WO2015056960A1 - Sensitizing composition using electromagnetic waves for thermal therapy of cancers, and cancer therapy using same - Google Patents

Sensitizing composition using electromagnetic waves for thermal therapy of cancers, and cancer therapy using same Download PDF

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WO2015056960A1
WO2015056960A1 PCT/KR2014/009641 KR2014009641W WO2015056960A1 WO 2015056960 A1 WO2015056960 A1 WO 2015056960A1 KR 2014009641 W KR2014009641 W KR 2014009641W WO 2015056960 A1 WO2015056960 A1 WO 2015056960A1
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cancer
ions
electromagnetic waves
iron
thermal therapy
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PCT/KR2014/009641
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French (fr)
Korean (ko)
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김현진
홍성출
정혜종
이희관
조해국
유재각
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주식회사 지니스
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Priority claimed from KR1020140109008A external-priority patent/KR101536325B1/en
Application filed by 주식회사 지니스 filed Critical 주식회사 지니스
Priority to EP14853591.7A priority Critical patent/EP3058949B1/en
Priority to CN201480057376.4A priority patent/CN105682677B/en
Priority to US15/029,726 priority patent/US20160310594A1/en
Priority to JP2016524018A priority patent/JP6404920B2/en
Priority to RU2016118565A priority patent/RU2016118565A/en
Priority to CA2927528A priority patent/CA2927528C/en
Publication of WO2015056960A1 publication Critical patent/WO2015056960A1/en
Priority to US17/219,479 priority patent/US20210236637A1/en
Priority to US17/981,080 priority patent/US11752210B2/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/40Transferrins, e.g. lactoferrins, ovotransferrins

Definitions

  • the present invention relates to a sensitizer composition for cancer thermal therapy using electromagnetic waves and a method for treating cancer using the same, and more particularly, to a sensitizer composition for cancer thermal therapy increasing cancer sensitivity when treating cancer using electromagnetic waves and cancer treatment using the same. It is about a method.
  • cancer therapies that have been developed to date are merely extending the survival of cancer patients rather than treating them fundamentally. Therefore, there is an urgent need to develop effective cancer treatments with fewer side effects.
  • Surgical therapy is the best treatment for early cancer, but it is difficult to expect a good therapeutic effect if the cancer has spread to other tissues.
  • Radiation therapy and anticancer drug therapy are known to not only lower cancer treatment effects but also affect normal tissues, causing various side effects such as gastrointestinal disorders, decreased immune function, loss of appetite, systemic weakness and hair loss.
  • heat treatment chemotherapy (Wust et al., The Lancet Oncology, 2002, 3: 487-497) .
  • Heat therapy chemotherapy is a chemotherapy that treats cancer by raising the temperature of the cancer tissue and surroundings to 42 ° C. or more using the characteristics of cancer cells lacking heat adaptation ability.
  • the surrounding normal cells can survive the heat shock, but the cancer cells are unable to adapt to high fever and die because of poor heat adaptation ability.
  • various methods such as the use of ultrasonic waves, heat transfer by contact, and electromagnetic waves have been developed.
  • Electromagnetic waves are waves that occur as the electric and magnetic fields change over time;
  • Gamma rays, X-rays, ultraviolet rays, visible light, infrared rays, microwaves, and radio waves are all electromagnetic waves.
  • the electromagnetic waves pass through a polar material, the electromagnetic waves generate heat by stimulating the molecular movement of the polar material.
  • all electromagnetic waves can be used for heat therapy chemotherapy.
  • 13.56 MHz radio The high frequency of frequency is the most commonly used.
  • Electromagnetic waves used in "cancer heat treatment using electromagnetic waves” generate heat through a process called dielectric heating.
  • Water molecules which make up most of the human body, have a dipole moment due to an asymmetric bond between oxygen and hydrogen atoms. Due to the dipole moment of water molecules, water molecules exposed to electromagnetic waves during the "thermal cancer therapy using electromagnetic waves” rotate their molecules by the frequency of electromagnetic waves, and they push, pull or collide with each other. Heat is generated in the tissue. If electromagnetic waves can be exposed only to cancer cells, cancer cells can be efficiently killed because of the characteristics of cancer cells that lack heat adaptation ability. However, exposing electromagnetic waves only to cancer cells without exposing them to normal cells is almost impossible due to the structural and physical limitations of the body.
  • cancer heat treatment using electromagnetic waves is not as effective as cancer treatment drugs or radiation therapy.
  • carcinoma therapy is used only as an adjuvant to chemotherapy or radiation therapy, rather than to treat cancer alone.
  • the best way to enhance the therapeutic efficacy of cancer thermal therapy using electromagnetic waves is to administer cancer thermal therapy using electromagnetic waves after administering a sensitizer for thermal therapy.
  • the thermal sensitizers attempted to date are nanoparticles based on metal components such as gold and iron oxide (International Patent Application No. 2009-091597, International Patent Application No. 2012-036978, International Patent Application No. 2012-177875, United States Patent No. 6561039, Korea Patent No. 0802139).
  • Metals react very well with electromagnetic waves and generate high heat. Therefore, after the metal component is accumulated in cancer cells instead of accumulating in normal cells, radiation of electromagnetic waves may maximize cancer treatment efficacy.
  • a technology for selectively delivering metal components to cancer tissues has not been developed, and the concept of "sensitizer for cancer thermal therapy using electromagnetic waves" has not been realized.
  • Metal nanoparticles such as gold or iron oxides do not have a selectivity for cancer tissues and accumulate in cancer tissues as well as normal tissues. When electromagnetic waves are processed, damage to normal tissues is generated by heat generation in all regions where nanoparticles are located. do. In addition, metal nanoparticles do not decompose or release in vivo, so there is a problem of low safety. Therefore, nanoparticles based on these metal components failed to be commercialized as sensitizers for thermotherapy, and there are no cases of sensitizers for thermotherapy commercialized worldwide.
  • the transferrin protein In human serum, about 70% of the transferrin protein is present as apotransferrin, which is not yet bound to iron ions, and about 30% of the transferrin is known to be iron-transferred apottransferrin, ie monoferric transferrin or diferric transferrin. Huebers et al., 1981, Proc. Natl. Acad. Sci. 78: 2572-2576. Therefore, the blood has a large amount of aporttransferin that can bind to the metal ions from the outside at any time.
  • Transferrin with non-covalently bound metal ions is carried along the blood and binds to the transferrin receptor to enter the cell through endocytosis to deliver metal ions, and the metal ions are separated. Transferrin, or apotransferrin, exits the cell through exocytosis and binds to metal ions, thereby circulating metabolic processes. Transferrin receptors, which play the most important role in the transfer of metal ions bound to transferrin into cells, are known to be overexpressed in cancer cells than in normal cells.
  • Cancer cells strongly absorb metal ions present in the blood because they absolutely require enzymes that use metal ions as coenzymes during cell metabolism. As described above, since metal ions do not exist independently in the blood but in the form of binding to transferrin, the metal ions that cancer cells absorb from the blood are substantially metal ions bound to transferrin. Iron, which is carried by transferrin, is used as an essential carrier and regulator of various enzymes that perform various functions of dividing cells such as DNA synthesis, cell division cycle, and metabolic regulation. Because these enzymes play a key role in metabolism, cancer cells have a strong ability to receive transferrin because they need large amounts of iron to maintain fast metabolism. In other words, cancer cells require more iron than normal cells, and the receptor for transferrin, a protein that delivers iron, is overexpressed.
  • transferrin as a target for cancer targeting, but there have been no reports of using "non-covalently bonded aprotransferrin" (transferrin) as a thermal sensitizer.
  • the present inventors recognized the characteristics of cancer cells that strongly absorb metal ions in the blood and the metals that react sensitively to electromagnetic waves.
  • the present inventors can administer metal ions instead of metals or metal compounds to cancer patients as sensitizers for cancer thermal therapy using electromagnetic waves.
  • the inventors of the present invention show that the treatment of cancer by improving the cancer treatment as described above is possible even by administering "non-covalently bound apattransferrin with a metal ion" to a cancer patient as a sensitizing agent for thermotherapy and performing "cancer thermotherapy using electromagnetic waves". The facts were confirmed and the present invention was completed.
  • the present invention provides a sensitizer composition for cancer thermal therapy using electromagnetic waves.
  • the sensitizer is characterized in that it is selected from the group consisting of a metal ion, a metal ion bond, a non-covalently bonded aprotransferrin and a metal ion-non-covalently bonded aprotransferrin derivative.
  • the metal ion is iron (iron), manganese (manganese), zinc (zinc), copper (copper), magnesium (magnesium), bismuth (bismuth), ruthenium (ruthenium) Selected from the group consisting of titanium ions, gallium ions, indium ions, vanadyl ions, chromium ions, aluminum ions, and plutonium ions It is characterized by.
  • the metal ion combination is dextran, sucrose, gluconate, sorbitol, polysaccharide, citrate, citrate to metal ions.
  • Carboxymaltose, perumoxytol, isomaltoside, maltose, starch, cellulose, chloride, sulfate, fumarate (fumurate) and albumin (albumin) is characterized in that the binder selected from the group comprising a non-covalent bond.
  • the atransferrin or the atransferrin derivative is characterized in that the human or mammalian-derived serum protein or recombinant protein.
  • the sensitizer is characterized in that the concentration of 0.01 ⁇ 100mg / ml.
  • the sensitizer composition for cancer thermal therapy is characterized in that it further comprises a pharmaceutically acceptable carrier.
  • the present invention also provides a kit for cancer thermal therapy, comprising a sensitizer composition for cancer thermal therapy using electromagnetic waves and a device for irradiating electromagnetic waves.
  • the present invention also provides a method for treating cancer, comprising the steps of: (a) administering the sensitizer composition for cancer thermal therapy to animals other than humans, thereby increasing susceptibility to cancer treatment; and (b) treating electromagnetic waves. do.
  • the sensitizer composition for cancer thermal therapy is characterized in that the administration of 1 ⁇ 250mg / kg dose.
  • the electromagnetic wave is selected from the group consisting of gamma rays, X-rays, ultraviolet rays, visible rays, infrared rays, microwaves and radio waves.
  • the cancer treatment method is one selected from the group consisting of chemotherapy, radiation therapy, biological therapy, immunotherapy and photodynamic therapy It is characterized by a combination of the above treatment methods.
  • the sensitizer composition for cancer thermal therapy according to the present invention has a cancer target orientation, thereby selectively transferring metal ions to cancer tissues, and thus, heat generation is increased in cancer tissues in which metal ions are accumulated during cancer thermal therapy using electromagnetic waves. Can maximize cancer treatment efficacy of cancer thermal therapy using.
  • Example 1 is a photograph measured by the thermal imaging camera the temperature of the aqueous solution of aforetransferrin before and after electromagnetic wave treatment according to Experimental Example 3 of the present invention.
  • the graph shows the difference between and temperature change.
  • Figure 2 is a graph showing the difference in the temperature change of the aqueous solution of aquatransferrin before and after electromagnetic wave treatment according to Experimental Example 3 of the present invention.
  • FIG. 3 is a photograph of a transferrin aqueous solution temperature before and after electromagnetic wave treatment according to Experimental Example 3 of the present invention measured by a thermal imaging camera.
  • Figure 5 is a photograph of the measurement of the transfer cell culture normal cell temperature before and after the electromagnetic wave treatment in accordance with Experimental Example 3 of the present invention.
  • FIG. 6 is a graph showing the difference in transfer cell culture normal cell temperature before and after electromagnetic wave treatment according to Experimental Example 3 of the present invention.
  • FIG. 7 is a photograph of a transferrin cultured cancer cell temperature before and after electromagnetic wave treatment according to Experimental Example 3 of the present invention measured by a thermal imaging camera.
  • Figure 8 is a graph showing the difference in transferin culture cancer cell temperature change before and after electromagnetic wave treatment according to Experimental Example 3 of the present invention.
  • FIG. 9 shows normal tissues and cancer tissue regions (arrows) during thermal treatment in which physiological saline (control) is respectively administered to a tumor-induced mouse (Tumor xenograft mouse) and electromagnetic waves are treated according to Example 1 of the present invention. It is a graph showing the difference between the temperature measured by the image camera and the temperature change (White bar: normal tissue, Black bar: cancer tissue).
  • FIG. 10 shows normal tissues and cancer tissue regions during thermal treatments in which thermal susceptibility agents (iron sucrose) are respectively administered to a tumor-induced mouse (Tumor xenograft mouse) and electromagnetic waves are treated according to Example 1 of the present invention.
  • Arrow is a graph showing the difference between the temperature measured with a thermal imaging camera (white bar: normal tissue, black bar: cancer tissue).
  • FIG. 11 shows normal tissues and cancer tissue regions (arrows) during thermal treatments in which a therapeutic agent for thermotherapy according to Example 1 of the present invention is administered to a cancer-induced mouse (Tumor xenograft mouse) and treated with electromagnetic waves.
  • a therapeutic agent for thermotherapy according to Example 1 of the present invention is administered to a cancer-induced mouse (Tumor xenograft mouse) and treated with electromagnetic waves.
  • Example 12 is a result of analyzing the size of the cancer tissue of the mouse treated with electromagnetic waves after administration of saline or metal ion conjugate to the tumor-induced mouse (Tumor xenograft mouse) according to Example 1 of the present invention (biofluorescence)
  • A untreated
  • B physiological saline
  • C iron gluconate
  • D iron sucrose
  • E iron carboxymaltose
  • F iron dextran
  • G iron starch
  • H transferrin
  • Example 13 is a biofluorescence analysis of cancer tissue size of mice treated with electromagnetic waves after administration of saline, iron sucrose, iron dextran and transferrin to a tumor-induced mouse (Tumor xenograft mouse) according to Example 1 of the present invention The result is.
  • the cancer tissue selectivity and cancer treatment of "cancer thermotherapy using electromagnetic waves” when using as a sensitizer capable of delivering metal ions only to cancer cells by using a target-oriented material for cancer tissue without toxicity and side effects It was expected to increase the efficacy.
  • the metal ions are transferred to cancer cells instead of the normal cells to increase the metal ion concentration of the cancer cells, followed by "cancer heat treatment using electromagnetic waves.”
  • cancer heat treatment using electromagnetic waves When the trial was performed, it was confirmed that the cancer treatment effect can be maximized.
  • transferrin is selectively delivered to cancer cells rather than normal cells by transferrin receptors overexpressed in cancer cells; (2) separation of metal ions bound to transferrin results in higher metal ion concentrations in cancer cells than normal cells; (3) increased metal ions in the cancer tissue during electromagnetic wave treatment further increase heat generation; (4) The death of cancer cells was increased by the generated heat, and as a result, it was confirmed that the cancer therapeutic efficacy of cancer thermal therapy using electromagnetic waves was maximized.
  • the metal ion introduced into the blood is excessively present in the blood. Since it is transferred to transferrin in combination with transferrin, it is confirmed that even if the metal ion is administered to cancer patients as a sensitizer for thermal therapy and cancer thermal therapy using electromagnetic waves, the same or better anticancer effect as described above can be obtained. It was.
  • the present invention relates to a sensitizer composition for cancer thermal therapy using electromagnetic waves.
  • the sensitizer is used to enhance cancer treatment efficacy during heat treatment, and has a characteristic of amplifying heat generation in cancer tissues while having a target orientation for cancer tissues when administered to the body.
  • the sensitizer may be selected from the group consisting of a metal ion, a metal ion bond, an apotransferrin non-covalently bonded to a metal ion, and an apotransferrin derivative non-covalently bound to a metal ion.
  • the metal ion is iron (iron), manganese (manganese), zinc (zinc), copper (copper), magnesium (magnesium), bismuth (bismuth), ruthenium (ruthenium) , Titanium ions, gallium ions, indium ions, vanadyl ions, chromium ions, aluminum ions, plutonium ions, etc. It is not limited to this.
  • the metal ion combination is dextran, sucrose, gluconate, sorbitol, polysaccharide, polysaccharide, citrate, carboxymaltose in metal ions. ), Perumoxytol, isomaltoside, maltose, starch, cellulose, chloride, sulfate, fumurate and albumin
  • the combination selected from the group containing (albumin) is non-covalently bound, and any combination that can be used as a medicament can be used without limitation, and may include iron dextran, iron sucrose, iron gluconate, iron carboxymaltose, iron isomaltoside, iron ferumoxytol , iron sorbitol, iron polysaccharide, ferric citrate, ferrous gluconate, ferrous sulfate, ferrous fumurate, magnesium chloride, gallium citrate, aluminum citrate, and the like.
  • the metal ions have a charge, they have polarity, that is, a dipole moment, and thus generate heat by amplifying molecular motion during electromagnetic wave treatment, and thus have a sensitizer characteristic that reacts more sensitively to electromagnetic waves than the metal itself.
  • non-covalently bonded aforttransferrin is a blood cell to cancer cells
  • selective delivery of cancer cells to the cancer cells by the overexpressed transferrin receptors will increase the concentration of metal ions delivered by the transferrin in the cancer cells
  • heat treatment using electromagnetic waves heat generation occurs intensively in cancer cells accumulated with metal ions, which minimizes damage to normal cells while intensively killing only cancer cells.
  • the transferrin is a protein widely distributed in blood, and refers to a metalloprotein that binds to metal ions such as iron and spins blood to transfer metal ions to cells having transferrin receptors.
  • the aforetransferin or aforetransferin derivative may be human or mammalian-derived serum protein or recombinant protein without particular limitation, as long as it has cancer targeting ability and is capable of binding with metal ions such as iron, manganese, and zinc.
  • the transferrin is preferably a transferrin in a form in which a metal ion is non-covalently bonded to an aprotransferrin, and as a iron-bound transferrin, iron-bound transferrin, monoferric transferrin, and diferric transferrin. , Holo-transferrin, ferric acetyl transferrin, and the like.
  • the transferrin binds to the transferrin receptor, which is overexpressed in cancer tissue, is delivered into cancer cells, and releases the bound metal ions into the cancer cells, thereby finally delivering metal ions selectively to the cancer tissue.
  • metal ions such as iron, manganese, and zinc are highly charged ions, they have a much stronger polarity than the dipole moment of water molecules. When metal ions with strong polarities are exposed to electromagnetic waves, molecular motion is amplified to maximize heat generation.
  • transferrin When the transferrin is administered to a cancer patient, 1) transferrin is selectively delivered to cancer cells rather than normal cells by a transferrin receptor overexpressed in cancer cells; 2) the concentration of metal ions in cancer cells is higher than that of normal cells due to the separation of metal ions bound to transferrin; 3) increased metal ions of the cancer tissue during electromagnetic wave treatment further increases heat generation; 4) The death of cancer cells is increased by the generated heat, which has the characteristics of cancer treatment efficacy.
  • the sensitizer composition for cancer thermal therapy is not particularly limited, but the concentration of the sensitizer is preferably 0.01-100 mg / ml. If the concentration is less than 0.01mg / ml there is an inconvenience to be administered in an excess volume, there is a problem that difficult to manufacture if it exceeds 100mg / ml.
  • the sensitizer composition for cancer thermal therapy may further include a pharmaceutically acceptable carrier or lubricant, wetting agent, emulsifier, suspending agent, preservative and the like.
  • the present invention relates to a cancer thermal therapy kit including the device for irradiating electromagnetic waves and the sensitizer composition for cancer thermal therapy in another aspect.
  • the sensitizer composition for cancer thermal therapy of the present invention treats various diseases related to cancer, such as gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, cervical cancer, etc. It can be used to, it can be included in the kit for cancer thermal therapy, including a device for irradiating electromagnetic waves.
  • the electromagnetic waves are waves generated as the electric and magnetic fields change with time, and may include gamma rays, X-rays, ultraviolet rays, visible rays, infrared rays, microwaves, radio waves, and the like. Phosphorus electromagnetic wave irradiation apparatus can be used.
  • the present invention provides a method for treating cancer, comprising the steps of: (a) administering the sensitizer composition for cancer thermal therapy to animals other than humans to increase susceptibility to cancer treatment; and (b) treating electromagnetic waves. It is about.
  • the sensitizer composition for cancer thermal therapy is a metal ion, a metal ion conjugate, a covalently bonded aforttransferrin, or a metal covalently bonded aforttransferrin derivative in a solution such as water, physiological saline suitable for injection 0.01 It is preferable to dissolve it at a concentration of ⁇ 100 mg / ml.
  • the dosage of the sensitizer composition for cancer thermal therapy is preferably in the range of 0.1 to 50 mg / kg in the case of metal ions and metal ion conjugates, and the atransferrin and non-covalently bonded metal ions In the case of derivatives, the dosage range of 0.1 to 200 mg / kg is preferred.
  • cancer thermal therapy using the electromagnetic wave it is preferable to perform cancer thermal therapy using electromagnetic waves within 1 to 48 hours after administering the sensitizer composition for cancer thermal therapy.
  • thermotherapy therapy using the electromagnetic wave can be easily determined and used by known thermotherapy therapy.
  • a treatment that is irradiated for 30 to 60 minutes with a thermal therapy device that outputs 13.56 MHz high frequency can be performed twice or more for four weeks or more per week.
  • Cancer treatment method can improve the therapeutic effect of cancer by using in conjunction with or in combination with existing chemotherapy.
  • Existing chemotherapy may be exemplified by chemotherapy, radiation therapy, biological therapy, immunotherapy and photodynamic therapy.
  • Oral or injectable metal ions for human consumption are in the form of conjugates combined with salts or conjugated polymers such as carbohydrates or proteins.
  • salts bound with metal ions include citrate, chloride, sulfate, fumurate, and the like.
  • Ferrous sulfate, ferrous fumarate, and the like are combined. (ferrous fumurate), ferrous gluconate and the like can be exemplified.
  • Carbohydrates bound to metal ions include gluconate (monosaccharide), sucrose (disaccharide), maltose (maltose), isomaltoside (polymal), carboxymaltose, and dextran (dextran) ), Starch, cellulose (saccharides) such as cellulose (saccharides) and the like, as the protein bound to the metal ions (transferrin, albumin, etc.) can be exemplified.
  • the ferrous sulfate solution which is a metal ion and salt combination
  • the exothermic ability of the metal ion binder was confirmed by measuring the temperature after treating the electromagnetic wave.
  • iron gluconate, magnesium sucrose, iron sucrose, iron isomaltoside, iron carboxymaltose, iron dextran, and iron starch complex solutions which are metal ions and carbohydrate combinations, are prepared as follows to treat the electromagnetic wave and then measure the temperature to generate the exothermic ability of the metal ion binders. It was confirmed.
  • Ferrous sulfate solution was prepared by dissolving 1 g of FeSO 4 7H 2 O in 10 ml of distilled water, stirring for 30 minutes or more, passing through a 0.22 ⁇ m filter, and diluting with sterile distilled water.
  • Iron gluconate solution sodium ferric gluconate complex
  • Sanofi Ferrlecit product was used by diluting Sanofi Ferrlecit product with sterile distilled water.
  • Magnesium sucrose solution was prepared by passing 83.6mg of MgCl 2 H 2 O and 150mg of sucrose in 10ml of distilled water, stirring for 30 minutes or more, passing through a 0.22 ⁇ m filter, and diluting with distilled water.
  • iron sucrose solution (ferric hydroxide sucrose complex) is first prepared the sucrose aqueous solution into the sugar 100mg in 50ml distilled water and continue stirring the addition of 5M NaOH was dissolved in 1ml 90 °C then put FeCl 3 0.9g in 50ml distilled water for 20 min or more Stir-dissolved 0.01M FeCl 3 aqueous solution was added to an aqueous solution of sucrose at 90 ° C., and then adjusted to pH 12 by dropping 5M NaOH solution. Next, after reacting for 2 hours at 80 °C centrifuged for 5 minutes at 5,000 rpm to obtain a ferric hydroxide sucrose complex, washed with distilled water and dried.
  • Iron isomaltoside complexes, iron carboxymaltose complexes, and iron starch complexes were also prepared by changing only carbohydrates from the method of preparing iron sucrose.
  • the metal ion conjugate was prepared in 10 mg / ml concentration of metal ions, and 0.1 ml was dispensed into 3 wells in a 96-well plate. At this time, 0.1ml of distilled water was used as a control. After exposing the 96-well plate at high frequency thermal cancer treatment device (EHY-2000, Oncothermia) for 5 minutes at 100W energy dose, the temperature was measured by thermal imaging camera (E60, Korea Rental, Korea) after 5 minutes. The difference in temperature after the change is shown in Table 1.
  • Table 1 delta Temp. (°C) control 0.5 ferrous sulfates 3.3 magnesium sucrose 5.2 iron sucrose 4.7 iron gluconate 5.7 iron isomaltoside 3.2 iron carboxymaltose 5.8 iron dextran 6.4 iron starch 4.5
  • transferrin binding capacity (Unsaturated Iron-Binding Capacity, UIBC) was measured according to the concentration of ferric iron (FeIII +) as follows.
  • FeCl 3 (Sigma Aldrich, USA) was added to 400 ml of distilled water to melt an aqueous solution of iron and stirred for at least 20 minutes. Then, 5 M NaOH solution was dropped dropwise to pH 9. When reddish brown precipitate was observed, the mixture was stirred at 90 ° C. for 2 hours, centrifuged at 5,000 rpm for 5 minutes to obtain precipitated ferric hydroxide, washed with distilled water, and dried. Ferric hydroxide in powder form was dissolved in distilled water to prepare ferric hydroxide solution of 1, 10, 50, 200, 500g / dL concentration.
  • Apotransferrin (Sigma Aldrich, USA) was added to the aqueous solution of iron ions at each concentration to 200 mg / dL, mixed with vortex for 1 minute, and apotransferrin reacted with iron ions at 37 ° C. for 30 minutes.
  • Ferrozine colorimetric method was used to measure the unsaturated iron-binding capacity of apoptransferin.
  • Ferrous chloride was prepared at 500 g / dL concentration in Hydroxylamine hydrochloride as an iron standard, and a reaction solution of apotransferrin and iron ions was prepared in the experimental group.
  • 2 ml of 0.5 M Tris buffer (pH 8) was dispensed into all test tubes.
  • 1 ml of distilled water, 0.5 ml of distilled water and 0.5 ml of iron standard, and 0.5 ml of apotransferrin and iron ion reaction solution and 0.5 ml of iron standard in test tube were mixed with vortex for 1 minute.
  • Absorbance A1 was measured after the spectrophotometer value was zero at 560 nm. Next, 50 ml of Ferrozine Hydroxylamine hydrochloride solution at a concentration of 16.6 mM was added, and then mixed with vortex for 1 minute. All test tubes were incubated at 37 ° C. for 10 minutes, and then absorbance A2 was measured at 560 nm. Absorbance A 560 at 560 nm was calculated by subtracting absorbance A1 from absorbance A2, and is shown in Table 2.
  • Unsaturated Iron-binding capacity was calculated by the following equation.
  • the temperature was measured after treating electromagnetic apattransferrin and iron-bonded apattransferrin solution with iron.
  • the iron ion aqueous solution was reacted with the aprotransferrin as follows. 3.6 g of FeCl 3 (Sigma Aldrich, USA) was added to 400 ml of distilled water and stirred for more than 20 minutes to dissolve. Then, 5 M NaOH solution was dropped dropwise to pH 9. When the reddish brown precipitate was observed, the mixture was incubated with stirring at 90 ° C. for 2 hours, followed by centrifugation at 5,000 rpm for 5 minutes to obtain precipitated ferric hydroxide, washed with distilled water, and dried.
  • FeCl 3 Sigma Aldrich, USA
  • Ferric hydroxide in powder form was dissolved in distilled water to prepare a ferric hydroxide solution at a concentration of 100 ⁇ g / dL.
  • Apottransferrin was added to 500 mg / dL in an aqueous solution of iron ions, mixed with vortex for 1 minute, and the aforetransferrin and iron ions were reacted at 37 ° C. for 30 minutes.
  • the iron ion-coupled aprotransferrin solution was diluted to 0, 0.04, 0.2, 1, and 5 mg / ml concentrations, and 0.1 ml of each concentration was prepared by dispensing in a 96-well plate.
  • thermotransfer camera (E60, Korea) exposed the temperature before and after exposing the APOTHERPIN aqueous solution plate and iron ion-coupled APOTHERPIN (transferrin) aqueous solution plate at 100W energy dose for 3 minutes in high frequency thermal cancer treatment device (EHY-2000, Oncothermia). Rental, Korea) and the difference in temperature change is shown in FIGS. 1 to 4.
  • the temperature change before and after the electromagnetic wave treatment of the aqueous solution of aprotransferrin was maintained at less than 3 °C at all treatment concentrations, while before and after the electromagnetic wave treatment of the aqueous solution of aprotransferrin (transferrin) with iron ions
  • the temperature change was found to increase 4.4 °C at 1mg / ml and 10.9 °C was increased at 5mg / ml.
  • Iron ion-coupled apattransferrin (transferrin) aqueous solution was added to 0, 0.04, 0.2, 1 or 5mg / ml in each of the prepared normal cell line and cancer cell line plates, and then cultured in a 37 ° C. CO 2 incubator for 4 hours.
  • the plate that completed transferrin and cell culture was washed with DMEM medium to remove transferrin that did not enter the cells.
  • each plate was exposed to an energy dose of 100 W for 3 minutes in a high-frequency thermal cancer treatment device (EHY-2000, Oncothermia), and then the temperature change was measured by a thermal imaging camera (E60, Korea Rental, Korea), and the results are shown in FIG. 5. It is shown to -8.
  • the temperature change before and after the electromagnetic wave treatment in the normal cell line was maintained at about 7 °C at all treatment concentrations, while in the cancer cell line the temperature change before and after the electromagnetic wave treatment was increased by 11.9 °C at 1mg / ml , 5mg / ml was confirmed to increase by 12.6 °C.
  • a tumor xenograft animal model was prepared as follows. After culturing the lung cancer cell line NCI-H460-luc2 (Califer Life Sciences), 5 ⁇ 10 6 cells were injected subcutaneously in 6-8 week old female BALB / c athymic nude mice (multiple science). Next, a cancer x-graft animal model was prepared by growing cancer tissues over 100 mm 3 for 10 days.
  • Established tumor xenograft BALB / c athymic nude mice were diluted with 0.2 mg / ml of magnesium sucrose, iron sucrose, and iron dextran aqueous metal ions prepared in Experiment 1, and then injected 0.1 ml intravenously to a dose of 1 mg / kg. .
  • 1 g of each tissue was removed for ICP-MS measurement, ground in an ice bath to a tissue grinder, and 1 ml of the pulverized liquid was dried at -60 ° C. and 7 ⁇ m Hg for 24 hours. 2 ml of 6N HCl was added to the dried powder, which was placed in a closed glass reactor and incubated in a 55 ° C. incubator.
  • each sample was vortexed, centrifuged at 1,000 rpm for 15 minutes, the supernatant was dried with nitrogen gas, and again, 1 ml of 0.01 N HCl was added and vortexed, followed by centrifugation at 1,000 rpm for 15 minutes. After the supernatant was recovered, the concentration of metal ions in normal and cancerous tissues was measured by inductively coupled plasma mass spectrometry (ICP-MS; Varian 800-MS, Palo Alto, US).
  • ICP-MS inductively coupled plasma mass spectrometry
  • Table 3 shows the results of measuring the concentration of metal ions accumulated in normal tissues and cancer tissues by ICP-MS after magnesium sucrose was administered to cancer-induced mice (Tumor xenograft mice).
  • magnesium sucrose administration increased the magnesium ion concentration of cancer tissue by more than 2.4 times.
  • Table 4 shows the results of measuring the concentration of metal ions accumulated in normal tissue and cancer tissue by ICP-MS after iron sucrose was administered to tumor xenograft mice.
  • the iron ion concentration of cancer tissue increased more than 3.3 times when iron sucrose was administered, which was higher than the major organs such as liver, kidney, heart, stomach and brain.
  • Table 5 shows the results of measuring the concentration of metal ions accumulated in normal tissues and cancer tissues by ICP-MS after iron dextran was administered to cancer-induced mice (Tumor xenograft mice).
  • the iron ion concentration of cancer tissues increased more than 3.4 times compared to the control group when iron dextran was administered, which was significantly higher than that of major organs such as liver, kidney, heart, stomach and brain.
  • the iron-bound transferrin aqueous solution was administered to rats, and the concentration of metal ions in normal tissues and cancer tissues was measured.
  • An aqueous solution of apottransferrin (transferrin) in which iron ions were bound was prepared at 4 mg / ml, followed by 0.1 ml intravenous injection at a 16 mg / kg dose. After 24 hours, each tissue was collected in the same manner as in Experimental Example 5, and the concentration of metal ions was measured by Inductively coupled plasma mass spectrometry (ICP-MS; Varian 800-MS, Palo Alto, US).
  • ICP-MS Inductively coupled plasma mass spectrometry
  • Table 6 shows the concentration of iron ions accumulated in normal tissues and cancer tissues by ICP-MS after administration of "iron ion-bound atransferrin" (transferrin) to cancer-induced mice (Tumor xenograft mice). One result.
  • the iron ion concentration of iron ion-coupled atransferrin increased more than 3.2 times the concentration of iron ions in cancer tissues compared to the control group, which was found in major organs such as liver, kidney, heart, stomach and brain. It was significantly higher than the increase rate.
  • Example 1 Administration of sensitizer for thermal therapy and cancer thermal therapy using electromagnetic waves
  • Cancer cells have poor metabolic control instead of rapidly receiving the nutrients needed for rapid cell division in order to continue abnormal division.
  • cancer cells overexpress the transferrin receptor and receive a lot of iron required for cell division, but are poor in heat regulation and are relatively sensitive to high fever compared to normal cells. Therefore, if heat is concentrated only on cancer cells, selective killing of cancer cells is possible.
  • Transferrin which has a target orientation to cancer cells, delivers iron to cancer cells intensively through transferrin receptors overexpressed in cancer cells.
  • Example 1 the use of a metal ion conjugate with excellent temperature rise in the experimental example was used as a sensitizer for heat treatment, thereby confirming the possibility of anticancer efficacy in the heat treatment of a tumor xenograft animal model.
  • the lung cancer cell line NCI-H460-luc2 (Califer Life Sciences) was cultured, and then 5 ⁇ 10 6 cells were subcutaneously injected into 6-8 week old female BALB / c athymic nude mice (multiple science). After injection, the tumor tissue was grown for 10 days to grow 100 mm 3 or more to produce a tumor xenograft animal model to study the therapeutic efficacy of the cancer.
  • iron sucrose which is a sensitizer composition for thermal therapy
  • 0.1 ml intravenous was injected with a 0.2 mg / ml iron sucrose aqueous solution to a dose of 1 mg / kg in the established tumor xenograft mice.
  • the transferrin aqueous solution of 5 mg / ml concentration to 20 mg / kg dose to the established tumor xenograft mice 0.1 ml intravenous injection.
  • Physiological saline was administered to the control group. After 4 hours of administration, high-frequency thermal cancer treatment device (EHY-2000, Oncothermia) was irradiated with an energy dose of 100W for 3 minutes, and then the temperature of normal tissue and cancer tissue was taken with a thermal imaging camera (E60, Korea Rental, Korea). The results are shown in FIGS. 9 to 11.
  • EHY-2000, Oncothermia high-frequency thermal cancer treatment device
  • the temperature in the normal tissue and the cancer tissue before and after the electromagnetic wave treatment both increased by about 1 °C level was no difference.
  • the temperature change before and after electromagnetic wave treatment was 1 °C for normal tissues and 1.9 °C for cancer tissues.
  • the temperature change before and after electromagnetic wave treatment was also different.
  • the use of the metal ion combination as a sensitizer for heat treatment confirmed the possibility of cancer treatment during heat treatment.
  • NCI-H460-luc2 Califer Life Sciences
  • 5 ⁇ 10 6 cells were injected subcutaneously in 6-8 week old female BALB / c athymic nude mice (multiple science).
  • a tumor xenograft animal model was prepared to study the therapeutic efficacy of cancer by growing cancer tissues over 100 mm 3 while growing for 10 days.
  • the metal ion monosaccharide conjugate iron gluconate
  • the metal ion disaccharide conjugate iron sucrose
  • metal ion in the tumor xenograft mouse Oligosaccharide conjugates (iron isomaltoside), metal ion polysaccharide conjugates (iron carboxymaltose, iron dextran, iron starch) were injected 0.1 ml intravenously to a 1 mg / kg dose.
  • the iron ion-coupled aprotransferrin (transferrin), a sensitizer composition for thermotherapy was injected 0.1 ml intravenously to a dose of 20 mg / kg in the established tumor xenograft mice.
  • the high-frequency heat cancer treatment device (EHY-2000, Oncothermia) underwent heat treatment for 10 minutes with 100W energy dose three times a week for 4 weeks. At this time, the untreated group and the saline treated group proceeded to the control group. In the last week, bioluminescence imaging was performed to analyze cancer tissue size.
  • D-luciferin (Xenogen, USA) was intraperitoneally injected into mice at a concentration of 150 mg luciferin / kg / d to luminesce luciferase-expressing cancer cell line NCI-H460-luc2, and with isoflurane gas After inhalation anesthesia mixed with oxygen, the cancer cells luminesced with Xenogen imager (IVIS 200) were superimposed and analyzed using Igor Pro imaging analysis software, and the results are shown in FIG. 12.
  • Example 12 is untreated (A), saline (B), iron gluconate (C), iron sucrose (D), iron carboxymaltose in cancer-induced mouse (Tumor xenograft mouse model animal) according to Example 1 of the present invention (E), iron dextran (F), iron starch (G), and transferrin (H) were administered and biofluorescence was analyzed for the size of the cancerous tissue in the mouse model after radiofrequency thermal treatment.
  • iron dextran and iron sucrose groups 0.1 ml intravenous injection of 0.2 mg / ml iron dextran or iron sucrose, respectively, and in the transferrin group, 0.1 ml intravenous injection of 5 mg / ml transferrin aqueous solutions.
  • the untreated group and physiological saline administration group was progressed to the control group.
  • the experimental group administered iron dextran as a sensitizer and the experimental group administered iron sucrose as a sensitizer it was confirmed that the growth rate of cancer was clearly decreased during the heat treatment using electromagnetic waves.
  • the transferrin-administered group it was found that the anticancer efficacy was markedly suppressed and reduced in size, and disappeared completely after 4 weeks at the end of the test.
  • the sensitizer composition for cancer thermal therapy according to the present invention, only cancer cells can selectively accumulate metal components, and thus can be the most ideal anti-cancer therapy with significantly improved cancer treatment efficacy without pain and side effects. It is expected to be widely used, and can be used in combination with other cancer treatment methods such as chemotherapy and radiation therapy, thereby increasing the cure potential of cancer.

Abstract

The present invention relates to a sensitizing composition using electromagnetic waves for the thermal therapy of cancers, and cancer therapy using same. More particularly, the present invention relates to a sensitizing composition using electromagnetic waves to increase sensitivity for cancer treatment, and cancer therapy using same. The sensitizing composition using electromagnetic waves for the thermal therapy of cancers according to the present invention includes metal ions, a metal ion binding material, "apotransferrin to which metal ions are non-covalently bonded" (transferrin), or an apotransferrin inducer to which metal ions are non-covalently bonded. Since the sensitizing composition for the thermal therapy of cancers according to the present invention has target selectivity for cancers when administered to the human body, metal ions are selectively delivered to cancer tissue. Therefore, heat generation in cancer tissue in which the metal ions are accumulated is increased when thermal therapy of cancers using electromagnetic waves is performed. Thus, it is possible to maximize the cancer treating efficacy of the thermal therapy of cancers using electromagnetic waves. Further, since it is possible to significantly increase cancer treating efficacy without pain and side effects, the present invention is expected to be widely used for anticancer treatment. Also, since it is possible to combine other cancer therapies such as chemotherapy and radiotherapy, the probability of complete recovery from cancer can be increased.

Description

전자기파를 이용한 암 온열치료용 감작제 조성물 및 이를 이용한 암 치료 방법 Sensitizer composition for cancer thermal therapy using electromagnetic waves and cancer treatment method using the same
본 발명은 전자기파를 이용한 암 온열치료용 감작제 조성물 및 이를 이용한 암 치료 방법에 관한 것으로, 더욱 상세하게는 전자기파를 이용하여 암 치료시 감수성을 증가시키는 암 온열치료용 감작제 조성물 및 이를 이용한 암 치료 방법에 관한 것이다.The present invention relates to a sensitizer composition for cancer thermal therapy using electromagnetic waves and a method for treating cancer using the same, and more particularly, to a sensitizer composition for cancer thermal therapy increasing cancer sensitivity when treating cancer using electromagnetic waves and cancer treatment using the same. It is about a method.
최근 현대 의학의 급격한 발달로 암에 대한 조기진단이 가능해지고 동시에 수술, 방사선, 그리고 항암제 약물요법 등과 같은 다양한 암 치료 방법이 개발되어 암을 극복할 수 있는 가능성이 보이기 시작하고 있다. 하지만 현재까지 개발된 암 치료법들은 암을 근본적으로 치료하기보다는 단순히 암환자들의 생존기간을 연장시키는 정도에 불과하다. 따라서 효과적이면서도 부작용이 적은 암 치료 방법을 개발할 필요성이 절실하다. Recently, with the rapid development of modern medicine, early diagnosis of cancer becomes possible, and various cancer treatment methods such as surgery, radiation, and anticancer drug therapy have been developed, and thus, the possibility of overcoming cancer is beginning to be shown. However, cancer therapies that have been developed to date are merely extending the survival of cancer patients rather than treating them fundamentally. Therefore, there is an urgent need to develop effective cancer treatments with fewer side effects.
암 치료에서 보편적으로 수행되고 있는 치료법으로는 수술요법, 항암제 약물요법, 그리고 방사선 치료법이 있다. 수술요법은 조기 암에는 가장 좋은 치료방법이나 암이 다른 조직으로 전이된 경우에는 좋은 치료 효과를 기대하기가 어렵다. Therapies commonly used in cancer treatment include surgery, chemotherapy, and radiation. Surgical therapy is the best treatment for early cancer, but it is difficult to expect a good therapeutic effect if the cancer has spread to other tissues.
방사선 치료법과 항암제 약물요법은 암 치료 효과가 낮을 뿐 아니라, 정상조직에도 영향을 미쳐 위장장애, 면역기능 저하, 식욕부진, 전신쇠약, 탈모 등 다양한 부작용들을 야기시키는 것으로 알려졌다. 이러한 기존 암치료 방법의 한계점들을 보완하기 위하여 현재 여러 종류의 암 치료 방법들이 개발되고 있으며, 그 중 대표적인 것이 열 치료 항암요법이다(Wust et al., The Lancet Oncology, 2002, 3:487-497).Radiation therapy and anticancer drug therapy are known to not only lower cancer treatment effects but also affect normal tissues, causing various side effects such as gastrointestinal disorders, decreased immune function, loss of appetite, systemic weakness and hair loss. In order to supplement the limitations of the existing cancer treatment methods, several types of cancer treatment methods are currently being developed, and among them, heat treatment chemotherapy (Wust et al., The Lancet Oncology, 2002, 3: 487-497) .
암 세포의 고유한 특성중 하나는 열 적응 능력이 정상 세포에 비해 현저히 떨어진다는 것이다(Wust et al., The Lancet Oncology, 2002, 3:487-497). 열 치료 항암요법은 이처럼 열 적응 능력이 결여된 암세포의 특성을 이용하여 암 조직 및 주변의 온도를 42℃ 이상으로 올려 암을 치료하는 항암요법이다. 열 치료 항암요법 과정에서 암 조직의 온도를 올리면, 주변 정상 세포는 열 충격에 저항하여 생존할 수 있지만 암 세포는 열 적응 능력이 떨어지기 때문에 고열에 적응하지 못하고 사멸한다. 열 치료 항암요법 과정에서 암 조직의 온도를 높이기 위해 초음파를 이용하는 방법, 접촉에 의한 열전달 방법, 전자기파를 이용하는 방법 등과 같은 여러 가지 방법들이 개발되었다. 하지만, 현재 열치료 항암요법에서 가장 일반적이고 효과적인 방법은 전자기파를 조사하여 암 조직에 열을 발생시키는 전자기파를 이용한 암 온열치료이다(유럽공개특허 제2174689호, 미국등록특허 제4323056호, 국제특허출원 제2002-172198호, 한국등록특허 제1125200호, 국제특허출원 제2010-043372호, 국제특허출원 제2009-013630호). One of the unique characteristics of cancer cells is that their ability to adapt to heat is significantly lower than that of normal cells (Wust et al., The Lancet Oncology, 2002, 3: 487-497). Heat therapy chemotherapy is a chemotherapy that treats cancer by raising the temperature of the cancer tissue and surroundings to 42 ° C. or more using the characteristics of cancer cells lacking heat adaptation ability. When the temperature of cancer tissue is raised during the heat treatment chemotherapy, the surrounding normal cells can survive the heat shock, but the cancer cells are unable to adapt to high fever and die because of poor heat adaptation ability. In order to increase the temperature of cancer tissue during the heat treatment chemotherapy, various methods such as the use of ultrasonic waves, heat transfer by contact, and electromagnetic waves have been developed. However, at present, the most common and effective method of heat therapy chemotherapy is cancer thermal therapy using electromagnetic waves that generate heat to cancer tissues by irradiating electromagnetic waves (European Patent No. 2174689, US Patent No. 4323056, International Patent Application) 2002-172198, Korean Patent No. 1125200, International Patent Application No. 2010-043372, International Patent Application No. 2009-013630).
전자기파(electromagnetic waves)는 전기장과 자기장이 시간에 따라 변하면서 발생하는 파동으로서; 감마선, X-선, 자외선, 가시광선, 적외선, 마이크로웨이브, 라디오 전파 등이 모두 전자기파이다. 전자기파가 극성물질을 통과하면 전자기파는 극성물질의 분자운동을 자극하여 열을 발생하기 때문에 기본적으로 모든 전자기파는 열 치료 항암요법에 활용되어질 수 있지만, 현재 "전자기파를 이용한 암 온열치료"에서는 13.56 MHz radio frequency의 고주파를 가장 보편적으로 사용하고 있다. Electromagnetic waves are waves that occur as the electric and magnetic fields change over time; Gamma rays, X-rays, ultraviolet rays, visible light, infrared rays, microwaves, and radio waves are all electromagnetic waves. When electromagnetic waves pass through a polar material, the electromagnetic waves generate heat by stimulating the molecular movement of the polar material. Basically, all electromagnetic waves can be used for heat therapy chemotherapy. However, in current "thermal cancer therapy using electromagnetic waves," 13.56 MHz radio The high frequency of frequency is the most commonly used.
"전자기파를 이용한 암 온열치료"에 이용되는 전자기파는 유전가열(dielectric heating)이라는 과정을 통해 열을 발생시킨다. 인체의 대부분을 구성하는 물 분자는 산소와 수소원자 간 비대칭적 결합으로 쌍극자 모멘트를 가지게 된다. 이 물 분자의 쌍극자 모멘트 때문에, "전자기파를 이용한 암 온열치료"시 전자기파에 노출된 물 분자가 전자기파의 진동수만큼 분자회전을 거듭하면서 분자들끼리 밀고 당기거나 충돌을 거듭하게 되며, 그 결과 전자기파가 노출된 조직에서 열이 발생하는 것이다. 암 세포에만 전자기파를 노출시킬 수 있다면, 열 적응 능력이 부족한 암세포의 특성 때문에 효율적으로 암세포를 사멸시킬 수 있다. 하지만 전자기파를 정상세포에는 노출시키지 않고 암 세포에만 노출시키는 것은 신체의 구조적물리적 한계로 인해 거의 불가능하다. 따라서, 전자기파와 같은 물리적 파장을 암 조직에 방사하여 암을 치료하는 것은 한계가 있으며, "전자기파를 이용한 암 온열치료"는 항암제 약물요법이나 방사선요법 보다는 암 치료 효능이 뛰어나지 않아, "전자기파를 이용한 암 온열치료"가 단독으로 암 치료에 쓰이기보다는 항암제 약물요법이나 방사선요법에 보조적 수단 정도로만 활용되고 있다. Electromagnetic waves used in "cancer heat treatment using electromagnetic waves" generate heat through a process called dielectric heating. Water molecules, which make up most of the human body, have a dipole moment due to an asymmetric bond between oxygen and hydrogen atoms. Due to the dipole moment of water molecules, water molecules exposed to electromagnetic waves during the "thermal cancer therapy using electromagnetic waves" rotate their molecules by the frequency of electromagnetic waves, and they push, pull or collide with each other. Heat is generated in the tissue. If electromagnetic waves can be exposed only to cancer cells, cancer cells can be efficiently killed because of the characteristics of cancer cells that lack heat adaptation ability. However, exposing electromagnetic waves only to cancer cells without exposing them to normal cells is almost impossible due to the structural and physical limitations of the body. Therefore, there is a limitation in treating cancer by radiating physical wavelengths such as electromagnetic waves to cancer tissue, and "cancer heat treatment using electromagnetic waves" is not as effective as cancer treatment drugs or radiation therapy. "Thermal therapy" is used only as an adjuvant to chemotherapy or radiation therapy, rather than to treat cancer alone.
전자기파를 이용한 암 온열치료의 치료 효능을 높일 수 있는 가장 좋은 방법은, 온열치료용 감작제를 투여한 후 전자기파를 이용한 암 온열치료를 시행하는 것이다. The best way to enhance the therapeutic efficacy of cancer thermal therapy using electromagnetic waves is to administer cancer thermal therapy using electromagnetic waves after administering a sensitizer for thermal therapy.
현재까지 시도된 온열치료용 감작제는 금, 산화철 등과 같은 금속 성분에 기초한 나노입자이다 (국제특허출원 제2009-091597호, 국제특허출원 제2012-036978호, 국제특허출원 제2012-177875호, 미국등록특허 제6541039호, 한국등록특허 제0802139호). 금속은 전자기파와 매우 잘 반응하여 고열을 발생시킨다. 따라서 금속 성분이 정상세포에는 축적되지 않고 암세포에만 축적되도록 한 후, 전자기파를 방사하면 암 치료 효능이 극대화 될 수 있을 것이다. 하지만 현재까지 금속 성분을 암 조직에만 선택적으로 전달할 수 있는 기술은 개발되지 못했고, "전자기파를 이용한 암 온열치료용 감작제"라는 개념 또한 구현되지 못한 실정이다. The thermal sensitizers attempted to date are nanoparticles based on metal components such as gold and iron oxide (International Patent Application No. 2009-091597, International Patent Application No. 2012-036978, International Patent Application No. 2012-177875, United States Patent No. 6561039, Korea Patent No. 0802139). Metals react very well with electromagnetic waves and generate high heat. Therefore, after the metal component is accumulated in cancer cells instead of accumulating in normal cells, radiation of electromagnetic waves may maximize cancer treatment efficacy. However, until now, a technology for selectively delivering metal components to cancer tissues has not been developed, and the concept of "sensitizer for cancer thermal therapy using electromagnetic waves" has not been realized.
금이나 산화철과 같은 금속나노입자는 암 조직에 대한 선택성이 없어 암 조직뿐 아니라 정상 조직에도 축적되게 되는데, 이 때 전자기파를 처리하면 나노입자가 위치한 모든 부위에서 열 발생에 의해 정상 조직에도 손상을 일으키게 된다. 또한 금속나노입자는 생체에서 분해되거나 방출되지 않아 안전성이 낮은 문제가 있다. 따라서 이러한 금속성분에 기초한 나노입자는 온열치료용 감작제로 상용화에 실패하였고, 아직까지 전 세계적으로 온열치료용 감작제가 상용화된 사례는 없다.Metal nanoparticles such as gold or iron oxides do not have a selectivity for cancer tissues and accumulate in cancer tissues as well as normal tissues. When electromagnetic waves are processed, damage to normal tissues is generated by heat generation in all regions where nanoparticles are located. do. In addition, metal nanoparticles do not decompose or release in vivo, so there is a problem of low safety. Therefore, nanoparticles based on these metal components failed to be commercialized as sensitizers for thermotherapy, and there are no cases of sensitizers for thermotherapy commercialized worldwide.
사람을 포함한 모든 생명체에는 다양한 종류의 금속 성분이 체내 필수 구성성분으로 존재한다. 생명체의 체내에 존재하는 금속성분은 금속 그 자체로 존재하지 않고 대부분 이온 상태로 존재하여 생명유지에 필요한 다양한 기능을 수행한다. 마그네슘, 망간, 철 등과 같은 금속이온들은 생명유지를 위해 인체가 섭취해야할 필수 영양분이다. 인체로 흡수된 금속이온들은 혈액에서 독립적으로 존재하지 않고 트랜스페린(transferrin)이라는 금속이온 전달 단백질과 결합한 상태로 존재한다. 철 이온과 아직 결합되어 있지 않은 아포트랜스페린(apotransferrin)에 철 이온이 하나 결합되면 모노페릭 트랜스페린(monoferric transferrin), 두 개 결합되면 디페릭 트랜스페린(diferric transferrin) 또는 홀로트랜스페린 (holo-transferrin)이라고 한다. 인간의 혈청에서 약 70%의 트랜스페린 단백질은 철 이온과 아직 결합되어 있지 않은 아포트랜스페린(apotransferrin)으로 존재하고 나머지 30% 정도의 트랜스페린은 철 이온이 결합된 아포트랜스페린 즉 monoferric transferrin 또는 diferric transferrin인 것으로 알려져 있다(Huebers et al., 1981, Proc. Natl. Acad. Sci. 78:2572-2576). 따라서 혈액에는 언제든지 외부에서 유입되는 금속이온과 결합할 수 있는 아포트랜스페린이 다량 존재한다.In all living things, including humans, various kinds of metals exist as essential components of the body. Metallic components present in the body of life do not exist in the metal itself, but mostly exist in ionic state to perform various functions necessary for life maintenance. Metal ions such as magnesium, manganese and iron are essential nutrients for humans to consume. Metal ions absorbed into the human body do not exist independently in the blood but in combination with a metal ion transfer protein called transferrin. When one iron ion is bound to apotransferrin that is not yet bound to iron ions, it is called monoferric transferrin, and two are called diferric transferrin or holo-transferrin. In human serum, about 70% of the transferrin protein is present as apotransferrin, which is not yet bound to iron ions, and about 30% of the transferrin is known to be iron-transferred apottransferrin, ie monoferric transferrin or diferric transferrin. Huebers et al., 1981, Proc. Natl. Acad. Sci. 78: 2572-2576. Therefore, the blood has a large amount of aporttransferin that can bind to the metal ions from the outside at any time.
"금속이온이 비공유 결합된 아포트랜스페린"(트랜스페린)은 혈액을 따라 운반되다가 트랜스페린 수용체(transferrin receptor)와 결합하여 엔도사이토시스(endocytosis)를 통해 세포 내로 들어가 금속이온을 전달하고, 금속이온이 떨어져 나간 트랜스페린, 즉 아포트랜스페린(apotransferrin)은 엑소사이토시스(exocytosis)를 통해 세포 밖으로 나가 다시 금속이온과 결합함으로써 대사 과정이 순환하게 된다. 트랜스페린에 결합된 금속이온을 세포 내로 전달하는데 가장 중요한 역할을 하는 트랜스페린 수용체는 정상 세포에서보다 암 세포에서 과발현되는 것으로 알려졌다. "Atransferrin with non-covalently bound metal ions" (transferrin) is carried along the blood and binds to the transferrin receptor to enter the cell through endocytosis to deliver metal ions, and the metal ions are separated. Transferrin, or apotransferrin, exits the cell through exocytosis and binds to metal ions, thereby circulating metabolic processes. Transferrin receptors, which play the most important role in the transfer of metal ions bound to transferrin into cells, are known to be overexpressed in cancer cells than in normal cells.
암세포는 세포대사 과정에서 금속이온을 조효소로 하는 효소를 절대적으로 필요로 하므로 혈액에 존재하는 금속이온을 강하게 흡수한다. 앞서 설명했듯이 혈액에는 금속이온이 독립적으로 존재하는 것이 아니라 트랜스페린과 결합한 형태로 존재하기 때문에 암세포가 혈액에서 흡수하는 금속이온은 실질적으로 트랜스페린에 결합된 금속이온이다. 트랜스페린이 운반하는 철은 DNA 합성, 세포분열 주기, 대사 조절 등 분열하는 세포의 다양한 기능을 수행하는 각종 효소들의 필수적 보인자 및 조절인자로 쓰인다. 이들 효소들은 대사과정에 핵심 역할을 수행하기 때문에 암세포는 빠른 대사과정을 유지하기 위해 철이 다량 필요하여 트랜스페린을 강력하게 받아들이는 특성이 있다. 즉, 암세포는 철이 정상 세포보다 많이 필요하여, 철을 전달하는 단백질인 트랜스페린에 대한 수용체가 과발현되어 있고, 그 결과 혈액 내 트랜스페린이 암조직으로 잘 전달되는 특성을 가지는데, 이를 트랜스페린의 암 표적지향성이라고 한다. 이와 같은 트랜스페린의 암 표적지향성을 이용하여 트랜스페린을 부착시킨 항암제 나노입자들이 공지되어 있다 (미국공개특허 제2009-0181048호, 유럽공개특허 제2216341호, 유럽공개특허 제1369132호). Cancer cells strongly absorb metal ions present in the blood because they absolutely require enzymes that use metal ions as coenzymes during cell metabolism. As described above, since metal ions do not exist independently in the blood but in the form of binding to transferrin, the metal ions that cancer cells absorb from the blood are substantially metal ions bound to transferrin. Iron, which is carried by transferrin, is used as an essential carrier and regulator of various enzymes that perform various functions of dividing cells such as DNA synthesis, cell division cycle, and metabolic regulation. Because these enzymes play a key role in metabolism, cancer cells have a strong ability to receive transferrin because they need large amounts of iron to maintain fast metabolism. In other words, cancer cells require more iron than normal cells, and the receptor for transferrin, a protein that delivers iron, is overexpressed. As a result, transferrin in blood is well transmitted to cancer tissues. It is called. Anticancer agent nanoparticles to which transferrin has been attached by using the cancer target orientation of transferrin are known (US Patent Publication No. 2009-0181048, European Patent Publication No. 22116341, and European Patent Publication No. 1369132).
이처럼, 트랜스페린을 암 표적 지향물질로 이용한 예는 있으나, 아직까지 "금속이온이 비공유 결합된 아포트랜스페린"(트랜스페린)을 온열치료용 감작제로 이용한다는 보고는 없었다. As such, there have been examples of using transferrin as a target for cancer targeting, but there have been no reports of using "non-covalently bonded aprotransferrin" (transferrin) as a thermal sensitizer.
이에, 혈액의 금속이온을 강하게 흡수하는 암세포의 특성과 전자기파에 민감하게 반응하는 금속의 특성을 인지한 본 발명자들은 전자기파를 이용한 암 온열치료 감작제로 금속 혹은 금속화합물 대신 금속이온을 암 환자에게 투여할 경우; 1) 혈액에 주입된 금속이온은 혈액에 과량 존재하는 아포트랜스페린과 결합하여 트랜스페린화되고; 2) 금속이온이 비공유 결합된 아포트랜스페린은 암세포에 과발현된 트랜스페린 수용체에 의해 암세포에 선택적으로 전달되므로 트랜스페린에 의해 전달되는 금속이온 농도가 암세포에서 높아질 것이고; 3) 여기에 전자기파를 이용한 온열치료를 시행하면 금속이온이 축적된 암세포에서 열 발생이 집중적으로 일어나 정상세포에 대한 손상을 최소화하면서 암세포만 집중적으로 사멸시킬 수 있다는 사실을 확인하고 본 발명을 완성하였다. 또한 본 발명자들은 "금속이온이 비공유 결합된 아포트랜스페린"을 온열치료용 감작제로 암 환자에게 투여하고 "전자기파를 이용한 암 온열치료"를 시행하여도 상기에서 기술한 바와 같이 암 치료가 혁신적으로 개선된다는 사실을 확인하고 본 발명을 완성하였다. Accordingly, the present inventors recognized the characteristics of cancer cells that strongly absorb metal ions in the blood and the metals that react sensitively to electromagnetic waves. Thus, the present inventors can administer metal ions instead of metals or metal compounds to cancer patients as sensitizers for cancer thermal therapy using electromagnetic waves. Occation; 1) Metal ions injected into the blood are transferred to the atransferrin in excess of the presence of blood in the blood; 2) Aforetransferin with non-covalently bound metal ions is selectively delivered to cancer cells by transferrin receptors overexpressed in cancer cells, so that the metal ion concentration delivered by transferrin will be increased in cancer cells; 3) When the heat treatment using electromagnetic waves is performed, heat generation occurs intensively in cancer cells in which metal ions are accumulated, thereby confirming that only cancer cells can be intensively killed while minimizing damage to normal cells. . In addition, the inventors of the present invention show that the treatment of cancer by improving the cancer treatment as described above is possible even by administering "non-covalently bound apattransferrin with a metal ion" to a cancer patient as a sensitizing agent for thermotherapy and performing "cancer thermotherapy using electromagnetic waves". The facts were confirmed and the present invention was completed.
본 발명의 목적은 암 조직에만 금속이온을 선택적으로 전달함으로써, "전자기파를 이용한 암 온열치료" 시 암 조직에만 열 발생이 극대화되어 암을 치료할 수 있는 암 온열치료용 감작제 조성물, 이를 포함하는 암 온열치료용 키트 및 이를 이용하는 암 치료방법을 제공하는데 있다.It is an object of the present invention to selectively deliver metal ions only to cancer tissues, thereby maximizing the generation of heat only in cancer tissues during "cancer heat treatment using electromagnetic waves", a sensitizer composition for cancer thermal therapy that can treat cancer, including cancer It is to provide a kit for heat treatment and a cancer treatment method using the same.
상기 목적을 달성하기 위하여, 본 발명은 전자기파를 이용한 암 온열치료용 감작제 조성물을 제공한다.In order to achieve the above object, the present invention provides a sensitizer composition for cancer thermal therapy using electromagnetic waves.
본 발명에 있어서, 상기 감작제는 금속이온, 금속이온 결합물, 금속이온이 비공유 결합된 아포트랜스페린 및 금속이온이 비공유 결합된 아포트랜스페린 유도체로 구성된 군으로부터 선택되는 것을 특징으로 한다.In the present invention, the sensitizer is characterized in that it is selected from the group consisting of a metal ion, a metal ion bond, a non-covalently bonded aprotransferrin and a metal ion-non-covalently bonded aprotransferrin derivative.
본 발명에 있어서, 상기 금속이온은 철(iron) 이온, 망간(manganese) 이온, 아연(zinc) 이온, 구리(copper) 이온, 마그네슘(magnesium) 이온, 비스무트(bismuth) 이온, 루테늄(ruthenium) 이온, 티타늄(titanium) 이온, 갈륨(gallium) 이온, 인듐(indium) 이온, 바나딜(vanadyl) 이온, 크로미움(chromium) 이온, 알루미늄(aluminum) 이온 및 플루토늄(plutonium) 이온으로 구성된 군으로부터 선택되는 것을 특징으로 한다.In the present invention, the metal ion is iron (iron), manganese (manganese), zinc (zinc), copper (copper), magnesium (magnesium), bismuth (bismuth), ruthenium (ruthenium) Selected from the group consisting of titanium ions, gallium ions, indium ions, vanadyl ions, chromium ions, aluminum ions, and plutonium ions It is characterized by.
본 발명에 있어서, 상기 금속이온 결합물은 금속이온에 덱스트란(dextran), 수크로즈(sucrose), 글루코네이트(gluconate), 소르비톨(sorbitol), 폴리사카라이드(polysaccharide), 시트레이트(citrate), 카복시말토스(carboxymaltose), 페루목시톨(ferumoxytol), 이소말토사이드(isomaltoside), 말토스(maltose), 전분(starch), 셀룰로오스(cellulose), 클로라이드(chloride), 설페이트(sulfate), 푸마레이트(fumurate) 및 알부민(albumin)을 포함하는 군으로부터 선택되는 결합물이 비공유 결합되어 있는 것을 특징으로 한다.In the present invention, the metal ion combination is dextran, sucrose, gluconate, sorbitol, polysaccharide, citrate, citrate to metal ions. Carboxymaltose, perumoxytol, isomaltoside, maltose, starch, cellulose, chloride, sulfate, fumarate (fumurate) and albumin (albumin) is characterized in that the binder selected from the group comprising a non-covalent bond.
본 발명에 있어서, 상기 아포트랜스페린 또는 아포트랜스페린 유도체는 인간 또는 포유동물 유래 혈청 단백질이거나 재조합 단백질인 것을 특징으로 한다.In the present invention, the atransferrin or the atransferrin derivative is characterized in that the human or mammalian-derived serum protein or recombinant protein.
본 발명에 있어서, 상기 감작제는 0.01~100mg/ml 농도인 것을 특징으로 한다.In the present invention, the sensitizer is characterized in that the concentration of 0.01 ~ 100mg / ml.
본 발명에 있어서, 상기 암 온열치료용 감작제 조성물은 약학적으로 허용 가능한 담체를 추가로 포함하는 것을 특징으로 한다.In the present invention, the sensitizer composition for cancer thermal therapy is characterized in that it further comprises a pharmaceutically acceptable carrier.
본 발명은 또한, 전자기파를 이용한 암 온열치료용 감작제 조성물 및 전자기파를 조사하는 장치를 포함하는 암 온열치료용 키트를 제공한다.The present invention also provides a kit for cancer thermal therapy, comprising a sensitizer composition for cancer thermal therapy using electromagnetic waves and a device for irradiating electromagnetic waves.
본 발명은 또한, (a) 인간을 제외한 동물에 상기 암 온열치료용 감작제 조성물을 투여하여 암 치료에 대한 감수성을 증가시키는 단계 및 (b) 전자기파를 처리하는 단계를 포함하는 암 치료방법을 제공한다.The present invention also provides a method for treating cancer, comprising the steps of: (a) administering the sensitizer composition for cancer thermal therapy to animals other than humans, thereby increasing susceptibility to cancer treatment; and (b) treating electromagnetic waves. do.
본 발명에 있어서, 상기 암 온열치료용 감작제 조성물은 1~250mg/kg 용량으로 투여하는 것을 특징으로 한다.In the present invention, the sensitizer composition for cancer thermal therapy is characterized in that the administration of 1 ~ 250mg / kg dose.
본 발명에 있어서, 상기 전자기파는 감마선, X-선, 자외선, 가시광선, 적외선, 마이크로웨이브 및 라디오 전파로 구성된 군으로부터 선택되는 것을 특징으로 한다.In the present invention, the electromagnetic wave is selected from the group consisting of gamma rays, X-rays, ultraviolet rays, visible rays, infrared rays, microwaves and radio waves.
본 발명에 있어서, 상기 암 치료방법은 화학 치료(chemotherapy), 방사선 치료(radiation therapy), 생물학적 치료(biological therapy), 면역치료(immunotherapy) 및 광역동 치료(photodynamic therapy)로 구성된 군으로부터 선택되는 하나 이상의 치료방법을 병행하는 것을 특징으로 한다.In the present invention, the cancer treatment method is one selected from the group consisting of chemotherapy, radiation therapy, biological therapy, immunotherapy and photodynamic therapy It is characterized by a combination of the above treatment methods.
본 발명에 따른 암 온열치료용 감작제 조성물은 암 표적지향성을 가지고 있어 금속이온을 암조직에 선택적으로 전달시키므로, 전자기파를 이용한 암 온열치료시 금속이온이 축적된 암조직에서 열 발생이 증가되어 전자기파를 이용한 암 온열치료의 암 치료 효능을 극대화시킬 수 있다.The sensitizer composition for cancer thermal therapy according to the present invention has a cancer target orientation, thereby selectively transferring metal ions to cancer tissues, and thus, heat generation is increased in cancer tissues in which metal ions are accumulated during cancer thermal therapy using electromagnetic waves. Can maximize cancer treatment efficacy of cancer thermal therapy using.
도 1은 본 발명의 실험예 3에 따라 전자기파 처리 전과 후의 아포트랜스페린 수용액 온도를 열화상카메라로 측정한 사진이다. 과 온도 변화 차이를 나타낸 그래프이다. 1 is a photograph measured by the thermal imaging camera the temperature of the aqueous solution of aforetransferrin before and after electromagnetic wave treatment according to Experimental Example 3 of the present invention. The graph shows the difference between and temperature change.
도 2는 본 발명의 실험예 3에 따라 전자기파 처리 전과 후의 아포트랜스페린 수용액 온도 변화 차이를 나타낸 그래프이다. Figure 2 is a graph showing the difference in the temperature change of the aqueous solution of aquatransferrin before and after electromagnetic wave treatment according to Experimental Example 3 of the present invention.
도 3은 본 발명의 실험예 3에 따라 전자기파 처리 전과 후의 트랜스페린 수용액 온도를 열화상카메라로 측정한 사진이다. FIG. 3 is a photograph of a transferrin aqueous solution temperature before and after electromagnetic wave treatment according to Experimental Example 3 of the present invention measured by a thermal imaging camera.
도 4는 본 발명의 실험예 3에 따라 전자기파 처리 전과 후의 트랜스페린 수용액 온도 변화 차이를 나타낸 그래프이다. 4 is a graph showing the difference in the temperature change of the transferrin aqueous solution before and after the electromagnetic wave treatment according to Experimental Example 3 of the present invention.
도 5는 본 발명의 실험예 3에 따라 전자기파 처리 전과 후의 트랜스페린 배양 정상세포 온도를 열화상카메라로 측정한 사진이다.Figure 5 is a photograph of the measurement of the transfer cell culture normal cell temperature before and after the electromagnetic wave treatment in accordance with Experimental Example 3 of the present invention.
도 6은 본 발명의 실험예 3에 따라 전자기파 처리 전과 후의 트랜스페린 배양 정상세포 온도 변화 차이를 나타낸 그래프이다.6 is a graph showing the difference in transfer cell culture normal cell temperature before and after electromagnetic wave treatment according to Experimental Example 3 of the present invention.
도 7은 본 발명의 실험예 3에 따라 전자기파 처리 전과 후의 트랜스페린 배양 암세포 온도를 열화상카메라로 측정한 사진이다.FIG. 7 is a photograph of a transferrin cultured cancer cell temperature before and after electromagnetic wave treatment according to Experimental Example 3 of the present invention measured by a thermal imaging camera.
도 8은 본 발명의 실험예 3에 따라 전자기파 처리 전과 후의 트랜스페린 배양 암세포 온도 변화 차이를 나타낸 그래프이다.Figure 8 is a graph showing the difference in transferin culture cancer cell temperature change before and after electromagnetic wave treatment according to Experimental Example 3 of the present invention.
도 9는 본 발명의 실시예 1에 따라 생리식염수(control)를 암이 유도된 마우스(Tumor xenograft mouse)에 각각 투여하고 전자기파를 처리하는 온열치료시, 정상 조직과 암 조직 부위(화살표)를 열화상카메라로 측정한 사진과 온도 변화 차이를 나타낸 그래프이다 (White bar: 정상 조직, Black bar: 암 조직). FIG. 9 shows normal tissues and cancer tissue regions (arrows) during thermal treatment in which physiological saline (control) is respectively administered to a tumor-induced mouse (Tumor xenograft mouse) and electromagnetic waves are treated according to Example 1 of the present invention. It is a graph showing the difference between the temperature measured by the image camera and the temperature change (White bar: normal tissue, Black bar: cancer tissue).
도 10은 본 발명의 실시예 1에 따라 온열치료용 감작제(iron sucrose)를 암이 유도된 마우스(Tumor xenograft mouse)에 각각 투여하고 전자기파를 처리하는 온열치료시, 정상 조직과 암 조직 부위(화살표)를 열화상카메라로 측정한 사진과 온도 변화 차이를 나타낸 그래프이다 (White bar: 정상 조직, Black bar: 암 조직). FIG. 10 shows normal tissues and cancer tissue regions during thermal treatments in which thermal susceptibility agents (iron sucrose) are respectively administered to a tumor-induced mouse (Tumor xenograft mouse) and electromagnetic waves are treated according to Example 1 of the present invention. Arrow) is a graph showing the difference between the temperature measured with a thermal imaging camera (white bar: normal tissue, black bar: cancer tissue).
도 11은 본 발명의 실시예 1에 따라 온열치료용 감작제(transferrin)를 암이 유도된 마우스(Tumor xenograft mouse)에 각각 투여하고 전자기파를 처리하는 온열치료시, 정상 조직과 암 조직 부위(화살표)를 열화상카메라로 측정한 사진과 온도 변화 차이를 나타낸 그래프이다 (White bar: 정상 조직, Black bar: 암 조직). FIG. 11 shows normal tissues and cancer tissue regions (arrows) during thermal treatments in which a therapeutic agent for thermotherapy according to Example 1 of the present invention is administered to a cancer-induced mouse (Tumor xenograft mouse) and treated with electromagnetic waves. ) Is a graph showing the difference between the temperature measured with a thermal imaging camera (white bar: normal tissue, black bar: cancer tissue).
도 12는 본 발명의 실시예 1에 따라 암이 유도된 마우스(Tumor xenograft mouse)에 식염수 또는 금속이온 결합물을 투여한 후 전자기파를 처리한 마우스의 암조직 크기를 바이오형광으로 분석한 결과이다((A):무처리, (B):생리식염수, (C):iron gluconate, (D):iron sucrose, (E):iron carboxymaltose, (F):iron dextran, (G):iron starch, (H):transferrin). 12 is a result of analyzing the size of the cancer tissue of the mouse treated with electromagnetic waves after administration of saline or metal ion conjugate to the tumor-induced mouse (Tumor xenograft mouse) according to Example 1 of the present invention (biofluorescence) (A): untreated, (B): physiological saline, (C): iron gluconate, (D): iron sucrose, (E): iron carboxymaltose, (F): iron dextran, (G): iron starch, ( H): transferrin).
도 13은 본 발명의 실시예 1에 따라 암이 유도된 마우스(Tumor xenograft mouse)에 식염수, iron sucrose, iron dextran 및 transferrin을 투여한 후 전자기파를 처리한 마우스의 암조직 크기를 바이오형광으로 분석한 결과이다. 13 is a biofluorescence analysis of cancer tissue size of mice treated with electromagnetic waves after administration of saline, iron sucrose, iron dextran and transferrin to a tumor-induced mouse (Tumor xenograft mouse) according to Example 1 of the present invention The result is.
본 발명에서는 독성 및 부작용이 없으면서, 암 조직에 대한 표적지향성이 있는 물질을 이용하여 금속 이온을 암 세포에만 전달할 수 있는 감작제로 이용할 경우, "전자기파를 이용한 암 온열치료"의 암조직 선택성과 암치료 효능을 높일 수 있을 것으로 예측하였다. In the present invention, the cancer tissue selectivity and cancer treatment of "cancer thermotherapy using electromagnetic waves" when using as a sensitizer capable of delivering metal ions only to cancer cells by using a target-oriented material for cancer tissue without toxicity and side effects It was expected to increase the efficacy.
즉, 암 조직에 대한 표적지향성이 있고, 독성 문제가 없는 생체유래 물질을 이용하여 금속 이온을 정상 세포가 아닌 암세포에만 전달시켜 암세포의 금속이온 농도를 증가시킨 후, "전자기파를 이용한 암 온열치료"를 시행할 경우, 암 치료 효능을 극대화시킬 수 있다는 것을 확인하고자 하였다. In other words, by using a bio-derived material that is targeted to cancer tissue and does not have toxicity problems, the metal ions are transferred to cancer cells instead of the normal cells to increase the metal ion concentration of the cancer cells, followed by "cancer heat treatment using electromagnetic waves." When the trial was performed, it was confirmed that the cancer treatment effect can be maximized.
이에 본 발명에서는 "금속이온과 비공유 결합된 아포트랜스페린"(트랜스페린)을 "전자기파를 이용한 암 온열치료"용 감작제로 암 이식 동물모델(tumor xenograft mouse model)에 혈관주사로 투여한 후, 전자기파를 처리하였다. 그 결과, (1) 암세포에 과발현된 트랜스페린 수용체에 의해 트랜스페린이 정상세포보다 암세포에 선택적으로 전달되고; (2) 트랜스페린에 결합되어 있던 금속이온의 분리로 정상세포보다 암세포 내 금속이온 농도가 높아지고; (3) 전자기파 처리 시 암조직의 증가된 금속이온이 열 발생을 더욱 증가시키고; (4) 발생된 열에 의해 암세포의 사멸이 증가되어 결과적으로 전자기파를 이용한 암 온열치료의 암 치료 효능이 극대화 된 것을 확인할 수 있었다. Therefore, in the present invention, after administering "Aforttransferrin (transferrin) non-covalently coupled with metal ion" (transferrin) to the tumor xenograft mouse model as a sensitizer for "cancer thermotherapy using electromagnetic waves," electromagnetic waves are treated. It was. As a result, (1) transferrin is selectively delivered to cancer cells rather than normal cells by transferrin receptors overexpressed in cancer cells; (2) separation of metal ions bound to transferrin results in higher metal ion concentrations in cancer cells than normal cells; (3) increased metal ions in the cancer tissue during electromagnetic wave treatment further increase heat generation; (4) The death of cancer cells was increased by the generated heat, and as a result, it was confirmed that the cancer therapeutic efficacy of cancer thermal therapy using electromagnetic waves was maximized.
또한 본 발명에서는 "금속이온과 비공유 결합된 아포트랜스페린"(트랜스페린) 대신 금속이온을 감작제로 암 이식 동물모델(tumor xenograft mouse model)에 투여하여도 혈액으로 유입된 금속이온이 혈액에 과량 존재하는 아포트랜스페린과 결합하여 트랜스페린화되므로, 금속이온을 온열치료용 감작제로 암 환자에게 투여하고 전자기파를 이용한 암 온열치료를 시행하여도, 상기에서 기술한 효과와 동일하거나 더 좋은 항암 효과를 얻을 수 있다는 것을 확인하였다.In addition, in the present invention, even when a metal ion is used as a sensitizer to a tumor xenograft mouse model instead of "aport transferrin covalently bonded to a metal ion" (transferrin), the metal ion introduced into the blood is excessively present in the blood. Since it is transferred to transferrin in combination with transferrin, it is confirmed that even if the metal ion is administered to cancer patients as a sensitizer for thermal therapy and cancer thermal therapy using electromagnetic waves, the same or better anticancer effect as described above can be obtained. It was.
따라서, 본 발명은 일 관점에서, 전자기파를 이용한 암 온열치료용 감작제 조성물에 관한 것이다.Therefore, in one aspect, the present invention relates to a sensitizer composition for cancer thermal therapy using electromagnetic waves.
상기 감작제란 온열치료시 암치료 효능을 높여주기 위해 사용되는 것으로, 체내에 투여시 암조직에 대한 표적지향성을 가지면서 암조직에서 열 발생을 증폭시켜 주는 특징을 가진다. The sensitizer is used to enhance cancer treatment efficacy during heat treatment, and has a characteristic of amplifying heat generation in cancer tissues while having a target orientation for cancer tissues when administered to the body.
상기 감작제는 금속이온, 금속이온 결합물, 금속이온이 비공유 결합된 아포트랜스페린 및 금속이온이 비공유 결합된 아포트랜스페린 유도체로 구성된 군으로부터 선택될 수 있다.The sensitizer may be selected from the group consisting of a metal ion, a metal ion bond, an apotransferrin non-covalently bonded to a metal ion, and an apotransferrin derivative non-covalently bound to a metal ion.
본 발명에 있어서, 상기 금속이온은 철(iron) 이온, 망간(manganese) 이온, 아연(zinc) 이온, 구리(copper) 이온, 마그네슘(magnesium) 이온, 비스무트(bismuth) 이온, 루테늄(ruthenium) 이온, 티타늄(titanium) 이온, 갈륨(gallium) 이온, 인듐(indium) 이온, 바나딜(vanadyl) 이온, 크로미움(chromium) 이온, 알루미늄(aluminum) 이온, 플루토늄(plutonium) 이온 등을 예시할 수 있으나, 이에 한정되지는 않는다.In the present invention, the metal ion is iron (iron), manganese (manganese), zinc (zinc), copper (copper), magnesium (magnesium), bismuth (bismuth), ruthenium (ruthenium) , Titanium ions, gallium ions, indium ions, vanadyl ions, chromium ions, aluminum ions, plutonium ions, etc. It is not limited to this.
상기 금속이온 결합물은 금속이온에 덱스트란(dextran), 수크로즈(sucrose), 글루코네이트(gluconate), 소르비톨(sorbitol), 폴리사카라이드(polysaccharide), 시트레이트(citrate), 카복시말토스(carboxymaltose), 페루목시톨(ferumoxytol), 이소말토사이드(isomaltoside), 말토스(maltose), 전분(starch), 셀룰로오스(cellulose), 클로라이드(chloride), 설페이트(sulfate), 푸마레이트(fumurate) 및 알부민(albumin)포함하는 군으로부터 선택되는 결합물이 비공유 결합된 것으로써, 약제로 사용할 수 있는 결합물이라면 제한없이 이용될 수 있으며, iron dextran, iron sucrose, iron gluconate, iron carboxymaltose, iron isomaltoside, iron ferumoxytol, iron sorbitol, iron polysaccharide, ferric citrate, ferrous gluconate, ferrous sulfate, ferrous fumurate, magnesium chloride, gallium citrate, aluminium citrate 등을 예시할 수 있으나 이에 한정되지 않는다. The metal ion combination is dextran, sucrose, gluconate, sorbitol, polysaccharide, polysaccharide, citrate, carboxymaltose in metal ions. ), Perumoxytol, isomaltoside, maltose, starch, cellulose, chloride, sulfate, fumurate and albumin The combination selected from the group containing (albumin) is non-covalently bound, and any combination that can be used as a medicament can be used without limitation, and may include iron dextran, iron sucrose, iron gluconate, iron carboxymaltose, iron isomaltoside, iron ferumoxytol , iron sorbitol, iron polysaccharide, ferric citrate, ferrous gluconate, ferrous sulfate, ferrous fumurate, magnesium chloride, gallium citrate, aluminum citrate, and the like.
상기 금속이온은 전하를 가지므로 극성, 즉 쌍극자모멘트를 가지고 있어 전자기파 처리시 분자운동이 증폭되어 열이 발생하게 되므로, 금속 자체보다 전자기파에 민감하게 반응하는 감작제 특성을 가진다.Since the metal ions have a charge, they have polarity, that is, a dipole moment, and thus generate heat by amplifying molecular motion during electromagnetic wave treatment, and thus have a sensitizer characteristic that reacts more sensitively to electromagnetic waves than the metal itself.
상기 감작제로 금속이온 또는 금속이온 결합물을 암 환자에 투여시, 1) 혈액에 주입된 금속이온은 혈액에 과량 존재하는 아포트랜스페린과 결합하여 트랜스페린화되고; 2) 트랜스페린은 암세포에 과발현된 트랜스페린 수용체에 의해 암세포에 선택적으로 전달되므로 트랜스페린에 의해 전달되는 금속이온 농도가 암세포에서 높아질 것이고; 3) 여기에 전자기파를 이용한 온열치료를 시행하면 금속이온이 축적된 암세포에서 열 발생이 집중적으로 일어나 정상세포에 대한 손상을 최소화하면서 암세포만 집중적으로 사멸시키는 특징을 가진다.When a metal ion or a metal ion combination is administered to a cancer patient with the sensitizer, 1) the metal ion injected into the blood is transferred and combined with apoptransferrin present in the blood; 2) transferrin is selectively delivered to cancer cells by transferrin receptors that are overexpressed in cancer cells so that the metal ion concentration delivered by transferrin will be elevated in cancer cells; 3) When heat treatment using electromagnetic waves is performed here, heat generation occurs intensively in cancer cells accumulated with metal ions, and thus only cancer cells are intensively killed while minimizing damage to normal cells.
상기 감작제로 금속이온이 비공유 결합된 아포트랜스페린 및 금속이온이 비공유 결합된 아포트랜스페린 유도체를 암 환자에 투여시, 1) "금속이온이 비공유 결합된 아포트랜스페린"(트랜스페린)은 혈액을 따라 돌다가 암세포에 과발현된 트랜스페린 수용체에 의해 암세포에 선택적으로 전달되므로 트랜스페린에 의해 전달되는 금속이온 농도가 암세포에서 높아질 것이고; 2) 여기에 전자기파를 이용한 온열치료를 시행하면 금속이온이 축적된 암세포에서 열 발생이 집중적으로 일어나 정상세포에 대한 손상을 최소화하면서 암세포만 집중적으로 사멸시키는 특징을 가진다.When the sensitizer is administered non-covalently bonded apottransferrin and metal ions non-covalently bonded aforetransferin derivative to cancer patients, 1) "non-covalently bonded aforttransferrin" (transferrin) is a blood cell to cancer cells Selective delivery of cancer cells to the cancer cells by the overexpressed transferrin receptors will increase the concentration of metal ions delivered by the transferrin in the cancer cells; 2) When heat treatment using electromagnetic waves is carried out, heat generation occurs intensively in cancer cells accumulated with metal ions, which minimizes damage to normal cells while intensively killing only cancer cells.
상기 트랜스페린은 혈액에 많이 분포하는 단백질로, 철과 같은 금속이온과 결합하고 혈액을 돌다가 트랜스페린 수용체를 가지고 있는 세포들에게 금속이온을 전달하는 역할을 하는 메탈로프로테인(metalloprotein)을 의미한다.The transferrin is a protein widely distributed in blood, and refers to a metalloprotein that binds to metal ions such as iron and spins blood to transfer metal ions to cells having transferrin receptors.
상기 아포트랜스페린 또는 아포트랜스페린 유도체는 암 표적지향성을 갖고, 철, 망간, 아연 등의 금속이온과 결합이 가능한 것이라면 특별한 제한없이 인간 또는 포유동물 유래 혈청 단백질이거나 재조합 단백질인 것을 사용할 수 있다.The aforetransferin or aforetransferin derivative may be human or mammalian-derived serum protein or recombinant protein without particular limitation, as long as it has cancer targeting ability and is capable of binding with metal ions such as iron, manganese, and zinc.
상기 트랜스페린은 아포트랜스페린에 금속이온이 비공유 결합된 형태의 트랜스페린이 바람직하며, 철 이온과 결합된 형태의 트랜스페린(iron-bound transferrin)으로는 모노페릭 트랜스페린(monoferric transferrin), 디페릭 트랜스페린(diferric transferrin), 홀로트랜스페린(holo-transferrin), 페릭 아세틸 트랜스페린(ferric acetyl transferrin) 등을 예시할 수 있다.The transferrin is preferably a transferrin in a form in which a metal ion is non-covalently bonded to an aprotransferrin, and as a iron-bound transferrin, iron-bound transferrin, monoferric transferrin, and diferric transferrin. , Holo-transferrin, ferric acetyl transferrin, and the like.
상기 트랜스페린은 특히 암조직에 과발현되어 있는 트랜스페린 수용체와 결합하여 암세포 안으로 전달되고, 결합하고 있던 금속이온을 암세포 내로 유리시켜서, 결국 암조직에 선택적으로 금속이온을 전달할 수 있는 것을 특징으로 한다. In particular, the transferrin binds to the transferrin receptor, which is overexpressed in cancer tissue, is delivered into cancer cells, and releases the bound metal ions into the cancer cells, thereby finally delivering metal ions selectively to the cancer tissue.
철, 망간, 아연 등과 같은 금속이온은 강한 전하를 띈 이온이므로 물 분자의 쌍극자모멘트 보다 훨씬 강력한 극성을 갖는데, 강한 극성을 가진 금속이온이 전자기파에 노출되면 분자운동이 증폭되어 열 발생이 극대화된다. Since metal ions such as iron, manganese, and zinc are highly charged ions, they have a much stronger polarity than the dipole moment of water molecules. When metal ions with strong polarities are exposed to electromagnetic waves, molecular motion is amplified to maximize heat generation.
상기 트랜스페린은 암 환자에 투여시, 1) 암세포에 과발현된 트랜스페린 수용체에 의해 트랜스페린이 정상세포보다 암세포에 선택적으로 전달되고; 2) 트랜스페린에 결합되어 있던 금속이온의 분리로 정상세포보다 암세포 내 금속이온 농도가 높아지고; 3) 전자기파 처리 시 암조직의 증가된 금속이온이 열 발생을 더욱 증가시켜; 4) 발생한 열에 의해 암세포의 사멸이 증가하게 되어 암치료 효능을 가지는 특징을 가진다.When the transferrin is administered to a cancer patient, 1) transferrin is selectively delivered to cancer cells rather than normal cells by a transferrin receptor overexpressed in cancer cells; 2) the concentration of metal ions in cancer cells is higher than that of normal cells due to the separation of metal ions bound to transferrin; 3) increased metal ions of the cancer tissue during electromagnetic wave treatment further increases heat generation; 4) The death of cancer cells is increased by the generated heat, which has the characteristics of cancer treatment efficacy.
상기 암 온열치료용 감작제 조성물은 특별히 제한되지는 않으나, 감작제의 농도가 0.01~100mg/ml인 것이 바람직하다. 상기 농도가 0.01mg/ml 미만인 경우에는 과량의 부피로 투여해야 하는 불편함이 있고, 100mg/ml을 초과할 경우 제조가 어려운 문제점이 있다.The sensitizer composition for cancer thermal therapy is not particularly limited, but the concentration of the sensitizer is preferably 0.01-100 mg / ml. If the concentration is less than 0.01mg / ml there is an inconvenience to be administered in an excess volume, there is a problem that difficult to manufacture if it exceeds 100mg / ml.
본 발명에 있어서, 상기 암 온열치료용 감작제 조성물은 약학적으로 허용 가능한 담체나 윤활제, 습윤제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.In the present invention, the sensitizer composition for cancer thermal therapy may further include a pharmaceutically acceptable carrier or lubricant, wetting agent, emulsifier, suspending agent, preservative and the like.
본 발명은 다른 관점에서 상기 암 온열치료용 감작제 조성물 및 전자기파를 조사하는 장치를 포함하는 암 온열치료용 키트에 관한 것이다.The present invention relates to a cancer thermal therapy kit including the device for irradiating electromagnetic waves and the sensitizer composition for cancer thermal therapy in another aspect.
본 발명의 암 온열치료용 감작제 조성물은 암과 관련된 다양한 질병, 예를 들어 위암, 폐암, 유방암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 방광암, 결장암, 자궁경부암 등을 치료하는데 이용될 수 있으므로, 전자기파를 조사하는 장치를 포함하는 암 온열치료용 키트에 포함될 수 있다.The sensitizer composition for cancer thermal therapy of the present invention treats various diseases related to cancer, such as gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, cervical cancer, etc. It can be used to, it can be included in the kit for cancer thermal therapy, including a device for irradiating electromagnetic waves.
상기 전자기파(electromagnetic waves)는 전기장과 자기장이 시간에 따라 변하면서 발생하는 파동으로서, 감마선, X-선, 자외선, 가시광선, 적외선, 마이크로웨이브, 라디오 전파 등을 예시할 수 있으며, 본 발명에서는 통상적인 전자기파 조사 장치를 이용할 수 있다.The electromagnetic waves are waves generated as the electric and magnetic fields change with time, and may include gamma rays, X-rays, ultraviolet rays, visible rays, infrared rays, microwaves, radio waves, and the like. Phosphorus electromagnetic wave irradiation apparatus can be used.
본 발명은 또 다른 관점에서 (a) 인간을 제외한 동물에 상기 암 온열치료용 감작제 조성물을 투여하여 암 치료에 대한 감수성을 증가시키는 단계 및 (b) 전자기파를 처리하는 단계를 포함하는 암 치료방법에 관한 것이다.In another aspect, the present invention provides a method for treating cancer, comprising the steps of: (a) administering the sensitizer composition for cancer thermal therapy to animals other than humans to increase susceptibility to cancer treatment; and (b) treating electromagnetic waves. It is about.
상기 암 온열치료용 감작제 조성물은 상기 금속이온, 금속이온 결합물, 금속이온이 비공유 결합된 아포트랜스페린, 또는 금속이온이 비공유 결합된 아포트랜스페린 유도체를 주사에 적합한 물, 생리식염수 등의 용액에 0.01~100mg/ml 농도로 녹여 사용하는 것이 바람직하다.The sensitizer composition for cancer thermal therapy is a metal ion, a metal ion conjugate, a covalently bonded aforttransferrin, or a metal covalently bonded aforttransferrin derivative in a solution such as water, physiological saline suitable for injection 0.01 It is preferable to dissolve it at a concentration of ˜100 mg / ml.
전자기파를 이용한 암 온열치료의 효과를 위해서는 암 온열치료용 감작제 조성물의 투여 용량은 금속이온 및 금속이온 결합물의 경우에는 0.1~50mg/kg 범위가 바람직하며, 금속이온이 비공유 결합된 아포트랜스페린 및 그 유도체의 경우에는 0.1~200mg/kg 용량 범위가 바람직하다.For the effect of cancer thermal therapy using electromagnetic waves, the dosage of the sensitizer composition for cancer thermal therapy is preferably in the range of 0.1 to 50 mg / kg in the case of metal ions and metal ion conjugates, and the atransferrin and non-covalently bonded metal ions In the case of derivatives, the dosage range of 0.1 to 200 mg / kg is preferred.
상기 전자기파를 이용한 암 온열치료의 바람직한 효과를 위해서는 암 온열치료용 감작제 조성물을 투여한 후 1~48시간 이내에 전자기파를 이용한 암 온열치료를 시행하는 것이 바람직하다. For the desirable effect of cancer thermal therapy using the electromagnetic wave, it is preferable to perform cancer thermal therapy using electromagnetic waves within 1 to 48 hours after administering the sensitizer composition for cancer thermal therapy.
상기 전자기파를 이용한 암 온열치료 요법은 공지의 온열치료 요법에 의해 용이하게 결정되어 사용될 수 있다. 예를 들어, 13.56 MHz 고주파를 출력하는 온열치료기로 30~60분간 조사하는 치료를 1주일에 2회 이상 4주 이상 시행할 수 있다. Cancer thermotherapy therapy using the electromagnetic wave can be easily determined and used by known thermotherapy therapy. For example, a treatment that is irradiated for 30 to 60 minutes with a thermal therapy device that outputs 13.56 MHz high frequency can be performed twice or more for four weeks or more per week.
본 발명에 따른 암 치료방법은 기존의 항암요법과 연계 또는 병행하여 사용함으로써 암의 치료 효과를 개선할 수 있다. 기존의 항암요법으로는 화학 치료(chemotherapy), 방사선 치료(radiation therapy), 생물학적 치료(biological therapy), 면역치료(immunotherapy) 및 광역동 치료(photodynamic therapy) 등을 예시할 수 있다.Cancer treatment method according to the present invention can improve the therapeutic effect of cancer by using in conjunction with or in combination with existing chemotherapy. Existing chemotherapy may be exemplified by chemotherapy, radiation therapy, biological therapy, immunotherapy and photodynamic therapy.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실험예 1. 금속이온의 발열능 평가Experimental Example 1. Evaluation of the exothermic ability of the metal ion
인체로 섭취되는 경구용 또는 주사제용 금속이온은 염과 결합된 결합물 형태이거나 탄수화물(carbohydrate) 또는 단백질과 같은 고분자와 결합된 결합물 형태이다. 금속이온과 결합되는 염으로는 시트레이트(citrate), 클로라이드(chloride), 설페이트(sulfate), 푸마레이트(fumurate) 등을 예시할 수 있으며 이로 인한 결합물로는 황산제일철(ferrous sulfate), 푸마르산제일철(ferrous fumurate), 글루코네이트제일철(ferrous gluconate) 등을 예시할 수 있다. Oral or injectable metal ions for human consumption are in the form of conjugates combined with salts or conjugated polymers such as carbohydrates or proteins. Examples of salts bound with metal ions include citrate, chloride, sulfate, fumurate, and the like. Ferrous sulfate, ferrous fumarate, and the like are combined. (ferrous fumurate), ferrous gluconate and the like can be exemplified.
금속이온과 결합되는 탄수화물로는 단당류인 글루코네이트(gluconate), 이당류인 수크로즈(sucrose), 말토스(maltose), 다당류인 이소말토사이드(isomaltoside), 카르복시말토스(carboxymaltose), 덱스트란(dextran), 스타치(starch), 셀룰로오스(cellulose) 등의 사카라이드(saccharides)가 있고, 금속이온과 결합되는 단백질로는 트랜스페린(transferrin), 알부민(albumin) 등을 예시할 수 있다.Carbohydrates bound to metal ions include gluconate (monosaccharide), sucrose (disaccharide), maltose (maltose), isomaltoside (polymal), carboxymaltose, and dextran (dextran) ), Starch, cellulose (saccharides) such as cellulose (saccharides) and the like, as the protein bound to the metal ions (transferrin, albumin, etc.) can be exemplified.
본 실험예에서는 금속이온과 염 결합물인 ferrous sulfate 용액을 다음과 같이 준비하여 전자기파를 처리한 후 온도를 측정함으로써 금속이온 결합물의 발열능을 확인하였다. 또한 금속이온과 탄수화물 결합물인 iron gluconate, magnesium sucrose, iron sucrose, iron isomaltoside, iron carboxymaltose, iron dextran, iron starch complex 용액을 다음과 같이 준비하여 전자기파를 처리한 후 온도를 측정함으로써 금속이온 결합물의 발열능을 확인하였다. In the present experimental example, the ferrous sulfate solution, which is a metal ion and salt combination, was prepared as follows, and the exothermic ability of the metal ion binder was confirmed by measuring the temperature after treating the electromagnetic wave. In addition, iron gluconate, magnesium sucrose, iron sucrose, iron isomaltoside, iron carboxymaltose, iron dextran, and iron starch complex solutions, which are metal ions and carbohydrate combinations, are prepared as follows to treat the electromagnetic wave and then measure the temperature to generate the exothermic ability of the metal ion binders. It was confirmed.
ferrous sulfate 용액은 FeSO47H2O 1g을 증류수 10ml에 넣고 30분 이상 저어서 녹인 후 0.22㎛ 필터를 통과시켜 제조한 다음, 멸균 증류수로 희석하여 사용하였다.Ferrous sulfate solution was prepared by dissolving 1 g of FeSO 4 7H 2 O in 10 ml of distilled water, stirring for 30 minutes or more, passing through a 0.22 μm filter, and diluting with sterile distilled water.
iron gluconate 용액은 (sodium ferric gluconate complex)은 Sanofi사의 Ferrlecit 제품을 멸균 증류수로 희석하여 사용하였다.Iron gluconate solution (sodium ferric gluconate complex) was used by diluting Sanofi Ferrlecit product with sterile distilled water.
magnesium sucrose 용액은 MgCl2H2O 83.6mg와 sucrose 150mg을 증류수 10ml에 넣고 30분 이상 저어서 녹인 후 0.22㎛ 필터를 통과시켜 제조한 다음, 증류수를 이용하여 희석하여 사용하였다. Magnesium sucrose solution was prepared by passing 83.6mg of MgCl 2 H 2 O and 150mg of sucrose in 10ml of distilled water, stirring for 30 minutes or more, passing through a 0.22㎛ filter, and diluting with distilled water.
iron sucrose 용액 (ferric hydroxide sucrose complex)은 먼저 설탕 100mg을 증류수 50ml에 넣고 90℃에서 녹인 후 계속 저으면서 5M NaOH 1ml을 첨가하여 sucrose 수용액을 제조한 다음, FeCl3 0.9g을 증류수 50ml에 넣고 20분 이상 저어서 녹인 0.01M FeCl3 수용액을 90℃의 sucrose 수용액에 첨가한 후, 5M NaOH 용액을 방울씩 떨어뜨리며 pH 12로 조정하였다. 다음으로, 80℃에서 2시간 동안 반응시킨 후 5,000 rpm으로 5분간 원심분리하여 ferric hydroxide sucrose complex를 얻어내고 증류수로 세척하고 건조시킨 후 사용하였다. iron sucrose solution (ferric hydroxide sucrose complex) is first prepared the sucrose aqueous solution into the sugar 100mg in 50ml distilled water and continue stirring the addition of 5M NaOH was dissolved in 1ml 90 ℃ then put FeCl 3 0.9g in 50ml distilled water for 20 min or more Stir-dissolved 0.01M FeCl 3 aqueous solution was added to an aqueous solution of sucrose at 90 ° C., and then adjusted to pH 12 by dropping 5M NaOH solution. Next, after reacting for 2 hours at 80 ℃ centrifuged for 5 minutes at 5,000 rpm to obtain a ferric hydroxide sucrose complex, washed with distilled water and dried.
iron isomaltoside complex, iron carboxymaltose complex와 iron starch complex 역시 iron sucrose를 제조한 방법에서 탄수화물만 바꾸어서 제조하여 준비하였다. Iron isomaltoside complexes, iron carboxymaltose complexes, and iron starch complexes were also prepared by changing only carbohydrates from the method of preparing iron sucrose.
상기의 금속이온 결합물을 금속이온 10 mg/ml 농도로 준비한 후 0.1ml을 96-well plate에 3 well씩 분주하였다. 이때, 대조군으로는 증류수 0.1ml을 사용하였다. 96-well plate를 고주파온열암치료기(EHY-2000, Oncothermia)에서 100W의 energy dose로 5분간 노출시킨 후 5분 후 온도를 열화상카메라(E60, 한국렌탈, 한국)로 측정하고, 전자기파 처리 전과 후의 온도 변화 차이를 표 1에 나타내었다. The metal ion conjugate was prepared in 10 mg / ml concentration of metal ions, and 0.1 ml was dispensed into 3 wells in a 96-well plate. At this time, 0.1ml of distilled water was used as a control. After exposing the 96-well plate at high frequency thermal cancer treatment device (EHY-2000, Oncothermia) for 5 minutes at 100W energy dose, the temperature was measured by thermal imaging camera (E60, Korea Rental, Korea) after 5 minutes. The difference in temperature after the change is shown in Table 1.
표 1
delta Temp. (℃)
control 0.5
ferrous sulfates 3.3
magnesium sucrose 5.2
iron sucrose 4.7
iron gluconate 5.7
iron isomaltoside 3.2
iron carboxymaltose 5.8
iron dextran 6.4
iron starch 4.5
Table 1
delta Temp. (℃)
control 0.5
ferrous sulfates 3.3
magnesium sucrose 5.2
iron sucrose 4.7
iron gluconate 5.7
iron isomaltoside 3.2
iron carboxymaltose 5.8
iron dextran 6.4
iron starch 4.5
표 1로부터, 금속이온 탄수화물 복합체에 고주파를 처리하면 대조군인 증류수 처리군보다 온도가 3~6℃ 이상 상승하는 것을 확인할 수 있었다.From Table 1, when the high-frequency treatment to the metal ion carbohydrate complex, it was confirmed that the temperature rises 3 ~ 6 ℃ or more than the distilled water treatment group as a control.
실험예 2. 금속이온의 아포트랜스페린 결합능 평가Experimental Example 2 Evaluation of Apottransferin Binding Capacity of Metal Ions
금속이온의 아포트랜스페린 결합능을 평가하기 위해 철 이온(ferric iron, FeIII+)의 농도에 따라 트랜스페린 결합능(Unsaturated Iron-Binding Capacity, UIBC)을 다음과 같이 측정하였다. In order to evaluate the apoptransfer binding capacity of the metal ion, transferrin binding capacity (Unsaturated Iron-Binding Capacity, UIBC) was measured according to the concentration of ferric iron (FeIII +) as follows.
먼저 철 이온 수용액을 만들기 위해 FeCl3(Sigma Aldrich, USA) 3.6g을 증류수 400ml에 넣고 20분 이상 저어서 녹인 후 5M NaOH 용액을 방울씩 떨어뜨리며 계속 저으면서 pH 9로 맞추었다. 적갈색의 침전물이 보이면 90℃에서 2시간 동안 저어준 후 5,000rpm으로 5분간 원심분리하여 침전된 ferric hydroxide를 얻어내고 증류수로 세척한 후 건조시켰다. 분말형태의 ferric hydroxide을 증류수에 녹여 1, 10, 50, 200, 500g/dL 농도의 철 이온 수용액(ferric hydroxide solution)을 준비하였다. 각 농도의 철 이온 수용액에 apotransferrin(Sigma Aldrich, USA)을 200mg/dL이 되도록 첨가하고 vortex로 1분간 섞은 후 37℃에서 30분간 apotransferrin과 철 이온을 반응시켰다.First, 3.6 g of FeCl 3 (Sigma Aldrich, USA) was added to 400 ml of distilled water to melt an aqueous solution of iron and stirred for at least 20 minutes. Then, 5 M NaOH solution was dropped dropwise to pH 9. When reddish brown precipitate was observed, the mixture was stirred at 90 ° C. for 2 hours, centrifuged at 5,000 rpm for 5 minutes to obtain precipitated ferric hydroxide, washed with distilled water, and dried. Ferric hydroxide in powder form was dissolved in distilled water to prepare ferric hydroxide solution of 1, 10, 50, 200, 500g / dL concentration. Apotransferrin (Sigma Aldrich, USA) was added to the aqueous solution of iron ions at each concentration to 200 mg / dL, mixed with vortex for 1 minute, and apotransferrin reacted with iron ions at 37 ° C. for 30 minutes.
아포트랜스페린의 철 이온 결합능(Unsaturated Iron-binding capacity)를 측정하기 위해 Ferrozine colorimetric method를 이용하였다. iron standard로는 Ferrous chloride를 Hydroxylamine hydrochloride에 500 g/dL 농도로 준비하고, 실험군에는 apotransferrin과 철 이온의 반응액을 준비하였다. 먼저 0.5M의 Tris buffer(pH8) 2ml을 모든 시험관에 분주하였다. 다음 blank 시험관에는 증류수 1ml, standard 시험관에는 증류수 0.5ml과 iron standard 0.5ml, 그리고 test 시험관에는 apotransferrin과 철 이온의 반응액 0.5ml과 iron standard 0.5ml을 넣은 후 vortex로 1분간 섞었다. Ferrozine colorimetric method was used to measure the unsaturated iron-binding capacity of apoptransferin. Ferrous chloride was prepared at 500 g / dL concentration in Hydroxylamine hydrochloride as an iron standard, and a reaction solution of apotransferrin and iron ions was prepared in the experimental group. First, 2 ml of 0.5 M Tris buffer (pH 8) was dispensed into all test tubes. In the next blank test tube, 1 ml of distilled water, 0.5 ml of distilled water and 0.5 ml of iron standard, and 0.5 ml of apotransferrin and iron ion reaction solution and 0.5 ml of iron standard in test tube were mixed with vortex for 1 minute.
560nm에서 spectrophotometer 값을 제로로 한 후 흡광도 A1을 측정하였다. 다음 16.6mM 농도의 Ferrozine Hydroxylamine hydrochloride 용액을 50l씩 넣은 후 vortex로 1분간 섞었다. 모든 시험관을 37℃에서 10분간 배양한 후, 560nm에서 흡광도 A2를 측정하였다. 560nm에서의 흡광도 A560는 흡광도 A2에서 흡광도 A1을 뺀 값으로 계산하고, 표 2에 나타내었다. Absorbance A1 was measured after the spectrophotometer value was zero at 560 nm. Next, 50 ml of Ferrozine Hydroxylamine hydrochloride solution at a concentration of 16.6 mM was added, and then mixed with vortex for 1 minute. All test tubes were incubated at 37 ° C. for 10 minutes, and then absorbance A2 was measured at 560 nm. Absorbance A 560 at 560 nm was calculated by subtracting absorbance A1 from absorbance A2, and is shown in Table 2.
Unsaturated Iron-binding capacity (UIBC)는 다음 식으로 계산되었다.Unsaturated Iron-binding capacity (UIBC) was calculated by the following equation.
UIBC = [standard conc.]-[standard conc.] × Test A560/Standard A560 UIBC = [standard conc.]-[Standard conc.] × Test A 560 / Standard A 560
표 2
iron conc.(㎍/dL) 0 10 50 200 500
UIBC (㎍/dL) 500 498 481 332 119
TABLE 2
iron conc. (㎍ / dL) 0 10 50 200 500
UIBC (µg / dL) 500 498 481 332 119
표 2에서와 같이, 철 이온과 아포트랜스페린의 혼합용액에서는 철 이온이 아포트랜스페린과 결합하여 monoferric 및 diferric 트랜스페린으로 되면서 철 이온과의 결합능(Unsaturated Iron-Binding Capacity, UIBC)이 500㎍/dL에서 119㎍/dL으로 감소하는 것을 확인할 수 있었다. As shown in Table 2, in the mixed solution of iron ions and apottransferrin, iron ions are combined with apoptransfer and become monoferric and diferric transferrins, and the binding ability with iron ions (UIBC) is 119 at 500 µg / dL. It was confirmed that the decrease to μg / dL.
실험예 3: 금속이온이 비공유 결합된 아포트랜스페린의 온도 발열능 평가Experimental Example 3: Evaluation of the temperature exothermic ability of the apattransferrin non-covalently bound
"금속이온이 비공유 결합된 아포트랜스페린"(트랜스페린)의 발열능을 평가하기 위하여 철이 결합되어 있지 않은 아포트랜스페린 및 철이 결합되어 있는 아포트랜스페린 수용액에 전자기파를 처리한 후 온도를 측정하였다. 철 이온이 결합되어 있지 않은 아포트랜스페린(Sigma Aldrich, USA) 수용액을 0, 0.04, 0.2, 1, 5mg/ml 농도로 희석한 후 각 농도별로 0.1ml을 96-well plate에 분주하여 준비하였다. In order to evaluate the exothermic ability of the "non-covalently bonded apattransferrin" (transferrin), the temperature was measured after treating electromagnetic apattransferrin and iron-bonded apattransferrin solution with iron. An aqueous solution of apottransferrin (Sigma Aldrich, USA), to which iron ions were not bound, was diluted to 0, 0.04, 0.2, 1, and 5 mg / ml, and 0.1 ml of each concentration was prepared by dispensing in a 96-well plate.
철 이온이 결합된 아포트랜스페린을 준비하기 위해서는 다음과 같이 철 이온 수용액과 아포트랜스페린을 반응시켰다. FeCl3(Sigma Aldrich, USA) 3.6g을 증류수 400ml에 넣고 20분 이상 저어서 녹인 후 5M NaOH 용액을 방울씩 떨어뜨리며 계속 저으면서 pH 9로 맞추었다. 적갈색의 침전물이 보이면 90℃에서 2시간 동안 저어주면서 배양한 후 5,000 rpm으로 5분간 원심분리하여 침전된 ferric hydroxide를 얻어내고 증류수로 세척한 후 건조시켰다. 분말형태의 ferric hydroxide을 증류수에 녹여 100㎍/dL 농도의 철 이온 수용액(ferric hydroxide solution)을 준비하였다. 철 이온 수용액에 아포트랜스페린을 500mg/dL이 되도록 첨가하고 vortex로 1분간 섞은 후 37℃에서 30분간 아포트랜스페린과 철 이온을 반응시켰다. 철 이온이 결합된 아포트랜스페린 용액을 0, 0.04, 0.2, 1, 5mg/ml 농도로 희석한 후 각 농도별로 0.1ml을 96-well plate에 분주하여 준비하였다. In order to prepare a fortran transferrin combined with iron ions, the iron ion aqueous solution was reacted with the aprotransferrin as follows. 3.6 g of FeCl 3 (Sigma Aldrich, USA) was added to 400 ml of distilled water and stirred for more than 20 minutes to dissolve. Then, 5 M NaOH solution was dropped dropwise to pH 9. When the reddish brown precipitate was observed, the mixture was incubated with stirring at 90 ° C. for 2 hours, followed by centrifugation at 5,000 rpm for 5 minutes to obtain precipitated ferric hydroxide, washed with distilled water, and dried. Ferric hydroxide in powder form was dissolved in distilled water to prepare a ferric hydroxide solution at a concentration of 100 µg / dL. Apottransferrin was added to 500 mg / dL in an aqueous solution of iron ions, mixed with vortex for 1 minute, and the aforetransferrin and iron ions were reacted at 37 ° C. for 30 minutes. The iron ion-coupled aprotransferrin solution was diluted to 0, 0.04, 0.2, 1, and 5 mg / ml concentrations, and 0.1 ml of each concentration was prepared by dispensing in a 96-well plate.
아포트랜스페린 수용액 plate 및 철 이온이 결합된 아포트랜스페린(트랜스페린) 수용액 plate를 고주파온열암치료기(EHY-2000, Oncothermia)에서 100W의 energy dose로 3분간 노출시키기 전과 후의 온도를 열화상카메라 (E60, 한국렌탈, 한국)로 측정하고, 그 온도 변화 차이를 도 1~4에 나타내었다. A thermotransfer camera (E60, Korea) exposed the temperature before and after exposing the APOTHERPIN aqueous solution plate and iron ion-coupled APOTHERPIN (transferrin) aqueous solution plate at 100W energy dose for 3 minutes in high frequency thermal cancer treatment device (EHY-2000, Oncothermia). Rental, Korea) and the difference in temperature change is shown in FIGS. 1 to 4.
도 1~4에 도시된 바와 같이, 아포트랜스페린 수용액의 전자기파 처리 전후의 온도변화는 모든 처리 농도에서 3℃ 미만으로 유지된 반면, 철이온이 결합되어 있는 아포트랜스페린(트랜스페린) 수용액의 전자기파 처리 전후의 온도변화는 1mg/ml에서 4.4℃ 상승하고 5mg/ml에서는 10.9℃ 상승한 것을 확인할 수 있었다.As shown in Figures 1 to 4, the temperature change before and after the electromagnetic wave treatment of the aqueous solution of aprotransferrin was maintained at less than 3 ℃ at all treatment concentrations, while before and after the electromagnetic wave treatment of the aqueous solution of aprotransferrin (transferrin) with iron ions The temperature change was found to increase 4.4 ℃ at 1mg / ml and 10.9 ℃ was increased at 5mg / ml.
실험예 4: 금속이온이 비공유 결합된 아포트랜스페린의 in vitro 암세포 온도 상승능 평가Experimental Example 4: Evaluation of in vitro cancer cell temperature synergism of non-covalently bound apoptransferin
금속이온이 비공유 결합된 아포트랜스페린에 의한 온도 상승능을 in vitro 세포실험으로 평가하였다. 트랜스페린 수용체가 과발현되어 있는 암 세포주 NCI-H460 (Califer Life Sciences)를 배양한 후 1×103 cells/ml 농도의 세포현탁액 0.1ml을 96-well plate에 분주하고 37℃ CO2 배양기에서 12시간 배양하였다. 또한 대조군으로 인간 정상 세포 stromal cell를 배양하여 3×103 cells/ml 농도 0.1ml을 96-well plate에 분주하고 37℃ CO2 배양기에서 12시간 배양하였다. In vitro cell experiments were carried out to evaluate the temperature rise by non-covalently bound aprotransferin. After incubating cancer cell line NCI-H460 (Califer Life Sciences) overexpressing transferrin receptor, 0.1 ml of 1 × 10 3 cells / ml cell suspension was dispensed in 96-well plate and incubated for 12 hours in 37 ° C CO 2 incubator. It was. In addition, human normal cell stromal cells were cultured as a control group, and 0.1 ml of 3 × 10 3 cells / ml concentration was dispensed into a 96-well plate and incubated for 12 hours in a 37 ° C. CO 2 incubator.
준비된 정상세포주 plate와 암세포주 plate 각각에 철 이온이 결합된 아포트랜스페린(트랜스페린) 수용액을 0, 0.04, 0.2, 1 또는 5mg/ml이 되도록 첨가한 후 37℃ CO2 배양기에서 4시간 배양하였다. 트랜스페린과 세포 배양을 완료한 plate를 DMEM 배지로 세척하여 세포 내로 유입되지 않은 트랜스페린을 제거하였다. 다음으로 각 plate를 고주파온열암치료기(EHY-2000, Oncothermia)에서 100W의 energy dose로 3분간 노출시킨 후 온도 변화를 열화상카메라(E60, 한국렌탈, 한국)로 측정하고, 그 결과를 도 5~8에 나타내었다. Iron ion-coupled apattransferrin (transferrin) aqueous solution was added to 0, 0.04, 0.2, 1 or 5mg / ml in each of the prepared normal cell line and cancer cell line plates, and then cultured in a 37 ° C. CO 2 incubator for 4 hours. The plate that completed transferrin and cell culture was washed with DMEM medium to remove transferrin that did not enter the cells. Next, each plate was exposed to an energy dose of 100 W for 3 minutes in a high-frequency thermal cancer treatment device (EHY-2000, Oncothermia), and then the temperature change was measured by a thermal imaging camera (E60, Korea Rental, Korea), and the results are shown in FIG. 5. It is shown to -8.
도 5~8에 도시된 바와 같이, 정상세포주에서 전자기파 처리 전후의 온도변화는 모든 처리 농도에서 7℃ 내외로 유지된 반면, 암세포주의 경우 전자기파 처리 전후의 온도변화는 1mg/ml에서 11.9℃ 상승하고, 5mg/ml에서는 12.6℃ 상승한 것을 확인할 수 있었다.As shown in Figures 5 to 8, the temperature change before and after the electromagnetic wave treatment in the normal cell line was maintained at about 7 ℃ at all treatment concentrations, while in the cancer cell line the temperature change before and after the electromagnetic wave treatment was increased by 11.9 ℃ at 1mg / ml , 5mg / ml was confirmed to increase by 12.6 ℃.
즉, 도 5~8로부터 트랜스페린을 투여한 후, 전자기파를 처리할 경우 트랜스페린의 농도에 따른 온도 상승이 정상세포주보다 암세포주에 더 선택적임을 확인할 수 있었다.That is, after administering transferrin from Figures 5 to 8, it was confirmed that the temperature rise according to the concentration of transferrin is more selective to cancer cell lines than normal cell lines when electromagnetic waves are treated.
실험예 5: 금속이온의 in vivo 암조직 축적 평가Experimental Example 5: Evaluation of in vivo cancer tissue accumulation of metal ions
금속이온을 투여시 실제로 암조직에 축적되는지를 in vivo 동물실험으로 평가하기 위해 먼저 암 이식(tumor xenograft) 동물모델을 다음과 같이 제작하였다. 폐암 세포주 NCI-H460-luc2(Califer Life Sciences)을 배양한 후, 5×106의 세포를 6~8주령의 암컷 BALB/c 무흉선 누드 마우스(athymic nude mouse)(다물사이언스)의 피하에 주사한 다음, 10일 정도 키우면서 암 조직이 100mm3 이상 자라도록 하여 암 이식(tumor xenograft) 동물모델을 제작하였다. In order to evaluate whether the metal ions are actually accumulated in the cancer tissue by in vivo animal experiments, first, a tumor xenograft animal model was prepared as follows. After culturing the lung cancer cell line NCI-H460-luc2 (Califer Life Sciences), 5 × 10 6 cells were injected subcutaneously in 6-8 week old female BALB / c athymic nude mice (multiple science). Next, a cancer x-graft animal model was prepared by growing cancer tissues over 100 mm 3 for 10 days.
확립된 tumor xenograft BALB/c 무흉선 누드 마우스에 실험예 1에서 제조된 magnesium sucrose, iron sucrose, iron dextran 금속이온 수용액을 0.2mg/ml 농도로 희석한 후 1mg/kg 용량이 되도록 0.1ml 정맥 주사하였다. 24시간 경과 후 ICP-MS 측정을 위해서 각 조직을 1g 떼어내어 얼음 수조에서 조직 분쇄기로 갈은 후 분쇄액 1 ml을 -60℃, 7㎛Hg 진공 상태로 24시간동안 건조시켰다. 건조된 분말에 2ml의 6N HCl을 첨가한 후 밀폐된 유리반응기 안에 넣고 55℃ 배양기에서 배양시켰다. 12시간 이상 경과한 후 각 시료를 vortex하고, 1,000rpm으로 15분간 원심분리 시켜, 상층액을 질소 가스로 건조시키고, 다시 0.01N HCl 1ml을 넣고 vortex한 후 1,000rpm으로 15분간 원심분리하였다. 상층액을 회수한 후 정상 조직과 암 조직에서의 금속이온의 농도를 Inductively coupled plasma mass spectrometry (ICP-MS; Varian 800-MS, Palo Alto, US)로 측정하였다. Established tumor xenograft BALB / c athymic nude mice were diluted with 0.2 mg / ml of magnesium sucrose, iron sucrose, and iron dextran aqueous metal ions prepared in Experiment 1, and then injected 0.1 ml intravenously to a dose of 1 mg / kg. . After 24 hours, 1 g of each tissue was removed for ICP-MS measurement, ground in an ice bath to a tissue grinder, and 1 ml of the pulverized liquid was dried at -60 ° C. and 7 μm Hg for 24 hours. 2 ml of 6N HCl was added to the dried powder, which was placed in a closed glass reactor and incubated in a 55 ° C. incubator. After 12 hours or more, each sample was vortexed, centrifuged at 1,000 rpm for 15 minutes, the supernatant was dried with nitrogen gas, and again, 1 ml of 0.01 N HCl was added and vortexed, followed by centrifugation at 1,000 rpm for 15 minutes. After the supernatant was recovered, the concentration of metal ions in normal and cancerous tissues was measured by inductively coupled plasma mass spectrometry (ICP-MS; Varian 800-MS, Palo Alto, US).
표 3는 magnesium sucrose를 암이 유도된 마우스(Tumor xenograft mouse)에 투여한 후, 정상 조직과 암 조직 부위에 축적된 금속이온의 농도를 ICP-MS로 측정한 결과이다. Table 3 shows the results of measuring the concentration of metal ions accumulated in normal tissues and cancer tissues by ICP-MS after magnesium sucrose was administered to cancer-induced mice (Tumor xenograft mice).
표 3
Saline Mg-Sucrose Fold increase
Tumor 58.5±24.8 137.7±74.1 2.4
Liver 195.4±12.6 145.1±98.5 0.7
Muscle 193.5±7.9 378.4±212.3 2.0
Spleen 200.5±2.1 419.2±115.2 2.1
Brain 126.8±8.7 113.9±11.6 0.9
TABLE 3
Saline Mg-Sucrose Fold increase
Tumor 58.5 ± 24.8 137.7 ± 74.1 2.4
Liver 195.4 ± 12.6 145.1 ± 98.5 0.7
Muscle 193.5 ± 7.9 378.4 ± 212.3 2.0
Spleen 200.5 ± 2.1 419.2 ± 115.2 2.1
Brain 126.8 ± 8.7 113.9 ± 11.6 0.9
표 3에 나타낸 바와 같이, magnesium sucrose 투여시 암 조직의 magnesium ion 농도가 2.4배 이상 증가하였다. As shown in Table 3, magnesium sucrose administration increased the magnesium ion concentration of cancer tissue by more than 2.4 times.
표 4는 iron sucrose을 암이 유도된 마우스(Tumor xenograft mouse)에 투여한 후, 정상 조직과 암 조직 부위에 축적된 금속이온의 농도를 ICP-MS로 측정한 결과이다. Table 4 shows the results of measuring the concentration of metal ions accumulated in normal tissue and cancer tissue by ICP-MS after iron sucrose was administered to tumor xenograft mice.
표 4
Saline Fe-Sucrose Fold increase
Tumor 13.5±1.34 44.4±3.9 3.29
Liver 74.5±7.5 168.2±37.3 2.26
Kidney 56.9±7.5 59.8±4.1 1.05
Heart 32.2±1.8 99.2±2.8 3.08
Muscle 29.5±4.2 28.9±22.1 0.98
Stomach 27.8±3.8 83.1±27.7 2.99
Brain 22.5±3.7 46.2±8.6 2.05
Table 4
Saline Fe-Sucrose Fold increase
Tumor 13.5 ± 1.34 44.4 ± 3.9 3.29
Liver 74.5 ± 7.5 168.2 ± 37.3 2.26
Kidney 56.9 ± 7.5 59.8 ± 4.1 1.05
Heart 32.2 ± 1.8 99.2 ± 2.8 3.08
Muscle 29.5 ± 4.2 28.9 ± 22.1 0.98
Stomach 27.8 ± 3.8 83.1 ± 27.7 2.99
Brain 22.5 ± 3.7 46.2 ± 8.6 2.05
표 4에 나타낸 바와 같이, iron sucrose 투여시 암 조직의 iron ion 농도가 3.3배 이상 증가하였으며, 이는 간, 신장, 심장, 위, 뇌 등 주요 장기보다 높았다. As shown in Table 4, the iron ion concentration of cancer tissue increased more than 3.3 times when iron sucrose was administered, which was higher than the major organs such as liver, kidney, heart, stomach and brain.
표 5는 iron dextran을 암이 유도된 마우스(Tumor xenograft mouse)에 투여한 후, 정상 조직과 암 조직 부위에 축적된 금속이온의 농도를 ICP-MS로 측정한 결과이다. Table 5 shows the results of measuring the concentration of metal ions accumulated in normal tissues and cancer tissues by ICP-MS after iron dextran was administered to cancer-induced mice (Tumor xenograft mice).
표 5
Saline Fe-Dextran Fold increase
Tumor 13.5±1.34 46.8±6.6 3.47
Liver 74.5±7.5 115.5±17.3 1.55
Kidney 56.9±7.5 59.8±15.6 1.05
Heart 32.2±1.8 92.1±5.8 2.86
Muscle 29.5±4.2 65.7±30.4 2.23
Stomach 27.8±3.8 54.1±12.8 1.95
Brain 22.5±3.7 36.4±3.4 1.62
Table 5
Saline Fe-Dextran Fold increase
Tumor 13.5 ± 1.34 46.8 ± 6.6 3.47
Liver 74.5 ± 7.5 115.5 ± 17.3 1.55
Kidney 56.9 ± 7.5 59.8 ± 15.6 1.05
Heart 32.2 ± 1.8 92.1 ± 5.8 2.86
Muscle 29.5 ± 4.2 65.7 ± 30.4 2.23
Stomach 27.8 ± 3.8 54.1 ± 12.8 1.95
Brain 22.5 ± 3.7 36.4 ± 3.4 1.62
표 5에 나타낸 바와 같이, iron dextran 투여시 암 조직의 철 이온의 농도가 대조군에 비해 3.4배 이상 증가하였으며, 이는 간, 신장, 심장, 위, 뇌 등 주요 장기에서의 증가율보다 현저히 높았다. As shown in Table 5, the iron ion concentration of cancer tissues increased more than 3.4 times compared to the control group when iron dextran was administered, which was significantly higher than that of major organs such as liver, kidney, heart, stomach and brain.
실험예 6: 금속이온이 비공유 결합된 아포트랜스페린의 in vivo 암조직 축적능 평가Experimental Example 6 Evaluation of In Vivo Cancer Tissue Accumulation Capacity of Apattransferin with Non-Covalently Bonded Metal Ion
"금속이온이 비공유 결합된 아포트랜스페린"(트랜스페린)의 암 조직 축적능을 평가하기 위하여 철이 결합되어 있는 트랜스페린 수용액을 쥐에 투여한 후 정상 조직과 암 조직에서의 금속이온의 농도를 측정하였다. 철 이온이 결합된 아포트랜스페린(트랜스페린) 수용액을 4mg/ml로 준비한 후 16mg/kg 용량으로 0.1ml 정맥 주사하였다. 24시간 경과 후 실험예 5와 동일한 방법으로 각 조직을 채취하여 금속이온의 농도를 Inductively coupled plasma mass spectrometry (ICP-MS; Varian 800-MS, Palo Alto, US)로 측정하였다. In order to evaluate the cancer tissue accumulation ability of the "non-covalently bound apattransferrin" (transferrin), the iron-bound transferrin aqueous solution was administered to rats, and the concentration of metal ions in normal tissues and cancer tissues was measured. An aqueous solution of apottransferrin (transferrin) in which iron ions were bound was prepared at 4 mg / ml, followed by 0.1 ml intravenous injection at a 16 mg / kg dose. After 24 hours, each tissue was collected in the same manner as in Experimental Example 5, and the concentration of metal ions was measured by Inductively coupled plasma mass spectrometry (ICP-MS; Varian 800-MS, Palo Alto, US).
표 6은 "철 이온이 결합된 아포트랜스페린"(트랜스페린)을 암이 유도된 마우스(Tumor xenograft mouse)에 투여한 후, 정상조직과 암조직 부위에 축적된 철 이온의 농도를 ICP-MS로 측정한 결과이다. Table 6 shows the concentration of iron ions accumulated in normal tissues and cancer tissues by ICP-MS after administration of "iron ion-bound atransferrin" (transferrin) to cancer-induced mice (Tumor xenograft mice). One result.
표 6
Saline Transferrin Fold increase
Tumor 13.5±1.34 43.9±9.7 3.25
Liver 74.5±7.5 94.7±8.1 1.27
Lung 28.3±6.1 43.9±17.9 1.55
Kidney 56.9±7.5 41.2±1.7 0.72
Heart 32.2±1.8 70.1±12.3 2.18
Muscle 29.5±4.2 25.7±5.2 0.87
Stomach 27.8±3.8 46.4±14.9 1.67
Brain 22.5±3.7 28.4±3.5 1.26
Table 6
Saline Transferrin Fold increase
Tumor 13.5 ± 1.34 43.9 ± 9.7 3.25
Liver 74.5 ± 7.5 94.7 ± 8.1 1.27
Lung 28.3 ± 6.1 43.9 ± 17.9 1.55
Kidney 56.9 ± 7.5 41.2 ± 1.7 0.72
Heart 32.2 ± 1.8 70.1 ± 12.3 2.18
Muscle 29.5 ± 4.2 25.7 ± 5.2 0.87
Stomach 27.8 ± 3.8 46.4 ± 14.9 1.67
Brain 22.5 ± 3.7 28.4 ± 3.5 1.26
표 6에 나타낸 바와 같이, 철 이온이 결합된 아포트랜스페린(트랜스페린) 투여시 암조직의 철 이온의 농도가 대조군에 비해 3.2배 이상 증가하였으며 이는 간, 신장, 심장, 위, 뇌 등 주요 장기에서의 증가율보다 현저히 높았다. As shown in Table 6, the iron ion concentration of iron ion-coupled atransferrin (transferrin) increased more than 3.2 times the concentration of iron ions in cancer tissues compared to the control group, which was found in major organs such as liver, kidney, heart, stomach and brain. It was significantly higher than the increase rate.
실시예 1: 온열치료용 감작제의 투여 및 전자기파를 이용한 암 온열치료 Example 1: Administration of sensitizer for thermal therapy and cancer thermal therapy using electromagnetic waves
암세포는 비정상적인 분열을 계속하기 위해 급속한 세포분열에 필요한 영양성분들을 급격히 받아들이는 대신 대사 조절능이 떨어진다. 실제 암세포는 트랜스페린 수용체를 과발현시켜 세포분열에 필요한 철을 많이 받아들이지만, 열 조절능이 떨어져 정상 세포에 비해 고열에 상대적으로 민감한 것으로 알려져 있다. 따라서 암세포에만 집중적으로 열을 가하면 암세포의 선택적 사멸이 가능하다. 암세포에 대한 표적지향성을 가지는 트랜스페린은 암 세포에 과발현된 트랜스페린 수용체를 통해 암세포에 철을 집중적으로 전달하는데 이때 암세포에 전자기파를 처리시 온도 상승에 의한 암세포 사멸이 가능할 것으로 예측되었다. Cancer cells have poor metabolic control instead of rapidly receiving the nutrients needed for rapid cell division in order to continue abnormal division. In fact, cancer cells overexpress the transferrin receptor and receive a lot of iron required for cell division, but are poor in heat regulation and are relatively sensitive to high fever compared to normal cells. Therefore, if heat is concentrated only on cancer cells, selective killing of cancer cells is possible. Transferrin, which has a target orientation to cancer cells, delivers iron to cancer cells intensively through transferrin receptors overexpressed in cancer cells.
본 실시예 1에서는, 실험예에서 우수한 온도 상승능이 확인된 금속이온 결합물을 온열치료용 감작제로 사용하여 암 이식(tumor xenograft) 동물모델의 온열치료시 항암 효능 가능성을 확인하였다. In Example 1, the use of a metal ion conjugate with excellent temperature rise in the experimental example was used as a sensitizer for heat treatment, thereby confirming the possibility of anticancer efficacy in the heat treatment of a tumor xenograft animal model.
이를 위해 폐암 세포주 NCI-H460-luc2(Califer Life Sciences)을 배양한 후, 5×106의 세포를 6~8주령의 암컷 BALB/c 무흉선 누드 마우스(athymic nude mouse)(다물사이언스)의 피하에 주사한 다음, 10일 정도 키우면서 암 조직이 100mm3 이상 자라도록 하여 암의 치료 효능을 연구하기 위한 암 이식(tumor xenograft) 동물모델을 제작하였다.To this end, the lung cancer cell line NCI-H460-luc2 (Califer Life Sciences) was cultured, and then 5 × 10 6 cells were subcutaneously injected into 6-8 week old female BALB / c athymic nude mice (multiple science). After injection, the tumor tissue was grown for 10 days to grow 100 mm 3 or more to produce a tumor xenograft animal model to study the therapeutic efficacy of the cancer.
다음으로 온열치료용 감작제 조성물인 iron sucrose을 실험예 1의 방법으로 준비한 후 확립된 tumor xenograft 마우스에 1mg/kg 용량이 되도록 0.2mg/ml 농도의 iron sucrose 수용액을 0.1ml 정맥 주사하였다. Next, iron sucrose, which is a sensitizer composition for thermal therapy, was prepared by the method of Experimental Example 1, and then 0.1 ml intravenous was injected with a 0.2 mg / ml iron sucrose aqueous solution to a dose of 1 mg / kg in the established tumor xenograft mice.
또한 온열치료용 감작제 조성물인 "철 이온이 결합된 아포트랜스페린"(트랜스페린)을 실험예 3의 방법으로 준비한 후 확립된 tumor xenograft 마우스에 20mg/kg 용량이 되도록 5 mg/ml 농도의 transferrin 수용액을 0.1ml 정맥 주사하였다. In addition, after preparing the iron sensitized sensitizer composition for iron therapy "transferrin" (transferrin) by the method of Experiment 3, the transferrin aqueous solution of 5 mg / ml concentration to 20 mg / kg dose to the established tumor xenograft mice 0.1 ml intravenous injection.
대조군에는 생리식염수를 투여하였다. 투여하고 4시간 경과 후, 고주파온열암치료기(EHY-2000, Oncothermia)에서 100W의 energy dose로 3분간 조사한 다음 정상 조직과 암 조직의 온도를 열화상카메라(E60, 한국렌탈, 한국)로 촬영하고, 그 결과를 도 9~11에 나타내었다.Physiological saline was administered to the control group. After 4 hours of administration, high-frequency thermal cancer treatment device (EHY-2000, Oncothermia) was irradiated with an energy dose of 100W for 3 minutes, and then the temperature of normal tissue and cancer tissue was taken with a thermal imaging camera (E60, Korea Rental, Korea). The results are shown in FIGS. 9 to 11.
도 9~11에 도시된 바와 같이, 대조군의 경우 전자기파 처리 전후 정상조직과 암조직에서의 온도가 모두 1℃ 정도 상승한 수준으로 차이가 없었다. 그러나, iron sucrose 투여군의 경우 전자기파 처리 전후의 온도변화가 정상조직은 1℃, 암조직은 1.9℃로 차이가 있었으며, 철 이온이 결합된 아포트랜스페린(트랜스페린) 투여군의 경우 역시 전자기파 처리 전후의 온도변화가 정상조직은 1℃, 암조직은 2℃로 차이가 있었다. 즉 도 9~11로부터 온열치료용 감작제를 투여한 쥐의 암 조직에 전자기파를 처리할 경우 암 조직에 전달된 철 이온의 발열에 따라 정상조직보다 암조직에서 온도 상승이 증가함을 확인할 수 있었다.As shown in Figures 9-11, in the control group, the temperature in the normal tissue and the cancer tissue before and after the electromagnetic wave treatment both increased by about 1 ℃ level was no difference. However, in the iron sucrose group, the temperature change before and after electromagnetic wave treatment was 1 ℃ for normal tissues and 1.9 ℃ for cancer tissues.In the case of the iron-transported apottransferin (transferrin) group, the temperature change before and after electromagnetic wave treatment was also different. There was a difference in normal tissue to 1 ℃ and cancer tissue to 2 ℃. In other words, when the electromagnetic waves are treated in the cancer tissues of the rats to which the sensitizer for thermal therapy is administered from FIGS. .
다음으로, 상기 금속이온 결합물을 온열치료용 감작제로 이용하여 온열치료시 암 치료 가능성을 확인하였다. 폐암 세포주 NCI-H460-luc2 (Califer Life Sciences)을 배양한 후, 5×106의 세포를 6~8주령의 암컷 BALB/c 무흉선 누드 마우스(athymic nude mouse)(다물사이언스)의 피하에 주사한 다음, 10일 정도 키우면서 암 조직이 100mm3 이상 자라도록 하여 암의 치료 효능을 연구하기 위한 암 이식(tumor xenograft) 동물모델을 제작하였다.Next, the use of the metal ion combination as a sensitizer for heat treatment confirmed the possibility of cancer treatment during heat treatment. After culturing the lung cancer cell line NCI-H460-luc2 (Califer Life Sciences), 5 × 10 6 cells were injected subcutaneously in 6-8 week old female BALB / c athymic nude mice (multiple science). Next, a tumor xenograft animal model was prepared to study the therapeutic efficacy of cancer by growing cancer tissues over 100 mm 3 while growing for 10 days.
다음으로 온열치료용 감작제 조성물인 금속이온 결합물을 실험예 1의 방법으로 준비한 후 확립된 tumor xenograft 마우스에 금속이온 단당류 결합물(iron gluconate), 금속이온 이당류 결합물(iron sucrose), 금속이온 올리고당 결합물(iron isomaltoside), 금속이온 다당류 결합물(iron carboxymaltose, iron dextran, iron starch)을 1mg/kg 용량이 되도록 0.1ml 정맥 주사하였다. Next, after preparing the metal ion conjugate which is a sensitizer composition for heat treatment by the method of Experimental Example 1, the metal ion monosaccharide conjugate (iron gluconate), the metal ion disaccharide conjugate (iron sucrose), and metal ion in the tumor xenograft mouse established Oligosaccharide conjugates (iron isomaltoside), metal ion polysaccharide conjugates (iron carboxymaltose, iron dextran, iron starch) were injected 0.1 ml intravenously to a 1 mg / kg dose.
또한 온열치료용 감작제 조성물인 철 이온이 결합된 아포트랜스페린(트랜스페린)을 실험예 3의 방법으로 준비한 후 확립된 tumor xenograft 마우스에 20mg/kg 용량이 되도록 0.1ml 정맥 주사하였다. In addition, after preparing the method of Experiment 3, the iron ion-coupled aprotransferrin (transferrin), a sensitizer composition for thermotherapy, was injected 0.1 ml intravenously to a dose of 20 mg / kg in the established tumor xenograft mice.
4시간 경과 후 고주파온열암치료기(EHY-2000, Oncothermia)에서 100W의 energy dose로 10분간 온열치료하는 과정을 주 3회 4주간 진행하였다. 이때, 무처리군과 생리식염수 처리군을 대조군으로 진행하였다. 마지막 주에 암 조직 크기를 분석하기 위해서는 바이오형광 이미징(bioluminescence imaging)를 수행하였다. 루시페라아제(luciferase) 발현 암 세포주인 NCI-H460-luc2를 발광시키기 위해 D-luciferin(Xenogen, USA)을 150mg luciferin/kg/d의 농도로 쥐에 복강주사하고, 이소플루레인 가스(isoflurane gas)와 산소를 혼합해 흡입마취시킨 후, Xenogen imager(IVIS 200)로 발광된 암세포를 중첩 촬영하고, Igor Pro imaging analysis software를 이용해 분석하고, 그 결과를 도 12에 나타내었다.After 4 hours, the high-frequency heat cancer treatment device (EHY-2000, Oncothermia) underwent heat treatment for 10 minutes with 100W energy dose three times a week for 4 weeks. At this time, the untreated group and the saline treated group proceeded to the control group. In the last week, bioluminescence imaging was performed to analyze cancer tissue size. D-luciferin (Xenogen, USA) was intraperitoneally injected into mice at a concentration of 150 mg luciferin / kg / d to luminesce luciferase-expressing cancer cell line NCI-H460-luc2, and with isoflurane gas After inhalation anesthesia mixed with oxygen, the cancer cells luminesced with Xenogen imager (IVIS 200) were superimposed and analyzed using Igor Pro imaging analysis software, and the results are shown in FIG. 12.
도 12는 본 발명의 실시예 1에 따라 암이 유도된 마우스(Tumor xenograft mouse model animal)에 무처리 (A), 생리식염수 (B), iron gluconate (C), iron sucrose (D), iron carboxymaltose (E), iron dextran (F), iron starch (G) 및 transferrin (H)을 투여하고 고주파 온열치료 후 마우스 모델의 암조직 크기를 바이오형광으로 분석한 결과이다. 12 is untreated (A), saline (B), iron gluconate (C), iron sucrose (D), iron carboxymaltose in cancer-induced mouse (Tumor xenograft mouse model animal) according to Example 1 of the present invention (E), iron dextran (F), iron starch (G), and transferrin (H) were administered and biofluorescence was analyzed for the size of the cancerous tissue in the mouse model after radiofrequency thermal treatment.
도 12에 도시된 바와 같이, 무처리군(A) 및 생리식염수 처리군(B)과는 달리 금속이온 결합물 투여군(C~H)의 경우 온열치료 후 암의 크기가 분명히 줄어드는 것을 확인할 수 있었다. 특히 iron sucrose (D), iron dextran (F) 및 transferrin (H) 처리군에서는 온열치료를 통한 암 치료 효과가 가장 우수하였다.As shown in Figure 12, unlike the untreated group (A) and physiological saline treated group (B) in the case of the metal ion conjugated administration group (C ~ H) was confirmed that the size of the cancer after the heat treatment clearly reduced. . In particular, heat treatment was the most effective cancer treatment in iron sucrose (D), iron dextran (F) and transferrin (H) treatment groups.
끝으로, 온열치료 감작제로 사용시 항암 효과가 가장 우수한 것으로 확인된 iron sucrose, iron dextran 및 transferrin을 온열치료용 감작제로 사용하여 전자기파를 이용한 온열치료시 암의 완치 가능성 여부를 확인하였다. 이를 위해 tumor xenograft BALB/c 무흉선 누드 마우스에 생리식염수, iron dextran, iron sucrose, 및 철 이온이 결합된 아포트랜스페린(transferrin) 수용액을 격일로 주 3회 정맥 주사하고 4시간 경과 후 고주파온열암치료기(EHY-2000, Oncothermia)에서 100W의 energy dose로 30분 이상 온열치료하는 과정을 4주간 진행하면서 1주일 간격으로 암 조직의 크기를 모니터링하였다. iron dextran과 iron sucrose 투여군에는 각각 0.2mg/ml 농도의 iron dextran 또는 iron sucrose 수용액을 0.1ml 정맥 주사하였고, transferrin 투여군에는 5mg/ml 농도의 transferrin 수용액을 0.1ml 정맥 주사하였다. 이때, 무처리군과 생리식염수 투여군을 대조군으로 진행하였다.Finally, iron sucrose, iron dextran, and transferrin, which were found to have the best anti-cancer effects when used as a thermotherapy sensitizer, were used as sensitizers for thermotherapy, and it was confirmed whether the cancer could be cured during heat treatment using electromagnetic waves. To this end, tumor xenograft BALB / c athymic nude mice were injected intravenously with saline, iron dextran, iron sucrose, and iron ion-coupled afferent transferrin solution three times a week every other day, and then 4 hours later. (EHY-2000, Oncothermia) The cancer tissue size was monitored at weekly intervals during the 4 weeks of thermal therapy for 30 minutes at 100W energy dose. In the iron dextran and iron sucrose groups, 0.1 ml intravenous injection of 0.2 mg / ml iron dextran or iron sucrose, respectively, and in the transferrin group, 0.1 ml intravenous injection of 5 mg / ml transferrin aqueous solutions. At this time, the untreated group and physiological saline administration group was progressed to the control group.
암 조직 크기를 분석하기 위해서는 바이오형광 이미징(bioluminescence imaging)를 1주 간격으로 수행하고, Igor Pro imaging analysis software를 이용해 분석한 다음, 그 결과를 도 13에 나타내었다.To analyze cancer tissue size, bioluminescence imaging was performed at weekly intervals, analyzed using Igor Pro imaging analysis software, and the results are shown in FIG. 13.
도 13에 도시된 바와 같이, 최초 암 조직의 크기와 4주 후 암 조직의 크기를 바이오형광 이미지로 비교한 결과, 무처리 대조군에 비해 생리식염수 처리군의 경우 초기에는 다소 암의 성장을 저해하는 듯 보였으나 시간이 경과하면서 효능이 감소하였다. iron dextran을 감작제로 투여한 실험군과 iron sucrose를 감작제로 투여한 실험군의 경우 전자기파를 이용한 온열치료시 암의 성장 속도가 분명히 줄어드는 것을 확인할 수 있었다. 트랜스페린 투여군의 경우에는 뚜렷하게 암의 성장을 억제하고 크기를 감소시키다가 시험 종료 시점인 4주 후에는 완전히 사라지는 놀라운 항암 효능을 확인할 수 있었다. As shown in Figure 13, the size of the first cancer tissue compared with the size of the cancer tissue after 4 weeks as a result of biofluorescence image, the saline treated group compared to the untreated control group initially inhibited the growth of the cancer slightly It seemed to be less effective over time. In the experimental group administered iron dextran as a sensitizer and the experimental group administered iron sucrose as a sensitizer, it was confirmed that the growth rate of cancer was clearly decreased during the heat treatment using electromagnetic waves. In the case of the transferrin-administered group, it was found that the anticancer efficacy was markedly suppressed and reduced in size, and disappeared completely after 4 weeks at the end of the test.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
본 발명에 따른 암 온열치료용 감작제 조성물을 이용하면 암세포만 선택적으로 금속성분을 축적시킬 수 있어, 고통과 부작용이 없으면서도 암 치료 효능이 현저히 향상된 가장 이상적인 항암 치료법이 될 수 있어, 항암 치료에 널리 이용될 것으로 기대되며, 화학 요법, 방사선 요법 등 다른 암 치료방법과도 병용 가능하므로, 암의 완치 가능성을 높일 수 있다.By using the sensitizer composition for cancer thermal therapy according to the present invention, only cancer cells can selectively accumulate metal components, and thus can be the most ideal anti-cancer therapy with significantly improved cancer treatment efficacy without pain and side effects. It is expected to be widely used, and can be used in combination with other cancer treatment methods such as chemotherapy and radiation therapy, thereby increasing the cure potential of cancer.

Claims (12)

  1. 전자기파를 이용한 암 온열치료용 감작제 조성물.Sensitizer composition for cancer thermal therapy using electromagnetic waves.
  2. 제1항에 있어서, 상기 감작제는 금속이온, 금속이온 결합물, 금속이온이 비공유 결합된 아포트랜스페린 및 금속이온이 비공유 결합된 아포트랜스페린 유도체로 구성된 군으로부터 선택되는 것을 특징으로 하는 전자기파를 이용한 암 온열치료용 감작제 조성물.According to claim 1, wherein the sensitizer is cancer using electromagnetic waves, characterized in that selected from the group consisting of a metal ion, a metal ion bond, a non-covalently bonded aprotransferrin and a metal covalently bonded aprotransferrin derivative Sensitizer composition for heat treatment.
  3. 제2항에 있어서, 상기 금속이온은 철(iron) 이온, 망간(manganese) 이온, 아연(zinc) 이온, 구리(copper) 이온, 마그네슘(magnesium) 이온, 비스무트(bismuth) 이온, 루테늄(ruthenium) 이온, 티타늄(titanium) 이온, 갈륨(gallium) 이온, 인듐(indium) 이온, 바나딜(vanadyl) 이온, 크로미움(chromium) 이온, 알루미늄(aluminum) 이온 및 플루토늄(plutonium) 이온으로 구성된 군으로부터 선택되는 것을 특징으로 하는 암 온열치료용 감작제 조성물.The method of claim 2, wherein the metal ions are iron ions, manganese ions, zinc ions, copper ions, magnesium ions, bismuth ions, ruthenium Selected from the group consisting of ions, titanium ions, gallium ions, indium ions, vanadyl ions, chromium ions, aluminum ions and plutonium ions A sensitizer composition for cancer thermal therapy, characterized in that it becomes.
  4. 제2항에 있어서, 상기 금속이온 결합물은 금속이온에 덱스트란(dextran), 수크로즈(sucrose), 글루코네이트(gluconate), 소르비톨(sorbitol), 폴리사카라이드(polysaccharide), 카복시말토스(carboxymaltose), 페루목시톨(ferumoxytol), 이소말토사이드(isomaltoside), 시트레이트(citrate), 클로라이드(chloride), 설페이트(sulfate), 푸마레이트(fumurate), 말토스(maltose), 전분(starch), 셀룰로오스(cellulose) 및 알부민(albumin)을 포함하는 군으로부터 선택되는 결합물이 비공유 결합되어 있는 것을 특징으로 하는 암 온열치료용 감작제 조성물.The method of claim 2, wherein the metal ion complex is dextran, sucrose, gluconate, sorbitol, polysaccharide, carboxymaltose to metal ions. ), Perumoxytol, isomaltoside, citrate, chloride, sulfate, fumurate, maltose, starch, A sensitizer composition for cancer thermal therapy, wherein the binder selected from the group consisting of cellulose and albumin is non-covalently bound.
  5. 제2항에 있어서, 상기 아포트랜스페린 또는 아포트랜스페린 유도체는 인간 또는 포유동물 유래 혈청 단백질이거나 재조합 단백질인 것을 특징으로 하는 암 온열치료용 감작제 조성물.The sensitizer composition for cancer thermal therapy according to claim 2, wherein the apoptransferrin or the atransferrin derivative is a human or mammalian-derived serum protein or a recombinant protein.
  6. 제1항에 있어서, 상기 감작제는 0.01~100mg/ml 농도인 것을 특징으로 하는 암 온열치료용 감작제 조성물.The method of claim 1, wherein the sensitizer sensitizer composition for cancer thermal therapy, characterized in that the concentration of 0.01 ~ 100mg / ml.
  7. 제1항에 있어서, 약학적으로 허용 가능한 담체를 추가로 포함하는 것을 특징으로 하는 암 온열치료용 감작제 조성물.The method of claim 1, wherein the sensitizer composition for cancer thermal therapy, characterized in that it further comprises a pharmaceutically acceptable carrier.
  8. 제1항의 전자기파를 이용한 암 온열치료용 감작제 조성물 및 전자기파를 조사하는 장치를 포함하는 암 온열치료용 키트.The cancer thermal therapy kit comprising a device for irradiating electromagnetic waves and a sensitizer composition for cancer thermal therapy using electromagnetic waves of claim 1.
  9. (a) 인간을 제외한 동물에 제1항의 암 온열치료용 감작제 조성물을 투여하여 암 치료에 대한 감수성을 증가시키는 단계; 및(a) increasing the susceptibility to cancer treatment by administering the sensitizer composition for cancer thermal therapy of claim 1 to animals other than humans; And
    (b) 전자기파를 처리하는 단계를 포함하는 암 치료방법.(b) treating the cancer comprising electromagnetic wave treatment.
  10. 제9항에 있어서, 상기 암 온열치료용 감작제 조성물이 금속이온 또는 금속이온 결합물인 경우에는 0.1~50mg/kg 용량으로, 금속이온이 비공유 결합된 아포트랜스페린 또는 그 유도체인 경우에는 0.1~200mg/kg 용량으로 투여하는 것을 특징으로 하는 암 치료방법.10. The method according to claim 9, wherein the cancer sensitizer composition for the treatment of cancer is 0.1 to 50 mg / kg when the metal ion or the metal ion conjugate is used, and 0.1 to 200 mg / kg when the metal ion is a non-covalently bonded aprotransferrin or a derivative thereof. Cancer treatment method characterized in that administered in a kg dose.
  11. 제9항에 있어서, 상기 전자기파는 감마선, X-선, 자외선, 가시광선, 적외선, 마이크로웨이브 및 라디오 전파로 구성된 군으로부터 선택되는 것을 특징으로 하는 암 치료방법.The method of claim 9, wherein the electromagnetic wave is selected from the group consisting of gamma rays, X-rays, ultraviolet rays, visible rays, infrared rays, microwaves, and radio waves.
  12. 제9항에 있어서, 화학 치료(chemotherapy), 방사선 치료(radiation therapy), 생물학적 치료(biological therapy), 면역치료(immunotherapy) 및 광역동 치료(photodynamic therapy)로 구성된 군으로부터 선택되는 하나 이상의 치료방법을 병행하는 것을 특징으로 하는 암 치료방법.10. The method of claim 9, wherein at least one treatment method is selected from the group consisting of chemotherapy, radiation therapy, biological therapy, immunotherapy and photodynamic therapy. Cancer treatment method, characterized in that parallel.
PCT/KR2014/009641 2013-10-16 2014-10-14 Sensitizing composition using electromagnetic waves for thermal therapy of cancers, and cancer therapy using same WO2015056960A1 (en)

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