WO2019157714A1 - Copolymère séquencé amphiphile, nanoparticule le contenant, composition médicinale de celui-ci et procédé d'inhibition de la liaison de l'acide ribonucléique à l'antigène humain r l'utilisant - Google Patents

Copolymère séquencé amphiphile, nanoparticule le contenant, composition médicinale de celui-ci et procédé d'inhibition de la liaison de l'acide ribonucléique à l'antigène humain r l'utilisant Download PDF

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WO2019157714A1
WO2019157714A1 PCT/CN2018/076848 CN2018076848W WO2019157714A1 WO 2019157714 A1 WO2019157714 A1 WO 2019157714A1 CN 2018076848 W CN2018076848 W CN 2018076848W WO 2019157714 A1 WO2019157714 A1 WO 2019157714A1
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amphiphilic block
block copolymer
protein
hur
pblg
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PCT/CN2018/076848
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English (en)
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Jeng-Shiung Jan
Yu-Fon Chen
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Jan Jeng Shiung
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10—Alpha-amino-carboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/40—Polyamides containing oxygen in the form of ether groups

Definitions

  • the present invention relates to an amphiphilic block copolymer, a nanoparticle containing the same and its application. More particularly, the present invention relates to an amphiphilic block copolymer specifically binding to a ribonucleic acid binding region on the human antigen R (HuR) protein, a nanoparticle containing the same, a medicinal composition and a method of inhibiting ribonucleic acid binding to HuR protein.
  • HuR human antigen R
  • the binding ability of the aforementioned polymer to HuR protein is limited; therefore, it is observed that the polymer is unfavorable to improve conditions such as cell necrosis around the opening of the hepatic blood vessel, cytoplasmic vacuolization, splenomegaly after induced inflammation, assessment (erythema, thickness and scaling) after ear psoriasis treatment in a mouse acute hepatitis model, such that there is still room for the improvement in the inflammation treatment effect overall.
  • most drugs influence normal physiologic metabolism of other cells when they enter the body in positions of inflammation or cancer tissue.
  • an aspect of the present invention provides an amphiphilic block copolymer, in which the amphiphilic block copolymer specifically binds to human antigen R (HuR) protein.
  • Human antigen R Human antigen R
  • nanoparticle including the aforementioned amphiphilic block copolymer.
  • Another aspect of the present invention provides a method of inhibiting HuR protein binding to ribonucleic acid (RNA) , in which the aforementioned nanoparticle specifically binds to a RNA binding region on the HuR protein.
  • RNA ribonucleic acid
  • Yet another aspect of the present invention provides a medicinal composition of inhibiting RNA binding to HuR protein, which includes the nanoparticle and a pharmaceutically acceptable carrier.
  • an amphiphilic block copolymer which has a molecular structure as shown in formula (I) :
  • Y of the formula (I) can be a hydrophilic polymer chain, which can include but be not limited to polyethylene glycol derivative group, polypeptide derivative group and polysaccharide derivative group;
  • R can be a hydrophobic polymer chain, and the hydrophobic polymer chain can be amino acid group having phenyl group or modified by phenyl group or its derivative; and
  • n is an integer of 3 to 40, and the amphiphilic block copolymer specifically binds to a RNA binding region on HuR protein.
  • the aforementioned amino acid group having the phenyl group can be L-tryptophanyl and L-phenylalanyl groups.
  • the amino acid group modified by the phenyl group or its derivative can include but be not limited to carbobenzyloxy-L-lysyl, ⁇ -benzyl-glutamic, ⁇ -benzyl-L-aspartic, O-benzyl-L-tyrosyl, O-benzyl-L-threonyl, S-benzyl-cysteinyl, O-benzyl-serinyl and Z, Z-L-arginyl groups.
  • n can be an integer of 3 to 20. In an example, n can be an integer of 5 to 20.
  • amphiphilic block copolymer which has a molecular structure as shown in formula (I-1) :
  • m of the formula (I-1) can be an integer of 45 to 356
  • n can be an integer of 5 to 20
  • the amphiphilic block copolymer specifically binds to a RNA binding region on HuR protein.
  • n can be an integer of 5 to 8.
  • a nanoparticle including any one of the aforementioned amphiphilic block copolymers, in which an average particle size range of the nanoparticle is 50 nm to 500 nm.
  • a method of inhibiting ribonucleic acid binding to HuR protein in which the aforementioned nanoparticle specifically binds to a RNA binding region on the HuR protein.
  • a medicinal composition of inhibiting RNA binding to HuR protein which includes the aforementioned nanoparticle and a pharmaceutically acceptable carrier.
  • an amount of the nanoparticles can be 20%to 80%.
  • the medicinal composition can be an oral medicinal composition or an external medicinal composition.
  • the amphiphilic block copolymer the nanoparticle containing the same, the medicinal composition and the method of inhibiting the RNA binding to HuR protein of the present invention, in which the amphiphilic block copolymer can specifically bind to the RNA binding region on the HuR protein, for effectively inhibiting the RNA binding to the HuR protein, and further being applied to the medicinal composition of HuR-related diseases.
  • Figs. 1A to 1F are stereograms of predicted molecule surface models of an amphiphilic block copolymers of one embodiment of the present invention, RNA, MS-444 and quercetin (Q) coupling with human antigen R (HuR) protein.
  • Fig. 1G is a bar chart of affinity and binding energy between an amphiphilic block copolymer of one embodiment of the present invention and HuR protein.
  • Fig. 1H is a hydrogen-bond distribution diagram of an amphiphilic block copolymer of one embodiment of the present invention and HuR protein.
  • Fig. 1I is a native-PAGE result between an amphiphilic block copolymer of one embodiment of the present invention and HuR protein.
  • Fig. 2A is immunofluorescence staining images of distributions of HuR protein (HuR) , an amphiphilic block copolymer of an embodiment of the present invention (PEG) , cell nucleus (DAPI) and merged image of the aforementioned results (Merge) in a macrophage model induced by LPS.
  • HuR HuR protein
  • PEG amphiphilic block copolymer of an embodiment of the present invention
  • DAPI cell nucleus
  • Merge merged image of the aforementioned results in a macrophage model induced by LPS.
  • Fig. 2B is an immunoblot analysis result of HuR protein (HuR) in cell nucleus (N) and cytoplasm (C) and an amphiphilic block copolymer (PEG) of an embodiment of the present invention in a macrophage model induced by LPS.
  • HuR HuR protein
  • N cell nucleus
  • C cytoplasm
  • PEG amphiphilic block copolymer
  • Figs. 2C to 2F are immunoblot analysis results of an amphiphilic block copolymer of various embodiments of the present invention against inflammation-related cytokines induced by LPS in a macrophage model induced by LPS.
  • Fig. 3A is liver tissue section pictures of amphiphilic block copolymers of various embodiments of the present invention inhibiting HuR protein in a mouse hepatitis model induced by LPS/GalN.
  • Figs. 3B to 3E are transaminase activity (Fig. 3B) , amounts of cytokines (Fig. 3C) , pathological grade (Fig. 3D) and survival rate (Fig. 3E) of amphiphilic block copolymers of various embodiments of the present invention inhibiting related inflammation in a mouse hepatitis model induced by LPS/GalN.
  • Figs. 3F to 3G are results of amphiphilic block copolymers of various embodiment of the present invention inhibiting splenomegaly in a mouse hepatitis model induced by LPS/GalN.
  • Figs. 4A to 4C are results of amphiphilic block copolymers of an embodiment of the present invention inhibiting psoriasis-related cytokines in a macrophage model induced by imiquimod (IMQ) .
  • Figs. 5A to 5D are skin appearance (Fig. 5A) and clinical scores (Fig. 5B-5D) resulted from the amphiphilic block copolymers of an embodiment of the present invention in a mouse psoriasis model induced by IMQ.
  • Figs. 6A to 6C are ear skin tissue staining section graphs (Fig. 6A) , bar charts of thickness of ear epidermis (Fig. 6B) and layers of ear epidermis (Fig. 6C) of amphiphilic block copolymers of an embodiment of the present invention inhibiting psoriasis symptom in a mouse psoriasis model induced by IMQ.
  • Fig. 7A is an immunohistochemically stained skin sections showing HuR protein distribution in a mouse psoriasis model induced by IMQ.
  • Figs. 7B to 7C are bar charts of amphiphilic block copolymers of an embodiment of the present invention inhibiting psoriasis related cytokines VEGF (Fig. 7B) and IL-23 (Fig. 7C) in a mouse psoriasis model induced by IMQ.
  • Fig. 7D is an immunohistochemically stained skin sections showing amphiphilic block copolymers of an embodiment of the present invention distributed in skin, liver and spleen detected by anti-PEG antibody in a mouse psoriasis model induced by IMQ.
  • Figs. 7E to 7F are bar charts of amphiphilic block copolymers of an embodiment of the present invention inhibiting serum ALT (Fig. 7E) and AST (Fig. 7F) in a mouse psoriasis model induced by IMQ.
  • Fig. 8 is results of amphiphilic block copolymers of an embodiment of the present invention inhibiting cytokine protein array of ear skin in a mouse psoriasis model induced by IMQ.
  • Term “a” , “one” and “the” of the single form used in the present invention include the plural form, unless limited otherwise in the specification.
  • Data range for example, 10%to 11%of A, includes upper limit and lower limit (i.e. 10% ⁇ A ⁇ 11%) , unless limited otherwise; if the data range doesn’t define lower limit, for example, lower than 0.2%of B or below 0.2%of B, which indicates the lower limit can be 0 (i.e. 0% ⁇ B ⁇ 0.2%) .
  • the terms herein are only helpful to describe and understand the present invention rather than limiting the present invention.
  • the present invention provides an amphiphilic block copolymer, in which the amphiphilic block copolymer herein indicates the one specifically binds to a ribonucleic acid binding region on human antigen R (HuR) protein, and can compete the ribonucleic acid binding region with the ribonucleic acid (RNA) .
  • a structure formula of the suitable amphiphilic block copolymer can be shown as formula (I) :
  • Y group of the formula (I) can be a hydrophilic polymer chain, which includes but is not limited to polyethylene glycol derivative group, polypeptide derivative group and polysaccharide derivative group.
  • the polypeptide derivative groups can be pegylated polypeptides, in which examples can include but be not limited to pegylated poly-L-lysine, such as poly ⁇ N-2- [2- (2-methoxyethoxy) ethoxy] acetyl-lysine ⁇ ; pegylated poly-L-glutamate, such as poly ⁇ - [2- (2-methoxyethoxy) ethoxy] esteryl-L-glutamate ⁇ ; polysarcosine; poly (L-lysine) ; poly (L-glutamic acid) ; poly (L-arginine) ; and the polypeptide derivative group can be L-form, D-form or its combination.
  • pegylated poly-L-lysine such as poly ⁇ N-2- [2- (2-methoxyethoxy) ethoxy] acetyl-lysine ⁇
  • pegylated poly-L-glutamate such as poly ⁇ - [2- (2-me
  • the polysaccharide derivative group can be oligosaccharides or glycans, in which examples can include but be not limited to alginic acid, amylose, cellulose, cellulose trimester, chitosan, curdlan gel, dextrin, ⁇ -cyclodextrin, dextran, heparin, hyaluronan, maltoheptaose, maltodextrin, ovomucoid, trimethylcellulose, pullulan, schizophyllan, succinoglycan, xanthan, xyloglucan and so on.
  • examples can include but be not limited to alginic acid, amylose, cellulose, cellulose trimester, chitosan, curdlan gel, dextrin, ⁇ -cyclodextrin, dextran, heparin, hyaluronan, maltoheptaose, maltodextrin, ovomucoid,
  • R group of the formula (I) can be a hydrophobic polymer chain, such as amino acid group having phenyl group or modified by phenyl group or its derivative.
  • the amino acid group with phenyl group can include but be not limited to L-tryptophanyl (as shown in formula II-7) or L-phenylalanyl (as shown in formula II-5) group.
  • the amino acid group modified by phenyl group or its derivative can include but be not limited to carbobenzyloxy-L-lysyl (as shown in formula II-1) , ⁇ -benzyl-glutamic (as shown in formula II-2) , ⁇ -benzyl-L-aspartic (as shown in formula II-3) , O-benzyl-L-tyrosyl (as shown in formula II-4) , O-benzyl-L-threonyl (as shown in formula II-6) , S-benzyl-cysteinyl (as shown in formula II-8) , O-benzyl-serinyl (as shown in formula II-9) or Z, Z-L-argininyl (as shown in formula II
  • n of the formula (I) can be an integer between 3 and 40, thereby endowing the block copolymer with amphiphilic property having both hydrophilic property and hydrophobic property.
  • the n is better to be an integer between 3 and 20, and is preferably an integer between 5 and 8.
  • structure formula of the amphiphilic block copolymer can be 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 (abbreviated as 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 20
  • the amphiphilic block copolymers in a solution can self-assemble to nanoparticles (also called as nanocomposites or micelle) .
  • nanoparticles also called as nanocomposites or micelle
  • an average particle size of the obtained nanoparticles can be 50 nm to 500 nm, such as 100 nm, 200 nm, 300 nm, 400 nm or 500 nm, but it is not limited to the aforementioned examples.
  • an average particle size of the obtained nanoparticles can be 50 nm to 150 nm.
  • HuR target RNAs can specifically bind to a binding site of the HuR protein and form a complex, so as to stabilize target RNAs, up-regulate or repress translational expression of the target RNAs.
  • the HuR-related disease discussed herein refers to the disease due to stabilizing target RNAs, up-regulating or repressing its translational expression of the target RNAs, increasing HuR protein expression or enabling HuR protein to move into cytoplasm after the HuR target RNAs bind to the HuR protein and form the complex.
  • the amphiphilic block copolymer itself or the nanoparticle containing the same of the present invention can specifically bind to HuR protein and compete the same or neighboring binding region with target RNAs, interfering, decreasing and/or blocking the binding of the target RNAs or anti-HuR antibodies to the HuR protein, leading unstable target RNAs, up-regulated or repressed translational expression, decreased HuR protein expression and so on, thereby reducing or even treating the HuR-related diseases.
  • the amphiphilic block copolymer which has higher binding ability between the amphiphilic block copolymer and the HuR protein than the one between the RNA with the HuR protein, can sufficiently compete the RNA binding region on the HuR protein with the RNA.
  • examples of target RNAs stabilized by HuR protein can include but be not limited to mRNA of c-Fos, cyclin-dependent kinase (cdk) inhibitive factor p21, cyclins A2, B1, D1, E1, inducible nitric oxide synthase (iNOS) , granulocyte-macrophage-colony stimulating factor (GM-CSF) , eukaryotic initiation factor-4E (eIF-4E) , murine double minute2 (mdm2) , vascular endothelial growth factor (VEGF) , transforming growth factor- ⁇ , sirtuin 1 (SIRT1) , tumor necrosis factor- ⁇ , B-cell leukemia-2 (Bcl-2) , myeloid leukemia cell differentiation protein-1 (mcl-1) , oncostatin M (OSM) , cyclooxygenase-2 (COX-2) , ⁇ -glutamyl
  • RNAs up-regulating its translational expression by HuR protein can include but be not limited to mRNA of cyclin A2, prothymosin ⁇ (ProT ⁇ ) , hypoxia-inducible factor-1 ⁇ (HIF-1 ⁇ ) , Bcl-2, VEGF, thrombospondin-1 (TSP-1) , MKP-1, p53, cationic amino acid transporter (CAT-1) , intrinsic cellular caspase inhibitor (XIAP) , cytochrome c and so on.
  • ProT ⁇ prothymosin ⁇
  • HIF-1 ⁇ hypoxia-inducible factor-1 ⁇
  • Bcl-2 hypoxia-inducible factor-1 ⁇
  • VEGF thrombospondin-1
  • TSP-1 thrombospondin-1
  • MKP-1 MKP-1
  • p53 cationic amino acid transporter
  • CAT-1 cationic amino acid transporter
  • XIAP intrinsic cellular caspase inhibitor
  • examples of the target RNAs repressing its translational expression by HuR protein can include but be not limited to mRNA of p27, IGF-1 R, thrombomodulin (TM) , Wnt5a, c-Myc and so on.
  • HuR protein can increase inflammatory factors expression, repress anti-inflammatory factors expression, and etc. Additionally, HuR protein can induce growth, immune escape, invasion and transition of cancer cells, angiogenesis and so on.
  • the amphiphilic block copolymer itself or the nanoparticle formed by the same can further apply in medicinal composition to treat the HuR-related disease, such as inflammation (e.g. hepatitis, psoriasis and so on) and cancer, but the application of the present invention is not limited to the examples herein.
  • the nanoparticles can apply in treating another HuR-related disease.
  • the medicinal composition can include but be not limited to the nanoparticles and a pharmaceutically acceptable carrier.
  • the carrier can be water, Vaseline or any other ointment, which can apply to skin, depending on demand. For example, if the composition is applied to skin, Vaseline can be selected.
  • the carrier can be a carrier mainly based on water, such as physiological buffer solution, and the medicinal composition can be delivered through oral or injection.
  • an amount of the nanoparticles when the nanoparticle applied in the medicinal composition, based on a total amount of the medicinal composition is 100%, an amount of the nanoparticles can be 20%to 80%, such as 20%, 30%, 40%, 50%, 60%, 70%or 80%, but it’s not limited thereto.
  • an amount of the carrier can be 0.1%to 20%, such as 0.1%, 0.5%, 0.7%, 1.5%, 2%, 5%, 10%, 15%or 20, but it’s not limited thereto.
  • Embodiment 1 Evaluate binding ability between amphiphilic block copolymers and HuR protein
  • RNA U-rich RNA sequence rich was AUUUUUAUUUU, with total 11 nucleotides
  • MS-444 quercetin
  • HuR protein HuR protein
  • RNA binding region on the HuR protein also called as RNA-recognition motif, RRM
  • Fig. 1B to Fig. 1F portion of surface model of RNA binding region on the HuR protein (also called as RNA-recognition motif, RRM) was shown in Fig. 1B to Fig. 1F.
  • the aforementioned RNA was a U-rich RNA ligand, which had a sequence of AUUUUUAUUUU, with total 11 nucleotides.
  • An interatomic distance in Fig. 1B to Fig. 1F was less than
  • affinity (pKD) of the amphiphilic block copolymer, MS-444 and quercetin (Q) were predicted by X-score software, binding energy of the amphiphilic block copolymer, MS-444 and quercetin (Q) were scored by HotLig software, and the two results were shown in Fig. 1G and Table 2.
  • hydrogen-bond distribution between the amphiphilic block copolymer and the HuR protein was predicted by Ligplot software, and the results was shown in Fig. 1H.
  • Figs. 1A to 1F showed stereograms of predicted molecule surface models of the amphiphilic block copolymers of one embodiment of the present invention, RNA, MS-444 and quercetin (Q) coupling with the HuR protein, in which the amphiphilic block copolymers were oligomers of L-glutamic acid having polyphenyl group (PBLG 5 , PBLG 7 ) , while MS-444 and quercetin (Q) were used as control molecules.
  • PBLG 5 , PBLG 7 polyphenyl group
  • MS-444 and quercetin (Q) were used as control molecules.
  • MS-444 referred to an article titled as “Identification and mechanistic characterization of low-molecular-weight inhibitors for HuR” , which was authored by Nicole-Claudia Meisner et al.
  • the HuR protein includes N-terminal domain 101 (Fig. 1A and Fig. 1B) , C-terminal domain 103 (Fig. 1A and Fig. 1B) and a RNA binding region 105 (dark grey area in Fig. 1B) , and PBLG 5 and PBLG 7 could bind to the RNA binding region on the HuR protein (Fig. 1E and Fig. 1F) .
  • the control molecules MS-444 and quercetin (Q) could also bind to the RNA binding region on the HuR protein, which is shown in Fig. 1C and Fig. 1D, respectively.
  • Fig. 1G showed a bar chart of affinity and binding energy between the amphiphilic block copolymer of one embodiment of the present invention and HuR protein
  • Table 1 showed data of respective bar in Fig. 1G.
  • higher Xscore showed the prediction of stronger affinity between both
  • higher HotLig score in negative value showed the prediction of higher binding energy between both.
  • binding energy of the amphiphilic block copolymer of the present invention and the HuR protein in descending order is 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, which represented that binding abilities between the amphiphilic block copolymers PBLG 3 to PBLG 8 of the present invention and the HuR protein are better than that of quercetin (Q) and MS-444.
  • binding energy between ligand and receptor increases with increasing molecular weight of the ligand, but the results of Fig. 1G and Table 1 show that PBLG 7 and PBLG 6 are better than PBLG 8 and PBLG 5 , in which the influence of structure flexibility of the ligand itself and/or space dimension of binding region of the receptor cannot be excluded.
  • Fig. 1H a hydrogen-bond distribution diagram of the amphiphilic block copolymer of one embodiment of the present invention and HuR protein, in which bold line structure formula represents PBLG 7 , dotted lines represent potential hydrogen-bonds, and spoked arcs represent non-bonded contacts on the HuR protein.
  • results of Fig. 1H show that the amphiphilic block copolymer PBLG 7 of the present invention has more hydrogen-bonds and non-bonded (or hydrophobic) contacts with the HuR protein, which indicated stronger binding ability between both; therefore the amphiphilic block copolymer PBLG 7 of the present invention is able to compete the RNA binding region on the HuR protein with the RNA, and is expected to be an inhibitor against the HuR protein.
  • the antibodies used are mouse polyclonal antibody against amino acids 1-101 of HuR protein (abcam, Cambridge, MA) , mouse monoclonal antibody against amino acids 1-180 of HuR protein, and mouse monoclonal antibody against amino acids 1-326 of HuR protein (the manufacturer of the latter two is Santa Cruz Biotechnology Inc., CA) , respectively. Moreover, signal of bands were detected by commercial enhanced chemiluminescence (ECL) kit, and the result was shown in Fig. 1I.
  • ECL enhanced chemiluminescence
  • Fig. 1I which showed that a native-PAGE result between the amphiphilic block copolymer of one embodiment of the present invention and the HuR protein, in which based on a concentration of free HuR protein without adding the amphiphilic block copolymer PBLG 5 as 100%.
  • symbol “*” referred to p ⁇ 0.05
  • symbol “**” referred to p ⁇ 0.01
  • symbol “***” referred to p ⁇ 0.001.
  • Example 2 evaluate effect of amphiphilic block copolymers on forming nanoparticles
  • Results of Table 2 showed that the PBLG 3 to PBLG 8 could afford the nanoparticle with smaller average size, and the average particle size was 50 nm to 150 nm.
  • Example 3 evaluate effect of amphiphilic block copolymers on inhibiting inflammation
  • in vitro cell model was established by macrophage cell line Raw264.7 (deposited in Bioresource Collection and Research Center, Hsinchu, Taiwan, and serial number of deposit: BCRC 60001; or deposited in China Center for Type Culture Collection, Wuhan, China, and serial number of deposit: CCTCC GDC0143) .
  • macrophage cell line Raw264.7 was cultured in DMEM culture medium [including 10%fetal bovine serum (FBS) and 50 ⁇ g/mL gentamicin] at 37°C and 5%CO 2 in saturated relative humidity environment.
  • FBS fetal bovine serum
  • macrophage cell line Raw264.7 was seeded in 6-well cell culture plate with 2 ⁇ 10 5 cells per well, and was treated with LPS (Sigma-Aldrich, St. Louis, MO; 0.25 ⁇ g/mL) for 24 hours after being pretreated with 100 ⁇ M amphiphilic block copolymer PBLG 5 for 1 hour. Afterwards, the cells were fixed by methanol for 10 minutes, and then blocked by 5%low-fat milk with 0.3%Triton-x100. The cells reacted with primary antibody at 4°C overnight after washing with PBS for 3 times, in which the primary antibody included mouse anti-HuR antibody (Santa Cruz Biotechnology Inc. ) and rabbit anti-PEG antibody (Abcam plc.
  • LPS Sigma-Aldrich, St. Louis, MO; 0.25 ⁇ g/mL
  • the samples reacted with secondary antibody at room temperature for 2 hours after washing with PBS for 3 times, in which the secondary antibody included Texas red-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG (both are from Jackson ImmunoResearch Inc. ) .
  • the samples were performed cell nucleus staining with 4’, 6-diamidino-2-phenylindole (0.1 ⁇ g/mL) after washing with PBS for 3 times. After 30 minutes, the cells were observed using confocal microscopy with multiphoton laser scanning microscope (Olympus FV1000 MPE) combined with 60x water-immersion objective lens, and the results was shown in Fig. 2A.
  • Fig. 2A showed immunofluorescence staining image of distributions of HuR protein (HuR) , an amphiphilic block copolymer of an embodiment of the present invention (PEG) , cell nucleus (DAPI) and merged image of the aforementioned results (Merge) in a macrophage model induced by LPS.
  • HuR HuR protein
  • PEG amphiphilic block copolymer of an embodiment of the present invention
  • DAPI cell nucleus
  • Merge merged image of the aforementioned results
  • 2B showed an immunoblot analysis result of HuR protein (HuR) in cell nucleus (N) and cytoplasm (C) and an amphiphilic block copolymer (PEG) of an embodiment of the present invention in a macrophage model induced by LPS, in which band fluorescence intensity of the HuR protein (HuR) or the amphiphilic block copolymer (PEG) of cell nucleus (N) or cytoplasm (C) without treating with LPS was set as 1.00 to calculate relative values of each group, while ⁇ -actin was used as an internal control.
  • amounts of the amphiphilic block copolymer PBLG 5 in cytoplasm detected by anti-PEG antibody are 1.16 times of amount of that in cell nucleus (treated with LPS for 0.5 hour) and 1.43 times of amount of that in cell nucleus (treated with LPS for 1.5 hours) .
  • macrophage cell line Raw264.7 was seeded into 6-well cell culture plate with 2 ⁇ 10 5 cells per well, and was treated with LPS (Sigma-Aldrich, St. Louis, MO; 0.25 ⁇ g/mL) for 24 hours after being pretreated with 100 ⁇ M amphiphilic block copolymer PBLG 5 , PBLG 20 , PBLG 40 , dextrin-b-PBLG 5 , PEG 45 -b-PBLG 20 , PEG 245 -b-PBLG 20 for 1 hour.
  • LPS Sigma-Aldrich, St. Louis, MO; 0.25 ⁇ g/mL
  • cell lysate was collected and analyzed by SDS-PAGE, and expressions of cellular factors involving in signal transduction pathway of LPS-induced inflammation were detected by individual antibodies, in which the cellular factors included pNF ⁇ Bp65, pAKT, pERK, pJNK, pp38, iNOS, COX2, procaspase-3, caspase-3 and so on, while ⁇ -actin was used as an internal control group.
  • the antibodies against pNF ⁇ Bp65, pAKT, pERK, pJNK, pp38 and caspase-3 were purchased from Danvers, MA.
  • the antibodies against iNOS, COX2, HuR and ⁇ -actin were purchased from Santa Cruz Biotechnology, Inc.
  • nitric oxide (NO) in cell culture medium was detected by commercial Griess analysis kit (Sigma-Aldrich) , and results are shown in Fig. 2C to Fig. 2F.
  • Figs. 2C to 2F which showed immunoblot analysis results of an amphiphilic block copolymer of various embodiments of the present invention against inflammation-related cytokines induced by LPS in a macrophage model induced by LPS.
  • symbol “*” referred to p ⁇ 0.05
  • symbol “**” referred to p ⁇ 0.01
  • symbol “***” referred to p ⁇ 0.001.
  • amphiphilic block copolymer PBLG 5 could inhibit production of iNOS and COX2 after treating with LPS for 24 hours. It is conjectured that influence stage of the amphiphilic block copolymer could be translational expression of inflammation-related gene, but not contact between LPS and TLR4-related protein on cellular membrane.
  • amphiphilic block copolymers in different length did effects on anti-inflammation and apoptosis of macrophage cell of inflammation induced by LPS without leading to normal cells apoptosis.
  • dextrin-b-PBLG 5 As shown in results of Fig. 2E, after replacing hydrophilic group of amphiphilic block copolymer PEG 245 -b-PBLG 5 to dextrin, dextrin-b-PBLG 5 also did effects on anti-inflammation and apoptosis of macrophage cell of inflammation induced by LPS without leading to normal cells apoptosis.
  • mice After C57BL/6 male mice was induced acute hepatitis by LPS/GalN [LPS (10 ⁇ g/kg) + D-GalN (400 mg/kg) ] , the amphiphilic block copolymer PBLG 5 , PBLG 20 , PBLG 40 (20 mg/kg) or quercetin (50 mg/kg) were intraperitoneally (i.p. ) administrated to mice.
  • Mice of positive control group were administrated with saline without drug in the same amount after being induced acute hepatitis by LPS/GalN. Afterwards, blood samples of the mice were collected to analyze transaminase activity of blood plasma and cytokine expression.
  • Reitman-Frankel method was used for the blood samples to detect activities of alanine transaminase (ALT) and aspartate transaminase (AST) in serum, thereby evaluating level of liver damage.
  • ALT alanine transaminase
  • AST aspartate transaminase
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IL-6 interleukin-6
  • biopsies of liver and spleen of the mice were collected, fixed by 4%formalin and embedded by paraffin.
  • the sections of the biopsies were stained by hematoxylin-eosin (HE) and immunohistochemically stained by mouse monoclonal antibody against amino acids 1-326 of HuR protein (Santa Cruz Biotechnology Inc., CA) .
  • Fig. 3A showed liver tissue section picture of amphiphilic block copolymers of various embodiments of the present invention inhibiting HuR protein in mouse hepatitis model induced by LPS/GalN.
  • treating effect of the amphiphilic block copolymers PBLG 5 was better than that of PBLG 20
  • treating effect of PBLG 20 was better than that of PBLG 40
  • liver tissue section of the mice of the positive control group (acute hepatitis induced by LPS/GalN and administrated with saline) showed cytoplasmic vacuolization (HE dyes was difficult to stain with color, pictures of a first line and a second line were microscopic image magnified 100 times and 200 times, respectively) .
  • liver tissue section of treating with the amphiphilic block copolymer showed that conditions such as cytoplasmic vacuolization and cell necrosis around the opening of the hepatic blood vessel indeed decreased obviously.
  • Figs. 3B to 3E which showed transaminase activity (Fig. 3B) , amounts of cytokines (Fig. 3C) , pathological grade (Fig. 3D) and survival rate (Fig. 3E) of amphiphilic block copolymers of various embodiment of the present invention inhibiting related inflammation in a mouse hepatitis model induced by LPS/GalN.
  • symbol “*” referred to p ⁇ 0.05
  • symbol “**” referred to p ⁇ 0.01
  • symbol “***” referred to p ⁇ 0.001.
  • all of the amphiphilic block copolymers PBLG 5 , PBLG 20 , and PBLG 40 could decrease inflammatory transaminase activity and amounts of cytokines (TNF- ⁇ and IL-6) , reduce score of pathological grade, and increase survival rate of hepatitis mice.
  • Figs. 3F to 3G mouse acute hepatitis leaded to higher weight (Fig. 3F, the saline group) and bigger size (Fig. 3G, the saline group) of spleen, after administration with the amphiphilic block copolymers PBLG 5 , PBLG 20 , and PBLG 40 , weight and size of spleen can decrease obviously, and even approach to normal range value.
  • macrophage cell line Raw264.7 was seeded in 96-well cell culture plate with 2 ⁇ 10 4 cells per well, and was treated with IMQ (10 ⁇ g/mL) for 18 hours after being pretreated with 25 ⁇ M to 100 ⁇ M amphiphilic block copolymer PBLG 5 for 1 hour. Afterwards, cell supernatants was collected and amounts of TNF- ⁇ and IL-23 were detected by using ELISA kit (R&D) , in which results were shown in Fig. 4A to Fig. 4B.
  • Figs. 4A to 4C which showed psoriasis-related cytokines inhibited by an amphiphilic block copolymers of an embodiment of the present invention in a macrophage model induced by imiquimod (IMQ) , which included results of TNF- ⁇ (Fig. 4A) , IL-23 (Fig. 4B) , procaspase-3 and caspase-3 (Fig. 4C) , while GADPH of Fig. 4C was used as an internal control group.
  • IMQ imiquimod
  • the amphiphilic block copolymer PBLG 5 could inhibit amounts of psoriasis-related cytokines TNF- ⁇ and IL-23 in the macrophage model induced by IMQ, and there was dose-dependent relationship between dosages of the amphiphilic block copolymer and amounts of inhibiting cytokines.
  • the amphiphilic block copolymer PBLG 5 did an effect on causing apoptosis for macrophage cell of psoriasis induced by IMQ, and there was dose-dependent relationship between dosages of the amphiphilic block copolymer and an increasing amount of procaspase; however, it did not lead to apoptosis of normal cells.
  • psoriasis animal model was established by BALB/c male mice (8 to 10 weeks old) , which were purchased from National Laboratory Animal Center (Taipei, Taiwan) , and the experimentation was carried out according to guidelines for Institutional Animal Care and Use Committee of National Cheng Kung University (Tainan, Taiwan) and rules of Taiwan Animal Protection Act.
  • mice After psoriasis of the BALB/c male mice were induced by IMQ (two ears of each mouse were treated with a total amount of 13.88 mg/kg every day) for consecutive 5 days, the amphiphilic block copolymer PBLG 5 (20 mg/kg or 100 mg/kg) or quercetin (50 mg/kg) was intraperitoneally (i. p. ) administrated to mice. Mice of positive control group were administrated with saline without drug in the same amount after psoriasis induced by IMQ. After 5 days treatment, mouse ear skin appearance (Fig. 5A) and psoriasis area and severity index (PASI) (Fig. 5B to Fig. 5D) were observed to evaluate treatment effect of the amphiphilic block copolymer PBLG 5 .
  • Fig. 5A mouse ear skin appearance
  • PASI psoriasis area and severity index
  • Figs. 5A to 5D which showed skin appearance (Fig. 5A) and clinical scores (Figs. 5B-5D) resulted from the amphiphilic block copolymers of an embodiment of the present invention in a mouse psoriasis model induced by IMQ.
  • Fig. 5A skin appearance
  • Figs. 5B-5D clinical scores
  • Fig. 5D amphiphilic block copolymers of an embodiment of the present invention in a mouse psoriasis model induced by IMQ.
  • Fig. 5B severity of mouse ear appearance was evaluated by erythema (Fig. 5B) , thickness (Fig. 5C) and scaling (Fig. 5D) according to PASI, and the severity was assessed as asymptomatic (o) , mild (1) , moderate (2) , 3 severe (3) or very severe (4) .
  • Figs. 6A to 6C which showed ear skin tissue staining section graphs (Fig. 6A) , bar charts of thickness of ear epidermis (Fig. 6B) and layers of ear epidermis (Fig. 6C) of amphiphilic block copolymers of an embodiment of the present invention inhibiting psoriasis symptom in a mouse psoriasis model induced by IMQ.
  • Fig. 7A was an immunohistochemically stained skin sections showing HuR protein distribution in a mouse psoriasis model induced by IMQ.
  • HuR protein expression in the skin of psoriasis mouse could be significantly inhibited by administration with 100 mg/kg of the amphiphilic block copolymer PBLG 5 .
  • Figs. 7B to 7C showed bar charts of amphiphilic block copolymers of an embodiment of the present invention inhibiting psoriasis related cytokines VEGF (Fig. 7B) and IL-23 (Fig. 7C) in a mouse psoriasis model induced by IMQ. Each group of experiments was repeated for at least three times.
  • Fig. 7D showed an immunohistochemically stained skin sections showing amphiphilic block copolymers of an embodiment of the present invention distributed in skin, liver and spleen detected by anti-PEG antibody in a mouse psoriasis model induced by IMQ.
  • amphiphilic block copolymer PBLG 5 could distribute in skin, liver and spleen.
  • the blood samples of the psoriasis mouse induced by IMQ could use Reitman-Frankel method to detect activities of ALT and AST in serum, in which results were shown in Fig. 7E and Fig. 7F.
  • Figs. 7E and 7F show results of amphiphilic block copolymers of an embodiment of the present invention inhibiting transaminase activities in a mouse psoriasis model induced by IMQ.
  • Fig. 8 showed results of amphiphilic block copolymers of an embodiment of the present invention inhibiting cytokine protein array of ear skin in a mouse psoriasis model induced by IMQ.
  • ear skin protein lysate of psoriasis mouse was analyzed by a commercial cytokine protein array (R&D) , in which protein lysate was obtained from ear skin of three mice.
  • R&D commercial cytokine protein array
  • Various groups administrated with saline were shown in lower-left area of Fig. 8, and various groups administrated with the amphiphilic block copolymer PBLG 5 were shown in lower-right area of Fig.
  • Table 3 shows dot concentrations of cytokines of Fig. 8 by using Image J software and optical densitometry method, and all data was normalized by the positive control groups, in which the p-value with less than 0.05 was presented in bold italic type, referring that the individual protein corresponding to such p-value had statistically significant difference.
  • amphiphilic block copolymer of the present invention could specifically bind to a RNA binding region on the HuR protein, and could also interfere, decrease and/or block RNA or anti-HuR antibody binding to the HuR protein. It indeed had potential to apply in medicinal composition for HuR-related diseases, such as drugs for psoriasis and hepatitis.
  • amphiphilic block copolymer with a specific structure, specific dosage or a specific evaluation method so as to describe an amphiphilic block copolymer, a nanoparticle containing the same, a composition and a method of inhibiting RNA binding to HuR protein. It will be apparent to those skilled in the art that the present invention is not limited within, the present invention can also use the amphiphilic block copolymers with another structures, another dosages or another evaluation methods without departing from the scope or spirit of the invention.
  • amphiphilic block copolymer and/or the nanoparticles of the present invention into existing forms of topical drugs, forms of oral drugs or forms of injecting drugs in 20%to 80%effective dosage so as to inhibit RNA or anti-HuR antibody binding to the HuR protein, and furtherly apply in medicinal composition of HuR-related diseases.
  • advantages of the amphiphilic block copolymer, the nanoparticle containing the same, the medicinal composition and the method of inhibiting the RNA binding to the HuR protein are that the amphiphilic block copolymer can specifically bind to the RNA binding region on the HuR protein, effectively inhibit the RNA binding to the HuR protein, and furtherly apply in medicinal composition of HuR-related diseases.

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Abstract

La présente invention concerne un copolymère séquencé amphiphile, une nanoparticule le contenant, une composition et un procédé d'inhibition de la liaison de l'acide ribonucléique (ARN) à la protéine d'antigène humain R (HuR). Le copolymère séquencé amphiphile peut se lier spécifiquement à une région de liaison à l'ARN sur la protéine HuR. La nanoparticule comprenant le copolymère séquencé amphiphile peut être appliquée à une composition médicinale et à un procédé d'inhibition de la liaison de l'ARN à la protéine d'antigène humain R (HuR) l'utilisant.
PCT/CN2018/076848 2018-02-14 2018-02-14 Copolymère séquencé amphiphile, nanoparticule le contenant, composition médicinale de celui-ci et procédé d'inhibition de la liaison de l'acide ribonucléique à l'antigène humain r l'utilisant WO2019157714A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114129721A (zh) * 2021-10-12 2022-03-04 江苏省农业科学院 双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013006173A1 (fr) * 2011-07-07 2013-01-10 Empire Technology Development Llc Co-polymères à blocs fluorés
CN104856952A (zh) * 2015-05-13 2015-08-26 江南大学 一种聚(L-谷氨酸)-b-聚乙二醇载药纳米胶束的制备
CN107446095A (zh) * 2017-07-05 2017-12-08 中山大学 一种三嵌段peg‑pbla‑paapba聚合物及其制备方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013006173A1 (fr) * 2011-07-07 2013-01-10 Empire Technology Development Llc Co-polymères à blocs fluorés
CN104856952A (zh) * 2015-05-13 2015-08-26 江南大学 一种聚(L-谷氨酸)-b-聚乙二醇载药纳米胶束的制备
CN107446095A (zh) * 2017-07-05 2017-12-08 中山大学 一种三嵌段peg‑pbla‑paapba聚合物及其制备方法和应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114129721A (zh) * 2021-10-12 2022-03-04 江苏省农业科学院 双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯及其应用
CN114129721B (zh) * 2021-10-12 2024-05-07 江苏省农业科学院 双亲性咪喹莫特嫁接γ-聚谷氨酸月桂酯及其应用

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