Hydrophilic-hydrophobic copolymer loaded with short-chain fatty acid esters

Abstract

The present invention provides derivatives of short-chain fatty acids, which can exert the inherent physiological functions of short-chain fatty acids. The present invention provides block or graft copolymers, which contain: a hydrophobic segment containing repeating units of short-chain fatty acid esters that can be hydrolyzed by esterases in vivo; and a hydrophilic segment containing poly(ethylene glycol) chains. These copolymers are effective for the treatment or management of various diseases or disorders including cancer.

Description

Technical Field

[0001] The present invention relates to a block copolymer or a graft copolymer containing a hydrophobic moiety carrying a short-chain fatty acid ester and a poly(ethylene glycol) chain as a hydrophilic moiety, and its use as a nanomedicine material. Background Art

[0002] It has been reported that short-chain fatty acids including acetic acid, propionic acid, and butyric acid have various physiological functions such as immunosuppressive ability, inhibition of liver fibrosis, obesity inhibitory ability, and anticancer ability depending on their chain lengths (for example, refer to Non-Patent Document 1). Short-chain fatty acids having the above physiological functions are produced from sugars by intestinal flora, but are not necessarily sufficient, and sufficient supply is expected.

[0003] However, short-chain fatty acids are difficult to handle because not only their administration methods are limited due to their solubility or odor, but also they have a fast metabolism due to their low molecular weight, and their physiological functions change due to association. Therefore, they are provided in the form of some supplements, etc., but it can be said without exaggeration that there is no effective administration method or preparation that can exert the physiological functions inherently possessed by short-chain fatty acids.

[0004] Prior Art Documents

[0005] Non-Patent Documents

[0006] Non-Patent Document 1: Takashi Sakata, Hirofumi Ichikawa, Physiological Activity of Short-Chain Fatty Acids, Journal of the Japanese Oil Chemists' Society, Vol. 46, 1205-1212 (1997). Summary of the Invention

[0007] Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a method for effectively administering and delivering a short-chain fatty acid to a living body.

[0009] Means for Solving the Problems

[0010] Heretofore, the present inventors have designed and proposed a polymerized drug system in which a drug to be delivered is carried on a polymer compound having associativity and capable of self-assembling in an aqueous medium to change the delivery characteristics of the drug (for example, WO2009 / 133647, WO2016 / 052463). The present inventors hypothesized that if any polymerized drug system could also be successfully utilized for the above purpose, it would contribute to solving the above problems, and repeated studies were conducted.

[0011] As a result, it was confirmed that nanoparticles or nano-sized polymeric micelles formed by self-assembly through the association in water of block copolymers or graft copolymers containing or supporting a hydrophobic moiety carrying one or more short-chain fatty acid esters and poly(ethylene glycol) chains can cure or correct the drawbacks or defects in the delivery of the above-mentioned short-chain fatty acids.

[0012] Therefore, as the main solutions provided by the present invention, the following solutions can be listed:

[0013] Solution 1: A hydrophilic-hydrophobic copolymer, the hydrophilic-hydrophobic copolymer comprising the following (1) and (2):

[0014] (1) A hydrophobic chain segment derived from the repeating unit represented by Formula I,

[0015]

[0016] In the formula, R is -(C=O)R 1 or a hydrogen atom, and R 1 is an unsubstituted or substituted straight-chain or branched alkyl group having 1 to 7 carbon atoms (when substituted, the substituent is an unsubstituted or substituted phenyl group, and the substituent of the substituted phenyl group is one or more halogens, hydroxyl groups, or methoxy groups). Here, even if a hydrogen atom is present, it is 30% or less, preferably 20% or less, more preferably 10% or less, and most preferably 0% of n, and n is an integer of 5 to 1000, preferably 10 to 1000, more preferably 15 to 1000, and 30 to 1000; and

[0017] (2) A hydrophilic chain segment containing a poly(ethylene glycol) chain, the hydrophilic chain segment being the following (i) or (ii):

[0018] (i) A hydrophilic chain segment represented by Formula IIa,

[0019]

[0020] In the formula, A is an unsubstituted or substituted C1-C 12 alkoxy group, and when substituted, the substituent is a formyl group, an R'R”CH- group, a phenylamino group or a phenethylamino group, a phenyl group, or a methoxyphenyl group. Here, R' and R” are independently C1-C4 alkoxy groups or R' and R” together are -OCH2CH2O-, -O(CH2)3O-, or -O(CH2)4O-, and m is an integer of 2 to 500, preferably 10 to 300, and more preferably 20 to 200; or

[0021] (ii) A hydrophilic chain segment derived from the repeating unit represented by Formula IIb,

[0022]

[0023] In the formula, R a is a hydrogen atom or a carboxyl group,

[0024] When R a is a carboxyl group, X is C(=O)O or C(=O)NH, or when R a is a hydrogen atom, X is O or NH.

[0025] B is A-CH2CH2, and A and m are as defined above respectively.

[0026] y is an integer from 1 to 300, preferably from 2 to 150, and more preferably from 5 to 100.

[0027] The hydrophobic segment in (1) and the hydrophilic segment in (2)(i) exist in block form respectively.

[0028] Each member of each repeating unit containing the hydrophobic segment in (1) and the hydrophilic segment in (2)(ii) exists randomly with each other.

[0029] Scheme 2: The hydrophilic-hydrophobic copolymer of Scheme 1, which forms nanoparticles or nano-sized polymeric micelles through self-assembly by association in water.

[0030] Scheme 3: The hydrophilic-hydrophobic copolymer of Scheme 1 or 2, wherein the copolymer containing the hydrophobic segment from the repeating unit represented by formula (I) in (1) and the hydrophilic segment of formula IIa in (2)(i) is a block copolymer represented by formula BC:

[0031]

[0032] In the formula, A, R, m, and n are as defined above respectively, L1 represents a direct bond or a divalent linking group, Z is a hydrogen atom, SH, SC(=S)-Ph, SC(=S)OCH2CH3, a hydroxyl group, a C1-C6 alkoxy group, or an aryl-C1-C2 alkoxy group.

[0033] The copolymer containing the hydrophobic segment from the repeating unit represented by formula (I) in (1) and the hydrophilic segment of formula IIb in (2)(ii) is a graft copolymer represented by formula GC:

[0034]

[0035] In the formula, R, R a , B, X, m, n, and y are as defined above respectively.

[0036] Scheme 4: Nanoparticles formed from the hydrophilic-hydrophobic copolymer of any one of Schemes 1 to 3 in an aqueous medium.

[0037] Scheme 5: A pharmaceutical preparation, which is formed by including the hydrophilic-hydrophobic copolymer of any one of Schemes 1 to 3 or the nanoparticles of Scheme 4 as an active ingredient.

[0038] Scheme 6: The pharmaceutical preparation of Scheme 5, which is used for the prevention or treatment of cancer, obesity inhibition, ulcerative colitis, prevention or treatment of non-alcoholic fatty liver (or inhibition of liver fibrosis), prevention or treatment of diabetes, enhancement of radiation in radiotherapy, or prevention or treatment of hyperammonemia.

[0039] Scheme 7: The hydrophilic-hydrophobic copolymer of any one of Schemes 1 to 3, which is used for the prevention or treatment of cancer, obesity inhibition, ulcerative colitis, prevention or treatment of non-alcoholic fatty liver, prevention or treatment of diabetes, enhancement of radiation in radiotherapy, or prevention or treatment of hyperammonemia.

[0040] Scheme 8: The nanoparticles of Scheme 4, which are used for the prevention or treatment of cancer, obesity inhibition, ulcerative colitis, prevention or treatment of non-alcoholic fatty liver (or inhibition of liver fibrosis), prevention or treatment of diabetes, enhancement of radiation in radiotherapy, or prevention or treatment of hyperammonemia.

[0041] Scheme 9: A method of administering the hydrophilic-hydrophobic copolymer of any one of Schemes 1 to 3 to a patient in need thereof for the prevention or treatment of cancer, obesity inhibition, ulcerative colitis, prevention or treatment of non-alcoholic fatty liver (or inhibition of liver fibrosis), prevention or treatment of diabetes, enhancement of radiation in radiotherapy, or prevention or treatment of hyperammonemia.

[0042] Scheme 10: A method of administering any hydrophilic-hydrophobic copolymer of Scheme 4 to a patient in need thereof for the prevention or treatment of cancer, obesity inhibition, ulcerative colitis, prevention or treatment of non-alcoholic fatty liver, prevention or treatment of diabetes (or inhibition of liver fibrosis), enhancement of radiation in radiotherapy, inhibition of liver fibrosis, or prevention or treatment of hyperammonemia.

[0043] Advantages of the Invention

[0044] When the hydrophilic-hydrophobic copolymer or its nanoparticles or polymeric micelles of the present invention are administered to mammals including humans, they are delivered to the local part of the organism, and the short-chain fatty acid ester bond in its local hydrophobic segment undergoes enzymatic hydrolysis to slowly release the corresponding short-chain fatty acid, which can eliminate or alleviate the problems associated with the administration of short-chain fatty acids themselves. Therefore, it is possible to provide: a copolymer that can effectively exert various physiological functions inherently possessed by short-chain fatty acids locally or systemically in the organism of mammals, as well as its nanoparticles and pharmaceutical preparations. Brief Description of the Drawings

[0045] Figure 1 The 1H NMR spectrum of N684 obtained in Production Example 3 1 .

[0046] Figure 2 The 1H NMR spectrum of N731 obtained in Production Example 4 1 .

[0047] Figure 3 The 1H NMR spectrum of N721 obtained in Production Example 5 1 .

[0048] Figure 4 The 1H NMR spectrum of N741 obtained in Production Example 6 1 .

[0049] Figure 5 The dynamic light scattering spectra of N684, N731, and N721 obtained in Production Example 7

[0050] Figure 6 The dynamic light scattering spectrum of N741 obtained in Production Example 8

[0051] Figure 7 Illustration of the cytotoxicity of the vinyl ester nanoparticles (N684, N731, N721) in Test Example 1

[0052] Figure 8 Illustration of the cytotoxicity of the vinyl ester nanoparticles (N741) in Test Example 2

[0053] Figure 9 Illustration of the change in body weight of the mice in each test group in Test Example 3. The data corresponds to the data of the samples diluted 5-fold, 10-fold, 20-fold, and 40-fold from the left

[0054] Figure 10 Illustration of the results of confirmation of the number of lung cancer metastases (macroscopic observation) in Test Example 3. The data corresponds to the data of the samples of water (control), 5-fold dilution, 10-fold dilution, 20-fold dilution, 40-fold dilution, 80-fold dilution, and 160-fold dilution

[0055] Figure 11 Illustration of the results of measuring the number of micrometastases that could not be macroscopically observed in the confirmation of the number of lung cancer metastases in Test Example 3

[0056] Figure 12 Alternative photograph (left) of the H&E staining of the lung tissue map in Test Example 3 and illustration (right) of the number of metastatic cancers obtained from the figure

[0057] Figure 13 ​​​​​​​​​​​​​Alternative photograph of the H&E staining of the lung tissue diagram in Test Example 3 (left) and diagram of the area of metastatic cancer obtained from the figure (right).

[0058] Figure 14 Diagram of the ALT, AST, LDH, and ALB levels in the blood in Test Example 3.

[0059] Figure 15 Alternative photograph of the H&E staining of the duodenum in Test Example 3 (left) and diagram of the villus length in each test group (right).

[0060] Figure 16 Alternative photograph of the H&E staining of the jejunum in Test Example 3 (left) and diagram of the villus length in each test group (right).

[0061] Figure 17 Alternative photograph of the H&E staining of the ileum in Test Example 3 (left) and diagram of the villus length in each test group (right).

[0062] Figure 18 Alternative photograph of the H&E staining of the large intestine tissue in Test Example 3 (left) and diagram of the villus length in each test group (right).

[0063] Figure 19 Diagram of the body weight change in Test Example 4.

[0064] Figure 20 Diagram of the disease activity index (DAI) in Test Example 5.

[0065] Figure 21 Diagram of the white blood cell count of each experimental animal in each treatment group (including control) in Test Example 5.

[0066] Figure 22 Diagram of the liver and spleen weights of each experimental animal in each treatment group (including control) in Test Example 6.

[0067] Figure 23 Diagram of the sample consumption during the test and the body weight change of the experimental animals in Test Example 7.

[0068] Figure 24 Diagram of the sample consumption and the body weight change of the experimental animals in Test (1) of Test Example 8.

[0069] Figure 25 Diagram of the results of the glucose tolerance test in Test (2) of Test Example 8.

[0070] Figure 26 Diagram of the organ weights at the end time point of the test in Test (3) of Test Example 8. ​​​​​​​​​​​​​

[0071] Figure 27 Alternative photograph of the figure showing the histological analysis results of the H and E stained intestine in Test (4) of Test Example 8.

[0072] Figure 28 Alternative photograph of the figure showing the histological analysis results of the H and E stained pancreas in Test (5) of Test Example 8.

[0073] Figure 29 Graph showing the effects of radiation exposure on body weight change and volume change in Test (1) of Test Example 9.

[0074] Figure 30 Graph showing the effects of radiation exposure on the volume change of cancer in Test (2) of Test Example 9.

[0075] Figure 31 Graph showing the data of the cancer growth curve in Test (1) of Test Example 10.

[0076] Figure 32 Graph showing the weight of cancer at the end time point of the test in Test (2) of Test Example 10.

[0077] Figure 33 Graph showing the body weight change of the test animals in Test (3) of Test Example 10.

[0078] Figure 34 Alternative photograph of the figure showing the growth inhibitory effect of spheroid (sphere) cancer cells related to the radiation enhancement effect in Test Example 11.

[0079] Figure 35 Graph showing the weights of the liver and spleen in Test (1) of Test Example 12.

[0080] Figure 36 Graph showing the histological analysis results of the HE stained liver in Test (2) of Test Example 12.

[0081] Figure 37 Graph showing the histological analysis results of the MT stained liver in Test (3) of Test Example 12.

[0082] Figure 38 Of N821 obtained in Production Example 9 1 1H NMR spectrum.

[0083] Figure 39 Representation of the size distribution of the nanoparticles (Ph-BNP, N832) obtained in Production Example 10.

[0084] Figure 40 ​​​​​​​​​​​​​​Illustration of the biochemical test results of the blood in Experiment (1) of Test Example 13.

[0085] Figure 41 Alternative photograph of the histological analysis diagram of the HE-stained liver of the blood in Experiment (2) of Test Example 13.

[0086] Figure 42 Illustration of the results of the pharmacokinetic experiment (1) of Test Example 14.

[0087] Figure 43 Illustration of the results of the pharmacokinetic experiment (2) of Test Example 14. Detailed implementation mode

[0088] Unless otherwise specified, the terms and the like described in connection with the present invention are used as terms having the meanings or contents commonly used in the technical field. Generally, the following additional explanations can be made for the present invention.

[0089] As described above, short-chain fatty acids can be produced from sugars by the intestinal flora of mammals, typically including acetic acid, propionic acid, butyric acid (or butyric acid), but may also include, depending on the circumstances: branched-chain fatty acids such as isobutyric acid or isovaleric acid, which are sometimes produced by the degradation of proteins containing certain branched-chain amino acids; and straight-chain or branched-chain fatty acids with 7 carbon atoms that sometimes exhibit functions similar to those of these fatty acids. Therefore, in the copolymer disclosed in the present specification, when R in the hydrophobic chain segment represented by Formula I for providing a hydrophobic domain is -(C=O)R 1 in the case of, there is no limitation on R 1 and examples include: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, heptyl, pentyl, 3-methylbutyl, etc. These groups can be substituted, and the substituent in the case of substitution is preferably an unsubstituted or substituted phenyl group that can be bonded to the carbon atom at the unbonded end, and the substituent of the substituted phenyl group can be one or more halogens, hydroxyl groups, or methoxy groups. In addition, the alkyl moiety denoted as C x -C xx such as alkoxy refers to a straight-chain or branched-chain alkyl group with x to xx carbon atoms. R can be a hydrogen atom, and in this case, it is 30% or less, preferably 20% or less, more preferably 10% or less, and most preferably 0% (absent) of the total number of repeating units of n. Such a degree of preference is for the copolymer to more reliably form a hydrophobic domain or region when associating in water for self-assembly.

[0090] In the copolymer disclosed in the present specification, the segment represented by Formula IIa for providing a hydrophilic segment provides a hydrophilic block. On the other hand, it can become a member of a hydrophilic-hydrophobic block copolymer with the hydrophobic chain segment represented by Formula I as the hydrophobic block, or​​​

[0091] Each repeating unit represented by Formula IIb that provides a hydrophilic segment can be a member of a hydrophilic-hydrophobic random copolymer that can randomly exist with each hydrophobic segment represented by Formula I. The expression "randomly with each other" can alternately exist under appropriate circumstances, for example, when n in Formula I and y in Formula IIb are approximate values. In addition, for example, when n in Formula I:y in Formula IIb is 30 or more:1, the hydrophobic segments can substantially be in a form where multiple segments form a block.

[0092] These block copolymers and random copolymers can be copolymers as long as multiple copolymers associate in water and self-assemble to form so-called core-shell type nanoparticles or polymer micelles in which the core contains hydrophobic segments and the shell contains hydrophilic segments, and can include other members. It is not limited to these. As typical examples of the copolymers disclosed in this specification, the copolymers represented by the above-mentioned Formula BC or Formula GC can be cited. For example, when L1 in Formula BC represents a divalent linking group, the divalent linking group generally refers to a group having a maximum of 34, preferably 18, more preferably a maximum of 10 carbons, and optionally containing oxygen and nitrogen atoms. As such a linking group, specifically, the following groups can be cited:

[0093] This group is selected from the groups represented by the following structural formulas, or selected from -(CH2) c S-, -CO(CH2) c S-, -(CH2) c NH-, -(CH2) c CO-, -CO-, -OCOO-, -CONH-, where, respectively and independently: b is an integer from 2 to 6, and c is an integer from 1 to 5.

[0094]

[0095] Regarding the present invention, the nano or nano-size in the nanoparticles or nano-sized polymer micelles means that when performing dynamic light scattering measurement (DLS) of the nanoparticles or polymer micelles in water, the average diameter is in the nano range. Generally, the average diameter is in the range of about 10 nm to about 2000 nm, preferably about 10 nm to about 500 nm, more preferably about 25 nm to about 200 nm.

[0096] The above-mentioned hydrophilic-hydrophobic copolymer can be manufactured according to its own known manufacturing method with reference to the defined chemical structure. It can be conveniently manufactured according to any of the following methods.

[0097] Conveniently, the copolymer represented by the typical Formula BC defined as above in (1) and above in (2)(i) is manufactured as follows with reference to the above-mentioned WO2009 / 133647, WO2016 / 052463.

[0098] The following method can be used: First, prepare a poly(ethylene glycol) (PEG) segment, i.e., a PEG derivative represented by the following structural formula:

[0099] Formula A-1:

[0100] or

[0101] Formula A-2:

[0102]

[0103] (A, L, and m in the above formula are defined as described above),

[0104] Then, prepare a short-chain fatty acid ester of vinyl alcohol corresponding to the repeating unit of Formula I, and add the latter to the former through a living radical polymerization reaction mediated by reversible exchange chain transfer. At this time, if L-SC(=S)Ph in A-1 is L-SC(=S)OCH2CH3 described later, the target copolymer can be obtained more efficiently. It should be noted that when A has a phenylamino or phenethylamino as a substituent, this substituent can be introduced through a reductive amination reaction of the formyl group of the compound corresponding to Formula IIa having a formyl group as a substituent with the corresponding amine.

[0105] A typical graft copolymer represented by Formula GC can be manufactured as follows: Prepare a short-chain fatty acid ester of vinyl alcohol corresponding to the repeating unit of Formula I and a polymerizable unsaturated monomer bearing a carboxyl group or its protected group or a halogen atom (Cl, Br, etc.), such as maleic anhydride or vinyl chloride, and carry out radical polymerization of them in the presence of a radical initiator. In the anhydride unit or vinyl chloride unit of the resulting random copolymer, for example, lithiation or other metal alcoholization of a hydroxyl group at one end, or conversion of the hydroxyl group at this one end into an amino group (NH2), and use the resulting poly(ethylene glycol) derivative to graft the poly(ethylene glycol) chain via an ester or amide bond or an ether (-O-) or -NH-. Thus, it can be manufactured. However, during the above polymerization reaction, if a chain transfer agent such as methyl(phenyl)aminodithiocarbamic acid cyanomethyl ester or cyanomethyl methyl(phenyl)aminodithiocarbonate is used, the molecular weight of the synthesized polymer can be reduced, and the operation of the subsequent grafting reaction can be simplified. Of course, the target copolymer can also be manufactured without using such a chain transfer agent.

[0106] The above-mentioned nanoparticles or polymeric micelles are prepared as follows: Since the copolymer according to the present invention is amphiphilic, after preparing an aqueous solution containing a water-soluble organic solvent such as N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), dialysis is carried out in water through a dialysis membrane with a certain molecular weight cut-off, causing the copolymer itself to associate to form micelles, whereby they can be prepared. The micelles or nanoparticles formed by such an operation can be separated, for example, by freeze-drying, centrifugation, etc., and obtained in the form of the resulting solid matter.

[0107] The nanoparticles and nano-sized polymeric micelles provided by such an operation can be solubilized or uniformly dispersed in an aqueous medium (an aqueous solution that may contain physiological saline or a pH regulator as required), and provided in the form of the resulting solution or liquid preparation, and thus can be made into oral preparations in various forms including parenteral preparations. For example, when provided in the form of an oral preparation, the nanoparticles of the present invention can also be made into tablets, pills, and granules using excipients and diluents commonly used in this technical field. There is no limitation on the excipient or diluent, and examples include sodium carboxymethyl cellulose, crystalline cellulose, hypromellose, sodium lauryl sulfate, magnesium stearate, polyethylene glycol (Macrogol) 4000, titanium oxide, etc. commonly used in this technical field.

[0108] A pharmaceutical preparation containing such nanoparticles as an active ingredient can exert the physiological functions inherently possessed by the above-mentioned short-chain fatty acids locally in the organism of a mammal including a human to which the nanoparticles are delivered. The optimal dose of such a pharmaceutical preparation varies depending on the disease or administration method for the purpose of treatment, and thus the dose cannot be uniquely determined, but the dose can be determined by a professional doctor based on data obtained through small-scale clinical trials, etc. Detailed implementation mode

[0109] Hereinafter, in order to avoid making the description complicated, typical examples of the present invention will be specifically described, but the present invention is not limited to these examples.

[0110] Production Example 1: Synthesis of CH3O-(CH2CH2O) m -CH2PhCH2Br (N686)

[0111] In 100 g (20 mmol) of commercially available CH3O-(CH2CH2O) mTo 200 mL of tetrahydrofuran (THF) and 14.4 mL (23 mmol) of butyllithium (1.6 M - hexane) were added to -H (MW = 5,000), and then 25 g (95 mmol) of α,α'-dibromoxylene was added, and the reaction was carried out at 50 °C for 2 days. After precipitation in 2-propanol (IPA), the precipitate was dried under reduced pressure. The resulting pale yellow polymer was dissolved in methanol, centrifuged, and α,α'-dibromoxylene was removed in the form of a precipitate. A methanol solution was added to IPA, and the resulting white precipitate was dried under reduced pressure to obtain the target product (N686) (yield 104 g).

[0112] Production Example 2: CH3O-(CH2CH2O) m -CH2PhCH2SC(=S)OCH2CH3 (N717) Synthesis

[0113] 20 g of N686 synthesized in Production Example 1 was dissolved in 100 mL of ethanol, 7 g of potassium ethyl xanthate (CH3CH2OC(=S)SK) was added, and the reaction was carried out at room temperature for 10 minutes. After centrifuging to separate the precipitate, ethanol was distilled off under reduced pressure. The residue was dissolved in chloroform, washed with water, the chloroform layer was separated, dehydrated over anhydrous sodium sulfate, filtered, precipitated in IPA, centrifuged, and dried under reduced pressure to obtain the target product (N717) (yield 15 g).

[0114] Production Example 3: CH3O-(CH2CH2O) m -CH2PhCH2[CH2CH(OC(=O)CH3)] n SC(=S)OCH2CH3 (N684) Synthesis: 1 g of N717 synthesized in Production Example 2, 15 mg of azobisisobutyronitrile, and 6.6 g of vinyl acetate were added to a flask. After purging with nitrogen for 5 minutes, the reaction was carried out at 60 °C for 1 day. The resulting target product was dissolved in THF, precipitated in IPA, and dried under reduced pressure to obtain 2.6 g of the target product (N684). The 1 1H NMR spectrum is shown in Figure 1 .

[0115] Production Example 4: CH3O-(CH2CH2O) m -CH2PhCH2[CH2CH(OC(=O)CH2CH3)] n SC(=S)OCH2CH3 (N731) Synthesis

[0116] 5 g of N717 synthesized in Production Example 2, 75 mg of azobisisobutyronitrile, and 7 g of vinyl propionate were added to a flask. After purging with nitrogen for 5 minutes, the reaction was carried out at 60 °C for 2 days. The resulting target product was dissolved in THF, precipitated in IPA, and dried under reduced pressure to obtain 9.3 g of the target product. The1 The 1H NMR spectrum is shown in Figure 2 .

[0117] Production Example 5: CH3O-(CH2CH2O) m -CH2PhCH2[CH2CH(OC(=O)CH2CH2CH3)] n Synthesis of SC(=S)OCH2CH3 (N721): 5 g of N717 synthesized in Example 2, 75 mg of azobisisobutyronitrile, and 10 g of vinyl butyrate were added to a flask. After purging with nitrogen for 5 minutes, the mixture was reacted at 60 °C for 2 days. The resulting target product was dissolved in THF, precipitated in IPA, and dried under reduced pressure to obtain 12.3 g of the target product (N721). The 1 1H NMR spectrum of N721 is shown in Figure 3 .

[0118] Production Example 6: Synthesis of graft copolymer (N741)

[0119]

[0120] 75 mg of azobisisobutyronitrile, 5.7 g of vinyl butyrate, 100 mg of maleic anhydride, and 200 mg of cyanomethyl methyl(phenyl)carbamodithioate (PhN(CH3)C(=S)SCH2CN) were added to a flask. After purging with nitrogen for 5 minutes, the mixture was reacted at 60 °C for 2 days. The resulting target product was dissolved in THF, and after taking a small sample, the previously prepared THF solution of CH3O-(CH2CH2O) m OLi* was added as described below and stirred for 30 minutes. The reaction solution was poured into IPA, and the precipitate was dried to obtain 6 g of the target product (N741). The 1H NMR spectrum of N741 is shown in Figure 4 .

[0121] * A THF solution of CH3O-(CH2CH2O) m OLi was prepared by adding 5 g of CH3O-(CH2CH2O) m OH (MW = 5,000) and 0.6 mL of butyllithium to 20 mL of THF.

[0122] Production Example 7: Preparation of self-assembled particles 1

[0123] 50 mg each of the polymers (N684, N731, N721) synthesized above were collected, dissolved in 1 mL of DMF, 1 mL of water was added, and the mixture was placed in a dialysis membrane (MWCO = 3.5 kDa) and dialyzed in 2 L of water. The dialysis water was changed 3 times every half day, and then dynamic light scattering measurement was performed to confirm the formation of particles with an average size of 30 - 100 nm (refer to Figure 5 ).

[0124] Production Example 8: Preparation of Self-Assembled Particles 2

[0125] Except for using the graft polymer (N741) synthesized in Production Example 6, the operation of Production Example 7 was repeated, and it was confirmed that particles with an average particle diameter of 153 nm were formed (refer to Figure 6 ).

[0126] Test Example 1: Cytotoxicity 1

[0127] On each 96-well plate seeded with 1 × 10 4 HepG2 cells, various samples of the self-assembled particles prepared in Production Example 7 were added. After culturing for 24 hours, a WST solution was added, and the UV absorption at 450 nm was measured 2 hours later. When compared with the control, it was confirmed that almost no toxicity was generated at the measured concentrations ( Figure 7 : showing the cytotoxicity of each nanoparticle of N684, N731, and N721).

[0128] Test Example 2: Cytotoxicity 2

[0129] Except for using the self-assembled particles prepared in Production Example 8, the operation of Test Example 1 was repeated to evaluate the cytotoxicity of N741. The test results are shown in Figure 8 . From Figure 8 judgment: The nanoparticles of N741 did not show cytotoxicity in this test system.

[0130] Test Example 3: Cancer Metastasis Inhibition Effect

[0131] A group of 5 - 7 five-week-old BALB / c male mice were allowed to freely ingest the various samples described in the following administration groups of GP1 - GP6. After 2 days, 1 × 10 4 B16F10 / B16F10 melanoma cells (obtained from the RIKEN cell bank) were injected into the tail vein, and the sample water was continuously ingested. Dissection was performed on the 11th day, and the number of cancers adhering (upright) to the lungs was confirmed by macroscopic observation. The results are shown in Figure 10 , and in addition, the weight changes of the experimental animals during the test are shown in Figure 9 .

[0132] According to Figure 10 , almost no cancer metastasis number inhibition effect was found in short-chain fatty acids. In contrast, the GP5 group of propionic acid nanoparticles showed extremely high cancer metastasis inhibition. Figure 11 Shows the results of measuring the number of microscopic metastases that cannot be observed with the naked eye under a microscope. Similar to Figure 10 , it was confirmed that propionic acid nanoparticles (abbreviated as PEG-b-PVPro in the figure) extremely inhibited cancer metastasis.

[0133] Figure 12H&E staining of lung tissue (left) and the number of metastatic cancers obtained from the image (right) are shown. Compared with the control, a significant decrease in the number of cancers was observed in propionic acid nanoparticles and butyric acid nanoparticles. Figure 13 H&E staining of lung tissue (left) and the area of ​​metastatic cancer obtained from the image (right) are shown. Compared with the control and low molecular weight fatty acids, a significant reduction in the area of ​​cancer was observed in propionic acid nanoparticles and butyric acid nanoparticles. Figure 14 The levels of ALT, AST, LDH, and ALB in the blood were displayed. It was confirmed that almost no malfunction was found in the liver or other organs in all cases.

[0134] Figures 15 - 18 H&E staining of small and large intestine tissues is shown. Shortening of villi was found in the low molecular weight fatty acid group according to the location, confirming the presence of damage.

[0135] Sample Administration Group

[0136] GP1: healthy group (n=5);

[0137] GP2: melanoma administration group (n=7);

[0138] GP3: melanoma administration group / 30 mM propionic acid free intake group (n=7);

[0139] GP4: melanoma administration group / 30 mM butyrate free intake group (n=7);

[0140] GP5: melanoma administration group / 30 mM N731 free intake group (n=7);

[0141] GP6: melanoma administration group / 30 mM N721 free intake group (n=7).

[0142] Test Example 4: Dieting Effect

[0143] Solid feed D12492 (60% fat, super high fat feed) purchased from EPS Yixin Co., Ltd. was administered to 5 4-week-old C57BL / 6J mice (male) in a group, and the samples described below were administered to each administration group of GP1 to GP3, and the body weight was measured. Figure 19 .

[0144] Depend on Figure 19 It was confirmed that the butyric acid granule group (N721) significantly suppressed the increase in body weight compared with the tap water group and the propionic acid granule group (N731).

[0145] Sample Administration Group

[0146] GP1: free access to tap water;

[0147] GP2: Free intake of N731 (5 mg / mL);

[0148] GP3: Free intake of N721 (5 mg / mL).

[0149] Test Example 5: Effect on ulcerative colitis

[0150] A group of 7 male 7-week-old ICR mice were allowed to freely intake 4% dextran sulfate sodium (DSS), and the following samples were orally administered once a day through a sonde. After 10 days, the disease activity index (DAI) was measured, and the blood was evaluated. As Figure 20 shown, for the ulcerative colitis model, butyric acid nanoparticles (denoted as BNP in the figure) significantly decreased the DAI, and a therapeutic effect was observed. In addition, as Figure 21 shown, a significant increase in the white blood cell count was found in the ulcerative colitis model, whereas it was significantly inhibited in BNP.

[0151] Sample Administration Group

[0152] GP1: Free intake of tap water + tap water (0.65 mL);

[0153] GP2: Intake of 4% DSS + tap water (0.65 mL);

[0154] GP3: Intake of 4% DSS + butyric acid (2.32 mg / mL, 0.65 mL);

[0155] GP4: Intake of 4% DSS + CNP (PEG-b-polystyrene) (10 mg / mL, 0.65 mL);

[0156] GP5: Intake of 4% DSS + BNP (PEG-b-poly(vinyl butyrate)) (10 mg / mL, 0.65 mL).

[0157] Test Example 6: Effect on non-alcoholic steatohepatitis (NASH)

[0158] Forty-nine 5-week-old C57BL / 6J male mice were given solid feed A06071302 purchased from EPS Shinyo Co., Ltd. (choline-deficient high-fat feed, reduced methionine, added 0.1% methionine) by free intake. After 4 weeks, they were randomly divided into groups of 7, and the following samples described in each dosing group of GP1 - GP7 were given by free intake. Data analysis was performed after 8 weeks. Figure 22 The liver and spleen weights are shown. It was confirmed that the NASH group hypertrophied due to inflammation, whereas hypertrophy was significantly inhibited in the propionic acid particle dosing group.

[0159] Sample Administration Group

[0160] GP1: Normal solid feed (Oriental Yeast MF);

[0161] GP2: Solid feed A06071302;

[0162] GP3: Solid feed A06071302 + butyric acid (65 mM);

[0163] GP4: Solid feed A06071302 + propionic acid (50 mM);

[0164] GP5: Solid feed A06071302 + butyric acid nanoparticles (10 mg / mL, polymer concentration 1 mM, butyric acid equivalent to 65 mM);

[0165] GP6: Solid feed A06071302 + propionic acid nanoparticles (10 mg / mL, polymer concentration 1 mM, propionic acid equivalent to 50 mM);

[0166] GP7: Solid feed A06071302 + polystyrene nanoparticles (10 mg / mL).

[0167] Test Example 7: Second inhibitory effect on cancer metastasis

[0168] A group of 5 - 7 male C57BL / 6J mice, 7 - 8 weeks old, were obtained from Charles River Japan, Inc. (Yokohama). These mice were housed under specific pathogen - free conditions, with a 12 - hour dark / light cycle, and controlled temperature (23 ± 1°C) and humidity (50 ± 5%) while freely consuming a standard solid feed. The mice were randomly divided into the following administration groups of GP1 - GP6 and freely consumed the recorded samples. One day later, 2.5×10 5 B16F10 / B16F10 melanoma cells (obtained from the RIKEN cell bank) were injected into the tail vein at a concentration of 200 μL per injection. From 1 day before this tail vein injection until 11 days as the test endpoint, the mice were continuously given the respective samples in the form of free drinking water. On the 11th day, plasma and other organs were collected and appropriately stored in preparation for the following various further analyses.

[0169] The changes in the sample consumption and the body weight of the experimental animals during the test are shown in Figure 23 . For other tests, test results substantially equivalent to those observed or confirmed in Test Example 3 were obtained.

[0170] Sample Administration Group

[0171] GP1: Healthy group (n = 5);

[0172] GP2: Melanoma drug-administered group (n = 7);

[0173] GP3: Melanoma drug-administered group / 30 mM propionic acid free intake group (n = 7);

[0174] GP4: Melanoma drug-administered group / 30 mM butyric acid free intake group (n = 7);

[0175] GP5: Melanoma drug-administered group / 30 mM PNP (particles from N731: refer to Production Example 7) free intake group (n = 7);

[0176] GP6: Melanoma drug-administered group / 30 mM BNP (particles from N721: refer to Production Example 7) free intake group (n = 7).

[0177] Test Example 8: Effects on anti-diabetes test

[0178] Regarding the management and use of animals in this test and all tests using experimental animals disclosed in this specification, they were strictly conducted in accordance with the guidelines of the University of Tsukuba regarding such management.

[0179] One group of 7 male C57BL / 6J mice at 7 - 8 weeks old was obtained from Charles River Japan Co., Ltd. (Yokohama). These mice were raised under pathogen-free conditions, with a 12-hour dark / light cycle, and at a controlled temperature of (23 ± 1°C) and humidity of (50 ± 5%) while freely consuming standard solid feed. The mice were randomly divided into the following drug-administered groups of GP1 - GP6, and were allowed to freely drink and ingest the recorded various samples until 36 days. One day later, a glucose tolerance test was conducted. During the test, the body weight of each mouse and the consumption of the samples were monitored every other day. After that, the samples were replaced with drinking water until the end time point of the test (day 40).

[0180] Sample Administration Group

[0181] GP1: Exenatide (traditional anti-diabetes drug), subcutaneous injection group at 1 μg (days 1 - 4) and 2 μg (days 5 - 36) per day;

[0182] GP2: 60 mM BNP (particles from N721: refer to Production Example 7) free intake group;

[0183] GP3: 60 mM PNP (particles from N721: refer to Production Example 7) free intake group;

[0184] GP4: 30 mM butyric acid free intake group;

[0185] GP5: The group with free intake of 30 mM propionic acid;

[0186] GP6: The control group (free water intake).

[0187] (1) The sample consumption in this experiment was calculated as the amount per mouse (mL), and the body weight of the mice was recorded as the mean ± SD value, as shown respectively in Figure 24 .

[0188] (2) The glucose tolerance test was conducted to evaluate the therapeutic effect of short-chain fatty acids in controlling the metabolism of administered glucose. After 16 hours of overnight restraint, glucose (2 g / kg) was orally administered to the mice. Around 1 hour before and after glucose administration, 10 μL of blood was collected from the tail vein and mixed with physiological saline containing heparin (50 units / mL) at a volume (v:v) ratio of 1:1. The glucose concentration in the diluted blood was measured using FUJIDRY-CHEM 7000V (Fujifilm). The final blood glucose concentration was calculated by the following formula.

[0189] Final glucose concentration (mg / mL) = [Glucose] 60分钟 - [Glucose] 0分钟

[0190] The data as the mean ± SEM (n = 7) values are shown in Figure 25 .

[0191] Student's t-test, tails (2) and type (2), was performed to determine the statistical error between the means (P < 0.05 was considered statistically significant).

[0192] For the diabetic model mice, no significant differences were found in the propionic acid administration group, butyric acid administration group, and propionic acid nanoparticle (PNP) administration group. In contrast, in the exenatide and BNP administration groups for diabetes drugs, the glucose concentration decreased significantly, indicating an improvement in pancreatic function.

[0193] (3) The weights of the organs at the end time point of the experiment

[0194] After dissecting the mice, the weights of the removed spleen, kidney, and liver were quickly measured, and the results are shown in Figure 26 , and these organs were used for further histological analysis and preserved in 10% neutral buffer. Student's t-test was the same as above.

[0195] In these results, an increase in spleen weight was found in the propionic acid administration group, and an increase in liver weight was confirmed in the butyric acid administration group and exenatide administration group. This indicates organ inflammation. On the other hand, in the BNP administration group, the spleen, kidney, and liver were all comparable to the control and showed no toxicity.

[0196] (4) Histological analysis of the intestine by H&E staining

[0197] The organs of the dissected mice were quickly placed in 10% neutral formalin solution and fixed by soaking for 1 day. After that, they were replaced with 70% ethanol solution for paraffin embedding. All the paraffin-embedded organs were processed into tissue sections with a thickness of 5 μm and stained with hematoxylin / eosin (H / E) by a conventional method. The tissue sections were dehydrated with high-concentration alcohol, washed with xylene, and then examined under a microscope (biorevo, BZ-9000, Keyence). The villus length was measured using Image J software (Image J software) (NIH). The results are shown in Figure 29 . The minimum and maximum range values, upper and lower quartiles, and median of the quantitative data: duodenum (n = 130 - 132), jejunum (n = 96 - 185), ileum (n = 109 - 138), colon (n = 37 - 47) are shown in box plots. The t-test is the same as above.

[0198] It was shown that: in the exenatide administration group, the villi of the duodenum, jejunum, ileum, and large intestine were significantly shortened, showing strong side effects. The propionic acid and butyric acid administration groups also showed the same tendency. On the other hand, it was found that there was no shortening of the villi in the BNP administration group and PNP administration group, and there was no damage to the digestive tract.

[0199] (5) Histological analysis of the pancreatic tissue by H&E staining

[0200] The organs of the dissected mice were quickly placed in 10% neutral formalin solution and fixed by soaking for 1 day. After that, they were replaced with 70% ethanol solution for paraffin embedding. According to the conventional method, all the paraffin-embedded viscera were processed into tissue sections with a thickness of 5 μm and stained with hematoxylin / eosin (H / E) by a conventional method. The tissue sections were dehydrated with high-concentration alcohol, washed with xylene, and then fixed for microscopic examination (biorevo, BZ-9000, Keyence). The area of the islets of Langerhans was determined using Image J software (Image J software) (NIH). The results are shown in Figure 30 . The minimum and maximum range values, upper and lower quartiles, and median (n = 10 - 20) of the quantitative data are shown in box plots. The t-test is the same as above.

[0201] In the diabetic model mice and the propionic acid administration group, butyric acid administration group, and PNP administration group, the size of the islets of Langerhans was reduced, while no atrophy of the islets of Langerhans was found in the BNP administration group and exenatide administration group, indicating that the function of the islets of Langerhans was maintained.

[0202] Test Example 9: As one of the effects of the radiation enhancer

[0203] A group of 5 C57BL / 6J mice, 5 - 7 weeks old, obtained in the same manner as in the above tests, were raised under the same conditions. On the outer side of the right leg of the mice, 0.076×10 6 melanoma B16F10 cells per 100 μL (serum - free DMEM) were subcutaneously injected (7 days before irradiation). After 1 week of cancer proliferation, the mice were randomly divided into the following administration groups. One day before irradiation and immediately after irradiation, BNP (500 mg / kg) was administered intraperitoneally (i.p.) to the mice to confirm the radiation enhancement effect (GP3 and GP4 respectively). The irradiation conditions were set as 10 Gy, 150 kV, 20 mA, Al filtration, and 330 mm distance.

[0204] Sample Administration Group

[0205] GP1: Cancer control group;

[0206] GP2: Cancer + irradiation (IR: 10 Gy) group;

[0207] GP3: BNP 500 mg / kg (1 day before irradiation) group;

[0208] GP4: BNP 500 mg / kg (0 days after irradiation) group.

[0209] (1) After irradiation, cancer proliferation and body weight were tracked until the end - point of the experiment (8 days). The results are shown in Figure 31 . Day 0 is the data before irradiation, and day 8 is the data after irradiation (the end - point of the experiment).

[0210] Similar to the non - administered group, there was no change in body weight in the BNP - administered group, and no toxicity was found.

[0211] (2) At the end - point of the experiment, plasma and other organs were collected and properly preserved for further analysis. The size of the cancer was measured using calipers, and the volume of the cancer was calculated using the following equation.

[0212] Volume of cancer = 0.52×length×width 2

[0213] The results are shown in Figure 30 . The t - test was the same as in Test 8(2).

[0214] There was a tumor growth inhibitory effect in the 10 Gy irradiation group. Even when BNP was administered before and after X - ray irradiation, the tumor size after 8 days was significantly smaller than that of the BNP - non - administered group, confirming the radiation enhancement effect.

[0215] Test Example 10: Second Effect as a Radiation Enhancer

[0216] A group of 5 or 7 C57BL / 6J mice, 5 - 7 weeks old, which were used for the same tests as above, were raised under the same conditions. 0.076×10 6 melanoma B16F10 cells per 100 μL (serum-free DMEM) were subcutaneously injected into the outer side of the right leg of the mice (9 days before irradiation). When the volume of the cancer reached 350 - 560 mm 3 , the mice were randomly divided into the following administration groups. The irradiation conditions were set as 10 Gy, 150 kV, 20 mA, Al + Cu (0.5 mm + 0.1 mm) filtration, and 330 mm interval.

[0217] The cancer volume was calculated in the same manner as above.

[0218] Sample Administration Group

[0219] GP1: Healthy (saline) group (n = 5);

[0220] GP2: Cancer control group (n = 7);

[0221] GP3: Cancer + irradiation (IR (5 Gy)) group (n = 7);

[0222] GP4: Butyric acid (i.p.; 250 mg / kg) (6 hours before irradiation) + IR group (n = 7);

[0223] GP5: Butyric acid (i.p.; 500 mg / kg) (24 hours before irradiation) + IR group (n = 7);

[0224] GP6: BNP (i.p.: 500 mg / kg) (6 hours before irradiation) + IR group (n = 7);

[0225] GP7: BNP (i.p.: 500 mg / kg) (12 hours before irradiation) + IR group (n = 7).

[0226] (1) Data on the cancer proliferation curve based on 5 Gy irradiation after administration of butyric acid and NP in a melanoma xenograft model are shown in Figure 33 .

[0227] Butyric acid and BNP were administered before radiation exposure. The tumor growth inhibitory effect increased in the order of irradiated group < butyric acid administration group 6 hours before < BNP administration group 6 hours before = butyric acid administration group 24 hours before < BNP administration group 24 hours before.

[0228] (2) Data on the weight of cancer at the end time point of the experiment (6th day after irradiation) are shown as mean ± SEM values (n = 2 - 7) in Figure 32 . The t-test was the same as in Experiment 8(5).

[0229] The results of tumor weight were as follows: The BNP administration group 24 hours ago and the butyric acid administration group 6 hours ago significantly reduced the tumor size. (3) Data on the body weight change of mice as mean ± SD values (n = 7) are shown in Figure 35 . The t-test was the same as above.

[0230] A decrease in body weight was found in the butyric acid administration group, resulting in toxicity. On the other hand, no decrease in body weight was seen in the BNP administration group, and no toxicity occurred.

[0231] Test Example 11: In vitro evaluation of the radiation enhancement effect of BNP evaluated by spheroid assay

[0232] Melanoma B16F10 cells were treated with 50 mM butyric acid for 7 hours and then irradiated with 2 Gy (Al + Cu (0.5 mm + 0.1 mm) filter). After stopping for 1 hour at 37°C, the medium was removed, and the samples were washed several times to remove them, and then fresh medium was supplemented. After culturing the treated cells for 24 hours, they were cultured in 0.6% methylcellulose and DMEM (1:1) for 3 days to form spheroids. Images were taken by direct microscopy, and the spheroids were measured using Image J software (NIH). Grouping was performed, and then the cells were treated with the sample for 7 hours after 2 Gy irradiation. The data are presented as mean ± SEM values (n = 32 - 37). These results are shown in Figure 38 . The t-test was the same as above.

[0233] Cell culture:

[0234] Mouse melanoma B16F10 cells were purchased from a cell bank (RIKEN, Japan). These cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; L-glutamine, 1 g / L glucose, sodium bicarbonate, Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum and an antibiotic mixture of 100 ng / mL penicillin-streptomycin-neomycin in a humidified environment of 5% CO2 at 37°C. The results are shown in Figure 34 .

[0235] A growth inhibitory effect on spheroid cancer cells was clearly confirmed in the BNP administration group.

[0236] Test Example 12: The second effect on non-alcoholic steatohepatitis (NASH)

[0237] Forty-two 5-week-old male C57BL / 6J mice were given solid feed A06071302 (choline-deficient high-fat feed, reduced methionine, 0.1% methionine added) purchased from EPS Co., Ltd. by free intake. After 4 weeks, they were randomly divided into 1 group of 7 mice, and the following 6 groups, namely the Healthy group (not given solid feed A06071302), the NASH group, the butyric acid administration group (BA), the propionic acid administration group (PA), the PNP administration group, and the BNP administration group, were given the respective samples described by free intake (the PA in the PA and PNP groups was prepared at a concentration of 65 mM, and the BA in the BA and BNP groups was prepared at a concentration of 50 mM). Data analysis was performed after 8 weeks.

[0238] (1) Liver and spleen weights are shown in Figure 35 . It was confirmed that the NASH group showed hypertrophy due to inflammation. In contrast, hypertrophy was significantly inhibited in the propionic acid granule administration group.

[0239] Here, in order to compare the differences in the amount of fat accumulated in the liver and the degree of splenomegaly corresponding to the inflammation in the liver, the weights of the liver and spleen taken out after dissecting the mice were measured, and the average weight of each group was calculated (n = 7 for each group). The error bars for the average liver weight value and the average spleen weight value of each group were the standard deviation values (n = 7 for each group). A t-test was performed to study whether there were significant differences statistically between the mean values, and P < 0.05, indicating a significant difference statistically.

[0240] Regarding the liver weight (left figure), compared with the Healthy group, the liver weight of the NASH group was significantly heavier, indicating inflammation. On the other hand, compared with the NASH group, the liver weight of the PNP administration group was significantly lighter, suppressing inflammation.

[0241] Similarly, regarding the spleen weight (left figure), compared with the Healthy group, it increased significantly in the NASH group and decreased significantly in the PNP administration group.

[0242] (2) Histological analysis of the liver stained with HE

[0243] The liver of the dissected mice was quickly placed in a 10% neutral buffered formalin solution and soaked for 1 day for fixation. Then, it was replaced with a 70% ethanol solution for paraffin embedding. After paraffin embedding, all the livers were processed into tissue sections with a thickness of 5 μm and stained with hematoxylin / eosin (HE). All the stained tissue sections were digitalized for image data through a microscope (All-in-One fluorescence microscope, BZ-X710, Keyence), and then the ratio of the oil droplet tissue area to the liver tissue area was calculated using Image J (NIH) (n = 7 for each group). The results are shown in Figure 36The figure shows error bars representing the mean and standard deviation of the oil droplet tissue area for each group. A t-test was performed to study whether there were significant differences statistically among the means, with P < 0.05, indicating a significant difference statistically.

[0244] Regarding the amount of oil droplets in the liver, it increased significantly in the NASH group (fatty liver state) compared to the healthy group. On the other hand, the amount of oil droplets in the PNP-administered group was significantly less compared to the NASH group.

[0245] (3) Histological analysis of the liver stained with MT

[0246] The liver of the dissected mouse was quickly placed in a 10% neutral buffered formalin solution and soaked for 1 day for fixation. Then, it was replaced with a 70% ethanol solution for paraffin embedding. After paraffin embedding, all the livers were processed into tissue sections with a thickness of 5 μm and stained with Masson's trichrome (MT). All the stained tissue sections were digitalized for image data through a microscope (All-in-One fluorescence microscope, BZ-X710, Keyence), and then the ratio of the fibrotic tissue area to the liver tissue area was determined using Image J (NIH) (n = 7 for each group). The results are shown in Figure 42 The figure shows error bars representing the mean and standard deviation of the fibrotic tissue area for each group. A T-test was performed to study whether there were significant differences statistically among the means, with p < 0.05, indicating a significant difference statistically.

[0247] Regarding the amount of liver fibrosis, it was significantly more in the NASH group compared to the healthy group. On the other hand, the amount of liver fibrosis in the PNP-administered group was significantly less compared to the NASH group.

[0248] Production Example 9: CH3O-(CH2CH2O) m -CH2PhCH2[CH2CH2(OC(C=O)CH2CH2CH2Ph) n Synthesis of SC(=S)OCH2CH3 (N821)

[0249] 1.5 g of N817 synthesized in the same manner as N717 synthesized in Production Example 2 (this application), 45 mg of azobisisobutyronitrile, and 2 g of vinyl 4-phenylbutyrate were added to a flask. After purging with nitrogen for 5 minutes, the reaction was carried out at 60 °C for 1 day. The obtained target product was dissolved in THF, precipitated in isopropanol (IPA), and dried under reduced pressure to obtain 2.8 g of the target product (N821). The 1 1H NMR spectrum of N821 is shown in Figure 38 .

[0250] Production Example 10: Preparation of self-assembled particles (Ph-BNP)

[0251] Separate 50 mg of the polymer (NN821) synthesized in Production Example 9, dissolve it in 1 mL of DMF, add 1 mL of water, place it in a dialysis membrane (MWCO = 3.5 KDa), and perform dialysis in 2 L of water. Exchange the dialysis water 3 times every half day, and then perform dynamic light scattering measurement. It can be confirmed that particles with an average size of 67 nm are formed. The size distribution of PEG-b-poly(vinyl 4-phenylbutyrate) nanoparticles (Ph-BNP, N832) as the result of dynamic scattering measurement is shown in Figure 39 .

[0252] Test Example 13: Anti-ammonemia effect

[0253] Twenty-four 6-week-old C57BL / 6N male mice were randomly divided into 4 groups, and the samples were orally administered via a sonde (once a day, 1.22 mmol - 4PBA / kg) for 4 days. In the administration groups 2GP - 4GP, acute liver dysfunction and hyperammonemia were induced by intraperitoneal administration of acetaminophen (acetyl-p-aminophenol; APAP; 300 mg / kg) on the 4th day, and dissection was performed on the 5th day for evaluation.

[0254] Sample Administration Group

[0255] 1GP: Normal saline;

[0256] 2GP: APAP + water;

[0257] 3GP: APAP + 4-phenylbutyric acid (200 mg / kg, 1.22 mmol - 4PBA / kg);

[0258] 4GP: APAP + Ph-BNP (200 mg / kg, 1.22 mmol - 4PBA / kg).

[0259] (1) Biochemical examination of blood

[0260] Just after cardiac blood collection on the 5th day at the start of the experiment, a colorimetric slide was used, and the blood ammonia concentration, plasma aspartate aminotransferase (AST), and plasma alanine aminotransferase (ALT) levels, which are liver function indicators, were measured separately using an automatic biochemical analyzer for animals. The results (oral administration effect of Ph-BNP on the APAP acute liver injury model) are shown in Figure 40 .

[0261] As can be seen from the figure: 4-Ph-BNP significantly reduces the blood ammonia concentration. In addition, the AST and ALT levels also significantly decrease, which helps the recovery of liver function.

[0262] (2) Histological analysis of the liver stained with HE

[0263] The liver of the dissected mouse was stained according to the above-mentioned HE staining method for organs or viscera. The results are shown in Figure 41 . As can be seen from the figure: In the APAP administration group, strong disorders appeared in the liver, but the damage was inhibited in Ph-BNP. In the figure, Healthy corresponds to the healthy group of mice and the APAP administration group of mice, APAP + PBA corresponds to the administration group of APAP and 4-phenylbutyric acid, and APAP + Ph-BNP corresponds to the administration group of APAP and Ph-BNP (nanoparticles according to the present invention).

[0264] Test Example 15: Pharmacokinetic Test

[0265] According to the methods of the above-mentioned Production Examples 1 to 8, with corresponding changes made according to the conventional method, iodine was introduced into the phenyl groups of PEG-b-poly(vinyl butyrate) (refer to the left formula below) and PEG-b-poly(vinyl 4-phenylbutyrate) (refer to the right formula below) having a benzene ring at the α-terminus of PEG by the chloramine method 125 I. The unreacted iodine was separated by purifying twice using a PD-10 column.

[0266] (1)

[0268] After 24 hours, blood, liver, and digestive tract were collected from the mice that had freely ingested N932 from the water supply bottle, and the γ-ray intensity was measured using a scintillation detector. The results are shown in Figure 42 (left). It was confirmed from the figure that: N932 was almost completely confined to the digestive tract.

[0269] On the other hand, N930 was forcibly orally administered by Sonde, and the γ-ray intensity of the main organs was measured after 24 hours. The results are shown in Figure 42 (right). It was confirmed from this figure that: In addition to the digestive tract, it was widely distributed in blood, liver, kidney, etc., and 4-phenylbutyric acid was hydrolyzed in the digestive tract and ingested into the circulatory system. (2)

[0271] Forty-five 7-week-old ICR male mice were randomly divided into the following 3 groups. Regarding the administration groups: The dosages of 2GP and 3GP were determined such that the contained phenylbutyric acid reached 200 mg / kg (1.22 mmol - 4PBA / kg). All the mice were administered various samples almost simultaneously, and then 1 mL of blood was collected by cardiac puncture at each endpoint (30 minutes, 1 hour, 2 hours, 4 hours, 12 hours, 16 hours, 24 hours, these 7 endpoints), and plasma was separated. In addition, the liver, the targeted organ, was removed from the mice, and the content of phenylbutyric acid monomer was quantified by high performance liquid chromatography / mass spectrometry (LC / MS). The measurement results are shown inFigure 43 As can be seen from these figures: PBA is completely metabolized in the body within 4 hours. In contrast, 4-Ph-BNP continuously releases phenylbutyric acid monomers in the blood and liver, and its release has not ended even at the 24-hour time point. In addition, when calculating the area under the concentration-time curve (AUC), it is also found that compared with PBA, 4-Ph-BNP increases by about 3 times in the blood and about 15 times in the liver.

[0272] Sample Administration Group

[0273] 1GP: Normal saline (control, 3 animals);

[0274] 2GP: 4-Phenylbutyric acid (7 end points at 30 minutes, 1 hour, 2 hours, 4 hours, 12 hours, 16 hours, and 24 hours × 3 animals);

[0275] 3GP: Ph-BNP (7 end points at 30 minutes, 1 hour, 2 hours, 4 hours, 12 hours, 16 hours, and 24 hours × 3 animals).

Claims

1. Use of a hydrophilic-hydrophobic copolymer in the preparation of a medicament for preventing or treating cancer, suppressing obesity, preventing or treating ulcerative colitis, preventing or treating non-alcoholic fatty liver, preventing or treating diabetes, enhancing radiation in radiotherapy, or preventing or treating hyperammonemia. The hydrophilic-hydrophobic copolymer comprises the following (1) and (2): (1) A hydrophobic segment derived from the repeating unit represented by formula I, wherein, R is -(C=O)R 1 or a hydrogen atom, and R 1 is ethyl, propyl, isopropyl, butyl, isobutyl, heptyl, pentyl or 3-methylbutyl, and these groups may be substituted, and when substituted, the substituent is unsubstituted or substituted phenyl, and the substituent of the substituted phenyl is one or more halogens, hydroxyl groups, methoxy groups; even if R is a hydrogen atom, the number of such hydrogen atoms is 30% or less of n, where n is an integer of 5 to 1000; and (2) A hydrophilic segment containing a poly(ethylene glycol) chain represented by formula IIa. In the formula, A is unsubstituted or substituted C1-C 12 alkoxy group, and when it is substituted, the substituents are formyl group, a group of formula R ’ R ” CH-group, or phenylamino group or phenethylamino group, wherein R ’ and R ” are independently C1-C4 alkoxy groups, and m is an integer of 10 to 500; The hydrophobic segment of (1) and the hydrophilic segment of (2) exist in block form respectively. The copolymer containing the hydrophobic segment derived from the repeating unit represented by formula (I) of (1) and the hydrophilic segment of formula IIa of (2) is a block copolymer represented by formula BC: wherein, A, R, m, n are as defined above respectively, L1 represents a direct bond or a divalent linking group, and Z is a hydrogen atom, SH, SC(=S)-Ph, SC(=S)OCH2CH3, a hydroxyl group, a C1-C6 alkoxy group or an aryl-C1-C2 alkoxy group.

2. The application according to claim 1, wherein, The hydrophilic-hydrophobic copolymer forms nanoparticles or nano-sized polymeric micelles by self-assembly upon association in water.

3. The application according to claim 1 or 2, wherein The hydrophilic-hydrophobic copolymer forms nanoparticles in an aqueous medium.

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