JP2008106024A - Proteinaceous medicine-containing sustained release particulate composition and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sustained release particulate composition which is suppressed in the excessive release of a proteinaceous medicine in the early stage of administration, keeps stable sustained release and hardly causes the local disorder of administration. <P>SOLUTION: The composition comprises porous hydroxyapatite derivative particulates and a proteinaceous medicine, wherein the content of the proteinaceous medicine is 40-80 wt.% of the saturated adsorption weight to the porous hydroxyapatite derivative particulates. The method for producing the composition comprises mixing the hydroxyapatite derivative particulates containing the proteinaceous medicine in an amount of 40-80 wt.% of the saturated absorption weight into the porous hydroxyapatite derivative particulates with a water-soluble bivalent metal compound aqueous solution, agitating, freeze-drying to prepare a powder, mixing the powder with an aqueous solution or suspension of a water-affinitive polymer disappearing in vivo, agitating and freeze-drying or vacuum-drying to prepare a powder. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a sustained-release fine particle composition of a protein drug based on fine particles of a porous hydroxyapatite derivative that slowly disappears in vivo and a method for producing the same.

  Long-term sustained-release injection preparations of protein drugs have been studied based on polylactic acid / glycolic acid (PLGA) (for example, Patent Documents 1 and 2, Non-Patent Documents 1, 2, and 3). reference). In addition, sustained-release microcapsules based on PLGA containing human growth hormone (hGH) are used for treatment in the United States (see, for example, Non-Patent Document 4). PLGA is a bioerodible base that gradually disappears upon hydrolysis in vivo, and has favorable properties as a base for injections. In general, when producing a sustained-release preparation using PLGA, an organic solvent for dissolving it is used. On the other hand, many protein drugs are water-soluble, and an organic solvent solution and an aqueous solution are used to produce a sustained-release fine particle preparation using PLGA.

  When these two solvents are used simultaneously, the protein drug is denatured and inactivated. Such a decrease in activity not only impairs the effectiveness but also has a risk of adversely affecting the living body. In addition, in a sustained-release fine particle formulation of a water-soluble proteinaceous drug, it is inevitable that a temporary excessive release occurs at the initial stage (immediately after administration). In addition, there is a report on a sustained release fine particle formulation of hGH (protein drug) using hydroxyapatite (see Non-Patent Documents 5, 6, 7 and Patent Document 3, for example). These are two-component systems, and the particle diameter of hydroxyapatite is as large as 40 to 80 μm or 200 μm, and administration with a thin injection needle is difficult. Furthermore, the sustained release effect in vivo is unknown.

Further, the amount of hGH contained in hydroxyapatite was 1% or less and was small. Patent Literature 3 and Non-Patent Literature 7 describe that pores present in porous hydroxyapatite fine particles are filled with a biologically active agent, human serum protein, mucopolysaccharide, and added with divalent metal ions to embolize or be water-soluble. Is provided, and a method for producing a sustained-release composition by plugging the outer layer of the fine particles by adding sodium carbonate or sodium hydrogen carbonate or an aqueous carbonate ion solution is provided. The content of the sex drug is about 1% and the sustained release period is about 7 days. For clinical use, the dosage of the preparation must be increased, so there is a problem to be solved from a practical viewpoint. there were.

  In general, in a sustained-release preparation for injection of a protein drug, a base having an in-vivo disappearance function that disappears from the living body after the release of the drug after administration must be selected. For the production of protein drugs, simultaneous use of an organic solvent that is not miscible with water and an aqueous solution must be avoided to avoid denaturation of the protein drug. In order to avoid the concern that the dosage of the preparation becomes too large and administration with a thin injection needle becomes difficult, the drug adsorption amount in the fine particle preparation is preferably at least 5% or more. Further, in many cases, since the microparticle preparation is repeatedly administered, it is preferable to pass through a thin injection needle in order to reduce the burden on the patient, and the sustained release period is at least one week or more and side effects. It is necessary to minimize the initial excessive release that can cause the above.

Already, such a problem has been submitted by using a fine particle of a porous hydroxyapatite derivative as a base and using an appropriate amount of a water-soluble divalent metal compound (see Patent Document 3 and Patent Document 4). It was done. That is, these techniques have both in vivo disappearance and sustained release functions, and release the drug contained at a substantially constant rate over a week or more, and the amount of adsorbed drug is 5% or more. In addition, a sustained-release fine particle formulation for injection of a protein drug and a method for producing the same have been clarified. A technique has also been proposed in which a sustained release of a drug is sustained for a longer time by coating to impart a preferable sustained release (see Patent Document 5).
JP 10-231252 A US Patent 5656297 International public relations WO2004 / 112827 A1 International public relations WO2004 / 112751 A1 International public relations WO2005 / 082405 A1 OL Johnson et al .: Nature Medicine, 2: 795-799 (1996). M. Takenaga et al .: J. Pharmacy Pharmacology, 54: 1189-1194 (2002). S. Takada et al .: J. Controlled Release, 88: 229-242 (2003). NDA21-075 J. Guicheux et al .: J. Biomedical Materials Research, 34: 165-170 (1997). H. Gautier et al .: J. Biomedical Materials Research, 40: 606-613 (1998). Y. Mizushima et al: J. Controlled Release, 110: 260-265 (2006).

  As described above, it is well known that excessive release at the initial stage of administration is inevitable in the sustained-release fine particle preparation for injection of protein drugs, but it has an inhibitory effect to suppress the initial release as much as possible. Valence metal compounds are used as a known technique. However, when a zinc compound having an excellent effect among divalent metal compounds is used, contrary to the suppression of initial excessive release, there is a possibility of causing an injury locally in some cases as the dose increases. Therefore, in order to obtain a safer sustained-release preparation, it is necessary to suppress the initial excessive release and to reduce the local injury to the human body as much as possible.

  Under these circumstances, the results of further investigations aimed at minimizing excessive release at the initial stage of administration for various protein drugs and obtaining stable sustained release have resulted in that the porous hydroxyapatite derivative fine particles are inherently proteinaceous. Even when the amount of the divalent metal compound added is reduced by adsorbing the proteinaceous drug in an amount less than the maximum amount capable of adsorbing (containing) the drug to the porous hydroxyapatite derivative in a weakly acidic state, excessive release at the initial stage of administration It was found that a sustained-release fine-particle composition containing a protein drug that suppresses the above, further maintains a stable sustained-release property, and has few local injuries to administration can be obtained.

  Accordingly, an object of the present invention is to obtain a sustained-release fine particle composition that suppresses excessive release at the beginning of administration of a protein drug, maintains stable sustained-release properties, and has little local injury to the administration.

  In order to achieve the above object, the sustained release fine particle composition containing a protein drug of the present invention is a composition comprising porous hydroxyapatite derivative fine particles and a protein drug, and the adsorbed amount (content of the protein drug) ) Comprises 40 to 80% by weight of the saturated adsorption weight on the porous hydroxyapatite derivative fine particles.

  The protein drug-containing sustained-release fine particle composition of the present invention is a composition comprising porous hydroxyapatite derivative fine particles, a protein drug and a water-affinity bioerodible polymer compound, the protein drug A protein drug-containing sustained-release fine particle composition, wherein the adsorbed amount (content) is 40 to 80% by weight of the saturated adsorption weight to the porous hydroxyapatite derivative fine particles.

  The protein drug-containing sustained-release fine particle composition of the present invention is based on a porous hydroxyapatite derivative fine particle as a base, from a protein drug, a water-soluble divalent metal compound, and a water-affinity bioerodible polymer compound. The protein drug adsorption amount (content) is 40 to 80% by weight of the saturated adsorption weight (saturation content weight) to the porous hydroxyapatite derivative fine particles.

  The protein drug-containing sustained-release fine particle composition of the present invention comprises a protein drug, a water-soluble divalent metal compound, and a water-affinity bioerodible polymer compound based on porous hydroxyapatite derivative fine particles. It is a composition, and the water-soluble divalent metal compound is zinc chloride, and the content of zinc chloride is 2 to 15% by weight based on the composition.

The porous hydroxyapatite derivative fine particles constituting the protein drug-containing sustained release fine particle composition described herein are compounds in which a part of calcium atoms, which are constituents of porous hydroxyapatite, are substituted with zinc atoms. At this time, the substitution rate (molar ratio) of zinc atoms is preferably 1 to 20% (molar) of calcium atoms by molar ratio. In other words, the ratio of the number of calcium atoms and the number of zinc atoms in the porous hydroxyapatite derivative fine particles is preferably 99: 1 to 4: 1.
For example, derivative fine particles in which 0.5 of 10 calcium atoms are substituted with zinc atoms are represented by the molecular formula of Ca9.5Zn0.5 (PO4) 6 (OH) 2. The general formula is Ca (10-x) Znx (PO4) 6 (OH) 2, where x is preferably 0.1-2.0. Represented as HAp-Zn-0.5 and HAp-Zn-x, respectively. The zinc atom substituted with the calcium atom is present in the crystal lattice and therefore does not elute when suspended in water.

  The nanocrystals of the hydroxyapatite derivative are synthesized by a wet method in which a phosphoric acid aqueous solution containing a predetermined amount of zinc chloride is dropped into an aqueous suspension of calcium hydroxide. This reaction is a neutralization reaction between an acid and an alkali, and apatite nanocrystals containing zinc having a size of about 40 nm are directly precipitated. The zinc-containing hydroxyapatite nanocrystal suspension thus obtained is sprayed and dried by a spray drying method to obtain porous fine particles in which a large number of nanoparticles are aggregated. Furthermore, the porous fine particles are produced by firing at a high temperature (see Toshiyuki Ikoma et al., PHARM TECH JAPAN 21: 2088-2090 (2005)).

  The porous hydroxyapatite derivative fine particles are prepared as described above and then fired at a high temperature. The lower the firing temperature, the easier it is to dissolve in water and the faster the disappearance rate. The treatment temperature is from room temperature to 800 ° C, preferably from 150 to 600 ° C. When it is baked at 800 ° C. or higher, it will not disappear in vivo. Moreover, when it processes at 100 degrees C or less, particle | grains will aggregate easily and normal injection administration will become difficult. The average particle size is preferably 50 μm or less. However, if it is too small, there is a concern that the encapsulation rate of the protein drug is lowered, and 0.1 to 50 μm is preferable, and 0.5 to 30 μm is more preferably used. In addition, when a proteinaceous drug is contained in the porous hydroxyapatite derivative fine particles, the protein drug adsorption amount (content) in the porous hydroxyapatite derivative fine particles is preferably 4 to 25%.

  The water-soluble divalent metal compound used for suppressing the initial excessive release of the protein drug is preferably a zinc compound, and examples thereof include zinc chloride, zinc acetate and zinc carbonate. Of these, zinc chloride is preferably used. In addition to zinc chloride, sodium carbonate or sodium bicarbonate may be used in combination. The amount used varies depending on the protein drug encapsulated, but in general, a range of 2 to 25% by weight of the porous hydroxyapatite derivative fine particle composition is preferably used. A more preferred range is 2 to 15% by weight.

  For the water-affinity block copolymer used to give sustained release, in vivo disappearance polymer compounds are good, and as a result of various studies, hydrophilic polyethylene glycol (PEG) is used to avoid denaturation of protein drugs. It was concluded that a block copolymer in which polylactic acid (PLA) or polylactic acid / glycolic acid (PLGA) is bonded to is preferable, and that the block copolymer is more preferably a block copolymer composed of PLA or PLGA-PEG-PLA or PLGA. . By introducing this hydrophilic group, it is possible to treat fine particles of a porous hydroxyapatite derivative containing a proteinaceous drug in an aqueous system. Protein treatment can be avoided if treated only with aqueous systems.

  This block copolymer bonding mode may be a compound in which PLA or PLGA is ester-bonded to hydroxyl groups at both ends of PEG (triblock copolymer), or a compound in which an ester bond is formed at one end of PEG. In the case of a single terminal ester, the other terminal is preferably protected with an alkoxy group or the like, but a functional group such as an amino group or a carboxyl group may be bonded thereto. The ratio of PEG to PLA or PLGA at both ends or one end of the block copolymer is preferably 20 to 90% by weight, more preferably 25 to 65% by weight. The molecular weight of the block copolymer is preferably 3,000 to 20,000, and more preferably 5,000 to 12,000. The block copolymer which is a water-affinity bioerodible polymer compound is used in the range of 3 to 100% by weight of the porous hydroxyapatite derivative fine particles, preferably in the range of 5 to 30%.

  The protein drug is applied to a compound having a molecular weight of 5,000 or more. For example, human growth hormone (hGH), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), IGF-1, NK4, VEGF, NGF, BDNF, BMP, adiponectin, interferons (IFN-α, IFN) -β), interleukins (IL-2, IL-4, IL-5, etc.), erythropoietin (EPO), granulocyte stimulating factor (G-CSF, GM-CSF), insulin, ANP, TNF-α, An antibody etc. are mentioned. The amount of saturated and encapsulated porous porous apatite or a derivative thereof is generally 5 to 50% of the apatite.

  The method for producing a sustained-release fine particle composition containing a protein drug according to the present invention comprises the porous hydroxyapatite derivative fine particles containing 40 to 80% by weight of the saturated adsorption weight of the protein drug in the porous hydroxyapatite derivative fine particles. Is mixed with a water-soluble divalent metal compound aqueous solution, stirred, and then lyophilized. The resulting powder is mixed with an aqueous biocompatible polymer aqueous solution or suspension, stirred, lyophilized, or vacuum-dried. It consists of making it powder by drying.

  In addition, the method for producing a sustained-release fine particle composition containing a protein drug of the present invention comprises the porous hydroxyapatite derivative fine particles containing a protein drug in an amount of 40 to 80% by weight of the saturated adsorption weight, followed by further freezing. The powder obtained by drying is mixed with an aqueous solution or suspension of an aqueous biocompatible polymer, and after stirring, freeze-dried or vacuum-dried to obtain a powder.

  The method for producing a sustained-release fine particle composition containing a protein drug of the present invention comprises porous hydroxyapatite derivative fine particles and 40 to 80% by weight of the protein drug saturated adsorption weight of the porous hydroxyapatite derivative fine particles. Mix with the protein drug aqueous solution or buffer, add the water-soluble divalent metal compound, stir, and freeze-dry the resulting powder with the water-soluble bioerodible polymer aqueous solution or suspension. Then, after stirring, the powder is formed by freeze drying or vacuum drying.

  The method for producing a sustained-release fine particle composition containing a protein drug of the present invention comprises porous hydroxyapatite derivative fine particles and 40 to 80% by weight of the protein drug saturated adsorption weight of the porous hydroxyapatite derivative fine particles. The protein obtained by mixing the protein drug aqueous solution or buffer solution, adding 2 to 15% by weight of zinc chloride aqueous solution to the composition, stirring and freeze-drying, and the water-soluble bioerodible polymer An aqueous solution or a suspension is mixed, and after stirring, freeze-dried or vacuum-dried to form a powder.

  Further, the method for producing a sustained release fine particle composition containing a protein drug of the present invention, a pH of 4.0 to 6 containing a protein drug of 40 to 80% by weight of the saturated adsorption weight of the protein drug of the porous hydroxyapatite derivative fine particles. .9 weakly acidic aqueous solution or buffer and porous hydroxyapatite derivative fine particles are mixed, and 2 to 15% by weight of zinc chloride aqueous solution is added to the composition. After stirring, the powder obtained by freeze-drying and PLA -PEG-PLA is mixed with a 20 (W / V)% acetone aqueous solution or suspension, stirred, and then freeze-dried or vacuum-dried to form a powder.

  The method for producing a sustained-release fine particle composition containing a protein drug according to the present invention comprises mixing porous hydroxyapatite derivative fine particles with 2 to 5% by weight of zinc chloride aqueous solution and stirring the porous hydroxyapatite derivative. It was obtained by mixing a weakly acidic aqueous solution or buffer solution having a pH of 4.0 to 6.9 containing 40 to 80% by weight of a protein drug saturated adsorption weight of fine particles, stirring, and freeze-drying. The powder and PLA-PEG-PLA 20 (W / V)% acetone aqueous solution or suspension are mixed, and after stirring, freeze-dried or vacuum-dried to obtain a powder.

  However, generally it consists of the following operations. The porous hydroxyapatite derivative fine particle is a weakly acidic solution (preferably having a pH of 3 to 7, more preferably 4.) containing 40 to 80% by weight of the protein drug saturated adsorption weight of the porous hydroxyapatite derivative fine particle. 0 to 6.9), and the mixture is stirred to adsorb the protein drug, and the hydroxyapatite derivative fine particles adsorbed with the protein drug are collected by centrifugation or the like. Further, the collected protein drug-adsorbed hydroxyapatite derivative fine particles and the divalent metal compound aqueous solution are mixed and stirred if necessary, and the hydroxyapatite derivative fine particles adsorbed with the protein drug are infiltrated with the divalent metal compound aqueous solution. . Subsequently, the precipitate part obtained by filtration is vacuum-dried or freeze-dried to obtain powder fine particles containing a proteinaceous drug. If necessary, mix this powder microparticles with an aqueous solution or suspension of a water-compatible bioerodible polymer compound or a solution containing a water-miscible solvent (eg, acetone, ethanol, etc.) The powder is prepared by adding a stabilizer and freeze-drying or vacuum-drying accordingly.

  When actually administered, the fine powder particles are dispersed in a suitable dispersion medium and injected subcutaneously or intramuscularly. The particle diameter of the finally obtained sustained-release fine particle composition may be passed through an injection needle used for normal administration. In fact, the thinner the injection needle, the less fear the patient has. Therefore, it is preferable to pass a needle thinner than 25G according to the international standard representing the thickness of the injection needle. The average particle diameter of the sustained-release fine particles satisfying these is 0.5 to 50 μm. In addition, the sustained release period of the proteinaceous drug is generally preferably one week or longer, although it depends on the type of drug.

  According to the present invention, the initial release amount can be reduced, the effective concentration of the drug can be maintained for a longer period, the protein drug is not denatured, and the protein drug has a preferable sustained release property that suppresses the local inflammation as much as possible. The sustained-release fine particle composition and the production method thereof could be provided. That is, in the step of encapsulating the drug in the porous hydroxyapatite derivative fine particles, 30 to 90%, more preferably 40 to 80% by weight of the amount of the protein drug saturated and adsorbed (maximum adsorption) to the porous hydroxyapatite derivative fine particles. A weakly acidic aqueous solution or buffer solution (preferably pH = 3 to 7, more preferably 4.0 to 6.9) containing a corresponding amount of drug and porous hydroxyapatite derivative fine particles are mixed, and a zinc compound aqueous solution, water is mixed. The present inventors have found a new method characterized in that a sustained release fine particle composition containing a protein drug is obtained by treatment with an affinity polymer compound. In addition, after the water-soluble divalent metal compound is adsorbed to the porous hydroxyapatite derivative fine particles in advance during the above production process, it contains 40 to 80% by weight of the protein drug saturated adsorption amount of the porous hydroxyapatite derivative fine particles. It may be mixed with a weakly acidic solution or buffer solution, and then treated with a water-affinity polymer compound.

  The sustained-release fine particle composition containing a protein drug obtained by the present invention provides a sustained release of a protein drug over at least 7 days, a small initial excess release, and a blood concentration after the second day after subcutaneous administration. 2 to 10 times higher than the sustained-release fine particle composition in which the drug is saturated and adsorbed, and when these compositions are administered, the water-soluble divalent metal compound compared to the preparation prepared by the prior art The present inventors have found that even when the blending amount is reduced to about one third, the excessive release is small and the sustained release property can be sufficiently maintained, thereby eliminating the local injury caused by the metal compound. The resulting composition passed through a 25G or 27G needle. Finally, it was possible to prepare a microparticle preparation that was sustained-released by lyophilization, and the encapsulated protein drug was also very stable.

  The in vitro and in vivo sustained release test results comparing the protein drug-containing sustained release fine particle composition obtained in Examples with the control composition are shown below, but are not limited to this example.

[Test Example 1]
Weigh 100 mg of hydroxyapatite derivative fine particles (HAp-Zn-0.5, a derivative in which 0.5 atom of 10 calcium atoms is replaced with zinc atom, the same applies below), which is baked at 400 ° C, in a plastic container with a lid. Consists of 2.18 mL of human growth hormone (hGH) aqueous solution (10.07 mg / mL) prepared by desalting using 10 columns (Amersham Pharmacia), 2.22 mL of purified water and 1.10 mL of 100 mM ammonium acetate buffer. The mixture was mixed with 5 mL, stirred for 5 minutes, centrifuged at 3,000 rpm for 3 minutes, 10 mL of purified water was added to the resulting precipitate, stirred for 1 minute, and then centrifuged at 3,000 rpm for 3 minutes. The obtained precipitate and 20 mg / mL zinc chloride aqueous solution (Wako Pure Chemicals, Osaka) 0.68 mL (zinc chloride is equivalent to 100 μmol per 100 mg of HAp-Zn-0.5) are mixed, and 1 with a touch mixer. After stirring for minutes, it was lyophilized. The obtained freeze-dried powder and PLA-PEG-PLA 20 (W / V)% acetone aqueous solution 0.5 mL were mixed, stirred well with a touch mixer, and then freeze-dried. PLA-PEG-PLA having a molecular weight of 11,400 and a PEG ratio of 45.6 mol% was used. When the hGH content of the obtained hGH-containing fine particle composition was quantified using Micro BCA Protein Assay kit (Pierce), the adsorbed amount of hGH was 21.3 mg (63% of the saturated adsorbed amount), and the hGH content in the composition was 13%.
Prepared in the same manner as the sustained-release fine particle composition using 3.8 mL of hGH solution (10.07 mg / mL), 0.6 mL of purified water and 5 mL of a mixture of 1.10 mL of 100 mM ammonium acetate buffer as a control formulation. The hGH-containing sustained-release fine particle composition was prepared through the final freeze-drying step. The adsorption amount of hGH was 33.9 mg (saturated adsorption amount), and the hGH content in the composition was 19%.

  The in vitro release properties of the prepared hGH-containing sustained release fine particle compositions were compared. 2.5 mg of each hGH-containing sustained-release fine particle composition sample thus obtained is precisely weighed, 0.25 mL of water is added thereto, centrifuged at 8,000 rpm for 3 minutes, and 1/10 phosphate buffered physiology is added to the precipitate. After adding 0.25 mL of saline (PBS) and incubating at 37 ° C., the supernatant was collected over time, and the amount of hGH released in the supernatant was quantified by gel filtration HPLC (TOSO G2000SW-xl). The elution rate was calculated. The results are shown in Table 1. As for the elution rate of the composition containing 63% of the saturated adsorption amount, elution of hGH in PBS was suppressed as compared with the saturated adsorption composition prepared as a control.

Suspended hGH-containing sustained-release fine particle composition was suspended in 0.5% CMC-Na, 0.1% Tween 80, and 4 male SDs were immunosuppressed by administration of tacrolimus (Astellas Pharma Inc., Tokyo). 10.5 mg / kg (30 IU / kg) was administered subcutaneously to the back of the rat. In addition, tacrolimus was administered subcutaneously in the dorsal region at a dosage of 0.1 mg / rat 3 days before and 1 days before administration of the preparation and 0.1 mg / rat every other day after the start of preparation administration.
After administration of the composition, blood is collected from the tail vein every 4, 8 or 12 hours, and every 1 or 2 days thereafter, and the blood concentration of hGH is E-test “TOSOH” using a fully automatic EIA device AIA-600II (Tosoh Corporation). Measurement was performed using II (HGH). Table 2 shows the results of hGH concentration in rat blood of this result and the hGH-containing sustained-release fine particle composition (control 2 composition, hGH is a saturated adsorption amount) prepared by the prior art of Example 4 of Patent Document 3.
The composition containing 63% of the saturated adsorption amount has the same amount of zinc chloride even though zinc chloride is 13.6% by weight of the porous hydroxyapatite derivative fine particle weight (1/3 of the control 2 composition). However, compared with the control 1 composition in which the adsorption amount is saturated, the excessive release at the initial stage of administration is suppressed, and the blood concentration transition after the third day of administration is also 40.8% by weight. It was as good as the two compositions and showed favorable sustained release properties.

On the other hand, regarding the administration local state, the composition containing 63% of the saturated adsorption amount and the control 1 composition were both less in the amount of zinc chloride than the control 2 composition. In the two compositions, weak inflammation was observed at all administered sites due to the presence of 3 times the amount of zinc chloride.
For reference, the blood concentration transition in Table 2 is shown in the figure (Table 3).

[Test Example 2]
Weighing 200 mg of hydroxyapatite derivative fine particles (HAp-Zn-0.5) calcined at 400 ° C. into a plastic container with a lid, and interferon-α prepared by desalting using this and a PD-10 column (Amersham Pharmacia) IFN-α) was mixed with 20 mL of 20 mM aqueous ammonium acetate (1 mg / mL), stirred for 5 minutes, and then centrifuged at 3,000 rpm for 3 minutes. The resulting precipitate and 10 mL of purified water were mixed and stirred for 1 minute, and then centrifuged at 3,000 rpm for 3 minutes. Mix the precipitated precipitate with 1.34 mL of 20.4 mg / mL zinc chloride aqueous solution (Wako Pure Chemicals, Osaka) (zinc chloride is equivalent to 100 μmol per 100 mg of HAp-Zn-0.5), and touch mixer And then lyophilized. The obtained freeze-dried powder was mixed with 1 mL of PLA-PEG-PLA 20 (W / V)% acetone aqueous solution, stirred well with a touch mixer, and then freeze-dried to obtain a powdery sample 1 composition. . Note that PLA-PEG-PLA having a molecular weight of 11,400 and a PEG ratio of 45.6 mol% was used. When the IFN-α content in the obtained IFN-α-containing fine particle composition was quantified using a Micro BCA Protein Assay kit (Pierce), the amount of IFN-α adsorbed to this composition was 14.5 mg (of the saturated adsorption amount). 72%), and the IFN-α content in the composition was 8.9%.

  Using 20 mL of an IFN-α 20 mM ammonium acetate aqueous solution (1 mg / mL) prepared in the same manner, treatment and preparation were performed in the same manner as the sample 1 composition to obtain a powdery composition sample 2 composition. The obtained composition had an IFN-α adsorption amount of 9.9 mg (49% of the saturated adsorption amount), and the IFN-α content in the composition was 6.2%.

  As a control, weigh 150 mg of HAp-Zn-0.5 calcined at 400 ° C. into a plastic container with a lid, mix this with 37.5 mL of IFN-α 20 mM ammonium acetate aqueous solution (1 mg / mL), and treat the powder in the same manner. Obtained. This was mixed with 0.75 mL of a 20% (W / V) acetone aqueous solution of PLA-PEG-PLA and processed in the same manner to prepare a powdery sample 3 composition. The amount of IFN-α adsorbed in this composition was 20 mg in a saturated adsorption state, and the IFN-α content in the composition was 12.4%.

Each prepared IFN-α-containing sustained-release fine particle composition was suspended in 0.5% CMC-Na, 0.1% Tween 80, and 4 groups were administered immunosuppression by administration of tacrolimus (Astellas Pharma Inc., Tokyo). 12 mg / kg was administered subcutaneously to the back of male SD rats. As for tacrolimus, 0.1 mg / rat was administered subcutaneously on the back 3 days before and after administration of the composition, and 0.1 mg / rat every other day after the start of administration of the composition.
After administration of the composition, blood was collected from the tail vein at 4, 8, and 12 hours, and thereafter every 1 or 2 days, and the blood concentration of IFN-α was measured by ELISA. The results are shown in Table 4. A composition containing 72% or 49% of the saturated adsorption amount has a low initial excessive release compared to a control in which the adsorption amount is saturated, and the blood IFN-α concentration after the second day of administration is high. Sustained and more preferable sustained release.
For reference, the blood concentration transition in Table 4 is shown in the figure (Table 5).

Claims (21)

A composition comprising porous hydroxyapatite derivative fine particles and a protein drug, wherein the protein drug adsorption amount is 40 to 80% by weight of the saturated adsorption weight to the porous hydroxyapatite derivative fine particles. A sustained-release fine particle composition containing a protein drug. A composition comprising a porous hydroxyapatite derivative fine particle, a protein drug and a water-affinity bioerodible polymer compound, wherein the adsorption amount of the protein drug is equal to the saturated adsorption weight of the porous hydroxyapatite derivative fine particle. A protein drug-containing sustained-release fine particle composition comprising 40 to 80% by weight. A composition comprising a porous drug based on porous hydroxyapatite derivative fine particles, a protein drug, a water-soluble divalent metal compound, and a water-affinity bioerodible polymer compound, wherein the adsorption amount of the protein drug is A protein drug-containing sustained-release fine particle composition comprising 40 to 80% by weight of a saturated adsorption weight to porous hydroxyapatite derivative fine particles. A composition comprising a protein drug based on porous hydroxyapatite derivative fine particles, a water-soluble divalent metal compound, and a water-compatible bioerodible polymer compound, wherein the water-soluble divalent metal compound is zinc chloride. A protein drug-containing sustained-release fine particle composition characterized in that the content of zinc chloride is 2 to 15% by weight based on the composition. The porous hydroxyapatite derivative fine particles are porous hydroxyapatite derivative fine particles obtained by substituting a part of calcium atoms, which are constituents of porous hydroxyapatite, with zinc atoms. 10. A sustained-release fine particle composition containing the protein drug according to item. The protein drug-containing sustained-release fine particle composition according to claim 5, wherein the ratio of the number of calcium atoms and the number of zinc atoms in the porous hydroxyapatite derivative fine particles is 99: 1 to 4: 1. . The protein drug-containing sustained-release fine particle composition according to any one of claims 1 to 5, wherein the porous hydroxyapatite derivative fine particles have an average particle size of 0.5 to 30 µm. The protein drug-containing sustained-release fine particle composition according to any one of claims 1 to 5, wherein the porous hydroxyapatite derivative fine particles are treated at 150 ° C to 600 ° C. The protein drug-containing sustained release according to any one of claims 1 to 5, wherein the amount of protein drug adsorbed in the porous hydroxyapatite derivative fine particles is 4 to 25% by weight based on the composition. Fine particle composition. The protein-containing sustained-release fine particle composition according to any one of claims 3 and 4, wherein the water-soluble divalent metal compound is a zinc compound. The protein drug-containing sustained-release fine particle composition according to claim 10, wherein the zinc compound is zinc chloride, zinc acetate or zinc carbonate. The protein drug according to any one of claims 2 to 4, wherein the water-affinity bioerodible polymer compound is a block copolymer comprising polyethylene glycol and polylactic acid or polylactic acid / glycolic acid. Contained sustained release fine particle composition. The block copolymer composed of polyethylene glycol and polylactic acid or polylactic acid / glycolic acid is a triblock copolymer composed of polylactic acid or polylactic acid / glycolic acid-polyethylene glycol-polylactic acid or polylactic acid / glycolic acid. Item 13. A sustained-release fine particle composition containing a protein drug according to Item 12. 14. The weight average molecular weight of the polylactic acid or a polyblock copolymer composed of polylactic acid / glycolic acid-polyethylene glycol-polylactic acid or polylactic acid / glycolic acid is 5,000 to 12,000. A sustained-release fine particle composition containing a protein drug. The protein drug-containing sustained-release fine particles according to claim 12, wherein, in the block copolymer comprising polyethylene glycol and polylactic acid or polylactic acid / glycolic acid, the weight ratio of polyethylene glycol is 20 to 90% by weight. Composition The porous hydroxyapatite derivative fine particles obtained by adsorbing 40 to 80% by weight of the saturated adsorption weight of the protein drug in the porous hydroxyapatite derivative fine particles are mixed with the water-soluble divalent metal compound aqueous solution, stirred and then lyophilized. A protein drug-containing sustained release, wherein the powder obtained by mixing with an aqueous solution or suspension of an aqueous biocompatible polymer is stirred and then freeze-dried or vacuum-dried. For producing a fine particle composition. The powder obtained by adsorbing 40 to 80% by weight of the saturated adsorption weight of the protein drug in the porous hydroxyapatite derivative fine particles, stirring, and further freeze-drying is added to the aqueous hydrophilic bioerodible polymer aqueous solution or suspension. A method for producing a sustained-release fine particle composition containing a protein drug, which is mixed with a turbid liquid, stirred, and then freeze-dried or vacuum-dried to obtain a powder. A porous hydroxyapatite derivative fine particle and the protein drug aqueous solution or buffer containing 40 to 80% by weight of the protein drug saturated adsorption weight of the porous hydroxyapatite derivative fine particle are mixed to form a water-soluble divalent metal compound. After adding, stirring, and freeze-drying, the powder obtained by mixing with an aqueous biocompatible polymer aqueous solution or suspension is mixed into the powder, and after stirring, freeze-dried or vacuum-dried to form a powder. A method for producing a sustained-release fine particle composition containing a protein drug. The porous hydroxyapatite derivative fine particles are mixed with the protein drug aqueous solution or buffer containing 40 to 80% by weight of the protein drug saturated adsorption weight of the porous hydroxyapatite derivative fine particles. Add a weight percent aqueous solution of zinc chloride, stir and freeze-dry the powder obtained by mixing the water-affected biodissolvable polymer aqueous solution or suspension, then stir and freeze-dry or vacuum-dry. A method for producing a sustained-release fine particle composition containing a protein drug, characterized in that the powder is made into a powder. Mixing a weakly acidic aqueous solution or buffer solution having a pH of 4.0 to 6.9 containing a protein drug of 40 to 80% by weight of the saturated adsorption weight of the protein of the porous hydroxyapatite derivative fine particles with the porous hydroxyapatite derivative fine particles. Then, a powder obtained by adding 2 to 15% by weight of zinc chloride aqueous solution to the composition, stirring and freeze-drying, and polylactic acid / glycolic acid-polyethylene glycol-polylactic acid or polylactic acid / glycolic acid A method for producing a sustained-release fine particle composition containing a protein drug, which comprises mixing a triblock copolymer with an aqueous acetone solution or suspension, stirring, and freeze-drying or vacuum-drying to obtain a powder. The porous hydroxyapatite derivative fine particles and the composition are mixed with 2 to 15% by weight of zinc chloride aqueous solution, and after stirring, the protein having a saturated drug adsorption amount of 40 to 80% by weight of the porous hydroxyapatite derivative fine particles. A powder obtained by mixing a weakly acidic aqueous solution or buffer solution having a pH of 4.0 to 6.9 containing a drug and stirring and freeze-drying, and polylactic acid / glycolic acid-polyethylene glycol-polylactic acid or polylactic acid A sustained-release fine-particle composition containing a protein drug, which is mixed with an acetone aqueous solution or suspension of a triblock copolymer composed of lactic acid / glycolic acid, stirred and then freeze-dried or vacuum-dried to form a powder. Manufacturing method.