JP6443907B2 - Accumulation promoter for promoting accumulation of contrast agent in tumor - Google Patents

Description

  The present invention relates to an accumulation promoting agent that promotes accumulation of a contrast agent in a tumor and enables imaging of the tumor. The present invention also relates to a tumor imaging kit and a tumor imaging agent using the accumulation accelerator.

  In recent years, the survival rate of cancer patients has been on the rise due to advances in cancer drugs and treatment methods, but cancer is still the number one cause of death in Japan, and even today, more than 300,000 people a year Died of cancer. When diagnosing cancer or formulating a treatment plan, it is important to perform an imaging diagnosis of the tumor and accurately identify the location and size of the tumor. Furthermore, during cancer surgery, tumor imaging is also effective in accurately discriminating tumors from normal tissues and accurately removing tumors.

  Conventionally, fluorescence imaging, X-ray imaging, MRI, PET, SPECT and the like are known as methods for imaging a tumor. Among such methods for imaging a tumor, fluorescence imaging has an advantage that a subject can be easily imaged in a short time with less burden on the subject (Non-patent Document 1). In fluorescence imaging of tumors, a fluorescent contrast agent that emits fluorescence when irradiated with excitation light of a specific wavelength is accumulated in the tumor, and the body is irradiated with excitation light from the outside to detect the fluorescence emitted from the fluorescent contrast agent in the body. The tumor is imaged.

  Conventionally, it has been known that 5-aminolevulinic acid, liposomal indocyanine green, or the like can be used as a fluorescent contrast agent for fluorescent imaging of tumors. 5-aminolevulinic acid has the property of being able to selectively accumulate in tumors and emitting fluorescence upon excitation with ultraviolet light. Indocyanine green itself has no action of accumulating in a tumor, but can be accumulated in a tumor by forming into a liposome, and can emit fluorescence by excitation with infrared light.

  On the other hand, since cancer is a serious disease, there is no spare time for requests for improvement in diagnostic accuracy and treatment technology. Furthermore, in research and development, techniques for easily and accurately discriminating tumors from normal tissues in laboratory animals are indispensable, and improvements in tumor detection techniques are strongly demanded. In order to follow such a demand, in the imaging technique of tumors, it is required to improve resolution performance capable of distinguishing tumors from normal tissues. In order to improve the resolution performance in the tumor imaging technique, it is effective to increase the selective accumulation ability of the contrast agent in the tumor. Conventionally, a drug delivery system for selectively delivering a drug to a tumor has been developed. By applying a carrier used in the drug delivery system to a contrast agent, the ability of the contrast agent to accumulate in the tumor is developed. It is also possible to improve. However, when a carrier used in a drug delivery system is used, there are drawbacks such as complicated preparation of a contrast medium, and a contrast medium trapped in the liver or kidney, resulting in noise.

  Against the background of such a conventional technique, development of a technique for easily accumulating a contrast agent in a tumor is eagerly desired.

Lipspn R. L. et al., J. Natl. Cancer Inst., 26, 1-11 (1961)

  An object of the present invention is to provide an accumulation promoting agent that enables imaging of a tumor by accumulating a contrast agent in a tumor by a simple technique. Another object of the present invention is to provide a tumor imaging kit and a tumor imaging agent using the accumulation promoter.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors can promote the accumulation of the contrast agent in the tumor by administering carbonate apatite together with the contrast agent, so that the imaging of the tumor with high accuracy is possible. Found that it would be possible. The present invention has been completed by further studies based on such knowledge.

That is, this invention provides the invention of the aspect hung up below.
Item 1. An accumulation promoter that promotes accumulation of a contrast agent in a tumor, comprising carbonate apatite as an active ingredient.
Item 2. Item 2. The accumulation accelerator according to Item 1, wherein the carbonate apatite is nanoparticles having an average particle size of 50 nm or less.
Item 3. Item 3. The accumulation accelerator according to Item 1 or 2, wherein the contrast agent is a fluorescent contrast agent.
Item 4. Item 4. The accumulation accelerator according to Item 3, wherein the fluorescent contrast agent is indocyanine green.
Item 5. Item 5. The accumulation promoter according to any one of Items 1 to 4, further comprising albumin.
Item 6. A tumor imaging kit comprising: a first preparation containing a contrast agent; and a second preparation containing the accumulation promoter according to any one of Items 1 to 5.
Item 7. A tumor imaging agent comprising a contrast agent and the accumulation promoter according to any one of Items 1 to 5.

  According to the present invention, accumulation of a contrast agent in a tumor can be promoted by a simple technique of administering carbonate apatite, and the tumor can be visualized and imaged. Therefore, according to the present invention, it is possible to perform cancer diagnosis, planning of cancer treatment, confirmation of tumor during cancer operation, detection of cancer in experimental animals, etc. with high accuracy by a simple method. Can greatly contribute to technological progress in the field of diagnosis and treatment.

  In the present invention, it is considered that the effect of promoting the accumulation of the contrast agent in the tumor is achieved based on the following mechanism of action. The interstitial fluid pressure of the tumor tissue is higher than that of the normal tissue, and this is considered to be one of the major causes that the contrast agent is difficult to penetrate into the deep part of the tumor. On the other hand, as shown in Example 2 described later, it has been found that carbonate apatite has an action of reducing the interstitial fluid pressure of tumor tissue. This decrease in the interstitial fluid pressure of the tumor tissue allows the contrast agent to penetrate deep into the tumor tissue, which is considered to be a factor in promoting the accumulation of the contrast agent in the tumor. It is done. Of course, the present invention is not construed as being limited to the above-mentioned mechanism of action.

The result of having measured the particle size and form of the carbonate apatite nanoparticle (sCA (1) particle) obtained by the manufacture example with the scanning probe microscope is shown. The result of having measured the particle size and the form of the carbonate apatite nanoparticle (sCA (2) particle) obtained by the manufacture example with the scanning probe microscope is shown. In Example 1, it is a figure which shows the result of having imaged by indocyanine green being administered to the tumor model mouse on various conditions, and visualizing the tumor. In Example 1, it is a figure which shows the result of administering indocyanine green on various conditions to the tumor model mouse, and measuring the accumulation state of indocyanine green in each organ and tumor.

1. Accumulation accelerator The accumulation accelerator of the present invention is used for the purpose of promoting the accumulation of a contrast agent in a tumor, and is characterized by containing carbonate apatite as an active ingredient. Hereinafter, the accumulation promoter of the present invention will be described in detail.

The composition of “carbonate apatite” used in the present invention is known per se. Carbonate apatite has a chemical structure in which a hydroxyl group (OH-) of hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ) is partially substituted with a carbonate group (CO ₃ ^2- ). _{Ca 10-m X m (PO} 4) 6 (CO 3) can be represented by _1-n Y n. Here, X may be any element that can partially replace Ca in the carbonate apatite, and examples thereof include Sr, Mn, and a rare earth element. m is a positive number of usually 0 or more and 1 or less, preferably 0 or more and 0.1 or less, more preferably 0 or more and 0.01 or less, and still more preferably 0 or more and 0.001 or less. Y is a unit that can partially replace CO ₃ in carbonate apatite, and examples thereof include OH, F, and Cl. n is usually a positive number of 0 or more and 0.1 or less, preferably 0 or more and 0.01 or less, more preferably 0 or more and 0.001 or less, and further preferably 0 or more and 0.0001 or less. .

  In addition, the carbonate apatite used in the present invention is preferably a nanoparticle having an average particle size of 50 nm or less in order to be administered in vivo and accumulate the anticancer agent in the tumor tissue. The lower limit of the average particle diameter of the carbonate apatite nanoparticles is not particularly limited as long as the desired effect described above can be obtained, but is, for example, 1 nm or more, preferably 3 nm or more, more preferably 5 nm or more. On the other hand, the upper limit of the average particle diameter of the carbonate apatite nanoparticles is more preferably 40 nm or less, still more preferably 30 nm or less, still more preferably 20 nm or less, and even more preferably 10 nm or less.

  The average particle diameter of the carbonate apatite nanoparticles is measured by observing with a scanning probe microscope, as shown in the examples described later. When measuring the average particle size, confirm the measurement site with a CCD camera. If there are huge particles that are clearly unsuitable for measurement using a scanning probe microscope (for example, a particle size of 5 μm or more), Is removed from the measurement target range. In this document, the particle size means the particle size of an independent particle that can be recognized as a separate particle when measured with a scanning probe microscope. Therefore, when a plurality of particles are aggregated, the aggregate is determined as one particle.

  The form of the accumulation promoting agent of the present invention is not particularly limited, but efficiently promotes accumulation of the contrast agent in the tumor while suppressing reaggregation of the carbonate apatite particles and maintaining the state of the average particle size. From this point of view, a dispersed liquid is preferable.

In the accumulation promoter of the present invention, the concentration of the carbonate apatite is not particularly limited, and may be set as appropriate so that the dose described later can be satisfied in consideration of the administration method and the like. For example, when the accumulation accelerator of the present invention is a dispersion, the concentration of the carbonate apatite is 1 × 10 ⁸ to 1 × 10 ¹² pieces / ml, preferably 1 × 10 ⁹ to 1 × 10 ¹¹ pieces / ml. ml, more preferably 1 × 10 ^{10 to} 5 × 10 ¹⁰ pieces / ml, more preferably 3 × 10 ^{9 to} 3 × 10 ¹⁰ pieces / ml, and even more preferably 6 × 10 ^{9 to} 1.5 × 10 ¹⁰ pieces / ml. Is mentioned.

  When the accumulation accelerator of the present invention is a dispersion, the solvent for dispersing the carbonate apatite is not particularly limited as long as it is pharmaceutically acceptable and can disperse the carbonate apatite. Examples include physiological saline and other buffer solutions.

  The method for producing carbonate apatite having the average particle diameter described above is not particularly limited, and specifically, a step of preparing a dispersion in which carbonate apatite particles are dispersed in a pharmaceutically acceptable solvent, and the dispersion A method of undergoing a process of ultrasonic vibration treatment for the liquid is mentioned.

  The carbonate apatite particles can be obtained according to a known method. For example, by preparing and incubating an aqueous solution containing calcium ions, phosphate ions, and bicarbonate ions, carbonate apatite particles including a drug having a tumor action can be produced. The concentration of each ion in the aqueous solution is not particularly limited as long as carbonate apatite particles are formed, and can be appropriately set with reference to the following.

  The calcium ion concentration in the aqueous solution is usually 0.1 mM or more, preferably 0.5 mM or more, more preferably 1 mM or more. The upper limit of the calcium ion concentration is usually 1 M or less, preferably 100 mM or less, more preferably 10 mM or less.

  The phosphate ion concentration in the aqueous solution is usually 0.1 mM or more, preferably 0.5 mM or more, more preferably 1 mM or more. The upper limit of the phosphate ion concentration is usually 1 M or less, preferably 100 mM or less, more preferably 10 mM or less.

  The bicarbonate ion concentration in the aqueous solution is usually 1.0 mM or more, preferably 5 mM or more, more preferably 10 mM or more. The upper limit of the bicarbonate ion concentration is usually 10M or less, preferably 1M or less, and more preferably 100 mM or less.

The source of calcium ions, phosphate ions, and hydrogen carbonate ions is not particularly limited as long as these ions can be supplied in the aqueous solution. For example, salts of these ions can be added to the aqueous solution. Specifically, CaCl ₂ can be used as the calcium ion source, NaH ₂ PO ₄ .2H ₂ O can be used as the phosphate ion source, and NaHCO ₃ can be used as the carbonate ion source.

  The mixing order of each ion source is not particularly limited, and the aqueous solution may be prepared in any mixing order as long as carbonate apatite particles are obtained. For example, a first solution containing calcium ions is prepared, and a second solution containing phosphate ions and bicarbonate ions is prepared separately, and the first solution and the second solution are mixed. An aqueous solution can be prepared.

The aqueous solution for producing the carbonate apatite particles may contain components other than the above-mentioned ion supply sources as long as the purpose is not impaired. For example, Ca or CO ₃ in the carbonate apatite may be partially substituted by adding fluorine ions, chlorine ions, Sr, Mn, or the like to the composition in the aqueous solution. However, the addition amount of fluorine ions, chlorine ions, Sr, and Mn is preferably in a range that does not significantly affect the pH solubility and particle size range of the composite particles to be formed. Moreover, the aqueous solution for producing carbonate apatite particles can also be prepared using various media and buffers for cell culture.

  Carbonate apatite particles can be obtained by adjusting the pH of the aqueous solution containing each of the above ions to a range of 6.0 to 9.0 and incubating for a certain time. The pH of the aqueous solution when forming the carbonate apatite particles is preferably 7.0 or more, more preferably 7.1 or more, still more preferably 7.2 or more, still more preferably 7.3 or more, particularly Preferably it is 7.4 or more, most preferably 7.5 or more. On the other hand, the pH of the aqueous solution when forming the carbonate apatite particles is preferably 8.5 or less, more preferably 8.0 or less.

  The temperature condition of the aqueous solution when forming the carbonate apatite particles is usually 10 ° C. or higher, preferably 25 ° C. or higher, more preferably 37 ° C. or higher. On the other hand, the upper limit of the temperature condition is usually 80 ° C. or lower, preferably 70 ° C. or lower.

  The incubation time of the aqueous solution for forming the carbonate apatite particles is usually 1 minute to 24 hours, preferably 10 minutes to 1 hour. The presence or absence of particle formation can be confirmed by observing under a microscope, for example.

  Thus, a dispersion containing carbonate apatite particles is formed, and the carbonate apatite particles have an average particle size of more than 50 nm. Therefore, the carbonate apatite particles having the average particle diameter described above can be obtained by refining the carbonate apatite particles to make the average particle diameter 50 nm or less.

  Further, as described above, the carbonate apatite particles can be obtained by dissolving substances serving as various ion sources in a solvent such as water, a medium, or a buffer. The dispersion of carbonate apatite particles thus obtained is obtained. Is not necessarily suitable for administration to a living body (intravascular administration) from the viewpoint of osmotic pressure, buffer capacity, sterility, and the like. Therefore, in order to replace the solvent in which the carbonate apatite particles are dispersed with a solvent suitable for administration to a living body (for example, physiological saline), the carbonate apatite particles are usually separated from the solvent by centrifugation and collected. An operation to replace the solvent is necessary. However, when such an operation is performed, the carbonate apatite particles are aggregated and the particles become enormous, so that the state changes to a state unsuitable for administration to a living body. Therefore, by replacing the dispersion medium of the aggregated carbonate apatite particles with a pharmaceutically acceptable solvent suitable for administration to a living body, and performing a refinement process described later, carbonate apatite nano particles having a desired average particle diameter are obtained. The particles can be obtained in a dispersed state in a pharmaceutically acceptable solvent.

  As a method for reducing the average particle size of the carbonate apatite particles to 50 nm or less, ultrasonic vibration treatment is preferably used. Here, the ultrasonic vibration treatment is not a treatment in which an ultrasonic vibrator such as an ultrasonic crusher or a homogenizer used for so-called microbial cell crushing is directly brought into contact with a sample, but generally a precision instrument or a test. This is a process using an ultrasonic cleaner in which an ultrasonic vibrator and a cleaning tank used for cleaning tubes and the like are integrated. A liquid (for example, water) is placed in a cleaning tank (water tank) of an ultrasonic cleaner, and a container (for example, made of plastic) containing a dispersion liquid in which carbonate apatite particles are dispersed in a pharmaceutically acceptable solvent. The tube is floated and ultrasonic waves are applied to the dispersion through the liquid in the manner of washing the precision instrument. Thereby, the carbonate apatite particles can be refined simply and efficiently.

  An apparatus that can be used for ultrasonic vibration treatment is capable of applying ultrasonic vibration to a container containing carbonate apatite particles indirectly through a solvent such as water, like the ultrasonic cleaner. If there is no particular limitation. From the viewpoint of versatility and ease of handling, it is preferable to use an ultrasonic cleaner equipped with an ultrasonic vibrator and a thermostatic bath.

  The conditions of the ultrasonic vibration treatment are not particularly limited as long as the conditions can be controlled to a desired average particle diameter. For example, the temperature of a water tank can be suitably selected from the temperature of 5-45 degreeC, Preferably it is 10-35 degreeC, More preferably, it is 20-30 degreeC. The high frequency output of the ultrasonic vibration treatment can be appropriately set, for example, in the range of 10 to 500 W, preferably 20 to 400 W, more preferably 30 to 300 W, and still more preferably 40 to 100 W. The oscillation frequency is usually 10 to 60 Hz, preferably 20 to 50 Hz, and more preferably 30 to 40 Hz. The ultrasonic vibration treatment period is, for example, 30 seconds to 30 minutes, preferably 1 to 20 minutes, more preferably 3 to 10 minutes.

  The type of container containing a dispersion containing carbonate apatite particles used when performing ultrasonic vibration treatment is not limited as long as the average particle diameter of carbonate apatite particles can be reduced to a desired range, It can be suitably selected according to the volume of the dispersion and the purpose of use. For example, a plastic tube having a capacity of 1 to 1000 ml can be used.

  The ultrasonic vibration treatment may be performed by adding albumin to a dispersion containing carbonate apatite particles. This is because by performing ultrasonic vibration treatment in an environment in which albumin and carbonate apatite particles coexist, carbonate apatite nanoparticles having a finer particle diameter can be obtained, and reaggregation of the particles can also be suppressed. Because. In addition, when albumin is contained, reaggregation of the refined carbonate apatite nanoparticles can be suppressed. When adding albumin to a dispersion containing carbonate apatite particles, the amount of albumin added is not particularly limited, but from the viewpoint of refining the carbonate apatite particles and / or suppressing reaggregation, for example, 0.1 to 500 mg / ml, preferably 1 to 100 mg / ml, more preferably 1 to 10 mg / ml. Thus, albumin added to make carbonate apatite particles fine can be administered in vivo together with the carbonate apatite particles in a state of being contained in the accumulation accelerator of the present invention.

  The accumulation promoter of the present invention is used for the purpose of promoting the accumulation of a contrast agent in a tumor. In the present invention, the type of contrast agent that promotes accumulation in the tumor is not particularly limited as long as it is non-cytotoxic and can contrast an in vivo tissue, and the contrast agent itself accumulates in the tumor. It doesn't matter whether or not you have the ability Even if the contrast agent itself does not have the ability to accumulate in a tumor, it can be accumulated in the tumor and imaged by the accumulation promoter of the present invention. Of course, even a contrast agent capable of accumulating in a tumor can be further promoted to accumulate in the tumor by the accumulation promoter of the present invention. Specific examples of contrast agents include fluorescent contrast agents, X-ray contrast agents, MRI contrast agents, PET contrast agents, SPECT contrast agents, and the like. Among these contrast agents, a fluorescent contrast agent is suitable as an application target of the accumulation accelerator of the present invention because it has a high effect of promoting accumulation in tumors and has a small burden on the subject.

  Specific examples of the fluorescent contrast agent include indocyanine green (ICG), fluorescein, quantum dots, near-infrared fluorescent dyes (Cy5.5, Cy7, AlexaFluoro, etc.), and other fluorescent substances. Indocyanine green is a fluorescent contrast agent that emits fluorescence around a wavelength of 835 nm upon receiving excitation light around a wavelength of 805 nm. 5-Aminolevulinic acid is a fluorescent contrast agent that emits fluorescence around a wavelength of 620 nm upon receiving excitation light around a wavelength of 380 nm. Fluorescein is a fluorescent contrast agent that receives excitation light having a wavelength of about 494 nm and emits fluorescence having a wavelength of about 521 nm. Among these fluorescent contrast agents, indocyanine green is preferable from the viewpoint of promoting the accumulation in the tumor more efficiently. Conventionally, indocyanine green has been formulated and used as a fluorescent contrast agent so that it can be easily introduced into cells by encapsulating it in liposomes. However, according to the present invention, indocyanine green is incorporated into commercially available nanoparticles such as liposomes. Even if it is used as it is without being encapsulated, it is possible to efficiently promote the accumulation in the tumor.

  In addition, the cancer that is imaged by accumulating the contrast agent in the accumulation accelerator of the present invention is not particularly limited as long as it is a solid cancer, and specifically, colon cancer, colon cancer, stomach cancer, rectal cancer. Liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, prostate cancer, cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, oral cancer and the like.

  The administration method of the accumulation promoter of the present invention is not particularly limited, and may be systemic administration or local administration. The accumulation promoting agent of the present invention has an excellent effect of promoting the accumulation of a contrast agent in a tumor even when administered by systemic administration. Therefore, systemic administration is a preferable administration method. Specific examples of systemic administration include intravascular (intraarterial or intravenous) administration, subcutaneous administration, subcutaneous administration, intraperitoneal administration, etc., preferably intravascular administration, more preferably arterial and intravenous administration. . Intravascular administration includes continuous infusion as well as intravascular injection.

  Further, the administration method of the contrast agent accumulated in the tumor by the accumulation accelerator of the present invention is not particularly limited, and is appropriately set according to the type of the contrast agent, the site of the tumor, etc. May be the same as or different from each other. Even if the administration method of the contrast agent is different from the administration method of the accumulation promoter of the present invention, the accumulation of the contrast agent in the tumor can be promoted by the accumulation promoter of the present invention. Specific examples of the administration method of the contrast agent include intraperitoneal administration, intravascular (intraarterial or intravenous) administration, subcutaneous administration, subcutaneous administration, etc., preferably intraperitoneal administration. Intravascular administration includes continuous infusion as well as intravascular injection.

  The dosage of the accumulation promoting agent of the present invention is determined as appropriate according to the type of contrast medium targeted for promoting accumulation in the tumor, the patient's sex, age, symptom, etc., and thus cannot be determined generally. However, what is necessary is just about 10 mg-1 g / kg (body weight) per time in conversion of carbonate apatite amount, for example.

  Further, the dose of the contrast agent may be appropriately determined according to the tumor site, patient sex, age, symptoms, and the like. For example, if indocyanine green is used as a contrast agent, the dose may be about 5 to 30 mg per adult.

  Further, the administration timing of the accumulation promoter of the present invention is not particularly limited, and may be, for example, within 24 hours before or after the contrast agent administration, or within 12 hours before or after the contrast agent administration. And within 8 hours at the same time or before and after the administration of the contrast agent is more preferable. Further, from the viewpoint of avoiding the aggregation of the carbonate apatite particles before the administration of the accumulation accelerator of the present invention, it is preferable to administer immediately after the ultrasonic vibration treatment. For example, administration within 1 minute, preferably within 30 seconds after ultrasonic vibration treatment is preferred. However, as described above, when the aggregation of carbonate apatite nanoparticles is suppressed by adding albumin, it can be administered after several minutes to several tens of minutes after the ultrasonic vibration treatment.

  By administering the contrast agent and the accumulation promoting agent of the present invention, the contrast agent is accumulated in the tumor. Thereafter, the tumor can be imaged by a known method according to the type of contrast agent used. For example, in the case of using a fluorescent contrast agent, the fluorescence can be emitted from the fluorescent contrast agent by irradiating excitation light, so that the tumor can be imaged by observing the fluorescence.

  Tumor imaging using the accumulation promoter of the present invention can be used for cancer diagnosis and confirmation of progression, discrimination between tumor and normal tissue during cancer surgery, imaging of cancer micrometastasis, confirmation of cancer in experimental animals, etc. Preferably used.

2. Tumor Imaging Kit, Tumor Imaging Agent The tumor imaging kit of the present invention is used when a tumor is imaged by administering the accumulation promoting agent by the same or different administration method as the contrast agent. 1 formulation and the 2nd formulation containing the said accumulation promoter are characterized by the above-mentioned.

  The tumor imaging agent of the present invention is used when treating the cancer by administering the accumulation promoting agent by the same administration method as the contrast agent, and contains the anticancer agent and the enhancing agent in the same preparation. It is characterized by that. In the tumor imaging agent of the present invention, the accumulation accelerator and the contrast agent may be contained in the same preparation as mutually independent substances (that is, the accumulation accelerator and the contrast agent are mixed). An agent may be included in a state of being included in the accumulation accelerator.

  The contrast agent included in the accumulation accelerator can be obtained, for example, by the following method. First, in the method for producing carbonate apatite particles described above, a desired amount (for example, about 20 to 1600 μM) of contrast is added to an aqueous solution (an aqueous solution containing calcium ions, phosphate ions, and hydrogen carbonate ions) for producing carbonate apatite particles. A dispersion of carbonate apatite particles containing a contrast agent is prepared by containing the agent and incubating under the conditions described above. Subsequently, the dispersion liquid is subjected to ultrasonic vibration treatment under the above-described conditions, so that the carbonate apatite particles encapsulating the contrast agent are refined and adjusted to the average particle diameter.

  The configuration and usage of the tumor imaging kit and tumor imaging agent of the present invention are as described in the column of “1. Accumulation accelerator”.

  Hereinafter, the present invention will be described with reference to examples. However, the present invention is not construed as being limited to the following examples.

Production example: Production of sonicated carbonate apatite (sCA) particles (1) Production of carbonate apatite nanoparticles (sCA) using DMEM solution In 100 ml of distilled water, 1.35 g of DMEM powder and 0.37 g of NaHCO ₃ was added in sequence to dissolve completely, and the pH was adjusted to 7.5 using 1N HCl. This DMEM solution (100 ml) was filtered through a 0.2 μm diameter filter, mixed with 4 μl of CaCl ₂ (1M) per 1 ml of DMEM solution, and incubated in a 37 ° C. water bath for 30 minutes. Thereafter, the solution is centrifuged at 15000 rpm × 5 minutes, and the obtained pellet is dispersed in an aqueous solution that can be administered to cells or living bodies such as distilled water, cell culture solution, or physiological saline to obtain a carbonate apatite particle dispersion. Was subjected to ultrasonic vibration treatment for 10 minutes to obtain carbonate apatite nanoparticles (hereinafter referred to as “sCA (1) particles”). The ultrasonic vibration treatment uses a water bath having an ultrasonic vibration function to float a carbonate apatite particle dispersion in a plastic container on water set at 20 ° C. under conditions of a high frequency output of 55 W and an oscillation frequency of 38 kHz. 10 minutes. When measuring the particle size using a microscope, the sCA (1) particles were dispersed in distilled water after centrifugation. When used for cell experiments, it is not necessary to perform the above centrifugation, or the sCA (1) particles were dispersed in a cell culture medium such as a DMEM solution after the centrifugation. When used for animal experiments, sCA (1) particles were dispersed in physiological saline after centrifugation.

(2) Production of carbonate apatite nanoparticles (sCA) using buffer In 100 ml of distilled water, 0.37 g of NaHCO ₃ , 90 μl of NaH ₂ PO ₄ .2H ₂ O (1M), and 180 μl of CaCl ₂ (1M ) Were added and dissolved in this order, and the pH was adjusted to 7.5 with 1N HCl. This was filtered through a 0.2 μm diameter filter. After mixing ₄ μl of CaCl ₂ (1M) per 1 ml of the obtained buffer (hereinafter also referred to as “Buffer A”), incubating in a water bath at 37 ° C. for 30 minutes, and then rotating at 15000 rpm × 5 minutes. The pellet obtained is dispersed in an aqueous solution that can be administered to cells or living bodies such as distilled water, cell culture solution or physiological saline to obtain a carbonate apatite particle dispersion, which is subjected to ultrasonic vibration treatment for 10 minutes. To obtain carbonate apatite nanoparticles (hereinafter referred to as “sCA (2) particles”). The ultrasonic vibration treatment uses a water bath having an ultrasonic vibration function to float a carbonate apatite particle dispersion in a plastic container on water set at 20 ° C. under conditions of a high frequency output of 55 W and an oscillation frequency of 38 kHz. 10 minutes. When measuring the particle size using a microscope, the sCA (1) particles were dispersed in distilled water after centrifugation. When used for cell experiments, sCA (1) particles were dispersed in a cell culture medium such as a DMEM solution after centrifugation. When used for animal experiments, sCA (1) particles were dispersed in physiological saline after centrifugation.

(3) Measurement of particle diameter and form of carbonate apatite nanoparticles (sCA) The particle diameter and form of sCA (1) particles and sCA (2) particles prepared in Example 1 were measured using microcantilevers (OMCL-AC240TS-RS, Measurement was performed using a scanning probe microscope (SPM-9500, manufactured by Shimadzu Corporation) equipped with Olympus in dynamic mode. The measurement was performed twice within 30 seconds after the ultrasonic vibration treatment. About 10 μl of the sample aqueous solution was dropped on the surface of the cover glass, and after vacuum drying for 5 minutes, a smooth surface was selected with a CCD camera, and measurement was performed in the range of 1 to 5 square μm. The results are shown in Table 1 below. Moreover, the graph which shows the distribution of the number of the two-dimensional analysis image obtained about the measurement range and the size of particle | grains is shown to FIG. FIG. 1 shows sCA (1) particles, and FIG. 2 shows sCA (2) particles. From these results, it was confirmed that the particle size of the carbonate apatite particles can be reduced to 10 nm or less by ultrasonic vibration treatment.

Example 1: Tumor imaging in tumor model mice Tumor imaging with indocyanine green A human colon adenocarcinoma cell line HT29 was subcutaneously injected into the right and left backs of 7-week-old BALB / cA nude mice (CLEA Japan, Inc.). A model mouse having a tumor was prepared. When the tumor reached a size of about 10 mm, the mice were randomly divided into 4 groups shown in Table 2, and drug administration was performed in the manner shown in Table 2. After administration of indocyanine green, the fluorescence emitted by indocyanine green was measured over time using an IVIS spectrum CT apparatus (PerkinElmer), and in vivo images were acquired. In addition, 24 hours after administration of indocyanine green, lungs, heart, liver, spleen, kidney and tumor were taken out, and the fluorescence emitted by indocyanine green was measured using an IVIS spectrum CT apparatus (PerkinElmer). The image was acquired.

  FIG. 3 shows in vivo images 12 hours and 24 hours after administration of indocyanine green and the results of measuring the fluorescence intensity of indocyanine green in the tumor over time. FIG. 4 shows ex vivo images of the lung, heart, liver, spleen, kidney, and tumor 24 hours after administration of indocyanine green, and the results of measurement of indocyanine green fluorescence intensity in each organ. From these results, it was found that indocyanine green tumors were administered when both indocyanine green and carbonate apatite particles were administered, compared to when indocyanine green was administered alone, and when indocyanine green and atelocollagen were administered. The amount of accumulation in the tumor was large, and the tumor could be imaged more effectively. Furthermore, when both indocyanine green and carbonate apatite particles were administered, the accumulation of indocyanine green in normal organs did not increase compared to when indocyanine green was administered alone.

Example 2: Reduction of interstitial fluid pressure in tumor tissue Human colon cancer cells HT29 strain (5 × 10 ⁶ cells) were subcutaneously applied to the left and right sides of 7-week-old BALB / cA nude mice (manufactured by CLEA Japan, Inc.). The mouse subcutaneous solid tumor model was prepared by injection. When the diameter of the tumor reached about 10 mm, the sCA (2) particles prepared above (amount capable of encapsulating 40 μg of siRNA; equivalent to an amount prepared from 50 ml of buffer A) were administered via the tail vein. 2.5 to 4 hours after administration of the sCA (2) particles, the interstitial fluid pressure in the tumor was measured using an in vivo pressure catheter measurement system with a 1.6 Fr pressure catheter (transonic science, Inc) (measurement target tumor). Number 3, measurement location = 39). As a control, the interstitial fluid pressure in the tumor before administration of the sCA (2) particles was also measured (number of measurement target tumors: 5, measurement location = 30).

  The obtained results are shown in Table 3. From this result, it was clarified that the carbonate apatite particles have an action of reducing the interstitial fluid pressure in the tumor. Based on this result, the accumulation enhancement of the fluorescent contrast agent observed in the mouse subcutaneous solid tumor model in Example 1 in the tumor is contributed by the action of reducing the interstitial fluid pressure in the tumor by the carbonate apatite particles. It has been suggested.

Claims (7)

An integrated accelerating agent for accelerating accumulation in tumor imaging agents, including by the carbonated apatite as an active ingredient, does not contain a contrast agent, integrated accelerator.   The accumulation promoter according to claim 1, wherein the carbonate apatite is nanoparticles having an average particle size of 50 nm or less.   The accumulation promoting agent according to claim 1 or 2, wherein the contrast agent is a fluorescent contrast agent.   The accumulation accelerator according to claim 3, wherein the fluorescent contrast agent is indocyanine green.   The accumulation promoter according to any one of claims 1 to 4, further comprising albumin.   A tumor imaging kit comprising a first preparation containing a contrast agent and a second preparation containing the accumulation promoter according to any one of claims 1 to 5. A tumor imaging agent comprising a contrast agent and the accumulation accelerator according to any one of claims 1 to 5 in a mixed state .