WO2008041704A1 - Controlled release preparation - Google Patents

Abstract

Disclosed is a controlled release preparation comprising a proteinaceous substance (other than an osteoclastogenesis inhibitory factor or an analogue, mutant or modified form thereof) and a porous particle comprising a calcium phosphate nanocrystal and a polysaccharide, wherein the proteinaceous substance is bound to the whole inner surface of the pore of the porous particle via a bivalent metal ion.

Description

 Specification

 Sustained release formulation

 Technical field

 [0001] The present invention provides a sustained-release preparation comprising a protein-based drug (excluding osteoclast formation inhibitory factor, its analogs, mutants and modifications thereof) bound to the entire pore inner surface of the porous particles. Related.

 Background art

 [0002] For sustained-release preparations of physiologically active substances such as proteins, a method of directly embedding a physiologically active substance with a polymer is widely used.

[0003] So far, sustained-release preparations using apatite, a kind of calcium phosphate, have been reported (Non-patent Documents 1 and 2). Non-patent document 1 reports that antibiotics are adsorbed and released slowly, while non-patent document 2 reports that adsorbed proteins are released by dissolution of calcium according to the size of the crystals. Furthermore, Non-Patent Document 3 reports sustained release of human growth hormone using apatite particles having a specific surface area of 3 m ² / g to 22 m7 g and a particle diameter of 200 nm to 500 nm. Non-Patent Document 4 reports that about 1% of human growth hormone was adsorbed on a particle having a particle size of 40 01 to 80 01 and sustainedly released. However, the sustained-release preparations using apatite reported in these publications were not in the form of actively releasing the drug from calcium phosphate.

 [0004] In addition, W. Paul et al. Have reported many techniques for sustained release of proteins from calcium phosphate granules (Non-Patent Documents 5 to 8). In these reports, a coating using a biodegradable polymer such as polylactic acid (PLA) or polyethylene butyl acetate (PEVA) is performed for sustained release. The particles used are 200 to 1000 m in size and are dense bodies fired at 1100 ° C.

[0005] Further, sustained-release preparations of injectable proteins using apatite particles (excluding Balta) are disclosed in, for example, Patent Documents;! -2 and Non-Patent Document 9. In Patent Document 1, the pores present in porous hydroxyapatite fine particles are filled with a biologically active agent, human serum protein, mucopolysaccharide, and blocked by adding divalent metal ions. A sustained release composition characterized by the above is disclosed.

[0006] In addition, Patent Document 2 discloses a sustained release property of human growth hormone characterized by comprising a porous apatite derivative and human growth hormone and a water-soluble divalent metal compound contained in the porous apatite derivative. A particulate formulation is disclosed.

[0007] In Non-Patent Document 9, porous apatite particles are impregnated with a protein such as interferon α, and are slowly released by adding zinc ions!

[0008] Patent Document 3 discloses a composite particle of a calcium compound and glycosaminodarican produced by a spray dry method and a production method thereof. However, although Patent Document 3 describes that the composite particle is a useful material as a bone filler, a cell scaffold material, and a chromatographic material, it is used for sustained-release preparations. There is no mention of what can be done.

 Patent Document 1: JP 2004-75662 A

 Patent Document 2: JP-A-2005-8545

 Patent Document 3: JP 2004-236895 A

 Non-Patent Document l¾ l: K. Yamamura et al., “Joumal of Biomedical Materials Research J, 1992 Cattle, Vol. 26, .1053-1064

 Non-patent literature 2: Tsuji Matsumoto et al., “Biomaterials”, 2004, Vol. 25, .3807-3812 Non-patent literature 3: Tsuji Gautier et al., “Joumal of Biomedical Materials Research J, 1998, No.

Volume 40, .606-613

 Non-Patent Document 4: Guicheux et al., “Joumal of Biomedical Materials Research J, 1997, 34, p.165-170

 Non-Patent Document 5: W. Paul and CP. Sharma, "Journal of Materials Science Letters", 199, 7, Vol. 16, .2050-2051

 Non-Patent Document 6: W. Paul and CP. Sharma, “Journal of materials Science-materials in meditinej, 1999, Vol. 10, p.383-388

 Non-Patent Document 7: W. Paul et al., "Journal of Biomedical Materials Research J, 2002, 61st, .660-662

Non-Patent Document 8: W. Paul and CP. Sharma, “Journal of Biomaterials Applications J 2003 Year, Volume 17, .253-264

 Non-Patent Document 9: Mizushima et al., “Journal of Controlled Release J, 2006, Vol. 110, No. 2, p.260-265

 Disclosure of the invention

 Problems to be solved by the invention

[0009] In view of the above situation, the present invention is to provide a sustained-release preparation of a protein-based drug.

 Means for solving the problem

 [0010] As a result of diligent investigations such as a preparation method for releasing a drug from composite porous particles composed of calcium phosphate nanocrystals and polysaccharides produced by the same method as in Patent Document 3, a protein-based drug is gradually released. The composite porous particles are optimal as a base material to be used, and when the sustained release is further achieved, zero-order release with controlled initial burst is achieved by using divalent metal ions, and the present invention is completed. It came to do.

 [0011] The present invention includes the following.

 [0012] (1) Sustained release containing porous particles containing protein-based drugs (excluding osteoclast formation inhibitors, their analogs, mutants and their modifications), calcium phosphate nanocrystals and polysaccharides The sustained-release preparation, characterized in that the protein-based drug and the whole pore inner surface of the porous particle are bound via a divalent metal ion!

 [0013] (2) The sustained-release preparation according to (1), characterized in that calcium phosphate nanocrystals and polysaccharides are uniformly dispersed in the porous particles.

 [0014] (3) It is characterized in that it contains a divalent metal selected from the group consisting of magnesium, zinc, strontium and barium together with the calcium phosphate strength S, calcium, or in place of a part of calcium. (2) The sustained release preparation according to (1) or (2).

 [0015] (4) The sustained-release preparation according to any one of (1) to (3), wherein the molar specific force of calcium to phosphorus in the calcium phosphate is Sl.3 to 3.4. .

 [0016] (5) The sustained-release preparation according to any one of (1) to (4), wherein the calcium phosphate contains a carbonate group.

[0017] (6) The major axis force of the calcium phosphate nanocrystal is SIX 10 ² nm or less, The sustained release preparation according to any one of (1) to (5).

[0018] (7) The sustained-release preparation according to (6), wherein the major axis is ¹ nm to l X 10 ² nm.

[0019] (8) The polysaccharide of (1) to (7), wherein the polysaccharide is selected from the group consisting of chondroitin sulfate, hyaluronic acid, heparin and heparan sulfate. /, Displacement or sustained release preparation according to 1.

 [0020] (9) The sustained-release preparation according to (8), wherein the polysaccharide is chondroitin sulfate or hyaluronic acid.

 [0021] (10) The sustained-release preparation according to any one of (1) to (9) above, wherein the content of the polysaccharide is 4 to 10% by weight or less.

[0022] (11) wherein the content Ca 10% to 1 10 ¹ wt% Dearu characterized, (10) SL placement of sustained-release preparations.

 [0023] (12) The diameter force of the porous particles is S I X lO ^ m or less, (1) to (; 11

The sustained release preparation according to 1).

[0024] (13) The sustained-release preparation according to (12), wherein the diameter is from 101 to 1 10 ² 01.

[0025] (14) the you wherein the specific surface area of the porous particles is ^{5 X 10m 2 / g~2 X 10} 2 m 2 / g, (1) ~; a / (13),! The sustained-release preparation according to claim 1.

(15) The porous particles have a porosity power X 10% to 8 X 10%, (1)

The sustained release preparation according to any one of to ( ₁₄ ).

[0027] (16) The in vivo absorption of the porous particles is within 6 months, (1) to (1

The sustained release preparation according to 1)!

[0028] (17) The sustained release preparation according to any one of (1) to (; 16), wherein the porous particles are produced by spray drying.

[0029] (18) The divalent metal ion is selected from the group consisting of zinc ion, magnesium ion, calcium ion, strontium ion, norium ion and copper ion. ) To (; 17)! /, Any one of the sustained release preparations.

[0030] (19) The sustained-release preparation according to (18), wherein the divalent metal ion is a zinc ion. [0031] (20) A step of subjecting a suspension containing calcium phosphate nanocrystals and polysaccharides to spray drying to obtain porous particles containing calcium phosphate nanocrystals and polysaccharides, and the porous particles comprising: Impregnating a solution containing a protein-based drug (excluding osteoclast formation inhibitory factor, its analogs, mutants and modifications thereof) and supporting the protein-based drug on the entire inner surface of the porous particle; and A divalent metal ion-containing solution is added to the porous particles carrying the protein-based drug, thereby binding the protein-based drug and the entire pore inner surface of the porous particle via the divalent metal ions. A process for producing a sustained-release preparation, comprising a step.

 The invention's effect

 [0032] According to the present invention, there is provided a sustained-release preparation that stably and contains a large amount of a protein drug. In the sustained-release preparation according to the present invention, the binding property between the porous particles and the protein drug is improved by the divalent metal ion, and the protein drug is stably added for a certain period with almost no initial burst. Slow release.

 [0033] This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2006-271077, which is the basis for the priority of the present application.

 Brief Description of Drawings

 [0034] [FIG. 1] FIG. 1 shows BSA release behavior when the zinc ion addition amount in the binding treatment is changed.

 [Fig. 2] Fig. 2 shows the Hb release behavior when the amount of zinc ion added in the binding process is varied.

 [Fig. 3] Fig. 3 shows the cyt c release behavior when the zinc ion addition amount in the binding treatment is changed.

 [Figure 4] Figure 4 shows the release behavior of CAT.

 FIG. 5 shows the release behavior of BSA.

 FIG. 6 shows the release behavior of Hb.

 FIG. 7 shows the release behavior of Try.

 FIG. 8 shows the release behavior of Pap.

FIG. 9 shows the release behavior of cyt c. FIG. 10 shows the release behavior of LYZ.

 FIG. 11 shows HST release behavior.

 FIG. 12 shows the release behavior of each protein ((a) BSA, (b) cyt c, (c) Hb) when the amount of zinc ion added in the binding treatment is changed.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0035] Hereinafter, the present invention will be described in detail.

 [0036] The sustained-release preparation according to the present invention comprises a protein-based drug (excluding osteoclast formation inhibitory factor, its analogs, mutants and modifications thereof), calcium phosphate nanocrystals and polysaccharides. It contains porous particles. In the sustained-release preparation according to the present invention, the protein drug is supported on the entire inner surface of the pores of the porous particles. In the sustained-release preparation according to the present invention, the protein drug and the entire pore inner surface of the porous particle are bonded via a divalent metal ion.

 [0037] Here, protein-based drugs (excluding osteoclast formation inhibitory factor, its analogs, mutants and modifications thereof) include osteoclastogenesis inhibitory factor, its analogs, mutants and the same. Proteins (including polypeptides and peptides) other than these modified substances themselves or any drug containing the protein may be used. For example, protein-based drugs include those that can bind to the inner surface of the pores of a porous particle via a carboxyl group present in the protein side chain of the drug and / or those that can coordinately bond via an amino group. Can be mentioned. Specifically, examples of protein drugs include erythropoietin (EPO), granulocyte colony stimulating factor (G_CSF), granulocyte-macrophage colony stimulating factor (GM_CSF), thrombopoietin, interferon α, interferon β, interferon γ , Urokinase, tissue plasminogen activator (t_PA), interleukin-11 (IL-11), anti-TNF-α antibody, fibroblast growth factor (FGF), epidermal growth factor (EGF), hepatocytes Growth factor (HGF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), leptin, neutrophin-3 (NT-3), superoxide dismutase (SOD), insulin, human growth hormone, bone formation Factor (BMP group) and the like.

 [0038] On the other hand, calcium phosphate nanocrystals are tricalcium phosphate (Ca (PO))

 3 4 2

Lucium (Ca H (PO) · 5Η Ο), apatite compound, etc., which is smaller than the magnitude force _μ η This is a general term for a crystal. An apatite compound is a general formula Ca (PO X) Y (wherein

 10 4 6 2

 X represents a carbonate group or deficiency, and Y represents a hydroxyl group, a carbonate group, a halogen group or a deficiency), and hydroxyapatite, carbonate apatite, fluorapatite, chlorapatite, and the like. In addition, calcium phosphate may contain one or more divalent metals such as magnesium, zinc, strontium and barium, together with calcium or in place of calcium. In addition, the molar ratio of calcium to phosphorus in calcium phosphate is, for example, 1 · 3 to 3 · 4, preferably 1 · 5 to 3 · 0.

[0039] Regarding the production of calcium phosphate nanocrystals, for example, when producing hydroxyapatite nanocrystals, they can be produced by a wet method and cannot be carried out according to the following formula (H. Aoki, " Medical Applications of hydroxyapatitej, 1994, IshiyaKU iiuroAmerica, Inc., Tokyo, St.Lousi) _D

[Chemical 1] lOCa (OH) ₂ + 6H ₃ (P0 ₄ ) ₃ → Ca ₁₀ (P0 ₄ ) ₆ (OH) ₂

[0040] The major axis of the calcium phosphate nanocrystals thus produced is, for example, 1 X 10 ² nm or less, preferably 1 nm to 1 X 10 ² nm, particularly preferably 1 X 10 nm to 5 X 10 nm.

[0041] In the present invention, examples of the polysaccharide include chondroitin sulfate, hyaluronic acid, heparin and heparan sulfate, and chondroitin sulfate and hyaluronic acid are preferable. Incidentally, the polysaccharide content of sustained-release preparation according to the present invention, for example, 4 X 10 wt% or less, preferably 4 10 wt% to 1 ^10-1% by weight, particularly preferably from 1% to 2 10 weight %. When chondroitin sulfate is used as the polysaccharide, for example, one having a molecular weight of 10 kDa to 40 kDa, preferably 15 kDa to 35 kDa is used.

 [0042] The sustained-release preparation according to the present invention is produced from the protein drug, calcium phosphate nanocrystal, and polysaccharide described above.

[0043] In the method for producing a sustained-release preparation according to the present invention (hereinafter referred to as "the present method"), first, a suspension containing calcium phosphate nanocrystals and a polysaccharide is subjected to spray drying. Porous particles containing calcium phosphate nanocrystals and polysaccharides. In the porous particles, it is preferable that the calcium phosphate nanocrystals and the polysaccharide are uniformly dispersed. Yes. The amount of the polysaccharide with respect to the calcium phosphate nanocrystal is, for example, 1% by weight to 210% by weight, preferably 2% by weight to 110% by weight with respect to the calcium phosphate nanocrystal. The pH of the suspension is adjusted to, for example, 5 to 9, preferably 6 to 8. Furthermore, the suspension containing calcium phosphate nanocrystals and polysaccharides is aged for, for example, 0 to 12 hours (preferably 1 to 4 hours) in order to promote complexing while continuing the stirring operation. After that, it is used for spray drying. Here, aging means that the final pH is kept constant in the range of 7.5 to 8.0, and the surface state of the crystals obtained by complexing is kept stable by maintaining almost the same pH as the living body. Specifically, aging is carried out by setting the final pH using a neutralization reaction of strong alkali (calcium hydroxide) and strong acid (phosphoric acid).

[0044] The spray drying increases the force S and surface area that 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. For this purpose, the suspension is made into fine droplets of about 1 m to 5 × 10 ² m, blown into hot air of 1 × 10 ² ° C. to 3 × 10 ² ° C. and dried.

[0045] The diameter of the porous particles obtained after spray drying in this way is, for example, 1 X 10 ² µm or less, preferably 1 ^ 01 to 1 10 ² ^ 01, particularly preferably 1 ^ 01 to 3 10 ^ 01. The porous particles may, for example, specific surface area ^{5 X 10m 2 / g~2 X 10} 2 m 2 / g, a porosity of 3 X 10% ~8 X 10% . If the specific surface area of the porous particles is less than X 10 m ² / g, a sufficient amount of protein drug cannot be supported, and the sustained release force over the intended release period cannot be achieved. On the other hand, when the porosity power of the porous particles is less than ¾ X 10%, the tannic drug cannot be uniformly supported inside the particles, and when it exceeds 8 X 10%, the porous particles Breaks during formulation.

 The in vivo absorption of the porous particles obtained after spray drying is, for example, within 6 months, preferably 1/2 month to 3 months, particularly preferably 1 month to 2 months.

[0047] Next, in this method, the porous particles obtained after spray drying are impregnated in a protein drug-containing solution to be loaded (for example, in the form of an aqueous solution containing a protein drug to be loaded). Then, they are uniformly dispersed by mixing, and the protein drug is supported on the entire inner surface of the pores of the porous particles. Amount of protein based drugs bearing subject, for example a multi-porous particles lmg per ^{1 X 10- 1 ^ g~2 X 10} 2 ^ g, preferably to ^{1 ^ g~ 1 X 10 2 ^} g. Many The mixing of the porous particles and the protein drug to be supported is performed, for example, by inverting and stirring for 1 hour to 12 hours (preferably 3 hours to 6 hours).

 [0048] Further, after mixing, the mixture containing the porous particles and the protein drug to be loaded is subjected to centrifugation, and the supernatant (supernatant) is removed. At this stage, it can be subjected to a binding treatment with divalent metal ions without performing centrifugation.

 [0049] Evaluation of the loading of the protein-based drug to be loaded on the entire inner surface of the porous particle was performed by using a specific antibody against the supernatant (supernatant) removed after the centrifugation described above. It can be performed by subjecting it to an immunological technique (for example, ELISA) and measuring the amount of the protein-based drug to be loaded remaining in the supernatant. As a result of this evaluation, it was confirmed that the protein drug was sufficiently loaded on the entire inner surface of the porous particle by confirming that the protein drug to be loaded was slightly retained and did not remain in the supernatant. IJ refusing power S

 [0050] Next, a divalent metal ion-containing solution is added to the obtained mixture containing porous particles carrying a protein drug and subjected to a binding treatment. By this step, the protein drug and the porous particles carrying the protein drug are bound.

 [0051] In the present invention, the term "bond" means that the calcium phosphate surface and a protein drug are supported / bonded by coordination, ion, covalent bond, etc. via a metal ion, or a polysaccharide and a protein. It means inducing a bond between functional groups of a systemic drug, or inducing a bond between the calcium phosphate surface, a protein system drug and a polysaccharide. According to the binding, the protein drug and the entire pore inner surface of the porous particle are bound via the divalent metal ion.

[0052] As the divalent metal ion used for bonding, a force zincion including zinc ion, magnesium ion, calcium ion, strontium ion, norlium ion and copper ion is optimal. Divalent metal I protein-based drugs for carrying porous particles O emissions amount can be, for example, a protein-based drug carrying porous particles lmg per 1 X 10- ³ mg~lmg, preferably 1 X 10- ² mg~l Let X 10—g.

[0053] In the binding treatment, the divalent metal ion-containing solution is added to the mixture containing the porous particles carrying the protein drug, and then stirred by overturning. Then, after stirring by overturning, centrifuge the mixture Use for separation and remove supernatant (supernatant). Furthermore, after the residue obtained by removing the supernatant is washed with, for example, purified water, the precipitate is subjected to lyophilization or vacuum drying, for example, for 1 hour to 24 hours (preferably 1 hour to 12 hours). In this way, it is possible to obtain the sustained-release preparation according to the present invention with the force S.

 [0054] The sustained-release preparation according to the present invention exhibits a long-term sustained-release property of at least 7 days (for example, 7 days to 180 days, particularly 14 days to 30 days) with respect to the protein drug carried.

Yes

 [0055] Further, the sustained-release preparation of the present invention can be used alone or in combination with a pharmaceutically acceptable additive. The disease to be treated by the sustained-release preparation according to the present invention is appropriately determined according to the action and effect of the protein drug carried on the porous particles in the sustained-release preparation according to the present invention.

 [0056] The sustained-release preparation of the present invention can be prepared in various dosage forms and administered systemically or locally orally or parenterally. When orally administering the sustained-release preparation according to the present invention, it is formulated into tablets, capsules, granules, powders, pills, liquids for internal use, suspensions, emulsions, syrups, etc. It may be a dry product that is redissolved in When the sustained-release preparation of the present invention is administered parenterally, it is formulated into an intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, etc. In the case of pharmaceutical preparations, they are provided in the form of unit dose ampoules or multiple dose containers.

 [0057] These various preparations include excipients, extenders, binders, wetting agents, disintegrating agents, lubricants, surfactants, dispersants, buffers, preservatives, and solubilizing agents that are usually used in preparations. In addition, preservatives, flavoring agents, soothing agents, stabilizers, tonicity agents and the like can be appropriately selected and produced by conventional methods.

 [0058] The route of administration of the sustained-release preparation according to the present invention is a force that can be appropriately determined according to the use, for example, oral, intraperitoneal, intravenous, intraarterial, transdermal, subcutaneous, intramuscular, etc. Are listed. The administration route also includes direct administration near the affected area.

[0059] The dosage of the sustained-release preparation according to the present invention can be appropriately determined according to the age, sex, symptom, administration route, number of administrations, etc. of the administration subject. For example, an effective amount of a protein-based drug product encompassed, doses ranging from ^{1 X 10- 2 mg~l X 10 2} mg per body weight lkg per dose It is preferable to administer the drug several times a day to several times a month.

[0060] As described above, according to the sustained-release preparation of the present invention, a specific protein drug can be sustained-released stably for a certain period of time.

[0061] Further, the sustained-release preparation according to the present invention, unlike the sustained-release composition described in Patent Document 1, contains human serum protein as a base material (that is, not as a supported protein drug). In addition, it is not necessary to fill the pores of the particles with mucopolysaccharides.

 Example

 [0062] Hereinafter, the ability to explain the present invention in more detail with reference to examples. The technical scope of the present invention is not limited to these examples.

[Example 1] Protein-based drug release test of hydroxyapatite / chondroitin sulfate complex (hereinafter referred to as “HAp / ChS”) porous particles (corresponding to porous particles in the present invention)

 In this example, a protein-based drug release test of HAp / ChS porous particles was performed. More

In addition, we tried to control the release characteristics of protein drugs by formulation conditions using zinc ions.

 [0064] Therefore, changes in release behavior when (1) the amount of zinc ion added and (2) the molecular weight of chondroitin sulfate (hereinafter referred to as “ChS” and! /) Were changed were examined.

 Regarding (1), it has been clarified how much zinc ions can be added in practical use to suppress the initial burst by changing the amount of zinc ions added.

 [0066] On the other hand, with regard to (2), the ChS elution rate from the HAp / ChS porous particles in PBS varies depending on the molecular weight. In addition, in the adsorption characteristics, the maximum adsorption amount of protein and the adsorption equilibrium constant differ depending on the molecular weight of ChS (H. Watanabe, T. Ikoma, P. nen, J. Tanaka, Fl ransactions of the Materials Research society of Jap anj, vols. 31 (2) (2006) 341-344). Based on these facts, it is expected that the release characteristics of protein drugs may change due to changes in the molecular weight of ChS. Therefore, we examined the effect of ChS molecular weight change on the release characteristics of each protein drug.

 [0067] 1. Sample preparation

 1-1. Synthesis of HAp / ChS porous particles

0.25 mol / l calcium hydroxide in which ChS (molecular weight 15 kDa, 20 kDa or 35 kDa) is dispersed A 0.15 mol / l phosphoric acid aqueous solution was added dropwise to the suspension, and the final pH was adjusted to 7.5 to 8.0 to prepare a HAp / C hS gel. The amount of ChS added was 2% by weight with respect to the ideal hydroxyapatite (hereinafter referred to as “HAp”) weight. After aging for 12 hours, HAp / ChS porous particles were obtained with a spray dryer (trade name: Mini Spray Dryer B-290 Buchi).

 [0068] The obtained HAp / ChS porous particles were subjected to particle size distribution measurement. As a result, the obtained spherical HAp / ChS porous particles had a distribution of 1 μm to 20 μm and an average particle diameter of 4 μm.

 [0069] 1-2. Protein drugs to be loaded

 There are 8 proteins with different isoelectric points: Catalase (hereinafter referred to as “CAT”, molecular weight (MW): 240 kDa, isoelectric point (pi): 4.5), urine serum albumin (Hereinafter referred to as “BSA”, ring '.66 kDa, pi: 4 · 8), hemoglobin (hereinafter referred to as “Hb”, ring' .65 kDa, pi: 6.8-7.0), trypsinogen (hereinafter referred to as “Try”) , Ring '.24kDa, pi: 9 • 3), papain (hereinafter referred to as “Pap”, ring' .24kDa, pi: 8 · 8_9 · 5), cytochrome C (hereinafter referred to as “cyt c”, MW: 12.4) kDa, pi: 10.2), lysozyme (hereinafter referred to as “LYZ”, MW: 14 kDa, pi: 10.5), and histone (hereinafter referred to as “HST”, MW: 12 kDa, pi: 11.0).

 [0070] 1-3. Sample preparation

 200ml particles (pure HAp, ^^ / (¾5 (151 ^ &: 2wt%) porous particles, HAp / ChS (20kDa: 2) in 5ml each protein aqueous solution (800 g / ml, dissolved in 10% PBS) (Wt%) porous particles or HAp / ChS (35 kDa: 2 wt%) porous particles) was added and allowed to invert for 4 hours at room temperature, that is, up to 20 g of protein was supported per mg of particles.

 [0071] For unbound particles (without binding treatment), after 4 hours of inversion stirring, centrifugation (3500 rpm,

 10 minutes), the supernatant was removed, and the precipitate was freeze-dried overnight.

[0072] On the other hand, for the bound particles (with binding treatment), after 4 hours of overturning stirring, add lml, 2ml or 3ml zinc chloride aqueous solution (10mg / ml, adjusted to pH 5.5 with 0.1M HC1 aqueous solution) The binding process was performed by inversion for 2 hours. That is, the added amount of zinc chloride was changed to 10 mg, 20 mg, and 30 mg, respectively. According to the binding treatment, each protein and the whole pore inner surface of the porous particle are bound via zinc ions. [0073] After stirring by inverting for 2 hours, the supernatant was separated by centrifugation (3500 rpm, 10 minutes), and the precipitate was freeze-dried overnight. The amount of adsorption was calculated by quantifying the protein concentration of the supernatant after centrifugation with ADV01 (Advanced Protein Assay Reagent). Only three types of BSA, Hb, and cyt c were examined for release behavior due to changes in the amount of zinc ion added.

 [0074] 2. Release test

 The sample formulated in 1-3 above (HAp / ChS porous particles carrying each protein) was measured for lOmg, immersed in 5 ml of PBS, and stirred by inversion at 36.5 ° C. Preliminary experiments indicate that protein aggregation occurs when the rotational speed of rotary stirring is too high. It is also known that protein adsorbs on the wall of the container. Therefore, the rotation speed was set to the lowest speed, and the test was performed using a protein low adsorption centrifuge tube (manufactured by Sumitomo Bakelite).

 [0075] The release test was conducted for a period of 1 hour to 1 week. The stirred sample was subjected to centrifugation, the supernatant was extracted, and the amount released was quantified by ADV01.

[0076] 3. Results

 3-1. Adsorption rate of each protein to each HAp / ChS porous particle

 Table 1 below shows the protein adsorption rate for each HAp / ChS porous particle.

[table 1]

//: / O1 £ / J AV

S'6L Z'L6 6 "66 9" 86 ε6% S (^ OTS8) ^T iO / ^d VH

L'99 6 · 6 6 * 66 Vl 0Ί6 0 · 68 9'69% δ (Β (back) S O / dVH

^ OL V 6 6 * 66 8 '6 Z'ZQ ^ • 88 ⁰ / oS (^ OTST) S¾0 / ^d VH

9Έ8 Z'9 6 * 6668 Z'L L 'Q f'16

 JLSH ΖΑΊ died vsa ¾. 、 / Be ^ \:

(%) * Recruitment ^

[0077] In Table 1, each abbreviation is as follows.

 [0078] “HAp”: hydroxyapatite only, “HAp / ChS (15 kDa) 2%”: HAp / ChS (molecular weight: 15 kDa, 2 wt%) porous particles (without binding treatment), “HAp / ChS (20 kDa) ) 2% ”: HAp / ChS (molecular weight: 20 kDa, 2% by weight) porous particles (without binding treatment),“ HAp / ChS (35 kDa) 2% ”: HAp / ChS (molecular weight: 35 kDa, 2% by weight) porous Particle (without binding treatment), “HAp + 3Zn”: hydroxyapatite only (with 3 ml zinc chloride aqueous solution), “HAp / ChS (15kDa) 2% + 3Zn”: HAp / ChS (molecular weight: 15kDa, 2wt%) porous particles (with binding treatment with 3ml zinc chloride aqueous solution), "HAp / ChS (20kDa) 2% + 3Zn": HAp / ChS (molecular weight: 20kDa, 2wt%) porous particles ( "HAp / ChS (35kDa) 2% + 3Zn": HA p / ChS (Molecular weight: 35kDa, 2wt%) Porous particles (With 3ml zinc chloride aqueous solution) )

 In the following results of this example, the abbreviations of the sample names are also the same (however, Γ + lZnj is “binding treatment with 1 ml of zinc chloride aqueous solution” and “+ 2Zn” is “with 2 ml of zinc chloride aqueous solution”) "With join processing").

[0079] As described in 1-3 above, since 4 mg of each protein is added to 200 mg of each HAp / ChS porous particle, 20 mg of protein is adsorbed at a maximum per lmg of particle. In the following release test results, the ratio of the amount of protein released relative to the amount carried is expressed as the release rate.

 [0080] 3-2. Effects of changes in zinc ion addition on protein release characteristics

 Figures 1 to 3 show the protein release behavior when the amount of zinc ion added in the binding process is changed. Figures 1, 2, and 3 show the release curves for BSA, Hb, and cyt c, respectively.

[0081] As shown in the figures;! To 3, the initial burst was reduced as the zinc ion addition amount increased. However, with regard to BSA (Fig. 1), the amount released with time increased as the amount of zinc ion added increased, and the total amount released for one week was almost the same regardless of the amount of zinc ion added. On the other hand, with regard to Hb (Fig. 2), the initial burst could be completely suppressed when the amount of zinc chloride added was 20 mg or more. For unbound particles, the amount of release increased over time after the initial burst. Regarding cyt c (Fig. 3), there was a slight increase in the amount of released particles over time for the bound particles. [0082] 3-3. Release behavior of proteins with different isoelectric points

 3-3-1. Release behavior of acidic protein (CAT, BSA, Hb)

 Figures 4-6 show the release behavior of CAT, BSA, and Hb, respectively. 4 to 6, (a) shows the protein release behavior from the unbound particles, and (b) shows the protein release behavior from the bound particles.

 [0083] (1) Regarding CAT (Fig. 4)

 Unbound particles: about 3% was released within 1 hour after immersion, and then an unstable increase in the amount released was observed. The release behavior was dependent on ChS molecular weight!

[0084] Binding particles: The initial release amount was suppressed to less than 1%, and after that, an almost linear increase in the release amount was observed. Pure HAp had a slightly lower release compared to other HAp / ChS porous particles.

[0085] (2) Regarding BSA (Figure 5)

 Unbound particles: An initial burst occurred and no subsequent increase in release was observed. Release amount increased with increasing molecular weight of Ch S. Moreover, pure HAp released less than HAp / ChS porous particles.

 [0086] Binding particles: The release amount after 1 hour of immersion could be suppressed, but the release amount increased with time. The release behavior was dependent on ChS molecular weight!

[0087] (3) Regarding Hb (Fig. 6)

 Unbound particles: About 30% of the release was observed 1 hour after immersion. After one week, the release increased to about 60%.

[0088] Binding particles: All the released amounts after 1 hour of immersion were less than 1%. After one week, it gradually increased to about 2%.

[0089] 3-3-2. Basic protein release behavior (Try, Pap, cyt c, LYZ, HST)

 Figures 7 to 11 show the release behavior of Try, Pap, cyt c, LYZ, and HST, respectively. 7 to 11, (a) shows the protein release behavior from the unbound particles, and (b) shows the protein release behavior from the bound particles.

[0090] (1) Regarding Try (Fig. 7)

Unbound particles: More than 60% of initial burst occurred 1 hour after immersion. After that, the amount released decreased. When performing the release test, rotate slowly and keep the container in protein. The one with low quality adsorption was used. Therefore, protein aggregation and adsorption to the container wall cannot be considered, and it is thought that the protein was re-adsorbed to the particles.

[0091] Bound particles: The initial burst could hardly be suppressed. After that, as with unbound, the amount released decreased. This is also considered to be caused by re-adsorption to the particles.

[0092] (2) Regarding Pap (Figure 8)

 Unbound particles: Force released after 1 hour was less than 10%. Since the rate of increase in the amount released thereafter was high, it was released to nearly 100% in one week.

[0093] Bound particles: Similar to unbound particles, the release after 1 hour of immersion was less than 10%, but the release rate after 1 week increased to nearly 100%. However, only pure HAp was about 50%

Yes

 [0094] (3) cyt c (Figure 9)

 Unbound particles: An initial burst with a release rate of about 40% occurred, and the subsequent increase / decrease in the increase rate showed an unstable tendency.

[0095] Binding particles: An increase rate of about 1 to 2% after 1 hour of immersion, and an increase to about 10% after 1 week.

[0096] (4) Regarding LYZ (Fig. 10)

 Unbound particles: An initial burst of 50% or more was observed, and there was no time dependence of the amount released

. The amount of release decreased as the ChS molecular weight increased. Pure HAp released less than other HAp / ChS porous particles.

[0097] Bound particles: Pure HAp was removed !, and the initial burst could be slightly reduced by binding. Furthermore, the release amount increased linearly with time. The release amount decreased as the ChS molecular weight increased.

[0098] (5) Regarding HST (Figure 11)

 Unbound particles: The amount released after 1 hour of immersion was about 15 to 20%. Since then, the amount released has decreased. This is also thought to be re-adsorbed to the particles as in the case of Try. HAp / ChS (molecular weight: 35 kDa, 2% by weight) porous particles had the lowest released amount.

[0099] Bonded particles: The initial burst could be reduced to 10% or less by bonding. Subsequent releases decreased slightly. [0100] 4. Discussion

 The binding treatment with zinc ions could reduce the initial burst of all nine proteins. However, the degree of reduction varied depending on the protein, and did not depend on the isoelectric point.

 [0101] In particular, a protein that can almost completely suppress the initial burst can be expected to have a sustained release effect in vivo as well as control of the biodegradability of the particles.

[0102] [Example 2] Change in release behavior of protein drugs when the amount of zinc ion added in the binding treatment is changed

 In Example 1, it was verified that the initial burst was suppressed by the binding treatment using zinc ions during the formulation process. However, considering practical application, it is desirable that the amount of zinc ion added is as small as possible. Therefore, in this example, changes in the release behavior of protein drugs when the amount of zinc ion added in the binding treatment was changed were examined.

 [0103] 1. Sample preparation

 The same HAp / ChS porous particles as in Example 1 were used. On the other hand, BSA, cyt c, and Hb were used as protein drugs to be loaded.

 An 800 μg / ml protein (BSA, cyt c, or Hb) aqueous solution was prepared using 10% PBS as a solvent. 200 mg of HAp / ChS (molecular weight: 15 kDa, 2% by weight) porous particles were immersed in 5 ml of each aqueous solution and adsorbed by rotating and stirring for 4 hours.

 [0105] After rotating and stirring for 4 hours, add lml, 2ml and 3ml of 30mg / ml zinc chloride aqueous solution (adjusted to pH5.5 by adding 0.1M HC1 aqueous solution) and rotate for 2 hours to combine. (The sample names are called “HAp / ChS (15kDa) 2% + lZn”, “HAp / ChS (15kDa) 2% + 2Zn”, “HAp / ChS (15kDa) 2% + 3Zn”). ). According to the binding treatment, each protein and the whole pore inner surface of the porous particle are bound via zinc ions.

 [0106] After rotating and stirring for 2 hours, the supernatant was separated by centrifugation, and the precipitate was freeze-dried overnight.

 [0107] As in Example 1, a sample of unbound particles (HAp / ChS (15 kDa) 2%) not subjected to binding treatment was also prepared.

[0108] 2. Release test Each sample prepared in 1 was weighed 10 mg, immersed in 5 ml of PBS, gently stirred at 36.5 ° C., and rotated and stirred.

 [0109] The release test was conducted for a period of 1 hour to 1 week. The amount released was quantified by centrifuging the centrifuge tube and measuring the protein concentration in the supernatant. Protein quantification was done with ADV01.

 [0110] 3. Results

 Figure 12 shows each protein ((a) when the amount of zinc ion added in the binding treatment is changed.

The release behavior of BSA, (b) cyt c, (c) Hb) is shown.

[0111] In both BSA (Fig. 12 (a)) and cyt c (Fig. 12 (b)), the initial release amount decreased as the zinc ion addition amount increased. With regard to cyt c, there was no increase in the amount released over time. With regard to BSA, the amount released in one week was almost the same regardless of the amount of zinc ion added.

[0112] For Hb (Fig. 12 (c)), the initial release amount was not measured. Also, HAp / ChS (15kDa) 2% + 2Zn almost reduced the release amount! /.

 For HAp / ChS (15 kDa) 2% + 2Zn and HAp / ChS (15 kDa) 2% + 3Zn, almost no Hb was released for one week.

[0113] All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

The scope of the claims  [I] protein drugs (excluding osteoclast formation inhibitors, their analogs, mutants and their modifications),  A sustained-release preparation comprising porous particles containing calcium phosphate nanocrystals and polysaccharides, characterized in that the protein drug and the entire inner surface of the pores of the porous particles are bound via a divalent metal ion. The sustained-release preparation.  [2] The sustained-release preparation according to claim 1, wherein the porous particles are uniformly dispersed with calcium phosphate nanocrystals and polysaccharides. [3] The dibasic metal selected from the group consisting of magnesium, zinc, strontium, and barium, together with the calcium phosphate strength S, calcium, or in place of a part of calcium. Or the sustained-release preparation according to 2. [4] The sustained-release preparation according to any one of claims 1 to 3, wherein the molar specific force of calcium to phosphorus in the calcium phosphate is 1 · 3 to 3 · 4. [5] The sustained-release preparation according to any one of [1] to [4] above, wherein the calcium phosphate contains a carbonate group. [6] The sustained release preparation according to any one of claims 1 to 5, wherein the calcium phosphate nanocrystal has a major axis force of SIX 10 ² nm or less. 7. The sustained-release preparation according to claim 6, wherein the major axis is 1 to 1 × 10 ² . [8] The slow force according to any one of claims 1 to 7, wherein the polysaccharide is selected from the group consisting of chondroitin sulfate, hyaluronic acid, heparin and heparan sulfate. Release formulation.  [9] The sustained-release preparation according to claim 8, wherein the polysaccharide is chondroitin sulfate or hyaluronic acid.  [10] The sustained-release preparation according to [1], wherein the content of the polysaccharide is 4 × 10% by weight or less; [II], wherein the content is 4 X 10 wt% to 1 X 10- ¹ wt%, 10. Symbol mounting of sustained-release preparations. [12] The diameter force of the porous particle is SIX 10 ² m or less, The claim; V, slippery or sustained release preparation according to 1. [13] The sustained-release preparation according to claim 12, wherein the diameter is 1 am to l × 10 ² m. [14], wherein the specific surface area of the porous particles is ^{5 X 10m 2 / g~2 X 10} 2 m 2 / g,請Motomeko 1; any force 13, Xu according item 1 Release formulation. [15] The porosity of the porous particles is 3 × 10% to 8 × 10%, Any force to _14, sustained-release preparation according (1). [16] The sustained-release preparation according to [1], wherein the porous particles are absorbed in vivo within 6 months. [17] The sustained-release preparation according to [1], wherein the porous particles are produced by spray drying. [18] The divalent metal ion is selected from the group consisting of zinc ion, magnesium ion, calcium ion, strontium ion, norium ion and copper ion. Any one of 17 powers, sustained-release preparation according to 1. [19] The sustained-release preparation according to claim 18, wherein the divalent metal ion is a zinc ion.  [20] A step of subjecting the suspension containing calcium phosphate nanocrystals and polysaccharides to spray drying to obtain porous particles containing phosphoric acid ricum nanocrystals and polysaccharides;  The porous particles are impregnated in a solution containing a protein-based drug (excluding osteoclast formation inhibitory factor, its analogs, variants, and modifications thereof), and the protein-based drug is added to the entire pore inner surface of the porous particles. A process of supporting the  A divalent metal ion-containing solution is added to the porous particles carrying the protein-based drug, thereby binding the protein-based drug and the entire pore inner surface of the porous particle via the divalent metal ions. Process,  A method for producing a sustained-release preparation, comprising: