阅读说明：本技术 分支型分解性聚乙二醇键合物 (Branched degradable polyethylene glycol conjugate ) 是由 吉冈宏树 大坂间顺规 羽村美华 稻叶高德 西山伸宏 松井诚 武元宏泰 野本贵大 孙小 于 2020-03-26 设计创作，主要内容包括：本发明提供一种键合有下式(A)所示的在细胞内进行分解的分支型分解性聚乙二醇衍生物的生物相关物质。(式中,各符号如本说明书中所定义。)。(The present invention provides a microorganism to which a branched degradable polyethylene glycol derivative represented by the following formula (A) which degrades in cells is bondedAnd (4) related substances. (wherein each symbol is as defined in the specification).)

1. A bio-related substance to which a degradable polyethylene glycol derivative represented by the following formula (A) is bonded:

wherein n is 45 to 950, W is an oligopeptide of 5 to 47 residues with a symmetric structure with glutamic acid as the center, a is 2 to 8, D is a biologically related substance, L¹And L²Each independently is a 2-valent spacer group, and b is 1 to 40.

2. The bio-related substance according to claim 1, wherein b is 1.

3. The bio-related substance according to claim 1 or 2, wherein the oligopeptide of W having a symmetrical structure centering on glutamic acid is an oligopeptide having the following structure of W1, W2 or W3:

wherein Glu is a glutamic acid residue, and Z is a degradable oligopeptide of 2 to 5 residues consisting of neutral amino acids excluding cysteine.

4. The biorelevant substance according to claim 3, wherein the decomposing oligopeptide of Z is an oligopeptide having glycine as a C-terminal amino acid.

5. The biorelevant substance according to claim 3 or 4, wherein the degradable oligopeptide of Z is an oligopeptide with at least 1 hydrophobic neutral amino acids having a hydropathic index of 2.5 or more.

6. The biologically-relevant substance according to any one of claims 1 to 5, wherein the molecular weight of 1 molecule of the degradable polyethylene glycol derivative is 20,000 or more.

7. The bio-related substance according to any one of claims 1 to 6, wherein L¹Is a urethane bond, an amide bond, an ether bond, a thioether bond, a secondary amino group, a carbonyl group, a urea bond, a triazole group, a bond of maleimide and a mercapto group, or an oxime bond; or alkylene groups with or without these bonds and/or groups.

8. The biorelevant of any one of claims 1-7 wherein L is²Is a compound containing a group selected from alkylene; or an alkylene group of at least one bond and/or group of a urethane bond, an amide bond, an ether bond, a thioether bond, a secondary amino group, a carbonyl group, and a urea bond.

9. The bio-related substance according to any one of claims 1 to 8, wherein the bio-related substance D is a hormone, a cytokine, an antibody, an aptamer or an enzyme.

Technical Field

The present invention relates to a biologically relevant substance to which a branched degradable polyethylene glycol derivative which degrades in a cell is bonded.

Background

In general, when a drug using a biologically relevant substance such as a hormone, a cytokine, an antibody, or an enzyme is administered into a living body, the drug is rapidly excreted from the living body by glomerular filtration in the kidney or by uptake by macrophages in the liver, spleen, or the like. Therefore, the blood half-life is often shortened, and it is often difficult to obtain a sufficient pharmacological effect. In order to solve this problem, attempts have been made to chemically modify biologically relevant substances with a hydrophilic polymer such as a sugar chain or polyethylene glycol, or albumin. As a result, the blood half-life of the biologically-relevant substance can be prolonged due to the increase in molecular weight, the formation of a hydrated layer, and the like. It is also known that modification with polyethylene glycol can reduce the toxicity or antigenicity of biologically-relevant substances and improve the solubility of drugs that are poorly water-soluble.

Since the bio-related substance modified with polyethylene glycol is covered with a hydrated layer formed by hydrogen bonding of ether bond of polyethylene glycol and water molecule, the size of the molecule is increased, so that glomerular filtration in kidney can be avoided. Further, it is known that the interaction between opsonin and the cell surface constituting each tissue is reduced, and migration into each tissue is reduced. Polyethylene glycol is known to be an excellent material capable of extending the blood half-life of a biologically-relevant substance, and its property is that the larger the molecular weight, the higher the effect. Studies on bio-related substances modified with polyethylene glycol having a high molecular weight of 4 ten thousand or more have been conducted so far, and the blood half-life thereof can be remarkably prolonged.

Polyethylene glycol is used as the best standard among modifying agents for the improvement of the properties of bio-related substances, and a plurality of polyethylene glycol modifying agents are now on the market and used in medical sites. On the other hand, the European Medicines Agency (EMA) of 2012 reported that when a biologically-relevant substance modified with polyethylene glycol having a molecular weight of 4 ten thousand or more was administered to animals for a long period of time at a predetermined administration amount or more, cavitation occurred in cells of a part of the tissue (non-patent document 1). Currently, considering that there is no report that the generation of cavitation itself adversely affects the human body, and that the dose used in the above-mentioned report of EMA is extremely higher than the dose usually applied in the medical field, it can be said that there is no problem in the safety of the therapeutic preparation modified with polyethylene glycol having a molecular weight of 4 ten thousand or more which is currently manufactured and sold. However, treatment regimens employing high amounts and prolonged administration of polyethylene glycol modified formulations to patients in the treatment of very specific diseases (e.g., dwarfism, etc.) can also be envisioned. Therefore, there is expected to be a potential need for the development of polyethylene glycol-modified preparations that do not produce vacuoles in cells, and that can be applied in such special situations.

In non-patent document 2, when polyethylene glycol in an amount larger than the amount of a conventional polyethylene glycol-modified preparation is administered alone to an animal for a long period of time, no cavitation was observed at a molecular weight of 2 ten thousand, and the occurrence of cavitation was observed at a molecular weight of 4 ten thousand. As one means for suppressing cavitation, reduction of the molecular weight of polyethylene glycol is conceivable, but there is a problem that the blood half-life of a bio-related substance cannot be sufficiently improved if the molecular weight is reduced.

There are reported examples of techniques for promoting the decomposition of high molecular weight polyethylene glycol into low molecular weight polyethylene glycol in vivo and excretion from the kidney. Patent document 1 describes a polyethylene glycol derivative having a thioether bond or a peptide bond site that can be cleaved in vivo. It is described that the polyethylene glycol derivative is decomposed in vivo to a molecular weight suitable for excretion from the kidney. However, no data on specific breakdown was shown at all, nor was it shown that excretion from the kidney promoted such data. Further, there is no description about vacuoles of cells.

Patent document 2 describes a polyethylene glycol derivative having an acetal moiety that can be hydrolyzed in a low pH environment in vivo. It is described that the polyethylene glycol derivative is decomposed in vivo to a molecular weight suitable for excretion from the kidney. However, there is no data specifically promoting excretion from the kidney, and there is no description about vacuole of cells. Further, it is known that these acetal moieties capable of hydrolysis are also slowly decomposed in blood, and it is not expected that the half-life of the modified bio-related substance in blood will be sufficiently improved.

On the other hand, there are reported examples of polyethylene glycol derivatives into which degradable oligopeptides are introduced for effective drug release, hydrogels that decompose in vivo, and the like.

Non-patent document 3 describes polyethylene glycol derivatives having an oligopeptide portion decomposed by an enzyme. Among them, it is reported that an oligopeptide is introduced as a linker (linker) between an anticancer agent and polyethylene glycol, and the oligopeptide is decomposed by an enzyme specifically expressed around a tumor, thereby efficiently releasing the anticancer agent. The purpose of this is to release the anticancer agent, and not to impart degradability to polyethylene glycol for the purpose of suppressing cell vacuoles.

Non-patent document 4 describes a hydrogel using a cross-linking molecule having an oligopeptide portion decomposed by an enzyme and a multibranched polyethylene glycol derivative. Among them, oligopeptides are used as crosslinking molecules for linking the multibranched polyethylene glycol derivatives, and further, the hydrogel can be imparted with degradability by enzymes. The purpose of this is to prepare a degradable hydrogel, and not to impart degradability to polyethylene glycol for the purpose of suppressing cell vacuolation.

Patent document 3 describes a branched polyethylene glycol derivative having an oligopeptide as a skeleton. Wherein the oligopeptide is used as a basic skeleton of the polyethylene glycol derivative, rather than imparting degradability through an enzyme. The oligopeptide is characterized by containing amino acids having an amino group or a carboxyl group in a side chain, such as lysine and aspartic acid, and is intended to synthesize a branched polyethylene glycol derivative using these amino acids for the reaction. Polyethylene glycol derivatives that are not intended to inhibit vacuolation of cells.

Further, polyethylene glycol derivatives used for modification of biologically relevant substances generally include straight-chain type and branched type, and non-patent document 5 describes that branched type significantly extends the blood half-life of biologically relevant substances as compared with straight-chain type. The polyethylene glycol modified preparations on the market are mostly branched. However, there has been no report in this field of bio-related substances modified with branched polyethylene glycol derivatives that inhibit cell vacuole so far.

As described above, there is a need for a bio-related substance modified with a branched high molecular weight polyethylene glycol derivative, which is stable in blood, can improve the blood half-life of the modified bio-related substance, and can be specifically degraded in cells to inhibit the generation of vacuoles in the cells.

Documents of the prior art

Patent document

Patent document 1: japanese Kokai publication No. 2009-527581

Patent document 2: international publication No. 2005/108463

Patent document 3: international publication No. 2006/088248

Non-patent document

Non-patent document 1: EMA/CHMP/SWP/647258/2012

Non-patent document 2: daniel G.Rudmann, et al., Toxicol. Pathol.,41,970-

Non-patent document 3: france sco M Veronese, et al, Bioconjugate chem, 16,775-784(2005)

Non-patent document 4: jiyuan Yang, et al, Marcomol biosci, 10(4),445-

Non-patent document 5: yulia Vugmeysterang, et al, Bioconjugate chem.,23,1452-

Disclosure of Invention

Problems to be solved by the invention

The problem of the present invention is to provide a biologically relevant substance to which a branched polyethylene glycol derivative having a high molecular weight that does not cause cavitation of cells is bonded. More specifically, it is intended to provide a biologically-relevant substance which is stable in blood in vivo and is modified with a degradable polyethylene glycol derivative which is degraded in cells to improve the half-life in blood.

Means for solving the problems

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have invented a biologically relevant substance having a branched degradable polyethylene glycol derivative to which an oligopeptide that degrades intracellularly is bonded.

That is, the present invention is as follows.

[1] A bio-related substance to which a degradable polyethylene glycol derivative represented by the following formula (A) is bonded:

[ solution 1]

(wherein n is 45 to 950 and W is glutamic acid-centeredOligopeptides with 5-47 residues and a is 2-8, D is a biological related substance, L¹And L²Each independently is a 2-valent spacer group, and b is 1 to 40).

[2] A bio-related substance to which a degradable polyethylene glycol derivative represented by the following formula (1) is bonded:

[ solution 2]

(wherein n is 45 to 950, W is an oligopeptide of 5 to 47 residues having a symmetrical structure with glutamic acid as the center, a is 2 to 8, D is a biologically relevant substance, and L¹And L²Each independently a 2-valent spacer group).

[3] The bio-related substance according to [1] or [2], wherein the oligopeptide of W having a symmetrical structure centering on glutamic acid is an oligopeptide having a structure of W1, W2 or W3 below.

[ solution 3]

[ solution 4]

[ solution 5]

(wherein Glu is a glutamic acid residue, and Z is a decomposable oligopeptide of 2 to 5 residues consisting of neutral amino acids excluding cysteine.)

[4] The bio-related substance according to [3], wherein the degradable oligopeptide of Z is an oligopeptide having glycine as a C-terminal amino acid.

[5] The bio-related substance according to any one of [3] or [4], wherein the degradable oligopeptide of Z is an oligopeptide having at least 1 hydrophobic neutral amino acid having a hydropathic index of 2.5 or more.

[6] The bio-related substance according to any one of [1] to [5], wherein the molecular weight of 1 molecule of the degradable polyethylene glycol derivative is 20,000 or more.

[7]According to [1]～[6]The bio-related substance of any one of, wherein L¹Is a urethane bond, an amide bond, an ether bond, a thioether bond, a secondary amino group, a carbonyl group, a urea bond, a triazole group, a bond of maleimide and a mercapto group, or an oxime bond; or an alkylene group that may contain such bonds and/or groups.

[8]According to [1]～[7]The biologically-relevant substance of any one of, wherein L²Is a compound containing a group selected from alkylene; or an alkylene group of at least one bond and/or group of a urethane bond, an amide bond, an ether bond, a thioether bond, a secondary amino group, a carbonyl group, and a urea bond.

[9] The biologically-relevant substance according to any one of [1] to [8], wherein the biologically-relevant substance D is a hormone, a cytokine, an antibody, an aptamer, or an enzyme.

Effects of the invention

The biologically-relevant substance of the present invention is stable in blood in vivo and is modified with a branched degradable polyethylene glycol derivative having an oligopeptide in the structure which is degraded by an intracellular enzyme. Therefore, the bio-related substance is stable in blood and has a blood half-life equivalent to that of a bio-related substance modified with a conventional non-degradable polyethylene glycol derivative. Further, when the bio-related substance is taken into cells, the oligopeptide site of the degradable polyethylene glycol derivative is rapidly degraded, thereby suppressing the generation of vacuoles in the cells. The oligopeptide constituting the degradable polyethylene glycol derivative has a symmetrical structure with glutamic acid as the center, and the same degradable oligopeptide Z is bonded to the ends of all polyethylene glycol chains. Therefore, polyethylene glycol decomposition products generated during intracellular decomposition have the same molecular weight and the same structure, and have the characteristic of uniform discharge from tissues and cells.

Drawings

FIG. 1 shows Compound (p3) (I) of example 1 ₂NH- ₂E(FG-200ME)) GPC analysis result (2).

FIG. 2 shows the compound (p3) (recovered from the cells in the cell lysis test in example 8 _{2 2}NH-E(FG-200ME)) GPC analysis result (2).

FIG. 3 shows the compound (p13) ((p 13)) of example 5 ₂NH- ₂E{E(FG-100ME)} ₂ ) GPC analysis result (2).

FIG. 4 shows the compound (p13) (recovered from the cells in the cell lysis test in example 8 _{2 2}NH-E{E(FG-100ME)} ₂ ) GPC analysis result (2).

FIG. 5 shows the result of RPLC analysis of the conjugate (1) with salmon calcitonin of example 9, methoxy PEG40 kDa-sCT.

FIG. 6 shows the results of MALDI-TOF-MS analysis of the compound (p8) obtained in example 3 and the conjugate (1) obtained in example 9.

FIG. 7 shows the MALDI-TOF-MS analysis results of methoxy PEG aldehyde 40kDa and methoxy PEG40kDa-sCT obtained in example 9.

FIG. 8 shows the results of SDS-PAGE analysis of the conjugate (1) with salmon calcitonin and methoxy PEG40kDa-sCT from example 9 (left panel: CBB staining; right panel: iodine staining).

FIG. 9 shows the result of RPLC analysis of methoxy PEG40kDa-hGH conjugated with human growth hormone (2) of example 10.

FIG. 10 shows the result of MALDI-TOF-MS analysis of the conjugate (2) with human growth hormone of example 10.

FIG. 11 shows the MALDI-TOF-MS analysis result of methoxy PEG aldehyde 40kDa-hGH in human growth hormone of example 10.

FIG. 12 shows the SDS-PAGE analysis of the conjugate (2) with human growth hormone of example 10 and methoxy PEG40kDa-hGH (left panel: CBB staining; right panel: iodine staining).

FIG. 13 shows images of sections of the choroid plexus of mice chronically dosed with methoxy PEG amine 40kDa of example 13 (arrows indicate vacuoles).

FIG. 14 shows the chronic administration of Compound (p3) (I) of example 13 _{2 2}NH-E(FG-200ME)) Images of sections of mouse brain choroid plexus.

FIG. 15 shows the chronic administration of PBS, methoxy PEG amine 40kDa, methoxy PEG amine 20kDa, compound (p3) ((p 3)) _{2 2}NH-E(FG-200ME)) Images of sections of mouse brain choroid plexus (stained portions show accumulation of PEG).

FIG. 16 shows the results of evaluation of physiological activities (calcium concentration in blood) of salmon calcitonin and PEGylated salmon calcitonin in example 12.

Detailed Description

The present invention is described in detail below.

The biologically relevant substance to which the degradable polyethylene glycol derivative is bonded according to the present invention is represented by the following formula (a).

[ solution 6]

(wherein n is 45 to 950, W is an oligopeptide of 5 to 47 residues having a symmetrical structure with glutamic acid as the center, a is 2 to 8, D is a biologically relevant substance, L¹And L²Each independently is a 2-valent spacer group, and b is 1 to 40. )

The molecular weight of the polyethylene glycol derivative 1 molecule bonded to the bio-related substance of formula (A) of the present invention is usually 4,000 to 160,000, preferably 10,000 to 120,000, and more preferably 20,000 to 80,000. In a preferred embodiment of the present invention, the molecular weight of the polyethylene glycol derivative 1 of formula (a) of the present invention is 20,000 or more. The molecular weight referred to herein means the number average molecular weight (Mn).

N in the formula (A) is the number of repeating units of polyethylene glycol, and is usually 45 to 950, preferably 110 to 690, and more preferably 220 to 460.

In the formula (a), a is the number of polyethylene glycol chains bonded to the oligopeptide, and is usually 2 to 8, preferably 2 or 4 or 8, and more preferably 2 or 4.

The number of molecules of the degradable polyethylene glycol derivative bonded to the biologically relevant substance, b in the formula (A), is usually 1 to 40, preferably 1 to 20, and more preferably 1 to 10.

If the number of molecules of the polyethylene glycol derivative bonded to the biologically-relevant substance is increased, effects such as an increase in blood half-life and a decrease in antigenicity are obtained, but the activity of the biologically-relevant substance may be decreased. On the other hand, it is known that, in some biologically relevant substances such as enzymes, even when a plurality of polyethylene glycol derivatives are bonded, the activity is not reduced.

L in the formula (A)¹And L²Each independently represents a 2-valent spacer, and these spacers are not particularly limited as long as they are groups capable of forming a covalent bond, and L is¹Preferably an amide bond, an ether bond, a thioether bond, a urethane bond, a secondary amino group, a carbonyl group, a urea bond, a triazole group, a bond of maleimide and a mercapto group, or an oxime bond; or an alkylene group that may contain such bonds and/or groups.

In addition, L²Preferably an alkylene group; or an alkylene group containing at least one bond and/or group selected from an amide bond, an ether bond, a thioether bond, a urethane bond, a secondary amino group, a carbonyl group, and a urea bond. L is₂Preferably a repeating unit bonded to the polyethylene glycol at a carbon atom.

L¹And L²Particularly preferred modes of (b) are shown in the following group (I). In addition, 2 to 5 spacer groups of group (I) may also be combined. As the 2-valent spacer, an ester bond and a carbonate bond are not suitable because they are slowly decomposed in blood in vivo.

Group (I):

[ solution 7]

In the formulae (z1) to (z20), s represents an integer of 0 to 10, preferably an integer of 0 to 6, and more preferably an integer of 0 to 3. In addition, in the formulae (z2) to (z20), s may be the same or different. L is¹When an asymmetric 2-valent spacer group, withThe bonding position of the adjacent other group is not particularly limited, and if the right side of the spacer group represented by the above formula in the group (I) represents the bonding position with W and the left side represents the bonding position with D, two bonding positions may be adopted, the left side representing the bonding position with W and the right side representing the bonding position with D. Likewise, L²In the case of asymmetric 2-valent spacers, if the spacer of the above group (I) is represented by the formula₂CH₂When the bonding position of W and the bonding position of W are indicated on the left side, the bonding position of W and OCH can be indicated on the left side₂CH₂And the right side shows two bonding positions at the bonding position with W.

As L in formula (A)¹The group (I) preferably has groups represented by (z3), (z6), (z7) to (z20), more preferably groups represented by (z6), (z9), (z10), (z12), (z14), (z16), (z18) or (z20), and still more preferably groups represented by (z10), (z12), (z16) or (z 20).

As L in formula (A)²Preferred are groups of group (I) represented by (z1), (z2), (z3), (z4), (z5), (z6), (z7) or (z8), and more preferred is a group represented by (z3) or (z 5).

W in formula (a) is not particularly limited as long as it is an oligopeptide of 5 to 47 residues having a symmetrical structure with glutamic acid as the center, which is stable in blood in vivo and is decomposed by intracellular enzymes, and the amino acids constituting the oligopeptide are preferably neutral amino acids excluding cysteine, except glutamic acid constituting the center. The oligopeptide having a symmetric structure with glutamic acid as the center is a compound in which the same peptide is bonded to the carboxyl group at the α -position and the carboxyl group at the γ -position of glutamic acid, and the peptide paired with glutamic acid is an oligopeptide having a symmetric structure. The oligopeptide generally has a composition ratio of the number of neutral amino acids to the number of glutamic acids (number of neutral amino acids/number of glutamic acids) of 2 to 10, preferably 2 to 8, and more preferably 2 to 6. The amino acids constituting W are substantially in the L form.

A particularly preferred mode of W is shown in the following group (II).

Group (II):

[ solution 8]

[ solution 9]

[ solution 10]

(wherein Glu is a glutamic acid residue, and Z is a decomposable oligopeptide of 2 to 5 residues consisting of neutral amino acids excluding cysteine.)

Z in (w1) to (w3) is preferably an amino acid having an amino group or a carboxyl group in a side chain, specifically an oligopeptide composed of a neutral amino acid not containing lysine, aspartic acid, or glutamic acid. In the synthesis of the branched degradable polyethylene glycol derivative of formula (a) of the present invention, when the polyethylene glycol derivative and the oligopeptide as raw materials are bonded by reaction, the carboxyl group at the C-terminal end of the oligopeptide is used in the condensation reaction with the polyethylene glycol derivative. However, when the oligopeptide has an amino acid having an amino group or a carboxyl group in a side chain, a side reaction between oligopeptides occurs by a condensation reaction, and the polyethylene glycol derivative is not introduced into a target carboxyl group at the C-terminal end but is introduced into the carboxyl group in the side chain as an impurity.

Since such impurities are difficult to remove by a purification step such as ordinary extraction or crystallization, it is desirable to use oligopeptides composed of amino acids having no amino group or carboxyl group in the side chain in order to obtain a target product with good purity. The amino acids constituting Z are alpha-amino acids and are furthermore substantially in the L-form.

Cysteine as a neutral amino acid has a thiol group, and forms a disulfide bond with another thiol group, and Z in (w1) to (w3) is preferably an oligopeptide composed of a neutral amino acid excluding cysteine.

In addition, Z in (w1) to (w3) is preferably an oligopeptide having glycine as a C-terminal amino acid. When the carboxyl group at the C-terminal is reacted with a polyethylene glycol derivative, it is basically necessary to activate the carboxyl group at the C-terminal with a condensing agent or the like. It is known that amino acids other than glycine are easily epimerized in the activation step, and that stereoisomers are produced as by-products. By using achiral glycine as the C-terminal amino acid of the oligopeptide, a high-purity target product free from a by-product of a stereoisomer can be obtained.

Further, Z in (w1) to (w3) is a hydrophobic neutral amino acid having a hydropathic index of 2.5 or more, specifically preferably an oligopeptide having at least 1 of phenylalanine, leucine, valine and isoleucine, and more preferably an oligopeptide having phenylalanine. The quantitation achieved by Kate (Kyte) and Doolittle (Doolittle) indicates the hydropathic index (hydropathicity index) of the hydrophobicity of amino acids, with larger values indicating more hydrophobic amino acids (Kyte J & Doolittle RF,1982, J Mol Biol,157: 105-.

Z in (w1) to (w3) is not particularly limited as long as it is stable in blood in vivo and has the ability to be decomposed by intracellular enzymes, and is an oligopeptide of 2 to 5 residues comprising neutral amino acids excluding cysteine, and specific examples thereof include glycine-phenylalanine-leucine-glycine, glycine-phenylalanine-glycine, glycine-leucine-glycine, valine-citrulline-glycine, valine-alanine-glycine, phenylalanine-glycine and the like, and preferably glycine-phenylalanine-leucine-glycine, glycine-glycine, phenylalanine-glycine, glycine-serine-glycine, cysteine-glycine, and the like, Glycine-phenylalanine-glycine, valine-citrulline-glycine, valine-alanine-glycine or phenylalanine-glycine, more preferably glycine-phenylalanine-leucine-glycine, glycine-phenylalanine-glycine, valine-citrulline-glycine or phenylalanine-glycine, and still more preferably glycine-phenylalanine-leucine-glycine or phenylalanine-glycine.

D in formula (a) is a biologically relevant substance, and is not particularly limited, but is a substance relevant to diagnosis, cure, alleviation, treatment, or prevention of a disease in a human or other animal. Specifically, the protein or peptide includes proteins, peptides, nucleic acids, cells, viruses, and the like, and examples of suitable proteins or peptides include hormones, cytokines, antibodies, aptamers, enzymes, and the like.

More specifically, the cytokine includes interferons I, II, III, and interleukins that regulate immunity, tumor necrosis factor, receptor antagonists thereof, and the like. As the growth factor, erythropoietin as a hematopoietic growth factor, Granulocyte Colony Stimulating Factor (GCSF) as a stimulating factor, and the like are exemplified, and as the blood coagulation factor, factor V, factor VII, factor VIII, factor IX, factor X, factor XII, and the like are exemplified. Examples of hormones include calcitonin, insulin, an analog thereof, Exenatide (Exenatide), GLP-1, somatostatin, human growth hormone, and the like. The antibody may be a complete antibody, the antibody fragment may be Fab or svFV, the aptamer may be a DNA aptamer or an RNA aptamer, and the enzyme may be superoxide dismutase or uricase. Ideally, these proteins have low stability in blood and are modified with polyethylene glycol to increase blood half-life.

Suitable proteins include interferon, interleukin, erythropoietin, GCSF, factor VIII, factor IX, human growth hormone, antibody fragment, and the like, more preferably human growth hormone, interferon, GCSF, erythropoietin, or antibody fragment (particularly Fab), and still more preferably human growth hormone or GCSF.

Suitable peptides include insulin, bivalirudin, Teriparatide, Exenatide, Enfuvirtide, Degarelix, Mifamurtide, Nesiritide, Goserelin, Glatiramer, Octreotide, Lanreotide, Icatibant, cetibant, Ziconotide, Pramlintide, romidepsin, calcitonin, oxytocin, leuprolide, Leuprorelin, glucagon, more preferably insulin, Exenatide, calcitonin (in particular calcitonin).

One of preferred embodiments of the bio-related substance represented by formula (a) is a bio-related substance represented by the following formula (1) wherein b is 1.

[ solution 11]

(wherein n, W, a, D, L¹And L²Respectively, the same as above. )

One of preferred embodiments of formula (1) is a bio-related substance represented by formula (2) below, wherein W is W1 and a is 2.

[ solution 12]

(wherein Glu, Z, n, D, L¹And L²The same as above. )

One of the preferable embodiments of formula (1) is a bio-related substance represented by formula (3) below, wherein W is W2 and a is 4.

[ solution 13]

(wherein Glu, Z, n, D, L¹And L²The same as above. )

One of the preferable embodiments of formula (1) is a bio-related substance represented by formula (4) below, wherein W is W3 and a is 8.

[ solution 14]

(wherein Glu, Z, n, D, L¹And L²And aboveThe above meanings are the same. )

The bio-related substance of the formula (a) of the present invention can be obtained by reacting a biodegradable polyethylene glycol derivative represented by the following formula (5) with a bio-related substance.

[ solution 15]

(wherein X is a functional group capable of reacting with a biologically relevant substance, W, a, n, L¹And L²The same as above. )

X in formula (5) is not particularly limited as long as it is a functional group that reacts with a functional group present in a biologically relevant substance such as a physiologically active protein, peptide, antibody, or nucleic acid to be chemically modified to form a covalent bond. Examples thereof include "Harris, J.M. Poly (Ethylene Glycol) Chemistry; plenum Press, New York,1992(J.M. Harris, polyethylene glycol chemistry; Proelainan Press: New York, 1992), "Hermanson, G.T. bioconjugate Techniques,2nd ed.; academic Press: San Diego, CA,2008(G.T. Hermanson, Biocoupling technology, second edition; Academic Press: San Diego, Calif.; 2008) "and" PEGylated Protein Drugs: Basic Science and Clinical Applications; veronese, f.m., ed.; birkhauser: Basel, Switzerland,2009 (Pegylated protein drugs: basic science and clinical applications, F.M. Werlensted, Bukhaus Press: Basel, Switzerland, 2009) "and the like.

The "functional group reactive with the bio-related substance" represented by X in formula (5) is not particularly limited as long as it is a functional group capable of chemically bonding to a functional group such as an amino group, a thiol group, an aldehyde group, a carboxyl group, an unsaturated bond, or an azide group of the bio-related substance.

Specific examples thereof include an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxide group, a carboxyl group, a mercapto group, a maleimide group, a substituted maleimide group, a hydrazide group, a dithiopyridyl group, a substituted sulfonate group, and a vinyl groupSulfonyl, amino, oxyamino (H)₂N-O-group), an iodoacetamido group, an alkylcarbonyl group, an alkenyl group (e.g., allyl group, vinyl group), an alkynyl group, a substituted alkynyl group (e.g., alkynyl group substituted with a hydrocarbon group having 1 to 5 carbon atoms described later), an azido group, an acrylic group, a sulfonyloxy group (e.g., alkylsulfonyloxy group), an α -haloacetyl group, etc., preferably an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxide group, a maleimide group, a substituted maleimide group, a vinylsulfonyl group, an acrylic group, a sulfonyloxy group (e.g., alkyl-sulfonyloxy group having 1 to 5 carbon atoms), a substituted sulfonate group, a carboxyl group, a mercapto group, a dithiopyridyl group, an α -haloacetyl group, an alkynyl group, a substituted alkynyl group (e.g., alkynyl group having 2 to 5 carbon atoms substituted with a hydrocarbon group having 1 to 5 carbon atoms described later), etc.), Allyl, vinyl, amino, oxyamino, hydrazide and azide groups, more preferably active ester groups, active carbonate groups, aldehyde groups, maleimide groups, oxyamino groups and amino groups, particularly preferably aldehyde groups, maleimide groups and oxyamino groups.

In other suitable embodiments, the functional group X can be classified into group (III), group (IV), group (V), group (VI), group (VII), and group (VIII) described below.

Group (III): functional group capable of reacting with amino group of bio-related substance

Examples thereof include groups represented by the following (a), (b), (c), (d), (e), (f), (g), (j) or (k).

Group (IV): functional group capable of reacting with thiol group of bio-related substance

Examples thereof include groups represented by the following (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k) or (l).

Group (V): examples of the functional group capable of reacting with the aldehyde group of the bio-related substance include groups represented by the following (h), (m), (n) or (p).

Group (VI): examples of the functional group capable of reacting with a carboxyl group of a bio-related substance include groups represented by the following (h), (m), (n) or (p).

Group (VII): examples of the functional group capable of reacting with an unsaturated bond of a bio-related substance include groups represented by the following (h), (m), or (o).

Group (VIII): examples of the functional group capable of reacting with an azide group of a bio-related substance include the following groups (l).

[ solution 16]

In the functional group (j), W in the formula₁Represents a halogen atom such as a chlorine atom (Cl), a bromine atom (Br), or an iodine atom (I), and is preferably Br or I, more preferably I.

In addition, in the functional group (e) and the functional group (l), Y in the formula¹And Y³Each independently represents a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms, preferably a hydrocarbon group having 1 to 5 carbon atoms. The hydrocarbon group having 1 to 5 carbon atoms includes, specifically, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, and the like, and preferably methyl or ethyl.

In addition, in the functional group (k), Y in the formula²Specifically, the alkyl group may include methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, hexyl, nonyl, vinyl, phenyl, benzyl, 4-methylphenyl, trifluoromethyl, 2,2, 2-trifluoroethyl, 4- (trifluoromethoxy) phenyl, and the like, and preferably methyl, vinyl, 4-methylphenyl, or 2,2, 2-trifluoroethyl.

The active ester group means an ester group having an alkoxy group with a high leaving ability. Examples of the alkoxy group having a high leaving ability include alkoxy groups derived from nitrophenol, N-hydroxysuccinimide, pentafluorophenol and the like. The active ester group is preferably an ester group having an alkoxy group derived from N-hydroxysuccinimide.

The activated carbonate group means a carbonate group having an alkoxy group with a high leaving ability. Examples of the alkoxy group having a high leaving ability include alkoxy groups derived from nitrophenol, N-hydroxysuccinimide, pentafluorophenol and the like. The active carbonate group is preferably a carbonate group having an alkoxy group derived from nitrophenol or N-hydroxysuccinimide.

The substituted maleimide group is a maleimide group in which a hydrocarbon group is bonded to a carbon atom of one of the double bonds of the maleimide group. Specific examples of the hydrocarbon group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, and a tert-butyl group, and a methyl group or an ethyl group is preferable.

The substituted sulfonate group means a sulfonate group in which a hydrocarbon group which may contain a fluorine atom is bonded to a sulfur atom of the sulfonate group. Specific examples of the hydrocarbon group which may contain a fluorine atom include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a tert-butyl group, a hexyl group, a nonyl group, a vinyl group, a phenyl group, a benzyl group, a 4-methylphenyl group, a trifluoromethyl group, a 2,2, 2-trifluoroethyl group, a 4- (trifluoromethoxy) phenyl group and the like, and a methyl group, a vinyl group, a 4-methylphenyl group or a 2,2, 2-trifluoroethyl group is preferable.

The branched degradable polyethylene glycol derivative used for the bio-related substance of the present invention can be produced, for example, by the following steps.

[ solution 17]

(in the step (A), PEG is a polyethylene glycol chain, peptide is an oligopeptide, Pro is a protecting group, and L³Is a spacer group with a valence of 2.)

In the step, PEG is a polyethylene glycol chain, and the molecular weight is in the range of 2000 to 42000, as defined by n, which is the number of repeating units of the above-mentioned polyethylene glycol, that is, n is 45 to 950.

The peptide in the step (a) is an oligopeptide having the same meaning as Z. In this step, an oligopeptide in which the amino group at the N-terminal is protected with a protecting group is used.

Pro in the step is a protecting group, and the protecting group is a component which prevents or inhibits the reaction of a functional group capable of undergoing a specific chemical reaction in a molecule under a certain reaction condition. Protecting groups vary depending on the type of functional group that is protected to be capable of chemical reaction, the conditions used, and the presence of other functional groups or protecting groups in the molecule. Specific examples of protecting groups can be found in many common books, such as "Wuts, p.g.m.; greene, T.W.protective Groups in Organic Synthesis,4th ed.; Wiley-Interscience: new York,2007(P.G.M. Wus; T.W. Green, protective group in organic Synthesis, fourth edition; Wiley-Interscience: New York, 2007) ". Further, the functional group protected by a protecting group can regenerate its original functional group by deprotection, that is, chemical reaction, using reaction conditions suitable for the respective protecting groups. Typical deprotection conditions for protecting groups are as described in the above mentioned documents.

L in the process³Is related to the above-mentioned L¹And L²Spacer group with 2 valences of the same meaning.

Reaction a is a step of bonding a carboxyl group of an oligopeptide having an N-terminal amino group protected by a protecting group to an amino group of a polyethylene glycol derivative having a methoxy group at one terminal thereof by a condensation reaction to obtain a polyethylene glycol derivative (1).

The protective group for the amino group at the N-terminal of the oligopeptide is not particularly limited, and examples thereof include an acyl-based protective group and a carbamate-based protective group, and specifically include a trifluoroacetyl group, a 9-fluorenylmethoxycarbonyl (Fmoc) group, a tert-butoxycarbonyl group, and the like.

The condensation reaction is not particularly limited, but is preferably a reaction using a condensing agent. As the condensing agent, a carbodiimide-based condensing agent such as Dicyclohexylcarbodiimide (DCC) or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) may be used alone, or a reagent such as N-hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBt), or 1-hydroxy-7-azabenzotriazole (HOAt) may be used together. Furthermore, condensing agents such as HATU, HBTU, TATU, TBTU, COMU, and DMT-MM, which are more reactive, may also be used. In addition, a base such as triethylamine or dimethylaminopyridine may be used to accelerate the reaction.

Impurities generated by the side reaction in the reaction, remaining oligopeptides and condensing agents which are not consumed in the reaction, and the like are preferably purified and removed. The purification is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[ solution 18]

Deprotection B is a step of deprotecting the protecting group of polyethylene glycol derivative (1) obtained in reaction a to obtain polyethylene glycol derivative (2). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use oligopeptide and L³Without decomposition of the spacer group having a valence of 2. The present step may be carried out as a part of the step of reaction a.

Impurities and the like generated by side reactions in the deprotection reaction are preferably purified and removed. The purification is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[ solution 19]

Reaction C is a step of bonding the amino group of the polyethylene glycol derivative (2) obtained by deprotection B to two carboxyl groups of the glutamic acid derivative whose amino group is protected by a protecting group by a condensation reaction to obtain a branched polyethylene glycol derivative (3) having a structure in which 2 degradable polyethylene glycol chains are linked by a glutamic acid residue.

As in the case of the above reaction A, a reaction using a condensing agent is preferable, and a base such as triethylamine or dimethylaminopyridine may be used to accelerate the reaction.

The protecting group of the amino group of glutamic acid is not particularly limited, and examples thereof include an acyl-based protecting group and a carbamate-based protecting group, and specifically include trifluoroacetyl group, 9-fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl, and the like.

Impurities produced by the side reaction in the reaction, polyethylene glycol derivatives remaining without being consumed in the reaction, and the like are preferably purified and removed. The purification is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[ solution 20]

Deprotection D is a step of deprotecting the protecting group of the polyethylene glycol derivative (3) obtained in reaction C to obtain a polyethylene glycol derivative (4). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use oligopeptide and L³Without decomposition of the spacer group having a valence of 2. The present step may be performed as a part of the step of reaction C.

Impurities and the like generated by side reactions in the deprotection reaction are preferably purified and removed. The purification is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[ solution 21]

Reaction E is a step of bonding the amino group of the polyethylene glycol derivative (4) obtained by deprotection D to two carboxyl groups of a glutamic acid derivative whose amino group is protected by a protecting group by a condensation reaction to obtain a branched polyethylene glycol derivative (5) having a structure in which 4 degradable polyethylene glycol chains are linked by a glutamic acid residue.

It can be reacted and purified under the same conditions as in the above reaction C.

As a method for removing polyethylene glycol impurities having different molecular weights or functional groups from the polyethylene glycol derivative (5), purification techniques described in Japanese patent laid-open Nos. 2014-208786 and 2011-79934 can be used.

[ solution 22]

Deprotection F is the reaction of the polyethylene glycol derivative obtained in reaction E(5) Deprotecting the protecting group (2) to obtain a polyethylene glycol derivative (6). As the deprotection reaction, a conventionally known method can be used, but it is necessary to use oligopeptide and L³Without decomposition of the spacer group having a valence of 2. Can be reacted and purified under the same conditions as the above deprotection D. The present step may be carried out as a part of the step of the reaction E.

[ solution 23]

Reaction G is a step of bonding the amino group of the polyethylene glycol derivative (6) obtained by deprotection F to two carboxyl groups of a glutamic acid derivative whose amino group is protected by a protecting group by a condensation reaction to obtain a branched polyethylene glycol derivative (7) having a structure in which 8 degradable polyethylene glycol chains are linked by a glutamic acid residue.

It can be reacted and purified under the same conditions as in the above reaction C.

[ solution 24]

Deprotection H is a step of deprotecting the protecting group of the polyethylene glycol derivative (7) obtained in reaction G to obtain a polyethylene glycol derivative (8). Can be reacted and purified under the same conditions as the above-mentioned deprotection F. The present step may be carried out as a part of the step of the reaction G.

By carrying out the above-mentioned reaction A, deprotection B, reaction C and deprotection D, a 2-branched degradable polyethylene glycol derivative (4) can be obtained. The 4-branched degradable polyethylene glycol derivative (6) can be obtained by continuing the reaction E and deprotection F using the 2-branched degradable polyethylene glycol derivative (4) as a starting material. Further, by continuing the reaction G and deprotection H, 8-branched degradable polyethylene glycol derivative (8) can be obtained.

The polyethylene glycol derivatives (4), (6) and (8) obtained in deprotection D, deprotection F and deprotection H each have an amino group, and can be converted into various functional groups by using the amino group.

The step of converting the terminal amino group of the polyethylene glycol derivative into another functional group is not particularly limited, and basically, it can be easily converted into various functional groups by using a compound having an active ester group capable of reacting with an amino group, a general reaction reagent such as an acid anhydride or an acid chloride.

For example, when it is desired to convert an amino group at the terminal of a polyethylene glycol derivative into a maleimide group, a target substance can be obtained by reacting the maleimide group with the following reagent.

[ solution 25]

For example, when it is desired to convert an amino group at the terminal of a polyethylene glycol derivative into a carboxyl group, the target compound can be obtained by reacting the carboxyl group with succinic anhydride or glutaric anhydride.

For example, when it is desired to convert an amino group at the terminal of a polyethylene glycol derivative into a hydroacid group, the target compound is obtained by a condensation reaction with a ring-opened product of a cyclic ester such as caprolactone.

These reagents are low molecular weight reagents, and have a large solubility difference from polyethylene glycol derivatives of high molecular weight polymers, and therefore can be easily removed by a common purification method such as extraction or crystallization.

The degradable polyethylene glycol obtained through the above-mentioned steps is required to be stable in blood and to have a property of being degraded only in cells. In order to appropriately evaluate the performance, for example, the stability of degradable polyethylene glycol in blood and the degradability in cells can be evaluated by the following tests.

In these evaluations, all the evaluation samples were tested in a lump for polyethylene glycol derivatives having one amino group, taking into account the influence of the kind of functional group of the polyethylene glycol derivative.

The test method for evaluating the stability of the degradable polyethylene glycol derivative in blood is not particularly limited, and examples thereof include a test using serum of a mouse, a rat, a human, and the like. Specifically, the degradation rate can be evaluated by dissolving a polyethylene glycol derivative in serum to a concentration of 1 to 10mg/mL, incubating the resulting solution at 37 ℃ for 96 hours, collecting the polyethylene glycol derivative contained in the serum, and measuring GPC. The decomposition rate was calculated based on the% peak area of the GPC main fraction of the polyethylene glycol derivative before the stability test and the% peak area of the GPC main fraction of the polyethylene glycol derivative after the stability test. Specifically, the following formula is used.

Decomposition rate ═ peak area before test% peak area after test ÷ peak area before test ÷ peak area% before test x 100

For example, if the peak area% of the GPC main fraction of the degradable polyethylene glycol derivative before the stability test is 95% and the peak area% of the GPC main fraction after the test is 90%, the degradation rate is calculated as follows.

Decomposition rate (95-90) ÷ 95 × 100 ═ 5.26 (%)

Since the target blood half-life cannot be obtained when the degradable polyethylene glycol derivative is degraded in blood, the degradation rate after 96 hours in the stability test is preferably 10% or less, and more preferably 5% or less.

The test method for evaluating the intracellular degradability of the degradable polyethylene glycol derivative is not particularly limited, and examples thereof include a test in which cells are cultured using a medium containing the degradable polyethylene glycol derivative. The cells or the medium used herein are not particularly limited, and specifically, the decomposition rate can be evaluated by dissolving the polyethylene glycol derivative in the RPMI-1640 medium to a concentration of 1 to 20mg/mL, culturing the macrophage RAW264.7 at 37 ℃ for 96 hours using the medium, recovering the polyethylene glycol derivative in the cells, and measuring GPC. The decomposition rate can be calculated using the% peak area of the GPC main fraction of the polyethylene glycol derivative before and after the test, similarly to the stability test.

For example, if the peak area% of the GPC main fraction using the decomposable polyethylene glycol derivative before the decomposition test of cells is 95%, and the peak area% of the GPC main fraction after the test is 5%, the decomposition rate is calculated as follows.

Decomposition rate (95-5) ÷ 95 × 100 ═ 94.7 (%)

Since vacuoles of target cells cannot be suppressed if the degradable polyethylene glycol derivative cannot be efficiently degraded in cells, the degradation rate after 96 hours in the degradability test is preferably 90% or more, and more preferably 95% or more.

The method for bonding the obtained degradable polyethylene glycol derivative to the biologically relevant substance is not particularly limited, and for example, "Hermanson, g.t. bioconjugate Techniques,3rd ed.; academic Press: San Diego, CA,2013(G.T. Hermanson, bioconjugate technology, third edition; Academic Press: San Diego, Calif., 2013), "Mark, Sonny S.bioconjugate protocols, strategies and methods; 2011 (Mark, Sanny S., bioconjugation protocols, strategies and methods; 2011) ". Among these, for example, when a side chain amino group of a lysine residue such as a protein or a peptide which is a bio-related substance is used as a target (target), a polyethylene glycol derivative having an activated ester group or an activated carbonate group is used. In addition, when a thiol group of a cysteine residue such as a protein or a peptide which is a biologically-relevant substance is used as a target, a polyethylene glycol derivative having a maleimide group or an iodoacetamide group is used. Since the amount of free cysteine residues contained in the natural bio-related substance is extremely small, the method can further selectively bond polyethylene glycol to the bio-related substance. Further, as a method for generating or introducing a thiol group, there are a method of cleaving a disulfide bond of a bio-related substance, a method of introducing a cysteine residue by modifying a bio-related substance by genetic engineering, and the like, and a polyethylene glycol derivative capable of bonding a desired number of thiol groups to a target site of a bio-related substance by combining these techniques is known.

Next, when an amino group at the N-terminus of a protein, a peptide, or the like, which is a biologically-relevant substance, is targeted, a polyethylene glycol derivative having an aldehyde group is used. Specifically, the polyethylene glycol derivative can be selectively bonded to the amino group at the N-terminus of a protein or peptide by using a polyethylene glycol derivative having an aldehyde group and a suitable reducing agent in a buffer solution at low pH.

These bio-related substances to which polyethylene glycol derivatives are bonded can be purified by dialysis, Gel Permeation Chromatography (GPC), Ion Exchange Chromatography (IEC), and the like, which are known as general methods. In addition, the obtained biologically-relevant substance can be generally evaluated by an analysis method such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), polyacrylamide gel electrophoresis (SDS-PAGE), and reverse phase chromatography (RPLC).

The method for evaluating the physiological activity of the bio-related substance to which the degradable polyethylene glycol derivative is bonded is not particularly limited, and for example, the method can be evaluated by measuring the blood sugar concentration in the case of insulin or the calcium concentration in the case of calcitonin, and the like, by periodically collecting blood from an animal after administration and measuring the substance in the blood with a suitable analyzer. Specifically, in the case of insulin, the decrease in glucose concentration after administration can be monitored using a glucose assay kit, and in the case of calcitonin, the decrease in calcium concentration after administration can be monitored using a calcium assay kit for evaluation.

The test method for evaluating the blood half-life and in vivo distribution of the bio-related substance to which the degradable polyethylene glycol derivative is bonded is not particularly limited, and examples thereof include a test in which the bio-related substance is labeled with a radioisotope or a fluorescent substance, administered to a mouse or a rat, and monitored.

It is considered that the lytic peptide introduced into the polyethylene glycol derivative imparts degradability in the cell to polyethylene glycol, but the peptide structure may change the in vivo kinetics of the biologically relevant substance to which polyethylene glycol is bonded. Therefore, in order to confirm the influence of the introduced peptide structure on the in vivo kinetics, it is necessary to compare the blood half-life and the in vivo distribution thereof with those of a biologically relevant substance modified with a polyethylene glycol derivative having the same molecular weight and not having degradability. Specifically, a polyethylene glycol derivative having no degradability and a degradable polyethylene glycol derivative are bonded to each of the bio-related substances labeled with a radioisotope, and the obtained 2 bio-related substances are administered to mice, and the radiation doses of blood and organs are measured at a plurality of time points to perform quantitative measurement.

The test method for evaluating the blood half-life and in vivo distribution of the degradable polyethylene glycol derivative is not particularly limited, and examples thereof include a test in which the degradable polyethylene glycol derivative is labeled with a radioisotope or a fluorescent substance, administered to a mouse or a rat, and monitored.

It is considered that the lytic peptide introduced into the polyethylene glycol derivative imparts degradability to polyethylene glycol in a cell, but the pharmacokinetics of polyethylene glycol may be changed due to the peptide structure. Therefore, in order to confirm the influence of the introduced peptide structure on pharmacokinetics, it is necessary to compare the half-life in blood and the distribution in vivo thereof with polyethylene glycol derivatives having the same molecular weight and not having degradability. Specifically, a polyethylene glycol derivative labeled with a radioisotope and not degradable and a degradable polyethylene glycol derivative are administered to mice, and the radiation dose of blood and organs is measured at a plurality of time points to perform quantitative measurement.

The test method for evaluating the vacuolation inhibition of cells by a degradable polyethylene glycol derivative is not particularly limited, and examples thereof include a test in which the drug is administered to a mouse or a rat for a long period of time, at a high frequency, and at a high dose, and a slice image of an organ or organ that is thought to be likely to generate vacuolation is confirmed, as described in non-patent document 2.

Specifically, the polyethylene glycol derivative is dissolved in physiological saline to a concentration of 10 to 250mg/mL, and the administration is continued 3 times a week, 4 weeks or more and 20 to 100 μ L per week through the mouse tail vein, whereby paraffin sections of the brain choroid plexus, spleen or the like of organs considered to be likely to cause cavitation are prepared and stained, and then the section images are confirmed by a pathological method to evaluate the cavitation inhibition.

In addition, in this evaluation, the amount of polyethylene glycol to be administered is required to be excessively larger than the amount of polyethylene glycol generally used in the art.

Non-patent document 2 describes that vacuolation of cells by high molecular weight polyethylene glycol is related to accumulation of polyethylene glycol in tissues. The test method for evaluating the accumulation property in the cells of the degradable polyethylene glycol derivative is not particularly limited, and the evaluation can be performed by using a slice image prepared in the same manner as the above-described vacuole evaluation. The evaluation of the accumulation of polyethylene glycol can be carried out by confirming stained slice images of the brain choroid plexus, spleen, etc. of organs thought to be susceptible to vacuolation by a pathological method.

In addition, in this evaluation, the amount of polyethylene glycol to be administered is required to be excessively larger than the amount of polyethylene glycol generally used in the art.

Examples

Obtained in the examples below¹H-NMR was obtained from JNM-ECP400 or JNM-ECA600, manufactured by JEOLDATUM, Inc. Used in the assaySample tube, use of D in deuterated solvents₂O or CDCl containing Tetramethylsilane (TMS) as internal standard₃And d₆-DMSO. The molecular weight and amine purity of the obtained polyethylene glycol derivative were calculated using liquid chromatography (GPC and HPLC). The liquid chromatography system used was "HLC-8320 GPC EcoSEC" manufactured by Tosoh corporation and "ALLIANCE" manufactured by WATERS corporation for GPC. Analytical conditions for GPC and HPLC are shown below.

GPC analysis (molecular weight measurement)

Standard polymer: molecular weight determination by GPC analysis was performed using polyethylene glycols having molecular weights of 8,000, 20,000, 50,000, and 100,000 as standard polymers.

A detector: differential refractometer

A chromatographic column: ultrahydrogel 500 and 250 (manufactured by WATERS Co., Ltd.)

Mobile phase: 100mM Acetate buffer (Acetate buffer) + 0.02% NaN₃(pH5.2)

Flow rate: 0.5mL/min

Sample amount: 5mg/mL, 20. mu.L

Temperature of the column: 30 deg.C

HPLC analysis (determination of amine purity)

A detector: differential refractometer

A chromatographic column: TSKgel SP-5PW (manufactured by Tosoh corporation)

Mobile phase: 1mM Sodium phosphate buffer (Sodium phosphate buffer) (pH6.5)

Flow rate: 0.5mL/min

Injection amount: 5mg/mL, 20. mu.L

Temperature of the column: 40 deg.C

[ example 1]

Compound (p3) (p) _{2 2}NH-E(FG-200ME)) Synthesis of (2)

[ solution 26]

[ example 1-1]

[ solution 27]

L-phenylalanyl-glycine (Fmoc-Phe-Gly) protected to the N-terminus by 9-fluorenylmethoxycarbonyl (Fmoc group) (0.267g, 6.0X 10^-4mol, manufactured by Dubian chemical Co., Ltd.) and methoxy PEG having propylamino group at the terminal (6.0g, 2.8X 10)^-4To "SUNBRIGHT MEPA-20T" manufactured by Nichikoku K.K., 21,120 mol, was added dehydrated N, N' -dimethylformamide (60g), and the mixture was dissolved at 30 ℃ with heating. Then, diisopropylethylamine (192. mu.L, 1.2X 10) was added^-3mol, manufactured by Kanto chemical Co., Ltd.) and (1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) (0.321g, 7.5X 10)^-4mol, manufactured by Sigma-Aldrich corporation), and allowed to react at room temperature under a nitrogen atmosphere for 1 hour. After completion of the reaction, the reaction mixture was diluted with chloroform (600g), and saturated sodium bicarbonate was added theretoThe solution (240g) was stirred at room temperature for 15 minutes for washing. After separating the aqueous layer and the organic layer, a saturated aqueous sodium bicarbonate solution (240g) was added to the organic layer again, and the mixture was stirred at room temperature for 15 minutes to wash the mixture, thereby recovering the organic layer. To the obtained organic layer (chloroform solution), magnesium sulfate (2.4g) was added, and the mixture was stirred for 30 minutes to dehydrate, followed by suction filtration using an aleurite funnel on which Oplite was spread on a 5A filter paper. The filtrate obtained was concentrated at 40 ℃, ethyl acetate (240g) was added to the concentrate, and the mixture was stirred until uniform, followed by addition of hexane (120g), and stirring was carried out at room temperature for 15 minutes to precipitate a product. After the precipitate was recovered by suction filtration using a 5A filter paper, it was dissolved in ethyl acetate (240g) again, and hexane (120g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (120g), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p1) ((p 1))ME-200GF-Fmoc). The yield was 5.1 g.

¹H-NMR(d₆-DMSO):1.62ppm(m,2H,-CO-NH-CH₂- ₂CH-CH₂-O-(CH₂-CH₂-O)n-CH₃)，2.80ppm(m,1H,-NH-CO-CH- ₂CH-C₆H₅)，3.04ppm(m,1H,-NH-CO-CH-CH ₂ -C₆H₅)，3.10ppm(m,2H,-CO-NH- ₂CH-CH₂-CH₂-O-(CH₂-CH₂-O)n-CH₃)，3.24ppm(s,3H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n- ₃CH) 3.48ppm (m, about 1,900H, -CO-NH-CH)₂-CH₂-CH₂-O-( ₂CH-CH ₂-O)n-CH₃)，4.20ppm(m,4H)，7.33ppm(m,9H)，7.66ppm(m,4H,Ar)，7.88ppm(d,2H,Ar)，8.27ppm(t,1H)

[ examples 1-2]

[ solution 28]

Into example 1-1The resulting ME-200GF-Fmoc (4.9g, 2.3X 10)^-4mol) was added to N, N' -dimethylformamide (29.4g), and the mixture was dissolved at 30 ℃ with heating. Piperidine (1.55g, 1.8X 10) was added^-2mol, Wako pure chemical industries, Ltd.) was reacted at room temperature under a nitrogen atmosphere for 2 hours. After completion of the reaction, ethyl acetate (300g) was added thereto and stirred until uniform, and hexane (150g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. After the precipitate was recovered by suction filtration using a 5A filter paper, it was dissolved in ethyl acetate (300g) again, hexane (150g) was added thereto, and the mixture was stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (150g), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p2) (R) ₂ME-200GF-NH). The yield was 3.9 g.

¹H-NMR(d₆-DMSO):1.62ppm(m,2H,-CO-NH-CH₂- ₂CH-CH₂-O-(CH₂-CH₂-O)n-CH₃) 1.64ppm (width, 1H), 2.59ppm (dd,1H, -NH-CO-CH- ₂CH-C₆H₅)，2.98ppm(dd,1H,-NH-CO-CH- ₂CH-C₆H₅)，3.10ppm(q,2H,-CO-NH- ₂CH-CH₂-CH₂-O-(CH₂-CH₂-O)n-CH₃)，3.24ppm(s,3H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n ₃-CH) 3.48ppm (m, about 1,900H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃)，7.24ppm(m,6H,-NH-CO-CH-CH₂- _{6 5}CH-NH-), 7.73ppm (t,1H), 8.12ppm (Wide, 1H)

[ examples 1 to 3]

[ solution 29]

L-glutamic acid (Fmoc-Glu-OH) protected by Fmoc group to the N-terminus (16.0mg, 4.3X 10)^-5mol, Bian chemical industryManufactured by Kokai Co., Ltd.) and ME-200GF-NH obtained in example 1-2₂(2.0g，1.0×10^-4mol) was added with dehydrated N, N' -dimethylformamide (10g), and the mixture was dissolved by heating at 30 ℃. Then, diisopropylethylamine (19.2. mu.L, 1.1X 10) was added^-4mol, manufactured by Kanto chemical Co., Ltd.) and 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholine hydrochloride n hydrate (DMT-MM) (39.0mg, 1.1X 10)^-4mol, Wako pure chemical industries, Ltd.) was reacted at room temperature under a nitrogen atmosphere for 1 hour. Then, piperidine (0.5g, 5.9X 10) was added^-3mol, Wako pure chemical industries, Ltd.) was reacted at room temperature under a nitrogen atmosphere for 2 hours. After completion of the reaction, the reaction mixture was diluted with toluene (80g), and hexane (40g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. After the precipitate was recovered by suction filtration using a 5A filter paper, it was dissolved in toluene (80g) again, and hexane (40g) was added thereto, followed by stirring at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (40g), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p3) (b) _{2 2}NH-E(FG-200ME)). The yield was 1.6 g. The molecular weights are shown in table 1. HPLC: the amine purity was 92%.

¹H-NMR(d₆-DMSO):1.54ppm(m,2H,-NH-CO-CH(NH₂)- ₂CH-CH₂-)，1.62ppm(m,4H,-CO-NH-CH₂- ₂CH-CH₂-)，1.97ppm(m,2H,-NH-CO-CH(NH₂)-CH₂- ₂CH-)，2.74ppm(dd,1H,-CO-NH-CH- ₂CH-C₆H₅)，2.81ppm(dd,1H,-CO-NH-CH- ₂CH-C₆H₅)，3.11ppm(m,11H)，3.24ppm(s,6H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n- ₃CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃)，4.49ppm(m,1H,-CO-NH-CH-CH₂-C₆H₅)，4.57ppm(m,1H,-CO-NH-CH-CH₂-C₆H₅)，7.25ppm(m,10H,-CO-NH-CH-CH₂- _{6 5}CH)，7.74ppm(m,2H)，8.44ppm(m,2H)，8.61ppm(m,2H)

[ example 2]

Compound (p4) (p) ₂MA-E(FG-200ME)) Synthesis of (2)

[ solution 30]

The compound (p3) (200mg, 5.0X 10) obtained in example 1 was added^-6mol) was dissolved in acetonitrile (160mg) and toluene (1.0 g). Then, N-methylmorpholine (10mg, 1.0X 10) was added^-5mol, manufactured by Kanto chemical Co., Ltd.) and 3-Maleimidopropionic acid N-hydroxysuccinimide ester (8.0mg, 3.0X 10)^-5mol, manufactured by osaka synthetic organic chemistry research institute corporation), and reacted at room temperature under a nitrogen atmosphere and under light shielding for 6 hours. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (50g) containing 2, 6-di-t-butyl-p-cresol (BHT) (10mg), and hexane (25g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (25g) containing BHT (5mg), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p4) ((25 g)) ₂MA-E(FG-200ME)). The yield was 137 mg. The molecular weights are shown in table 1. The purity of maleimide is 90%, (¹H-NMR)。

¹H-NMR(d₆-DMSO):1.62ppm(m,6H)，1.99ppm(m,2H,-NH-CO-CH(NH₂)-CH₂- ₂CH-)，2.34ppm(m,2H,-NH-CO- ₂CH-CH₂-maleimide), 2.75ppm (dd,1H, -CO-NH-CH- ₂CH-C₆H₅)，2.82ppm(dd,1H,-CO-NH-CH- ₂CH-C₆H₅)，3.11ppm(m,11H)，3.24ppm(s,6H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n- ₃CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃)，4.04ppm(m,2H,-NH-CO-CH_{2 2}-CHMaleimide), 4.49ppm (m,2H, -CO-NH-CH-CH₂-C₆H₅)，6.98ppm(s,2H,-CO-CH-CH-CO-)，7.25ppm(m,10H,-CO-NH-CH-CH₂- _{6 5}CH)，7.69ppm(dt,2H)，8.04ppm(d,1H)，8.29ppm(dd,2H)，8.41ppm(dt,2H)

[ example 3]

Compound (p8) (p) ₂AL-E(FG-200ME)) Synthesis of (2)

[ solution 31]

[ example 3-1]

Compound (p5) (p) ₂HO-E(FG-200ME)) Synthesis of (2)

[ solution 32]

Mixing epsilon-caprolactone (114mg, 1.0X 10)^-3mol, manufactured by Tokyo chemical industry Co., Ltd.) was dissolved in 1N NaOH (0.8mL, 8.0X 10^-4mol, manufactured by Kanto chemical Co., Ltd.) was reacted for 2 hours to prepare an aqueous solution of 6-hydroxycaproic acid (0.88M). Further, the compound (p3) (2.0g, 5.0X 10) obtained in example 1 was added^-5mol) was dissolved in acetonitrile (8.0 g). Then, the above 6-hydroxycaproic acid aqueous solution (114. mu.L, 1.0X 10)^-4mol), diisopropylethylamine (20. mu.L, 1.2X 10)^{- 4}mol, manufactured by Kanto chemical Co., Ltd.) and DMT-MM (21mg, 6.0X 10)^-5mol, Wako pure chemical industries, Ltd.) was added to the acetonitrile solution of (p3), and reacted for 1 hour at room temperature under a nitrogen atmosphere. After completion of the reaction, the reaction mixture was concentrated at 40 ℃ and chloroform (24g) was added to the obtained concentrate to dissolve it. Saturated aqueous sodium bicarbonate (10g) was added thereto, and the mixture was stirred at room temperature for 15 minutes to wash the mixture. Separating the aqueous layer from the organic layer, and converting the separated aqueous layer to the organic layerSaturated aqueous sodium bicarbonate (10g) was added to the layer, and the mixture was stirred at room temperature for 15 minutes to wash the layer, and the organic layer was collected. To the obtained organic layer (chloroform solution), magnesium sulfate (1.2g) was added, and the mixture was stirred for 30 minutes to dehydrate, followed by suction filtration using an aleurite funnel in which Oplite was spread on a 5A filter paper. The obtained filtrate was concentrated at 40 ℃, toluene (50g) was added to the concentrate and stirred until uniform, then hexane (25g) was added thereto, and stirred at room temperature for 15 minutes to precipitate a product. After the precipitate was recovered by suction filtration using a 5A filter paper, it was dissolved in toluene (50g) again, and hexane (25g) was added thereto, followed by stirring at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (10g) containing BHT (2mg), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p5) (10g) ₂HO-E(FG-200ME)). The yield was 1.5 g.

¹H-NMR(CDCl₃):1.37ppm(m,2H,HO-CH₂-CH₂- ₂CH-CH₂-CH₂-CO-NH-)，1.55ppm(m,4H,HO-CH₂- ₂CH-CH₂- ₂CH-CH₂-CO-NH-)，1.77ppm(m,4H,-CO-NH-CH₂- ₂CH-CH₂-O-(CH₂-CH₂-O)n-CH₃)，1.85ppm(m,1H)，2.01ppm(m,2H,HO-CH₂-CH₂-CH₂-CH₂- ₂CH-CO-NH-)，3.01ppm(m,1H)，3.24ppm(m,8H)，3.38ppm(s,6H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n- ₃CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃)，4.03ppm(m,4H)，4.14ppm(m,1H)，4.48ppm(m,2H,-CO-NH-CH-CH₂-C₆H₅) 6.95ppm (Wide, 1H), 7.00ppm (Wide, 1H), 7.26ppm (m,10H, -CO-NH-CH-CH)₂- _{6 5}CH) 7.66ppm (Wide, 1H), 8.29ppm (Wide, 1H)

[ examples 3-2]

Compound (p6) (p) ₂SC-E(FG-200ME)) Synthesis of (2)

[ solution 33]

The compound (p5) (500mg, 1.3X 10) obtained in example 3-1 was added^-5mol) was dissolved in dichloromethane (3.5 g). Then, N' -disuccinimidyl carbonate (51mg, 2.0X 10) was added^-4mol, manufactured by Tokyo chemical industry Co., Ltd.) and pyridine (24. mu.L, 3.0X 10)^-4mol, manufactured by Kanto chemical Co., Ltd.), and reacted at room temperature under a nitrogen atmosphere for 8 hours. After completion of the reaction, the reaction solution was washed with 5% saline, magnesium sulfate (0.1g) was added thereto, and the mixture was stirred at 25 ℃ for 30 minutes, followed by suction filtration using a Fukusan funnel in which Oplite was spread on a 5A filter paper. The obtained filtrate was concentrated, and toluene (50g) was added to the concentrate to dissolve it, followed by addition of hexane (25g) and stirring at room temperature for 15 minutes to precipitate a product. After the precipitate was recovered by suction filtration using a 5A filter paper, it was dissolved in toluene (50g) again, and hexane (25g) was added thereto, followed by stirring at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (25g) containing BHT (5mg), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p6) ((25 g)) ₂SC-E(FG-200ME)). The yield was 286 mg. Purity of activated carbonate 92% ((¹H-NMR)。

¹H-NMR(CDCl₃) 1.38ppm (m,2H, succinimide-OCO-CH)₂-CH₂- ₂CH-CH₂-CH₂-CO-NH-), 1.59ppm (m,2H, succinimide-OCO-CH)₂-CH₂-CH₂- ₂CH-CH₂-CO-NH-), 1.75ppm (m,6H), 1.85ppm (m,1H), 2.13ppm (m,2H, succinimide-OCO-CH)₂-CH₂-CH₂-CH₂- ₂CH-CO-NH-)，2.83ppm(s,4H,-CO- _{2 2}CH-CH-CO-)，3.01ppm(m,1H)，3.19ppm(m,6H)，3.38ppm(s,6H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n- ₃CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃) 4.03ppm (m,3H), 4.18ppm (m,1H), 4.31ppm (t,2H, succinimide-OCO- ₂CH-CH₂-CH₂-CH₂-CH₂-CO-NH-)，4.50ppm(m,2H,-CO-NH-CH-CH₂-C₆H₅) 6.98ppm (Wide, 1H), 7.15ppm (Wide, 1H), 7.26ppm (m,10H, -CO-NH-CH-CH)₂- _{6 5}CH) 7.81ppm (Wide, 1H), 8.37ppm (Wide, 1H)

[ examples 3 to 3]

Compound (p7) (p) ₂DE-E(FG-200ME)) Synthesis of (2)

[ chemical 34]

The compound (p6) (250mg, 6.3X 10) obtained in example 3-2 was added^-6mol) in chloroform (2 g). Then, 1-amino-3, 3-diethoxypropane (10. mu.L, 6.3X 10) was added^-5mol, manufactured by ACROS ORGANICS corporation), at room temperature under a nitrogen atmosphere for 3 hours. After completion of the reaction, the reaction mixture was diluted with toluene (25g), and hexane (12.5g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using a 5A filter paper to collect the precipitate, followed by washing with hexane (12.5g) containing BHT (2.5mg), suction filtration was performed using a 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p47) ((p 47)) ₂DE-E(FG-200ME)). The yield was 185 mg.

¹H-NMR(CDCl₃):1.20ppm(t,6H,( ₃CH-CH₂-O)₂-CH-)，1.32ppm(m,2H,(CH₃-CH₂-O)₂-CH-CH₂-CH₂-NH-COO-CH₂-CH₂- ₂CH-CH₂-CH₂-CO-NH-)，1.58ppm(m,2H,(CH₃-CH₂-O)₂-CH-CH₂-CH₂-NH-COO-CH₂-CH₂-CH₂- ₂CH-CH₂-CO-NH-)，1.76ppm(m,4H,-CO-NH-CH₂- ₂CH-CH₂-O-(CH₂-CH₂-O)n-CH₃)，1.82ppm(m,2H,(CH₃-CH₂-O)₂-CH- ₂CH-CH₂-NH-COO-CH₂-CH₂-CH₂-CH₂-CH₂-CO-NH-)，2.11ppm(m,2H,(CH₃-CH₂-O)₂-CH-CH₂-CH₂-NH-COO-CH₂- ₂CH-CH₂-CH₂-CH₂-CO-NH-)，2.16ppm(m,1H)，2.70ppm(m,1H)，3.06ppm(m,2H)，3.25ppm(m,11H)，3.38ppm(s,6H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n- ₃CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃)，4.02ppm(m,8H)，4.17ppm(m,1H)，4.51ppm(m,2H,-CO-NH-CH-CH₂-C₆H₅)，4.55ppm(t,1H,(CH₃-CH₂-O)₂-CH-), 5.36ppm (Width, 1H), 6.47ppm (Width, 1H), 6.98ppm (Width, 2H), 7.26ppm (m,10H, -CO-NH-CH-CH)₂- _{6 5}CH) 7.81ppm (Wide, 1H), 8.36ppm (Wide, 1H)

[ examples 3 to 4]

Compound (p8) (p) ₂AL-E(FG-200ME)) Synthesis of (2)

[ solution 35]

The compound (p7) (150mg, 3.8X 10) obtained in example 3-3 was added^-6mol) was dissolved in a phosphate buffer (2.25g) adjusted to pH 1.90 and reacted at room temperature under a nitrogen atmosphere for 3 hours. After the reaction, 0.1N aqueous sodium hydroxide (0.89g) was added to adjust the pH to 6.40, and then sodium chloride (0.56g) was added to dissolve the mixture. To the obtained solution, 0.1N aqueous sodium hydroxide solution (0.60g) was added dropwise to adjust the pH to 7.06, and then chloroform (3g) containing BHT (0.6mg) was added thereto, and the mixture was stirred at room temperature for 20 minutes to obtain a productAnd extracting to an organic layer. After the organic layer and the aqueous layer were separated and the organic layer was collected, chloroform (3g) containing BHT (0.6mg) was added again to the aqueous layer, and the mixture was stirred at room temperature for 20 minutes to extract the product into the organic layer. The combined organic layers obtained in the 1 st and 2nd extractions were concentrated at 40 ℃, and the obtained concentrate was diluted in toluene (30g), and hexane (15g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using a 5A filter paper to collect the precipitate, followed by washing with hexane (15g) containing BHT (3.0mg), suction filtration was performed using a 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p8), (p8)AL-E(FG- ₂200ME)). The yield was 84 mg. The molecular weights are shown in table 1. The aldehyde purity is 92%, (¹H-NMR)。

¹H-NMR(CDCl₃):1.32ppm(m,2H,CHO-CH₂-CH₂-NH-COO-CH₂-CH₂- ₂CH-CH₂-CH₂-CO-NH-)，1.57ppm(m,2H,CHO-CH₂-CH₂-NH-COO-CH₂-CH₂-CH₂- ₂CH-CH₂-CO-NH-)，1.76ppm(m,4H,-CO-NH-CH₂- ₂CH-CH₂-O-(CH₂-CH₂-O)n-CH₃)，1.82ppm(m,1H)，2.10ppm(m,2H,CHO-CH₂-CH₂-NH-COO-CH₂- ₂CH-CH₂-CH₂-CH₂-CO-NH-)，2.16ppm(m,1H)，2.71ppm(m,2H,CHO-C ₂H-CH₂-NH-COO-CH₂-CH₂-CH₂-CH₂-CH₂-CO-NH-)，3.02ppm(m,1H)，3.26ppm(m,8H)，3.38ppm(s,6H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n- ₃CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃)，4.01ppm(m,4H)，4.16ppm(m,1H)，4.49ppm(m,2H,-CO-NH-CH-CH₂-C₆H₅) 5.59ppm (Wide, 1H), 6.36ppm (Wide, 1H), 6.93ppm (Wide, 2H), 7.08ppm (Wide, 1H), 7.26ppm (m,10H, -CO-NH-CH-CH)₂- _{6 5}CH) 7.80ppm (Width, 1H)) 8.37ppm (width, 1H), 9.79ppm (s,1H,CHO-CH₂-CH₂-NH-COO-)

[ example 4]

Compound (p9) (p) _{2 2}NHO-E(FG-200ME)) Synthesis of (2)

[ solution 36]

The compound (p5) (300mg, 7.5X 10) obtained in example 3-1 was added^-6mol) was dissolved in toluene (2.4g) at 30 ℃ with heating and azeotropically dehydrated under reduced pressure. Then, the concentrate was dissolved in chloroform (2.4g), and N-hydroxyphthalimide (7.3mg, 4.5X 10) was added^-5mol, Wako pure chemical industries, Ltd.), triphenylphosphine (35mg, 1.4X 10^{- 4}mol, manufactured by Kanto chemical Co., Ltd.) and diisopropyl azodicarboxylate (22. mu.L, 1.1X 10)^-4mol, manufactured by ACROS ORGANICS corporation), and reacted at room temperature under a nitrogen atmosphere for 4 hours. After completion of the reaction, methanol (9.1. mu.L) was added to the reaction solution, stirred at 25 ℃ for 30 minutes, and concentrated at 40 ℃. The concentrate was diluted and azeotroped in toluene (3.0g), then the concentrate was dissolved in toluene (1.5g), and ethylenediamine monohydrate (24. mu.L, 3.0X 10)^-4mol, manufactured by Kanto chemical Co., Ltd.), and reacted at room temperature under a nitrogen atmosphere for 1 hour. After completion of the reaction, the reaction mixture was diluted with toluene (50g), and hexane (25g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (20g), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p9) ((p 9)) _{2 2}NHO-E(FG-200ME)). The yield was 156 mg. The molecular weights are shown in table 1. HPLC: the purity of the amine oxide is 91%.

¹H-NMR(CDCl₃):1.32ppm(m,2H,H₂N-O-CH₂-CH₂- ₂CH-CH₂-CH₂-CO-NH-)，1.56ppm(m,4H,H₂N-O-CH₂- ₂CH-CH₂- ₂CH-CH₂-CO-NH-)，1.76ppm(m,4H,-CO-NH-CH₂- ₂CH-CH₂-O-(CH₂-CH₂-O)n-CH₃)，1.85ppm(m,1H)，2.10ppm(m,2H,H₂N-O-CH₂-CH₂-CH₂-CH₂- ₂CH-CO-NH-)，2.17ppm(m,1H)，3.01ppm(m,1H)，3.24ppm(m,8H)，3.38ppm(s,6H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n- ₃CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃)，4.03ppm(m,2H)，4.17ppm(m,1H)，4.49ppm(m,2H,-CO-NH-CH-CH₂-C₆H₅) 5.37ppm (Width, 2H), 6.40ppm (Width, 1H), 6.95ppm (Width, 2H), 7.12ppm (Width, 1H), 7.26ppm (m,10H, -CO-NH-CH-CH)₂- _{6 5}CH) 7.74ppm (Wide, 1H), 8.31ppm (Wide, 1H)

[ example 5]

Compound (p13) (p) _{2 2 2}NH-E{E(FG-100ME)}) Synthesis of (2)

[ solution 37]

[ example 5-1]

Compound (p10) (p)ME-100GF-Fmoc) Synthesis of (2)

[ solution 38]

L-phenylalanyl-glycine (Fmoc-Phe-Gly) (533mg, 1.2X 10) having its N-terminal protected by Fmoc group was used in the same manner as in example 1-1^-3mol, manufactured by Dubian chemical Co., Ltd.) and methoxy PEG having propylamino group at the terminal (9.9g, 1.0X 10^-3mol, number average molecular weight of 9,896, "SUNBRIGHT MEPA-10T" manufactured by Nichii oil Co., Ltd.)To obtain the above compound (p10) ((p 10))ME-100GF-Fmoc). The yield was 9.2 g.

¹H-NMR(d₆-DMSO):1.62ppm(m,2H,-CO-NH-CH₂- ₂CH-CH₂-O-(CH₂-CH₂-O)n-CH₃)，2.80ppm(m,1H,-NH-CO-CH- ₂CH-C₆H₅)，3.04ppm(m,1H,-NH-CO-CH- ₂CH-C₆H₅)，3.10ppm(m,2H,-CO-NH- ₂CH-CH₂-CH₂-O-(CH₂-CH₂-O)n-CH₃)，3.24ppm(s,3H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n- ₃CH) 3.48ppm (m, about 900H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃)，4.20ppm(m,4H)，7.33ppm(m,9H)，7.66ppm(m,4H,Ar)，7.88ppm(d,2H,Ar)，8.27ppm(t,1H)

[ examples 5-2]

Compound (p11) (p) ₂ME-100GF-NH) Synthesis of (2)

[ solution 39]

The compound (p10) (9.2g, 4.6X 10) obtained in example 5-1 was used in the same manner as in example 1-2^-4mol) to obtain the above compound (p11), (p11) ₂ME-100GF-NH). The yield was 8.7 g.

¹H-NMR(d₆-DMSO):1.62ppm(m,2H,-CO-NH-CH₂- ₂CH-CH₂-O-(CH₂-CH₂-O)n-CH₃) 1.64ppm (width, 1H), 2.59ppm (dd,1H, -NH-CO-CH- ₂CH-C₆H₅)，2.98ppm(dd,1H,-NH-CO-CH- ₂CH-C₆H₅)，3.10ppm(q,2H,-CO-NH- ₂CH-CH₂-CH₂-O-(CH₂-CH₂-O)n-CH₃)，3.24ppm(s,3H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n- ₃CH) 3.48ppm (m, about 900H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃)，7.24ppm(m,6H,-NH-CO-CH-CH₂- _{6 5}CH-NH-), 7.73ppm (t,1H), 8.12ppm (Wide, 1H)

[ examples 5 to 3]

Compound (p12) (p) _{2 2}NH-E(FG-100ME)) Synthesis of (2)

[ solution 40]

L-glutamic acid (Fmoc-Glu-OH) (135mg, 3.7X 10) having its N-terminus protected by Fmoc group was used in the same manner as in example 1-3^-4mol, manufactured by Biffman chemical Co., Ltd.) and the compound (p11) (8.5g, 8.5X 10) obtained in example 5-2^-4mol) as a starting material, successively subjected to a reaction and deprotection to obtain the above-mentioned compound (p12) ((p 12) ₂NH- ₂E(FG-100ME)). The yield was 6.6 g. HPLC: the amine purity was 95%.

¹H-NMR(d₆-DMSO):1.54ppm(m,2H,-NH-CO-CH(NH₂)- ₂CH-CH₂-)，1.62ppm(m,4H,-CO-NH-CH₂- ₂CH-CH₂-)，1.97ppm(m,2H,-NH-CO-CH(NH₂)-CH₂- ₂CH-)，2.74ppm(dd,1H,-CO-NH-CH- ₂CH-C₆H₅)，2.81ppm(dd,1H,-CO-NH-CH- ₂CH-C₆H₅)，3.11ppm(m,11H)，3.24ppm(s,6H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n- ₃CH) 3.64ppm (m, about 1,800H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃)，4.49ppm(m,1H,-CO-NH-CH-CH₂-C₆H₅)，4.57ppm(m,1H,-CO-NH-CH-CH₂-C₆H₅)，7.25ppm(m,10H,-CO-NH-CH-CH₂- _{6 5}CH)，7.74ppm(m,2H)，8.44ppm(m,2H)，8.61ppm(m,2H)

[ examples 5 to 4]

Compound (p13) (p) _{2 2 2}NH-E{E(FG-100ME)}) Synthesis of (2)

[ solution 41]

L-glutamic acid (Fmoc-Glu-OH) (15.2mg, 4.1X 10) having its N-terminus protected by Fmoc group was used in the same manner as in example 1-3^-5mol, manufactured by Biffman chemical Co., Ltd.) and the compound (p12) (2.0g, 1.0X 10) obtained in example 5-3^-4mol) as a starting material, successively subjected to a reaction and deprotection to obtain the above-mentioned compound (p13) ((p 13) ₂NH- _{2 2}E{E(FG-100ME)}). The yield was 1.2 g. The molecular weights are shown in table 1. HPLC: the amine purity was 94%.

¹H-NMR(d₆-DMSO):1.62ppm(m,14H)，2.00ppm(m,6H,-NH-CO-CH(NH₂)-CH₂- ₂CH-)，2.78ppm(m,4H)，3.11ppm(m,14H)，3.24ppm(s,16H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n- ₃CH) 3.64ppm (m, about 3,600H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃)，4.19ppm(m,2H)，4.51ppm(m,4H)，7.25ppm(m,20H,-CO-NH-CH-CH₂- _{6 5}CH)，7.71ppm(m,4H)，7.89ppm(m,1H)，8.45ppm(m,9H)

[ example 6]

Compound (p16) (p) _{2 2}NH-E(GFLG-200ME)) Synthesis of (2)

[ solution 42]

[ example 6-1]

Compound (p14) (p)ME-200GLFG-Fmoc) Synthesis of (2)

[ solution 43]

Using L-glycyl-phenylalanyl-leucyl-glycine (Fmoc-Gly-Phe-Leu-Gly) (66mg, 1.1X 10) having its N-terminal protected by Fmoc group, the preparation was carried out in the same manner as in example 1-1^-4mol, manufactured by Dubian chemical industries Co., Ltd.) and methoxy PEG having propylamino group at the terminal (1.5g, 7.1X 10)^-5mol, number average molecular weight 21,120, "sunberg MEPA-20T" manufactured by japan oil co., ltd., as a raw material, to obtain the above compound (p14) ((mME-200GLFG- Fmoc). The yield was 1.2 g.

¹H-NMR(CDCl₃):0.89ppm(d,3H,-NH-CO-CH-CH₂-CH( ₃CH)₂)、0.91ppm(d,3H,-NH-CO-CH-CH₂-CH( ₃CH)₂)，1.53ppm(m,2H,-NH-CO-CH- ₂CH-CH(CH₃)₂)，1,70ppm(m,1H,-NH-CO-CH-CH₂-CH(CH₃)₂)，1.80ppm(m,2H,-CO-NH-CH₂- ₂CH-CH₂-O-(CH₂-CH₂-O)n-CH₃)，3.10ppm(dd,1H,-NH-CO-CH- ₂CH-C₆H₅)，3.18ppm(dd,1H,-NH-CO-CH- ₂CH-C₆H₅) 3.33ppm (m,7H), 3.74ppm (m, about 1,900H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃) 4.31ppm (width, 1H), 4.55ppm (t,1H, -NH-CO-CH-CH₂-C₆H₅) 6.91ppm (Wide, 1H), 7.00ppm (Wide, 1H), 7.28ppm (m,5H, -NH-CO-CH-CH)₂- _{6 5}CH) 7.33ppm (t,2H, Ar), 7.41ppm (m,3H, Ar), 7.73ppm (m,3H, Ar), 7.89ppm (d,2H, Ar), 7.98ppm (Width, 1H)

[ example 6-2]

Compound (p15) (p) ₂ME-200GLFG-NH) Synthesis of (2)

[ solution 44]

The compound (p14) (1.2g, 5.7X 10) obtained in example 6-1 was used in the same manner as in example 1-2^-5mol) to obtain the above compound (p15), (p15) ₂ME-200GLFG-NH). The yield was 1.0 g.

¹H-NMR(CDCl₃):0.89ppm(d,3H,-NH-CO-CH-CH₂-CH( ₃CH)₂)、0.91ppm(d,3H,-NH-CO-CH-CH₂-CH( ₃CH)₂)，1.53ppm(m,2H,-NH-CO-CH- ₂CH-CH(CH₃)₂)，1,70ppm(m,1H,-NH-CO-CH-CH₂-CH(CH₃)₂)，1.80ppm(m,2H,-CO-NH-CH₂- ₂CH-CH₂-O-(CH₂-CH₂-O)n-CH₃)，3.10ppm(dd,1H,-NH-CO-CH- ₂CH-C₆H₅)，3.18ppm(dd,1H,-NH-CO-CH- ₂CH-C₆H₅) 3.33ppm (m,7H), 3.74ppm (m, about 1,900H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃) 4.31ppm (width, 1H), 4.55ppm (t,1H, -NH-CO-CH-CH₂-C₆H₅) 6.91ppm (Wide, 1H), 7.00ppm (Wide, 1H), 7.28ppm (m,5H, -NH-CO-CH-CH)₂- _{6 5}CH) 7.98ppm (Wide, 1H)

[ examples 6 to 3]

Compound (p16) (p) _{2 2}NH-E(GFLG-200ME)) Synthesis of (2)

[ solution 45]

In accordance with and implementExample 1-3 same preparation method, L-glutamic acid (Fmoc-Glu-OH) having N-terminus protected by Fmoc group (8.3mg, 2.3X 10^-5mol, manufactured by Biffman chemical Co., Ltd.) and the compound (p15) (1.0g, 4.8X 10) obtained in example 6-2^-5mol) as a starting material, successively subjected to a reaction and deprotection to obtain the above-mentioned compound (p16) ((p 16) ₂NH-E ₂(GFLG-200ME)). The yield was 0.5 g. The molecular weights are shown in table 1. HPLC: the amine purity was 90%.

¹H-NMR(CDCl₃):0.89ppm(d,6H,-NH-CO-CH-CH₂-CH( ₃CH) ₂)、0.91ppm(d,6H,-NH-CO-CH-CH₂-CH( ₃CH)₂)，1.53ppm(m,4H,-NH-CO-CH-CH ₂ -CH(CH₃)₂)，1,70ppm(m,2H,-NH-CO-CH-CH₂-CH( ₃CH)₂)，1.77ppm(m,4H,-CO-NH-CH₂- ₂CH-CH₂-O-(CH₂-CH₂-O)n-CH₃)，1.85ppm(m,1H)，3.01ppm(m,1H)，3.24ppm(m,8H)，3.38ppm(s,6H,-CO-NH-CH₂-CH₂-CH₂-O-(CH₂-CH₂-O)n- ₃CH) 3.64ppm (m, about 3,800H, -CO-NH-CH)₂-CH₂-CH₂-O-( _{2 2}CH-CH-O)n-CH₃)，4.03ppm(m,4H)，4.14ppm(m,1H)，4.48ppm(m,2H,-CO-NH-CH-CH₂-C₆H₅) 6.95ppm (Wide, 1H), 7.00ppm (Wide, 1H), 7.26ppm (m,10H, -CO-NH-CH-CH)₂- _{6 5}CH) 7.66ppm (Wide, 2H), 8.29ppm (Wide, 2H)

Comparative example 1

Compound (p18) (p) ₂LY-400NH) Synthesis of (2)

[ solution 46]

Comparative examples 1 to 1

Compound (p17) (p)LY-400BO) Synthesis of (2)

[ solution 47]

2-branched polyethylene glycol activated ester of lysine skeleton (3.0g, 7.5X 10) used in polyethylene glycol modifier on the market^-5mol, "SUNBRIGHT LY-400 NS" manufactured by Nichikoku K.K., having a number average molecular weight of 39,700 was dissolved in toluene (15g) at 40 ℃ under heating, and N- (tert-butoxycarbonyl) -1, 2-diaminoethane (48. mu.L, 3.0X 10)^-4mol, manufactured by Tokyo chemical industry Co., Ltd.) was reacted at 40 ℃ for 1 hour under a nitrogen atmosphere. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (12g), and hexane (14g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. After the precipitate was recovered by suction filtration using a 5A filter paper, it was dissolved in ethyl acetate (27g) again, and hexane (14g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using 5A filter paper to collect the precipitate, followed by washing with hexane (30g), suction filtration was performed using 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p17) (b)LY-400BO). The yield was 2.7 g.

¹H-NMR(CDCl₃):1.37ppm(m,2H)，1.43ppm(s,9H,-CH₂-NH-CO₂-C-( ₃CH)₃)，1.51ppm(m,2H)，3.15ppm(m,2H)，3.38ppm(s,6H,-O-(CH₂-CH₂-O)n- ₃CH) 3.65ppm (m, about 3,650H, -O- (R-) - (R)) _{2 2}CH-CH-O)n-CH₃)，4.21ppm(m,4H)

Comparative examples 1 and 2

Compound (p18) (p) ₂LY-400NH) Synthesis of (2)

[ solution 48]

The compound (p17) (1.0g, 2.5X 10) obtained in comparative example 1-1^-6mol) was dissolved in ion-exchanged water (4.0g), and methanesulfonic acid (57. mu.L, 8.8) was added×10^-4mol, manufactured by Kanto chemical Co., Ltd.), at 40 ℃ under a nitrogen atmosphere for 6 hours. After the reaction, the reaction mixture was diluted with ion-exchanged water (6.0g), a 1N aqueous solution (0.9g) of sodium hydroxide was added to adjust the pH to 12, and then sodium chloride (2.5g) was added to dissolve the mixture. Chloroform (10g) containing BHT (1.0mg) was added to the obtained solution, and the mixture was stirred at room temperature for 20 minutes to extract the product into an organic layer. The organic layer and the aqueous layer were separated, the organic layer was recovered, and then concentrated at 40 ℃ to obtain a concentrate, which was diluted with toluene (30g), and hexane (15g) was added thereto and stirred at room temperature for 15 minutes to precipitate a product. Suction filtration was performed using a 5A filter paper to collect the precipitate, followed by washing with hexane (15g) containing BHT (3.0mg), suction filtration was performed using a 5A filter paper, and vacuum drying was performed to obtain the above-mentioned compound (p18), (p18) ₂LY-400NH). The yield was 0.7 g. The molecular weights are shown in table 1. HPLC: the amine purity was 97%.

¹H-NMR(CDCl₃):1.37ppm(m,2H)，1.51ppm(m,2H)，3.15ppm(m,2H)，3.38ppm(s,6H,-O-(CH₂-CH₂-O)n- ₃CH) 3.65ppm (m, about 3,650H, -O- (R-) - (R)) _{2 2}CH-CH-O)n-CH₃)，4.21ppm(m,4H)

[ Table 1]

	Sample name	Molecular weight (Mn)
			Example 1	Compound (p3)	42，417
Example 2	Compound (p4)	42，534
			Example 3	Compound (p8)	42，334
Example 4	Compound (p9)	42，190
			Example 5	Compound (p13)	38，234
Example 6	Compound (p16)	42，398
			Comparative example 1	Compound (p18)	39，654

[ example 7]

Stability test in serum

To a 1.5mL microcentrifuge tube, 1mL of mouse or human serum was added, and each polyethylene glycol derivative was added to a concentration of 5.0 mg/mL. After incubation at 37 ℃ for 96 hours, 200. mu.L of the sample was taken, acetonitrile was added thereto, and the mixture was stirred for 1 minute with a vortex mixer (vortex) to precipitate proteins in serum, which was centrifuged to collect the supernatant. Next, hexane was added to the recovered solution to remove hydrophobic substances such as fatty acids, and the mixture was stirred with a vortex mixer for 1 minute, centrifuged, and the lower layer was recovered. The solution was concentrated under vacuum, and the polyethylene glycol derivative was recovered from the serum. Then, GPC analysis was performed to calculate the decomposition rate of the decomposable polyethylene glycol derivative.

The decomposition rate was calculated as follows:

decomposition rate ═ peak area of 40kDa before assay% — peak area of 40kDa after assay%/(% peak area of 40kDa before assay) × 100 ×

The results are shown in table 2 below.

[ Table 2]

According to Table 2, no decomposition was observed in the serum of the compounds (p3), (p13) and (p16) which are decomposable polyethylene glycol derivatives, as in the case of the compound (p18) which is a non-decomposable polyethylene glycol derivative and methoxy PEG amine 40 kDa. That is, the degradable polyethylene glycol derivative is stable in blood.

[ example 8]

Disintegration test Using cells

Using 10mL of the culture medium RPMI-1640 (10% FBS Pn/St), 10X 10 was inoculated into a 100mm dish⁶The cells were cultured at 37 ℃ for 24 hours in RAW264.7, then replaced with a medium containing various polyethylene glycol derivatives dissolved therein to a concentration of 10mg/mL, and cultured at 37 ℃ for 96 hours. After the culture, the cells were dissolved in a 1% SDS solution, diluted with Phosphate Buffered Saline (PBS), acetonitrile was added thereto, and the mixture was stirred with a vortex mixer for 1 minute to precipitate the protein in the cell lysate, and after centrifugation, the supernatant was collected. Next, hexane was added to the recovered solution to remove hydrophobic substances such as fatty acids, and the mixture was stirred with a vortex mixer for 1 minute, centrifuged, and the lower layer was recovered. The solution was concentrated under vacuum, and the polyethylene glycol derivative was recovered from the cells.

In addition, in order to confirm the decomposition in the culture medium for cell culture, only in the dissolved various polyethylene glycol derivatives to the concentration of 10mg/mL culture medium at 37 degrees C were cultured for 96 hours, according to the same operation of polyethylene glycol derivatives recovery.

Then, GPC analysis of each of the collected polyethylene glycol derivatives was performed, and the decomposition rate of the decomposable polyethylene glycol derivative was calculated according to the same calculation formula as in example 7.

The results are shown in table 3 below. Furthermore, GPC spectra of the compounds (p3) and (p13) before and after the cell experiment are shown in fig. 1 and fig. 2, and fig. 3 and fig. 4, respectively.

[ Table 3]

From Table 3, it was confirmed that the compounds (p3) and (p16) which are degradable polyethylene glycol derivatives are efficiently degraded in cells (degradation rate 99%) and degraded from 4 to 2 million in molecular weight. In addition, in the compound (p13), decomposition from 4 to 1 million in molecular weight was observed at a decomposition rate of 99%. Since these degradable polyethylene glycol derivatives do not decompose in the medium used for cell culture, they were confirmed to be specifically degraded in the cells. On the other hand, no intracellular degradation was observed in both the compound (p18) which is a non-degradable polyethylene glycol derivative and methoxy PEG amine 40 kDa.

[ example 9]

PEGylation of salmon calcitonin (sCT)

The amino acid sequence:

CSNLSTCVLG KLSQELHKLQ TYPRTNTGSG TP (Serial number: 1)

Salmon calcitonin (sCT) (0.5mg, 1.5X 10)^-7mol, manufactured by PH Japan K.K.) was dissolved in 100mM sodium acetate buffer solution (pH5.0), and the compound (p8) obtained in example 3 or methoxy PEG aldehyde 40kDa (18mg, 4.5X 10)^-7mol) and 2-methylpyridine borane (2.0X 10) as reducing agent^-6mol), the concentration of sCT was adjusted to 1.0mg/mL, and the reaction was carried out at 4 ℃ for 24 hours. Then, the reaction mixture was dialyzed against 10mM sodium acetate buffer (pH5.0) and purified by ion exchange chromatography using HiTrap SP HP (5mL, manufactured by GE Healthcare Co.) to obtain sCT-E (FG-200ME)₂Or methoxy PEG40kDa-sCAnd T. The molar yields were 36% and 49%, respectively.

RPLC analysis

The device comprises the following steps: "ALLIANCE" manufactured by WATERS Inc "

A detector: UV (280nm)

A chromatographic column: inertsil WP 300C 18(GL Sciences company)

Mobile phase A: 0.05% TFA-H₂O

Mobile phase B: 0.05% TFA-ACN

Gradient: the sequence of B30% (0min), B40% (5min), B50% (15min), B100% (16min) and B100% (20min) was changed

Flow rate: 1.0mL/min

Temperature of the column: 40 deg.C

The purity of the pegylated sCT was calculated under the RPLC analysis conditions described above. The results are shown in FIG. 5.

sCT-E(FG-200ME)₂RPLC purity of (a): 99 percent

RPLC purity of methoxy PEG40 kDa-sCT: 99 percent

MALDI-TOF-MS analysis

The device comprises the following steps: "autoflex 3" manufactured by Bruker corporation "

Sample preparation: 0.5mg/mL, PBS solution

Matrix: saturated solution of alpha-cyano-4-hydroxycinnamic acid (CHCA) (0.01% TFA-H)₂O：ACN＝2：1)

The sample (1. mu.L) and the substrate (19. mu.L) were mixed, and 1. mu.L was spotted on the target plate.

The molecular weights of PEG and PEGylated sCT of the starting material were determined under the conditions of MALDI-TOF-MS analysis described above.

In FIG. 6, the starting compound (p8) and sCT-E (FG-200ME) are shown in combination₂The result of MALDI-TOF-MS (Dimethylacryloylmethane-TOF-MS) of (1).

sCT-E(FG-200ME)₂Molecular weight of (a): 46,405

Molecular weight of compound (p 8): 43,136

In FIG. 7, the results of MALDI-TOF-MS showing methoxy PEG aldehyde 40kDa and methoxy PEG40kDa-sCT as raw materials were combined.

Molecular weight 46,427 of methoxy PEG40kDa-sCT

Methoxy PEG aldehyde 40kDa molecular weight 43,303

From fig. 7, it was confirmed that the molecular weight of the pegylated sCT was increased by almost only a part of the molecular weight of the sCT as compared with the molecular weight of the PEG derivative as the raw material.

SDS-PAGE analysis

The kit comprises: NuPAGE (registered trademark) Bis-Tris Precast Gel (Gel concentration 4-12%)

Dyeing liquid: coomassie brilliant blue solution (CBB solution) or iodine staining solution (BaCl)₂+I₂Solutions)

The PEGylated sCT was evaluated under the conditions recommended for the SDS-PAGE kit. The results are shown in FIG. 8. According to fig. 8, in the pegylated sCT, a band was observed by CBB staining selective for proteins and peptides, and further, a band was also observed by iodine staining for polyethylene glycol. A band was observed at both stains, and it was confirmed that the polyethylene glycol derivative was bonded to sCT.

[ example 10]

PEGylation of human growth hormone (hGH)

The amino acid sequence:

MFPTIPLSRL FDNAMLRAHR LHQLAFDTYQ EFEEAYIPKE QKYSFLQNPQ TSLCFSESIP TPSNREETQQ KSNLELLRIS LLLIQSWLEP VQFLRSVFAN SLVYGASDSN VYDLLKDLEE GIQTLMGRLE DGSPRTGQIF KQTYSKFDTN SHNDDALLKN YGLLYCFRKD MDKVETFLRI VQCRSVEGSC GF (SEQ ID NO: 2) human growth hormone (hGH) (0.4mg, 1.8X 10^-8mol, manufactured by Shonandoah Biotechnology Co., Ltd.) was dissolved in 100mM sodium acetate buffer solution (pH5.5), and the compound obtained in example 3 (p8) or methoxy PEG aldehyde 40kDa (3.6mg, 9.0X 10)^-8mol) and sodium cyanoborohydride (9.0X 10) as reducing agent^-7mol), the hGH concentration was adjusted to 1.0mg/mL, and the reaction was allowed to proceed at 25 ℃ for 24 hours. Then, the reaction solution was dialyzed against 10mM sodium acetate buffer solution (pH4.7) and purified by ion exchange chromatography using HiTrap SP HP (5mL, manufactured by GE Healthcare Co.) to obtain hGH-E (FG-200ME)₂Or methoxy PEG40 kDa-hGH. The molar yields were 28% and 32%, respectively.

RPLC analysis

The device comprises the following steps: "ALLIANCE" manufactured by WATERS Inc "

A detector: UV (280nm)

A chromatographic column: inertsil WP 300C 18(GL Sciences company)

Mobile phase A: 0.1% TFA-H₂O

Mobile phase B: 0.1% TFA-ACN

Gradient: the procedure was changed in the order of B40% (0min), B80% (25min), B90% (27min) and B40% (27.1min)

Flow rate: 1.0mL/min

Temperature of the column: 25 deg.C

The purity of the PEGylated hGH was calculated under the RPLC analysis conditions described above. The results are shown in FIG. 9.

hGH-E(FG-200ME)₂RPLC purity of (a): 90 percent of

RPLC purity of methoxy PEG40 kDa-hGH: 97 percent

MALDI-TOF-MS analysis

The device comprises the following steps: "autoflex 3" manufactured by Bruker corporation "

Sample preparation: 0.5mg/mL, PBS solution

Matrix: cinnamic acid (SA) saturated solution (0.01% TFA-H)₂O：ACN＝2：1)

The sample (1. mu.L) and the substrate (19. mu.L) were mixed, and 1. mu.L was spotted on the target plate

The molecular weight of the PEGylated hGH was determined under the MALDI-TOF-MS analysis conditions described above. The results are shown in fig. 10 and 11.

hGH-E(FG-200ME)₂Molecular weight of (a): 65,584

Molecular weight of methoxy PEG40 kDa-hGH: 65,263

From fig. 10 and fig. 11, it was confirmed that the molecular weight of the pegylated hGH was increased substantially only partially compared to the molecular weight of the PEG derivative of the starting material (refer to the lower panels of fig. 6 and fig. 7).

SDS-PAGE analysis

The kit comprises: NuPAGE (registered trademark) Bis-Tris Precast Gel (Gel concentration 4-12%)

Dyeing liquid: coomassie brilliant blue solution (CBB solution) or iodine staining solution (BaCl)₂+I₂Solutions)

Evaluation of PEGylated hGH was performed according to the conditions recommended for the SDS-PAGE kit described above. The results are shown in FIG. 12. According to FIG. 12, in PEGylated hGH, a band was observed under CBB staining for selective staining of proteins and peptides, and further, in iodine staining for staining of polyethylene glycol, a band was also observed. In addition, a band was observed at both stains, and it was confirmed that the polyethylene glycol derivative was bonded to hGH.

[ example 11]

PEGylation of Granulocyte Colony Stimulating Factor (GCSF)

The amino acid sequence:

TPLGPASSLP QSFLLKCLEQ VRKIQGDGAA LQEKLCATYK LCHPEELVLL GHSLGIPWAP LSSCPSQALQ LAGCLSQLHS GLFLYQGLLQ ALEGISPELG PTLDTLQLDV ADFATTIWQQ MEELGMAPAL QPTQGAMPAF ASAFQRRAGG VLVASHLQSF LEVSYRVLRH LAQP Granulocyte Colony Stimulating Factor (GCSF) (0.1mg, 5.3X 10) (SEQ ID NO: 3)^-9mol, manufactured by PeproTech Co., Ltd.) was dissolved in 10mM sodium acetate buffer solution (pH4.6, containing 5% sorbitol), and the compound obtained in example 3 (p8) and sodium cyanoborohydride (5.3X 10) as a reducing agent were added^-7mol), the GCSF concentration was adjusted to 2.0mg/mL, and the reaction was carried out at 4 ℃ for 24 hours. Then, the reaction mixture was diluted with 10mM sodium acetate buffer (pH4.6) and purified by ion exchange chromatography using HiTrap SP HP (5mL, manufactured by GE Healthcare Co.) to obtain GCSF-E (FG-200ME)₂. The molar yield was 41%.

RPLC analysis

The device comprises the following steps: "ALLIANCE" manufactured by WATERS Inc "

A detector: UV (280nm)

A chromatographic column: inertsil WP 300C 18(GL Sciences company)

Mobile phase A: 0.1% TFA-H₂O

Mobile phase B: 0.1% TFA-ACN

Gradient: the respective contents were changed in the order of B40% (0min), B70% (25min), B90% (27min) and B40% (29min)

Flow rate: 1.0mL/min

Temperature of the column: 40 deg.C

The purity of the pegylated GCSF was calculated under the RPLC analysis conditions described above.

GCSF-E(FG-200ME)₂RPLC purity of (a): 97 percent

MALDI-TOF-MS analysis

The device comprises the following steps: "autoflex 3" manufactured by Bruker corporation "

Sample preparation: 0.5mg/mL, PBS solution

Matrix: cinnamic acid (SA) saturated solution (0.01% TFA-H)₂O：ACN＝2：1)

The sample (1. mu.L) and the substrate (19. mu.L) were mixed, and 1. mu.L was spotted on the target plate

The molecular weight of the PEGylated GCSF was determined under the MALDI-TOF-MS analysis conditions described above. GCSF-E (FG-200ME)₂Molecular weight of (a): 62,199

From the above MALDI-TOF-MS analysis results, it was confirmed that the molecular weight of PEGylated GCSF was increased by almost only a part of the molecular weight of GCSF as compared with the molecular weight of the PEG derivative of the raw material.

SDS-PAGE analysis

The kit comprises: NuPAGE (registered trademark) Bis-Tris Precast Gel (Gel concentration 4-12%)

Dyeing liquid: coomassie brilliant blue solution (CBB solution) or iodine staining solution (BaCl)₂+I₂Solutions)

Evaluation of the PEGylated GCSF was performed according to the conditions recommended for the SDS-PAGE kit. From the results of the SDS-PAGE analysis described above, a band was observed in the PEGylated GCSF by CBB staining which selectively stains proteins and peptides, and a band was also observed in the PEG-stained iodine staining. In addition, a band was observed at both stains, confirming that the polyethylene glycol derivative was bonded to GCSF.

[ example 12]

Evaluation of physiological Activity of PEGylated Salmon calcitonin (sCT)

sCT-E (FG-200ME) as an sCT to which a degradable polyethylene glycol derivative having a molecular weight of 4 ten thousand was bonded, obtained in example 9₂And 4 groups of methoxy PEG40kDa-sCT bonded with non-degradable methoxy PEG40kDa, unmodified sCT and PBS, and their physiological activities were evaluated by animal experiments. The mouse breed was Balb/c (8 weeks old, male), PEGylated sCT solution and unmodifiedThe sCT solution was prepared in PBS such that the concentration of sCT became 8.0. mu.g/mL, and the dose of sCT became 40. mu.g/kg. Plasma was collected from the blood collected from the mice at 1, 6, and 24 hours, and the calcium concentration was measured using calcium E-Test Wako (manufactured by Fuji film and Wako pure chemical industries, Ltd.). The results are shown in FIG. 16.

According to fig. 16, all scts significantly reduced calcium concentration compared to the PBS cohort. An increase in calcium concentration was observed in the unmodified sCT 6 hours after administration, but the sCT-E (FG-200ME)₂And methoxy PEG40kDa-sCT continued to maintain lower calcium concentrations. It was confirmed that the half-life of sCT in blood was increased by pegylation, and that the physiological activity was maintained without being affected.

[ example 13]

Vacuole formation evaluation test by animal experiment

Compound (p3) NH which is a decomposable polyethylene glycol derivative having an amino group at the end and a molecular weight of 4 ten thousand₂-E(FG-200ME)₂And a non-decomposable methoxy PEG amine of 40kDa, and subjected to blank cannon formation evaluation by an animal experiment. The mouse breed is Balb/c (8 weeks old, male), polyethylene glycol solution using physiological saline to prepare polyethylene glycol derivatives to a concentration of 100mg/mL, via the tail vein of the mouse 20 u L. Continuous administration was continued 3 times a week for 4 weeks, and after completion of administration, mice were fixed by perfusion with 4% paraformaldehyde aqueous solution to prepare paraffin sections. Vacuole formation in brain choroid plexus epithelial cells was assessed by HE staining and immunostaining with anti-PEG antibodies. The immunostaining was carried out using an immunostaining Kit (Bond reference Polymer Detection Kit, manufactured by Leica) and an anti-PEG antibody (B-47 antibody, manufactured by Abcam). The images of the choroid plexus brain sections subjected to immunostaining with anti-PEG antibody are shown in FIG. 13 (methoxy PEG amine 40kDa) and FIG. 14 (NH)₂-E(FG-200ME)₂) In (1).

As a result, NH as a decomposable polyethylene glycol₂-E(FG-200ME)₂Vacuole formation was significantly inhibited compared to methoxy PEG amine 40 kDa.

The amount of polyethylene glycol administered in this example was finally estimated to be an optimized amount for evaluating vacuolation, and was very large compared with the amount of polyethylene glycol generally administered in this technical field.

[ example 14]

Evaluation test of accumulation Property of polyethylene glycol Using animal experiment

Compound (p3) NH Using a decomposable polyethylene glycol derivative having an amino group at the end and having a molecular weight of 4 million₂-E(FG-200ME)₂And non-degradable methoxy PEG amine 20kDa, methoxy PEG amine 40kDa and PBS as control, and the accumulation of polyethylene glycol was evaluated by animal experiments. The mouse breed is Balb/c (8 weeks old, male), polyethylene glycol solution using physiological saline to prepare polyethylene glycol derivatives to a concentration of 62.5mg/mL, via mouse tail vein administration of 100 u L. Continuous administration was continued 3 times a week for 4 weeks, and after completion of administration, mice were fixed by perfusion with 4% paraformaldehyde aqueous solution to prepare paraffin sections. Immunostaining with anti-PEG antibody was performed to evaluate the accumulation in the brain choroid plexus epithelial cells. The image of each section of the choroid plexus after immunostaining is shown in fig. 15.

According to FIG. 15, the mouse choroid plexus section administered with PBS containing no polyethylene glycol was not stained, whereas the section was confirmed to be extensively stained brown in 40kDa, which is a nondegradable methoxy PEG amine. The stained portion indicates accumulation of PEG. On the other hand, in NH which is a decomposable polyethylene glycol₂-E(FG-200ME)₂The sections of (a) were less stained brown, showing the same accumulation as 20kDa, methoxy PEG amine with a molecular weight of half of that. As a result, the degradable polyethylene glycol significantly inhibited the accumulation of polyethylene glycol in the tissue due to its degradability, compared to the non-degradable methoxy PEG amine 40kDa, which was the same molecular weight.

The amount of polyethylene glycol administered in this example was finally optimized for evaluation of the accumulation property, and was very large compared with the amount of polyethylene glycol generally administered in this technical field.

Industrial applicability

The degradable polyethylene glycol derivative of the present invention is a high molecular weight polyethylene glycol derivative that does not cause cavitation of cells, and can be effectively used for modification of biologically relevant substances, and is stable in blood in vivo and degraded in cells.

The present application is based on Japanese patent application No. 2019-069450 (application date: 2019, 3, 29), the contents of which are incorporated herein in their entirety.

Sequence listing

<110> Nissan oil Co., Ltd (NOF CORPORATION)

National university OF French Dongjing university OF Industrial university (TOKYO INSTITUTE OF TECHNOLOGY)

<120> branched degradable polyethylene glycol conjugate

<130> 093018

<150> JP2019-069450

<151> 2019-03-29

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