WO2020116640A1 - Drug conjugate, polymer conjugate, and drug delivery composition - Google Patents

Abstract

The present invention provides a technology for efficiently delivering drugs to cells or mitochondria. This drug conjugate includes a drug and a target-seeking domain bound to the drug, wherein the target-seeking domain includes a phosphocholine group represented by formula (I).

Description

Drug conjugate, polymer conjugate and drug delivery composition

The present invention relates to a drug conjugate and a polymer conjugate containing a phosphocholine group, and a drug delivery composition containing the polymer conjugate.

Generally, when a drug is systemically administered by oral or intravenous injection, the drug accumulates not only in the affected part of the drug but also in normal tissues. As a result, side effects due to drug administration are observed, and it may be necessary to change or interrupt the treatment method. On the other hand, a drug delivery system (DDS) that selectively transports a drug to an affected area has been developed for the purpose of reducing side effects.

　To increase the amount of drug accumulated in the affected area, the development of ligand molecules that direct the affected area is underway. In the past, research focused on increasing cell uptake, but in recent years, there has been much interest in drug delivery to mitochondria in cells, and it is difficult to cure drug delivery of proteins and nucleic acids to mitochondria. Treatment of sexual disorders is expected. Regarding drug delivery into cells and mitochondria, for example, techniques utilizing triphenylphosphonium as a ligand molecule have been investigated (for example, Non-Patent Documents 1 to 3).

However, in the above technology, control of biodistribution is insufficient, and development of a technology for efficiently delivering a drug to cells or mitochondria is desired.

Mol. Pharmaceutics, 2014, 11(8), 2640-2649. Chem. Commun. , 2017, 53, 8790-8793 J. Am. Chem. Soc. , 2015, 137(18), 5930-5938

The present invention has been made to solve the above problems, and its main purpose is to provide a technique for efficiently delivering a drug into cells or mitochondria.

The inventors of the present invention have conducted extensive studies to solve the above problems, and by introducing a targeting site containing a phosphocholine group into a drug to be delivered or a carrier carrying the drug or a component thereof, cancer cells and mitochondria The inventors have found that the directivity to can be imparted, and have completed the present invention.

　本発明の別の局面によれば、薬物送達用ポリマーと該薬物送達用ポリマーに結合された標的指向性部位とを含む、ポリマー複合体であって、該標的指向性部位が、上記式（Ｉ）で表されるホスホコリン基を含む、ポリマー複合体が提供される。
　１つの実施形態において、上記薬物送達用ポリマーが、親水性ポリマーセグメントと疎水性ポリマーセグメントとを含む。
　１つの実施形態において、上記ポリマー複合体には、さらに薬物が結合されている。
　本発明のさらに別の局面によれば、上記ポリマー複合体を含む、薬物送達用組成物が提供される。
　１つの実施形態において、上記薬物送達用組成物は、薬物をさらに含む。
　本発明のさらに別の局面によれば、送達対象化合物と、上記式（Ｉ）で表されるホスホコリン基を有する化合物と、を結合させる工程を含む、標的指向性を有する化合物の製造方法が提供される。
　本発明のさらに別の局面によれば、送達対象物を上記式（Ｉ）で表されるホスホコリン基を含む標的指向性部位で修飾することを含む、送達対象物に細胞又はミトコンドリアへの指向性を付与する方法が提供される。
　１つの実施形態において、上記送達対象物が、リポソーム、高分子ミセル、ポリイオンコンプレックス、ポリプレックス、リポプレックス、リポポリプレックス、無機金属粒子、脂質ナノ粒子及びゲルから選択される。
　本発明はまた、細胞又はミトコンドリアへの指向性を有する化合物の製造のための、上記式（Ｉ）で表されるホスホコリン基又は上記式（Ｉ）で表されるホスホコリン基を有する化合物の使用に関する。
　本発明のさらに別の局面によれば、標的指向性部位を含み、標識物質によって標識化された検出試薬であって、該標的指向性部位が、上記式（Ｉ）で表されるホスホコリン基を含む、検出試薬が提供される。 That is, according to one aspect of the present invention, a drug complex comprising a drug and a targeting site bound to the drug, wherein the targeting site is represented by the following formula (I): Drug conjugates are provided that include a phosphocholine group.

According to another aspect of the present invention, there is provided a polymer conjugate comprising a drug delivery polymer and a targeting moiety bound to the drug delivery polymer, wherein the targeting moiety is represented by the above formula (I The polymer conjugate containing the phosphocholine group represented by these is provided.
In one embodiment, the drug delivery polymer comprises a hydrophilic polymer segment and a hydrophobic polymer segment.
In one embodiment, the polymer conjugate further has a drug bound thereto.
According to still another aspect of the present invention, there is provided a composition for drug delivery, which comprises the polymer conjugate.
In one embodiment, the drug delivery composition further comprises a drug.
According to still another aspect of the present invention, there is provided a method for producing a compound having targeting property, which comprises a step of binding a compound to be delivered and a compound having a phosphocholine group represented by the above formula (I). To be done.
According to still another aspect of the present invention, the target of delivery is directed to cells or mitochondria, which comprises modifying the target of delivery with a targeting site containing a phosphocholine group represented by the above formula (I). A method of granting is provided.
In one embodiment, the delivery target is selected from liposomes, polymeric micelles, polyion complexes, polyplexes, lipoplexes, lipopolyplexes, inorganic metal particles, lipid nanoparticles and gels.
The present invention also relates to the use of a compound having a phosphocholine group represented by the above formula (I) or a compound having a phosphocholine group represented by the above formula (I) for the production of a compound having a directivity to cells or mitochondria. ..
According to still another aspect of the present invention, there is provided a detection reagent comprising a targeting site and labeled with a labeling substance, wherein the targeting site has a phosphocholine group represented by the above formula (I). Detection reagents are provided that include.

In designing a DDS carrier, generally, a positive charge is favorable for electrostatic interaction with a negatively charged cell membrane, and a hydrophobic property is preferable because it has high affinity with a hydrophobic cell membrane surface. it is conceivable that. Triphenylphosphonium, which has been used as the conventional targeting site, is in line with the design policy, is hydrophobic, and has a positive charge. On the other hand, according to the present invention, the charge is neutralized as a whole, and by utilizing a hydrophilic phosphocholine group as the targeting site, the targeting site is controlled while controlling the biodistribution. Good cell uptake and directivity to mitochondria can be imparted to the bound drug or carrier. This is because the phosphocholine group whose charge is neutralized can reduce non-specific interactions with normal tissues and proteins and can utilize the phospholipid and phospholipid derivative uptake functions of cells and mitochondria. Conceivable.

It is a graph which shows the result of a cell uptake test of PIC micelle, a competition test, and an inhibition test. It is a graph which shows the localization level to the mitochondria of PIC micelles. 3 is a graph showing cell viability and IC ₅₀ . It is a graph which shows the gene expression efficiency of mRNA inclusion micelle. Fig. 6 is a graph showing the cellular uptake level of albumin PC conjugate. 3 is a graph showing the localization level of albumin PC conjugate in mitochondria. It is a TEM observation image which shows transfer of gold nanoparticles loaded with PC to mitochondria.

Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited to these embodiments. Further, the respective embodiments can be combined appropriately.

A. Drug Conjugate A drug conjugate (conjugate) in one embodiment of the present invention comprises a drug and a targeting site bound to the drug, and the targeting site is represented by the following formula (I). Containing a phosphocholine group.

The drug is not particularly limited, and a drug having a desired activity can be used. Preferably, a drug that is desired to be delivered intracellularly, for example to mitochondria, is used. In the present specification, a drug refers to a substance having some physiological activity. The physiological activity possessed by the drug may be any physiological activity that can function as an active ingredient of a drug, and examples thereof include antitumor activity, immunostimulatory activity, antiviral activity, antibacterial activity, and anti-inflammatory activity. The drug may be a protein such as an enzyme, hormone, vaccine, antibody, mRNA, pDNA, antisense, ribozyme, siRNA, decoy nucleic acid, nucleic acid such as aptamer, and high molecular drug such as polysaccharide.

The drug and the targeting moiety may be directly bonded or indirectly bonded via a linker moiety.

（式ＩＩにおいて、Ｌ^０は、単結合又は二価の原子団を表し、Ｄは、薬物の残基を表し、ｓは、１～２０００の整数を表す。） In one embodiment, the drug conjugate of the present invention can be represented by the following formula (II).

(In Formula II, L ⁰ represents a single bond or a divalent atomic group, D represents a drug residue, and s represents an integer of 1 to 2000.)

The divalent atomic group that can be represented by L ⁰ is not particularly limited as long as the effects of the present invention can be obtained. The divalent atomic group is, for example, an atomic group formed by a reaction of a compound having a functional group capable of binding with a functional group of a drug, which will be described later, and a targeting site, and the drug. And the residue of the compound of interest. The divalent atomic group includes, for example, a linear or branched alkylene group having 1 to 6 carbon atoms, —COO—, —CONH—, —NH—, —CO—, —O—, —S—, and these It can be any combination. The number of atoms in the main chain of the divalent atomic group can be, for example, 1 to 20, preferably 1 to 15, and more preferably 1 to 10.

S represents the number of targeting sites bound to the drug (the number of bonds per molecule). s can be appropriately selected depending on the chemical structure, three-dimensional structure, molecular weight, etc. of the drug. For example, when the drug has a large molecular weight (such as when the drug is a macromolecular drug), s may be 2 or more, for example, 2 to 200, or 2 to 100 or 2 to 50.

The above drug conjugate may be prepared by any suitable method. For example, by using a compound having a functional group capable of binding to a functional group of a drug and a targeting site, by reacting the functional group of the compound with the functional group of the drug, a targeting site can be obtained. A drug complex bound with a drug can be obtained. In this case, the functional group possessed by the drug may be intrinsic in the drug or may be additionally introduced. Specific examples of the combination of the functional groups include, for example, thiol group and (meth)acryloyl group, thiol group and maleimide group, thiol group and thiol group, thiol group and carboxyl group, (meth)acryloyl group and hydroxyl group, ( Examples thereof include a (meth)acryloyl group and an amino group, a carboxyl group and an amino group, a carboxyl group and a hydroxyl group, and an amino group and a hydroxyl group.

As the compound having the functional group capable of binding to the functional group of the drug and the targeting site, any appropriate compound having the functional group and the phosphocholine group can be used. Specific examples include 2-(meth)acryloyloxyethylphosphorylcholine, 3-(meth)acryloyloxypropylphosphorylcholine, 4-(meth)acryloyloxybutylphosphorylcholine, 6-(meth)acryloyloxyhexylphosphorylcholine, 10-(meth) Acryloyloxydecylphosphorylcholine, ω-(meth)acryloyl(poly)oxyethylenephosphorylcholine, 2-(meth)acrylamidoethylphosphorylcholine, 3-(meth)acrylamidopropylphosphorylcholine, 4-(meth)acrylamidobutylphosphorylcholine, 6-(meth) Examples thereof include acrylamidohexylphosphorylcholine, 10-(meth)acrylamidedecylphosphorylcholine, and ω-(meth)acrylamide(poly)oxyethylenephosphorylcholine.

B. Polymer Conjugate According to another aspect of the present invention, comprising a drug delivery polymer and a targeting moiety bound to the drug delivering polymer, the targeting moiety is represented by the above formula (I). Polymer conjugates comprising phosphocholine groups are provided.

As the polymer for drug delivery, any suitable polymer applicable in the field of DDS can be used, and for example, a conventionally known known polymer for drug delivery can be preferably used.

Examples of the known drug delivery polymer include a chargeable polymer capable of forming a polyion complex (PIC) by associating with a charged drug by electrostatic interaction, and a block copolymer capable of forming a drug-encapsulating polymer micelle. Preferable examples include, biocompatible polymers capable of forming microparticles capable of supporting a drug, and water-soluble polymers used for modifying a delivery target.

B-1. Complex Using Chargeable Polymer As the chargeable polymer capable of forming PIC by associating with a drug having a charge by the electrostatic interaction, a cationic polymer and an anionic polymer may be used depending on the kind of charge as the polymer. It is divided into

The above cationic polymer is a polymer having a cationic group and having a positive charge at physiological pH. The cationic polymer may have some anionic groups as long as the cationic properties of the polymer as a whole are not hindered.

The cationic polymer may be composed of a single repeating unit, and may contain two or more kinds of repeating units in any combination and ratio. The cationic polymer may be a polymer containing an amino group in the main chain or a polymer containing an amino group in the side chain.

Examples of polymers containing an amino group in the main chain include polyethyleneimine.

Examples of the polymer having an amino group in the side chain include a polyamino acid containing an amino acid having an amino group in the side chain as a monomer unit or a derivative thereof. Examples of the polyamino acid containing an amino acid having an amino group in the side chain as a monomer unit or a derivative thereof include polyaspartamide, polyglutamide, polylysine, polyarginine, polyhistidine, and derivatives thereof. By reacting polyaspartic acid (or polyglutamic acid) with 1,5-diaminopentane, poly(Asp-AP) (or poly(Asp-AP)) in which aminopentane (AP) is introduced into the side chain carboxylic acid of aspartic acid (glutamic acid) (Glu-AP)) and polyaspartic acid (or polyglutamic acid) are reacted with DET (H ₂ NCH ₂ CH ₂ NH-CH ₂ CH ₂ NH ₂ ) to give a side chain carboxylic acid of aspartic acid (or glutamic acid). Poly(Asp-DET) (or poly(Glu-DET)) in which DET is introduced is preferably used.

The polyamino acid or its derivative containing an amino acid having an amino group in its side chain as a monomer unit may further contain an uncharged amino acid having a hydrophobic group in its side chain as a monomer unit, if necessary. Examples of the uncharged amino acid having a hydrophobic group in its side chain include amino acids having a solubility of 5 g or less in 100 g of water at 25° C., and more preferably 4 g or less. Examples of such amino acids include non-polar natural amino acids such as leucine, isoleucine, phenylalanine, methionine, and tryptophan, and hydrophobic derivatives of amino acids having a hydrophobic group introduced into their side chains. The hydrophobic derivative of an amino acid is preferably a hydrophobic derivative of an acidic amino acid such as aspartic acid or glutamic acid. Examples of the introduced hydrophobic group include a saturated or unsaturated linear or branched aliphatic hydrocarbon group having 6 to 27 carbon atoms, an aromatic hydrocarbon group having 6 to 27 carbon atoms, or a cholesterol residue. It can be preferably exemplified.

For a more detailed description of a polyamino acid or a derivative thereof containing an amino acid having an amino group in a side chain as a monomer unit, WO 2006/085664, WO 2010/093036, WO 2011/105402, etc. (the teachings of these applications are (Incorporated herein by reference).

The above anionic polymer is a polymer having an anionic group and having a negative charge at physiological pH. The anionic polymer may have some cationic groups as long as the anionicity of the polymer as a whole is not impaired.

The anionic polymer may be composed of a single repeating unit, and may contain two or more kinds of repeating units in any combination and ratio. Examples of the anionic polymer include a polymer containing a monomer unit containing a carboxyl group, a polymer containing a monomer unit containing a sulfate group, a polymer containing a monomer unit containing a phosphoric acid group, and the like. The monomer unit containing a carboxyl group is preferably an amino acid containing a carboxyl group in its side chain, and examples thereof include aspartic acid and glutamic acid.

-For the above-mentioned charged polymer, the targeting site is bound to any appropriate site. For example, it may be attached to one or both ends of the polymer, or it may be introduced into a side chain. The number of targeting moieties bound to the polymer (the number of bonds per molecule of the polymer) is not limited as long as the effect of the present invention can be obtained, and may be 1 or 2 or more. The binding between the polymer and the targeting moiety is performed by using a compound having a functional group capable of binding to a functional group of the polymer and the targeting moiety, and linking the drug according to the item A with the targeting moiety. It can be performed in the same manner as the coupling.

B-2. Complex Using Block Copolymer The block copolymer capable of forming the polymer micelle capable of encapsulating the drug is typically a block copolymer containing a hydrophilic polymer segment and a hydrophobic polymer segment, and preferably these It is a block copolymer in which segments are connected in series.

As the polymer constituting the hydrophilic polymer segment, polyethylene glycol, polypropylene glycol, poly(2-oxazoline), polysaccharide, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, polymethacrylamide, polyacrylic acid ester, polymethacrylic acid ester, etc. And polyethylene glycol can be preferably used. The hydrophilic polymer segment may be linear or branched.

As a polymer that constitutes the hydrophobic polymer segment, the block copolymer is more hydrophilic than the hydrophilic polymer segment to the extent that it can form micelles with the hydrophilic polymer segment facing outward and the hydrophobic polymer segment facing inward in an aqueous solvent. A polymer having a low hydrophilicity is also selected. Examples of such polymers include polyglycolic acid (PGA), polylactic acid (PLA) and its copolymers (PLGA), polyamino acids and their derivatives, polyethers and their derivatives, and polyamino acids and their derivatives. , Polyethers and their derivatives can be preferably used.

As the polyamino acid, one or more amino acids selected from amino acids containing an amino group in the side chain, uncharged amino acids containing a hydrophobic group in the side chain, amino acids containing a carboxyl group in the side chain, or A polyamino acid containing a derivative as a monomer unit or a derivative thereof can be preferably used. Examples of the amino acid having an amino group in its side chain include basic amino acids such as lysine, arginine, histidine, ornithine, and amino acid derivatives having an amino group introduced into the side chain of acidic amino acids such as aspartic acid and glutamic acid. The uncharged amino acid containing a hydrophobic group in the side chain and the amino acid containing a carboxyl group in the side chain are as described above. As the polyamino acid, the chargeable polymer described in the section B-1 can also be used.

The above-mentioned polyether includes polyglycidyl ether having a side chain structure.

Specific examples of the block copolymers are described in WO2007/099660, WO2007/099661, WO2010/093036, WO2012/096399, WO2014/133172, WO2015/170757, etc. Incorporated by reference).

With respect to the block copolymer, the targeting site can be bound to any appropriate site, and is preferably bound to the end on the hydrophilic polymer segment side. The number of targeting moieties attached to the polymer is not limited as long as the effects of the present invention can be obtained, and may be 1 or 2 or more. The polymer and the targeting moiety can be bound by the same method as the method described in Section A for binding the drug and the targeting moiety.

A polymer conjugate having a structure in which a targeting site is bound to a block copolymer can be represented by, for example, the formula: ZA ¹ -B ¹ (wherein Z is a phosphocholine represented by the formula (I)). Represents a group, A ¹ represents a hydrophilic polymer segment, and B ¹ represents a hydrophobic polymer segment).

（上記式中、
Ｚは、式（Ｉ）で表されるホスホコリン基を表し；
Ｌ^１、Ｌ^２、Ｌ^３、及びＬ^４はそれぞれ独立して、二価の連結基を表し；
Ｒ^１は、水素原子、未置換又は置換された直鎖もしくは分枝の炭素数１～１２のアルキル基あるいは未置換又は置換された直鎖もしくは分枝の炭素数１～２４のアルキルカルボニル基を表し；
Ｒ^２は、水酸基、オキシベンジル基、－Ｏ－Ｒ^２ａ又はＮＨ－Ｒ^２ｂ基を表し、ここでＲ^２ａ又はＲ^２ｂはそれぞれ独立して、未置換又は置換された直鎖もしくは分枝の炭素数１～１２アルキル基を表し；
Ｒ^３ａ、Ｒ^３ｂ、Ｒ^４ａ及びＲ^４ｂは、相互に独立して、メチレン基又はエチレン基を表し；
Ｒ^５ａ及びＲ^５ｂは、相互に独立して、－Ｏ－又はＮＨ－を表し；
Ｒ^６ａ及びＲ^６ｂは、相互に独立して、水素原子又は疎水性有機基を表し；
Ｒ^７ａ及びＲ^７ｂは、相互に独立して、下記の基：
－ＮＨ－（ＣＨ_２）_ｐ１－〔ＮＨ－（ＣＨ_２）_ｑ１－〕_ｒ１ＮＨ_２　　　（ｉ）；
－ＮＨ－（ＣＨ_２）_ｐ２－Ｎ〔－（ＣＨ_２）_ｑ２－ＮＨ_２〕_２　　　（ｉｉ）；
－ＮＨ－（ＣＨ_２）_ｐ３－Ｎ｛〔－（ＣＨ_２）_ｑ３－ＮＨ_２〕〔－（ＣＨ_２）_ｑ４－ＮＨ－〕_ｒ２Ｈ｝　　　（ｉｉｉ）；
－ＮＨ－（ＣＨ_２）_ｐ４－Ｎ｛－（ＣＨ_２）_ｑ５－Ｎ〔－（ＣＨ_２）_ｑ６－ＮＨ_２〕_２｝_２　　　（ｉｖ）；及び
－ＮＨ－（ＣＨ_２）_ｐ５－ＮＨ_２　　　（ｖ）
からなる群の同一もしくは異なる基から選ばれ、
ここで、ｐ１～ｐ５、ｑ１～６、及びｒ１～ｒ２は、それぞれ相互に独立して、１～５の整数であり；
　Ｒ^８は、リシン、オルニチン、アルギニン、ホモアルギニン、及びヒスチジンからなる群より選択されるアミノ酸の側鎖を表し；
　ｋは、２０～２０，０００の整数を表し；
　ａ、ｂ、ｃ、ｄ、及びｅは、それぞれ独立して、０～４００の整数であり；
　ただし、５≦ａ＋ｂ＋ｃ＋ｄ＋ｅ≦４００の関係を満たし；
　上記各アミノ酸繰り返し単位の結合順は任意であり；
　Ｒ^６ａ、Ｒ^６ｂ、Ｒ^７ａ、Ｒ^７ｂ及びＲ^８は、ポリマー分子内のアミノ酸繰り返し単位毎に任意に選択可能である。） Specific examples of the polymer composite are shown in the following formula (III) or (IV).

(In the above formula,
Z represents a phosphocholine group represented by the formula (I);
L ¹ , L ² , L ³ , and L ⁴ each independently represent a divalent linking group;
R ¹ is a hydrogen atom, an unsubstituted or substituted straight-chain or branched alkyl group having 1 to 12 carbon atoms, or an unsubstituted or substituted straight-chain or branched alkylcarbonyl group having 1 to 24 carbon atoms. Representation;
R ² represents a hydroxyl group, an oxybenzyl group, —O—R ^2a or NH—R ^2b group, wherein each of R ^{2a and} R ^2b independently represents an unsubstituted or substituted straight or branched carbon atom. Represents an alkyl group of 1 to 12;
R ^3a , R ^3b , R ^4a and R ^4b each independently represent a methylene group or an ethylene group;
R ^5a and R ^5b each independently represent -O- or NH-;
R ^6a and R ^6b independently of each other represent a hydrogen atom or a hydrophobic organic group;
R ^7a and R ^7b , independently of each other, are the following groups:
-NH-(CH ₂ ) _p1 -[NH-(CH ₂ ) _q1 -] _r1 NH ₂ (i);
-NH- _{(CH 2)} p2 -N _{[- (CH 2)} q2 -NH _{2] 2} (ii);
-NH-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NH ₂ ][-(CH ₂ ) _q4 -NH-] _r2 H} (iii);
-NH-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NH ₂ ] ₂ } ₂ (iv); and -NH-(CH ₂ ) _p5- NH ₂ ( v)
Selected from the same or different groups of the group consisting of
Here, p1 to p5, q1 to 6, and r1 to r2 are each independently an integer of 1 to 5;
R ⁸ represents a side chain of an amino acid selected from the group consisting of lysine, ornithine, arginine, homoarginine, and histidine;
k represents an integer of 20 to 20,000;
a, b, c, d, and e are each independently an integer of 0 to 400;
However, the relationship of 5≦a+b+c+d+e≦400 is satisfied;
The binding order of the above-mentioned amino acid repeating units is arbitrary;
R ^6a , R ^6b , R ^7a , R ^7b and R ⁸ can be arbitrarily selected for each amino acid repeating unit in the polymer molecule. )

L ¹ and L ³ are each independently, for example, a linear or branched alkylene group having 1 to 6 carbon atoms, —COO—, —CONH—, —NH—, —CO—, —O—, —S. -, and any combination thereof. The number of main chain atoms of the divalent linking group can be, for example, 1 to 20, preferably 1 to 15, and more preferably 1 to 10. Specific examples include -SCH ₂ CHCOH-, -SS-, -SCO-, -OCH ₂ CHCOH-, -NHCH ₂ CHCOH-, -NHCOO-, -CH ₂ CH ₂ SCH ₂ CH(CH ₃ )COOCH ₂ CH. _2- and the like.

The ^{L 2} is, for ^{example, -NH -, - O -,} - O-L 2a -NH -, - CO -, - CH 2 -, and ^{-NH- O-L 2a -S-L} 2a ( here, L ^2a is independently an alkylene group having 1 to 6 carbon atoms).

The above L ⁴ may be, for example, a linking group selected from —OCO—L ^4a —CO—, and NHCO—L ^4a —CO— (wherein L ^4a is an alkylene group having 1 to 6 carbon atoms).

Examples of the linear or branched alkyl group having 1 to 12 carbon atoms, which is defined by the groups of R ¹ , R ^2a and R ^2b , include, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n -Butyl group, sec-butyl group, tert-butyl group, n-hexyl group, decyl group, undecyl group and the like can be mentioned.

The straight-chain or branched alkyl moiety having 1 to 12 carbon atoms in the straight-chain or branched alkylcarbonyl group having 1 to 24 carbon atoms defined by the group of R ¹ can be referred to the above-mentioned examples. Examples of the alkyl moiety having 13 or more carbon atoms include a tridecyl group, a tetradecyl group, a pentadecyl group, a nonadecyl group, a docosanyl group, and a tetracosyl group.

With respect to the above alkyl group or alkyl moiety, the substituent in the case of “substituted” is not limited, but is a C _1-6 alkoxy group, an aryloxy group, an aryl C _1-3 oxy group, a cyano group, a carboxyl group. Group, amino group, C _1-6 alkoxycarbonyl group, C _2-7 acylamido group, tri-C _1-6 alkylsiloxy group, siloxy group, silylamino group, or an acetalized formyl group, formyl group, chlorine or Halogen atoms such as fluorine can be mentioned. Here, for example, a display such as C _1-6 means that the carbon number is 1 to 6.

The hydrophobic organic group defined by the groups of R ^6a and R ^6b is, for example, a saturated or unsaturated linear or branched aliphatic hydrocarbon group having 6 to 27 carbon atoms, or an aryl group having 6 to 27 carbon atoms. A group or a residue derived from an aralkyl group or a sterol.

The R ^7a and R ^7b groups are preferably, independently of one another, a group of formula (i) or (v). In formula (i), it is preferable that p1 and q1 are each independently 2 or 3, and more preferably 2. On the other hand, r1 is preferably an integer of 1 to 3.

The above R ⁸ group is preferably a side chain of lysine or ornithine.

“A”, “b”, “c”, “d”, and “e” representing the number of repetitions of each amino acid residue are each independently an integer of preferably 0 to 300, more preferably an integer of 0 to 250. The polymerization degree (a+b+c+d+e) of the polyamino acid is preferably an integer of 10 to 300, more preferably an integer of 20 to 250.

K, which represents the repeating number of ethylene glycol, preferably represents an integer of 40 to 2,000, more preferably 45 to 1,000.

The polymer complex represented by the above formula (III) or (IV) can be obtained by, for example, purifying polyethylene glycol having a functional group at the α-terminal and a desired polyamino acid as they are, or if necessary, after purifying so as to narrow the molecular weight distribution. It can be formed by preparing a block copolymer by coupling by a known method, and utilizing a functional group at the α-terminal of the block copolymer to cause condensation or addition reaction of a compound having a targeting site. The binding of the targeting site may be performed after the micelle formation described below.

In addition, for example, for the polymer complex represented by the formula (III), polyethylene glycol having a functional group at the α-terminal and an amino group at the ω-terminal is prepared, and β-benzyl-L-aspartate is introduced from the amino terminal. , Γ-benzyl-L-glutamate, Nε-ZL-lysine, N-carboxylic acid anhydride (NCA) of a protected amino acid is polymerized, and if necessary, the side chain of the obtained polyamino acid is depolymerized. A block copolymer is prepared by protecting and/or introducing groups (i) to (v), and the functional group at the α-terminal of the block copolymer is used to condense or add a compound having a targeting moiety. It can be formed by reacting. The binding of the targeting site may be performed after the micelle formation described below. In addition, when synthesizing a cationic polyamino acid, a structural change (for example, formation of an imide ring by dealcoholization of an amino acid ester residue) may occur in a part of the amino acid ester residue during the synthesis due to a nucleophilic attack of polyamine. Although it may occur, in the present specification, the case where the block copolymer contains a residue that undergoes such a structural change is also included in the above formulas (III) and (IV). In addition, some NH groups and NH ₂ groups in the cationic amino acid residue may be converted to salts (mainly hydrochloride) due to the use of acid (mainly hydrochloric acid) in the synthetic process. In the text, even when the block copolymer contains such a structure, it is included in the above formulas (III) and (IV).

（上記式中、
Ｚは、式（Ｉ）で表されるホスホコリン基を表し；
Ｌ^５及びＬ^６はそれぞれ独立して、単結合又は二価の連結基を表し；
Ｒ^９は、水素原子、未置換又は置換された直鎖もしくは分枝の炭素数１～１２のアルキル基あるいは未置換又は置換された直鎖もしくは分枝の炭素数１～２４のアルキルカルボニル基を表し；
Ｒ^１０は、アルキレン基又はエステル結合を介した１級アミンを表し；
ｍは、５～２０，０００の整数を表し；
ｎは、２～５，０００の整数を表す。） Another specific example of the above polymer composite is shown in the following formula (V).

(In the above formula,
Z represents a phosphocholine group represented by the formula (I);
L ⁵ and L ⁶ each independently represent a single bond or a divalent linking group;
R ⁹ represents a hydrogen atom, an unsubstituted or substituted straight-chain or branched alkyl group having 1 to 12 carbon atoms, or an unsubstituted or substituted straight-chain or branched alkylcarbonyl group having 1 to 24 carbon atoms. Representation;
R ¹⁰ represents a primary amine through an alkylene group or an ester bond;
m represents an integer of 5 to 20,000;
n represents an integer of 2 to 5,000. )

Regarding R ⁹ described above, the same explanation as for R ¹ defined by the formula (III) is applied.

（上記式中、Ｒ^１１は水素原子、又は下記式（ＶＩＩＩ）

（式中、Ｒ^１２は炭素数１～２０の直鎖状若しくは分岐状の炭化水素基、置換基を有していてもよいフェニル基、又は置換基を有していてもよい複素環式官能基を表し、ｗは１～５の整数を表す。）で示される基を表し、ｘ、ｙはそれぞれ独立して、１～５の整数を表す。） Examples of the primary amine having an alkylene group or an ester bond defined for R ¹⁰ include those represented by the following formula (VI) or (VII).

(In the above formula, R ¹¹ is a hydrogen atom or the following formula (VIII)

(In the formula, R ¹² is a linear or branched hydrocarbon group having 1 to 20 carbon atoms, a phenyl group which may have a substituent, or a heterocyclic functional group which may have a substituent. Group, w represents an integer of 1 to 5, and x and y each independently represent an integer of 1 to 5. )

Examples of the heterocyclic functional group which may have a substituent defined for R ¹² above include an indolyl ring, a pyrrolidone group, a furan ring, a pyridine ring, a morpholine ring, an epoxy ring, a purine ring and a pyrimidine ring. Can be mentioned.

In one embodiment, x is, for example, 1, 2 or 3, and can be, for example, 1 or 2, and also 1, for example.

In one embodiment, y and w are each independently, for example, 1, 2 or 3, and can be, for example, 1 or 2, and also 1, for example. R ¹² can also be, for example, an indolyl ring.

L ⁵ is, for example, a linear or branched alkylene group having 1 to 6 carbon atoms, —COO—, —CONH—, —NH—, —CO—, —O—, —S—, or any of these. It can be a combination. The number of main chain atoms of the divalent linking group can be, for example, 1 to 20, preferably 1 to 15, and more preferably 1 to 10. Specific examples include —NHCOCH ₂ CH ₂ —, —CH ₂ CH ₂ NHCH ₂ CH ₂ —, —CH ₂ CH ₂ SCH ₂ CH ₂ —, and —CH ₂ CH ₂ OCH ₂ CH ₂ —.

The above L ⁶ is, for example, a single bond or —CH ₂ CH ₂ O—.

M is preferably an integer of 40 to 2,000, more preferably 45 to 1,000. Further, n represents an integer of preferably 10 to 300, more preferably 20 to 250.

The polymer complex represented by the above formula (V) is prepared, for example, by preparing polyethylene glycol having a functional group at the α-terminal, epichlorohydrin, 1,2-epoxy-5-hexene, 1-allyl-2,3. -Epoxy ring-opening polymerization using epoxy group-containing monomers such as epoxypropane, epibromohydrin, 3,4-epoxy-1-butane, 1,2-epoxy-9-decene, 2,3-epoxypropylpropargyl ether To obtain a block copolymer of polyethylene glycol and a polyglycidyl chain having a side chain, and utilizing the functional group at the α-terminal of the obtained block copolymer, condensation or addition of a compound having a targeting site It can be obtained by a method including reacting and introducing a primary amine into a side chain of a polyglycidyl chain of a block copolymer having a targeting site added thereto.

In one embodiment, the polymer conjugate may have a drug attached to it. The drug may be introduced at the side chains and/or the ends of the hydrophobic polymer segment of the block copolymer. The number of drugs bound to the polymer is not limited as long as the effects of the present invention can be obtained, and can be, for example, 1 to 200, preferably 2 to 100.

The polymer conjugate to which a drug is bound can be represented by, for example, the formula: ZA ² -B ² (-D) (wherein Z represents a phosphocholine group represented by the formula (I), A ² represents the hydrophilic polymer segment, B ² represents the hydrophobic polymer segment, and D represents the residue of the drug). Specific examples include an embodiment in which a drug is bound to the hydrophobic polymer side chain of the polymer complex represented by the above formulas (III) to (V).

For example, when the block copolymer has a carboxyl group in the side chain of the hydrophobic polymer segment (in formulas (III) and (IV), R ^5a ═O, R ^6a ═H and/or R ^5b ═O, R ^6b ═H In this case, by reacting the drug having a hydroxyl group with the carboxyl group, the drug can be bound to the side chain of the hydrophobic polymer segment of the block copolymer via an ester bond.

In addition, for example, a block copolymer having an ester group on the side chain of a hydrophobic polymer segment and a block copolymer having an amide bond formed by a reaction between a drug which is an amine or a carboxyl group on the side chain of the hydrophobic polymer segment, and an amino group The drug can be attached to the hydrophobic polymer segment side chain of the block copolymer by an amide bond formed by the reaction with the drug having a group.

B-3. Complex using biocompatible polymer Examples of the biocompatible polymer capable of forming the fine particles capable of supporting the drug include polyglycolic acid (PGA), polylactic acid (PLA) and copolymers thereof (PLGA). , Poly ε-caprolactone, chitosan and the like. For these polymers, the targeting site is attached to any suitable site. For example, it may be attached to one or both ends of the polymer, or it may be introduced into a side chain. The number of targeting moieties attached to the polymer is not limited as long as the effects of the present invention can be obtained, and may be 1 or 2 or more. The binding between the polymer and the targeting moiety is performed by using a compound having a functional group capable of binding to a functional group of the polymer and the targeting moiety, and linking the drug according to the item A with the targeting moiety. It can be performed in the same manner as the coupling.

B-4. Complex Using Water-Soluble Polymer The above-mentioned water-soluble polymer is used for modification of a delivery target for the purpose of solubilization, sustained release, improvement of blood retention, avoidance of enzymatic degradation, and the like. Examples of such water-soluble polymers include polyethylene glycol, polypropylene glycol and copolymers thereof, soluble proteins such as albumin, polysaccharides, etc. Among them, polyethylene glycol can be preferably used.

The target substance to be modified may be a drug or a carrier carrying a drug such as liposome, micelle, gel, gold nanoparticle and the like. For example, modification with polyethylene glycol (PEG modification) is well known in the art, and PEGylated proteins such as PEGylated interferon and PEG-modified liposomes can be obtained.

-For the above water-soluble polymer, the targeting site is bound to any appropriate site. For example, it may be attached to one or both ends of the polymer, or it may be introduced into a side chain. The number of targeting moieties bound to the polymer (the number of bonds per molecule of the polymer) is not limited as long as the effect of the present invention can be obtained, and may be 1 or 2 or more. The binding between the polymer and the targeting moiety is performed by using a compound having a functional group capable of binding to a functional group of the polymer and the targeting moiety, and linking the drug according to the item A with the targeting moiety. It can be performed in the same manner as the coupling.

A polymer complex using a water-soluble polymer that modifies a carrier such as a liposome has a targeting site containing a phosphocholine group represented by the above formula (I) at one end and a carrier surface at the other end. It is preferable to have a group capable of interacting. If modifying liposomes, the polymer conjugate of the formula: may be represented by Z-E-R ^X (wherein, Z is represents a phosphocholine group represented by the formula (I), E is a water-soluble polymer segment the stands, R ^X represents a hydrophobic group derived from phospholipids, long chain fatty acids, sterols, etc.).

C. Drug Delivery Composition According to still another aspect of the present invention, there is provided a drug delivery composition comprising the polymer conjugate according to the item B. In one embodiment, the drug delivery composition comprises particles formed by the polymer conjugate. The average particle size of the particles may vary depending on the polymer complex used, but is, for example, 1000 nm or less, preferably 400 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, or 80 nm or less, for example, 20 nm or more or 30 nm or more. The average particle diameter of the particles can be measured using a commercially available dynamic light scattering (DLS) measuring device.

In one embodiment, the drug delivery composition may further include a drug in addition to the polymer conjugate. By including the drug, the drug can be carried (eg, encapsulated) in the particles, and the drug can be efficiently delivered into cells and further into mitochondria. This embodiment is particularly useful when the drug is not bound to the polymer conjugate, but it is also possible to use the drug-bound polymer conjugate in combination with the drug.

In one embodiment, the drug delivery composition may further comprise a drug delivery polymer that does not contain a targeting moiety in addition to the polymer conjugate. As the drug delivery polymer containing no targeting moiety, the known drug delivery polymers described in the above section B can be used. The combination of polymers can be appropriately selected according to the purpose, and, for example, the block copolymer having the target-directing moiety attached thereto (ie, the polymer complex described in the section B-2) and the target-directing moiety are bound thereto. It can be used in combination with a non-charged polymer. In the drug delivery composition, the content ratio (molar ratio) of the polymer complex and the drug delivery polymer containing no targeting site is, for example, 5:95 to 95:5, or 10:90 to 90:. It can be 10.

When using a polymer complex containing a charged polymer and a targeting moiety, mixing the polymer complex with a drug having an opposite charge to the charged polymer in an optionally buffered aqueous solution. Thus, they can be bound or aggregated by electrostatic interaction to form a particulate PIC. When the complex of the cationic polymer and the anionic drug are used, the ratio of the number of moles (N) of the cationic group derived from the cationic polymer to the number of moles (A) of the anionic group derived from the anionic drug in the composition (N) /A ratio) can be, for example, 1 or more, preferably 3 or more, and more preferably 5 to 100. On the other hand, when an anionic polymer complex and a cationic drug are used, the ratio of the number of moles of the anionic polymer-derived anion group (A) to the number of moles of the cationic group derived from the cationic drug (N) in the composition. The (A/N ratio) can be, for example, 1 or more, preferably 3 or more, and more preferably 5 to 100.

When a polymer complex containing a block copolymer and a targeting site is used, a polymer micelle can be formed by adding the polymer complex to a buffered aqueous solution as necessary and stirring. Alternatively, the polymer composite is dissolved and mixed in an organic solvent, and the homogenized solution is distilled off under reduced pressure to prepare a polymer film, and water is added to the obtained polymer film and mixed for self-assembly. Thereby, a polymeric micelle can be formed. When the drug is included in the polymer micelle, the micelle may be formed in the presence of the drug. The N/A ratio or A/N ratio in the composition when the block copolymer and the drug having opposite charges are used is as described for the charged polymer. In addition, if necessary, a cross-linking structure may be formed between the hydrophobic polymer segments by using a cross-linking agent or by utilizing a functional group in the side chain. Regarding the method for preparing the polymer micelles, WO2007/099660, WO2007/099661, WO2010/093036, WO2012/096399, WO2014/133172, WO2015/170757 and the like can be referred to.

When a polymer complex containing a biocompatible polymer and a targeting moiety is used, a polymer spherical crystallization method such as emulsion solvent diffusion (ESD) method in water, an emulsion solvent evaporation method, a phase separation method, a phase transition method, etc. Particles carrying the drug can be formed.

When using a polymer complex containing a water-soluble polymer and a targeting moiety, a carrier whose surface is modified with the polymer complex can be obtained by mixing with a carrier in an aqueous solution optionally buffered. A drug can be included in advance in the carrier. For example, a liposome is typically a vesicle formed by a bilayer membrane of phospholipids, can be appropriately prepared by those skilled in the art, and a drug can be included therein.

D. Method of using drug conjugate or drug delivery composition The above drug conjugate or drug delivery composition is administered to an individual in need of administration of a drug. In one embodiment, the drug is a drug that is desired to be taken up intracellularly, preferably a drug that is desired to be delivered to the mitochondria.

The individual to be administered includes, for example, humans or non-human mammals. The administration method may be oral administration or parenteral administration. Parenteral administration is preferable, and subcutaneous administration, intramuscular administration, intravenous administration, intraperitoneal administration, intrathecal administration and the like can be illustrated.

According to the drug complex or the composition for drug delivery of the present invention, a drug can be efficiently delivered into cells and further localized in mitochondria.

E. According to another aspect of the present invention, according to another aspect of the present invention, a targeting property including a step of binding a compound to be delivered and a compound having a phosphocholine group represented by the formula (I) Methods of making the compounds having are provided. The targeting property imparted to the compound to be delivered is the intracellular targeting, more specifically, the mitochondrial targeting.

The compound to be delivered includes a drug or a polymer for drug delivery. Specific examples thereof include the drug described in the section A and the known polymer for drug delivery described in the section B.

Any appropriate method can be selected as a method for binding the compound to be delivered and the compound having the phosphocholine group represented by the formula (I). For example, a compound having a functional group capable of reacting with the functional group of the compound to be delivered and a phosphocholine group represented by the formula (I) is selected, and the functional group of the compound and the functional group of the compound to be delivered are selected. Any method of reacting can be used. Specific examples of such a compound include the phosphocholine group-containing compound used in the production of the drug conjugate described in the section A.

F. Method for imparting mitochondrial tropism to delivery target According to another aspect of the present invention, the delivery target is modified with a targeting site containing a phosphocholine group represented by the above formula (I). , A method of imparting a tropism to a cell or mitochondria to a delivery target is provided.

The delivery target may be a drug or a carrier carrying the drug. The drug-carrying carrier is not limited as long as it can be applied to DDS, and includes liposomes, polymer micelles, polyion complexes, polyplexes, lipoplexes, lipopolyplexes, inorganic metal particles, lipid nanoparticles, gels and the like. Can be mentioned. These carriers are widely known in the DDS field and can be easily prepared by those skilled in the art.

The method for modifying the carrier with the targeting moiety containing the phosphocholine group represented by the formula (I) includes binding the targeting moiety containing the phosphocholine group represented by the formula (I) to the surface of the carrier. Is mentioned. In this case, the bond is not limited to a covalent bond, and may be a non-covalent bond caused by an intermolecular force such as electrostatic interaction or hydrophobic interaction. The method for modifying the drug is as described in Section A.

Modification of the carrier by covalent bonding is, for example, a method of previously binding a target-directing site to a component of the carrier and forming a carrier using the component to which the target-directing site is bound, or a functional group. By forming a carrier so that the functional group is exposed to the surface of the carrier using a component having, and then reacting with a compound having a functional group capable of reacting with the functional group and a targeting site. .. Specific examples of the former include, for example, a method of forming a carrier by using the polymer composite described in the section B, and more specifically, a polymer micelle formed by using the polymer composite described in the section B-2. There is a method of doing. Specific examples of the latter include a hydrophilic polymer segment and a hydrophobic polymer segment, and a functional group (hydroxyl group, carboxyl group, amino group, aldehyde group, thiol group, maleimide group, etc.) at the end of the hydrophilic polymer segment side. A method of forming a polymer micelle using the block copolymer having the following, and then reacting with a compound having a functional group capable of reacting with the functional group and a targeting site.

Modification of the carrier by non-covalent bonding can be performed, for example, by forming the carrier and then allowing the carrier to interact with a compound having a group capable of interacting with its surface and a targeting site. As a specific example, a compound having a chargeable carrier (for example, a chargeable liposome, a polyion complex) and a charged group having a charge opposite to the carrier and a targeting site (for example, at one end A method of electrostatically binding a polymer complex according to the item B-4, which has a charged group and a phosphocholine group at the other end, a carrier having a hydrophobic surface (for example, liposome, lipid nanoparticle, lipoplex) And a compound having a hydrophobic group and a targeting site (eg, PEG having a hydrophobic group at one end and a phosphocholine group at the other end) is used as a carrier with the hydrophobic group as an anchor. Examples include a method of fixing on the surface.

According to the above method, a DDS carrier having a targeting site containing a phosphocholine group on its surface can be obtained.

G. Detection Reagent According to yet another aspect of the present invention, it is a detection reagent containing a targeting site and labeled with a labeling substance, wherein the targeting site is a phosphocholine represented by the above formula (I). A detection reagent is provided that includes a group. Since the phosphocholine group represented by the above formula (I) exhibits directivity to mitochondria, it is possible to suitably observe mitochondria in cells or individuals by using the above detection reagent.

Any appropriate label may be used as the labeling substance depending on the purpose. Specifically, fluorescent labels, luminescent substance labels, radiolabels, enzyme labels and the like can be preferably exemplified. Examples of the fluorescent label include fluorescent dyes such as Alexa compounds, Cy3, Cy5, phycoetinin, phycocyanin, allophycocyanin, FITC, rhodamine, and lanthanide, or derivatives of these fluorescent dyes, or fluorescent proteins such as green fluorescent protein ( GFP) or the like or a mutant thereof can be used. In the case of labeling with a luminescent substance, a chemiluminescent substance such as luminol, fluoroscein and rhodamine B, or a bioluminescent substance such as luciferin and iocrine may be used. In the case of radiolabeling, radioisotopes such as ³³ P, ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I, ³² P, ¹³¹ I and the like can be used. When labeling with an enzyme, β-galactosidase, β-lactamase, GUS, mustard peroxidase, alkaline phosphatase, luciferase or the like can be used.

The detection reagent can be obtained, for example, by binding a labeling substance and a compound having a phosphocholine group represented by the formula (I). Further, for example, the detection reagent can also be obtained by binding the labeling substance and the compound having the phosphocholine group represented by the formula (I) to other carriers such as polymers and metal particles.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples. In the description of the following examples, the phosphocholine group may be abbreviated as "PC".

[Production Example 1] Synthesis of PC-PEG-PAsp PC-PEG-PAsp was synthesized according to the scheme shown below.

First, SH-PEG-NH ₂ (molecular weight: 10 kDa) was dissolved in ethanol (20 mg/mL), and 2-methacryloyloxyethylphosphocholine (MPC, 10 equivalents) was added. After bubbling argon for 15 minutes, diisopropylamine (DIPA, 10 equivalents) was added, and the mixture was reacted at 30° C. for 48 hours. Ethanol was removed by vacuum drying, the polymer was dissolved in pure water, dialyzed against pure water (MWCO: 3,500), and then freeze-dried to obtain PC-PEG-NH ₂ . From ¹ H-NMR in deuterated chloroform, it was confirmed that the introduction rate of PC was 98%.

Next, PC-PEG-PBLA was synthesized by the NCA polymerization method using PC-PEG-NH ₂ as an initiator. Specifically, PC-PEG-NH ₂ is dissolved in dichloromethane (DCM, 67 mg/mL), and a mixed solvent of DCM and N,N-dimethylformamide (DMF) (DCM:DMF=10:1, 67 mg/mL) is used. β-benzyl-L-aspartic acid-N-carboxylic acid anhydride (BLA-NCA, 100 equivalents) dissolved in (mL) was added, and the mixture was reacted at 35° C. for 3 days. PC-PEG-PBLA was obtained by adding the reaction solution to an excess amount of diethyl ether and drying under reduced pressure. ¹ H-NMR in heavy DMSO confirmed that the degree of polymerization of PBLA chains was 76.

Finally, PC-PEG-PAsp was synthesized by deprotecting the benzyl ester of the side chain structure of the PBLA chain of PC-PEG-PBLA. Specifically, an aqueous sodium hydroxide solution (0.5 M, 380 equivalents) was added to PC-PEG-PBLA, and the mixture was reacted at room temperature for 1 hour. PC-PEG-PAsp was obtained by lyophilization after dialysis against pure water (MWCO: 6,000-8,000). From ¹ H-NMR in heavy water, it was confirmed that the benzyl group was completely deprotected. A control polymer (MeO-PEG-PAsp) having a methoxy group at the terminal and no PC was also synthesized in the same manner by the NCA polymerization method using MeO-PEG-NH ₂ as an initiator and deprotection of the benzyl ester.

[Production Example 2] Synthesis of Homo-P (Asp-AP) Homo-PBLA was synthesized by the NCA polymerization method using N-butylamine as an initiator. Specifically, BLA-NCA (100 equivalents) dissolved in a mixed solvent of DCM and DMF (DCM:DMF=10:1, 67 mg/mL) was added to N-butylamine and reacted at 35° C. for 3 days. Homo-PBLA was obtained by adding the reaction solution to an excess amount of diethyl ether and drying under reduced pressure. ¹ H-NMR in heavy DMSO confirmed that the degree of polymerization of PBLA chains was 65.

Next, Homo-P (Asp-AP) was synthesized by aminolysis reaction of benzyl ester having a side chain structure of Homo-PBLA. Specifically, Homo-PBLA was dissolved in DCM (40 mg/mL), benzene (4 mg/mL) was added, and the mixture was freeze-dried. The polymer was dissolved in N-methyl-2-pyrrolidone (NMP), added to diaminopentane (DAP, 6,500 equivalents) in NMP, and reacted at 12 degrees for 1 hour. After neutralization with hydrochloric acid, dialysis against pure water (MWCO: 3,500) was performed, and freeze-drying gave Homo-P (Asp-AP). From ¹ H-NMR in heavy water, it was confirmed that the benzyl group was completely deprotected and aminopentane was introduced. If necessary, the obtained Homo-P (Asp-AP) was labeled with Cy5 or Cy3 fluorescence and used in the experiment.

[Production Example 3] Preparation of polyion complex (PIC) micelles PC-PEG-PAsp and Homo-P (Asp-AP) were each dissolved in 10 mM phosphate buffer (pH 7.4), and the carboxyl group of PAsp chain PC primary PIC micelles were prepared by mixing so that the molar ratio of the primary amine of the P(Asp-AP) chain was 1. Further, non-PC-loaded PIC micelles were prepared in the same manner as above except that a control polymer (MeO-PEG-PAsp) was used instead of PC-PEG-PAsp as the control PIC micelle. Next, 10 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to the carboxyl group of PAsp was added to the PIC micelle, and the mixture was reacted overnight at room temperature. The size and polydispersity (PDI) of PIC micelles after purification by ultrafiltration (MWCO: 100,000) and EDC crosslinking were measured by dynamic light scattering (DLS). Further, the zeta potential of the PIC micelle was measured by the electrophoretic light scattering (ELS) method. The results are shown in Table 1.

≪Cell uptake test of PC mounted PIC micelle≫
Human pancreatic adenocarcinoma cells (BxPC3) were seeded in a 96-well plate (5,000 cells/well, medium: 100 μL). After 24 hours, the medium was exchanged, and PC-loaded PIC micelles fluorescently labeled with Cy5 and control non-PC-loaded PIC micelles fluorescently labeled with Cy3 were added (fluorescence intensity of Cy3 and Cy5: 1,000). After washing for 1, 6, and 24 hours with PBS, nuclear staining with Hoechst was performed. Fluorescence derived from Cy5, Cy3 and Hoechst was measured by confocal laser scanning microscopy, and the fluorescence intensity was quantified by ImageJ.

<<Competition test for cell uptake of PC-loaded PIC micelles>>
A competition test for cell uptake of PC-loaded PIC micelles was performed using 2-methacryloyloxyethylphosphocholine (MPC). Specifically, human pancreatic adenocarcinoma cells (BxPC3) were seeded in a 96-well plate (5,000 cells/well, medium: 100 μL). After 24 hours, the medium was exchanged, PC-loaded PIC micelles fluorescently labeled with Cy5 and control PIC micelles fluorescently labeled with Cy3 were added (fluorescence intensity of Cy3 and Cy5: 1,000), and MPC was used for the competitive experiment group. Solution (PBS, 10 mM) was added. After washing for 1, 6, and 24 hours with PBS, nuclear staining with Hoechst was performed. Fluorescence derived from Cy5, Cy3 and Hoechst was measured by confocal laser scanning microscopy, and the fluorescence intensity was quantified by ImageJ.

<<Inhibition test of cell uptake of PC-loaded PIC micelles>>
PC loading was carried out in the same manner as the above competition test except that a thimerosal solution (PBS, 10 nM), which is an inhibitor of phospholipid transfer protein (PLTP), was added instead of the MPC solution (PBS, 10 mM). An experiment for inhibiting the cellular uptake of PIC micelles was performed.

The results of the cell uptake test, competition test, and inhibition test of the above PIC micelles are summarized in Fig. 1.

As shown in FIG. 1, the experimental group to which PC-loaded PIC micelles were added alone showed a significantly higher fluorescence intensity than the experimental group to which non-PC-loaded PIC micelles were added, which resulted in an increase in cell uptake. I understand. In addition, in the experimental group to which MPC was added and the experimental group to which thimerosal was added, such an increase in the amount of cell uptake was suppressed, and therefore, the increase in the amount of cell uptake caused by the interaction between PC and PLTP on the cell surface. Suggest that it is due to increased endocytosis.

<<Evaluation of intracellular distribution of PC-loaded PIC micelles by mitochondrial staining>>
Human pancreatic adenocarcinoma cells (BxPC3) were seeded in a 96-well plate (5,000 cells/well, medium: 100 μL). After 24 hours, the medium was exchanged and the mitochondria were stained with the product name “CellLight Mitochondria-GFP” (2 μL/well). After 24 hours, a PC5-loaded PIC micelle fluorescently labeled with Cy5 and a non-PC-loaded PIC micelle fluorescently labeled with Cy3 were added (fluorescence intensity of Cy3 and Cy5: 1,000), and the product name “CellLight Mitochondria-GFP” (2 μL /Well) was further added. After washing for 1, 6, and 24 hours with PBS, nuclear staining with Hoechst was performed. Fluorescence derived from Cy5, Cy3, GFP and Hoechst was measured by confocal laser microscopy, and the co-localization ratio of Cy5 and GFP or Cy3 and GFP (ratio of mitochondria co-localized with micelle to total mitochondria) was measured It was quantified by ImageJ. The results are shown in Figure 2.

As shown in FIG. 2, PC-loaded PIC micelles were localized in mitochondria with a co-localization ratio several times higher than control non-PC-loaded PIC micelles.

[Production Example 4] Synthesis of PC-PEG-P (Asp-simvastatin) PC-PEG-P (Asp-simvastatin) was synthesized according to the scheme shown below.

Specifically, PC-PEG-PAsp-Ac was dissolved in DMF (10 mg/mL) containing 10 mM LiCl, and EDC (345 equivalent), dimethylaminopyridine (DMAP, 345 equivalent) and simvastatin (345 equivalent) were added. did. After reacting at room temperature for 24 hours, it was added to an excess amount of diethyl ether and dried under reduced pressure to obtain PC-PEG-P(Asp-simvastatin)-Ac. The introduction rate of simvastatin calculated from ¹ H-NMR in heavy DMF was 86%. The results are shown in Table 2.
Further, a simvastatin-binding polymer without PC (MeO-PEG-P(Asp-simvastatin)-Ac) was obtained in the same manner as above except that MeO-PEG-PAsp was used instead of PC-PEG-PAsp. It was

[Production Example 5] Preparation of PC-loaded simvastatin-encapsulating micelle PC-PEG-P(Asp-simvastatin)-Ac was dissolved in methanol (10 mg/mL), and methanol was removed by vacuum drying to obtain a polymer thin film. Pure water (1 mg/mL) was added to the obtained polymer thin film, and micelle was sonicated at room temperature for 30 minutes to prepare micelles encapsulating PC-loaded simvastatin. In addition, a non-PC-loaded simvastatin-encapsulating micelle was prepared in the same manner as described above, except that MeO-PEG-P(Asp-simvastatin)-Ac was used instead of PC-PEG-P(Asp-simvastatin)-Ac. .. After purification with a filter, the size and PDI of micelles were measured by the DLS method, and the zeta potential of the micelles was measured by the ELS method. The results are shown in Table 2.

≪Toxicity test of PC-loaded simvastatin-encapsulating micelles≫
Human pancreatic adenocarcinoma cells (BxPC3) were seeded in a 96-well plate (5,000 cells/well, medium: 100 μL). After 24 hours, the medium was exchanged, and a PC-loaded simvastatin-encapsulated micelle solution, a non-PC-loaded simvastatin-encapsulated micelle solution or a simvastatin solution was added. After 48 hours, the plate was washed with PBS and Cell Counting Kit-8 (CCK-8, 10 μL/well) was added. After 30 minutes, the absorbance was measured with a plate reader, and the cell viability and IC ₅₀ were calculated. Results are shown in FIG.

As shown in FIG. 3, the PC-loaded simvastatin-encapsulated micelle solution was significantly lower than the simvastatin solution, and exhibited a slightly higher cytotoxicity than the non-PC-loaded simvastatin-encapsulated micelle solution. From this, it is understood that the cytotoxicity of simvastatin is remarkably suppressed by micellization, and that the cytotoxicity of simvastatin can be exerted even when micelle is formed by the increase of cell uptake due to the addition of PC.

　ＮＨ_２－ＰＥＧ－ｐｏｌｙ（Ｇｌｙｃｅｒｏｌ）（ＰＥＧ分子量：１２ｋＤａ、Ｇｌｙｃｅｒｏｌ重合度：８０）を純水（３０ｍｇ／ｍＬ）に溶解し、ＭＰＣ（１０当量）、ＥＤＣ（１０当量）、Ｎ－ヒドロキシスクシンイミド（ＮＨＳ－ＯＨ、１０当量）を加えた。室温で２４時間の反応後、純水に対する透析（ＭＷＣＯ：６，０００－８，０００）を行い、凍結乾燥によりＰＣ－ＰＥＧ－ｐｏｌｙ（Ｇｌｙｃｅｒｏｌ）を得た。重水中での^１Ｈ－ＮＭＲよりＰＣの導入率を算出したところ、７３％であった。 [Production Example 6] Synthesis of PC-PEG-PGTrp The following PC-PEG-PGTrp was synthesized. The specific procedure is as follows.

NH ₂ -PEG-poly(Glycerol) (PEG molecular weight: 12 kDa, Glycerol polymerization degree: 80) was dissolved in pure water (30 mg/mL), and MPC (10 equivalents), EDC (10 equivalents), N-hydroxysuccinimide ( NHS-OH, 10 eq) was added. After 24 hours of reaction at room temperature, dialysis against pure water (MWCO: 6,000-8,000) was carried out, and freeze-dried to obtain PC-PEG-poly(Glycerol). The PC introduction rate was calculated from ¹ H-NMR in heavy water to find that it was 73%.

PC-PEG-poly(Glycerol) was dissolved in DMF (10 mg/mL) and Fmoc-NH-tryptophan-OH (810 eq), EDC (810 eq) and DMAP (810 eq) were added. After the reaction at room temperature for 24 hours, it was added to an excess amount of diethyl ether and dried under reduced pressure to obtain PC-PEG-P (Glycidyl Tryptophan (Fmoc)). The introduction rate of Fmoc-NH-tryptophan-OH was calculated from ¹ H-NMR in deuterated DMSO, and it was 88%.

PC-PEG-poly(Glycidyl Tryptophan (Fmoc)) was dissolved in DMF (10 mg/mL) containing 20% piperidine and reacted at room temperature for 24 hours. After the reaction, it was added to an excess amount of diethyl ether and dried under reduced pressure to obtain PC-PEG-PGTrp. Deprotection of the Fmoc group was confirmed by ¹ H-NMR in heavy water. Further, a polymer without PC (MeO-PEG-PGTrp) was obtained in the same manner as above except that MeO-PEG-poly(Glycerol) was used instead of PC-PEG-poly(Glycerol).

[Production Example 7] Preparation of PC-loaded mRNA micelle PC-PEG-PGTrp and mRNA (Gaussia luciferase (GLuc)) were dissolved in 10 mM Hepes buffer (pH 7.3), and primary amine (N) of PGTrp chain and mRNA were dissolved. The mixture was carried out so that the molar ratio (N/P) of the phosphate groups (P) in the mixture was 3 to prepare PC-loaded mRNA micelles. Further, non-PC-loaded mRNA micelles were prepared in the same manner as above except that MeO-PEG-PGTrp was used instead of PC-PEG-PGTrp. The size of mRNA micelles and PDI were measured by the DLS method. Further, the association number of mRNA molecules per one micelle was measured by the fluorescence correlation spectroscopy (FCS) method. The results are shown in Table 3.

<<Gene expression efficiency of PC-loaded mRNA micelles>>
Human pancreatic adenocarcinoma cells (BxPC3) were seeded in a 96-well plate (5,000 cells/well). After 24 hours, the medium was exchanged and a GLuc mRNA micelle solution (50 μg/well) was added. After 24 hours, the supernatant was added to the plate, and the luminescence intensity was measured by a luminometer after addition of luciferin. The results are shown in Fig. 4.

As shown in FIG. 4, PC-loaded mRNA micelles showed significantly higher gene expression efficiency than non-PC-loaded mRNA micelles. It is suggested that the gene expression efficiency was also increased by the increase in cell uptake due to the addition of PC.

[Production Example 8] Synthesis of albumin PC conjugate Alexa647-modified albumin was dissolved in 10 mM phosphate buffer (pH 7.4, 5 mg/mL), and 100 mM tris(2-carboxyethyl)phosphine (TCEP, 1 mL) was added. .. After reacting for 30 minutes at room temperature, MPC (350 equivalents) and DIPA (350 equivalents) were added and reacted at room temperature for 48 hours. After the reaction, the product was purified by an ultrafiltration method (MWCO: 3,000) and PC was introduced into albumin. From the quantification of thiol groups by Ehlmann's reagent, 10 molecules of PC were introduced per molecule of albumin.

≪Cell uptake test of albumin PC conjugate≫
Human pancreatic adenocarcinoma cells (BxPC3) were seeded in a 96-well plate (5,000 cells/well). The medium was exchanged after 24 hours, and Alexa647-modified albumin PC conjugate solution was added. Further, Alexa647-modified albumin was added to the control group. After 6 and 24 hours, the cells were washed with PBS and subjected to nuclear staining with Hoechst. Fluorescence derived from Alexa647 and Hoechst was measured by confocal laser microscopy, and the fluorescence intensity was quantified by ImageJ. Results are shown in FIG.

As shown in FIG. 5, the experimental group to which the albumin PC conjugate was added showed significantly higher fluorescence intensity than the experimental group to which albumin alone was added. This indicates that the cellular uptake of albumin PC conjugate was increased.

<<Evaluation of intracellular distribution of albumin PC conjugate by mitochondrial staining>>
Human pancreatic adenocarcinoma cells (BxPC3) were seeded in a 96-well plate (5,000 cells/well, medium: 100 μL). After 24 hours, the medium was exchanged and the mitochondria were stained with the product name “CellLight Mitochondria-GFP” (2 μL/well). After 24 hours, Alexa647-modified albumin PC conjugate solution or Alexa647-modified albumin (control) was added (fluorescence intensity: 1,000), and the product name "CellLight Mitochondria-GFP" (2 μL/well) was further added. After 6 and 24 hours, the cells were washed with PBS and subjected to nuclear staining with Hoechst. Fluorescence derived from Alexa647, GFP and Hoechst was measured by confocal laser scanning microscopy, and the colocalization ratio of Alexa647 and GFP was quantified by ImageJ. Results are shown in FIG.

As shown in FIG. 6, the albumin PC conjugate was localized in mitochondria with a higher co-localization ratio than albumin alone. This shows that the drug can be efficiently delivered into mitochondria by binding PC to the drug.

[Production Example 9] Preparation of PC-loaded gold nanoparticles <<Synthesis of PC-PEG-lipoic acid>>
PC-PEG-NH ₂ (10 kDa, 30 mg) prepared in the same manner as in Production Example 1 was dissolved in DMSO (4 mL), and α-lipoic acid (20 equivalents, 12 mg) and 1-ethyl-3-(3- Dimethylaminopropyl)carbodiimide (20 equivalents, 12 mg) and N-hydroxysuccinimide (20 equivalents, 7 mg) were added, and the mixture was stirred at 35° C. overnight. Then, the reaction solution was put into a dialysis bag (MWCO: 3,500 Da) and dialyzed against pure water overnight. The polymer obtained after freeze-drying was confirmed by ¹ H-NMR in deuterated chloroform to have an introduction rate of α-lipoic acid into the polymer of 77%.

<<Preparation of PC-loaded gold nanoparticles>>
A PC-PEG-lipoic acid solution (0.01 mg/mL, PBS, 17 μL) was added to gold nanoparticles (4.0×10 ¹¹ particles/mL, solution: 50 μL), and the mixture was reacted overnight at room temperature. After that, impurities were removed by a filter (pore size: 220 nm), and the size and polydispersity were measured by the dynamic light scattering method (DLS), and it was 86 nm (PDI=0.17).

<<Confirmation of migration of PC-loaded gold nanoparticles to mitochondria>>
BxPC3 cells (human pancreatic adenocarcinoma cells) were seeded in a 6-well plate (1×10 ⁶ cells/well, medium: 2 mL). After 24 hours, 1×10 ¹⁰ PC-loaded gold nanoparticles prepared by the above procedure were added to each well (2 mL) and incubated for 24 hours (the number of PC-loaded gold nanoparticles was determined by the dynamic light scattering method (DLS). ) Was measured). The cells were trypsinized, collected by centrifugation, and prefixed and postfixed with a prefix solution (mixed solution of 4% paraformaldehyde, 0.1M PBS, 25% glutaraldehyde at 5:4:1). (1:1 mixed solution of 2% osmic acid and 0.1 PBS) was used for post-fixation. Then, ascending ethanol dehydration was performed, and after embedding with Epon resin, an ultrathin section was prepared by cutting with a diamond cutter to a thickness of 1 μm. The obtained ultrathin section was stained with a uranium staining solution (uranium acetate, 50% alcohol solution), and then Reynolds lead staining solution (2.66 g of lead nitrate, 3.52 g of sodium citrate, 16 mL of 1M NaOH). Was dyed for 10 minutes each and subjected to transmission electron microscope (TEM) observation. The TEM observation image is shown in FIG. 7.

FIG. 7A is a cross-sectional image of BxPC3 cells, FIG. 7B is an enlarged image of a portion surrounded by a square in FIG. 7A, and mitochondria are imaged in the central portion. .. As shown in FIG. 7( b ), a black spot based on the gold nanoparticles having PC groups bound thereto was observed inside the mitochondria, which indicates that the PC-loaded gold nanoparticles were taken up into the cells and then inside the mitochondria. It was confirmed that it was moved to.

The present invention can be preferably used in the DDS field, for example.

Claims (11)

A drug conjugate comprising a drug and a targeting site attached to the drug, comprising:
The drug conjugate, wherein the targeting moiety comprises a phosphocholine group represented by the following formula (I).

A polymer conjugate comprising a drug delivery polymer and a targeting moiety attached to the drug delivery polymer, comprising:
The polymer conjugate in which the targeting moiety contains a phosphocholine group represented by the following formula (I).

The polymer conjugate according to claim 2, wherein the drug delivery polymer comprises a hydrophilic polymer segment and a hydrophobic polymer segment. The polymer complex according to claim 2 or 3, further comprising a drug bound thereto. A composition for drug delivery, comprising the polymer complex according to any one of claims 2 to 4. The composition for drug delivery according to claim 5, further comprising a drug. A method for producing a compound having targeting property, which comprises a step of binding a compound to be delivered and a compound having a phosphocholine group represented by the following formula (I).

A method for imparting cell or mitochondrial directivity to a delivery target, which comprises modifying the delivery target with a targeting site containing a phosphocholine group represented by the following formula (I).

The method according to claim 8, wherein the delivery target is selected from liposomes, polymeric micelles, polyion complexes, polyplexes, lipoplexes, lipopolyplexes, inorganic metal particles, lipid nanoparticles and gels. Use of a compound having a phosphocholine group represented by the following formula (I) or a phosphocholine group represented by the following formula (I) for the production of a compound having directivity to cells or mitochondria.

A detection reagent containing a targeting site, which is labeled with a labeling substance,
The detection reagent, wherein the targeting moiety comprises a phosphocholine group represented by the following formula (I).

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