Triblock copolymer and use therefor

Abstract

Provided is a triblock copolymer represented by General Formula (I):
CNR-PEG-CNR  (1)
or a polycation thereof, wherein each CNR is independently a polymer segment having a repeating unit containing as part of a pendant group a cyclic nitroxide radical that binds to the polymer main chain via a linking group having at least one imino group or ether bond, and PEG is a segment containing poly(ethylene glycol).

Claims

1. A composition comprising: a polyion complex comprising a polyanion selected from the group consisting of poly(acrylic acid), poly(methacrylic acid), poly(sulfonic acid), DNA, RNA and anionic proteins, and a triblock copolymer or salt thereof of following formula: ##STR00005## wherein each L.sub.1 is independently selected from the group consisting of a single bond, —S—(CH.sub.2).sub.c—, —S—(CH.sub.2).sub.cCO—, —(CH.sub.2).sub.cS— and —CO(CH.sub.2).sub.cS—, in which c is an integer from 1 to 5; each L.sub.2 is independently —C.sub.1-6 alkylene-NH—(C.sub.1-6 alkylene).sub.q-, in which q is the integer 0 or 1; and at least 50% of the total number n of R in the formula independently represent residues of cyclic nitroxide radical compounds selected from the group consisting of 2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl, 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-yl, 2,2,5,5-tetramethylpyrroline-1-oxyl-3-yl, 2,4,4-trimethyl-1,3-oxyazolidine-3-oxyl-2-yl, 2,4,4-trimethyl-1,3-thiazolidine-3-oxyl-2-yl and 2,4,4-trimethyl-imidozolidine-3-oxyl-2-yl, with other R if any representing hydrogen atoms, halogen atoms or hydroxy groups, and m is an integer from 20 to 5,000 while each n is independently an integer from 3 to 1,000, wherein the polyion complex is in the form of a polyion complex micelle in Na.sub.2HPO.sub.4 buffer (0.1M) at pH 6.28, wherein said composition forms a gel when injected into a living body and said gel is retained in a specific area of the body selected from the group consisting of a periodontal pocket, a cancer lesion and a site affected by arthritis; and wherein the composition further provides at least one property selected from the group consisting of suppressing inflammation, scavenging active oxygen, and regulating fluidity.

2. A method for suppressing inflammation, comprising administering the composition according to claim 1 to a patient requiring elimination of active oxygen.

3. The composition according to claim 1, further comprising a physiologically acceptable diluent or excipient.

4. The composition according to claim 1, wherein the polyanion is selected from the group consisting of poly(acrylic acid), poly(methacrylic acid), and poly(sulfonic acid).

5. The composition according to claim 1, wherein in the triblock copolymer m is an integer from 20 to 1,000.

6. The composition according to claim 1, wherein in the triblock copolymer m is an integer from 50 to 200.

7. The composition according to claim 1, wherein in the triblock copolymer each n is independently an integer from 3 to 100.

8. The composition according to claim 1, wherein in the triblock copolymer each n is independently an integer from 3 to 50.

9. The composition according to claim 1, wherein in the triblock copolymer R is selected from the groups represented by the following formulae: ##STR00006## in which R′ is a methyl group, and groups represented by these formulae constitute at least 80% of the total number n of R.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a conceptual diagram showing the formation of a triblock copolymer flower micelle of the invention, and the formation of an injectable gel in a local area of the body in a suitable environment.

(2) FIG. 2 shows the results of size exclusion chromatography (SEC) measurement and NMR spectrum measurement of a PCMS-b-PEG-b-PCMS triblock copolymer obtained in Manufacturing Example 1.

(3) FIG. 3 shows SEC measurement and NMR spectrum measurement results for a PMNT-b-PEG-b-PMNT triblock copolymer obtained in Manufacturing Example 2.

(4) FIG. 4 shows purification results for the same PMNT-b-PEG-b-PMNT triblock copolymer.

(5) FIG. 5 shows the results of dynamic light scattering analysis of polyion complex micelles with different molar ratios obtained in Manufacturing Example 3, plotted against changes in the pH values of the polyion complex micelles.

(6) FIG. 6 is a photograph in place of a drawing illustrating the gelling state of concentrated polyion complex micelles (r=1:1, 60 mg/ml).

(7) FIG. 7 is a photograph in place of a drawing illustrating in vivo gelling of polyion complex micelles (r=1:1).

(8) FIG. 8 is a graph showing body weight changes in mice after injection of a polyion complex.

(9) FIG. 9 is an imaging photograph in place of a drawing illustrating local retention of a polyion complex gel (RIG).

(10) FIG. 10 is a graph of (quantified) local retention of a polyion complex gel (RIG).

(11) FIG. 11 is a graph of the protective effects of a polyion complex gel (RIG) in a carrageenan-induced inflammation model.

EXAMPLES

(12) The invention is explained in more detail below with specific examples, but the intent is not to limit the present invention to these specific examples.

Synthesis of Polychloromethylstyrene-b-Polyethylene Glycol-b-Polychloromethylstyrene (PCMS-b-PEG-b-PCMS) Triblock Copolymer

(13) PCMS-b-PEG-b-PCMS was synthesized according to the following synthesis scheme 1:

(14) ##STR00004##

(15) Poly(ethylene glycol) having thiol groups at both ends (HS-PEG-SH) (Mn: 10,000; 0.115 mmol, 1.15 g) was added to a reaction container. Next, the reaction container was vacuumized, and the operation of blowing in nitrogen gas was repeated three times to produce a nitrogen atmosphere in the reaction container. A 1.89 mg/mL azobisisobutyronitrile/toluene (0.115 mmol/10 ml) solution and chloromethylstyrene (8.63 mmol, 1.22 mL) were added to the reaction container, heated to 60° C., and agitated for 24 hours. The reaction mixture was washed three times with diethyl ether, which is a good solvent for poly(chloromethylstyrene) homopolymer, and then freeze-dried with benzene to obtain a white power. 1.45 mg was obtained for a yield of 91.6%. FIG. 2 shows the results of size exclusion chromatography (SEC) measurement and NMR spectrum analysis of the resulting PCMS-b-PEG-b-PCMS triblock copolymer.

Synthesis of Triblock Copolymer Having 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)

(16) (1) Triblock copolymer of Formula (II) with —CH.sub.2—NH— as L (PMNT-b-PEG-b-PMNT)

(17) PCMS-b-PEG-b-PCMS (Mn: 13,800; 1.45 g, 0.105 mmol) was added to a reaction container. 4-amino-TEMPO (2.34 g, 13.69 mmol) was then dissolved in 12 mL of dimethylsulfoxide (DMSO), added to the reaction container, and agitated for 24 hours at room temperature. After completion of the reaction, the reaction solution was added to a dialysis membrane (Spectra/Por, molecular weight cutoff size 3,500, Spectrum Medical Industries Inc., Houston, Tex.), and dialysis was performed with 2 L of methanol. The methanol was exchanged 8 times every 2 hours, and the mixture was evaporated to and freeze-dried with benzene. The yield was 92.1%.

(18) The results of .sup.1H NMR measurement confirmed 100% reaction of the chloromethyl groups and introduction of TEMPO (FIG. 3). The SEC results also showed that there were no side-reactions, and the chromatogram was similar to that of PCMS-b-PEG-b-PCMS. When this was sampled with SEC and the electron spin resonance (ESR) signals of each fraction were measured, the ESR signal for unreacted amino-TEMPO had disappeared, indicating that the unreacted amino-TEMPO had been completely removed by purification (FIG. 4).

(19) (2) Triblock copolymer of Formula (II) with —CH.sub.2—O— as L.sub.2

(20) The target triblock copolymer was manufactured by repeating operations similar to those described under (1) above except that 4-hydroxy-TEMPO was used instead of 4-amino-TEMPO, and dimethylformamide (DMF) dissolved in NaH was used for the reaction solution.

Design of Polyion Complex Micelle

(21) PMNT-b-PEG-b-PMNT triblock polymer powder was dissolved in a 0.1 M HCl aqueous solution, the amino groups of the PMNT chains were completely protonated, and the polymer was collected by water-based freeze-drying. Next, the PMNT-b-PEG-b-PMNT triblock polymer and polyacrylic acid (PAA; Mn: 5,000) were each dissolved in Na.sub.2HPO.sub.4 buffer (0.1 M, pH 6.28), to prepare an anionic PAA aqueous solution and a cationic PMNT-b-PEG-b-PMNT aqueous solution with a concentration of 5 mg/ml. The PMNT-b-PEG-b-PMNT triblock polymer aqueous solution was dropped with agitation into the PAA aqueous solution, to prepare a polyion complex micelle. Four different polyion complex micelles were prepared with molar ratios r of PMNT-b-PEG-b-PMNT to PAA of 4:1, 2:1, 1:1 and 1:2 (molar ratio r=[moles of activated carboxyl groups of PAA]/[moles of activated amino groups of PMNT-b-PEG-b-PMNT]). The zeta potentials of the resulting polyion complex micelles were measured (see Table 1 below). Furthermore, polyion complex micelle solutions of each molar ratio were divided into 6 groups, and the pH values of each were varied from 4 to 8 with 0.01 M HCl/NaOH. When the average particle diameter of the resulting polyion complex micelles was measured by dynamic light scattering (DLS), they were found to be monomodal particles with an average diameter of 46 to 80 nm (see FIG. 5).

(22) TABLE-US-00001 TABLE 1 Polyion complex micelle zeta potential measurement Molar Ratio r Zeta potential (mV) PAA −4.33 PMNT-PEG-PMNT +3.76 4:1 −3.73 2:1 −2.24 1:1 −0.119 1:2 +1.43

Design of Injectable Gel

(23) The polyion complex micelle solutions (5 mg/ml) prepared above with various molar ratios and pH values (molar ratio r=4:1, 2:1, 1:1; pH 4 to 8) were concentrated by centrifugal evaporation, the ion strength was adjusted to 150 mM, and gelling was investigated by the test tube inversion method in a water bath at 37° C. An investigation of optimal pH and concentration conditions relative to the molar ratio of polyion complex micelles irreversibly gelled under conditions of ion strength 150 mM, temperature 37° C. revealed that gelling occurred under conditions of 40 mg/ml, pH 5 when the molar ratio r was 4:1, 50 mg/ml, pH 4 to 5 when the molar ratio r was 2:1, and 60 mg/ml, pH 6 to 6 when the molar ratio r was 1:1 (see Table 2 below and FIG. 6).

(24) TABLE-US-00002 TABLE 2 Gelling conditions for polyion complex micelles at 37° C., ion strength 150 mM Molar ratio r 4:1 2:1 1:1 Concentration 40 50 60 (mg/ml) pH 4 Gel (25° C.) Injectable gel Liquid pH 5 Injectable gel Injectable gel Injectable gel pH 6 Gel (25° C.) Liquid Injectable gel pH 7 Precipitate (25° C.) — Gel (25° C.) pH 8 Precipitate (25° C.) Precipitate Precipitate (25° C.) (25° C.)

(25) <Test 1> In Vivo Gel Formation

(26) Polyion complexes (5 mg/ml, 9 ml) with a 1:1 molar ratio r of PMNT-b-PEG-b-PMNT to PAA were prepared in accordance with Manufacturing Example 3 and divided into two groups, the pH was adjusted to 5 and 6 with a 0.1 M HCl aqueous solution, and the solutions were concentrated to 50 mg/ml by centrifugal evaporation. When 100 μl of each of the concentrated micelle solutions with the respective pH values was injected subcutaneously to the left thighs of mice, gel formation was confirmed inside the mouse bodies, and tissue adhesiveness was also observed (Table 3, FIG. 7). However, gelling was not confirmed when the molar ratio was 2:1 (pH 4, 5, 6).

(27) TABLE-US-00003 TABLE 3 In vivo gelling results for polyion complex micelles Molar ratio r 2:1 1:1 Concentration 50 60 (mg/ml) pH 4 5 6 5 6 In vivo gelling Δ Δ Δ ◯ ◯

(28) <Test 2> Injectable Gel Toxicity Test

(29) To evaluate the in vivo toxicity of the gel, a 60 mg/ml polyion complex was prepared with a molar ratio r of 1:1 and a pH of 6, 100 μl and 50 μl were injected subcutaneously into the left thighs and right hind paws, respectively, of 10 mice, and changes in the body weight of the mice were recorded over the course of 4 weeks. The results showed that the body weights of the mice rose gradually, and the survival rate was 100% (see FIG. 8). These results show that the toxicity of an injectable gel developed by the present invention is extremely low.

(30) <Test 3> Local Retention of Injectable Gel (Imaging by L-Band Electron Spin Resonance)

(31) Three mice were used in each group. 70 μl of a solution of the nitroxide radical-containing polyion complex micelles used in Test 1 (PMNT-b-PEG-b-PMNT to PAA molar ratio r=1:1 (RIG). pH 6, 60 mg/ml) was injected subcutaneously into the left hind paws of three mice. For the control groups, 70 μl each of a low-molecular-weight nitroxide radical compound (4-aminoTEMPO; 5.45 mg/ml), a micelle solution of a nitroxide radical-containing polymer (polymer micelles formed by dialysis from a PEG-b-PMNT amphiphilic block copolymer) (RNP.sup.N; 60 mg/ml, and a solution mixed with low-molecular-weight 4-aminoTEMPO physically encapsulated in polyion complex micelles without nitroxide radicals (formed by mixing 4-aminoTEMPO with TEMPO-free flower micelles (nRIG) formed using a polymer with amino ethanol introduced instead of TEMPO, and gelling as is to encapsulate the 4-aminoTEMPO inside the gel) (nRIG+4-aminoTEMPO; molar ratio r=1:1, pH 6, 60 mg/ml) was injected into the right hind paws of three mice. Local retention of nitroxide radicals in the mouse paws was imaged by L-band electron spin resonance (ESR) using mice immediately after administration. In the case of the low-molecular-weight 4-aminoTEMPO solution, the ESR signal disappeared from the mouse paws within 30 minutes after administration. Although retention was slightly improved with the RNP.sup.N solution containing micelles of 4-aminoTEMPO, the signal disappeared after an hour. Similarly, retention was only observed for up to an hour due to dispersion with the nRIG-containing gel mixed with 4-aminoTEMPO. By contrast, when RIG was gelled in the soles of mice injected with the polyion complex micelle solution of the present invention, a strong ESR signal was detected through a 5-hour period during measurement. Thus, a gel formed from the polyion complex micelles of the present invention is confirmed to have excellent local retention. Photographs are included in place of drawings illustrating the imaging (see FIG. 9).

(32) Thus, it has been confirmed that evaluation methods using non-invasive imaging are ordinarily suited to evaluating residual drug quantities and for evaluating the degree of oxidative stress.

(33) <Test 4> Local Retention of Injectable Gel (Quantified by X-Band Electron Spin Resonance)

(34) Three mice (n=3) were used in each of the four groups (4-aminoTEMPO, RNP.sup.N, RIG, nRIG+4-aminoTEMPO). 50 μl each of the low-molecular-weight nitroxide radical compound 4-aminoTEMPO (5.45 mg/ml), a solution of nitroxide radical-containing polymer micelles (RNP.sup.N; 60 mg/ml), a solution of nitroxide radical-containing polyion complex micelles (RIG: molar ratio r=1:1, pH 6, 60 mg/ml), and a solution of low-molecular-weight 4-aminoTEMPO physically mixed with polyion complex micelles without nitroxide radicals (nRIG+4-aminoTEMPO: molar ratio r=1:1, pH 6, 60 mg/ml) was injected subcutaneously into the right hind paws of the mice. The mice were dissected at different intervals (0 h, 1 h, 3 h, 12 h, 24 h, 72 h) after administration, and a homogenized solution of the tissue of the collected right hind paws with potassium ferricyanide (200 mN) added as an oxidizing agent was subjected to X-band electron spin resonance to quantify local retention at each time interval. The result is shown in FIG. 10.

(35) With the low-molecular-weight compound (4-aminoTEMPO) and the mixed nRIG gel with 4-aminoTEMPO (nRIG+4-aminoTEMPO), the gel was eliminated by dispersion from the administration site within 1 hour. In the case of the polymer micelles consisting of a TEMPO-containing diblock polymer (RNP), the gel was eliminated from the administration site within 12 hours, while with RIG 40% of the gel remained even after 72 hours. These results confirm that RIG exhibits excellent local retention properties.

(36) <Test 5> Protective Effect of Injectable Gel in Carrageenan-Induced Arthritis Model

(37) Five mice (n=5) were used in each of 9 groups (Control, RIG, nRIG, Saline@Carr, RIG@Carr, nRIG@Carr, 4-amino-TEMPO@Carr, RNP.sup.N@Carr, nRIG+4-amino-TEMPO@Carr).

(38) The tested groups are defined as follows.

(39) Control: discussed below

(40) RIG: TEMPO-containing gel formed in vivo after administration of TEMPO-containing flower micelles

(41) nRIG: Gel without TEMPO formed in vivo after administration of flower micelles containing no TEMPO

(42) Saline@Carr: Carrageenan administered 18 hours after administration of physiological saline

(43) RIG@Carr: Carrageenan administered 18 hours after administration of TEMPO-containing flower micelles

(44) nRIG@Carr: Carrageenan administered 18 hours after administration of flower micelles containing no TEMPO

(45) 4-amino-TEMPO@Carr: Carrageenan administered 18 hours after administration of 4-amino-TEMPO

(46) RNP.sup.N@Carr: Carrageenan administered 18 hours after administration of TEMPO-containing polymer micelles

(47) nRIG+4-amino-TEMPO@Carr: Carrageenan administered 18 hours after administration of TEMPO-free flower micelles encapsulating 4-amino-TEMPO

(48) After the mice had fasted for 6 hours, the pain hypersensitivity of the hind paws of normal mice was evaluated in a heat stimulus test (51° C.) using a hot plate. The time taken until the mice began to lick, pull or shiver their hind paws on the hot plate was given as the Paw Withdrawal Latency (PWL). 50 μl each of nitroxide radical-containing polyion complex micelles for forming an RIG gel (RIG; molar ratio r=1:1, pH 6, 60 mg/ml), nitroxide radical-free polyion complex micelles for forming an nRIG gel (nRIG; molar ratio r=1:1, pH 6, 60 mg/ml), saline, a low-molecular-weight nitroxide radical compound solution (4-aminoTEMPO; 5.45 mg/ml), a nitroxide radical-containing polymer micelle solution (RNP.sup.N; 60 mg/ml), and a solution of low-molecular-weight 4-aminoTEMPO physically mixed with nitroxide radical-free polyion complex micelles (n-RIG+4-aminoTEMPO; molar ratio r=1:1, pH 6, 60 mg/ml) was then injected subcutaneously into the right hind paws of mice. 18 hours later, 50 μl of 5% carrageenan buffer was injected subcutaneously into the right hind paws of the mice in the Saline@Carr, RIG@Carr, nRIG@Carr, 4-amino-TEMPO@Carr, RNP.sup.N@Carr, and nRIG+4-aminoTEMPO@Carr groups (n=5). Nothing was administered to the Control group (n=5). 6 hours after carrageenan administration, the heat stimulus test was performed again using the hot plate. The time difference in Paw Withdrawal Latency (PWL) after subtraction of the PWL time after occurrence of inflammation from the PWL time before occurrence of inflammation is shown on the vertical axis of the to graph. The greater the time difference, the greater the degree of inflammation. The collected tissue of the mouse right hind paws was then homogenized, and the amount of inflammatory cytokines (TNF-α and IL-1β) and myeloperoxidase (MPO) activity as a marker of neutrophil infiltration were measured with an ELISA kit in the supernatant collected by centrifugation. The results are shown in FIG. 11.

(49) As shown in the figure, increased MPO activity was confirmed in the blood and locally in the paws of the group receiving only nRIG without nitroxide radicals. This activity was significantly constrained in the RIG group. These results suggest that RIG itself does not cause inflammation. In the Saline@Carr, nRIG@Carr, 4-amino-TEMPO@Carr, RNP.sup.N@Carr and nRIG+4-aminoTEMPO@Carr groups, MPO activity caused by carrageenan was not significantly suppressed, but in the RIG@Carr group MPO activity was significantly suppressed.

(50) Similarly, the RIG gel significantly suppressed the occurrence of the inflammatory cytokines TNF-α and IL-1β, effectively preventing carrageenan-induced pain hypersensitivity. These results suggest that the RIG gel effectively eliminates active oxygen produced by neutrophils and macrophages.

INDUSTRIAL APPLICABILITY

(51) The triblock copolymer of the present invention can be used not only as an active component of a composition for forming a gel when injected into a living body, but also as an in vivo active oxygen scavenger because it carries cyclic nitroxide radicals in the polymer. Thus, the present invention can be used in medical manufacturing and the like.