Gold nanoparticle-containing pharmaceuticals

Abstract

The present invention pertains to a gold nanoparticle-containing medicine and a treatment for proliferative diseases using said medicine. The present invention also pertains to: a gold nanoparticle-containing medicine that is bound to an alpha ray-emitting isotope; and a treatment for proliferative diseases using said medicine.

Description

[Technical Field]

[0001] This invention relates to a pharmaceutical containing gold nanoparticles and the treatment of proliferative disorders using the same. This invention also relates to a pharmaceutical containing gold nanoparticles bound to an alpha-emitting radionuclide and the treatment of proliferative disorders using the same. [Background technology]

[0002] Treatment for malignant tumors, including brain tumors, involves surgery, chemotherapy with systemic administration of antitumor drugs, and radiation therapy using gamma rays. These treatments are often ineffective when used alone, and it is important to employ a multidisciplinary approach combining as many treatment methods as possible, but each has its own drawbacks. Surgical treatment is highly invasive, and systemic chemotherapy has very strong systemic side effects. Radiation therapy using gamma rays presents problems due to its relatively long range and resulting radiation exposure. Recently, treatments involving the direct administration of conventional antitumor drugs to malignant tumors have been developed, but their effectiveness has not yet been established.

[0003] It is known that the cytotoxicity of radiation varies depending on its ray quality. Alpha rays have a very high line energy transfer rate (LET) compared to gamma rays and beta rays, which are conventionally used in treatment (Non-Patent Literature 1), and have particularly high antitumor effects. In addition, alpha rays have a very short range and affect only a narrow area. Therefore, it is necessary to diffuse alpha-emitting radionuclides within the lesion while preventing their distribution to normal tissue. Conventionally, attempts to use alpha-emitting radionuclides in the treatment of proliferative diseases such as tumors have been proposed, such as attaching targeting molecules that specifically bind to or have affinity for target tumor cells (Non-Patent Literature 1, 2). However, since such methods require selecting the optimal targeting molecule for each tumor, there is a need for more versatile technology to apply alpha-emitting radionuclides to the treatment of proliferative diseases.

[0004] The inventors have previously demonstrated for the first time that AuNP(At)-PEG, which is gold nanoparticles (AuNP) conjugated with the alpha-emitting radionuclide astatine-211 (At-211) and polyethylene glycol (PEG), is useful for direct administration to gliomas. Because the alpha rays of AuNP(At)-PEG have a very short range, the diffusibility of the particles within tumor tissue is crucial, and therefore, adjusting the particle size is paramount. However, optimization of this particle size has not been performed to date, and its effectiveness when administered to living organisms has not been demonstrated. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Royal Society of Chemistry,2017,Vol.4,pp.41024-41032 [Non-Patent Document 2] Nanomaterials,2019,Vol.9,doi.org / 10.3390 / nano9040632,pp.1-15 [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide a pharmaceutical product with excellent therapeutic effects by optimizing the size of gold nanoparticles to regulate their diffusion characteristics in tumor cells and the like. [Means for solving the problem]

[0007] The inventors focused on non-toxic gold nanoparticles (AuNPs) and added polyethylene glycol (PEG) to ensure diffusibility in tissue fluid. As a result of diligent research into particle size to achieve high diffusion within tissues and non-diffusivity throughout the body, they have succeeded in creating the first locally administered therapeutic agent for gliomas that does not require the use of selectively targeted molecules for specific cells. This agent allows alpha-emitting radionuclides to remain locally within tumors for extended periods, exerting a strong antitumor effect on nearby malignant cells while preventing the drug from diffusing into organs throughout the body, thus achieving no side effects.

[0008] In one embodiment, the present invention utilizes the above-mentioned gold nanoparticles (AuNP) to directly administer alpha-emitting radionuclides to proliferative diseases and obtain a therapeutic effect. Furthermore, in one embodiment, the present invention provides a pharmaceutical agent for treating proliferative diseases, characterized by containing gold nanoparticles having a particle size of 0.5 to 110 nanometers bound to At-211, and being administered topically. In one embodiment, the surface of the gold nanoparticles may be modified with molecules selected from polyethylene glycol, polyether, polyol, polyethyleneimine, silica gel, peptide, antibody, protein, lipid, complex lipid, glycan, complex carbohydrate, terpene, terpenoid, and virus-like particles. In one embodiment, the surface modification includes a molecule that does not have a targeting molecule for a specific cell bound to it, and in yet another embodiment, the surface modification does not include a targeting molecule for a specific cell. In one embodiment, the molecule not bound to a targeting molecule for a specific cell may be polyethylene glycol having an average molecular weight of 2,000 to 20,000. In one embodiment, the particle size of the gold nanoparticles may be 0.5 to 13 nm. In one embodiment, the pharmaceutical product of the present invention can be administered by local administration selected from the group consisting of injection into a lesion, superselective administration into an artery nourishing the lesion, and intracavitary dispersal. In one embodiment, the proliferative disease is a malignant tumor, which may be a solid tumor. In one embodiment, the solid tumor may be selected from brain tumors, endocrine tumors, prostate cancer, head and neck cancer, oral cancer, breast cancer, gynecological cancer, skin cancer, pancreatic cancer, and gastrointestinal cancer.

[0009] In another embodiment, the present invention provides gold nanoparticles having a particle size of 0.5 to 110 nanometers, bound to At-211, and surface-modified with polyethylene glycol that is not bound to a targeting molecule for specific cells. In one embodiment, the gold nanoparticles of the present invention may further include surface modification with a targeting molecule for specific cells.

[0010] In another aspect, the present invention provides the use of gold nanoparticles having a particle size of 0.5 to 110 nanometers for manufacturing a topically administered pharmaceutical for treating proliferative disorders. In one embodiment, the gold nanoparticles are bound to At-211. In one embodiment, the surface of the gold nanoparticles may be modified with molecules selected from polyethylene glycol, polyether, polyol, polyethyleneimine, silica gel, peptide, antibody, protein, lipid, complex lipid, glycan, complex carbohydrate, terpene, terpenoid, and virus-like particles. In one embodiment, the surface of the gold nanoparticles may be modified with polyethylene glycol having a molecular weight of 2,000 or more.

[0011] In another aspect, the present invention provides a method for treating proliferative diseases by topically administering gold nanoparticles having a particle size of 0.5 to 110 nanometers, bound to At-211. In one embodiment, the surface of the gold nanoparticles may be modified with molecules selected from polyethylene glycol, polyether, polyol, polyethyleneimine, silica gel, peptide, antibody, protein, lipid, complex lipid, glycan, complex carbohydrate, terpene, terpenoid, and virus-like particles. In one aspect, the surface of the gold nanoparticles may be modified with polyethylene glycol having a molecular weight of 2,000 or more. In one aspect, the local administration can be selected from the group consisting of injection into a lesion, super-selective administration into an artery nourishing the lesion, and intraluminal spraying. In one aspect, the proliferative disease is a malignant tumor and may be a solid cancer. In one aspect, the solid cancer is selected from brain tumors, endocrine tumors, prostate cancer, head and neck cancer, oral cancer, breast cancer, gynecological cancer, skin cancer, pancreatic cancer, and digestive tract cancer.

[0012] In another aspect, the present invention also provides (1) A step of preparing At-211-conjugated gold nanoparticles having different particle diameters of 0.5 to 110 nanometers; (2) A step of administering each of the At-211-conjugated gold nanoparticles having the respective particle diameters into a proliferative disease tissue in vivo; (3) A step of confirming the alpha-ray distribution in the administered proliferative disease tissue and the whole-body alpha-ray distribution; (4) A step of selecting a particle diameter based on the alpha-ray distribution in the proliferative disease tissue and the whole-body alpha-ray distribution. A method for selecting the particle diameter of At-211-conjugated gold nanoparticles that is optimal for treating a proliferative disease by local administration is provided, which includes the above steps.

[0013] In one aspect, in addition to the step (4), it may further include (5) a step of evaluating the body weight change of the administered animal and / or the growth inhibitory effect on the proliferative disease tissue. In one aspect, the administration into the proliferative disease tissue can be selected from the group consisting of injection into the central part of the tissue, super-selective administration into an artery nourishing the tissue, and drug spraying into the cavity where the tissue is present. In one aspect, the proliferative disease tissue in vivo may be a heterologous proliferative disease tissue transplanted into a subject. In one aspect, the surface of the gold nanoparticles may be modified with a molecule selected from hydrocarbon polymers such as polyethylene glycol, polyether, and polyol, polyethyleneimine, silica gel, peptide, antibody, protein, lipid, complex lipid, sugar chain, complex carbohydrate, terpene, terpenoid, and virus-like particles. In one aspect, the surface of the gold nanoparticles may be modified with polyethylene glycol having a molecular weight of 2,000 or more. In one aspect, the proliferative disease is a malignant tumor and may be a solid cancer. In one aspect, the solid cancer is selected from brain tumor, endocrine tumor, prostate cancer, head and neck cancer, oral cancer, breast cancer, gynecological cancer, skin cancer, pancreatic cancer, and digestive tract cancer.

[0014] Also, in another aspect of the present invention, (1) A step of preparing At-211-conjugated gold nanoparticles having different particle diameters of 0.5 to 110 nanometers, (2) A step of administering the At-211-conjugated gold nanoparticles having each particle diameter into a proliferative disease tissue in vivo, (3) A step of confirming the alpha-ray distribution in the administered proliferative disease tissue and the whole-body alpha-ray distribution, (4) A step of selecting a particle diameter based on the alpha-ray distribution in the proliferative disease tissue and the whole-body alpha-ray distribution A method for producing At-211-conjugated gold nanoparticles having an optimal particle diameter for treating a proliferative disease by local administration is provided, which includes the above steps.

[0015] In one aspect, in addition to the step (4), the method may include (5) a step of evaluating the body weight change of the administered animal and / or the growth inhibitory effect on the proliferative disease tissue. In one aspect, the administration into the proliferative disease tissue can be selected from the group consisting of injection into the central part of the tissue, super-selective administration into the artery nourishing the tissue, and drug spraying into the cavity where the tissue is present. In one aspect, the proliferative disease tissue in vivo may be a heterologous proliferative disease tissue transplanted into a subject. In one embodiment, the surface of the gold nanoparticles may be modified with molecules selected from hydrocarbon polymers such as polyethylene glycol, polyethers, and polyols, polyethyleneimines, silica gel, peptides, antibodies, proteins, lipids, complex lipids, glycans, complex carbohydrates, terpenes, terpenoids, and virus-like particles. In one embodiment, the surface of the gold nanoparticles may be modified with polyethylene glycol having a molecular weight of 2,000 or more. In one embodiment, the proliferative disease is a malignant tumor, which may be a solid tumor. In one embodiment, the solid tumor is selected from brain tumors, endocrine tumors, prostate cancer, head and neck cancer, oral cancer, breast cancer, gynecological cancer, skin cancer, pancreatic cancer, and gastrointestinal cancer. [Effects of the Invention]

[0016] The alpha-emitting radionuclide-containing nanoparticles of the present invention possess excellent intratumor tissue diffusion and low systemic diffusion, effectively suppressing the proliferation of proliferative diseases while exhibiting low risk of damaging other organs. Furthermore, because there is little need to consider radiation exposure, extremely high doses can be repeatedly administered to proliferative disease tissue, providing a pharmaceutical product that exhibits a high inhibitory effect on the proliferation of proliferative disease tissue. [Brief explanation of the drawing]

[0017] [Figure 1] Figure 1 shows the results of scintigraphy analysis after administration of 120 nm AuNP(At)PEG to a subcutaneous glioma model rat. [Figure 2] Figure 2 shows the results of scintigraphy analysis after administration of 30 nm AuNP(At)PEG to a subcutaneous glioma model rat. [Figure 3] Figure 3 shows the results of scintigraphy analysis after administration of AuNP(I-123)PEG to a subcutaneous glioma model rat. [Figure 4]Figure 4 shows the results of autoradiography performed on subcutaneous glioma model rats after administration of 120nm AuNP(At)PEG or 30nm AuNP(At)PEG. [Figure 5] Figure 5 shows the results of autoradiography performed on subcutaneous glioma model rats after administration of PEG-unmodified 30nm AuNP(At). [Figure 6] Figure 6 shows the changes in tumor size in subcutaneous glioma model rats after administration of AuNP(At)PEG at 5 nm, 13 nm, 30 nm, or 120 nm. [Figure 7] Figure 7 shows the changes in body weight in subcutaneous glioma model rats after administration of AuNP(At)PEG at 5 nm, 13 nm, 30 nm, or 120 nm. [Figure 8] Figure 8 shows the mass of tumor tissue removed 42 days after administration of AuNP(At)PEG at 5 nm, 13 nm, 30 nm, or 120 nm in subcutaneously transplanted glioma model rats. [Figure 9] Figure 9 shows the results of scintigraphy analysis performed 4 hours, 19 hours, and 42 hours after injection of 5 nma mAuNP(At)PEG particles into a subcutaneous glioma model rat. [Figure 10] Figure 10 shows the changes in tumor size in subcutaneously transplanted renal cell carcinoma model mice after administration of AuNP(At)PEG or AuNPPEG (unlabeled). [Figure 11] Figure 11 shows the changes in body weight in subcutaneous transplanted renal cell carcinoma model mice after administration of AuNP(At)PEG or AuNP-PEG (unlabeled). [Figure 12] Figure 12 shows the mass of tumor tissue removed 40 days after administration to subcutaneous transplanted renal cell carcinoma model mice using AuNP(At)PEG or AuNP-PEG (unlabeled). [Figure 13]Figure 13 is a schematic diagram showing the structure of 5nm PEG-AuNP(At)-c[RGDfK(C)](5nm mPEG-S-AuNP[211At]-c[RGDfK(C)]). [Figure 14] Figure 14 shows the results of scintigraphy in a rat model of subcutaneously transplanted glioma. The results are shown 9 and 14 hours after selective intra-arterial administration of 5nm PEG-AuNP(At)-c[RGDfK(C)] to the artery supplying the tumor transplant area, followed by ligation of the left femoral artery. The arrows indicate the location of the subcutaneously transplanted tumor in the thigh. [Figure 15] Figure 15 shows the changes in body weight in a subcutaneous glioma model rat. It shows the changes in body weight of rats after selective intra-arterial administration of 5nm PEG-AuNP(At)-c[RGDfK(C)]. IA-1 and IA-2 show data from rats administered 5nm PEG-AuNP(At)-c[RGDfK(C)], while Ctl-1 and Ctl-2 show data from control rats that underwent only ligation of the left femoral artery without drug administration. [Figure 16] Figure 16 shows the changes in tumor volume in a subcutaneous glioma model rat. It shows the changes in tumor volume after selective intra-arterial administration of 5nm PEG-AuNP(At)-c[RGDfK(C)]. IA-1 and IA-2 show data from rats administered with 5nm PEG-AuNP(At)-c[RGDfK(C)], while Ctl-1 and Ctl-2 show data from control rats that did not receive the drug. [Figure 17] Figure 17 shows the results of scintigraphy in a mouse model of intraperitoneal glioma. It shows the results of scintigraphy 9 hours and 14 hours after intraperitoneal administration of 5nm PEG-AuNP(At)-c[RGDfK(C)]. [Figure 18]Figure 18 shows the survival curves in a mouse model of intraperitoneal glioma transplantation. The survival curves are shown after intraperitoneal administration of 5nm PEG-AuNP(At)-c[RGDfK(C)] two weeks after tumor transplantation (Group A). ​​"At" represents mice administered with 5nm PEG-AuNP(At)-c[RGDfK(C)] (n=3). "Ctl" represents mice administered with physiological saline as a control (n=2). [Figure 19] Figure 19 shows the changes in body weight in a mouse model of intraperitoneal glioma transplantation. It shows the changes in body weight after intraperitoneal administration of 5nm PEG-AuNP(At)-c[RGDfK(C)] (Group B) one week after tumor transplantation. "At" represents the average for mice administered 5nm PEG-AuNP(At)-c[RGDfK(C)] (n=3). "Ctl" represents the average for mice administered physiological saline as a control (n=3). [Figure 20] Figure 20 shows fluorescence imager results of tumors in intraperitoneal transplanted glioma model mice. It shows the results of fluorescence imager observation of tumor characteristics after intraperitoneal administration of 5nm PEG-AuNP(At)-c[RGDfK(C)] (Group B) one week after tumor transplantation. Data from three weeks after tumor transplantation and 11 days after drug administration are also shown. The left column, "At Administration," shows the results for mice administered 5nm PEG-AuNP(At)-c[RGDfK(C)], while the right column, "Control," shows the results for mice administered physiological saline as a control. [Figure 21] Figure 21 shows fluorescence imager results of tumors in a mouse model of intraperitoneal glioma transplantation. It shows the results of fluorescence imager observation of tumor characteristics after intraperitoneal administration of 5nm PEG-AuNP(At)-c[RGDfK(C)] (Group B) one week after tumor transplantation. Data from 4 weeks after tumor transplantation and 18 days after drug administration are shown. The left column, "At administration," shows the results for mice administered 5nm PEG-AuNP(At)-c[RGDfK(C)], and the right column, "Control," shows the results for mice administered physiological saline as a control. [Figure 22]Figure 22 shows images of tumors in a mouse model of intraperitoneal glioma transplantation. The images show tumors excised 32 days after transplantation and 23 days after drug administration, following intraperitoneal administration of 5nm PEG-AuNP(At)-c[RGDfK(C)] one week after intraperitoneal tumor transplantation (Group B). The "At administration group" (At-1, At-2, At-3) shows images of tumors excised from mice administered 5nm PEG-AuNP(At)-c[RGDfK(C)], while the "Control group" (Ctl-1, Ctl-2, Ctl-3) shows images of tumors excised from mice administered physiological saline as a control. [Figure 23] Figure 23 shows fluorescence imaging results of tumors in a mouse model of intraperitoneal glioma transplantation. The images show fluorescence imaging results of tumors excised 32 days after transplantation and 23 days after drug administration, following intraperitoneal administration of 5nm PEG-AuNP(At)-c[RGDfK(C)] one week after intraperitoneal tumor transplantation (Group B). The "At administration group" (At-1, At-2, At-3) shows fluorescence imaging results of tumors excised from mice administered 5nm PEG-AuNP(At)-c[RGDfK(C)], while the "Control group" (Ctl-1, Ctl-2, Ctl-3) shows fluorescence imaging results of tumors excised from mice administered physiological saline as a control. [Figure 24] Figure 24 shows the tumor mass in a mouse model of intraperitoneal glioma transplantation. It shows the tumor mass excised 32 days after transplantation and 23 days after drug administration, following intraperitoneal administration of 5nm PEG-AuNP(At)-c[RGDfK(C)] one week after intraperitoneal tumor transplantation (Group B). "At" represents the average tumor mass (n=3) excised from mice administered 5nm PEG-AuNP(At)-c[RGDfK(C)], and "Control" represents the average tumor mass (n=3) excised from mice administered physiological saline as a control. [Modes for carrying out the invention]

[0018] Embodiments of the present invention will be described below. The following description is merely illustrative, and the scope of the present invention is not limited to this description. The invention may be modified and implemented as appropriate without impairing its spirit.

[0019] When At-211 is used as an alpha-emitting radionuclide, it accumulates rapidly in the thyroid gland if administered directly, as it has properties similar to iodine. To retain the drug within the tumor, the distribution of the drug can be controlled using AuNPs. Specifically, AuNP-PEG can be produced, purified, and then labeled with At-211 to produce At-211AuNP(At)-PEG. The size of AuNPs can be appropriately adjusted according to the purpose. In one embodiment, when administering At-211AuNP(At)-PEG to a malignant tumor, an appropriate amount of At-211AuNP(At)-PEG (hereinafter referred to as nanoparticles) can be filled into a syringe and slowly injected into the malignant tumor through a needle. The nanoparticles injected into the tumor diffuse within the tumor. This diffusion is better with smaller particles, but it is known that the capillaries of malignant tumors have higher permeability to substances compared to those of normal tissue, and if the particle size is small enough, it will diffuse throughout the body via the capillaries.

[0020] On the other hand, when the particle size is around 100 nm, diffusion within the tumor deteriorates, and sufficient effect cannot be obtained. It was found that by using a particle size of around 30 nm or less, diffusion within the tumor is good, and the nanoparticles do not flow into the capillaries that perfuse the tumor, nor do they diffuse into other organs outside the tumor. The nanoparticles that remain within the tumor continue to irradiate tumor cells with alpha rays, decaying with a half-life of approximately 7 hours, and the radioactivity disappears. Tumor cells are damaged by alpha rays with a single injection, and their proliferative capacity is lost or significantly reduced.

[0021] (definition) In this specification, when multiple numerical ranges are given, a range consisting of any combination of the lower and upper limits of those ranges is also meant.

[0022] In this specification, "alpha-emitting radionuclides" means radionuclides that emit alpha particles, and these can be used in mixtures. Examples of alpha-emitting radionuclides that can be used include, but are not limited to, At-211, Ac-225, and Ra-223. It is preferable to use At-211 as the alpha-emitting radionuclide.

[0023] At-211 is a radioactive isotope of astatine (At), an element belonging to the halogen group. At-211 decays into stable lead (207Pb) by emitting high-energy alpha particles that are cytotoxic. The half-life of At-211 is 7.2 hours. In other words, because At-211 has a short lifespan and high cytotoxicity, it can efficiently destroy tumor tissue when used in tumor treatment.

[0024] These radioactive nuclides can be produced using known methods. For example, AT-211 is produced by a (α,2n) nuclear reaction using Bi-209 as the target material with an accelerator such as a cyclotron, purified by a dry process (distillation), and then dissolved in water to be supplied as an AT-211 aqueous solution.

[0025] In this specification, “proliferative disorder” means a disorder involving the unwanted proliferation of one or more subsets of cells in a multicellular organism. Proliferative disorders can occur in a variety of animals, including humans. As used herein, “proliferative disorder” includes benign tumors, malignant tumors and other proliferative disorders. Examples of “proliferative disorders” include, but are not limited to, hematopoietic disorders (e.g., myeloproliferative disorders) and malignant tumors (e.g., brain tumors, prostate cancer, head and neck cancer, oral cancer, breast cancer, pancreatic cancer and gastrointestinal cancer).

[0026] In this specification, "tumor" means a mass of tissue that grows excessively and autonomously against the control of the body. "Tumor" includes "benign tumors" that do not have pathologically malignant findings and "malignant tumors" that invade surrounding tissues or metastasize.

[0027] (Method for synthesizing gold nanoparticles according to the present invention) The gold nanoparticles (AuNP) of the present invention can be prepared by known methods. For example, the method described in J. Phys. Chem. C 2011, vol.115, pp.45024506 can be used, but is not limited thereto. The size of the gold nanoparticles can be arbitrarily set by methods well known to those skilled in the art, and the size of the manufactured particles can be measured. For example, microscopy using a transmission electron microscope (TEM) can be used, but is not limited thereto. When the gold nanoparticles of the present invention are used as a therapeutic agent for malignant tumors, the size of the gold nanoparticles can be appropriately selected based on the type and condition of the malignant tumor, the desired pharmacological effect, etc. Preferably, the size of the gold nanoparticles is adjusted so that they show sufficient diffusion within the tumor tissue, but do not flow into the capillaries that perfuse the tumor, and do not spread widely to other organs outside the tumor. In one embodiment, the size of the gold nanoparticles is preferably 0.5 nm or larger, 0.6 nm or larger, 0.7 nm or larger, 0.8 nm or larger, 0.9 nm or larger, 1 nm or larger, 2 nm or larger, 3 nm or larger, 4 nm or larger, 5 nm or larger, 6 nm or larger, 7 nm or larger, 8 nm or larger, 9 nm or larger, 10 nm or larger, 11 nm or larger, 12 nm or larger, 13 nm or larger, 14 nm or larger, 15 nm or larger, 20 nm or larger, 25 nm or larger, or 30 nm or larger. In one embodiment, the size of the gold nanoparticles is preferably 110 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, or 30 nm or less. In one embodiment, the size of the gold nanoparticles is preferably 1.0 nm to 110 nm. In another embodiment, the size of the gold nanoparticles is preferably 5 nm to 30 nm.

[0028] The binding of gold nanoparticles to alpha-emitting radionuclides can be carried out by known methods. In particular, gold is known to form stable bonds with halogen elements (Dziawer L et al. RSC advances, 2017, Vol.7, pp.41024-41032), and when using a halogen alpha-emitting radionuclide such as At-211, the gold nanoparticles and the alpha-emitting radionuclide can be bound by mixing the gold nanoparticles with the halogen alpha-emitting radionuclide.

[0029] (Modification of gold nanoparticles according to the present invention) The behavior of gold nanoparticles (AuNPs) in vivo can be regulated by modifying their surface. For example, by modifying the surface of gold nanoparticles (AuNPs) with polyethylene glycol, sugars, peptides (proteins), or other polymers, functions such as aggregation inhibition, cell targeting, cell membrane permeability promotion, and enhanced cell membrane adsorption can be imparted to gold nanoparticles in tumor tissue. Such modifications of gold nanoparticles can be made using, but are not limited to, hydrocarbon polymers such as polyethylene glycol, polyethers, and polyols, polyethyleneimines, silica gel, peptides, antibodies, proteins, lipids, complex lipids, glycans, complex carbohydrates, terpenes, terpenoids, and virus-like particles. Preferably, the gold nanoparticles of the present invention are modified with polyethylene glycol (PEG). The PEG used in this invention can be various polymers obtained by condensation polymerization of ethylene oxide and water, having various structures known to be used in biomaterials. This includes, but is not limited to, PEG without chemically reactive end groups, monofunctional PEG with one chemically reactive end group, bifunctional PEG with two chemically reactive end groups, linear PEG, multi-armed PEG, and PEG with reactive end groups such as N-hydroxysuccinimide ester groups, thiol groups, and carboxyl groups (Drug Delivery System, 2015, Vol.30, No.4, pp.390-392). Multiple types of modifications can also be combined as long as they do not inhibit each other's functions.

[0030] The size of the polymer used to modify the gold nanoparticles of the present invention can be appropriately determined by those skilled in the art depending on the type and condition of the tumor to be treated, the desired effect, etc. For example, when using PEG, any polymer greater than or equal to diethylene glycol can be used, and its molecular weight is not particularly limited, but for example, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1,000 or more, 1,500 or more, 2,000 or more, 2,500 or more, 3,000 or more, 3,500 or more, 4 It may be 1,000 or more, 4,500 or more, 5,000 or more, or 6,000 or more, and it may be 7,000 or less, 8,000 or less, 9,000 or less, 10,000 or less, 11,000 or less, 12,000 or less, 13,000 or less, 14,000 or less, 15,000 or less, 16,000 or less, 17,000 or less, 18,000 or less, 19,000 or less, or 20,000 or less. More specifically, PEGs with molecular weights of approximately 100-20,000, 200-19,000, 300-18,000, 400-17,000, 500-16,000, 1,000-15,000, 2,000-12,000, 3,000-10,000, 4,000-9,000, 5,000-8,000, 5,500-7,000, and 6,000 can be used. The molecular weight of the PEG can be adjusted depending on the modifications used in combination. Such modifications can be carried out by known methods (e.g., Gold Bull. 2011, vol. 44, pp. 99-105).

[0031] (The gold nanoparticle carrier of the present invention) When administering the gold nanoparticles of the present invention to cells, various substances that regulate cellular uptake can be used in combination. For example, gold nanoparticles can be efficiently delivered into cells by encapsulating them within viral particles that can pass through the cell membrane. Furthermore, modifying the surface of the gold nanoparticles with compounds that cause a cationic charge, such as polyethyleneimine, improves the cell permeability of the gold nanoparticles. Additionally, modifying the surface with antibodies that induce cell-specific uptake improves the uptake of gold nanoparticles into cells. As targeted molecules to improve uptake into specific cells, molecules that bind to substances specifically present in proliferative disease cells, for example, antibodies or antigen-binding fragments that use proteins specifically expressed in proliferative disease cells as antigens, such as antibodies or antigen-binding fragments that use CD19, EpCAM, CD20, CD45, EGFR, HER2, and CDH17 as antigens; ligands or fragments that bind to receptors specifically expressed in proliferative disease cells, for example, substance P or fragments that are ligands for the NK1 receptor expressed in gliomas, for example, peptides consisting of 5 to 11 amino acids at the N-terminus; and other peptides with tumor-targeting function, such as the cyclic peptide c[RGDfK(C)] (Vivitide (Kentucky, USA)) with tumor-targeting function. The above targeted molecules can be directly bound to alpha-emitting radionuclide-bound gold nanoparticles, or they can be bound via the above surface-modified molecules or other carriers.

[0032] (Pharmaceutical compositions and therapeutic methods) The gold nanoparticles bound to the alpha-emitting radionuclide of the present invention are provided as a pharmaceutical for treating proliferative diseases and the like. The pharmaceutical containing the gold nanoparticles bound to the alpha-emitting radionuclide of the present invention is effective in treating various proliferative diseases and can be applied to the treatment of malignant tumors such as brain tumors, prostate cancer, head and neck cancer, oral cancer, breast cancer, and gastrointestinal cancer. The gold nanoparticles bound to the alpha-emitting radionuclide of the present invention have excellent diffusion properties within tumor tissue while having little migration outside the tumor tissue. Therefore, even in the treatment of malignant tumors with many blood vessels, such as brain tumors, it is possible to effectively treat malignant tumors while suppressing radiation exposure to other organs. The present invention also provides a method for treating proliferative diseases and the like using gold nanoparticles bound to alpha-emitting radionuclides.

[0033] The treatment of the above-mentioned malignant tumors includes suppressing the progression, regression, elimination, suppression of metastasis, and prevention of recurrence of the primary malignant tumor.

[0034] The alpha-emitting radionuclide-bound gold nanoparticles of the present invention can be administered to humans or other mammals, such as mice, rats, rabbits, sheep, pigs, cattle, cattle, dogs, and monkeys. The administration route of the composition containing the gold nanoparticles of the present invention can be appropriately selected by those skilled in the art, and the composition is provided in dosage forms suited to the administration route. In one embodiment, the alpha-emitting radionuclide-bound gold nanoparticles of the present invention are preferably administered locally to lesions such as tumors. In this case, they can be administered by injection into the lesion using a needle or the like (intratumor administration). For example, while observing with an ultrasound image, a liquid formulation containing alpha-emitting radionuclide-bound gold nanoparticles in a volume equal to the tumor volume can be injected into the center of the tumor tissue over a period of 1 minute.

[0035] In addition to the intratumor tissue injection described above, other methods obvious to those skilled in the art can be used for local administration. For example, gold nanoparticles can be superselectively administered into the artery nourishing the lesion using a catheter or the like (superselective intra-arterial administration). This superselective intra-arterial administration can utilize catheter-based techniques, which have seen remarkable development in recent years. This administration method makes it possible to deliver the drug thoroughly and exclusively to the lesion tissue without directly invading the lesion tissue. Furthermore, gold nanoparticles can also be administered to lesions disseminated in cavities such as tumor resection cavities, abdominal cavities, and thoracic cavities by scattering them into the cavity (intra-cavitary administration). This intra-cavitary administration allows for the delivery of high concentrations of the drug to disseminated lesions. This administration method is useful for prophylactic administration to prevent recurrence in resection cavities after surgery.

[0036] By selecting the optimal particle size for gold nanoparticles, these locally administered alpha-emitting radionuclide-bound gold nanoparticles do not require the use of specific targeting molecules for proliferative disease cells. Once they reach the proliferative disease tissue, they exhibit a uniform distribution within the tissue, while outflow into surrounding capillaries outside the tissue is minimized. Therefore, it is possible to obtain an effective inhibitory effect on proliferative disease cells while suppressing systemic radiation exposure.

[0037] The alpha-emitting nuclide-bound gold nanoparticles used in the pharmaceutical composition or therapeutic treatment of the present invention may or may not be modified by the surface modification described above, to the extent that it does not contradict the objectives of the present invention. Preferably, the alpha-emitting nuclide-bound gold nanoparticles are surface-modified with a hydrocarbon polymer such as polyethylene glycol (PEG). The alpha-emitting nuclide-bound gold nanoparticles used in the pharmaceutical composition or therapeutic treatment of the present invention can suppress whole-body radiation exposure while obtaining an excellent inhibitory effect on the proliferation of proliferative disease cells without using targeted molecules that have affinity for specific proliferative disease cells, such as antibodies, other proteins, peptides, or small molecule compounds. In one embodiment, the alpha-emitting nuclide-bound gold nanoparticles used in the pharmaceutical composition or therapeutic treatment of the present invention are surface-modified with a hydrocarbon polymer that does not have a targeted molecule bound to it.

[0038] In one embodiment, the alpha-emitting nuclide-bound gold nanoparticles used in the pharmaceutical composition or therapeutic method of the present invention are surface-modified with a hydrocarbon polymer that does not have a targeting molecule bound to it, and the same or different hydrocarbon polymer to which the targeting molecule is bound. The proportion of the hydrocarbon polymer that does not have a targeting molecule bound to it can be adjusted within a range that does not contradict the purpose of the present invention. The proportion of the hydrocarbon polymer that does not have a targeting molecule bound to it may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total number of molecules of the hydrocarbon polymer used for surface modification.

[0039] (Method for selecting particle size of alpha-emitting radionuclide-bound gold nanoparticles) In one embodiment, the present invention provides a method for selecting the optimal particle size of alpha-emitting radionuclide-bound gold nanoparticles for treating proliferative disorders by topical administration. The method comprises the steps of (1) preparing alpha-emitting radionuclide-bound gold nanoparticles having different particle sizes ranging from 0.5 to 110 nanometers; (2) administering each of the alpha-emitting radionuclide-bound gold nanoparticles having each particle size into proliferative disorder tissue in vivo; (3) confirming the alpha distribution in the administered proliferative disorder tissue and the whole-body alpha distribution; and (4) selecting the particle size based on the alpha distribution in the proliferative disorder tissue and the whole-body alpha distribution.

[0040] The above method can be carried out using model animals that have proliferative disease tissue, tumor-bearing model animals, or other model animals in which proliferative disease tissue has been transplanted and grown. The evaluation of the alpha radiation distribution within the proliferative disease tissue and the whole-body alpha radiation distribution can be carried out using methods known to those skilled in the art. For example, the evaluation can be carried out by image analysis using scintigraphy or autoradiography. It is preferable to select a gold nanoparticle size that shows a relatively uniform alpha radiation distribution within the proliferative disease tissue while showing a whole-body alpha radiation distribution with low levels of alpha radiation outside the proliferative disease tissue.

[0041] The uniformity of alpha radiation distribution within proliferative disease tissue and the alpha radiation distribution level outside proliferative disease tissue can be determined by a person skilled in the art using statistical indicators known to them, according to the desired effect. For example, the uniformity of alpha radiation distribution within proliferative disease tissue can be visually evaluated by the radioactivity distribution obtained by scintigraphy or autoradiography. Alternatively, texture analysis can be performed, using values ​​such as entropy as indicators, and a lower value indicates higher uniformity. In these evaluations, higher uniformity is preferable. The alpha radiation distribution level outside proliferative disease tissue can be evaluated by, for example, measuring the radioactivity of each organ using scintigraphy or SPECT. The radioactivity values ​​of each organ can be measured from the images. It is preferable that the measured values ​​are close to zero. If radioactivity is measured, the exposure dose is calculated using dedicated software such as OLINDA / EXM, and it is preferable that the exposure dose is low. Based on the above measurements and evaluations, we select a particle size for gold nanoparticles that yields a higher uniformity of alpha radiation distribution within proliferative disease tissue and a lower alpha radiation distribution level outside the proliferative disease tissue.

[0042] In the method of the present invention, in addition to step (4) above, (5) the change in body weight of the administered animals and / or the inhibitory effect on the proliferation of proliferative disease tissue may be evaluated, and the particle size may be selected based on these evaluations. The inhibitory effect on the proliferation of proliferative disease tissue can be evaluated, for example, by comparing the change in volume of proliferative disease tissue after administration of alpha-coupled gold nanoparticles and / or the mass of proliferative disease tissue after a certain period of time has elapsed since administration with a control.

[0043] The administration of the aforementioned proliferative disease tissue can be selected from the group consisting of injection into the central part of the tissue, superselective administration into the arteries that nourish the tissue, and dispersal of the drug into the lumen where the tissue is located. The in vivo proliferative disease tissue may be xenoproliferative disease tissue transplanted into the subject.

[0044] The surface of the gold nanoparticles may be modified with molecules selected from hydrocarbon polymers such as polyethylene glycol, polyethers, and polyols, polyethyleneimines, silica gel, peptides, antibodies, proteins, lipids, complex lipids, glycans, complex carbohydrates, terpenes, terpenoids, and virus-like particles. In one embodiment, the surface modification may be carried out with polyethylene glycol having a molecular weight of 2,000 to 20,000.

[0045] The proliferative disorder is a malignant tumor, and may be a solid tumor. The solid tumor may be selected from brain tumors, endocrine tumors, prostate cancer, head and neck cancer, oral cancer, breast cancer, gynecological cancer, skin cancer, pancreatic cancer, and gastrointestinal cancer.

[0046] The alpha-emitting radionuclide-bound gold nanoparticles having the particle size selected by the method of the present invention exhibit a uniform distribution within the tissue once they reach the proliferative disease tissue, while their outflow into the surrounding capillaries outside the tissue is minimized. Therefore, the method of the present invention makes it possible to select a particle size of gold nanoparticles that possesses excellent properties as an active ingredient in pharmaceuticals that suppresses systemic radiation exposure while obtaining a sufficient inhibitory effect on proliferative disease cells.

[0047] (Method for producing alpha-emitting radionuclide-bound gold nanoparticles with selected particle size) In one embodiment, the present invention provides a method for producing alpha-emitting radionuclide-bound gold nanoparticles having an optimal particle size for treating proliferative diseases by topical administration. The method includes a step of selecting a particle size based on a method for selecting the particle size of alpha-emitting radionuclide-bound gold nanoparticles, and producing alpha-emitting radionuclide-bound gold nanoparticles using gold nanoparticles having the selected particle size. The alpha-emitting radionuclide-bound gold nanoparticles produced by the method of the present invention exhibit a uniform distribution within proliferative disease tissue once they reach it, while their outflow into surrounding capillaries outside the tissue is minimized. Therefore, the method of the present invention makes it possible to produce gold nanoparticles with excellent properties as an active ingredient in pharmaceuticals that suppresses systemic radiation exposure while obtaining a sufficient inhibitory effect on proliferative disease cells.

[0048] The present invention will be described in more detail using the following examples, but the scope of the present invention is not limited to the following examples. [Examples]

[0049] (1) Synthesis of AuNP(At)PEG Tetrachloroauro(III) tetrahydrate (Kishida Chemical Co., Ltd. (Osaka, Japan)) was used as a raw material for the synthesis of gold nanoparticles. Poly(ethylene glycol) methyl ether thiol (M) was used for PEG labeling of the gold nanoparticles. n We used 6,000 (Sigma-Aldrich Co, LLC (St. Louis, USA)). The quality of the synthesized PEG-labeled gold nanoparticles was confirmed by transmission electron microscopy (TEM) imaging (JEM-2100, JEOL Ltd. (Tokyo, Japan)). The radioactivity of AuNP(At)PEG was measured using a germanium semiconductor detector (BE-2020, Mirion Technologies (Canberra), Inc. (Connecticut, USA)).

[0050] The following aqueous AE solution was used for the synthesis of AuNP. Aqueous solution A: Prepared by adding 8 mL of water to 2 mL of tetrachloroaurate(III) acid (0.17% w / v) aqueous solution. Solution B: Prepared by mixing 0.5 mL of ascorbic acid (1% w / v) aqueous solution and 0.25 mL of trisodium citrate (0.88% w / v) aqueous solution, and adding 9.25 mL of water. Aqueous solution C was prepared by adding 2 mL of water to 8 mL of tetrachloroaurate(III) acid (0.17% w / v) aqueous solution. Solution D: Prepared by mixing 2 mL of ascorbic acid (1% w / v) aqueous solution with 1 mL of trisodium citrate (0.88% w / v) aqueous solution and adding 7 mL of water. AuNP(At)PEG was administered to rats and mice after being appropriately diluted with physiological saline.

[0051] (1-1) Preparation of 5nm AuNP The 5nm AuNP was purchased from Sigma-Aldrich Co., LLC (St. Louis, USA).

[0052] (1-2) Synthesis of 13nm AuNP 2.5 mL of tetrachloroaurate(III) acid (0.17% w / v) aqueous solution was mixed with 47.5 mL of water. After adding the water, the mixture was heated to over 100°C while stirring. After heating, 2 mL of an aqueous solution of trisodium citrate (0.88% w / v) and citric acid (0.05% w / v) was added, and the mixture was stirred at the same temperature for another 5 minutes. After stirring, the mixture was returned to room temperature to obtain 13 nm AuNPs. TEM imaging confirmed that the average particle size of the AuNPs was 13 nm (13.1 ± 1.4 nm).

[0053] (1-3) Synthesis of 30nm AuNP 5 mL of 13 nm AuNP aqueous solution was prepared by adding 15 mL of water. While stirring the prepared solution, aqueous solutions A and B were simultaneously added from separate syringes at a flow rate of 0.25 mL / min. After adding the solutions, the temperature was raised to over 100°C while stirring, and the mixture was stirred at the same temperature for another 30 minutes. After stirring, the mixture was returned to room temperature to obtain 30 nm AuNP. TEM imaging confirmed that the average particle size of the AuNP was 30 nm (30.8 ± 2.7 nm).

[0054] (1-4) Synthesis of 60nm AuNP 5 mL of 30 nm AuNP aqueous solution was prepared by adding 15 mL of water. While stirring the prepared solution, aqueous solutions A and B were simultaneously added from separate syringes at a flow rate of 0.25 mL / min. After adding the solutions, the temperature was raised to over 100°C while stirring, and the mixture was stirred at the same temperature for another 30 minutes. After stirring, the mixture was returned to room temperature to obtain 60 nm AuNP. TEM imaging confirmed that the average particle size of the AuNP was 60 nm.

[0055] (1-5) Synthesis of 120nm AuNP While stirring 20 mL of a 60 nm AuNP aqueous solution, aqueous solutions C and D were simultaneously added from separate syringes at a flow rate of 0.25 mL / min. After adding the solutions, the temperature was raised to over 100°C while stirring, and the mixture was stirred at the same temperature for another 30 minutes. After stirring, the mixture was returned to room temperature to obtain 120 nm AuNP. TEM imaging confirmed that the average particle size of the AuNP was 120 nm (120.7 ± 13.3 nm).

[0056] (1-6) PEG modification of 5nm, 13nm, 30nm, and 120nm AuNPs Poly(ethylene glycol)methyl etherthiol (M) was added to 5nm, 13nm, 30nm, and 120nm AuNP aqueous solutions, respectively. n 6,000) was added to achieve a final concentration of 0.1 mg / mL. The mixture was then stirred at room temperature for 2 hours. After stirring, the 5 nm AuNP-PEG aqueous solution was ultrafiltered (10,000 G, 10 min) and distilled water was added. This procedure was repeated a total of three times to obtain a 5 nm AuNP-PEG aqueous solution from which impurities had been removed. For the 13 nm AuNP-PEG aqueous solutions, 30 nm AuNP-PEG aqueous solutions, and 120 nm AuNP-PEG aqueous solutions, AuNP-PEG was precipitated by centrifugation (10,000 G, 1 hour). The supernatant was then removed by decantation, and the same amount of distilled water as the removed solution was added. This procedure was repeated a total of two times to obtain 13 nm AuNP-PEG aqueous solutions, 30 nm AuNP-PEG aqueous solutions, and 120 nm AuNP-PEG aqueous solutions from which impurities had been removed. TEM imaging confirmed that the AuNP surface was modified with PEG.

[0057] (1-7) At-211 labeling of AuNP-PEG (At-211 labeling of 5nm AuNP-PEG) A 5nm AuNP-PEG aqueous solution was mixed with an At-211 aqueous solution and shaken at room temperature for 15 minutes. After shaking, the 5nm AuNP(At)PEG(5nm mPEG-S-AuNP[ 211 An aqueous solution of [At] (approximately 42.3 MBq / mL) was obtained.

[0058] (At-211 labeling of 13nm AuNP-PEG) An At-211 aqueous solution was added to a 13nm AuNP-PEG aqueous solution and shaken at room temperature for 15 minutes. After shaking, 13nm AuNP(At)PEG(13nm mPEG-S-AuNP[ 211 An aqueous solution of [At] (approximately 40.7 MBq / mL) was obtained.

[0059] (At-211 labeling of 30nm AuNP-PEG) At-211 aqueous solution was added to a 30nm AuNP-PEG aqueous solution and shaken at room temperature for 15 minutes. After shaking, 30nm AuNP(At)PEG(30nm mPEG-S-AuNP[ 211 An aqueous solution of [At] (approximately 39.0 MBq / mL) was obtained.

[0060] (At-211 labeling of 120nm AuNP-PEG) An At-211 aqueous solution was added to a 120nm AuNP-PEG aqueous solution and shaken at room temperature for 15 minutes. After shaking, the 120nm AuNP(At)PEG(120nm mPEG-S-AuNP[ 211 An aqueous solution of [At] (approximately 39.9 MBq / mL) was obtained.

[0061] The mass concentrations of AuNP(At)-PEG aqueous solutions of each size were measured using ICP-OES (Optima 8300, Perkin Elmer Inc. (Waltham, USA)), and the particle concentrations were calculated. Using these values, the particle concentrations in the AuNP(At)-PEG aqueous solutions administered to in vitro and in vivo experimental models were adjusted to generally equalize the administered radiation doses. In the in vitro experiments, the concentration was adjusted with distilled water, and in the in vivo experiments, the concentration was adjusted with physiological saline. In the in vitro experiments, administration was performed by serial dilution as described below, and in the in vivo experiments, preparations were made and administered such that the dose per animal was the radiation dose shown in Table 1 below.

Table 1

[0062] (1 - 8) Synthesis of 5nm PEG-AuNP(At)-c[RGDfK(C)] An aqueous solution in which poly(ethylene glycol) methyl ether thiol (M n 350) (Biochempeg Scientific Inc. (Massachusetts, USA)) and c[RGDfK(C)] (Vivitide (Kentucky, USA)) were dissolved was added to the 5nm AuNP aqueous solution. The final concentrations of poly(ethylene glycol) methyl ether thiol (M n 350) and c[RGDfK(C)] were adjusted to 0.1 mM each. Thereafter, stirring was performed at room temperature for 2 hours. After stirring, ultrafiltration (10000 G, 10 minutes) was performed on the 5nm PEG-AuNP-c[RGDfK(C)] aqueous solution, and distilled water was added. This operation was performed 3 times in total to obtain a 5nm PEG-AuNP-c[RGDfK(C)] aqueous solution from which impurities had been removed. An At-211 aqueous solution was added to the 5nm PEG-AuNP-c[RGDfK(C)] aqueous solution, and shaking was performed at room temperature for 15 minutes. After shaking, 5nm PEG-AuNP(At)-c[RGDfK(C)] (5nm mPEG-S-AuNP 211 At]-c[RGDfK(C)]) (approximately 58.5 MBq / mL) was obtained.

[0063] (2) In vitro cytotoxicity test (2-1) Tumor cells C6 glioma cells and PANC-1 cells (TACC (Virginia, USA)) were used. These cells were cultured in DMEM medium (Fujifilm Wako Pure Chemical Corporation (Osaka, Japan)) containing 10% fetal bovine serum (Gibco™, Life Technologies, Carlsbad, (CA USA)) and 1% penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Corporation (Osaka, Japan)) at 37°C with 5% CO2 added in a humidified incubator. (2-2) Test Method C6 glioma cells (2 x 10 in 100 μL medium 4 Cells / well) and PANC-1 cells (1 × 10 in 100 μL of medium) 4 Cells were seeded in a 96-well plate and cultured for 1 day. AuNP-PEG (without radioactive nuclide) and AuNP(At)-PEG with different particle sizes were synthesized, and test solutions were prepared so that the radiation dose per 1 mL of AuNP(At)-PEG with each particle size was approximately the same. The prepared test solutions were serially diluted and added to each well (25 μL / well) to obtain a radiation dose of 0 to 1 MBq / mL. After 24 hours of incubation, cell viability was measured using a Cell Counting Kit 8 (CCK8) (Dojin Chemical Research Institute, (Kumamoto, Japan)) with a 450 nm microplate reader.

[0064] (2-3) Results When C6 glioma cells treated with AuNP-PEG without attached radionuclides of 5 nm, 13 nm, 30 nm, or 120 nm were cultured for 24 hours, there was no effect on cell viability even at high concentrations. In other words, AuNP-PEG without attached radionuclides was not toxic regardless of particle size or concentration. On the other hand, a significant decrease in viability was observed in C6 glioma and PANC-1 cells treated with 120 nm AuNP(At)-PEG, which has a particle size of 120 nm and is attached to At-211, at a radiation dose of 1 MBq / mL. When C6 gliomas were cultured with AuNP-PEG for 24 hours, AuNP(At)-PEG at 5 nm, 13 nm, 30 nm, and 120 nm wavelengths were internalized into C6 glioma cells. However, intracellular internalization of AuNP(At)-PEG at 5 nm, 13 nm, and 30 nm was not observed unless the concentration was higher compared to AuNP(At)-PEG at 120 nm. On the other hand, only AuNP(At)-PEG at 120 nm precipitated in solution, suggesting that the local concentration of AuNP(At)-PEG around cells increased in the well. From these results, it was inferred that AuNP-PEG and AuNP(At)-PEG are taken up into cells in a concentration-dependent manner. Similar results were obtained in experiments performed on PANC-1 cells instead of C6 glioma cells, suggesting that the cytotoxicity of AuNP(At)-PEG is independent of cell type.

[0065] (3) Animal models (3-1) rat A male nude rat (F344 / NJcl-rnu / rnu (CLEA Japan, Inc. (Tokyo, Japan))) (7 weeks old) was used. (3-2) Mouse Male nude mice (BALB / C Slc-nu / nu (Japan SLC, Inc. (Tokyo, Japan))) (5 ​​weeks old) were used.

[0066] (3-3) Tumor cells As glioma cells, we used C6 glioma cells, a rat glioma cell line obtained by introducing N-nitrosomethylurea (obtained from RIKEN BRC). The C6 glioma cells were cultured in MEM medium (Sigma-Aldrich Japan, (Tokyo, Japan)) supplemented with 10% fetal bovine serum in a humidified incubator at 37°C with 5% CO2 added.

[0067] Human pancreatic cancer cells PANC-1 were used as the pancreatic cancer cells (obtained from the American Type Culture Collection). The PANC-1 cells were cultured in RPMI1640 medium containing L-glutamine and phenol red (Wako Pure Chemical Industries, Ltd. (Tokyo, Japan)), 10% thermoactivated fetal bovine serum, and 1% penicillin-streptomycin.

[0068] (3-4) Transplantation of tumor cells into rats 0.9 x 10^7 C6 glioma cells were mixed with 50 μl of matrigel (Corning (New York, USA)) in 50 μl MEM and transplanted bilaterally into the subcutaneous tissue of rats under anesthesia using 2.0% isoflurane in oxygen.

[0069] Human pancreatic cancer PANC-1 cells (1.0 × 10^7 cells / 50 μl) were mixed with 50 μl of matrigel (Corning (New York, USA)) and transplanted subcutaneously into the left shoulder of mice.

[0070] (3-5) Administration of AuNP Rats (n=11) 13 days after C6 glioma cell transplantation underwent intratumoral drug administration under anesthesia using 2.0% isoflurane in oxygen. Physiological saline (n=3), 120 nm diameter AuNP(At)PEG + physiological saline (n=4), and 30 nm diameter AuNP(At)PEG + physiological saline (n=4) were prepared in volumes equal to the tumor volume and slowly administered into each tumor over approximately 1 minute. Administration was performed using a linear probe on an ultrasound device (ProSound α6, Hitachi-Aloka Medical, Ltd. (Tokyo, Japan)) with the needle tip of a syringe (Myjector 29G, Terumo Co. Ltd. (Tokyo, Japan)) positioned at the center of the tumor. Regarding the radiopharmaceuticals, the administered radioactivity per tumor was 1.4 ± 0.5 MBq. After administration, an alpha-ray survey meter (TCS-232B, Hitachi Co. Ltd. (Tokyo, Japan)) was used to confirm that there was no contamination from the backflow of the radioactive drug onto the skin.

[0071] In a study in which rats were administered 5nm, 13nm, 30nm, and 120nm AuNP(At)PEG, 12 rats (n=12) 13 days after C6 glioma cell transplantation were anesthetized with 2.0% isoflurane in oxygen and administered intratumoral drugs. Physiological saline (3 rats, 6 tumors), 30nm AuNP-PEG + physiological saline without radionuclide (3 rats, 6 tumors), 120nm AuNP(At)-PEG + physiological saline (4 rats, 8 tumors), 30nm AuNP(At)-PEG + physiological saline (4 rats, 8 tumors), 13nm AuNP(At)-PEG + physiological saline (3 rats, 6 tumors), and 5nm AuNP(At)-PEG + physiological saline (3 rats, 6 tumors) were prepared in quantities equal to the tumor volume. Similarly, physiological saline (3 animals, 6 tumors) and 30 nma NP-PEG + physiological saline without added radionuclide (3 animals, 6 tumors) were prepared for control animals. These test solutions were administered slowly over approximately 1 minute. Administration was performed using a linear probe on an ultrasound device (ProSound α6, Hitachi-Aloka Medical, Ltd. (Tokyo, Japan)) and by placing the needle tip of a syringe (Myjector 29G, Terumo Co. Ltd. (Tokyo, Japan)) at the center of the tumor. Regarding the radiopharmaceutical, the administered radioactivity per tumor at the time of administration was 1.4 ± 0.4 MBq (Table 1). After administration, an alpha-ray survey meter (TCS-232B, Hitachi Co. Ltd. (Tokyo, Japan)) was used to confirm that there was no contamination from backflow of the radiopharmaceutical onto the skin.

[0072] (3-6) Autoradiography Rats administered with AuNP(At)PEG with an average diameter of 120 nm (n=1) and rats administered with AuNP(At)PEG with a diameter of 30 nm (n=1) had all their tumors removed the day after administration and rapidly frozen at -80°C. After tumor removal, the rats were euthanized with an excess dose of isoflurane. The sections were thinly sliced ​​to a thickness of approximately 30 μm using a cryostat (CryoStar NX70, Thermo Scientific Inc. (MA, USA)) and mounted on glass slides. The frozen sections were then rapidly dried with a dryer and kept in contact with an imaging plate for approximately 1 hour. Imaging was performed using a nonconfocal variable-mode laser scanner (Typhoon FLA 7000, GE Healthcare Life Sciences (Buckinghamshire, England)).

[0073] (3-7) Scintigraphy analysis Scintigraphy was performed on rats administered AuNP(At)PEG 4 hours after administration and again from 19 to 21 hours after administration. Scintigraphy was performed using a gamma camera (E.cam, Siemens Healthcare (Erlangen, Germany)) equipped with a low-energy, high-resolution, parallel-hole collimator. Rats were anesthetized with 2.0% isoflurane in oxygen, fixed to a bed in a prone position, and imaging was performed for 10 minutes at 4 hours and for 30 minutes from 19 hours onward to obtain 128 × 128 matrix size anterior and posterior planar images.

[0074] (3-8) Statistics Statistical calculations were performed using SPSS 17.0. Tumor mass comparisons were performed using one-way ANOVA and Levene's test, followed by a post-hoc test using Tukey's HSD test.

[0075] All experiments were conducted in accordance with the Osaka University Animal Experiment Regulations, with the approval of the Osaka University Animal Experiment Committee. The results of the experiments are reported in accordance with the ARRIVE (Animal Research: Reporting in Vivo Experiments) guidelines.

[0076] (4) Injection of AuNP(At)PEG into subcutaneous glioma (4-1) Scintigraphy analysis Bilateral tumors in C6 glioma cell-transplanted rats were injected with either saline alone, 120nm AuNP(At)PEG + saline, or 30nm AuNP(At)PEG + saline. Scintigraphy analysis was performed 4 and 19 hours after administration. The results for 120nm AuNP(At)PEG (Figure 1) and 30nm AuNP(At)PEG (Figure 2) are shown. No signals were detected in non-tumor areas with either 120nm AuNP(At)PEG or 30nm AuNP(At)PEG. On the other hand, 120nm AuNP(At)PEG accumulated in a punctate manner within the tumor tissue (Figure 1), while 30nm AuNP(At)PEG was observed to diffuse within the tumor tissue (Figure 2).

[0077] As a reference experiment, Figure 3 shows the results when AuNP(I-123)PEG, which is similar AuNP-PEG conjugated with the gamma-ray radionuclide I-123, was administered. The binding of I-123 to AuNP is unstable, and 1.5 hours after injection into the subcutaneous tumor on the left (arrow), I-123 detaches relatively quickly from AuNP, enters the bloodstream, and is distributed throughout the body (Figure 3).

[0078] (4-2) Autoradiography Tumor tissue was removed from rats injected with 120nm AuNP(At)PEG or 30nm AuNP(At)PEG the day after injection, and autoradiography was performed. The results are shown in Figure 4. 120nm AuNP(At)PEG was concentrated in the tumor periphery (Figure 4B), while 30nm AuNP(At)PEG was found to be distributed throughout the entire tumor (Figure 4A). Furthermore, 30nm AuNP(At) that was not modified with PEG showed a slightly uneven distribution within the tumor (Figure 5).

[0079] (4-3) Tumor growth inhibitory effect The antitumor effects of 5nm, 13nm, 30nm, and 120nm AuNP(At)PEG were compared using a subcutaneous C6 glioma model in rats. After injecting tumors with saline alone, or with 5nm, 13nm, 30nm, and 120nm AuNP(At)PEG in saline, rats were observed for 38 or 39 days. During this period, body weight and tumor size were measured under anesthesia using 2.0% isoflurane in oxygen. The tumors were also excised 40 days after administration, and their mass was measured. After the experiment, the animals were euthanized by administering an excess dose of isoflurane. Figure 6 shows the change in tumor size, Figure 7 shows the change in body weight, and Figure 8 shows a comparison of the mass of the removed tumors.

[0080] When comparing tumor sizes in rats administered AuNP(At)PEG of different particle sizes, it was found that among these AuNP(At)PEGs, rats administered with smaller particle sizes showed less tumor size increase, with rats administered with 5nm AuNP(At)PEG showing the smallest increase in tumor size (Figure 6). There were no significant differences in body weight changes (Figure 7). When the mass of tumor tissue removed from rats 40 days after administration was measured, rats administered with smaller particle sizes had smaller tumor masses, with rats administered with 5nm AuNP(At)PEG showing the smallest tumor mass (Figure 8). In rats administered with 30nm AuNP-PEG without At-211, the tumor mass was almost the same as that of rats administered with physiological saline. From these results, it was confirmed that AuNP(At)PEG particles exhibit excellent antitumor effects due to their small particle size of 5nm. Regarding 5 nmA uNP(At)PEG particles, scintigraphy analysis was performed at 4 hours, 19 hours, and 42 hours after injection of 5 nmA uNP(At)PEG particles into bilateral subcutaneous tumors, as in (3-1) above, and the results shown in Figure 9 were obtained. From these results, it was confirmed that AuNP(At)PEG particles with a small particle size of 5 nm remain within the tumor tissue, and diffusion outside the tumor tissue is limited. While not bound by theory, it is thought that AuNP(At)PEG particles with a small particle size of 5 nm possess excellent diffusion characteristics within tumor tissue, while their diffusion from tumor tissue is limited, resulting in superior antitumor effects. This experimental result confirms that, even without using targeting molecules specific to proliferative disease cells, it is possible to produce alpha-ray gold nanoparticles with extremely excellent properties as radioactive therapeutic agents—uniform distribution within tumor tissue while limiting diffusion outside the tumor tissue—by adjusting the particle size.

[0081] (4-4) Inhibitory effect on tumor growth in pancreatic cancer tissue Human pancreatic cancer PANC-1 cells were transplanted into the left shoulder of 12 nude mice (BALB / cSlc-nu / nu) (male, 5 weeks old). Fourteen days after transplantation, 6 mice were administered 13 nm AuNP(At)mPEG, and 6 mice were administered unlabeled 13 nm AuNPmPEG as a control. The administration method was the same as in rats. Tumor size and mouse body weight were measured for 39 days after administration. Forty days after transplantation, the tumors were removed and the mice were euthanized.

[0082] Scintigraphy confirmed that, within 42 hours after administration, the radioactive nuclide was not distributed outside the tumor. Furthermore, the tumor proliferative capacity was significantly reduced in mice administered AuNP(At)mPEG compared to the control group (Figure 10). On the other hand, the body weight of the control mice was significantly lower compared to the group administered AuNP(At)mPEG (p=0.006) (Figure 11). When comparing the mass of the excised tumors, the group administered AuNP(At)mPEG had significantly smaller tumor mass compared to the control group (Figure 12) (p=0.006). This experiment confirmed that local administration of AuNP(At)mPEG according to the present invention is also effective in the treatment of pancreatic cancer.

[0083] (5) Selective intra-arterial administration to subcutaneous transplantation models As a preliminary C6 experiment, four 8-week-old nude rats (F344 / NJcl-rnu / rnu (CLEA Japan, Inc. (Tokyo, Japan))) were used to clarify the arterial supply. A catheter was inserted under direct visualization into a branch of the left femoral artery of the rats, and an appropriate amount of diagnogreen (indocyanine green) was injected. By visually evaluating the staining of the tissue, it was confirmed that the subcutaneous tissue of the medial distal left thigh was supplied by the artery in question. C6 cells were then transplanted into the subcutaneous tissue of the medial left thigh where staining was observed.

[0084] Fourteen days after transplantation, the groin was incised under isoflurane anesthesia, and a catheter was placed distally into a branch of the femoral artery under direct visualization. Subsequently, 0.07 ml / 100g of a drug containing 0.3 ml of Dormicum (midazolam 1.5 mg), 0.3 ml of Seraktal (xylazine 6 mg), and 0.4 ml of Betorphar (butorphanol tartrate 0.8 mg) was administered intramuscularly to induce anesthesia. Then, two rats were given a bolus of Adenoscan 0.7 ml (adenosine 2 mg) via the tail vein to temporarily lower their pulse rate and blood pressure and reduce blood flow velocity. Immediately afterward, 3.3 MBq of 5 nm PEG-AuNP(At)-c[RGDfK(C)] dissolved in 0.3 ml of physiological saline was slowly administered (over approximately 1.5 minutes) via the catheter placed in the femoral artery. 5nm PEG-AuNP(At)-c[RGDfK(C)](5nm mPEG―S-AuNP[ 211 The structure of At]-c[RGDfK(C)]) is shown in Figure 13.

[0085] After administration, the catheter was removed and the femoral artery was ligated. In the control group, two rats underwent surgery to ligate the femoral artery and then closed the wound. Nine and fourteen hours after administration, the systemic distribution of the drug was evaluated by scintigraphy in the rats that received At. At all time points, the RI accumulated in the tumor, liver, and spleen, but no significant accumulation was observed in other organs (Figure 14). Subsequently, changes in body weight and tumor volume were observed (Figures 15 and 16), and no significant difference in body weight was observed between the rats that received At and the control group (Figure 15). On the other hand, in the observation of tumor volume, it was confirmed that tumor growth was suppressed in the rats that received At compared to the control group (Figure 16). This experiment revealed that selective intra-arterial administration of 5nm PEG-AuNP(At)-c[RGDfK(C)] suppresses tumor growth. These results demonstrate that gold nanoparticles having a specific particle size, bound to the alpha-emitting radionuclide of the present invention, exhibit excellent therapeutic effects for proliferative diseases when superselectively administered into arteries nourishing lesions.

[0086] (6) Experiments on intraperitoneal administration to a peritoneal dissemination model Six male nude mice (BALB / C Slc-nu / nu (Japan SLC, Inc, (Tokyo, Japan))) at 8 weeks old (Group A) and six at 9 weeks old (Group B) were introduced into 1 × 10 C6 cells containing a fluorescent protein gene. 7 The cells were transplanted intraperitoneally. In group A, 14 days after transplantation, and in group B, 7 days after transplantation, 3 mice in each group were intraperitoneally administered 0.98±0.19 MBq / 0.2 ml of 5 nm PEG-AuNP(At)-c[RGDfK(C)] using a 26G needle. 3 control animals in each group were intraperitoneally administered 0.2 ml of physiological saline using a 26G needle.

[0087] The systemic distribution of the drug was evaluated by scintigraphy 9 and 14 hours after administration (Figure 17). At all time points, the RI was localized in the peritoneal cavity and no distribution to other organs was observed. Tumor engraftment was not observed in one control animal in group A, so this animal was excluded. Tumor engraftment was observed in all other animals. The mean time of death in group A was 10.5 days after administration for controls and 15.3 days after administration for At (Figure 18). In group B, all animals survived until 32 days after transplantation (23 days after administration). Regarding group B, weight changes and observation of the peritoneal cavity with a fluorescence imager revealed that the control animals showed weight gain compared to the drug-treated animals, which was thought to be due to increased ascites (Figure 19).

[0088] Four weeks after transplantation (17 days after administration), control animals showed palpable tumors in the peritoneal cavity, and fluorescence was observed in the area using a fluorescence imager. On the other hand, in At-administered animals, no tumors were palpable, or only small nodules were palpable, and mild fluorescence was observed in the area. Tumor excision was performed 32 days after transplantation (23 days after administration), and when the mass of the excised tumors was compared, the control animals had a larger tumor extent and mass compared to At-administered animals (Figures 20-24). Furthermore, although heterogeneous, fluorescence was observed in the excised tumors (Figure 23). This experiment revealed that intraperitoneal administration of 5nm PEG-AuNP(At)-c[RGDfK(C)] can suppress the progression of C6 peritoneal dissemination lesions. These results indicate that gold nanoparticles with a specific particle size bound to the alpha-emitting radionuclide of the present invention exhibit excellent therapeutic effects on proliferative diseases when administered by intracavitary dispersal.

[0089] The experimental results described above demonstrate that various proliferative diseases can be safely and effectively treated by topically administering gold nanoparticles having a specific particle size bound to an alpha-emitting radionuclide, as described in the present invention. It has been confirmed that when gold nanoparticles with a particle size below this range are used, leakage outside the tumor tissue may occur. In one embodiment, the present invention provides the following. [Item 1] A pharmaceutical product for treating proliferative disorders, characterized by containing gold nanoparticles having a particle size of 0.5 to 110 nanometers bound to At-211, and being administered topically. [Item 2] The pharmaceutical product described in item 1, wherein the surface of gold nanoparticles is modified with molecules selected from polyethylene glycol, polyether, polyol, polyethyleneimine, silica gel, peptide, antibody, protein, lipid, complex lipid, glycan, complex carbohydrate, terpene, terpenoid, and virus-like particles. [Item 3] The pharmaceutical product according to item 2, wherein the surface modification includes molecules that do not have a targeting molecule bound to a specific cell. [Item 4] The pharmaceutical product according to item 3, wherein the surface modification is a polyethylene glycol having an average molecular weight of 2,000 to 20,000, where the molecule not bound to a targeting molecule for specific cells is a polyethylene glycol. [Item 5] The pharmaceutical product according to any one of items 1 to 4, wherein the particle size of the gold nanoparticles is 0.5 to 13 nanometers. [Item 6] A pharmaceutical product as described in any one of items 1 to 5, wherein local administration is selected from the group consisting of infusion into the lesion, superselective administration into the artery nourishing the lesion, and intracavitary dispersal. [Item 7] A pharmaceutical product according to any one of items 1 to 6, wherein the proliferative disorder is selected from the group consisting of brain tumors, endocrine tumors, prostate cancer, head and neck cancer, oral cancer, breast cancer, gynecological cancer, skin cancer, pancreatic cancer, and gastrointestinal cancer. [Item 8] Gold nanoparticles having a particle size of 0.5 to 110 nanometers, bound to At-211, and surface-modified with polyethylene glycol that is not bound to a targeting molecule for specific cells. [Item 9] (1) A step of preparing At-211 bound gold nanoparticles having different particle sizes of 0.5 to 110 nanometers, (2) A step of administering At-211 bound gold nanoparticles having each of the particle sizes described above into proliferative disease tissue in vivo. (3) A step of confirming the alpha radiation distribution in the proliferative disease tissue to which the dose was administered, and the whole-body alpha radiation distribution. (4) A step of selecting particle size based on the alpha radiation distribution in proliferative disease tissue and the whole-body alpha radiation distribution. A method for producing At-211-bound gold nanoparticles having an optimal particle size for treating proliferative disorders by topical administration.

Claims

1. A pharmaceutical for treating proliferative disorders, characterized by containing gold nanoparticles having a particle size of 0.5 to 110 nanometers bound to At-211, and being administered topically, wherein the gold nanoparticles do not contain surface modifications including targeting molecules for specific cells, the surface of the gold nanoparticles is modified with polyethylene glycol, and the pharmaceutical is administered by injection into tumor tissue.

2. The pharmaceutical product according to claim 1, wherein the surface of gold nanoparticles is further modified with molecules selected from polyethers, polyols, polyethyleneimines, silica gel, peptides, antibodies, proteins, lipids, complex lipids, glycans, complex carbohydrates, terpenes, terpenoids, and virus-like particles.

3. The pharmaceutical product according to claim 1, wherein the molecule that modifies the surface of the gold nanoparticles is polyethylene glycol having an average molecular weight of 2,000 to 20,000.

4. The pharmaceutical product according to any one of claims 1 to 3, wherein the particle size of the gold nanoparticles is 0.5 to 13 nanometers.

5. The pharmaceutical product according to any one of claims 1 to 4, wherein the proliferative disease is selected from the group consisting of brain tumors, endocrine tumors, prostate cancer, head and neck cancer, oral cancer, breast cancer, gynecological cancer, skin cancer, pancreatic cancer, and gastrointestinal cancer.

6. Gold nanoparticles having a particle size of 0.5 to 110 nanometers, bound to At-211, and containing surface modification with polyethylene glycol that is not bound to a targeting molecule for specific cells, and gold nanoparticles that do not contain surface modification containing a targeting molecule for specific cells.

7. (1) A step of confirming the alpha radiation distribution within the proliferative disease tissue and the whole-body alpha radiation distribution of At-211 bound gold nanoparticles having different particle sizes ranging from 0.5 to 110 nanometers, which do not contain surface modifications including targeting molecules for specific cells, and which are administered into the target proliferative disease tissue in vivo. (2) A step of selecting particle size based on the alpha radiation distribution in proliferative disease tissue and the whole-body alpha radiation distribution. A method for producing At-211 bound gold nanoparticles having an optimal particle size for treating the target proliferative disease by topical administration, comprising: The method described above, wherein the confirmation of alpha radiation distribution within proliferative disease tissue includes confirmation of the uniformity of alpha radiation distribution within proliferative disease tissue.

Images