Hydrophilic-hydrophobic copolymer containing (ethylene glycol) chain and poly(branched-chain amino acid) chain, and use for said copolymer

Abstract

Disclosed are: a hydrophilic-hydrophobic diblock copolymer or a nano-sized polymeric micelle containing said hydrophilic-hydrophobic diblock copolymer, said copolymer containing a poly(branched amino acid) segment represented by formula AA in which R is a residue for forming leucine, isoleucine, or valine and m is an integer of 3-200, and a poly(ethylene glycol) segment represented by formula BB in which n is an integer of 5-1,000; and a use for said copolymer / micelle in improving athletic ability and the like. When the hydrophilic-hydrophobic copolymer or the polymeric micelle is administered to a mammal such as a human, an improvement in athletic ability is achieved which cannot be realized by using a corresponding low-molecular leucine, isoleucine, or valine.

Description

Hydrophilic-hydrophobic copolymers containing (ethylene glycol) chains and poly(branched amino acid) chains and uses thereof

[0001] The present invention relates to a hydrophilic-hydrophobic copolymer comprising a poly(ethylene glycol) chain segment and a poly(branched-chain amino acid) segment, and uses thereof. More specifically, the present invention relates to the copolymer, in which the branched-chain amino acid is any amino acid selected from leucine, isoleucine, and valine, or to nano-sized polymeric micelles or nanoparticles containing the copolymer, and uses thereof for the purpose of improving athletic performance, etc.

[0002] Branched-chain amino acids (hereinafter sometimes referred to as "BCAA") are a collective term for the essential amino acids leucine, isoleucine, and valine. BCAAs have been reported to activate muscle synthesis, inhibit muscle breakdown, or act as an energy source, and are expected to improve athletic performance, such as increasing muscle mass or strength (Wolfe, Robert R. Journal of the International Society of Sports Nutrition, 14.1 (2017): 30, sometimes referred to as Non-Patent Document 1, see). From another perspective, BCAA supplementation has been used to reduce delayed onset muscle soreness (DOMS) after exercise training, promote protein metabolism, inhibit muscle damage, and induce anabolic muscle activity (see Michael V. Fedewa, et al., International Journal for Vitamin and Nutrition Research, (2019), 89(5-6), 348-356, sometimes referred to as Non-Patent Document 2).

[0003] However, because BCAAs are small molecules, they have low in vivo retention and cannot maintain an effective concentration in the body for a long period of time after administration or ingestion. Therefore, the effect of administering or ingesting BCAAs on improving exercise performance in humans has not been demonstrated (Doma, Kenji, et al., Appl. Physiol. Nutr. Metab. 46.11 (2021): 1303-1313, sometimes referred to as Non-Patent Document 3, see). Meanwhile, in order to solve the problems associated with the use of ornithine, an amino acid, in the medical field due to its low molecular weight, some of the present inventors, including Nagasaki, have provided so-called AB diblock copolymers and ABA triblock copolymers containing an ornithine segment (A) in which the δ-amino group of ornithine is protected and an ethylene glycol segment (B), and have discovered that ornithine's inherent physiological activity (e.g., preventive or therapeutic effects on liver dysfunction) can be effectively exerted in vivo, and have also proposed their use in the medical field (see WO 2022 / 102608 A1, sometimes referred to as Patent Document 1). From this perspective, we searched for specific examples of similarly polymerized BCAAs, and found a report on the synthesis of an ABA triblock copolymer containing an L-leucine segment (A) and an ethylene glycol segment (B) as a polymerized form of L-leucine, and the effect of the concentration on the viscosity of the aqueous solution (Tatsuro Nakano, Yutaka Tanaka, Research Reports, Graduate School of Engineering, University of Fukui, Vol. 60, March 2012, pp. 35-40). This report discusses the physical properties of the ABA triblock copolymer, such as the solubility, transparency, and gel-forming properties of the copolymer aqueous solution. However, the triblock copolymer described in Non-Patent Document 3 does not state or suggest that it can be used in the medical or nutritional supplement fields.

[0004] An object of the present invention is to provide a means for overcoming the above-mentioned problems associated with BCAAs due to their small molecular weight when used in treated animals (mammals including humans). Specifically, an object of the present invention is to provide a BCAA-based preparation, construct, or formulation that can be effectively administered to the living body of a treated animal, thereby improving athletic performance, etc.

[0005] To solve these problems, the present inventors have conducted research to provide a preparation or construct that allows sustained release of BCAAs in vivo, thereby maintaining the effective concentration of BCAAs in vivo for a long period of time. As a result, they discovered that nano-sized polymeric micelles or nanoparticles can be prepared by incorporating BCAAs into a polymer (polymerization) and combining it with hydrophilic and biocompatible poly(ethylene glycol) (hereinafter sometimes referred to as "PEG") to form a hydrophilic-hydrophobic diblock copolymer, and then self-assembling this copolymer in an aqueous medium to produce nano-sized polymeric micelles or nanoparticles that can solve the above-mentioned objectives or problems in an extremely safe manner. Surprisingly, as described above, this hydrophilic-hydrophobic diblock copolymer exhibits a significantly excellent effect of improving athletic performance, as well as a related effect of suppressing various organ damage in treated animals and, independently, a suppression effect of fat accumulation, including subcutaneous fat, in treated animals. However, the ABA triblock copolymer described in Non-Patent Document 4 is not known to be capable of exhibiting these effects.

[0006] In addition to this method, some of the present inventors, including Toriumi, have reported that excess reactive oxygen species (sometimes referred to as "ROS") generated during strenuous or unaccustomed exercise can be continuously removed by using a self-assembling nanoparticle antioxidant (including a copolymer containing a PEG chain segment and a poly(TEMPO) chain segment) that can be selectively distributed in the gastrointestinal tract, thereby improving exercise performance (see DOI: 10.1002 / advs.202301159 Adv. Sci. 2023. 10. 2301158 or JP 2020-186191 A).

[0007] However, the hydrophilic-hydrophobic diblock copolymer disclosed in the present application and the latter copolymer differ from each other in structure and mechanism of action in the body, and even when the purpose is to improve athletic performance, they provide completely different means.

[0008] Therefore, the main features or subjects disclosed herein include, but are not limited to, the following: 1. A hydrophilic-hydrophobic block copolymer represented by the following formula (I) or (II):

[0009] In each formula, A １ and A ２ are each independently unsubstituted or substituted C １ -C １２ The substituents when substituted are a formyl group, a group of formula R ’ R ” CH—, phenylamino, or phenethylamino, where R ’ and R ” is independently C １ -C ４ Alkyloxy or R ’ and R ” Let's get together -OCH ２ CH ２ O-, -O(CH ２ ) ３ O- or -O(CH ２ ) ４ O-, represented by L １ represents a single bond or a linking group; ２ represents a linking group, and each R is independently CH ２ CH (CH ３ ) ２ , CH (CH ３ ) CH ２ CH ３ and CH(CH ３ ) ２ and Z is selected from one or more groups represented by the following formula: １ represents a hydrogen atom or an amino-protecting group; Z ２represents an OH or carboxy protecting group, m1 and m2 are each independently an integer of 3 to 200, preferably 5 to 150, and more preferably 5 to 100, and each n1 and n2 is each independently an integer of 5 to 1000, preferably 10 to 500, and more preferably 10 to 300. 2. A polymeric micelle comprising the copolymer described in 1 above and having an average particle size in the nanosize range. 3. A formulation for improving the athletic performance of a subject animal, comprising as an active ingredient the copolymer described in 1 above or a polymeric micelle comprising said copolymer and having an average particle size in the nanosize range. 4. The formulation described in 3 above, wherein the improvement in athletic performance is an improvement in the muscle mass or muscle strength of the subject animal or an improvement in running performance. 5. A method for improving the athletic performance of a subject animal in need thereof, comprising the step of administering to said subject animal an effective amount of the copolymer described in 1 above or a polymeric micelle comprising said copolymer and having an average particle size in the nanosize range. 6. 6. The copolymer according to 1 above or polymeric micelles comprising said copolymer and having an average particle size in the nanosize range, for use with the purpose of improving the athletic ability of a target animal. 7. Use of the copolymer according to 1 above or polymeric micelles comprising said copolymer and having an average particle size in the nanosize range, for improving the athletic ability of a target animal. 8. A formulation for suppressing exercise-induced liver, kidney, or skeletal muscle damage in a target animal, comprising as an active ingredient the copolymer according to 1 above or polymeric micelles comprising said copolymer and having an average particle size in the nanosize range. 9. A method for suppressing exercise-induced liver, kidney, or skeletal muscle damage in a target animal, comprising the step of administering to a target animal in need thereof an effective amount of the copolymer according to 1 above or polymeric micelles comprising said copolymer and having an average particle size in the nanosize range. 10. The copolymer according to 1 above or polymeric micelles comprising said copolymer and having an average particle size in the nanosize range, for use with the purpose of suppressing exercise-induced liver, kidney, or skeletal muscle damage in a target animal. 11. 2. Use of the copolymer according to 1 above or a polymeric micelle containing the copolymer and having an average particle size in the nanosize range, for suppressing exercise-induced damage to the liver, kidney, or skeletal muscle of a treated animal.12. A preparation for suppressing fat accumulation in a target animal, comprising as an active ingredient the copolymer described in 1 above or polymeric micelles comprising the copolymer and having an average particle size in the nanosize range. 13. A method for suppressing fat accumulation in a target animal, comprising the step of administering to a target animal in need thereof an effective amount of the copolymer described in 1 above or polymeric micelles comprising the copolymer and having an average particle size in the nanosize range. 14. The copolymer described in 1 above or polymeric micelles comprising the copolymer and having an average particle size in the nanosize range, for use with the purpose of suppressing fat accumulation in a target animal. 15. Use of the copolymer described in 1 above or polymeric micelles comprising the copolymer and having an average particle size in the nanosize range, for suppressing fat accumulation in a target animal.

[0010] The hydrophilic-hydrophobic copolymer of multiple molecules described in 1 above or polymeric micelles containing the copolymer can be provided in an aqueous medium as core-shell nanoparticles with an average particle size of nanometer size, which have a core primarily composed of poly(branched-chain amino acid) segments and a shell primarily composed of poly(ethylene glycol). Therefore, the nanoparticles can be dissolved in an aqueous medium. Therefore, they are suitable for use in treated animals (humans and other mammals) via various administration routes or in various ingestion modes. Furthermore, even when orally administered and exposed to harsh in vivo environments, the nanoparticles exhibit significant effects in treated animals, including improved athletic performance, compared to the corresponding low-molecular-weight BCAAs. Detailed Description of the Invention

[0011] Unless otherwise specified, terms used herein are to be construed as having the meanings commonly used in the art. <Block copolymer and polymeric micelle prepared therefrom>

[0012] Nano-sized polymeric micelles and nanoparticles are used interchangeably.

[0013] Theoretically, when polymeric micelles are formed or used in a lipophilic solvent, they may be nanoparticles containing primarily poly(ethylene glycol) segments as shown in formula (I) or (II) as a core and primarily poly(branched-chain amino acid) segments as a shell. However, polymeric micelles suitable for the applications of the present invention, particularly pharmaceutical applications, are, conversely, nanoparticles that are molecular assemblies with an average particle size of nanometers, formed by self-assembly or association of multiple molecules of the copolymer of formula (I) or (II) as a core and primarily poly(ethylene glycol) segments as a shell in an aqueous medium (containing water, and optionally containing a buffer such as a phosphate buffer, salt, or a water-miscible organic solvent).

[0014] Without being bound by theory, the latter polymeric micelles are provided as so-called core-shell nanoparticles in an aqueous medium, where the hydrophobic poly(branched amino acid) segments primarily associate via hydrophobic interactions to form a core, while the flexible, hydrophilic, and biocompatible poly(ethylene glycol) segments primarily form a shell. Nano-sized nanoparticles refer to nano-sized particles with an average particle size measured by dynamic light scattering (DLS) in an aqueous medium, which is within the nano-size range, but is not limited to, 10 nm to 500 nm, 10 nm to 300 nm, 10 nm to 200 nm, 10 nm to 150 nm, or 10 nm to 60 nm.

[0015] Therefore, due to these sizes and biocompatibility, even when the latter polymeric micelles are orally administered to mammals including humans, it can be understood that the poly(branched-chain amino acid) segments are less susceptible to strong acidity or direct action by biological enzymes (peptidases, etc.), resulting in improved retention in the gastrointestinal tract. Furthermore, without being bound by theory, on the other hand, the hydrophilic and biocompatible poly(ethylene glycol) segments are easily hydrated and mobile, allowing various endogenous enzymes with hydrolytic activity to access the poly(branched-chain amino acid) segments that form the core, subject to certain appropriate constraints. Therefore, by cleaving the peptide bonds within the segments, and optionally the ester bonds or amide bonds contained in the linking groups, branched-chain amino acids (BCAAs) can be sustainedly released from the nanoparticles or copolymers. Therefore, for example, when orally administered, it can be understood that in vivo, BCAAs enter the bloodstream from the gastrointestinal tract for a long period of time, and are delivered to muscles, thereby achieving improvements in muscle mass, muscle strength, and running performance.

[0016] Therefore, the hydrophilic-hydrophobic block copolymer disclosed herein can achieve the object of the present invention as long as it can form the aforementioned core-shell type polymeric micelles or nanoparticles in an aqueous medium. Specific examples of such a block copolymer include the hydrophilic-hydrophobic block copolymer represented by formula (I) or (II) described in Subject 1 above.

[0017] L in formula (I) or formula (II) １ or L ２ The linking group referred to in 、 It can be defined as a divalent group, generally containing up to 34, preferably 18, more preferably up to 10 carbon atoms, and optionally oxygen and nitrogen atoms, and having at least one group or moiety capable of forming an amide bond or an ester bond within the linking group. １ is -(CH ２ ) ａ -NH- or -(CH ２ ) ａ represents —O—, and L ２ Ha-(CH ２ )ｂ -C(=O)- or -C(=O)-(CH ２ ) ｂ represents —C(═O)—, where a is an integer of 1 to 6, and b is an integer of 1 to 6. As can be understood from the description of formula (I) or formula (II) above, these linking groups are incorporated into the respective formulas in the direction described therein.

[0018] Z in formula (I) or formula (II) １ and Z ２ The amino protecting group and carboxy protecting group referred to in the above formula (I) are not limited to the following groups. For example, the amino protecting group is C １ - ２１ Alkylcarbonyl, substituted C １－４ Alkylcarbonyl, unsubstituted or substituted C ３ - ７ represents a cycloalkylcarbonyl, an unsubstituted or substituted arylcarbonyl, or an unsubstituted or substituted 5- or 6-membered heteroarylcarbonyl, wherein １－４ The substituents of the alkylcarbonyl are halogen atoms, hydroxyl, carboxyl, 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 cholesterol residues, and when these substituents are substituted, the substituents are C １－４ Alkyl, C １－４ Alkyloxy, hydroxyl, carboxyl, cyano, nitro, halogen atoms, or mono- or di-C １－４ It can be alkylamino.

[0019] On the other hand, the carboxy-protecting group may be represented by the corresponding "represents oxy" instead of "represents carbonyl" as in the amino-protecting group.

[0020] In Formula (I) or Formula (II), the branched amino acid-derived units constituting each repeating unit in the poly(branched-chain amino acid) segment, which is composed of m repeating units, have one asymmetric center and can adopt the so-called L-type or D-type relative configuration. As described above, the copolymer can form, in an aqueous medium, core-shell polymeric micelles of Formula (I) or Formula (II), in which the hydrophobic core is primarily composed of poly(branched-chain amino acid) segments, while the hydrophilic shell is primarily composed of poly(ethylene glycol) segments. Therefore, when a hydrophobic drug is coexisted in a system that forms such polymeric micelles, nanoparticles containing the drug can be provided. Therefore, in addition to the above-mentioned uses, the hydrophilic-hydrophobic copolymer disclosed herein can also be used as a carrier for delivering drugs into the body.

[0021] Therefore, the branched-chain amino acids (leucine, isoleucine, or valine, or a mixture of one or more of these amino acids) constituting each of the repeating units have industrial applicability regardless of whether they can be classified as D or L. However, for applications such as those described in Subjects 4 and 5, it is preferred that less than 50%, preferably 70% or less, and more preferably 90% or less of the m repeating units derived from branched amino acids in the poly(branched-chain amino acid) segment represented by the formulas above are D-type, with the remainder being L-type.

[0022] Each of A and Z １ , Z ２ When defining each group or part thereof, C ａ -C ｚ -Alkyl means a straight or branched alkyl having a to z carbon atoms, and may be, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, undecyl, etc. <Production of Copolymer>

[0023] In producing the block copolymer represented by Formula (I) or Formula (II) described in Subject 1, for example, a polypeptide is prepared in which either the C-terminus or the N-terminus of the corresponding poly(branched amino acid) segment may be protected with a known carboxy-protecting group or amino-protecting group, and a poly(ethylene glycol) derivative is prepared in which the α-terminus of the corresponding poly(ethylene glycol) segment may be protected and the ω-terminus may be modified with an aminoalkyl or carboxyalkyl. Some of the polypeptides and poly(ethylene glycol) derivatives prepared in this manner are known and commercially available, or can be obtained by making appropriate modifications to known products as necessary.

[0024] The desired copolymer can be produced by condensing the two thus obtained components, if necessary, using a condensation promoter commonly used in the art. Specifically, the block copolymer represented by formula (I) or formula (II) may be a block copolymer in which either the N-terminus of the polypeptide corresponding to the poly(branched-chain amino acid) segment is protected with, for example, a benzyloxycarbonyl group (Z group) or the N-terminus of the poly(ethylene glycol) corresponding to the poly(ethylene glycol) segment is protected with an aminoalkyl [(CH ２ ) a NH ２ where a is the integer.] is condensed in the presence of a known appropriate condensing agent to obtain a polypeptide in which the C-terminus, instead of the N-terminus of the polypeptide corresponding to the poly(branched-chain amino acid) segment, is protected with, for example, a t-butyloxy group, and a polypeptide in which either end of the poly(ethylene glycol) segment may be protected, and the other or both ends are protected with a carboxyalkyl [(CH ２ ) aCOOH, where a is the above-mentioned integer. In the case where the branched amino acids in the poly(branched amino acid) segment of the polypeptide are composed of multiple types of amino acids, for example, a polypeptide produced by ring-opening polymerization of a mixture of multiple types of N-carboxyanhydrides (NCAs) corresponding to the branched amino acids can be used.

[0025] Such a block copolymer, particularly that represented by formula (I), can be more conveniently produced by the synthetic route outlined in the following Reaction Scheme 1. <Preparation of polymeric micelles>

[0026] The polymeric micelles or nanoparticles, or the block copolymer represented by formula (I) or formula (II), can be prepared by dissolving the polymeric micelles or nanoparticles, or the block copolymer represented by formula (I) or formula (II), in a good solvent for the copolymer, such as a mixed solvent of chloroform and trifluoroacetic acid, followed by dialysis against a water-soluble organic solvent, such as N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), and then dialyzing the copolymer in the dialyzed DMF or DMSO against distilled water, thereby causing the copolymers to associate with each other to form micelles. The polymeric micelles or nanoparticles thus formed can be obtained as a separated or isolated solid by, for example, freeze-drying, centrifugation, etc. <Preparation or formulation containing polymeric micelles>

[0027] The polymeric micelles or nanoparticles thus prepared can be provided as a solution or liquid formulation dissolved or uniformly dispersed in an aqueous medium (containing water, and optionally containing a buffer such as a phosphate buffer, salt, or a water-miscible organic solvent), and can be used in various forms, including parenteral formulations and oral formulations. Formulations containing the copolymer or polymeric micelle as an active ingredient may be in any form, as long as the copolymer or polymeric micelle exerts its effects systemically or locally on the target animal to which it is delivered. For example, when provided as an oral formulation, the nanoparticles of the present invention can be provided as tablets, pills, or granules using excipients and diluents commonly used in the art. Examples of excipients and diluents include, but are not limited to, chlorocarmellose sodium, crystalline cellulose, hypromellose, sodium lauryl sulfate, magnesium stearate, macrogol 4000, titanium oxide, and the like, which are commonly used in the art. <Uses of Copolymers or Polymeric Micelles>

[0028] The aspects of the use include those described in the above-mentioned themes 3 to 15, and the following explanations can be added regarding the terms and matters mentioned therein.

[0029] The "subject animal" is not limited in any way as long as it is an animal that can achieve the intended purpose in the applications disclosed in this application, and may include mammals such as humans, children, adults, livestock, pets, racehorses, etc.

[0030] "Improving athletic performance" means improving at least some or all of the physiological functions that BCAAs themselves are understood to potentially possess, including an improvement in muscle mass or strength or running performance of the treated animal. On the other hand, symptoms such as, but not limited to, a reduction or alleviation of delayed onset muscle soreness (DOMS), which is exercise-induced muscle damage that occurs after exercise, may be observed.

[0031] "Exercise-induced" means that the injury is caused or induced by physical activity, primarily for health or enjoyment, and specifically, see the experimental schedules shown in Test Examples 1 to 5. Exercise-induced injury may also be a factor in causing or inducing subclinical muscle damage, which typically manifests as symptoms ranging from mild tenderness and stiffness to severe, debilitating pain that limits movement (see Non-Patent Document 2). Such symptoms can be confirmed in Test Example 12.

[0032] The phrase "suppressing exercise-induced damage to the liver, kidney, or skeletal muscle" referred to in Aspects 8 to 11 means that the "copolymer or polymeric micelle containing the copolymer" disclosed in the present application can protect the liver, kidney, or skeletal muscle from such damage caused by exercise.

[0033] The fat accumulation in "suppressing fat accumulation in a treated animal" or "suppressing fat accumulation in a treated animal" mentioned in Topics 12 to 15 mainly refers to the accumulation of subcutaneous fat or visceral fat, and specifically, by bringing about such an effect, it is possible to expect a preventive or therapeutic effect on lifestyle-related diseases, visceral diseases, and the like caused by fat accumulation.

[0034] The "effective amount" referred to in Topics 5, 9, 13, etc. cannot be uniformly specified because the optimal dose varies depending on the treatment target, species, and condition of the treated animal, and the type of formulation, but can be determined by a specialist based on data obtained through the test examples described below or small-scale clinical trials, etc. Although not limited thereto, a typical single or divided dose may be about 3.8 μmol to about 762 μmol per kg of body weight, preferably about 38 μmol / kg / day to about 381 μmol / kg / day, in terms of the BCAA dose in the copolymer or polymeric micelle.

[0035] PEG-Ms and PEG-NH obtained in Production Example 1, respectively ２ of １ 1 H NMR spectra of NCA-leucine, NCA-isoleucine, and NCA-valine obtained in Preparation Examples 2 to 4, respectively. １1 H NMR spectra of PEG-b-PLeu, PEG-b-PIle, and PEG-b-PVal obtained in Preparation Examples 5 to 7, respectively. １ 1H NMR spectrum. TFA in the figure means trifluoroacetic acid. The results of dynamic light scattering (DLS) measurement of each polymeric micelle solution obtained in Production Examples 8 to 10 are shown. The results of circular dichroism (CD) measurement of each polymeric micelle solution obtained in Production Examples 8 to 10 are shown. １９ 1 shows the results of F-NMR measurements (internal standard: trifluoroethanol). 2 shows the experimental schedules carried out in Test Examples 1 to 3. 3 shows a graphical representation of the results of a grip test at each administration concentration of leucine in Test Example 1. 4 shows a graphical representation of the results of a grip test at each week after leucine intake in Test Example 1. 5 shows a graphical representation of the results of a grip test with isoleucine administration in Test Example 2. 6 shows a graphical representation of the results of a grip test with valine administration in Test Example 3. 7 shows a graphical representation of the results of an all-out test with leucine in Test Example 1. 8 shows a graphical representation of the results of an all-out test with isoleucine in Test Example 2. 9 shows a graphical representation of the results of an all-out test with valine in Test Example 3. 10 shows the experimental schedule carried out in Test Example 4. 11 shows a graphical representation of the results of evaluating the weight of skeletal muscle of test individuals in Test Example 4. 12 shows a graphical representation of the results of evaluating mRNA in skeletal muscle by PCR in Test Example 5. 13 shows a graphical representation of the evaluation results of the body weight, food intake, and survival of each test individual in Test Examples 6 to 8. 1 is a graph showing a photograph of a stained section of the soleus muscle (left) and the cross-sectional area of ​​the soleus muscle (right) in Test Example 9. FIG. 1 is a graph showing the measurement results of muscle glycogen in Test Example 10. FIG. 1 is a graph showing the measurement results of various blood system factors in Test Example 11. FIG. 1 is a graph showing the measurement results of various organ damage markers in Test Example 12. FIG. 1 is a graph showing the measurement results of body fat, subcutaneous fat, and visceral fat of test animals in Test Example 13.

[0036] The subject matter disclosed in this specification or the present invention will be described in more detail below, but this does not limit the scope of the subject matter or the present invention in any way. <Synthesis example of a typical block copolymer>

[0037] Although not limited thereto, the synthesis can typically be carried out according to the following reaction scheme 1.

[0038] The synthesis can be broadly divided into the following steps: ２ synthesis of branched-chain amino acid N-carboxyanhydrides (NCA-BCAAs) as monomers; and synthesis of PEG-b-PBCAAs by extending the poly-BCAA chain from the PEG terminal using these.

[0039] More specific synthesis examples are described in the following production examples. Production Example 1: PEG-Ms, PEG-NH ２ Manufacturing

[0040] Using a 35°C water bath, 10 g (2 mmol) of commercially available mono-terminated methoxy polyethylene glycol (MeO-PEG-OH; molecular weight 5000) was dissolved in 25 mL of chloroform. At room temperature, 1.39 mL (10 mmol) of triethylamine (TEA) and 0.77 mL (10 mmol) of methanesulfonyl chloride (MsCl) were added and incubated for 5 hours. The reaction solution was precipitated in 600 mL of isopropanol (IPA) pre-cooled to -30°C, and the precipitate was collected by centrifugation. The supernatant was discarded, and the precipitate was dissolved in a small amount of methanol. Reprecipitation and centrifugation were performed again in 600 mL of cold IPA (this procedure was repeated four times). The IPA was removed by vacuum drying, and PEG-Ms was collected (yield: 9.65 g, yield: 96.5%, incorporation rate: 100%).

[0041] 9 g of PEG-Ms was dissolved in 70 mL of 28% aqueous ammonia and allowed to react at room temperature for 3 days. The reaction solution was evaporated under reduced pressure, and the residue was dissolved in a small amount of methanol. The above-mentioned IPA reprecipitation and centrifugation were repeated four times. The IPA was removed by drying under reduced pressure, and PEG-NH ２ was recovered (yield: 7.3 g, yield: 81.2%, introduction rate: 100%).

[0042] The above PEG-Ms and PEG-NH ２ of １The NMR absorption spectra obtained by H NMR (nuclear magnetic resonance) measurement are shown in Figure 1. Production Examples 2 to 4: Production of NCA-BCAA

[0043] 15 g (60.4 mmol) of N-Boc-leucine monohydrate was dried overnight under reduced pressure and dissolved in 120 mL of ultra-dehydrated tetrahydrofuran (THF) under a nitrogen atmosphere. A mixture of 47.9 mL (302 mmol) of (1S)-(-)-α-pinene, 10.9 mL (151 mmol) of thionyl chloride, and 30 mL of ultra-dehydrated THF was added and reacted under ice cooling (15 minutes of ice bath stirring, followed by 3 hours at room temperature). The reaction solution was concentrated under reduced pressure, and the residue was poured into 1.5 L of hexane to precipitate. The precipitate was filtered, dissolved in THF, and reprecipitated by adding 1 L of hexane (overnight at -30°C). The precipitate was collected, dissolved in a small amount of THF, and recrystallized using hexane (twice). The collected material was dried under reduced pressure, and NCA-leucine was recovered (yield: 7.07 g, 74.7%).

[0044] The above process was repeated except that N-Boc-leucine hydrate was replaced with N-Boc-isoleucine hydrate and N-Boc-valine hydrate in equivalent stoichiometric amounts (Preparation Examples 3 and 4, respectively). NCA-isoleucine and NCA-valine were recovered from isoleucine and valine in similar yields to those from leucine.

[0045] The recovered NCA-leucine, NCA-isoleucine and NCA-valine １ The H NMR absorption spectra are shown in Figure 2. Preparation Examples 5 to 7: Preparation of PEG-b-PBCAA

[0046] PEG-NH synthesized in Production Example 1 ２ 3 g (0.6 mmol) of the above and 4.9 g (34 mmol) of NCA-leucine synthesized in Production Example 2 were each dissolved in ultra-dehydrated N,N-dimethylformamide (DMF) under a nitrogen atmosphere (total concentration of 350 mM relative to NCA-leucine), and the NCA-leucine solution was added to PEG-NH ２The reaction solution was added to the solution and polymerized for 3 days at 30° C. The reaction solution was precipitated with methyl tert-butyl ether, and the precipitate was collected. This procedure was repeated twice, and then PEG-b-PLeu was collected by drying under reduced pressure (yield: 5.03 g, degree of leucine polymerization: 49).

[0047] The above process was repeated (Production Examples 6 and 7, respectively), except that NCA-isoleucine and NCA-valine recovered in Production Examples 3 and 4 were used in place of NCA-leucine in equivalent stoichiometric amounts, and PEG-b-PIle and PEG-b-PVal were recovered in yields and degrees of polymerization similar to those of PEG-b-PLeu, respectively.

[0048] The recovered PEG-b-PLeu, PEG-b-PIle, and PEG-b-PVal １ The H NMR absorption spectra are shown in Figure 3. Production Examples 8 to 10: Preparation of branched-chain amino acid nanoparticles

[0049] 4 g of PEG-b-PLeu synthesized in Production Example 5 was dissolved in 53.2 mL of trifluoroacetic acid and 120 mL of chloroform, and the solution was sealed in a dialysis membrane with a molecular weight cutoff of 3500 Da. The solution was dialyzed against DMF for one day. After exchanging the DMF and continuing dialysis for another day, the solution was dialyzed against distilled water. The distilled water was exchanged every few hours, a total of eight times over two days, and then a nanoparticle solution (Nano) containing polyleucine as a single component was prepared. Ｌｅｕ The concentration was adjusted by appropriate concentration and dilution.

[0050] The above process was repeated (Production Examples 9 and 10), except that PEG-b-PIle and PEG-b-PVal synthesized in Production Examples 6 and 7 were used in place of PEG-b-PLeu in equivalent stoichiometric amounts, to prepare isoleucine nanoparticle solutions (Nano Ｉｌｅ ) and valine nanoparticle solution or polymeric micelle solution (Nano Ｖａｌ ) were collected, respectively.

[0051] The dynamic light scattering (DLS) measurement results for each polymeric micelle solution are shown in Figure 4, and the circular dichroism (CD) measurement results for each polymeric micelle solution are shown in Figure 5. Ｌｅｕ represents the α-helical structure, and Nano Ｉｌｅ, Nano Ｖａｌ Each of the polymeric micelle solutions has a β-sheet structure. １９ The results of F-NMR measurement (internal standard: trifluoroethanol) are shown in Figure 6. It was also confirmed that no trifluoroacetic acid used in the production of the nanoparticles remained. Test Examples 1 to 3: Verification of the athletic performance improving effect of branched-chain amino acid nanoparticles

[0052] The effect of branched-chain amino acid nanoparticles on improving athletic performance was examined using commercially available mice (C57BL6 / J, male, 6 weeks old) (8 mice per group). To evaluate athletic performance, a grip test was conducted to measure the grip strength of the mice, and an all-out test was conducted to evaluate running performance based on the running time after the mice were forced to run until exhaustion. Evaluations were conducted by a third party blinded to the groups. The experiment was conducted according to the experimental schedule shown in Figure 7. After one week of environmental acclimation, the mice were placed in water, leucine, and nanoparticles. Ｌｅｕ The rats were divided into three groups and given each compound with free access to water. Ｌｅｕ Groups were prepared by feeding 1.88, 3.75, and 7.5 mmol / L of leucine equivalent. A grip test was performed once a week. After three weeks of feeding, the animals underwent five days of running training, one day of rest, and three hours of fasting. Then, an all-out test was performed at a speed of 28 m / min. Two days after the all-out test, the animals were sacrificed under anesthesia.

[0053] The above leucine group and Nano Ｌｅｕ Instead of the group, the isoleucine group and Nano Ｉlｅ Alongside the valine group and Nano Ｖａｌ The above procedure was repeated except that the 1000-2000 group was used (Test Examples 2 and 3). Similar experiments were also conducted for these alternative test examples, in which the concentration of each amino acid was adjusted to 7.5 mmol / L and inoculated, and the motor ability was evaluated.

[0054] The results of the grip test at each leucine administration concentration are shown in Figure 8 (the results of the grip test at each leucine administration concentration, in the figure, ■: control, ▲: leucine, ◆: Nano). Ｌｅｕ and ＊P < 0.05; ＊＊ *P<0.01 vs. control, *P<0.05, *P<0.01 vs. leucine are shown.) and Figure 9 (results of grip test at each week after intake, in the figure, ■: control, ▲: leucine, ◆: Nano Ｌｅｕ , ＊ P < 0.05; ＊＊ *P<0.01 vs. control, *P<0.05, *P<0.01 vs. leucine.) As shown in Figures 8 and 9, the leucine group did not show a significant improvement in grip strength compared to the control group, but the Nano Ｌｅｕ A significant improvement in grip strength was confirmed in each group at each administration concentration and each week after intake.

[0055] Leucine group and Nano Ｌｅｕ Instead of the groups, the isoleucine group and Nano Ｉｌｅ Group, Valine Group and Nano Ｖａｌ The above-mentioned grip test was carried out for the isoleucine group and the Nano group (Test Examples 2 and 3, respectively). Ｉｌｅ The results of the grip test when the group was administered are shown in Figure 10 (in the figure, ■: control, ▲: isoleucine, ◆: Nano Ｉｌｅ , ＊ *P < 0.05 vs. control, *P < 0.05 vs. isoleucine) and the valine group and Nano Ｖａｌ The results of the grip test when the group was administered are shown in Figure 11 (in the figure, ■: control, ▲: valine, ◆: Nano). Ｖａｌ , ＊ P < 0.05; ＊＊ *P<0.01 vs. control, *P<0.01 vs. valine).

[0056] 10 and 11, similar to the test results for leucine, low molecular weight isoleucine and valine did not significantly improve grip strength, Ｉｌｅ , Nano Ｖａｌ It can be seen that there is a significant improvement in grip strength.

[0057] The results of the all-out test in Test Example 1 are shown in FIG. ＊ P < 0.05; ＊＊P < 0.01, r: Pearson's correlation coefficient). As shown in Figure 12, the leucine group did not show a significant increase in the limit running time compared to the control group, but the Nano Ｌｅｕ A significant and dose-dependent increase in running time was observed in the group.

[0058] The results of the all-out tests in Test Examples 2 and 3 are shown in Figures 13 and 14, respectively. As in the results of Test Example 1, the low molecular weight isoleucine and valine do not significantly extend the limit running time. Ｉｌｅ , Nano Ｖａｌ It can be seen that the running time is significantly extended. Test Example 4: Evaluation of muscle mass by branched-chain amino acid nanoparticles

[0059] Using a different individual from Test Examples 1 to 3, an experiment was conducted in which the individual was run for 80 minutes according to the experimental schedule shown in Figure 15. The conditions up to the 3-hour fasting were the same as in the all-out test of Test Examples 1 to 3 (Figure 7), and the individual was made to run for 80 minutes at a speed of 28 m / min, and immediately after the run, the individual was sacrificed under anesthesia.

[0060] The weight of the skeletal muscle of each subject in each group (7 animals per group) was evaluated. Ｌｅｕ The test results are shown in Figure 16 (in the figure, ＊ P < 0.05; ＊＊ P < 0.01). The figure shows that the leucine group did not show a significant increase in skeletal muscle mass compared to the control group, but the Nano Ｌｅｕ A significant increase in skeletal muscle mass was observed in the Nano group. ＢＣＡＡ Evaluation of activation of muscle synthesis and mitochondrial biogenesis and inhibition of muscle degradation by

[0061] The mRNA in the skeletal muscle collected in Test Example 4 was evaluated by PCR. The evaluation results are shown in Figure 17 (in the figure, ＊ P < 0.05; ＊＊ P < 0.01). From the figure, Nano Ｌｅｕ It is confirmed that the group significantly increases mTOR, which promotes muscle synthesis, and suppresses 4EBP-1. ＬｅｕIt was confirmed that Foxo3a, which promotes muscle breakdown, was significantly suppressed in the Nano group. Ｌｅｕ The group also showed a significant increase in SIRT1 and NRF-1, which are involved in mitochondrial biogenesis, and SIRT3, which is involved in the energy system. ＢＣＡＡ Toxicity assessment with continuous administration

[0062] Evaluation was carried out using test individuals from Test Examples 1 to 3 and Test Example 4. The body weight, food intake, and survival status of each individual were measured during the period from the start of rearing to rest as shown in the experimental schedules in Figures 7 and 15, and toxicity was evaluated. The results of each evaluation are shown in Figure 18. From the graph, Nano mice were fed with concentrations of 1.88, 3.75, and 7.5 mmol / L in terms of leucine. Ｌｅｕ The survival of all individuals in the group was confirmed. Ｌｅｕ No significant change in food intake was observed in the group compared to the control group and the leucine group. Ｌｅｕ No significant effect on weight gain was observed in the group compared to the control group and the leucine group. Ｌｅｕ No toxicity was observed due to administration.

[0063] Also, Nano Ｌｅｕ Instead, Nano Ｉｌｅ and Nano Ｖａｌ The same toxicity evaluation test as described above was carried out using each of the Nano Ｌｅｕ Test results were almost equivalent to those observed for Nano, and no toxicity was observed. ＢＣＡＡ Evaluation of changes in skeletal muscle cross-sectional area due to administration of

[0064] This test was carried out using the Nano Ｌｅｕ The purpose of this study was to evaluate the muscle cross-sectional area involved in muscle strength to support the effect of administration of α-glucan on improving grip strength.

[0065] The skeletal muscle (soleus muscle) collected in Test Example 4 was fixed in 10% neutral buffered formalin, and sections were prepared perpendicular to the tissue to a thickness of 10 μm, followed by hematoxylin-eosin staining (HE staining). The stained sections were observed using an all-in-one fluorescence microscope (BZ-X710), and the cross-sectional area of ​​the skeletal muscle was evaluated using Image J, an image analysis software. <Results>

[0066] The photograph of the stained section of the skeletal muscle and the cross-sectional area are shown in FIG. 19 (*P<0.05, **P<0.01). Ｌｅｕ A significant increase in muscle cross-sectional area was confirmed in the Nano group. This result is considered to have had an effect on muscle strength enhancement. ＢＣＡＡ Evaluate the effect of administration of on muscle glycogen increase

[0067] In this test, the Nano shown in FIG. Ｌｅｕ The purpose of this study was to evaluate glycogen in the muscles involved in running performance to support the effect of administration of α-glucan on extending the limit running time.

[0068] The skeletal muscle (gastrocnemius muscle) collected in Test Example 4 was weighed and placed in a centrifuge tube containing 300 μL of 30% (W / V) potassium hydroxide solution and incubated in a boiling water bath for 20 minutes. The centrifuge tube was then cooled using an ice bath, and 30 μL of saturated sodium sulfate solution was added for neutralization. 500 μL of 95% (V / V) ethanol was added, and the mixture was incubated in an ice bath for 5 minutes to precipitate glycogen. The precipitate was collected by centrifugation (1200 × g, 5 minutes, 4°C) and dissolved in distilled water (50 μL). 75 μL of distilled water and 100 μL of 1.2 M HCl solution were added to 25 μL of the sample dissolved in distilled water, and the mixture was incubated in a boiling water bath for 2 hours. 300 μL of 0.4 M sodium hydroxide solution was added for neutralization, and a glycogen hydrolyzate solution was prepared. A glycogen standard solution was also hydrolyzed using the same method. Glucose Measurement Kit (Lab Assay) ＴＭGlucose in the hydrolyzed sample solution was measured according to the protocol of the manufacturer (Glucose, Fujifilm Wako Pure Chemical Industries, Ltd.), and the amount of glycogen was calculated from a calibration curve using a glycogen standard solution and normalized by the tissue weight. <Results>

[0069] The results of measuring glycogen in the skeletal muscle are shown in Figure 20 (*P<0.05, **P<0.01). From Figure 20, it was confirmed that muscle glycogen decreased due to running. Ｌｅｕ It was confirmed that the group significantly maintained muscle glycogen. This result is considered to have an effect on enhancing running performance. ＢＣＡＡ Evaluation of toxicity following administration of

[0070] In this test, Nano Ｌｅｕ To evaluate the inhibitory effect of erythrocyte motile damage by erythrocyte motility, blood cell systems were measured.

[0071] The whole blood collected in Test Example 4 was used for measurement using an automated animal hemocytometer (Celltac α MEK6558, manufactured by Nihon Kohden). <Results>

[0072] The results of the blood cell measurement are shown in Figure 21 (*P<0.05, **P<0.01). Ｌｅｕ No effects of administration were observed (similar results were observed for platelet count (PLT) and mean corpuscular volume (MCV)). In addition, a significant decrease in red blood cell count and hemoglobin was confirmed after running. Ｌｅｕ It was confirmed that Nano maintains the red blood cell count and hemoglobin level even after exercise (similar results were observed for mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC)). Ｌｅｕ It has been shown that this compound does not cause side effects on the blood system and protects red blood cells from exercise, and is considered to have an effect on enhancing running performance. ＢＣＡＡ Evaluation of the inhibitory effect of administration of exercise-induced damage to various organs

[0073] In this test, Nano ＬｅｕTo evaluate the toxicity and the inhibitory effect of administration of the compound on various organ damage caused by exercise, damage markers in various organs were measured.

[0074] Using the plasma collected in Test Example 4, various damage markers were measured using an animal biochemistry automatic analyzer (Fuji DryChem NX600, manufactured by FUJIFILM) and according to the instructions on the measurement slide. The measurement items included alanine aminotransferase (ALT) and aspartate aminotransferase kit (AST) as liver damage markers, lactate dehydrogenase (LDH) and creatine kinase (CK) as skeletal muscle damage markers, and creatinine (CRE) and urea nitrogen (BUN) as kidney damage markers. <Results>

[0075] The measurement results of each organ damage marker are shown in Figure 22 (*P<0.05, **P<0.01). From Figure 22, it was confirmed that damage had occurred in the liver and kidneys from the significant increases in ALT, AST, CRE, and BUN due to running. Ｌｅｕ The group was found to be protected from exercise-induced kidney damage (a tendency to decrease in AST, CRE, and BUN). ＢＣＡＡ Evaluation of changes in fat in test animals due to administration of

[0076] In this test, Nano Ｌｅｕ In order to verify the changes in fat in the test animals due to the administration of the compound, fat was evaluated by CT scan.

[0077] The animals used in Test Example 4 were subjected to CT scanning using a small animal CT scanner (LaTheta LCT-100, manufactured by Hitachi Aloka Medical) on the resting schedule shown in Figure 15, and body fat, subcutaneous fat, and visceral fat were measured. <Results>

[0078] The results of fat measurement by CT scan are shown in Figure 23 (*P<0.05, **P<0.01). Ｌｅｕ It was confirmed that intake of leucine significantly reduced body fat, subcutaneous fat, and visceral fat. This is due to the metabolic activation effect of leucine, and it is expected to have preventive and therapeutic effects on lifestyle-related diseases and liver diseases caused by fat accumulation.

[0079] Nanoparticles made from copolymers based on branched chain amino acid polymers ＢＣＡＡ ) can be taken orally without invasiveness and easily, and it is effective in increasing muscle mass and strength, improving running performance, and other athletic performance improvements. Therefore, it is expected that branched-chain amino acids will not only repair and increase muscle mass and improve muscle strength in athletes, but also suppress the decline in motor function associated with aging, such as frailty and sarcopenia, and improve the health of the elderly. In addition, branched-chain amino acids have been reported to have therapeutic effects on gastrointestinal diseases, liver diseases, dementia, etc., so Nano ＢＣＡＡ is expected to be used as a therapeutic agent for these diseases.

Claims

1. A hydrophilic-hydrophobic block copolymer represented by the following formula (I) or (II): In each formula, A １ and A ２ are each independently unsubstituted or substituted C １ -C １２ The substituents when substituted are a formyl group, a group of formula R ’ R ” CH—, phenylamino, or phenethylamino, where R ’ and R ” is independently C １ -C ４ Alkyloxy or R ’ and R ” Let's get together -OCH ２ CH ２ O-, -O(CH ２ ) ３ O- or -O(CH ２ ) ４ O-, represented by L １ represents a single bond or a linking group; ２ represents a linking group, and each R is independently CH ２ CH (CH ３ ) ２ , CH (CH ３ ) CH ２ CH ３ and CH(CH ３ ) ２ and Z is selected from one or more groups represented by １ represents a hydrogen atom or an amino-protecting group; Z ２ represents an OH or carboxy protecting group; m1 and m2 are each independently an integer from 3 to 200; and n1 and n2 are each independently an integer from 5 to 1000.

2. A polymeric micelle comprising the copolymer of claim 1 and having an average particle size in the nano-size range.

3. A preparation for improving athletic performance, comprising as an active ingredient the copolymer according to claim 1 or polymeric micelles containing said copolymer and having an average particle size within the nano-size range.

4. The preparation according to claim 3, wherein the improvement in athletic ability is an improvement in muscle mass or muscle strength or an improvement in running performance.

5. A method for improving the athletic performance of an animal in need of treatment, comprising the step of administering to said animal an effective amount of the copolymer described in claim 1 or a polymeric micelle containing said copolymer and having an average particle size in the nanosize range.

6. The copolymer according to claim 1 or a polymeric micelle containing said copolymer and having an average particle size in the nano-size range, for use in improving the athletic performance of a treated animal.

7. Use of the copolymer according to claim 1 or polymeric micelles containing said copolymer and having an average particle size in the nano-size range, for improving the athletic performance of a treated animal.

8. A preparation for suppressing exercise-induced liver, kidney, or skeletal muscle damage in a treated animal, comprising as an active ingredient the copolymer according to claim 1 or polymeric micelles containing said copolymer and having an average particle size within the nanosize range.

9. A method for suppressing exercise-induced damage to the liver, kidneys, or skeletal muscles of an animal in need of treatment, comprising the step of administering to said animal an effective amount of the copolymer described in claim 1 or a polymeric micelle containing said copolymer and having an average particle size within the nanosize range.

10. The copolymer according to claim 1 or a polymeric micelle containing said copolymer and having an average particle size in the nano-size range, for use in suppressing exercise-induced liver, kidney or skeletal muscle damage in a treated animal.

11. Use of the copolymer according to claim 1 or a polymeric micelle containing said copolymer and having an average particle size in the nano-size range, for suppressing exercise-induced damage to the liver, kidney or skeletal muscle of a treated animal.

12. A preparation for suppressing fat accumulation in a treated animal, comprising as an active ingredient the copolymer according to claim 1 or polymeric micelles containing said copolymer and having an average particle size within the nano-size range.

13. A method for inhibiting fat accumulation in an animal, comprising the step of administering to said animal an effective amount of the copolymer of claim 1 or a polymeric micelle comprising said copolymer and having an average particle size in the nanosize range.

14. The copolymer according to claim 1 or a polymeric micelle comprising said copolymer and having an average particle size in the nano-size range, for use in suppressing fat accumulation in a treated animal.

15. Use of the copolymer according to claim 1 or polymeric micelles comprising said copolymer and having an average particle size in the nano-size range, for suppressing fat accumulation in a treated animal.

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