of the present invention have been described above in detail. It can be produced by appropriately performing (A1), step (B), step (C), step (D), step (E), step (F), and step (G).
  • pH-responsive lipid derivative represented by the structure represented by formula (i) and the pH-responsive lipid derivative represented by the structure represented by formula (1) are described above in detail.
  • formula (i), formula (1), formulas (1-1), formulas (47), formulas (36), and formulas (46) are expanded and described in that order.
  • pH-responsive lipid derivative of the present invention (specifically, pH-responsive lipid derivative (i), pH-responsive lipid derivative (1), and pH-responsive lipid derivative (1-1)) is used for drug delivery.
  • pH-responsive lipid derivative (1) will be described as a representative example of pH-responsive lipid derivative (i) of the present invention.
  • a person skilled in the art can similarly use the pH-responsive lipid derivative (i) and the pH-responsive lipid derivative (1-1) as transport carriers for drug delivery based on the description.
  • the transport carrier for drug delivery of the present invention contains the pH-responsive lipid derivative represented by the formula (1), and has a phospholipid or a derivative thereof, or a lipid other than sterol or phospholipid as a basic constituent.
  • Examples include lipid nanoparticles such as
  • the form of the lipid nanoparticles according to the present invention is not particularly limited. Examples include solid lipid nanoparticles, amorphous layered structures, and the like. Preferred lipid nanoparticles according to the present invention are spherical micelles, solid lipid nanoparticles and liposomes.
  • the lipid nanoparticles according to the present invention are particles having a structure in which the hydrophilic portion of the pH-responsive lipid derivative is arranged toward the aqueous phase side of the interface.
  • the lipid portion is hydrophobic and the polymer portion containing the pH-responsive functional group is hydrophilic, so that the pH-responsive portion is exposed on the surface of the lipid nanoparticle. Therefore, the lipid nanoparticles according to the present invention have pH responsiveness like pH-responsive lipid derivatives. That is, it has the property of becoming electrically neutral in a neutral environment (pH 7.4) and increasing in cationicity under weakly acidic conditions (pH 6.5) around tumor tissue.
  • lipids other than the pH-responsive lipid derivative of the present invention can be lipids used for forming general lipid nanoparticles.
  • lipids include, for example, phospholipids, neutral lipids, cationic lipids, sterols, saturated or unsaturated fatty acids, and the like. These can be used singly or in combination of two or more.
  • Phospholipids include phosphatidylcholine, glycerophospholipid, sphingophospholipid, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, and specifically dioleylphosphatidylethanolamine (DOPE) and dioleylphosphatidine. acid (DOPA) and the like.
  • DOPE dioleylphosphatidylethanolamine
  • DOPA dioleylphosphatidylethanolamine
  • sterols examples include animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrosterol, desmosterol and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol and brassicasterol; Microorganism-derived sterols such as zymosterol and ergosterol are included.
  • Preferred phospholipids include DOPE and preferred sterols are cholesterol.
  • Neutral lipids include glycerolipids and sphingolipids.
  • Fatty acid residues in these glycerolipids or sphingolipids are not particularly limited, and examples thereof include saturated or unsaturated fatty acid residues having 12 to 24 carbon atoms.
  • Specific examples include acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, and lignoceric acid.
  • cationic lipids examples include DODAC (dioctadecyldimethylammonium chloride), DOTMA (N-[2,3-bis(oleoyloxy)propyl]-N,N,N-trimethylammonium chloride N-[2 ,3-bis(oleoyloxy)propyl]-N,N,N-trimethylammonium chloride), DDAB (didodecyldimethylammonium bromide), DOTAP (1,2-dioleyloxy-3-trimethylammonium propane, 1,2 -dioleoyloxy-3-trimethylammonium propane), DC-Chol (3 ⁇ -[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol 3 ⁇ -N-(N',N',-dimethylaminoethane)-carbamoyl cholesterol), DMRIE (1,2-dimyristoyloxypropyl-3-dimethylhydroxy
  • Fatty acids include caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, arachidic acid, behenic acid, erucic acid, lignoceric acid, and derivatives thereof. is mentioned.
  • the blending ratio of lipids other than the pH-responsive lipid derivative of the present invention it is possible to use the blending ratio used when forming general lipid nanoparticles. can.
  • the pH-responsive lipid derivative represented by the formula (1) is preferably blended in an amount of 0.1 to 30 mol % of the total lipid at the time of production of the lipid nanoparticles. It is preferably blended in an amount of 5 to 20 mol %. If it is less than 0.1 mol %, the proportion of the pH-responsive portion on the surface of the lipid nanoparticle becomes small, and there is a risk that it will not be sufficiently cationic for accumulation and uptake into tumor tissue under weakly acidic conditions. If it is more than 30 mol %, it may become difficult to produce stable lipid nanoparticles.
  • the average particle size is preferably 500 nm or less, more preferably 400 nm or less, and even more preferably 200 nm or less.
  • the particle size distribution is preferably 0.5 or less, more preferably 0.45 or less, and most preferably 0.4 or less.
  • the average particle size of lipid nanoparticles means the number average particle size measured by dynamic light scattering (DLS). Measurement by the dynamic light scattering method can be performed using a commercially available DLS device or the like.
  • the lipid nanoparticles of the present invention are neutral at pH 7.2 to 7.6 and contain pH-responsive lipid derivatives that change to cationic at pH 6.0 to 6.6.
  • the surface charge (zeta potential) of also changes in response to pH changes.
  • the surface charge of lipid nanoparticles at each pH can be measured using a commercially available zeta potential measurement device.
  • the zeta potential of the lipid nanoparticles of the present invention at pH 7.4 varies depending on the constituents, the drug to be encapsulated, the content, etc., but it is preferably electrically neutral.
  • lipid nanoparticles in the range of -10 mV to +10 mV are mainly used.
  • the pH responsiveness of the lipid nanoparticles of the present invention can be calculated from the difference between the zeta potential value at pH 6.5 and the zeta potential value at pH 7.4.
  • the lipid nanoparticles of the present invention exhibit electrical neutrality in a neutral environment of pH 7.4, they exhibit stealth properties against blood components and normal tissues, and exhibit minute pH changes (pH 6.5) around tumor tissues. Cationicity increases in response to Since the cell surface has a negative charge, an increase in cationicity is expected to improve the efficiency of accumulation and uptake into tumor tissue. As a result, the low-molecular-weight drug or nucleic acid drug encapsulated in the transport carrier can be released around the tumor tissue or efficiently introduced into the tumor cells.
  • the zeta potential difference (Q) of the lipid nanoparticles is less than 2.5 mV, the amount of zeta potential displacement due to pH changes is small, so it is expected that the accumulation in tumor tissue and the efficiency of uptake into cells will be improved. there is a risk that it will not.
  • the lipid nanoparticles of the present invention exhibit a positive zeta potential at pH 6.5. Since the cell surface has a negative charge, a positive value promotes electrostatic interactions and further promotes cellular uptake.
  • the method for producing lipid nanoparticles according to the present invention is not particularly limited, and any method available to those skilled in the art can be adopted.
  • all the lipid components are dissolved in an organic solvent such as chloroform, dried under reduced pressure using an evaporator or spray-dried using a spray dryer to form a lipid film.
  • It can be produced by adding and further emulsifying with an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure injection emulsifier.
  • an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure injection emulsifier.
  • It can also be produced by a well-known method for producing liposomes, for example, a reversed-phase atmospheric pressure method.
  • extrusion extrusion filtration
  • aqueous solvent is not particularly limited. be able to.
  • These aqueous solvents (dispersion media) can stably disperse lipid nanoparticles. good.
  • the concentration of the aqueous solvent is not particularly limited, it is preferably 2 to 20% (W/V), more preferably 5 to 10% (W/V), for example, in an aqueous sugar solution.
  • the polyhydric alcohol aqueous solution 1 to 5% (W/V) is preferable, and 2 to 2.5% (W/V) is more preferable.
  • the buffer concentration is preferably 5 to 50 mM, more preferably 10 to 20 mM.
  • the concentration of the lipid membrane structure in the aqueous solvent is not particularly limited, but the total concentration of lipids in the aqueous solvent is preferably 0.1 to 500 mM, more preferably 1 to 100 mM.
  • lipid nanoparticles dispersed in the aqueous solvent can be mentioned as methods for further drying the lipid nanoparticles dispersed in the aqueous solvent.
  • aqueous solvent an aqueous sugar solution, preferably an aqueous sucrose solution or an aqueous lactose solution, can be used as described above. If the lipid nanoparticles dispersed in an aqueous solvent are produced and then dried, the lipid nanoparticles can be stored for a long period of time.
  • an aqueous drug solution is added to the dried lipid nanoparticles, the lipid mixture is efficiently hydrated, and the drug can be efficiently retained in the lipid nanoparticles.
  • the lipid nanoparticles according to the present invention are particularly useful as carriers for delivering medicinal ingredients to tumor tissue.
  • the component to be encapsulated inside the lipid nanoparticles of the present invention is not particularly limited as long as it has a size that can be encapsulated. of substances can be encapsulated.
  • the component to be encapsulated in the lipid nanoparticles according to the present invention is preferably one or more selected from the group consisting of low-molecular-weight drugs, nucleic acids, and peptides.
  • Nucleic acids include, for example, antisense oligonucleotides, siRNA, miRNA, shRNA, mRNA, nucleic acid aptamers, decoy nucleic acids, ribozymes, CpG oligonucleic acids and the like.
  • Low-molecular-weight drugs include drugs with a molecular weight of approximately 1000 or less.
  • a small molecule drug may be, for example, an anti-cancer drug, a contrast agent.
  • Anticancer agents include, for example, paclitaxel, doxorubicin, cisplatin, gemcitabine and the like.
  • Peptides include antibody drugs such as Herceptin, Avastin, and Cyramza.
  • pBLG Poly( ⁇ -benzyl L-glutamic acid) (hereinafter abbreviated as pBLG) was measured using a column: PL gel manufactured by Agilent (trade name “MIXED-D”) (combining two columns), column temperature: 40°C, Sample concentration: 0.2% by weight, injection volume: 100 ⁇ L, eluent: N-methyl-2-pyrrolidone added with 10 mM LiBr, flow rate: 0.6 ml/min, detector: differential refractometer (RI), standard : Measurement was performed under the conditions of polymethyl methacrylate.
  • MIXED-D trade name “MIXED-D”
  • Poly (glutamic acid diethylenetriaminecarboxylic acid) (hereinafter abbreviated as pGlu (DET-Car)) and poly (glutamic acid diethylenetriaminesulfonic acid) (hereinafter abbreviated as pGlu (DET-Sul)) are measured using a column: manufactured by Tosoh Corporation.
  • Two types of TSK gel (trade name "TSKgel G3000PW XL " and trade name "TSKgel G5000PW XL ”) were combined, column temperature: 40°C, sample concentration: 0.2% by weight, injection volume: 100 ⁇ L, eluent: 500 mM. Measurement was performed under the conditions of 10 mM HEPES buffer (pH 7.4) to which NaCl was added as described above, flow rate: 0.4 ml/min, detector: differential refractometer (RI), standard: polyethylene glycol.
  • RI differential refractometer
  • Diethylenetriamine (hereinafter abbreviated as DET, 474.9 g, 4.6 mol, compound 1) was dissolved in 500 mL of methanol under ice-cooling, and t-butyl acrylate (5.9 g, 46 mol, compound Various solutions were prepared by dissolving 2) using 270 mL of methanol. While stirring the DET solution, the t-butyl acrylate solution was added dropwise over 3 hours while maintaining the temperature at 15-25°C. After completion of the dropwise addition, the mixture was stirred at 15-25° C. for 1 hour and then distilled off under reduced pressure to obtain 109.7 g of a yellow transparent oil. The obtained oil was subjected to column purification using a silica gel column to obtain 66.0 g of DET-CartBu (compound 3) as colorless and transparent oil.
  • DET Diethylenetriamine
  • BLG-N-carboxyanhydride (hereinafter abbreviated as BLG-NCA, 8.2 g, 31 mmol, compound 5) was added to 45 mL of N,N-dimethylformamide (hereinafter abbreviated as DMF, weight of BLG-NCA) under a nitrogen atmosphere. 5.5 times the volume of BLG-NCA) and 45 mL of dichloromethane (hereinafter abbreviated as DCM, 5.5 times the volume of the weight of BLG-NCA). To the resulting solution, 900 mg of 11-azido-3,6,9-trioxaundecan-1-amine (0.9 g, 4.1 mmol, compound 4) was added and stirred at 30° C.
  • DMF N,N-dimethylformamide
  • DCM dichloromethane
  • THF tetrahydrofuran
  • EtOH ethanol
  • DMSO dimethyl sulfoxide
  • TMS trimethylsilane
  • AZ-pBLG20 (3.0 g, 13.6 mmol of benzyl groups in the polymer, compound 6) and 2-hydroxypyridine (3.9 g, 41 mmol, 3 times the amount of benzyl groups in the polymer)
  • the compound 3 synthesized in Production Example 1 (15.7 g, 68 mmol, 5 times the number of moles of benzyl groups in the polymer) and 15 mL of THF were uniformly dissolved and reacted at 50 ° C. for 21 hours. .
  • Trimethylsilylpropionic acid (TMSP) was added to 0.65% dihydrochloric acid in water. 1.0 mL of the deuterated solvent was added to 10 mg of the target product, and the dissolved sample was used to confirm the structure by 1 H-NMR analysis, confirming that it was AZ-pGlu (DET-C2-Car) 20. .
  • a specific calculation method in AZ-pGlu (DET-Car) 20 is given below as an example.
  • the final target polymer of each production example or example may be referred to as the polymer of production example ⁇ or the polymer of example ⁇ .
  • AZ-pGlu(DET-C2-Car)20 is the polymer of Preparation 2.
  • the structure was confirmed by 1 H-NMR analysis in the same manner as in Production Example 2, and confirmed to be AZ-pBLG30.
  • the structure was confirmed by 1 H-NMR analysis in the same manner as in Production Example 2, and confirmed to be AZ-pGlu(DET-C2-Car)30.
  • the degree of polymerization is calculated from the integrated value of the peak (4.30-4.50 ppm) derived from the ⁇ -position of the amide group of the polypeptide. As a result, it was confirmed that the degree of polymerization was equivalent to 32.
  • the structure was confirmed by 1 H-NMR analysis in the same manner as in Production Example 2, and it was confirmed to be AZ-pBLG100.
  • the degree of polymerization is calculated from the integrated value of the peak (4.30-4.50 ppm) derived from the ⁇ -position of the amide group of the polypeptide. As a result, it was confirmed that the degree of polymerization was equivalent to 98.
  • AZ-pBLG100 synthesized in Production Example 4 50 mg, the number of moles of benzyl groups in the polymer 0.23 mmol, compound 6) and 2-hydroxypyridine (107 mg, 1.1 mmol, relative to the number of moles of benzyl groups in the polymer DET-C3-Car tBu (852 mg, 3.5 mmol, 15 times the number of moles of benzyl groups in the polymer, compound 10) and 5 mL of THF were uniformly dissolved at 50°C. The reaction was allowed to proceed for 21 hours.
  • TMSP trimethylsilylpropionic acid
  • the degree of polymerization is calculated from the integrated value of the peak (4.30-4.50 ppm) derived from the ⁇ -position of the amide group of the polypeptide. As a result, it was confirmed that the degree of polymerization was equivalent to 98.
  • TFE 2,2,2-trifluoroethanol
  • 18.4 g, 183.9 mmol 2-chloroethanesulfonyl chloride
  • the resulting filtrate was washed with 1M HCl and ion-exchanged water, and the organic layer was dried over magnesium sulfate.
  • the filtrate was distilled off under reduced pressure to obtain 14.5 g of vinylsulfonic acid 2,2,2-trifluoroethyl ester (compound 13) as a colorless transparent oil.
  • AZ-pBLG100 synthesized in Production Example 4 50 mg, the number of moles of benzyl groups in the polymer 0.23 mmol, compound 6) and 2-hydroxypyridine (107 mg, 1.1 mmol, relative to the number of moles of benzyl groups in the polymer compound 14 (1.02 g, 3.5 mmol, 15 times the amount of benzyl groups in the polymer) and 5 mL of THF, and reacted at 50° C. for 21 hours. .
  • the structure was confirmed by 1 H-NMR analysis in the same manner as in Production Example 4, and confirmed to be AZ-pGlu(DET-C2-Sul)100.
  • the degree of polymerization is calculated from the integrated value of the peak (4.30-4.50 ppm) derived from the ⁇ -position of the amide group of the polypeptide. As a result, it was confirmed that the degree of polymerization was equivalent to 98.
  • a white yellow powder was obtained by performing the same operation as in Production Example 6 except that DET-C3-Sul TFE (1.06 g, 3.4 mmol, compound 19) was used instead of DET-C2-Sul TFE (compound 14). 53.1 mg of AZ-pGlu(DET-C3-Sul)100 (Compound 20) was obtained.
  • the structure was confirmed by 1 H-NMR analysis in the same manner as in Production Example 4, and confirmed to be AZ-pGlu(DET-C3-Sul)100.
  • the degree of polymerization is calculated from the integrated value of the peak (4.30-4.50 ppm) derived from the ⁇ -position of the amide group of the polypeptide. As a result, it was confirmed that the degree of polymerization was equivalent to 98.
  • the degree of polymerization is calculated from the integrated value of the peak (4.30-4.50 ppm) derived from the ⁇ -position of the amide group of the polypeptide. As a result, it was confirmed that the degree of polymerization was equivalent to 33.
  • 11-azido-3,6,9-trioxaundecane-1-amine (0.6 g, 2.5 mmol) and triethylamine (480 ⁇ L, 3.4 mmol) were added and allowed to stir for an additional 24 hours. After completion of the reaction, distillation was carried out under reduced pressure to obtain a brown viscous substance, which was column-purified to obtain 1.1 g of colorless transparent viscous oil AZ-EG 3 -Ph-DiBocPr (compound 28).
  • the structure was confirmed by 1 H-NMR analysis in the same manner as in Production Example 2, and confirmed to be AZ-Ph-(pBLG30)2.
  • the peak (3.65-3.80 ppm) derived from the initiator is used as the standard value (14H), and the degree of polymerization is calculated from the integrated value of the peak (4.30-4.50 ppm) derived from the amino group ⁇ -position of the polypeptide. As a result, it was confirmed that the degree of polymerization was equivalent to 29.
  • AZ-pBLG30 (50 mg, 6.91 ⁇ mol, compound 6) synthesized in Production Example 3, acetic anhydride (3.5 mg, 0.035 mmol, 5 times the number of moles of the polymer), dimethylaminopyridine (0. 04 mg, 0.35 ⁇ mol, 0.05 times the number of moles of the polymer) was uniformly dissolved in 0.5 mL of dichloromethane and reacted at room temperature for 16 hours. Completion of the reaction was confirmed by the disappearance of the ninhydrin coloration derived from the starting material on the TLC spot. After completion of the reaction, reprecipitation purification using THF and ethanol was performed, followed by drying to obtain 37 mg of white powder of terminally acetylated AZ-pBLG30.
  • the structure was confirmed by 1 H-NMR analysis in the same manner as in Production Example 2, and it was confirmed to be terminally acetylated AZ-pGlu(DET-C2-Car)30.
  • the degree of polymerization is calculated from the integrated value of the peak (4.30-4.50 ppm) derived from the ⁇ -position of the amide group of the polypeptide. As a result, it was confirmed that the degree of polymerization was equivalent to 32.
  • the degree of polymerization is calculated from the integrated value of the peak (4.30-4.50 ppm) derived from the ⁇ -position of the amide group of the polypeptide. As a result, it was confirmed that the degree of polymerization was equivalent to 98.
  • 4-chlorobutanesulfonic acid 2,2 was obtained in the same manner as in Production Example 7, except that 4-chlorobutanesulfonyl chloride (15.0 g, 78.5 mmol, compound 37) was used instead of compound 16. , 2-trifluoroethyl ester (compound 38) was obtained in an amount of 16.3 g.
  • the structure was confirmed by 1 H-NMR analysis in the same manner as in Production Example 2, and confirmed to be AZ-pGlu(DET-C4-Sul)100.
  • the degree of polymerization is calculated from the integrated value of the peak (4.30-4.50 ppm) derived from the ⁇ -position of the amide group of the polypeptide. As a result, it was confirmed that the degree of polymerization was equivalent to 98.
  • the structure was confirmed by 1 H-NMR analysis in the same manner as in Production Example 2 except that deuterated chloroform was used as the measurement solvent, and it was confirmed to be DSGE-pBLG30.
  • the degree of polymerization was calculated from the integrated value of the peak (3.80-4.30 ppm) derived from the amide group ⁇ -position of the polypeptide. , the degree of polymerization was confirmed to be equivalent to 31.
  • DSGE-pBLG30 (0.9 g, compound 43) was used instead of compound 6, and the deprotection reaction was performed at 50°C using 6N hydrochloric acid solution.
  • 0.8 g of DSGE-pGlu(DET-C2-Car)30 (compound 44) was obtained as a whitish yellow powder.
  • the peak (0.80-0.95 ppm) derived from the methyl group at the lipid terminal is used as the standard value (6H), and the degree of polymerization is calculated from the integrated value of the peak (4.30-4.50 ppm) derived from the ⁇ -position of the amide group of the polypeptide. was calculated, it was confirmed that the degree of polymerization was equivalent to 31.
  • the structure was confirmed by 1 H-NMR analysis in the same manner as in Example 1-1, and it was confirmed to be DSGE-pBLG100.
  • the degree of polymerization was calculated from the integrated value of the peak (3.80-4.30 ppm) derived from the amide group ⁇ -position of the polypeptide. , the degree of polymerization was confirmed to be equivalent to 98.
  • DSGE-pGlu (DET-C2 -Car) 100 (compound 44) was obtained in an amount of 1.30 g.
  • the peak (0.80-0.95 ppm) derived from the methyl group at the lipid terminal is used as the standard value (6H), and the degree of polymerization is calculated from the integrated value of the peak (4.30-4.50 ppm) derived from the ⁇ -position of the amide group of the polypeptide. was calculated, it was confirmed that the degree of polymerization was equivalent to 98.
  • a heavy solvent was prepared by adding trimethylsilylpropionic acid (TMSP) and adding 20 mg of 20% deuterated hydrochloric acid and 0.4 mL of THF per 0.6 mL of heavy water. 0.6 mL of the deuterated solvent was added to 5 mg of the desired product, and the dissolved sample was used to confirm the structure by 1 H-NMR analysis. )20.
  • TMSP trimethylsilylpropionic acid
  • DPPE-DBCO-AZ-pGlu as a whitish-yellow powder was obtained in the same manner as in Example 1-3 except that DPPE-DBCO (184 mg, 188 ⁇ mol, Compound 49) was used instead of DSPE-EG 4 -DBCO. 519 mg of (DET-C2-Car)20 (compound 50) was obtained.
  • a white yellow powder of DSGE-DBCO-AZ-pGlu was obtained in the same manner as in Example 1-4 except that DSGE-DBCO (111 mg, 126 ⁇ mol, compound 51) was used instead of DSPE-EG 4 -DBCO. 666 mg of (DET-C2-Car)30 (compound 52) was obtained.
  • the structure was confirmed by 1 H-NMR analysis in the same manner as in Example 1-3, and confirmed to be DSPE-DBCO-AZ-pGlu(DET-C2-Car)20.
  • 27 mg of terminally acetylated DSPE-DBCO-AZ-pGlu(DET-C2-Car)30 (compound 57) as white yellow powder was obtained by the same procedure as in Example 1-3 except that
  • the structure was confirmed by 1 H-NMR analysis in the same manner as in Production Example 2, and it was confirmed to be terminally acetylated DSGE-pGlu(DET-C2-Car)30.
  • the peak (0.80-0.95 ppm) derived from the methyl group at the lipid terminal is used as the standard value (6H), and the degree of polymerization is calculated from the integrated value of the peak (4.30-4.50 ppm) derived from the ⁇ -position of the amide group of the polypeptide. was calculated, it was confirmed that the degree of polymerization was equivalent to 31.
  • Example 2-1 Preparation of solid lipid nanoparticles using DSPE-DBCO-AZ-pGlu(DET-C2-Car)20 First, 90% t-BuOH aqueous solution was used to prepare a 5 mM cholesterol solution and a 5 mM DOTAP solution. , 5 mM DOPE solutions were prepared, respectively. Then, the above cholesterol solution (45 ⁇ L), DOTAP solution (52.5 ⁇ L), DOPE solution (52.5 ⁇ L) and 90% t-BuOH aqueous solution (50 ⁇ L) were added to a 1.5 mL tube.
  • siRNA siGL3, sense: 5'-CUUACGCUGAGUACUUCGAdTdT-3' (SEQ ID NO: 1), antisense: 5'-UCGAAGUACUCAGCGUAAGdTdT-3' (SEQ ID NO: 2), manufactured by Hokkaido System Science Co., Ltd.
  • aqueous solution (20 ⁇ L)
  • 1.2 mM polymer aqueous solution (20 ⁇ L) of Example 1-3
  • pure water 10 ⁇ L
  • the prepared mixed solutions were stirred and mixed, and the resulting solution was added dropwise to 20 mM HEPES buffer (pH 7.4, 2 mL) with stirring to form solid lipid nanoparticles.
  • the resulting solid lipid nanoparticles were purified by ultrafiltration (Amicon Ultra-15 (MWCO 50 kDa) manufactured by Merck Co., Ltd.) using a PBS buffer.
  • Example 2-2 Preparation of solid lipid nanoparticles using DSPE-DBCO-AZ-pGlu(DET-C2-Car) 20 2.0 mM Example 1-3 Except for changing to the aqueous polymer solution (20 ⁇ L) , solid lipid nanoparticles were prepared in the same manner as in Example 2-1.
  • Example 2-3 Preparation of solid lipid nanoparticles using DSPE-DBCO-AZ-pGlu(DET-C2-Car)30 1.0 mM instead of the aqueous polymer solution in Example 1-3, 1.0 mM Solid lipid nanoparticles were prepared in the same manner as in Example 2-1, except that the aqueous polymer solution (20 ⁇ L) of Example 1-4 was used.
  • Example 2-4 Preparation of solid lipid nanoparticles using DSPE-DBCO-AZ-pGlu(DET-C2-Car) 30 2.0 mM
  • Example 1-4 Except for changing to the aqueous polymer solution (20 ⁇ L) , solid lipid nanoparticles were prepared in the same manner as in Example 2-3.
  • Example 2-5 Preparation of solid lipid nanoparticles using DSPE-DBCO-AZ-pGlu(DET-C2-Car) 30 3.0 mM Example 1-4 Except for changing to the aqueous polymer solution (20 ⁇ L) , solid lipid nanoparticles were prepared in the same manner as in Example 2-3.
  • Example 2-6 Preparation of solid lipid nanoparticles using DSPE-DBCO-AZ-pGlu(DET-C2-Car) 30 4.0 mM Example 1-4 Except for changing to the aqueous polymer solution (20 ⁇ L) , solid lipid nanoparticles were prepared in the same manner as in Example 2-3.
  • Example 2-7 Preparation of solid lipid nanoparticles using DSPE-DBCO-AZ-pGlu(DET-C2-Car)30 -3' (SEQ ID NO: 3), antisense: 5'-Alexa647-UCGAAGUACUCAGCGUAAGdTdT-3' (SEQ ID NO: 4), manufactured by Gene Design Co., Ltd.) The same method as in Example 2-3. prepared solid lipid nanoparticles.
  • Example 2-8 Preparation of solid lipid nanoparticles using DSPE-DBCO-AZ-pGlu(DET-C2-Car)30 5), antisense: 5'-UAUUUAAgGAGGGUGAuCUUU-3' (SEQ ID NO: 6), manufactured by Genedesign), was used to prepare solid lipid nanoparticles in the same manner as in Example 2-3.
  • siPLK1 sequences lower case letters indicate that the base portion is the same, but the sugar portion is modified with 2′-O-methylation.
  • Example 2-1 Preparation of solid lipid nanoparticles using DSPE-PEG5k (SUNBRIGHT DSPE-050CN, manufactured by NOF Corporation)
  • Example 1-3 Polymer aqueous solution (20 ⁇ L) instead of 2.0 mM DSPE- Solid lipid nanoparticles were prepared in the same manner as in Example 2-1, except that the PEG5k aqueous solution (20 ⁇ L) was used.
  • Example 2-5 Preparation of Solid Lipid Nanoparticles Using DSPE-PEG5k Instead of the aqueous polymer solution (20 ⁇ L) in Example 1-3, 1.0 mM DSPE-PEG5k aqueous solution (20 ⁇ L) was used, and the siRNA used was siPLK1 ( sense: 5'-AGAuCACCCuCCUuAAAuAUU-3' (SEQ ID NO: 5), antisense: 5'-UAUUUAAgGAGGGUGAuCUUU-3' (SEQ ID NO: 6), manufactured by Gene Design Co., Ltd.)
  • siPLK1 sense: 5'-AGAuCACCCuCCUuAAAuAUU-3' (SEQ ID NO: 5), antisense: 5'-UAUUUAAgGAGGGUGAuCUUU-3' (SEQ ID NO: 6), manufactured by Gene Design Co., Ltd.
  • Example 2-1 solid lipid nanoparticles were prepared in a similar manner.
  • the relative diffusion coefficient is a value calculated from (diffusion coefficient of polymer in the presence of heparin) ⁇ (diffusion coefficient of polymer in the absence of heparin). That is, when the relative diffusion coefficient is less than 1, it indicates that the anionic heparin interacts with the polymer, suggesting that the polymer is cationic.
  • a relative diffusion coefficient of less than 0.95 is defined as cationic. All the polymers exhibited cationic properties with a relative diffusion coefficient of less than 0.95 at pH 6.0 to pH 6.5. Furthermore, it was confirmed that the polymers of Production Examples 4 to 7 showed a relative diffusion coefficient of 0.95 or more at pH 7.4, and that their cationic properties almost disappeared. This result indicates that the polymer responds to minute pH changes. On the other hand, the polymers of Comparative Examples 1-1 and 1-2 had a relative diffusion coefficient of less than 0.95 even at pH 7.4, demonstrating that they maintain their cationic properties.
  • the particles of Examples 2-2 and 2-7 were 9.0 mV and 4.9 mV, respectively, at pH 7.4, whereas they were 14.8 mV and 19.2 mV, respectively, at pH 6.5. From this result, it was found that the particles obtained from the pH-responsive lipid derivative of the present invention became cationic in response to minute changes in pH.
  • the particles of Comparative Examples 2-2 and 2-4 were 0.2 mV and 4.2 mV, respectively, at pH 7.4, whereas they were -2.9 mV and 2.3 mV at pH 6.5. From this result, it was found that the particles obtained from conventional PEG lipids that did not contain the pH-responsive lipid derivative of the present invention maintained neutrality regardless of pH.
  • siRNA Encapsulation Efficiency The siRNA encapsulation efficiency in particles of Examples 2-2, 2-7, Comparative Examples 2-2 and 2-4 was evaluated by a fluorometric method using a RiboGreen reagent. bottom. The fluorescence intensity derived from the RiboGreen reagent in the particle solutions prepared in Examples 2-2, 2-7 and Comparative Examples 2-2 and 2-4, and the fluorescence intensity derived from the RiboGreen reagent in a system in which each particle was disrupted by Triton X treatment The amount of siRNA encapsulation was calculated by comparing the fluorescence intensity. Table 3 shows the siRNA encapsulation efficiency.
  • the siRNA encapsulation efficiency was 93.0% in Example 2-2, 95.0% in Example 2-7, 98.4% in Comparative Example 2-2, and 90.0% in Comparative Example 2-4. .
  • the results show that both the particles obtained from the pH-responsive lipid derivative of the present invention and the particles obtained from the conventional PEG lipid have approximately the same siRNA encapsulation efficiency.
  • Example 3 Cellular uptake test of siRNA The efficiency of uptake of siRNA into cells using the particles of Example 2-2 and Comparative Example 2-2 was evaluated. Each particle was separately prepared using Cy5-labeled siRNA according to the method described in each example. A549 cells were plated at 50,000 cells/well (24-well plate) and cultured overnight. Each particle encapsulating Cy5-labeled siRNA was applied thereto (final siRNA concentration: 100 nM), incubated for 4 hours in a pH 7.4 or pH 6.5 serum medium, and the amount of siRNA uptake was evaluated using a flow cytometer. . The results are shown in FIG.
  • Example 2-2 In the cells to which the particles of Example 2-2 were applied, a greater amount of Cy5-derived fluorescence was observed at pH 6.5 than at pH 7.4. Furthermore, the particles of Example 2-2 show an equivalent amount of siRNA uptake at pH 7.4 as compared with the particles of Comparative Example 2-2, while the amount of siRNA uptake at pH 6.5 increases by about 40%. Gave.
  • the particles containing the pH-responsive lipid derivative of the present invention became cationic in response to minute changes in pH, as compared with particles using conventional PEG lipids. It was shown that the amount of uptake was improved.
  • Example 4 Pharmacokinetics test of siRNA Tumor accumulation of siRNA using the particles of Example 2-7 and Comparative Example 2-4 was evaluated.
  • Tumor-bearing model mice were prepared by subcutaneously transplanting mouse colon cancer cell line CT26 into BALB/c mice (5 ⁇ 10 5 cells/mouse). After the tumor size increased to about 200 mm 3 , each particle was injected into the tail vein (dosage: 0.5 mg/kg siRNA equivalent). Tumors were excised 1, 6 and 24 hours after administration. Tumors were homogenized using Lysis buffer and centrifuged. Next, the supernatant was taken out and adjusted to a concentration of 3% sodium lauryl sulfate and 60% t-BuOH.
  • the concentration of siRNA in the resulting solution was determined using a microplate reader (SPARK TKS01, manufactured by TECAN) (excitation wavelength: 630 nm, fluorescence wavelength: 690 nm). The results are shown in FIG. The statistically significant difference was confirmed using a 2-way ANOVA with Tukey's multiple comparisons method, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001. It was determined to be significant.
  • siRNA was detected at all times after 1, 6, and 24 hours from administration, whereas in the particles of Comparative Example 2-4, siRNA was not detected after 6 hours from administration. rice field.
  • the particles containing the pH-responsive lipid derivative of the present invention became cationic in response to minute changes in pH when reaching the tumor tissue, compared with particles using conventional PEG lipids. As a result, it was shown that it stays around the tumor tissue, that is, it has the property of accumulating in the tumor tissue. In addition, since siRNA was detected in tumor tissue even after 6 and 24 hours, it was shown to have blood retention.
  • Example 5 Antitumor effect using siPLK1
  • the antitumor effect of siPLK1 was evaluated using the particles of Example 2-8 and Comparative Example 2-5.
  • Tumor-bearing model mice were prepared by subcutaneously transplanting human ovarian cancer cell line SKOV3-luc cells into Balb/c nude mice (5 ⁇ 10 6 cells/mouse). When the tumor size reached about 25 mm 3 , the tumors were randomized and divided into three groups: the particle-administered group of Example 2-8, the particle-administered group of Comparative Example 2-5, and the control group, and injected into the tail vein. .
  • the single dose was 2.5 mg/kg in terms of siPLK1, and was administered once every two days (9 times in total).
  • a transport carrier capable of achieving an introduction system capable of safely and efficiently delivering low-molecular drugs, nucleic acid drugs, etc. to the vicinity of tumor tissue and transfecting tumor cells. It is possible to provide a pH-responsive transport carrier effective as a pH-responsive lipid derivative useful for forming such a transport carrier.
  • the pH-responsive transport carrier of the present invention has the property of becoming electrically neutral in a neutral environment (pH 7.4) and becoming cationic under weakly acidic conditions (pH 6.5) around tumor tissue. Therefore, it exhibits stealth properties against blood components and normal tissues, which are in a neutral environment in vivo, and improves the efficiency of accumulation and uptake into tumor tissues in response to minute changes in pH around tumor tissues. do. As a result, the low-molecular-weight drug or nucleic acid drug encapsulated in the transport carrier can be released in the vicinity of the tumor tissue or efficiently introduced into the tumor tissue cells.
  • the pH-responsive lipid derivative of the present invention is composed of an amino acid polymer contained in vivo. Therefore, after delivery of low-molecular-weight drugs and nucleic acid drugs, they are degraded in vivo and do not accumulate in vivo.
  • This application is based on Japanese Patent Application No. 2021-113189 (filing date: July 7, 2021) filed in Japan, the contents of which are hereby incorporated by reference.

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