Protein aggregation inhibitors

Abstract

By using a polymer compound represented by formula IV and a production method thereof, provided is a novel protein aggregation inhibitor that exhibits a protein aggregation inhibitory effect when added at a low concentration and, after once added, that can be easily separated from the protein to be protected.

Description

[Technical Field]

[0001] The present invention relates to a protein aggregation inhibitor. [Background technology]

[0002] In recent years, expectations for protein pharmaceuticals, including antibody drugs and enzyme preparations, have been rising. A major issue in the development and practical application of protein pharmaceuticals, particularly antibody drugs and enzyme preparations, is stability. Stability issues can be broadly categorized into two types: inactivation during long-term storage and aggregation during purification. Inactivation during long-term storage often manifests as aggregation due to freezing. Therefore, the stability issue can be understood as the prevention of protein aggregation, both during long-term storage and during purification.

[0003] Trehalose (Non-Patent Document 1), polyethylene glycol (Non-Patent Document 2), arginine (Non-Patent Document 3), and polysulfobetaine (Non-Patent Document 4, Patent Document 1) have been reported as compounds that are effective in inhibiting protein aggregation. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. WO2018 / 003909 [Non-patent literature]

[0005] [Non-Patent Document 1] Tomita S., Nagasaki Y., Shiraki K., Biotechnol Bioengineer., 2012, 109, 2543-2552. [Non-patent document 2] Zhao X., et al., PNAS, 2006, 103, 17707-17712. [Non-patent document 3] Muraoka T., Adachi K., et al., Angenwangte Chem. Int. Ed., 2013, 125, 2490-2494. [Non-patent document 4] Rajan R, Matsumura K., J. Mater. Chem. B, 2015,3, 5683-5689. Summary of the Invention [Problem to be solved by the invention]

[0006] According to the investigations of the present inventors, the compounds having a protein aggregation inhibitory effect disclosed in Non-Patent Documents 1 to 4 and Patent Document 1 have the disadvantages that they need to be added at high concentrations in order to exert a sufficient protein protection effect, and once added, they cannot be easily separated and removed.

[0007] It would be highly desirable if there were a compound that exhibited a protein aggregation inhibitory effect when added at a low concentration and that could be easily separated from the protein to be protected once added.

[0008] Therefore, an object of the present invention is to provide a novel protein aggregation inhibitor that exhibits a protein aggregation-inhibiting effect when added at a low concentration and that can be easily separated from the protein to be protected once added. [Means for solving the problem]

[0009] The present inventors have conducted extensive research into protein aggregation inhibitors and have discovered that the above-mentioned object can be achieved by the protein aggregation inhibitor described below, thereby arriving at the present invention.

[0010] Therefore, the present invention includes the following (1) and the following. (1) A method for producing a polymer compound represented by formula IV by polymerizing a trithiocarbonate compound represented by formula I, a sugar monomer compound represented by formula II, and a zwitterionic monomer compound represented by formula III: R1-S-(C=S)-S-R2 (Formula I) R3-R7-C(R4)=CH2 (Formula II) CH2=C(R6)-R8―R5 (Formula III) (Formula IV) TIFF0007755878000001.tif66147 (In Formula I, R1 is a monovalent group of a polymer chain having a modified or unmodified polyethylene structure, a polymer chain having a modified or unmodified polystyrene structure, or a polymer chain having a modified or unmodified polycaprolactone structure, R2 is a C6 to C24 alkyl group, In Formula II, R3 is a reducing or non-reducing monovalent radical of a monosaccharide or disaccharide; R4 is a hydrogen atom or a methyl group; In Formula III, R5 is a monovalent radical of a zwitterionic compound having a quaternary ammonium cation and a sulfonic acid group; R6 is a hydrogen atom or a methyl group; R7 is a divalent group selected from -(C=O)-O-, -(C=O)-NH-, and -(O=S=O)- (provided that R3 is bonded to -(C=O)-O-R3, -(C=O)-NH-R3, and -(O=S=O)-R3); R8 is a divalent group selected from -(C=O)-O-, -(C=O)-NH-, and -(O=S=O)- (wherein R5 is bonded to R5 at -(C=O)-O-R5, -(C=O)-NH-R5, and -(O=S=O)-R5); In formula IV, R1, R3, R4, R5, R6, R7, and R8 are all the groups described above; x is the average degree of polymerization of the repeating units and is 10 to 500; y is the average degree of polymerization of the repeating units and is 10 to 1000; -r- indicates that the repeating units on both sides of -r- are randomly copolymerized to form a random copolymer, -b- indicates that the repeating units on both sides of -b- are block copolymerized to form a block copolymer, m is the number of repeating units and is 1).

[0011] (2) The production method according to (1), wherein the trithiocarbonate compound represented by formula I is a compound represented by the following formula Ia or Ib: Formula Ia: TIFF0007755878000002.tif43150 Formula Ib: TIFF0007755878000003.tif56109 (in formula Ia, n is the average degree of polymerization of the repeating units and is 10 to 100; R2 is the same group as R2 in formula I, In formula Ib: l is the average degree of polymerization of the repeating units and is 10 to 100; R2 is the same group as R2 in formula I).

[0012] (3) The method according to any one of (1) to (2), wherein the glycomonomer compound represented by formula II is a compound represented by the following formula IIa: Formula IIa: TIFF0007755878000004.tif63117

[0013] (4) The method according to any one of (1) to (3), wherein the zwitterionic monomer compound represented by formula III is a compound represented by the following formula IIIa: Formula IIIa: CH2=CH-(C=O)-NH―R51-[N(R52)2]+ -R53-SO3 - (In formula IIIa, R51 is a C1-C4 alkylene group; R52 is a C1-C4 alkyl group, R53 is a C1-C4 alkylene group.

[0014] (5) The method according to any one of (1) to (4), wherein the zwitterionic monomer compound represented by formula III is a compound represented by the following formula IIIb: Formula IIIb: TIFF0007755878000005.tif35128

[0015] (6) The method according to any one of (1) to (5), wherein the polymer compound represented by formula IV is a compound represented by the following formula IVa: Formula IVa: TIFF0007755878000006.tif87138 (in formula IVa, R1, x, y, m, -b-, and -r- are all the same as R1, x, y, m, -b-, and -r- in formula IV).

[0016] (7) The method according to any one of (1) to (6), wherein the polymer compound represented by formula IV is a compound represented by the following formula IVb or IVc: Formula IVb: TIFF0007755878000007.tif59150 formula IVc: TIFF0007755878000008.tif79145 (in formula IVb, n is the average degree of polymerization of the repeating units and is 10 to 100; x, y, m, -b-, and -r- are all the same as x, y, m, -b-, and -r- in formula IV, In formula IVc: l is the average degree of polymerization of the repeating units and is 10 to 100; x, y, m, -b-, and -r- are all the same as x, y, m, -b-, and -r- in formula IV).

[0017] (8) A method for producing micelles consisting of a polymer compound represented by formula IV, by dispersing a polymer compound represented by formula IV produced by the production method according to any one of (1) to (7) in an aqueous solution.

[0018] (9) A polymer compound represented by formula IV according to (1), formula IVa according to (6), or formula IVb or formula IVc according to (7).

[0019] (10) A micelle comprising the polymer compound according to (9).

[0020] (11) A protein aggregation inhibitor comprising a micelle made of the polymer compound according to (9) or the polymer compound according to (10).

[0021] (12) The protein aggregation inhibitor according to (11), which is a protein aggregation inhibitor that can be separated from the protein to be protected after being mixed with the protein to be protected to inhibit aggregation.

[0022] (13) A method for inhibiting protein aggregation, comprising the step of mixing a micelle comprising the polymer compound according to (9) or the polymer compound according to (10) with an aqueous protein solution.

[0023] (14) A method for producing an aggregation-suppressed aqueous protein solution, comprising the step of mixing a micelle comprising the polymer compound according to (9) or the polymer compound according to (10) with an aqueous protein solution.

[0024] (15) (14) A step of obtaining an aqueous protein solution by separating the polymer compound or micelles comprising the polymer compound from the aggregation-suppressed aqueous protein solution produced by the production method according to (14). A method for producing an aqueous protein solution, comprising:

[0025] The polymer compound of the present invention can be used as a protein aggregation inhibitor, a protein structure protector, a protein activity protector, or a protein function protector.

[0026] Furthermore, since the polymer compound of the present invention can be used without exhibiting cytotoxicity, the present invention also relates to a medicine and a pharmaceutical composition comprising the polymer compound as an active ingredient.

[0027] The present invention also relates to a method for inhibiting protein aggregation, a method for protecting protein structure, a method for protecting protein activity, and a method for protecting protein function, each of which comprises a step of mixing micelles composed of the above-mentioned polymer compound with an aqueous protein solution.

[0028] The present invention also relates to a method for producing an aqueous protein solution in which aggregation is inhibited, a method for producing an aqueous protein solution in which structure is protected, a method for producing an aqueous protein solution in which activity is protected, or a method for producing an aqueous protein solution in which function is protected, which comprises the step of mixing micelles comprising the polymer compound with an aqueous protein solution.The present invention also relates to a method for producing an aqueous protein solution, which comprises the step of obtaining an aqueous protein solution by separating the polymer compound or micelles comprising the polymer compound from the aqueous protein solution produced by these methods. [Effects of the Invention]

[0029] The present invention provides a novel protein aggregation inhibitor, which exerts its effect when added at a low concentration and can be easily separated from the protein to be protected once added. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 is an explanatory diagram showing the procedure for synthesizing trehalose methacrylate. [Figure 2A] FIG. 2A is an explanatory diagram showing the procedure for synthesizing PCL-CTA. [Figure 2B] FIG. 2B is an explanatory diagram showing the procedure for synthesizing PS-CTA. [Figure 3A] FIG. 3A is an explanatory diagram showing the procedure for synthesizing PCL micelles. [Figure 3B] FIG. 3B is an explanatory diagram showing the procedure for synthesizing PS micelles. [Figure 4] FIG. 4 is a graph showing the change in UV absorption of LDH solution over time. [Figure 5] FIG. 5 is a graph showing the LDH aggregation inhibitory ability (2 mg / mL) of each additive. [Figure 6] FIG. 6 is a graph showing the concentration dependency of the LDH aggregation inhibitory activity of M1. [Figure 7A] FIG. 7A is a graph comparing the LDH activity maintenance by each micelle at each concentration. [Figure 7B] FIG. 7B is a graph comparing the maintenance of LDH activity by PS micelles (M4) at various concentrations. [Figure 8A] FIG. 8A is a graph of the CD spectrum of LDH heated at 37° C. with each micelle. [Figure 8B] FIG. 8B is a graph showing the results of maintaining the secondary structure of LDH evaluated from CD spectra. [Figure 9] Figure 9 shows an image of the appearance of the solution before centrifugation (left side of Figure 9 ), an image of the appearance of the solution (supernatant and precipitate) after centrifugation (center of Figure 9 ), and an image of the appearance of the solution redispersed after centrifugation (right side of Figure 9 ) from a centrifugation experiment using PCL micelles. [Figure 10] FIG. 10 is a graph showing the change in UV absorbance (350 nm) when the supernatant recovered from each micelle by centrifugation was re-incubated at 37° C. to re-condense the supernatant. [Figure 11]FIG. 11 is a graph showing the enzyme activity of LDH in the supernatant recovered by centrifugation after incubation at 37° C. [Figure 12] FIG. 12 is a graph comparing the inhibitory effect on the aggregation of LDH caused by freezing and thawing in terms of the remaining rate of LDH enzyme activity when each type of micelle was used. [Figure 13] FIG. 13 is a graph showing the aggregation rate of insulin (100 μM) heated at 37° C. together with each micelle. [Figure 14] FIG. 14 is a graph of the CD spectrum of insulin (10 μM) heated at 37° C. together with each micelle (2 mg / mL). [Figure 15] FIG. 15 is a graph comparing the cytotoxicity against non-cancer cells (mouse fibroblast L929) with the cell viability when each micelle was used. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention will be described in detail below with reference to specific embodiments, but the present invention is not limited to the specific embodiments described below.

[0032] [Production of the polymer compound of the present invention] The polymer compound of the present invention can be produced by polymerizing a trithiocarbonate compound represented by Formula I, a sugar monomer compound represented by Formula II, and a zwitterionic monomer compound represented by Formula III. The polymer compound of the present invention can be obtained as a polymer compound represented by Formula IV.

[0033] R1-S-(C=S)-S-R2 (Formula I)

[0034] R3-R7-C(R4)=CH2 (Formula II)

[0035] CH2=C(R6)-R8―R5 (Formula III)

[0036] (Formula IV) TIFF0007755878000009.tif66147

[0037] Trithiocarbonate Compounds of Formula I In a preferred embodiment, in Formula I: R1 is a monovalent group of a polymer chain having a modified or unmodified polyethylene structure, a polymer chain having a modified or unmodified polystyrene structure, or a polymer chain having a modified or unmodified polycaprolactone structure, and is preferably a monovalent group of a polymer chain having a modified or unmodified polystyrene structure or a polymer chain having a modified or unmodified polycaprolactone structure. The monovalent group of the polymer chain refers to a monovalent group generated by losing one hydrogen atom from the polymer chain.

[0038] In a preferred embodiment, in Formula I: R2 is a C6 to C24 alkyl group, preferably a C8 to C16 alkyl group, and more preferably a C10 to C14 alkyl group.

[0039] [Trithiocarbonate Compounds of Formula Ia and Formula Ib] In a preferred embodiment, the trithiocarbonate compound of formula I can be a compound represented by formula Ia or Ib:

[0040] Formula Ia: TIFF0007755878000010.tif43150

[0041] Formula Ib: TIFF0007755878000011.tif56109

[0042] In a preferred embodiment, in formula Ia: n is the average degree of polymerization of the repeating units and can be in the range of 10 to 100, preferably in the range of 20 to 80.

[0043] In a preferred embodiment, in formula Ia, R2 can be a group as described above in formula I.

[0044] In a preferred embodiment, in formula Ib: l is the average degree of polymerization of the repeating units, and can be in the range of 10 to 100, preferably in the range of 20 to 80.

[0045] In a preferred embodiment, in formula Ib, R2 can be a group as described above in formula I.

[0046] [Sugar monomer compound of formula II] In a preferred embodiment, in Formula II: R3 is a reducing or non-reducing monovalent radical of a monosaccharide or disaccharide. Examples of such monosaccharides include glucose, fructose, galactose, and mannose. Examples of such disaccharides include trehalose, sucrose, maltose, and lactose.

[0047] In a preferred embodiment, in Formula II: R4 is a hydrogen atom or a methyl group, preferably a methyl group.

[0048] In a preferred embodiment, in Formula II: R7 is a divalent group selected from -(C=O)-O-, -(C=O)-NH-, and -(O=S=O)-, provided that these divalent groups are bonded to R3 at the positions -(C=O)-O-R3, -(C=O)-NH-R3, and -(O=S=O)-R3, respectively.

[0049] [Sugarmonomer compound of formula IIa] In a preferred embodiment, the glycomonomer compound of formula II can be a compound of formula IIa:

[0050] Formula IIa: TIFF0007755878000012.tif63117

[0051] [Sugarmonomer compound of formula IIb] In a preferred embodiment, the glycomonomer compound of formula II can be a compound of formula IIb: R3-O-(C=O)-C(R4)=CH2 (Formula IIb)

[0052] In formula IIb, R3 and R4 can each be a group as described above.

[0053] Zwitterionic Monomeric Compounds of Formula III In a preferred embodiment, in Formula III: R5 can be a monovalent radical of a zwitterionic compound having a quaternary ammonium cation and a sulfonic acid group.

[0054] In a preferred embodiment, in Formula III: R6 can be a hydrogen atom or a methyl group.

[0055] In a preferred embodiment, in Formula III: R8 can be a divalent group selected from -(C=O)-O-, -(C=O)-NH-, and -(O=S=O)-, provided that these divalent groups are bonded to R5 at positions -(C=O)-O-R5, -(C=O)-NH-R5, and -(O=S=O)-R5, respectively.

[0056] Zwitterionic Monomeric Compounds of Formula IIIa In a preferred embodiment, the zwitterionic monomeric compound of formula III can be a compound of formula IIIa:

[0057] Formula IIIa: CH2=CH-(C=O)-NH―R51-[N(R52)2] + -R53-SO3 -

[0058] In a preferred embodiment, in formula IIIa: R51 can be, for example, a C1 to C4 alkylene group, preferably a C2 to C4 alkylene group, preferably a C3 to C4 alkylene group, or a C2 to C3 alkylene group.

[0059] In a preferred embodiment, in formula IIIa: R52 can be, for example, a C1 to C4 alkyl group, preferably a C1 to C3 alkyl group, and more preferably a C1 to C2 alkyl group.

[0060] In a preferred embodiment, in formula IIIa: R53 can be, for example, a C1 to C4 alkylene group, preferably a C2 to C4 alkylene group, preferably a C3 to C4 alkylene group, or a C2 to C3 alkylene group.

[0061] Zwitterionic Monomeric Compounds of Formula IIIb In a preferred embodiment, the zwitterionic monomeric compound of formula III can be a compound of formula IIIb:

[0062] Formula IIIb: TIFF0007755878000013.tif35128

[0063] Zwitterionic Monomeric Compounds of Formula IIIc In a preferred embodiment, the zwitterionic monomeric compound of formula III can be a compound of formula IIIc: CH2=CH-(C=O)-NH―R5 (formula IIIc)

[0064] In formula IIIc, R5 can be a group as described above.

[0065] Polymer Compound of Formula IV In a preferred embodiment, in Formula IV: R1, R3, R4, R5, R6, R7, and R8 can all be the groups described above.

[0066] In a preferred embodiment, in Formula IV: x is the average degree of polymerization of the repeating units and can be in the range of 10 to 1,000, preferably in the range of 50 to 500.

[0067] In a preferred embodiment, in Formula IV: y is the average degree of polymerization of the repeating units and can be in the range of 10 to 1,000, preferably 50 to 500.

[0068] In a preferred embodiment, in Formula IV: The ratio of x to y can be, for example, in the range of 100:25 to 100:1000, or in the range of 100:40 to 100:600, preferably in the range of 100:50 to 100:500, preferably in the range of 100:50 to 100:200, or in the range of 100:90 to 100:150.

[0069] In a preferred embodiment, there is one R group in formula IV, and x and y have the ratio of the above values, i.e., the ratio of R:x:y can be in the range of 1:100:25 to 1:100:1000.

[0070] In a preferred embodiment, in Formula IV: -r- indicates that the repeating units written on both sides of -r- are randomly copolymerized to form a random copolymer.

[0071] In a preferred embodiment, in Formula IV: -b- indicates that the repeating units written on both sides of -b- are block copolymerized to form a block copolymer.

[0072] In a preferred embodiment, in Formula IV: m is the number of repeating units and is 1.

[0073] [Polymer Compound of Formula IVa] In a preferred embodiment, the polymeric compound of formula IV can be a compound of formula IVa:

[0074] Formula IVa: TIFF0007755878000014.tif87138

[0075] In a preferred embodiment, in Formula IVa: R1, x, y, m, -b-, and -r- are all groups as defined above in formula IV, or symbols as defined above.

[0076] [Polymer Compound of Formula IVb] In a preferred embodiment, the polymer compound represented by formula IV can be a compound represented by formula IVb:

[0077] Formula IVb: TIFF0007755878000015.tif59150

[0078] In a preferred embodiment, in formula IVb: n is the average degree of polymerization of the repeating units and can be in the range of 10 to 100, preferably in the range of 20 to 80.

[0079] In a preferred embodiment, in formula IVb: x, y, m, -b-, and -r- are all groups or symbols having the above-mentioned meanings in formula IV.

[0080] In Formula IVb, terminal groups derived from the monomers are present at both ends of the polymer, but these are omitted in accordance with the usual practice of polymer notation.

[0081] [Polymer Compound of Formula IVc] In a preferred embodiment, the polymeric compound of formula IV can be a compound of formula IVc:

[0082] Formula IVc: TIFF0007755878000016.tif79145

[0083] In a preferred embodiment, in Formula IVc: l is the average degree of polymerization of the repeating units, and can be in the range of 10 to 100, preferably in the range of 20 to 80.

[0084] In a preferred embodiment, in Formula IVc: x, y, m, -b-, and -r- are all groups or symbols having the above-mentioned meanings in formula IV.

[0085] In Formula IVc, terminal groups derived from the monomers are present at both ends of the polymer, but these are omitted in accordance with the usual practice of polymer notation.

[0086] [Polymerization reaction] In a preferred embodiment, the polymer compound of the present invention represented by Formula IV can be produced by polymerizing a trithiocarbonate compound represented by Formula I, a sugar monomer compound represented by Formula II, and a zwitterionic monomer compound represented by Formula III. This polymerization reaction proceeds via reversible addition / fragmentation chain transfer polymerization (RAFT polymerization). The trithiocarbonate compound represented by Formula I serves as a macro-chain transfer agent instead of a RAFT agent. In this case, the sugar monomer compound represented by Formula II and the zwitterionic monomer compound represented by Formula III are used as monomers, and random copolymerization of the sugar monomer compound represented by Formula II and the zwitterionic monomer compound represented by Formula III occurs from the end of the trithiocarbonate compound represented by Formula I, thereby producing the polymer compound of the present invention represented by Formula IV as a block copolymer.

[0087] In a preferred embodiment, to promote such a polymerization reaction, a solvent such as dimethyl sulfoxide or dimethylformamide can be used, and an additive such as azobisisobutyronitrile or 4,4'-azobis(4-cyanovaleric acid) can be used. The polymerization reaction can be carried out under conditions known as radical polymerization conditions.

[0088] [Micelle formation] In a preferred embodiment, the polymer compound of the present invention represented by Formula IV can be dispersed in an aqueous solution to form micelles through self-association. Dispersion in an aqueous solution can be achieved, for example, by synthesizing the polymer compound and then replacing the solvent with an aqueous solution by dialysis or other means, or by lyophilizing the synthesized polymer compound and then dispersing it in an aqueous solution.

[0089] [Protein aggregation suppression] The micelles formed by the polymeric compounds of formula IV of the present invention exhibit excellent protein aggregation inhibition at very high concentrations, which can be achieved simply by mixing the target protein with the micelles in an aqueous solution.

[0090] That is, according to the present invention, the aggregation-inhibiting effect is exerted on proteins that would normally aggregate due to the procedures during purification or that would aggregate under storage conditions after purification, thereby achieving the excellent effect of enabling the purification of proteins that have been difficult to purify in the past and the storage of proteins under conditions that have been difficult to store in the past.

[0091] [Concentration for inhibiting aggregation] In a preferred embodiment, the protein aggregation inhibitory effect can be exerted by dispersing the polymer compound of the present invention represented by formula IV in an aqueous solution at a concentration of, for example, 0.1 to 10 mg / L, for example, 0.2 to 5 mg / L, for example, 0.5 to 5 mg / L, or for example, 1 to 3 mg / mL.

[0092] [Protein structure protection] In a preferred embodiment, the micelles formed by the polymer compound of the present invention represented by formula IV exhibit an excellent protein aggregation-inhibiting effect, i.e., protein aggregation is inhibited under macroscopic observation, while at the same time exhibit an excellent structure-protecting effect, i.e., the protein structure is maintained under microscopic observation. This excellent structure-protecting effect can be detected, for example, by CD (Circular Dichroism) spectroscopy, which will be described later in the Examples.

[0093] [Protein activity protection] In a preferred embodiment, the micelles formed by the polymer compound of the present invention represented by formula IV exhibit an excellent protein aggregation-inhibiting effect, i.e., the aggregation of proteins is inhibited by visual observation, while at the same time exhibiting an excellent activity-protecting effect, i.e., the protein maintains its activity and function, as demonstrated, for example, by measuring the LDH enzyme activity described in the Examples below.

[0094] [Inhibition and protection against freezing aggregation] In a preferred embodiment, the excellent aggregation-inhibiting effect, structure-protecting effect, activity-protecting effect, and function-protecting effect also exert a protective effect on LDH enzyme activity against aggregation that occurs during freezing and thawing, as will be described later in the Examples, and the aggregation-inhibiting effect, structure-protecting effect, activity-protecting effect, and function-protecting effect are exerted during freezing and freezing and thawing.

[0095] [Effects on various proteins] In a preferred embodiment, the excellent aggregation-inhibiting, structural-protecting, activity-protecting, and functional-protecting effects of the compounds of the present invention on proteins are not limited to LDH, as will be described later in the Examples. This demonstrates that the compounds of the present invention exert similar protective effects on a wide range of proteins. As is well known, protein aggregation is one of the major causes of protein structural changes and inactivation. Therefore, the results of the Examples described later demonstrate that the compounds of the present invention, which protect proteins by inhibiting aggregation, exert similar protective effects on a wide range of proteins. Examples of proteins for which such protective effects are particularly expected include antibodies, insulin, lactate dehydrogenase, alkaline phosphatase, acetylcholinesterase, ascorbate oxidase, and alcohol dehydrogenase.

[0096] [Cytotoxicity] In a preferred embodiment, this excellent protein protection effect can be exerted without exhibiting cytotoxicity, as will be described later in the Examples. Therefore, the compounds of the present invention are not limited to being used as molecular biological research tools, but can also be used as drugs and pharmaceuticals by administering them to cells, tissues, organs, and living organisms.

[0097] [Separation from the protected protein] In a preferred embodiment, micelles formed by the polymer compound of the present invention represented by Formula IV exhibit excellent protein aggregation inhibitory activity when mixed with a protein to be protected, and can be easily separated from the mixed protein to be protected. Surprisingly, the separated protein to be protected maintains almost the same function and activity as before mixing with the micelles, and at the same time, the separated micelles can be reused to inhibit protein aggregation. Such excellent separation properties are highly advantageous in that they do not impose any restrictions on the subsequent use of the protected protein, and also in that the separated micelles can be reused.

[0098] In a preferred embodiment, the protein to be protected mixed with the micelles can be separated by known separation means that take advantage of the molecular weight of the micelles. Examples of such separation means include filtration and centrifugation. In a preferred embodiment, centrifugation can be performed by applying centrifugal force at an acceleration of, for example, 5,000 to 30,000 g, preferably 10,000 to 20,000 g, for example, 1 to 120 minutes or 10 to 60 minutes. In a preferred embodiment, solid-liquid separation by centrifugation results in the precipitation of the micelles and the supernatant of the protein to be protected. [Example]

[0099] The present invention will be described in detail below with reference to examples. The present invention is not limited to the examples illustrated below. In the examples, "%" and "parts" indicate % by weight and parts by weight, respectively, unless otherwise specified.

[0100] [Experimental Example 1] 1. Synthesis of micelles [1.1 Synthesis of trehalose methacrylate] Anhydrous trehalose was dried in vacuum at 105°C for 2 days. Dried trehalose (2 mmol) was dissolved in anhydrous dimethylformamide (DMF, 2 mmol), and 4 mmol of triethylamine and 2 mmol of methacrylic anhydride were added. The mixture was reacted under nitrogen at 25°C for 24 hours. The reaction mixture was quenched with diethyl ether at 4°C, washed with hexane and diethyl ether, and the precipitate was collected. The synthesis procedure is shown in Figure 1.

[0101] [1.2 Synthesis of macro chain transfer agents (PCL-CTA, PS-CTA)] One mmol of the RAFT agent (4-cyano-4-[(dodecylsulfanylthiocarbonyl)-sulfanyl]pentanol) was dissolved in 1 mmol of DMF, 30 mmol of ε-caprolactone was added, and a catalytic amount of tin(II) 2-ethylhexanoate (0.1 wt%) was added. The reaction was allowed to proceed for 16 hours under a nitrogen atmosphere. The product was then reprecipitated with 1:1 (v / v) methanol:diethyl ether (PCL-CTA). The synthesized compound, which contains both a polycaprolactone (PCL) moiety and a chain transfer agent (CTA) moiety, is referred to as PCL-CTA. The synthesis procedure is shown in Figure 2A.

[0102] One mmol of the RAFT agent (4-cyano-4-[(dodecylsulfanylthiocarbonyl)-sulfanyl]pentanol) was dissolved in 1 mmol of DMF, and 30 mmol of styrene and 0.03 mmol of azobisisobutyronitrile (AIBN) were added. The mixture was allowed to react for 16 hours under a nitrogen atmosphere. The product was then reprecipitated with 1:1 (v / v) methanol:diethyl ether (PS-CTA). The synthesized compound, which contains both a polystyrene (PS) moiety and a chain transfer agent (CTA) moiety, is referred to as PS-CTA. The synthesis procedure is shown in Figure 2B.

[0103] NMR analysis revealed that the number average degree of polymerization of PCL was 69, and that of PS was 45.

[0104] [1.3 Synthesis of micelles] PCL-CTA or PS-CTA and V-501 (initiator) were added to a round-bottom flask, and a solution of trehalose methacrylate and SPB monomer dissolved in dimethyl sulfoxide (DMSO) was added dropwise. The reaction mixture was allowed to stand at 70°C for 24 hours under a nitrogen atmosphere. The reaction mixture was then dialyzed against distilled water using a dialysis membrane (MWCO 100 kDa) to remove unreacted materials, SPB homopolymer, etc. In this manner, three types of PCL micelles (M1–M3 in Table 1) and one type of PS micelle (M4 in Table 2) were synthesized. The synthesis procedure is shown in Figures 3A and 3B. The properties of the synthesized micelles are shown in Tables 1 and 2.

[0105] [Table 1]

[0106] [Table 2]

[0107] In Table 1, R is PCL-RAFT Agent, i.e., PCL-CTA. C1 is Poly-SPB (sulfobetaine polymer). C2 is TrMA (trehalose methacrylate).

[0108] In Table 2, R is PS-RAFT Agent, i.e., PS-CTA. C1 is Poly-SPB (sulfobetaine polymer). C2 is TrMA (trehalose methacrylate).

[0109] As shown in Table 1, M1 is a polymer in which 100-mer SPB and 100-mer trehalose are randomly polymerized onto a PCL block; M2 is a polymer in which 200-mer SPB and 100-mer trehalose are polymerized onto a PCL block; and M3 is a polymer in which 100-mer SPB and 500-mer trehalose are polymerized onto a PCL block.

[0110] [Experimental Example 2] [2. Protein aggregation suppression effect experiment] [LDH (lactate dehydrogenase) aggregation inhibition] [Time course of LDH aggregation compared with each micelle and polymer] A given concentration of micelles or polymer was mixed with 0.2 mg / mL LDH (PBS solution), and the mixture was stirred at 37°C for 30 minutes. The absorbance at 350 nm was measured using a UV-visible spectrophotometer (UV-1800, Shimadzu). This allowed for the evaluation of the increase in absorbance due to aggregation. SPB homopolymer was used as a control.

[0111] The results are shown in Figure 4. Figure 4 is a graph showing the change in UV absorption of the LDH solution over time. The concentration of the polymer or micelles was 2 mg / mL.

[0112] As shown in Figure 4, an increase in absorbance due to aggregation was confirmed in the control group and the system without added polymer. On the other hand, no increase in absorbance was observed in any of the micelles M1, M2, and M3. The initial high absorbance in the micellar system is due to the turbidity of the micelles.

[0113] [Comparison of LDH aggregation inhibitory ability of each micelle and polymer] Figure 5 is a graph showing the LDH aggregation inhibitory activity (2 mg / mL) of each additive. Figure 5 also shows the aggregation rate calculated from the rate of increase in UV absorbance when each polymer and trehalose were added to LDH at a concentration of 2 mg / mL. The aggregation rate was 0.02% for M1, 22.7% for M2, and 32.4% for M3, respectively, demonstrating particularly high activity in M1.

[0114] As shown in Figure 5, these micelles M1 to M3 all exhibited superior aggregation-inhibiting properties compared to Poly-SPB and trehalose. In particular, M1 exhibited extremely superior aggregation-inhibiting properties, in other words, LDH protection effects. These aggregation-inhibiting properties were effective even when added at an ultralow concentration of just 0.2% (2 mg / mL).

[0115] [Concentration dependence of the LDH aggregation inhibitory activity of M1 micelles] Figure 6 is a graph showing the concentration dependence of M1's ability to inhibit LDH aggregation. The conditions, other than the M1 micelle concentration, were the same as those shown in Figure 5 above. It was found that even at 1 mg / mL, M1 had a high aggregation inhibitory activity of 16.6%. Furthermore, when added at 0.2% (2 mg / mL), M1 demonstrated an LDH aggregation inhibitory activity that almost completely inhibited aggregation.

[0116] [Maintaining LDH activity with each micelle] A substrate stock solution (pH 7.0) was prepared by mixing 200 μL of 63 mM NADH in PBS and 500 μL of 10 mM sodium pyruvate in PBS, and then diluting to 50 mL. Next, a given concentration of micelles was mixed with a 20.7 mU / mL LDH solution (PBS) and incubated at 37°C for 1 hour. 5 μL of this solution was mixed with 195 μL of the substrate stock solution in a 96-well plate. The absorbance at 340 nm was measured for 20 minutes using a Tecan Infinite 200 PRO M Nano+ microplate reader. The enzyme activity was calculated from the rate of absorbance decrease, with the untreated sample being set at 100%. The remaining enzyme activity was calculated as the percentage of enzyme activity. The results are shown in Figures 7A and 7B.

[0117] Figure 7A is a graph comparing the LDH activity retention of each micelle at various concentrations. Figure 7A shows the values ​​for M1 (left end), M2 (center), and M3 (right end) for each concentration. As shown in Figure 7A, all M1 to M3 exhibited high residual activity, even at very low concentrations of 1 to 2 mg / mL. As shown in Figure 7A, high residual activity was observed at all concentrations. In particular, M1 exhibited high residual activity rates: 60% at 0.5 mg / mL, 66% at 0.75 mg / mL, 76% at 1 mg / mL, 88% at 1.5 mg / mL, and 94% at 2 mg / mL. Furthermore, M2 exhibited high residual activity rates: 63% at 0.5 mg / mL, 68% at 0.75 mg / mL, 73% at 1 mg / mL, 87% at 1.5 mg / mL, and 89% at 2 mg / mL. Furthermore, M3 showed high residual activity rates of 50% at 0.75 mg / mL, 63% at 1 mg / mL, 75% at 1.5 mg / mL, and 82% at 2 mg / mL.

[0118] Figure 7B is a graph comparing the LDH activity maintenance by PS micelles (M4) at various concentrations. As shown in Figure 7B, high residual activity was observed at all concentrations. In particular, high residual activity rates of 61% at 0.75 mg / mL and 72% at 1 mg / mL were observed.

[0119] [CD spectrum measurement] CD spectroscopy was performed to evaluate the secondary structural changes of LDH. Each micelle was added to a 0.1 mg / mL LDH solution (PBS) at a concentration of 2 mg / mL, and the mixture was placed in a JASCO-820 spectrometer cell. The mixture was then incubated at 15°C under a nitrogen atmosphere for 30 minutes, followed by incubation at 37°C for 1 hour. The results are shown in Figure 8A and Figure 8B.

[0120] Figure 8A is a graph of the CD spectrum of LDH heated at 37°C with each micelle. Figure 8B is a graph showing the results of maintaining the secondary structure of LDH evaluated from the CD spectrum. Incubated LDH shows the results of an experiment in which similar incubation was performed without adding micelles. Native LDH shows the results of an experiment in which no micelles were added and the LDH was not incubated at 37°C for 1 hour.

[0121] Figure 8B is a bar graph showing the content ratio of each structure based on the results of CD spectrum measurement. In Figure 8B, the first section from the top of each column shows the content ratio of "Unordered," the second section shows the content ratio of "Turns," the third section shows the content ratio of "Strand," and the fourth section (the bottom of each column) shows the content ratio of "Helix."

[0122] As shown in Figures 8A and 8B, it was found that the micelles-added system maintained almost the same structure as before the heat treatment, compared to the additive-free LDH system.

[0123] [Examination of separation ability of each micelle] An LDH solution (LDH 0.2 mg / mL) was incubated in the presence of each micelle (2 mg / mL) at 37°C for 30 minutes, and then centrifuged at 16,000 rpm (13,100 g) for 90 minutes to separate the supernatant and precipitate.

[0124] The protein concentrations in the supernatants were measured by the Bradford method, and the recovery rates were 91.55 ± 2.83% for M1, 92.75 ± 1.87% for M2, and 94.87 ± 1.29% for M3, indicating that there was almost no loss of protein due to adsorption to micelles.

[0125] Furthermore, the recovered micelles were reusable. It was also confirmed that the recovered LDH from the supernatant aggregated when re-incubated at 37°C for 1 hour. These findings were confirmed by the experiments described below.

[0126] [Redispersibility of micelles after centrifugation] Figure 9 shows an image of the appearance of the solution before centrifugation (left side of Figure 9), an image of the appearance of the solution (supernatant and precipitate) after centrifugation (center of Figure 9), and an image of the appearance of the solution redispersed after centrifugation (right side of Figure 9) from a centrifugation experiment using PCL micelles. As shown in the image on the right side of Figure 9, the micelles had high dispersibility and could be redispersed even after centrifugation.

[0127] [Maintenance of the aggregation ability of LDH recovered by centrifugation] The LDH in the supernatant recovered after centrifugation became cloudy when re-incubated at 37°C, indicating that it still retained agglutination ability. The results of this experiment are shown in Figure 10.

[0128] Figure 10 is a graph showing the change in UV absorbance (350 nm) when the supernatant recovered after adsorption to each micelle by centrifugation was re-incubated at 37°C and re-condensed. The upper curve in the graph of Figure 10 shows the change in absorbance due to LDH in the supernatant recovered by centrifugation, and the lower curve in the graph of Figure 10 shows the change in absorbance due to LDH that had not been previously incubated at 37°C.

[0129] [LDH enzyme activity recovered by centrifugation] After incubation at 37°C, the supernatant was collected by centrifugation and the LDH enzyme activity was confirmed. The experiment was carried out under the same conditions as the experiment to determine the maintenance of LDH activity by each micelle. The results are shown in Figure 11.

[0130] Figure 11 is a graph showing the enzymatic activity of LDH in the supernatant recovered by centrifugation after incubation at 37°C. The horizontal axis of Figure 11 represents the concentration of each PCL micelle. The vertical axis of Figure 11 represents the residual amount of LDH enzymatic activity, which is expressed as a relative value when the unheated LDH activity is set to 100%. The graph in Figure 11 shows the LDH enzymatic activity when M1 (left end), M2 (center), and M3 (right end) were used as micelles for each concentration of PCL micelles. As shown in Figure 11, LDH exhibited high residual activity in all micelles.

[0131] [Experimental Example 3] [Inhibitory effect on freezing-induced aggregation of LDH] A given concentration of micelles was mixed with a 20.7 mU / mL LDH solution (PBS), frozen for 24 hours in a -20°C freezer, and thawed at room temperature. This process was repeated 15 times. 5 μL of this solution was mixed with 195 μL of substrate stock solution on a 96-well plate. The absorbance at 340 nm was measured for 20 minutes using a microplate reader (Tecan Infinite 200 PRO M Nano+). The enzyme activity was calculated from the rate of absorbance decrease. The residual enzyme activity was calculated based on the untreated sample (100%). The results are shown in Figure 12.

[0132] Figure 12 is a graph comparing the LDH activity maintained by each micelle at each concentration. Figure 12 shows the values ​​for M1 (left end), M2 (center), and M3 (right end) for each concentration. As shown in Figure 12, while activity was almost completely lost in a system without added micelles (0 mg / mL), high residual activity was observed for all of M1 to M3 even at very low concentrations of 1 to 2 mg / mL.

[0133] [Experimental Example 4] [Inhibitory effect on insulin aggregation] Figure 13 shows the aggregation rate of insulin (100 μM) heated with each micelle at 37°C for 1 hour. Each micelle was mixed to a final concentration of 1 mL, with the concentrations shown on the horizontal axis in Figure 13. Figure 13 shows values ​​for M1 (left end), M2 (center), and M3 (right end) for each concentration. As shown in Figure 13, while aggregation occurred almost 100% in the system without added micelles (0 mg / mL), it was found that all of M1 to M3 showed high inhibitory ability even at very low concentrations of 0.25 to 2 mg / mL.

[0134] Figure 14 shows the CD spectra of insulin (10 μM) heated at 37°C with each micelle (2 mg / mL). Insulin-heat shows the results of an experiment in which the same incubation was carried out without adding micelles. Insulin Native shows the results of an experiment in which the micelles were not added and the incubation was not carried out for 1 hour at 37°C. In the case of insulin-heat, the curve indicating the higher-order structure has changed significantly, whereas the system with M1-M3 added has a structure close to that of insulin native.

[0135] [Experimental Example 5] [Toxicity testing] The cytotoxicity of each micelle was examined using mouse fibroblast L929 cells. L929 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% serum at 37°C under a 5% CO2 environment. 1,000 cells were seeded in a 96-well plate and cultured for 72 hours. Each micelle was added to the medium at a predetermined concentration. After 24 hours, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to the medium at 100 μg / mL. After 4 hours, the medium was discarded, and 100 μL of dimethyl sulfoxide was added to extract the dye. The absorbance at 540 nm was measured. The absorbance of the system without micelles was set as 100% viability, and the viability was calculated from the absorbance at each concentration.

[0136] Figure 15 shows the values ​​of M1 (left end), M2 (center), and M3 (right end) for each concentration. Even at 10 mg / mL, which is five times the concentration of 2 mg / mL, which had the highest aggregation inhibitory effect, high cell viability was maintained, demonstrating the low toxicity of the micelles. [Industrial Applicability]

[0137] The present invention provides a novel protein aggregation inhibitor, and is an industrially useful invention.

Claims

1. A method for producing a polymer compound represented by formula IV by polymerizing a trithiocarbonate compound represented by formula I, a sugar monomer compound represented by formula II, and a zwitterionic monomer compound represented by formula III: R1-S-(C=S)-S-R2 (Formula I) R3-R7-C (R4)=CH ２ (Formula II) CH ２ =C(R6)-R8-R5 (Formula III) (Formula IV) (In Formula I, R1 is a monovalent group of a polymer chain having a modified or unmodified polyethylene structure, a polymer chain having a modified or unmodified polystyrene structure, or a polymer chain having a modified or unmodified polycaprolactone structure, R2 is a C6 to C24 alkyl group; In Formula II, R3 is a reducing or non-reducing monovalent radical of a monosaccharide or disaccharide; R4 is a hydrogen atom or a methyl group; In Formula III, R5 is a monovalent radical of a zwitterionic compound having a quaternary ammonium cation and a sulfonic acid group; R6 is a hydrogen atom or a methyl group; R7 is a divalent group selected from -(C=O)-O-, -(C=O)-NH-, and -(O=S=O)- (provided that R7 is bonded to R3 at -(C=O)-O-R3, -(C=O)-NH-R3, and -(O=S=O)-R3); R8 is a divalent group selected from -(C=O)-O-, -(C=O)-NH-, and -(O=S=O)- (provided that R5 is bonded to at -(C=O)-O-R5, -(C=O)-NH-R5, and -(O=S=O)-R5); In Formula IV, R1, R3, R4, R5, R6, R7, and R8 are all the groups described above; x is the average degree of polymerization of the repeating units, which is 10 to 500; y is the average degree of polymerization of the repeating units, which is 10 to 1000; -r- indicates that the repeating units on both sides of -r- are randomly copolymerized to form a random copolymer, -b- indicates that the repeating units on both sides of -b- are block copolymerized to form a block copolymer, m is the number of repeating units and is 1).

2. The method according to claim 1, wherein the trithiocarbonate compound represented by formula I is a compound represented by the following formula Ia or Ib: Formula Ia: Formula Ib: (In formula Ia, n is the average degree of polymerization of the repeating units and is 10 to 100; R2 is the same group as R2 in formula I, In formula Ib, l is the average degree of polymerization of the repeating units, which is 10 to 100; R2 is the same group as R2 in formula I).

3. The method according to claim 1, wherein the sugar monomer compound represented by formula II is a compound represented by the following formula IIa: Formula IIa:

4. 2. The method of claim 1, wherein the zwitterionic monomeric compound of formula III is a compound of formula IIIa: Formula IIIa: CH ２ =CH-(C=O)-NH―R51-[N(R52) ２ ] ＋ -R53-SO ３ － (In Formula IIIa, R51 is a C1-C4 alkylene group; R52 is a C1-C4 alkyl group; R53 is a C1-C4 alkylene group).

5. 2. The method of claim 1, wherein the zwitterionic monomeric compound of formula III is a compound of formula IIIb: Formula IIIb:

6. The method according to claim 1, wherein the polymer compound represented by formula IV is a compound represented by the following formula IVa: Formula IVa: (In formula IVa, R1, x, y, m, -b-, and -r- are all the same as R1, x, y, m, -b-, and -r- in formula IV).

7. The method according to claim 1, wherein the polymer compound represented by formula IV is a compound represented by the following formula IVb or IVc: Formula IVb: Formula IVc: (In formula IVb, n is the average degree of polymerization of the repeating units and is 10 to 100; x, y, m, -b-, and -r- are all the same as x, y, m, -b-, and -r- in formula IV, In formula IVc, l is the average degree of polymerization of the repeating units, which is 10 to 100; x, y, m, -b-, and -r- are all the same as x, y, m, -b-, and -r- in formula IV).

8. A method for producing micelles comprising a polymer compound represented by formula IV, by dispersing the polymer compound represented by formula IV produced by the production method according to any one of claims 1 to 7 in an aqueous solution.

9. A polymeric compound of formula IV according to claim 1, formula IVa according to claim 6, or formula IVb or formula IVc according to claim 7.

10. A micelle comprising the polymer compound according to claim 9.

11. A protein aggregation inhibitor comprising the polymer compound according to claim 9.

12. The protein aggregation inhibitor according to claim 11, which is a protein aggregation inhibitor that can be separated from a protein to be protected after being mixed with the protein to be protected to inhibit aggregation.

13. A method for inhibiting protein aggregation, comprising the step of mixing the polymer compound of claim 9 with an aqueous protein solution.

14. A method for producing an aggregation-suppressed aqueous protein solution, comprising the step of mixing the polymer compound according to claim 9 with an aqueous protein solution.

15. a step of obtaining an aqueous protein solution by separating the polymer compound or micelles comprising the polymer compound from the aggregation-inhibited aqueous protein solution produced by the production method according to claim 14; A method for producing an aqueous protein solution, comprising:

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