JP2019123773A - Modified styrene-maleic anhydride copolymer and use thereof - Google Patents

Abstract

To provide a support that enables effective drag delivery or a material for use in the modification of a biological contact base material surface.SOLUTION: The present invention provides a copolymer of maleic anhydride and styrene, which is a copolymer in which, via a maleic anhydride unit, a poly (ethylene glycol) chain as a graft chain, and an action compound residue selected from cyclic nitroxide radical, boron-containing compound, iodine-containing compound and trialkoxysilylalkyl amine as a pendant group are covalently linked; and a use thereof.SELECTED DRAWING: None

Description

  The invention relates to modified styrene-maleic anhydride copolymers and their use, in particular to poly (ethylene glycol) grafted styrene-maleic anhydride copolymers and their use as nanomedicine materials.

  In recent years, the creation of new drugs has shifted from synthetic substances centered on low molecular weight organic compounds to natural products and proteins, and a paradigm shift of drug targets is taking place. However, when proteins or nucleic acids such as molecular targeting drugs are used in medicine, they are degraded by endogenous enzymes, so they have not only a very short bioavailability, but also various antigenicity and renal excretion. The problem is inevitable. Davis et al. Of Rutgers University, U.S. are a novel idea of covalently attaching polyethylene glycol (PEG) to a protein, and showed avoidance of its antigenicity and prolongation of retention (see Non-Patent Document 1). Despite the reckless attempt to covalently bond synthetic polymers to delicate proteins, the first macromolecularized drug was born, and more than 10 PEGylated proteins are currently marketed. However, since the protein and PEG are directly reacted, problems such as a significant decrease in activity and an increase in cost remain.

  In Japan, in 1986, Maeda et al discovered an EPR effect in which a macromolecular substance is passively accumulated in cancer (see Non-Patent Document 2 or Patent Document 1). That is, blood vessels in solid cancer tissue are rougher in structure than blood vessels in normal tissue, and not only low molecular weight substances but also high molecular weight substances easily leak from blood vessels to cancer tissues. The low molecular substance leaked into the cancer tissue is recovered by diffusion and capillary lymphatic vessels, whereas the macromolecule is poorly diffusive and the capillary lymphatic system can not be collected in time. For this reason, it accumulates in cancer tissue for a long time. As a result, macromolecular substances were selectively retained and accumulated in solid cancer tissues, and this was named as the EPR effect. Therefore, Maeda et al. Bound an anticancer drug, neocarcinostatin, to a poly (styrene-co-maleic anhydride) copolymer, and was approved and sold as the world's first polymeric anticancer drug, Smanx, in 1993. Although the tumor accumulation effect of the polymerizing drug was confirmed, the dispersion stability was poor, and only catheterization from the artery was approved and did not reach widespread use. Even so, many attempts have been made to use poly (styrene-co-maleic anhydride) copolymers as carriers for pharmaceutical compounds, including other anti-cancer agents (eg, for paclitaxel conjugates Non-Patent Document 3).

The present inventors, including Nagasaki, have developed antioxidative nanomedicines by dramatically extending the bioavailability of the high molecular weight drug compared with the small molecule and significantly changing the distribution of internal organs. We have been promoting (see Patent Document 2) and have been developing a medicine (see prior patent below). That is, excess reactive oxygen species (ROS) derived from oxygen cause various diseases. Although natural products such as vitamins C and E and various synthetic antioxidants have been developed and studied to eliminate ROS involved in such diseases, almost all clinical results show “good” results. There is nothing out of it. The present inventors have noted that these low molecular weight antioxidants nonspecifically spread into the body, destroying the redox reaction in the mitochondria of normal cells and inducing side effects. We have created an antioxidative ability in the self-assembled nanoparticles shown in FIG. 19 of Patent Document 2, and have advanced the design of a new nanomedicine that selectively removes ROS involved in diseases without entering normal cells and their mitochondria. . As a result, for example, a high molecular weight antioxidant (TEMPOL) completely suppresses mitochondrial damage even at a 10 mM high concentration condition of 3 mM at which the freshly produced zebrafish are completely destroyed by a low molecular weight antioxidant (TEMPOL), It was confirmed that they survive almost 100% (Non-patent Document 4). Thus, it has been confirmed that RNP, which overcomes the disadvantages of the conventional antioxidants, has a remarkable therapeutic effect on various diseases involving ROS. However, the antioxidative nanoparticles by the conventional block copolymer require high vacuum conditions for the synthesis, and there is a drawback that the synthesis is expensive. In addition, it is difficult to introduce a ligand, and there is a drawback such as easy aggregation during particle preparation. Unlike conventional low-molecular-weight drugs and protein drugs, there is a need for materials that are easy to design, suppress nonspecific nucleic acids, and provide materials that can be stably dispersed in order to reduce long bioavailability and side effects.

  The poly (styrene-co-maleic anhydride) copolymer utilized by Maeda et al. Mentioned above is a synthetic polymer that has been known for a long time, and its cyclic anhydride residue easily reacts with a nucleophile. For example, a living body or a living body which appears to be a reaction product of the above-mentioned copolymer and TEMPOL (4-hydroxy-TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) and 4-amino-TEMPO) It can be suggested that it can be used to modify the surface in contact with the substance (see Patent Document 3).

EP 0087957 A1 WO 2009/133647 A1 JP, 2011-78706, A

Abuchowski et al. Biolog. Chem. , 1977, 10: 252 (11): 3582-3586. Matsumura, Maeda, Cancer Research, 46, 6387-6392 (1986) ACS Appl. Mater. Interfaces 2015, 7, 26530-26548 Vong, et al. , Mol. Pharm. , 13, 9, 3091 (2016)

  Poly (styrene-co-maleic anhydride) copolymer (hereinafter sometimes abbreviated as PSMA) is not a problem when it is used for surface modification etc., but the copolymer and its biological activity Conjugates with substances, such as anticancer agents, have problems such as poor dispersion stability when administered to humans as a medicine as described above. In addition, although the polymeric anticancer agent containing the half ester unit of the drug derived from the maleic anhydride unit of said PSMA and the half ester unit of polyethyleneglycol is generally described in patent document 1, it describes concretely Not only is it not described, but generally it does not describe what kind of properties they have. In addition, since these macromolecularized anticancer agents are fixed to the polymer skeleton through covalent bonds and the like, it is necessary for the drug to be released from the macromolecularized anticancer agent at the target site in order to exert medicinal effects. The inventors of the present invention have found that the dispersion stability of a polymerized physiologically active substance in an in vivo or in vitro aqueous medium has been a serious issue to be addressed in the development of a block copolymer as described in Patent Document 2. In the case of using a poly (styrene-co-maleic anhydride) copolymer, it was set as a problem to be improved that I improve.

The object of the present invention is to introduce (or graft) a water-soluble polymer containing poly (ethylene glycol) into PSMA, and to select the drug (s) to be introduced into PSMA, etc. In addition, it is possible to improve the dispersion stability which has been a problem in the prior art, and further, a drug-modified conjugate which covalently bonds the specific drug to the polymer skeleton without covalently bonding or ion binding the drug to the polymer skeleton. It was solved by having confirmed that it could be enclosed or included in micelles (nanoparticles) formed from coalescence.

Therefore, according to the present invention, the invention of the following aspect is provided.
Aspect 1: A copolymer comprising each repeating unit represented by the following formula (I):

During the ceremony
x + y is an integer of 5-1400, n is an integer of 5-1400, x + y: n is in a ratio of 1: 1-5, x: y is in a ratio of 1-20: 1, x: y is in the ratio of 1 to 60: 1.
(1) In L-PEG-A in the repeating unit to which the subscript y is added
L is O or NH, and PEG is represented by the following formula:

Here, p is an integer of 1 to 6, and q is an integer of 5 to 500,
A represents an A1: unsubstituted or substituted C ₁ -C ₁₂ alkoxy group, and the substituent when substituted is a formyl group, a formula R ^a R ^b CH- (wherein R ^a and R ^b are independent of each other) _and, C 1 -C ₄ alkoxy or ^{R 1} and ^{R 2} are -OCH ₂ CH ₂ O together _{-, - O (CH 2)} 3 O- or -O _{(CH 2)} represents a 4 O-. Or A2:

And the repeating unit accounts for 2% to 15% of the total units of the copolymer represented by the formula (I).
(2) In the repeating unit to which the subscript x is attached,
(A) Either one of R ₁ or R ₂ is
a1:

Represented by, where
TEMPO is 2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl, 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-yl, 2,2,5,5 -Tetramethylpyrroline-1-oxyl-3-yl, 2,4,4-trimethyl-1,3-oxazolidine-3-oxyl-2-yl, 2,4,4-trimethyl-1,3-thiazolidine-3 A residue of a cyclic nitroxide radical compound selected from the group consisting of oxyl-2-yl and 2,4,4-trimethyl-imidazolidine-3-oxyl-2-yl;
a2:

Residues represented by any of
a3:

Residues represented by any of
a4:

Wherein R ₃ is a C _1-3 alkyl group and r is an integer of 2 to 6;
A residue selected from the group consisting of
The other is OH, or (b) R ₁ and R ₂ together represent -O- and form a cyclic anhydride residue, or (c) R ₁ and R ₂ are each OH Represents
And in the repeat unit to which x is attached,
(I) containing only one residue of any one of a1 to a4 in the unit including (a),
(Ii) a unit comprising any one residue of a1 to a4 in the unit comprising (a) and any one of the units comprising (b) and (c) are randomly present independently of each other, Do the units containing any one of a1 to a4 in the units containing (a) occupy 15% to 60% of the total number of repeating units with x, or
(Iii) a unit comprising any one of a1 to a4 in the unit comprising (a), a unit comprising (b) and a unit comprising (c) are independently and randomly present, Or a unit containing any one residue of a1 to a4 in the unit containing) accounts for 15% to 60% of the total number of repetitive units attached with x,
(Iv) Among the units containing (a), the unit containing the residue of a4, the unit containing any one of the residues a1 to a3, and the unit containing any one of (b) and (c) Are both independently and randomly, and does the unit containing any one residue of a1 to a3 in the unit containing (a) occupy 15% to 60% of the total number of repeating units given x? ,
Among units including (v) (a), a unit including the residue of a4 and a unit including any one residue of a1 to a3 and a unit including (b) and a unit including (c) A unit which exists independently and randomly and which includes any one residue of a1 to a3 in the unit including (a) occupies 15% to 60% of the total number of repeating units to which x is attached. Aspect 2: The copolymer according to aspect 1, wherein the unit containing (a) is a unit containing a residue of a1.
Aspect 3: The copolymer described in aspect 1, wherein the unit containing (a) is a unit containing a residue of a2.
Aspect 4: The copolymer described in aspect 1, wherein the unit containing (a) is a unit containing a residue of a3.
Aspect 5: The copolymer according to aspect 1, wherein the unit containing (a) is a unit containing a residue of a4.
Aspect 6: The copolymer according to aspect 1, wherein the unit containing (a) is a unit containing a residue of a1 and a residue of a4.
Aspect 7: The copolymer according to any one of the aspects 1 to 6, wherein in the repeating unit to which the subscript y is attached, A in L-PEG-A is a formula of A2

Copolymer which is a group represented by
Aspect 8: The copolymer according to any one of aspects 1 to 7, wherein TEMPO is

In the above formula, R 'is a methyl group,
The copolymer which is any residue represented by
Aspect 9: A copolymer according to any one of the aspects 1 to 8, which is present as nanoparticles having an average particle size in the nanosize, as measured by dynamic light scattering in an aqueous medium. .
Aspect 10: A complex comprising the copolymer described in any of aspects 1 to 8 and benzalkonium chloride.
Aspect 11: A medical composition comprising the copolymer described in any of aspects 1 to 8 and an anticancer agent.
Aspect 12: The medical composition according to claim 11, wherein the anticancer agent is paclitaxel or sorafenib.
Aspect 13: A copolymer represented by the following formula (II). The

During the ceremony
Either one of R _{1-1 and} R _2-1 is
a1:

Represented by, where
TEMPO is 2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl, 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-yl, 2,2,5,5 -Tetramethylpyrroline-1-oxyl-3-yl, 2,4,4-trimethyl-1,3-oxazolidine-3-oxyl-2-yl, 2,4,4-trimethyl-1,3-thiazolidine-3 A residue of a cyclic nitroxide radical compound selected from the group consisting of oxyl-2-yl and 2,4,4-trimethyl-imidazolidine-3-oxyl-2-yl;
a2:

Residues represented by any of
a3:

Residues represented by any of
a4:

Wherein R ₃ is a C _1-3 alkyl group and r is an integer of 2 to 6;
A residue selected from the group consisting of
The other is OH, and the cyclic anhydride residue moiety in the formula (II) may be hydrolyzed to 10% of m1 and opened.
And m1 + m2 is an integer of 5-1400, n is an integer of 5-1400, m1 + m2: n is in a ratio of 1: 1-5.
Aspect 14: In the repeating unit to which the subscript m2 is attached, either one of R _{1-1 and} R _2-1 is
a1:

The copolymer according to aspect 13, which is represented by

  According to the present invention, a novel drug or material having high dispersion stability in an aqueous medium or body fluid, for in vivo delivery such as a physiologically active substance or a diagnostic drug, or for modifying a surface in contact with a living body, It is possible to provide a novel material which can also be used as a precursor of the material.

Graphic representation of measurement results of size-resolved chromatography (SEC) of SMAPo (n = 2) obtained in Production Example 1 ¹ H-NMR spectrum of said SMA Po (n = 2) ¹ H-NMR spectrum of the polymer (SMA ^TN ) obtained in Preparation Example 4 ¹ H-NMR spectrum of the polymer (SMA ^TN ) obtained in Preparation Example 5 ¹ H-NMR spectrum of the polymer (SMA ^TN ) obtained in Preparation Example 6 ¹ H-NMR spectrum of the polymer (pSMA ^TN ) obtained in Preparation Example 7 ¹ H-NMR spectrum of the polymer (pSMA ^TN ) obtained in Preparation Example 8 Graphic representation of the SEC measurement results of the polymer (SMAPo ^TN (n = 2)) obtained in Preparation Example 9 ¹ H-NMR spectrum of said SMA Po ^TN (n = 2) ¹ H-NMR spectrum of the polymer (SMAPo ^TN (n = 2)) obtained in Preparation Example 10 ¹ H-NMR spectrum of the polymer (pSMAPo ^TN (n = 2)) obtained in Preparation Example 11 ¹ H-NMR spectrum of the polymer (SMAPo ^B (n = 2)) obtained in Preparation Example 12 ¹ H-NMR spectrum of the polymer (SMAPo ^I (n = 2)) obtained in Preparation Example 13 TOF-MS spectrum of the polymer (SMA ^CHO-Ph Po (n = 2)) obtained in Production Example 14 ¹ H-NMR spectrum of said SMA ^CHO-Ph Po (n = 2) Graphical representation of the SEC measurement results of said SMA ^CHO-Ph Po (n = 2) TOF-MS spectrum of the polymer (SMA ^CHO-Ph Po (n = 2)) obtained in Preparation Example 16 ¹ H-NMR spectrum of the polymer obtained in Preparation Example 16 Graphic representation of SEC measurement results of the polymer obtained in Production Example 16 Graphic representation of measurement results of dynamic light scattering of the SMA Po ^TN nanoparticle solution obtained in Production Example 17 Graphical representation of the measurement results of the zeta (ζ) potential of the nanoparticles Graphic representation of measurement results of particle size of composite prepared in Production Example 18 Graph display of zeta potential measurement result of the complex Graphical display of measurement results of scattering intensity (count rate) of the complex Graphic representation of measurement results of particle strength distribution dependent on ionic strength of composite in Test Example 1 Graphical display of measurement results of scattering intensity in Test Example 1 Graphic representation of measurement results of particle size distribution of the complex obtained in Production Example 19 Graphic representation of measurement results of particle size distribution dependent on ionic strength of the complex obtained in Production Example 19 Graphic representation of measurement results of measurement of scattering intensity depending on ion intensity of the complex obtained in Production Example 19 Graphic representation of measurement results of particle size distribution of the complex obtained in Production Example 20 Graphic representation of measurement results of particle strength distribution dependent on ionic strength of the complex obtained in Production Example 20 Graphic representation of measurement results of measurement of scattering intensity depending on ion intensity of the complex obtained in Production Example 20 Graphic representation of measurement results (particle size distribution) of dynamic light scattering of the SMA Po ^I nanoparticle solution obtained in Production Example 21 Graph display of organ distribution measurement results of each nanoparticle 30 minutes after oral administration of each sample to test animals in Test Example 2 Graphical representation of organ distribution measurement results of each nanoparticle 4 hours after the administration Graphic representation of detection results for suppression of superoxide formed by kneading each polymer onto polystyrene surface in Test Example 3 Graphic display of measurement results of particle size distribution by dynamic light scattering analysis of drug (sorafenib) -containing silica-containing nanoparticles in Test Example 4 A photograph as an alternative to the figure showing the evaluation result as a contrast agent in a tumor-bearing mouse of a contrast medium carrying polymer (SMAPo ^I ) in Test Example 5 Graphical display of measurement results of particle size distribution by dynamic scattering light analysis of paclitaxel-encapsulated SMAPo ^TN nanoparticles in Test Example 6 Graphic representation of the evaluation results of the stability of the polymer coated on collagen-coated beads in Test Example 7 (1). Graphic representation of the evaluation results of the antithrombotic ability of the polymer coated on collagen-coated beads in Test Example 7 (2) The photograph instead of the figure showing the state after washing | cleaning of a column following formation of the thrombus by coating in Experiment 7 (2). Graphical representation of the evaluation results of the protective effect of the polymer (SMAPo ^TN ) during cryopreservation of animal cells in Test Example 8 ¹ H-NMR spectrum of N 618 obtained in Production Example 22 ¹ H-NMR spectrum of N623 obtained in Production Example 23 Graph display of the result of substrate surface modification (impartation of blood compatibility) test by N563 of Test Example 9 Graphic representation of SEC measurement results of polymer SMA ^TN obtained in Production Example 24 ¹ H-NMR Spectrum of Polymer SMAP _n1 ^TN Obtained in Production Example 25 ¹ H-NMR spectrum of polymer SMAP _n3 ^TN obtained in Preparation Example 26 Graphic representation of SEC measurement results of polymer SMA ^TN obtained in Production Example 27 Graphic representation of SEC measurement results of polymer SMAP _n1.5 ^TN (and control PEG-NH ₂ ) obtained in Preparation Example 28 Graphic representation of measurement results of dynamic light scattering of nanoparticles produced from SMA ^TN in Production Example 29 Graphic representation of measurement results of dynamic light scattering of nanoparticles produced from SMAP _n1 ^TN in Production Example 29 Graphic representation of measurement results of dynamic light scattering of nanoparticles produced from SMAP _n3 ^TN in Production Example 29 Graphic representation of measurement results of dynamic light scattering of nanoparticles produced from SMA ^TN in Production Example 30 Graphic representation of measurement results of dynamic light scattering of nanoparticles produced from SMAP _n1.5 ^TN in Production Example 30 Graphical representation of the results measured for SMA ^TN response in Test Example 10 Graphical representation of the results measured for SMAP _n1.5 ^TN response in Test Example 10 Graphic representation of measurement results of dynamic scattering intensity over time of SMA ^TN in Test Example 11. Signal change due to TEMPO with time of SMA ^TN in Test Example 11 Graphic representation of measurement results of dynamic scattering intensity over time of SMAP _n1.5 ^TN in Test Example 11. Signal change due to TEMPO over time of SMAP _n1.5 ^TN in Test Example 11

Detailed Description of the Invention

  Hereinafter, the present invention will be described in more detail.

  In the copolymer represented by the above formula (I), the characteristic point compared with the prior art is not limited, but generally, two or more poly ( Not only that the ethylene glycol) (abbreviated as PEG) chain is in a grafted state, but also if the composition containing the copolymer includes a drug such as an anticancer drug, In some cases, certain physiologically active substances or agents that can contribute to the stability of the composition itself during drug delivery of the composition to the living body are covalently bonded as a pendant group. The copolymer according to the present invention may be used in an aqueous medium (pure water, body fluid, an aqueous solution buffered with a suitable buffer, an aqueous solution containing a water-soluble organic solvent, etc.) itself or with the aid of a cationic compound etc. By self-assembly, nano-sized micelles (or particles) can be formed. Such a copolymer is an anti-cancer drug (sorafenib, camptothecin) in forming nano-sized micelles, ie, micelles within the nano size range, for example, 10 nm to 800 nm, preferably 15 nm to 200 nm. Drugs such as paclitaxel, anticancer platinum complexes, etc.) can be effectively or stably encapsulated or encapsulated in the micelles without the covalent bond to the copolymer.

  In the definition of (a) in the repeating unit to which the subscript x of the formula (I) is attached, the formula of a1:

The residue of the cyclic nitroxide radical compound in the inside is caused by the production and existence of excess active oxygen etc. in the living body by utilizing the redox function that the nitroxide radical can exert in the living environment when polymerized. It is known to be useful for preventing or treating a suspected disease or the like (see, for example, Patent Document 2 mentioned above).

Although not limited by theory, the copolymer represented by the formula (I) according to the present invention is different from the block copolymer described in Patent Document 2 in that it has the above-mentioned characteristics, It has properties superior to those of the block copolymer. For example, by selecting the number of PEG chains, it is possible to control the average particle size of the nanosized micelles or particles and to provide nanomicelles with improved stability in an aqueous medium. Furthermore, for example, R ₁ and R ₂ as defined in (b) in the repeating unit to which x is attached are the same as the cyclic anhydride residue formed by representing -O- together For example, a protein such as collagen without adversely affecting the function of the residue defined in (a), for example, the redox function of the nitroxide radical attributed to the residue of the cyclic nitroxide radical compound when the residue contains TEMPO The surface of the medical device or the like coated with can be modified or modified.

  In the definition of (a), the formula of a2

The residue represented by any one of the above is a residue functioning in boron neutron capture therapy (BNCT), and the copolymer of the formula (I) in the covalently bonded form of the residue is They exhibit similar properties, except that they function in boron neutron capture therapy, not the redox function mentioned previously.

  In the definition of (a), the expression of a3

The residue represented by any of the above is a residue functioning as an X-ray contrast agent, and the copolymer of the formula (I) in the covalently bonded form of the residue is the same as described above under a1. It exerts the same characteristics except that it functions not as a redox function but as an X-ray contrast agent. In addition, such a residue also serves, for example, to track the site where the copolymer stays or accumulates in vivo.

  In the definition of (a), the expression of a4

The copolymer of the formula (I), which is represented by and in the covalently bonded form of the residue, is optionally obtained via the trialkoxysilyl group of the residue, optionally in the presence of tetraalkoxysilane or nanosized silica. For example, a nano-sized so-called core-shell type micelle having a silica-containing core and a shell mainly composed of a PEG chain because crosslinks can be formed via so-called sol-gel reaction at 10 ° C. to 30 ° C. Can be produced. Such micelles can adsorb or contain the above-mentioned anticancer agent etc. at the silica-containing core site during or after their formation. In addition to the characteristics derived from such silica, the unit having the residue exhibits equivalent characteristics other than the redox function described above for a1.

The copolymer of the formula (I) has at least the functions described for a1 to a4 described above, and in addition to the functions described for these a2 to a4, it is defined for (b) described above in relation to a1 And R ₁ and R ₂ together have a function to be exerted based on a cyclic anhydride residue formed by representing -O-. The functions of a1 to a4 described above are useful for delivering an anticancer drug or the like to a target as described above, and when appropriate (for example, when half ester or half amide is hydrolyzed in the target region) In addition, the anticancer agent or the like can be covalently bonded as a pendant group of the copolymer via the cyclic anhydride residue defined in the above (b). The cyclic anhydride residue as defined in (b) may be in the state of being hydrolyzed and opened as in whole or in part as defined in (c), but all or 80% or more remaining It is preferred that the group is as defined in (b).

  In the copolymer of the formula (I), when the residue of a4 and any one of a1 to a3 are combined and coexist, the function of a4 described above and each function described above for each a1 to a3 It is exhibited in combination. Therefore, when the anticancer agent etc. described above is included in the micelle formed by the copolymer to enhance the stability in the aqueous medium, or when adjusting the average particle size of the micelle, etc. Preferably, any one of the residues a1 to a3 is used in combination.

  In the copolymer of the formula (I), x + y and n are not limited, but random copolymers obtained by radical copolymerization of maleic anhydride and styrene, preferably alternating copolymers, each repeating unit Can be derived from the number of units that make up the The unit is not limited as long as it exerts each unit of the unit defined in (a) and the unit defined in (b), but x + y can be an integer of 5 to 1400, n Can be an integer of 5 to 1400, and x + y: n can be in a ratio of 1: 1 to 5, preferably 1: 1 to 3, more preferably 1: 2. Such x + y can be in the ratio of 1 to 60: 1, preferably 1 to 40: 1. Furthermore, y may be 2 or more when L is O, and 1 or more when NH.

In the copolymer of formula (I), L-PEG- in the repeating unit to which the subscript y is added
PEG in A has the formula

In which p is an integer of 1 to 6, preferably 2 to 4, particularly 2 and q is an integer of 5 to 500, preferably 10 to 300, more preferably 20 to 150 it can. In addition, unless otherwise indicated, a formula or a part or group in the present specification is described as having the indicated directionality.

On the other hand, A represents an unsubstituted or substituted C ₁ -C ₁₂ alkoxy group as defined in A ₁ , and the substituent when substituted is a formyl group, a formula R ^a R ^b CH— (wherein R ^a and ^{R b} are _{independently,} C 1 -C ₄ alkoxy or ^{R 1} and ^{R 2} are -OCH ₂ CH ₂ O together _{-, - O (CH 2)} 3 O- or -O (CH ₂ Or ₄ ) O-)) or a formula defined in A2

It can be a specific group represented by

  When a protected formyl group as defined in A1 or a specific protected formyl group as defined in A2 is present at the end of the PEG chain, a targeting ligand etc. is introduced at the end of the PEG chain via the group. can do. In the latter case, in particular, it easily bonds to amino groups on the surface of mucous membranes and organs, which can contribute to the improvement of the local retention of the copolymer of the formula (I) in vivo.

  When the copolymer of formula (I) is OH (forming a carboxyl group) which is derived from the group defined for (a), (b) and (c) above or defined as the other, A polyion complex (PIC) can be formed together with a quaternary ammonium or amine compound which is a counter ion compound of a carboxyl group. Such PIC can exist as a stable PIC micelle in an aqueous medium, and can be filled or encapsulated with the above-mentioned anticancer drug etc. during formation of the micelle. Although not limited thereto, as quaternary ammonium compounds, benzalkonium, 1,2-dimyristoyl-3-trimethylammonium propane (chloride), 1,2-dioleyl-3-trimethylammonium propane (chloride), There can be mentioned 1,2-dioleole-3-trimethylammonium propane (methyl sulfate), chitosan, poly (2-triethylaminoethyl chloride of methacrylic acid) and the like.

The micelles formed by the above-mentioned copolymer, silica-containing micelles, PIC micelles are those in the case where dynamic light scattering (DLS) is measured for the aqueous solution of micelles according to the number of PEG chains to be grafted, the content of silica, etc. It can be obtained as micelle particles having an average particle size of about 10 nm to about 600 nm, preferably about 15 nm to about 150 nm.

  The micelles thus obtained can be separated by separation means such as centrifugation, and can be stored as a dry composition by lyophilization, and can be reconstituted in an aqueous medium as required. Such a dry composition can be provided as an aqueous solution of PIC micelles, optionally including a physiologically acceptable diluent or excipient. Such diluent may be sterile water, physiological saline, a solution containing a physiologically acceptable buffer, etc., and as an excipient, for example, sorbitol, dextrin, glucose, mannitol, amino acid ( For example, it can be glycine, isoleucine, valine, methionine, glutamic acid, etc. Such aqueous solutions can be administered to mammals, particularly humans, via veins or arteries in need thereof, and the aqueous solutions or dry compositions can be directly administered to the local area of tumor. The effective dose is not limited because the optimum value varies depending on the type of drug contained in the copolymer or the inclusion of the copolymer, the condition of the patient, etc., but results of further clinical tests etc. with reference to test examples etc. described later It can be determined by a specialist based on

  The copolymer of the formula (I) can be obtained as a random copolymer, preferably an alternating copolymer, which can be obtained by radical copolymerization of maleic anhydride and styrene described above, but is not limited thereto, and is commercially available. The graft chain represented by L-PEG-A of the formula (I), a1 defined in (a), a cyclic anhydride residue or maleic anhydride ring residue or part of an available raw material copolymer It is possible to obtain the compound corresponding to any one of a2, a3 and a4 by reacting them simultaneously or in tandem with each other in a so-called nucleophilic reaction known per se.

  The copolymer of the formula (I) is, for example, a raw material copolymer and a compound corresponding to a graft chain represented by L-PEG-A and / or a1, a2, a3 or a4 defined for (a) The compound can be produced by reacting a compound corresponding to any one residue in an anhydrous organic solvent, optionally in the presence of an alkali or the like at room temperature for several hours to 24 hours. When the compound is a hydroxy compound, it can be made into an alcoholate of an alkali metal in advance during the reaction. On the other hand, when the compound is an amine compound, the reaction can be allowed to proceed without the need for alcoholate formation treatment and the like. Such organic solvents are not limited, but aprotic solvents, preferably dipolar aprotic solvents such as tetrahydrofuran (THF), dimethylformamide (DMF), acetonitrile (AN), dimethylsulfoxide (DMSO) Can be mentioned. The reaction between the compound corresponding to the graft chain represented by L-PEG-A and the copolymer depends on the number of the compound residues to be introduced into the cyclic anhydride residue of the copolymer, It can implement by adjusting the addition amount of the former compound. For example, the graft chain represented by about two or more L-PEG-A can be obtained as a raw material copolymer by appropriately selecting so that the equivalent of the compound is 2 or more per equivalent of the cyclic anhydride residue. Can be introduced. When the residue of any one of a1, a2, a3 and a4 defined for (a) is introduced into the cyclic anhydride residue, the equivalent ratio of the former and the latter is 20 or more to 1: If the selection is made such that the former residue can be introduced to substantially all cyclic anhydride residues, and the ratio is selected to be 10 or less to 1: 1, the introduction ratio is usually less than 100%, for example, 10 It can be up to 60%. The remaining cyclic anhydride residue can be introduced into the cyclic anhydride residue by further reacting the additional compound containing the compound other than the above compounds nucleophilically. In addition, the cyclic anhydride residue can be opened by hydrolysis to form a dicarboxylic residue.

  Thus, formula (II):

In formula, R < _1-1 >, R < _2-1 >, m <1>, m <2>, n are as having defined above.
The copolymer of formula (I) can also be provided by nucleophilically reacting a compound corresponding to the graft chain represented by L-PEG-A or a PEG derivative with the copolymer represented by From such a point of view, the copolymer represented by the formula (II) is a precursor of the copolymer of the formula (I) and, itself, using the unit of cyclic anhydride residue It can also be used as a coating material for surfaces of medical devices and the like.

  Hereinafter, the present invention will be more specifically described with reference to specific examples, but the scope of the present invention is not intended to be limited to these examples.

Preparation Example 1: Synthesis of SMAPo (n = 2) (N 557)
Combined with

Dissolve 38 g of a commercially available poly (styrene-co-maleic anhydride) copolymer (PSMA, molecular weight 7,500, styrene: maleic anhydride unit ratio = 2: 1) (abbreviated as PSMA) in 100 mL of anhydrous tetrahydrofuran (THF) I did. After taking 50 g of commercially available one-end methoxy, other-end hydroxy poly (ethylene glycol) (MW = 5,000, abbreviated as MeO-PEG-OH) into a 200 mL eggplant-type flask and drying under reduced pressure at 80 ° C. for 1 day, It was dissolved in 200 mL of dry THF, and 6.5 mL of commercial butyllithium (hexane solution, 1.6 M) was added to convert the PEG end to an alcoholate. This solution was added to the previous PSMA THF solution and allowed to react at room temperature for 1 day. The reaction solution was poured into 1 L of isopropyl alcohol, and a precipitate was obtained by centrifugation (9,000 rpm, 2 minutes). The supernatant was discarded, the precipitate was dispersed in hexane, and the precipitate was purified by centrifugation (twice). The polymer was then recovered by vacuum drying (76 g). The measurement results of size fractionation chromatography (SEC) of the polymer obtained above are shown in FIG. 1, and the ¹ H-NMR spectrum is shown in FIG.

Preparation Example 2: Synthesis of SMA Po (n = 5) (N 605 I)
Combined with tel)
A commercially available poly (styrene-co-maleic anhydride) copolymer (PSMA, molecular weight: 7,500, styrene: maleic anhydride unit ratio = 2: 1) (abbreviated as PSMA) 7.6 g was dissolved in 50 mL of anhydrous THF . 25 g of a commercially available one-end methoxy, other-end hydroxy poly (ethylene glycol) (MW = 5,000, abbreviated as MeO-PEG-OH) is weighed into a 100 mL eggplant type flask and dried under reduced pressure at 80 ° C. for 1 day It was dissolved in 200 mL of dry THF, and 3.3 mL of commercial butyllithium (hexane solution, 1.6 M) was added to convert the PEG end to an alcoholate. This solution was added to the previous PSMA THF solution and allowed to react at room temperature for 1 day. The reaction solution was poured into 1 L of isopropyl alcohol, and a precipitate was obtained by centrifugation (9,000 rpm, 2 minutes). The supernatant was discarded, the precipitate was dispersed in hexane, and the precipitate was purified by centrifugation (twice). The polymer was then recovered by vacuum drying (30 g). The structure of the obtained polymer is referred to that of Production Example 1.

Preparation Example 3: Synthesis of SMA Po (n = 10) (N 605 II) (10 PEGs are PSMA
Bound by ester)
The same method as in Production Example 2 was carried out except using 3.8 g of PSMA (molecular weight: 7,500, styrene: maleic anhydride unit ratio = 2: 1) (yield: 30 g). The structure of the obtained polymer is referred to that of Production Example 1.

Preparation Example 4: Synthesis of TEMPO-introduced PSMA (SMA ^TN , N547)

9 g of a commercially available poly (styrene-co-maleic anhydride) copolymer (PSMA, molecular weight 7,500, styrene: maleic anhydride unit ratio = 2: 1) (abbreviated as PSMA) was dissolved in 50 mL of anhydrous THF. 5 g of commercially available 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl NH ₂ -TEMPO) was dissolved in 15 mL of THF, added to the above PSMA / THF solution, and stirred at room temperature for 2 hours. The reaction solution was poured into 1 L of ether to obtain a precipitate. The supernatant was discarded, and the precipitate was dispersed in ether and purified by filtration (twice). The polymer was then recovered by vacuum drying (14 g). According to electron spin resonance (ESR) spectroscopy of the obtained polymer, the TEMPO introduction rate was 14 per polymer. The ¹ H-NMR spectrum of the obtained polymer is shown in FIG. In addition, one part or all part of the maleic anhydride residue in said Formula can be hydrolyzed.

Preparation Example 5 Synthesis of TEMPO-introduced PSMA (SMA ^TN , N568) Part 2
A target product was synthesized by carrying out the same method as in Production Example 4 except that 7 g of PSMA, molecular weight 230 KDa, styrene: maleic anhydride unit ratio = 1: 1) was dissolved in 50 mL of anhydrous THF (11 g). According to electron spin resonance (ESR) spectrum measurement, the TEMPO introduction rate was 464 per polymer. The ¹ H-NMR spectrum of the obtained polymer is shown in FIG.

Production Example 6: Synthesis of TEMPO-introduced PSMA (SMA ^TN , N569) Part 3
The target product was obtained in the same manner as in Production Example 5 except that anhydrous N, N-dimethylformamide (DMF) was used instead of anhydrous THF to obtain the desired product (7 g). According to electron spin resonance (ESR) spectrum measurement, the TEMPO introduction rate was 468 per polymer. The ¹ H-NMR spectrum of the obtained polymer is shown in FIG.

Preparation Example 7: Synthesis of pSMA ^TN (N572) (part of maleic anhydride of PSMA is NH ₂
-Introduced TEMPO)

Production Example except that 10 g of a commercially available poly (styrene-co-maleic anhydride) copolymer (PSMA, molecular weight: 1,600, styrene: maleic anhydride unit ratio = 1.3: 1) was dissolved in 100 mL of anhydrous THF The same method as in 4 was followed to synthesize the desired product (14.6 g). According to electron spin resonance (ESR) spectroscopy, the TEMPO introduction rate was 2.6 per polymer (4.4 residual maleic anhydride). The ¹ H-NMR spectrum of the obtained polymer is shown in FIG.

Preparation Example 8 Synthesis of pSMA ^TN (N 574) Part 2
Production Example except that 15 g of a commercially available poly (styrene-co-maleic anhydride) copolymer (PSMA, molecular weight: 1,600, styrene: maleic anhydride unit ratio = 1.3: 1) was dissolved in 100 mL of anhydrous THF The same method as in 7 was followed to synthesize the desired product (19.5 g). According to electron spin resonance (ESR) spectroscopy, the TEMPO introduction rate was 2.2 per polymer (4.8 residual maleic anhydride). The ¹ H-NMR spectrum of the obtained polymer is shown in FIG.

Preparation Example 9 Synthesis of SMA Po ^TN (n = 2, N564) (2 PEGs
With NH ₂ -TEMPO introduced to the remaining maleic anhydride ring by ter

20 g of SMAPo synthesized in Preparation Example 1 was dissolved in 150 mL of anhydrous THF. Dissolve ₅ g of NH ₂ -TEMPO in 15 mL of anhydrous THF, add to the above solution and stir at room temperature for 2 hours. The reaction solution was poured into 1 L of ether, and the precipitate was collected by filtration under reduced pressure, and dried under reduced pressure to obtain the desired product (yield 25 g). The measurement results of SEC of the polymer obtained above are shown in FIG. 8, and the ¹ H-NMR spectrum is shown in FIG. According to the measurement results of SEC, the introduced amount of TEMPO was 16 per chain and 25 for carboxyl group per chain.

Preparation Example 10: Synthesis of SMA Po ^TN (n = 2, N 607) (two PEGs were introduced into PSMA
In addition, NH ₂ -TEMPO was introduced into the remaining maleic anhydride ring by bonding.
)) (One-step synthesis)
38 g of a commercially available poly (styrene-co-maleic anhydride) copolymer (PSMA, molecular weight 7,500, styrene: maleic anhydride unit ratio = 2: 1) (abbreviated as PSMA) was dissolved in 100 mL of anhydrous THF. After taking 50 g of commercially available one-end methoxy and other-end hydroxy polyethylene glycol (MW = 5,000, MeO-PEG-OH) in a 200 mL eggplant type flask and drying under reduced pressure at 80 ° C. for 1 day, dry THF 200 mL The solution was dissolved, and 6.5 mL of commercially available butyl lithium (hexane solution, 1.6 M) was added to alcoholize the PEG end. This solution was added to the previous PSMA THF solution and allowed to react at room temperature for 2 hours. ₂₀ g of NH ₂ -TEMPO is dissolved in 60 mL of anhydrous THF, added to the above solution and stirred at room temperature for 2 hours. The reaction solution was poured into 1 L of ether, and the precipitate was collected by filtration under reduced pressure, and dried under reduced pressure. According to ESR, the amount of TEMPO introduced was 16.5. The ¹ H NMR spectrum of the obtained polymer is shown in FIG.

Preparation Example 11 Synthesis of pSMAPoTN (n = 2, N563) (2 PEGs are converted to PSMA)
Bond with ester and introduce NH ₂ -TEMPO to a part of the remaining maleic anhydride ring
Inserted)

5 g of SMAPo (N 557) synthesized in Preparation Example 1 was dissolved in 50 mL of anhydrous THF. 0.6 g of NH ₂ -TEMPO was dissolved in 3 mL of anhydrous THF, added to the above solution, and stirred at room temperature for 2 hours. The reaction solution was poured into 400 mL of ether, and the precipitate was recovered by filtration under reduced pressure, and dried under reduced pressure. The ¹ H-NMR spectrum of the obtained polymer is shown in FIG. ^From the measurement results of ¹ H-NMR and ESR, it was determined that x = 15, y = 7.6, z = 2 (yield 5.6 g).

Preparation Example 12 Synthesis of SMA Po ^B (n = 2, N559) (2 PEGs
(A boron compound introduced into the remaining maleic anhydride ring)

5 g of SMAPo (N 557) synthesized in Preparation Example 1 was dissolved in 50 mL of anhydrous THF. 2 g of 4-aminophenylboronic acid pinacol ester was dissolved in 20 mL of anhydrous THF, 6 mL of butyllithium was added (1.6 M) and stirred at room temperature for 10 minutes, then added to the above solution and stirred at room temperature for 12 hours. The reaction solution was poured into 1 L of ether, and the precipitate was recovered by filtration under reduced pressure and dried under reduced pressure to recover (yield: 4.1 g). According to ICP-MS determination, the boron content was 6.9 pieces / polymer. The ¹ H NMR spectrum of the obtained polymer is shown in FIG.

Preparation Example 13 Synthesis of SMAPo ^I (n = 2, N561) (2 PEGs
With iodine compound attached to the remaining maleic anhydride ring bonded with a ter)

2.5 g of SMAPo (N 557) synthesized in Preparation Example 1 was dissolved in 30 mL of anhydrous DMF. After 1.73 g of 2,4-diiodoaniline was dissolved in 20 mL of anhydrous THF, 3 mL of butyllithium was added (1.6 M) and stirred at room temperature for 10 minutes, then added to the above solution and stirred at room temperature for 5 hours. The reaction solution was poured into 500 mL of 2-propanol, and the precipitate was collected by vacuum filtration and dried under reduced pressure to recover the target polymer (yield: 2.1 g). The iodine content was 1.8 / polymer by ICP-MS. The ¹ H-NMR spectrum of the obtained polymer is shown in FIG.

Preparation Example 14 Synthesis of SMA ^CHO-Ph Po (n = 2, N606I) Part 1 (PEG End)
Of SMAPo with benzylacetal group introduced

Under nitrogen, 0.45 g of 4- (dimethoxymethyl) benzyl alcohol was dissolved in 50 mL of anhydrous THF in a 200 mL eggplant flask, and 3 mL of a THF solution (1.05 M) of potassium naphthalene (K-Naph) was added. Thereafter, 12.5 g of cooled ethylene oxide (EO) was added, and the mixture was stirred for 1 day under water cooling (referred to as solution A). 5 g of commercially available PSMA (molecular weight 7,500, styrene: maleic anhydride unit ratio = 2: 1) was dissolved in 40 g of anhydrous THF to make solution B, 11 g of solution A and 18 g of solution B were mixed and reacted for 5 hours . The reaction solution was poured into 1 L of 2-propanol, the precipitate was collected by filtration under reduced pressure, and dried under reduced pressure (yield: 4.2 g). The TOF-MS spectrum of the obtained polymer is shown in FIG. 14, the ¹ H-NMR spectrum is shown in FIG. 15, and the SEC measurement results are shown in FIG.

Preparation Example 15 Synthesis of SMA ^CHO-Ph Po (n = 5, N606 II) Part 2
43 g of the solution A prepared in Production Example 14 and 27 g of the solution B were mixed, and stirred at room temperature for 5 hours. The reaction solution was poured into 1 L of 2-propanol, and the precipitate was collected by filtration under reduced pressure, and dried under reduced pressure to obtain the target polymer (yield: 7 g).

Preparation Example 16 Synthesis of SMA ^CHO-Ph Po (n = 2, N604): Part 3 (PEG Chain 2K
Da)
Under nitrogen, 0.54 g of 4- (dimethoxymethyl) benzyl alcohol was dissolved in 30 mL of anhydrous THF in a 100 mL recovery flask, and 3.3 mL of a potassium naphthalene solution (1.05 M) was added. Thereafter, 7 g of cooled ethylene oxide (EO) was added, and the mixture was stirred for 1 day under water cooling. 2.3 g of commercially available PSMA (molecular weight 7,500, styrene: maleic anhydride unit ratio = 2: 1) was dissolved in 10 g of anhydrous THF, mixed with the above solution, and reacted for 5 hours. The reaction solution was poured into 1 L of 2-propanol, and the precipitate was recovered by filtration under reduced pressure, and dried under reduced pressure to obtain a target polymer (yield: 8.2 g). The TOF-MS spectrum of the obtained polymer is shown in FIG. 17, the ¹ H-NMR spectrum is shown in FIG. 18, and the SEC measurement results are shown in FIG.

Production Example ^17: SMAPo TN to 50mg of ^SMAPo TN (N564) synthesized in Preparation Production Example 9 of nanoparticles (N564) was dissolved in DMF and 10 mL, water 2L using a dialysis membrane of molecular cutoff 3.6KDa Hemodialysis was performed. After changing the dialysis water twice every 12 hours, it was concentrated by a centrifugal evaporator to 10 mg / mL. Dynamic light scattering and zeta potential of the solution were measured. The measurement results of dynamic light scattering are shown in FIG. 20, and the measurement results of zeta (タ) potential are shown in FIG.

Preparation Example 18 Complex Preparation of SMAPo ^TN (N564) and BKCl 0.28 g of benzalkonium chloride (BKCl) (10% aqueous solution) was mixed with 3.7 mL of milli Q water (solution A). 70 mg of SMA Po ^TN (N564) was added and mixed with 2 mL of milli Q water and 1 M of 0.1 M NaOH (solution B). Solutions A and B were mixed as shown in Table 1 below to prepare a complex.

The measurement results of the particle size, zeta potential and scattering intensity (count rate) of the composite are shown in FIG. 22, FIG. 23 and FIG. 24, respectively.

Test Example 1: Complex of SMAPo ^TN (N 564) and benzalkonium chloride (BKCl)
Ionic Intensity Stability Dynamic light scattering and scattering by adding NaCl to the complex (50:50) of SMAPo ^TN (N 564) and benzalkonium chloride free (BKCl) prepared in Preparation Example 18 to set the ionic strength to 60 to 500 mM The strength etc. were measured. The ion intensity-dependent particle size distribution of the complex is shown in FIG. 25 and the scattering intensity is shown in FIG.

Preparation example 19: Complex of SMAPo ^B (N 586) and benzalkonium chloride (BKCl)
Preparation and characterization: A solution was prepared in the same manner as in Preparation Example 18 except that SMA Po ^B (N559) was used, and a complex was prepared. Measurement of particle size distribution and scattering intensity was performed. The measurement results are shown in FIG. 27, FIG. 28 and FIG. 29, respectively.

Preparation Example 20: Complex of SMAPo ^I (N561) and benzalkonium chloride (BKCl)
Preparation and Determination of Properties A solution was prepared by the same method as in Preparation Example 19 except that SMAPo ^I (N561) obtained in Preparation Example 13 was used, and a complex was prepared, and the particle size distribution as described in Test Example 1 , And measurement of particle size distribution and scattering intensity depending on ion intensity. The measurement results are shown in FIG. 30, FIG. 31 and FIG. 32, respectively.

Preparation Example 21 Nanoparticle Preparation of SMA Po ^I (n = 2, N561) 20 mg of N561 synthesized in Preparation Example 13 was dissolved in 4 mL of methanol, 4 mL of milli-Q water was added, and the mixture was evaporated. When it became half volume, 4 mL of water was added and evaporated again. After repeating this twice, the total particle size was 4 mL and the particle size was measured by dynamic light scattering. The measurement results are shown in FIG. The figure shows that the average particle size is 11 nm in volume distribution.

Test Example 2: Organ distribution by oral administration of each sample Samples N547 (PEG-free SMA ^TN ), N 564 (SMA Po ^TN , n = 2) and N 606 I (SMA ^ace-Ph Po) synthesized in Production Examples 4, 9 and 15, respectively. 1.5 g of each ^TN (n = 2) was dissolved in 15 mL of DMF and dialyzed against 2 L of dialysis water for 2 days. The final concentrations were 30 mg / mL, 23 mg / mL and 26 mg / mL, respectively. 0.5 mL of each of these solutions were orally administered to ICR mice, and organs were collected after 30 minutes and 4 hours, and the distribution was measured by an electron spin resonance apparatus. The organ distribution measurement results of each nanoparticle 30 minutes after and 4 hours after administration are shown in FIGS. 34 and 35. From these results, the above three nanoparticles did not enter the blood at all by oral administration. Also, N547 without PEG showed little accumulation in the small intestine, but N564 accumulated well in the small intestine. Nanoparticles having a benzacetal end tend to have a long survival in the stomach.

Test Example ^{3: SMA TN (N547) (} 100mg / ml) of a toluene solution of styrene kneading of the polymer to polystyrene surfaces (100mg / ml), pSMAPo TN (N563) (100mg / ml) or ^SMAPo TN (N564) ( A 100 mg / ml) toluene solution was mixed in a ratio of 1: 1, and 100 μL each was added to a 96 well white plate, dried under vacuum, the toluene was removed, and cast on the plate surface. The cast with only styrene was performed similarly as comparison object. Hypoxanthine and xanthine oxidase were added and the generated superoxide was detected by MPEC (Ato). The results are shown in FIG. It was confirmed that the light emission amount of those obtained by kneading N563 and N564 into styrene was reduced as compared with the case of using only styrene, and that superoxide was eliminated.

Test Example 4 Drug Encapsulation of Sorafenib (Anticancer Drug) in pSMAPo ^TN (N563) (1) 22 mg of pSMAPo ^TN (n = 2, N563) obtained in Production Example 11 in 1 mL of DMF in a glass vial It was completely dissolved and mixed with 21 μL of 3-aminopropyltrimethoxysilane (d = 0.946). Then, 2.2 mg of sorafenib (Sorafenib), 21 μL of tetraethyl orthosilicate (TEOS) and 30 μL of aqueous ammonia (NH ₃ , 28%) were successively added to the solution. After 2 hours of mixing, 500 μL of milli-Q water was added to the solution and mixed for another 5 minutes. Thereafter, dialysis was performed on 2 L of water using a dialysis membrane with a molecular weight cut off of 3.6 KDa. The water was replaced after 12 hours and recovered after another 24 hours. The solution was subjected to dynamic light scattering analysis by mixing it in PBS buffer (pH 7.2 (20 mM PO ₄ ^3- )) at a ratio of 1: 1, and the particle size distribution was determined. The obtained result is shown in FIG.
(2) The amount of drug contained was tested as follows. The sample was mixed with Tris (50:50) in aceto and left in an ultrasonic bath for 2 hours and then filtered using a 0.2 μm pore filter. The amount of drug was determined by testing the absorption wavelength at 254 nm using high performance liquid chromatography (HPLC). The results are shown in Table 2.

Test Example 5 Evaluation of Contrast Agent in Tumor-Bearing Mouse Using SMA Po ^I (N561) N561 is dissolved in dimethylsulfoxide (DMSO) at a concentration of 125 mg / ml and 100 μL is injected in tumor-bearing mouse (Balb / c 5 weeks old) The cancer was locally administered, and imaging was performed with ALOKA LaTheta LCT-100. As a comparative object, iomeron (Eisai) was topically administered at an equivalent iodine content. A photograph as a substitute for a diagram showing measurement results is shown in FIG. In the case of N561, it was confirmed that detection was possible in the tumor part even after 30 minutes (the same arrow).

Test Example 6 Drug Sealing of Paclitaxel (PTX, Anticancer Drug) to SMA Po ^TN (N 564)
PTX was added 10% (4 mg) and 20% (8 mg) of the polymer weight to input N 564 (40 mg), respectively, and mixed in a 1 mL DMF glass vial for 3 hours. Thereafter, the mixture was dialyzed against 2 L of ultrapure water using a dialysis membrane with a molecular weight cut off of 3.6 kDa, water was changed after 12 hours, and recovered after another 12 hours. After removing the unencapsulated drug with a 0.2 μm filter, the particle size distribution was measured by dynamic scattered light analysis. The results are shown in FIG. Thereafter, the drug loading was examined as follows. After freeze-drying the sample and dissolving it in acetonitrile (1 mg / mL), the amount of PTX drug was quantified by examining wavelength absorption at 227 nm using high performance liquid chromatography (HPLC). The results are shown in Table 3 below.

Test Example 7 Coating of polymer on plastic bead column (collagen coat) pSMAPo ^TN (N563), commercially available plastic bead column (http: //dev.medicalonline) coated with collagen in methanol solution (50 mg / mL) of SMAPo ^TN (N 564) .Jp / index / product / eid / 45155) was coated. The coating conditions are as follows.

<Coating conditions>
(Mar 1): Plastic beads as it is (Mar 2): N 563 coating (Mar 3): N 564 coating

Group I: Air-dried after sample coating II: Air-dried after sample coating, repeated coating air-dried again Group III: Sealed for 1 week after sample coating

(1) The columns (circle 2) and (circle 3) prepared as described above were washed three times with a phosphate buffer. The results of measuring the ESR intensity of the washing solution are shown in FIG. From the figure (circle 3) 40, in the case of N564 ((circle 3) series, sample with no or less residual maleic anhydride (MA)), the coating polymer is exfoliated after each washing, while N563 (MA It is found that almost no elution occurs in the case of apparently remaining, and a stable coating was achieved. See FIG.
(2) Inject 500 μL of mouse whole blood without anticoagulant such as heparin into the prepared column
Let stand for 0 minutes. Thereafter, it was washed with 1 μL of phosphate buffer solution. The results of measurement of thrombus formation by coating are shown in FIG. 41, and a photograph as a substitute for a diagram showing the state of the column after washing is shown in FIG. In the collagen-coated column, a very large amount of thrombus was formed, and it was almost impossible to wash. While N564 coating still retains considerable thrombus, N563 almost completely suppresses thrombus formation, resulting in a very high performance surface coating material.

Test Example 8 Protective effect of SMAPo ^TN (N 564) on cryopreservation of animal cells After culturing bovine aortic endothelial cells (BAEC) in 150 mm petri dishes and reaching confluence, the cells are dispersed in 10 ⁷ cells / mL in culture medium. , 1 mL each was dispensed into the vial. After adding 1% of DMSO and a predetermined amount of SMA Po ^TN (N564) and pipetting, it was quick frozen in a -80 ° C freezer. Two days later, the vial was placed in 37 ° C. hot water, quickly thawed, and 4 mL of 37 ° C. DMEM medium and 1 mL of cell suspension were placed in 15 mL falcon. 50 μL of the diluted cell suspension and 50 μL of a commercially available 0.4 w / v% trypan blue staining solution were taken in a 1.5 mL tube, mixed, and the number of living cells was counted by a hemocytometer. The results are shown in FIG. From the figure, it can be seen that the addition of N564 significantly improves the cell viability. In general, about 5 to 10% of dimethyl sulfoxide (DMSO) is added to the culture solution, and cells of about 10 ⁵ to 10 ⁶ cells / mL are cryopreserved at -80 ° C and thawed when used again. When considering regenerative medicine and organ preservation, it is desirable to reduce the amount of DMSO as much as possible and freeze in large quantities.

Production Example 22 Production of N 618 2.5 g of SMAPo (N 557) synthesized in Production Example 1 was dissolved in 25 mL of anhydrous THF. A solution of 1.0 g of NH ₂ -CH ₂ Ph-I (4-iodobenzylamine) in 25 mL of DMF was added to the above solution and stirred at room temperature for 2 hours. 400 mL of reaction solution
The precipitate was collected by filtration under reduced pressure and dried under reduced pressure. The amount of introduced NH-CH ₂ Ph-I in the polymer obtained by ICP analysis was 18. ^{The 1} H-NMR spectrum is shown in FIG. (Yield 2.5 g).

Preparation Example 23 Preparation of N623 2.5 g of SMAPo (N 557) synthesized in Preparation Example 1 was dissolved in 25 mL of anhydrous THF. NH ₂ -CH ₂ Ph-B (pinacol):

1.0 g of 25 mL DMF solution was added to the above solution and stirred at room temperature for 2 hours. The reaction solution was purified by dialysis against water through a dialysis membrane (molecular weight cut off 2,500), and dialyzed to obtain the title polymer. The ¹ H-NMR spectrum of the obtained polymer is shown in FIG.

Test Example 9: Modification of substrate surface by N563 (impartation of blood compatibility)
300 μL of a styrene solution (100 mg / mL toluene) and a mixed solution of styrene / N 563 (a solution obtained by mixing the above styrene solution and N 563 solution (supernatant obtained by centrifuging a 10 mg / mL toluene solution) in 1: 1) And allowed to dry to form a thin film. 100 μL of whole blood obtained from the mouse was added and allowed to stand for 5 minutes, followed by washing 5 times with physiological saline. The adsorbed protein component was dissolved in RIPA buffer, and the amount of adsorption was measured by the BCA method. The results are shown in FIG. From the figure, it was confirmed that the thin film to which N563 was added had a smaller amount of protein adsorption.

Production Example 24: Synthesis of TEMPO-introduced SMA (SMA ^TN ) (4)
200 mg of a commercially available poly (styrene-co-maleic anhydride) copolymer (PSMA, molecular weight: 7,500, styrene: maleic anhydride unit ratio = 2: 1) (abbreviated as PSMA) was dissolved in 5 mL of anhydrous DMF. 171 mg of commercially available 4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl NH _2- TEMPO) was dissolved in 0.85 mL of DMF, added to the above PSMA / DMF solution, and stirred at room temperature for 1 day . The reaction solution was placed in a dialysis membrane (molecular weight cut off 3500) and dialyzed against distilled water for 24 hours to remove DMF and residual NH ₂ -TEMPO. The polymer after dialysis was recovered by freezing the solution with liquid nitrogen and lyophilizing under reduced pressure (503 mg). According to electron spin resonance (ESR) spectroscopy of the obtained polymer, the TEMPO introduction rate was 21 per polymer. The measurement result of the size differential chromatography (SEC) of the obtained polymer is shown in FIG.

Production Example 25: TEMPO · PEG-introduced SMAP (SMAP _n1 ^TN ) (1 PEG
It was amino-linked to SMA and TEMPO was introduced to the remaining maleic anhydride.
Synthesis of

Commercially available poly (styrene-co-maleic anhydride) copolymer (PSMA, molecular weight 7,500, styrene: maleic anhydride unit ratio = 2: 1) (abbreviated as PSMA) 373 mg and NH ₂ -PEG relative to PSMA A SMAP _n1 ^TN was synthesized (732 mg) in the same manner as in Production Example 9, except that a molar ratio of 1: 1 (246 mg) was used to dissolve in 5 mL of anhydrous DMF. According to electron spin resonance (ESR) spectroscopy of the obtained polymer, the TEMPO introduction rate was 17 per polymer. The ¹ H-NMR spectrum of the obtained polymer is shown in FIG.

Production Example 26: TEMPO · PEG-introduced SMAP (SMAP _n3 ^TN ) (3 PEGs
It was amino-linked to SMA and TEMPO was introduced to the remaining maleic anhydride.
Of commercially available poly (styrene-co-maleic anhydride) copolymer (PSMA, molecular weight 7,500, styrene: maleic anhydride unit ratio = 2: 1) (abbreviated as PSMA) 200 mg and NH ₂ -PEG SMAP _n3 ^TN was synthesized (536 mg) in the same manner as in Production Example 9 except that the compound was dissolved in 5 mL of anhydrous DMF at a molar ratio of 1: 3 (399 mg) to PSMA. According to electron spin resonance (ESR) spectroscopy of the obtained polymer, the TEMPO introduction rate was 16 per polymer. The measurement result of the ¹ H-NMR spectrum of the obtained polymer is shown in FIG.

Production Example 27: Synthesis of TEMPO-introduced SMA (SMA ^TN ) (5)
300 mg of a commercially available poly (styrene-co-maleic anhydride) copolymer (PSMA, molecular weight 7,500, styrene: maleic anhydride unit ratio = 2: 1) (abbreviated as PSMA) was dissolved in 10 mL of anhydrous THF. 200 mg of commercially available 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl NH _2- TEMPO) was dissolved in 1.2 mL of THF, added to the above PSMA / THF solution, and stirred at room temperature for 3 days . The reaction solution was poured into 1 L of ether to obtain a precipitate. The supernatant was discarded, and the precipitate was dispersed in ether and purified by filtration (twice). The polymer was then recovered by vacuum drying (397 mg). According to electron spin resonance (ESR) spectroscopy of the obtained polymer, the TEMPO introduction rate was 17 per polymer. The measurement result of the size differential chromatography (SEC) of the obtained polymer is shown in FIG.

Production Example 28: TEMPO · PEG-introduced SMAP (SMAP _n1.5 ^TN ) (1.5 P
Amino-bond EG to PSMA and introduce TEMPO to the remaining maleic anhydride
Commercially available poly (styrene-co-maleic anhydride) copolymer (PSMA, molecular weight 7,500, styrene: maleic anhydride unit ratio = 2: 1) (abbreviated as PSMA) 300 mg and NH ₂ SMAP _n1.5 ^TN was synthesized in the same manner as in Production Example 25 except that -PEG was dissolved in 10 mL of anhydrous THF at a molar ratio of 1: 1.5 (300 mg) with respect to PSMA ( 720 mg). According to electron spin resonance (ESR) spectroscopy of the obtained polymer, the TEMPO introduction rate was 21.5 per polymer. The measurement result of the size differential chromatography (SEC) of the obtained polymer is shown in FIG.

Preparation Example 29 Preparation of Nanoparticles of SMA ^TN and SMAPn ^TN (n = 1 and 3) 20 mg of three samples SMA ^TN and SMAPn ^TN (n = 1, 3) synthesized in Preparation Examples 24, 25 and 26, respectively. It was dissolved in 2 mL of methanol and dialyzed against 2 L of water using a dialysis membrane with a molecular weight cut off of 3.5 KDa. The dialysis water was replaced every several hours, and the final concentration was adjusted to about 5 mg / mL, and the dynamic light scattering of the solution was measured. The measurement results of dynamic light scattering are shown in FIGS. 52, 53 and 54, respectively.

Preparation ^30: SMA ^TN, SMAPn ^TN 2 single sample ^SMA TN synthesized respectively prepared in Production Example 27 and 28 of ^{nanoparticles,} the 20mg of SMAPn TN (n = 1.5) of 2mL of (n = 1.5) It was dissolved in methanol and dialyzed against 2 L of water using a dialysis membrane with a molecular weight cut off of 3.5 KDa. The dialysis water was replaced every several hours, and the final concentration was adjusted to about 5 mg / mL, and the dynamic light scattering of the solution was measured. The measurement results of dynamic light scattering are shown in FIGS.

Test Example 10: SMA ^TN (Reaction Solvent DMF) and SMAP _n1.5 ^TN (Reaction Solvent THF)
Evaluation of pH response of TEMPO Based on the above-mentioned micelle preparation method, the final micelle concentration is approximately 10 mg / mL against SMA ^TN (reaction solvent DMF) and SMAP _n1.5 ^TN (reaction solvent THF) with almost equal TEMPO introduction rates. The micelles were prepared to evaluate the responsiveness to pH. The buffer is prepared to have various pHs by adding 0.1 M and 0.01 M HCl and 0.1 M NaOH to phosphate buffered saline (PBS) buffer, and then to 300 μL of the prepared micelles. 300 μL was added and stirred for several seconds to measure the dynamic scattering intensity. The measurement results for SMA ^TN and SMAP _n1.5 ^TN are shown in FIGS. 57 and 58, respectively. As a result of the scattering intensity according to the volume, as a result, SMA ^TN not introduced with PEG showed a tendency to aggregate remarkably at around pH = 2, while SMAP _n1.5 ^TN introduced with 1.5 PEG chains It was found that the particle size was maintained around 30 nm until pH = 3.

Test Example 11: ^{SMA TN} (reaction solvent DMF) after PBS dropping the _{SMAP N1.5} ^TN (anti
Evaluation of the stability of the reaction solvent THF) With respect to SMA ^TN (reaction solvent DMF) and SMAP _{n 1.5} ^TN (reaction solvent THF), micelles are adjusted to a final micelle concentration of about 10 mg / mL based on the above-mentioned micelle preparation method. Prepared and assessed the stability of micelles after PBS instillation. After 500 μL of pH = 7.7 PBS was added to 500 μL of the prepared micelles and stirred, dynamic scattering intensity and ESR measurement were performed. In addition, it stored at 37.4 degreeC which is biological body temperature using a constant temperature water tank, and measured time progress. The results for SMA ^TN are shown in FIGS. 59 and 60, respectively, and the results for SMAP _n1.5 ^TN are shown in FIGS. 61 and 62, respectively. The SMA ^TN without PEG introduction did not show any significant change in particle size or scattering intensity over time, but the TEMPO signal was clearly seen after PBS addition. SMAP _n1.5 ^TN introduced with 1.5 PEG chains increased in particle size and scattering intensity with the passage of time, and TEMPO signal gradually became clear, so it should be compatible with the PBS solvent containing ions Was suggested.

  The present invention can be used in the pharmaceutical or diagnostic industry.

Claims (14)

Copolymer comprising each repeating unit represented by the following formula (I):
During the ceremony
x + y is an integer of 5 to 1400, n is an integer of 5 to 1400, x + y: n is in a ratio of 1: 1 to 5 and x: y is in a ratio of 1 to 60: 1.
(1) In L-PEG-A in the repeating unit to which the subscript y is added
L is O or NH, and PEG is represented by the following formula:
Here, p is an integer of 1 to 6, and q is an integer of 5 to 500,
A represents an A1: unsubstituted or substituted C ₁ -C ₁₂ alkoxy group, and the substituent when substituted is a formyl group, a formula R ^a R ^b CH- (wherein R ^a and R ^b are independent of each other) _and, C 1 -C ₄ alkoxy or ^{R 1} and ^{R 2} are -OCH ₂ CH ₂ O together _{-, - O (CH 2)} 3 O- or -O _{(CH 2)} represents a 4 O-. Or A2:
And the repeating unit accounts for 2% to 15% of the total units of the copolymer represented by the formula (I).
(2) In the repeating unit to which the subscript x is attached,
(A) Either one of R ₁ or R ₂ is
a1:
Represented by, where
TEMPO is 2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl, 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-yl, 2,2,5,5 -Tetramethylpyrroline-1-oxyl-3-yl, 2,4,4-trimethyl-1,3-oxazolidine-3-oxyl-2-yl, 2,4,4-trimethyl-1,3-thiazolidine-3 A residue of a cyclic nitroxide radical compound selected from the group consisting of oxyl-2-yl and 2,4,4-trimethyl-imidazolidine-3-oxyl-2-yl;
a2:
Residues represented by any of
a3:
Residues represented by any of
a4:
Wherein R ₃ is a C _1-3 alkyl group and r is an integer of 2 to 6;
A residue selected from the group consisting of
The other is OH, or (b) R ₁ and R ₂ together represent -O- and form a cyclic anhydride residue, or (c) R ₁ and R ₂ are each OH Represents
And in the repeat unit to which x is attached,
(I) containing only one residue of any one of a1 to a4 in the unit including (a),
(Ii) a unit comprising any one residue of a1 to a4 in the unit comprising (a) and any one of the units comprising (b) and (c) are randomly present independently of each other, Do the units containing any one of a1 to a4 in the units containing (a) occupy 15% to 60% of the total number of repeating units with x, or
(Iii) a unit comprising any one of a1 to a4 in the unit comprising (a), a unit comprising (b) and a unit comprising (c) are independently and randomly present, A in the unit including)
Or a unit containing any one residue of 1 to a4 accounts for 15% to 60% of the total number of repetitive units attached with x,
(Iv) Among the units containing (a), the unit containing the residue of a4, the unit containing any one of the residues a1 to a3, and the unit containing any one of (b) and (c) Are both independently and randomly, and does the unit containing any one residue of a1 to a3 in the unit containing (a) occupy 15% to 60% of the total number of repeating units given x? ,
Among units including (v) (a), a unit including the residue of a4 and a unit including any one residue of a1 to a3 and a unit including (b) and a unit including (c) A unit which exists independently and randomly and which includes any one residue of a1 to a3 in the unit including (a) occupies 15% to 60% of the total number of repeating units to which x is attached. The copolymer according to claim 1, wherein the unit containing (a) is a unit containing a residue of a1. The copolymer according to claim 1, wherein the unit containing (a) is a unit containing a residue of a2. The copolymer according to claim 1, wherein the unit containing (a) is a unit containing a residue of a3. The copolymer according to claim 1, wherein the unit containing (a) is a unit containing a residue of a4. The copolymer according to claim 1, wherein the unit containing (a) is a unit containing a residue of a1 and a unit containing a4 residues. The copolymer according to any one of claims 1 to 6, wherein in the repeating unit to which the subscript y is attached, A in L-PEG-A has a formula of A2
Copolymer which is a group represented by The copolymer according to any one of claims 1 to 7, wherein TEMPO is
In the above formula, R 'is a methyl group,
The copolymer which is any residue represented by 9. A copolymer as claimed in any one of the preceding claims, wherein the copolymer is present as nanoparticles having an average particle size of nanosize when measuring dynamic light scattering in an aqueous medium. A complex comprising the copolymer according to any one of claims 1 to 8 and benzalkonium chloride. A medical composition comprising the copolymer according to any one of claims 1 to 8 and an anticancer agent. The medical composition according to claim 11, wherein the anticancer agent is paclitaxel or sorafenib. The copolymer represented by following Formula (II).
During the ceremony
Either one of R _{1-1 and} R _2-1 is
a1:
Represented by, where
TEMPO is 2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl, 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-yl, 2,2,5,5 -Tetramethylpyrroline-1-oxyl-3-yl, 2,4,4-trimethyl-1,3-oxazolidine-3-oxyl-2-yl, 2,4,4-trimethyl-1,3-thiazolidine-3 A residue of a cyclic nitroxide radical compound selected from the group consisting of oxyl-2-yl and 2,4,4-trimethyl-imidazolidine-3-oxyl-2-yl;
a2:
Residues represented by any of
a3:
Residues represented by any of
a4:
Wherein R ₃ is a C _1-3 alkyl group and r is an integer of 2 to 6;
A residue selected from the group consisting of
The other is OH, and the cyclic anhydride residue moiety in the formula (II) may be hydrolyzed to 10% of m1 and opened.
And m1 + m2 is an integer of 5-1400, n is an integer of 5-1400, m1 + m2: n is in a ratio of 1: 1-5. In the repeating unit to which the subscript m2 is attached, either one of R _{1-1 and} R _2-1 is
a1:
The copolymer according to claim 13 represented by