Embolism material for blood vessel, preparation method therefor and use thereof in preparation of drugs

Abstract

Provided are the use of poly(N-isopropylacrylamide-co-butyl methacrylate) in preparing an embolism material for blood vessels, an embolism material for blood vessels and the use thereof in preparation of drugs. The embolism material for blood vessels comprises poly(N-isopropylacrylamide-co-butyl methacrylate) and a dispersion medium consisting of an electrolyte, a contrast agent, a pH regulator and water. The concentrations of the polymer, electrolyte and contrast agent are respectively 5-30 mg/ml, 0.1-30 mg/ml and 100-350 mg/ml based on iodine. The embolism material for blood vessels is suitable for embolization therapy of tumors in hypervascular and parenchymal visceral organs.

Claims

1. A blood vessel embolic material comprising from 5 to 30 mg of poly(N-isopropylacrylamide-co-butyl methacrylate) crosslinked with N,N′-methylenebis(acrylamide), from 100 to 350 mg of an iodine-containing polyol in terms of iodine, and a dispersion medium; wherein the dispersion medium comprises from 0.1 to 30 mg of an electrolyte, and 0.1 to 10 mg of an iodine-free polyol; wherein all amounts are per mililiter of the blood vessel embolic material; and wherein the poly(N-isopropylacrylamide-co-butyl methacrylate) crosslinked with N,N′-methylenebis(acrylamide), is prepared by radical polymerization of a mixture comprising N-isopropylacrylamide, n-butyl methacrylate, and N,N′-methylenebis(acrylamide).

2. The blood vessel embolic material according to claim 1, wherein the electrolyte is at least one selected from the group consisting of sodium chloride, sodium hydroxide, calcium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, and calcium disodium edetate.

3. The blood vessel embolic material according to claim 1, wherein the poly(N-isopropylacrylamide-co-butyl methacrylate) crosslinked with N,N′-methylenebis(acrylamide) has an intrinsic viscosity of 40 to 100 ml/g.

4. The blood vessel embolic material according to claim 1, wherein the dispersion medium further comprises a contrast agent, a pH regulator, and water.

5. The blood vessel embolic material according to claim 1, wherein the poly(N-isopropylacrylamide-co-butyl methacrylate) crosslinked with N,N′-methylenebis(acrylamide) is spherical.

6. The blood vessel embolic material according to claim 1, comprising: from 10 to 20 mg of the poly(N-isopropylacrylamide-co-butyl methacrylate) crosslinked with N,N′-methylenebis(acrylamide), from 0.1 to 30 mg of the electrolyte, and from 150 to 240 mg of the iodine-containing polyol in terms of iodine, per milliliter of the blood vessel embolic material.

7. The blood vessel embolic material according to claim 4, having a pH of 6.5 to 8.0.

8. The blood vessel embolic material according to claim 4, wherein the contrast agent includes an iodine-containing polyol, wherein the iodine-containing polyol is at least one of iohexol, ioversol, iopamidol, and iobitridol.

9. The blood vessel embolic material according to claim 4, wherein the pH regulator includes hydrochloric acid.

10. The blood vessel embolic material according to claim 5, wherein the iodine-free polyol includes at least one selected from the group consisting of tromethamine, mannitol, Tween-80, poly(ethylene glycol) 200, poly(ethylene glycol) 400, and poly(ethylene glycol) 600.

11. The blood vessel embolic material according to claim 1, further comprising a chemotherapeutic agent.

12. The blood vessel embolic material according to claim 4, wherein the chemotherapeutic agent is at least one selected from the group consisting of doxorubicin hydrochloride, epirubicin hydrochloride, mitomycin C and fluorouracil.

13. A method of treating a solid tumor in a patient in need thereof, comprising injecting a composition to an artery supplying the tumor, wherein the composition comprises the blood vessel embolic material according to claim 1.

Description

DESCRIPTION OF FIGURES

(1) FIG. 1 shows a graph comparing the viscosity of the blood vessel embolic material of Example 1, the blood vessel embolic material of Example 2, a domestic lipiodol and a super-liquefied lipiodol.

(2) FIG. 2 shows the in vitro release profile of Experimental example 1.

(3) FIG. 3 shows Digital Subtraction Angiography (DSA) effect images of Experimental example 2.

(4) FIG. 4 shows the DSA contrast images at four examination time points of Experimental example 3.

(5) FIG. 5 shows the gross histopathological photographs at the four examination time points of Experimental example 3.

(6) FIG. 6 shows MR effect images before and after interventional treatment of Experimental example 4.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(7) The poly(N-isopropylacrylamide-co-butyl methacrylate) was prepared by thermally initiated radical polymerization of N-isopropylacrylamide as a monomer, N,N′-dimethylenebisacrylamide as a crosslinking agent, and butyl methacrylate as a comonomer by a soap-free emulsion polymerization method (see the Master's thesis of Huazhong University of Science and Technology, “Preparation, Characterization and Related Biological Evaluation of High Concentration Poly(N-isopropylacrylamide-co-butyl methacrylate) Nanogel Dispersion for Interventional Therapy for Liver Cancer”, LIAO Yuanxia, page 12).

Example 1

(8) Step 1: At 0 to 30° C., 10.0 g of poly(N-isopropylacrylamide-co-butyl methacrylate) having an intrinsic viscosity of 68 ml/g and 259 g of iohexol powder as a contrast agent were added to 400 ml water and stirred well, to obtain a solution containing poly(N-isopropylacrylamide-co-butyl methacrylate) and the contrast agent. To this solution, 1.25 g of calcium chloride as an electrolyte and 0.3 g of mannitol as an iodine-free polyol were added, made up to 500 ml, and mixed well.

(9) Step 2: the pH of the solution obtained in Step 1 was adjusted to 6.5 to 8.0 with hydrochloric acid as a pH regulator, so as to obtain a blood vessel embolic material containing poly(N-isopropylacrylamide-co-butyl methacrylate) at a mass/volume concentration of 20 mg/ml, calcium chloride at a mass/volume concentration of 2.5 mg/ml, mannitol at a mass/volume concentration of 0.6 mg/ml, and iohexol at a mass/volume concentration of 240 mg/ml in terms of iodine.

(10) The amount by mass and intrinsic viscosity of poly(N-isopropylacrylamide-co-butyl methacrylate), the amount by mass of the contrast agent, the amount by mass of the electrolyte, and the amount by mass of the iodine-free polyol of Examples 2 to 10 are shown in Table 1, and the rest of the preparation process was the same as that of Example 1.

Examples 2 to 10

(11) TABLE-US-00001 TABLE 1 List of mass and intrinsic viscosity of poly(N-isopropylacrylamide-co-butyl methacrylate)(abbreviated as Polymer), contrast agent mass, electrolyte mass and iodine-free polyol mass of Examples 2 to 10. Polymer Polymer intrinsic Iodine-free polyol Example mass viscosity Contrast agent mass Electrolyte mass mass 2  2.5 g  40 ml/g 102 g of iopamidol 1.25 g of calcium disodium 0.35 g of mannitol, edetate 0.30 g of tromethamine 3  7.5 g  60 ml/g 159 g of ioversol 1.25 g of calcium hydroxide 0.35 g of mannitol, 0.30 g of tromethamine 4 12.5 g  80 ml/g 259 g of iohexol 0.75 g of disodium hydrogen 0 g phosphate, 0.50 g of calcium disodium edetate 5 15.0 g 100 ml/g 357 g of iopamidol 0.75 g of sodium hydroxide, 0 g 0.50 g of sodium dihydrogen phosphate 6  5.0 g  68 ml/g 259 g of iohexol 0.50 g of sodium chloride 1.00 g of Tween-80 7 10.0 g  68 ml/g 259 g of iohexol 0.05 g of disodium hydrogen 2.50 g of mannitol phosphate 8 10.0 g  68 ml/g 259 g of iohexol 1.00 g of sodium chloride, 1.00 0.05 g of g of calcium chloride, 0.5 g of polyethylene glycol sodium dihydrogen phosphate 600 9  5.0 g  68 ml/g 259 g of iohexol 3.00 g of sodium chloride, 1.00 0.05 g of g of calcium chloride, 1.00 g polyethylene glycol of calcium disodium edetate 200 10  7.5 g  68 ml/g 259 g of iohexol 8.00 g of sodium chloride 5.00 g of 7.00 g of calcium disodium tromethamine edetate

Example 11

(12) In this example, doxorubicin hydrochloride was added to the blood vessel embolic material of Example 1. Specifically, 2 ml to ml was taken from 5 ml of the blood vessel embolic material of Example 1 with a 10 ml syringe, and injected into 50 mg of doxorubicin hydrochloride for injection (Haizheng Pfizer Pharmaceutical Co., Ltd.). The mixture was shaken for 5 minutes at room temperature on a vortexer, and then allowed to stand for half an hour. The subnatant was taken to obtain the blood vessel embolic material of Example 11.

Comparative Example 1

(13) A blood vessel embolic material containing no electrolyte or iodine-free polyol was prepared as follows.

(14) Step 1: At 0 to 30° C., 10.0 g of poly(N-isopropylacrylamide-co-butyl methacrylate) having an intrinsic viscosity of 68 ml/g and 259 g of iohexol powder as a contrast agent were added to 500 ml water and stirred well.

(15) Step 2: the pH of the solution obtained in Step 1 was adjusted to 6.5 to 8.0 with hydrochloric acid as a pH regulator, so as to obtain a blood vessel embolic material containing poly(N-isopropylacrylamide-co-butyl methacrylate) at a mass/volume concentration of 20 mg/ml and the contrast agent iohexol at a mass/volume concentration of 240 mg/ml in terms of iodine.

Comparative Example 2

(16) A blood vessel embolic material containing linear poly(N-isopropylacrylamide-co-butyl methacrylate) was prepared as follows.

(17) Step 1: 2.263 g of N-isopropylacrylamide, 0.032 g of sodium dodecyl sulfate and 0.168 ml of butyl methacrylate were dissolved in 160 ml of purified water under stirring. Under N.sub.2 protection, the temperature was raised to 70° C., and a 10 ml aqueous solution of a 9.5 mg/ml potassium persulfate was added and allowed to react for 4.5 hours. The reaction solution was purified by dialysis and lyophilized to obtain the linear polymer of poly(N-isopropylacrylamide-co-butyl methacrylate) as a solid for use.

(18) Step 2: 10.0 g of the solid for use in Step 1 and 259 g of iohexol powder as a contrast agent were weighed and added to 400 ml water and mixed well, so as to obtain a solution containing the linear poly(N-isopropylacrylamide-co-butyl methacrylate) and the contrast agent. Subsequently, to this solution, 1.25 g of calcium chloride as an electrolyte and 0.3 g of mannitol as an iodine-free polyol were added, made up to 500 ml, and mixed well.

(19) Step 3: the pH of the solution obtained in Step 2 was adjusted to 6.5 to 8.0 with hydrochloric acid as a pH regulator, so as to obtain a blood vessel embolic material containing the poly(N-isopropylacrylamide-co-butyl methacrylate) linear polymer at a mass/volume concentration of 20 mg/ml, calcium chloride at a mass/volume concentration of 2.5 mg/ml, mannitol at a mass/volume concentration of 0.6 mg/ml, and iohexol at a mass/volume concentration of 240 mg/ml in terms of iodine.

(20) Table 2 shows the test results of the performance of the blood vessel embolic materials of Examples 1 to 10 and Comparative Examples 1 and 2.

(21) TABLE-US-00002 TABLE 2 Performance indices of the blood vessel embolic materials of Examples 1 to 10 and Comparative Examples 1 and 2. In vitro Gelation Free iodine Viscosity gelation Modulus temperature Escaping Dissolution concentration No. (mPa .Math. s) time (S) (gel/sol) (° C.) ratio or not (μg/g) Example 1 <35 50~60 >100 31.5~35.5 <10% No ≤60 Example 2 <35 70~80 >100 31.5~35.5 <10% No ≤60 Example 3 <35 50~60 >100 31.5~35.5 <10% No ≤60 Example 4 <35 50~60 >100 31.5~35.5 <10% No ≤60 Example 5 <35 40~50 >100 31.5~35.5 <10% No ≤60 Example 6 <35 50~65 >100 31.5~35.5 <10% No ≤60 Example 7 <35 50~65 >100 31.5~35.5 <10% No ≤60 Example 8 <35 50~65 >100 31.5~35.5 <10% No ≤60 Example 9 <35 50~65 >100 31.5~35.5 <10% No ≤60 Example 10 <35 50~65 >100 31.5~35.5 <10% No ≤60 Comparative 16.8 >180 >100 — >80% Yes ≤60 example 1 Comparative 56.7 15~20 >100 34.1 <10% No ≤60 example 2

(22) The test results shown in Table 2 demonstrate that the blood vessel embolic material according to the present invention has a reasonable gelation time, regulatable gelation kinetics, suitable gel strength, resistance to blood flow scouring, and a performance superior to lipiodol. It can permanently embolize target vessels, has an ideal visualization performance, and avoids problems such as safety risks caused by operations such as blending with the contrast agent right before clinical use and insufficient clarity after visualization.

(23) At the same time, it can be seen from the test result of Comparative example 1 that when the blood vessel embolic material does not contain an electrolyte, its in vitro gelation time is too long, so that it is easy to dissolve, cannot well cast the ends of blood vessels, and the escaping ratio is high, allowing easy mis-occlusion.

(24) At the same time, it can be seen from the test result of Comparative example 2 that when the poly(N-isopropylacrylamide-co-butyl methacrylate) of the blood vessel embolic material is a linear polymer, it has a viscosity significantly higher than that of Example 1 and relatively poor fluidity under the same conditions. Moreover, its in vitro gelation time is too short, which means a short operation time in clinical use, which increases the difficulty in operation by clinicians. During the interventional therapy, there is not enough time for it to enter and cast the ends of blood vessels.

(25) FIG. 1 shows a comparison of shear thinning and viscosity of the blood vessel embolic material of Example 1, the blood vessel embolic material of Comparative example 2, a domestic lipiodol, and a super-liquefied lipiodol.

(26) It can be seen from the results of the viscosity test in Table 2 and the viscosity curve in FIG. 1 that both the blood vessel embolic material composition of Example 1 and the blood vessel embolic material composition formulated with a linear polymer in Comparative example 2 have a shear thinning property. However, under the same conditions, the viscosity of the blood vessel embolic material according to the present invention is significantly lower than that of the blood vessel embolic material composition formulated with the linear polymer, and is also significantly lower than that of the domestic lipiodol and is comparable to that of the super-liquefied lipiodol.

Experimental Example 1

(27) A simulation experiment for the in vitro release of the blood vessel embolic material according to Example 11 was performed using a drug eluting device which can well simulate the distribution and release of the drug released in vivo from transcatheter arterial chemoembolization (TACE).

(28) The drug eluting device includes a glass release cell, a peristaltic pump, an oil bath, a thermometer, a conical flask, a beaker, and connecting pipes. The glass release cell is a customized glass cell which is 2 cm in diameter and 2 cm in height and has its top closed with a glass stopper. Both ends of the glass release cell can each be connected with a connecting pipe. The conical flask was placed in the oil bath, and then the conical flask, the peristaltic pump, the glass release cell, and the beaker were sequentially connected through connecting pipes.

(29) The entire device was placed in a dark environment. In the experiment, the temperature of the oil bath was adjusted to 37° C., the flow rate of the peristaltic pump was adjusted to 10 rpm, then the oil bath was opened, 0.01 M PBS as a release liquid was injected into the conical flask which was then placed in the oil bath, and the temperature was monitored with the thermometer to ensure that the temperature of release liquid was 37° C. Thereafter, the glass stopper was removed from the glass release cell. 50 μL of the blood vessel embolic material loaded with doxorubicin according to Example 11 was pipetted and injected into the glass release cell, and spread over the bottom of the release cell. The glass release cell was then covered with the glass stopper. Subsequently, the peristaltic pump was started to provide power. When the liquid flowed into the release cell, timing was started. Samples were taken from the beaker at regular intervals to determine the fluorescence intensity of the samples. The cumulative release rate of doxorubicin hydrochloride was calculated according to the following formulae.
Cumulative release S.sub.n=C.sub.1×V.sub.1+C.sub.2×V.sub.2+ . . . +C.sub.n×V.sub.n
M.sub.Dox=V.sub.gel×ρ.sub.Dox
Cumulative release rate (%)=S.sub.n/M.sub.Dox×100
wherein, n means the n.sup.th hour, V.sub.n is the volume released in the n.sup.th hour, and C.sub.n is the concentration of doxorubicin hydrochloride in the release liquid in the n.sup.th hour. M.sub.Dox is the content of doxorubicin hydrochloride in the doxorubicin hydrochloride-loaded gel. V.sub.gel is the volume of the doxorubicin hydrochloride-loaded gel added in the release cell. ρ.sub.Dox is the concentration of doxorubicin hydrochloride in the doxorubicin hydrochloride-loaded gel.

(30) FIG. 2 shows the 7-day in vitro release profile of Experimental example 1. The results show that the blood vessel embolic material according to the present invention can be used in combination with a chemotherapeutic agent and has the ability to slowly release the medicament in a controlled manner.

Experimental Example 2

(31) The blood vessel embolic material of Example 1 was used for interventional embolization of the renal artery of normal rabbits.

(32) FIG. 3 shows the Digital Subtraction Angiography (DSA) effect images of the interventional embolization of the renal artery of normal rabbits using the blood vessel embolic material according to Experimental example 2.

(33) FIGS. 3-A1 to 3-A3 are angiographic DSA images before the interventional embolization of rabbit renal artery. FIGS. 3-B1 to 3-B3 are the respective contrast examination images after the interventional embolization, wherein the amount of blood vessel embolic material used in 3-B1 was 0.5 ml, the amount of blood vessel embolic material used in 3-B2 was 1.0 ml, and the amount of blood vessel embolic material used in 3-B3 was 2.0 ml. In the figures, a indicates renal peripheral blood vessels, b and c indicate small arterial blood vessels, and d and e indicate large arterial blood vessels.

(34) The results shown in FIG. 3 indicate that different levels of renal artery embolization can be achieved by controlling the amount of blood vessel embolic material. In clinical practice, doctors can choose the amount of blood vessel embolic material to be used according to the tumor site. The operability of blood vessel embolic material in clinical practice is further improved.

Experimental Example 3

(35) The blood vessel embolic material of Example 1 was used for interventional embolization of the right posterior renal artery of normal rabbits.

(36) 30 healthy Japanese White Rabbits which had been dry-fasted for 12 hours before the experiment were subjected to femoral artery intubation. After successful intubation, renal artery intubation was performed. Thereafter, renal artery embolization (RAE) was performed, followed by renal artery extubation, femoral artery extubation, and postoperative management. At each of four time points, i.e., one week, one month, two months, and three months after RAE, five post-embolization rabbits were randomly selected for contrast examination. Pathological observations were performed after four contrast examinations.

(37) FIG. 4 shows DSA contrast examination images at the four examination time points of Experimental example 3. FIGS. 4-A to 4-D are the DSA contrast examination images at 1 week, 1 month, 2 months, and 3 months, respectively.

(38) The results show that no recanalization of the right renal artery or formation of collateral circulation was observed at the four preset examination time points, i.e. 1 week, 1 month, 2 months, and 3 months. This indicates that the blood vessel embolic material according to the present invention enables effective and long-term embolization of the targeted blood vessels.

(39) FIG. 5 shows the gross histopathological photographs at the four examination time points of Experimental example 3. FIGS. 5-A to 5-D are gross histopathological photographs corresponding to the examinations at 1 week, 1 month, 2 months, and 3 months, respectively.

(40) The results show that, at later examination time points, the embolized kidney gradually shrank, while the contralateral kidney enlarged in compensation to different extents. This indicates that the blood vessel embolic material according to the present invention enables effective embolization of the targeted blood vessels.

Experimental Example 4

(41) The blood vessel embolic material of Example 1, an equal amount of lipiodol+gelatin sponge, and an equal amount of saline were each used for interventional treatment of VX2 tumor-bearing rabbits. Specifically, after successful femoral artery intubation, renal artery intubation was performed, followed by extubation and postoperative management. MRI examination was performed one week after the operation.

(42) FIG. 6 shows the MR images before and after the interventional treatment. FIGS. 6-A to 6-C correspond to the MR images with the blood vessel embolic material of Example 1, the lipiodol+gelatin sponge, and the saline, before and after the interventional treatment, respectively.

(43) The results show that, the liver tumors in each treatment group displayed a trend of growth, accompanied by central and peripheral irregular regional necrosis. Among them, tumor necrosis after treatment by embolization with the blood vessel embolic material of Example 1 and with the equal amount of lipiodol+gelatin sponge was more obvious, mostly manifested in flaky necrosis in the central region of the tumors. This indicates that the composition according to the present invention has a good embolization effect on liver tumors.