WO2023100795A1

WO2023100795A1 - Protein aggregation inhibitor

Info

Publication number: WO2023100795A1
Application number: PCT/JP2022/043739
Authority: WO
Inventors: 和明松村; ロビンラジャン
Original assignee: 国立大学法人北陸先端科学技術大学院大学
Priority date: 2021-11-30
Filing date: 2022-11-28
Publication date: 2023-06-08
Also published as: JPWO2023100795A1

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

protein aggregation inhibitor

The present invention relates to protein aggregation inhibitors.

In recent years, expectations for protein drugs, including antibody drugs and enzyme preparations, have increased. A major problem in the development and practical use of protein drugs, especially antibody drugs and enzyme preparations, is the problem of stability. The problems of stability can be broadly classified into inactivation during long-term storage and aggregation during purification. Since deactivation during long-term storage often manifests itself as aggregation due to freezing, the problem of stability can be grasped as a problem of inhibition of protein aggregation during long-term storage and purification.

Examples of compounds effective in suppressing protein aggregation include 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). has been reported.

International publication WO2018/003909

According to studies by the present inventors, the compounds with protein aggregation inhibitory effects disclosed in Non-Patent Documents 1 to 4 and Patent Document 1 need to be added at a high concentration in order to exhibit a sufficient protein-protecting effect. However, once added, it is not possible to separate and remove easily.

It would be very preferable if there was a compound that exerts the effect of inhibiting protein aggregation even when added at a low concentration and that can be easily separated from the protein to be protected once added.

Therefore, an object of the present invention is to provide a novel protein aggregation inhibitor that exhibits an effect of inhibiting protein aggregation even when added at a low concentration, and that can be easily separated from the protein to be protected once added. It is in.

The inventor has conducted extensive research on protein aggregation inhibitors. Then, the inventors have found that the above object can be achieved by the protein aggregation inhibitor described below, and have arrived at the present invention.

Therefore, the present invention includes the following (1) and below.
(1)
A trithiocarbonate compound represented by Formula I, a sugar monomer compound represented by Formula II, and a zwitterionic monomer compound represented by Formula III are polymerized to obtain a polymer compound represented by Formula IV. How to manufacture:

R1-S-(C=S)-S-R2 (Formula I)

R3-R7-C(R4)= _CH2 (Formula II)

_CH2 =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-C24 alkyl group,
In Formula II,
R3 is a reducing or non-reducing, mono- or disaccharide monovalent group;
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)-, where R3 is - (C=O)-O-R, -(C=O)-NH-R, and -(O=S=O)-R3),
R8 is a divalent group selected from -(C=O)-O-, -(C=O)-NH-, and -(O=S=O)-, where R5 is - (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 unit and is 10 to 500,
y is the average degree of polymerization of the repeating unit and is 10 to 1000,
-r- indicates that the repeating units described on both sides of -r- are randomly copolymerized to form a random copolymer,
-b- indicates that the repeating units described on both sides of -b- are block copolymerized to form a block copolymer,
m is the repeating number of the repeating unit and is 1).

(2)
The process according to (1), wherein the trithiocarbonate compound represented by Formula I is a compound represented by Formula Ia or Formula Ib below:
Formula Ia:

Formula Ib:

(In Formula Ia,
n is the average degree of polymerization of the repeating unit 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 unit and is 10 to 100,
R2 is the same group as R2 in Formula I).

(3)
The production method according to any one of (1) to (2), wherein the sugar monomer compound represented by formula II is a compound represented by the following formula IIa:
Formula IIa:

(4)
The production method according to any one of (1) to (3), wherein the zwitterionic monomeric compound represented by Formula III is a compound represented by the following 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)
The production method according to any one of (1) to (4), wherein the zwitterionic monomeric compound represented by Formula III is a compound represented by the following Formula IIIb:
Formula IIIb:

(6)
The production method according to any one of (1) to (5), wherein the polymer compound represented by Formula IV is a compound represented by Formula IVa below:
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 production method according to any one of (1) to (6), wherein the polymer compound represented by Formula IV is a compound represented by Formula IVb or Formula IVc below:
Formula IVb:

Formula IVc:

(In formula IVb,
n is the average degree of polymerization of the repeating unit and is 10 to 100,
x, y, m, -b-, -r- are all the same as x, y, m, -b-, -r- in formula IV,
In Formula IVc,
l is the average degree of polymerization of the repeating unit 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).

(8)
By dispersing the polymer compound represented by Formula IV produced by the production method according to any one of (1) to (7) in an aqueous solution, a micelle composed of the polymer compound represented by Formula IV is produced. how to.

(9)
A polymeric compound of formula IV as described in (1), formula IVa as described in (6), or formula IVb or IVc as described in (7).

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

(11)
A protein aggregation inhibitor comprising a micelle comprising the polymer compound according to (9) or the polymer compound according to (10).

(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.

(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.

(14)
A method for producing an aggregation-suppressed aqueous protein solution, comprising the step of mixing the polymer compound according to (9) or micelles comprising the polymer compound according to (10) 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-suppressed protein aqueous solution produced by the production method according to (14);
A method for producing an aqueous protein solution, comprising:

The polymer compound of the present invention can be used as a protein aggregation inhibitor, protein structure-protecting agent, protein activity-protecting agent, or protein function-protecting agent.

In addition, since the polymer compound of the present invention can be used without exhibiting cytotoxicity, the present invention also resides in pharmaceuticals and pharmaceutical compositions containing the polymer compound as an active ingredient.

In addition, the present invention provides a method for inhibiting protein aggregation, a method for protecting protein structure, a method for protecting protein activity, and a protein function, which comprise the step of mixing a micelle comprising the polymer compound with an aqueous protein solution. There is also a way to protect the

Further, the present invention provides a method for producing an aqueous protein solution whose aggregation is inhibited, a method for producing an aqueous protein solution whose structure is protected, and an aqueous protein solution whose activity is protected, comprising the step of mixing a micelle comprising the polymer compound with an aqueous protein solution. and a method for producing a functionally protected aqueous protein solution. The present invention also provides a method for producing an aqueous protein solution, comprising 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.

The present invention provides a novel protein aggregation inhibitor. INDUSTRIAL APPLICABILITY The protein aggregation inhibitor of the present invention exerts its effect even when added at a low concentration, and can be easily separated from the protein to be protected once added.

FIG. 1 is an explanatory diagram showing the procedure for synthesizing trehalose methacrylate. FIG. 2A is an explanatory diagram showing the procedure for synthesizing PCL-CTA. FIG. 2B is an explanatory diagram showing the procedure for synthesizing PS-CTA. FIG. 3A is an explanatory diagram showing a procedure for synthesizing PCL micelles. FIG. 3B is an explanatory diagram showing the procedure for synthesizing PS micelles. FIG. 4 is a graph showing the time change of UV absorption of L and LDH solutions. FIG. 5 is a graph showing the LDH aggregation inhibitory ability (2 mg/mL) of each additive. FIG. 6 is a graph showing the concentration dependence of the ability of M1 to suppress LDH aggregation. FIG. 7A is a graph comparing LDH activity maintenance by each micelle at each concentration. FIG. 7B is a graph comparing LDH activity maintenance by each concentration of PS micelle (M4). FIG. 8A is a graph of the CD spectra of LDH heated at 37° C. with each micelle. FIG. 8B is a graph showing the results of maintaining the secondary structure of LDH evaluated from the CD spectrum. FIG. 9 shows an image of the appearance of the solution before centrifugation (left side of FIG. 9) and an image of the appearance of the solution (supernatant and precipitate) after centrifugation (Fig. 9 middle) and an image of the appearance of the redispersed solution after centrifugation (right side of FIG. 9). FIG. 10 is a graph showing the change in UV absorbance (350 nm) when the supernatant collected by centrifugation after adsorption to each micelle was re-incubated at 37° C. and recondensed. FIG. 11 is a graph showing the enzymatic activity of LDH in the supernatant recovered by centrifugation after incubation at 37°C. FIG. 12 is a graph comparing the inhibitory effect of LDH on freeze-thaw aggregation with the residual rate of LDH enzymatic activity when each micelle was used. FIG. 13 is a graph showing the aggregation rate of insulin (100 μM) heated at 37° C. with each micelle. FIG. 14 is a graph of CD spectra of insulin (10 μM) heated at 37° C. with each micelle (2 mg/mL). FIG. 15 is a graph comparing the cytotoxicity against non-cancer cells (mouse fibroblast L929) by cell viability when using each micelle.

The present invention will be described in detail below with specific embodiments. The present invention is not limited to the specific embodiments given below.

[Production of the polymer compound of the present invention]
The polymer compound of the present invention is 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. be able to. The polymeric compounds of the present invention can be obtained as polymeric compounds of Formula IV.

　R1-S-(C=S)-S-R2　(Formula I)

R3-R7-C(R4)= _CH2 (Formula II)

_CH2 =C(R6)-R8-R5 (formula III)

(Formula IV)

[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, preferably It is a monovalent group of a polymer chain having a modified or unmodified polystyrene structure, a polymer chain having a modified or unmodified polycaprolactone structure. A monovalent group of a polymer chain refers to a monovalent group resulting from the loss of one hydrogen atom from the polymer chain.

In a preferred embodiment, in Formula I,
R2 is a C6-C24 alkyl group, preferably a C8-C16 alkyl group, more preferably a C10-C14 alkyl group.

[Trithiocarbonate compounds of Formula Ia and Formula Ib]
In a preferred embodiment, the trithiocarbonate compound of Formula I can be a compound of Formula Ia or Formula Ib below.

Formula Ia:

Formula Ib:

In a preferred embodiment, in Formula Ia,
n is the average degree of polymerization of the repeating unit, and can be in the range of 10-100, preferably in the range of 20-80.

In a preferred embodiment, in Formula Ia, R2 can be the group described above in Formula I.

In a preferred embodiment, in Formula Ib,
l is the average degree of polymerization of repeating units, and can be in the range of 10-100, preferably in the range of 20-80.

In a preferred embodiment, in formula Ib, R2 can be the group described above in formula I.

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

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

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 defined as -(C=O)-O-R3, -(C=O)-NH-R3, and -(O=S=O)-R3 positions, respectively. join with .

[Sugar monomer compound of formula IIa]
In a preferred embodiment, the glycomonomer compound of Formula II can be a compound of Formula IIa below.

Formula IIa:

[Sugar monomer compound of formula IIb]
In a preferred embodiment, the glycomonomer compound represented by Formula II can be a compound represented by Formula IIb below.
R3-O-(C=O)-C(R4)= _CH2 (Formula IIb)

In Formula IIb, R3 and R4 can each be the groups described above.

[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.

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

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 defined as -(C=O)-O-R5, -(C=O)-NH-R5, and -(O=S=O)-R5, respectively. Join in position.

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

Formula IIIa:
CH ₂ =CH-(C=O)-NH-R51-[N(R52) ₂ ] ⁺ -R53-SO ₃ ^-

In a preferred embodiment, in Formula IIIa,
R51 can be, for example, a C1-C4 alkylene group, preferably a C2-C4 alkylene group, preferably a C3-C4 alkylene group, or a C2-C3 alkylene group.

In a preferred embodiment, in Formula IIIa,
R52 can be, for example, a C1-C4 alkyl group, preferably a C1-C3 alkyl group, preferably a C1-C2 alkyl group.

In a preferred embodiment, in Formula IIIa,
R53 can be, for example, a C1-C4 alkylene group, preferably a C2-C4 alkylene group, preferably a C3-C4 alkylene group, or a C2-C3 alkylene group.

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

Formula IIIb:

[Zwitterionic Monomeric Compounds of Formula IIIc]
In a preferred embodiment, the zwitterionic monomeric compound of Formula III can be a compound of Formula IIIc below.
_CH2 =CH-(C=O)-NH-R5 (Formula IIIc)

In Formula IIIc, R5 can be the group described above.

[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.

In a preferred embodiment, in Formula IV,
x is the average degree of polymerization of the repeating unit, and can be in the range of 10-1000, preferably in the range of 50-500.

In a preferred embodiment, in Formula IV,
y is the average degree of polymerization of the repeating unit, and may range from 10 to 1,000, preferably from 50 to 500.

In a preferred embodiment, in Formula IV,
x and y, the ratio of x:y is, for example, in the range of 100:25 to 100:1000, alternatively in the range of 100:40 to 100:600, preferably in the range of 100:50 to 100:500, preferably 100: It can be in the range of 50-100:200, alternatively in the range of 100:90-100:150.

In a preferred embodiment, there can be one R1 group in Formula IV and x and y can be in the ratio of the above numerical values. That is, by way of example, the ratio of R1:x:y can range from 1:100:25 to 1:100:1000.

In a preferred embodiment, in Formula IV,
-r- indicates that repeating units described on both sides of -r- are randomly copolymerized to form a random copolymer.

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.

In a preferred embodiment, in Formula IV,
m is the repeating number of the repeating unit and is 1;

[Polymer Compound of Formula IVa]
In a preferred embodiment, the polymeric compound represented by Formula IV can be a compound represented by Formula IVa below.

Formula IVa:

In a preferred embodiment, in Formula IVa,
R1, x, y, m, -b-, and -r- are all the groups described above in Formula IV, or symbols having the meanings described above.

[Polymer compound of formula IVb]
In a preferred embodiment, the polymeric compound represented by Formula IV can be a compound represented by Formula IVb below.

Formula IVb:

In a preferred embodiment, in formula IVb,
n is the average degree of polymerization of the repeating unit, and can be in the range of 10-100, preferably in the range of 20-80.

In a preferred embodiment, in formula IVb,
All of x, y, m, -b-, and -r- are the groups described above in Formula IV, or symbols having the meanings described above.

In formula IVb, terminal groups derived from monomers are present at both ends of the polymer, but these descriptions are omitted in accordance with the general notation of polymers.

[Polymer Compound of Formula IVc]
In a preferred embodiment, the polymeric compound of Formula IV can be a compound of Formula IVc below.

Formula IVc:

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

In a preferred embodiment, in Formula IVc,
All of x, y, m, -b-, and -r- are the groups described above in Formula IV, or symbols having the meanings described above.

In formula IVc, terminal groups derived from monomers are present at both ends of the polymer, but these descriptions are omitted in accordance with the general notation of polymers.

[Polymerization reaction]
In a preferred embodiment, the polymeric compound of the present invention represented by Formula IV comprises a trithiocarbonate compound represented by Formula I, a sugar monomer compound represented by Formula II, and a glycomonomer compound represented by Formula III. Zwitterionic monomeric compounds can be produced by polymerization reactions. This polymerization reaction proceeds by Reversible Addition/Fragmentation Chain Transfer Polymerization (RAFT polymerization). The trithiocarbonate compounds of Formula I are macro chain transfer agents that replace RAFT agents. In this case, a sugar monomeric compound of formula II and a zwitterionic monomeric compound of formula III are used as monomers to convert from the terminal trithiocarbonate compound of formula I to formula II A polymer compound of the present invention represented by Formula IV is obtained as a block copolymer by random copolymerization of the sugar monomer compound represented by and the zwitterionic monomer compound represented by Formula III.

In a preferred embodiment, solvents such as dimethyl sulfoxide and dimethylformamide can be used to carry out such polymerization reactions, and additives such as azobisisobutyronitrile, 4,4' - Azobis(4-cyanovaleric acid) can be used. As conditions for this polymerization reaction, conditions known as radical polymerization conditions can be used.

[Formation of micelles]
In a preferred embodiment, the polymeric compound of Formula IV of the present invention is capable of self-associating to form micelles by dispersing it in an aqueous solution. Dispersion in an aqueous solution can be carried out, for example, by replacing the solvent with an aqueous solution by means of dialysis after synthesis of the polymer compound, or, for example, by freeze-drying the synthesized polymer compound and then dispersing it in the aqueous solution. can be done by

[Inhibition of protein aggregation]
The micelles formed by the polymeric compounds of the present invention represented by Formula IV exhibit excellent protein aggregation inhibitory efficacy at very high concentrations. The protein aggregation inhibitory effect can be exhibited by a simple means of mixing the protein to be protected with micelles in an aqueous solution.

That is, according to the present invention, by exerting the effect of suppressing aggregation on proteins that have already aggregated due to operations during purification and proteins that aggregate under storage conditions after purification, It achieves excellent effects of purifying proteins that have been difficult to purify and preserving proteins under conditions that have been difficult to preserve.

[Concentration for suppressing aggregation]
In a preferred embodiment, the protein aggregation inhibitory effect is achieved by adding the polymer compound of formula IV according to the invention in the range of, for example, 0.1 to 10 mg/L, such as in the range of 0.2 to 5 mg/L, such as It can be exerted by dispersing it in an aqueous solution at a concentration in the range of 0.5-5 mg/L, eg 1-3 mg/mL.

[Protein structure protection]
In a preferred embodiment, the micelle formed by the polymer compound of the present invention represented by Formula IV exhibits an excellent protein aggregation-inhibiting effect in which protein aggregation is inhibited in macroscopic observation, and at the same time, protein aggregation is inhibited in microscopic observation. It is accompanied by an excellent structural protection effect that the structure is maintained even in the observation of This excellent structure-protecting effect can be detected, for example, by CD (Circular Dichroism) spectrum measurement described later in Examples.

[Protection of protein activity]
In a preferred embodiment, the micelle formed by the polymer compound of the present invention represented by formula IV exhibits an excellent protein aggregation-inhibiting effect in that protein aggregation is inhibited in observation of the appearance, and at the same time, the protein is It is accompanied by an excellent activity-protecting effect that maintains activity and function. This excellent activity-protecting effect and function-protecting effect are as shown, for example, by the measurement of LDH enzyme activity described later in Examples.

[Inhibition and protection against aggregation by freezing]
In a preferred embodiment, the excellent aggregation-inhibiting effect, structure-protecting effect, activity-protecting effect and function-protecting effect, for example, on LDH enzyme activity and on aggregation occurring during freeze-thaw, as described later in Examples. , and exerts a protective effect, and exhibits effects of aggregation suppression, structure protection, activity protection and function protection during freezing and freezing and thawing.

[Effects on various proteins]
In preferred embodiments, the excellent aggregation-inhibiting effect, structure-protecting effect, activity-protecting effect and function-protecting effect that the compound of the present invention exerts on proteins is limited to, for example, LDH, as described later in Examples. However, it is also effective against insulin. This indicates that a similar protective effect is exerted on a wide range of proteins in general. As is well known, protein aggregation is one of the major causes of structural changes and deactivation of proteins. The results of the examples described later show that the protective effect is exhibited. Examples of proteins for which such a protective effect is particularly expected include antibodies, insulin, lactate dehydrogenase, alkaline phosphatase, acetylcholinesterase, ascorbic acid oxidase, and alcohol hydrogenase.

[Cytotoxicity]
In preferred embodiments, this superior protein-protective effect can be exerted without exhibiting cytotoxicity, as will be described later in the Examples. Therefore, the compounds of the present invention can be used not only as molecular biological research tools but also as drugs and medicines by administering them to cells, tissues, organs and living organisms.

[Separation from protein to be protected]
In a preferred embodiment, the micelle formed by the polymer compound of the present invention represented by Formula IV exhibits excellent protein aggregation-inhibiting action when mixed with a protein to be protected. It can be easily separated from the protein. Surprisingly, the isolated protein to be protected maintains almost the same function and activity as compared to before mixing with micelles, and at the same time, the isolated micelles are effective in suppressing protein aggregation. can be reused for Such excellent separability is very advantageous in that it does not impose any restrictions on the subsequent use of the protected protein, and is also very advantageous in that the separated micelles can be reused. is.

In a preferred embodiment, the protein to be protected mixed with the micelle can be separated by a known separation means that utilizes the molecular weight of the micelle. Examples of such separation means include filter filtration and centrifugation. In a preferred embodiment, centrifugation can be performed by applying centrifugal force at an acceleration of, for example, 5000 to 30000 g, preferably 10000 to 20000 g, for 1 to 120 minutes, or 10 to 60 minutes. . In a preferred embodiment, when solid-liquid separation is performed by centrifugation, micelles are precipitated, and the protein to be protected can be obtained as a supernatant.

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

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

[1.2 Synthesis of macro chain transfer agents (PCL-CTA, PS-CTA)]
1 mmol of 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. , under a nitrogen atmosphere for 16 hours. Reprecipitation recovery was performed with 1:1 (v/v) methanol:diethyl ether (PCL-CTA). The synthesized compound has a polycaprolactone (PCL) moiety and a chain transfer agent (CTA) moiety, and thus is denoted as PCL-CTA. The procedure for this synthesis is shown in Figure 2A.

RAFT agent (4-Cyano-4-[(dodecylsulfanylthiocarbonyl)-sulfanyl]pentanol) 1 mmol dissolved in DMF 1 mmol, styrene 30 mmol added, azobisisobutyronitrile (AIBN) 0.03 mmol added, under nitrogen atmosphere The reaction was allowed to proceed for 16 hours. Reprecipitation recovery was performed with 1:1 (v/v) methanol:diethyl ether (PS-CTA). Since the synthesized compound has a polystyrene (PS) portion and a chain transfer agent (CTA) portion, it is denoted as PS-CTA. The procedure for this synthesis is shown in FIG. 2B.

According to NMR, PCL had a number average degree of polymerization of 69, and PS had a number average degree of polymerization of 45.

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

In Table 1, R is PCL-RAFT Agent, ie PCL-CTA. C1 is Poly-SPB (sulfobetaine polymer). C2 is TrMA (trehalose methacrylate).

In Table 2, R is PS-RAFT Agent, ie PS-CTA. C1 is Poly-SPB (sulfobetaine polymer). C2 is TrMA (trehalose methacrylate).

As shown in Table 1, M1 is obtained by randomly polymerizing 100-mers of SPB and trehalose with respect to the PCL block, and M2 is obtained by polymerizing 200-mers of SPB and 100-mers of trehalose with respect to the PCL block. M3 is obtained by polymerizing 100 mers of SPB and 500 mers of trehalose with respect to the PCL block.

[Experimental example 2]
[2. Protein aggregation inhibitory effect experiment]
[LDH (lactate dehydrogenase) aggregation suppression]
[Time change of LDH aggregation comparing each micelle and polymer]
0.2 mg/mL LDH (PBS solution) was mixed with micelles or polymers at a predetermined concentration, and the absorbance at 350 nm was measured with an ultraviolet-visible spectrophotometer (UV-1800, Shimadzu) while stirring at 37°C for 30 minutes. . This assessed the increase in absorbance caused by aggregation. SPB homopolymer was used as a control group.

The results are shown in Figure 4. FIG. 4 is a graph showing changes in UV absorption of LDH solutions over time. The polymer or micelle concentration was 2 mg/mL.

As shown in Fig. 4, an increase in absorbance due to aggregation was confirmed in the control group and the system to which no polymer was added. On the other hand, no increase in absorbance was observed in any of the micelles M1, M2 and M3. The reason why the initial absorbance is high in the micellar system is that the micelles are cloudy.

[Comparison of LDH aggregation inhibitory ability of each micelle and polymer]
FIG. 5 is a graph showing the LDH aggregation inhibitory ability (2 mg/mL) of each additive. FIG. 5 shows the results of calculating the aggregation rate of a system in which each polymer and trehalose were added to LDH at a concentration of 2 mg/mL from the rate of increase in UV absorbance. Aggregation rate was 0.02% for M1, and 22.7% and 32.4% for M2 and M3, respectively, indicating very high activity especially for M1.

As shown in FIG. 5, these micelles M1 to M3 all exhibited superior aggregation inhibitory properties compared to Poly-SPB and trehalose. In particular, M1 showed a very good anti-aggregation property, in other words an LDH protective effect. These aggregation-inhibiting properties were effective even at an ultra-low concentration of only 0.2% (2 mg/mL).

[Concentration dependence of LDH aggregation inhibitory ability of M1 micelles]
FIG. 6 is a graph showing the concentration dependence of the ability of M1 to suppress LDH aggregation. Conditions other than the M1 micelle concentration were the same as in FIG. It was found to have a high aggregation inhibitory activity of 16.6% even at 1 mg/mL. Moreover, when M1 was added at 0.2% (2 mg/mL), it exhibited LDH aggregation-inhibiting ability, in which aggregation was almost completely suppressed.

[Maintenance of LDH activity by each micelle]
200 μL of NADH (63 mM) PBS solution and 500 μL of sodium pyruvate (10 mM) PBS solution were mixed and brought up to 50 mL to make a pH 7.0 substrate stock solution. Next, micelles of a predetermined concentration were mixed with a 20.7 mU/mL LDH solution (PBS) and incubated at 37° C. for 1 hour. Take 5 μL of this solution, mix with 195 μL of the substrate stock solution on a 96-well plate, measure the absorbance at 340 nm for 20 minutes with a microplate reader (Tecan Infinite 200 PRO M Nano+), calculate the enzyme activity from the rate of decrease in absorbance, The residual enzyme activity was calculated with the untreated case as 100%. The results are shown in FIGS. 7A and 7B.

FIG. 7A is a graph comparing LDH activity maintenance by each micelle at each concentration. In FIG. 7A, the values of M1 (left end), M2 (middle), and M3 (right end) are shown for each density. As shown in FIG. 7A, even at very low concentrations of 1-2 mg/mL, all of M1-M3 exhibited high residual activity. As shown in FIG. 7A, it was found that high residual activity was exhibited at any concentration. Specifically, M1 was 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. It showed a high residual activity rate. M2 was 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. It showed a high residual activity rate. M3 also showed a high residual activity rate of 50% at 0.75 mg/mL, 63% at 1 mg/mL, 75% at 1.5 mg/mL, and 82% at 2 mg/mL.

FIG. 7B is a graph comparing LDH activity maintenance by each concentration of PS micelles (M4). As shown in FIG. 7B, it was found that high residual activity was exhibited at any concentration. In particular, it showed a high residual activity rate of 61% at 0.75 mg/mL and 72% at 1 mg/mL.

[CD spectrum measurement]
CD spectrum was measured to evaluate the secondary structure change of LDH.
Each micelle was added to an LDH solution with a final concentration of 0.1 mg/mL (PBS) at a concentration of 2 mg/mL, introduced into a JASCO-820 spectrometer cell, and kept at 15° C. under a nitrogen gas atmosphere for 30 minutes. , 37° C. for 1 hour, and then measured. The results are shown in FIGS. 8A and 8B.

FIG. 8A is a graph of the CD spectrum of LDH heated at 37° C. together with each micelle. FIG. 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 the same incubation experiment without the addition of micelles. Native LDH indicates the results of an experiment in which micelles were not added and incubation was not performed at 37°C for 1 hour.

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

As shown in FIGS. 8A and 8B, it was found that the micelle-added system substantially maintained the structure before the treatment even after the heat treatment, compared to the additive-free LDH.

[Examination of separability of each micelle]
After incubating an LDH solution (LDH 0.2 mg/mL) in the presence of each micelle (2 mg/mL) at 37° C. for 30 minutes, it was centrifuged at 16000 rpm (13100 g) for 90 minutes to separate the supernatant and the precipitate.

When the protein concentration in the supernatant was measured by the Bradford method, the recoveries were 91.55 ± 2.83% for M1, 92.75 ± 1.87% for M2, and 94.87 ± 1.29% for M3. , and almost no loss of protein due to adsorption to micelles was observed.

In addition, the collected micelles were reusable. It was also confirmed that LDH in the collected supernatant aggregated by incubating again at 37° C. for 1 hour. These were confirmed by experiments described later.

[Redispersibility of centrifuged micelles]
FIG. 9 shows an image of the appearance of the solution before centrifugation (left side of FIG. 9) and an image of the appearance of the solution (supernatant and precipitate) after centrifugation (FIG. 9) from the centrifugation experiment using PCL micelles. middle), and an image of the appearance of the redispersed solution after centrifugation (right side of FIG. 9). As shown in the image on the right side of FIG. 9, the micelles were highly dispersible and could be dispersed again after centrifugation.

[Maintenance of aggregation ability of LDH recovered by centrifugation]
LDH in the supernatant recovered by centrifugation became cloudy when incubated again at 37°C, indicating that the aggregation ability remained. The results of this experiment are shown in FIG.

FIG. 10 is a graph showing the change in UV absorbance (350 nm) when the supernatant collected by centrifugation after adsorption to each micelle is reincubated at 37° C. and recondensed. The upper curve of the graph in FIG. 10 shows the change in absorbance due to LDH in the supernatant collected by centrifugation, and the lower curve of the graph in FIG. Shows change in absorbance.

[Enzyme activity of LDH recovered by centrifugation]
After incubating at 37° C., the enzymatic activity of LDH in the supernatant recovered by centrifugation was confirmed. The experiment was conducted under the same conditions as the experiment for maintaining LDH activity with each micelle. The results are shown in FIG.

FIG. 11 is a graph showing the enzymatic activity of LDH in the supernatant recovered by centrifugation after incubation at 37°C. The horizontal axis in FIG. 11 indicates the concentration of PCL micelles. The vertical axis of FIG. 11 represents the residual amount of LDH enzymatic activity, which is a relative value when the unheated LDH activity is taken as 100%. The graph in FIG. 11 shows the LDH enzymatic activity when M1 (leftmost), M2 (middle) and M3 (rightmost) are used as micelles for each concentration of PCL micelles. As shown in FIG. 11, LDH showed high residual activity in all micelles.

[Experimental example 3]
[Inhibitory effect on aggregation due to freezing of LDH]
A micelle of a predetermined concentration was mixed with a 20.7 mU/mL LDH solution (PBS), left in a −20° C. freezer for 24 hours to freeze, and then thawed at room temperature, which was repeated 15 times. Take 5 μL of this solution, mix with 195 μL of the substrate stock solution on a 96-well plate, measure the absorbance at 340 nm for 20 minutes with a microplate reader (Tecan Infinite 200 PRO M Nano+), calculate the enzyme activity from the rate of decrease in absorbance, The residual enzyme activity was calculated with the untreated case as 100%. The results are shown in FIG.

FIG. 12 is a graph comparing LDH activity maintenance by each micelle at each concentration. In FIG. 12, the values of M1 (left end), M2 (middle), and M3 (right end) are shown for each density. As shown in FIG. 12, the system without the addition of micelles (0 mg/mL) almost lost the activity, whereas even at a very low concentration of 1 to 2 mg/mL, high residual activity was observed in all of M1 to M3. found to show.

[Experimental example 4]
[Inhibitory effect on insulin aggregation]
FIG. 13 is the aggregation rate of insulin (100 μM) heated at 37° C. for 1 hour with each micelle. Each micelle was mixed so that the final concentration was the concentration on the horizontal axis of FIG. 13 to make 1 mL. In FIG. 13, the values of M1 (left end), M2 (middle), and M3 (right end) are shown for each density. As shown in FIG. 13, the system without the addition of micelles (0 mg/mL) showed almost 100% aggregation, whereas even at extremely low concentrations of 0.25-2 mg/mL, any of M1-M3 It was found to exhibit high inhibitory ability.

FIG. 14 is a CD spectrum graph of insulin (10 μM) heated at 37° C. together with each micelle (2 mg/mL). Insulin-heat shows the results of the same incubation experiment without the addition of micelles. Insulin Native indicates the results of an experiment in which micelles were not added and incubation was not performed at 37°C for 1 hour. Insulin-heat shows a large change in the curve showing the higher-order structure, whereas the M1-M3 addition system has a structure close to that of insulin native.

[Experimental example 5]
[Toxicity test]
Cytotoxicity of each micelle was examined using mouse fibroblast L929. L929 was cultured in 10% serum-containing Dulbecco's Modified Eagle's Medium (DMEM) at 37° C. under 5% CO ₂ environment. 1,000 cells were seeded in a 96-well plate, cultured for 72 hours, and 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 100 μg/mL, the medium was discarded after 4 hours, and 100 μL of dimethyl sulfoxide was added. The dye was extracted by addition and the absorbance at 540 nm was measured. The survival rate was calculated from the absorbance at each concentration, assuming that the absorbance of the system to which micelles were not added was 100%.

FIG. 15 shows the values of M1 (left end), M2 (middle), and M3 (right end) for each density. Even at 10 mg/mL, which is five times the concentration of 2 mg/mL at which the anticoagulant effect was high, the survival rate of the cells was maintained at a high level, indicating low toxicity of the micelles.

The present invention provides a novel protein aggregation inhibitor. The present invention is an industrially useful invention.

Claims

A trithiocarbonate compound represented by Formula I, a sugar monomer compound represented by Formula II, and a zwitterionic monomer compound represented by Formula III are polymerized to obtain a polymer compound represented by Formula IV. How to manufacture:

R1-S-(C=S)-S-R2 (Formula I)

R3-R7-C(R4)= CH2 (Formula II)

CH2 =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-C24 alkyl group,
In formula II,
R3 is a reducing or non-reducing, mono- or disaccharide monovalent group;
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)-, where R3 is - (C=O)-O-R, -(C=O)-NH-R, and -(O=S=O)-R3),
R8 is a divalent group selected from -(C=O)-O-, -(C=O)-NH-, and -(O=S=O)-, where R5 is - (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 unit and is 10 to 500,
y is the average degree of polymerization of the repeating unit and is 10 to 1000,
-r- indicates that the repeating units described on both sides of -r- are randomly copolymerized to form a random copolymer,
-b- indicates that the repeating units described on both sides of -b- are block copolymerized to form a block copolymer,
m is the repeating number of the repeating unit and is 1).
2. The process according to claim 1, wherein the trithiocarbonate compound of Formula I is a compound of Formula Ia or Formula Ib:
Formula Ia:

Formula Ib:

(In Formula Ia,
n is the average degree of polymerization of the repeating unit 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 unit and is 10 to 100,
R2 is the same group as R2 in Formula I).
The production method according to any one of claims 1 and 2, wherein the sugar monomer compound represented by formula II is a compound represented by the following formula IIa:
Formula IIa:
A process according to any one of claims 1 to 3, 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).
A process according to any one of claims 1 to 4, wherein the zwitterionic monomeric compound of formula III is a compound of formula IIIb:
Formula IIIb:
A process according to any one of claims 1 to 5, wherein the polymeric compound of formula IV is a compound of 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).
The process according to any one of claims 1 to 6, wherein the polymeric compound represented by formula IV is a compound represented by formula IVb or formula IVc:
Formula IVb:

Formula IVc:

(In formula IVb,
n is the average degree of polymerization of the repeating unit and is 10 to 100,
x, y, m, -b-, -r- are all the same as x, y, m, -b-, -r- in formula IV,
In Formula IVc,
l is the average degree of polymerization of the repeating unit 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).
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. .
A polymer compound represented by Formula IV according to claim 1, Formula IVa according to claim 6, or Formula IVb or Formula IVc according to claim 7.
A micelle made of the polymer compound according to claim 9.
A protein aggregation inhibitor comprising a micelle comprising the polymer compound according to claim 9 or the polymer compound according to claim 10.
The protein aggregation inhibitor according to claim 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.
A method for inhibiting protein aggregation, comprising the step of mixing the polymer compound according to claim 9 or a micelle comprising the polymer compound according to claim 10 with an aqueous protein solution.
A method for producing an aggregation-suppressed aqueous protein solution, comprising the step of mixing the polymer compound according to claim 9 or a micelle comprising the polymer compound according to claim 10 with an aqueous protein solution.
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: