JP5842367B2 - Anticancer sensitizer - Google Patents

Description

  The present invention relates to an anticancer agent sensitizer.

  High concentrations of nitric oxide (NO) are known to induce apoptosis. Using this phenomenon, cancer is treated by administering S-nitroso group-containing HSA (SNO-HSA) obtained by adding a large amount of NO to human serum albumin (HSA) by chemical modification to cancer cells. An attempt has been reported (Patent Document 1). Since HSA is an endogenous substance, even if SNO-HSA is administered into the body, there are few side effects and nitroso groups are also stably present, so SNO-HSA is expected as an effective anticancer agent. However, about 10 mg / mL or more of SNO-HSA is required to effectively treat cancer.

  By the way, in cancer pharmacotherapy with anticancer agents, there are known attempts to reduce the side effects of each drug by using multiple anticancer agents with different side effects in combination, as in multi-drug combination therapy, and to reduce the amount of each drug used. ing.

JP 2009-57318 A

  An object of the present invention is to provide an anticancer agent sensitizer which is excellent in safety and stability and enhances the anticancer agent sensitivity of cancer cells at a low concentration.

  As a result of intensive studies to solve the above problems, the present inventors have found that S-nitroso group-containing human serum albumin has a high anticancer agent sensitizing action and completed the present invention.

  The anticancer agent sensitizer of the present invention contains S-nitroso group-containing albumin as an active ingredient.

  In one embodiment, the S-nitroso group-containing albumin is contained at a concentration of 2 to 10 μM.

  The present invention also provides a pharmaceutical composition comprising the anticancer agent sensitizer and the anticancer agent.

  In one embodiment, the S-nitroso group-containing albumin contained in the anticancer agent sensitizer is administered to a human in an amount of 2 to 10 mg / kg · dose.

  In one embodiment, the anticancer agent sensitizer and the anticancer agent are administered simultaneously or separately.

  In one embodiment, the anticancer agent is doxorubicin.

  In one embodiment, the anticancer agent is applied to at least one selected from the group consisting of malignant lymphoma, lung cancer, gastrointestinal cancer, breast cancer, bladder tumor, osteosarcoma, brain tumor and skin cancer.

  ADVANTAGE OF THE INVENTION According to this invention, the anticancer agent sensitizer which is excellent in safety | security and stability, and enhances the anticancer agent sensitivity of a cancer cell at low concentration can be provided. By combining the anticancer agent sensitizer and the anticancer agent of the present invention, a pharmaceutical composition having a high anticancer activity can be provided.

It is a graph which shows the growth inhibitory effect of dx with respect to K562 and K562 / dx. It is a graph which shows the growth inhibitory effect of dx and / or SNO-HSA with respect to K562 and K562 / dx. It is a graph which shows the growth inhibitory effect of SNO-HSA with respect to K562 and K562 / dx. It is a graph which shows the result of having measured the progress of cancer of the K562 (a) and K562 / dx (b) origin cancer-bearing mouse which administered dx and / or SNO-HSA with time. It is a photograph of TUNEL staining showing cells in which HE staining and apoptosis were induced in cancer tissues of K562 and K562 / dx-derived tumor-bearing mice administered with dx and / or SNO-HSA. It is a graph which shows the growth inhibitory effect of dx and / or SNO-HSA and / or ODQ with respect to K562 and K562 / dx. It is a graph which shows the effect of SNO-HSA with respect to accumulation | storage of dx in the cell of K562 and K562 / dx. It is a graph which shows the effect of SNO-HSA with respect to the expression of P-gp in the cell of K562 and K562 / dx. It is a graph which shows the effect of SNO-HSA with respect to the expression of HIF-1 (alpha) in the cell of K562.

  The anticancer agent sensitizer of the present invention contains S-nitroso group-containing albumin (SNO-HSA) as an active ingredient.

  The SNO-HSA of the present invention refers to albumin into which at least one, preferably two or more, more preferably three or more S-nitroso groups are introduced.

  The albumin in the present invention may be natural albumin or albumin produced by a gene recombination method.

  Natural albumin refers to albumin present in the body of humans and mammals, and also includes ovalbumin and muscle albumin (myogen).

  As long as albumin produced by a gene recombination method has a function equivalent to that of natural albumin, one or more amino acids in the amino acid sequence of natural albumin are substituted, deleted, inserted and / or added. The albumin which consists of is also included. For example, albumin in which the 410th arginine in the amino acid sequence of natural albumin is substituted with cysteine can be mentioned.

  The SNO-HSA of the present invention may be natural SNO-HSA or SNO-HSA obtained by artificially introducing an S-nitroso group into albumin.

  The S-nitroso group is introduced into albumin by replacing a sulfur atom present in an amino acid constituting albumin, for example, a sulfur atom in a thiol group with a nitroso group. Examples of amino acids containing a thiol group include cysteine, cystine, and methionine.

  Naturally, albumin contains 35 cysteines, 34 of which form disulfide bonds because of the secondary structure of albumin. Therefore, for example, when an S-nitroso group is introduced into natural albumin via a cysteine thiol group, the S-nitroso group can be introduced into one cysteine not involved in disulfide bonds.

  The method for artificially introducing the S-nitroso group into the thiol group of albumin is not particularly limited, and examples include methods known in the art. For example, a method using a nitrosation reagent (nitrite ion, isoamyl nitrite, n-butyl nitrite, etc.) can be mentioned.

  It is also possible to artificially introduce a thiol group into albumin and introduce an S-nitroso group into this thiol group. The method for artificially introducing a thiol group into albumin is not particularly limited, and includes methods known in the art. For example, the method of introduce | transducing a thiol group into the epsilon-amino group of a lysine using a thiolation reagent (2-iminothiolane or its salt etc.) is mentioned.

  The administration route of the anticancer agent sensitizer of the present invention is not particularly limited, and examples thereof include intradermal, subcutaneous, intramuscular, intraperitoneal, transdermal, transmucosal, oral, and inhalation, and preferably a parenteral administration route. More preferably, it is a route of administration by injection.

  The dosage form of the anticancer agent sensitizer of the present invention is not particularly limited, and examples thereof include injections, patches, patches, eye drops, nasal drops, sprays, tablets, capsules, troches, sublingual tablets, Examples include creams, lotions, and powders.

  In particular, in the case of an injection, it may be a solution or suspension containing SNO-HSA. Further, for example, a pH adjuster, electrolyte, saccharide, vitamins, pharmacologically acceptable salt or You may add suitably the additive normally used in the said field | areas, such as a fatty acid and / or an amino acid.

  In the case of a solution or suspension, for example, water (water for injection) prescribed by the Japanese Pharmacopoeia, physiological saline, various buffer solutions (for example, phosphate buffer solution) and the like can be used.

  Any pH regulator may be used as long as it is generally used as a pH regulator for injections. Examples thereof include organic acids such as citric acid, tartaric acid, acetic acid and lactic acid, inorganic acids such as hydrochloric acid and phosphoric acid, and inorganic bases such as sodium hydrogen carbonate, sodium carbonate and sodium hydroxide.

  As the electrolyte, various water-soluble salts conventionally used for infusion can be used. For example, water-soluble salts (eg, chlorides, sulfates, acetates) of various inorganic components (eg, sodium, potassium, calcium, magnesium, phosphorus) required to maintain the biological function and electrolyte balance of body fluids Gluconate, lactate).

  As the saccharide, those conventionally used for various infusions can be used. Examples thereof include monosaccharides such as glucose, fructose and galactose, disaccharides such as lactose and maltose, polyhydric alcohols such as glycerol, sugar alcohols such as xylitol, sorbitol and mannitol, dextrans such as dextran 40 or dextran 80, and sucrose. It is done.

  As the vitamins, various water-soluble / fat-soluble vitamins can be used. For example, vitamin A, provitamin A, vitamin D, provitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, niacin, vitamin B6 group, pantothenic acid, biotin, myo-inositol, choline, folic acid, vitamin B12, Vitamin C, vitamin P or vitamin U can be mentioned.

  Examples of the pharmacologically acceptable salt or fatty acid include alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, formic acid, acetic acid, oxalic acid, tartaric acid, maleic acid, citric acid, and caprylic acid. Organic acids such as succinic acid and malic acid, and organic bases such as trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, and dicyclohexylamine.

  Examples of amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Can be mentioned. An amino acid derivative such as N-acetylmethionine may be added.

  The anticancer agent sensitizer of the present invention can be produced according to a usual pharmaceutical method. That is, the pH is about 4.5 to 8.7 in water, various buffer solutions, generally available infusion solutions (for example, general amino acid infusion solutions, electrolyte infusion solutions) or aqueous solutions containing the same components. It can be produced by diluting and / or dissolving SNO-HSA.

  The concentration of SNO-HSA contained in the anticancer agent sensitizer of the present invention is usually 0.5 to 10 μM, preferably 2 to 10 μM.

  Although it does not specifically limit as an anticancer agent used together with the anticancer agent sensitizer of this invention, For example, actinomycin D, camptothecin, cisplatin, doxorubicin, etoposide, 5-fluorouracil, mitomycin C, cyclophosphamide is mentioned. The cancer to which the anticancer agent is applied is not particularly limited, and examples thereof include malignant lymphoma, lung cancer, gastrointestinal cancer, breast cancer, bladder tumor, osteosarcoma, brain tumor and skin cancer.

  The administration of these anticancer agents alone induces the expression of P-glycoprotein (P-gp) having a drug efflux function in cancer cells, and the cancer cells acquire anticancer drug resistance by evacuating the anticancer drug out of the cells. SNO-HSA, which is an active ingredient of the inventive anticancer agent sensitizer, overcomes anticancer drug resistance by inhibiting the expression of P-gp in cancer cells and inhibiting the extracellular excretion of anticancer agents in cancer cells.

  The present invention also provides a pharmaceutical composition comprising the anticancer agent sensitizer and the anticancer agent. The dosage form, production method, and the like of the pharmaceutical composition of the present invention are not particularly limited, and conform to the anticancer agent sensitizer of the present invention.

  In the administration of the pharmaceutical composition of the present invention, the anticancer agent sensitizer has an active ingredient SNO-HSA in an amount of usually 1 to 10 mg / kg.times., Preferably 2 to 10 mg / kg.times. To be administered. When the SNO-HSA is administered at a dose of 10 mg / kg · dose to a human, the anticancer agent sensitizer is usually administered 1 to 2 times / week, preferably 2 times / week.

  The administration method of the pharmaceutical composition of the present invention is not particularly limited, and the anticancer agent sensitizer and the anticancer agent may be administered simultaneously, or the anticancer agent sensitizer and the anticancer agent may be administered separately. When administered separately, the anticancer agent sensitizer may be administered first, the anticancer agent may be administered later, the anticancer agent may be administered first, and the anticancer agent sensitizer may be administered later. The interval between the administration of the anticancer agent sensitizer and the administration of the anticancer agent is usually within 2 to 6 hours, preferably within 2 to 3 hours.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited by these Examples.

(Reference Example 1: In vitro test)
Human chronic myelogenous leukemia cell line (K562) and K562 anti-cancer drug doxorubicin (dx) resistant cell line (K562 / dx) (Kumamoto University School of Medicine and Pharmacy, Cell Functional Molecular Analysis) was prepared. SIGMA) was cultured in the presence for 60 days, and the surviving cell line was designated K562 / dx.) Was suspended in the medium (RPMI-1640 / 10% FCS / antibiotic-antifungal mixed solution; Nacalai Tesque, Inc.). It became cloudy and 1 × 10 ⁴ cells were seeded in each well of a 96-well plate. Then, dx (final concentrations: 0.5 μM, 1 μM, 5 μM, 10 μM and 50 μM) was added to each well, and the cells were cultured at 37 ° C. for 24 hours in a 5% CO ₂ incubator. Next, 10 μL of WST-8 reagent (Dojin Chemical Co., Ltd.) was added to each hole, and the absorbance of each hole was measured at 450 nm after 2-3 hours. The ratio (%) of the control (without dx addition) to the absorbance was calculated as the survival rate. The results are shown in FIG.

  As is apparent from FIG. 1, 1 to 50 μM dx inhibited the growth of K562, but hardly inhibited the growth of 562 / dx. From this, it was confirmed that K562 / dx has dx resistance.

(Preparation Example 1: Preparation of SNO-HSA)
By the method described in Example 1 of Patent Document 1, a sterile aqueous solution having a concentration of 1 mM of S-nitroso group-containing albumin (SNO-HSA) into which about 7 S-nitroso groups were introduced per molecule of albumin was prepared.

(Example 1: In vitro test)
K562 and K562 / dx were suspended in a medium (RPMI-1640 / 10% FCS / antibiotic-antifungal mixed solution), and 1 × 10 ⁴ cells were seeded in each well of a 96-well plate. Next, SNO-HSA prepared in Preparation Example 1 (final concentrations: 0 μM (no addition), 0.5 μM, 5 μM, and 10 μM) was added to each hole, and the cells were placed in a 5% CO ₂ incubator at 37 ° C. Cultured for 2 hours. Furthermore, dx (final concentrations: 0 μM (no addition) and 5 μM) was added to each well, and the cells were cultured at 37 ° C. for 24 hours in a 5% CO ₂ incubator. Next, 10 μL of WST-8 reagent was added to each hole, and after 2 to 3 hours, the absorbance of each hole was measured at 450 nm. The ratio (%) to the absorbance of the control (no SNO-HSA added, no dx added) was calculated as the survival rate. The results are shown in FIG.

  As is apparent from FIG. 2, 5 μM dx hardly inhibited the proliferation of K562 / dx, but the combination of 5 μM dx and 0.5 to 10 μM SNO-HSA was dependent on the concentration of SNO-HSA. Inhibited the growth of K562 / dx.

(Comparative Example 1: In vitro test)
K562 and K562 / dx were suspended in a medium (RPMI-1640 / 10% FCS / antibiotic-antifungal mixed solution), and 1 × 10 ⁴ cells were seeded in each well of a 96-well plate. Subsequently, SNO-HSA (final concentrations: 0.5 μM, 5 μM, 10 μM, 25 μM and 50 μM) was added to each well, and the cells were cultured at 37 ° C. for 24 hours in a 5% CO ₂ incubator. Next, 10 μL of WST-8 reagent was added to each hole, and after 2 to 3 hours, the absorbance of each hole was measured at 450 nm. The ratio (%) of the control (no addition of SNO-HSA) to the absorbance was calculated as the survival rate. The results are shown in FIG.

  As is clear from FIG. 3, 0-10 μM SNO-HSA hardly inhibited the growth of K562 and K562 / dx, whereas 10-50 μM SNO-HSA increased the growth of K562 / dx in a concentration-dependent manner. Inhibited.

  2 and 3, it was found that 0.5 to 10 μM SNO-HSA overcomes the d562 resistance of K562 / dx and restores the anticancer effect of dx.

(Example 2: In vivo test)
2 × 10 ⁷ cells each of K562 and K562 / dx were suspended in 0.1 mL of medium (RPMI-1640 / 10% FCS / antibiotic-antimycotic mixed solution) and Matrigel® (Becton, Dickinson and Company) (Product made) After mixing with 0.1 mL, BALB / cA Jcl-nu / nu mice (4 weeks old, female) were transplanted subcutaneously in the hind limbs to produce tumor-bearing mice.

  For tumor-bearing mice derived from K562 or K562 / dx, dx (4 mg / kg; intraperitoneal injection), SNO-HSA (2 mg / kg; intravenous injection), or dx (4 mg / kg; intraperitoneal injection) / SNO- HSA (2 mg / kg; intravenous injection) was administered, and the volume of cancer was measured over time. The results are shown in FIG.

  As is apparent from FIG. 4, administration of dx hardly inhibited the progression of cancer in K562 / dx-derived tumor-bearing mice, but combined administration of dx and SNO-HSA resulted in administration of K562 / dx-derived tumor-bearing mice. Inhibited cancer progression. From this, it was found that SNO-HSA overcomes dx resistance of K562 / dx-derived tumor-bearing mice and restores the anticancer effect of dx.

  Next, a cancer tissue was taken out from the mouse 42 days after administration of the anticancer agent, a tissue section was prepared, and cells in which apoptosis was induced were analyzed using a TUNEL staining kit (In Situ Cell Death Detection Kit, Fluorescein, Boehringer Mannheim; Roche). ). The results are shown in FIG.

  As is clear from FIG. 5, administration of dx alone did not induce apoptosis in K562 / dx-derived tumor-bearing mice, but combined administration of dx and SNO-HSA caused apoptosis in K562 / dx-derived tumor-bearing mice. Induced. From this, it was found that SNO-HSA overcomes d562 resistance of K562 / dx and restores the anticancer effect of dx.

(Example 3: Mechanism analysis 1 of overcoming dx tolerance by SNO-HSA)
K562 / dx was suspended in the medium, and 1 × 10 ⁴ cells were seeded in each well of a 96-well plate. Each hole was then filled with SNO-HSA (final concentrations: 0 μM (no addition) and 10 μM) and 1H- [1,2,4] oxadiazole [4,3-a] quinoxalin-1-one (ODQ) (SIGMA (Final concentrations: 0 μM (no addition), 0.01 μM, 1 μM and 100 μM), and the cells were cultured in a 5% CO ₂ incubator at 37 ° C. for 2 hours. Furthermore, dx (final concentrations: 0 μM (no addition) and 5 μM) was added to each well, and the cells were cultured at 37 ° C. for 24 hours in a 5% CO ₂ incubator. Next, 10 μL of WST-8 reagent was added to each hole, and after 2 to 3 hours, the absorbance of each hole was measured at 450 nm. The ratio (%) to the absorbance of the control (no SNO-HSA added, no ODQ added, no dx added) was calculated as the survival rate. The results are shown in FIG.

  As is clear from FIG. 6, since the ODQ, which is an inhibitor of the cGMP pathway, inhibited the overcoming of d562 resistance of K562 / dx by SNO-HSA, the cGMP pathway was necessary for overcoming d562 resistance of K562 / dx by SNO-HSA. It was suggested to be involved.

(Example 4: Mechanism analysis 2 of overcoming dx resistance by SNO-HSA)
K562 and K562 / dx were suspended in the medium, and 5 × 10 ⁵ cells were seeded in each well of a 96-well plate. Subsequently, SNO-HSA (final concentrations: 0 μM (no addition), 0.5 μM, 5 μM and 10 μM) was added to each well, and the cells were cultured at 37 ° C. for 2 hours in a 5% CO ₂ incubator. Furthermore, dx (final concentration: 5 μM) was added to each well, and the cells were cultured at 37 ° C. for 24 hours in a 5% CO ₂ incubator. Next, cells were collected from each hole, and fluorescence of dx accumulated in the cells was analyzed by flow cytometry (manufactured by Becton Dickinson; excitation wavelength: 488 nm; measurement wavelength: 585 nm). The ratio (%) of K562 (no SNO-HSA added, dx 5 μM) to the fluorescence intensity was calculated as the fluorescence unit. The results are shown in FIG.

  As is clear from FIG. 7, since the combined use of 5 μM dx and 0.5 to 10 μM SNO-HSA induced dx accumulation in K562 / dx cells depending on the concentration of SNO-HSA, SNO- It was suggested that intracellular accumulation of dx is involved in overcoming d562 resistance of K562 / dx by HSA.

(Example 5: Mechanism analysis 3 of overcoming dx tolerance by SNO-HSA)
K562 and K562 / dx were suspended in the medium, and 1 × 10 ⁴ cells were seeded in each well of a 96-well plate. Subsequently, SNO-HSA (final concentrations: 0 μM (no addition), 5 μM and 10 μM) was added to each well, and the cells were cultured at 37 ° C. for 24 hours in a 5% CO ₂ incubator. Next, cells were collected from each well, and the expression level of intracellular P-glycoprotein (P-gp) was analyzed by Western blotting. For detection of P-gp, an anti-P-gp mouse antibody (Santa Cruz Biotechnology; sc-59591) was used. The ratio (%) to the intensity of the band of K562 / dx (no addition of SNO-HSA) was calculated as a fluorescence unit. The results are shown in FIG.

  As is clear from FIG. 8, since 5-10 μM SNO-HSA inhibited the expression of P-gp at K562 / dx depending on the concentration of SNO-HSA, the dx resistance of K562 / dx by SNO-HSA It was suggested that inhibition of P-gp expression was involved in overcoming the problem.

(Example 6: Mechanism analysis 4 of overcoming dx resistance by SNO-HSA)
K562 was suspended in the medium, and 1 × 10 ⁴ cells were seeded in each well of a 96-well plate. SNO-HSA (final concentrations: 0 μM (no addition), 5 μM and 10 μM) is then added to each well and the cells are placed in a 5% CO ₂ incubator at 37 ° C. with an oxygen concentration of 20% (normal oxygen concentration) or The cells were cultured for 24 hours under the condition of 0.1% (low oxygen concentration). Next, cells were collected from each hole, and the expression level of hypoxia inducible factor (HIF-1α) in the cells was analyzed by Western blotting. For detection of HIF-1α, an anti-HIF-1α mouse antibody (BD Transduction Laboratories; 610958) was used. The ratio (%) with respect to the band density of K562 (no addition of SNO-HSA, low oxygen concentration) was calculated as a fluorescence unit. The results are shown in FIG.

  As is clear from FIG. 9, 5-10 μM SNO-HSA inhibited the expression of HIF-1α in K562 under hypoxia depending on the concentration of SNO-HSA. It was suggested that HIF-1α expression inhibition, that is, improvement of hypoxic conditions was involved in overcoming dx resistance.

  As described above, SNO-HSA inhibits the extracellular discharge of dx by inhibiting the expression of P-gp having a drug excretion function through inhibition of the expression of HIF-1α in K562 / dx resistant to dx. It was suggested to overcome dx resistance.

  ADVANTAGE OF THE INVENTION According to this invention, the anticancer agent sensitizer which is excellent in safety | security and stability, and enhances the anticancer agent sensitivity of a cancer cell at low concentration can be provided. By combining the anticancer agent sensitizer and the anticancer agent of the present invention, a pharmaceutical composition having a high anticancer activity can be provided.

Claims (7)

  An anticancer agent sensitizer containing S-nitroso group-containing albumin as an active ingredient.   The anticancer agent sensitizer according to claim 1, wherein the S-nitroso group-containing albumin is contained at a concentration of 2 to 10 µM.   A pharmaceutical composition comprising the anticancer agent sensitizer according to claim 1 or 2 and an anticancer agent.   The pharmaceutical composition according to claim 3, wherein the S-nitroso group-containing albumin contained in the anticancer agent sensitizer is administered to a human in an amount of 2 to 10 mg / kg · dose.   The pharmaceutical composition according to claim 3 or 4, wherein the anticancer agent sensitizer and the anticancer agent are administered simultaneously or separately.   The pharmaceutical composition according to any one of claims 3 to 5, wherein the anticancer agent is doxorubicin.   The said anticancer agent is applied to at least 1 sort (s) selected from the group which consists of malignant lymphoma, lung cancer, digestive organ cancer, a breast cancer, a bladder tumor, an osteosarcoma, a brain tumor, and skin cancer. A pharmaceutical composition as described in 1. above.

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