Copolymer containing poly(ethylene glycol) segment and repeating unit having n-acylethanolamine or homologue or analog thereof in side chain, nanoparticles of said copolymer, and use thereof

Abstract

A copolymer of the present invention contains a repeating unit having oleylethanolamide or a homologue or analog thereof in a side chain and a poly(ethylene glycol) segment and is represented by formula (BC) (in the formula, A represents a C1-12 alkyloxy, etc., L1 represents a phenylene group, etc., R represents a hydrogen atom, etc., A represents S(=S)OCH2CH3, etc., LMD represents O-CH2CH(RO)NHC(=O)RL, C(=O)RL represents a residue resulting from removal of an HO group from oleic acid, m represents an integer of 5-500, and n represents an integer of 3-500) or formula (GC) (in the formula, L2 represents OCH2CH2, etc., PEG represents (CH2CH2O)m, m, A, and LMD are as described in formula (BC), x+y is an integer of 5-1400, z is an integer of 5-1400, x+y:z is 1:1-5, and x:y is 1:1-300).

Description

Copolymer containing repeating units having N-acylethanolamine or its homologue or analogue in the side chain and poly(ethylene glycol) segments, nanoparticles of said copolymer, and uses thereof

[0001] The present invention relates to a copolymer containing a repeating unit having a lipid mediator, N-acylethanolamine, typically oleylethanolamide (OEA) or a homolog or analog thereof, at its side chain and a poly(ethylene glycol) (PEG) segment, as well as nanoparticles of the copolymer and pharmaceutical uses thereof. This application claims priority based on Japanese Patent Application No. 2023-143094, filed September 4, 2023, the contents of which are incorporated herein by reference.

[0002] For example, OEA is a strong ligand for peroxisome proliferator-activated receptor α (PPAR-α) and a transcription factor that upregulates the expression of genes related to lipid breakdown and inflammation (Non-Patent Document 1). Due to this effect, OEA is a potential treatment option for non-alcoholic fatty liver disease (NAFLD) and metabolic diseases (Non-Patent Document 2). OEA homologs, such as palmitoylethanolamide, stearoylethanolamide, linoleylethanolamide, arachidonoylethanolamide (anandamide), and docosahexaenoylethanolamide (sinabutanamide), share biological activities with OEA and are known to have one or more effects selected from anti-inflammatory, appetite suppressant, anticonvulsant, neuroprotective, and analgesic effects (Non-Patent Document 3).

[0003] Furthermore, for example, OEA is naturally produced in the small intestine and is involved in regulating satiety. However, its biological effects are reduced due to degradation by two intracellular lipolytic enzymes, fatty acid amide hydrolase and N-acylethanolamine hydrolyzing amidase, making it difficult to maintain an appropriate physiological level, limiting its use as a pharmaceutical. Therefore, attempts have been made to modify the structure of OEA to increase its resistance to enzymatic hydrolysis, and (Z)-(R)-9-octadecenamide, N-(2-hydroxyethyl, 1-methyl) (KDS-5104), an analogue in which a methyl group has been introduced at the 1-position of the N-(2-hydroxyethyl) of OEA, has been proposed (Non-Patent Document 4). A wide range of compounds including the above-mentioned homologues or analogues has also been proposed (Patent Document 1). Furthermore, OEA or its homologues or analogues are fatty acid ethanolamides, which are hydrophobic compounds with low molecular weights. Therefore, they are insoluble in aqueous solutions and are degraded by the above-mentioned lipolytic enzymes, resulting in low bioavailability (Non-Patent Document 5). In some preclinical studies, animals injected intraperitoneally with OEA showed movement disorders and discomfort (Non-Patent Document 6).

[0004] On the other hand, some of the present inventors have reported the use of short-chain fatty acid-containing block copolymers (referred to as poly(ethylene glycol)-b-poly(vinyl ester)) as a means for enabling precise delivery of drugs to target sites in a controlled manner in drug therapy using low molecular weight compounds, such as short-chain fatty acids (SCFAs) (Patent Document 2). In this case, SCFAs are conjugated via an ester bond between their carboxyl groups and the hydroxyl groups of poly(vinyl alcohol).

[0005] WO 2002 / 080860WO 2021 / 024906

[0006] Tutunchi H, et al., Obesity Reviews, 2019; 20(7): 1057-1069Li Long, et al., Journal of Pharmacological Sciences, 2015; 127(3): 244-250E.D. et al,. J Pharmacol Exp Ther. 2006; 318(2):563-570Nielsen MJ, et al., J Lipid Res. 2004; 45(6): 1027-1029Fedele S, et al., Physiol Rep. 2018; 6(3):e13517

[0007] The present invention aims to provide uniform polymeric micelles or nanoparticles that retain biological effectiveness while avoiding the problems or toxicity associated with the direct use of OEA and the like.

[0008] The present invention includes the following aspects: Aspect 1: A copolymer comprising a repeating unit having oleylethanolamide (OEA) or a homolog or analog thereof in a side chain and a poly(ethylene glycol) (PEG) segment, the copolymer being represented by the following formula (BC) or (GC):

[0009]

[0010] In formula (BC), A is OH, unsubstituted or substituted C １ -C １２ Alkyloxy, or unsubstituted or substituted C ６ -C １２ aryloxy, and when substituted, the substituents are C １ -C ４ Alkyl, C １ -C ４ Alkoxy, C ６ -C １２ aryl, formyl, or a group of formula R １ R ２ CH- (where R １ and R ２ is independent, C１ -C ４ Alkoxy or R １ and R ２ Let's get together -OCH ２ CH ２ O-, -O(CH ２ ) ３ O- or -O(CH ２ ) ４ O—; １ teeth,

[0011]

[0012] or -(CH ２ ) ｃ S- and -CO(CH ２ ) ｃ S-, wherein, independently at each occurrence, b is an integer from 2 to 6, and c is an integer from 1 to 5 (wherein L １ When the meanings of the bonds differ depending on the directionality of the bond, they are to be bonded in the order shown. For example, -(CH ２ ) ｃ In the case of S-, methylene (CH ２ ) is bonded to the oxygen atom in the unit with the subscript m, and the sulfur atom is bonded to the first methylene in the unit with the subscript n), R represents a hydrogen atom or methyl, Z represents a hydrogen atom, SH, S(C=S)-Ph, S(=S)OCH ２ CH ３ , hydroxyl, C １ -C ６ Alkyloxy, or C ６ -C １２ Aryl-C １ -C ２ alkyloxy, and LMD is O—CH ２ CH (R Ｏ )NHC(=O)R Ｌ represents R Ｏ is a hydrogen atom or methyl, and C(=O)R Ｌrepresents a residue resulting from the removal of an HO group from a fatty acid selected from palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, and docosahexaenoic acid; m is an integer of 5 to 500, or 10 to 300, or 20 to 200; and n is an integer of 3 to 500, or 5 to 500, or 10 to 200.

[0013]

[0014] In formula (GC), each repeating unit with subscripts x, y, and z is present randomly, and L ２ is CH ２ CH ２ O or CH ２ CH ２ PEG represents (CH ２ CH ２ O) ｍ wherein m is as defined for formula (BC), A is as defined for formula (BC), LMD is as defined for formula (BC), x+y is an integer of 5 to 1400, or an integer of 5 to 300, or an integer of 5 to 50, z is an integer of 5 to 1400, or an integer of 5 to 300, or an integer of 5 to 50, x+y:z is in a ratio of 1:1 to 5, and x:y is in a ratio of 1:1 to 300, or a ratio of 1:1 to 60. Aspect 2: The copolymer according to Aspect 1, represented by formula (BC). Aspect 3: In LMD in formula (BC) or formula (GC), C(═O)R Ｌ is a residue resulting from the removal of an HO group from oleic acid. Aspect 4: A nano-sized polymeric micelle or nanoparticle formed in an aqueous medium from the copolymer according to any one of Aspects 1 to 3. Aspect 5: A pharmaceutical preparation comprising, as an active ingredient, the copolymer according to any one of Aspects 1 to 3 or the polymeric micelle or nanoparticle according to Aspect 4. Aspect 6: A pharmaceutical preparation according to Aspect 5 for treating non-alcoholic fatty liver disease, treating obesity, or for anti-inflammation.

[0015] The copolymer of Aspect 1 exhibits amphiphilicity due to the inclusion of hydrophobic repeating units bearing N-acylethanolamine (including analogs in which the methylene hydrogen atom in the ethanol moiety is replaced with methyl) in the side chain via an ester bond that can be hydrolyzed by in vivo enzymes, and hydrophilic PEG segments. The nano-sized polymeric micelles or nanoparticles formed from the copolymer in aqueous media are easily dispersible in aqueous media or aqueous environments, making them suitable for various administration routes, including oral and parenteral. The polymeric micelles or nanoparticles thus formed contain N-acylethanolamine in their cores and PEG segments in their shells, limiting their exposure to N-acylethanolamine-degrading enzymes in vivo. Meanwhile, the N-acylethanolamine bonded to the backbone of the copolymer via an ester bond interacts with esterases, due in part to the high affinity between the ester bond and esterases, gradually releasing the N-acylethanolamine from the copolymer. This not only suppresses the toxicity induced by direct injection of N-acylethanolamine, but also improves the bioavailability and efficacy of N-acylethanolamine, thereby enhancing its efficacy in pharmaceutical therapy.

[0016] 1 is a conceptual diagram of the formation of nanoparticles disclosed herein. １ 1H-NMR spectrum. ... １ 1H-NMR spectrum. 1D shows the results of gel filtration chromatography of PSMA-g-OEA(PEG) and PEG-OH prepared in Preparation Example 6. 1E shows the results of gel filtration chromatography of nanoparticles (Nano ＯＥＡ 1 is a diagram showing the results of measurement by DLS of the size of nanoparticles (SMAPo) prepared in Production Example 8. ＯＥＡ 1 is a diagram showing the results of measurement of the size of nano particles by DLS method. ＯＥＡ1 shows the results of a stability evaluation test of Nano HCl in Test Example 1, which was incubated at different temperatures for a predetermined period of time. ＯＥＡ 1 shows the results of a stability evaluation test of Nano HCl in Test Example 1, which was incubated for a predetermined period of time in a 150 mM NaCl aqueous solution. ＯＥＡ 1 shows the results of a stability evaluation test of Nano in Test Example 2, which was incubated for a predetermined period of time in the presence of 45 mg / mL BSA. ＯＥＡ or microscopic images of BAECs after OEA treatment (0 μM is Nano ＯＥＡ In Test Example 2, Nano ＯＥＡ 10 is a graph showing the survival rate of BAEC after exposure to OEA or OEA. ＯＥＡ 1 is a graph showing the results of an in vitro release test of Nano in Test Example 4. ＯＥＡ 4 is a schematic diagram of an in vivo toxicity test of Nano 100 mg / kg / day. FIG. 5 is a graph showing changes in mouse body weight over one week measured in Test Example 4. FIG. 6 is a graph showing plasma ALT levels measured in Test Example 4. FIG. 7 is a graph showing plasma BUN levels measured in Test Example 4. FIG. 8 is a graph showing white blood cell (WBC) levels measured in Test Example 4. FIG. 9 is a graph showing platelet (PLT) levels measured in Test Example 4. FIG. 10 is a graph showing red blood cell (RBC) levels measured in Test Example 4. FIG. 11 is a graph showing hemoglobin (HGB) levels measured in Test Example 4. FIG. 12 is a graph showing locomotor activity recorded in an open field test performed in Test Example 4. FIG. 13 is a graph showing the effect of Nano 100 mg / kg / day on NAFLD in Test Example 5. ＯＥＡ 1 is a graph showing the values ​​of AST in plasma measured in Test Example 5. 2 is a graph showing the values ​​of ALT in plasma measured in Test Example 5. 3 is a graph showing the values ​​of SMAPo on NAFLD in Test Example 6. ＯＥＡ 1 is a graph showing the values ​​of AST in plasma measured in Test Example 6. FIG. 2 is a graph showing the values ​​of ALT in plasma measured in Test Example 6. FIG. 3 is a graph showing the values ​​of AST in plasma measured in Test Example 6.

[0017] The copolymer of the present invention comprises repeating units having oleylethanolamide (OEA) or a homolog or analog thereof in the side chain and poly(ethylene glycol) (PEG) segments, and is represented by formula (BC) or formula (GC).

[0018]

[0019] In formula (BC), A is OH, unsubstituted or substituted C １ -C １２ Alkyloxy, or unsubstituted or substituted C ６ -C １２ aryloxy, and when substituted, the substituents are C １ -C ４ Alkyl, C １ -C ４ Alkoxy, C ６ -C １２ aryl, formyl, or a group of formula R １ R ２ CH- (where R １ and R ２ is independent, C １ -C ４ Alkoxy or R １ and R ２ Let's get together -OCH ２ CH ２ O-, -O(CH ２ ) ３ O- or -O(CH ２ ) ４ Among these, A represents an unsubstituted or substituted C １ -C １２ It is preferably alkyloxy. １ teeth,

[0020]

[0021] or -(CH ２ ) ｃ S- and -CO(CH ２ ) ｃ S-, and each independently, b is an integer of 2 to 6, and c is an integer of 1 to 5. １When the meanings of - and - are different depending on the direction of the bond, they are to be bonded in the order of the description. For example, -(CH ２ ) ｃ In the case of S-, methylene (CH ２ ) is bonded to the oxygen atom in the unit with the subscript m, and the sulfur atom is bonded to the first methylene in the unit with the subscript n. １ is preferably a group represented by the following formula:

[0022]

[0023] R represents a hydrogen atom or methyl, and is preferably a hydrogen atom.

[0024] Z is a hydrogen atom, SH, S(C=S)-Ph, S(=S)OCH ２ CH ３ , hydroxyl, C １ -C ６ Alkyloxy, or C ６ -C １２ Aryl-C １ -C ２ represents alkyloxy, S(=S)OCH ２ CH ３ It is preferable that:

[0025] LMD is O-CH ２ CH (R Ｏ )NHC(=O)R Ｌ Represents R Ｏ represents a hydrogen atom or methyl, and is preferably a hydrogen atom. Ｌ represents a residue in which an HO group has been removed from a fatty acid selected from palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid and docosahexaenoic acid, and is preferably a residue in which an HO group has been removed from oleic acid.

[0026] m is an integer of 5 to 500, or 10 to 300, or 20 to 200, and more preferably an integer of 100 to 150. n is an integer of 3 to 500, or 5 to 500, or 10 to 200.

[0027]

[0028] In formula (GC), each repeating unit with subscripts x, y, and z occurs randomly. ２ is CH ２ CH ２ O or CH ２ CH ２ represents NH, and CH ２ CH ２ Preferably, PEG is (CH ２ CH ２ O) ｍ and m is as defined for formula (BC). A is as defined for formula (BC). LMD is as defined for formula (BC). x+y is an integer of 5 to 1400, or an integer of 5 to 300, or an integer of 5 to 50. z is an integer of 5 to 1400, or an integer of 5 to 300, or an integer of 5 to 50. x+y:z is in a ratio of 1:1 to 5. x:y is in a ratio of 1:1 to 300, or a ratio of 1:1 to 60.

[0029] The copolymer is preferably a copolymer represented by formula (BC).

[0030] The nano-sized polymeric micelles or nanoparticles of the present invention are formed from the copolymer in an aqueous medium.

[0031] Terms used herein, particularly technical terms, have the meanings commonly used in the art unless otherwise defined.

[0032] The terms "alkyl," by themselves or as part of another substituent, mean, unless structurally inconsistent with the compound or portion thereof in which they are used, a straight-chain, branched-chain, or cyclic saturated hydrocarbon radical, or combinations thereof, having the specified number of carbon atoms (e.g., C １ -C １２Alkyl means alkyl having 1 to 12 carbon atoms, examples of which include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers thereof (e.g., n-pentyl, n-hexyl, n-heptyl, n-octyl, etc.).

[0033] The terms "nano-sized" or "nano" are not limited to nano-sized particles, but refer to particles of, for example, 5 nm to 500 nm, preferably 10 nm to 100 nm, within the nano-sized range. Nano-sized polymeric micelles or nanoparticles are used interchangeably. The size is the average diameter determined by dynamic light scattering (DLS) in an aqueous medium (water or saline).

[0034] Hereinafter, nano-sized polymeric micelles or nanoparticles will be referred to primarily as nanoparticles. Figure 1 shows a conceptual diagram of nanoparticle formation when a typical N-acylethanolamine, OEA, is used. The copolymers disclosed herein can be classified as amphiphilic block copolymers represented by formula (BC) and amphiphilic graft copolymers represented by formula (GC). Without being bound by theory, both of these can be provided in aqueous media as so-called core-shell nanoparticles, which have a core region primarily containing hydrophobic OEA (or OEA residues) and a shell primarily containing hydrophilic PEG segments. Here, the aqueous medium refers to an aqueous solution containing a water-soluble organic solvent, such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or methanol (MeOH). Therefore, it is understood that the OEA residue carried on the side chain is resistant to degradation in vivo by two intracellular lipolytic enzymes, fatty acid amide hydrolase and N-acylethanolamine hydrolyzing amidase, while the ester bond carrying the OEA residue on the side chain is hydrolyzed by esterase in vivo, releasing OEA from the copolymer and allowing the inherent biological activity of OEA to be exhibited. In the above and below, in this specification, OEA and OEA residue may be used as interchangeable technical terms, and in such cases, the residue refers to the state in which the H from the hydroxyl (OH) of the ethanolamine in the OEA molecule has been eliminated. Furthermore, the residue has the same meaning for N-acylethanolamines other than OEA.

[0035] The copolymers also include those containing repeating units having (or carrying) an N-acylethanolamine residue in the side chain other than OEA (amide of oleic acid (18:1 ω-9) and ethanolamine), and the residue is derived from palmitoylethanolamide (amide of palmitic acid (16:0) and ethanolamine), stearoylethanolamide (amide of stearic acid (18:0) and ethanolamine), linoleylethanolamide (amide of linoleic acid (18:2 ω-6) and ethanolamine), arachidonoylethanolamide (anandamide) (amide of arachidonic acid (20:4 ω-6) and ethanolamine), or docosahexaenoylethanolamide (sinabutanamide) (amide of docosahexaenoic acid (22:6 ω-3) and ethanolamine), and all of these copolymers have a common structure in which a common ethanolamine moiety is conjugated via an amide bond to a fatty acid classified as a long-chain fatty acid. Therefore, it would be easy for a person skilled in the art to understand that these residues in the copolymer or nanoparticles formed therefrom behave similarly to OEA with respect to in vivo enzymes (fatty acid amide hydrolase, N-acylethanolamine hydrolyzing amidase, esterase, etc.). Therefore, it is reasonable to assume that copolymers represented by formula (BC) or formula (GC) containing repeating units having N-acylethanolamine residues other than OEA in their side chains, as well as nanoparticles formed therefrom, behave similarly to those containing OEA with respect to in vivo enzymes including the aforementioned enzymes.Therefore, in the copolymer, a copolymer containing palmitoylethanolamide, stearoylethanolamide, linoleylethanolamide, arachidonoylethanolamide (anandamide), or docosahexaenoylethanolamide (sinabutanamide) instead of OEA can also exhibit the biological activities that these N-acylethanolamines themselves have in vivo, such as anti-inflammation, analgesia, anticonvulsant, neuroprotection, appetite reduction, etc. for palmitoylethanolamide; appetite reduction, anti-inflammation, etc. for stearoylethanolamide; appetite reduction, neuroprotection, etc. for linoleylethanolamide; neurotransmission, appetite stimulation, analgesia, anti-anxiety, etc. for anandamide; and neurogenesis, anti-inflammation, etc. for sinabutanamide. The biological activities of these OEA or its homologues are specifically described in the aforementioned Patent Documents 1 and 2 regarding OEA, as well as in the aforementioned Non-Patent Document 3, ED Mock, et al., Progress in Lipid Research 89 (2023) 101194: 1-24, and the documents cited therein, and the biological activities of analogues are specifically described in the aforementioned Non-Patent Document 4, Astarita G, et al., J Pharmacol Exp Ther. 2006; 318(2): 563-570, which can be referenced. These documents are incorporated herein by reference, and their disclosures are incorporated herein by reference.

[0036] <Production of Copolymer Represented by Formula (BC) or Formula (GC)> The copolymer represented by Formula (BC) or Formula (GC) can typically be provided based on Production Example 4 or Production Example 6, respectively, which will be described later. However, the present invention is not limited thereto.

[0037] Copolymer represented by formula (BC): In general, the repeating unit with the subscript m is prepared by preparing commercially available poly(ethylene glycol) having as narrow a molecular weight distribution as possible, protecting the α-terminal OH group with an appropriate protecting group as necessary, and modifying the ω-terminal OH group so as to conform to the definition of L1 in formula (BC), and then using the resulting copolymer.

[0038] L １When the OH group is alkylene, for example, a dihalogenated xylylene is reacted with the OH terminal of A-PEG to introduce a halogenated xylylene group, followed by reaction with potassium ethylxanthate to produce a xanthate. The resulting xanthate can then be polymerized by reversible addition-fragmentation chain transfer (RAFT) polymerization with an ester of N-acylethanolamine and (meth)acrylic acid (acrylic acid or methacrylic acid) corresponding to the repeating unit with the subscript n. The ω-terminal residue thus formed can be converted to SH, OH, or H, as necessary, by hydrolysis, oxidation, reduction, or the like. For specific conditions for such RAFT polymerization, see WO 2016 / 05246 A1, in which a styrene derivative is reacted with the xanthate thus produced.

[0039] L １ But -(CH ２ ) ｃ S- or -CO(CH ２ ) ｃ When it is S-, if necessary, a corresponding sulfur atom-containing compound in which the SH group is protected is introduced to the OH terminal of A-PEG, and then the SH group is liberated, and then the above-mentioned acrylic acid ester is subjected to radical chain transfer polymerization to form a compound in which L1 in formula (BC) is -(CH ２ ) ｃ S- or -CO(CH ２ ) ｃ A block copolymer having an S- group can be obtained. Specific reaction conditions for such chain transfer polymerization can be found in WO 2009 / 133647 A1, which describes the introduction of an SH group at the end of PEG followed by reaction with a styrene derivative.

[0040] When the copolymer represented by formula (BC) is produced as described above and treated as necessary, the LMD residues in the formula may be hydrolyzed and converted to OH groups within a range that meets the objectives of the present invention (forming stable polymeric micelles and exhibiting the desired biological activity attributable to the LMD). When the copolymer represented by formula (BC) is produced as described above and treated as necessary, the LMD residues in the formula may be hydrolyzed and converted to OH groups within a range that meets the objectives of the present invention (forming stable polymeric micelles and exhibiting the desired biological activity attributable to the LMD). Therefore, copolymers in which up to about 10% of the n LMD residues are converted to OH groups are also included within the scope of the present invention.

[0041] Copolymer represented by formula (GC): The copolymer represented by formula (GC) can be prepared by adding A-(CH) to the acid anhydride moiety (unit with x) of a copolymer (some of which are commercially available) in which the subscripts x and y in formula (GC) are in the form of an acid anhydride, which can be used as a starting material. ２ CH ２ O) ｍ X, where X is CH ２ CH ２ OH or CH ２ CH ２ NH ２ The PEG derivative is added under conditions known per se, and before and after the addition, the acid anhydride moiety (unit marked with y) is converted to AcNH(CH ２ ) ２ These PEG derivatives can be prepared by adding OH (Ac is a fatty acid residue corresponding to the acyl in N-acylethanolamine) under known conditions. Some of these PEG derivatives are commercially available, and others can be prepared in the same manner as commercially available products.

[0042] When an analogue in which a methyl group is introduced at the 1-position of the N-(2-hydroxyethyl) of the aforementioned N-acylethanolamine is used, the analogue in which the methyl group is introduced may be prepared based on the method for introducing the methyl group into OEA described in Non-Patent Document 4.

[0043] Preparation of nanoparticles of copolymers represented by formula (BC) or formula (GC) The copolymers represented by formula (BC) or formula (GC) are amphiphilic and can form self-associated polymeric micelles or nanoparticles by dissolving them in an aqueous solution containing a water-soluble organic solvent, such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or methanol (MeOH), and then dialysis of the resulting solution against water through a dialysis membrane with a specific molecular weight cutoff. The nanoparticles thus formed can be separated from the dispersion or dissolution medium by, for example, centrifugation or microfiltration, and can be handled as the separated product.

[0044] The copolymer represented by formula (BC) or (GC) has N-acylethanolamine introduced into the side chain of the copolymer through the formation of an ester bond utilizing the hydroxyl group of the ethanol moiety, and by containing a repeating unit having such a side chain and a PEG segment, it is possible to form uniform nanoparticles that retain biological effectiveness while avoiding the problems or toxicity associated with the direct use of OEA, etc.

[0045] <Pharmaceutical Preparations> The pharmaceutical preparations of the present invention contain, as an active ingredient, the copolymer represented by formula (BC) or formula (GC) prepared as described above, or nanoparticles composed of said copolymer. The pharmaceutical preparations are prepared so as to exert the known or future confirmed medicinal effects of the N-acylethanolamine, the bioactive ingredient contained therein, and can be provided as pharmaceutical preparations for treating (preventing or treating) symptoms, diseases, or disorders for which said medicinal effects are expected to be exerted. Among these, the pharmaceutical preparations are preferably used as pharmaceutical preparations for the treatment of non-alcoholic fatty liver disease, obesity, or anti-inflammation.

[0046] In addition to the copolymer or nanoparticles, the pharmaceutical preparations may contain, if appropriate, a pharmaceutically acceptable carrier and, if necessary, other biologically active ingredients. Because the copolymer or nanoparticles can be dissolved or uniformly dispersed in an aqueous medium (water, saline, etc.), these pharmaceutical preparations can be easily formulated for parenteral (subcutaneous, intramuscular, or intravenous) administration, and can also be formulated for oral, rectal, or topical administration. In practice, the copolymer or nanoparticles can be thoroughly mixed with the carrier according to conventional pharmaceutical compounding techniques to form the active ingredient. The carrier can take a variety of forms depending on the form of formulation desired for administration (e.g., oral or parenteral (intravenous, etc.)).

[0047] When the pharmaceutical preparation is an oral liquid preparation, it may be, for example, a suspension, elixir, solution, etc., and may contain a conventional pharmaceutical medium (e.g., water, glycol, oil, alcohol, flavoring, preservative, coloring agent, etc.). When the pharmaceutical preparation is an oral solid preparation, it may be, for example, a powder, hard capsule, soft capsule, tablet, etc., and may contain a carrier (e.g., starch, sugar, microcrystalline cellulose, diluent, granule, lubricant, binder, disintegrant, etc.). For example, when the pharmaceutical preparation is a tablet, it may contain a binder (e.g., tragacanth gum, acacia, corn starch, or gelatin), an excipient (e.g., disodium phosphate), a disintegrant (e.g., corn starch, potato starch, alginic acid), a lubricant (e.g., sodium stearate), and a sweetener (e.g., sucrose, lactose, or saccharin).

[0048] When the pharmaceutical preparation is a parenteral preparation, a solution or suspension of the active ingredient of the pharmaceutical preparation can be prepared in water appropriately mixed with a surfactant (e.g., hydroxypropylcellulose).Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof in oil.For example, injectable preparations can be sterile aqueous solutions or dispersions, or sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions.In any case, the form must be fluid enough to be easily injectable.Therefore, the carrier can be a solvent or dispersion medium, and can include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils, as long as they do not adversely affect the stability of the nanoparticles.

[0049] Such pharmaceutical formulations preferably contain at least 0.1% by weight of the copolymer or nanoparticles as an active ingredient, calculated as the N-acylethanolamine contained therein. The percentage of the active ingredient in these pharmaceutical formulations can vary, but is preferably from about 2% to about 60% by weight based on the total weight of the pharmaceutical formulation. The copolymer or nanoparticles are effective over a wide range of dosages, for example, from about 10 to about 1000 mg, from about 100 to about 500 mg, or from about 1 to about 100 mg for adults. The copolymer or nanoparticles can be used in dosages of about 0.05 to about 100 mg / day, more preferably from about 0.1 to about 100 mg / day. The optimal dosage depends on the mode of administration, the desired therapy, the dosage form, the subject to be treated, the subject's weight, and the experience of the attending physician.

[0050] In order to avoid complicating the explanation, the present invention will be specifically explained below mainly in relation to OEA among N-acylethanolamines, but the present invention is not limited to these explanations.

[0051] Production example 1: CH ３ O-(CH ２ CH ２ O) １１３ -CH ２ PhCH ２Br(MeO-PEG-CH ２ PhCH ２ Preparation of commercially available CH ３ O-(CH ２ CH ２ O) １１３ -H (MeO-PEG-OH, MW=5000, 50 g, 10 mmol) was dehydrated under reduced pressure in an oil bath at 110°C. After 12 hours, tetrahydrofuran (THF, 100 mL), butyllithium (BuLi, 7.2 mL, 11.5 mmol, 1.6 M in hexane), and α,α'-dibromo-p-xylene (12.5 g, 47.5 mmol) were added sequentially under nitrogen. The reaction mixture was precipitated in cold isopropyl alcohol (IPA) to obtain a crude polymer. To remove unreacted α,α'-dibromo-p-xylene, the crude polymer was dissolved in methanol (MeOH) and centrifuged. The supernatant was precipitated with cold IPA to obtain purified MeO-PEG-CH ２ PhCH ２ After obtaining Br, it was dried under reduced pressure.

[0052] Production example 2: CH ３ O-(CH ２ CH ２ O) １１３ -CH ２ PhCH ２ SC(=S)OCH ２ CH ３ Preparation of (MeO-PEG-xanthate) MeO-PEG-CH obtained according to Preparation Example 1 ２ PhCH ２ Br (10 g) was dissolved in ethanol (50 mL), and then potassium ethyl xanthate (0.35 g) was added and stirred at room temperature for 10 minutes. The precipitate was then removed by centrifugation, and the ethanol was removed by evaporation. The remaining polymer was further purified by extraction with chloroform and water to remove potassium ions. The polymer-containing chloroform layer was separated and dried over anhydrous sodium sulfate. Finally, the sodium sulfate was filtered off, and the solution was poured into IPA, followed by centrifugation and drying under reduced pressure to obtain MeO-PEG-xanthate.

[0053] Production example 3: CH ３ (CH２ ) ６ CH ２ CH=CHCH ２ (CH ２ ) ４ CH ２ CH ２ C(=O)NHCH ２ CH ２ OC(=O)CH=CH ２ Preparation of OEA acrylate: OEA acrylate was prepared by reacting commercially available OEA (20 g, 62 mmol) with acryloyl chloride (5.1 mL, 63 mmol) in chloroform using triethylamine (TEA, 9 mL) to trap the resulting acid. The reaction mixture was stirred at 0°C for 1 day. The reaction mixture was extracted with water, aqueous NaOH (0.5 M, 10 mL), and HCl (0.5 M, 10 mL). The chloroform layer was then separated and dried over sodium hydrogen sulfate. Finally, the mixture was filtered, and the chloroform was evaporated to obtain the product OEA acrylate (yield: 25.7 g).

[0054] Production Example 4: Production of PEG-b-POEA

[0055]

[0056] MeO-PEG-xanthate (5 g) obtained according to Preparation Example 2, isobutyronitrile (250 mg), and OEA acrylate (5 g) obtained according to Preparation Example 3 were reacted at 60°C under a nitrogen atmosphere to carry out reversible addition-fragmentation chain transfer (RAFT) polymerization. After 2 days, the reaction mixture was precipitated in hexane and then dried under reduced pressure to obtain PEG-b-POEA (yield: 7.5 g). １ The H-NMR spectrum is shown in Figure 2A. The results of gel filtration chromatography of the obtained PEG-b-POEA (Mn=7300, Mw / Mn=1.5) and PEG-OH (5 kDa, Mn=4700, Mw / Mn=1.1) are shown in Figure 2B.

[0057] Preparation Example 5: Preparation of PSMA-g-PEG

[0058]

[0059] Commercially available MeO-PEG-OH (MW = 5,000, 50 g, 10 mmol) was dried under vacuum in an oil bath at 110° C. for 3 days. Then, to convert MeO-PEG-OH to MeO-PEG-OLi, tetrahydrofuran (THF, 500 mL) and butyllithium (BuLi, 6.25 mL, 10 mmol, 1.6 M in hexane) were added under a nitrogen atmosphere.

[0060] Separately, commercially available poly(styrene-co-maleic anhydride) copolymer (PSMA, molecular weight 7,500, styrene:maleic anhydride unit ratio = 2:1, 75 g, 250 mmol) was dissolved in 200 mL of anhydrous tetrahydrofuran (THF) under a nitrogen atmosphere. The previously obtained MeO-PEG-OLi was then added and reacted at room temperature for 2 hours. The reaction solution was poured into 1 L of isopropyl alcohol, and a precipitate was obtained by centrifugation (9,000 rpm, 2 minutes). The copolymer (76 g) was then recovered by drying under reduced pressure. The product PSMA-g-PEG was then obtained by precipitation in diethyl ether and dried under reduced pressure.

[0061] Preparation Example 6: Preparation of PSMA-g-OEA(PEG)

[0062]

[0063] Commercially available OEA (2.8 g, 8.5 mmol) was dissolved in THF (20 mL), and BuLi (5.3 mL, 8.5 mmol, 1.6 M in hexane) was added at 0°C to convert OEA to OEA-Li. Separately, PSMA-g-PEG (5 g) obtained according to Preparation Example 5 was dissolved in THF, followed by the addition of OEA-Li. The reaction mixture was further stirred for 3 hours at room temperature. The reaction mixture was dialyzed against MeOH for 4 days. After dialysis, MeOH was removed by evaporation, and the product PSMA-g-OEA(PEG) was obtained by benzene lyophilization. The resulting PSMA-g-OEA(PEG) １ The H-NMR spectrum is shown in Figure 3A. The results of gel filtration chromatography of PEG-OH (5 kDa, Mn = 4700, Mw / Mn = 1.1) and the resulting PSMA-g-OEA(PEG) (Mn = 20500, Mw / Mn = 1.2) are shown in Figure 3B.

[0064] Production example 7: Nano ＯＥＡ PEG-b-POEA (100 mg) obtained according to Preparation Example 4 was completely dissolved in DMF (1 mL), water was added, and the whole solution was then dialyzed against water (2 L) through a dialysis membrane (MWCO = 3500) for 3 days. The external dialysate was changed three times a day. ＯＥＡ The size and zeta potential of the nanoparticles were measured by dynamic light scattering (DLS). The results are shown in Figure 4 and Table 1.

[0065]

[0066] From FIG. 4 and Table 1, Nano ＯＥＡ had an average size (hydrodynamic diameter) of about 31 nm and a slight negative charge.

[0067] Production Example 8: SMAPo ＯＥＡ Preparation of PSMA-g-OEA(PEG) (100 mg) obtained according to Preparation Example 6 was completely dissolved in MeOH (1 mL), followed by dialysis (MWCO = 3500) against MeOH (2 L) for 1 day and then against water (2 L) for 2 days. ＯＥＡ The size and zeta potential of the particles were measured by DLS. The results are shown in Figure 5 and Table 2.

[0068]

[0069] From Figure 5 and Table 2, SMAPo ＯＥＡ had an average size (hydrodynamic diameter) of about 20 nm and a slight negative charge.

[0070] Test Example 1: Nano ＯＥＡ Stability evaluation of Nano ＯＥＡ The stability of Nano was investigated at different temperatures, with NaCl, and bovine serum albumin (BSA). ＯＥＡ (5 mg / mL) was incubated at 4°C (storage conditions) for 20 days, at room temperature for 20 days, and at 37°C (physiological conditions) for 2 days, and then the size was measured by DLS. The results are shown in Figure 6A. ＯＥＡNanoparticles (5 mg / mL) were incubated with a physiological concentration of NaCl (150 mM) at room temperature, and the size was measured by DLS at predetermined time points. The results are shown in Figure 6B. ＯＥＡ Nanoparticles (5 mg / mL) were incubated with BSA (45 mg / mL) at room temperature, and then their sizes were measured by DLS at predetermined time points. The results are shown in Figure 6C. As shown by the intensity distribution curves in Figures 6A-6C, Nanoparticles (5 mg / mL) were incubated with BSA (45 mg / mL) at room temperature, and their sizes were measured by DLS at predetermined time points. ＯＥＡ was stable under all test conditions.

[0071] Test Example 2: Nano ＯＥＡ Cytotoxicity Evaluation of Nano Bovine aortic endothelial cells (BAEC) were seeded in a 96-well plate at 8000 cells / well. After 24 hours at 37°C, ＯＥＡ Or OEA was added to BAEC at equivalent concentrations of 0 to 200 μM. After 24 hours of incubation at 37°C, the medium was removed and supplemented with fresh medium (100 μL) containing WST reagent (10 μL). After further incubation at 37°C for 4 hours, cytotoxicity was evaluated. Microscopic images of BAEC after incubation are shown in Figure 7A. From these microscopic images, it was clear that Nano ＯＥＡ Cells treated with were compared with control cells (Nano ＯＥＡ No significant changes in cell morphology or cell number were observed compared to the control cells (which differed only in the absence of OEA and OEA treatment). However, the OEA-treated cells showed a significant decrease in cell number and a significant change in morphology compared to the control cells. Cell viability was also quantified as the difference in absorbance at 450 nm between treated and control cells, and the results are shown in Figure 7B. From Figure 7B, it can be seen that at 100-200 μM, Nano ＯＥＡ However, it was confirmed that OEA exerts strong toxicity on BAEC.

[0072] Test Example 3: Nano ＯＥＡ In vitro release of Nano ＯＥＡ (4 mL, 18 mg / mL) was mixed with pig liver esterase (1 mg, 19 U) and incubated at 37°C with gentle shaking (200 rpm). ＯＥＡ(4 mL, 18 mg / mL) was used as a control. At predetermined time points (0, 1, 3, 6, 12, 24, and 48 hours), 100 mL of the incubated mixture was collected and immediately frozen in liquid nitrogen to stop the enzyme reaction. The amount of released OEA in the collected mixture was measured using an LC-MS / MS system.

[0073] The results are shown in Figure 8. From Figure 8, Nano ＯＥＡ It was confirmed that the compound can continuously release OEA in the presence of esterase.

[0074] Test Example 4: Nano ＯＥＡ As shown in Figure 9A, Nano ＯＥＡ Or OEA was administered intraperitoneally (i.p.) every other day at 50 mg-OEA / kg-BW or 100 mg-OEA / kg-BW. Because OEA is poorly soluble, it was dissolved in a vehicle solution containing 5% propylene glycol, 5% Tween 80, and 90% saline. Similarly, C57BL / 6J mice (male, 7 weeks old) were intraperitoneally (i.p.) every other day with a vehicle solution containing 5% propylene glycol, 5% Tween 80, and 90% saline, or with water. After each administration, an open field test was performed to observe the locomotor activity of the mice. Specifically, after administration, mice were isolated in an open chamber (30 × 30 × 30 cm), and a digital camera was placed above the chamber to record their movements. The results of analyzing the total distance traveled by the mice over a 10-minute period are shown in Figure 9I. Furthermore, the mouse body weights were recorded daily, and the results are shown in Figure 9B. After 7 days, the mice were euthanized, and whole blood and plasma samples were collected to measure hematological parameters and organ function markers, respectively. The measured plasma ALT levels are shown in Figure 9C, BUN levels in Figure 9D, white blood cell (WBC) levels in Figure 9E, platelet (PLT) levels in Figure 9F, red blood cell (RBC) levels in Figure 9G, and hemoglobin (HGB) levels in Figure 9H.

[0075] From the results of Figures 9B to 9I, Nano ＯＥＡNo side effects were observed in healthy mice when OEA was administered at 50 and 100 mg / kg-BW. However, the results in Figures 9G and 9H confirmed that mice administered 100 mg / kg-OEA showed a decrease in red blood cell (RBC) and hemoglobin (HGB) counts. Furthermore, the results in Figure 9I showed that severe motor impairment was observed only in the OEA-administered group, and that the Nano ＯＥＡ The importance of using

[0076] Test Example 5: Nano for NAFLD ＯＥＡ Hepatoprotective Effects of i.p. Administration of 100mg ... Water / CDAHFD(+) group: 13 mice were intraperitoneally injected with water as a sample and were given CDAHFD for one week from the day after the start of intraperitoneal injection. Vehicle / CDAHFD(+) group: 13 mice were intraperitoneally injected with the vehicle solution as a sample and were given CDAHFD for one week from the day after the start of intraperitoneal injection. Nano ＯＥＡ CDAHFD(+) group: 13 mice were given 50 mg-OEA / kg-BW of Nano ＯＥＡwas intraperitoneally injected into 13 mice, and CDAHFD was administered for one week from the day after the start of intraperitoneal injection. OEA / CDAHFD(+) group: 13 mice were intraperitoneally injected with OEA at 50 mg-OEA / kg-BW as a sample, and CDAHFD was administered for one week from the day after the start of intraperitoneal injection. After one week, the mice were euthanized, and plasma was collected for measurement of liver damage markers. The measured plasma AST values ​​are shown in Figure 10B, and the ALT values ​​are shown in Figure 10C. These results indicate that liver damage was induced in mice that had been given CDAHFD for one week, as ALT and AST in plasma were significantly higher. Compared to OEA, the same dose of Nano ＯＥＡ It was confirmed that intraperitoneal injection of the compound significantly alleviated liver damage.

[0077] Test Example 6: SMAPo for NAFLD ＯＥＡ Hepatoprotective Effects of Oral Administration (As shown in Figure 11A), C57BL / 6J male mice (7 weeks old) were intraperitoneally injected with the sample four times every other day. In addition, some mice were fed CDAHFD instead of a normal diet for one week starting the day after the intraperitoneal injection of the sample. Mice were divided into six groups: Water / CDAHFD(-) group: Five mice were intraperitoneally injected with water as the sample and fed a normal diet; Vehicle / CDAHFD(-) group: Five mice were intraperitoneally injected with a vehicle solution containing 5% propylene glycol, 5% Tween 80, and 90% saline as the sample and fed a normal diet; Water / CDAHFD(+) group: 13 mice were intraperitoneally injected with water as the sample and fed CDAHFD for one week starting the day after the intraperitoneal injection. Vehicle / CDAHFD(+) group: 13 mice were intraperitoneally injected with the vehicle solution as a sample, and were given CDAHFD for one week from the day after the start of the intraperitoneal injection. ＯＥＡ CDAHFD(+) group: 13 mice were given 80 mg-OEA / kg-BW of SMAPo ＯＥＡThe mice were intraperitoneally injected with OEA and were fed CDAHFD for one week from the day after the start of intraperitoneal injection. OEA / CDAHFD(+) group: 13 mice were intraperitoneally injected with OEA at 80 mg-OEA / kg-BW as a sample, and were fed CDAHFD for one week from the day after the start of intraperitoneal injection. After one week, the mice were euthanized and plasma was collected for measurement of liver damage markers. The measured plasma AST values ​​are shown in Figure 11B, and the plasma ALT values ​​are shown in Figure 11C. From these results, it was confirmed that SMAPo ＯＥＡ Oral administration of the compound showed a decrease in ALT and AST levels compared to the water / CDAHFD(+) group.

[0078] The copolymers of formula (BC) and formula (GC) provided by the present invention and the nanoparticles formed from the copolymers can be used as active ingredients in pharmaceutical preparations, and therefore can be used in the manufacturing industry, particularly in the pharmaceutical manufacturing industry.

Claims

1. A copolymer comprising a repeating unit having oleylethanolamide or a homologue or analogue thereof in the side chain and a poly(ethylene glycol) segment, said copolymer being represented by the following formula (BC) or formula (GC). In formula (BC), A is OH, unsubstituted or substituted C １ -C １２ Alkyloxy, or unsubstituted or substituted C ６ -C １２ aryloxy, and the substituents when substituted are C １ -C ４ Alkyl, C １ -C ４ Alkoxy, C ６ -C １２ Aryl, formyl, or a group of formula R １ R ２ CH- (where R １ and R ２ is independent, C １ -C ４ Alkoxy or R １ and R ２ Let's get together - OCH ２ CH ２ O-, -O(CH ２ ) ３ O- or -O(CH ２ ) ４ O—; １ teeth, or -(CH ２ ) ｃ S- and -CO(CH ２ ) ｃ S-, wherein, independently, b is an integer from 2 to 6, c is an integer from 1 to 5, R represents a hydrogen atom or methyl, Z represents a hydrogen atom, SH, S(C=S)-Ph, S(=S)OCH ２ CH ３ , Hydroxyl, C １ -C ６ Alkyloxy or C ６ -C １２ Aryl-C １ -C ２ alkyloxy; LMD is O-CH ２ CH(R Ｏ )NHC(=O)R Ｌ represents R Ｏ is a hydrogen atom or methyl, and C(=O)R Ｌ represents a residue resulting from the removal of an HO group from a fatty acid selected from palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, and docosahexaenoic acid; m is an integer of 5 to 500; and n is an integer of 3 to 500. In formula (GC), each repeating unit with subscripts x, y, and z is present randomly, ２ is CH ２ CH ２ O or CH ２ CH ２ NH, PEG represents (CH ２ CH ２ O) ｍ wherein m is as defined for formula (BC), A is as defined for formula (BC), LMD is as defined for formula (BC), x+y is an integer from 5 to 1400, z is an integer from 5 to 1400, the ratio of x+y:z is 1:1-5, and x:y is 1:1-300.

2. The copolymer according to claim 1, represented by formula (BC).

3. C(═O)R in LMD in the formula (BC) or the formula (GC). Ｌ The copolymer according to claim 1 , wherein is a residue of an HO group removed from oleic acid.

4. Nano-sized polymeric micelles or nanoparticles formed in an aqueous medium from the copolymer of claim 1.

5. A pharmaceutical preparation comprising the copolymer according to claim 1 or the polymeric micelle or nanoparticle according to claim 4 as an active ingredient.

6. C(=O)R in LMD in formula (BC) or formula (GC) Ｌ The pharmaceutical formulation according to claim 5 , wherein represents a residue resulting from the removal of an HO group from oleic acid, for the treatment of non-alcoholic fatty liver disease, or the treatment of obesity, or anti-inflammation.

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