Copolymers containing poly(ethylene glycol) and poly(l-amino acid derivatives), microparticles thereof, and use thereof in pharmaceutical compositions

Abstract

[Object] The present application aims to provide an ornithine assembly having low toxicity, effects even when administered orally, and high bioavailability. In addition, the present application aims to provide an ornithine assembly effective for the prevention or treatment of liver damage. [Solution] A condensate in which an acyl group or the like is introduced into the ornithine side chain amino group of polyethylene glycol-b-polyornithine is provided, and further, a new assembly independent of PIC is provided.

Description

TECHNICAL FIELD

[0001] The present application relates to a copolymer containing poly(ethylene glycol) and poly(L-amino acid derivative), and particularly to a copolymer containing a poly(ornithine) segment or a poly(aspartate), poly(acylated arginine) or poly(ornithine)-co-poly(aspartate) segment as the above-mentioned L-amino acid derivative, microparticles thereof, and use thereof in pharmaceutical compositions. BACKGROUND

[0002] In patients with liver damage such as cirrhosis, a condition such as hyperammonemia can sometimes occur due to abnormal urea cycle. If L-ornithine is administered to such a patient, it functions as a substrate for urea production, and furthermore, it has been reported that the enzymes of the urea cycle including carbamoyl phosphate synthetase (CPS), ornithine transcarbamoylase (OTC) are activated, as a result of which the urea cycle is improved and the ammonia level is reduced (Non-Patent Literature 1).

[0003] Thus, it has been reported that L-ornithine and a mixture of L-ornithine and L-aspartate (LOLA) have an effect of favorably reducing ammonia concentration.

[0004] On the other hand, in Non-Patent Literature 2, it was confirmed by a double-blind randomized placebo-controlled study that the above-mentioned amino acid mixture has a very weak effect of injection. It is considered that this is because the amount of water-soluble low-molecular-weight amino acid taken into the body is low and it is metabolized very quickly.

[0005] In order to solve this problem, the inventors have designed a copolymer of poly(ethylene glycol) and poly(arginine) (polycationic polymer) and proposed a polyion complex (PIC) obtained by combining this with a polyanion such as chondroitin sulfate (Patent Literature 1). In addition, regarding a tissue-engineered (assembled) drug (abbreviated as Nano ORN ) composed of a PIC to which ornithine is added, it was shown that the blood retention and liver accumulation were greatly improved, and furthermore, if Nano ORN is administered to mice with acute liver damage caused by p-acetylaminophenol (APAP), the ammonia in the blood, which had significantly increased due to APAP liver damage, was not only reduced to the normal level, but also the levels of AST and ALT, which are markers of liver function, were meaningfully reduced, and the liver function was improved (Non-Patent Literature 3). In addition, it was confirmed that the functional improvement effect was also brought about in NASH model mice. ORN

[0006] In this way, by adding L-ornithine to the assembled drug and administering it subcutaneously, the bioavailability of L-ornithine can be improved and a liver function improvement effect can be obtained.

[0007] ​On the other hand, Nano ORN Sometimes unstable to changes in ionic strength and pH, and free the polycation and produce toxicity, etc., thus not good for oral administration.

[0008] Prior Art Documents

[0009] Patent Documents

[0010] Patent Document 1: International Publication No. 2016-167333 pamphlet

[0011] Non-Patent Documents

[0012] Non-Patent Document 1: Nutr. Res. 28(11) 738 (2008)

[0013] Non-Patent Document 2: Gastroenterology 136(7) 2159 (2009)

[0014] Non-Patent Document 3: Poly(ornithine)-based self-assembling drug for recovery of hyperammonemia and damage in acute liver injury, Long B. Vong, Yota Ibayashi, Yaroslav Lee, Dai-Nghiep Ngo, Yuji Nishikawa, Yukio Nagasaki, Journal of Controlled Release, 310, 74-81 (2019) SUMMARY

[0015] Problems to be Solved by the Invention

[0016] To solve the above problems, an object of the present application is to provide an assembly of ornithine which is low in toxicity, has an effect even when administered orally, and has high bioavailability. In addition, an object is to provide an ornithine assembly which is effective for the prevention or treatment of liver damage.

[0017] Means for Solving the Problems

[0018] The present inventors et al. found that by newly introducing an acyl group or the like into the ornithine side chain amino group of poly(ethylene glycol)-b-poly(ornithine), a copolymer which can form a new assembly which does not require the formation of a PIC can be provided. In addition, it was also found that when the copolymer thus provided is made into a composition with a copolymer containing other poly(ethylene glycol) segments and poly(side chain-protected L-amino acid) segments, the properties, functions, or effects of the former copolymer can sometimes be enhanced.

[0019] Accordingly, the present application provides each of the following embodiments.

[0020] (1) A copolymer represented by the formula (I),

[0021]

[0022] in the formula,

[0023] A represents (i) or (ii) formula, wherein,

[0024] (i) is a hydrogen atom, an unsubstituted or substituted C1-C 12 alkyl group, an unsubstituted or substituted C1-C 12 alkoxy group, an unsubstituted or substituted aryl group, and the substituents in the case of substitution are C1-C4 alkyl group, C1-C4 alkoxy group, aryl group, formyl group, R 1 R 2 CH- (wherein, R 1 and R 2 are independently C1-C4 alkoxy group or R 1 and R 2 are combined to represent -OCH2CH2O-, -O(CH2)3O- or -O(CH2)4O-),

[0025] (ii) formula

[0026]

[0027] wherein,

[0028] L' independently represents a linking group,

[0029] Y and Y' independently represent a hydrogen atom, C 1-21 alkyl group, substituted C 1-4 alkyl group, unsubstituted or substituted C 3-7 cycloalkyl group, unsubstituted or substituted arylcarbonyl group, or unsubstituted or substituted 5- or 6-membered heteroarylcarbonyl group, wherein the substituents of the substituted C 1-4 alkylcarbonyl group are selected from the group consisting of halogen atom, hydroxy group, carboxy group, unsubstituted or substituted C 3-7 alkyl group, unsubstituted or substituted aryl group and unsubstituted or substituted 5- or 6-membered heteroaryl group, unsubstituted or substituted adamantyl group, unsubstituted or substituted cholesteric residue, and the substituents in the case of substitution can be C 1-4 alkyl group, C 1-4 alkoxy group, hydroxy group, carboxy group, cyano group, nitro group, halogen atom, or mono- or di-C 1-4 alkylamino group,

[0030] R 10 and R10 R' is a hydrogen atom or R 11 -(C=O)-, R 11 each independently a substituted or unsubstituted C1-C 21 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted C1-C 21 alkoxy, the substituents in the case where these substituents are substituted being C 1-4 alkyl, C 1-4 alkoxy, aryl,

[0031] m and m' are independently integers of 2 to 300,

[0032] n is an integer of 2 to 1,000,

[0033] m or m' of R 10 and R 10 of 20% or more are R 11 -(C=O)-.

[0034] (2) The copolymer according to the embodiment (1), wherein R 10 of formula (I) is R 11 -(C=O)-, m of R 10 of 100% are R 11 -(C=O)-.

[0035] (3) The copolymer according to the embodiment (1), wherein A is defined by (i).

[0036] (4) The copolymer according to the embodiment (1), wherein A is defined by (i), R 10 of formula (I) is R 11 -(C=O)-, m of R 10 of 100% are R 11 -(C=O)-.

[0037] (5) The copolymer according to the embodiment (1), wherein A is defined by (ii).

[0038] (6) The copolymer according to the embodiment 1, wherein A is defined by (ii), R 10 and R 10 of formula (I) are R 11 -(C=O)-, m and m' of R 10 and R 10 of 100% are R 11 -(C=O)-.

[0039] (7) An ornithine microparticle composed of the copolymer according to any one of the embodiments (1) to (6), and having an average particle diameter of 1 nanometer to 100 micrometers.

[0040] (8) A pharmaceutical composition comprising the copolymer contained in any one of embodiments (1) to (6) as an active ingredient and an additive.

[0041] (9) The pharmaceutical composition according to embodiment (8), wherein the pharmaceutical composition is an oral administration agent.

[0042] (10) A composition for the prevention or treatment of liver dysfunction, comprising, as an active ingredient, any copolymer of any one of embodiments 1 to 6 or ornithine microparticles of embodiment (7).

[0043] (11) A pharmaceutical composition comprising the copolymer included as an active ingredient in any one of embodiments (1) to (6) or the ornithine microparticles described in embodiment 7 and the copolymer represented by formula (IV),

[0044]

[0045] In the formula,

[0046] A ASP Represents hydrogen atoms, unsubstituted or substituted C1-C 12 Alkyl, unsubstituted or substituted C1-C 12 Alkoxy, unsubstituted or substituted aryl, and in the case of substitution, the substituents represent C1-C4 alkyl, C1-C4 alkoxy, aryl, formyl, or formula R. 1 R 2 CH-(wherein, R) 1 and R 2 Independently C1-C4 alkoxy or R 1 and R 2 The groups that combine to represent -OCH2CH2O-, -O(CH2)3O-, or -O(CH2)4O- are groups.

[0047] L ASP Indicates a linking group.

[0048] R ASP It is a hydrogen atom, a substituted or unsubstituted C1-C atom. 21 Alkyl, substituted or unsubstituted aryl groups, where the substituent is C when the substituent is substituted. 1-4 Alkyl, C 1-4 Alkoxy, aryl,

[0049] Y ASP Represents hydrogen atom, C 1-21 Alkyl carbonyl, substituted C 1-4 Alkyl carbonyl, unsubstituted or substituted C 3-7cycloalkylcarbonyl, unsubstituted or substituted arylcarbonyl, or unsubstituted or substituted 5- or 6-membered heteroarylcarbonyl, wherein the substituents of the substituted C 1-4 The substituents of the alkylcarbonyl group are selected from the group consisting of a halogen atom, a hydroxy group, a carboxy group, an unsubstituted or substituted C 3-7 cycloalkyl, unsubstituted or substituted aryl, and unsubstituted or substituted 5- or 6-membered heteroaryl, unsubstituted or substituted adamantyl, and unsubstituted or substituted cholesteric residue, and the substituents in the case of substitution of these substituents can be C 1-4 alkyl, C 1-4 alkoxy, a hydroxy group, a carboxy group, a cyano group, a nitro group, a halogen atom, or a mono- or di-C 1-4 alkylamino,

[0050] m A is an integer of 2 to 300,

[0051] n A is an integer of 2 to 1,000.

[0052] (12) A pharmaceutical composition comprising, as an effective ingredient, the copolymer contained in any one of embodiments (1) to (6) or the ornithine microparticle and the copolymer represented by formula (V) described in embodiment 7,

[0053]

[0054] in the formula,

[0055] A CO represents a hydrogen atom, an unsubstituted or substituted C1-C 12 alkyl, an unsubstituted or substituted C1-C 12 alkoxy, an unsubstituted or substituted aryl, the substituents in the case of substitution representing C1-C4 alkyl, C1-C4 alkoxy, aryl, formyl, a group of the formula R 1 R 2 CH- (wherein, R 1 and R 2 are independently C1-C4 alkoxy or a group of R 1 and R 2 are combined to represent -OCH2CH2O-, -O(CH2)3O- or -O(CH2)4O-,

[0056] n CO is an integer of 2 to 1,000,

[0057] L CO represents a linking group,

[0058] R ASP is a hydrogen atom, a substituted or unsubstituted C1-C 21Alkyl, substituted or unsubstituted aryl groups, where the substituent is C when the substituent is substituted. 1-4 Alkyl, C 1-4 Alkoxy, aryl,

[0059] m A It is an integer between 2 and 300.

[0060] R ORN It is a hydrogen atom or R 11 -(C=O)-,R 11 Each is independently either substituted or unsubstituted C1-C 21 Alkyl, substituted or unsubstituted aryl, substituted or unsubstituted C1-C 21 Alkoxy groups, where the substituent is C when these substituents are replaced. 1-4 Alkyl, C 1-4 Alkoxy, aryl,

[0061] m O It is an integer between 2 and 300.

[0062] Y CO Represents hydrogen atom, C 1-21 Alkyl carbonyl, substituted C 1-4 Alkyl carbonyl, unsubstituted or substituted C 3-7 Cycloalkyl carbonyl, unsubstituted or substituted aryl carbonyl, or unsubstituted or substituted 5 or 6-membered heteroaryl carbonyl, wherein the substituted C 1-4 The substituents of the alkyl carbonyl group are selected from halogen atoms, hydroxyl groups, carboxyl groups, and unsubstituted or substituted carbon atoms. 3-7 In the group consisting of cycloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted 5- or 6-membered heteroaryl, unsubstituted or substituted adamantyl, and unsubstituted or substituted cholesterol residues, the substituents can be C when these substituents are substituted. 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, carboxyl, cyano, nitro, halogen atom, or single or double carbon atom 1-4 Alkylamino,

[0063] Where, m A repeating units and m O Each amino acid in a repeating unit exists randomly to form another block.

[0064] (13) A pharmaceutical composition comprising, as an active ingredient, the copolymer contained in any one of embodiments (1) to (6) or the ornithine microparticles described in embodiment 7 and the copolymer represented by formula (VI),

[0065]

[0066] In the formula,

[0067] A ARG is a hydrogen atom, a substituted or unsubstituted C1-C 21 alkylcarbonyl group, a substituted or unsubstituted arylcarbonyl group, a substituted or unsubstituted C1-C 21 alkoxy group, and the substituents in the case where the substituents are substituted are C 1-4 alkyl group, a C 1-4 alkoxy group, an aryl group,

[0068] n G is an integer of 2 to 1,000,

[0069] L ARG represents a linking group,

[0070] R ARG and R ARG are independently a hydrogen atom or R 11 -(C=O)-, R 11 are each independently a substituted or unsubstituted C1-C 21 alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted C1-C 21 alkoxy group, and the substituents in the case where the substituents are substituted are C 1-4 alkyl group, a C 1-4 alkoxy group, an aryl group,

[0071] m G is an integer of 2 to 300,

[0072] Y ARG represents a hydrogen atom, a C 1-21 alkylcarbonyl group, a substituted C 1-4 alkylcarbonyl group, an unsubstituted or substituted C 3-7 cycloalkylcarbonyl group, an unsubstituted or substituted arylcarbonyl group, or an unsubstituted or substituted 5- or 6-membered heteroarylcarbonyl group, wherein the substituents of the substituted C 1-4 alkylcarbonyl group are selected from the group consisting of a halogen atom, a hydroxyl group, a carboxyl group, an unsubstituted or substituted C 3-7 cycloalkyl group, an unsubstituted or substituted aryl group, and an unsubstituted or substituted 5- or 6-membered heteroaryl group, an unsubstituted or substituted adamantyl group, an unsubstituted or substituted cholesteric residue, and the substituents in the case where the substituents are substituted can be C 1-4 alkyl group, a C 1-4 alkoxy group, a hydroxyl group, a carboxyl group, a cyano group, a nitro group, a halogen atom, or a mono- or di-C 1-4 alkylamino group.

[0073] (14) A copolymer represented by formula (IV-a),

[0074]

[0075] AASP represents a hydrogen atom, an unsubstituted or substituted C1-C4 alkyl group, an unsubstituted or substituted C1-C4 alkoxy group, an unsubstituted or substituted aryl group, a formyl group, a group of the formula 12 represents a hydrogen atom, an unsubstituted or substituted C1-C4 alkyl group, an unsubstituted or substituted C1-C4 alkoxy group, an unsubstituted or substituted aryl group, a formyl group, a group of the formula 12 represents a hydrogen atom, an unsubstituted or substituted C1-C4 alkyl group, an unsubstituted or substituted C1-C4 alkoxy group, an unsubstituted or substituted aryl group, a formyl group, a group of the formula 1 R 2 CH- (wherein, R 1 and R 2 are independently a C1-C4 alkoxy group or R 1 and R 2 taken together represent a group of -OCH2CH2O-, -O(CH2)3O- or -O(CH2)4O-,

[0076] L ASP represents a linking group,

[0077] Y ASP represents a C 1-21 alkyl group, a substituted or unsubstituted C1-C4 alkyl group, a substituted or unsubstituted C1-C4 alkoxy group, a substituted or unsubstituted aryl group, a formyl group, a group of the formula 1-4 alkyl group, a substituted or unsubstituted C1-C4 alkyl group, a substituted or unsubstituted C1-C4 alkoxy group, a substituted or unsubstituted aryl group, a formyl group, a group of the formula 3-7 cycloalkyl group, a substituted or unsubstituted aryl group and a substituted or unsubstituted 5- or 6-membered heteroaryl group, a substituted or unsubstituted adamantyl group, a substituted or unsubstituted cholesteric residue, the substituents in the case of substitution being a C 1-4 alkyl group, a substituted or unsubstituted C1-C4 alkyl group, a substituted or unsubstituted C1-C4 alkoxy group, a substituted or unsubstituted aryl group, a formyl group, a group of the formula 3-7 alkyl group, a substituted or unsubstituted C1-C4 alkyl group, a substituted or unsubstituted C1-C4 alkoxy group, a substituted or unsubstituted aryl group, a formyl group, a group of the formula 1-4 alkyl group, a C 1-4 alkoxy group, a hydroxy group, a carboxyl group, a cyano group, a nitro group, a halogen atom, or a mono- or di-C 1-4 alkyl group, a C

[0078] R ASP is a substituted or unsubstituted C1-C4 alkyl group, 21 alkyl group, a substituted or unsubstituted C1-C4 alkyl group, a substituted or unsubstituted C1-C4 alkoxy group, a substituted or unsubstituted aryl group, a formyl group, a group of the formula 1-4 alkyl group, a C 1-4 alkoxy group, an aryl group, but benzyl is excluded,

[0079] m A is an integer of 2 to 300,

[0080] n A is an integer of 2 to 1,000.

[0081] (15) A copolymer represented by the formula (V),

[0082]

[0083] wherein

[0084] A CO represents a hydrogen atom, an unsubstituted or substituted C1-C4 alkyl group, an unsubstituted or substituted C1-C4 alkoxy group, an unsubstituted or substituted aryl group, or a substituted case substituent represents a C1-C4 alkyl group, a C1-C4 alkoxy group, an aryl group, a formyl group, a group of the formula R 12 R 12 R 1 R 2 CH- (wherein, R 1 and R 2 are independently a C1-C4 alkoxy group or R 1 and R 2 are combined to represent a group of -OCH2CH2O-, -O(CH2)3O- or -O(CH2)4O-,

[0085] n CO is an integer of 2 to 1,000,

[0086] L CO represents a linking group,

[0087] R ASP is a hydrogen atom, a substituted or unsubstituted C1-C4 alkyl group, a substituted or unsubstituted aryl group, a substituted case substituent of the C1-C4 alkyl group, a C1-C4 alkoxy group, an aryl group, or a formyl group, 21 R 1-4 R 1-4 R

[0088] m A is an integer of 2 to 300,

[0089] R ORN is a hydrogen atom or R 11 -(C=O)-, R 11 are each independently a substituted or unsubstituted C1-C4 alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted C1-C4 alkoxy group, a substituted case substituent of the C1-C4 alkyl group, a C1-C4 alkoxy group, an aryl group, or a formyl group, 21 R 21 R 1-4 R 1-4 R

[0090] m O is an integer of 2 to 300,

[0091] Y CO represents a hydrogen atom, a C1-C4 alkyl group, a C1-C4 alkylcarbonyl group, a substituted C1-C4 alkylcarbonyl group, an unsubstituted or substituted C3-C7 cycloalkylcarbonyl group, an unsubstituted or substituted arylcarbonyl group, or an unsubstituted or substituted 5- or 6-membered heteroarylcarbonyl group, 1-21 R 1-4 R 3-7 R1-4 The substituents of the alkylcarbonyl group are selected from the group consisting of a halogen atom, a hydroxy group, a carboxy group, an unsubstituted or substituted C 3-7 unsubstituted or substituted aryl group and an unsubstituted or substituted 5- or 6-membered heteroaryl group, an unsubstituted or substituted adamantyl group, an unsubstituted or substituted cholesteric residue, and the substituents in the case where these substituents are substituted can be C 1-4 alkyl, C 1-4 alkoxy, a hydroxy group, a carboxy group, a cyano group, a nitro group, a halogen atom, or a mono- or di-C 1-4 alkylamino,

[0092] wherein m A is an integer of 2 to 1,000, O each of the amino acids in the m

[0093] (16) A copolymer represented by the formula (VI-a),

[0094]

[0095] in the formula,

[0096] A ARG is a hydrogen atom, an unsubstituted or substituted C1-C 21 alkylcarbonyl group, an unsubstituted or substituted arylcarbonyl group, an unsubstituted or substituted C1-C 21 alkoxy carbonyl group, and the substituents in the case where these substituents are substituted are C 1-4 alkyl, C 1-4 alkoxy, an aryl group,

[0097] n G is an integer of 2 to 1,000,

[0098] L ARG represents a linking group,

[0099] R ARG and R ARG are independently R 11 -(C=O)-, R 11 each independently an unsubstituted or substituted C1-C 21 alkyl group, an unsubstituted or substituted aryl group, an unsubstituted or substituted C1-C 21 alkoxy group, and the substituents in the case where these substituents are substituted are C 1-4 alkyl, C 1-4 alkoxy, an aryl group, but a benzyloxy carbonyl group and a tert-butyl oxy carbonyl group are excluded,

[0100] m G is an integer of 2 to 300,

[0101] YARG Represents hydrogen atom, C 1-21 Alkyl carbonyl, substituted C 1-4 Alkyl carbonyl, unsubstituted or substituted C 3-7 Cycloalkyl carbonyl, unsubstituted or substituted aryl carbonyl, or unsubstituted or substituted 5 or 6-membered heteroaryl carbonyl, wherein the substituted C 1-4 The substituents of the alkyl carbonyl group are selected from halogen atoms, hydroxyl groups, carboxyl groups, and unsubstituted or substituted carbon atoms. 3-7 In the group consisting of cycloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted 5- or 6-membered heteroaryl, unsubstituted or substituted adamantyl, and unsubstituted or substituted cholesterol residues, the substituents can be C when these substituents are substituted. 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, carboxyl, cyano, nitro, halogen atom, or single or double carbon atom 1-4 Alkylamino.

[0102] The effects of the invention

[0103] The assembled drug comprising the copolymer of the present invention as an active ingredient can be administered orally and used as a novel treatment for liver injury. In particular, by oral administration, it accumulates on the intestinal mucosa and is hydrolyzed by degrading enzymes, gradually releasing ornithine, thus resulting in high bioavailability of ornithine in vivo and a sustained effect. Furthermore, the assembled drug of the present invention exhibits low toxicity. Attached Figure Description

[0104] Figure 1 The compound synthesized in Preparation Example 4 1 H-NMR spectrum.

[0105] Figure 2 The compound synthesized in Preparation Example 5 1 H-NMR spectrum.

[0106] Figure 3 The compound synthesized in Preparation Example 6 1 H-NMR spectrum.

[0107] Figure 4 The compound synthesized in Preparation Example 7 1 H-NMR spectrum.

[0108] Figure 5 It is Nano ORN(iBu) The results of dynamic light scattering measurement.

[0109] Figure 6 It is Nano ORN(Z) Nano ORN(Me) Nano ORN(iBu)The results of pH-dependent determination of particle size (left) and scattering intensity (right) by dynamic light scattering meter.

[0110] Figure 7 The results show the effects of ornithine nanotissues on serum ammonia concentration (a), ALT (b), and AST in mice with acute liver injury caused by acetaminophen (APAP).

[0111] Figure 8 This is a hematoxylin-eosin (H&E) staining image of liver tissue in mice with acute liver injury due to APAP after administration of ornithine nanoassemblies.

[0112] Figure 9 This refers to the change in body weight after administration of the nanoassemblies to normal mice.

[0113] Figure 10 This refers to the changes in white blood cell and platelet counts after administration of the nanoassemblies to normal mice.

[0114] Figure 11 This is a graph showing the survival rate of nanoassemblies after administration to mice with acute liver injury caused by APAP.

[0115] Figure 12 yes 125 I-Tagged Nanoassemblies (Nano ORN(iBu) The dynamic analysis results after oral administration of )

[0116] Figure 13 It is various polyornithine nanoassemblies (Nano ORN The results of the cytotoxicity evaluation.

[0117] Figure 14 The compound synthesized in Preparation Example 16 1 H-NMR spectrum.

[0118] Figure 15 The compound synthesized in Preparation Example 17 1 H-NMR spectrum.

[0119] Figure 16 It is Nano ASP(Bz) Nano ASP(Me) Nano ASP / ORN The results of pH-dependent determination of particle size by dynamic light scattering meter.

[0120] Figure 17 It is Nano ASP(Bz) Nano ASP(Me) Nano ASP / ORNThe results of evaluation of blood ammonia concentration (a), ALT (b), and AST of the mice with acute liver injury induced by APAP.

[0121] Figure 18 The compound synthesized in Preparation Example 21 is 1 H-NMR spectrum.

[0122] Figure 19 The compound synthesized in Preparation Example 22 is 1 H-NMR spectrum.

[0123] Figure 20 Nano ARG(Ac) , Nano ARG(iBu) Dynamic light scattering measurement results.

[0124] Figure 21 The effect of acetylated arginine assembly on APAP-induced acute liver injury mice orally taken.

[0125] Figure 22 The effect of acetylated arginine assembly on non-alcoholic steatohepatitis (NASH) model mice. DETAILED DESCRIPTION

[0126] The technical terms used in the present specification are used as terms representing the meanings commonly used in the art, unless otherwise defined.

[0127] <copolymer>

[0128] The copolymer of the present application is represented by Formula (I) of Embodiment (1).

[0129] A in Formula (I) represents (i) or a group represented by (ii) formula,

[0130] wherein (i) is a hydrogen atom, an unsubstituted or substituted C1-C4 alkyl group, an unsubstituted or substituted C1-C4 alkoxy group, an unsubstituted or substituted aryl group, and the substituent group in the case of substitution is a C1-C4 alkyl group, a C1-C4 alkoxy group, an aryl group, a formyl group, a formula R 12 R 12 CH- (wherein, R 1 and R 2 are independently a C1-C4 alkoxy group or R 1 and R 2 are independently a C1-C4 alkoxy group or R 1 and R 2The group represented by L and / or L' in formula (I) can be an unbranched or branched alkyl group, and examples thereof include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, pentyl, hexyl, heptyl, nonyl, undecyl, tridecyl, heptadecyl, nonadecyl, and the like. Preferably, the alkyl group is selected from C

[0131] The linking group L and / or L' in formula (I) can be an unbranched or branched alkyl group, and examples thereof include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, pentyl, hexyl, heptyl, nonyl, undecyl, tridecyl, heptadecyl, nonadecyl, and the like. Preferably, the alkyl group is selected from C a -O-(CH2) a -O-(CH2) a -O-(CH2) a -O-(CH2) a -O-(CH2) a -O-(CH2) a -O-(CH2) 1-21 -O-(CH2) 1-4 -O-(CH2) 3-7 -O-(CH2)

[0132] Y and Y' in formula (I) each independently represent a hydrogen atom, an unsubstituted or substituted C 1-21 -O-(CH2) 1-4 -O-(CH2) 3-7 -O-(CH2)

[0133] The alkyl group as each of the above groups or a part of each of the above groups can be an unbranched or branched alkyl group, and although not limited, examples thereof include the corresponding alkyl groups among methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, pentyl, hexyl, heptyl, nonyl, undecyl, tridecyl, heptadecyl, nonadecyl, and the like. Preferably, the alkyl group is selected from C 1-6 -O-(CH2) 3-7The cycloalkyl group can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl. The aryl group can be phenyl, biphenyl, naphthyl. The 5- or 6-membered heteroaryl group is an unsaturated heterocyclic group containing 1 or 2 identical or different heteroatoms selected from oxygen, nitrogen and sulfur atoms, which can be thienyl, furanyl, pyranyl, pyrrolyl, isoxazole, pyrazole, imidazole, pyridyl, pyrazine, pyrimidine, and in addition, these heterocyclic rings can also be benzo-fused rings. As such fused rings, for example, isoindolyl, indolyl, quinolyl, isoquinolyl, quinoxalyl, quinazolyl, phenanthridyl can be mentioned.

[0134] substituted C 1-4 The substituents of the alkylcarbonyl group are selected from the group consisting of a halogen atom (Cl, F, Br, I), a hydroxy group, a carboxyl group, an unsubstituted or substituted C 3-7 The substituents of the cycloalkyl group, the unsubstituted or substituted aryl group and the unsubstituted or substituted 5- or 6-membered heteroaryl group, the unsubstituted or substituted adamantyl group, the unsubstituted or substituted cholesteric residue, in the case of substitution, can be C 1-4 alkyl, C 1-4 alkoxy, a hydroxy group, a carboxyl group, a cyano group, a nitro group, a halogen atom, or a mono- or di-C 1-4 alkylamino group. The last substituent is also applicable to the substituted C 3-7 cycloalkylcarbonyl group, a substituted arylcarbonyl group, or a substituted 5- or 6-membered heteroarylcarbonyl group.

[0135] In the above definition, the cholesteric residue can be a residue in which any one of the H of the carbon at the 22nd to 27th position of the cholesteric molecule is removed or a hydrocarbon chain containing any one of the carbons at the 22nd to 27th position is removed. As such a residue-substituted alkylcarbonyl group, for example, cholic acid, chenodeoxycholic acid can be mentioned. As Y, C 1-6 alkylcarbonyl group.

[0136] R 10 and R 10 ' are independently a hydrogen atom or R 11 -(C=0). Although not bound by theory, in the case where R 10 and R 10 ' contain R 11 -(C=0), the amide bond formed from the δ-amino group thereof and the polyornithine segment and the amide (or peptide) bond of the polyornithine main chain are each a target of enzymatic decomposition, and it can be considered that free ornithine is released, for example, it can be considered that the ornithine is decomposed by digestive enzymes in the digestive tract and gradually released.

[0137] R 11 is a substituted or unsubstituted C1-C 21 alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted C1-C 21alkyl, C 1-4 alkyl, C 1-4 alkyl, C 11 may be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, (may also be branched) pentyl, (may also be branched) hexyl, (may also be branched) heptyl, (may also be branched) octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, phenyl, naphthyl, benzyloxy, tert-butyloxy. These groups are preferable from the viewpoint of efficiency in assembling and forming ornithine microparticles from the copolymer. On the other hand, R 11 is preferably methyl, propyl, isopropyl, butyl, isobutyl, (may have a substituent) phenyl, (may have a substituent) benzyl.

[0138] For example, in the case where R 11 is methyl, it can be obtained by using, for example, acetic anhydride as the carboxylate in the condensation reaction of formula (II) described in the production of the copolymer described below. Similarly, in the case where R 11 is ethyl, for example, propionic anhydride is used, in the case where R 11 is propyl, for example, butyric anhydride is used, in the case where R 11 is isopropyl, for example, isobutyric anhydride is used, in the case where R 11 is butyl, for example, valeric anhydride is used, in the case where R 11 is isobutyl, for example, isovaleric anhydride is used, in the case where R 11 is phenyl, for example, benzoic anhydride is used, in the case where R 11 is benzyl, for example, diphenylacetic anhydride is used, and in this way, by changing the kind of carboxylic acid, the desired R 11 can be produced. By using a carboxylate or derivative having a substituent as the carboxylate, respectively, R 11 can also have a substituent.

[0139] From the viewpoint of stability of the particles formed from the copolymer, m and m' can independently be preferably an integer of 2 to 300, more preferably an integer of 15 to 150, and most preferably an integer of 15 to 100.

[0140] Similarly, n can be preferably an integer of 2 to 1000, more preferably an integer of 10 to 500, and most preferably an integer of 30 to 400.

[0141] As for m and m' of R 10 and R 10are independently, usually, 80%, can preferably be from 60%, more preferably from 30%, most preferably from 10%, and, especially, most preferably, all (100%) of m and m' are -R 11 -(C=O).

[0142] <Preparation of the copolymer>

[0143] Hereinafter, an example of a scheme for the synthesis of the copolymer is shown. The following is merely an example and the present application is not limited by this example.

[0144] The copolymer can contain corresponding PEG segments and polyornithine segments prepared by any method as long as the object of the present application is achieved. However, the copolymer is preferably one having a narrow molecular weight distribution and capable of self-assembly in an aqueous medium to form ornithine microparticles having an average diameter of an appropriate size.

[0145] A copolymer represented by the following formula (II) is prepared in advance, and then, in order to introduce an acyl group into the δ-amino group in the segment from L-ornithine in the formula, a compound such as a carboxylate can be added.

[0146]

[0147] [In the formula,

[0148] A represents (i)' or (ii)' formula, wherein,

[0149] (i)' is a hydrogen atom, an unsubstituted or substituted C1-C4 alkyl group, an unsubstituted or substituted C1-C4 alkoxy group, an unsubstituted or substituted aryl group, a formyl group, a formula R 12 alkyl group, an unsubstituted or substituted C1-C4 alkoxy group, an unsubstituted or substituted aryl group, a formyl group, a formula R 12 alkyl group, an unsubstituted or substituted C1-C4 alkoxy group, an unsubstituted or substituted aryl group, a formyl group, a formula R 1 R 2 CH- (wherein, R 1 and R 2 are independently a C1-C4 alkoxy group or R 1 and R 2 are combined to represent -OCH2CH2O-, -O(CH2)3O- or -O(CH2)4O-,

[0150] (ii)' formula

[0151]

[0152] L and L' independently represent a linking group,

[0153] Y and Y' independently represent a hydrogen atom, a C 1-21 alkylcarbonyl group, a substituted C1-4 alkylcarbonyl, unsubstituted or substituted C 3-7 cycloalkylcarbonyl, unsubstituted or substituted arylcarbonyl, or unsubstituted or substituted 5- or 6-membered heteroarylcarbonyl, wherein the substituents of the substituted C 1-4 the substituents of the alkylcarbonyl are selected from the group consisting of a halogen atom, a hydroxyl group, a carboxyl group, unsubstituted or substituted C 3-7 cycloalkyl, unsubstituted or substituted aryl and unsubstituted or substituted 5- or 6-membered heteroaryl, unsubstituted or substituted adamantyl, unsubstituted or substituted cholesteric residue, and the substituents of these substituents in the case of being substituted can be C 1-4 alkyl, C 1-4 alkoxy, a hydroxyl group, a carboxyl group, a cyano group, a nitro group, a halogen atom, or mono- or di-C 1-4 alkylamino,

[0154] m and m' are independently an integer of 2 to 300, and n is an integer of 2 to 1,000.

[0155] A typical example of such a preparation can be exemplified by the condensation reaction of the copolymer represented by the formula (II) with a carboxylic acid ester represented by the following scheme.

[0156]

[0157] As examples of the carboxylic acid ester, there can be exemplified a carboxylic acid halide, a carboxylic anhydride, a carboxylic azide, an active ester, etc., and specifically, there can be used acetic anhydride, propionic anhydride, isobutyric anhydride, butyric anhydride, oxalic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride, maleic anhydride, phthalic anhydride, benzoic anhydride, bisphenylacetic anhydride, etc.

[0158] Here, when A in the formula (II) in the above reaction scheme corresponds to A defined by the above (ii)', the δ-amino group in the segment derived from ornithine in which m' in the formula is recorded as a repeating unit undergoes the condensation reaction similarly to the δ-amino group of the repeating unit of the above m.

[0159] Further, in order to provide the copolymer of the present application having a narrow molecular weight distribution, the precursor copolymer represented by the formula (II) (in the case where A of the formula (II) is represented by (i)' of the formula) is preferably prepared according to the following synthesis scheme.

[0160]

[0161] Here, the compound 1 uses a commercially available or a compound prepared by a method complying with the method of preparation thereof, which has a molecular weight distribution as narrow as possible, and for this compound, an active ring-opening polymerization of the N-carboxyanhydride of ornithine having an amino group protected at the δ position is performed, and then, a self-known method of stopping the reaction using an active end modifier such as acetic anhydride is performed, thereby preparing the compound 2, and further, the amino protecting group in the poly(L-ornithine) segment is removed.

[0162] According to such a reaction treatment using the compound 1 as a starting material, a precursor copolymer having a poly(L-ornithine) segment with a molecular weight distribution as narrow as possible can be provided.

[0163] Therefore, the block copolymer represented by the formula (I) according to the present application, which is finally obtained, can provide a block copolymer having a molecular weight distribution of 1.01 to 1.20, preferably 1.01 to 1.06.

[0164] In this case, in the case where A of the formula (II) is defined by (ii)', as the starting material in the above reaction scheme, i.e., the compound 1, NH2CH2CH2-(OCH2CH2) n -NH2, each of the A portions of the compounds 2 to 4 can be a repeating unit corresponding to m repeating units. The triblock copolymer of the formula (II) in which A is defined by (ii)' thus obtained is represented by the following formula (III).

[0165]

[0166] In the formula, L, L', Y, Y', m, m', and n are the same as the definitions in the above formula (II).

[0167] In addition, as another example, the copolymer can also be synthesized starting from the above compound 1 and by a synthesis method represented by the following scheme.

[0168]

[0169] <Ornithine microparticle>

[0170] By allowing the copolymer represented by the formula (I) to assemble in water or an aqueous medium (may also contain a water-soluble organic solvent, a buffer), an ornithine microparticle can be prepared. Since the polyornithine segment containing R 10 and R 10 ' shows hydrophobicity, it is considered that the copolymer coagulates and self-assembles to form an ornithine microparticle having a polyornithine segment as a core and a PEG segment as a shell. For example, by dissolving the copolymer represented by the formula (I) in a polar organic solvent such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and dialyzing in water, an aqueous solution or a buffer, an ornithine microparticle can be obtained.

[0171] The ornithine microparticle (or assembly) is not particularly limited, but from the viewpoint of ease of administration or preparation, the average particle diameter is preferably 1 mm or less, preferably 1 nm to 1 mm, and more preferably 1 nm to 100 μm. Further, 10 nm to 200 nm, and most preferably 20 nm to 70 nm. The particle diameter can be measured by dynamic light scattering (DLS) in water or an aqueous medium, or the like.

[0172] Further, for example, by adding a solution of a polyanionic compound polymer such as chondroitin to compound 3 or formula (II) and forming a complex thereof, a polyelectrolyte complex particle (PIC) can be obtained. On the other hand, the ornithine microparticle of the present application is obtained by self-assembly of poly(ethylene glycol)-b-poly(ornithine) copolymer or a derivative thereof in an aqueous medium, and is characterized by not forming a complex with other polyanionic compound polymers. Thus, the ornithine microparticle of the present application is distinguished from a PIC.

[0173] Thus, preferably, the ornithine microparticle of the present application is characterized by containing poly(ethylene glycol)-b-poly(ornithine) polymer or a derivative thereof as a main component, and not containing a polyanionic polymer or the like. As examples of the polyanionic polymer, polyacrylic acid, polymethacrylic acid, polysulfonic acid, polyanionic polysaccharides, anionic proteins, and the like can be given, and more specifically, chondroitin sulfate, carrageenan, heparin, carboxymethyl dextran, xanthan gum, hyaluronic acid, polyaspartic acid, polyglutamic acid, polyacrylic acid, polymethacrylic acid, polyvinyl sulfate, polystyrene sulfonic acid, and the like can be given. However, the ornithine microparticle of the present application can contain an aqueous medium, a solvent, or the like as long as it does not adversely affect the stability of the particle.

[0174] The obtained ornithine microparticle can be isolated by a separation means such as centrifugation, and further, by freeze-drying, it can be stored as a dry composition, and as needed, it can be reconstituted in an aqueous medium. As needed, such a dry composition can be provided as an aqueous solution of the ornithine microparticle containing a physiologically acceptable diluent or excipient. Such a diluent can be a solution containing sterilized water, physiological saline, a physiologically acceptable buffer, or the like, and further, as an additive, for example, sorbitol, dextrin, glucose, mannitol, amino acids (e.g., glycine, isoleucine, valine, methionine, glutamic acid, and the like), and the like can be given.

[0175] Further, in other words, as described above, as one embodiment, the present application provides a method for producing an ornithine microparticle. That is, the present application provides a method for producing an ornithine microparticle, characterized by reacting a carboxylate compound with a copolymer represented by the above formula (II) to introduce a δ-amino group in a segment from ornithine to R 10substituents (but R 10 is R 11 -(C=O)-, R 11 is a substituted or unsubstituted C1-C 21 alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted C1-C 21 alkoxy group, and the substituents in the case of substitution of these substituents are C 1-4 alkyl groups, C 1-4 alkoxy groups, aryl groups), and the resulting copolymer is assembled in water.

[0176] <Further copolymers (including the case where a new compound is present) that can be used together with the above or below-described additives or independently as other additives>

[0177] The copolymer represented by formula (I) or ornithine microparticles from the copolymer can be provided as an additive together with the above-described additives or independently, as a pharmaceutical composition containing the following copolymer, for example, as a pharmaceutical composition for preventing or treating liver dysfunction.

[0178] • a copolymer represented by formula (IV):

[0179] In the formula, A ASP and L ASP are synonymous with the definitions of A(i) and L in formula (I), and specifically, the explanations for L in formula (I) can be applied. Y ASP is synonymous with the definition of Y in formula (I). Further, R ASP is a hydrogen atom, a substituted or unsubstituted C1-C 21 alkyl group, a substituted or unsubstituted aryl group, the substituents in the case of substitution of these substituents are C 1-4 alkyl groups, C 1-4 alkoxy groups, aryl groups, and the explanations for the above-described C1-C 21 alkyl groups and aryl groups can be applied to the explanations for R 11 in formula (I). Further, as a specific L ASP linking group, the specific explanations for L are synonymous.

[0180] m A is an integer of 2 to 300, preferably an integer of 15 to 150, more preferably an integer of 15 to 100,

[0181] n A is an integer of 2 to 1,000, preferably an integer of 10 to 500, more preferably an integer of 30 to 400.

[0182] The copolymer represented by formula (Ⅳ) is obtained by the following method: In the reaction schemes of compounds 1 to 4 described above, instead of the N-carboxylic anhydride of ornithine with a protected δ-amino group, a benzyl or alkylated N-carboxylic anhydride of aspartic acid, for example, is used, and its active ring-opening polymerization is performed on the omega (ω) terminal amino group of compound 1. This provides a compound in compound 2 where m protected ornithine units are replaced by protected aspartic acid units. The compound thus provided can be obtained, if desired, through known methods such as deprotection of the protecting group, esterification, or alkyl exchange reactions. Furthermore, in R... ASP In the case of hydrogen atoms or benzyl groups, compounds that are known to themselves may sometimes be included, but to the knowledge of the inventors, other compounds represented by formula (Ⅳ-a) are considered copolymers not described in the prior art literature. In addition, such copolymers can also autoassemble in an aqueous medium to form particles having the same size as the copolymer represented by formula (Ⅰ).

[0183] Since low molecular weight L-aspartic acid also has a similar effect to low molecular weight L-ornithine in expelling harmful ammonia from the body, the copolymer represented by formula (Ⅳ) can also increase the effect of the copolymer of formula (Ⅰ), or improve the stability of pharmaceutical compositions containing the copolymer of formula (Ⅰ) or its microparticles (assemblies).

[0184] • Copolymers represented by formula (V):

[0185] In the formula, A CO L CO n CO R ASP m A Each of them is related to A as defined in equation (Ⅳ) above. ASP L ASP n A R ASP m A Synonyms, A ORN R ORN m O and Y CO Each is synonymous with A(i), R, m, n, and Y as defined in equation (Ⅰ). In this copolymer, m... ASP Each unit and m ORN Each unit can exist randomly.

[0186] The copolymer represented by formula (V) is obtained by using, in place of the N-carboxyanhydride of δ-amino-protected ornithine in the reaction scheme of Compound 1 to Compound 4 described above, a mixture of the N-carboxyanhydride of the β-carboxyl of, for example, aspartic acid and the N-carboxyanhydride of δ-amino-protected ornithine, and allowing those N-carboxyanhydrides to actively ring-open polymerize at the omega (ω) terminal amino group of Compound 1 and further at the newly generated amino group, whereby a polymer in which m units of protected ornithine in Compound 2 are randomly replaced with protected aspartic acid units and protected ornithine units is provided, and the product can be obtained by deprotection of the protecting group per se, esterification, and / or acylation, and the like. In addition, the copolymer thus provided can be assembled in the same manner as the copolymer of formula (I).

[0187] In addition, the copolymer represented by formula (V) can have the same effects as the copolymer represented by formula (I) or (IV) and the assembly thereof.

[0188] • a copolymer represented by formula (VI):

[0189] In the formula, A ARG , L ARG , n G , m G , Y ARG each are synonymous with A ASP , L ASP , n A , m A , Y ARG defined for formula (IV) described above, and R ARG and R ARG are independently synonymous with R ASP defined for formula (IV) described above. The copolymer represented by formula (IV) can be prepared by reacting the copolymer represented by formula (IV) described in WO2016 / 167333A1 in which R ARG and R ARG are each H and Y ARG may be synonymous with Y ASP defined in formula (IV) with an anhydride or carboxylic acid capable of forming R ARG and R ARG '. The copolymer thus prepared can be assembled without using a polyanionic polymer. It was confirmed that these copolymers or assemblies thereof showed an antitumor effect against a lung cancer metastasis model mouse and a subcutaneously transplanted cancer model mouse by oral administration in addition to the effects (antitumor effect) described in WO2016 / 167333A1, and greatly reduced the adhesion of melanoma cells to the lung (data not published), and in addition to these, can have the same effects as the copolymer of formula (I) and the assembly thereof.

[0190] Composition for preventing or treating liver dysfunction

[0191] The ornithine microparticle (or assembly) formed of the copolymer of formula (I) of the present application is used as a composition for preventing or treating liver damage. That is, the present application provides, as one embodiment, a pharmaceutical composition using the ornithine microparticle of the present application as an effective ingredient for preventing or treating liver dysfunction.

[0192] Ornithine plays a function as a substrate for urea production in an organism, or activates enzymes of the urea cycle containing carbamoyl phosphate synthetase (CPS) or ornithine transcarbamylase (OTC). By these actions, an effect of preventing or treating liver damage can be expected.

[0193] The ornithine microparticle of the present application, once orally administered, is aggregated on the intestinal mucosa, the amide bond inside the polymer is hydrolyzed by gradual enzymatic decomposition and ornithine is released, thus effectively functioning in an organism, and has high bioavailability, as shown in the model animal experiment of the examples, and is very effectively used in the prevention or treatment of liver dysfunction.

[0194] Further, the so-called liver dysfunction includes viral hepatitis, drug- and alcohol-related liver disease, immune-mediated liver disease, metabolic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver failure, acute liver failure, hepatocellular carcinoma, and complications of liver transplantation, etc.

[0195] The ornithine microparticle or aqueous solution thereof in the pharmaceutical composition of the present application can be administered to a mammal in need of administration, particularly to a human, by oral administration. The pharmaceutical composition of the present application has low toxicity and is easily aggregated in the liver by injection, etc., and thus has the property of being hydrolyzed to release ornithine, and thus can also be administered directly to the vein, artery, subcutaneous, abdominal cavity, etc., and particularly more effectively functions in the intestine, and thus is more advantageously used as an oral administration agent.

[0196] As examples of the dosage form of the pharmaceutical composition, there can be mentioned liquid preparations (including internal water, suspensions, emulsions, syrups), solid preparations (including tablets, pills, sublinguals, capsules, drops, lozenges), granules, powders, and the like. Further, the solid preparation can also be prepared into a dosage form in which a well-known dosage form in the art is implemented, for example, sugar-coated tablets, gelatin-sealed tablets, enteric-coated tablets, film-coated tablets, double tablets, multi-layer tablets, as needed. As for the shape and size of each dosage form, it is included in the range of well-known dosage forms in the art, and can contain additives such as dissolving agents, pH adjusters, carriers, excipients, diluents, binders, disintegrants, lubricants, emulsifiers, extenders, coloring agents, flavoring agents, sweetening agents, stabilizers, preservatives, etc.

[0197] Further, the amount of administration, the number of administration are applied within the optimum concentration range according to the age, sex, state, the degree of disease of the patient, etc., but the amount of ornithine to be administered per 1 Kg of body weight is about 10 μmg to 1 g per day, more preferably 0.1 mg to 100 mg, which can be administered in 1 to 3 times or so, but the pharmaceutical combination of the present application is high in availability, so that the effect can be expected even if it is administered once a day.

[0198] The amount of administration, the method of administration, the number of administration are determined by the skilled in the art through animal experiments or experimental administration to humans, with reference to various literatures, etc.

[0199] Example

[0200] Hereinafter, the present application will be described in more detail by citing specific examples, but it is not intended to limit the present application to these examples.

[0201] Preparation Example 1: Synthesis of CH3O-(CH2CH2O) n -CH2CH2OSO2CH3 (N813)

[0202] CH3O-(CH2CH2O) n After adding THF (200 mL) and butyl lithium 14.4 mL (23 mmol, 1.6 M-hexane) to CH3O-(CH2CH2O) -H (MW=5,000, 60 g, 12 mmol), methylsulfonyl chloride (CH3SO2Cl) (1.5 mL, 2.3 g, 20 mmol) was added and reacted at 40°C for 1 day. After precipitating in 2-propanol (IPA), it was dissolved in methanol and precipitated. After repeating this operation twice, the precipitate was dried under reduced pressure to obtain the target (yield 60 g).

[0203] Preparation Example 2: Synthesis of CH3O-(CH2CH2O) n -CH2CH2NH2 (N819)

[0204] CH3O-(CH2CH2O) n -CH2CH2OSO2CH3 (60 g) was added with 28% ammonia water 600 mL, and after reacting at 50°C for 1 day, it was extracted with 50 mL of chloroform, and the chloroform phase was dehydrated with NaHSO4, filtered, and precipitated in 2-propanol (IPA), and the obtained precipitate was dissolved in methanol and precipitated. After repeating this operation twice, the precipitate was dried under reduced pressure to obtain the target (yield 54 g).

[0205] Preparation Example 3: Synthesis of L-Ornithine (Z)-N-Carboxyanhydride (L-Orn(Z)-NCA) (N831)

[0206] After Boc-ornithine (Z)-OH (Boc-Orn(Z)-OH, 15 g) was dissolved in 100 mL of THF and ice-cooled to 0°C, a solution of thionyl chloride (SOCl2, 10 mL, 16 g) in THF (50 mL) was added and reacted for 1 hour at 0°C and then for 3 hours at room temperature. The reaction solution was added to 1 L of hexane, the precipitate was filtered, dissolved in 100 mL of ethyl acetate, and then precipitated in 1 L of hexane, and filtered. This was repeated twice, and the resulting precipitate was dried under reduced pressure to obtain the target product (yield 10 g).

[0207] Preparation Example 4: CH3O-(CH2CH2O) n -CH2CH2NH-(COCH(CH2CH2CH2NH(Z))NH) m Synthesis of -H (N857)

[0208] N819 (5 g) obtained in Preparation Example 2 was dissolved in DMF 30 mL, a solution of N831 (5 g) obtained in Preparation Example 3 in DMF (20 mL) was added, and stirred at room temperature for two days. The solution was added to hexane: 2-propanol (8:2, 500 mL), the polymer was separated by centrifugation, and dried under reduced pressure to obtain the target product (yield 6.3 g).

[0209] The NMR measurement results of the obtained N857 are shown in Figure 1 .

[0210] Preparation Example 5: CH3O-(CH2CH2O) n -CH2CH2NH-(COCH(CH2CH2CH2NH2)NH) m Synthesis of -H (PEG-b-POrn); N967)

[0211] N833 (4.3 g) obtained in Preparation Example 4 was dissolved in trifluoroacetic acid 45 mL and ice-cooled to 0°C, and then 3% HBr (acetic acid solution, 15 mL) was added and reacted for 4 hours. This was added to 800 mL of cooled 2-propanol to obtain a precipitate, which was vacuum-dried to obtain the target product (yield 3.3 g).

[0212] The NMR measurement results of the obtained N967 are shown in Figure 2 .

[0213] Preparation Example 6: CH3O-(CH2CH2O) n -CH2CH2NH-(COCH(CH2CH2CH2NHC(=O)CH3)NH) mSynthesis of -C(=O)CH3(PEG-b-POrn(COCH3); N977)

[0214] N967 (1 g) obtained in Preparation Example 5 was dissolved in 1 mL of DMF, triethylamine 5 mL, acetic anhydride 10 mL were added, and reacted at room temperature for 1 hour. This was added to hexane:2-propanol (8:2, 500 mL), the polymer was separated by centrifugation, and dried under reduced pressure to obtain the target (1.0 g).

[0215] The results of the measurement of the NMR of the obtained N977 are shown in Figure 3 .

[0216] Preparation Example 7: CH3O-(CH2CH2O) n -CH2CH2NH-(COCH(CH2CH2CH2NHC(=O)CH(CH3)2)NH) m Synthesis of -C(=O)CH(CH3)2(PEG-b-POrn(COCH(CH3)2); N841)

[0217] The target polymer (1.4 g) was obtained in exactly the same manner as in Preparation Example 6, except that isobutyric anhydride was used instead of acetic anhydride.

[0218] The results of the measurement of the NMR of the obtained N841 are shown in Figure 4 .

[0219] Preparation Example 8: CH3O-(CH2CH2O) n -CH2H2NH-(COCH(CH2CH2CH2NH2)NH) m Preparation of polyion complex particles (Nano ORN ) of -H and chondroitin sulfate

[0220] PEG-b-POrn (N836, 1 g) prepared in the same manner as in Preparation Example 5 was dissolved in 20 mL of DMF, 20 mL of water was added, and put in a dialysis membrane (molecular weight cut off (MWCO) = 3.5 KDa), and dialyzed against 0.01 M HCl (2 L) for 24 hours. After that, dialysis was performed against 2 L of distilled · ion exchange water (DI water) for 48 hours (dialysis water was changed every 12 hours) to obtain an aqueous solution (85 mL). The total cationic charge amount in the aqueous solution was calculated, and a chondroitin sulfate solution (2.7 mg / mL, 115 mL) was added so that the ratio to the total anionic charge amount was 1:1. Also, by stirring the mixed solution for 30 minutes, polyion complex (PIC) particles (Nano ORN ) were obtained.

[0221] Preparation Example 9: Modulation of PEG-b-POrn(Z) assembly (Nano ORN(Z) ) Preparation Example 10: Modulation of PEG-b-POrn(COCH3) assembly (Nano

[0222] N833 (1 g) synthesized in Preparation Example 4 was dissolved in 20 mL of DMF, and after 20 mL of DI water was added, it was put into a dialysis membrane (molecular weight cut off (MWCO) = 12 KDa - 14 KDa), and dialysis was performed for 72 hours against 2 L of DI water (dialysis water was changed every 12 hours) to obtain an aqueous solution.

[0223] Preparation Example 10: Modulation of PEG-b-POrn(COCH3) assembly (Nano ORN(Me) ) Preparation Example 11: Modulation of PEG-b-POrn(COCH(CH3)2) assembly (Nano

[0224] Preparation was performed in exactly the same manner as in Preparation Example 9, except that N977 (1 g) synthesized in Preparation Example 6 was used.

[0225] Preparation Example 11: Modulation of PEG-b-POrn(COCH(CH3)2) assembly (Nano ORN(iBu) ) Preparation Example 11: Modulation of PEG-b-POrn(COCH(CH3)2) assembly (Nano

[0226] Preparation was performed in exactly the same manner as in Preparation Example 9, except that N977 (1 g) synthesized in Preparation Example 6 was used. ORN(iBu) Dynamic light scattering measurement results of the assembly (Nano Figure 5 ) are shown in

[0227] Test Example 1: Stability of nano assembly

[0228] Changes in the size of the nano assembly modulated in Preparation Examples 9 to 11 with pH are shown in Figure 6 . Until around pH 12, the size and scattering intensity of any particles were also stable and did not change. In addition, in Figure 6 , Figure 7 and other documents, there are cases where the XX part of Nano XX is described as Nano XX without being described as a superscript, but is synonymous with the superscript.

[0229] Test Example 2: Effect of oral administration of ornithine assembly to mice induced by acetylaminophenol (APAP)-induced acute liver injury

[0230] Six male C57BL / 6N mice were allowed free access to the following sample (groups 3 to 7 were adjusted to have an ornithine content of approximately 200 mg / kg). Three days after the start of administration, an APAP aqueous solution (15 mg / mL, 0.5 mL, 300 mg / kg BW) was administered intraperitoneally. Twenty-four hours later, blood and liver samples were collected. Whole blood was used to determine serum ammonia concentration, and plasma was used to determine liver injury markers AST and ALT. The liver was stained with hematoxylin and eosin and histologically diagnosed.

[0231] Group 1: Healthy Group

[0232] Group 2: APAP administration group

[0233] Group 3: APAP administration group / 2 mg / mL L-ornithine oral free uptake

[0234] Group 4: APAP administration group / 5 mg / mL Nano ORN Oral free intake

[0235] Group 5: APAP administration group / 5 mg / mL PEG-b-POrn(Z) assembly (Nano ORN(Z) Oral free intake

[0236] Group 6: APAP administration group / 5 mg / mL PEG-b-POrn(COCH3) assembly (Nano ORN(Me) Oral free intake

[0237] Group 7: APAP administration group / 5 mg / mL PEG-b-POrn(COCH(CH3)2) assembly (Nano ORN(iBu) Oral free intake

[0238] like Figure 7 As shown in a), compared to the healthy group, serum ammonia concentrations were significantly increased in the APAP-treated group, while low molecular weight ornithine did not reduce serum ammonia concentrations. Although Nano ORN There are some effects, but it has been confirmed that, with Nano ORN In comparison, the PEG-b-POrn(COCH(CH3)2) assembly (Nano) ORN(iBu) It effectively reduces blood ammonia levels.

[0239] Figure 7 Images b) and c) show the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the blood. Both levels showed a significant increase after APAP administration. This indicates liver injury. (Nano) ORN(PIC: ion pair complex), PEG-b-POrn(COCH3) assembly (Nano ORN(Me) ) and PEG-b-POrn(COCH(CH3)2) assembly (Nano ORN(iBu) ) were reduced.

[0240] Hematoxylin-eosin (H&E) staining of liver tissue sections of these experiments are shown in Figure 8 In the APAP administration group, necrosis of liver tissue was observed. Although the same tendency was shown in Nano ORN , Nano ORN(Z) , it was considerably suppressed in Nano ORN(Me) and Nano ORN(iBu)

[0241] Test Example 3: Effect of Ornithine Assembly on Oral Administration to C57 / BL Healthy Mice (Toxicity Evaluation)

[0242] To 6 C57BL / 6N mice (male) of Group 1, 0.5 mL of the following sample was forcibly administered orally (adjusted to approximately 200 mg / kg of ornithine content for Groups 2 to 7). Body weight was measured every day, and after 4 days from the start of administration, whole blood was used for measurement of blood cell count, and plasma was used for measurement of liver damage markers AST and ALT.

[0243] n = 6

[0244] Group 1: Healthy group

[0245] Group 2: 10 mg / mL of L-ornithine forcibly administered orally (probe)

[0246] Group 3: 20 mg / mL of PEG-b-Orn forcibly administered orally (probe)

[0247] Group 4: 20 mg / mL of Nano ORN forcibly administered orally (probe)

[0248] Group 5: 20 mg / mL of PEG-b-POrn(Z) assembly (Nano ORN(Z) ) forcibly administered orally (probe)

[0249] Group 6: 20 mg / mL of PEG-b-POrn(COCH3) assembly (Nano ORN(Me) ) forcibly administered orally (probe)

[0250] Group 7: 20 mg / mL of PEG-b-POrn(COCH(CH3)2) assembly (Nano ORN(iBu) ​) forced oral administration (probe)

[0251] Figure 9 The change in the body weight of the mice was shown. It was confirmed that PEG-b-POrn forced oral administration had a meaningful body weight reduction, showing toxicity. In addition, as shown in Figure 10 , it was seen that the white blood cell count increased and the red blood cell count decreased in the group administered with PEG-b-Orn and Nano ORN , but there was little change in the nano-assemblies, especially in Nano ORN(iBu) , showing no toxicity.

[0252] Test Example 4: Effect of Ornithine Assembly on Oral Administration to Acetaminophen (APAP)-induced Acute Liver Injury Mice (2)

[0253] Six C57BL / 6N mice (male) of Group 1 were forced orally administered with 0.5 mL of the following sample (adjusted to be approximately 200 mg / kg of ornithine content for Groups 3 to 5) using a probe. Three days after the start of administration, an APAP aqueous solution (15 mg / mL, 0.5 mL, 300 mg / Kg-BW) was intraperitoneally administered. Thereafter, the survival of the mice was confirmed until 14 days.

[0254] Group 1: Healthy group

[0255] Group 2: APAP administration group

[0256] Group 3: APAP administration group / 10 mg / mL of L-Ornithine + 10 mg / mL of L-Aspartate forced oral administration (probe)

[0257] Group 4: APAP administration group / 20 mg / mL of Nano ORN forced oral administration (probe)

[0258] Group 5: APAP administration group / 20 mg / mL of PEG-b-POrn (COCH(CH3)2) assembly (Nano ORN(iBu) forced oral administration (probe)

[0259] As shown in Figure 11 , the survival rate after 14 days was 33% in the APAP administration group, and in comparison, the survival rate was 50% in the group administered with Nano ORN , and the survival rate was 83% in the group administered with Nano ORN(iBu) , confirming that the survival rate was very high and the efficiency was high.

[0260] Reference Preparation Example 1: Synthesis of CH3O-C6H5-CH2O-(CH2CH2O) n -CH2CH2OSO2CH3 (N953(2))

[0261] To CH3OCH2CH2OH (1 mmol) in 50 mL of super dehydrated THF in a 100 mL flask, naphthalene potassium (1 mml) and oxirane (115 mmol) were added and after reacting for 1 day under water cooling, methyl sulfonyl chloride (5 mmol) was added. After 20 minutes, it was put into 500 mL of 2-propanol and after precipitating, it was dissolved in methanol and precipitated. After repeating this operation twice, the precipitate was dried under reduced pressure to obtain the target.

[0262] Reference Preparation Example 2: Synthesis of CH3O-C6H5-CH2O-(CH2CH2O) n -CH2CH2NH2(N956)

[0263] To CH3O-C6H5-CH2O-(CH2CH2O) n -CH2CH2OSO2CH3obtained in Experimental Example 1, 600 mL of 28% ammonia water was added and after reacting for 1 day at 50°C, it was extracted with 50 mL of chloroform and after dehydrating the chloroform phase with NaHSO4, filtering, precipitating it in 2-propanol (IPA), the obtained precipitate was dissolved in methanol and precipitated. After repeating this operation twice, the precipitate was dried under reduced pressure to obtain the target.

[0264] Reference Preparation Example 3: Synthesis of CH3O-C6H5-CH2O-(CH2CH2O) n -CH2CH2NH-(CO(CH2CH2CH2NH2)NH) m -H (PEG-b-POrn); N973)

[0265] N956 obtained in Reference Preparation Example 2 was dissolved in DMF and N-benzyl-aspartic acid NCA commercially available was added to the DMF solution and stirred at room temperature for two days. The solution was added to hexane: 2-propanol (8:2, 500 mL) and the polymer was separated by centrifugation and dried under reduced pressure to obtain the target.

[0266] Reference Preparation Example 4: Synthesis of CH3O-C6H5-CH2O-(CH2CH2O) n -CH2CH2NH-(CO(CH2CH2CH2NH2)NH) m -H (PEG-b-POrn); N973)

[0267] N962 obtained in Reference Preparation Example 3 was dissolved in trifluoroacetic acid and ice-cooled to 0°C, after which 3% HBr (acetic acid solution) was added and reacted for 4 hours. This was added to 800 mL of cooled 2-propanol to obtain a precipitate, and vacuum-dried to obtain the target product.

[0268] Preparation Example 12: Synthesis of CH3O-C6H5-CH2O-(CH2CH2O) n -CH2CH2NH-(CO(CH2CH2CH2NHC(=O)CH(CH3)2)NH) m -H (PEG-b-POrn(COCH(CH3)2); N978)

[0269] The target polymer was obtained in exactly the same manner as Preparation Example 7, except that N973 synthesized in Reference Preparation Example 4 was used instead of N841 (1.4 g).

[0270] Test Example 5: According to 125 Dynamic analysis of I-labeled PEG-b-POrn(COCH(CH3)2)

[0271] N978 synthesized in Preparation Example 125 was dissolved in DMSO, and a 2 L DI water was dialyzed for 72 hours (dialysis water was changed every 12 hours) to obtain an aqueous solution of the assembly. Thereafter, the phenyl group introduced to the end of the PEG chain was labeled with 125 I, and the radioisotope 125 I was mixed.

[0272] In addition, in order to investigate pharmacokinetics, 3 ICR mice each were prepared for respective endpoints of 0.5 h, 1 h, 2 h, 4 h, 24 h, for a total of 15 ICR mice. After the labeled assembly was forcibly orally administered through a probe, after each endpoint was killed, the heart, lung, digestive tract, liver, spleen, kidney, blood was extracted, and the radioactivity of each internal organ was measured using a gamma counter. As Figure 12 shown, 125 I-labeled Nano ORN(iBu) was localized to the digestive tract, and was hardly seen at all in the blood and internal organs.

[0273] Preparation Example 13: Synthesis of PEG-b-Orn(iBu)-TAMRA or PEG-b-Orn(iBu)-Cy5

[0274] The former of the target products was synthesized in exactly the same manner, except that 1 mg of commercially available TAMRA-N=C=S was added before the addition of isobutyric anhydride of Preparation Example 7, and it was reacted for 10 minutes.

[0275] The latter was synthesized in the same way as the former except that 1 mg of commercially available Cy5-NHS was added before the addition of isobutyric anhydride of Preparation Example 7 and reacted for 10 minutes.

[0276] Test Example 6: Nano Porn In vitro toxicity evaluation

[0277] BAEC (bovine aortic endothelial cells), L-929 (mouse fibroblast cells), and RAW264.7 (mouse macrophage cells) were prepared and 5-10 x 10 3 cells were seeded in each of 96-well plates. Different concentrations of Nano ORN(Z) , Nano ORN(Me) , Nano ORN(iBu) , and Nano ORN were added to each well. Cultured for 24 hours or 48 hours, after which MTT reagent was added. After 4 more hours, DMSO was added to solubilize the formazan crystals. The absorbance at a wavelength of 575 nm was measured.

[0278] The results are shown in Figure 13 . Nano ORN(Z) , Nano ORN(Me) showed high toxicity to L-929 cells, Nano ORN showed high toxicity to RAW264.7 macrophage cells, and in comparison, Nano ORN(iBu) showed almost no cytotoxicity, showing a very high level of safety.

[0279] Preparation Example 14: Synthesis of CH3O-(CH2CH2O) n -CH2H2NH-(COCH(CH2COOCH2Ph)NH) m -H (N865) (PEG-b-PBLA)

[0280]

[0281] N819 (10 g) synthesized according to Preparation Example 2 was dissolved in DMF 60 mL. Commercially available L-aspartic acid (CH2Ph)-N-carboxyanhydride (L-ASP(Z)-NCA) 10 g was dissolved in 40 mL of DMF. Both were mixed and reacted at room temperature for two days. The resulting polymer was precipitated in 2-propanol (IPA), dissolved in acetone, and precipitated in hexane. After repeating this operation twice, the precipitate was dried under reduced pressure to obtain the target (harvest 16 g; n = 114; m = 15.5).

[0282] Preparation Example 15: Synthesis of L-Aspartic acid(Me)-N-carboxyanhydride (L-ASP(Me)-NCA) (N871)

[0283] The commercially available L-Aspartic acid 4-methyl hydrochloride (15 g; 81.6 mmol) was dissolved in 90 mL of THF, and α-pinene (33 g; 252 mmol) and triphosgene 12.2 g (40.8 mmol) were added, and reacted at 50°C for 2 hours. The reaction solution was added to 1 L of hexane, and the precipitate was filtered and dissolved in 100 mL of 2-propanol, after which precipitation, filtration was performed in 1 L of hexane. This was repeated twice, and the obtained precipitate was dried under reduced pressure to obtain the target product (harvested amount 10.2 g).

[0284] Preparation Example 16: Synthesis of CH3O-(CH2CH2O) n -CH2H2NH-(COCH(CH2COOCH3)NH) m -H (PEG-b-PASP(Me)) (N869)

[0285]

[0286] N819 (5 g) synthesized according to Preparation Example 2 was dissolved in 30 mL of DMF. L-Aspartic acid (CH3)-N-carboxyanhydride (L-ASP(Me)-NCA) 5 g synthesized in Preparation Example 15 was dissolved in 20 mL of DMF. Both were mixed and reacted at room temperature for two days. The obtained polymer was precipitated in 2-propanol (IPA), after which it was dissolved in methanol and precipitated in ether. After repeating this operation twice, the precipitate was dried under reduced pressure to obtain the target product (harvested amount 6.4 g; n = 114; m = 10).

[0287] Preparation Example 17: Synthesis of PEG-b-P(Orn(Z)-co-Asp(CH2Ph)) (N877)

[0288]

[0289] PEG-NH2 (N866) (5 g; 1 mmol) synthesized according to Preparation Example 2 was dissolved in 30 mL of DMF.

[0290] L-Orn(Z)-NCA(N854) (6 g; 20 mmol) synthesized according to the preparation example and commercially available L-aspartic acid (CH2Ph)-N-carboxylic anhydride (L-ASP(Z)-NCA) (2.5 g; 10 mmol) were dissolved in 20 mL of DMF. The two were mixed and reacted at room temperature for two days. The resulting polymer was precipitated in 2-propanol (IPA) / hexane (1:9 v), then dissolved in acetone and precipitated in hexane. After repeating this process twice, the precipitate was dried under reduced pressure to obtain the target product (yield 8.8 g; n = 114; asparagine units = 15; ornithine units = 20).

[0291] Preparation Example 18: PEG-b-PBLA Assembly (Nano ASP(Bz) modulation

[0292] The N865 (1g) synthesized according to Preparation Example 14 was dissolved in 20mL of DMF, and 20mL of DI water was added. The solution was then placed in a dialysis membrane (molecular weight cutoff (MWCO) = 12KDa-14KDa) and dialyzed with 2L of DI water for 72 hours (the dialysis water was replaced every 12 hours) to obtain an aqueous solution containing the target assembly.

[0293] Preparation Example 19: PEG-b-PASP(Me) Assembly (Nano ASP(Me) modulation

[0294] Except for using N869 (1g) synthesized according to Preparation Example 16, the same operation as in Preparation Example 18 was repeated to obtain an aqueous solution containing the target assembly.

[0295] Preparation Example 20: PEG-bP(Orn(Z)-co-Asp(CH2Ph)) assembly (Nano ASP / ORN Modulation of (N978)

[0296] Except for using N877 (1g) synthesized according to Preparation Example 17, the same operation as in Preparation Example 18 was repeated to obtain an aqueous solution containing the target assembly.

[0297] Experimental Example 7: Nano ASP(Bz) Nano ASP(Me) Nano ASP / ORN Stability evaluation

[0298] The particle sizes of the particles prepared in Preparation Examples 16-18 were determined by dynamic light scattering.

[0299] Nano ASP(Bz) Stable nanoparticles of approximately 120-140 nm were formed within 24 hours up to pH 12.

[0300] Nano ASP(Me) At all pHs measured, assemblies of about 10 nm were formed up to 6 hours. After 24 hours, associations of about 100 nm were observed at pH 1 and 5.

[0301] Nano ASP / ORN At all pHs measured, assemblies of about 100 nm were formed up to 24 hours.

[0302] In addition, according to the results of the body weight changes of the acute liver injury mice to which each of the (probes) was orally administered, Nano ASP (Bz) , Nano ASP(Me) , Nano ASP / ORN PEG-b-POrn(Z) assemblies (Nano ORN(Z) ) and PEG-b-POrn(Me) assemblies (Nano ORN(Me) ) did not show substantial toxicity to mice in the same manner as the PEG-b-POrn(Z) assemblies (Nano

[0303] Test Example 8: Effects of oral administration of the samples of the following groups to APAP-induced acute liver injury mice

[0304] As the samples, the samples shown in the following groups were used, and the method of Test Example 2 was repeatedly performed except for this.

[0305] Group 1: Healthy group

[0306] Group 2: APAP administration group

[0307] Group 3: APAP administration group / L-Aspartate oral administration (probe) of 200 mg / kg

[0308] Group 4: APAP administration group / PEG-b-POrn(Z) assembly (Nano ORN(Z) ) oral administration (probe) of 200 mg / kg

[0309] Group 5: APAP administration group / PEG-b-POrn(Me) assembly (Nano ORN(Me) ) oral administration (probe) of 200 mg / kg

[0310] Group 6: APAP administration group / PEG-b-PBLA assembly (Nano ASP(Me) ) oral administration (probe) of 200 mg / kg

[0311] Group 7: APAP administration group / PEG-b-PAsp(Me) assembly (Nano ORN(Bz)Oral administration (probes)

[0312] Group 8: APAP administration group / PEG-b-PAsp(Me) assembly (Nano ASP / ORN Oral administration (probes)

[0313] As a result of the above test, these assemblies showed weight changes equivalent to the healthy group, showed almost no toxicity, and in addition, showed acute liver injury in mice as Figure 17 indicated by the blood ammonia levels, AST, ALT levels of acute liver injury mice.

[0314] Preparation Example 21: Synthesis of PEG-b-PArg(Ac)2(N1047)

[0315]

[0316] PEG-b-PArg (N1046; 2.4 g) synthesized in the same manner as Example 5 of PCT / JP2016 / 62062 A1 was dissolved in 50 mL of DMF. To this, acetic anhydride 30 mL was added, and after reacting at 50°C for 5 hours, it was reacted at room temperature for 1 day. The resulting polymer was precipitated in t-butyl methyl ether and washed with hexane. After repeating this operation twice, the precipitate was dried under reduced pressure to obtain the target (yield 2.4 g; n = 114; m = 28). The 1 H-NMR spectrum of the copolymer as the target is shown in Figure 18 .

[0317] Preparation Example 22: Synthesis of PEG-b-PArg(iBu)2(N1063)

[0318]

[0319] The target was obtained in exactly the same manner as Preparation Example 21, except that isobutyric anhydride was used instead of acetic anhydride (yield 2.4 g; n = 114; m = 28). The H-NMR spectrum of the copolymer as the target is shown in 1 Figure 19 .

[0320] Preparation Example 23: Assembly of PEG-b-PArg(Ac)2(Nano ARG(Ac) ) modulation

[0321] N1047 (1 g) synthesized according to Preparation Example 21 was dissolved in 20 mL of DMF, after adding DI water 20 mL, it was put into a dialysis membrane (molecular weight cut off (MWCO) = 12 KDa-14 KDa), and dialysis was performed for 72 hours against 2 L of DI water (dialysis water was changed every 12 hours) to obtain Nano Arg(Ac) ​of 132 nm and a zeta potential of +4.9 mV.

[0322] Preparation Example 24: Assembly of PEG-b-PARG(iBu)2(Nano Arg(iBu) ) in water

[0323] An assembly (average particle diameter: 44 nm, zeta potential: -2.2 mV) was prepared in the same manner as in Preparation Example 23, except that N1063 (1 g) synthesized in Preparation Example 22 was used.

[0324] Nano Arg(Ac) and Nano Arg(iBu) The results of measurement of the light scattering particle diameter in water are shown in Figure 20 .

[0325] Test Example 9: Effects of oral administration and intravenous injection of acetylated arginine assembly on mice with acute liver injury induced by p-acetamidophenol (APAP)

[0326] The method of Test Example 2 was repeated, except that acetylated arginine assembly was used instead of ornithine assembly of Test Example 2. The results are shown in Figure 21 . According to Figure 21 , acetylated polyarginine particles (Nano Arg(Ac) ) did not show effects in oral administration, but reduced the ammonia concentration in blood superiorly in intravenous injection.

[0327] Test Example 10: Effects of oral administration of acetylated arginine assembly on mice with nonalcoholic steatohepatitis (NASH) model

[0328] Six C57BL / 6J mice (male) of Group 1 were orally administered (0 days-8 days) with an aqueous solution of acetylated polyarginine particles (Nano Arg(Ac) ) ([Nano Arg(Ac) ] = 5 mg / mL) by free drinking water. One day after the start of oral administration, a choline-deficient methionine-reduced high-fat diet feed (CADHFD) was fed (1 day-8 days). Blood and liver were collected on the 8th day after the start of feeding of CADHFD. Plasma was used for determination of triglyceride (TG).

[0329] GP1: Healthy group

[0330] GP2: CADHFD feeding group

[0331] GP3: CADHFD feeding group / oral administration of an aqueous solution of acetylated polyarginine particles (Nano Arg(Ac) ) (free drinking water) at 5 mg / mL

[0332] GP4: CADHFD feeding group / or 1.5 mg / mL of L-arginine aqueous solution was orally administered (free drinking water)

[0333] The results are shown in Figure 22 Fig. 2. As can be seen from the figure, triglyceride was superiorly reduced by oral administration.

[0334] Industrial applicability

[0335] The copolymer of the present application, the ornithine microparticle, although not limited, can be used as, for example, a medicament for preventing or treating liver dysfunction, and can be at least industrially applicable in the pharmaceutical industry.

Claims

1. A pharmaceutical composition, characterized in that, Contains: ornithine microparticles represented by formula (I) below or composed of the above copolymers and having an average particle size of 1 nanometer to 100 micrometers; and copolymers represented by formula (IV) below. In the formula, A indicates: (i) Hydrogen atom, unsubstituted or substituted C1-C 12 Alkyl, unsubstituted or substituted aryl, wherein, in the case of substitution, the substituent is C1-C4 alkyl, C1-C4 alkoxy, aryl, formyl, or of formula R. 1 R 2 CH- groups, wherein R 1 and R 2 Independently C1-C4 alkoxy or R 1 and R 2 Combined into one, it can be represented as -OCH2CH2O-, -O(CH2)3O-, or -O(CH2)4O-; or (ii) L and L' independently represent linking groups. Y and Y' independently represent hydrogen atoms and C atoms. 1-21 Alkyl carbonyl, substituted C 1-4 Alkyl carbonyl, unsubstituted or substituted C 3-7 Cycloalkyl carbonyl, unsubstituted or substituted aryl carbonyl, or unsubstituted or substituted 5 or 6-membered heteroaryl carbonyl, wherein the substituted C 1-4 The substituents of the alkyl carbonyl group are selected from halogen atoms, hydroxyl groups, carboxyl groups, and unsubstituted or substituted carbon atoms. 3-7 In the group consisting of cycloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted 5- or 6-membered heteroaryl, unsubstituted or substituted adamantyl, and unsubstituted or substituted cholesterol residues, the substituent when these substituents are substituted is C. 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, carboxyl, cyano, nitro, halogen atom, or single or double carbon atom 1-4 Alkylamino, R 10 For R 11 -(C=O)-,R 10 'For hydrogen atoms or R 11 -(C=O)-, where R 11 Each is independently either substituted or unsubstituted C1-C 21 Alkyl, substituted or unsubstituted aryl, substituted or unsubstituted C1-C 21 Alkoxy groups, the above C1-C 21 When alkyl groups and the aforementioned aryl groups are substituted, the substituents are C. 1-4 Alkyl, C 1-4 Alkoxy, aryl, and the above C1-C 21 When the alkoxy group is substituted, the substituent is C. 1-4 Alkyl, C 1-4 Alkoxy m and m' are independent integers from 2 to 300. n is an integer from 2 to 1,000. In the formula, A ASP Represents hydrogen atoms, unsubstituted or substituted C1-C 12 Alkyl, unsubstituted or substituted aryl, where the substituent is C1-C4 alkyl, C1-C4 alkoxy, aryl, formyl, or of formula R 1 R 2 CH- groups, wherein R 1 and R 2 Independently C1-C4 alkoxy or R 1 and R 2 Combined into one, it can be represented as -OCH2CH2O-, -O(CH2)3O-, or -O(CH2)4O-. L ASP Indicates a linking group. R ASP It is a hydrogen atom, a substituted or unsubstituted C1-C atom. 21 Alkyl, substituted or unsubstituted aryl groups, where the substituent is C when the substituent is substituted. 1-4 Alkyl, C 1-4 Alkoxy, aryl, Y ASP Represents hydrogen atom, C 1-21 Alkyl carbonyl, substituted C 1-4 Alkyl carbonyl, unsubstituted or substituted C 3-7 Cycloalkyl carbonyl, unsubstituted or substituted aryl carbonyl, or unsubstituted or substituted 5 or 6-membered heteroaryl carbonyl, wherein the substituted C 1-4 The substituents of the alkyl carbonyl group are selected from halogen atoms, hydroxyl groups, carboxyl groups, and unsubstituted or substituted carbon atoms. 3-7 In the group consisting of cycloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted 5- or 6-membered heteroaryl, unsubstituted or substituted adamantyl, and unsubstituted or substituted cholesterol residues, the substituent when these substituents are substituted is C. 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, carboxyl, cyano, nitro, halogen atom, or single or double carbon atom 1-4 Alkylamino, m A It is an integer between 2 and 300. n A It is an integer between 2 and 1,000.

2. A pharmaceutical composition, characterized in that, Contains: ornithine microparticles represented by formula (I) below or composed of the above copolymers and having an average particle size of 1 nanometer to 100 micrometers; and copolymers represented by formula (V) below. In the formula, A indicates: (i) Hydrogen atom, unsubstituted or substituted C1-C 12 Alkyl, unsubstituted or substituted aryl, wherein, in the case of substitution, the substituent is C1-C4 alkyl, C1-C4 alkoxy, aryl, formyl, or of formula R. 1 R 2 CH- groups, wherein R 1 and R 2 Independently C1-C4 alkoxy or R 1 and R 2 Combined into one, it can be represented as -OCH2CH2O-, -O(CH2)3O-, or -O(CH2)4O-; or (ii) L and L' independently represent linking groups. Y and Y' independently represent hydrogen atoms and C atoms. 1-21 Alkyl carbonyl, substituted C 1-4 Alkyl carbonyl, unsubstituted or substituted C 3-7 Cycloalkyl carbonyl, unsubstituted or substituted aryl carbonyl, or unsubstituted or substituted 5 or 6-membered heteroaryl carbonyl, wherein the substituted C 1-4 The substituents of the alkyl carbonyl group are selected from halogen atoms, hydroxyl groups, carboxyl groups, and unsubstituted or substituted carbon atoms. 3-7 In the group consisting of cycloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted 5- or 6-membered heteroaryl, unsubstituted or substituted adamantyl, and unsubstituted or substituted cholesterol residues, the substituent when these substituents are substituted is C. 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, carboxyl, cyano, nitro, halogen atom, or single or double carbon atom 1-4 Alkylamino, R 10 For R 11 -(C=O)-,R 10 'For hydrogen atoms or R 11 -(C=O)-, where R 11 Each is independently either substituted or unsubstituted C1-C 21 Alkyl, substituted or unsubstituted aryl, substituted or unsubstituted C1-C 21 Alkoxy groups, the above C1-C 21 When alkyl groups and the aforementioned aryl groups are substituted, the substituents are C. 1-4 Alkyl, C 1-4 Alkoxy, aryl, and the above C1-C 21 When the alkoxy group is substituted, the substituent is C. 1-4 Alkyl, C 1-4 Alkoxy m and m' are independent integers from 2 to 300. n is an integer from 2 to 1,000. In the formula, A CO Represents hydrogen atoms, unsubstituted or substituted C1-C 12 Alkyl, unsubstituted or substituted aryl, where the substituent is C1-C4 alkyl, C1-C4 alkoxy, aryl, formyl, or of formula R 1 R 2 CH- groups, wherein R 1 and R 2 Independently C1-C4 alkoxy or R 1 and R 2 Combined into one, it can be represented as -OCH2CH2O-, -O(CH2)3O-, or -O(CH2)4O-. n CO It is an integer between 2 and 1,000. L CO Indicates a linking group. R ASP It is a hydrogen atom, a substituted or unsubstituted C1-C atom. 21 Alkyl, substituted or unsubstituted aryl groups, where the substituent is C when the substituent is substituted. 1-4 Alkyl, C 1-4 Alkoxy, aryl, m A It is an integer between 2 and 300. R ORN It is a hydrogen atom or R 11 -(C=O)-,R 11 Each is independently either substituted or unsubstituted C1-C 21 Alkyl, substituted or unsubstituted aryl, substituted or unsubstituted C1-C 21 Alkoxy groups, where the substituent is C when these substituents are replaced. 1-4 Alkyl, C 1-4 Alkoxy, aryl, m O It is an integer between 2 and 300. Y CO Represents hydrogen atom, C 1-21 Alkyl carbonyl, substituted C 1-4 Alkyl carbonyl, unsubstituted or substituted C 3-7 Cycloalkyl carbonyl, unsubstituted or substituted aryl carbonyl, or unsubstituted or substituted 5 or 6-membered heteroaryl carbonyl, wherein the substituted C 1-4 The substituents of the alkyl carbonyl group are selected from halogen atoms, hydroxyl groups, carboxyl groups, and unsubstituted or substituted carbon atoms. 3-7 In the group consisting of cycloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted 5- or 6-membered heteroaryl, unsubstituted or substituted adamantyl, and unsubstituted or substituted cholesterol residues, the substituent when these substituents are substituted is C. 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, carboxyl, cyano, nitro, halogen atom, or single or double carbon atom 1-4 Alkylamino, Where, m A repeating units and m O Each amino acid in a repeating unit exists randomly to form another block.

3. A pharmaceutical composition, characterized in that, Contains: ornithine microparticles represented by formula (I) below or composed of the above copolymers and having an average particle size of 1 nanometer to 100 micrometers; and copolymers represented by formula (VI) below. In the formula, A indicates: (i) Hydrogen atom, unsubstituted or substituted C1-C 12 Alkyl, unsubstituted or substituted aryl, wherein, in the case of substitution, the substituent is C1-C4 alkyl, C1-C4 alkoxy, aryl, formyl, or of formula R. 1 R 2 CH- groups, wherein R 1 and R 2 Independently C1-C4 alkoxy or R 1 and R 2 Combined into one, it can be represented as -OCH2CH2O-, -O(CH2)3O-, or -O(CH2)4O-; or (ii) L and L' independently represent linking groups. Y and Y' independently represent hydrogen atoms and C atoms. 1-21 Alkyl carbonyl, substituted C 1-4 Alkyl carbonyl, unsubstituted or substituted C 3-7 Cycloalkyl carbonyl, unsubstituted or substituted aryl carbonyl, or unsubstituted or substituted 5 or 6-membered heteroaryl carbonyl, wherein the substituted C 1-4 The substituents of the alkyl carbonyl group are selected from halogen atoms, hydroxyl groups, carboxyl groups, and unsubstituted or substituted carbon atoms. 3-7 In the group consisting of cycloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted 5- or 6-membered heteroaryl, unsubstituted or substituted adamantyl, and unsubstituted or substituted cholesterol residues, the substituent when these substituents are substituted is C. 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, carboxyl, cyano, nitro, halogen atom, or single or double carbon atom 1-4 Alkylamino, R 10 For R 11 -(C=O)-,R 10 'For hydrogen atoms or R 11 -(C=O)-, where R 11 Each is independently either substituted or unsubstituted C1-C 21 Alkyl, substituted or unsubstituted aryl, substituted or unsubstituted C1-C 21 Alkoxy groups, the above C1-C 21 When alkyl groups and the aforementioned aryl groups are substituted, the substituents are C. 1-4 Alkyl, C 1-4 Alkoxy, aryl, and the above C1-C 21 When the alkoxy group is substituted, the substituent is C. 1-4 Alkyl, C 1-4 Alkoxy m and m' are independent integers from 2 to 300. n is an integer from 2 to 1,000. In the formula, A ARG It is a hydrogen atom, a substituted or unsubstituted C1-C atom. 21 Alkyl carbonyl, substituted or unsubstituted aryl carbonyl, substituted or unsubstituted C1-C 21 Alkoxycarbonyl, where the substituent is C when these substituents are replaced. 1-4 Alkyl, C 1-4 Alkoxy, aryl, n G It is an integer between 2 and 1,000. L ARG Indicates a linking group. R ARG and R ARG 'Independently for R 11 -(C=O)-,R 11 Each is independently either substituted or unsubstituted C1-C 21 Alkyl, substituted or unsubstituted aryl groups, where the substituent is C when the substituent is substituted. 1-4 Alkyl, C 1-4 Alkoxy, aryl, m G It is an integer between 2 and 300. Y ARG Represents hydrogen atom, C 1-21 Alkyl carbonyl, substituted C 1-4 Alkyl carbonyl, unsubstituted or substituted C 3-7 Cycloalkyl carbonyl, unsubstituted or substituted aryl carbonyl, or unsubstituted or substituted 5 or 6-membered heteroaryl carbonyl, wherein the substituted C 1-4 The substituents of the alkyl carbonyl group are selected from halogen atoms, hydroxyl groups, carboxyl groups, and unsubstituted or substituted carbon atoms. 3-7 In the group consisting of cycloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted 5- or 6-membered heteroaryl, unsubstituted or substituted adamantyl, and unsubstituted or substituted cholesterol residues, the substituent when these substituents are substituted is C. 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, carboxyl, cyano, nitro, halogen atom, or single or double carbon atom 1-4 Alkylamino.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein, A is defined by (i).

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein, A is defined by (ii).

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein, It also contains additives.

7. The pharmaceutical composition according to claim 6, wherein, The above-mentioned pharmaceutical composition is an oral administration agent.

8. The pharmaceutical composition according to any one of claims 1 to 3, wherein, The above-mentioned pharmaceutical composition is used for the prevention or treatment of liver dysfunction.

9. A pharmaceutical composition, characterized in that, Contains: ornithine microparticles represented by formula (I) below or composed of the above copolymers and having an average particle size of 1 nanometer to 100 micrometers; and copolymers represented by formula (VI-a) below. In the formula, A indicates: (i) Hydrogen atom, unsubstituted or substituted C1-C 12 Alkyl, unsubstituted or substituted aryl, wherein, in the case of substitution, the substituent is C1-C4 alkyl, C1-C4 alkoxy, aryl, formyl, or of formula R. 1 R 2 CH- groups, wherein R 1 and R 2 Independently C1-C4 alkoxy or R 1 and R 2 Combined into one, it can be represented as -OCH2CH2O-, -O(CH2)3O-, or -O(CH2)4O-; or (ii) L and L' independently represent linking groups. Y and Y' independently represent hydrogen atoms and C atoms. 1-21 Alkyl carbonyl, substituted C 1-4 Alkyl carbonyl, unsubstituted or substituted C 3-7 Cycloalkyl carbonyl, unsubstituted or substituted aryl carbonyl, or unsubstituted or substituted 5 or 6-membered heteroaryl carbonyl, wherein the substituted C 1-4 The substituents of the alkyl carbonyl group are selected from halogen atoms, hydroxyl groups, carboxyl groups, and unsubstituted or substituted carbon atoms. 3-7 In the group consisting of cycloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted 5- or 6-membered heteroaryl, unsubstituted or substituted adamantyl, and unsubstituted or substituted cholesterol residues, the substituent when these substituents are substituted is C. 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, carboxyl, cyano, nitro, halogen atom, or single or double carbon atom 1-4 Alkylamino, R 10 For R 11 -(C=O)-,R 10 'For hydrogen atoms or R 11 -(C=O)-, where R 11 Each is independently either substituted or unsubstituted C1-C 21 Alkyl, substituted or unsubstituted aryl, substituted or unsubstituted C1-C 21 Alkoxy groups, the above C1-C 21 When alkyl groups and the aforementioned aryl groups are substituted, the substituents are C. 1-4 Alkyl, C 1-4 Alkoxy, aryl, and the above C1-C 21 When the alkoxy group is substituted, the substituent is C. 1-4 Alkyl, C 1-4 Alkoxy m and m' are independent integers from 2 to 300. n is an integer from 2 to 1,000. In the formula, A ARG It is a hydrogen atom, a substituted or unsubstituted C1-C atom. 21 Alkyl carbonyl, substituted or unsubstituted aryl carbonyl, substituted or unsubstituted C1-C 21 Alkoxycarbonyl, where the substituent is C when these substituents are replaced. 1-4 Alkyl, C 1-4 Alkoxy, aryl, n G It is an integer between 2 and 1,000. L ARG Indicates a linking group. R ARG and R ARG 'Independently for R 11 -(C=O)-,R 11 Each is independently either substituted or unsubstituted C1-C 21 Alkyl, substituted or unsubstituted aryl groups, where the substituent is C when the substituent is substituted. 1-4 Alkyl, C 1-4 Alkoxy, aryl, m G It is an integer between 2 and 300. Y ARG Represents hydrogen atom, C 1-21 Alkyl carbonyl, substituted C 1-4 Alkyl carbonyl, unsubstituted or substituted C 3-7 Cycloalkyl carbonyl, unsubstituted or substituted aryl carbonyl, or unsubstituted or substituted 5 or 6-membered heteroaryl carbonyl, wherein the substituted C 1-4 The substituents of the alkyl carbonyl group are selected from halogen atoms, hydroxyl groups, carboxyl groups, and unsubstituted or substituted carbon atoms. 3-7 In the group consisting of cycloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted 5- or 6-membered heteroaryl, unsubstituted or substituted adamantyl, and unsubstituted or substituted cholesterol residues, the substituent when these substituents are substituted is C. 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, carboxyl, cyano, nitro, halogen atom, or single or double carbon atom 1-4 Alkylamino.

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