JP2015120651A - Antitumor agent and method of producing the same - Google Patents
Antitumor agent and method of producing the same Download PDFInfo
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- JP2015120651A JP2015120651A JP2013264582A JP2013264582A JP2015120651A JP 2015120651 A JP2015120651 A JP 2015120651A JP 2013264582 A JP2013264582 A JP 2013264582A JP 2013264582 A JP2013264582 A JP 2013264582A JP 2015120651 A JP2015120651 A JP 2015120651A
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- antitumor agent
- lactobacillus plantarum
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Abstract
Description
本発明は、乳酸菌の加熱処理菌体を用いた抗腫瘍剤及びその製造方法に関する。 The present invention relates to an antitumor agent using a heat-treated microbial cell and a method for producing the same.
乳酸菌などを用いた多くのプロバイオティクス製品やプレバイオティクス製品について抗腫瘍作用を有することが報告されている。経口投与での抗腫瘍効果が認められている乳酸菌製剤として、抗悪性腫瘍剤OK-432（商品名：ピシバニール）がよく知られている（非特許文献１）。OK-432は、ストレプトコッカス・ピオゲネス（Streptococcus pyogenes）AIII Su株をペニシリン及びH2O2処理後に凍結乾燥した製剤である。OK-432の胃がんを対象とした薬効投与量域は0.1〜2mg/ヒト個体/1回/日（一週間に1〜7回経口投与）でありその幅は約20倍と考えられている。 Many probiotic products and prebiotic products using lactic acid bacteria have been reported to have antitumor activity. An anti-neoplastic agent OK-432 (trade name: Picibanil) is well known as a lactic acid bacteria preparation that has been confirmed to have an antitumor effect by oral administration (Non-patent Document 1). OK-432 is a preparation obtained by freeze-drying Streptococcus pyogenes AIII Su strain after penicillin and H 2 O 2 treatment. The effective dose range of OK-432 for gastric cancer is 0.1-2 mg / human individual / day (1-7 times a week orally), and the width is considered to be about 20 times.
特許文献１には、ラクトバチルス・プランタラムCJLP 243株生菌体の凍結乾燥物をマウスに経口投与したことにより免疫増強されたことが開示され、その菌をアトピー、アレルギー、癌及び自己免疫疾患の予防又は治療に利用できる可能性が示唆されている。しかし、特許文献１では抗腫瘍効果は実証されていない。 Patent Document 1 discloses that immunization was enhanced by orally administering a lyophilized product of Lactobacillus plantarum CJLP 243 strain live cells to mice, and the bacteria were atopy, allergic, cancer and autoimmune diseases. It has been suggested that it may be used for the prevention or treatment of cancer. However, Patent Document 1 does not demonstrate an antitumor effect.
経口投与によって有効な抗腫瘍効果が得られることが判明している乳酸菌は多くはない。乳酸菌を用いた、経口投与でも高い抗腫瘍効果が得られる抗腫瘍剤の新たな製造方法の開発が求められている。 There are not many lactic acid bacteria that have been found to have effective antitumor effects by oral administration. Development of a new method for producing an antitumor agent using a lactic acid bacterium and capable of obtaining a high antitumor effect even by oral administration is required.
本発明は、乳酸菌を用いた効果的な抗腫瘍剤を提供することを課題とする。 An object of the present invention is to provide an effective antitumor agent using lactic acid bacteria.
本発明者らは、上記課題を解決するため鋭意検討を重ねた結果、ラクトバチルス・プランタラム菌体を比較的低温で加熱処理することにより、極めて広い投与量範囲（薬効投与量域）で強い腫瘍増殖抑制効果をもたらすことができることを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention are strong in an extremely wide dosage range (medicinal dosage range) by heat-treating Lactobacillus plantarum cells at a relatively low temperature. The present inventors have found that a tumor growth inhibitory effect can be brought about and have completed the present invention.
すなわち、本発明は以下を包含する。
[1] ラクトバチルス・プランタラム（Lactobacillus plantarum）菌体を６０〜８０℃で加熱することにより、増強された腫瘍増殖抑制効果を示す加熱処理菌体を調製することを含む、抗腫瘍剤の製造方法。
[2] ラクトバチルス・プランタラムが、ラクトバチルス・プランタラムALAL006株（受領番号NITE ABP-01754）である、上記[1]の方法。
[3] 前記加熱を５〜２５分間行う、上記[1]又は[2]の方法。
[4]菌体を前記加熱後、乾燥させることをさらに含む、上記[1]〜[3]の方法。
[5] 乾燥が凍結乾燥である、上記[4]の方法。
[6] 上記[1]〜[5]の方法によって製造される、ラクトバチルス・プランタラムの加熱処理菌体を含む抗腫瘍剤。
[7] 上記[6]の抗腫瘍剤を含む、飲食品。
[8] 上記[6]の抗腫瘍剤を含む、腫瘍増殖抑制用の医薬。
That is, the present invention includes the following.
[1] Production of an antitumor agent comprising preparing a heat-treated microbial cell exhibiting an enhanced tumor growth-inhibiting effect by heating Lactobacillus plantarum microbial cell at 60 to 80 ° C Method.
[2] The method of [1] above, wherein the Lactobacillus plantarum is Lactobacillus plantarum ALAL006 strain (reception number NITE ABP-01754).
[3] The method according to [1] or [2] above, wherein the heating is performed for 5 to 25 minutes.
[4] The method according to [1] to [3] above, further comprising drying the bacterial cells after the heating.
[5] The method according to [4] above, wherein the drying is freeze-drying.
[6] An antitumor agent comprising a heat-treated cell of Lactobacillus plantarum produced by the method of [1] to [5] above.
[7] A food and drink comprising the antitumor agent of [6] above.
[8] A medicament for suppressing tumor growth, comprising the antitumor agent of [6] above.
本発明に従えば、乳酸菌を用いて、広い薬効投与量域で強い腫瘍増殖抑制効果を有する抗腫瘍剤を簡便に製造することができる。 According to the present invention, an antitumor agent having a strong tumor growth inhibitory effect in a wide medicinal dose range can be easily produced using lactic acid bacteria.
以下、本発明を詳細に説明する。
本発明は、ラクトバチルス・プランタラム（Lactobacillus plantarum）菌体を比較的低温で加熱処理することにより得られる増強された腫瘍増殖抑制効果を示す加熱処理菌体を含む抗腫瘍剤及びその製造方法を提供する。
Hereinafter, the present invention will be described in detail.
The present invention relates to an antitumor agent comprising a heat-treated microbial cell having an enhanced tumor growth inhibitory effect obtained by heat-treating Lactobacillus plantarum microbial cells at a relatively low temperature, and a method for producing the same. provide.
本発明に係る抗腫瘍剤の製造には、ラクトバチルス・プランタラムの菌体（細胞）を用いる。限定するものではないが、好適なラクトバチルス・プランタラム菌株としては、ラクトバチルス・プランタラム（Lactobacillus plantarum）ALAL006株が挙げられる。ラクトバチルス・プランタラム（Lactobacillus plantarum）ALAL006株は、2013年11月21日付で、独立行政法人 製品評価技術基盤機構 特許微生物寄託センター（日本国千葉県木更津市かずさ鎌足2-5-8 122号室）に、受領番号NITE ABP-01754の下でブタペスト条約に基づき国際寄託されている。 Lactobacillus plantarum cells (cells) are used for the production of the antitumor agent according to the present invention. Non-limiting examples of suitable Lactobacillus plantarum strains include Lactobacillus plantarum strain ALAL006. The Lactobacillus plantarum ALAL006 strain was issued on November 21, 2013, by the National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (Kazusa Kamashi, Kisarazu City, Chiba, Japan, Room 2-5-8 122) ) Under the receipt number NITE ABP-01754 under the Budapest Treaty.
本発明では、ラクトバチルス・プランタラムの腫瘍増殖抑制効果を増強するため、ラクトバチルス・プランタラムの菌体を、比較的低温、具体的には60〜80℃、好ましくは65℃〜75℃、例えば68℃〜72℃で加熱する。ラクトバチルス・プランタラム菌体の加熱は、通常は上記の加熱温度で、5〜25分間、例えば10〜20分間行えばよいが、これらに限定するものではない。加熱処理は任意の方法で行うことができるが、例えば菌体を含む溶液を収容した容器を、溶液の温度が上記加熱温度で維持されるように湯浴中で加温してもよい。 In the present invention, in order to enhance the tumor growth inhibitory effect of Lactobacillus plantarum, the cells of Lactobacillus plantarum are relatively low temperature, specifically 60-80 ° C, preferably 65 ° C-75 ° C, For example, heating is performed at 68 ° C to 72 ° C. The Lactobacillus plantarum cells may be heated usually at the above heating temperature for 5 to 25 minutes, for example, 10 to 20 minutes, but is not limited thereto. The heat treatment can be performed by any method. For example, a container containing a solution containing bacterial cells may be heated in a hot water bath so that the temperature of the solution is maintained at the heating temperature.
ラクトバチルス・プランタラムは、上記の加熱処理前に培養してもよい。ラクトバチルス・プランタラムの培養は、通常の培養条件に従って実施することができる。培養には、ラクトバチルス・プランタラムを始めとする乳酸菌の培養に用いられる様々な培地を用いて行うことができる。そのような培地としては、例えば、MRS（de MAN, ROGOSA, SHARPE）培地、LBS培地、APT培地、トマトジュース寒天培地、牛乳培地、豆乳培地などが挙げられるが、これらに限定されない。培養は、以下に限定するものではないが、例えば、25〜45℃、好ましくは30〜40℃、例えば35〜39℃で好適に行うことができる。培養時間は、特に限定されないが、例えば5〜30時間行うことができる。 Lactobacillus plantarum may be cultured before the above heat treatment. Cultivation of Lactobacillus plantarum can be performed according to normal culture conditions. The culture can be performed using various media used for culturing lactic acid bacteria including Lactobacillus plantarum. Examples of such a medium include, but are not limited to, MRS (de MAN, ROGOSA, SHARPE) medium, LBS medium, APT medium, tomato juice agar medium, milk medium, and soy milk medium. Although culture is not limited to the following, it can be suitably performed, for example, at 25 to 45 ° C, preferably 30 to 40 ° C, such as 35 to 39 ° C. The culture time is not particularly limited, and can be performed, for example, for 5 to 30 hours.
培養後、培養液からラクトバチルス・プランタラムの菌体を回収し、生理食塩水等により洗浄した後に、加熱処理を行うことが好ましい。洗浄後の菌体を水や生理食塩水等に懸濁し、その懸濁液を加熱処理に供してもよい。 After culturing, it is preferable to recover the cells of Lactobacillus plantarum from the culture solution, wash with physiological saline, and then perform heat treatment. The washed cells may be suspended in water, physiological saline or the like, and the suspension may be subjected to heat treatment.
加熱処理後の菌体（又は菌体懸濁液）は、そのまま抗腫瘍剤に使用してもよいが、さらに乾燥させてから抗腫瘍剤に使用してもよい。乾燥は、凍結乾燥、減圧乾燥、風乾等の任意の乾燥法を用いて行うことができる。但し乾燥時の温度は上記加熱処理の温度（例えば60〜80℃）を超えないか又は下回ることが好ましい。乾燥は、非加熱条件下での乾燥がより好ましく、凍結乾燥がさらに好ましい。加熱処理後の菌体や乾燥させた加熱処理菌体については、さらに粉砕、顆粒化等の処理を施してもよい。 The cells (or cell suspension) after the heat treatment may be used as it is for an antitumor agent, but may be further dried before being used for an antitumor agent. Drying can be performed using any drying method such as freeze-drying, vacuum drying, and air drying. However, it is preferable that the temperature during drying does not exceed or falls below the temperature of the heat treatment (for example, 60 to 80 ° C.). As for drying, drying under non-heating conditions is more preferable, and freeze-drying is more preferable. The cells after the heat treatment and the dried heat-treated cells may be further subjected to treatment such as pulverization and granulation.
以上のようにして調製される、加熱処理を施した菌体又はそれに由来する組成物を、本発明では「加熱処理菌体」と称する。この加熱処理菌体は、通常は加熱死菌体である。本発明に係る加熱処理菌体は、強い腫瘍増殖抑制効果を示すことから、抗腫瘍剤に使用することができる。したがって本発明は、ラクトバチルス・プランタラム菌体を上記のように加熱することにより、ラクトバチルス・プランタラムの加熱処理菌体を調製することを含む、抗腫瘍剤を製造する方法も提供する。本発明はまた、そのような方法によって製造される、ラクトバチルス・プランタラムの加熱処理菌体を含む抗腫瘍剤も提供する。 In the present invention, the microbial cells subjected to heat treatment or a composition derived therefrom are referred to as “heat-treated microbial cells” prepared as described above. This heat-treated cell is usually a heat-killed cell. Since the heat-treated microbial cell according to the present invention exhibits a strong tumor growth inhibitory effect, it can be used as an antitumor agent. Therefore, the present invention also provides a method for producing an antitumor agent, which comprises preparing a heat-treated cell of Lactobacillus plantarum by heating the Lactobacillus plantarum cell as described above. The present invention also provides an antitumor agent comprising heat-treated cells of Lactobacillus plantarum produced by such a method.
本発明に係るラクトバチルス・プランタラムの加熱処理菌体及びそれを含む抗腫瘍剤は、その加熱処理前の生菌体と比較して、増強された腫瘍増殖抑制効果を示す。本発明において増殖抑制の対象となる「腫瘍」は、悪性腫瘍であっても良性腫瘍であってもよいが、悪性腫瘍が特に好ましい。悪性腫瘍としては、線維肉腫、リンパ腫、粘液肉腫、脂肪肉腫、軟骨肉腫、骨肉腫、横紋筋肉腫、平滑筋肉腫、血管肉腫、悪性リンパ腫、腺癌、扁平上皮癌、移行上皮癌などが挙げられるが、これらに限定されない。本発明において増殖抑制の対象となる腫瘍の具体例としては、例えば、肺癌、乳癌、胃癌、腎癌、大腸癌（直腸癌、結腸癌、盲腸癌）、頭頸部癌、脳腫瘍、肝癌、子宮癌（子宮頸癌、子宮体癌）、卵巣癌、甲状腺癌、前立腺癌、食道癌、膵癌、胆道癌、膀胱癌、悪性リンパ腫、骨髄腫、骨肉腫、ユーイング肉腫、皮膚癌、黒色腫、白血病などが挙げられるが、これらに限定されない。 The heat-treated microbial cell of Lactobacillus plantarum according to the present invention and the antitumor agent containing the same show an enhanced tumor growth inhibitory effect as compared to the live microbial cell before the heat treatment. In the present invention, the “tumor” subject to growth inhibition may be a malignant tumor or a benign tumor, but a malignant tumor is particularly preferable. Malignant tumors include fibrosarcoma, lymphoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, rhabdomyosarcoma, leiomyosarcoma, hemangiosarcoma, malignant lymphoma, adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, etc. However, it is not limited to these. Specific examples of tumors targeted for growth inhibition in the present invention include, for example, lung cancer, breast cancer, stomach cancer, kidney cancer, colon cancer (rectal cancer, colon cancer, cecal cancer), head and neck cancer, brain tumor, liver cancer, uterine cancer. (Cervical cancer, endometrial cancer), ovarian cancer, thyroid cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, bladder cancer, malignant lymphoma, myeloma, osteosarcoma, Ewing sarcoma, skin cancer, melanoma, leukemia, etc. However, it is not limited to these.
本発明における「腫瘍増殖抑制効果」は、例えば、後述の実施例に記載の移植腫瘍増殖抑制試験方法に従って評価することができる。簡単に説明すると、マウス（例えば、5週齢のBALB/c系統の雌マウス）に、本発明に係るラクトバチルス・プランタラムの加熱処理菌体を含む生理食塩水を一定期間（例えば3週間）にわたり定期的に（典型的には毎日１回）投与（例えば、経口投与）した後、適当量（例えば、1×106細胞）の腫瘍（例えばMeth-A腫瘍）をマウス鼠経部皮下に移植し、移植後も定期的に（例えば隔日で）加熱処理菌体の経口投与を継続する。腫瘍移植後、経時的に腫瘍のサイズを測定する。具体的には、腫瘍の長径と短径をノギスで計測し、その積の平方根を腫瘍サイズとして算出する。対照群として、本発明に係るラクトバチルス・プランタラムの加熱処理菌体の代わりに生理食塩水を投与して、同様に腫瘍を移植したマウスについても経時的に腫瘍サイズを測定する。このようにして腫瘍サイズを測定し、移植後一定期間経過後（例えば、移植の18日後）、ラクトバチルス・プランタラムの加熱処理菌体を投与したマウスにおいて、対照群と比較して、統計学的に有意に腫瘍サイズの増加率が低減されていれば、腫瘍増殖抑制効果を有すると評価できる。 The “tumor growth inhibitory effect” in the present invention can be evaluated, for example, according to the transplanted tumor growth inhibition test method described in Examples described later. Briefly, mice (for example, 5-week-old BALB / c female mice) are treated with physiological saline containing Lactobacillus plantarum heat-treated cells according to the present invention for a certain period (for example, 3 weeks). Regularly (typically once daily) over a period of time (eg, oral administration), then an appropriate amount (eg, 1 × 10 6 cells) of a tumor (eg, Meth-A tumor) is subcutaneously injected into the mouse vaginal region After the transplantation, oral administration of the heat-treated cells is continued regularly (for example, every other day). After tumor implantation, the size of the tumor is measured over time. Specifically, the major axis and minor axis of the tumor are measured with calipers, and the square root of the product is calculated as the tumor size. As a control group, physiological saline is administered in place of the heat-treated cells of Lactobacillus plantarum according to the present invention, and the tumor size is also measured over time for mice similarly transplanted with tumor. In this way, the tumor size was measured, and after a certain period of time after transplantation (for example, 18 days after transplantation), in mice administered with heat-treated cells of Lactobacillus plantarum, statistics were compared with the control group. If the increase rate of the tumor size is significantly reduced, it can be evaluated that it has a tumor growth inhibitory effect.
本発明に係るラクトバチルス・プランタラムの加熱処理菌体及びそれを含む抗腫瘍剤はまた、その腫瘍増殖抑制効果に関して、極めて幅広い薬効投与量域を有する。本発明において薬効投与量域とは、毒性を示すことなく腫瘍増殖抑制効果をもたらすのに有効である、薬剤の下限用量から上限用量までの範囲を意味する。本発明では、上記の移植腫瘍増殖抑制試験において、有害な影響が観察されることなく、対照群と比較して統計学的に有意な腫瘍サイズ増加率の低減をもたらした用量範囲は、薬効投与量域に含まれるものとする。薬効投与量域は、その幅に基づいて評価することができる。すなわち、薬効投与量域の下限用量に対する上限用量の倍率が大きい程、幅広い投与量で腫瘍増殖抑制効果をもたらすことができて有利である。本発明に係るラクトバチルス・プランタラムの加熱処理菌体及びそれを含む抗腫瘍剤は、以下に限定するものではないが、例えば、35倍以上、好ましくは45倍以上の幅の薬効投与量域を有するものであり得る。特に、低温加熱処理によって得られる本発明に係る加熱処理菌体及びそれを含む抗腫瘍剤は、低用量でも増強された腫瘍増殖抑制効果を発揮することができるため、投与量を低減することができて有利である。本発明に係る加熱処理菌体及びそれを含む抗腫瘍剤の投与量は、ラクトバチルス・プランタラムの加熱処理菌体の乾燥重量で、例えば0.001〜200mg/体重kg/日、例えば0.001〜50mg/体重kg/日又は0.001〜5mg/体重kg/日に低減することができる。 The heat-treated microbial cells of Lactobacillus plantarum according to the present invention and the antitumor agent containing the same also have a very wide medicinal dosage range with respect to the tumor growth inhibitory effect. In the present invention, the medicinal dose range means a range from the lower limit dose to the upper limit dose of a drug that is effective for producing a tumor growth inhibitory effect without showing toxicity. In the present invention, in the transplanted tumor growth inhibition test, the dose range that resulted in a statistically significant reduction in the tumor size increase rate compared to the control group without any adverse effects was observed. It shall be included in the quantity range. The medicinal dose range can be evaluated based on the range. That is, the larger the magnification of the upper limit dose relative to the lower limit dose in the medicinal dose range, the more advantageous it is that a tumor growth inhibitory effect can be achieved with a wider dose. The heat-treated bacterial cells of Lactobacillus plantarum according to the present invention and the antitumor agent containing the same are not limited to the following, but, for example, a medicinal dose range of 35 times or more, preferably 45 times or more It may have. In particular, the heat-treated microbial cells according to the present invention obtained by low-temperature heat treatment and the antitumor agent containing the same can exert an enhanced tumor growth inhibitory effect even at a low dose, so that the dose can be reduced. It is possible and advantageous. The dosage of the heat-treated cells according to the present invention and the antitumor agent containing the same is, for example, 0.001 to 200 mg / kg body weight / day, for example, 0.001 to 50 mg / day, based on the dry weight of the heat-treated cells of Lactobacillus plantarum. It can be reduced to body weight kg / day or 0.001-5 mg / kg body weight / day.
本発明に係るラクトバチルス・プランタラムの加熱処理菌体及びそれを含む抗腫瘍剤はまた、サイトカイン（例えば、TNF-α、IFN-γ及びIL-12）の産生を増強することができる。本発明に係るラクトバチルス・プランタラムの加熱処理菌体及びそれを含む抗腫瘍剤はまた、サイトカイン、特にTNF-αを産生するM1マクロファージの活性化を顕著に促進することができる。本発明に係るラクトバチルス・プランタラムの加熱処理菌体及びそれを含む抗腫瘍剤は、さらに、サイトカイン、特にIL-12を産生するT細胞の活性化を顕著に促進することができる。本発明に係るラクトバチルス・プランタラムの加熱処理菌体及びそれを含む抗腫瘍剤は、そのようなサイトカインの増強により、全身免疫を増強することもできる。 The heat-treated bacterium of Lactobacillus plantarum according to the present invention and the antitumor agent containing the same can also enhance the production of cytokines (eg, TNF-α, IFN-γ and IL-12). The heat-treated cell of Lactobacillus plantarum according to the present invention and the antitumor agent containing the same can also significantly promote the activation of M1, macrophages that produce cytokines, particularly TNF-α. The heat-treated cell of Lactobacillus plantarum according to the present invention and the antitumor agent containing the same can further significantly promote the activation of T cells that produce cytokines, particularly IL-12. The Lactobacillus plantarum heat-treated microbial cell and the antitumor agent containing the same according to the present invention can enhance systemic immunity by enhancing such cytokines.
本発明に係るラクトバチルス・プランタラムの加熱処理菌体及びそれを含む抗腫瘍剤は、予防的な事前の投与により、腫瘍増殖を効果的に抑制することができる。したがって本発明に係るラクトバチルス・プランタラムの加熱処理菌体及びそれを含む抗腫瘍剤は、予防的な投与により、体内で発生した腫瘍細胞の増殖を効果的に抑制できることから、腫瘍（好ましくは癌）の発生を阻止又は遅らせる上で有効である。また本発明に係るラクトバチルス・プランタラムの加熱処理菌体及びそれを含む抗腫瘍剤は、腫瘍増殖を効果的に抑制することにより、腫瘍（好ましくは癌）の進行を遅らせ、化学療法、放射線療法や外科手術等との併用治療の治療成績を向上させる上で有効である。さらに本発明に係るラクトバチルス・プランタラムの加熱処理菌体及びそれを含む抗腫瘍剤は、腫瘍増殖を効果的に抑制することにより、癌の転移を阻止又は遅らせる上でも有効である。 The heat-treated bacterial cells of Lactobacillus plantarum according to the present invention and the antitumor agent containing the same can effectively suppress tumor growth by preventive prior administration. Therefore, the heat-treated bacterial cells of Lactobacillus plantarum according to the present invention and the antitumor agent containing the same can effectively suppress the growth of tumor cells generated in the body by prophylactic administration, and thus tumors (preferably It is effective in preventing or delaying the occurrence of cancer. The Lactobacillus plantarum heat-treated bacterial body and the antitumor agent containing the same according to the present invention effectively suppress tumor growth, thereby delaying the progression of tumor (preferably cancer), chemotherapy, radiation It is effective in improving the therapeutic results of combined treatment with therapy and surgery. Furthermore, the heat-treated bacterial cells of Lactobacillus plantarum according to the present invention and the antitumor agent containing the same are effective in inhibiting or delaying cancer metastasis by effectively suppressing tumor growth.
本発明に係る抗腫瘍剤を被験体に投与することにより、被験体における腫瘍増殖を効果的に抑制し、かつ全身免疫を始めとする宿主免疫反応を増強することができる。本発明に係る抗腫瘍剤の投与対象となる被験体は、ヒト、家畜、愛玩動物、実験（試験）動物等を含む任意の哺乳動物である。特に好ましい被験体の例として、腫瘍（例えば癌）の発生リスクが高い環境に曝されている哺乳動物、腫瘍（例えば癌）の素因を有する哺乳動物、腫瘍（例えば癌）に罹患している哺乳動物、腫瘍（例えば癌）の再発又は転移のリスクがある哺乳動物等が挙げられるが、これらに限定されない。 By administering the antitumor agent according to the present invention to a subject, it is possible to effectively suppress tumor growth in the subject and enhance a host immune response including systemic immunity. The subject to which the antitumor agent according to the present invention is administered is any mammal including humans, domestic animals, pets, experimental (test) animals and the like. Examples of particularly preferred subjects include mammals that are exposed to an environment where the risk of developing a tumor (eg, cancer) is high, mammals that are predisposed to tumors (eg, cancer), and mammals suffering from a tumor (eg, cancer) Examples include, but are not limited to, animals, mammals that are at risk of tumor recurrence or metastasis, and the like.
本発明に係る抗腫瘍剤の投与量は、投与対象となる被験体の年齢及び体重、投与経路、投与回数により異なり、当業者の裁量によって広範囲に変更することができる。例えば、本発明に係る抗腫瘍剤を経口的に投与する場合には、本発明に係るラクトバチルス・プランタラムの加熱処理菌体の乾燥重量で、通常は0.001〜1000mg/体重kg/日、典型的には0.001〜200mg/体重kg/日、例えば0.001〜50mg/体重kg/日又は0.001〜5mg/体重kg/日となる量で投与することが好ましい。ヒトの場合、本発明に係るラクトバチルス・プランタラムの加熱処理菌体の乾燥重量で、例えば0.1mg〜20g/日、典型的には0.1mg〜200mg/日、より低用量では0.1mg〜50mg/日又は0.1mg〜1mg/日の投与が好ましい。本発明に係る抗腫瘍剤に含まれる加熱処理菌体は、上述のように幅広い薬効投与量域を有する。本発明に係る抗腫瘍剤は、単回投与でもよいが、反復的に複数回投与することが好ましく、例えば、5〜72時間の間隔で反復的に投与することが好ましいが、これに限定されない。本発明は、本発明に係るラクトバチルス・プランタラムの加熱処理菌体を含む抗腫瘍剤を上記のような被験体に投与することを含む、腫瘍増殖を抑制する方法も提供する。本発明はまた、本発明に係るラクトバチルス・プランタラムの加熱処理菌体を含む抗腫瘍剤を上記のような被験体に投与することを含む、腫瘍（例えば癌）の治療又は予防方法も提供する。このような腫瘍（例えば癌）の治療又は予防方法では、化学療法、放射線療法や外科手術等を併用することが好ましい。ここで腫瘍は、良性腫瘍又は悪性腫瘍（癌）である。本発明において腫瘍の「予防」とは、検出可能な腫瘍の発生を阻止若しくは遅延させること、又は腫瘍細胞の転移を阻止若しくは遅延させることを意味する。本発明において腫瘍の「治療」とは、腫瘍サイズの増加を阻止若しくは遅延させるか又は腫瘍サイズを低減させる（腫瘍の消滅を含む）ことを意味する。 The dose of the antitumor agent according to the present invention varies depending on the age and weight of the subject to be administered, the route of administration, and the number of administrations, and can be widely changed at the discretion of those skilled in the art. For example, when the antitumor agent according to the present invention is administered orally, the dry weight of the heat-treated bacterial cell of Lactobacillus plantarum according to the present invention, usually 0.001 to 1000 mg / kg body weight / day, typically Specifically, the dose is preferably 0.001 to 200 mg / kg body weight / day, for example, 0.001 to 50 mg / kg body weight / day or 0.001 to 5 mg / kg body weight / day. In the case of humans, the dry weight of the heat-treated cells of Lactobacillus plantarum according to the present invention is, for example, 0.1 mg to 20 g / day, typically 0.1 mg to 200 mg / day, and 0.1 mg to 50 mg at lower doses. / Day or 0.1 mg to 1 mg / day is preferred. The heat-treated microbial cells contained in the antitumor agent according to the present invention have a wide medicinal dosage range as described above. The antitumor agent according to the present invention may be administered once, but is preferably repeatedly administered multiple times, for example, it is preferably repeatedly administered at intervals of 5 to 72 hours, but is not limited thereto. . The present invention also provides a method for suppressing tumor growth, comprising administering an antitumor agent comprising a heat-treated bacterial cell of Lactobacillus plantarum according to the present invention to the subject as described above. The present invention also provides a method for treating or preventing a tumor (for example, cancer), which comprises administering an antitumor agent comprising a heat-treated bacterial cell of Lactobacillus plantarum according to the present invention to the subject as described above. To do. In such a method for treating or preventing a tumor (for example, cancer), it is preferable to use chemotherapy, radiation therapy, surgery, or the like in combination. Here, the tumor is a benign tumor or a malignant tumor (cancer). In the present invention, “prevention” of a tumor means preventing or delaying the development of a detectable tumor, or preventing or delaying metastasis of tumor cells. In the present invention, “treatment” of a tumor means preventing or delaying an increase in tumor size or reducing a tumor size (including tumor disappearance).
本発明は、本発明に係るラクトバチルス・プランタラムの加熱処理菌体又はそれを含む抗腫瘍剤を含む飲食品も提供する。本発明において「飲食品」とは、限定するものではないが、飲料及び食品を包含する。 This invention also provides the food-drinks containing the heat-processed microbial cell of the Lactobacillus plantarum which concerns on this invention, or the antitumor agent containing it. In the present invention, “food or drink” includes, but is not limited to, beverages and foods.
本発明に係る飲食品は、本発明に係るラクトバチルス・プランタラムの加熱処理菌体又はそれを含む抗腫瘍剤に加えて、食品分野で通常用いられる他の食材や食品添加物を含んでもよい。本発明に係る飲食品は、例えば、水、タンパク質、糖質、脂質、ビタミン類、ミネラル類、有機酸、有機塩基、果汁、フレーバー類等を含んでもよい。本発明に係る飲食品はまた、食品サプリメント等の調剤に使用される製剤補助剤（例えば、希釈剤、賦形剤、保存剤、結合剤、崩壊剤、滑沢剤、着色剤、矯味矯臭剤、溶解補助剤、懸濁化剤、コーティング剤等）を含んでいてもよい。本発明に係る飲食品は、固体、液体、懸濁液、ペースト、ゲル状、粉末、顆粒、カプセル等の任意の食品形態であってよい。 The food and drink according to the present invention may contain other foods and food additives usually used in the food field, in addition to the heat-treated cells of Lactobacillus plantarum according to the present invention or an antitumor agent containing the same. . The food and drink according to the present invention may contain, for example, water, protein, carbohydrate, lipid, vitamins, minerals, organic acid, organic base, fruit juice, flavors and the like. The food and drink according to the present invention also includes formulation adjuvants (eg, diluents, excipients, preservatives, binders, disintegrants, lubricants, colorants, flavoring agents) used in preparations such as food supplements. , Dissolution aids, suspending agents, coating agents, etc.). The food and drink according to the present invention may be in any form of food such as solid, liquid, suspension, paste, gel, powder, granule, capsule and the like.
本発明に係る飲食品は、菓子、サプリメント、惣菜、調味料、清涼飲料、冷凍食品、流動食、病者用食品等の任意の形態の飲食品であってよい。本発明に係る飲食品は、機能性食品であってもよい。本発明において「機能性食品」は、生体に対して一定の機能性を有する食品を意味し、例えば、特定保健用食品（条件付きトクホ[特定保健用食品]を含む）及び栄養機能食品を含む保健機能食品、特別用途食品、栄養補助食品、健康補助食品、サプリメント製品（例えば、錠剤、被覆錠、糖衣錠、カプセル及び液剤などの各種剤形のもの）及び美容食品（例えばダイエット食品）などのいわゆる健康食品全般を包含する。本発明の機能性食品はまた、コーデックス（FAO/WHO合同食品規格委員会）の食品規格に基づく健康強調表示（Health claim）が適用される健康食品を包含する。本発明に係る飲食品は、腫瘍増殖抑制用の飲食品（例えば、機能性食品）であってもよい。 The food and drink according to the present invention may be any form of food and drink such as confectionery, supplements, side dishes, seasonings, soft drinks, frozen foods, liquid foods, and foods for the sick. The food or drink according to the present invention may be a functional food. In the present invention, “functional food” means food having a certain functionality with respect to a living body, and includes, for example, food for specified health use (including conditional tokuho [food for specified health use]) and nutritional function food. So-called functional health foods, special-purpose foods, dietary supplements, health supplements, supplement products (for example, tablets, coated tablets, dragees, capsules, liquids, etc.) and beauty foods (for example, diet foods) Includes all health foods. The functional food of the present invention also includes a health food to which a health claim based on the food standards of Codex (FAO / WHO Joint Food Standards Committee) is applied. The food / beverage products according to the present invention may be a food / beverage product for inhibiting tumor growth (for example, a functional food product).
本発明に係るラクトバチルス・プランタラムの加熱処理菌体又はそれを含む抗腫瘍剤の飲食品への配合量は特に限定されず、場合に応じて様々であってよい。具体的な配合量は、飲食品の種類や求める味や食感を考慮して、当業者が適宜定めることができる。一般的には、添加される加熱処理菌体の総量で、0.001〜99質量％、例えば0.1〜80質量％となるような配合量が用いられる。本発明に係る飲食品の一日の摂取量は、上記の抗腫瘍剤の投与量に従って定められるが、比較的低用量となる量が好ましい。 The compounding quantity to the food-drinks of the heat-treated microbial cell of the Lactobacillus plantarum which concerns on this invention or the antitumor agent containing it is not specifically limited, According to the case, it may be various. Specific blending amounts can be appropriately determined by those skilled in the art in consideration of the type of food and drink, the desired taste and texture. Generally, the blending amount is 0.001 to 99% by mass, for example, 0.1 to 80% by mass, based on the total amount of heat-treated cells to be added. The daily intake of the food and drink according to the present invention is determined according to the dose of the antitumor agent described above, but an amount that is relatively low is preferable.
本発明はまた、本発明に係るラクトバチルス・プランタラムの加熱処理菌体又はそれを含む抗腫瘍剤を含む医薬も提供する。本発明に係る医薬は、製剤分野において通常使用される任意の製剤補助剤を含んでもよい。製剤補助剤としては、製薬上許容される、不活性担体（固体又は液体担体）、賦形剤、界面活性剤、結合剤、崩壊剤、滑沢剤、矯臭剤、溶解補助剤、懸濁剤、コーティング剤、着色剤、矯味剤、保存剤、緩衝剤等の、様々な薬物担体又は添加剤を用いることができる。具体的には、水、他の水性溶媒、製薬上許容される有機溶媒、カルシウム、コラーゲン、ポリビニルアルコール、ポリビニルピロリドン、カルボキシビニルポリマー、アルギン酸ナトリウム、水溶性デキストラン、水溶性デキストリン、カルボキシメチルスターチナトリウム、ペクチン、キサンタンガム、アラビアゴム、カゼイン、ゼラチン、寒天、グリセリン、プロピレングリコール、ポリエチレングリコール、ワセリン、パラフィン、ステアリルアルコール、ステアリン酸、ヒト血清アルブミン、マンニトール、ソルビトール、ラクトースなどの他、リポゾームなどの人工細胞構造物等も挙げられる。製剤補助剤は、製剤の剤形に応じて適宜又は組み合わせて選択されうる。本発明に係る医薬はまた、適当量のビタミン、ミネラル、有機酸、糖類、アミノ酸、ペプチド類などを含んでもよい。 The present invention also provides a medicament comprising the heat-treated cell of Lactobacillus plantarum according to the present invention or an antitumor agent comprising the same. The medicament according to the present invention may contain any formulation adjuvant normally used in the pharmaceutical field. As formulation aids, pharmaceutically acceptable inert carriers (solid or liquid carriers), excipients, surfactants, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents Various drug carriers or additives such as coating agents, colorants, flavoring agents, preservatives, and buffering agents can be used. Specifically, water, other aqueous solvents, pharmaceutically acceptable organic solvents, calcium, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, Artificial cell structures such as pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, and liposomes A thing etc. are also mentioned. The formulation adjuvant can be selected appropriately or in combination depending on the dosage form of the formulation. The medicament according to the present invention may also contain appropriate amounts of vitamins, minerals, organic acids, sugars, amino acids, peptides and the like.
本発明に係る医薬は、錠剤、顆粒剤、散剤、丸剤、カプセル剤などの固形製剤、ジェル剤、又は液剤、懸濁剤、シロップ剤などの液体製剤等の剤形であってよい。液体製剤として用いる場合には、本発明の医薬組成物を使用する際に再溶解させることを意図した乾燥物として供給してもよい。本発明に係る医薬は、経口的又は非経口的に投与することができるが、特に経口的に投与することが好ましい。 The medicament according to the present invention may be in the form of solid preparations such as tablets, granules, powders, pills and capsules, gel preparations, or liquid preparations such as liquid preparations, suspensions and syrups. When used as a liquid preparation, when using the pharmaceutical composition of the present invention, it may be supplied as a dry product intended to be redissolved. The medicament according to the present invention can be administered orally or parenterally, but is preferably administered orally.
本発明に係る抗腫瘍剤を含む医薬は、高い腫瘍増殖抑制効果、及び全身免疫の増強効果を有する。したがって本発明は、腫瘍増殖抑制用の医薬も提供する。本発明に係る腫瘍増殖抑制用の医薬は、例えば、腫瘍（好ましくは癌）の予防又は治療用に用いることができる。 The medicament containing the antitumor agent according to the present invention has a high tumor growth inhibitory effect and a systemic immunity enhancing effect. Therefore, the present invention also provides a medicament for suppressing tumor growth. The medicament for suppressing tumor growth according to the present invention can be used, for example, for prevention or treatment of tumor (preferably cancer).
以下、実施例を用いて本発明をさらに具体的に説明する。但し、本発明の技術的範囲はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
［実施例１］ 組成物の調製
1白金耳量の植物発酵物由来乳酸菌ラクトバチルス・プランタラム（Lactobacillus plantarum）ALAL006株を、2mlのMRS（de MAN, ROGOSA, SHARPE）液体培地（組成：培地1L当たり、ペプトン10.0g、ラブ−レムコ末（肉エキス）8.0g、酵母エキス4.0g、ブドウ糖20.0g、モノオレイン酸ソルビタン1.0ml、リン酸水素二カリウム2.0g、酢酸ナトリウム三水和物5.0g、クエン酸トリアンモニウム2.0g、硫酸マグネシウム七水和物0.2g、及び硫酸マンガン四水和物0.05g、pH 6.2±0.2；関東科学株式会社、東京）に接種し、37℃で24時間培養して前培養液を調製した。この前培養液を1Lの本培養培地（上記と同じMRS液体培地）に接種（1×106〜1×107/ml）し、37℃で20時間培養した。培養終了後、冷却遠心機にて集菌し、生理食塩水にて洗浄後、菌体ペレットを滅菌蒸留水90mlに懸濁した。得られた菌懸濁液の30mlをそのまま凍結乾燥することにより生菌体乾燥物を調製した（サンプルP1）。また菌懸濁液の30mlを115℃にて15分、又は70℃にて15分加熱殺菌した後、凍結乾燥することにより、加熱死菌体を含む組成物（それぞれサンプルP2、P3）を得た。この加熱処理は、具体的には、菌懸濁液の30mlを100ml三角フラスコに入れ、オートクレーブ滅菌器にて115℃で15分加熱することにより、又は、菌懸濁液の30mlを100ml三角フラスコに入れ、湯浴中でマグネットスターラーにて撹拌しながら菌懸濁液の温度が70℃に達してから15分加熱することにより、実施した。凍結乾燥後の組成物の重量はいずれも培養液1L当たり2.2gであった。
[Example 1] Preparation of composition
1 Lactobacillus plantarum lactic acid bacterium Lactobacillus plantarum ALAL006 strain derived from a plant fermented product with a platinum loop volume of 2 ml of MRS (de MAN, ROGOSA, SHARPE) liquid medium (composition: 10.0 g peptone per liter of medium, Lab-Remco) Powder (meat extract) 8.0g, yeast extract 4.0g, glucose 20.0g, sorbitan monooleate 1.0ml, dipotassium hydrogen phosphate 2.0g, sodium acetate trihydrate 5.0g, triammonium citrate 2.0g, magnesium sulfate 0.2 g of heptahydrate and 0.05 g of manganese sulfate tetrahydrate, pH 6.2 ± 0.2; Kanto Kagaku Co., Ltd., Tokyo) were inoculated and cultured at 37 ° C. for 24 hours to prepare a preculture solution. This preculture was inoculated (1 × 10 6 to 1 × 10 7 / ml) into a 1 L main culture medium (the same MRS liquid medium as described above), and cultured at 37 ° C. for 20 hours. After completion of the culture, the cells were collected with a cooling centrifuge, washed with physiological saline, and the cell pellet was suspended in 90 ml of sterile distilled water. 30 ml of the obtained bacterial suspension was lyophilized as it was to prepare a dry cell of live cells (sample P1). In addition, a composition containing heated dead cells (samples P2 and P3, respectively) was obtained by sterilizing 30 ml of the bacterial suspension at 115 ° C for 15 minutes or 70 ° C for 15 minutes and then freeze-drying. It was. Specifically, 30 ml of the bacterial suspension is placed in a 100 ml Erlenmeyer flask and heated in an autoclave sterilizer at 115 ° C. for 15 minutes, or 30 ml of the bacterial suspension is added to the 100 ml Erlenmeyer flask. It was carried out by heating for 15 minutes after the temperature of the bacterial suspension reached 70 ° C. while stirring with a magnetic stirrer in a hot water bath. The weight of the composition after lyophilization was 2.2 g per liter of the culture solution.
［実施例２］ 移植腫瘍増殖抑制試験
5週齢のBALB/c系統の雌マウス（平均体重およそ16g）を対照群、P1投与群（0.2mg群、10mg群）、P2投与群（0.2mg群、10mg群）及びP3投与群（0.2mg群、10mg群）の各群にそれぞれ6匹ずつ割りつけた。投与群のマウスに与えるP1、P2及びP3懸濁液は、実施例１で調製したサンプルP1、P2及びP3のそれぞれについて10mg又は0.2 mgを生理食塩液0.2mLに混合することにより調製した。各投与群のマウスに、それぞれ指定された懸濁液（10mg又は0.2 mgのP1、P2又はP3/0.2mL）を3週間にわたり毎日１回ゾンデを用いて経口投与した。投与期間最終日の翌日に、マウス線維肉腫Meth-A（東北大学細胞センターより供与）1×106細胞をマウスの鼠経部皮下に移植した。移植後は隔日で、ゾンデを用いた各指定懸濁液の経口投与を継続した。対照群のマウスには、懸濁液に代えて生理食塩液（0.2mL）を用いること以外は、投与群と同じスケジュールで同様に経口投与及び移植を行った。腫瘍移植後、経時的に腫瘍の長径と短径をノギスで計測し、その積の平方根を腫瘍サイズとして算出し、そこから腫瘍増殖の推移を評価した。得られたデータについては、一元配置分散分析の後、Tukey又はDunnnetの多重比較検定を行った。統計的有意水準は5％未満とした。
[Example 2] Transplant tumor growth inhibition test
Five-week-old BALB / c strain female mice (average body weight approximately 16 g) were used as control group, P1 administration group (0.2 mg group, 10 mg group), P2 administration group (0.2 mg group, 10 mg group) and P3 administration group (0.2 6 animals were assigned to each group (mg group, 10 mg group). The P1, P2 and P3 suspensions given to the mice of the administration group were prepared by mixing 10 mg or 0.2 mg of each of the samples P1, P2 and P3 prepared in Example 1 with 0.2 mL of physiological saline. Mice of each administration group were orally administered with a designated suspension (10 mg or 0.2 mg of P1, P2 or P3 / 0.2 mL) once a day for 3 weeks using a sonde. The day after the last day of the administration period, mouse fibrosarcoma Meth-A (provided by Tohoku University Cell Center) 1 × 10 6 cells were transplanted subcutaneously into the vaginal region of the mouse. Every other day after transplantation, oral administration of each designated suspension using a sonde was continued. The mice in the control group were orally administered and transplanted in the same manner as in the administration group except that physiological saline (0.2 mL) was used instead of the suspension. After tumor transplantation, the major axis and minor axis of the tumor were measured over time with calipers, the square root of the product was calculated as the tumor size, and the transition of tumor growth was evaluated therefrom. The obtained data were subjected to Tukey or Dunnnet's multiple comparison test after one-way analysis of variance. Statistical significance level was less than 5%.
結果を図１に示す。図１Ａに示されるとおり、10mg/日の投与量の場合、P2、P3投与群では高い腫瘍増殖抑制効果が認められたが（対照群に対してp<0.01）、P1投与群では有意な腫瘍増殖抑制作用は認められなかった。一方、図１Ｂに示されるとおり、0.2mg/日の投与量の場合、P3投与群では高い腫瘍増殖抑制効果が認められたが（対照群に対してp<0.05)、P1投与群、P2投与群では有意な腫瘍増殖抑制作用は認められなかった。なお10mg/日及び0.2mg/日のいずれの投与量でも、マウスにおいて有害な影響は観察されなかった。 The results are shown in FIG. As shown in FIG. 1A, when the dose was 10 mg / day, a high tumor growth inhibitory effect was observed in the P2 and P3 administration groups (p <0.01 compared to the control group), but a significant tumor was observed in the P1 administration group. No growth inhibitory effect was observed. On the other hand, as shown in FIG. 1B, when the dose was 0.2 mg / day, a high tumor growth inhibitory effect was observed in the P3 administration group (p <0.05 compared to the control group), but the P1 administration group and the P2 administration There was no significant tumor growth inhibitory effect in the group. No adverse effects were observed in mice at any dose of 10 mg / day and 0.2 mg / day.
この結果は、生菌体（P1）では腫瘍増殖抑制効果が認められなかったにもかかわらず、加熱処理（P2、P3）することにより腫瘍増殖抑制効果が付与されたこと、さらに、比較的低温で加熱処理（P3）することにより、腫瘍増殖抑制効果がさらに増強され、高用量だけでなく低用量でも腫瘍増殖抑制効果を得られたことを示している。上記の結果は、サンプルP3の薬効投与量域が、少なくとも50倍の幅を有することも示している。すなわち、乳酸菌を低温加熱処理することにより、生菌体と比較して腫瘍増殖抑制効果を増強することができ、また腫瘍増殖抑制に有効な投与量（薬効投与量域）をより広範囲に拡張できることが示された。 This result shows that the tumor growth inhibitory effect was imparted by heat treatment (P2, P3) despite the fact that the live cell body (P1) did not show the tumor growth inhibitory effect. It was shown that the tumor growth inhibitory effect was further enhanced by the heat treatment (P3), and that the tumor growth inhibitory effect was obtained not only at a high dose but also at a low dose. The above results also show that the medicinal dose range of sample P3 has a width of at least 50 times. In other words, low-temperature heat treatment of lactic acid bacteria can enhance the tumor growth inhibitory effect compared to viable bacterial cells, and can expand the dose (effective dose range) effective for tumor growth inhibition to a wider range. It has been shown.
［実施例３］ 宿主免疫能の変化
サンプルP1、P2及びP3の投与による宿主免疫能の変化を抗腫瘍中和試験Winn assay（Saito M, et al., (1984) Int. J. Cancer, 33: p.271-276）を用いて評価した。
5週齢のBALB/c系統の雌マウスを対照群、P1投与群、P2投与群及びP3投与群の各群にそれぞれ6匹ずつ割りつけた。P1、P2及びP3投与群のマウスには、実施例２と同様にして調製した懸濁液（10mgのP1、P2又はP3/0.2mL/日）を3週間にわたり毎日１回ゾンデを用いて経口投与した。投与期間最終日の翌日に、Meth-A腫瘍1×106細胞をマウスの鼠経部皮下に移植した。対照群のマウスには、懸濁液に代えて生理食塩液（0.2mL）を用いること以外は、投与群と同じスケジュールで同様に経口投与及び移植を行った。腫瘍移植後20日目に各群それぞれのマウスから採取した脾細胞1×107細胞と新たなMeth-A腫瘍1×106細胞を混和（脾細胞数：腫瘍細胞数＝30：1）し、それぞれ新たな6匹の5週齢のBALB/cマウスの鼠経部皮下に移植した。さらに、Meth-A腫瘍単独移植群として、マウス脾細胞と混和することなくMeth-A腫瘍単独を、6匹の5週齢のBALB/c雌マウスの鼠経部皮下に移植した。これらの腫瘍移植後、経時的に腫瘍の長径と短径をノギスで計測し、その積の平方根を腫瘍サイズとして算出し、そこから腫瘍増殖の推移を評価した。得られたデータについては、一元配置分散分析の後、Tukeyの多重比較検定を行った。統計的有意水準は5％未満とした。
[Example 3] Changes in host immunity The changes in host immunity caused by administration of samples P1, P2 and P3 were determined by antitumor neutralization test Winn assay (Saito M, et al., (1984) Int. J. Cancer, 33 : p.271-276).
Six 5-week-old BALB / c female mice were assigned to each of the control group, the P1 administration group, the P2 administration group, and the P3 administration group. For mice in the P1, P2 and P3 groups, a suspension (10 mg of P1, P2 or P3 / 0.2 mL / day) prepared in the same manner as in Example 2 was orally administered once a day for 3 weeks using a sonde. Administered. The day after the last day of the administration period, Meth-A tumor 1 × 10 6 cells were implanted subcutaneously in the vagina of the mouse. The mice in the control group were orally administered and transplanted in the same manner as in the administration group except that physiological saline (0.2 mL) was used instead of the suspension. On the 20th day after tumor transplantation, spleen cells 1 × 10 7 cells collected from each group of mice and new Meth-A tumor 1 × 10 6 cells were mixed (spleen cell number: tumor cell number = 30: 1). Each was transplanted subcutaneously into the vagina of six new 5-week-old BALB / c mice. Further, as a Meth-A tumor single transplant group, Meth-A tumor alone was transplanted subcutaneously into the vagina of six 5-week-old BALB / c female mice without mixing with mouse spleen cells. After transplanting these tumors, the major axis and minor axis of the tumor were measured with a caliper over time, and the square root of the product was calculated as the tumor size, from which the transition of tumor growth was evaluated. The obtained data were subjected to Tukey's multiple comparison test after one-way analysis of variance. Statistical significance level was less than 5%.
結果を図２に示す。図２に示される通り、P1、P2、P3投与群では、Meth-A腫瘍単独移植群と比較して有意な腫瘍増殖抑制効果を示したが、対照群では有意差が認められなかった。またP3投与群では、対照群、P1群と比較して有意な腫瘍増殖抑制効果が認められた。
以上の結果は、低温加熱処理物（P3）の投与により、脾細胞中に強い腫瘍増殖抑制作用を有する免疫細胞群を誘導できたことを示している。
The results are shown in FIG. As shown in FIG. 2, the P1, P2, and P3 administration groups showed a significant tumor growth inhibitory effect as compared to the Meth-A tumor single transplant group, but no significant difference was observed in the control group. In addition, a significant tumor growth inhibitory effect was observed in the P3 administration group compared to the control group and the P1 group.
The above results indicate that administration of the low-temperature heat-treated product (P3) can induce an immune cell group having a strong tumor growth inhibitory effect in spleen cells.
［実施例４］ マウスパイエル氏板細胞におけるサイトカイン産生能に対する効果
経口投与した乳酸菌は、消化管のパイエル氏板の主にマクロファージに取り込まれ、宿主免疫を賦活すると考えられている（Hiramatsu Y., et al., Cytotechnology (2011) 63:307-317; Mowat A. M. and Bain C.C., J. Innate Immun., (2011) 3:550-564）。そこで、サンプルP1、P2及びP3がパイエル氏板の免疫細胞に及ぼす影響を、サイトカイン（TNF-α、IL-10、及びIFN-γ）産生誘導能に基づいて検討した。
[Example 4] Effect on cytokine-producing ability in mouse Peyer's plate cells Orally administered lactic acid bacteria are thought to be taken up mainly by macrophages in Peyer's plate of the digestive tract and activate host immunity (Hiramatsu Y., et al., Cytotechnology (2011) 63: 307-317; Mowat AM and Bain CC, J. Innate Immun., (2011) 3: 550-564). Therefore, the influence of samples P1, P2 and P3 on Peyer's plaque immune cells was examined based on the ability to induce cytokine (TNF-α, IL-10, and IFN-γ) production.
具体的には、6週齢のBALB/c系統の雌マウスのパイエル氏板を採取し、コラゲナーゼ処理（コラゲナーゼ1mg/ml、37℃で1時間）した後、5％ウシ胎児血清（FCS）添加RPMI培地にて細胞浮遊液（2.5×106細胞/ml）を調製した。得られた細胞浮遊液に、実施例１で調製したサンプルP1、P2、又はP3を最終濃度0.1μg/ml（TNF-α、IL-10測定群）又は1μg/ml（IFN-γ測定群）で添加し、5％CO2下、37℃で3日間（IFN-γ測定群）又は7日間（TNF-α測定群及びIL-10測定群）培養した後、上清中のサイトカイン濃度（TNF-α、IL-10、又はIFN-γ）をELISA法で測定した。測定結果に基づき、抗腫瘍作用を誘導するM1マクロファージ及び免疫抑制作用を誘導するM2マクロファージの活性化（Hiramatsu Y., et al., Cytotechnology (2011) 63:307-317; Mowat A. M. and Bain C.C., J. Innate Immun., (2011) 3:550-564）を、それぞれTNF-α産生能とIL-10産生能で評価した（図３）。同様に、測定結果に基づき、抗腫瘍作用を誘導するT細胞の活性化をIFN-γ産生能で評価した（図４）。 Specifically, Peyer's plaques of 6-week-old BALB / c female mice were collected, treated with collagenase (collagenase 1 mg / ml, 1 hour at 37 ° C.), and then added with 5% fetal calf serum (FCS) A cell suspension (2.5 × 10 6 cells / ml) was prepared in RPMI medium. In the obtained cell suspension, the sample P1, P2, or P3 prepared in Example 1 was added at a final concentration of 0.1 μg / ml (TNF-α, IL-10 measurement group) or 1 μg / ml (IFN-γ measurement group). And cultured at 37 ° C. for 3 days (IFN-γ measurement group) or 7 days (TNF-α measurement group and IL-10 measurement group) under 5% CO 2 , and then the cytokine concentration in the supernatant (TNF -α, IL-10, or IFN-γ) was measured by ELISA. Based on the measurement results, activation of M1 macrophages that induce antitumor action and M2 macrophages that induce immunosuppressive action (Hiramatsu Y., et al., Cytotechnology (2011) 63: 307-317; Mowat AM and Bain CC, J. Innate Immun., (2011) 3: 550-564) was evaluated by TNF-α production ability and IL-10 production ability, respectively (FIG. 3). Similarly, based on the measurement results, activation of T cells that induce antitumor effects was evaluated by IFN-γ production ability (FIG. 4).
図３Ａに示される通り、TNF-αはP3刺激によってのみ産生された。したがって、サンプルP3は抗腫瘍作用を誘導するM1マクロファージを強く誘導（活性化）することが示された。一方、IL-10産生能についてはサンプルP1〜P3間で違いが認められなかったことから、サンプルP3は免疫抑制作用を誘導するM2マクロファージの活性化に対しては影響を及ぼさないことが示された。さらに、図４に示される通り、P3刺激はIFN-γ産生能を増強させたことから、サンプルP3は抗腫瘍作用を誘導するT細胞を強く誘導（活性化）することが示された。 As shown in FIG. 3A, TNF-α was produced only by P3 stimulation. Therefore, sample P3 was shown to strongly induce (activate) M1 macrophages that induce antitumor effects. On the other hand, no difference was observed between samples P1 and P3 in terms of IL-10 production ability, indicating that sample P3 has no effect on the activation of M2 macrophages that induce immunosuppressive action. It was. Furthermore, as shown in FIG. 4, since P3 stimulation enhanced IFN-γ production ability, it was shown that sample P3 strongly induces (activates) T cells that induce antitumor action.
［実施例５］ マウス脾臓細胞におけるサイトカイン産生能に対する効果
サンプルP1、P2及びP3の投与が全身免疫に及ぼす影響を、脾臓細胞におけるサイトカイン（IL-12及びIFN-γ）産生能で評価した。IL-12はマクロファージや樹状細胞などから産生され、NK細胞の活性化やリンパ球からのIFN-γの産生を促進する。IFN-γには抗腫瘍作用やNK細胞活性化作用がある。いずれもNK細胞活性化を始めとする全身免疫の活性化において大きな役割を果たしている。
[Example 5] Effect on cytokine production ability in mouse spleen cells The effect of administration of samples P1, P2 and P3 on systemic immunity was evaluated by cytokine (IL-12 and IFN-γ) production ability in spleen cells. IL-12 is produced from macrophages and dendritic cells, and promotes activation of NK cells and production of IFN-γ from lymphocytes. IFN-γ has antitumor and NK cell activation effects. Both play a major role in the activation of systemic immunity including NK cell activation.
具体的には、まず、6週齢のBALB/c系統の雌マウスから採取した脾臓を、5％ウシ胎児血清（FCS）添加RPMI培地中で200メッシュのナイロンスクリーン上にのせ、1mlシリンジのプランジャーを用いて破砕して細胞浮遊液を調製することにより、脾臓細胞を調製した（2.5×106細胞/ml）。得られた脾臓細胞の細胞浮遊液に、実施例１で調製したサンプルP1、P2、又はP3を最終濃度1μg/ml（IL-12測定群）又は0.01μg/ml（IFN-γ測定群）で添加し、5％CO2下、37℃で72時間培養した後、上清中のサイトカイン濃度（IL-12、又はIFN-γ）をELISA法で測定した。 Specifically, first, the spleen collected from a 6-week-old BALB / c female mouse was placed on a 200-mesh nylon screen in RPMI medium supplemented with 5% fetal calf serum (FCS), and the 1 ml syringe plan Spleen cells were prepared by crushing using a jar to prepare a cell suspension (2.5 × 10 6 cells / ml). In the obtained spleen cell suspension, the sample P1, P2, or P3 prepared in Example 1 was added at a final concentration of 1 μg / ml (IL-12 measurement group) or 0.01 μg / ml (IFN-γ measurement group). After adding and culturing at 37 ° C. for 72 hours under 5% CO 2 , the cytokine concentration (IL-12 or IFN-γ) in the supernatant was measured by ELISA.
その結果、P3刺激はP1刺激の約5倍高いIL-12産生能（図５Ａ）、またP1刺激の約1.6倍高いIFN-γ産生能（図５Ｂ）を示した。この結果から、サンプルP3は、全身免疫を増強し、抗腫瘍効果をより高めたことが示された。 As a result, P3 stimulation showed IL-12 production ability (FIG. 5A) about 5 times higher than P1 stimulation and IFN-γ production ability (FIG. 5B) about 1.6 times higher than P1 stimulation. From this result, it was shown that sample P3 enhanced systemic immunity and further enhanced the antitumor effect.
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