POLY(ETHYLENE GLYCOL)-B-POLY(HALOMETHYLSTYRENE), DERIVATIVE THEREOF, AND METHOD FOR PRODUCING SAME

Abstract

[Problem] To provide a method for efficiently producing a poly(ethylene glycol)-b-poly(halomethylstyrene), a novel poly(ethylene glycol)-b-poly(halomethylstyrene) produced using the method, and a derivative thereof.

[Solution] The target novel copolymer can be provided by introducing a functional group, which enables reversible addition-fragmentation chain transfer (RAFT) polymerization, to the ω terminal of poly(ethylene glycol) and copolymerizing the resulting poly(ethylene glycol) with a halomethylstyrene.

Claims

1-11. (canceled)

12. A method for producing, based on a reaction scheme below, a block copolymer represented by formula (I) or a block copolymer wherein the phenyldithiocarbonyl group in formula (I), which may be substituted by (R).sub.a, is substituted by a hydrogen atom or a mercapto group, the method comprising a step of adding a styrene derivative represented by formula (7) to an inert solvent containing a poly(ethylene glycol) derivative represented by formula (6) and a radical reaction initiator and carrying out polymerization: ##STR00013## wherein, A denotes an unsubstituted or substituted C.sub.1-C.sub.12 alkoxy group, with the substituent group being a formyl group or a group represented by the formula R.sub.1R.sub.2CH— in cases where A is substituted, R.sub.1 and R.sub.2 each independently denote a C.sub.1-C.sub.4 alkoxy group or R.sub.1 and R.sub.2 together denote a —OCH.sub.2CH.sub.2O— group, a —O(CH.sub.2).sub.3O— group or a —O(CH.sub.2).sub.4O— group, or in cases where A is substituted, the substituent group is a group represented by the formula R.sub.3R.sub.4B-Ph-, where R.sub.3 and R.sub.4 each denote a hydroxy group or R.sub.3 and R.sub.4 together denote a —OC(CH.sub.3).sub.2C(CH.sub.3).sub.2O— group, and Ph denotes an o-phenylene group, m-phenylene group or p-phenylene group that may be substituted by a methyl group or methoxy group, L is m-xylylene or p-xylylene: X denotes chlorine, bromine or iodine, R groups each independently denote a methyl group or methoxy group, a is an integer between 0 and 3, m is an integer between 3 and 500, and n is an integer between 2 and 10,000.

13. A block copolymer represented by formula (I) or a block copolymer in which the phenyldithiocarbonyl group in formula (I), which may be substituted by (R).sub.a, is substituted by a hydrogen atom or a mercapto group: ##STR00014## in the formula, A denotes an unsubstituted or substituted C.sub.1-C.sub.12 alkoxy group, with the substituent group being a formyl group or a group represented by the formula R.sub.1R.sub.2CH— in cases where A is substituted, R.sub.1 and R.sub.2 each independently denote a C.sub.1-C.sub.4 alkoxy group or R.sub.1 and R.sub.2 together denote a —OCH.sub.2CH.sub.2O— group, a —O(CH.sub.2).sub.3O— group or a —O(CH.sub.2).sub.4O— group, or in cases where A is substituted, the substituent group is a group represented by the formula R.sub.3R.sub.4B-Ph-, where R.sub.3 and R.sub.4 each denote a hydroxy group or R.sub.3 and R.sub.4 together denote a —OC(CH.sub.3).sub.2C(CH.sub.3).sub.2O— group, and Ph denotes an o-phenylene group, m-phenylene group or p-phenylene group that may be substituted by a methyl group or methoxy group, L is m-xylylene or p-xylylene: X denotes chlorine, bromine or iodine, R groups each independently denote a methyl group or methoxy group, a is an integer between 0 and 3, m is an integer between 3 and 500, and n is an integer between 2 and 10,000.

14. The block copolymer of claim 13, wherein the phenyldithiocarbonyl group in formula (I), which may be substituted by (R).sub.a, is present in an unmodified form.

15. A block copolymer represented by formula (II) or a block copolymer in which the phenyldithiocarbonyl group in formula (II), which may be substituted by (R).sub.a, is substituted by a hydrogen atom or a mercapto group: ##STR00015## wherein, A denotes an unsubstituted or substituted C.sub.1-C.sub.12 alkoxy group, with the substituent group being a formyl group or a group represented by the formula R.sub.1R.sub.2CH— in cases where A is substituted, R.sub.1 and R.sub.2 each independently denote a C.sub.1-C.sub.4 alkoxy group or R.sub.1 and R.sub.2 together denote a —OCH.sub.2CH.sub.2O— group, a —O(CH.sub.2).sub.3O— group or a —O(CH.sub.2).sub.4O— group, or in cases where A is substituted, the substituent group is a group represented by the formula R.sub.3R.sub.4B-Ph-, where R.sub.3 and R.sub.4 each denote a hydroxy group or R.sub.3 and R.sub.4 together denote a —OC(CH.sub.3).sub.2C(CH.sub.3).sub.2O— group, and Ph denotes an o-phenylene group, m-phenylene group or p-phenylene group that may be substituted by a methyl group or methoxy group, L is m-xylylene or p-xylylene: R groups each independently denote a methyl group or methoxy group, a is an integer between 0 and 3, m is an integer between 3 and 500, n is an integer between 2 and 10,000, Z is selected from groups represented by the formulae below, which are covalently bonded via a —NH— group or —O— group: ##STR00016## or denotes a —P(═O)(OCH.sub.2CH.sub.3).sub.2 group or —P(═O)(OH).sub.2 group, with these groups accounting for at least 60% of the total number of Z groups, and the remaining Z groups, when present, being chlorine atoms, bromine atoms, iodine atoms or hydroxyl groups.

16. The block copolymer of claim 15, wherein the phenyldithiocarbonyl group in formula (I), which may be substituted by (R).sub.a, is present in an unmodified form.

17. The block copolymer of claim 15, wherein Z is selected from groups represented by the formulae below, which are covalently bonded via a —NH— group or —O— group. ##STR00017##

18. The block copolymer of claim 15, wherein Z denotes a —P(═O)(OCH.sub.2CH.sub.3).sub.2 group or a —P(═O)(OH).sub.2 group.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0032] FIG. 1 shows size exclusion chromatogram (SEC) of CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2Cl (1).

[0033] FIG. 2 shows proton nuclear magnetic resonance spectrum (.sup.1H-NMR) of CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2Cl (1).

[0034] FIG. 3 shows matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) spectrum of CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2Cl (1).

[0035] FIG. 4 shows size exclusion chromatogram (SEC) of CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2Br (2).

[0036] FIG. 5 shows proton nuclear magnetic resonance spectrum (.sup.1H-NMR) of CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2Br (2).

[0037] FIG. 6 shows matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) spectrum of CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2Br (2).

[0038] FIG. 7 shows proton nuclear magnetic resonance spectrum (.sup.1H-NMR) of (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2Cl (3).

[0039] FIG. 8 shows proton nuclear magnetic resonance spectrum (.sup.1H-NMR) of CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2SC(═S)Ph (4).

[0040] FIG. 9 shows matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) spectrum of CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2SC(═S)Ph (4).

[0041] FIG. 10 shows proton nuclear magnetic resonance spectrum (.sup.1H-NMR) of (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2SC(═S)Ph (5).

[0042] FIG. 11 shows proton nuclear magnetic resonance spectrum (.sup.1H-NMR) of CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2(CH.sub.2CH(PhCH.sub.2Cl)).sub.mSC(═S)Ph (6).

[0043] FIG. 12 shows proton nuclear magnetic resonance spectrum (.sup.1H-NMR) of CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2(CH.sub.2CH(PhCH.sub.2NH-TEMPO)).sub.mSC(═S)Ph (7) (after adding and reducing hydrazine).

[0044] FIG. 13 shows proton nuclear magnetic resonance spectrum (.sup.1H-NMR) of CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2(CH.sub.2CH(PhCH.sub.2O-TEMPO)).sub.mSC(═S)Ph (8) (after adding and reducing hydrazine).

[0045] FIG. 14 shows dynamic light scattering (DLS) spectrum of newRNP.sup.N (m=13).

[0046] FIG. 15 shows electron spin resonance spectrum of newRNP.sup.N 13).

[0047] FIG. 16 shows changes over time in concentration in blood following oral administration of newRNP.sup.N (m=30) and newRNP.sup.O (m=30).

[0048] FIG. 17 shows prothrombin time for oral administration of newRNP.sup.N (in =13).

[0049] FIG. 18 shows superoxide dismutase (SOD) production for oral administration of newRNP.sup.N (m 13).

[0050] FIG. 19 shows albumin production for oral administration of newRNP.sup.N (m=13).

[0051] FIG. 20 shows aspartate aminotransferase (AST) production for oral administration of newRNP.sup.N (m=13).

[0052] FIG. 21 shows alanine transaminase (ALT) production for oral administration of newRNP.sup.N (m=13).

[0053] FIG. 22 shows alkali phosphatase (ALP) production for oral administration of newRNP.sup.N (m 13).

[0054] FIG. 23 shows changes in blood concentration following intravenous injection of newRNP.sup.N and newRNP.sup.O.

[0055] FIG. 24 shows proton nuclear magnetic resonance spectrum (.sup.1H-NMR) of CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2(CH.sub.2CH(PhCH.sub.2P(═O)(OCH.sub.2CH.sub.3).sub.2).sub.mSC(═S)Ph (9).

[0056] FIG. 25 shows proton nuclear magnetic resonance spectrum (.sup.1H-NMR) of CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nCH.sub.2PhCH.sub.2(CH.sub.2CH(PhCH.sub.2P(═O)(OH).sub.2).sub.mSC(═S)Ph (10).

[0057] FIG. 26 shows GPC chart of block copolymer (1) obtained in Example 1.

[0058] FIG. 27 shows size exclusion chromatogram (SEC) of 4,4,5,5-tetramethyl-1,3,2-dioxaboranophenylmethoxy-(CH.sub.2CH.sub.2O).sub.nOH (Pre).

[0059] FIG. 28 shows proton nuclear magnetic resonance spectrum (.sup.1H-NMR) of 4,4,5,5-tetramethyl-1,3,2-dioxaboranophenylmethoxy-(CH.sub.2CH.sub.2O).sub.nOH (Pre).

[0060] FIG. 29 shows carbon nuclear magnetic resonance spectrum (.sup.13C-NMR) of 4,4,5,5-tetramethyl-1,3,2-dioxaboranophenylmethoxy-(CH.sub.2CH.sub.2O).sub.nOH (Pre).

WORKING EXAMPLES

[0061] The present invention will now be explained in greater detail.

Example 1: Synthesis of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.Cl (1)

[0062] (In the formula, Ph denotes a benzene ring.)

[0063] 50 g of a commercially available polyethylene glycol having a methoxy group at one terminal and a hydroxyl group at the other terminal (CH.sub.3O—(CH.sub.2CH.sub.2O).sub.nH, molecular weight 5000, manufactured by Fluka Chemical Corp.), 200 mL of tetrahydrofuran and 10 mL of a commercially available butyl lithium (1.6 M, hexane solution) were added to a 500 mL flask, 25 g of a,a′-dichloro-p-xylylene (ClCH.sub.2PhCH.sub.2Cl) was added to the flask, and a reaction was allowed to progress at 60° C. for 24 hours. The reaction mixture liquid was precipitated in 500 mL of cold 2-propanol, and the precipitate was subjected to centrifugal separation (4° C., 9000 rpm, 2 minutes) and then to vacuum drying. A size exclusion chromatogram, NMR spectrum and MALDI-TOF spectrum of the thus obtained (1) are shown in FIGS. 1, 2 and 3. The quantity obtained was 50 g, and the yield was 97%. In addition, a gel permeation chromatograph (GPC) is shown in FIG. 26.

Example 2: Synthesis of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.Br (2)

[0064] Synthesis was carried out in the same way as in Example 1, except that a,a′-dibromo-p-xylylene (BrCH.sub.2PhCH.sub.2Br) was used instead of the a,a′-dichloro-p-xylylene. A size exclusion chromatogram, NMR spectrum and MALDI-TOF spectrum of the thus obtained (2) are shown in FIGS. 4, 5 and 6. The quantity obtained was 50 g, and the yield was 97%.

Example 3: Synthesis of (CH.SUB.3.CH.SUB.2.O).SUB.2.CHCH.SUB.2.CH.SUB.2.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.Cl (3)

[0065] 100 mL of THF, commercially available 1,1-diethoxypropanol (0.9 g, 6 mmol) and potassium naphthalene (12 mL, 0.5 M) were added to a 500 mL flask to produce potassium 3,3-diethoxypropanoxide, after which 20 g (0.45 mol) of ethylene oxide was added and polymerization was allowed to progress at room temperature for 2 days. Following the polymerization, 25 g of a,a′-dichloro-p-xylylene (ClCH.sub.2PhCH.sub.2Cl) was added, and a reaction was allowed to progress at 60° C. for 24 hours. The reaction mixture liquid was precipitated in 500 mL of cold 2-propanol, and the precipitate was subjected to centrifugal separation (4° C., 9000 rpm, 2 minutes) and then to vacuum drying. A NMR spectrum of the thus obtained (3) is shown in FIG. 7. The quantity obtained was 20 g, and the yield was 95%.

Example 4: Synthesis of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.SC(═S)Ph (4)

[0066] 50 mL of THF, 5 mL of commercially available carbon disulfide and 10 mL of bromophenyl magnesium (3M, THF solution) were added to a 100 mL flask to produce dithiobenzoate magnesium bromide (PhC(═S)SMgBr). 50 g of (1) synthesized in Example 1 and 200 mL of THF were added to a separate 500 mL flask, the obtained THF solution of dithiobenzoate magnesium bromide was added to the flask, and a reaction was allowed to progress at 40° C. for 24 hours. The reaction mixture liquid was precipitated in 500 mL of cold 2-propanol, and the precipitate was subjected to centrifugal separation (4° C., 9000 rpm, 2 minutes) and then to vacuum drying. NMR and MALDI-TOF mass spectra of the thus obtained (4) are shown in FIGS. 8 and 9. The quantity obtained was 50 g, and the yield was 97%.

Example 5: Synthesis of (CH.SUB.3.CH.SUB.2.O).SUB.2.CHCH.SUB.2.CH.SUB.2.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.SC(═S)Ph (5)

[0067] Synthesis of (5) was carried out in the same way as in Example 4, except that 20 g of (3) was used instead of (1). A NMR spectrum of the thus obtained (5) is shown in FIG. 10. The quantity obtained was 20 g, and the yield was 97%.

Example 6: Synthesis of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.(CH.SUB.2.CH(PhCH.SUB.2.Cl)).SUB.m.SC(═S)Ph (6)

[0068] 20 g of (4), 120 mg of azobisisobutyronitrile (AIBN), 60 mL of m,p-chloromethylstyrene (CMS) and 200 mL of toluene were added to a 500 mL flask, nitrogen gas was flushed for 3 minutes, and polymerization was then allowed to progress at 70° C. for 12 hours. The reaction mixture liquid was precipitated in 500 mL of cold 2-propanol, and the precipitate was subjected to centrifugal separation (4° C., 9000 rpm, 2 minutes) and then to vacuum drying. A NMR spectrum of the thus obtained (6) is shown in FIG. 11. The quantity obtained was 40 g. The degree of polymerization (m) of PCMS segments was 30.

Example 7: Synthesis (2) of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.(CH.SUB.2.CH(PhCH.SUB.2.Cl)).SUB.m.SC(═S)Ph (6)

[0069] Synthesis was carried out in the same way as in Example 6, except that 30 mL of CMS was used. The quantity obtained was 32 g. The degree of polymerization (m) of PCMS segments was 13.

Example 8: Synthesis (3) of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.(CH.SUB.2.CH(PhCH.SUB.2.Cl)).SUB.m.SC(═S)Ph (6)

[0070] Synthesis was carried out in the same way as in Example 6, except that 120 mL of CMS was used. The quantity obtained was 50 g. The degree of polymerization (m) of PCMS segments was 42.

Example 9: Synthesis of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.(CH.SUB.2.CH(PhCH.SUB.2.NH-TEMPO)).SUB.m.SC(═S)Ph (7)

[0071] (TEMPO is 2,2,6,6-tetramethylpiperidine-1-oxyl.)

[0072] 10 g of (6) synthesized in Example 6, 100 mL of dimethylformamide (DMF) and 20 g of 4-amino-TEMPO were added to a 500 mL flask, and a reaction was allowed to progress at room temperature for 2 days. The reaction mixture liquid was precipitated in 500 mL of cold 2-propanol, and the precipitate was subjected to centrifugal separation (4° C., 9000 rpm, 2 minutes) and then to vacuum drying. A NMR spectrum of the thus obtained (7) is shown in FIG. 12. The quantity obtained was 10 g. The degree of TEMPO substitution was 80%.

Example 10: Synthesis (2) of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.(CH.SUB.2.CH(PhCH.SUB.2.NH-TEMPO)).SUB.m.SC(═S)Ph (7)

[0073] Synthesis was carried out in the same way as in Example 9, except that (6) synthesized in Example 7 was used instead of (6) synthesized in Example 6. The quantity obtained was 10 g. The degree of TEMPO substitution was 80%.

Example 11: Synthesis (3) of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.(CH.SUB.2.CH(PhCH.SUB.2.NH-TEMPO)).SUB.m.SC(═S)Ph (7)

[0074] Synthesis was carried out in the same way as in Example 9, except that (6) synthesized in Example 8 was used instead of (6) synthesized in Example 6. The quantity obtained was 10 g. The degree of TEMPO substitution was 80%.

Example 12: Synthesis of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.(CH.SUB.2.CH(PhCH.SUB.2.O-TEMPO)).SUB.m.SC(═S)Ph (8)

[0075] LiO-TEMPO was produced by adding 50 mL of THF, 10 g of 4-hydroxy-TEMPO and 40 mL of butyl lithium to a 200 mL flask and stirring. 10 g of (6) synthesized in Example 6, 100 mL of dimethylformamide (DMF) and the obtained LiO-TEMPO solution were added to a 500 mL flask, and a reaction was allowed to progress at room temperature for 2 days. The reaction mixture liquid was precipitated in 500 mL of cold 2-propanol, and the precipitate was subjected to centrifugal separation (4° C., 9000 rpm, 2 minutes) and then to vacuum drying. A NMR spectrum of the thus obtained (8) is shown in FIG. 13. The quantity obtained was 10 g. The degree of TEMPO substitution was 80%.

Example 13: Synthesis (2) of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.(CH.SUB.2.CH(PhCH.SUB.2.O-TEMPO)).SUB.m.SC(═S)Ph (8)

[0076] Synthesis was carried out in the same way as in Example 11, except that (6) synthesized in Example 7 was used instead of (6) synthesized in Example 6. The quantity obtained was 10 g. The degree of TEMPO substitution was 80%.

Example 14: Synthesis (3) of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.(CH.SUB.2.CH(PhCH.SUB.2.O-TEMPO)).SUB.m.SC(═S)Ph (8)

[0077] Synthesis was carried out in exactly the same way as in Example 11, except that (6) synthesized in Example 11 was used instead of (6) synthesized in Example 6. The quantity obtained was 10 g. The degree of TEMPO substitution was 80%.

Example 15: Preparation (1) of Redox Nanoparticles newRNP.SUP.N .that Disintegrate when the pH Decreases

[0078] 10 g of (7) synthesized in Example 9 was placed in a glass container and dissolved in 400 mL of methanol, and the obtained solution was dialyzed (at 25 mg/mL) with 10 L of water using a hollow fiber type dialysis module having a molecular cutoff of 3000 (mPES Midikros (registered trademark), Modules 3 kD IC 0.5 mm D06-E003-05-N). A light scattering spectrum and electron spin resonance spectrum of the obtained particle solution are shown in FIGS. 14 and 15.

Example 16: Preparation (2) of Redox Nanoparticles newRNP.SUP.N .that Disintegrate when the pH Decreases

[0079] Preparation was carried out in the same way as in Example 15, except that (7) synthesized in Example 10 was used instead of (7) synthesized in Example 9. (25 mg/mL)

Example 17: Preparation (3) of Redox Nanoparticles newRNP.SUP.N .that Disintegrate when the pH Decreases

[0080] Preparation was carried out in the same way as in Example 15, except that (7) synthesized in Example 11 was used instead of (7) synthesized in Example 9. (25 mg/mL)

Example 18: Preparation (1) of Redox Nanoparticles newRNP.SUP.O .that do not Disintegrate when the pH Decreases

[0081] Preparation was carried out in the same way as in Example 15, except that (8) synthesized in Example 12 was used instead of (7) synthesized in Example 9. (25 mg/mL)

Example 19: Preparation (2) of Redox Nanoparticles newRNP.SUP.O .that do not Disintegrate when the pH Decreases

[0082] Preparation was carried out in the same way as in Example 15, except that (8) synthesized in Example 13 was used instead of (7) synthesized in Example 9. (25 mg/mL)

Example 20: Preparation (3) of Redox Nanoparticles newRNP.SUP.O .that do not Disintegrate when the pH Decreases

[0083] Preparation was carried out in the same way as in Example 15, except that (8) synthesized in Example 14 was used instead of (7) synthesized in Example 9. (25 mg/mL)

Example 21: Preparation of Blank Micelles

[0084] 1 g of the PEG-b-PCMS synthesized in Example 6 (m=30) was dissolved in 160 mL of methanol, and the thus obtained solution was dialyzed (at 6.25 mg/mL) with 10 L of water using a hollow fiber type dialysis module having a molecular cutoff of 3000 (mPES Midikros (registered trademark), Modules 3 kD IC 0.5 mm D06-E003-05-N).

Example 22: Blood Uptake Evaluation and Safety for Oral Administration of newRNP.SUP.N

[0085] newRNP.sup.N prepared in Example 9 (=30) was evaluated through oral administration.

[0086] newRNP.sup.N was administered in the manner described below to 10 week old IGS mice (5 mice per group) weighing 38 g to 41 g (during the period from arrival to completion of the test, the mice were reared at room temperature (23° C. (±1° C.)) and a humidity of 50% using a 12 hours light-12 hours dark cycle, and the mice ingested food and water freely).

[0087] Group 1: Free administration of water

[0088] Group 2: Aqueous solution of newRNP.sup.N forcibly administered intragastrically once per day using a feeding tube (day 1: 1 mL at 10 mg/mL, day 2 onwards: 1 mL at 20 mg/mL).

[0089] Group 3: 5 mg/mL aqueous solution of newRNP.sup.N freely ingested instead of water via a water feeding bottle.

[0090] Group 4: 10 mg/mL aqueous solution of newRNP.sup.N freely ingested instead of water via a water feeding bottle.

[0091] Group 5: 20 mg/mL aqueous solution of newRNP.sup.N freely ingested instead of water via a water feeding bottle.

[0092] Group 6: 20 mg/mL aqueous solution of newRNP.sup.O freely ingested instead of water via a water feeding bottle.

[0093] The quantities of water ingested by Group 1 and Groups 3 to 6 did not vary, as shown in Table 1.

TABLE-US-00001 TABLE 1 Comparison of quantities of water ingested day Group 1 2 3 4 5 6 7 1 65 40 40 45 40 40 50 3 72.5 40 40 45 45 35 60 4 65 40 40 40 40 40 55 5 85 40 40 50 50 50 55 6 80 40 40 40 45 45 55 Note 1: Quantity of water ingested for cage of 5 mice (mL/d/cage)

[0094] When uptake into the blood was investigated by means of electron spin resonance spectra, it was confirmed that newRNP.sup.O was not taken up into the blood at all by administration group 6. However, the in-blood concentration increased up to day 4 and reached a constant level in group 3, in which forcible administration was carried out using a feeding tube. In the free ingestion groups, a tendency was seen for the in-blood concentration to increase in a concentration-dependent manner, and in the 10 mg/mL or higher groups, the same level as the forcible administration group was reached after 6 days (see FIG. 16).

Example 23: Effect on Acetaminophen-Induced Hepatotoxicity

[0095] newRNP.sup.N prepared in Example 16 (m=13) was evaluated through oral administration.

[0096] newRNP.sup.N was administered in the manner described below to 10 week old IGS mice (6 mice per group) weighing 38 g to g (during the period from arrival to completion of the test, the mice were reared at room temperature (23° C. (±1° C.)) and a humidity of 50% using a 112 hours light-112 hours dark cycle, and the mice ingested food and water freely).

[0097] Group 1: Untreated control (4 mice in this group only)

[0098] Group 2: 3 mg/kg of acetaminophen administered orally on the 7th day from the start of the test

[0099] Group 3: 1 mL of the blank micelles (6.25 mg/mL, 160 mg/kg) synthesized in Example 21 forcibly administered once per day via a feeding tube, 3 m g/kg of acetaminophen administered orally on the 7th day from the start of the test.

[0100] Group 4: 1 mL of newRNP.sup.N (6.25 mg/mL, 160 mg/kg) synthesized in Example 16 (m=13) forcibly administered once per day via a feeding tube, 3 mg/kg of acetaminophen administered orally on the 7th day from the start of the test.

[0101] Group 5: 170 mg/kg of 4-amino-TEMPO forcibly administered once per day via a feeding tube, 3 mg/kg of acetaminophen administered orally on the 7th day from the start of the test.

[0102] Group 6: 600 mg/kg of acetylcysteine forcibly administered once per day via a feeding tube, 3 mg/kg of acetaminophen administered orally on the 7th day from the start of the test.

[0103] The numbers of mice alive before and after administration of acetaminophen under these test conditions are shown in Table 2. When 3 mg/kg of acetaminophen was administered to IGS mice, the hepatopathic effect was too high, and two thirds of the mice died within 1 day (group 2). A similar tendency was seen with blank micelles (group 3), 4-amino-TEMPO (group 5) and acetylcysteine (group 6). With newRNP.sup.N, however, all the mice survived (group 4).

TABLE-US-00002 TABLE 2 Numbers of mice alive before and after administration of acetaminophen Group Before test After test 1 4 4 2 6 2 3 6 3 4 6 6 5 6 2 6 6 4

[0104] Prothrombin time, which is an indicator of hepatic function, was found to be extended for 4-amino-TEMPO and acetylcysteine, but was the same level as the control group for the newRNP.sup.N group (see FIG. 17). When superoxide dismutase (SOD) production was quantitatively determined, a significant decrease was observed in the acetaminophen administration group and other drug administration groups, but was the same level as the control group for the newRNP.sup.N group (see FIG. 18).

[0105] When the albumin quantity, which is an indicator of hepatic function, and AST, ALT and ALP enzyme levels were measured, low molecular weight 4-amino-TEMPO exhibited extremely high toxicity, but newRNP.sup.N exhibited extremely low hepatotoxicity (see FIGS. 19 to 22).

[0106] Therefore, the newRNP.sup.N of the present invention did not exhibit hepatotoxicity and exhibited suppression of acetaminophen-induced hepatopathy.

Example 24: Retention of newRNP.SUP.N .and newRNP.SUP.O .in Blood

[0107] newRNP.sup.N and newRNP.sup.O produced in Examples 15 to 20 were administered intravenously, and evaluated in terms of retention in blood. Retention of newRNP.sup.N and newRNP.sup.O in blood was evaluated in the manner described below in 10 week old IGS mice (5 mice per group) weighing 38 g to 41 g (during the period from arrival to completion of the test, the mice were reared at room temperature (23° C. (±1° C.)) and a humidity of 50% using a 12 hours light-12 hours dark cycle, and the mice ingested food and water freely).

[0108] Group 1: 4-amino-TEMPO (200 μL was administered intravenously at 10 mg/mL into the tail, 50 mg/kg)

[0109] Group 2: newRNP.sup.N (200 μL of a sample obtained in Example 15 (m=30) was administered intravenously at 25 mg/mL into the tail, 125 mg/kg)

[0110] Group 3: newRNP.sup.N (200 μL of a sample obtained in Example 16 (m=13) was administered intravenously at 25 mg/mL into the tail, 125 mg/kg)

[0111] Group 4: newRNP.sup.N (a sample obtained in Example 17 (m=42) was concentrated to 50 mg/mL, and 200 μL was then administered intravenously into the tail, 250 mg/kg)

[0112] Group 5: newRNP.sup.O (a sample obtained in Example 18 (m=30) was concentrated to 50 mg/mL, and 250 μL was then administered intravenously into the tail, 312.5 mg/kg)

[0113] Group 6: newRNP.sup.O (a sample obtained in Example 19 (m=13) was concentrated to 50 mg/mL, and 200 μL was then administered intravenously into the tail, 250 m g/kg)

[0114] Group 7: newRNP.sup.O (a sample obtained in Example 20 (m 42) was concentrated to 50 mg/mL, and 200 μL was then administered intravenously into the tail, 250 mg/kg)

[0115] Blood was sampled 5 minutes, 15 minutes, 30 minutes, 1 hour, 4 hours, 12 hours, 24 hours, 48 hours, 72 hours and 96 hours after administration and subjected to centrifugal separation, after which potassium ferricyanide was added to the supernatant liquid, and quantitative determination was carried out using an X band electron spin resonance apparatus.

[0116] FIG. 23 shows changes in the concentration of RNP in blood. For ease of understanding, data from Test Example 4 in WO 2009/133647 has been added as comparative data to FIG. 23. As shown in the diagram, newRNP produced in the present invention does not contain an ABA type block copolymer, unlike conventional RNPs, meaning that retention in blood was significantly improved. In particular, it was confirmed that newRNP.sup.O was retained for 4 days or longer following intravenous administration.

Example 25: Synthesis of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.(CH.SUB.2.CH(PhCH.SUB.2.P(═O)(OCH.SUB.2.CH.SUB.3.).SUB.2.)).SUB.m.SC(═S)Ph (9)

[0117] LiP(═O)(OCH.sub.2CH.sub.3).sub.2 was prepared by adding 20 mL of THF, 1.3 g of diethyl phosphite and 6.5 mL (10 mmol) of butyl lithium to a 200 mL flask and stirring. 3 g of (6) synthesized in Example 6, 50 mL of dimethylformamide (DMF) and the thus prepared LiP(═O)(OCH.sub.2CH.sub.3).sub.2 solution were added to a 500 mL flask, and a reaction was allowed to progress at room temperature for 2 days. The reaction mixture liquid was precipitated in 500 mL of cold 2-propanol, and the precipitate was subjected to centrifugal separation (4° C., 9000 rpm, 2 minutes) and then to vacuum drying. A NMR spectrum of the thus obtained (9) is shown in FIG. 24. The quantity obtained was 3 g. The degree of diethyl phosphate substitution was 80%.

Example 26: Synthesis of CH.SUB.3.O—(CH.SUB.2.CH.SUB.2.O).SUB.n.CH.SUB.2.PhCH.SUB.2.(CH.SUB.2.CH(PhCH.SUB.2.P(═O)(OH).SUB.2.)).SUB.m.SC(═S)Ph (10)

[0118] 20 mL of CHCl.sub.3, 2 g of (9) and 2 m of trimethylsilyl bromide were added to a 100 mL flask, a reaction was allowed to progress at 45° C. for 2 hours, after which the chloroform was distilled off, 80 mL of methanol was added, and a reaction was allowed to progress at room temperature for 15 hours. The solution was dialyzed with water and then vacuum dried. A NMR spectrum of the thus obtained (10) is shown in FIG. 25. The quantity obtained was 1.5 g. The degree of diethyl phosphate hydrolysis was 90%.

Example 27: Synthesis of 4,4,5,5-tetramethyl-1,3,2-dioxaboranophenylmethoxy-(CH.SUB.2.CH.SUB.2.O).SUB.n.OH (Pre)

[0119] 50 mL of THF, 1.67 g (7.1 mmol) of commercially available 2-(4-hydroxymethylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane and potassium naphthalene (8.0 mL, 0.9 M) were added to a 100 mL flask to produce a potassium alcoholate of 2-(4-hydroxymethylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane, after which 31 g (0.7 mol) of ethylene oxide was added, and polymerization was carried out at room temperature for 2 days. Following the polymerization, the reaction mixture liquid was precipitated in 700 mL of cold 2-propanol, and the precipitate was subjected to centrifugal separation (4° C., 9000 rpm, 2 minutes) and then to vacuum drying. SEC, .sup.1H-NMR and .sup.13C-NMR spectra of the obtained (Pre) are shown in FIGS. 27, 28 and 29. The quantity obtained was 28 g, and the yield was 90%.