JP5321772B2 - Medicinal drug substance containing magnetic particles - Google Patents

Description

  The present invention relates to a drug delivery system (hereinafter referred to as DDS) which is a drug delivery method in the medical technical field, CT (computed tomography) diagnosis and thermotherapy used in X-rays, MRI (magnetic resonance), etc. The present invention relates to a drug substance for use in a magnetic particle-containing medicine for the treatment of.

  More specifically, an object of the present invention is to improve performance such as directivity of delivery of the magnetic particle-containing drug to a diseased tissue or cell, contrast sensitivity at the time of diagnosis by CT, and heat generation at the time of thermotherapy. It is an active pharmaceutical ingredient containing magnetic particles.

  In recent years, magnetic particle-containing pharmaceuticals in which magnetic iron oxide fine particles are used as a magnetic substance and complexed with biocompatible substances such as phospholipids, proteins, and water-soluble polymers have been studied (Patent Documents 1 to 5 and the like).

  Further, in order to prepare a monodispersed aqueous solution of magnetic iron oxide fine particles, a method of coating the particle surface with a surface treatment agent such as a surfactant (Patent Document 6), a method of coating an inorganic substance such as Al and Si (Patent Document) 7) or a method of coating with an organometallic polymer (Patent Document 4) is known.

  However, although these use magnetic iron oxide particles, they mainly have a modification function added to the magnetic particles, and the powder properties such as the particle size and magnetic properties of the magnetic iron oxide fine particles and the magnetic particle-containing pharmaceuticals It is hard to say that the relationship between the characteristics and the characteristic factors has been fully elucidated.

  In particular, it is not easy to uniformly disperse and carry fine magnetic iron oxide particles in a biocompatible substance due to excessive magnetic aggregation of iron oxide particles. Iron particles have been used.

  In addition, magnetic iron oxide particles having a large particle size have a high possibility of iron oxide particles remaining in the body after treatment, and it is difficult to say that safety in use can be sufficiently secured.

  Therefore, magnetic particle-containing pharmaceutical raw materials that have homogeneous functionality, such as reagent delivery properties, contrast sensitivity, heat generation performance, etc., and that can generate magnetic particle-containing pharmaceuticals for diagnosis and treatment that can fully perform their functions with high reproducibility. There is a need for drug development.

Japanese Patent Laid-Open No. 3-128331 JP-A-4-52202 JP 7-122410 A JP-T 8-500700 Publication JP-A-11-106391 JP-A-1-4002 JP-A-5-310429

  As described above, in order to obtain a magnetic substance-containing drug having homogeneous characteristics with good reproducibility, the biocompatible substance and the magnetic iron oxide fine particles are uniformly dispersed and mixed at the time of pharmaceutical preparation of the magnetic particle-containing drug. Therefore, the magnetic iron oxide fine particles in the drug substance are required to be a monodispersed aqueous colloidal solution composed of fine and uniform magnetic iron oxide fine particles.

  However, when a surface treatment agent such as a surfactant is used to disperse the magnetic iron oxide fine particles, the used surface treatment agent remains, and these surface treatment agents are mixed in the obtained magnetic substance-containing medicine. There are problems such as affecting the safety to the living body and inhibiting mixing with the biocompatible substance. Furthermore, complicated treatment is required to remove the surface treatment agent.

  The present invention has been made in view of the above-mentioned conventional problems, and an object of the present invention is to provide a sterile colloidal aqueous solution of magnetic iron oxide fine particles having a uniform particle size without using a surface treatment agent. .

  As a result of intensive studies to solve the above-mentioned problems, the present inventor has focused on fine magnetic iron oxide particles as magnetic particles, and found the dispersion stability condition of a dispersed colloidal aqueous solution composed of superparamagnetic iron oxide particles.

  That is, the present invention relates to a magnetic particle-containing drug substance characterized in that it is a colloid sterile aqueous solution in which magnetic iron oxide fine particles having an average primary particle diameter of 5 to 30 nm and an aggregate particle diameter of 30 to 200 nm are dispersed. There is (Invention 1).

The present invention is also the above-mentioned medicinal drug substance containing magnetic particles, wherein the magnetic iron oxide fine particles have a saturation magnetization of 35 to 90 Am ² / kg and a coercive force of 0 to 6.0 kA / m (this book) Invention 2).

In addition, the present invention provides the above-described magnetic particle-containing pharmaceutical drug substance, wherein the magnetic iron oxide fine particles are a spinel structure composition MOFe ₂ O ₃ (M is a divalent metal) (Invention 3).

Further, in the present invention, M of the composition MOFe ₂ O ₃ (M is a divalent metal) is Fe and / or Mg (provided that the sum of Fe and Mg is 1 mol or less relative to 1 mol of Fe ₂ O ₃ ). The magnetic drug substance containing the magnetic particles according to the present invention (Invention 4).

  In addition, the present invention provides the drug substance containing magnetic particles according to any one of the above, wherein the concentration of the magnetic iron oxide fine particles in the colloid sterile aqueous solution is 5 to 50 mg / ml (Invention 5). .

  The present invention also provides the magnetic particle pharmaceutical raw material according to any one of the above, wherein the aqueous colloidal solution has a pH of 9.0 or higher, a zeta potential of −20 mV or lower, and an electric conductivity of 50 μS or higher. It is a drug (Invention 6).

  In addition, the present invention is a magnetic drug substance containing a magnetic particle, characterized in that it is a complex of the magnetic iron oxide fine particles described above and a phospholipid, polysaccharide, protein or dextrin (this invention) 7).

Since the magnetic substance-containing medicinal drug substance according to the present invention is a sterile colloidal aqueous solution of fine magnetic iron oxide particles, it is easy to produce a medicine comprising a composite in which magnetic particles are homogeneously dispersed in a biocompatible substance. Can be synthesized. Furthermore, since the liquid medium is a drug substance that does not contain a surfactant or the like, the influence on the safety to the living body is extremely small.
In addition, fine magnetic particles can impart a ferromagnetic function to the granulated particles by adjusting the aggregate state of the fine particles in the pharmaceutical granulation step.
In addition, the ultrafine particles can facilitate excretion from the body after administration.

  The configuration of the present invention will be described in more detail as follows.

  The average particle diameter of primary particles of the magnetic iron oxide fine particles in the present invention is 5 nm to 30 nm. When the primary particle size is less than 5 nm, the material is amorphous. When the primary particle size is more than 30 nm, the coercive force increases and excessive magnetic aggregation tends to occur, resulting in an aggregate particle size of 200 nm or more. Preferably it is 5-20 nm, More preferably, it is 10 nm or less with a small coercive force.

  The aggregated particle diameter of the magnetic iron oxide fine particles in the present invention is 30 to 200 nm. If the thickness is less than 30 nm, the exothermic property due to an alternating magnetic field is low when used for cancer thermotherapy, while if it exceeds 200 nm, a problem occurs in the elimination from the body after administration. Preferably it is 50 nm-150 nm.

  In the particle size distribution of the magnetic iron oxide fine particles in the present invention, the value of the coefficient of variation obtained by dividing the standard deviation value by the average particle size is preferably 10% or less, more preferably 8% or less. When it exceeds 10%, a problem occurs in the dispersibility in water, and the problem that the magnetic particles settle over time occurs.

The magnetic iron oxide fine particles in the present invention are spinel ferromagnets MO · Fe ₂ O ₃ (M is a divalent metal), and the composition when M is Fe is xFeO · Fe ₂ O _3. X represents the content of divalent iron, x = 1 is magnetite with FeO · Fe ₂ O ₃ , x = 0 is maghemite with γ-Fe ₂ O ₃ , and an intermediate (x = 0 to 1) spinel type Iron oxide is also magnetic iron oxide, and these superparamagnetic iron oxide particles are used.

The reason why Mg was selected in addition to Fe as M of the composition MO · Fe ₂ O ₃ (M is a divalent metal) of the magnetic iron oxide fine particles in the present invention is that Mg is biocompatible. These divalent metals can be selected and used according to the purpose.

The magnetic iron oxide fine particles in the present invention are preferably a superparamagnetic material, but the coercive force is preferably 0 to 6.0 kA / m. In the case of a large coercive force exceeding 6.0 kA / m, remanent magnetization occurs and magnetic aggregation is likely to occur. More preferably, it is 0.05-4.0 kA / m. The saturation magnetization is 35 to 90 Am ² / kg. When the saturation magnetization is less than 35 Am ² / kg, the magnetism is insufficient, and with spinel iron oxide particles, it is difficult to obtain a magnetization value exceeding 90 Am ² / kg. More preferably, it is 50-85 Am < ² > / kg.

The sterile aqueous solution in the present invention is an aqueous solution of a drug substance that is negative in both a toxicity test and an endotoxin test. Specifically, the viable cell count is less than 1 × 10 ⁻⁶ / UNIT, and the endotoxin is 5.0 EU / kg or less.

  The concentration of the magnetic iron oxide fine particles of the medicinal drug substance containing magnetic particles according to the present invention is preferably 5 to 50 mg / ml. If it exceeds 50 mg / ml, the effect of van der Waals force acting between the particles becomes large and aggregation is likely to occur, which is not preferable. If it is less than 5 mg / ml, the concentration is too thin to be practical. A preferred concentration is 10-40 mg / ml.

  The pH value of the medicinal drug substance containing magnetic particles according to the present invention is preferably 9.0 or more, more preferably 9.0 to 11.0.

  The zeta potential of the medicinal drug substance containing magnetic particles according to the present invention is preferably −20 mV or less, more preferably −30 mV or less.

  The electric conductivity of the magnetic drug substance containing medicinal drug according to the present invention is preferably 50 to 400 μS.

  In the present invention, a complex of magnetic iron oxide fine particles and phospholipids, polysaccharides, proteins, or dextrins can be used.

  Next, a method for producing a magnetic drug substance according to the present invention will be described.

  A monodispersed aqueous colloidal solution of magnetic iron oxide fine particles can be produced by the following three steps.

  That is, (1) after producing magnetic iron oxide fine particles, (2) purifying a colloidal aqueous solution of magnetic iron oxide fine particles by removing water-soluble by-product salts by-produced during the reaction from the reaction mother liquor by washing with a conventional method. 3) It can be obtained by replacing the dispersion medium of the purified colloidal aqueous solution with ultrapure water.

  The magnetic iron oxide fine particles in the present invention are obtained by an aqueous solution reaction using an iron salt aqueous solution and an alkali (referred to as a wet method), or a method in which iron oxide powder is heated and reduced in a reducing gas such as hydrogen (referred to as a dry method). Can be synthesized.

  In the method of synthesizing the magnetic iron oxide fine particles, synthesis is generally performed by a wet method called a coprecipitation method or an oxidation reaction of ferrous hydroxide colloid.

  In the coprecipitation method, when an alkaline aqueous solution is added to a mixed aqueous solution of 1 mol of ferrous salt aqueous solution Fe (II) and 2 mol of ferric salt aqueous solution Fe (III) while stirring, Fe (II) and 2Fe ( This is a reaction in which the coprecipitation reaction of III) occurs and magnetite particles, which are black spinel type magnetic iron oxide, are generated. In this reaction, when a divalent metal other than Fe, for example, Mg is added, spinel-type magnetic iron oxide fine particles containing Mg are obtained. In addition, since the size of the generated particles can be controlled by reaction conditions such as iron salt concentration and mixing temperature, magnetic iron oxide fine particles can be synthesized by combining these reaction conditions.

  The ferrous hydroxide colloid oxidation reaction method is that when an aqueous alkali solution is added to a ferrous salt aqueous solution, a ferrous hydroxide colloid is formed, and the aqueous solution containing the ferrous hydroxide colloid is heated and stirred. When oxygen-containing gas such as air is ventilated, magnetite particles, which are black magnetic iron oxide, are generated by oxidation reaction of ferrous hydroxide colloid. When a divalent metal other than Fe is added as in the coprecipitation method, spinel iron oxide particles containing the added metal are obtained. Moreover, magnetic iron oxide fine particles can be synthesized by controlling the reaction conditions in combination.

  The aqueous colloidal solution containing magnetic iron oxide fine particles may be washed according to a conventional method. For example, a method by repeated decantation, a method of passing through a membrane filter by pressurization, a method of filtering and washing with Nutsche, or solid-liquid separation with a centrifuge and then adding ultrapure water to redisperse The method can also be implemented.

  Then, the concentration of the aqueous colloidal solution is adjusted to 5 to 50 mg / ml with ultrapure water to obtain a magnetic particle-containing drug substance in which magnetic iron oxide fine particles are dispersed in sterile water.

  Furthermore, it can be used for various applications in the form of a complex of the obtained magnetic iron oxide fine particles and phospholipids, polysaccharides, proteins or dextrins. For example, DDS, X-ray and MRI (drug delivery methods) For example, for CT diagnosis and thermotherapy used in magnetic resonance.

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As a result of extensive research, the present inventor has focused on the superparamagnetism of fine magnetic iron oxide particles as magnetic particles, and has found the dispersion stability conditions of a dispersed colloidal aqueous solution composed of superparamagnetic iron oxide fine particles.

  Superparamagnets are manifested by ferromagnets with zero coercivity. That is, even if the ferromagnetic particles have a single magnetic domain structure, if the particles are large and magnetized by applying an external magnetic field and then released from the external magnetic field, residual magnetization occurs. It decreases to zero and finally magnetizes when an external magnetic field is applied, but no residual magnetization occurs after release from the external magnetic field. This phenomenon is due to thermal disturbance, and such ferromagnetic fine particles are said to be superparamagnetic.

The medicinal drug substance according to the present invention is a monodispersed colloidal aqueous solution that does not aggregate even when a permanent magnet having a surface magnetic flux of 10 mT (100 gauss) is brought close thereto and is stable for a long time. This seems to be a phenomenon contradictory to the fact that the saturation magnetization of the magnetic iron oxide fine particles is a ferromagnetic material of 50 to 90 Am ² / kg, but the saturation magnetization value is measured in a powder form of the magnetic iron oxide fine particles. Since it represents the magnetization value per unit weight of time, when this is converted into the magnetization value per particle, the finer the particle, the greater the total number of particles per unit weight, and the magnetization value per particle Is a small value.

  The superparamagnetic iron oxide fine particles are used as magnetic particles because iron oxide has biocompatibility, and the fine particles are more easily excreted from the living body.

  By the way, the magnetic properties as a drug containing magnetic particles are insufficient just by uniformly dispersing the magnetic iron oxide fine particles as the drug substance in the biocompatible substance. Since the iron fine particles are aggregated to form composite particles when granulated as a drug, they exhibit ferromagnetism. This phenomenon can be explained by the well-known phenomenon that even in superparamagnetic particles, when the particles are connected together in a rosary form, shape magnetic anisotropy occurs, the coercive force increases, and the material becomes ferromagnetic. That is, when superparamagnetic iron oxide particles are controlled in a secondary agglomeration state to a specific particle size and further uniformly dispersed and mixed with a biocompatible substance, the number of magnetic particles in the granulated particles and the aggregated form of the granulated particles It has been found that the magnetic properties are different and the magnetic properties necessary for a magnetic particle-containing pharmaceutical can be adjusted.

  For example, when the drug substance is used as a heating element of a pyrogen in cancer hyperthermia, for example, an aggregate composed of magnetic iron oxide fine particles is used as a core, and a bilayer membrane composed of phospholipids and cationic lipids is used as a shell. Liposomal composite particles can be generated and used.

  Hereinafter, the present invention will be described specifically by way of examples. However, the present invention is not limited to these examples.

  For structural analysis of the product, an X-ray diffractometer was used, and the average particle size of the primary particles was calculated from the half width of the X-ray diffraction line (311) using Scherrer's equation.

The particle size distribution was observed with a transmission electron microscope TEM. Furthermore, the average particle diameter and the standard deviation value were obtained by digitizer analysis, and the coefficient of variation was obtained from these values using the following equation.
Coefficient of variation (%) = (standard deviation) × 100 / (average particle diameter)

  The aggregated particle diameter was measured by a particle size distribution analyzer FPAR-1000 (manufactured by Otsuka Electronics Co., Ltd.) using a dynamic light scattering method.

  The specific surface area value was measured by the BET method.

The Fe ²⁺ content was measured by chelate titration method.

  The magnetic characteristics were measured using a vibrating sample magnetometer VSM in a magnetic field of 10 k / 4π kA / m.

  The product was subjected to sterility testing by membrane filter method and endotoxin testing for the presence of bacterial debris.

  The electrical conductivity of the product was measured using an electrical conductivity meter.

  The zeta potential was measured by ELS-6000 (manufactured by Otsuka Electronics).

Example 1
A 5000 ml reaction vessel equipped with a stirrer and a heating device was used, the raw iron salt and caustic soda were special grade reagents, and the water was ion-exchanged water.

(1) Step of synthesizing superparamagnetic iron oxide particles 75 ml of an aqueous ferric chloride solution having a concentration of 1.5 mol and 225 ml of an aqueous ferric chloride solution having a concentration of 1.0 mol are put into a reaction vessel, stirred and first After adjusting the mixed aqueous solution of iron and ferric salt, the temperature was raised by heating. When this mixed iron salt aqueous solution was heated to 60 ° C., it was added to the prepared mixed aqueous solution of 189 ml of caustic soda solution having a concentration of 6.0 mol and 11 ml of pure water while stirring. After the addition was complete, the temperature was maintained at 60 ° C. and stirring was continued for 60 minutes. The product was a black colloidal solution that was sensitive to magnets.
As a result of analyzing a powder obtained by collecting a part of the obtained aqueous colloid solution and freeze-drying the paste obtained by washing with water, the particle size of the primary particle is a particle powder having a spinel crystal structure with an average particle size of 11 nm. The coefficient of variation of the distribution was 7%. The magnetic iron oxide fine particles had a Fe ²⁺ content of 13.8 mol%, and the magnetic properties were superparamagnetic iron oxide particles having a saturation magnetization σs of 64 Am ² / kg and a coercive force Hc of 2.0 kA / m. . The aggregate particle diameter was 85 nm.

(2) Purification process of colloidal particles The resulting aqueous solution of black colloid contains soluble salts produced as a by-product of the synthesis reaction of black colloidal particles. Therefore, by-product salt is formed by decantation using ion-exchanged water. The black colloid aqueous solution was purified by washing with water.

(3) Refining process of drug substance containing magnetic substance (substitution with ultrapure water)
100 ml of the remaining black colloid aqueous solution collected in order to evaluate the physical properties of the product was collected with good stirring, and the colloid aqueous solution was subjected to solid-liquid separation using a centrifuge to remove the dispersion medium. Thereafter, the same amount of ultrapure water was injected and redispersed using an ultrasonic disperser. This was repeated as 5 cycles, and the dispersion medium of the colloidal aqueous solution was replaced with ultrapure water. Next, ultrapure water is added to the colloidal aqueous solution to adjust the colloidal particle concentration to 22 mg / ml, and 0.1 Z caustic soda aqueous solution is added while dispersing with an ultrasonic disperser to adjust the zeta potential to -55 mV. did. After 60 minutes, the ultrasonic disperser was stopped and the aqueous colloid solution was allowed to stand, and left for 360 minutes.
It was observed that no precipitation occurred in the obtained colloidal aqueous solution and that no magnetic aggregation occurred using a permanent magnet having a surface magnetic flux of 10 mT (100 gauss). This colloidal aqueous solution was subjected to a toxicity test and an endotoxin test, both were confirmed to be negative, and 150 ml of a monodispersed colloidal sterile aqueous solution of superparamagnetic iron oxide particles was produced.
The concentration of the magnetic iron oxide fine particles in the obtained aqueous colloidal solution was 20 mg / ml, the pH of the aqueous colloidal solution was 9.7, and the electric conductivity was 210 μS.

Example 2
In the synthesis step of superparamagnetic iron oxide particles in the above step (1), 150 ml of an aqueous ferrous chloride solution having a concentration of 0.5 mol, 200 ml of an aqueous solution of 0.5 mol of magnesium sulfate, and an aqueous solution concentration of 1.0 mol Except for using 225 mL of an aqueous ferric chloride solution, each of the steps (2) and (3) was performed under the same conditions as in Example 1, and a magnetic drug substance containing a drug substance containing magnetic iron oxide particles at a concentration of 20 mg / ml was used. Generated.

As a result of analyzing a powder obtained by freeze-drying a paste obtained by collecting a portion of the obtained aqueous colloid solution, washing and filtering with water, the particle size distribution of primary particles is a particle powder having a spinel crystal structure with an average particle diameter of 12 nm. The variation coefficient of was 7%. Further, it is magnetic iron oxide fine particles having an Fe ²⁺ content of 10 mol% and an Mg ²⁺ content of 3.5 mol%, and the magnetic properties are a saturation magnetization σs of 63 Am ² / kg and a coercive force Hc of 1.4 kA / m. The superparamagnetic iron oxide particles. The aggregate particle diameter was 125 nm.

In the same manner as in Example 1, the dispersion medium was replaced with ultrapure water, the concentration of the colloidal aqueous solution was diluted to 20 mg / ml, and at the same time, a 0.1 N aqueous sodium hydroxide solution was added to adjust the zeta potential to -45 mV. did. Furthermore, it was observed that no magnetic aggregation occurred using a permanent magnet having a surface magnetic flux density of 10 mT (100 gauss). The colloidal aqueous solution was endotoxin-checked to confirm that it was negative, and 150 ml of a monodispersed colloidal sterile aqueous solution of superparamagnetic iron oxide particles was produced.
The resulting aqueous colloidal solution had a pH of 9.8 and an electric conductivity of 250 μS.

Comparative Example A colloidal aqueous solution was prepared in the same manner as in Example 1. After purification and replacement with ultrapure water, a 0.1N aqueous sodium hydroxide solution was added to adjust the zeta potential to -15 mV.
The obtained aqueous colloidal solution had a pH of 8.1, an electric conductivity of 56 μS, and an aggregate particle size of 280 nm.

Since the magnetic substance-containing medicinal drug substance according to the present invention is a monodispersed, sterile aqueous solution of magnetic iron oxide fine particles, it is easy to synthesize a drug comprising a composite in which magnetic fine particles are uniformly dispersed in a biocompatible substance. In addition, in the pharmaceutical granulation step, the function of a ferromagnetic material can be imparted to the granulated particles by adjusting the aggregate state of the fine particles.
In addition, since the liquid medium is a drug substance that does not contain a surfactant or the like, and it is an ultrafine particle, it can be easily excreted from the body after administration, so that safety and metabolism after administration to the human body.・ We can provide drug substance that does not cause any problems with excretion.

Claims (2)

A colloid aseptic aqueous solution in which magnetic iron oxide fine particles having an average primary particle size of 5 to 30 nm and an aggregated particle size of 30 to 200 nm are dispersed, and the magnetic iron oxide fine particles are a composition having a spinel structure MOFe ₂ O ₃ (M is Fe and / or Mg of divalent metal (however, the sum of Fe and Mg is 1 mol or less with respect to 1 mol of Fe ₂ O ₃ ), the saturation magnetization is 35 to 90 Am ² / kg, and the coercive force is 0 to 0 6.0 kA / m, the coefficient of variation of primary particles is 10% or less, the concentration of magnetic iron oxide fine particles in the colloid sterile aqueous solution is 5 to 50 mg / ml, and the pH of the colloidal aqueous solution is 9.0 or more. and zeta potential -20mV or less, the magnetic particle-containing pharmaceutical drug substance electrical conductivity, characterized in der Rukoto than 50 [mu] S. Magnetic iron oxide particles and phospholipid of claim 1 Symbol placement, polysaccharides, magnetic particle-containing pharmaceutical drug, which is a complex of a protein or dextrins.