WO2008007796A1

WO2008007796A1 - Controlled-release drug preparation and method for producing the same

Info

Publication number: WO2008007796A1
Application number: PCT/JP2007/064047
Authority: WO
Inventors: Toshiyuki Ikoma; Tomohiko Yoshioka; Toru Tonegawa; Junzo Tanaka; Tetsuya Abe; Masataka Sakane; Naoyuki Ochiai
Original assignee: National Institute For Materials Science; University Of Tsukuba
Priority date: 2006-07-10
Filing date: 2007-07-10
Publication date: 2008-01-17
Also published as: JPWO2008007796A1; JP5273657B2

Abstract

It is intended to provide a drug-release particle which exhibits prolonged controlled release, has a sufficient strength even if it is implanted in the bone, and has high affinity for bone tissue. According to the invention, a controlled-release drug preparation characterized by containing a large number of porous calcium phosphate particles composed of an aggregate of calcium phosphate nanocrystals to the periphery of which a drug adheres by drying in a biocompatible polymer matrix, and a method for producing the same are provided.

Description

Drug sustained-release preparation and method for producing the same

Technical field

The present invention relates to a sustained-release pharmaceutical preparation that releases a therapeutic agent gradually and over a long period in a living body, and a method for producing the same. Light

Background art

In recent years, in order to increase the therapeutic effects of drugs and reduce side effects,

“Drug Delivery System (DDS) is being actively researched to deliver the required amount to the required site. The main role of DDS is to gradually release the drug at a constant rate over a period of time. Targeting (slow drug release) and selective transport to the target affected area (targeting drug) Various inorganic materials and polymer materials have been used so far for sustained drug release. For example, hydroxyapatite is slowly decomposed in vivo and has the property of binding or encapsulating various drugs, so it is used as a safe carrier for various drugs. .

On the other hand, a hydrogel obtained by bridging a biocompatible polymer compound such as sodium alginate collagen can also encapsulate the drug in the gel and can be slowly decomposed to release the drug, so that only the drug can be used. It is also used in food and cosmetics. Hybrid materials of both are also being developed.

As a report on the use of apatite tonoalginate complex as drug release particles, W.

Paul, CP Sharma, J. Mater. Sci. Lett., 16 (1997) 2050—2051 discloses the slow release of gentaraicin from hydroxyapatite / alginate composite mic mouth particles. . These composite microparticles are prepared by immersing 200-400 111 hydroxide particles in a gentamicin solution and drying to form a complex with the drug, mixing it with the alginate solution, and then uniaxially compressing it. However, the sustained release time of the drug is only about 24 hours. E. Krylova, A. Ivanov, V. Orlovski, G. In El-Registan, S. Barinov, J. Matei. Sci .: Mater. Med., 13 (2002) 87-90, apatite particles are mixed with an alginate solution, dropped into a hardened aqueous solution, dried and dried. The drug (Biocidel) dispersed in tanol is formulated by dripping it onto the surface of the dry particles. However, it has been confirmed that the sustained release time has been improved compared to the conventional method. . In M. Sivakumar, KP Rao, J. Biomed. Mater. Res., 65A (2003) 222-228, a mixed solution of hydroxyapatite and alginic acid derived from koji is dropped into a polymethyl methacrylate (PMMA) solution. After stirring, composite microparticles are produced by adding chlorinated chloride as a crosslinking agent. In this document, the drug is loaded onto the composite micro-mouth particles by immersing the particles in a phosphate buffer solution in which the drug is dissolved, and the sustained release time is zero-dimensional (approximately 3 days). ) Is displayed. In CC Ribeiro, CC Barrias, MA Barbosa, Biomater., 25 (2004) 4363-4373, the drug loading method is important for drug delivery (DDS) by the apatite nolginate complex. It has been reported that the method of adsorbing to the apatite particles and then combining with alginic acid has the best sustained release.

As described above, various methods have been tried to support the drug on the apatite Z-alginate complex, but the sustained release of the drug is at most several hours to several days. However, when the drug is an anticancer agent, such sustained release is not sufficient, and a sustained release over a long period of at least 14 days is required. Until now, research on sustained-release preparations of anticancer drugs has been conducted, and many of them are particles mainly made of polymer materials. Typically, microcapsule particles are encapsulated in a biodegradable polymer such as polylactic acid-dalycolic acid copolymer (PLGA), but it has the disadvantage of poor cell adhesion due to its hydrophobic nature. is there. Furthermore, many anticancer agents are poorly water-soluble, and it is difficult to prepare an aqueous solution and impregnate the composite. Disclosure of the invention

Therefore, the problem of the present invention is that long-term sustained release, which could not be expected in the past, is possible, and even if the sustained release drug is poorly water-soluble, such as an anticancer agent, It is an object of the present invention to provide a sustained-release pharmaceutical preparation capable of long-term sustained release and a method for producing the same. Further, the present invention further provides a material having a high affinity with bone tissue, preventing bone formation, and maintaining a material strength that can withstand bone grafting, even when cancer tissue having bone metastasis is targeted. At the same time, it is to provide a drug sustained-release preparation having a deformation characteristic that can be adapted to the shape of the bone defect site and a method for producing the same.

As a result of intensive studies to solve the above problems, the present inventors have provided the following inventions.

(1) Sustained-release pharmaceutical preparation, characterized in that a large number of porous calcium phosphate particles composed of aggregates of calcium phosphate nanocrystals, to which the drug is dried and fixed, are contained in a biocompatible polymer matrix .

(2) The drug sustained-release preparation according to (1), wherein the drug is an anticancer drug.

(3) The drug sustained-release preparation according to (1) or (2), wherein the drug is a poorly water-soluble drug.

(4) The sustained-release pharmaceutical preparation according to any one of (1) to (3), wherein the calcium phosphate contains a hydroxyl group, a carbonate group, or a halogen group.

(5) The calcium phosphate contains at least one or more divalent metals selected from the group consisting of magnesium, zinc, strontium, and barium together with calcium or as a part of calcium. And

The drug sustained-release preparation according to any one of (1) to (4).

(6) The biocompatible polymer is at least one selected from the group consisting of sodium alginate, locust bean gum, xanthan gum, dextran sodium, carrageenan, bectin, chitosan, hyaluronic acid, carboxymethylcellulose, and derivatives thereof. The sustained-release pharmaceutical preparation according to any one of (1) to (5), wherein

(7), wherein the size is 1 X 1 0- ¹ ~ 5 mm, the drug sustained-release preparation according to any one of (1) to (6).

(8) characterized in that said particle size of the calcium phosphate nanocrystals is 1~ 1 X 1 0 ³ nm, the drug sustained-release preparation according to any one of (7) (1).

(9) Spherical particles in which the porous calcium phosphate particles have a specific surface area of 30 m ² Zg or more The sustained-release drug preparation according to any one of (1) to (8), wherein

(10) The sustained-release drug preparation according to any one of (1) to (9), wherein the porous calcium phosphate particles have a particle size of 1 × 10 ² / m or less.

(1 1) A drug sustained-release medicament comprising the drug sustained-release preparation according to any one of (1) to (1 0).

(1 2) A method for producing a drug sustained-release preparation according to any one of (1) to (10), comprising the following steps.

(a) A step of spray-drying an aqueous dispersion of calcium phosphate nanocrystals and a drug to obtain porous calcium phosphate particles comprising aggregates of calcium phosphate nanocrystals with the drug heated and fixed around

(b) After the porous calcium phosphate particles are mixed and dispersed in a biocompatible polymer aqueous solution, the porous calcium phosphate particles are dropped into a cured aqueous solution, and the particles are contained in the biocompatible polymer matrix. Process for making affinity polymer particles

(c) A process for collecting and washing the biocompatible polymer particles.

(1 3) The production method according to (12), further comprising a step of drying the polymer particles after the step of collecting and washing the biocompatible polymer particles.

(14) The hardening aqueous solution is an aqueous solution of a salt of a divalent metal selected from the group consisting of magnesium, calcium, strontium, barium, and zinc.

The method described in (1 2) or (1 3). Brief Description of Drawings

Figure 1 shows a scanning electron microscope (SEM) photograph of each particle produced by spray-drying (sample A: no paglitaxel, sample B: paclitaxel 2.4%, sample C: paclitaxel 7.3%).

Figure 2 shows the particle size distribution measurement results of paclitaxel-containing (2.4%) apatite particles (Sample B).

Figure 3 shows the thermal analysis (TG-DTA) of each particle obtained by spray drying (sample A: no paclitaxel, sample B: 2.4% paclitaxel, sample C: 7.3% paclitaxel). Figure 4 shows the IR spectrum of each particle obtained by the spray drying process (Sample A: No Notaxel, Sample B: Paclitaxel 2.4%, Sample C: Paclitaxel 7.3%) o

Figure 5 shows photographs of the resulting particles before and after compression (left: before compression, right: after 90% compression).

Figure 6 shows the changes in the compressive strength of the particles produced as a result of the immersion time in the cured aqueous solution.

Figure 7 shows the correlation between the diameter and strength of the particles produced.

Figure 8 shows the change in compressive strength depending on the content of the particles in the produced particles.

Figure 9 shows the change in compressive strength of the produced particles with the concentration of alginic acid.

FIG. 10 shows the results of a drug elution test in PBS (+ Ca, Mg) to which Tween-80 added to the prepared paclitaxel-containing particle preparation was added.

Figure 1 1. Photographs of cells cultured with clitaxel-containing particles are shown ((a) —week, (b) week 4).

Figure 12 shows the results of lower limb motor function evaluation (B-B-B score) in the local treatment group and the control group.

Figure 13 shows the Kaplan-Meier survival curves for the local treatment group and the control mouth group. Figure 14 shows a histopathological section of a rat breast cancer spinal metastasis model (left figure: normal. Spinal canal, right figure: tissue invaded by tumor tissue).

Fig. 15 shows a cross-sectional view of the spine of a rat breast cancer spinal metastasis model (spine affected by tumor, H & E stained X40) (SC: spinal cord, VB: vertebral body, T: tumor). Bar = 500 / z m.

Figure 16 shows a SEM photograph and EDX analysis of a cross section of hydroxyapatite toalginic acid particles cross-linked with barium ions.

Figure 17 shows the expansion coefficient of hydroxyapatite toalginic acid particles crosslinked with barium ions.

Figure 18 shows the cumulative release of paclitaxel from hydroxide alginate and alginate particles crosslinked with barium ions. Hereinafter, the present invention will be described in detail. This application claims the priority of Japanese Patent Application No. 2006-189259 filed on Jul. 10, 2006. Description of the patent application The contents described in the above are included. The present invention is described in detail below.

(1) Drug sustained-release preparation

The drug sustained-release preparation of the present invention is that a large number of porous calcium phosphate particles composed of aggregates of calcium phosphate nanocrystals, to which the drug is dry-adhered around, are contained in the matrix of the biocompatible polymer. Features.

The drug sustained-release preparation of the present invention is granular, and its size is the amount of calcium phosphate mixed, the biocompatible polymer concentration, the diameter of the needle of the dropping device, the size of the droplet, the dropping method, and the hardening aqueous solution. Although it can be controlled by the type / concentration and curing time, etc., it can be visually observed, specifically 0.1 to 5 mm, preferably about 1 to 3 mm.

The above “size” means a value obtained by measuring the particle diameter of at least 5 gel particles and calculating the average.

The porous calcium phosphate particles included in the drug sustained-release preparation of the present invention are prepared by spray-drying an aqueous organic solvent dispersion suspension of calcium phosphate nanocrystals and a drug as described above, and firing is performed. Absent. Therefore, there is no decrease in the specific surface area by calcination, the specific surface area is 30111 ² Roh 8 or more, preferably 50 m ² Bruno g or more.

In the present invention, “calcium phosphate” is represented by the general formula C a ₁₀ (P0 ₄ X) 6 Y ₂ (X represents a carbonate group or a deficiency, and Υ represents a hydroxyl group, a carbonate group, a halogen group, or a deficiency). This concept includes the hydroxyl, carbonate, fluorine, and chlorine apatites. Further, at least one divalent metal selected from the group consisting of magnesium, zinc, strontium, and barium may be contained together with or in place of calcium.

The porous particle particles are spherical microparticles, and the particle diameter is 1 X 10 ² jum or less, preferably: ~ 5 X 10 μm.

Examples of biocompatible polymers include sodium alginate, locust bean gum, xanthan gum, dextran sodium, carrageenan, bectin, chitosan, hyaluronic acid, carboxymethylcellulose, and derivatives thereof. A polysaccharide selected from the group consisting of: 1 type of these or a combination of 2 types ^. Of these, sodium alginate is most preferred. The content of the drug in the drug sustained-release preparation of the present invention varies depending on the sustained-release ^{period, 1 X 1 0- 3 ~ 1} X 1 0 2 wt ^_0/0 for calcium phosphate by weight, preferably. 1 to 5 X 1 0% by weight.

In order that the drug sustained-release preparation of the present invention has sufficient strength, the content of the porous calcium phosphate particles in the particles is 1 to 5 X 10% by weight, preferably 1 X 10 to 3 For example, X 10% by weight, and the content of the biocompatible polymer may be 1 X 10 ⁻³ to 1 X 10% by weight, preferably 1 to 3% by weight.

The sustained release property of the drug sustained-release preparation of the present invention can be controlled by the type of biocompatible polymer, the concentration of the biocompatible high molecule, the apatite content, etc., and is at least 14 days, preferably 30 Sustained release over more than a day is possible.

(2) Method for producing drug sustained-release preparation

The drug sustained-release preparation of the present invention can be produced by the following method.

First, a water-organic solvent dispersion suspension of calcium phosphate nanocrystals and a drug is spray-dried, and the drug is dried and fixed around the crystal to produce porous calcium phosphate particles that are aggregates of the crystals.

The calcium phosphate nanocrystals used here have a particle size of 1 to 1 X 10

³ nm, preferably 1 X 10 to 1 X 10 ² nm, and the production thereof is preferably carried out by a wet method, for example, when producing hydroxyapatite nanocrystals. It can be carried out.

1 0 C a (OH) ₂ + 6 H ₃ (PO ₄ ) ₃ → C a ₁₀ (PO ₄ ) ₆ (OH) ₂

As the organic solvent for dispersing the drug, the type of the organic solvent used depending on the type of the drug and the ratio with water can be determined. For example, ethanol, methanol, etc. can be used as the organic solvent, The mixing ratio of water and organic solvent should be in the range of 1 X 10 ⁴ : 1 to 1: 1 X 10 ⁴ .

The type of the drug is not particularly limited, but a poorly water-soluble drug that is difficult to impregnate calcium phosphate particles in the form of an aqueous solution can also be used. Of these, Examples of poorly water-soluble anticancer agents include paclitaxel, adriamycin, camptothecin, cisplastin, daunomycin, pinorbin, methotrexate, mitomycin c, etoposide, gefitinib, irinotecan hydrochloride, topotecan hydrochloride, docetaxol, vinbrathol sulfate Vincristine sulfate, vindesine sulfate, teniposide, pinorelvin tartrate, busunolevane, carbocon, thiotepa, cyclophosphamide, melphalan, estramustine sodium phosphate, mechlorethamine oxide hydrochloride, ifosfamide, ramustine hydrochloride Bleomycin hydrochloride, pepromycin sulfate, dinostatin stimarate, actinomycin D, aclarubicin hydrochloride, doxorubicin hydrochloride, salt Idarubicin, amrubicin hydrochloride, daunorubicin hydrochloride, pyranolevicin, epenolevicin hydrochloride, valrubicin, mercaptopurine, fludarabine phosphate, cladribine, fluoracil, tegafur, cytarabine, gemcitabine hydrochloride, cytarabine , Oxalibratin, Xifluridine, Carmofur, Enositabine, Nedaplatin, Carboplatin, Fadrozole hydrochloride, Anastrozo Λ Exemestane, Bicanoletamide, Fu ^ / Tamido, Tamoxifen citrate, Toreminoin citrate, Pentatin L —Asparaginase, dacarbazine, procarbazine hydrochloride, mitoxantrone hydrochloride, sobuzoxane, trastuzumab, rituxi Mab, imatinib mesylate, 5-fluo-2, monodeoxyuridine, ascle, canolevocrine, quinolespan, krestin, picibanil, and derivatives thereof, but are not limited to these. Also. The above anticancer agents may be used singly or in combination of two or more.

Spray drying can be performed by a conventional method using a commercially available apparatus equipped with a two-fluid nozzle and a four-fluid nozzle such as Buchi, Yamato Kagaku, and Okawara Kogyo Co., Ltd. In order to increase the surface area, The liquid mixture is made into fine droplets of about 1 to 5 X 10 ² m, blown into hot air at 100 to 300 ° C., and dried.

Next, the obtained porous calcium phosphate spherical particles are mixed and dispersed in a biocompatible polymer aqueous solution, and then dropped into a cured aqueous solution to be granulated. In order for the particles to be produced to have sufficient strength, the particles are kept dripped in the aqueous curing solution for about 1 minute to 2 hours. It is preferable to immerse in the aqueous solution.

What is necessary is just to use the aqueous solution of the said polysaccharide as biocompatible polymer aqueous solution. Raw #: The concentration of the aqueous affinity polymer solution is sufficient so that the particles produced have sufficient strength.

1 X 10 ⁻³ to 1 X 10 wt%, preferably 1 to 3 wt%. The porous calcium phosphate particles are 1 to 5 10% by weight, preferably 1 X 10 to 3 X 10% by weight, based on the biocompatible polymer aqueous solution.

The aqueous hardening solution may be an aqueous solution of a divalent metal salt selected from the group consisting of magnesium, calcium, strontium, barium, and zinc. The salt may be either an inorganic acid salt or an organic acid salt. Specifically, calcium chloride, zinc chloride, barium chloride, strontium chloride, calcium acetate, zinc acetate, etc. can be used. Calcium chloride is particularly preferred. The concentration of the divalent metal ion in the curable aqueous solution is 1 X 10 ⁻³ to 5 mol 1, preferably 2 X 10 0 — ^{2 to 2} m o 1/1. Moreover, it is preferable to adjust the hardening aqueous solution to about pH 5 to 8 in advance.

By dispersing the porous calcium phosphate spherical particles in the biocompatible polymer aqueous solution, the biocompatible polymer is coated on the porous calcium phosphate spherical particles, and when this is dropped into the cured aqueous solution, the metal of the biocompatible polymer is added. A salt (for example, calcium alginate) is formed, forming a three-dimensional network structure and gelling.

When dropping a biocompatible polymer aqueous solution in which porous calcium phosphate spherical particles are dispersed into a hardened water solution, depending on the size of the target particle, pipette the microphone so that the dropped particles become fine particles. It is preferable to drop it with a syringe or syringe.

Collect and wash the final particles. The collection is performed by filtration or the like, and the recovered particles can be further classified as necessary to obtain particles having a desired particle size distribution.

In addition, a drying treatment after washing is preferable in terms of storage and sterilization. Drying is performed, for example, from 0 to: I 0 0 ° C. from 6 to: I for 2 hours.

(3) Drug sustained release medicine

The drug sustained-release preparation of the present invention exhibits a long-term sustained release of at least 14 days or more The In addition, the drug sustained-release preparation has a function of having sufficient strength even when transplanted into bone and having excellent affinity with bone tissue. Therefore, the drug sustained-release preparation of the present invention can be used alone or in combination with pharmaceutically acceptable additives as a drug sustained-release drug, particularly a drug sustained-release drug for cancer treatment. The types of cancer to be treated are not limited, but cancers that cause bone lesions, such as primary bone tumors such as osteosarcoma, chondrosarcoma, Ewing sarcoma, other organs / organs (lung, stomach, mammary gland) It is also effective for metastatic bone tumors in which cancer of the thyroid, kidney, prostate, etc. has metastasized to bone, and blood cancers such as myeloma and lymphoma.

The sustained-release drug of the present invention can be prepared in various dosage forms and administered systemically or locally orally or parenterally. When orally administering the medicament of the present invention, it is formulated into tablets, force capsules, granules, powders, pills, water for internal use, suspensions, emulsions, syrups, etc., or redissolved when used. It may be a dry product. In addition, when the pharmaceutical of the present invention is administered parenterally, it is formulated into intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, etc. Are provided in unit-dose samples or in multi-dose containers.

These various preparations include excipients, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, preservatives, A flavoring agent, a soothing agent, a stabilizer, a tonicity agent and the like can be appropriately selected and produced by a conventional method.

When the sustained-release medicament of the present invention contains an anticancer agent as a drug and is used for the above cancer treatment, the administration route is preferably administration by injection, and the dosage form is preferably injection. Examples of administration by injection include subcutaneous administration, intraperitoneal administration, arterial administration, intravenous administration and the like. Of these, arterial injection into the artery near the affected area or direct administration to the affected area is most preferable. In addition, when the sustained-release medicament of the present invention contains an anticancer agent as a drug and is used for the above-described cancer therapy, it may contain other drugs used for cancer therapy. Examples of such drugs include contrast media such as ribiodol or vascular occlusive agents, apoptosis inducers such as staurosporine, immunosuppressants such as steroids and cyclosporine, angiogenesis inhibitors, and narcotics such as morphine for pain relief. Can be illustrated.

The dosage of the medicament of the present invention is the age, sex, symptom, route of administration, number of times of administration. Varies and can vary widely. For example, the effective amount of drug included is: 0.1 mg / kg body weight at a time! A dose in the range of OO Omg can be selected, preferably administered once to several times a day. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by these.

(Example 1) Production and particle morphology of paclitaxel-containing porous apatite particles

A potassium suspension (1 liter) was added dropwise to a 0.25 mol / l calcium hydroxide suspension (2 liters) to synthesize an apatite suspension. The synthesized suspension

After drying at 120 ° C, the content of the buffer was measured, and the weight of the buffer per unit amount of suspension was calculated. 150mg of Pakri Taxel (Taxol; Wako Pure Chemical Industries, Ltd. Cat)

No. 163-18614, Lot No. CEG1585, molecular weight 853.91, assay 97% (HPLC)) was dispersed in 100 ml of ethanol and mixed with 500 ml of a suspension of a suspension (approximately 6 g of a suspension). The obtained apatite tonopaclitaxel / ^ ethanol suspension and the apatite ethanol suspension not containing paclitaxel were spray-dried with a spray dryer manufactured by Buchi Ne ± to produce porous spherical particles ( Sample A: no paclitaxel, sample B: containing 2.4% paclitaxel). Figure 1 shows a scanning electron microscope (SEM) photographic image of the particles produced. As is clear from the SEM image, all the particles produced were spherical, regardless of the mixing of paclitaxel. The particle diameter (diameter) measured from 100 particles is 3.31 ± 0.83 urn (maximum 6.1 μπι, minimum 1.9 / 1 m) for sample A (without paclitaxel), and 3.42 for sample B (contains 2.4% paclitaxel).

± 1 · 13μπι (maximum 9, l // m, minimum 1.8 / m). No change in particle morphology and particle size due to mixing of paclitaxel was observed. Figure 2 shows the particle size distribution measurement results for Sample B (containing paclitaxel 2.4%). It can be seen that the spray-dried particles are all distributed from 1 to 20 / zm. From this result, it is considered that particles of 10 zm or more aggregated during the measurement. The average particle size is 4.0 ± 0.2μηι for sample A (without paclitaxel), and the average particle size for sample Β (containing 2.4% paclitaxel).

It was 4.0 ± 0.2 μπι. Therefore, mixing paclitaxel makes a difference in the particle size distribution. Was not observed.

In addition, 300 mg of paclitaxel was dispersed in 50 ml of ethanol, and mixed with 250 ral (3 g of apatite content) of an apatite suspension synthesized in the same manner as described above. The mixed apatite paclitaxel / ethanol suspension was spray-dried with a spray dryer manufactured by Buchi under the same conditions as above to synthesize paclitaxel-containing (7.3%) porous apatite spherical particles (sample) C). Figure 1 also shows a scanning electron microscope (SEM) image of Sample C.

(Example 2) Paclitaxel content analysis

The paclitaxel content contained in each particle produced in Example 1 was quantified by UV method and thermal analysis method. Paclitaxel eluted by dispersing Sample B 13.2, 10.6, and 8 mg in 10 ml of purified water ethanol mixed solvent (50:50) was quantified at a wavelength of 230 nm. As a result, it was found that the content was 2.4 ± 0.2% per weight of the produced particles. Samples C 4.26, 6.03, and 7.19 mg were quantified in the same manner. As a result, it was found that 7.3 ± 0.4% was contained per particle weight.

Figure 3 shows the results of thermal analysis measurement. As a result, in the thermal analysis of the single apatite (sample A), a gentle weight loss up to 1200 ° C was observed. The weight loss was 11.6 wt%, and almost no exotherm was observed. In samples containing paclitaxel (samples B and C), exothermic peaks were observed at 242, 347, and 455 ° C. The weight loss to 1200 ° C was 13.6% (Sample B) and 19.6% (Sample C), respectively. The paclitaxel content was calculated by subtracting the weight loss (sample B—sample A, sample C one sample A). As a result, the paclitaxel content was 2.0 wt% (Sample B) and 8.0 wt% (Sample C). These results were in good agreement with the UV measurements.

(Example 3) FT-IR analysis of paclitaxel-containing porous apatite spherical particles Fig. 4 shows the results of FT-IR analysis of paclitaxel-containing porous apatite spherical particles prepared in Example 1. Absorption peaks due to paclitaxel are 1742, 1711, 1577,

Observed at 1347, 1316, 1276, 1244, 907, 773, and 708cnT ¹ , respectively. It is also clear that these absorption peak intensities increase due to the difference in paclitaxel content. It was easy. Further, 1490, 1453, absorption peaks assignable to carbonate groups SSCNTs ¹ was observed. This indicates that the carbonate group is substituted with a hydroxyl group or a phosphate group in the apatite structure.

(Example 4) Production of particles and strength properties due to immersion time in aqueous curing solution

A 1% aqueous alginate solution was prepared using sodium alginate (manufactured by Wako Pure Chemical Industries, Ltd .; viscosity 500-600 cps (lw / v%. 20 ° C), pH 7.3)). After mixing sodium alginate mechanically with purified water using Cell Master (As one), centrifuge for 10 minutes at 12,000 rpm (Tomy Seye) to remove insoluble matter and degas Went. The pure porous apatite spherical particles prepared in Example 1 (Sample A) lg were mixed with lOral aqueous alginate solution. This mixed solution was dropped into a hardening aqueous solution (100 ml of 2 mol / 1 calcium chloride solution) using a pipette with a microphone (200 1). The obtained particles were all spherical. The time-series strength change (compression test) was examined by changing the time of immersion in the cured aqueous solution to 10 minutes, 1, 2, 4, and 24 hours. Using a texture analyzer, the probe was pushed into purified water at a speed of 0.1 lram / s and compressed 90%. Figure 5 shows the outer shape of the particles produced and the morphology of the particles after the compression test.

The average particle diameter of the 25 particles produced (5 particles per time series) was also calculated using a texture analyzer. The average value was 2.04 ± 0.03 mm (maximum particle diameter: 2.07 mm, minimum particle diameter: 1.90 mm). In addition, Fig. 6 shows the time-series changes in strength when strain stresses of 10, 20, and 30% are applied. Particles with sufficient strength to be obtained even after immersion for 10 minutes were obtained. The compressive strength increased slightly by changing the time of immersion in the curable aqueous solution.

(Example 5) Strength property change of particle depending on particle diameter

In the same manner as in Example 4, pure porous abatite spherical particles (sample A) lg were mixed with 1% alginate aqueous solution lOral. This mixed solution was dropped into a hardening aqueous solution similar to that in Example 4 and immersed for 10 minutes to produce 50 particles in which only the particle diameter was changed. The compressive strength test was performed under the same conditions as in Example 4. Figure 7 shows the correlation between particle diameter and strength. The horizontal axis is normalized by 2mm diameter, and the vertical axis is normalized by minimum strength. Indicates the value. The diameter of the particles produced is in the range of 1.47 to 2.69 mm. As shown in Fig. 7, the correlation between diameter and intensity can be approximated by a linear function. As a result of regression curve analysis, Y = l.0073ΧR = 0.95. It was clear that the material strength was dependent on the diameter.

(Example 6) Change in strength properties of particles depending on the content of the particles

Pure porous apatite spherical particles (Sample A) of 5 10, 20, 30, and 40 wt% were mixed with the 1% alginate aqueous solution prepared in the same manner as in Example 4, and the resulting cured aqueous solution was the same as in Example 4. Dropped and dipped for 10 minutes to produce particles. The obtained particles were washed and similarly subjected to a compressive strength test using a texture analyzer. The size of the particles prepared at each concentration was 1.81 ± 0.03, 2.02 ± 0.04, 2.11 ± 0.04, 2.21 ± 0.01. 2.13 ± 0.03 mm. In order to correct the intensity depending on the size, the plot was made with the diameter corrected to 2 mm (Fig. 8). It was found that the strength increased linearly with increasing the content of the ferrite.

(Example 7) Strength property change of particles due to alginate concentration

1 2 10 for each 3% aqueous alginate solution. Pure porous sphere spherical particles (sample A) were mixed, dropped into a cured aqueous solution similar to that in Example 4, and immersed for 10 minutes to produce particles. Similarly, a compressive strength test was conducted and normalized by 2 diameters. The results are shown in Fig. 9. The% display in the figure is the distortion value. As the alginic acid concentration increased, the load carrying strength clearly increased.

(Example 8) Strength property change of particles due to difference in curing aqueous solution

Under the same mixing and dropping conditions as in Example 4, particles were prepared by immersing in each aqueous solution of hardening (calcium chloride aqueous solution, strontium chloride aqueous solution, and barium chloride aqueous solution) for 10 minutes. The compressive strength test was performed under the same conditions as in Example 4. The diameter was scaled to 2 in order to correct the strength due to size. The load required for 90% strain of the particles prepared with each aqueous curable solution was 1016 ± 77 1303 ± 45 1584 ± 126 g. It was clear that the strength of the particles could be increased by the aqueous curing solution. (Example 9) Sustained drug release test of the particle preparation of the present invention

Using the paclitaxel-containing porous apatite spherical particles (containing paclitaxel 7.3%) prepared in Example 1, particles (alginic acid concentration 1%; 10 wt% apatite with respect to the alginate aqueous solution) were prepared in the same manner as in Example 4. Was obtained as a preparation of the present invention. The resulting particle preparation was subjected to a paclitaxel elution test using water / ethanol (50-50). One particle formulation was added to 5 ml of solution and allowed to stand for up to 12 hours. The supernatant was collected and subjected to UV measurement (wavelength: 230 nm). As a result, a minimum of 6 hours was required for all paclitaxel to elute from the particle formulation. On the other hand, when 1.3 mg of paclitaxel was immersed in 4 ml of solution, all paclitaxel eluted within just 1 hour. Therefore, it was clear that coating of paclitaxel-containing porous apatite spherical particles with alginate gel was effective for sustained drug release.

Similarly, using the paclitaxel-containing porous apatite spherical particles (containing paclitaxel 2.4%) prepared in Example 1, particles (alginic acid concentration: 1%; with respect to aqueous alginate solution) in the same manner as in Example 4. 10wt%, 20% containing). The amount of paclitaxel contained in the obtained particles (diameter about 2mm) is water / ethanol.

5 particles were added to (50/50), and calculation was performed from the UV measurement (wavelength: 230 nm) of the supernatant. The 10 and 20 wt% 7-partite particle formulations contained 0.035 mg and 0.08 mgZ paclitaxel, respectively. Add 1 particle formulation to 20 ml of phosphate buffer solution (PBS) containing 0.25 mM calcium, 0.15 mM magnesium and 0.1 lwt% Tween_80. The mixture was stirred for 24 hours. The supernatant was collected by centrifugation and UV measurement (wavelength: 230 nm) was performed. The results are shown in FIG. In both cases, the sustained release amount was about 20% to 30% in 24 hours.

(Example 10) Efficacy of dried particle preparation for cultured cancer cells

The CRL-1666 breast cancer cell line (Rockville, MA, USA) was used as the cultured cancer cells. The cells are Dulbecco Modified Eagle Medium supplemented with 10% fetal bovine serum and 80.5 pg / ral streptomycin and 80.5 U / ral penicill in and 1% L-glutaraine. All products are from Gibco Invitrogen ™ 5% C0 ₂ at 37 ° C (purchased from Corp, CA, USA) Incubation was performed under humidified pressure conditions. The medium was changed approximately every 3 days at a concentration of 10 ⁶ cel ls / ml.

On the other hand, particles containing 7.3% of paclitaxel prepared in Example 9 (alginate concentration: 1%; containing 10 wt% apatite with respect to alginate aqueous solution) were dried at room temperature for 24 hours to obtain a dried product. This dried body was co-cultured with the above cultured cells. Under the microscope, cell reduction was observed from the second day, and the effect was maintained even after 4 weeks. Figure 11 shows a picture of cell culture ((a) -week, (b) week 4).

(Example 1 1) In vivo efficacy of the particle preparation of the present invention

(1) Rat breast cancer spinal metastasis model

CRL-1666 breast cancer cell line (Rockville, MA, USA) was used. Cells were Dulbecco Modified Eagle Medium supplemented with 10% fetal bovine serum, 80.5 pg / ml streptomycin, and 80.5 U / ml penici ll in and 1% L-glutaraine (all products from Gibco Invitrogen ™ Corp , CA, and cultured under conditions of 5% C0 ₂ humidified pressure at 37 ° C for at purchased from USA). The medium was changed approximately every 3 days at a concentration of 10 ⁶ cel ls / ml. Confluent cells were detached with 0.01 M EDTA, and proliferating cells were removed from the ventral skin of 8-week-old female Fischer 344 rats (weight 150-180 g, Charles River Laboratories, JAPAN). Injecting 1 × 10 ⁶ cells to produce solid tumor pieces for transplantation. The tumor mass on the 10th day after injection was removed and placed in sterile physiological saline to a size of 1 X 1 X 1 ram. A CRL-1666 solid tumor fragment was transplanted into the lumbar spine of a rat to obtain a rat breast cancer spinal metastasis model.

The obtained rat breast cancer spinal metastasis model was divided into a control group and a local treatment group. The rats in the control group closed the bone hole containing the tumor pieces with a paclitaxel-free particle preparation. In the local treatment group, particles containing paclitaxel 2.4% prepared in Example 9 (alginate concentration 1%, containing 10% apatite with respect to alginate aqueous solution) were administered. The following tests were performed, and the control group and the local treatment group were compared using a log-rank test. For statistical evaluation, P <0.05 was considered significant.

(2) Lower limb motor function evaluation

Basso-Beattie-Bresnahan locomotor rating scale

(B-B-B score) was used every day. BBB score is based on a rat gait of 21 points It is a score. Rats were placed in a 57 x 38 x 30 cm plastic enclosure and observed for 4 minutes. In the early stages of tumor growth, rotation of toes and heels is observed, corresponding to 20 to 14 points. When a neurological deficit symptom appears, a trauma or lameness appears, which corresponds to 13 to 8 points. When spinal cord compression progresses, continuous movement of the lower limbs is not observed, which corresponds to 7 to 0 points. All tests were done in a double blind study. The control group died of paralysis of the lower extremities in about 2 weeks, as reported previously. The average BBB scale at 15 days after surgery is 5.2 days for the control group and 16.4 days for the local treatment group (Fig.

1 2).

(3) Paralysis occurrence period

The period until the occurrence of paralysis was expressed as a Kaplan-Meier curve as a disease-free time, and was compared between the control group and the local treatment group. The mean time to onset of paralysis was 11.8 and 20.8 days in the control mouth group and local treatment group, respectively, and was significantly prolonged in the local administration group (P = 0.006).

(4) Survival period

The survival rate of each group was expressed by Kaplan-Meier curve, and the average survival time was compared between the control group and the local treatment group. The mean survival times in the control mouth group and the local administration group were 14.0 days and 21.6 days, respectively, while the local administration group had a statistically significant increase in survival time of up to 32 days (P = 0.014, Figure 1 3).

As described above, paclitaxel-containing apatite gel preparations significantly prolonged the occurrence of spinal paralysis and improved survival in a rat breast cancer spinal transition model.

(5) Average tumor weight (tumor size)

Evaluation was made by distinguishing extra- and intra-osseous tumors. Extraosseous tumors were evaluated by extravertebral soft tissue growth, and the maximum diameter (a) X minimum diameter (b) of the tumor was determined, and the tumor weight (mg) was calculated from the formula 0.5ab ² *. All cases of extraskeletal lesions (extravertebral soft tissue) Tumor weight is shown in Table 1 below. The mean tumor weights in the control mouth group and the local treatment group at the end of the experiment were 6.14 g and 6.09 g, respectively, and there was no statistically significant difference. table 1

(6) Microscopic observation of intraosseous lesions

The incised spine and tumor were fixed with 10% formalin solution, decalcified, and embedded in paraffin. A 3 μm section obtained by horizontally cutting the spinal column containing the tumor was stained with hematoxylin-eosin (H & E staining). The degree of vertebral body invasion and occupancy in the spinal canal was evaluated. The H & E-stained transverse section of the spine showed a strong infiltration of the tumor, which occupied the spinal canal extensively and pressed the spinal cord (Figures 14 and 15).

(7) X-ray evaluation

To evaluate osteolytic lesions at the end of the experiment, X-ray evaluation was performed using the SOFTEX CSM-2 type (SOFTEX CO., Tokyo JAPAN) X-ray system. The development machine used was Konica SRX-101. Rats have a small vertebral body, making it difficult to evaluate osteolytic lesions. Evaluation of locomotor function using the B-B-B scale reflected tumor growth more qualitatively and spatiotemporally.

(Example 1 2) In vivo efficacy of the dried particle preparation of the present invention

Example 11 Rat breast cancer spinal metastasis model (130-180 g Fischer)

344 rats (8-week-old female) were prepared and classified into the control group and the systemic treatment group (5 mg / Kg paclitaxel was administered from the tail vein on days 1, 7, and 14) and the local treatment group. The topical treatment group included particles containing paclitaxel 7.2 wt% (alginate concentration 1%; algin A dried product containing 10 wt% apatite with respect to the acid aqueous solution was used. The evaluation method was performed in the same manner as in Example 11.

(1) Lower limb motor function evaluation

Rats in the control group began to paralyze on average 9 days and died on average 14-75 days. In the systemic treatment group and the local treatment group, paralysis began to occur on averages of 10.4 days and 12.8 days, respectively, and died on averages of 14.22 days and 17.45 days, respectively.

(2) Paralysis occurrence period

When the treatment group (systemic treatment group and local treatment group) was compared with the control group using univariate analysis, the time to paralysis in the local treatment group was significant compared to 12.8 days compared to 9 days in the control group. (P-0.05, P = 0.044). On the other hand, there was no statistically significant difference in the systemic treatment group. The degree of paralysis was divided into 3 weeks using the AN0VA analysis of variance and Fischer's PLSD test, divided into 1 week from the start of the experiment, 1 week to 2 weeks, and after 2 weeks. There was no significant difference between the 3 groups at 1 week, but the progression of paralysis was significantly delayed in the systemic administration group and the local administration group compared with the control mouth group from 1 to 2 weeks (P = 0.046 and P = 0.039). From the second week onwards, the progression of paralysis in the local treatment group was significantly delayed compared to the control and systemic treatment groups (P = 0.0028 and P = 0.0025). As described above, the period until the onset of paralysis was significantly prolonged in the local administration group, and there were no cases of toxicity of anticancer drugs as observed in the systemic treatment group.

(3) Survival period and weight change

There was no statistically significant difference between the treatment group (systemic treatment group and local treatment group) and the control group using univariate analysis (control group 14.75 days, systemic treatment group 14. 22 days, local treatment group 17. 45 days; P values in systemic treatment group and local treatment group were 0.78 and 0.15, respectively, compared to control group.

The average weight of the rats at the start of the experiment was 144.lg (129-181g). The change in body weight of each group was evaluated using a paired t test for rats whose body weight at 2 weeks could be measured. In the systemic treatment group, there was no statistically significant difference between the start of the experiment and 2 weeks in the control group, but the local administration group tended to have less weight change at 2 weeks. On the other hand, the body weight of the whole body treatment group and the control group tended to decrease compared to the time when the experiment was started (control group 3.2 g, whole body treatment group 5.3 g, local treatment Treatment group 0.18g; p-value in the control group and local treatment group is 0.62 0.

(Example 1 3)

Sodium alginate (Wako Pure Chemical Industries, Ltd.;. The viscosity 500_600cps (lw / v% 20 ° C), P H7 3) was dissolved in distilled water in a concentration of l 3% (w / v) , was prepared alginate solution. To this aqueous solution of alginate, 5 40% (w / w) paclitaxel-containing porous apatite spherical particles (containing 2.6% paclitaxel) prepared in the same manner as in Example 1 were uniformly mixed at room temperature. . Hydroxyapatite toalginic acid mixture was dropped into a hardening aqueous solution (2.0 M barium chloride solution) using a micropipette (200 / zl) to prepare a spherical gel having a size of 2.02.8 mm. The resulting hydroxyapatite toalginic acid spherical gel was dried for at least 24 hours to obtain firm hydroxyapatite toalginic acid particles. This particle was named AxHy-Ba (where x and y represent alginate concentration and potassium content, respectively).

The internal structure of the particles was sputter coated with platinum and then examined with a scanning electron microscope (SEM) and EDX. Mechanical properties were examined using a texture analyzer (Stable Micro Systems, UK). The compressive strength was measured using a cylindrical probe (5.0mra diameter) with a speed of 0.1 / s. The expansion rate of the particles was measured in D-PBS according to the following formula.

Expansion coefficient = (W.-W _d ) / W _d

(Where W. and W _d indicate the weight of the expanded and dry particles, respectively)

The behavior of paclitaxel release was examined at 37 ° C in D-PBS containing 1% Tween_80. The amount of paclitaxel was quantified by high performance liquid chromatography (HPLC) in which the mobile phase consisted of acetonitrile and water (volume ratio 60 40), and the flow rate was l. O ml / min. Paclitaxel was detected at 227 nm using a UV detector.

Figure 16 shows SEM photographs and EDX analysis of the cross section of hydroxide apatite toalginic acid particles cross-linked with barium ions. The SEM photograph of A1H40-Ba showed that the mic mouth particles of the hydroxyapatite having a particle size of 1 10 jura were uniformly dispersed in the particles. Hydroxide microparticles did not appear on A1H05-Ba and A3H05-Ba. On the other hand, in the EDX analysis, all of the types of particles examined were subjected to hydroxide activity. Uniform dispersion of the mouthpiece particles was shown. Valium ions cross-linked with alginic acid were also uniformly dispersed in the particles. These results support that the obtained hydroxyapatite monoalginate particles are uniform regardless of their composition.

In addition, the compressive strength of hydroxyapatite-alginate particles increased in the order of AlH40_Ba <AlH05-Ba A3H05-Ba. This result shows that the compressive strength increases as the ratio of alginic acid to hydroxylapatite increases. The ratio of alginic acid to hydroxyapatite was more influenced by compressive strength than hydroxyapatite content. In previous studies by the inventors, it has been found that hydroxyapatite toalginic acid particles tend to collapse due to alginate when compressed. On the other hand, it was found that the hydroxyapatite-alginate particles obtained in the present invention became firm as the gel was dehydrated.

Figure 17 shows the expansion coefficient of hydroxyapatite toalginic acid particles crosslinked with barium ions. The expansion characteristics differed depending on the composition of the particles. The higher the ratio of alginic acid to hydroxyapatite, the better the expansion. This result was thought to be due to the strong hydration properties of alginic acid.

Figure 18 shows the cumulative release of paclitaxel from hydroxyapatite to alginate particles crosslinked with barium ions. Paclitaxel was gradually released from the particles into the culture medium. The release behavior of A1H05-Ba and A3H05-Ba was the same. Since the expansion coefficient of A3H05-Ba is higher than that of A1H05-Ba (Fig. 17), it was found that the release behavior of paclitaxel is not related to the expansion coefficient of the particles.

The release behavior from A1H20-Ba was almost the same for A1H05-Ba and A3H05_Ba. After 5 hours, A1H05-Ba and A3H05_Ba did not release paclitaxel, whereas A1H20-Ba still had a lot of release. The release of paclitaxel. Therefore, the release behavior of paclitaxel was considered to depend on the amount of hydroxyapatite to alginate particles supported. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety. Industrial applicability

The drug sustained-release preparation of the present invention is excellent in sustained-release property and can maintain its effect for at least 14 days. Such a sustained release effect over a long period of time of the drug sustained-release preparation of the present invention is that the drug is dried and fixed around the calcium phosphate nanocrystals, and porous calcium phosphate particles that are aggregates thereof. This is considered to be achieved by incorporating a large number of bioaffinity in a biocompatible high molecular matrix. In other words, compared to the conventional case where the drug is simply adsorbed around the nanocrystal, the adhesion of the drug is increased by drying and fixing around the nanocrystal, so that the drug is not separated but the crystal is not separated. More parts are released into the body due to disintegration. In addition, by incorporating a large number of such nanocrystal aggregates in the biocompatible polymer matrix, the collapse of the calcium phosphate nanocrystals in vivo can be mitigated.

In addition, the drug sustained-release preparation of the present invention has sufficient strength even when transplanted into bone, has excellent affinity with the bone tissue, and does not change the sustained-release effect. Furthermore, in manufacturing such sustained-release pharmaceutical preparations, the spray-dry method has been used to fix even poorly water-soluble anticancer agents to calcium phosphate nanocrystals. Then, the possibility of use for diseases for which sustained release action could not be utilized can be greatly expanded. Therefore, the drug sustained-release preparation of the present invention is very useful as a sustained-release pharmaceutical for treating bone metastasis cancer.

Claims

1. A sustained-release pharmaceutical preparation, characterized in that a large number of porous calcium phosphate particles composed of aggregates of calcium phosphate nanocrystals to which a drug is fixed by drying are fixedly contained in a biocompatible polymer matrix.

2. The sustained-release drug preparation according to claim 1, wherein the drug is an anticancer drug.

3. The drug sustained-release preparation according to claim 1 or 2, wherein the drug is a poorly water-soluble drug.

4. The sustained-release pharmaceutical preparation according to any one of claims 1 to 3, wherein the calcium phosphate contains a hydroxyl group, a carbonic acid group, or a halogen group.

5. The calcium phosphate is mixed with calcium or a part of the calcium.

The drug gradual agent according to any one of claims 1 to 4, characterized by containing at least one divalent metal selected from the group consisting of magnesium, zinc, strontium, and barium. Release formulation.

6. The biocompatible polymer is selected from the group consisting of sodium alginate, locust bean gum, xanthan gum, dextran rum, carrageenan, bectin, chitosan, hyaluronic acid, strong noroxymethylcellulose, and derivatives thereof. The drug sustained-release preparation according to any one of claims 1 to 5, which is at least one kind of polysaccharide.

7. Wherein the size is ^{1 X 1 0- 1 ~ 5 mm} , the drug sustained-release preparation according to any 6 Neu deviation from claim 1.

8., Characterized in that the particle size of said calcium phosphate nanocrystals is ^{1 ~ 1 X 1 0 3 nm} , the drug sustained-release preparation according to any one of claims 1 to 7.

9. The sustained-release pharmaceutical preparation according to any one of claims 1 to 8, wherein the porous calcium phosphate particles are spherical particles having a specific surface area of 3 O m ² / g or more.

10. The sustained-release pharmaceutical preparation according to any one of claims 1 to 9, wherein the porous calcium phosphate particles have a particle diameter of 1 X 10 ² m or less.

1 1. Sustained drug release comprising the drug sustained release preparation according to claim 1. Sex medicine.

1 2. The method for producing a sustained-release pharmaceutical preparation according to any one of claims 1 to 10, comprising the following steps.

(a) A step of spray-drying an aqueous dispersion of an organic solvent with calcium phosphate nanocrystals and a drug to obtain porous calcium phosphate particles comprising aggregates of calcium phosphate nanocrystals with the drug heated and fixed around

(b) The above-mentioned porous calcium phosphate particles are mixed and dispersed in a biocompatible polymer aqueous solution, then dropped into a cured aqueous solution, and the particles are contained in a biocompatible polymer matrix and have a biocompatible size of a visible size. Of making conductive polymer particles

(c) Step of collecting and washing the biocompatible polymer particles

1 3. The production method according to claim 12, further comprising a step of drying the polymer particles after the step of collecting and washing the biocompatible polymer particles.

14. The method according to claim 12 or 13, wherein the aqueous hardening solution is an aqueous solution of a salt of a divalent metal selected from the group consisting of magnesium, calcium, strontium, barium, and zinc. .