TW201912163A - Amphiphilic block copolymer, nanoparticle containing the same, medicinal composition thereof and method of inhibiting ribonucleic acid binding to human antigen r using the same - Google Patents

Abstract

The present invention relates to an amphiphilic block copolymer, a nanoparticle containing the same, a composition and a method of inhibiting ribonucleic acid (RNA) binding to human antigen R (HuR) protein. The amphiphilic block copolymer can specifically bind to a RNA binding region on the HuR protein. The nanoparticle including the amphiphilic block copolymer can be applied to a medicinal composition and a method of inhibiting RNA binding to human antigen R (HuR) protein using the same.

Description

 Amphoteric block copolymer, nano-containing particle and pharmaceutical composition thereof and method for inhibiting binding of ribonucleic acid to human antigen R protein  

The present invention relates to an amphiphilic block copolymer, a nanoparticle containing the same, and an application thereof, in particular to an amphiphilic block copolymer which specifically binds to a ribonucleic acid binding region of a human antigen R protein, including Nanoparticles and pharmaceutical compositions and methods for inhibiting the binding of ribonucleic acid to a human antigen R protein.

Chronic inflammation and malignant tumors have always been a disease of high medical resources worldwide. Traditional anti-inflammatory drugs and chemotherapy drugs mostly cause unnecessary side effects. Recently, various biopolymers (such as peptides) have been studied to selectively inhibit inflammation or induce cancer cell death, and have little effect on normal cells. For example, in the article "Identification and mechanistic characterization of low-molecular-weight inhibitors for HuR" published by Nicole-Claudia Meisner et al., 2007, Nature Chemical Biology, No. 3, pp. 508-515, MS-444 is disclosed; Min-Ju Chae et al., "Chemical inhibitors destabilize HuR binding to the AU-rich element of TNF-α mRNA", published in the journal Exp. Mol. Med., Vol. 41, No. 11, pp. 824-831, 2009. Revealing quercetin; both MS-444 and quercetin have a certain degree of binding ability to HuR protein, inhibiting the binding of HuR protein to ribonucleic acid (RNA), and inhibiting inflammation and cancer-related genes. which performed.

However, in the disease pattern-related experiments in mice, the above-mentioned macromolecules have limited binding ability to human antigen R protein, and thus the acute hepatic inflammation pattern in mice has been shown to improve liver vascular cell necrosis, cytoplasmic cavity, and inflammatory disease. The degree of splenomegaly and dry ear treatment (redness, thickening and desquamation) is not ideal, resulting in a general room for improvement in the overall treatment of inflammation. In addition, most drugs affect the normal physiological metabolism of other cells when they enter the site of inflammation or cancer tissue.

In view of the above, it is necessary to provide a drug molecule that inhibits binding of a human antigen R protein to a ribonucleic acid to enhance a therapeutic effect against a specific inflamed tissue/cell or cancer tissue/cell, and to solve the above problems.

Accordingly, one aspect of the present invention provides an amphiphilic block copolymer wherein the amphiphilic block copolymer specifically binds to a human antigen R protein.

Another aspect of the invention provides a nanoparticle comprising the amphoteric block copolymer described above.

Yet another aspect of the invention provides a method of inhibiting binding of a human antigen R protein to a ribonucleic acid. A ribonucleic acid binding region that specifically binds the aforementioned nanoparticle to a human antigen R protein is included.

A further aspect of the invention provides a pharmaceutical composition for inhibiting binding of a human antigen R protein to a ribonucleic acid comprising the above-described nanoparticle and a pharmaceutically acceptable carrier.

According to the above aspect of the invention, an amphiphilic block copolymer having a molecular structure represented by the following formula (I) is proposed:

In the above embodiments, the Y group of the formula (I) may be a hydrophilic polymeric chain, which may include, but is not limited to, a polyethylene glycol-derived group, a polypeptide-derived group or a polysaccharide-derived group; a hydrophobic polymeric chain, and the hydrophobic polymeric chain may, for example, be a phenyl-containing amino acid group, or an amino acid group modified with a phenyl group or a derivative thereof; and n is an integer from 3 to 40, and the two sexes The block copolymer is specifically bound to the ribonucleic acid binding region of the human antigen R protein.

In an embodiment of the invention, the phenyl group-containing amino acid group may be, for example, an L-tryptophan acid group or an L-phenylalanine group.

In an embodiment of the invention, the amino acid group modified by the phenyl group or a derivative thereof may include, but is not limited to, carbobenzyloxy (Cbz; Z)-L-isoamino acid group, γ- Benzyl-L-glutamic acid group, β-benzyl-L-aspartate, O-benzyl-L-tyrosine, O-benzyl-L-threonate , S-benzyl-cysteinyl group, O-benzyl-serine group, and Z, ZL-arginine group.

In an embodiment of the invention, the above n may be, for example, an integer of 3 to 20. In an example, the above n may be, for example, an integer of 5 to 20.

According to the above aspect of the invention, there is further proposed an amphiphilic block copolymer having the molecular structure represented by the following formula (I-1):

In the above embodiments, m of the formula (I-1) may be, for example, an integer of 45 to 245, n may be, for example, an integer of 5 to 20, and the amphiphilic block copolymer is a ribonucleic acid which specifically binds to the human antigen R protein. Binding area.

In an embodiment of the invention, the above m may be, for example, an integer of 45 to 112, and n may be, for example, an integer of 5 to 8.

According to another aspect of the present invention, there is provided a nanoparticle comprising the amphoteric block copolymer of any one of the above, wherein the nanoparticle has a particle size ranging from 50 to 500 nm.

According to still another aspect of the present invention, a method for inhibiting binding of a human antigen R protein to a ribonucleic acid is provided, comprising: specifically binding the above-described nanoparticle to a ribonucleic acid binding region of a human antigen R protein.

According to still another aspect of the present invention, a pharmaceutical composition for inhibiting binding of a human antigen R protein to ribonucleic acid is provided, comprising the above-described nanoparticle and a pharmaceutically acceptable carrier. In one embodiment, the content of the nanoparticles may be, for example, 20% to 80% based on 100% of the total content of the pharmaceutical composition.

In an embodiment of the invention, the pharmaceutical composition may be, for example, an oral pharmaceutical composition or a pharmaceutical composition for external use.

The use of the amphiphilic block copolymer, the nanoparticle-containing particle and the pharmaceutical composition of the present invention and a method thereof for inhibiting binding of a ribonucleic acid to a human antigen R protein, wherein the amphiphilic block copolymer is specifically bound to a human antigen The ribonucleic acid binding region of the R protein is effective for inhibiting the binding of ribonucleic acid to the human antigen R protein, and is more applicable to a pharmaceutical composition of a HuR-related disease.

101‧‧‧N terminal domain

103‧‧‧C terminal domain

105‧‧‧ribbonucleic acid binding region

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

1A to 1F are molecular pairing perspective views showing an amphiphilic block copolymer and a human antigen R protein according to an embodiment of the present invention.

Fig. 1G is a bar graph showing the affinity and bonding energy between the amphiphilic block copolymer and the human antigen R protein according to an embodiment of the present invention.

Fig. 1H is a diagram showing the hydrogen bond distribution of the amphiphilic block copolymer and the human antigen R protein according to an embodiment of the present invention.

[Fig. 1I] shows the results of the original colloidal electrophoresis between the amphiphilic block copolymer and the human antigen R protein according to an embodiment of the present invention.

[Fig. 2A] shows the distribution of HuR protein (HuR) in the LPS-induced macrophage mode, the distribution of the amphiphilic block copolymer (PEG), the nuclear distribution (DAPI) of one embodiment of the present invention, and the combination of the above results. Immunofluorescence staining.

[Fig. 2B] shows the immunoblots of the HuR protein (HuR) in the nucleus (N) and the cytoplasm (C) and the amphiphilic block copolymer (PEG) of an embodiment of the present invention in the LPS-induced macrophage mode. Analysis results.

[Fig. 2C] to [Fig. 2F] show the results of immunoblotting analysis of inflammatory factors related to LPS by the amphiphilic block copolymer of several embodiments of the present invention in the LPS-induced macrophage mode.

[Fig. 3A] shows a liver tissue section diagram of the HuR protein of the amphiphilic block copolymer of several embodiments of the present invention in the LPS/GalN-induced mouse hepatitis model.

[Fig. 3B] to [Fig. 3E] show that the amphiphilic block copolymer of several embodiments of the present invention inhibits hepatitis-related transaminase activity in the LPS/GalN-induced mouse hepatitis model (Fig. 3B), cytokines Content (Fig. 3C), pathological grade (Fig. 3D) and survival (Fig. 3E).

[Fig. 3F] to [Fig. 3G] show the results of inhibition of spleen enlargement by the amphiphilic block copolymer of several embodiments of the present invention in the LPS/GalN-induced mouse hepatitis mode.

[Fig. 4A] to [Fig. 4C] show that the amphiphilic block copolymer of one embodiment of the present invention inhibits cognac-related cytokine results in a mode in which imiquimod (IMQ) induces macrophages.

[Fig. 5A] to [Fig. 5D] show the appearance of the amphiphilic block copolymer on the ear skin of the mouse (Fig. 5A) and the clinical score (Fig. 5B to Fig. 5B) in the mode of IMQ-induced mouse cognac. Figure 5D) results.

[Fig. 6A] to [Fig. 6C] show the staining of the ear skin tissue staining of the amphiphilic block copolymer according to an embodiment of the present invention in the mode of IMQ-induced mouse dryness (Fig. 6A), the thickness of the ear skin. (Fig. 6B) and a bar graph of the number of layers of the epidermis (Fig. 6C).

[Fig. 7A] shows a skin immunohistochemical staining section of the HuR protein distribution in the mode of IMQ-induced mouse cognac.

[Fig. 7B] to [Fig. 7C] show that the amphiphilic block copolymer of one embodiment of the present invention inhibits the cognac-associated cytokine VEGF (Fig. 7B) and IL-23 (Fig. 7C) in the mode of IMQ-induced mouse cognac. Straight bar chart.

[Fig. 7D] shows a skin immunohistochemical staining profile in which the amphiphilic block copolymer of one embodiment of the present invention is distributed in the skin, liver and spleen by a PEG antibody in a mode in which IMQ induces mouse dryness.

[Fig. 7E] to [Fig. 7F] show a bar graph of inhibition of serum ALT (Fig. 7E) and AST (Fig. 7F) by the amphiphilic block copolymer of one embodiment of the present invention in the mode of IMQ-induced mouse cognac.

[Fig. 8] shows the protein matrix results of the serotonin inhibiting the cytokine of the ear skin of the amphiphilic block copolymer according to an embodiment of the IMQ-induced mouse cognac.

The singular forms "a", "the", and "the" The range of values (such as 10% to 11% of A) includes upper and lower limits (ie, 10% ≦A≦11%) unless otherwise specified; if the value range does not define a lower limit (such as less than 0.2% of B) , or B) below 0.2%, the lower limit may be 0 (ie 0% ≦ B ≦ 0.2%). The above terms are used to illustrate and understand the present invention and are not intended to limit the invention.

The present invention provides an amphiphilic block copolymer, wherein the amphiphilic block copolymer described herein refers to a ribonucleic acid binding region that specifically binds to a human antigen R protein and can compete with ribonucleic acid (RNA) for the ribonucleic acid binding region. By. In one embodiment, the structural formula of a suitable amphoteric block copolymer can be, for example, represented by the following formula (I):

In one exemplary embodiment, the Y group of formula (I) can be a hydrophilic polymeric chain including, but not limited to, a polyethylene glycol-derived group, a polypeptide-derived group, or a polysaccharide-derived group.

Specifically, the above polypeptide-derived group may be pegylated polypeptides, and specific examples thereof may include, but are not limited to, pegylated poly-L-lysine. For example, poly(N-2-[2-(2-methoxyethoxy)ethoxy]ethoxylated) [poly(N-2-[2-(2-methoxyethoxy)ethoxy]acetyl-) Lysine)]; pegylated poly-L-glutamate, such as poly(γ-(2-(2-methoxyethoxy))ethoxy]ester-L -glutamic acid group [poly(γ-(2-(2-methoxyethoxy)ethoxy)esteryl-L-glut amate)]; glycolated poly-L-isoamino acid group and poly-L-glutamic acid group ( Glycolated poly-L-lysine and poly-L-glutamate); polysarcosine; poly(L-lysine); poly-L-glutamic acid group [poly(L-) Glutamic acid)]; polyarginine poly(L-arginine); and the amino acid of the aforementioned polypeptide-derived group may be L-form, D-form or a combination thereof.

Next, examples of the above polysaccharide-derived group may be oligosaccharides or glycans, and specific examples thereof may include, but are not limited to, algionic acid, amylose, and cellulose. , cellulose triester, chitosan, curdlan gum, dextrin, β-cyclodextrin, dextran, Heparin, hyaluronan, maltoheptaose, maltodextrin, ovomucoid, trimethylcellulose, pullulan, Schizophyllan, succinoglycan, xanthan, xyloglucan, and the like.

In an exemplary embodiment, the R group of the above formula (I) may be a hydrophobic polymer chain such as a phenyl group-containing amino acid group or an amino acid group modified with a phenyl group or a derivative thereof.

As stated, examples of the above phenyl group-containing amino acid group may include, but are not limited to, L-tryptophan acid groups (as shown in Formula II-7) or L-phenylalanine groups (such as Formula II-5). Show). Examples of the above amino acid group modified with a phenyl group or a derivative thereof may include, but are not limited to, a carbobenzyloxy (Cbz; Z)-L-isoamino acid group (as shown in Formula II-1), γ-benzyl-L-glutamic acid group (as shown in formula II-2), β-benzyl-L-aspartate (as shown in formula II-3), O-benzyl group -L-tyrosine group (as shown in formula II-4), O-benzyl-L-threonine group (as shown in formula II-6), S-benzyl-cystyl group ( As shown in Formula II-8), O-benzyl-seramine group (as shown in Formula II-9) or Z,ZL-arginine group (as shown in Formula II-10). Further, the figure number * of the formula II-1 to formula II-10 represents a bond point of the R group.

In an exemplary embodiment, n of the formula (I) may be, for example, an integer of from 3 to 40 to impart both hydrophilic and hydrophobic amphoteric properties to the block copolymer, whereas n is preferably an integer of from 3 to 20, An integer of 5 to 8 is preferred.

In some embodiments, the amphoteric block copolymer may have a structural formula of PEG _45~245 - b -PBLG _3~40 , such as PEG ₁₁₂ - b -PBLG ₃ (abbreviated as PBLG ₃ ), PEG ₁₁₂ - b -PBLG ₄ (abbreviated as _{PBLG 4), PEG 112 - b} -PBLG 5 ( abbreviated as PBLG _5; formula _{III-1), PEG 112 -} b -PBLG 6 ( abbreviated as _{PBLG 6), PEG 112 - b} -PBLG 7 ( referred to as is PBLG _7; formula _{III-2), PEG 112 -} b -PBLG 8 ( abbreviated as _{PBLG 8), PEG 45 - b} -PBLG 20, PEG 112 - b -PBLG 20 ( abbreviated as PBLG _20), PEG ₂₄₅ - b -PBLG ₂₀ , PEG ₁₁₂ - b -PBLG ₄₀ (referred to as PBLG ₄₀ ), etc.:

In an embodiment of the invention, the amphiphilic block copolymer is self-assemblable into a nanoparticle (also known as a nanocomposite or a microcell) in solution. In the above embodiment, the obtained nanoparticles may have an average particle diameter of 50 to 500 nm, for example, 100 nm, 200 nm, 300 nm, 400 nm or 500 nm, but are not limited to the above. When the above amphoteric block copolymer is PBLG ₃ to PBLG ₈ , the obtained nanoparticles may have an average particle diameter of 50 to 150 nm.

It is known that HuR target RNAs (especially mRNA) can specifically bind to the binding site of HuR protein to form a complex, which can stabilize the target ribonucleic acid and its translation performance. It is up-regulated or repressed. The HuR protein-associated disease described herein refers to a HuR target ribonucleic acid that binds to a HuR protein to form a complex, and the target ribonucleic acid is stable, its translational expression is increased or inhibited, and the HuR protein expression is increased or It is possible to enter diseases caused by cytoplasm and the like.

However, the amphiphilic block copolymer of the present invention or the nanoparticle containing the same may specifically bind to the HuR protein and compete with the target ribonucleic acid for the same or similar binding position, thereby interfering with, reducing and/or hindering the target ribonucleic acid. Binding to a HuR protein with a HuR protein or an anti-HuR antibody, the target ribonucleic acid becomes unstable, the amount of translational expression cannot be up-regulated or repressed, and the HuR protein expression decreases, thereby slowing down Even treat diseases associated with HuR proteins. In some embodiments of the invention, the amphoteric block copolymer has a greater binding capacity to the HuR protein than the ribonucleic acid to the HuR protein and competes with the ribonucleic acid in the ribonucleic acid binding region of the HuR protein.

In the above examples, specific examples of the target ribonucleic acid stabilized by the HuR protein may include, but are not limited to, c-Fos, cyclin-dependent kinase (cdk) inhibitor p21, and cyclins A2. B1, D1, E1), inducible nitric oxide synthase (iNOS), granulocyte-macrophage-colony stimulating factor (GM-CSF), eukaryotic initiation factor ; eIF)-4E, murine double minute (mdm) 2, vascular endothelial growth factor (VEGF), transforming growth factor-β, sirtuin 1 1; SIRT1), tumor necrosis factor-α, B-cell leukemia (Bcl)-2, myeloid leukemia cell differentiation protein (Mcl)-1, tumor suppressor On-cost M (OSM), cyclooxygenase (COX)-2, γ-glutamylcysteine synthetase heavy subunit (γ-GCSh), transport Survival of motor neuron (SMN), SH2D1A, Gregulatory G-protein signaling 4 (RGS4), parathyroid hormone-related protein (PTHrP), Fas Ligand (FasL), myogenin, MyoD, acetylcholinesterase (AChE), p53, dysplastic Ras homolog member I[aplasia Ras homolog member I (DIRAS3) ;ARHI], nitric oxide/soluble guanylyl cyclase (sGC), urokinase plasminogen activator (uPA) and its receptor Body (uPAR), neurofibromatosis type 1 (NF1), von Hippel-Lindau protein (pVHL), toll-like receptor 4 (TLR4), zinc Refers to the transcription factor Snail (Snail), matrix metalloprotease (MMP)-9, c-Fms, mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) Interferon (in) Terferon; IFN)-γ, HuR protein itself, mRNA of interleukin (IL)-3, IL-4, IL-6 and IL-8.

In the above examples, specific examples of the ribonucleic acid which is up-regulated by the HuR protein may include, but are not limited to, cyclin A2, prothymosin α (ProTα), hypoxia-inducible factor-1α. (hypoxia-inducible factor-1α; HIF-1α), Bcl-2, VEGF, thrombospondin-1 (TSP-1), MKP-1, p53, cationic amino acid transporter-1 ( Cationic amino acid transporter; CAT-1), mRNA of intrinsic cellular caspase inhibitor XIAP, cytochrome c, and the like.

In the above examples, specific examples of the target ribonucleic acid which inhibits the amount of translational expression by the HuR protein may include, but are not limited to, mRNAs of p27, IGF-1R, thrombomodulin (TM), Wnt5a, c-Myc and the like.

In the above examples, related diseases due to an increase in the amount of HuR protein expression include an inflammatory response and cancer. HuR protein can increase the expression of pro-inflammatory factors and inhibit the expression of anti-inflammatory factors. In addition, HuR protein can promote cancer cell growth, immune escape, invasion and migration, and angiogenesis.

In one embodiment, the amphoteric block copolymer itself or the formation of nanoparticles can be further applied to a pharmaceutical composition for treating diseases associated with HuR proteins, such as inflammatory reactions (eg, hepatitis, cognac, etc.) and cancer. However, the application of the present invention is not limited to the above, and the above nanoparticles can be applied to treat other diseases associated with HuR proteins.

In an embodiment of the invention, the pharmaceutical composition may include, but is not limited to, the above-described nanoparticles and a pharmaceutically acceptable carrier. When the above pharmaceutical composition is for external use, the carrier may be, for example, water, petrolatum or any ointment which can be applied to the skin, depending on the need, for example, if the composition is applied to the skin, Vaseline may be selected. When the pharmaceutical composition is a pharmaceutical composition for internal use, the carrier may be a water-based carrier such as a physiological buffer and administered orally or by injection.

In an embodiment of the present invention, when the nanoparticle is applied to a pharmaceutical composition, the content of the nanoparticle may be 20% to 80%, for example, 20%, based on 100% of the total content of the pharmaceutical composition. 30%, 40%, 50%, 60%, 70% or 80%, but is not limited to this. In the above embodiment, the carrier may be contained in an amount of 0.1% to 20%, for example, 0.1%, 0.5%, 0.7%, 1.5%, 2%, 5%, based on 100% of the total content of the pharmaceutical composition. 10%, 15% or 20%, but not limited to this.

The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. Retouching.

Example 1. Evaluation of the binding of the amphiphilic block copolymer to the human antigen R protein

1. Evaluation of the effect of amphiphilic block copolymers binding to HuR protein

The following examples simulate a amphiphilic block copolymer, RNA (U-rich RNA sequence of UAUUUUAUUUU, a total of 11 nucleotides) according to an embodiment of the present invention using a commercially available molecular docking software (e.g., Dock 5.1). Molecular pairing stereograms of MS-444 and quercetin (Q) and HuR protein (recombinant human HuR/ELAVL1 protein; PDB code: 4ED5), and using the 3D pattern of UCSF Chimera software export molecule, the ribbon of HuR protein The model is shown in Figure 1A. The local surface model of the ribonucleic acid binding region (also known as RNA-recognition motif; RRM) of the HuR protein is shown in Figures 1B-1F. The RNA used above is a U-rich RNA ligand having a sequence of AUUUUUAUUUU of 11 nucleotides. The distance between the atoms of Figures 1B to 1F is less than 5 angstroms (Å).

Secondly, the affinity of the amphiphilic block copolymer, MS-444 and quercetin (Q) (pKD) was predicted by X-score software, and the amphoteric block copolymer, MS-444 and quercetin were scored by HotLig software. The bonding energy of Q) is shown in Fig. 1G and Table 2. In addition, the hydrogen bond distribution between the amphiphilic block copolymer and the human antigen R protein was predicted using Ligplot software, and the results are shown in Fig. 1H.

Please refer to FIG. 1A to FIG. 1F , which are schematic diagrams showing molecular pairing of amphiphilic block copolymer, RNA, MS-444 and quercetin (Q) and human antigen R protein according to an embodiment of the present invention, wherein amphoteric block copolymerization The material was an oligomer of L-glutamic acid group (PBLG ₅ , PBLG ₇ ) of polyphenyl group, and MS-444 and quercetin (Q) were used as a control compound. MS-444 is described in "Identification and mechanistic characterization of low-molecular-weight inhibitors for HuR" by Nicole-Claudia Meisner et al., 2007, Journal of Nature Chemical Biology, No. 3, pp. 508-515. Quercetin (Q) is described in Min-Ju Chae et al., 2009, Journal of Exp Mol Med., Vol. 41, No. 11, pp. 824-831, "Chemical inhibitors destabilize HuR binding to the AU-rich element of TNF- α mRNA" article.

The results of FIGS. 1A to 1F show that the HuR protein includes an N-terminal domain 101 (FIG. 1A, FIG. 1B), a C-terminal domain 103 (FIG. 1A, FIG. 1B), and ribose. The nucleic acid binding region 105 (dark gray region of Fig. 1B), PBLG ₅ and PBLG ₇ can bind to the ribonucleic acid binding region of the HuR protein (Fig. 1E, Fig. 1F) for binding. The control compound MS-444 and quercetin (Q) can also bind to the ribonucleic acid binding region of the HuR protein as shown in Figures 1C and 1D, respectively.

Please refer to FIG. 1G and Table 1. FIG. 1G is a bar graph showing the affinity and bonding energy between the amphiphilic block copolymer and the human antigen R protein according to an embodiment of the present invention, and Table 1 shows each of FIG. 1G. The value of the strip. In Table 1, the larger the value of Xscore, the stronger the affinity between the two; the larger the negative value of HotLig score, the higher the bond energy between the two.

From the results of FIG. 1G and Table 1, it is shown that the bond energy of the amphiphilic block copolymer of the present invention and HuR protein is from PBLG ₇ (7-mer)>PBLG ₆ (6-mer)>PBLG ₈ (8). -mer)>PBLG ₅ (5-mer)>PBLG ₄ (4-mer)>PBLG ₃ (3-mer)>Q>MS-444, representing PBLG ₃ to PBLG ₈ of the amphiphilic block copolymer of the present invention HuR protein has better binding ability than quercetin (Q) and MS-444. In general, the binding energy between the ligand and the acceptor increases with the increase of the molecular weight of the ligand, but the results of Figure 1G and Table 1 show that PBLG ₇ and PBLG _{6 are} superior to PBLG ₈ and PBLG ₅ , not excluded. It is affected by the elasticity of the structure of the ligand itself and/or the size of the binding zone of the receptor.

Next, referring to FIG. 1H, there is shown a hydrogen bond distribution diagram between the amphiphilic block copolymer and the human antigen R protein according to an embodiment of the present invention, wherein the purple thick line structure represents PBLG ₇ and the green dotted line represents potential hydrogen. The keys, while the black spokeed arcs represent non-bonded contacts on the HuR protein.

From the results of FIG. 1H, the PBLG ₇ and HuR proteins of the amphiphilic block copolymer of the present invention have more hydrogen bonds (9 in total) and non-bonded (or hydrophobic) contacts, indicating that both It has strong binding ability and can compete with RNA for the ribonucleic acid binding region of HuR protein, which is expected to be an inhibitor of HuR protein.

2. Evaluation of the effect of amphiphilic block copolymer on the recognition of HuR protein antibody

In this example, PBLG ₅ of the amphiphilic block copolymer of the present invention was subjected to ultrasonic shock for 3 minutes, and then reacted with HuR protein (Novus Biologicals, LLC, US; 0.4 μg) at 37 ° C for 3 hours. Thereafter, native-polyacrylamide gel electrophoresis (native-PAGE) is performed, and the amount (%) of free HuR protein is detected by immunoblotting using an antibody, and PBLG is not added. _{The 5} HuR protein of the reaction was 100%. The antibodies used above are mouse polyclonal antibodies (abcam, Cambridge, MA) against the 1st to 1st amino acid of HuR, and mouse monoclonal antibodies against the 1-280 amino acids of HuR, respectively. Mouse monoclonal antibody against the 1-326 amino acid of HuR (the latter two manufacturers are Santa Cruz Biotechnology Inc., CA). The signals of the respective ribbons were detected using a commercially available enhanced chemiluminescence (ECL) kit, and the results are shown in Fig. 1I.

Please refer to FIG. 1I, which shows the results of the original colloidal electrophoresis between the amphiphilic block copolymer and the human antigen R protein according to an embodiment of the present invention, wherein the free HuR protein is PBLG ₅ without the addition of the amphiphilic block copolymer. The concentration is taken as 100%. The figure number * of Fig. 1I represents p < 0.05, the figure number ** represents p < 0.01, and the figure number *** represents p < 0.001.

From the results of FIG. 1I, when the amount of PBLG ₅ added to the amphiphilic block copolymer of the present invention is increased, the antibody is the same as the antibody of the 1st to 1st amino acid against HuR, and the first of the anti-HuR. The lighter signal of the HuR protein detected by the -280 amino acid antibody and the antibody against the 1-36 amino acid of HuR) is lighter, indicating that the binding ability of PBLG ₅ and HuR protein is superior. The recognition of the antibody against the HuR protein can be hindered, and the PBLG ₅ of the amphiphilic block copolymer of the present invention can indirectly inhibit, hinder or interfere with the binding of the HuR protein to the RNA, thereby inhibiting the HuR protein-related diseases such as an inflammatory reaction.

Example 2: Evaluating the efficacy of amphoteric block copolymers to form nanoparticles

The following examples are given in Table 2, which shows the average aggregated particle size of the amphiphilic block copolymer self-assembled into nanoparticles in aqueous solution according to several embodiments of the present invention.

From the results of Table 2, it was revealed that nano particles having a smaller average particle diameter with PBLG ₃ to PBLG ₈ having an average particle diameter of 50 to 150 nm were obtained.

Example 3: Evaluating the efficacy of the amphiphilic block copolymer in inhibiting the inflammatory response

1. To evaluate the effect of amphiphilic block copolymers on inhibiting the inflammatory response induced by LPS

This example is an evaluation of the effect of the amphiphilic block copolymer on the inflammatory reaction after the lipopolysaccharide (LPS) caused an excessive expression of the HuR protein by a cell assay.

In this example, the macrophage cell line Raw264.7 (stored in the Bioresource Conservation and Research Center of the Hsinchu Food Research Institute of Taiwan, the registration number: BCRC 60001; or deposited in the China National Culture Collection of Wuhan, China) , accession number: CCTCC GDC0143) establish in vitro cell mode. In general, the macrophage cell line Raw264.7 was cultured in DMEM medium (containing 10% fetal bovine serum (FBS) and 50 μg/mL gentamicin) at 37 ° C, 5% CO. ₂ and culture in a relatively saturated humidity environment.

In the experiment, the macrophage cell line Raw264.7 was implanted into a 6-well culture plate at 2×10 ⁵ cells per well, pretreated with 100 μM amphiphilic block copolymer PBLG _{5 for} 1 hour, and then LPS (Sigma- Aldrich, St. Louis, MO; 0.25 μg/mL) was treated for 24 hours. Thereafter, the cells were fixed with methanol for 10 minutes, and then blocked with 5% low-fat milk containing 0.3% Triton-x100. After the above sample was washed three times with PBS, it was reacted with the primary antibody at 4 ° C until overnight, wherein the primary antibody included mouse anti-HuR antibody (Santa Cruz Biotechnology, Inc.) and rabbit anti-PEG antibody (Abcam plc.) or isotype control group. (isotype control) IgG (Santa Cruz Biotechnology Inc., CA). The above sample was washed three times with PBS and reacted with a secondary antibody for 2 hours at room temperature, wherein the secondary antibody included a texas red-conjugated goat anti-mouse IgG and a FITC-conjugated goat. Anti-rabbit IgG (FITC-conjugated goat anti-rabbit IgG (both of which are Jackson ImmunoResearch Inc.). The above sample was washed three times with PBS, using 4',6-diamidino-2-phenylindole (4 ',6-diamidino-2-phenylindole; 0.1 μg/mL) for nuclear staining. After 30 minutes, a multiphoton laser scanning microscope (Olympus FV1000 MPE) was used with conjugated coke microscopy. The cells were observed by a doubling (60 x) water-immersion objective lens, and the results are shown in Fig. 2A.

Please refer to FIG. 2A and FIG. 2B. 2A shows the HuR protein distribution (HuR), the distribution of the amphiphilic block copolymer (PEG), the nuclear distribution (DAPI) of the embodiment of the present invention, and the immunofluorescence combined with the above results in the LPS-induced macrophage mode. Light staining map. 2B shows the results of immunoblotting analysis of the HuR protein (HuR) in the nucleus (N) and the cytoplasm (C) and the amphiphilic block copolymer (PEG) according to an embodiment of the present invention in the LPS-induced macrophage mode. , wherein the fluorescence intensity of the HuR protein (HuR) or the amphiphilic block copolymer (PEG) of the nucleus (N) or the cytoplasm (C) measured by LPS treatment is 1.00, and the correlation values of each group are calculated, and β - Actin (beta-actin) is the internal control group.

From the results of Fig. 2A and Fig. 2B, after 1.5 hours of LPS treatment, the antibody detection using anti-HuR protein showed that the HuR protein decreased in the nucleus of macrophages and increased in the cytoplasm.

2A and 2B, after treatment with LPS, the anti-PEG antibody was used to detect the content of the amphiphilic block copolymer PBLG ₅ in the cytoplasm, which was 1.16 times of the nuclear content (0.5 hour for LPS treatment) and 1.43 times (LPS treatment for 1.5 hours).

The above image observation and the results of FIG. 2B are shown in FIG. 2A, and the distribution of the HuR protein and the amphiphilic block copolymer in the cytoplasm is correlated, which indirectly proves that the amphiphilic block copolymer of the present invention is permeable to PBLG _{5 .} Competitive binding, which in turn inhibits, blocks or interferes with the binding of antibodies against HuR to HuR proteins.

2. Evaluate the effect of amphiphilic block copolymers on the inhibition of LPS-induced inflammation-related signaling pathways

In this example, the macrophage cell line Raw264.7 was implanted into a 6-well culture dish at 2x10 ⁵ cells per well, first using 100 μM amphiphilic block copolymers PBLG ₅ , PBLG ₂₀ , PBLG ₄₀ , dextrin After pretreatment of -b-PBLG ₅ , PEG ₄₅ -b-PBLG ₂₀ and PEG ₂₄₅ -b-PBLG _{20 for} 1 hour, it was treated with LPS (Sigma-Aldrich; 0.25 μg/mL) for 24 hours. Thereafter, the collected cell lysate is analyzed by SDS-PAGE, and the expression of the relevant cytokine of the inflammatory-related signaling pathway is detected by the antibody, wherein the relevant cytokines include pNFκBp65, pAKT, pERK, pJNK, pp38, iNOS, COX2, procaspase-3 and caspase-3, and β-actin was used as an internal control group. Anti-pNFκBp65, pAKT, pERK, pJNK, pp38 and caspase-3 anti-systems were purchased from Danvers, MA. Antibodies against iNOS, COX2, HuR, and β-actin were purchased from Santa Cruz Biotechnology, Inc.

Next, the concentration of nitric oxide (NO) in the cell culture solution was detected using a commercially available Griess analysis kit (Sigma-Aldrich), and the results are shown in Fig. 2C to Fig. 2F.

2C to 2F, which show the results of immunoblot analysis of inflammatory factors associated with LPS by the amphiphilic block copolymer of several embodiments of the present invention in the LPS-induced macrophage mode. The figure number * of Fig. 2F represents p < 0.05, the figure number ** represents p < 0.01, and the figure number *** represents p < 0.001.

From the results of Fig. 2C, the amphiphilic block copolymer PBLG ₅ could not significantly inhibit the production of pNFκBp65, pAKT, pERK, pJNK, and pp38 after 0.5 hours of treatment with macrophages, but after 24 hours of LPS treatment, the amphiphilic mosaic The segment copolymer PBLG ₅ can inhibit the production of iNOS and COX2. It is speculated that the level of influence of the amphiphilic block copolymer is the translation of the inflammatory-related gene, but not the contact of LPS with the TLR4-related protein on the cell membrane.

From the results of Fig. 2D, it was revealed that amphiphilic block copolymers of different lengths have anti-inflammatory and apoptosis-inducing effects on LPS-induced inflammatory macrophages, but do not cause apoptosis of normal cells.

From the results of Fig. 2E, after the amphoteric block copolymer, the hydrophilic end of PEG ₂₄₅ -b-PBLG ₅ was replaced with dextrin, dextrin-b-PBLG ₅ also had an inflamed macrophage for LPS. Anti-inflammatory and cause apoptosis, but does not cause apoptosis in normal cells.

The results of Fig. 2F show that the amphiphilic block copolymers PBLG ₅ , PBLG ₂₀ , PBLG ₄₀ , PEG ₄₅ -b-PBLG ₂₀ and PEG ₂₄₅ -b-PBLG ₂₀ can inhibit nitric oxide after macrophage treatment by LPS. (NO), this result shows that the hydrophilic terminal of the amphiphilic block copolymer of the present invention is substituted with a different species or length, or the length of the hydrophobic end PBLG is changed, and is resistant to macrophage cells caused by LPS. Inflammation and apoptosis.

3. Evaluation of the effect of amphiphilic block copolymer on hepatitis inhibition

This example evaluates the effect of the amphiphilic block copolymer on suppressing hepatitis using the LPS/GalN induced mouse hepatitis model. C57BL/6 male mice (8-10 weeks old) were purchased from the National Experimental Animal Center of the National Experimental Research Institute (Taipei, Taiwan) and followed the guidelines of the National Laboratory for Animal Care and Use of the National Cheng Kung University and the Taiwan Animal Protection Act. The relevant regulations are carried out.

C57BL/6 male mice were induced with amphoteric block copolymerization by intraperitoneal injection (intraperitoneal; ip) after LPS/GalN (LPS [10 μg/kg] + D-GalN [400 mg/kg]) induced acute hepatitis for 1 hour. PBLG ₅ , PBLG ₂₀ , PBLG ₄₀ (20 mg/kg) or quercetin (50 mg/kg). In the positive control group, after LPS/GalN induced acute hepatitis, an equal amount of physiological saline containing no drug was administered. Thereafter, blood samples of the mice were collected, and plasma transaminase activity and cytokine expression were analyzed.

The above blood samples can be used to detect the serum adenine aminotransferase (ALT) and aspartate aminotransferase (AST) activity by Reitman-Frankel method to assess the degree of liver damage. . In addition, serum tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were measured using an ELISA kit (R&D) to assess inflammation-related cytokines.

In addition, a live tissue sample of the liver and spleen of the above mouse was fixed with 4% formalin and embedded in paraffin, stained with hematoxylin-eosin (HE), and anti-HuR protein was used. The mouse monoclonal antibody (Santa Cruz Biotechnology Inc., CA) of 1-32 amino acids was subjected to immunohistochemical staining.

Referring to Figure 3A, it is shown that the amphiphilic block copolymers of several embodiments of the present invention inhibit liver tissue histograms of HuR proteins in the LPS/GalN induced mouse hepatitis mode.

From the results of FIG. 3A, the amphoteric block copolymer PBLG ₅ is superior to PBLG ₂₀ , and the therapeutic effect of PBLG ₂₀ is superior to that of PBLG ₄₀ . Secondly, the results of liver tissue sections of mice in the control group (LPS/GalN-induced acute hepatitis, administered with physiological saline) showed that the cytoplasm was hollowed out (HE dyes are more difficult to stain, lines 1 and 2, Each microscope was magnified 100 times and 200 times), and the blood vessels in the liver were necrotic (photographs in lines 3 and 4, 100 times and 200 times each). In contrast, liver tissue sections treated with the amphiphilic block copolymers PBLG ₅ , PBLG ₂₀ , and PBLG ₄₀ showed that cytoplasmic vacuolation and hepatic vascular cell necrosis were indeed significantly reduced.

3B to 3E, which show that the amphiphilic block copolymers of several embodiments of the present invention inhibit hepatitis-associated transaminase activity in the LPS/GalN-induced mouse hepatitis model (Fig. 3B), cytokines Content (Fig. 3C), pathological grade (Fig. 3D) and survival (Fig. 3E). The figure number * of Figs. 3B to 3E represents p < 0.05, the figure number ** represents p < 0.01, and the figure number *** represents p < 0.001.

From the results of FIG. 3B to FIG. 3E, the amphiphilic block copolymers PBLG ₅ , PBLG ₂₀ and PBLG ₄₀ can reduce hepatitis-related transaminase activity and cytokines (TNF-α and IL-6) levels, and reduce pathological grading. Score and improve the survival rate of hepatitis mice.

Referring to Figures 3F to 3G, the results show that the amphiphilic block copolymers of several embodiments of the present invention inhibit splenomegaly in the LPS/GalN-induced mouse hepatitis mode. The figure number * of Fig. 3F represents p < 0.05, the figure number ** represents p < 0.01, and the figure number *** represents p < 0.001. Each group of experiments was repeated at least three times.

The results from Fig. 3F to Fig. 3G show that the acute hepatitis in mice causes the weight of the spleen to become heavier (Fig. 3F, physiological saline group) and the size becomes larger (Fig. 3G, physiological saline group), and the amphiphilic block is administered. After the copolymers PBLG ₅ , PBLG ₂₀ and PBLG ₄₀ , the weight and size of the spleen can be significantly reduced, even approaching the normal range.

4. Evaluation of the effect of the amphiphilic block copolymer on inhibiting cognac

In this embodiment, the macrophage cell line Raw264.7 is implanted into a 96-well culture dish at 2×10 ⁴ cells per well, and is pretreated with 25 μM to 100 μM amphiphilic block copolymer PBLG _{5 for} 1 hour, and then IMQ (10 μg/mL) was treated for 18 hours. Thereafter, cell supernatants were collected, and TNF-α and IL-23 contents were measured using an ELISA kit (R&D), and the results are shown in FIGS. 4A to 4B. In addition, the collected cell lysate was analyzed by SDS-PAGE, and then the antibody was used to detect the expression of procaspase-3 and caspase-3 by IMQ, and GAPDH was used as the internal control group, and the result is shown in Fig. 4C.

4A to 4C, which show that in the mode of imiquimod (IMQ)-induced macrophages, the amphiphilic block copolymer of one embodiment of the present invention inhibits cognac-related cytokine results, respectively. The results of TNF-α (Fig. 4A), IL-23 (Fig. 4B), apoptotic zymogen-3, and apoptotic enzyme-3 (Fig. 4C), while GAPDH of Fig. 4C is an internal control group.

From the results of FIG. 4A to FIG. 4B, in the mode of IMQ-induced macrophage dryness, the amphiphilic block copolymer PBLG ₅ can inhibit the content of cytokines TNF-α and IL-23, and the amphiphilic block copolymer. There is a dose-dependent relationship between the amount of use and the amount of cytokine inhibition.

From the results of Fig. 4C, the amphiphilic block copolymer PBLG ₅ has an effect of causing apoptosis to macrophages which cause dryness of IMQ, and there is a dose between the amount of the amphiphilic block copolymer and the increase in the amount of apoptotic enzyme. Dependence, but does not cause apoptosis in normal cells.

The results of the above cell tests were confirmed in the cognac mode. In this example, BALB/c male mice (8 to 10 weeks old) were used to establish a cognac animal model purchased from the National Experimental Animal Center of the National Experimental Research Institute (Taipei, Taiwan) and followed by the National Animal Science Laboratory Animal Care and Use. The main points established by the committee and the relevant provisions of the Taiwan Animal Protection Law are carried out.

BALB/c male mice were induced to dry codon by IMQ (13.88 mg/kg per mouse per day), and the amphiphilic block copolymer PBLG _{5 was} injected intraperitoneally (ip) by intraperitoneal injection (ip). (20 mg/kg or 100 mg/kg) or quercetin (50 mg/kg). After the mice in the positive control group were induced to dry up by IMQ, an equal amount of physiological saline containing no drug was administered. After 5 days of treatment, the appearance of the amphiphilic block copolymer PBLG _{5 was} evaluated after observing the appearance of the mouse ear skin (Fig. 5A) and the poriasis area severity index (PASI) (Fig. 5B to Fig. 5D). .

Please refer to FIG. 5A to FIG. 5D, which show the appearance of the amphiphilic block copolymer on the ear skin of the mouse ( FIG. 5A ) and the clinical score ( FIG. 5B to FIG. 5B in the mode of IMQ-induced mouse cognac. Figure 5D) results. Each group of experiments was repeated at least three times. After IMQ induced mouse cognac for 10 days, the severity of redness (erythema; Fig. 5B), skin thickening (Fig. 5C) and scaling (Fig. 5D) of the mouse ear appearance was evaluated according to PASI, where 0 Represents asymptomatic, 1 represents mild, 2 represents moderate, 3 represents severe, and 4 represents extremely severe.

From the results of FIG. 5A to FIG. 5D, the administration of the amphoteric block copolymer PBLG ₅ can significantly improve the clinical symptoms of cognac and the amphoteric block copolymerization compared to the dry control group (IMQ treatment, administration of physiological saline). There is a dose-dependent relationship between the amount of substance used and the degree of improvement, and it is indeed effective.

6A to 6C are diagrams showing the staining of the ear skin tissue staining of the amphiphilic block copolymer according to an embodiment of the present invention in the mode of IMQ-induced mouse cognac ( FIG. 6A ) and the thickness of the epidermis of the ear ( FIG. 6A to FIG. 6C ). 6B) and the bar graph of the number of layers of the epidermis (Fig. 6C).

From the results of Fig. 6A to Fig. 6C, it is shown that the administration of the amphoteric block copolymer PBLG ₅ can significantly improve the clinical symptoms of epidermal thickening caused by dryness, and there is a dose dependency between the amount of amphiphilic block copolymer used and the degree of improvement. It does have curative effect.

5. Evaluate the effect of amphiphilic block copolymers on the inhibition of HuR and cognac-related message transmission pathways

Please refer to Figure 7A, which shows a skin immunohistochemical stained section of the HuR protein distribution in the mode of IMQ-induced mouse cognac.

From the results of Fig. 7A, the administration of 100 mg/kg of the amphiphilic block copolymer PBLG ₅ significantly inhibited the expression of HuR protein in the skin of dried mice compared to the control group (IMQ treatment, physiological saline). .

Referring to Figures 7B to 7C, it is shown that in the mode of IMQ-induced mouse cognac, the amphiphilic block copolymer of one embodiment of the present invention inhibits the cognac-associated cytokine VEGF (Fig. 7B) and IL-23 (Fig. 7C). Straight bar chart. Each group of experiments was repeated at least three times.

The results from 7B to 7C show that the administration of the amphoteric block copolymer PBLG ₅ can significantly inhibit the expression of VEGF and IL-23 compared to the dry control group (IMQ treatment, physiological saline). There is a dose-dependent relationship between the amount of block copolymer used and the degree of inhibition of VEGF and IL-23, and it is indeed effective.

Referring to Figure 7D, it is shown that in the mode of IMQ-induced mouse cognac, a skin immunohistochemical staining profile of the amphiphilic block copolymer of one embodiment of the present invention in the skin, liver and spleen is detected by PEG antibody.

From the results of Fig. 7D, the amphoteric block copolymer PBLG _{5 was} distributed to the skin, liver and spleen.

The above-mentioned IMQ-induced dry blood samples of mice can be tested for serum ALT and AST activity by the Reitman-Frankel method, and the results are shown in Fig. 7E and Fig. 7F.

Referring to Figures 7E and 7F, the results show that the amphiphilic block copolymer inhibits transaminase activity in an embodiment of the IMQ-induced mouse cognac.

From the results of Fig. 7E and Fig. 7F, the amphiphilic block copolymer PBLG ₅ can reduce transaminase ALT (Fig. 7E) and AST (Fig. 7F) compared to the dry control group (IMQ treatment, administration of physiological saline). Activity.

Please refer to Figure 8 and Table 3. Fig. 8 is a graph showing the results of inhibition of cytokine protein matrix of the ear skin by the amphiphilic block copolymer of one embodiment of the present invention in the mode of IMQ-induced mouse cognac. This example is to analyze the ear skin protein lysate of the mouse ear, using a commercially available cytokine protein array (R&D), each protein lysate is composed of three mouse ears. Get it from the skin. The lower left area of Fig. 8 is the group in which physiological saline is administered, and the lower right area of Fig. 8 is the group in which the amphiphilic block copolymer PBLG ₅ is administered, and the green frame represents the positive control group (IMQ induced, physiological saline is administered). The red box represents the negative control group (no physiological saline is induced by IMQ). Table 3 shows the results of the cytokine of Figure 8 using software ImageJ and densitometry to measure the concentration of dots, and then normalized by the positive control group, wherein the p value of less than 0.05 is expressed in bold italics, representing Corresponding proteins have statistically significant differences.

The results from Fig. 8 and Table 3 show that the amphoteric block copolymer PBLG ₅ can significantly reduce CXCL13, CD54, IL-1α, IL-1β, IL compared to the dry control group (IMQ treatment, administered with physiological saline). -1ra, IL-16, CCL3, CXCL12, CXCL1, M-CSF, CCL2, TREM-1, etc.

From the above results, the amphiphilic block copolymer of the present invention can specifically bind to the ribonucleic acid binding region of HuR protein, and can interfere with, reduce and/or hinder the binding of RNA or anti-HuR antibody to HuR protein, and has potential application. A pharmaceutical composition for a HuR-related disease, such as a drug for treating dryness, hepatitis, and the like.

In summary, the present invention exemplifies the amphoteric block copolymer, the specific dosage form, or the specific evaluation mode of the specific structure, and describes the amphiphilic block copolymer, the nanoparticle containing the same, and the pharmaceutical composition of the present invention. It is a method for inhibiting the binding of ribonucleic acid to a human antigen R protein, but it is to be understood by those skilled in the art that the present invention is not limited thereto, and the present invention is also within the spirit and scope of the present invention. It can be carried out using amphiphilic block copolymers of other structures, other dosage forms or other evaluation methods. For example, any of the ordinary skill in the art to which the present invention pertains can effectively treat the amphiphilic block copolymer and/or nanoparticle of the present invention from 20% to 80% for the disease and/or symptom to be applied. The dose is added to an existing external pharmaceutical dosage form, an oral pharmaceutical dosage form or an injectable pharmaceutical dosage form, and an antibody that inhibits RNA or anti-HuR binds to the HuR protein, and is further applied to a pharmaceutical composition of a HuR-related disease.

It can be seen from the above examples that the amphiphilic block copolymer, the nanoparticle-containing particles and the pharmaceutical composition of the present invention and the method for inhibiting the binding of ribonucleic acid to the human antigen R protein have the advantages of the amphoteric block copolymer. It specifically binds to the ribonucleic acid binding region of the human antigen R protein, and effectively inhibits the binding of ribonucleic acid to the human antigen R protein, thereby being applied to a pharmaceutical composition of a HuR-related disease.

While the invention has been described above in terms of several embodiments, it is not intended to limit the scope of the invention, and the invention may be practiced in various embodiments without departing from the spirit and scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.

Claims (11)

An amphoteric block copolymer having the molecular structure represented by the following formula (I): Wherein the Y group is a hydrophilic polymeric chain, and the hydrophilic polymeric chain is selected from the group consisting of a polyethylene glycol-derived group, a polypeptide-derived group or a polysaccharide-derived group; The base is a hydrophobic polymeric chain, and the hydrophobic polymeric chain is a phenyl group-containing amino acid group, or an amino acid group modified with a phenyl group or a derivative thereof; and the n is an integer of 3 to 40, and The amphiphilic block copolymer is specifically bound to a ribonucleic acid binding region of a human antigen R protein.  The amphoteric block copolymer according to claim 1, wherein the phenyl group-containing amino group is an L-tryptophan group or an L-phenylalanine group.    The amphoteric block copolymer according to claim 1, wherein the amino acid group modified by the phenyl group or a derivative thereof is selected from the group consisting of carbobenzyloxy (Cbz; Z)-L. - lysine group, γ-benzyl-L-glutamic acid group, β-benzyl-L-aspartate group, O-benzyl-L-tyrosine group, O-benzoic acid A group consisting of a group consisting of a lysine-L-threonate group, an S-benzyl-cysteinyl group, an O-benzyl-serine group, and a Z, ZL-arginine group.    The amphoteric block copolymer of claim 1, wherein n is an integer from 3 to 20.    The amphoteric block copolymer of claim 1, wherein n is an integer from 5 to 20.   An amphoteric block copolymer having the molecular structure represented by the following formula (I-1): Wherein m is an integer from 45 to 245, the n is an integer from 5 to 20, and the amphiphilic block copolymer is specifically bound to a ribonucleic acid binding region of a human antigen R protein.  The amphoteric block copolymer of claim 6, wherein m is an integer from 45 to 112, and n is an integer from 5 to 8.    A nanoparticle comprising the amphiphilic block copolymer according to any one of claims 1 to 7, wherein the nanoparticle has an average particle diameter of 50 to 500 nm.    A pharmaceutical composition for inhibiting binding of a human antigen R protein to a ribonucleic acid, comprising: the nanoparticle of claim 8; and a pharmaceutically acceptable carrier, and wherein the composition is based on the pharmaceutical composition The total content of the substance is 100%, and the content of the nanoparticles is from 20% to 80%.    The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is an oral pharmaceutical composition or a topical pharmaceutical composition.    A method of inhibiting binding of a human antigen R protein to a ribonucleic acid, comprising binding a nanoparticle as described in claim 8 to a ribonucleic acid binding region of the human antigen R protein.