JP2008120838A - Method for preparation of medicinal substance-polymer complex utilizing coordinate bond - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple method for preparing a medicinal substance-polymer complex. <P>SOLUTION: A medicine unstable in vivo is coordinatively bonded to a polymer carrier through metal ion but not chemical bond. A medicine having a chelating ability and a carrier including the medicine or diagnostic agents and a high molecular material having chelating ability are mixed together in the presence of metal ion thereby medicinal substance-high polymer complex having extremely marked expression of the drug action is obtained. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a drug-polymer complex preparation comprising a therapeutic agent or diagnostic agent by a coordinate bond via a metal ion and a polymer, and to modification of the pharmacokinetics of the drug by complexing with the polymer and its slowdown. It relates to release and enhancement of its action.

  In general, a drug has a short half-life in vivo, and an expected medicinal effect cannot be obtained only by administration as an aqueous solution. Therefore, to improve the in vivo stability of the drug (for example, prolong the blood life of the drug), formulate the drug so that it can be gradually released over a certain period of time (drug release) Is desirable. In addition, only drugs that reach the target site of action while maintaining activity among the drugs administered in vivo show therapeutic and diagnostic effects, and the remaining part becomes invalid or unnecessary in some cases It is known to cause side effects. Therefore, it is necessary to effectively perform a drug treatment method by selectively allowing a drug to act on a target site (drug targeting). For these purposes, a drug delivery system modification of a drug using a polymer material is currently being performed. The same is true for diagnostics. That is, targeting of a drug to a target site to be diagnosed further enhances the diagnostic effect. Therefore, if targeting ability can be imparted to a drug, the dosage of the drug for expressing the pharmacological action of the therapeutic agent and the effect of the diagnostic agent can be reduced, and the therapeutic agent by large dose is particularly problematic. Reduction of side effects of existing drugs can also be expected.

  To modify drug pharmacokinetics using a carrier, it is necessary to know the fate of the carrier itself. For example, the biodistribution of synthetic and natural water-soluble polymers with no special affinity for living bodies is strongly influenced by their molecular weight and electric charge, and the drug can be activated to specific organs by modifying water-soluble polymers with sugar chains. Have been reported so far (eg, Takakura, Y., etc., Pharm. Res, 7, p339, 1990, Yamaoka, T., etc., Drug Delivery, 1, p75, 1993, and Takakura). Ydv et al., Adv. Drug Delivery Rev., Vol. 19, p377, 1996, etc.). In addition, it is reported that liposomes and the like that are often used as drug carriers are changed in biodistribution by modifying the surface thereof with a water-soluble polymer (for example, Woodle, MC, etc.). Biochim. Biophys. Acta, 1113, p171, 1992, and Kohei Hara, targeting therapy, Pharmaceutical Journal, p125, 1985, etc.). Furthermore, using the anatomical difference between cancer tissue and normal tissue, passive targeting of a therapeutic agent to the cancer tissue using a carrier such as a polymer compound and a liposome has been attempted (for example, Maeda, H. et al., Crit. Rev. Therap. Drug Carrier Sys., 6, p193, 1989, etc.).

  As described above, a drug is identified by appropriately combining a polymer material or liposome as a drug carrier and a drug, or a drug-containing liposome, a drug-containing polymer micelle, or a drug-containing polymer fine particle and a polymer material. It is thought that it can be targeted to a site. Already, a great number of reports have been published on the complexation of these polymer materials and drugs, or polymer materials and the above-mentioned drug-containing carriers. However, in conventional complex preparation, a chemical binding reaction is almost always performed between the drug and the polymer material. The problem of decreased activity is always present. This is considered to be one of the reasons for delaying commercialization, despite the many reports that the effectiveness of drugs is enhanced by complexing with polymers. Therefore, there has been a demand for a method for preparing a complex by simply combining a drug and a polymer without performing such a chemical bonding reaction.

  There have been many reports of sustained release of therapeutic agents using biodegradable or non-degradable polymer materials (eg, Hitoshi Sezaki, Development of Drugs, Volume 13, Drug delivery methods, Yodogawa Shoten, 1988). For example, there is an attempt to gradually release a drug by mixing a drug with a polymer material such as glycolic acid-lactic acid copolymer, which is a typical biodegradable and absorbable polymer. However, since these polymers are oil-soluble, there has been a problem in slow release of water-soluble drugs due to lack of compatibility. Therefore, as one of the solutions, an attempt to use a polymer hydrogel that decomposes and absorbs in vivo as a matrix for sustained drug release (for example, Biobotz, WR, etc., BioconjugateChcm., 6). Volume, p332, 1995). However, since the sustained release period of the drug in this case is basically determined by the self-diffusibility of the drug in the hydrogel, it is difficult to change the sustained release over a wide range. For this reason, in particular, in a polymer hydrogel having a small injectable size, the sustained release of the drug is practically impossible. Therefore, an approach to control the sustained release of the drug released along with the decomposition by immobilizing the drug in the hydrogel by some method and controlling the degradability of the hydrogel matrix is realistic. There are two methods for immobilizing drugs on hydrogels: chemical bonding and physical bonding. In the former method, matrix fragments are chemically bonded to the released drug. The latter immobilization method is preferred. An example of the immobilization of a drug through the latter physical bond is an attempt to utilize electrostatic interaction between the drug and the hydrogel matrix, and the protein drug from the injectable particulate hydrogel matrix Sustained release has been reported (Tabata, Y. et al., Proc. 4th Japan International SAMP Symposium, p25, 1995).

  A method called metal affinity chromatography, in which a substance interacts with a chelating ligand immobilized on a carrier by a binding force (coordination binding force) via a metal ion, and the substance is separated and purified by the strength of the interaction. (For example, Porath, J., etc., Nature, 258, p598, 1975, etc.). For example, since peptides, proteins, nucleic acids, and the like themselves have a chelating ability for metal ions, the substances are adsorbed on the carrier simply by flowing those substances through an affinity column that has been chelated to the metal ions in advance. Next, the adsorbed substance is easily desorbed from the support by flowing solutions having different pH or ionic strength through the column. In this case, the affinity between the column and the substance varies depending on the combination of the substance and the metal ion. According to this method, it has been reported that various physiologically active proteins have already been separated and purified, and that the biological activity of the protein after desorption is retained (for example, Victor, G. et al., J. Biol. Chem., 252, p 5934, 1977, etc.).

  In British Patent 1972 1-388-580, there is a description that the metal affinity binding force is used for immobilizing a drug and its sustained release, which is for a water-insoluble polymer hydrogel. There is no suggestion of applications using water-soluble polymer substances having a function (for example, extending the blood life of drugs, targeting drugs, etc.). Furthermore, in this patent, it is possible to inject injection into the body by homogenizing a polymer hydrogel prepared by the method of simultaneously mixing three substances of a polymer substance having chelating ability, a drug, and a metal ion. There is a description that it is. However, a polymer hydrogel having an injectable size prepared in advance (for example, a particulate, microgel, and fluid solid polymer hydrogel) can be used as a drug in the presence of metal ions. There is no description that mixing makes it possible to easily produce a polymer hydrogel having a drug immobilized thereon. Examples of applicable drugs include drugs having a molecular weight of 3,000 or more, such as hormones, enzymes, RNA for inducing interferon, etc. In the present invention, these drugs are included as described later. Alternatively, the application can be expanded to other therapeutic agents, drug carriers including therapeutic agents, and diagnostic agents.

  There are reports on making polymer hydrogels via metal ions (for example, Bonaccorsi, F., etc., Int. J. Polymeric Mater., 18, p165, 1992, etc.). There is no suggestion of drug immobilization and its application to sustained release.

Regarding diagnostic agents, chelating ligands such as diethylenetriaminepentaacetic acid (hereinafter referred to as DTPA) residues are introduced into water-soluble polymers such as polyethylene glycol, dextran, or serum albumin, and then gadolinium is chelated, followed by nuclear magnetism. There is a report that it is used as a sensitizer of a resonance image (hereinafter referred to as MRI) (for example, Dessor, TS, etc., J. Magnetic Resonance Imaging, Vol. 4, p467, 1994, etc.), and coordinate binding has already been diagnosed. Applied to the field. However, this is a report that simply introduces a metal ion having a high MRI sensitizing effect into a water-soluble polymer. -It is not described that a sensitized metal-polymer complex is prepared and used for treatment simultaneously with diagnosis.
British Patent 1972 1-388-580 Takakura, Y .; Pharm. Res, Volume 7, p339, 1990 Yamaoka, T .; Drug Delivery, Volume 1, p75, 1993 Takakura, Y .; Adv. Drug Delivery Rev. , 19, p377, 1996 Woodle, M.M. C. Biochim. Biophys. Acta, 1113, p171, 1992, Kohei Hara, Targeting Therapy, Medicinal Journal, p125, 1985 Maeda, H .; Crit. Rev. Therap. Drug Carrier Sys. , 6, p193, 1989 Edited by Hitoshi Sezaki, Development of pharmaceuticals, Volume 13, Drug delivery method, Yodogawa Shoten, 1988 Gombotz, W.M. R. BioconjugateChcm. , Volume 6, p332, 1995 Tabata, Y. et al. Proc. 4th Japan International SAMP Symposium, p25, 1995 Porath, J. et al. Nature, 258, p598, 1975, etc. Victor, G.M. J. et al. Biol. Chem. 252, p5934, 1977 Bonaccorsi, F.M. Int. J. et al. PolymerMatter. , 18, p165, 1992 Dessor, T .; S. J. et al. Magnetic Resonance Imaging, Volume 4, p467, 1994

  The present invention provides a simple method for producing a drug-polymer complex by binding a labile drug in vivo to a polymer carrier instead of chemically binding it via a metal ion. To do. The drug-polymer complex prepared by utilizing these coordination bonds is similar to the drug-polymer complex prepared by the conventional chemical bonding method, and extends the blood life of the drug and its targeting. As well as slow release of the drug.

  As a result of intensive studies to solve the above problems, the present inventors have found that a polymer material having a chelate ligand or a polymer material having a chelating ability itself and a drug in the presence of a metal ion. By simply mixing, it is easy to obtain a complex of drug and polymer, and the resulting complex has properties such as modification of drug pharmacokinetics and sustained release of drug. As in the case of the drug-polymer complex produced by the above method, the present inventors have found that the drug effect expression is extremely advantageous and completed the present invention.

  The technical configuration of the present invention will be described below. The drug-polymer complex preparation of the present invention can be obtained by the following method. DTPA acid anhydride is added to a dimethylformamide solution of a polymer having an amino group or a hydroxyl group, and a DTPA residue having chelating ability is introduced into the polymer. After the reaction, dialysis against water is performed to remove DTPA not bound to the polymer, and lyophilization yields a polymer having a DTPA residue of the metal chelate ligand. Next, these DTPA residue-introduced polymers and metal ions are mixed and stirred at room temperature. By subjecting the mixture to gel filtration, unbound metal ions are separated and removed, and a water-soluble polymer having a metal affinity binding force is produced. This water-soluble polymer is put into an aqueous solution containing a protein drug. The obtained mixture is subjected to gel filtration to separate and remove the unbound drug to obtain a water-soluble protein drug-polymer complex. A water-soluble protein drug-polymer complex can be similarly obtained by simultaneously mixing a protein drug and a metal ion with a DTPA residue-introduced polymer and stirring at room temperature. On the other hand, a polymer hydrogel particle having a metal chelate ligand by chemically crosslinking a polymer having a DTPA residue or by introducing a DTPA residue into a previously prepared crosslinked polymer hydrogel particle Is made. By immersing these polymer hydrogel particles in a metal ion aqueous solution to form a metal chelate, free metal ions are removed, and polymer hydrogel particles having a metal affinity binding force are produced. Next, these polymer hydrogel particles are put into an aqueous solution containing a protein drug. By washing the obtained hydrogel with water, the unbound drug is separated and removed to obtain polymer hydrogel particles on which the protein drug is immobilized.

  The drug used in the present invention is not related to the molecular weight and type as long as it has a chelating ability. For example, low molecular therapeutic agents such as anticancer agents, antibacterial agents, anti-inflammatory agents, peptides and protein drugs such as interferons, interleukins, various enzyme drugs, various differentiation factors, and growth factors, muramyl dipeptide or poly Drugs having an action of stimulating immunity such as I-C, RNA for interferon induction, and nucleic acid drugs used for gene therapy such as plasmids and antisense DNA can be used. From the viewpoint of the stability of the obtained drug-polymer complex, it is preferable to use a high molecular weight therapeutic agent as the drug. The present invention is not limited to therapeutic agents alone, but also to drug-containing carriers such as drug-containing liposomes, polymer micelles, or polymer microparticles, and diagnostic agents such as ultrasound contrast agents and MRI sensitizers. The composite preparation method is applicable.

  The polymer material used in the present invention is not particularly limited, and examples thereof include polyethylene glycol (hereinafter referred to as PEG), polyethyleneimine, polyallylamine, or nucleic acid, which itself has a chelating ability for metals. However, if the polymer material has a weak chelating ability or does not have it, it can be used for the purpose of the present invention by positively introducing a chelating ligand into the molecule. Examples of polymers having no or weak chelating ability include polyvinyl alcohol, polyamino acids, polyacrylic acid, and polysaccharides. These polymers introduce metal chelate residues into their side chains. Can be used in the present invention. Furthermore, by introducing a metal chelate residue at the end of the PEG molecule, it is possible to make a complex with a simple drug using only the end of PEG. In the present invention, these polymer materials can be used in two forms of an aqueous solution or a hydrogel obtained by chemical crosslinking. The former is used for conjugation with drugs or drug-containing carriers, and is intended to extend their blood life and target specific organs. On the other hand, the latter is used as a carrier for immobilizing drugs. A drug is immobilized by utilizing a metal affinity binding force to a polymer hydrogel having an injectable shape made of a biodegradable and absorbable polymer material prepared in advance. As the hydrogel body decomposes, the drug is gradually released.

  The chelate ligand that is chemically introduced into the polymer material has a chemical structure that coordinates a metal ion such as carboxyl group, carbonyl group, cyano group, amino group, imidazole group, thiol group, or hydroxyl group. If it is a thing, it will not specifically limit. For example, iminodiacetic acid, nitrilotriacetic acid, ethylenediaminetetraacetic acid (hereinafter referred to as EDTA), DTPA, triethylenetetraminehexaacetic acid, N-hydroxyethylethylenediaminotriacetic acid, ethylene glycol diethyl etherdiaminetetraacetic acid (hereinafter referred to as EGDTA), and ethylenediaminetetraacetic acid Examples include complex type ligands such as propionic acid, diethylenetriamine, triethylenetetramine, and α-amino acids (such as glutamic acid, lysine, aspartic acid, cystine, histidine, and tyrosine). Various methods are conceivable for the introduction reaction of these chelate ligands into the polymer. For example, in the case of a polymer having a hydroxyl group in the molecule, a chemical bond is formed between the hydroxyl group and the amino group of the chelate ligand by a cyanuric chloride method, a cyanogen bromide method, or an epichlorohydrin method. In addition to these, a binding reaction by the periodate oxidation method is also effective for polysaccharides. After the hydroxyl group is activated with carbonylimidazole, it can be bonded to the carboxyl group or amino group of the ligand. For polymers with carboxyl groups, carbodiimide, N-hydroxysuccinimide and carbodiimide, binding reactions between carboxyl groups and ligand amino groups using ethyl chlorocarbonate, etc., for polymer materials with amino groups Can also be applied to the chelating ligand polymer materials such as the bonding reaction between the carboxyl group of the ligand using carbodiimide and the like, and the bonding reaction between the ligand and amino group using cyanuric chloride, etc. It is useful for the introduction reaction. In addition to the above-described binding reaction, DTPA acid anhydride and aminobenzyl or isothiocyanobenzyl derivative of EDTA can easily react with the amino group or hydroxyl group of the polymer just by mixing, and chelate ligand This is preferable because it can be introduced into a polymer chain. In addition, it is also effective to introduce a 2,2'-dipyridine or 1,10-phenanthroline residue excellent in chelating ability into a polymer. As described above, in addition to the method of directly introducing a ligand residue having a chelating ability into a polymer chain, a spacer can also be introduced between the chelate coordination residue and the polymer. For example, N1, N1-bis (3-aminopropyl) 1,3-propyldiamine, bis (3-aminopropyl) amine, aliphatic diamine having different carbon chain length, or lysine having amino groups at both ends An ester derivative or the like is introduced into the polymer chain. Thereafter, a chelate ligand is introduced into an unreacted one-terminal amino group, and a polymer material into which the chelate ligand is introduced via a spacer is prepared. In addition to the examples described above, other spacer molecules having a chemically reactive functional group such as a hydroxyl group or a carboxyl group at both ends of the molecule can be used. Coordination residue-introduced polymers can be synthesized. The compounding ratio of the ligand / polymer material is not particularly limited, but a compounding ratio of 1/1000 to 1/10 is preferable.

  The metal to be used is not particularly limited as long as it is safe for the living body, and the chelate coordination residue introduced into the polymer such as transition and alkaline earth metal having a valence of 2 or higher. Any group that forms a stable chelate with the group may be used. From the viewpoint of the stability of the metal chelate and its toxicity, copper, zinc, manganese, iron or the like is preferable. There are many reports on the biotoxicity of these metals (for example, copper and hygiene, published by Japan Copper Center, or Yoshinori Itokawa, Diabetes Frontier, Vol. 3, p425, 1992, etc.), and they are considered extremely toxic. The LD50 value of copper is> 4,000 mg / kg (mouse oral). The amount of copper used for the purpose of this study is about 1 to 1.5 mg / kg, which is 1/1000 or less compared to literature values. By the way, it is known that the Japanese take 0.78 to 2.54 mg / kg of copper every day, and the biotoxicity of the metal ions used is not considered to be a problem. These metals are used in the preparation of drug-polymer complexes as chlorides, sulfates or acetates.

  Conditions for forming a metal chelate by mixing a polymer substance having a chelating ability or a chemically introduced chelate ligand with a metal ion are not particularly limited, but the polymer substance to be used The concentration is preferably 0.1 to 50 wt%, and the mixing temperature is preferably 4 to 50 ° C. The pH of the solution is preferably 3-10. If necessary, the pH of the aqueous reaction solution can be adjusted by adding sodium hydroxide or an aqueous hydrogen chloride solution. The amount of metal ion added is preferably 0.1 to 10 times the amount of the chelate ligand. The mixing conditions of the drug and the polymer substance having metal affinity binding force in which metal chelate is formed are not particularly limited, but the amount of drug added is 1 to 10 times the metal chelate-forming ligand, etc. The amount is desirable. The mixing temperature is preferably 4 to 50 ° C., and the pH of the solution is preferably 3 to 10. If necessary, the pH of the aqueous reaction solution can be adjusted by adding sodium hydroxide or an aqueous hydrogen chloride solution.

  The drug-polymer complex used may be either water-soluble or water-insoluble. The shape is not particularly limited, such as an aqueous solution, a microgel, a particle, or a fluid solid. In particular, the former three are suitable for uses such as prolonging the blood life of drugs or drug-containing carriers and targeting them to specific organs. The latter two water-insoluble complexes are suitable for sustained drug release. That is, a drug immobilized on a water-insoluble polymer carrier via metal ions is mainly released by decomposition of the carrier itself. Along with the decomposition of the carrier, the drug is released from the carrier and released together with the polymer fragment solubilized in water. The drug release pattern can be controlled by the degradation pattern of the polymer carrier.

  The drug-polymer complex preparation of the present invention may be used as it is, or may be used for assay, treatment, or diagnosis by dissolving or dispersing in a diluent such as a buffer solution, physiological saline or injection solvent. it can. Further, after lyophilization, it may be used after being dissolved or dispersed in a diluent at the time of use.

The drug-polymer complex preparation using the metal chelate coordination bond of the present invention has the following excellent characteristics.
1) A complex can be prepared by simply mixing a drug and a polymer.
2) The affinity between a drug and a polymer can be adjusted by a combination of the drug, metal ion, and chelate ligand.
3) It is applicable not only to a drug alone but also to a preparation comprising a drug and a carrier.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited to a following example.

  3.3, 6.6, 13.2, 26.5, 66.2 and 132.4 mg in 10 ml dimethyl sulfoxide solution containing 110 mg pullulan (molecular weight 200,000, manufactured by Tokyo Chemical Industry Co., Ltd.) DTPA acid anhydride (manufactured by Dojindo Laboratories Co., Ltd.) and 4.97 mg of 4-dimethylaminopyridine (manufactured by Nacalai Tesque Co., Ltd.) were added. This mixed solution was stirred at 40 ° C. for 24 hours to introduce a DTPA residue into the hydroxyl group of pullulan. After the reaction, dialysis against water for 2 days and lyophilization were performed to obtain a DTPA residue-introduced pullulan. When the introduction rate of the DTPA residue was determined by conductivity titration, the introduction rate was 0.062, 0.12, 0.22, 0.34, 0.53 as the amount of DTPA acid anhydride was increased. , And 0.73 μmole / mg pullulan. The molecular weight change of pullulan before and after the reaction was measured by gel permeation chromatography (manufactured by Tosoh Corporation, column: TSKgel3000PWXL + TSKgel6000PWXL, column temperature: 40 ° C., eluent: 0.2 M phosphate buffer solution (pH 6.8), elution rate: 0.9 ml / Min, detector: differential refractometer), the molecular weight of pullulan due to DTPA introduction reaction was not observed. This result shows that no cross-linking reaction between the pullulan molecules by the DTPA acid anhydride occurs, and this reaction yields a pullulan having a DTPA residue introduced in the side chain.

  A DTPA residue was introduced in the same manner as in Example 1 except that the water-soluble polymer used was polyvinyl alcohol (hereinafter referred to as PVA, manufactured by Unitika Ltd., weight average molecular weight 70,000, saponification degree 99.8%). PVA was produced. The DTPA acid anhydride added is 6.6, 13.2, 26.5, 42.4, and 53.0 mg. The DTPA residue introduction rate increased to 0.15, 0.21, 0.26, 0.39, and 0.62 μmole / mg PVA with an increase in the amount of DTPA acid anhydride added. No change in molecular weight of PVA due to DTPA residue introduction reaction was observed.

  5 mg of the DTPA residue-introduced pullulan prepared in Example 1 (DTPA residue introduction rate: 0.062 μmole / mg) was dissolved in an aqueous copper sulfate solution having a concentration of 0.125 mg / ml. After stirring for 30 minutes, the mixture was gel-filtered (Sephacryl-S200, 0.5 cm inner diameter, 36 cm long column, eluent PB, elution rate 0.33 ml / min) to separate and remove free copper ions from the reaction system. . The copper chelate amount and sugar mass (pullulan amount) of DTPA residue-introduced pullulan chelating copper ions were quantified by the atomic absorption method and the anthrone-sulfuric acid method, respectively. According to the elution curve, the elution position of pullulan coincided with the elution position of copper ions. The recovery rate of DTPA residue-introduced pullulan after gel filtration was 95%, and the chelate binding rate of copper ions was 1.20 / DTPA residue. In addition, when the original pullulan in which the DTPA residue was not introduced was similarly dissolved in an aqueous copper sulfate solution, no binding of copper ions to the pullulan was observed. This indicates that the copper ion is chelated via a DTPA residue which is a metal ligand.

  0.22 g of biodegradable and absorbable amylopectin (manufactured by Nacalai Tesque Co., Ltd., purified from potato) was poured into 2 ml of water, and then an amylopectin aqueous solution was prepared by stirring at 80 ° C. for several minutes. This aqueous solution was added to 400 ml of olive oil (manufactured by Wako Pure Chemical Industries, Ltd.) and stirred at 25 ° C. and 450 rpm for 1 hour to prepare a W / 0 type emulsion. Next, acetone was added into the emulsion to precipitate and solidify amylopectin. Amylopectin particles were obtained by centrifugally washing the particles with acetone (25 ° C., 3000 rpm, 10 minutes). The amylopectin particles (66.6 mg) were put into 10 ml of an acetone / 1N sodium hydroxide equivalent mixed solution in which 10 mmole of ethylene glycol diglycidyl ether (Denacol EX-810, manufactured by Nagase Chemical Industries, Ltd.) was dissolved. After allowing the crosslinking reaction to take place at 30 ° C. for 3 hours under stirring conditions, the mixture was further treated at 40 ° C. for 2 hours in 10 ml of an equivalent mixed solution of acetone / 0.1N sodium hydroxide. The particles were centrifugally washed with a mixed solution of equal amounts of acetone / water and dimethyl sulfoxide, and finally the crosslinked hydrogel particles were swollen in dimethyl sulfoxide. When the water content of the crosslinked hydrogel particles was evaluated from the particle size change before and after the swelling operation in water at 37 ° C., it was 95.6%. Moreover, the particle diameter at the time of water swelling was 20-100 micrometers. 1.46 mg of DTPA anhydride and 10.0 mg of 4-dimethylaminopyridine are added to a dimethyl sulfoxide solution containing swollen crosslinked hydrogel particles and allowed to react at 40 ° C. for 1, 3, 6, 12, and 24 hours. It was. After completion of the reaction, the crosslinked hydrogel particles were thoroughly washed with water. When the introduction rate of DTPA residues into the crosslinked amylopectin hydrogel particles was determined by conductivity titration, the DTPA residue introduction rate was 0.063, 0.069, 0.088, 0.10, as the reaction time increased. And 0.12 μmole / mg dry particles. The water content of the hydrogel increased from 95.6 to 97.5% with the introduction of DTPA residues. There was almost no change in the particle size due to the DTPA residue introduction reaction.

  6 mg of the dry weight of crosslinked amylopectin hydrogel particles having a DTPA residue (DTPA residue introduction rate: 0.10 μmole / mg dry hydrogel) prepared in Example 4 was immersed in aqueous copper sulfate solutions having different concentrations, and then heated to 40 ° C. And left for 1 hour. When the copper ion concentration in the aqueous solution was quantified by chelate titration and Scatchard analysis was performed, the chelate rate of copper ions to the hydrogel particles was 1.50 / DTPA residue.

  Recombinant human interferon αA in a 5 mg / ml aqueous solution of the DTPA residue-introduced pullulan prepared in Example 1 (DTPA residue introduction rate: 0.062 μmole / mg pullulan) to a final concentration of 10 μg / ml / D (hereinafter referred to as IFN) aqueous solution (2 × 10 7 international units (IU) / 100 μg protein / ml) was added. Next, an aqueous zinc chloride solution was added to the mixed aqueous solution so that the final concentration was 1.9 mg / ml. The mixture was stirred at room temperature for 1 hour, and IFN was bound to DTPA residue-introduced pullulan via a zinc ion via a coordinate bond. The molecular weight change of IFN before and after the reaction was measured by gel permeation chromatography (manufactured by Tosoh Corporation, column: TSKgel3000PWXL + TSKgel6000PWXL. Column temperature: 40 ° C., eluent: 0.05M phosphate buffer solution (pH 6.8) + 0.3M sodium chloride aqueous solution, Elution rate: 0.9 ml / min, detector: Fluorescence spectrophotometer (excitation wavelength: 278 nm, emission wavelength: 348 nm)). As controls, IFN, DTPA residue introduced pullulan, and a mixture of IFN and DTPA residue introduced pullulan were used.

  The result is shown in FIG.

  By mixing IFN and DTPA residue-introduced pullulan in the presence of zinc ions, the fluorescence peak of IFN itself shifted to the short time side. However, no IFN peak migration was observed in the absence of zinc ions. Fluorescence absorption was not observed in the DTPA residue-introduced pullulan itself. Although the same examination was conducted using copper ions instead of zinc ions, no movement of the IFN peak due to the addition of copper ions was observed. These results show that IFN and DTPA residue-introduced pullulan are coordinated via zinc ions, and the molecular size of IFN is increased, and depending on the type of metal ion, DTPA residue-introduced pullulan and IFN It shows that the coordinative bond strength of. As shown in this example, it can be seen that a complex through a coordination bond via a metal ion can be easily formed by simply mixing a water-soluble polymer and a protein in water.

  The DTPA residue-introduced PVA prepared in Example 2 (DTPA residue introduction rate: 0.15 μmole / mg PVA) into an aqueous solution having a concentration of 5 mg / ml so that the final concentration becomes 10 μg / ml (hereinafter referred to as Lyz, Nacalai Tesque). An aqueous solution was supplied. Next, an aqueous copper sulfate solution was added to the mixed aqueous solution so that the final concentration was 1.9 mg / ml. The mixture was stirred at room temperature for 1 hour, and Lyz was bound to DTPA residue-introduced PVA by coordination bond via copper ions. Changes in the molecular weight of Lyz before and after the reaction were evaluated using the gel permeation chromatography described in Example 6. As controls, Lyz, DTPA residue-introduced PVA, and a mixture of Lyz and DTPA residue-introduced PVA were used. By mixing Lyz and DTPA residue-introduced PVA in the presence of copper ions, the fluorescence peak of Lyz itself shifted to the short time side. However, in the absence of copper ions, no movement of the Lyz peak was observed. These results indicate that Lyz and pullulan are bonded through a coordinate bond through zinc ions, and the molecular size of Lyz is increased. As described above, also in this case, it was found that a complex of both can be formed by simply mixing DTPA residue-introduced PVA and protein in water.

  Copper sulfate aqueous solutions with different concentrations of 20 mg of dry weight of crosslinked amylopetatin hydrogel particles having a DTPA residue (DTPA residue introduction rate: 0.10 μmole / mg dry hydrogel) prepared by the same method as in Example 4 After soaking, the solution was left at 40 ° C. for 1 hour. Thereafter, the hydrogel particles were thoroughly centrifuged with water to remove unchelated copper ions. When the amount of copper ion chelated to the hydrogel particles was evaluated by quantifying the copper ions in the aqueous solution by chelate titration, 0.0005, 0.001, 0.006, and 0.04 μmole / mg dried hydrogel particles Met. The DTPA residue-introduced crosslinked amylopectin hydrogel particles chelated with copper ions were immersed in 3.2 ml of an aqueous solution of bovine serum albumin (hereinafter referred to as BSA, manufactured by Wako Pure Chemical Industries, Ltd.) having a concentration of 1 mg / ml. Protein adsorption to the hydrogel particles was evaluated by quantifying the protein in the supernatant every hour. The protein was quantified by the Bio-rad Protein Assay method.

  The result is shown in FIG.

  In DTPA residue-introduced crosslinked amylopectin hydrogel particles chelating copper ions, the amount of protein adsorbed increased with time and the amount of copper ion chelate. On the other hand, protein adsorption to DTPA residue-introduced crosslinked amylopectin hydrogel particles not chelating copper ions was not observed. Similar protein adsorption was examined for crosslinked amylopectin hydrogel particles into which no DTPA residues were introduced, but protein adsorption was not observed in any hydrogel regardless of the presence or absence of copper ions. From the above results, it was found that the protein can be bound to the hydrogel particles by immersing the amylopectin hydrogel particles in which the metal ions are chelate-immobilized in the protein aqueous solution.

  To the 15 mg / ml aqueous solution of DTPA residue-introduced pullulan (DTPA residue introduction rate: 0.062 μmole / mg) prepared in Example 1, an IFN aqueous solution (2 × 107 international units (2 × 107 international units ( IU) / 100 μg protein / ml). Next, an aqueous zinc chloride solution was added to the mixture so that the final concentration was 18.2 mg / ml. The mixture was stirred at room temperature for 3 hours, and IFN was bound to DTPA residue-introduced pullulan via a zinc ion via a coordinate bond. Next, gel filtration of the mixture (Sephacryl-S200, 0.5 cm inner diameter, 36 cm long column, eluent 0.01 M phosphate buffer solution +0.25 N NaCl, elution rate 0.33 ml / min) was performed, and free zinc ions and Free IFN was separated and removed from the IFN-DTPA residue-introduced pullulan conjugate. When the protein amount (IFN amount) and sugar mass (pullulan amount) of the IFN-pullulan conjugate were quantified by the Bio-Rad Protein Assay method and the anthrone-sulfuric acid method, respectively, the recovery rate of pullulan was 95%. The amount of protein bound to was 56% of the charged protein amount. When the IFN activity of an IFN-pullulan conjugate was evaluated from its antiviral activity in vitro (Rubinstein, S. et al., J. Virol., 37, p755, 1981), the activity was 7.2 × 107 IU / mg conjugate. Yes, the recovery of IFN activity before and after the binding reaction was 36%.

  As an IFN concentration, 100 μl of a 20 μg / ml aqueous solution and 200 μl of 0.5 M phosphate buffer solution (pH 7.5) were mixed. 4 μl of Na125I (3.7 GBq / ml 0.1 M NaOH solution, manufactured by NEN Research Products) was added to the mixed solution. After adding 200 μl of 0.05 M phosphate buffer solution (pH 7.2) of chloramine T at a concentration of 0.2 mg / ml, radiolabeling reaction of IFN was performed at room temperature for 2 minutes. Next, 200 μl of 4 mg / ml sodium disulfite 0.5 M phosphate buffer (pH 7.2) was added to stop the reaction. The reaction was passed through a column packed with an anion exchange resin (Dowex1-X8) to separate and remove unlabeled 125I-. The 125I-labeled free IFN thus obtained (final concentration of 3.92 μg / ml) was 6.53 mg / ml of the DTPA residue-introduced pullulan (DTPA introduction rate: 0.062 μmole / mg) prepared in Example 1. It poured into the aqueous solution of concentration. Next, zinc chloride or copper sulfate aqueous solution was added to this mixed aqueous solution so that the final concentration was 370 μg / ml. The mixture was stirred at room temperature for 3 hours, and IFN was bound to DTPA residue-introduced pullulan by coordination bond via metal ions. 125I-labeled free IFN or 125I-labeled IFN-DTPA residue introduced pullulan conjugate was administered into the tail vein of Balb / c mice (9 weeks old, female, 3 mice / group). 30 minutes after administration, blood, heart, lungs, thymus, liver, spleen, kidney, gastrointestinal tract, thyroid, other parts, urine, feces, etc. are collected, and the radioactivity of each organ is measured with a gamma counter. Measured. As a control, a mixture of DTPA residue-introduced pullulan to which no metal ion was added and 125I-labeled IFN was used. The results are shown in Table 1.

  The organ with a clear difference in body distribution was the liver. In IFN mixed with DTPA residue-introduced pullulan in the presence of zinc ions, the amount accumulated in the liver is significantly higher than that of free IFN. In addition, mixing of both in the absence of zinc ions did not affect the accumulation of IFN in the liver. For organs other than the liver, no significant difference was observed between free IFN and IFN-pullulan conjugates. These results indicate that IFN targeting to the liver could be achieved by conjugating IFN via metal coordination bonds to pullulan that transfers IFN to the liver. This excellent liver targeting ability hydrogel shows the same result as when IFN and pullulan are combined by chemical bonding. This indicates that FNPA residue-introduced pullulan and IFN were simply mixed in an aqueous solution to modify IFN via metal coordination, resulting in a liver targeting effect. Yes. However, when copper ion was used as the metal ion, the targeting effect of IFN on the liver was not observed. Considering that the presence of copper ions could not shift the IFN peak of gel permeation chromatography to the high molecular weight side, the bond between IFN and DTPA residue-introduced pullulan via metal coordination bond is the type of metal ion used. It turns out that it is influenced by.

  DTPA residue-introduced PVA prepared in Example 2 (DTPA residue introduction rate: 0.15 μmole / mg PVA), Lyz as a protein, and copper ions were used in the same manner as in Example 6 via copper ions. Conjugates of Lyz and DTPA residue-introduced PVA were prepared, and the hemodynamics of these conjugates and free Lyz were examined. The blood half-life of free Lyz, a mixture of Lyz and DTPA residue-introduced PVA, and a conjugate of DPTA-PVA and Lyz via copper ions were about 30, 32, and 800 minutes, respectively. In this way, the blood half-life of Lyz was significantly prolonged by simply mixing the DTPA residue-introduced PVA in the presence of copper ions. On the other hand, when both were mixed in the absence of copper ions, no significant increase in the blood half-life of Lyz was observed. Even when the Lyz-PVA conjugate was prepared by the chemical bonding method, the increase in the blood half-life of Lyz was observed. Therefore, the Lyz molecule was coordinated by metal ions and DTPA residue-introduced PVA. As a result of the modification, it is thought that the blood kinetics of Lyz changed.

  10 mg of the dry weight of crosslinked amylopectin hydrogel particles having a DTPA residue (DTPA residue introduction rate: 0.10 μmole / mg dry hydrogel) prepared in the same manner as in Example 4 was added to a 2.5 μM copper sulfate aqueous solution. It was immersed in 2 ml and left at 40 ° C. for 1 hour. Thereafter, the hydrogel particles were thoroughly washed with water to remove unchelated copper ions and freeze-dried. When the copper ions in the aqueous solution were quantified by chelate titration, the amount of copper ions chelated to the hydrogel particles was 0.3 μmole / mg dry hydrogel particles. Next, 125I-labeled BSA was prepared in the same manner as in Example 10, except that BSA was used instead of IFN. 0.1 ml of this aqueous solution (concentration of 1 mg / ml) was dropped on the dried hydrogel particles, and BSA was impregnated into the gel. These 125I-labeled BSA-impregnated copper ion chelate cross-linked amylopectin hydrogel particles were injected and administered subcutaneously to the back of the mouse, and the weight of the hydrogel particles and the radioactivity remaining in the hydrogel particles were measured over time. The hydrogel particles were degraded in vivo over time, and the residual weights were 90, 73, 40, and 0% after 1, 4, 7, and 14 days, respectively. The radioactivity remaining in the hydrogel decreased to 85, 67, 43, 0% with time. On the other hand, when the same examination was performed using crosslinked amylopectin hydrogel particles having a DTPA residue not treated with copper ions, the residual weights were 89, 70 after 1, 4, 7, and 14 days after injection, respectively. 46, 0% and in vivo degradability, no difference was seen compared to the copper ion-treated hydrogel particle group. However, the residual radioactivity decreased to 20% of the initial impregnation amount 1 day after injection, and no activity was observed after 4 days. As described above, it is considered that the protein coordinated and fixed via copper ions was released from the hydrogel particles as the hydrogel was decomposed without deviating from the hydrogel particles even in vivo.

  The present invention provides a simple method for preparing a drug-polymer complex, characterized in that a drug unstable in vivo is coordinately bonded to a polymer carrier via a metal ion instead of a chemical bond. It is. The drug applied to this preparation method may be any of a therapeutic agent, a drug carrier including the therapeutic agent, and a diagnostic agent. A complex of a drug and a polymer can be easily obtained by mixing a polymer material having a chelate ligand or a polymer material having a chelating ability with the drug in the presence of a metal ion. Furthermore, the obtained complex is effective in the same manner as the drug-polymer complex produced by the conventional chemical bonding method through the extension of the blood life of the drug and its targeting, and the sustained release of the drug. The expression is extremely easy, and it can be said that the industrial applicability of the present invention is very large.

It is a figure which shows the change of the IFN molecular size by mixing with IFN and DTPA residue introduction | transduction pullulan in presence of a zinc ion. It is a figure which shows adsorption | suction of BSA to a DTPA residue introduction | transduction bridge | crosslinking amylopectin hydrogel particle.

Claims (4)

  A drug-polymer composite comprising a drug having a chelating ability and a polymer substance having a chelating ability or a polymer substance having a chelate ligand introduced in the presence of a metal ion. Preparation method of body preparation.   2. The preparation of a drug-polymer complex preparation according to claim 1, wherein the high-molecular substance having a chelating ability is water-soluble or injectable form such as microgel, particulate, or flowable solid. Method.   A drug-containing carrier such as a drug-containing liposome having chelating ability, a polymer micelle, or a polymer fine particle and the polymer substance according to claim 1 are mixed in the presence of a metal ion. A method for preparing a polymer composite preparation. A drug comprising: a diagnostic agent such as an ultrasound contrast agent or a nuclear magnetic resonance sensitizer containing a drug having a chelating ability and the polymer substance according to claim 1 mixed in the presence of a metal ion. -Preparation method of polymer composite preparation.

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