JP2019210219A - Antitumor agent, and pharmaceutical composition for treating and/or preventing cancer - Google Patents

Abstract

To provide a novel medical drug that stays in a tumor site after administration for a long period of time and shows continuous tumor suppressing effect.SOLUTION: An antitumor agent contains a resin composite 100 having a structure in which a plurality of platinum nanoparticles are immobilized on a resin particle as an active ingredient. In the resin composite 100, at least a part of the platinum nanoparticles 20 is three-dimensionally distributed on a surface layer part 60 of the resin particle 10, and may include contained platinum nanoparticles 30 completely contained into the resin particle 10, partially exposed platinum nanoparticles 40 having a site buried in the resin particle 10 and a site exposed outside from the surface of the resin particle 10, and surface-adsorbed platinum nanoparticles 50 adsorbed on the surface of the resin particle 10.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a medicament that can be used for the treatment and / or prevention of cancer.

  Platinum compounds such as cisplatin, oxaliplatin, and carboplatin are known to have a strong anticancer action and are used as cancer therapeutics (for example, Patent Document 1). However, platinum compounds have serious side effects such as nephrotoxicity and neurotoxicity. In an attempt to reduce such side effects, a colloid-stable conjugate in which a platinum compound and gold, silver, or platinum nanoparticles are bound by a linker is used as a drug delivery system to reduce the attack on normal cells. It has been proposed (for example, Patent Document 2).

  MagNaGel (trademark) cancer coated with polyethylene glycol-polycarboxylic acid copolymer with 5-10 nm size magnetic iron oxide particles as a core and bound with diaminoplatinum (II) [Diamminoplatinum (II)] Toxicity to cells and drug delivery systems have been studied (Non-patent Document 1). In addition, it has been reported that platinum nanoparticles capped with several nm in size capped with polyvinyl alcohol show DNA damage, and are expected to be applied as cancer therapeutic agents having a drug delivery function (for example, Non-Patent Document 2). Furthermore, when we examined the cytotoxicity of platinum nanoparticles (Bioplatin) synthesized using green technology, they induced apoptosis in cancer cells but did not show cytotoxicity in normal cells. It has been reported (for example, Non-Patent Document 3). In this report, platinum nanoparticles having a particle diameter of 137.5 nm are used, and it has not been elucidated whether the same characteristics are exhibited with respect to platinum nanoparticles having a particle diameter of several tens of nm.

Special table 2014-530219 gazette Special table 2012-512149 gazette

Sunderland et al., “Targeted Nanoparticles for Detecting and Treating Cancer”, DRUG DEVELOPMENT RESEACH 67: 70-93 (2006) Joao Conde et al., “Noble Metal Nanoparticles Application in Cancer”, JOURNAL OF DRUG DELIVERY, Vol.2012, Article ID 751075 Yogesh Bendale et al., "Evaluation of cytotoxic activity of platinum nanoparticles against normal and cancer cells and its anticancer potential through induction of apoptosis", Integrative Medicine Research 6, 141-148 (2017).

  In order to suppress the side effects caused by cancer drugs, it is important to reduce the aggressiveness to normal cells. For this purpose, a drug that can be locally administered, hardly circulates in the living body by blood flow, and has a property of staying at the tumor site is preferable.

  An object of the present invention is to provide a novel medicament which remains at a tumor site for a long period of time after administration and exhibits a continuous tumor suppressive action.

  As a result of intensive studies, the present inventors have found that a resin composite having a structure in which a plurality of platinum nanoparticles are immobilized on resin particles is useful as an antitumor agent that can solve the above-described problems. The present invention has been completed.

  That is, the present invention is an antitumor agent containing, as an active ingredient, a resin complex having a structure in which a plurality of platinum nanoparticles are immobilized on resin particles.

  In the antitumor agent of the present invention, the supported amount of the platinum nanoparticles may be in the range of 5 to 70% by weight with respect to the entire resin complex.

  The antitumor agent of the present invention may be a polymer particle in which the resin particles have a substituent in the structure capable of adsorbing platinum compound ions.

  In the antitumor agent of the present invention, the platinum nanoparticles may have an average particle size of 80 nm or less.

  In the antitumor agent of the present invention, the resin composite may have an average particle size in the range of 100 to 700 nm.

  In the antitumor agent of the present invention, the tumor to be treated may be a solid cancer.

  The pharmaceutical composition for the treatment and / or prevention of cancer of the present invention comprises a resin complex having a structure in which a plurality of platinum nanoparticles are immobilized on resin particles, a pharmaceutically acceptable carrier or medium, and , Containing.

  Since the antitumor agent of the present invention can be locally administered and has a localized storage property that stays at the tumor site for a long time after administration, it exhibits a continuous tumor suppressing effect. Further, since the platinum nanoparticles contained in the resin complex show little toxicity to normal cells, side effects can be suppressed. Therefore, the antitumor agent of the present invention broadens the options for cancer treatment and is useful as a novel therapeutic agent for cancer.

It is a schematic diagram which shows the structure of the cross section of the resin composite used for one embodiment of this invention. It is a figure which shows the influence of the antitumor proliferation with respect to the oral squamous cell carcinoma cell which concerns on the Example and comparative example of this invention. It is a figure which shows the result of the HE dye | staining which verified the influence on the proliferation of the oral squamous cell carcinoma cell in the cancer bearing mouse concerning the comparative example 1 of this invention pathologically. It is a photograph which shows the structure | tissue extracted from the cancer bearing mouse concerning the comparative example 1 of this invention. It is a figure which shows the result of the HE dye | staining which verified the influence on the proliferation of the oral squamous cell carcinoma cell in the cancer bearing mouse which concerns on Example 1 of this invention pathologically. It is a photograph which shows the structure | tissue extracted from the cancer bearing mouse which concerns on Example 1 of this invention.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate. The antitumor agent of the present embodiment contains a resin complex having a structure in which a plurality of platinum nanoparticles are immobilized on resin particles as an active ingredient. Here, “tumor” means a neoplasm that grows or proliferates regardless of whether it is malignant or benign, and includes precancerous and cancerous cells and tissues. In addition, “cancer” generally means a tumor (malignant tumor) with autonomous growth, metastasis and wetting. Note that “tumor” and “cancer” are not mutually exclusive, and the term “antitumor” includes the meaning of “anticancer”.

<Resin composite>
Next, the resin composite used in the present embodiment will be described in detail.
FIG. 1 is a schematic cross-sectional view of a resin composite that can be preferably used in an embodiment of the present invention. The resin composite 100 includes resin particles 10 and platinum nanoparticles 20, and has a structure in which a plurality of platinum nanoparticles 20 are immobilized on the resin particles 10.

  In the resin composite 100, a plurality of platinum nanoparticles 20 are immobilized on the resin particles 10. In the resin composite 100, at least a part of the platinum nanoparticles 20 is three-dimensionally distributed in the surface layer portion 60 of the resin particles 10. Here, the “surface layer portion” refers to a particle radius of 50 in the depth direction from the surface of the resin particle 10 with reference to the outermost end portion of the platinum nanoparticle 20 protruding outward from the surface of the resin particle 10. Means a range up to%. In addition, “three-dimensional distribution” means that the platinum nanoparticles 20 are present not only in the plane direction of the resin particles 10 but also in the depth direction (radial direction). In addition, the three-dimensional existence state (distribution) of the platinum nanoparticles 20 in the resin composite 100 can be measured as follows. First, the resin composite 100 is cut near its center to expose a cross section. Since the resin composite 100 is a fine particle, a plurality of resin composites 100 are cut and the particle diameter D1 of the resin composite 100 measured by scanning electron microscope (SEM) observation is measured. By selecting a cross section having a diameter close to the average (average particle diameter), it can be estimated that the section is cut near the center. Next, the number and distribution of platinum nanoparticles 20 appearing in the cross section are measured by observation with a scanning electron microscope (SEM). Further, by calculating the volume from the number of platinum nanoparticles 20 appearing in the cross section and the average (average particle diameter) of the particle diameter D3, and converting the volume into a weight ratio, the resin composite 100 is three-dimensional in the radial direction. The dispersion ratio can be calculated.

  In the resin composite 100, a part of the three-dimensionally distributed platinum nanoparticles 20 may be partially exposed from the surface of the resin particle 10, and the remaining part is encapsulated in the resin particle 10. May be. That is, the platinum nanoparticle 20 is a platinum nanoparticle completely encapsulated in the resin particle 10 (hereinafter also referred to as “encapsulated platinum nanoparticle 30”), a portion embedded in the resin particle 10 and the resin particle 10. Platinum nanoparticles having portions exposed to the outside from the surface (hereinafter also referred to as “partially exposed platinum nanoparticles 40”) and platinum nanoparticles adsorbed on the surface of the resin particles 10 (hereinafter referred to as “surface-adsorbed platinum nanoparticles”). Also referred to as “particle 50”).

  The encapsulated platinum nanoparticles 30 are all covered with a resin constituting the resin particles 10 on the entire surface thereof.

  The partially exposed platinum nanoparticles 40 are those in which 5% or more and less than 100% of the surface area is covered with the resin constituting the resin particles 10. From the viewpoint of durability, the lower limit is preferably 20% or more, more preferably 30% or more of the surface area. The partially exposed platinum nanoparticles 40 have a large physical adsorption force such as an anchor effect due to the embedded state in addition to a large contact area with the resin particles 10 as compared with the surface-adsorbed platinum nanoparticles 50. Hard to detach from. Therefore, the antitumor agent using the resin composite 100 has excellent durability and stability, and stays in the vicinity of the tumor cells for a long period after administration, thus providing a continuous tumor suppressing effect. .

  The surface-adsorbed platinum nanoparticles 50 are those in which more than 0% and less than 5% of the surface area is covered with the resin constituting the resin particles 10. Since the surface-adsorbed platinum nanoparticles 50 have a large surface area of the exposed portion, the tumor-suppressing action by platinum can be expressed at an early stage.

  In addition, the loading amount of the platinum nanoparticles 20 (the total of the encapsulated platinum nanoparticles 30, the partially exposed platinum nanoparticles 40, and the surface-adsorbed platinum nanoparticles 50) on the resin composite 100 is based on the weight of the resin composite 100. It is preferably 5% by weight to 70% by weight. If the loading is within this range, the resin composite 100 exhibits a practical tumor suppressing action. When the loading amount of the platinum nanoparticles 20 is less than 5% by weight, the tumor suppressing action tends to be weakened, and the efficiency with respect to the dose may be lowered. The supported amount of the platinum nanoparticles 20 is more preferably 15% by weight to 70% by weight.

  Further, 10 wt% to 90 wt% of the platinum nanoparticles 20 are preferably partially exposed platinum nanoparticles 40 and surface adsorbed platinum nanoparticles 50. If it is this range, the tumor suppression effect with respect to a tumor cell is fully securable. More preferably, 20 wt% to 80 wt% of the platinum nanoparticles 20 are partially exposed platinum nanoparticles 40 and surface adsorbed platinum nanoparticles 50, and from the viewpoint of durability, the surface adsorbed platinum nanoparticles 50 are 20 wt%. More preferably, it is as follows.

  Further, in order to exert a sufficient tumor suppressing action, 60 wt% to 100 wt% of the platinum nanoparticles 20 are present in the surface layer portion 60, and 5 wt% to 90 wt% of the platinum nanoparticles 20 existing in the surface layer portion 60. % Is preferably partially exposed platinum nanoparticles 40 or surface adsorbed platinum nanoparticles 50. In other words, it is preferable that 10 wt% to 95 wt% of the platinum nanoparticles 20 existing in the surface layer portion 60 are the encapsulated platinum nanoparticles 30. The definition of the “surface layer portion” is as described above, but platinum existing across the boundary portion (50% of the particle radius in the depth direction from the surface) inside the surface layer portion 60 inside the resin particle 10. All the nanoparticles 20 are counted as existing in the surface layer portion 60.

<Resin particles>
The resin particle 10 is preferably a polymer particle having a structure capable of adsorbing platinum compound ions in its structure. In particular, nitrogen-containing polymer particles are preferable. Nitrogen atoms in the nitrogen-containing polymer are preferable because they easily chemisorb anionic ions that are precursors of the platinum nanoparticles 20. In the present embodiment, the platinum compound ions adsorbed in the nitrogen-containing polymer are reduced to form the platinum nanoparticles 20, so that some of the generated platinum nanoparticles 20 are encapsulated platinum nanoparticles 30 or partially exposed platinum. Nanoparticles 40 are formed.

  The nitrogen-containing polymer is a resin having a nitrogen atom in the main chain or side chain, and examples thereof include polyamine, polyamide, polypeptide, polyurethane, polyurea, polyimide, polyimidazole, polyoxazole, polypyrrole, and polyaniline. Preferable are polyamines such as poly-2-vinylpyridine, poly-3-vinylpyridine, and poly-4-vinylpyridine. Since polyamine is considered to have high biocompatibility and low possibility of causing side effects, it is advantageous as a constituent component of the resin particle 10. Moreover, when it has a nitrogen atom in a side chain, for example, an acrylic resin, a phenol resin, an epoxy resin, etc. can be used widely.

  Moreover, since carboxylic acid etc. can adsorb | suck a cationic ion like an acrylic acid polymer, it is easy to adsorb | suck the cationic ion which is the precursor of the platinum nanoparticle 20, and forms the platinum nanoparticle 20. Is possible.

  On the other hand, in the case of resin particles other than polymers having a substituent capable of adsorbing platinum compound ions, such as polystyrene, it is difficult to adsorb anionic ions or cationic ions inside the resin. As a result, most of the generated platinum nanoparticles 20 become surface-adsorbed platinum nanoparticles 50. As described above, since the surface-adsorbed platinum nanoparticles 50 have a small contact area with the resin particles 10, the adhesive force between the resin and the platinum nanoparticles 20 is small, and the platinum nanoparticles 20 tend to be detached from the resin particles 10. And storage stability may be lowered.

  In addition, known polymer units exhibiting biocompatibility such as polyethylene glycol, polyethylene oxide, polylactide, polyglycolide, polycaprolactone, poly (butylene terephthalate), ethylene-vinyl acetate copolymer, and the like are known. It is preferable to introduce into the resin particles by this method.

<Platinum nanoparticles>
The platinum nanoparticles 20 are platinum or platinum alloy nanoparticles. Here, the platinum alloy is made of, for example, platinum and a metal species other than platinum. The platinum content in the platinum alloy is not particularly limited as long as the tumor suppressive action is exhibited by platinum. For example, the platinum content is preferably 10% by weight or more, more preferably 50% by weight or more. Here, as the metal species other than platinum, for example, gold, silver, palladium and the like are preferable, and an alloy containing two or more of these may be used.

  The average value of the particle diameter D3 (average particle diameter) of the platinum nanoparticles 20 measured by scanning electron microscope (SEM) observation is preferably, for example, 80 nm or less because the tumor suppression action increases as the particle diameter decreases. When the average particle diameter of the platinum nanoparticles 20 exceeds 80 nm, the tumor suppressive action tends to decrease. The lower limit of the average particle diameter of the platinum nanoparticles 20 is not particularly limited and may be less than 1 nm. The average particle diameter of the platinum nanoparticles 20 is preferably 1 nm or more and less than 70 nm, and more preferably 1 nm or more and less than 50 nm.

<Average particle diameter of resin composite>
The average value (average particle diameter) of the particle diameter D1 of the resin composite 100 is a particle capable of directly acting on the cell membrane since the expression of cell killing activity by the resin composite 100 is observed in the form of necrosis. The diameter is preferably 100 to 700 nm, for example. In addition, when the average particle diameter of the resin composite 100 is less than 100 nm, for example, the amount of platinum nanoparticles 20 supported tends to decrease, and thus the tumor suppressing action tends to be weakened. The average particle diameter of the resin composite 100 is more preferably 100 nm or more and less than 650 nm. Here, the particle diameter D1 of the resin composite 100 means a value obtained by adding the length of the protruding portion of the partially exposed platinum nanoparticles 40 or the surface-adsorbed platinum nanoparticles 50 to the particle diameter D2 of the resin particles 10. It can be measured by laser diffraction / scattering method, dynamic light scattering method, or centrifugal sedimentation method.

<Manufacture of resin composite>
The manufacturing method of the resin composite 100 is not particularly limited. For example, a solution containing platinum compound ions is added to a dispersion of resin particles 10 produced by an emulsion polymerization method to adsorb platinum compound ions to the resin particles 10 (hereinafter referred to as “platinum ion-adsorbing resin particles”). . Furthermore, by adding the platinum ion-adsorbing resin particles to the reducing agent solution, the platinum compound ions can be reduced to generate the platinum nanoparticles 20, and the resin composite 100 can be obtained.

Examples of the solution containing platinum compound ions include a chloroplatinic acid (H ₂ PtCl ₆ ) aqueous solution. A metal complex may be used in place of the platinum compound ion. In addition, as a solvent of the solution containing platinum compound ions, instead of water, water-containing alcohol or alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, t-butanol, hydrochloric acid, sulfuric acid, An acid such as nitric acid may be used.

  In addition, if necessary, the solution containing platinum compound ions, for example, water-soluble polymer compounds such as polyvinyl alcohol, surfactants, alcohols; ethers such as tetrahydrofuran, diethyl ether, diisopropyl ether; alkylene glycol, You may add additives, such as polyalkylene glycol, these monoalkyl ethers or dialkyl ethers, polyols, such as glycerol; Various water-miscible organic solvents, such as ketones, such as acetone and methyl ethyl ketone. Such an additive is effective in promoting the reduction reaction rate of platinum ions and controlling the size of the produced platinum nanoparticles 20.

A well-known thing can be used for a reducing agent. For example, sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid, sodium phosphinate, hydroquinone, hydrazine sulfate, formaldehyde And Rochelle salt. Of these, sodium borohydride, dimethylamine borane, and citric acid are preferable. A surfactant can be added to the reducing agent solution as necessary, and the pH of the solution can be adjusted. The pH can be adjusted with a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide.
Furthermore, the particle size of the platinum nanoparticles 20 to be generated can be controlled by adjusting the reduction rate of the platinum compound ions depending on the temperature of the reducing agent solution.

  Further, when the platinum compound ions in the platinum ion adsorbing resin particles are reduced to generate the platinum nanoparticles 20, the platinum ion adsorbing resin particles may be added to the reducing agent solution, or the reducing agent may be added to the platinum ion adsorbing resin particles. The former is preferable from the viewpoint of easy generation of the encapsulated platinum nanoparticles 30 and the partially exposed platinum nanoparticles 40.

  In order to maintain the dispersibility in water, the resin composite 100 obtained as described above is, for example, citric acid, poly-L-lysine, polyvinyl pyrrolidone, polyvinyl pyridine, polyvinyl alcohol, DISPERBYK194, DISPERBYK180, DISPERBYK184 ( A dispersing agent such as Big Chemie Japan may be added. Furthermore, the dispersibility can be maintained by adjusting the pH with a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide.

  Since the resin composite 100 having the above configuration has a tumor suppressing action, it is preferable as a pharmaceutical for cancer prevention and treatment. For example, an antitumor agent comprising the resin complex 100 as an active ingredient is widely applicable to cancer treatment, but is suitable for treatment of solid cancer among cancers. Examples of solid cancer include head and neck cancer (laryngeal cancer, pharyngeal cancer, tongue cancer, etc.), esophageal cancer, stomach cancer, colon cancer, duodenal cancer, lung cancer, liver cancer, biliary tract cancer. , Bladder cancer, penile cancer, testicular cancer, breast cancer, prostate cancer, renal pelvis / ureter tumor, ovarian cancer, cervical cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosis Examples include sarcomas, liposarcomas, and intraepithelial neoplasia. Among these solid cancers, for example, head and neck cancer (laryngeal cancer, pharyngeal cancer, tongue cancer, etc.), esophageal cancer, stomach cancer, colon cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer It is preferably applied to cancer and the like, and particularly preferably applied as a therapeutic agent for oral squamous cell carcinoma which is a kind of oral cancer.

<Pharmaceutical composition>
When the resin complex 100 is used for treatment and / or prevention of cancer, it can be used as a pharmaceutical composition containing the resin complex 100 and a pharmaceutically acceptable carrier or medium. Any pharmaceutically acceptable carrier or vehicle may be used without particular limitation as long as it is not toxic to the patient at the dosage and concentration used. Preferred examples of the carrier or medium include water, physiological saline, alcohol, phosphate, organic acid salt buffer, low molecular weight polypeptide, serum albumin, gelatin, immunoglobulin protein, etc., hydrophilicity such as polyvinylpyrrolidone. Examples include polymers, various amino acids, sugars and carbohydrates such as glucose, sucrose, mannitol, trehalose, sorbitol, mannose, and dextrin, chelating agents such as EDTA, salt-forming ions such as sodium and potassium, metal complexes, and the like.

  Moreover, it is possible to mix | blend an additive with the pharmaceutical composition of this invention if desired. Examples of the additive include excipients, binders, disintegrants, fluidizers, emulsifiers, antioxidants, preservatives, and stabilizers. These additives may be used alone or in combination of two or more.

  Moreover, you may mix | blend the other medicinal component which has an anticancer effect | action etc. with the pharmaceutical composition of this invention. The type of other medicinal components is not particularly limited as long as it does not affect the resin composite 100, but is preferably one that can be taken into tumor cells and exert a tumor suppressing effect. , Antimetabolites, anticancer antibiotics, topoisomerase inhibitors and the like. These medicinal ingredients can be used alone or in combination of two or more.

  The pharmaceutical composition of the present invention can be produced by a known production method. Specifically, it can be produced by mixing, dispersing, etc. the resin composite 100 in an arbitrary carrier or medium.

  The pharmaceutical composition of the present invention may be a single preparation containing the resin complex 100, the first preparation containing the resin complex 100 and, if necessary, a carrier and a medium, the carrier, the medium, and other components. A second preparation containing the same may be prepared separately, and a plurality of these preparations may be mixed and administered at the time of use, or may be administered simultaneously or sequentially.

<Method of administration>
The pharmaceutical composition of the present invention may be administered surgically by surgery, or may be administered parenterally or orally in the form of a formulation. Examples of dosage forms for parenteral administration include injections, ointments, plasters, poultices, lotions and the like. Examples of dosage forms for oral administration include tablets, capsules, powders, fine granules, granules, liquids, troches, and jelly. As a preferable dosage form of the pharmaceutical composition, a parenteral preparation is preferable, and an injection is more preferable. Further, by allowing the resin complex 100 to bind an antibody specific to the target tumor cell, oral administration or administration from blood vessels is possible.

  The dosage of the pharmaceutical composition of the present invention to a patient is appropriately determined in consideration of the patient's sex, age, weight, symptoms, administration method, frequency of administration, administration timing, and the like.

  In addition, after the administration of the pharmaceutical composition of the present invention, a treatment method for supplying external energy such as laser irradiation, radiation irradiation, high-frequency irradiation, etc. to the tumor tissue may be used in combination.

  EXAMPLES Next, although an Example demonstrates this invention concretely, this invention is not limited at all by these Examples. Unless otherwise specified in the following examples and comparative examples, various measurements and evaluations are as follows.

<Measurement of solid content concentration and metal loading>
Into a magnetic crucible, 1 g of the dispersion of the resin composite before concentration adjustment was placed and dried at 70 ° C. for 3 hours. The weight before and after drying was measured, and the solid content concentration was calculated by the following formula.

  Solid content concentration (% by weight) = [weight after drying (g) / weight before drying (g)] × 100

Further, the sample after the above drying treatment was further subjected to heat treatment at 500 ° C. for 5 hours, the weight before and after the heat treatment was measured, and the metal loading was calculated from the following formula.
Metal loading (% by weight) = [weight after heat treatment (g) / weight before heat treatment (g)] × 100

<Measurement of average particle diameter of resin particles and resin composite>
The measurement was performed using a disk centrifugal particle size distribution analyzer (CPS Disc Centrifuge DC24000 UHR, manufactured by CPS Instruments, Inc.). The measurement was performed with the resin particles or the resin composite dispersed in water.

<Measurement of average particle diameter of metal nanoparticles>
A substrate prepared by dropping the resin composite dispersion on a metallic mesh with a carbon support film was arbitrarily observed from an image observed with a field emission scanning electron microscope (FE-SEM; manufactured by Hitachi High-Technologies Corporation, SU-9000). The area average diameter of 100 metal nanoparticles was measured.

[Synthesis Example 1]
<Synthesis of resin particles (latex beads)>
After dissolving methyltri-n-octylammonium chloride (1 g) and 50 wt% polyethylene glycol methyl ether methacrylate aqueous solution (10 g) in 300 g of pure water, 2-vinylpyridine (48 g) and divinylbenzene (2 g) were added, The mixture was stirred at 150 rpm, 30 ° C. for 50 minutes, and then at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (0.5 g) dissolved in 18.0 g of pure water was added dropwise, stirred at 150 rpm and 60 ° C. for 5 hours, and then up to 40 ° C. By stirring with air cooling, latex beads having an average particle diameter of 430 nm were obtained. After sedimentation by centrifugation (13500 × g, 40 minutes), the supernatant was removed, and then the impurities were removed by redispersing in pure water. Thereafter, the concentration was adjusted to obtain a 10% by weight latex bead dispersion.

[Synthesis Example 2]
<Preparation of platinum nanoparticle-resin composite (Pt nanocomposite beads)>
After adding the latex beads (50 g) obtained in Synthesis Example 1 to 130 g of pure water, 400 mM chloroplatinic acid aqueous solution (55 g) was added and stirred at 30 ° C. for 3 hours. Thereafter, latex beads were precipitated by centrifugation (1700 × g, 30 minutes), and excess chloroplatinic acid was removed by removing the supernatant. Thereafter, the concentration was adjusted to prepare a 5.0 wt% platinum ion adsorbing resin particle dispersion.

  Next, after adding 5.0 wt% platinum ion adsorption resin particle dispersion (70 g) to 4760 g of pure water and stirring at 3 ° C., 132 mM dimethylamine borane aqueous solution (138 g) was added dropwise over 22 minutes. Pt nanocomposite beads having an average particle diameter of 450 nm were obtained by stirring at 3 ° C. for 1 hour and at 25 ° C. for 3 hours. Pt nanocomposite beads were precipitated by centrifugation, and the concentrated liquid from which the supernatant was removed was purified by dialysis, and then the concentration was adjusted to obtain a 1% by weight Pt nanocomposite bead dispersion. The average particle diameter of platinum nanoparticles in the produced Pt nanocomposite beads was 3.8 nm, and the supported amount of platinum was 38% by weight.

[Synthesis Example 3]
<Preparation of gold nanoparticle-resin composite (Au nanocomposite beads)>
After adding the latex beads (50 g) obtained in Synthesis Example 1 to 130 g of pure water, a 400 mM chloroauric acid aqueous solution (82 g) was stirred at 30 ° C. for 3 hours. Thereafter, latex beads were precipitated by centrifugation (1700 × g, 30 minutes), and excess chloroauric acid was removed by removing the supernatant. Thereafter, the concentration was adjusted to prepare a 5.0 wt% gold ion adsorption resin particle dispersion.

  Next, the 5.0 wt% gold ion adsorption resin particle dispersion (65 g) was added to 4780 g of pure water, and a 528 mM dimethylamine borane aqueous solution (20 g) was added dropwise over 2 minutes while stirring at 3 ° C. By stirring at 3 ° C. for 1 hour and at 25 ° C. for 3 hours, Au nanocomposite beads having an average particle diameter of 452 nm were obtained. Au nanocomposite beads were precipitated by centrifugation, and the concentrated liquid from which the supernatant was removed was purified by dialysis, and then the concentration was adjusted to obtain a 1 wt% Au nanocomposite bead dispersion. The average particle diameter of the gold nanoparticles in the produced Au nanocomposite beads was 23.2 nm, and the amount of gold supported was 50% by weight.

[Example 1]
Oral squamous cell carcinoma using Eagle's minimal essential medium (manufactured by WAKO) with fetal bovine serum (10% by weight), penicillin (100 units / liter) and streptomycin (100 mg / liter) Cell line HSC-3 M3 cells (JCRB cell bank, cell number JCRB1354) were cultured at a concentration of 1 × 10 ⁷ cells / ml in a cell flask under conditions of 37 ° C. and 5% CO ₂ for 24 hours.
After aspirating the cell supernatant, Pt nanocomposite beads (450R) obtained in Synthesis Example 2 (concentration with respect to the cell culture medium: 0.5 mg / ml) were added, and the mixture was incubated at 37 ° C. and 5% CO ₂ for 48 hours. Cultured.

As anesthesia to mice, three types of mixed anesthesia were used, in which domitol 0.75 ml + dolmicum 2 ml + betolfal 2.5 ml was added with 19.75 ml of distilled water for injection, and 0.1 ml per 10 g body weight was intraperitoneally administered. Under anesthesia, HSC-3 M3 cells (1 × 10 ⁷ cells) were injected into the back of a KSN mouse (male, 6 weeks old) with an injection needle to create a tumor-bearing mouse model. The mice were reared for 10 days and observed for tumor formation.
The back tumor was measured every two days, and the tumor volume was determined by the formula [(major axis) × (minor axis) ² ] / 2. The increase rate of the tumor volume from the observation start date (day 0) is shown in FIG.

<Histopathological verification>
The mice were then euthanized by cervical dislocation and the tumor removed. A section was prepared from the excised tumor tissue, stained with hematoxylin eosin (HE), and observed under a microscope. In HE staining, the nucleus is stained blue-purple and the cytoplasm and stroma are stained pink, which is used for observing the tissue structure and cell morphology. The result of HE staining is shown in FIG. 4A, and the whole image of the excised tissue is shown in FIG. 4B.

[Comparative Example 1]
Instead of Pt nanocomposite beads (450R) (concentration with respect to cell culture medium: 0.5 mg / ml), only cell culture medium was added and cultured at 37 ° C. under 5% CO ₂ for 48 hours. In vivo antitumor growth effect was verified by the same method as in Example 1. The increase rate of the tumor volume from the observation start date (day 0) is shown in FIG.
Further, for Comparative Example 1 using only the cell culture medium, histopathological verification was performed in the same manner as in Example 1. The result of HE staining is shown in FIG. 3A, and the whole image of the extracted tissue is shown in FIG. 3B.

[Comparative Example 2]
Instead of Pt nanocomposite beads (450R) (concentration with respect to cell culture medium: 0.5 mg / ml), Au nanocomposite beads (concentration with respect to cell culture medium: 0.5 mg / ml) obtained in Synthesis Example 3 were added. The in vivo antitumor growth effect was verified in the same manner as in Example 1 except that the cells were cultured for 48 hours under conditions of 37 ° C. and 5% CO ₂ . The increase rate of the tumor volume from the observation start date (day 0) is shown in FIG.

[Comparative Example 3]
Instead of Pt nanocomposite beads (450R) (concentration with respect to cell culture medium: 0.5 mg / ml), the latex beads obtained in Synthesis Example 1 (concentration with respect to cell culture medium: 0.5 mg / ml) were added, The in vivo antitumor growth effect was verified in the same manner as in Example 1 except that the cells were cultured for 48 hours at 37 ° C. and 5% CO ₂ . The increase rate of the tumor volume from the observation start date (day 0) is shown in FIG.

[Comparative Example 4]
Instead of Pt nanocomposite beads (450R) (concentration with respect to cell culture medium: 0.5 mg / ml), Pt nanocolloid (nanoplatinum dispersion; manufactured by Renaissance Energy Research, concentration with respect to cell culture medium: 0. 2 mg / ml) was added, and the in vivo antitumor growth effect was verified in the same manner as in Example 1 except that the cells were cultured at 37 ° C. under 5% CO ₂ for 48 hours. The increase rate of the tumor volume from the observation start date (day 0) is shown in FIG.

In the verification of the anti-tumor growth effect in vivo, as shown in FIG. 2, in the case of only the cell culture solution (Comparative Example 1), about 10 days in the rear of the mouse after breeding for 10 days by injecting HSC-3 M3 cells. A 7-fold increase in tumor was observed. In the Au nanocomposite bead treatment group (Comparative Example 2), the tumor growth of 10 times, in the treatment group of only latex beads (Comparative Example 3), the tumor growth of 3 times, in the Pt nanocolloid treatment group (Comparative Example 4), 5-fold tumor growth was observed.
On the other hand, in the Pt nanocomposite bead treated group (Example 1), almost no increase in the tumor volume was confirmed, and a marked tumor growth inhibitory effect was recognized as compared with the other 4 groups (Comparative Examples 1 to 4). It was. Moreover, from the observation of the back of the mouse after administration, it was confirmed that the Pt nanocomposite beads remained at the administration site and had a local reservoir.

Moreover, in the histopathological verification, in the group (Comparative Example 1) in which tumor formation was performed only with HSC-3 M3 cells, as shown in FIGS. 3A and 3B, a volume of 405 mm having a major axis of 10 mm and a minor axis of 9 mm. ³ tumors were formed, and in the histological image, formation of carcinoma with marked nuclear abnormalities, tumor parenchyma composed of HSC-3 M3 cancer cells, and tumor stroma composed of fibrous connective tissue Was recognized.
On the other hand, in the group (Example 1) in which tumor formation was performed with HSC-3 M3 cells supplemented with the Pt nanocomposite beads of the present invention, as shown in FIGS. 4A and 4B, subcutaneous administration was performed by administration of Pt nanocomposite beads. Pigmentation was observed, but no tumor formation was observed. Moreover, infiltration of the tumor cell was not recognized in the histology.

  As mentioned above, although embodiment of this invention was described in detail for the purpose of illustration, this invention is not restrict | limited by the said embodiment.

DESCRIPTION OF SYMBOLS 10 ... Resin particle | grains, 20 ... Platinum nanoparticle, 30 ... Encapsulated platinum nanoparticle, 40 ... Partially exposed platinum nanoparticle, 50 ... Surface adsorption platinum nanoparticle, 60 ... Surface layer part, 100 ... Resin composite

Claims (7)

  An antitumor agent comprising, as an active ingredient, a resin complex having a structure in which a plurality of platinum nanoparticles are immobilized on resin particles.   The antitumor agent according to claim 1, wherein the supported amount of the platinum nanoparticles is in the range of 5 to 70% by weight with respect to the entire resin complex.   The antitumor agent according to claim 1 or 2, wherein the resin particle is a polymer particle having a substituent capable of adsorbing platinum compound ions in its structure.   The antitumor agent according to any one of claims 1 to 3, wherein the platinum nanoparticles have an average particle size of 80 nm or less.   The antitumor agent according to any one of claims 1 to 4, wherein an average particle size of the resin complex is in a range of 100 to 700 nm.   The antitumor agent according to any one of claims 1 to 5, wherein the tumor to be treated is a solid cancer. A pharmaceutical composition for treating and / or preventing cancer, comprising a resin complex having a structure in which a plurality of platinum nanoparticles are immobilized on resin particles, and a pharmaceutically acceptable carrier or medium.