JP2015067560A - Cancer metastasis inhibitor comprising lactoferrin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new use of lactoferrin.SOLUTION: A cancer metastasis inhibitor comprises lactoferrin.

Description

  The present invention relates to a cancer metastasis inhibitor containing lactoferrin.

  Lactoferrin is a multifunctional protein of about 80 kD belonging to the iron transporter family, and is abundant in milk such as breast milk and milk, and secretions from mucous membranes such as tears and saliva. The functions of lactoferrin so far include antibacterial and antiviral effects (see Non-patent Documents 1 and 2, Patent Document 1, etc.), anti-tumor effects (see Patent Document 2, etc.), anti-pain effects (see Non-Patent Document 3, etc.) Immunomodulating action (see Patent Documents 3 and 4) has been reported.

JP-A-5-320068 JP 2001-0488804 A JP 2004-359647 A JP 2004-359648 A

Journal of Pediatrics, 94, 1: 1979 Nonnecke, B. J. & Smith, K.L .; Journal of Dairy Science, 67, 606: 1984 The Journal of veterinary medical science, 66, 2, 149: 2004

  The present invention has been made in view of the above-described current state of the prior art, and its main purpose is to provide a new use of lactoferrin.

  The present inventors have surprisingly found that lactoferrin has an action of suppressing metastasis of cancer cells, and further improved and completed the present invention.

That is, the present invention encompasses the subject matters described in the following sections.
Item 1. A cancer metastasis inhibitor containing lactoferrin.
Item 2. Item 2. The cancer metastasis inhibitor according to Item 1, wherein the cancer is epithelial cancer.
Item 3. Item 3. The cancer metastasis inhibitor according to Item 2, wherein the epithelial cancer is squamous cell carcinoma.
Item 4. Item 4. The cancer metastasis inhibitor according to Item 3, wherein the squamous cell carcinoma is oral squamous cell carcinoma.
Item 5. The cancer metastasis inhibitor according to any one of Items 1 to 4, wherein the metastasis suppression is bone infiltration suppression.
Item 6. Item 6. The cancer metastasis inhibitor according to any one of Items 1 to 5, comprising a liposome containing lactoferrin.
Item 7. An oral composition containing the cancer metastasis inhibitor according to any one of Items 1 to 6.

  The cancer metastasis inhibitor and oral composition of the present invention have an action of suppressing metastasis of cancer cells, and are particularly useful for the treatment of oral squamous cell carcinoma.

It is a figure which shows the observation result of the cancer cell in Example 1. FIG. 4 is a graph showing the migration inhibition rate of cancer cells in Example 1. FIG. 4 is a diagram showing a culture chamber used in Example 2. 6 is a view showing a micrograph of cells appearing on the lower surface of a chamber in Example 2. FIG. 6 is a graph showing the number of cells appearing on the lower surface of the chamber in Example 2. 6 is a graph showing changes over time in the number of cancer cells in Example 3. It is a figure which shows the result of the FACS analysis in Example 4. It is a figure which shows the measurement result of the tumor size in Example 5. FIG. FIG. 10 is a diagram showing the imaging results of micro CT in Example 5.

  Hereinafter, the present invention will be described in detail.

  The present invention relates to a cancer metastasis inhibitor containing lactoferrin.

  Although the origin of the lactoferrin used in the present invention is not limited, for example, lactoferrin obtained from mammals such as humans, cows, goats, sheep and horses can be used. Of these, bovine lactoferrin derived from bovine or human lactoferrin derived from human is preferable. Lactoferrin can be obtained, for example, by separating it from milk products such as milk or skim milk or whey of these animals by a conventional method. Moreover, in this specification, apolactoferrin and metal saturated lactoferrin are also contained in lactoferrin. Apolactoferrin can be obtained, for example, by deironating lactoferrin with citric acid or the like, and metal saturated lactoferrin can be obtained, for example, by chelating lactoferrin with a metal such as iron, copper, zinc or manganese. In addition, various lactoferrin produced in microorganisms, animal cells, animals, and the like by genetic engineering techniques may be used. In addition, lactoferrin includes a hydrolyzate of lactoferrin. The molecular weight of lactoferrin is not particularly limited, but is preferably about 1,000 to 200,000.

  Moreover, it is preferable that the lactoferrin used for this invention is the aspect contained in the liposome. In other words, in the present invention, it is preferable to use a liposome containing lactoferrin. When using liposomes containing lactoferrin, the effect of suppressing cancer cell metastasis is further enhanced. In the present invention, the liposome is a lipid vesicle having a bilayer formed by hydrating a lipid mainly composed of phospholipid with a sufficient amount of water. Liposomes can incorporate water-soluble drugs into the inner water layer and lipid-soluble drugs into the lipid bilayer, and they can be applied as drug carriers for DDS preparations for the purpose of drug targeting, sustained release, and reduction of side effects. Has been tried. Liposomes are also known to have high safety because they are composed of components of biological membranes.

  In general, liposomes are classified based on the number of lipid bilayers, and are classified into multilamellar liposomes (MLV) and unilamellar liposomes. Single-membrane liposomes are further classified into SUV (small unilamella vesicle), LUV (large unilamella vesicle), GUV (giant unilamella vesicle), etc. according to the size. Any of these may be sufficient as the liposome of this invention. Preferred is MLV. In the present invention, the size of the liposome is usually 30 to 100 nm, preferably 30 to 600 nm, more preferably 50 to 200 nm.

  In the liposome containing lactoferrin used in the present invention, lactoferrin is preferably enclosed in a space surrounded by the liposome membrane. However, lactoferrin may be contained as a liposome membrane constituent, or multilamellar liposomes may be used. It may be contained between the multiple membranes to be formed, or may be contained in a form in which lactoferrin is attached or bound to the outermost membrane of the liposome membrane.

  Liposomes containing lactoferrin can be produced by conventional methods. For example, a desired amount of lecithin and, if necessary, a desired amount of sterol are solubilized with an appropriate organic solvent such as ethanol, and the solvent is removed under reduced pressure to form a membrane lipid. A liposome encapsulating lactoferrin can be obtained by adding an aqueous solution containing a physiologically active substance and stirring the mixture at about 1000 to 3000 rpm for about 2 to 5 minutes to prepare a liposome suspension.

  In addition to this method, a desired amount of lecithin and, if necessary, a desired amount of sterol are dissolved in a small amount of ethanol, dispersed in an aqueous solution or buffer solution, preliminarily emulsified, and then dispersed at high pressure. Liposomes encapsulating lactoferrin can also be obtained by preparing a liposome suspension by forming a multilayer.

  If necessary, the obtained suspension may be subjected to an operation for removing lactoferrin in the liposome outer liquid, for example, an operation for dialyzing the obtained filtrate after filtering the suspension.

  The liposome suspension can be used in a liquid state, but can also be used as a lyophilized dry product. Liposomes can be in a form suitable for various oral intakes, including those obtained by tableting or encapsulating the dried product.

  The content of lactoferrin in the liposome containing lactoferrin is preferably about 10 to 99% by weight, more preferably about 20 to 95% by weight, and still more preferably about 30 to 90% by weight.

  As lecithin, for example, egg yolk lecithin, soybean lecithin, rape lecithin, corn lecithin, sunflower lecithin, peanut lecithin and the like can be used alone or in combination of two or more. In the present invention, these hydrogenated products can also be used. Lecithin is also called phosphatidylcholine or 1,2-diacylglycerol 3-phosphocholine, and generally fatty acids are bound to the 1-position and 2-position of glycerol. In the present invention, it is preferable to use lecithin in which an unsaturated fatty acid having 12 to 24 carbon atoms is bonded to both or one of the 1st and 2nd positions in addition to the above exemplified lecithin. It is particularly preferable to use lecithin having a saturated fatty acid having ˜24 and an unsaturated fatty acid having 12 to 24 carbon atoms bonded to the 2-position. Here, the saturated fatty acid and the unsaturated fatty acid may be either linear or branched. As a preferable unsaturated fatty acid, an unsaturated fatty acid having 16 to 18 carbon atoms can be used. In particular, lecithin having a large amount of oleic acid and linoleic acid bonded to the 2-position is preferred. Specifically, egg yolk lecithin and soybean lecithin are preferable.

  As sterols, sterols derived from animals such as cholesterol, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol; plants such as β-sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, ergostadienol, sitosterol, and brassicasterol Sterols derived from (phytosterol); sterols derived from microorganisms such as timosterol and ergosterol, and the like, can be used alone or in combination of two or more. Among these, cholesterol or phytosterol is preferably used.

  The molar ratio of lecithin and sterol in the liposome is preferably about 55:45 to 95: 5, more preferably about 60:40 to 90:10, and most preferably about 75:25 to 85:15. When the molar ratio is within these ranges, the stability of the liposome membrane is improved.

  The content of lecithin in the liposome containing lactoferrin is preferably about 1 to 80% by weight, more preferably about 3 to 65% by weight, and still more preferably about 5 to 50% by weight.

  The content of sterol in the liposome containing lactoferrin is preferably about 0 to 40% by weight, more preferably about 0.1 to 30% by weight, and further preferably about 1 to 20% by weight.

  The content of lecithin or sterol can be measured by a known method. For example, the lecithin content can be quantified by Fiske-Subbarow method, and the sterol content can be quantified by HPLC, colorimetric method or the like.

  Furthermore, the surface of the liposome containing lactoferrin can be coated, and this coating can also be used as an active ingredient. A preferable coating includes a coating with a polysaccharide containing a sulfate group. Examples of sulfate group-containing polysaccharides include fucoidan, carrageenan, agar, and heparin. The sulfate group-containing polysaccharide includes a sulfated polysaccharide that does not contain a sulfate group, and may be chondroitin sulfate, dermatan sulfate, or the like.

  As the sulfate group-containing polysaccharide, those having a molecular weight of about 5000 to 300,000 are preferably used. Among these sulfate group-containing polysaccharides, fucoidan and carrageenan can be preferably used, and fucoidan is particularly preferable.

  The amount of sulfate group-containing polysaccharide used is, for example, preferably about 10 to 500 parts by weight and more preferably about 20 to 200 parts by weight with respect to 100 parts by weight of lecithin contained in the liposome.

  The coating can be performed, for example, by adding a sulfate group-containing polysaccharide to a suspension containing liposomes containing lactoferrin and stirring at about 1000 to 3000 rpm for about 2 to 5 minutes. A plurality of liposomes may be contained in one coating film.

  The fact that the liposome is coated with the sulfate group-containing polysaccharide can be confirmed, for example, by changing the zeta potential of the liposome solution by adding and stirring the sulfate group-containing polysaccharide.

  In addition to lecithin and phytosterol, liposomes containing lactoferrin include antioxidants such as tocopherol and ascorbic acid, organic acids such as lactic acid and citric acid, lipids such as phosphatidylglycerol and phosphatidylethanolamine, chitosan, and fucoidan. Natural polymers such as hyaluronic acid, synthetic polymers such as polyethylene glycol and carboxyvinyl polymer, carbohydrates such as trehalose, lactulose and maltitol, polyols such as glycerin, and the like can be added.

  Moreover, in addition to lactoferrin, various substances can be encapsulated in the liposome containing lactoferrin as necessary. For example, chloramphenicol, fradiomycin sulfate, sodium edetate / cetrimide, epidihydrocholesterine, zinc chloride, decalinium chloride, benzethonium chloride, dexamethasone, triamcinolone acetonide, sodium fluoride, nonsteroidal anti-inflammatory drugs, adrenal cortex Steroids, gold thiomalate, auranofin, penicillamine, bucillamine, lobenzalit disodium, salazosulfapyridine, actarit, methotrexate, mizoribine, anti-TNF antibody, soluble TNF receptor, anti-interleukin 1 antibody, anti-interleukin 6 antibody , Estrogen preparations such as estradiol and estriol, bisphosphonate preparations such as raloxifene hydrochloride, alendronate, etidronate, risedronate, etc. Down, calcitonin, ipriflavone and the like.

  The content of lactoferrin in the cancer metastasis inhibitor and oral composition of the present invention is appropriately set according to the dosage form and the like, but is usually about 0.5 to 95% by weight, preferably about 0.8 to 80% by weight, more preferably It is about 1 to 50% by weight.

  The cancer metastasis inhibitor of this invention can be mix | blended with an oral composition, and utilization as a pharmaceutical composition is especially preferable among oral compositions.

  When the cancer metastasis inhibitor of the present invention is used as a pharmaceutical composition, a pharmaceutically acceptable carrier is blended with liposomes containing phytosterols as necessary, and liquids, tablets, granules, fine granules It can be used as a pharmaceutical composition in an orally administrable form such as a solid preparation such as a powder or a capsule containing the liquid or solid preparation. Examples of pharmaceutically acceptable carriers include excipients and diluents. The oral pharmaceutical composition can also contain various additives such as fragrances.

  Examples of carriers include maltitol, lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, methylcellulose, glycerin, sodium alginate, gum arabic, talc, calcium monohydrogen phosphate, calcium hydrogen phosphate , Sodium hydrogen phosphate, dipotassium phosphate, potassium dihydrogen phosphate, calcium dihydrogen phosphate, sodium dihydrogen phosphate, calcium sulfate, calcium lactate, cacao butter, etc., simple syrup, glucose solution, starch Liquid, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, cross polyvinyl pyrrolidone, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxy Tillcellulose, carboxyvinyl polymer, crystalline cellulose, powdered cellulose, crystalline cellulose / carmellose sodium, carboxymethylcellulose, shellac, methylcellulose, ethylcellulose, potassium phosphate, gum arabic powder, pullulan, dextrin, corn starch, pregelatinized starch, hydroxypropyl Binders such as starch, gelatin, xanthan gum, tragacanth, tragacanth powder, macrogol, dry starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearin Disintegrants such as acid monoglycerides, starch, lactose, etc., disintegration of sucrose, stearic acid, cocoa butter, hydrogenated oil, etc. Inhibitors, absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid, purified talc, stearate, Lubricants such as boric acid powder and polyethylene glycol can be used. Further, the tablets can be made into tablets with ordinary coatings as necessary, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, multilayer tablets and the like. Capsules are prepared by mixing the active ingredient with the various carriers exemplified above and filling them into hard gelatin capsules, soft capsules and the like.

  The liquid preparation may be an aqueous or oily suspension, solution, syrup, or elixir, and is prepared according to a conventional method using usual additives.

  Examples of flavoring agents or fragrances include mint oil, eucalyptus oil, cinnamon oil, fennel oil, clove oil, orange oil, lemon oil, rose oil, fruit flavor, mint flavor, peppermint powder, dl-menthol, and l-menthol. Etc.

  In the oral composition of the present invention, the lactoferrin content relative to the total amount of the oral composition is appropriately set according to the dosage form of the composition, but is usually about 0.5 to 95% by weight, preferably about 0.8 to 80% by weight. Preferably, it is about 1 to 50% by weight.

  The intake (dose) of the cancer metastasis inhibitor and oral composition of the present invention is such that the intake weight of lactoferrin is preferably about 10 mg to 10000 mg, more preferably about 50 mg to 2000 mg per adult day. is there.

  The cancer metastasis inhibitor of the present invention has an action of suppressing metastasis of cancer cells, and particularly has an action of suppressing migration and invasion of cancer cells. In addition, the type of cancer targeted by the cancer metastasis inhibitor of the present invention is preferably epithelial cancer, more preferably squamous cell carcinoma, particularly preferably oral squamous cell carcinoma. In particular, since the cancer metastasis inhibitor of the present invention has an action of suppressing bone invasion of oral squamous cell carcinoma, it is useful for the treatment of oral squamous cell carcinoma.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to the following example.

Example 1: Examination of migration inhibitory action on cancer cells A cell line derived from human squamous cell carcinoma (HOC313 cells) was treated with 10% fetal bovine serum (FBS) (Invitrogen, Grand Island, USA) and antibiotics (100 U / mL). Subculture was performed in Dulbecco's Modified Eagle Mediun (DMEM) containing penicillin-streptomycin) (Invitorgen) at 37 ° C. in the presence of 5% CO ₂ . Then, 6 × 10 ⁵ HOC313 cells were seeded on a 6 cm diameter culture plate, and after cell attachment, cultured in DMEM containing 10% FBS for 72 hours, and bovine lactoferrin (bLF) was administered at 1, 10 or 100 μg / mL It was divided into the lactoferrin administration group and the non-administration control group, and the migration inhibitory effect was examined. To examine the migration inhibitory action, the culture plate was linearly detached at a width of 1 cm, and the closure of the exfoliated site due to cell migration was observed over time. Fig. 1 shows the observation results of cells after 0, 6, 12, and 24 hours. In addition, the separation distance of the same part at each time was measured by microscopic image analysis, and the migration inhibition rate was calculated as a percentage of the separation distance after 6, 12, and 24 hours with respect to the separation time of 0 hours. Figure 2 shows a graph showing the migration inhibition rate.

  From the examination results, it was confirmed that in the lactoferrin administration group, the number of cells that migrated to the linear detachment portion was smaller than that in the control group. From these results, it was found that lactoferrin has an action of suppressing migration of cancer cells.

Example 2: Examination of Invasion Suppressive Effect on Cancer Cells A cell line derived from human squamous cell carcinoma (HOC313 cells) was administered with bovine lactoferrin (bLF) at 1, 10 or 100 μg / mL and in non-administered lactoferrin groups Divided into control groups and cultured in Dulbecco's Modified Eagle Mediun (DMEM) containing 10% fetal bovine serum (FBS) (Invitrogem, Grand Island, USA) and antibiotics (100U / mL penicillin-streptomycin) (Invitorgen) for 72 hours It was. Next, each group of cells was adjusted to 2 × 10 ⁴ cells, seeded in the Cell Culture Insert shown in FIG. 3, and 500 μL of serum-free medium was added to the outside of the Insert at 37 ° C. Culturing was performed in the presence of 5% CO ₂ . After 24 hours, the cells in the lower part of the culture chamber were fixed with formalin for 1 hour, and stained with hematoxylin 3G (Sakura Finetech Japan Co., Ltd.). Cell number was counted. Note that cells with high invasive ability break through Matrigel and move to the bottom of the chamber. Fig. 4 shows a photomicrograph of each group, and Fig. 5 shows a graph showing the number of cells counted.

  From the examination results, it was confirmed that the number of cells migrated to the lower part of the chamber was smaller in the lactoferrin administration group than in the control group. From these results, it was found that lactoferrin has an action of suppressing invasion of cancer cells.

Example 3: Examination of growth inhibitory action on cancer cells A cell line derived from human squamous cell carcinoma cells (HSC3 cells) was treated with 10% fetal bovine serum (FBS) (Invitrogem, Grand Island, USA) and antibiotics (100 U / Subculture was performed in RPMI1640 (Nacalai Tesque) containing mL penicillin-streptomycin) (Invitorgen) in the presence of 37 ° C. and 5% CO ₂ . Thereafter, 3 × 10 ³ HSC3 cells were seeded on a 24-well plate and divided into a lactoferrin-administered group administered with bovine lactoferrin (bLF) at 1, 10 or 100 μg / mL and a non-administered control group. Cultivation was performed in RPMI1640 containing antibiotics (100 U / mL penicillin-streptomycin) in the presence of 5% CO ₂ at 37 ° C., and the number of cells after 1, 2, 4 and 6 days was counted. Figure 6 shows a graph showing the measured number of cells.

  From the examination results, it was confirmed that the increase rate of cells was lower in the lactoferrin administration group than in the control group. From these results, it was found that lactoferrin has an action of suppressing the growth of cancer cells.

Example 4: Examination of apoptosis-inducing action on cancer cells A cell line derived from human squamous cell carcinoma cells (HSC3 cells) was isolated from 10% fetal bovine serum (FBS) (Invitrogem, Grand Island, USA) and antibiotics (100 U / Subculture was performed in RPMI1640 (Nacalai Tesque) containing mL penicillin-streptomycin) (Invitorgen) in the presence of 37 ° C. and 5% CO ₂ . Thereafter, 3 × 10 ³ HSC3 cells were seeded on a 24-well plate and divided into a lactoferrin-administered group administered with bovine lactoferrin (bLF) at 1, 10 or 100 μg / mL and a non-administered control group. The cells were cultured in RPMI 1640 containing antibiotics (100 U / mL penicillin-streptomycin) at 37 ° C. in the presence of 5% CO _{2 for} 72 hours. After 72 hours, cells were collected, double-stained with 7-AAD and Annexin V-PE using PE Annexin V Apotosis Detection kit I (Becton, Dickinson and Company, NJ, USA), and FACS Calibur ( (Becton, Dickinson and Company, NJ, USA). The result of FACS analysis is shown in FIG.

  From the examination results, it was confirmed that in the lactoferrin administration group, the ratio of the number of cells that fell into apoptosis was larger than that in the control group. From these results, it was found that lactoferrin has an effect of inducing apoptosis of cancer cells.

Example 5: Inhibition of bone invasion to oral squamous cell carcinoma
Eight-week-old male C3H mice (Nippon Charles River) were divided into lactoferrin dose groups (5 mice each) and control groups (5 mice) with a lactoferrin dose of 100 mg / kg / day or 500 mg / kg / day. Each test sample was orally administered for 1 week. The lactoferrin administered to the lactoferrin administration group was prepared by dispersing and dissolving 4% soy lecithin and 12% lactoferrin in distilled water, followed by preliminary emulsification and high-pressure homogenization to obtain an average particle size of 40-100 nm. Lactoferrin-encapsulated liposomes (SL-bLF), and the control group was ingested with distilled water only.

Thereafter, 1 × 10 ⁴ oral gingival cancer-derived cell line SCC VII was transplanted into the left masseter muscle under general anesthesia of 10% pentobarbital sodium. Tumor size was measured using vertical vernier calipers (Mitutoyo, Kawasaki) at 1 to 2 weeks, and the diameter of the tumor with an oval shape was measured vertically and horizontally, and an approximate value was calculated as an oval shape. Figure 8 shows the tumor size measurement results. Next, 4 weeks after transplantation, the head was removed as a lump, fixed with 4% periodate-lysine-parformaldehyde (PLP) fixative at 4 ° C for 24 hours, and photographed with micro CT (SkyScan, Toyo Technica). Went. Figure 9 shows the results of micro CT imaging.

  From the examination results, it was confirmed that the lactoferrin-administered group had a smaller tumor size than the control group, and that bone resorption of the cheekbone and mandible was significantly reduced. From these results, it was found that lactoferrin has the effect of suppressing the growth of oral squamous cell carcinoma and bone infiltration accompanied by bone resorption.

Claims (7)

A cancer metastasis inhibitor containing lactoferrin. The cancer metastasis inhibitor according to claim 1, wherein the cancer is epithelial cancer. The cancer metastasis inhibitor according to claim 2, wherein the epithelial cancer is squamous cell carcinoma. The cancer metastasis inhibitor according to claim 3, wherein the squamous cell carcinoma is oral squamous cell carcinoma. The cancer metastasis inhibitor according to any one of claims 1 to 4, wherein the metastasis suppression is bone infiltration suppression. The cancer metastasis inhibitor according to any one of claims 1 to 5, comprising a liposome containing lactoferrin. The oral composition containing the cancer metastasis inhibitor in any one of Claims 1-6.

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