JP6820567B2 - Cancer treatment - Google Patents
Cancer treatment Download PDFInfo
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- JP6820567B2 JP6820567B2 JP2019135796A JP2019135796A JP6820567B2 JP 6820567 B2 JP6820567 B2 JP 6820567B2 JP 2019135796 A JP2019135796 A JP 2019135796A JP 2019135796 A JP2019135796 A JP 2019135796A JP 6820567 B2 JP6820567 B2 JP 6820567B2
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- Prior art keywords
- cddp
- cells
- cancer
- cafca
- cisplatin
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Classifications
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/243—Platinum; Compounds thereof
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
- A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
本発明は、有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤と組み合わせたことを特徴とする癌治療剤及び癌治療方法に関する。
本出願は、参照によりここに援用されるところの日本特願2016-203793号及び日本特願2017-061056号の優先権を請求する。
The present invention relates to a cancer therapeutic agent and a cancer treating method, which are characterized in that caffeine citrate, which is an active ingredient, is combined with an anticancer agent, which is an active ingredient.
This application claims the priority of Japanese Patent Application No. 2016-203793 and Japanese Patent Application No. 2017-061056, which are incorporated herein by reference.
(骨肉腫)
骨肉腫は、主に10歳代若年者に好発する原発生悪性骨腫瘍として最も頻度の高いものであるが、発生原因は不明である。日本における発生頻度は100万人に対して1人の発生率(1年間に130人から260人)といわれている。かつて切断術が唯一の治療選択肢であった時代、骨肉腫の2年生存率は15〜20%であり、非常に予後の悪い疾患であった。
しかし、化学療法の導入・進歩や画像診断及び手術療法の発達に伴い、現在では5年生存率が70%以上に向上している(非特許文献1)。本発明者らは、「カフェイン併用化学療法」を開示している。この療法により、初診時に転移のない治療完遂例では、5年生存率は89%と飛躍的に向上したが、依然として予後の改善が不十分であるため、更なる治療効果の向上が必要である(非特許文献2)。また、再発例や初診時より転移を認めている例では、5年生存率は10〜30%であり、これは過去40年間、顕著な改善はされておらず、この点においても更なる治療効果の向上が必要であると考える(非特許文献3)。
(Osteosarcoma)
Osteosarcoma is the most common primary malignant bone tumor that occurs mainly in young people in their teens, but the cause is unknown. The frequency of occurrence in Japan is said to be 1 in 1 million (130 to 260 per year). In the days when amputation was once the only treatment option, the 2-year survival rate for osteosarcoma was 15-20%, a very poor prognosis.
However, with the introduction and progress of chemotherapy and the development of diagnostic imaging and surgical therapy, the 5-year survival rate has now improved to 70% or more (Non-Patent Document 1). The present inventors disclose "chemotherapy with caffeine". This therapy dramatically improved the 5-year survival rate of 89% in patients who completed treatment without metastasis at the first visit, but the prognosis is still insufficient, so further improvement in therapeutic effect is required. (Non-Patent Document 2). In addition, the 5-year survival rate is 10 to 30% in patients with recurrence or metastasis from the first visit, which has not been significantly improved in the past 40 years, and further treatment in this respect as well. It is considered necessary to improve the effect (Non-Patent Document 3).
(軟部肉腫)
軟部肉腫は、平滑筋肉腫、線維肉腫、脂肪肉腫、横紋筋肉腫等の様々な分類がある。それぞれが違う性質を有しており、希少性は非常に高い。骨外性Ewing肉腫、横紋筋肉腫を除いた非小円形細胞肉腫において、四肢発生で病期IIIに限ると、手術単独での10年生存率が35%程度と報告されている(非特許文献4)。さらに、高悪性度軟部肉腫進行例に対する化学療法の効果に関しては、奏効率で30〜40%と報告されており、軟部肉腫は骨肉腫と比較して、さらに予後の改善に難渋する疾患である(非特許文献5〜7)。
軟部肉腫の現在の標準的治療方法は、悪性度、腫瘍の種類及び患者の状態にもよるが、術前化学療法と手術(広範切除術)と術後化学療法との組合せを基本とする。前記の化学療法とは、シスプラチン及びドキソルビシン等を使用した多剤併用化学療法である。
先に述べた通り、当該治療方法は、治療効果が十分なものとは言えないが、軟部肉腫の希少性の高さゆえ、新規薬剤の開発が進みにくいといった問題点がある。
(Soft tissue sarcoma)
Soft tissue sarcoma is classified into various categories such as leiomyosarcoma, fibrosarcoma, liposarcoma, and rhabdomyosarcoma. Each has different properties and is extremely rare. In non-small round cell sarcoma excluding extraosseous Ewing sarcoma and rhabdomyosarcoma, it has been reported that the 10-year survival rate by surgery alone is about 35% when limited to stage III in limb development (non-patent). Document 4). Furthermore, the effect of chemotherapy on advanced cases of high-grade soft tissue sarcoma has been reported to be 30-40% in response rate, and soft tissue sarcoma is a disease that is more difficult to improve the prognosis than osteosarcoma. (Non-Patent Documents 5 to 7).
Current standard treatments for soft tissue sarcoma are based on a combination of preoperative chemotherapy, surgery (extensive resection), and postoperative chemotherapy, depending on malignancy, tumor type, and patient condition. The above-mentioned chemotherapy is a multidrug combination chemotherapy using cisplatin, doxorubicin and the like.
As described above, the therapeutic method does not have a sufficient therapeutic effect, but there is a problem that the development of a new drug is difficult to proceed due to the high rarity of soft tissue sarcoma.
以上の通り、様々な癌種に効果を発揮し、従来の化学療法よりも強力な治療効果を有する新たな治療剤が望まれていた。 As described above, a new therapeutic agent that is effective against various cancer types and has a stronger therapeutic effect than conventional chemotherapy has been desired.
本発明は、様々な癌種において、従来の化学療法よりも強力な治療効果を有する癌治療剤及び癌治療方法を提供することを課題とする。 An object of the present invention is to provide a cancer therapeutic agent and a cancer treatment method having a stronger therapeutic effect than conventional chemotherapy in various cancer types.
本発明者等は、上記課題を解決すべく、抗癌剤と組み合わせることで抗癌剤の治療効果を増強できる薬剤を鋭意検討した結果、「カフェインクエン酸塩と抗癌剤との組み合わせが、抗癌剤の治療効果を相乗的に高めること」を見出し、本発明を完成した。 In order to solve the above problems, the present inventors have diligently studied a drug that can enhance the therapeutic effect of the anticancer drug by combining with the anticancer drug. As a result, "the combination of caffeine citrate and the anticancer drug has the therapeutic effect of the anticancer drug. The present invention was completed with the finding of "synergistic enhancement".
すなわち、本発明は以下からなる。
1.有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤と組み合わせたことを特徴とする癌治療剤。
2.前記癌は、原発生悪性骨腫瘍、軟部肉腫、肺癌、胃癌、乳癌、子宮癌、大腸癌、皮膚癌、卵巣癌、膵癌、胆管癌又は肝細胞癌である、前項1に記載の癌治療剤。
3.前記抗癌剤が、シスプラチン、ゲムシタビン又はアドリアマイシンである前項1又は前項2に記載の癌治療剤。
4.前記癌は、骨肉腫である、前項1〜3のいずれか1に記載の癌治療剤。
5.前記癌は、軟部肉腫である、前項1〜3のいずれか1に記載の癌治療剤。
6.前記癌は、肺癌である、前項1〜3のいずれか1に記載の癌治療剤。
7.前記癌は、膵癌である、前項1〜3のいずれか1に記載の癌治療剤。
8.前記癌は、胃癌である、前項1〜3のいずれか1に記載の癌治療剤。
9.前記癌は、肝細胞癌である、前項1〜3のいずれか1に記載の癌治療剤。
10.前記癌は、子宮癌である、前項1〜3のいずれか1に記載の癌治療剤。
11.前記癌は、乳癌である、前項1〜3のいずれか1に記載の癌治療剤。
12.前記癌は、卵巣癌である、前項1〜3のいずれか1に記載の癌治療剤。
13.前記癌は、皮膚癌である、前項1〜3のいずれか1に記載の癌治療剤。
14.前記癌は、胆管癌である、前項1〜3のいずれか1に記載の癌治療剤。
15.有効成分であるカフェインクエン酸塩と有効成分である抗癌剤を癌患者に投与することを特徴とする癌治療方法。
16.前記癌患者は、原発生悪性骨腫瘍、軟部肉腫、肺癌、胃癌、乳癌、子宮癌、大腸癌、皮膚癌、卵巣癌、膵癌、胆管癌又は肝細胞癌患者である、前項15に記載の癌治療方法。
17.前記抗癌剤が、シスプラチン、アドリアマイシン又はゲムシタビンである前項15又は前項16に記載の癌治療方法。
18.有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤であるゲムシタビンと組み合わせたことを特徴とする軟部肉腫治療剤。
19.有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤であるゲムシタビンと組み合わせたことを特徴とする胃癌治療剤。
20.有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤であるゲムシタビンと組み合わせたことを特徴とする皮膚癌治療剤。
21.有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤であるゲムシタビンと組み合わせたことを特徴とする膵癌治療剤。
22.有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤であるアドリアマイシンと組み合わせたことを特徴とする胃癌治療剤。
23.有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤であるアドリアマイシンと組み合わせたことを特徴とする子宮癌治療剤。
24.有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤であるシスプラチンと組み合わせたことを特徴とする肺癌治療剤。
25.有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤であるシスプラチンと組み合わせたことを特徴とする乳癌治療剤。
26.有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤であるシスプラチンと組み合わせたことを特徴とする子宮癌治療剤。
27.有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤であるシスプラチンと組み合わせたことを特徴とする皮膚癌治療剤。
28.有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤であるシスプラチンと組み合わせたことを特徴とする卵巣癌治療剤。
29.有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤であるシスプラチンと組み合わせたことを特徴とする胆管癌治療剤。
That is, the present invention comprises the following.
1. 1. A cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with an anticancer agent, which is an active ingredient.
2. 2. The cancer therapeutic agent according to item 1 above, wherein the cancer is a primary malignant bone tumor, soft tissue sarcoma, lung cancer, gastric cancer, breast cancer, uterine cancer, colon cancer, skin cancer, ovarian cancer, pancreatic cancer, bile duct cancer or hepatocellular carcinoma. ..
3. 3. The cancer therapeutic agent according to the preceding item 1 or the preceding item 2, wherein the anticancer agent is cisplatin, gemcitabine or adriamycin.
4. The cancer therapeutic agent according to any one of items 1 to 3 above, wherein the cancer is osteosarcoma.
5. The cancer therapeutic agent according to any one of items 1 to 3 above, wherein the cancer is soft tissue sarcoma.
6. The cancer therapeutic agent according to any one of items 1 to 3 above, wherein the cancer is lung cancer.
7. The cancer therapeutic agent according to any one of items 1 to 3 above, wherein the cancer is pancreatic cancer.
8. The cancer therapeutic agent according to any one of items 1 to 3 above, wherein the cancer is gastric cancer.
9. The cancer therapeutic agent according to any one of items 1 to 3 above, wherein the cancer is hepatocellular carcinoma.
10. The cancer therapeutic agent according to any one of items 1 to 3 above, wherein the cancer is uterine cancer.
11. The cancer therapeutic agent according to any one of items 1 to 3 above, wherein the cancer is breast cancer.
12. The cancer therapeutic agent according to any one of items 1 to 3 above, wherein the cancer is ovarian cancer.
13. The cancer therapeutic agent according to any one of items 1 to 3 above, wherein the cancer is skin cancer.
14. The cancer therapeutic agent according to any one of items 1 to 3 above, wherein the cancer is bile duct cancer.
15. A cancer treatment method characterized by administering caffeine citrate, which is an active ingredient, and an anticancer agent, which is an active ingredient, to a cancer patient.
16. The cancer according to item 15, wherein the cancer patient is a primary malignant bone tumor, soft tissue sarcoma, lung cancer, gastric cancer, breast cancer, uterine cancer, colon cancer, skin cancer, ovarian cancer, pancreatic cancer, bile duct cancer or hepatocellular carcinoma. Method of treatment.
17. The cancer treatment method according to the preceding item 15 or the preceding item 16, wherein the anticancer agent is cisplatin, adriamycin or gemcitabine.
18. A therapeutic agent for soft tissue sarcoma, which is characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
19. A gastric cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
20. A skin cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
21. A pancreatic cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
22. A gastric cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with adriamycin, which is an anticancer agent, which is an active ingredient.
23. A uterine cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with adriamycin, which is an anticancer agent, which is an active ingredient.
24. A lung cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
25. A breast cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
26. A uterine cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
27. A skin cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
28. An ovarian cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
29. A therapeutic agent for cholangiocarcinoma, which is characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
本発明の癌治療剤は、少なくとも、カフェインクエン酸塩を、抗癌剤と組み合わせることにより、抗癌剤の治療効果を相乗的に増強できる相乗効果を有する。 The cancer therapeutic agent of the present invention has at least a synergistic effect that can synergistically enhance the therapeutic effect of the anticancer agent by combining caffeine citrate with the anticancer agent.
本発明は、「有効成分であるカフェインクエン酸塩を、有効成分である抗癌剤と組み合わせたことを特徴とする癌治療剤」並びに「有効成分であるカフェインクエン酸塩と有効成分である抗癌剤を癌患者に投与することを特徴とする癌治療方法」である。 The present invention is "a cancer therapeutic agent characterized by combining caffeine citrate as an active ingredient with an anticancer agent as an active ingredient" and "a caffeine citrate as an active ingredient and an anticancer agent as an active ingredient". Is a cancer treatment method characterized by administering to a cancer patient.
(カフェインクエン酸塩)
本発明において、カフェインクエン酸塩(クエン酸カフェイン)は、以下の式(1)で表される無水カフェイン並びに式(2)及び/又は式(2')で表されるクエン酸(クエン酸水和物を含む)を含有する薬剤である。ここで、式(2)及び式(2')で表されるクエン酸とは、式(2)で表されるクエン酸と式(2')で表されるクエン酸との混合物を意味する。
カフェインクエン酸塩中の無水カフェイン及びクエン酸の比率はモル比で、無水カフェイン:クエン酸=0.1:9.9〜9.9:0.1、好ましくは1:9〜9:1、より好ましくは1:1である。
カフェインクエン酸塩としては、市販のカフェインクエン酸塩を使用してもよい。例えば、ノーベルファーマ株式会社により商品名「レスピア(登録商標)」が市販されている。
また、無水カフェインに類似する化合物(無水カフェイン類似化合物)として、カフェイン水和物、安息香酸ナトリウムカフェイン(いわゆるアンナカ)等も使用できるほか、カフェインはキサンチン誘導体であるから、キサンチン類であるキサンチン系神経中枢刺激薬(フェネチリン、プロベントフェリン)、キサンチン系血管拡張薬(ペントキシフェリン、ナキシフェリン、ニコチン酸キサンチノール、ペンチフィリン、ロロフィリン、トナポフェリン)、キサンチン系利尿薬(テオブロミン、パマプロム)等も使用できる。
クエン酸に類似する化合物(クエン酸類似化合物)として、クエン酸カリウム、クエン酸リチウム、クエン酸銅、クエン酸鉄アンモニウム、及びそれらの水和物等も使用できる。
したがって、カフェインクエン酸塩に類似する薬剤として、上記の無水カフェインに類似する化合物及びクエン酸に類似する化合物の組合せも使用できる。
(Caffeine citrate)
In the present invention, caffeine citrate (caffeine citrate) is an anhydrous caffeine represented by the following formula (1) and citric acid represented by the formulas (2) and / or the formula (2'). It is a drug containing (including citric acid hydrate). Here, the citric acid represented by the formula (2) and the formula (2') means a mixture of the citric acid represented by the formula (2) and the citric acid represented by the formula (2'). ..
The ratio of anhydrous caffeine and citric acid in caffeine citrate is a molar ratio, anhydrous caffeine: citric acid = 0.1: 9.9 to 9.9: 0.1, preferably 1: 9 to 9. 1, more preferably 1: 1.
As the caffeine citrate, a commercially available caffeine citrate may be used. For example, the trade name "Respia (registered trademark)" is commercially available by Nobel Pharma Co., Ltd.
Further, as a compound similar to anhydrous caffeine (anhydrous caffeine-like compound), caffeine hydrate, sodium benzoate caffeine (so-called Annaka) and the like can be used, and since caffeine is a xanthine derivative, xanthines are used. Xanthine-based nerve center stimulants (phenetylin, proventferin), xanthine-based vasodilators (pentoxyferin, naxisferin, xanthinol nicotinate, pentiphyllin, lorophyllin, tonapoferin), xanthine-based diuretics (theobromine, pamaprom), etc. Can also be used.
As a compound similar to citric acid (citric acid-like compound), potassium citrate, lithium citrate, copper citrate, ammonium iron citrate, hydrates thereof and the like can also be used.
Therefore, as a drug similar to caffeine citrate, a combination of the above-mentioned compound similar to anhydrous caffeine and a compound similar to citric acid can also be used.
カフェインクエン酸塩は、癌種に対する効果や抗癌剤との併用に関する報告はないが、未熟児無呼吸発作治療剤等として利用されている。すなわち、カフェインクエン酸塩は、他の疾患で既に使用され、その安全性が十分に確立されている治療薬である。
このように、カフェインクエン酸塩は、すでに保険診療で処方されている薬剤であるため、早期臨床応用において、新規薬剤と比較すると有利である。
Caffeine citrate has not been reported to be effective against cancer types or used in combination with anticancer agents, but it is used as a therapeutic agent for apnea of prematurity in premature infants. That is, caffeine citrate is a therapeutic agent that has already been used in other diseases and its safety has been well established.
As described above, since caffeine citrate is a drug that has already been prescribed in medical insurance, it is advantageous in early clinical application as compared with a new drug.
(抗癌剤)
本発明の癌治療剤の有効成分としての抗癌剤は、シスプラチン、アドリアマイシン(ドキソルビシン)、イホスファミド又はゲムシタビンが好ましい。
(Anti-cancer drug)
As the anticancer agent as an active ingredient of the cancer therapeutic agent of the present invention, cisplatin, adriamycin (doxorubicin), ifosfamide or gemcitabine is preferable.
(シスプラチン)
シスプラチンは、DNA中のグアニン、アデニンのN−7位に結合し、DNA鎖内に架橋を形成することにより、DNAの複製を阻害する抗癌剤である。
シスプラチンは、市販されているものを使用できる。例えば、日本化薬株式会社より商品名「アイエーコール」として市販されている。
(アドリアマイシン)
アドリアマイシンは、腫瘍細胞のDNAの塩基対間に挿入し、DNA合成酵素及びRNA合成酵素を阻害することにより、DNA、RNA双方の生合成を抑制する抗癌剤である。
アドリアマイシンは、市販されているものを使用できる。例えば、協和発酵キリン株式会社より商品名「アドリアシン」として市販されている。
(イホスファミド)
イホスファミドは、DNA中のグアニンをアルキル化し、DNA二本鎖間に架橋を形成することにより、DNAの複製を阻害する抗癌剤である。
イホスファミドは、市販されているものを使用できる。例えば、塩野義製薬株式会社より商品名「イホマイド」として市販されている。
(ゲムシタビン)
ゲムシタビンは、腫瘍細胞のDNA合成において、構造の類似するシチジンと同様にDNA鎖に取り込まれ、DNA鎖伸長を停止させることにより、アポトーシスを誘導する抗癌剤である。
ゲムシタビンは、市販されているものを使用できる。例えば、日本イーライリリー株式会社より商品名「ジェムザール」として市販されている。
(Cisplatin)
Cisplatin is an anticancer agent that inhibits DNA replication by binding to the N-7 position of guanine and adenine in DNA and forming a crosslink in the DNA strand.
Commercially available cisplatin can be used. For example, it is marketed by Nippon Kayaku Co., Ltd. under the trade name "IACOL".
(Adriamycin)
Adriamycin is an anticancer agent that suppresses the biosynthesis of both DNA and RNA by inserting it between the base pairs of DNA in tumor cells and inhibiting DNA synthase and RNA synthase.
Commercially available adriamycin can be used. For example, it is commercially available from Kyowa Hakko Kirin Co., Ltd. under the trade name "Adriacin".
(Ifosfamide)
Ifosfamide is an anticancer agent that inhibits DNA replication by alkylating guanine in DNA and forming a crosslink between DNA double strands.
Commercially available ifosfamide can be used. For example, it is marketed by Shionogi Pharmaceutical Co., Ltd. under the trade name "Ihomide".
(Gemcitabine)
Gemcitabine is an anticancer drug that induces apoptosis in DNA synthesis of tumor cells by being incorporated into the DNA strand in the same manner as cytidine having a similar structure and stopping the elongation of the DNA strand.
Commercially available gemcitabine can be used. For example, it is marketed by Eli Lilly Japan K.K. under the trade name "Gemzar".
シスプラチンは、プラチナ製剤であり、同じプラチナ製剤、例えば、カルボプラチン、オキサリプラチン、ネダプラチン等も同様の効果が期待される。
ドキソルビシンは、抗癌抗生物質であり、同じ抗癌抗生物質、例えば、アクチノマイシンD、ピラルビシン、ダウノルビシン等も同様の効果が期待される。
イホスファミドは、アルキル化薬であり、同じアルキル化薬、例えば、シクロホスファミド、ブスルファン、ダカルバジン等も同様の効果が期待される。
ゲムシタビンは、代謝拮抗薬であり、同じ代謝拮抗薬も同様の効果が期待される。
上記以外にも、ビンクリスチンやエトポシドのような植物アルカロイド等を含めたDNAの合成を阻害するタイプの薬剤全てにおいて、同様の効果が期待される。
Cisplatin is a platinum preparation, and the same platinum preparation, for example, carboplatin, oxaliplatin, nedaplatin, etc. is expected to have the same effect.
Doxorubicin is an anticancer antibiotic, and the same anticancer antibiotics such as actinomycin D, pirarubicin, and daunorubicin are expected to have similar effects.
Ifosfamide is an alkylating agent, and the same alkylating agent, for example, cyclophosphamide, busulfan, dacarbazine and the like is expected to have the same effect.
Gemcitabine is an antimetabolite, and the same antimetabolite is expected to have the same effect.
In addition to the above, similar effects are expected for all types of drugs that inhibit DNA synthesis, including plant alkaloids such as vincristine and etoposide.
(癌種)
本発明の癌治療剤及び癌治療方法の対象の癌種は、原発生悪性骨腫瘍、軟部肉腫、肺癌、胃癌、乳癌、子宮癌、大腸癌、皮膚癌、卵巣癌、膵癌、胆管癌又は肝細胞癌等が挙げられる。
原発生悪性骨腫瘍としては、特に限定されないが、例えば、骨肉腫、軟骨肉腫、ユーイング(Ewing)肉腫、骨未分化多形肉腫等が挙げられる。
軟部肉腫としては、特に限定されないが、例えば、線維肉腫、滑膜肉腫、平滑筋肉腫、脂肪肉腫、横紋筋肉腫、未分化多形肉腫、骨外性骨肉腫、骨外性Ewing肉腫等が挙げられる。
肺癌としては、特に限定されないが、例えば、腺癌、扁平上皮癌、大細胞癌等の非小細胞肺癌、小細胞肺癌等が挙げられる。
子宮癌としては、特に限定されないが、例えば、子宮頸癌、子宮体癌等が挙げられる。
皮膚癌としては、特に限定されないが、例えば、悪性黒色腫、基底細胞癌、有棘細胞癌等が挙げられる。
なお、本明細書において挙げた癌種は、それらの癌が生じる臓器、器官における全ての扁平上皮癌も対象となる。例えば、胃癌には、胃における扁平上皮癌が含まれる。
(Cancer type)
The target cancer types of the cancer therapeutic agent and the cancer treatment method of the present invention are primary malignant bone tumor, soft tissue sarcoma, lung cancer, gastric cancer, breast cancer, uterine cancer, colon cancer, skin cancer, ovarian cancer, pancreatic cancer, bile duct cancer or liver. Examples include cell cancer.
The primary malignant bone tumor is not particularly limited, and examples thereof include osteosarcoma, chondrosarcoma, Ewing's sarcoma, and undifferentiated polymorphic bone tumor.
The soft tissue sarcoma is not particularly limited, and examples thereof include fibrosarcoma, synovial sarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma, undifferentiated polymorphic sarcoma, extraosseous osteosarcoma, and extraosseous Ewing sarcoma. Can be mentioned.
The lung cancer is not particularly limited, and examples thereof include non-small cell lung cancer such as adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, and small cell lung cancer.
The uterine cancer is not particularly limited, and examples thereof include cervical cancer and endometrial cancer.
The skin cancer is not particularly limited, and examples thereof include malignant melanoma, basal cell carcinoma, and spinous cell carcinoma.
In addition, the cancer types listed in the present specification also include all squamous cell carcinomas in the organs in which those cancers occur. For example, gastric cancer includes squamous cell carcinoma of the stomach.
(本発明の癌治療剤の投与方法又は癌治療方法)
本発明の癌治療剤の投与方法又は癌治療方法における投与量および投与計画は、個々の治療対象毎の所要量、治療方法、疾病または必要性の程度などに依存して調整できる。投与量は、具体的には年齢、体重、一般的健康状態、性別、食事、投与時間、投与方法、排泄速度、薬物の組合せ、および患者の病状などに応じて決めることができ、さらに、その他の要因を考慮して決定してもよい。
以下を例示することができるが特に限定されない。
(1)1回の投与量
カフェインクエン酸塩(3日間投与の合計):0.1〜10000 mg/m2体表面積、好ましくは1〜10000 mg/m2体表面積、より好ましくは50〜9000 mg/m2体表面積
抗癌剤:シスプラチン(1日間投与)は、0.1〜150 mg/m2体表面積、好ましくは10〜150 mg/m2体表面積、より好ましくは60〜120 mg/m2体表面積。ドキソルビシン(2日間投与の合計)は、0.1〜120 mg/m2体表面積、好ましくは10〜90 mg/m2体表面積、より好ましくは30〜60 mg/m2体表面積。イホスファミド(3〜5日間投与の合計)は、0.1〜30 mg/m2体表面積、好ましくは1〜20 mg/m2体表面積、より好ましくは4.5〜15 mg/m2体表面積。ゲムシタビン(1日間投与)は、0.1〜1000 mg/m2体表面積、好ましくは10〜900 mg/m2体表面積、より好ましくは675〜900 mg/m2体表面積。
(2)投与間隔
カフェインクエン酸塩:10週間1〜70回投与、好ましくは2日1〜2回投与、より好ましくは1日1回投与。詳しくは、1日〜10週間を1クールとして1〜30日間連続で1日1〜10回投与、好ましくは1〜6週間を1クールとして1〜15日間連続で1日1〜3回投与、より好ましくは2〜3週間を1クールとして3〜7日間連続で1日1回投与。
抗癌剤:1〜10週間1回投与、好ましくは2〜8週間1回投与、より好ましくは3〜6週間1回投与。
(3)併用投与
カフェインクエン酸塩と抗癌剤は同時投与、数時間の間隔を空けての投与、1日の間隔を空けての投与、数日の間隔を空けての投与、数週間の間隔を空けての投与、数か月の間隔を空けての投与のいずれでも良い。
また、カフェインクエン酸塩及び抗癌剤を、生理学的に許容されうる溶媒、担体、賦形剤、結合剤等と混合し、医薬組成物にした後に、該組成物を患者に投与してもよい。なお、本発明におけるカフェインクエン酸塩及び/又は抗癌剤は、プロドラッグ、またはそれらの薬理学的に許容される塩も対象とする。
溶媒としては、特に限定されないが、例えば、水、アルコール、生理食塩水等が挙げられる。
担体としては、特に限定されないが、例えば、酸、塩基、緩衝液、安定化剤、等張化剤、保存剤等が挙げられる。
賦形剤としては、特に限定されないが、例えば、充填剤、凝固剤、滑たく剤、崩壊剤、色素、甘味料、香料、コーティング剤等が挙げられる。
結合剤としては、特に限定されないが、例えば、水、エタノール、プロパノール、単シロップ、ブドウ糖液、デンプン液、ゼラチン液、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルスターチ、メチルセルロース、エチルセルロース、シェラック、リン酸カルシウム、ポリビニルピロリドン等が挙げられる。
さらに、カフェインクエン酸塩及び抗癌剤を別々に製剤化した場合において、別々に製剤化したものを使用時に希釈剤等を用いて混合して混合物にして後に、該混合物を患者に投与してもよい。
本発明の癌治療剤の投与方法又は癌治療方法では、経口的または非経口的に投与することができる。経口投与には、錠剤、カプセル、コーティング錠、トローチ、溶液または懸濁液などの液剤といった既知の投与用剤形を用いることができる。また、非経口投与は、注射による静脈内、筋肉内、または皮下への投与、スプレーやエアロゾルなどを用いた経鼻腔や口腔などの経粘膜投与、坐剤などを用いた直腸投与、パッチやリニメントやゲルなどを用いた経皮投与などを挙げることができる。好ましくは経口投与、経鼻腔投与、または注射による静脈内投与を挙げることができる。
(A method of administering a cancer therapeutic agent of the present invention or a method of treating cancer)
The dose and administration plan in the administration method or the cancer treatment method of the cancer therapeutic agent of the present invention can be adjusted depending on the required amount, the treatment method, the degree of disease or necessity for each individual treatment target, and the like. The dose can be specifically determined according to age, body weight, general health, gender, diet, administration time, administration method, excretion rate, drug combination, patient's medical condition, etc. It may be decided in consideration of the factors of.
The following can be exemplified, but the present invention is not particularly limited.
(1) Single dose Caffeine citrate (total of 3 days administration): 0.1 to 10000 mg / m 2 body surface area, preferably 1 to 10000 mg / m 2 body surface area, more preferably 50 to 9000 mg / m 2 body surface area anticancer: cisplatin (administration 1 day) is, 0.1 to 150 mg / m 2 body surface area, preferably 10 to 150 mg / m 2 body surface area, more preferably 60 to 120 mg / m Two body surface areas. Doxorubicin (total of 2-day administration) has a surface area of 0.1 to 120 mg / m 2 body, preferably 10 to 90 mg / m 2 body surface area, more preferably 30 to 60 mg / m 2 body surface area. Ifosfamide (total of 3-5 day doses) is 0.1 to 30 mg / m 2 body surface area, preferably 1 to 20 mg / m 2 body surface area, more preferably 4.5 to 15 mg / m 2 body surface area. .. Gemcitabine (administered one day) it is, 0.1 to 1000 mg / m @ 2 of body surface area, preferably 10 to 900 mg / m 2 body surface area, more preferably from 675 to 900 mg / m 2 body surface area.
(2) Administration interval Caffeine citrate: Administered 1 to 70 times for 10 weeks, preferably 1 to 2 times a day, more preferably once a day. Specifically, 1 to 10 weeks as one course, 1 to 10 times a day for 1 to 30 consecutive days, preferably 1 to 6 weeks as 1 course, 1 to 15 days in a row, 1 to 3 times a day. More preferably, it is administered once a day for 3 to 7 consecutive days with 1 course for 2 to 3 weeks.
Anti-cancer agent: Administer once for 10 to 10 weeks, preferably once for 2 to 8 weeks, more preferably once for 3 to 6 weeks.
(3) Combined administration Caffeine citrate and anticancer drug are administered simultaneously, at intervals of several hours, at intervals of one day, at intervals of several days, and at intervals of several weeks. It may be administered at intervals of several months or at intervals of several months.
In addition, caffeine citrate and an anticancer agent may be mixed with a physiologically acceptable solvent, carrier, excipient, binder, etc. to form a pharmaceutical composition, and then the composition may be administered to a patient. .. The caffeine citrate and / or anticancer agent in the present invention also includes prodrugs or pharmacologically acceptable salts thereof.
The solvent is not particularly limited, and examples thereof include water, alcohol, and physiological saline.
The carrier is not particularly limited, and examples thereof include acids, bases, buffers, stabilizers, tonicity agents, preservatives and the like.
Excipients are not particularly limited, and examples thereof include fillers, coagulants, slip agents, disintegrants, pigments, sweeteners, flavors, and coating agents.
The binder is not particularly limited, but is, for example, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shelac, calcium phosphate, polyvinyl. Examples include pyrrolidone.
Furthermore, when caffeine citrate and an anticancer agent are separately formulated, the separately formulated ones may be mixed with a diluent or the like at the time of use to form a mixture, and then the mixture may be administered to the patient. Good.
In the method for administering the therapeutic agent for cancer or the method for treating cancer of the present invention, it can be administered orally or parenterally. For oral administration, known dosage forms such as tablets, capsules, coated tablets, troches, solutions such as solutions or suspensions can be used. Parenteral administration includes intravenous, intramuscular, or subcutaneous administration by injection, transmucosal administration such as nasal cavity and oral cavity using spray or aerosol, rectal administration using suppository, patch or liniment. And percutaneous administration using gel or the like can be mentioned. Preferably, oral administration, nasal administration, or intravenous administration by injection can be mentioned.
以下に示す実施例によって本発明を具体的に説明するが、本発明はこれらに限定されるものではない。 The present invention will be specifically described with reference to the following examples, but the present invention is not limited thereto.
[骨肉腫、軟部肉腫、肺癌及び胃癌培養細胞におけるカフェインクエン酸塩のシスプラチン効果増強の評価]
以下の方法により、骨肉腫、軟部肉腫、肺癌、胃癌に対するカフェインクエン酸塩の抗癌剤効果増強を評価した。
[Evaluation of enhanced cisplatin effect of caffeine citrate in cultured cells of osteosarcoma, soft tissue sarcoma, lung cancer and gastric cancer]
The enhancement of the anticancer drug effect of caffeine citrate on osteosarcoma, soft tissue sarcoma, lung cancer, and gastric cancer was evaluated by the following method.
<材料>
・細胞株
骨肉腫としては、ヒト由来のHOS細胞(American Type Culture Collection(ATCC)より入手)を用いた。
軟部肉腫としては、ヒト由来のHT1080細胞(ATCCより入手)を用いた。
肺癌としては、ヒト由来のRERF−LC−AI細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、国立研究開発法人理科学研究所バイオリソースセンター(理研BRC)より入手)を用いた。
胃癌としては、ヒト由来のMKN45細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
・抗癌剤
シスプラチン(和光純薬工業株式会社より入手、以下、CDDPと称する場合がある)を用いた。
・併用薬
カフェインクエン酸塩(クエン酸カフェイン、ノーベルファーマ株式会社、商品名:レスピア(登録商標)、以下、cafCAと称する場合がある)、無水カフェイン(和光純薬工業株式会社、以下、cafと称する場合がある)又はクエン酸(和光純薬工業株式会社、以下、CAと称する場合がある)を用いた。
<Material>
-As the cell line osteosarcoma, human-derived HOS cells (obtained from the American Type Culture Collection (ATCC)) were used.
As the soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
As lung cancer, human-derived RERF-LC-AI cells (obtained from the National BioResource Center (RIKEN BRC), RIKEN via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As gastric cancer, human-derived MKN45 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
-The anticancer drug cisplatin (obtained from Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as CDDP) was used.
・ Concomitant drug caffeine citrate (caffeine citrate, Nobelpharma Co., Ltd., trade name: Respia (registered trademark), hereinafter sometimes referred to as cafCA), anhydrous caffeine (Wako Pure Chemical Industries, Ltd., hereinafter , Caf) or citric acid (Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as CA) was used.
<WST−8アッセイ>
(1)上記の各癌種培養細胞について、HOS細胞、RERF−LC−AI細胞、及びMKN45細胞は、Roswell Park Memorial Institute(RPMI)1640培地を、HT1080細胞は、ダルベッコ改変イーグル培地(DMEM)を用いて、それぞれ3000〜5000個/100 μLの濃度で、37℃、CO2 5%の条件で、24時間、96wellの培養プレートで培養した。
(2)培養後、各癌種培養細胞について、4つの群に分け、薬剤(CDDP、CDDP+caf、CDDP+CA、CDDP+cafCA)を100 μL添加した。各群に添加する薬剤のCDDP濃度は、水(滅菌精製水)にCDDPの粉末を溶かして高濃度のものを作成し、その後細胞に応じた培養液で希釈することで0.125、0.25、0.5、1.0、2.0 μMに調製した。各群の併用薬であるcaf及びCAの濃度は、水に粉末を溶かしてそれぞれ高濃度のものを作成し、その後細胞に応じた培養液で希釈することで一律0.5 mMに調製した。cafCAの濃度は、製品が液体であるので、そのまま細胞に応じた培養液で希釈し、caf、CAと同様の0.5 mMに調製した。対照として、一部のウェルには薬剤を添加しなかった(CDDP濃度0 μM)。
(3)薬剤投与後72時間後に、Cell Counting Kit−8(CCK−8)(Dojindo社)で発色し、2〜4時間後に450 nmの吸光度を測定した。対照の吸光度を1として、吸光度の比率から細胞生存率を決定した。
(4)(3)の結果について、Statcel 3(オーエムエス出版)で分散分析を行い、各CDDP濃度における4つの群間の細胞生存率の有意差を解析し、CDDP+cafCAの効果を検討した。
(5)CalcuSyn ver.2(BIOSOFT)にてcafCAのもたらす抗癌剤増強効果と、caf/CAのもたらす抗癌剤増強効果の相乗効果について解析した。解析には、Median Effect法及びIsobologram法を用いた(参照:Ting-Chao Chou, Cancer Res 2010; 70: 440-6.; Tallarida RJ, et al., Life Sci 1989; 45: 947-61.; Roering SC et al., J Pharmacol Exp Ther 1988; 247: 603-8.; Tallarida RJ, Chapman & Hall/CRC, Florida, USA, 2000.)。
0.125、0.25、0.5、1.0、2.0 μMの各CDDP濃度において、CDDP+cafにおけるcaf濃度と、CDDP+CAにおけるCA濃度とのコンビネーションを、CDDP+cafCAにおけるcafCA濃度として、Combination Index(CI)を求めた。
ここで、CDDPは、前記コンビネーションの前後で、同量・同濃度である。例えば、CDDP濃度1.0 μMの場合、CDDP(1.0 μM)+caf(0.5 μM)とCDDP(1.0 μM)+CA(0.5 μM)とのコンビネーションは、CDDP(1.0 μM)+cafCA(0.5 μM)である。
Median Effect法によるCIは、各濃度による細胞生存率から導かれた「細胞が50%生存するために必要と考えられるCDDP濃度(ED50)」を基準に、caf、CA、cafCAの各々の濃度およびその際の細胞生存率を対象として算出した数値である。
Isobologram法によるCIは、各CDDP濃度による細胞生存率から導かれた、CDDP+caf、CDDP+CA及びCDDP+cafCAの「各Effective dose(ED50、ED75、ED90)」を用いて算出した数値である。
CI<1のとき、相乗効果と判定した。なお、CI=1のときは、相加効果と判定し、CI>1のときは、拮抗効果と判定する。
<WST-8 Assay>
(1) For each of the above-mentioned cultured cells of cancer type, HOS cells, RERF-LC-AI cells, and MKN45 cells were prepared with Roswell Park Memorial Institute (RPMI) 1640 medium, and HT1080 cells were used with Dalveco modified Eagle's medium (DMEM). used at a concentration of 3000 to 5000 pieces / 100 [mu] L, respectively, 37 ° C., with CO 2 5% of the conditions, 24 hours and cultured in a culture plate 96 well.
(2) After culturing, each cancer type cultured cell was divided into four groups, and 100 μL of a drug (CDDP, CDDP + caf, CDDP + CA, CDDP + cafCA) was added. The CDDP concentration of the drug added to each group is 0.125, 0. By dissolving CDDP powder in water (sterile purified water) to prepare a high concentration, and then diluting with a culture solution suitable for the cells. Prepared to 25, 0.5, 1.0, 2.0 μM. The concentrations of caf and CA, which are concomitant drugs in each group, were uniformly adjusted to 0.5 mM by dissolving powder in water to prepare high concentrations of each, and then diluting with a culture solution suitable for the cells. Since the product is a liquid, the concentration of cafCA was diluted with a culture solution suitable for the cells as it was, and adjusted to 0.5 mM, which was the same as for caf and CA. As a control, no drug was added to some wells (CDDP concentration 0 μM).
(3) 72 hours after administration of the drug, color was developed with Cell Counting Kit-8 (CCK-8) (Dojindo), and the absorbance at 450 nm was measured 2 to 4 hours later. The cell viability was determined from the absorbance ratio, with the absorbance of the control being 1.
(4) The results of (3) were analyzed by analysis of variance with Statcel 3 (OMS Publishing), the significant difference in cell viability between the four groups at each CDDP concentration was analyzed, and the effect of CDDP + cafCA was examined.
(5) CalcuSyn ver. In 2 (BIOSOFT), the synergistic effect of the anti-cancer agent enhancing effect brought about by cafCA and the anti-cancer agent enhancing effect brought about by caf / CA was analyzed. The median effect method and the Isobolog method were used for the analysis (see: Ting-Chao Chou, Cancer Res 2010; 70: 440-6 .; Tallarida RJ, et al., Life Sci 1989; 45: 947-61 .; Roering SC et al., J Pharmacol Exp Ther 1988; 247: 603-8 .; Tallarida RJ, Chapman & Hall / CRC, Florida, USA, 2000.).
At each CDDP concentration of 0.125, 0.25, 0.5, 1.0, and 2.0 μM, the combination of the caf concentration at CDDP + caf and the CA concentration at CDDP + CA is defined as the cafCA concentration at CDDP + cafCA, and the combination index ( CI) was sought.
Here, CDDP has the same amount and concentration before and after the combination. For example, when the CDDP concentration is 1.0 μM, the combination of CDDP (1.0 μM) + caf (0.5 μM) and CDDP (1.0 μM) + CA (0.5 μM) is CDDP (1.0 μM). μM) + cafCA (0.5 μM).
The CI by the Median Effect method is based on the "CDDP concentration (ED50) considered necessary for cells to survive 50%" derived from the cell viability at each concentration, and the respective concentrations of caf, CA, and cafCA. It is a numerical value calculated for the cell viability at that time.
The CI by the Cisplatin method is a numerical value calculated using "Effective doses (ED50, ED75, ED90)" of CDDP + caf, CDDP + CA and CDDP + cafCA derived from the cell viability according to each CDDP concentration.
When CI <1, it was judged to be a synergistic effect. When CI = 1, it is determined to be an additive effect, and when CI> 1, it is determined to be an antagonistic effect.
<骨肉腫の結果>
1.細胞生存率
図1に、HOS細胞における4つの群の450 nmの吸光度に基づく細胞生存率を示す。図1から明らかなように、HOS細胞(骨肉腫)において、CDDP+cafCA群は、全てのCDDP濃度で、他の群と比較して、最も細胞生存率が低かった。
また、分散分析の結果、CDDP+cafCA群は、各シスプラチン濃度において、他の全ての群との間で有意差があった(p<0.01)。
よって、カフェインクエン酸塩は、シスプラチンと組み合わせて添加することにより、シスプラチンの抗癌剤効果を増強することが、0.125〜2.0 μMの全てのシスプラチン濃度において確認できた。
<Results of osteosarcoma>
1. 1. Cell viability Figure 1 shows the cell viability of the four groups in HOS cells based on the absorbance at 450 nm. As is clear from FIG. 1, in HOS cells (osteosarcoma), the CDDP + cafCA group had the lowest cell viability at all CDDP concentrations as compared with the other groups.
In addition, as a result of analysis of variance, the CDDP + cafCA group was significantly different from all other groups in each cisplatin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer agent effect of cisplatin when added in combination with cisplatin at all cisplatin concentrations of 0.125 to 2.0 μM.
2.Median Effect法
次に、Median Effect法によるHOS細胞における相乗効果の解析結果を、表1に示す。相乗効果の指標であるCIは、図1におけるCDDP濃度毎の細胞生存率に基づき、CDDP+cafとCDDP+CAのコンビネーションをCDDP+cafCAとして算出した。CI<1.0のとき、相乗効果が認められる。
表1から明らかなように、CDDP+cafCA群は、0.125〜2.0 μMの全てのシスプラチン濃度で、CI<1.0となり、CDDP+caf及びCDDP+CAに対して、相乗効果が認められた。
2. 2. Median Effect method Next, the analysis results of the synergistic effect in HOS cells by the Median Effect method are shown in Table 1. The CI, which is an index of synergistic effect, was calculated by calculating the combination of CDDP + caf and CDDP + CA as CDDP + cafCA based on the cell viability for each CDDP concentration in FIG. When CI <1.0, a synergistic effect is observed.
As is clear from Table 1, the CDDP + cafCA group had a CI <1.0 at all cisplatin concentrations of 0.125 to 2.0 μM, and a synergistic effect was observed for CDDP + caf and CDDP + CA.
3.Isobologram法
Isobologram法によるHOS細胞における相乗効果の解析結果を図5に示す。
図5〜図8のIsobologram法の解析結果のグラフにおいて、CDDP+cafCAの各Effective Doseを示す円形の破線が、それぞれ、CDDP+cafの各Effective Doseと、CDDP+CAの各Effective Doseとを結んだ曲線上にあれば、相加効果を示し、曲線より左下にあれば、相乗効果を示し、曲線より右上にあれば、拮抗効果を示す。例えば、CDDP+cafCAのED50を示す円形の破線が、CDDP+cafのED50と、CDDP+CAのED50とを結んだ曲線上にあれば、相加効果を示し、曲線より左下にあれば、相乗効果を示し、曲線より右上にあれば、拮抗効果を示す。ED75、ED90についても同様である。
本解析における「Effective Dose」とは、各癌腫培養細胞を減少させるために必要なCDDP濃度を示しており、CDDP濃度毎の細胞生存率から導かれる。例えば、ED50であれば、細胞をベースから50%減少させる(細胞生存率を50%にする)ために必要なCDDP濃度を意味し、ED75であれば、細胞を75%減少させる(細胞生存率を25%にする)ために必要なCDDP濃度、ED90であれば、細胞を90%減少させる(細胞生存率を10%にする)ために必要なCDDP濃度を意味する。
図5のグラフから明らかなように、CDDP+cafCAの各Effective Doseを示す円形の破線が、それぞれ、CDDP+caf及びCDDP+CAの各Effective Doseを結んだ曲線よりも左下に位置することから、相乗効果が認められた。
図5の表から明らかなように、CDDP+cafCA群は、50〜90%の全てのEffective Doseで、CI<1.0となり、CDDP+caf及びCDDP+CAに対して、相乗効果が認められた。
3. 3. Isobologram method The analysis result of the synergistic effect in HOS cells by the Isobologram method is shown in FIG.
In the graph of the analysis result of the Isobologram method of FIGS. 5 to 8, if the circular broken line indicating each Effective Dose of CDDP + cafCA is on the curve connecting each Effective Dose of CDDP + caf and each Effective Dose of CDDP + CA, respectively. , An additive effect is shown, a synergistic effect is shown if it is in the lower left of the curve, and an antagonistic effect is shown if it is in the upper right of the curve. For example, if the circular broken line indicating the ED50 of CDDP + cafCA is on the curve connecting the ED50 of CDDP + caf and the ED50 of CDDP + CA, it indicates an additive effect, and if it is on the lower left of the curve, it indicates a synergistic effect. If it is in the upper right, it shows an antagonistic effect. The same applies to ED75 and ED90.
“Effective Dose” in this analysis indicates the CDDP concentration required to reduce each carcinoma cultured cell, and is derived from the cell viability for each CDDP concentration. For example, ED50 means the CDDP concentration required to reduce cells by 50% from the base (cell viability to 50%), and ED75 means to reduce cells by 75% (cell viability). The CDDP concentration required to reduce the number of cells to 25%, and ED90 means the CDDP concentration required to reduce cells by 90% (to increase the cell viability to 10%).
As is clear from the graph of FIG. 5, the circular dashed line indicating each Effective Dose of CDDP + cafCA is located at the lower left of the curve connecting each Effective Dose of CDDP + caf and CDDP + CA, respectively, so that a synergistic effect was recognized. ..
As is clear from the table of FIG. 5, the CDDP + cafCA group had a CI <1.0 in all Effective Dose of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.
<軟部肉腫の結果>
1.細胞生存率
HT1080細胞における4つの群の450 nmの吸光度に基づく細胞生存率を図2に示す。図2から明らかなように、HT1080細胞(軟部肉腫)において、CDDP+cafCA群は、全てのCDDP濃度で、他の群と比較して、最も細胞生存率が低かった。
また、分散分析の結果、CDDP+cafCA群は、各シスプラチン濃度において、他の全ての群との間で有意差があった(p<0.01)。
よって、カフェインクエン酸塩は、シスプラチンと組み合わせて添加することにより、シスプラチンの抗癌剤効果を増強することが、0.125〜2.0 μMの全てのシスプラチン濃度において確認できた。
<Results of soft tissue sarcoma>
1. 1. Cell viability Figure 2 shows the cell viability based on the absorbance at 450 nm of the four groups in HT1080 cells. As is clear from FIG. 2, in HT1080 cells (soft tissue sarcoma), the CDDP + cafCA group had the lowest cell viability at all CDDP concentrations as compared with the other groups.
In addition, as a result of analysis of variance, the CDDP + cafCA group was significantly different from all other groups in each cisplatin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer agent effect of cisplatin when added in combination with cisplatin at all cisplatin concentrations of 0.125 to 2.0 μM.
2.Median Effect法
Median Effect法によるHT1080細胞における相乗効果の解析結果を、表1に示す。相乗効果の指標であるCIは、図2におけるCDDP濃度毎の細胞生存率に基づき、CDDP+cafとCDDP+CAのコンビネーションをCDDP+cafCAとして算出した。
表1から明らかなように、CDDP+cafCA群は、0.125〜2.0 μMの全てのシスプラチン濃度で、CI<1.0となり、CDDP+caf及びCDDP+CAに対して、相乗効果が認められた。
2. 2. Median Effect Method Table 1 shows the analysis results of the synergistic effect in HT1080 cells by the Median Effect method. The CI, which is an index of the synergistic effect, was calculated by calculating the combination of CDDP + caf and CDDP + CA as CDDP + cafCA based on the cell viability for each CDDP concentration in FIG.
As is clear from Table 1, the CDDP + cafCA group had a CI <1.0 at all cisplatin concentrations of 0.125 to 2.0 μM, and a synergistic effect was observed for CDDP + caf and CDDP + CA.
3.Isobologram法
Isobologram法によるHT1080細胞における相乗効果の解析結果を図6に示す。
図6のグラフから明らかなように、CDDP+cafCAの各Effective Doseを示す円形の破線が、それぞれ、CDDP+cafとCDDP+CAの各Effective Doseを結んだ曲線よりも左下に位置することから、相乗効果が認められた。
図6の表から明らかなように、CDDP+cafCA群は、50〜90%の全てのEffective Doseで、CI<1となり、CDDP+caf及びCDDP+CAに対して、相乗効果が認められた。
3. 3. Isobologram method The analysis result of the synergistic effect in HT1080 cells by the Isobologram method is shown in FIG.
As is clear from the graph of FIG. 6, the circular broken line indicating each Effective Dose of CDDP + cafCA is located at the lower left of the curve connecting each Effective Dose of CDDP + caf and CDDP + CA, respectively, so that a synergistic effect was recognized. ..
As is clear from the table of FIG. 6, the CDDP + cafCA group had a CI <1 in all Effective Dose of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.
<肺癌の結果>
1.細胞生存率
RERF−LC−AI細胞における4つの群の450 nmの吸光度に基づく細胞生存率を図3に示す。図3から明らかなように、RERF−LC−AI細胞(肺癌)において、CDDP+cafCA群は、全てのCDDP濃度で、他の群と比較して、最も細胞生存率が低かった。
また、分散分析の結果、CDDP+cafCA群は、各シスプラチン濃度において、他の全ての群との間で有意差があった(p<0.01)。
よって、カフェインクエン酸塩は、シスプラチンと組み合わせて添加することにより、シスプラチンの抗癌剤効果を増強することが、0.125〜2.0 μMの全てのシスプラチン濃度において確認できた。
<Results of lung cancer>
1. 1. Cell viability The cell viability based on the absorbance at 450 nm of the four groups in RERF-LC-AI cells is shown in FIG. As is clear from FIG. 3, in RERF-LC-AI cells (lung cancer), the CDDP + cafCA group had the lowest cell viability at all CDDP concentrations as compared with the other groups.
In addition, as a result of analysis of variance, the CDDP + cafCA group was significantly different from all other groups in each cisplatin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer agent effect of cisplatin when added in combination with cisplatin at all cisplatin concentrations of 0.125 to 2.0 μM.
2.Median Effect法
Median Effect法によるRERF−LC−AI細胞における相乗効果の解析結果を、表1に示す。相乗効果の指標であるCIは、図3におけるCDDP濃度毎の細胞生存率に基づき、CDDP+cafとCDDP+CAのコンビネーションをCDDP+cafCAとして算出した。
表1から明らかなように、CDDP+cafCA群は、0.125〜2.0 μMの全てのシスプラチン濃度で、CI<1.0となり、CDDP+caf及びCDDP+CAに対して、相乗効果が認められた。
2. 2. Median Effect Method Table 1 shows the analysis results of the synergistic effect in RERF-LC-AI cells by the Median Effect method. The CI, which is an index of the synergistic effect, was calculated by calculating the combination of CDDP + caf and CDDP + CA as CDDP + cafCA based on the cell viability for each CDDP concentration in FIG.
As is clear from Table 1, the CDDP + cafCA group had a CI <1.0 at all cisplatin concentrations of 0.125 to 2.0 μM, and a synergistic effect was observed for CDDP + caf and CDDP + CA.
3.Isobologram法
Isobologram法によるRERF−LC−AI細胞における相乗効果の解析結果を図7に示す。
図7のグラフから明らかなように、CDDP+cafCAの各Effective Doseを示す円形の破線が、それぞれ、CDDP+cafとCDDP+CAの各Effective Doseを結んだ曲線よりも左下に位置することから、相乗効果が認められた。
図7の表から明らかなように、CDDP+cafCA群は、50〜90%の全てのEffective Doseで、CI<1となり、CDDP+caf及びCDDP+CAに対して、相乗効果が認められた。
3. 3. Isobologram method The analysis result of the synergistic effect in RERF-LC-AI cells by the Isobologram method is shown in FIG.
As is clear from the graph of FIG. 7, the circular broken line indicating each Effective Dose of CDDP + cafCA is located at the lower left of the curve connecting each Effective Dose of CDDP + caf and CDDP + CA, respectively, so that a synergistic effect was recognized. ..
As is clear from the table in FIG. 7, the CDDP + cafCA group had a CI <1 in all Effective Dose of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.
<胃癌の結果>
1.細胞生存率
MKN45細胞における4つの群の450 nmの吸光度に基づく細胞生存率を図4に示す。図4から明らかなように、MKN45細胞(胃癌)において、CDDP+cafCA群は、全てのCDDP濃度で、他の群と比較して、最も細胞生存率が低かった。
また、分散分析の結果、CDDP+cafCA群は、各シスプラチン濃度において、他の全ての群との間で有意差があった(p<0.01)。
よって、カフェインクエン酸塩は、シスプラチンと組み合わせて添加することにより、シスプラチンの抗癌剤効果を増強することが、0.125〜2.0 μMの全てのシスプラチン濃度において確認できた。
<Results of gastric cancer>
1. 1. Cell viability Figure 4 shows the cell viability based on the absorbance at 450 nm of the four groups in MKN45 cells. As is clear from FIG. 4, in MKN45 cells (gastric cancer), the CDDP + cafCA group had the lowest cell viability at all CDDP concentrations as compared with the other groups.
In addition, as a result of analysis of variance, the CDDP + cafCA group was significantly different from all other groups in each cisplatin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer agent effect of cisplatin when added in combination with cisplatin at all cisplatin concentrations of 0.125 to 2.0 μM.
2.Median Effect法
Median Effect法によるMKN45細胞における相乗効果の解析結果を、表1に示す。相乗効果の指標であるCIは、図4におけるCDDP濃度毎の細胞生存率に基づき、CDDP+cafとCDDP+CAのコンビネーションをCDDP+cafCAとして算出した。
表1から明らかなように、CDDP+cafCA群は、0.125〜2.0 μMの全てのシスプラチン濃度で、CI<1.0となり、CDDP+caf及びCDDP+CAに対して、相乗効果が認められた。
2. 2. Median Effect Method Table 1 shows the analysis results of the synergistic effect in MKN45 cells by the Median Effect method. The CI, which is an index of the synergistic effect, was calculated by calculating the combination of CDDP + caf and CDDP + CA as CDDP + cafCA based on the cell viability for each CDDP concentration in FIG.
As is clear from Table 1, the CDDP + cafCA group had a CI <1.0 at all cisplatin concentrations of 0.125 to 2.0 μM, and a synergistic effect was observed for CDDP + caf and CDDP + CA.
3.Isobologram法
Isobologram法によるMKN45細胞における相乗効果の解析結果を図8に示す。
図8のグラフから明らかなように、CDDP+cafCAの各Effective Doseを示す円形の破線が、それぞれ、CDDP+cafとCDDP+CAの各Effective Doseを結んだ曲線よりも左下に位置することから、相乗効果が認められた。
図8の表から明らかなように、CDDP+cafCA群は、50〜90%の全てのEffective Doseで、CI<1となり、CDDP+caf及びCDDP+CAに対して、相乗効果が認められた。
3. 3. Isobologram method The analysis result of the synergistic effect on MKN45 cells by the Isobologram method is shown in FIG.
As is clear from the graph of FIG. 8, the circular broken line indicating each Effective Dose of CDDP + cafCA is located at the lower left of the curve connecting each Effective Dose of CDDP + caf and CDDP + CA, respectively, so that a synergistic effect was recognized. ..
As is clear from the table in FIG. 8, the CDDP + cafCA group had a CI <1 in all Effective Dose of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.
<総合評価>
以上の4種類の細胞株におけるMedian Effect法及びIsobologram法の実験結果をまとめると、図9の通りである。
図9から明らかなように、本発明の癌治療剤は、実験した全ての癌種において、CDDP+cafCA群は、Median Effect法及びIsobologram法の双方の解析により、CI<1.0となり、相乗効果を有することが確認された。
<Comprehensive evaluation>
FIG. 9 summarizes the experimental results of the Median Effect method and the Isobologram method in the above four types of cell lines.
As is clear from FIG. 9, the cancer therapeutic agent of the present invention has a synergistic effect in all the cancer types tested, with the CDDP + cafCA group having a CI <1.0 by analysis of both the Median Effect method and the Isobologram method. It was confirmed to have.
以上の結果より、本発明の癌治療剤において、シスプラチンとカフェインクエン酸塩との組合せは、シスプラチン単独、シスプラチンと無水カフェインとの組合せ、シスプラチンとクエン酸との組合せと比較して、有意にシスプラチンの抗癌剤効果をより増強すること、カフェインクエン酸塩の有する抗癌剤増強効果は、無水カフェインの有する抗癌剤増強効果と、クエン酸の有する抗癌剤増強効果との、単なる相加効果ではなく、相乗効果であることが明らかになった。 From the above results, in the cancer therapeutic agent of the present invention, the combination of cisplatin and caffeine citrate is significant as compared with cisplatin alone, cisplatin and anhydrous caffeine, and cisplatin and citric acid. Further enhancing the anticancer agent effect of cisplatin, the anticancer agent enhancing effect of caffeine citrate is not a mere additive effect of the anticancer agent enhancing effect of anhydrous caffeine and the anticancer agent enhancing effect of citric acid. It became clear that it was a synergistic effect.
[肝細胞癌、子宮癌、乳癌及び卵巣癌培養細胞におけるカフェインクエン酸塩のシスプラチン効果増強の評価]
肝細胞癌、子宮癌、乳癌及び卵巣癌に対するカフェインクエン酸塩の抗癌剤(シスプラチン)効果増強を評価した。以下の細胞株及び培地を用いた他は、実施例1と同様の材料及び方法により試験を行った。
[Evaluation of enhanced cisplatin effect of caffeine citrate in cultured cells of hepatocellular carcinoma, uterine cancer, breast cancer and ovarian cancer]
The enhancement of the anticancer agent (cisplatin) effect of caffeine citrate on hepatocellular carcinoma, uterine cancer, breast cancer and ovarian cancer was evaluated. The test was carried out using the same materials and methods as in Example 1 except that the following cell lines and media were used.
<材料>
・細胞株
肝細胞癌としては、ヒト由来のHepG2細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
子宮癌としては、ヒト由来のHela細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
乳癌としては、ヒト由来のMCF7細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
卵巣癌としては、ヒト由来のOVCAR−3細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
・培地
上記の各癌種培養細胞について、HepG2細胞、Hela細胞、MCF7細胞及びOVCAR−3細胞は、いずれもRPMI1640培地を用いた。
<Material>
-Cell line As hepatocellular carcinoma, human-derived HepG2 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As uterine cancer, human-derived Hela cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As breast cancer, human-derived MCF7 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As ovarian cancer, human-derived OVCAR-3 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
-Medium For each of the above-mentioned cancer type cultured cells, RPMI1640 medium was used for HepG2 cells, Hela cells, MCF7 cells and OVCAR-3 cells.
<結果>
1.細胞生存率
図10、図13、図16及び図19に、それぞれHepG2細胞、Hela細胞、MCF7細胞及びOVCAR−3細胞における4つの群(CDDP、CDDP+caf、CDDP+CA、CDDP+cafCA)の450 nmの吸光度に基づく細胞生存率を示す。図10、図13、図16及び図19から明らかなように、HepG2細胞(肝細胞癌)、Hela細胞(子宮癌)、MCF7細胞(乳癌)及びOVCAR−3細胞(卵巣癌)において、CDDP+cafCA群は、全てのCDDP濃度で、他の群と比較して、最も細胞生存率が低かった。
また、分散分析の結果、CDDP+cafCA群は、各シスプラチン濃度において、他の全ての群との間で有意差があった(p<0.01)。
よって、カフェインクエン酸塩は、シスプラチンと組み合わせて添加することにより、シスプラチンの抗癌剤効果を増強することが、0.125〜2.0 μMの全てのシスプラチン濃度において確認できた。
<Result>
1. 1. Cell viability Figure 10, 13, 16 and 19 are based on the absorbance at 450 nm of four groups (CDDP, CDDP + caf, CDDP + CA, CDDP + cafCA) in HepG2 cells, Hela cells, MCF7 cells and OVCAR-3 cells, respectively. Shows cell viability. As is clear from FIGS. 10, 13, 16 and 19, in HepG2 cells (hepatocellular carcinoma), Hela cells (uterine cancer), MCF7 cells (breast cancer) and OVCAR-3 cells (ovarian cancer), the CDDP + cafCA group. Had the lowest cell viability at all CDDP concentrations compared to the other groups.
In addition, as a result of analysis of variance, the CDDP + cafCA group was significantly different from all other groups in each cisplatin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer agent effect of cisplatin when added in combination with cisplatin at all cisplatin concentrations of 0.125 to 2.0 μM.
2.Median Effect法
次に、Median Effect法によるHepG2細胞、Hela細胞、MCF7細胞及びOVCAR−3細胞における相乗効果の解析結果を、図11、図14、図17及び図20に示す。相乗効果の指標であるCIは、図11、図14、図17及び図20におけるCDDP濃度毎の細胞生存率に基づき、CDDP+cafとCDDP+CAのコンビネーションをCDDP+cafCAとして算出した。CI<1.0のとき、相乗効果が認められる。
図11、図14、図17及び図20から明らかなように、CDDP+cafCA群は、0.125〜2.0 μMの全てのシスプラチン濃度で、CI<1.0となり、CDDP+caf及びCDDP+CAに対して、相乗効果が認められた。
2. 2. Median Effect Method Next, the analysis results of the synergistic effect on HepG2 cells, Hela cells, MCF7 cells and OVCAR-3 cells by the Median Effect method are shown in FIGS. 11, 14, 17 and 20. The CI, which is an index of the synergistic effect, was calculated by calculating the combination of CDDP + caf and CDDP + CA as CDDP + cafCA based on the cell viability for each CDDP concentration in FIGS. 11, 14, 17, and 20. When CI <1.0, a synergistic effect is observed.
As is apparent from FIGS. 11, 14, 17 and 20, the CDDP + cafCA group had a CI <1.0 at all cisplatin concentrations of 0.125 to 2.0 μM, relative to CDDP + caf and CDDP + CA. A synergistic effect was observed.
3.Isobologram法
Isobologram法によるHepG2細胞、Hela細胞、MCF7細胞及びOVCAR−3細胞における相乗効果の解析結果を図12、図15、図18及び図21に示す。
図12、図15、図18及び図21のグラフから明らかなように、CDDP+cafCAの各Effective Doseを示す円形の破線が、それぞれ、CDDP+caf及びCDDP+CAの各Effective Doseを結んだ曲線よりも左下に位置することから、相乗効果が認められた。
図12、図15、図18及び図21の表から明らかなように、CDDP+cafCA群は、50〜90%の全てのEffective Doseで、CI<1.0となり、CDDP+caf及びCDDP+CAに対して、相乗効果が認められた。
3. 3. Isobologram method The analysis results of the synergistic effect on HepG2 cells, Hela cells, MCF7 cells and OVCAR-3 cells by the Isobologram method are shown in FIGS. 12, 15, 18 and 21.
As is clear from the graphs of FIGS. 12, 15, 18 and 21, the circular broken line indicating each Effective Dose of CDDP + cafCA is located at the lower left of the curve connecting the Effective Dose of CDDP + caf and CDDP + CA, respectively. Therefore, a synergistic effect was recognized.
As is clear from the tables of FIGS. 12, 15, 18 and 21, the CDDP + cafCA group had a CI <1.0 at all Effective Dose of 50-90% and had a synergistic effect on CDDP + caf and CDDP + CA. Was recognized.
[骨肉腫、軟部肉腫、胃癌及び子宮癌培養細胞におけるカフェインクエン酸塩のアドリアマイシン効果増強の評価]
骨肉腫、軟部肉腫、胃癌及び子宮癌に対するカフェインクエン酸塩の抗癌剤(アドリアマイシン)効果増強を評価した。以下の細胞株、抗癌剤及び培地を用いた他は、実施例1と同様の材料により試験を行った。
[Evaluation of enhanced adriamycin effect of caffeine citrate in cultured cells of osteosarcoma, soft tissue sarcoma, gastric cancer and uterine cancer]
The enhancement of the anticancer agent (adriamycin) effect of caffeine citrate on osteosarcoma, soft tissue sarcoma, gastric cancer and uterine cancer was evaluated. The test was carried out using the same materials as in Example 1 except that the following cell lines, anticancer agents and media were used.
<材料>
・細胞株
骨肉腫としては、ヒト由来のHOS細胞(ATCCより入手)を用いた。
軟部肉腫としては、ヒト由来のHT1080細胞(ATCCより入手)を用いた。
胃癌としては、ヒト由来のMKN45細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
子宮癌としては、ヒト由来のHela細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
・抗癌剤
アドリアマイシン(和光純薬工業株式会社より入手、以下、ADMと称する場合がある)を用いた。
・培地
上記の各癌種培養細胞について、HOS細胞、MKN45細胞及びHela細胞は、RPMI1640培地を用いた。HT1080細胞は、DMEMを用いた。
<Material>
-As the cell line osteosarcoma, human-derived HOS cells (obtained from ATCC) were used.
As the soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
As gastric cancer, human-derived MKN45 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As uterine cancer, human-derived Hela cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
-The anticancer drug adriamycin (obtained from Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as ADM) was used.
-Medium For the above-mentioned cultured cells of each cancer type, RPMI1640 medium was used as HOS cells, MKN45 cells and Hela cells. DMEM was used for HT1080 cells.
<WST−8アッセイ>
工程(2)を下記の通りに変更した他は、実施例1の方法に準じて試験を行った。
(2)培養後、各癌種培養細胞について、4つの群に分け、薬剤(ADM、ADM+caf、ADM+CA、ADM+cafCA)を100 μL添加した。各群に添加する薬剤のADM濃度は、水(滅菌精製水)にADMの粉末を溶かして高濃度のものを作成し、その後細胞に応じた培養液で希釈することで12.5、25、50、100、200 nMに調製した。各群の併用薬であるcaf及びCAの濃度は、水に粉末を溶かしてそれぞれ高濃度のものを作成し、その後細胞に応じた培養液で希釈することで一律0.5 mMに調製した。cafCAの濃度は、製品が液体であるので、そのまま細胞に応じた培養液で希釈し、caf、CAと同様の0.5 mMに調製した。対照として、一部のウェルには薬剤を添加しなかった(ADM濃度0 μM)。
<WST-8 Assay>
The test was carried out according to the method of Example 1 except that the step (2) was changed as follows.
(2) After culturing, each cancer type cultured cell was divided into four groups, and 100 μL of a drug (ADM, ADM + caf, ADM + CA, ADM + cafCA) was added. The ADM concentration of the drug added to each group is 12.5, 25, by dissolving ADM powder in water (sterile purified water) to prepare a high concentration, and then diluting with a culture solution suitable for the cells. Prepared to 50, 100, 200 nM. The concentrations of caf and CA, which are concomitant drugs in each group, were uniformly adjusted to 0.5 mM by dissolving powder in water to prepare high concentrations of each, and then diluting with a culture solution suitable for the cells. Since the product is a liquid, the concentration of cafCA was diluted with a culture solution suitable for the cells as it was, and adjusted to 0.5 mM, which was the same as for caf and CA. As a control, no drug was added to some wells (ADM concentration 0 μM).
<結果>
1.細胞生存率
図22、図25、図28及び図31に、それぞれHOS細胞、HT1080細胞、MKN45細胞及びHela細胞における4つの群(ADM、ADM+caf、ADM+CA、ADM+cafCA)の450 nmの吸光度に基づく細胞生存率を示す。図22、図25、図28及び図31から明らかなように、HOS細胞(骨肉腫)、HT1080細胞(軟部肉腫)、MKN45細胞(胃癌)及びHela細胞(子宮癌)において、ADM+cafCA群は、全てのADM濃度で、他の群と比較して、最も細胞生存率が低かった。
また、分散分析の結果、ADM+cafCA群は、各アドリアマイシン濃度において、他の全ての群との間で有意差があった(p<0.01)。
よって、カフェインクエン酸塩は、アドリアマイシンと組み合わせて添加することにより、アドリアマイシンの抗癌剤効果を増強することが、12.5〜200 nMの全てのアドリアマイシン濃度において確認できた。
<Result>
1. 1. Cell viability Figure 22, FIG. 25, FIG. 28 and FIG. 31 show cell survival based on the absorbance at 450 nm of four groups (ADM, ADM + caf, ADM + CA, ADM + cafCA) in HOS cells, HT1080 cells, MKN45 cells and Hela cells, respectively. Shows the rate. As is clear from FIGS. 22, 25, 28 and 31, in HOS cells (osteosarcoma), HT1080 cells (soft tissue sarcoma), MKN45 cells (stomach cancer) and Hela cells (uterine cancer), the ADM + cafCA group is all At the ADM concentration of, the cell viability was the lowest compared with the other groups.
In addition, as a result of analysis of variance, the ADM + cafCA group was significantly different from all the other groups in each adriamycin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer agent effect of adriamycin when added in combination with adriamycin at all adriamycin concentrations of 12.5 to 200 nM.
2.Median Effect法
次に、Median Effect法によるHOS細胞、HT1080細胞、MKN45細胞及びHela細胞における相乗効果の解析結果を、図23、図26、図29及び図32に示す。相乗効果の指標であるCIは、図23、図26、図29及び図32におけるADM濃度毎の細胞生存率に基づき、ADM+cafとADM+CAのコンビネーションをADM+cafCAとして算出した。CI<1.0のとき、相乗効果が認められる。
図23、図26、図29及び図32から明らかなように、ADM+cafCA群は、12.5〜200 nMの全てのアドリアマイシン濃度で、CI<1.0となり、ADM+caf及びADM+CAに対して、相乗効果が認められた。
2. 2. Median Effect Method Next, the analysis results of the synergistic effect on HOS cells, HT1080 cells, MKN45 cells and Hela cells by the Median Effect method are shown in FIGS. 23, 26, 29 and 32. The CI, which is an index of the synergistic effect, was calculated by calculating the combination of ADM + caf and ADM + CA as ADM + cafCA based on the cell viability for each ADM concentration in FIGS. 23, 26, 29 and 32. When CI <1.0, a synergistic effect is observed.
As is clear from FIGS. 23, 26, 29 and 32, the ADM + cafCA group had a CI <1.0 at all adriamycin concentrations of 12.5 to 200 nM, a synergistic effect on ADM + caf and ADM + CA. Was recognized.
3.Isobologram法
Isobologram法によるHOS細胞、HT1080細胞、MKN45細胞及びHela細胞における相乗効果の解析結果を図24、図27、図30及び図33に示す。
図24、図27、図30及び図33のグラフから明らかなように、ADM+cafCAの各Effective Doseを示す円形の破線が、それぞれ、ADM+caf及びADM+CAの各Effective Doseを結んだ曲線よりも左下に位置することから、相乗効果が認められた。
図24、図27、図30及び図33の表から明らかなように、ADM+cafCA群は、50〜90%の全てのEffective Doseで、CI<1.0となり、ADM+caf及びADM+CAに対して、相乗効果が認められた。
3. 3. Isobologram method The analysis results of the synergistic effect on HOS cells, HT1080 cells, MKN45 cells and Hela cells by the Isobologram method are shown in FIGS. 24, 27, 30 and 33.
As is clear from the graphs of FIGS. 24, 27, 30 and 33, the circular broken line indicating each Effective Dose of ADM + cafCA is located at the lower left of the curve connecting each Effective Dose of ADM + caf and ADM + CA, respectively. Therefore, a synergistic effect was recognized.
As is clear from the tables of FIGS. 24, 27, 30 and 33, the ADM + cafCA group had a CI <1.0 at all Effective Dose of 50-90% and had a synergistic effect on ADM + caf and ADM + CA. Was recognized.
以上の結果より、本発明の癌治療剤において、アドリアマイシンとカフェインクエン酸塩との組合せは、アドリアマイシン単独、アドリアマイシンと無水カフェインとの組合せ、アドリアマイシンとクエン酸との組合せと比較して、有意にアドリアマイシンの抗癌剤効果をより増強すること、カフェインクエン酸塩の有する抗癌剤増強効果は、無水カフェインの有する抗癌剤増強効果と、クエン酸の有する抗癌剤増強効果との、単なる相加効果ではなく、相乗効果であることが明らかになった。 From the above results, in the cancer therapeutic agent of the present invention, the combination of adriamycin and caffeine citrate is significant as compared with the combination of adriamycin alone, adriamycin and anhydrous caffeine, and adriamycin and citric acid. In addition, the anticancer agent enhancing effect of adriamycin and the anticancer agent enhancing effect of caffeine citrate are not merely additive effects of the anticancer agent enhancing effect of anhydrous caffeine and the anticancer agent enhancing effect of citric acid. It became clear that it was a synergistic effect.
[骨肉腫、胃癌及び卵巣癌培養細胞におけるカフェインクエン酸塩のゲムシタビン効果増強の評価]
骨肉腫、胃癌及び卵巣癌に対するカフェインクエン酸塩の抗癌剤(ゲムシタビン)効果増強を評価した。以下の細胞株、抗癌剤及び培地を用いた他は、実施例1と同様の材料により試験を行った。
[Evaluation of enhanced gemcitabine effect of caffeine citrate in cultured cells of osteosarcoma, gastric cancer and ovarian cancer]
The enhancement of the anticancer drug (gemcitabine) effect of caffeine citrate on osteosarcoma, gastric cancer and ovarian cancer was evaluated. The test was carried out using the same materials as in Example 1 except that the following cell lines, anticancer agents and media were used.
<材料>
・細胞株
骨肉腫としては、ヒト由来のHOS細胞(ATCCより入手)を用いた。
胃癌としては、ヒト由来のMKN45細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
卵巣癌としては、ヒト由来のOVCAR−3細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
・抗癌剤
ゲムシタビン(和光純薬工業株式会社より入手、以下、GEMと称する場合がある)を用いた。
・培地
上記の各癌種培養細胞について、HOS細胞、MKN45細胞及びOVCAR−3細胞は、RPMI1640培地を用いた。
<Material>
-As the cell line osteosarcoma, human-derived HOS cells (obtained from ATCC) were used.
As gastric cancer, human-derived MKN45 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As ovarian cancer, human-derived OVCAR-3 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
-An anticancer drug gemcitabine (obtained from Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as GEM) was used.
-Medium For each of the above-mentioned cultured cells of cancer type, RPMI1640 medium was used as HOS cells, MKN45 cells and OVCAR-3 cells.
<WST−8アッセイ>
工程(2)を下記の通りに変更した他は、実施例1の方法に準じて試験を行った。
(2)培養後、各癌種培養細胞について、4つの群に分け、薬剤(GEM、GEM+caf、GEM+CA、GEM+cafCA)を100 μL添加した。各群に添加する薬剤のGEM濃度は、水(滅菌精製水)にGEMの粉末を溶かして高濃度のものを作成し、その後細胞に応じた培養液で希釈することで0.5、1、2、4、8 nMに調製した。各群の併用薬であるcaf及びCAの濃度は、水に粉末を溶かしてそれぞれ高濃度のものを作成し、その後細胞に応じた培養液で希釈することで一律0.5 mMに調製した。cafCAの濃度は、製品が液体であるので、そのまま細胞に応じた培養液で希釈し、caf、CAと同様の0.5 mMに調製した。対照として、一部のウェルには薬剤を添加しなかった(GEM濃度0 μM)。
<WST-8 Assay>
The test was carried out according to the method of Example 1 except that the step (2) was changed as follows.
(2) After culturing, each cancer type cultured cell was divided into four groups, and 100 μL of a drug (GEM, GEM + caf, GEM + CA, GEM + cafCA) was added. The GEM concentration of the drug added to each group is 0.5, 1, by dissolving GEM powder in water (sterile purified water) to prepare a high concentration, and then diluting with a culture solution suitable for the cells. Prepared to 2, 4, 8 nM. The concentrations of caf and CA, which are concomitant drugs in each group, were uniformly adjusted to 0.5 mM by dissolving powder in water to prepare high concentrations of each, and then diluting with a culture solution suitable for the cells. Since the product is a liquid, the concentration of cafCA was diluted with a culture solution suitable for the cells as it was, and adjusted to 0.5 mM, which was the same as for caf and CA. As a control, no drug was added to some wells (GEM concentration 0 μM).
<結果>
1.細胞生存率
図34、図37及び図40に、それぞれHOS細胞、MKN45細胞及びOVCAR−3細胞における4つの群(GEM、GEM+caf、GEM+CA、GEM+cafCA)の450 nmの吸光度に基づく細胞生存率を示す。図34、図37及び図40から明らかなように、HOS細胞(骨肉腫)、MKN45細胞(胃癌)及びOVCAR−3細胞(卵巣癌)において、GEM+cafCA群は、全てのGEM濃度で、他の群と比較して、最も細胞生存率が低かった。
また、分散分析の結果、GEM+cafCA群は、各ゲムシタビン濃度において、他の全ての群との間で有意差があった(p<0.01)。
よって、カフェインクエン酸塩は、ゲムシタビンと組み合わせて添加することにより、ゲムシタビンの抗癌剤効果を増強することが、0.5〜8.0 nMの全てのゲムシタビン濃度において確認できた。
<Result>
1. 1. Cell viability FIGS. 34, 37 and 40 show cell viability based on the absorbance at 450 nm of the four groups (GEM, GEM + caf, GEM + CA, GEM + cafCA) in HOS cells, MKN45 cells and OVCAR-3 cells, respectively. As is clear from FIGS. 34, 37 and 40, in HOS cells (osteosarcoma), MKN45 cells (gastric cancer) and OVCAR-3 cells (ovarian cancer), the GEM + cafCA group is the other group at all GEM concentrations. The cell viability was the lowest.
In addition, as a result of analysis of variance, the GEM + cafCA group was significantly different from all other groups in each gemcitabine concentration (p <0.01).
Therefore, it was confirmed that when caffeine citrate was added in combination with gemcitabine, the anticancer agent effect of gemcitabine was enhanced at all gemcitabine concentrations of 0.5 to 8.0 nM.
2.Median Effect法
次に、Median Effect法によるHOS細胞、MKN45細胞及びOVCAR−3における相乗効果の解析結果を、図35、図38及び図41に示す。相乗効果の指標であるCIは、図35、図38及び図41におけるGEM濃度毎の細胞生存率に基づき、GEM+cafとGEM+CAのコンビネーションをGEM+cafCAとして算出した。CI<1.0のとき、相乗効果が認められる。
図35、図38及び図41から明らかなように、GEM+cafCA群は、0.5〜8.0 nMの全てのゲムシタビン濃度で、CI<1.0となり、GEM+caf及びGEM+CAに対して、相乗効果が認められた。
2. 2. Median Effect Method Next, the analysis results of the synergistic effect on HOS cells, MKN45 cells and OVCAR-3 by the Median Effect method are shown in FIGS. 35, 38 and 41. The CI, which is an index of the synergistic effect, was calculated by calculating the combination of GEM + caf and GEM + CA as GEM + cafCA based on the cell viability for each GEM concentration in FIGS. 35, 38 and 41. When CI <1.0, a synergistic effect is observed.
As is clear from FIGS. 35, 38 and 41, the GEM + cafCA group had a CI <1.0 at all gemcitabine concentrations of 0.5 to 8.0 nM, synergistic effect on GEM + caf and GEM + CA. Admitted.
3.Isobologram法
Isobologram法によるHOS細胞、MKN45細胞及びOVCAR−3における相乗効果の解析結果を図36、図39及び図42に示す。
図36及び図39のグラフから明らかなように、GEM+cafCAの各Effective Doseを示す円形の破線が、それぞれ、GEM+caf及びGEM+CAの各Effective Doseを結んだ曲線よりも左下に位置することから、相乗効果が認められた。
図36、図39及び図42の表から明らかなように、GEM+cafCA群は、50〜90%の全てのEffective Doseで、CI<1.0となり、GEM+caf及びGEM+CAに対して、相乗効果が認められた。
3. 3. Isobologram method The analysis results of the synergistic effect on HOS cells, MKN45 cells and OVCAR-3 by the Isobologram method are shown in FIGS. 36, 39 and 42.
As is clear from the graphs of FIGS. 36 and 39, the circular broken line indicating each Effective Dose of GEM + cafCA is located at the lower left of the curve connecting each Effective Dose of GEM + caf and GEM + CA, respectively, so that the synergistic effect is obtained. Admitted.
As is clear from the tables of FIGS. 36, 39 and 42, the GEM + cafCA group had a CI <1.0 in all 50-90% of Effective Dose, and a synergistic effect was observed for GEM + caf and GEM + CA. It was.
以上の結果より、本発明の癌治療剤において、ゲムシタビンとカフェインクエン酸塩との組合せは、ゲムシタビン単独、ゲムシタビンと無水カフェインとの組合せ、ゲムシタビンとクエン酸との組合せと比較して、有意にゲムシタビンの抗癌剤効果をより増強すること、カフェインクエン酸塩の有する抗癌剤増強効果は、無水カフェインの有する抗癌剤増強効果と、クエン酸の有する抗癌剤増強効果との、単なる相加効果ではなく、相乗効果であることが明らかになった。 From the above results, in the cancer therapeutic agent of the present invention, the combination of gemcitabine and caffeine citrate is significant as compared with gemcitabine alone, gemcitabine and anhydrous caffeine, and gemcitabine and citric acid. To further enhance the anticancer agent effect of gemcitabine, the anticancer agent enhancing effect of caffeine citrate is not a mere additive effect of the anticancer agent enhancing effect of anhydrous caffeine and the anticancer agent enhancing effect of citric acid. It became clear that it was a synergistic effect.
[骨肉腫及び軟部肉腫培養細胞におけるカフェインクエン酸塩の抗癌剤による細胞増殖抑制効果の増強の評価]
以下の方法により、骨肉腫及び軟部肉腫に対するカフェインクエン酸塩の抗癌剤による細胞増殖抑制効果の増強を評価した。
[Evaluation of enhancement of cell growth inhibitory effect of caffeine citrate by anticancer drug in osteosarcoma and soft tissue sarcoma cultured cells]
The enhancement of the cell growth inhibitory effect of caffeine citrate on anticancer agents on osteosarcoma and soft tissue sarcoma was evaluated by the following method.
<材料>
・細胞株
骨肉腫としては、ヒト由来のHOS細胞(ATCCより入手)を用いた。
軟部肉腫としては、ヒト由来のHT1080細胞(ATCCより入手)を用いた。
・抗癌剤
シスプラチン(和光純薬工業株式会社より入手)を用いた。
・併用薬
カフェインクエン酸塩(cafCA、クエン酸カフェイン、ノーベルファーマ株式会社より入手)、無水カフェイン(caf、和光純薬工業株式会社より入手)又はクエン酸(CA、和光純薬工業株式会社より入手)を用いた。
・培地
HOS細胞は、RPMI1640培地を用い、HT1080細胞は、DMEMを用いた。
<Material>
-As the cell line osteosarcoma, human-derived HOS cells (obtained from ATCC) were used.
As the soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
-The anticancer drug cisplatin (obtained from Wako Pure Chemical Industries, Ltd.) was used.
-Concomitant drug caffeine citrate (cafCA, caffeine citrate, obtained from Nobelpharma Co., Ltd.), anhydrous caffeine (caf, obtained from Wako Pure Chemical Industries, Ltd.) or citric acid (CA, Wako Pure Chemical Industries, Ltd.) Obtained from the company) was used.
-Medium HOS cells used RPMI1640 medium, and HT1080 cells used DMEM.
<細胞増殖(EdU)アッセイ>
EdU(5−エチニル−2'−デオキシウリジン)とは、チミジンのヌクレオシド類似体(アナログ)であり、活発なDNA合成の間にDNAに取り込まれる。すなわち、EdUを検出することで、活発なDNA合成が起こっている増殖細胞を検出できる。
そこで、本実験は、以下の手順で、EdUを検出することにより、薬剤投与による細胞増殖能の変化を確認し、CDDP+cafCAによる細胞増殖抑制効果の増強を調べた。
(1)上記の各癌種培養細胞について、それぞれ3000〜5000個/100 μLの濃度で、上記の培地、37℃、CO2 5%の条件で、24時間、チャンバースライド(Lab−Tec(登録商標)、Thermo Scientific Fisher)で培養した。
(2)培養後、各癌種培養細胞について、5つの群(Non−drug、CDDP、CDDP+caf、CDDP+CA、CDDP+cafCA)に分け、培養液を入れ替えた。Non−drug群は0.5 mLの培養液を、薬剤投与群はCDDPが0.25 μM、caf、CA及びcafCAがそれぞれ0.5 mMとなるように調整した培養液を0.5 mL添加した。各群に添加する薬剤のCDDP濃度は、水(滅菌精製水)にCDDPの粉末を溶かして高濃度のものを作成し、その後細胞に応じた培養液で希釈することで0.25 μMに調製した。各群の併用薬であるcaf及びCAの濃度は、水に粉末を溶かしてそれぞれ高濃度のものを作成し、その後細胞に応じた培養液で希釈することで一律0.5 mMに調製した。cafCAの濃度は、製品が液体であるので、そのまま細胞に応じた培養液で希釈し、caf、CAと同様の0.5 mMに調製した。対照として、一部のウェルには薬剤を添加しなかった(Non−drug:0 μM)。
(3)薬剤添加後24時間後に、Click−iT(登録商標) Plus EdU Alexa Fluor(登録商標)555 Imaging Kit(Thermo Fisher Scientific社)、並びにNucBlue(商標) Live Cell Stain ReadyProbes(商標) reagent (molecular probes by life technologies)(Thermo Fisher Scientific社)を用いて、メーカーのマニュアルに従い、DNA合成を行っている細胞の核を染色した(Hoechst(Hoechst33342):核を検出、EdU検出試薬(Alexa Fluor(登録商標)555):DNA合成中の核を検出)。蛍光顕微鏡(BIOREVO:BZ−9000、KEYENCE社)により、Hoechstを光らせるためのフィルターはDAPI(励起波長:360±40 nm、ダイクロイックミラー波長:400 nm、吸収波長:460±50 nm)を、EdUを光らせるためのフィルターはTRITC(励起波長:540±25 nm、ダイクロイックミラー波長:565 nm、吸収波長:605±55 nm)を用い、各群の定点5視野を写真撮影した。Hoechst及びEdUの検出を行った。Non−drugと比較して、EdU検出シグナルが少なく(弱く)なるほど、細胞増殖を抑制しているといえる。
(4)(3)の写真の画面中の細胞をカウントした。なお、EdU発色率は以下の通りに算出した。EdU発色率=DNA合成細胞数(EdU検出細胞数)/細胞総数(Hoechst検出細胞数)。各群5視野のEdU発色率の平均値を算出し、Non−drugの平均値を1とした比率から、Statcel 3(オーエムエス出版)を用いて、各群のEdU発色率の有意差について分散分析した。
<Cell proliferation (EdU) assay>
EdU (5-ethynyl-2'-deoxyuridine) is a nucleoside analog of thymidine that is incorporated into DNA during active DNA synthesis. That is, by detecting EdU, proliferating cells in which active DNA synthesis is occurring can be detected.
Therefore, in this experiment, by detecting EdU by the following procedure, the change in cell proliferation ability due to drug administration was confirmed, and the enhancement of the cell proliferation inhibitory effect by CDDP + cafCA was investigated.
(1) For each cancer type cultured cells described above, at a concentration of 3000 to 5000 pieces / 100 [mu] L each, above the medium, 37 ° C., with CO 2 5% of the conditions, 24 hours, chamber slides (Lab-Tec (registered (Trademark), Thermo Scientific Fisher) was cultured.
(2) After culturing, each cancer type cultured cell was divided into 5 groups (Non-drug, CDDP, CDDP + caf, CDDP + CA, CDDP + cafCA), and the culture medium was replaced. The Non-drug group added 0.5 mL of the culture solution, and the drug-administered group added 0.5 mL of the culture solution adjusted so that the CDDP was 0.25 μM and the CDDP was 0.5 mM for each of caf, CA and cafCA. did. The CDDP concentration of the drug added to each group was adjusted to 0.25 μM by dissolving CDDP powder in water (sterile purified water) to prepare a high concentration, and then diluting with a culture solution suitable for the cells. did. The concentrations of caf and CA, which are concomitant drugs in each group, were uniformly adjusted to 0.5 mM by dissolving powder in water to prepare high concentrations of each, and then diluting with a culture solution suitable for the cells. Since the product is a liquid, the concentration of cafCA was diluted with a culture solution suitable for the cells as it was, and adjusted to 0.5 mM, which was the same as for caf and CA. As a control, no drug was added to some wells (Non-drug: 0 μM).
(3) Twenty-four hours after the addition of the drug, Click-iT (registered trademark) Plus EdU Alexa Fluor (registered trademark) 555 Imaging Kit (Thermo Fisher Scientific), and NucBlue (trademark) Live Cell Reagent (trademark) Live Cell Reagent Using probes by life technologies (Thermo Fisher Scientific), the nuclei of cells undergoing DNA synthesis were stained according to the manufacturer's manual (Hoechst (Hoechst 33342): nuclei detected, EdU detection reagent (AlexaFlu) Trademark) 555): Detects nuclei during DNA synthesis). With a fluorescence microscope (BIOREVO: BZ-9000, KEYENCE), the filter for illuminating Hoechst is DAPI (excitation wavelength: 360 ± 40 nm, dichroic mirror wavelength: 400 nm, absorption wavelength: 460 ± 50 nm), and EdU. A TRITC (excitation wavelength: 540 ± 25 nm, dichroic mirror wavelength: 565 nm, absorption wavelength: 605 ± 55 nm) was used as a filter for illuminating, and 5 fixed-point visual fields of each group were photographed. Hoechst and EdU were detected. It can be said that the smaller (weaker) the EdU detection signal is, the more the cell proliferation is suppressed as compared with Non-drug.
(4) The cells in the screen of the photograph of (3) were counted. The EdU color development rate was calculated as follows. EdU color development rate = number of DNA-synthesizing cells (number of EdU-detected cells) / total number of cells (number of Hoechst-detected cells). The average value of the EdU color development rate of the 5 fields of view in each group was calculated, and from the ratio with the average value of Non-drug as 1, using Statcel 3 (OMS Publishing), the significant difference in the EdU color development rate of each group was dispersed. analyzed.
<結果>
HOS細胞及びHT1080細胞における結果を、それぞれ図43及び図44に示す。
図43から明らかなように、HOS細胞(骨肉腫)において、CDDP+cafCA群は、他の群と比較して、最もHoechst検出細胞数に対するEdU検出細胞数の割合が少なかった。すなわち、CDDP+cafCA群は、他の群と比較して、最もHOS細胞(骨肉腫)の細胞増殖を抑制した。
図44から明らかなように、HT1080細胞(軟部肉腫)において、CDDP+cafCA群は、他の群と比較して、最もHoechst検出シグナルに対するEdU検出シグナルの割合が少なかった。すなわち、CDDP+cafCA群は、他の群と比較して、最もHT1080細胞(軟部肉腫)の細胞増殖を抑制した。
HOS細胞及びHT1080細胞における各群のEdU発色率の有意差の解析結果を図45に示す。
図45から明らかなように、CDDP+cafCA群は、HOS細胞及びHT1080細胞において、他の全ての群との間で有意差があった(p<0.01)。また、HOS細胞において、CDDP+cafCA群のEdU発色率は、Non−Drug群と比較して約51%、CDDP群と比較して約58%、CDDP+caf群と比較して約66%、CDDP+CA群と比較して約61%であり、大きく低下した。HT1080細胞において、CDDP+cafCA群のEdU発色率は、Non−Drug群と比較して約51%、CDDP群と比較して約56%、CDDP+caf群と比較して約72%、CDDP+CA群と比較して約57%であり、大きく低下した。
よって、カフェインクエン酸塩は、骨肉腫及び軟部肉腫において、シスプラチンと組み合わせて添加することにより、シスプラチンの細胞増殖抑制効果を増強することが確認できた。
<Result>
The results in HOS cells and HT1080 cells are shown in FIGS. 43 and 44, respectively.
As is clear from FIG. 43, in HOS cells (osteosarcoma), the ratio of the number of EdU-detected cells to the number of Hoechst-detected cells was the smallest in the CDDP + cafCA group as compared with the other groups. That is, the CDDP + cafCA group suppressed the cell proliferation of HOS cells (osteosarcoma) most as compared with the other groups.
As is clear from FIG. 44, in HT1080 cells (soft tissue sarcoma), the CDDP + cafCA group had the lowest ratio of the EdU detection signal to the Hoechst detection signal as compared with the other groups. That is, the CDDP + cafCA group suppressed the cell proliferation of HT1080 cells (soft tissue sarcoma) most as compared with the other groups.
The analysis result of the significant difference in the EdU color development rate of each group in HOS cells and HT1080 cells is shown in FIG.
As is clear from FIG. 45, the CDDP + cafCA group was significantly different from all other groups in HOS cells and HT1080 cells (p <0.01). In HOS cells, the EdU color development rate of the CDDP + cafCA group was about 51% as compared with the Non-Drug group, about 58% as compared with the CDDP group, about 66% as compared with the CDDP + caf group, and compared with the CDDP + CA group. It was about 61%, which was a large decrease. In HT1080 cells, the EdU color development rate of the CDDP + cafCA group was about 51% compared to the Non-Drug group, about 56% compared to the CDDP group, about 72% compared to the CDDP + caf group, and compared to the CDDP + CA group. It was about 57%, which was a large decrease.
Therefore, it was confirmed that caffeine citrate enhances the cell growth inhibitory effect of cisplatin when added in combination with cisplatin in osteosarcoma and soft tissue sarcoma.
以上の結果より、本発明の癌治療剤において、シスプラチンとカフェインクエン酸塩との組合せは、骨肉腫及び軟部肉腫において、シスプラチン単独、シスプラチンと無水カフェインとの組合せ、シスプラチンとクエン酸との組合せと比較して、有意にシスプラチンの細胞増殖抑制効果をより増強することが明らかとなった。 From the above results, in the cancer therapeutic agent of the present invention, the combination of cisplatin and caffeine citrate is the combination of cisplatin alone, cisplatin and anhydrous caffeine, and cisplatin and citric acid in osteosarcoma and soft sarcoma. It was revealed that the effect of cisplatin on suppressing cell proliferation was significantly enhanced as compared with the combination.
[骨肉腫及び軟部肉腫培養細胞におけるカフェインクエン酸塩の抗癌剤によるアポトーシス(細胞死)誘導効果の増強の評価]
以下の方法により、骨肉腫及び軟部肉腫に対するカフェインクエン酸塩の抗癌剤によるアポトーシス誘導効果の増強を評価した。
[Evaluation of enhancement of apoptosis (cell death) -inducing effect of caffeine citrate by anticancer drug in osteosarcoma and soft tissue sarcoma cultured cells]
The enhancement of the apoptosis-inducing effect of caffeine citrate on anticancer agents on osteosarcoma and soft tissue sarcoma was evaluated by the following method.
<材料>
・細胞株
骨肉腫としては、ヒト由来のHOS細胞(ATCCより入手)を用いた。
軟部肉腫としては、ヒト由来のHT1080細胞(ATCCより入手)を用いた。
・抗癌剤
シスプラチン(和光純薬工業株式会社より入手)を用いた。
・併用薬
カフェインクエン酸塩(cafCA、クエン酸カフェイン、ノーベルファーマ株式会社より入手)、無水カフェイン(caf、和光純薬工業株式会社より入手)又はクエン酸(CA、和光純薬工業株式会社より入手)を用いた。
・培地
HOS細胞は、RPMI1640培地を用い、HT1080細胞は、DMEMを用いた。
<Material>
-As the cell line osteosarcoma, human-derived HOS cells (obtained from ATCC) were used.
As the soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
-The anticancer drug cisplatin (obtained from Wako Pure Chemical Industries, Ltd.) was used.
-Concomitant drug caffeine citrate (cafCA, caffeine citrate, obtained from Nobelpharma Co., Ltd.), anhydrous caffeine (caf, obtained from Wako Pure Chemical Industries, Ltd.) or citric acid (CA, Wako Pure Chemical Industries, Ltd.) Obtained from the company) was used.
-Medium HOS cells used RPMI1640 medium, and HT1080 cells used DMEM.
<ミトコンドリア膜電位変化(TMRE)アッセイ>
TMRE(tetramethylrhodamine,ethyl ester)とは、膜電位の保たれているミトコンドリア内に蓄積される色素である。ミトコンドリア膜電位が消失すると、TMREは分散され、検出シグナル強度が弱くなる。アポトーシスでは、初期の段階として、ミトコンドリア外膜が破壊され、膜電位の消失が起こるため、TMREの蓄積は少なくなり、TMRE検出シグナル強度が弱くなる。
そこで、本実験は、以下の手順で、TMREを検出することにより、薬剤投与による膜電位の変化を確認し、CDDP+cafCAによるミトコンドリア膜電位の減弱・消失(アポトーシス誘導効果)の増強を調べた。
(1)上記の各癌種培養細胞について、それぞれ3000〜5000個/100 μLの濃度で、上記の培地、37℃、CO2 5%の条件で、24時間、チャンバースライド(Lab−Tec(登録商標)、Thermo Scientific Fisher)で培養した。
(2)培養後、各癌種培養細胞について、5つの群(Non−drug、CDDP、CDDP+caf、CDDP+CA、CDDP+cafCA)に分け、培養液を入れ替えた。Non−drug群は0.5 mLの培養液を、薬剤投与群はCDDPが0.25 μM、caf、CA及びcafCAがそれぞれ0.5 mMとなるように調整した培養液を0.5 mL添加した。各群に添加する薬剤のCDDP濃度は、水(滅菌精製水)にCDDPの粉末を溶かして高濃度のものを作成し、その後細胞に応じた培養液で希釈することで0.25 μMに調製した。各群の併用薬であるcaf及びCAの濃度は、水に粉末を溶かしてそれぞれ高濃度のものを作成し、その後細胞に応じた培養液で希釈することで一律0.5 mMに調製した。cafCAの濃度は、製品が液体であるので、そのまま細胞に応じた培養液で希釈し、caf、CAと同様の0.5 mMに調製した。対照として、一部のウェルには薬剤を添加しなかった(Non−drug:0 μM)。
(3)薬剤添加後24時間後に、TMRE−Mitochondrial Membrane Potential Assay Kit(アブカム社)を用いて、メーカーのマニュアルに従い、DNA合成を行っている細胞の核を染色した(Hoechst(Hoechst33342):核を検出、TMRE:膜電位の保たれているミトコンドリアを検出)。蛍光顕微鏡(BIOREVO:BZ−9000、KEYENCE社)によりHoechstを光らせるためのフィルターはDAPI(励起波長:360±40 nm、ダイクロイックミラー波長:400 nm、吸収波長:460±50 nm)を、TMREを光らせるためのフィルターはTRITC(励起波長:540±25 nm、ダイクロイックミラー波長:565 nm、吸収波長:605±55 nm)を用い、各群の定点5視野を写真撮影した。Hoechst及びTMREの検出を行った。Non−drugと比較して、TMRE検出シグナルが弱くなるほど、アポトーシスを誘導しているといえる。
(4)(3)の写真の画面中の細胞をカウントした。なお、1細胞あたりのTMRE輝度は以下の通りに算出した。1細胞あたりのTMRE輝度=TMRE輝度(検出シグナル強度)総和/細胞総数(Hoechst検出細胞数)。各群5視野の1細胞あたりのTMRE輝度の平均値を算出し、Non−drugの平均値を1とした比率から、Statcel 3(オーエムエス出版)を用いて、各群の1細胞あたりのTMRE輝度の有意差について分散分析した。
<Mitochondrial Membrane Potential Change (TMRE) Assay>
TMRE (tetramethylrhodamine, ester) is a dye that accumulates in mitochondria where the membrane potential is maintained. When the mitochondrial membrane potential disappears, TMRE is dispersed and the detection signal intensity is weakened. In apoptosis, as an early stage, the outer mitochondrial membrane is destroyed and the membrane potential is lost, so that TMRE accumulation is reduced and the TMRE detection signal intensity is weakened.
Therefore, in this experiment, the change in the membrane potential due to drug administration was confirmed by detecting TMRE by the following procedure, and the attenuation / disappearance (apoptosis-inducing effect) of the mitochondrial membrane potential by CDDP + cafCA was investigated.
(1) For each cancer type cultured cells described above, at a concentration of 3000 to 5000 pieces / 100 [mu] L each, above the medium, 37 ° C., with CO 2 5% of the conditions, 24 hours, chamber slides (Lab-Tec (registered (Trademark), Thermo Scientific Fisher) was cultured.
(2) After culturing, each cancer type cultured cell was divided into 5 groups (Non-drug, CDDP, CDDP + caf, CDDP + CA, CDDP + cafCA), and the culture medium was replaced. The Non-drug group added 0.5 mL of the culture solution, and the drug-administered group added 0.5 mL of the culture solution adjusted so that the CDDP was 0.25 μM and the CDDP was 0.5 mM for each of caf, CA and cafCA. did. The CDDP concentration of the drug added to each group was adjusted to 0.25 μM by dissolving CDDP powder in water (sterile purified water) to prepare a high concentration, and then diluting with a culture solution suitable for the cells. did. The concentrations of caf and CA, which are concomitant drugs in each group, were uniformly adjusted to 0.5 mM by dissolving powder in water to prepare high concentrations of each, and then diluting with a culture solution suitable for the cells. Since the product is a liquid, the concentration of cafCA was diluted with a culture solution suitable for the cells as it was, and adjusted to 0.5 mM, which was the same as for caf and CA. As a control, no drug was added to some wells (Non-drug: 0 μM).
(3) Twenty-four hours after the addition of the drug, the nuclei of cells undergoing DNA synthesis were stained using TMRE-Mitochondrial Membrane Potential Assay Kit (Abcam) according to the manufacturer's manual (Hoechst (Hoechst 33342): nuclei Detection, TMRE: Detects mitochondria whose membrane potential is maintained). The filter for illuminating Hoechst with a fluorescence microscope (BIOREVO: BZ-9000, KEYENCE) uses DAPI (excitation wavelength: 360 ± 40 nm, dichroic mirror wavelength: 400 nm, absorption wavelength: 460 ± 50 nm) to illuminate TMRE. A TRITC (excitation wavelength: 540 ± 25 nm, dichroic mirror wavelength: 565 nm, absorption wavelength: 605 ± 55 nm) was used as a filter for this purpose, and 5 fixed-point visual fields of each group were photographed. Hoechst and TMRE were detected. It can be said that the weaker the TMRE detection signal is, the more apoptosis is induced as compared with Non-drug.
(4) The cells in the screen of the photograph of (3) were counted. The TMRE brightness per cell was calculated as follows. TMRE brightness per cell = TMRE brightness (detection signal intensity) total / total number of cells (Hoechst detection cell number). The average value of TMRE brightness per cell in each group of 5 visual fields was calculated, and from the ratio with the average value of Non-drug as 1, using Statcel 3 (OMS Publishing), TMRE per cell in each group was used. Analysis of variance was performed on the significant difference in brightness.
<結果>
HOS細胞及びHT1080細胞における結果を、それぞれ図46及び図47に示す。
図46から明らかなように、HOS細胞(骨肉腫)において、CDDP+cafCA群は、他の群と比較して、最もHoechst検出細胞数に対するTMRE検出シグナル強度が弱かった。すなわち、CDDP+cafCA群は、他の群と比較して、最もHOS細胞(骨肉腫)のアポトーシスを誘導する可能性を確認した。
図47から明らかなように、HT1080細胞(軟部肉腫)において、CDDP+cafCA群は、他の群と比較して、最もHoechst検出細胞数に対するTMRE検出シグナル強度が弱かった。すなわち、CDDP+cafCA群は、他の群と比較して、最もHT1080細胞(軟部肉腫)のアポトーシスを誘導する可能性を確認した。
HOS細胞及びHT1080細胞における各群の1細胞あたりのTMRE輝度の有意差の解析結果を図48に示す。
図48から明らかなように、CDDP+cafCA群は、HOS細胞及びHT1080細胞において、他の全ての群との間で有意差があった(p<0.05又はp<0.01)。
また、HOS細胞において、CDDP+cafCA群の1細胞あたりのTMRE輝度は、Non−Drug群と比較して約51%、CDDP群と比較して約65%、CDDP+caf群と比較して約77%、CDDP+CA群と比較して約71%であり、大きく低下した。HT1080細胞において、CDDP+cafCA群の1細胞あたりのTMRE輝度は、Non−Drug群と比較して約35%、CDDP群と比較して約38%、CDDP+caf群と比較して約57%、CDDP+CA群と比較して約52%であり、大きく低下した。
よって、カフェインクエン酸塩は、骨肉腫及び軟部肉腫において、シスプラチンと組み合わせて添加することにより、ミトコンドリア膜電位が減弱・消失し、シスプラチンのアポトーシス誘導効果を増強する可能性を確認した。
<Result>
The results in HOS cells and HT1080 cells are shown in FIGS. 46 and 47, respectively.
As is clear from FIG. 46, in HOS cells (osteosarcoma), the CDDP + cafCA group had the weakest TMRE detection signal intensity with respect to the Hoechst detection cell number as compared with the other groups. That is, it was confirmed that the CDDP + cafCA group has the highest possibility of inducing apoptosis of HOS cells (osteosarcoma) as compared with other groups.
As is clear from FIG. 47, in HT1080 cells (soft tissue sarcoma), the CDDP + cafCA group had the weakest TMRE detection signal intensity with respect to the Hoechst detection cell number as compared with the other groups. That is, it was confirmed that the CDDP + cafCA group has the highest possibility of inducing apoptosis of HT1080 cells (soft tissue sarcoma) as compared with other groups.
FIG. 48 shows the analysis results of the significant difference in TMRE brightness per cell in each group of HOS cells and HT1080 cells.
As is clear from FIG. 48, the CDDP + cafCA group was significantly different from all other groups in HOS cells and HT1080 cells (p <0.05 or p <0.01).
In HOS cells, the TMRE brightness per cell in the CDDP + cafCA group was about 51% as compared with the Non-Drug group, about 65% as compared with the CDDP group, about 77% as compared with the CDDP + caf group, and CDDP + CA. It was about 71% compared to the group, which was a large decrease. In HT1080 cells, the TMRE brightness per cell in the CDDP + cafCA group was about 35% compared to the Non-Drug group, about 38% compared to the CDDP group, about 57% compared to the CDDP + caf group, and the CDDP + CA group. It was about 52% in comparison, which was a large decrease.
Therefore, it was confirmed that caffeine citrate may attenuate or eliminate the mitochondrial membrane potential and enhance the apoptosis-inducing effect of cisplatin when added in combination with cisplatin in osteosarcoma and soft tissue sarcoma.
以上の結果より、本発明の癌治療剤において、シスプラチンとカフェインクエン酸塩との組合せは、骨肉腫及び軟部肉腫において、シスプラチン単独、シスプラチンと無水カフェインとの組合せ、シスプラチンとクエン酸との組合せと比較して、有意にシスプラチンのアポトーシス誘導効果をより増強する可能性を確認した。 From the above results, in the cancer therapeutic agent of the present invention, the combination of cisplatin and caffeine citrate is the combination of cisplatin alone, cisplatin and anhydrous caffeine, and cisplatin and citric acid in osteosarcoma and soft sarcoma. It was confirmed that cisplatin may significantly enhance the apoptosis-inducing effect as compared with the combination.
[皮膚癌、膵癌及び胆管癌培養細胞におけるカフェインクエン酸塩のシスプラチン効果増強の評価]
皮膚癌、膵癌及び胆管癌に対するカフェインクエン酸塩の抗癌剤(シスプラチン)効果増強を評価した。以下の細胞株及び培地を用いた他は、実施例1と同様の材料及び方法により試験を行った。
[Evaluation of enhanced cisplatin effect of caffeine citrate in cultured cells of skin cancer, pancreatic cancer and cholangiocarcinoma]
The enhancement of the anticancer agent (cisplatin) effect of caffeine citrate on skin cancer, pancreatic cancer and cholangiocarcinoma was evaluated. The test was carried out using the same materials and methods as in Example 1 except that the following cell lines and media were used.
<材料>
・細胞株
皮膚癌としては、ヒト由来のA375細胞(DSファーマバイオメディカル株式会社より入手)を用いた。
膵癌としては、ヒト由来のPANC−1細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
胆管癌としては、ヒト由来のTFK−1細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
・培地
上記の各癌種培養細胞について、A375細胞、PANC−1細胞及びTFK−1細胞は、いずれもRPMI1640培地を用いた。
<Material>
-Cell line As skin cancer, human-derived A375 cells (obtained from DS Pharma Biomedical Co., Ltd.) were used.
As pancreatic cancer, human-derived PANC-1 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As bile duct cancer, human-derived TFK-1 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
-Medium For each of the above-mentioned cultured cells of cancer type, RPMI1640 medium was used for all of A375 cells, PANC-1 cells and TFK-1 cells.
<結果>
1.細胞生存率
図49、図50及び図51に、それぞれA375細胞、PANC−1細胞及びTFK−1細胞における4つの群(CDDP、CDDP+caf、CDDP+CA、CDDP+cafCA)の450 nmの吸光度に基づく細胞生存率を示す。図49、図50及び図51から明らかなように、A375細胞(皮膚癌)、PANC−1細胞(膵癌)及びTFK−1細胞(胆管癌)において、CDDP+cafCA群は、全てのCDDP濃度で、他の群と比較して、最も細胞生存率が低かった。
また、分散分析の結果、CDDP+cafCA群は、各シスプラチン濃度において、他の全ての群との間で有意差があった(p<0.01)。
よって、カフェインクエン酸塩は、シスプラチンと組み合わせて添加することにより、シスプラチンの抗癌剤効果を増強することが、0.125〜2.0 μMの全てのシスプラチン濃度において確認できた。
<Result>
1. 1. Cell viability Figure 49, 50 and 51 show the cell viability based on the absorbance at 450 nm of the four groups (CDDP, CDDP + caf, CDDP + CA, CDDP + cafCA) in A375 cells, PANC-1 cells and TFK-1 cells, respectively. Shown. As is clear from FIGS. 49, 50 and 51, in A375 cells (skin cancer), PANC-1 cells (pancreatic cancer) and TFK-1 cells (bile duct cancer), the CDDP + cafCA group was found at all CDDP concentrations and others. The cell viability was the lowest compared to the group of.
In addition, as a result of analysis of variance, the CDDP + cafCA group was significantly different from all other groups in each cisplatin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer agent effect of cisplatin when added in combination with cisplatin at all cisplatin concentrations of 0.125 to 2.0 μM.
2.Median Effect法
Median Effect法によるA375細胞、PANC−1細胞及びTFK−1細胞における相乗効果の解析を行った(図省略)。相乗効果の指標であるCIは、実施例1と同様に、CDDP濃度毎の細胞生存率に基づき、CDDP+cafとCDDP+CAのコンビネーションをCDDP+cafCAとして算出した。CI<1.0のとき、相乗効果が認められる。
CDDP+cafCA群は、0.125〜2.0 μMの全てのシスプラチン濃度で、CI<1.0となり、CDDP+caf及びCDDP+CAに対して、相乗効果が認められた。
2. 2. Median Effect method The synergistic effect on A375 cells, PANC-1 cells and TFK-1 cells was analyzed by the Median Effect method (not shown). For CI, which is an index of synergistic effect, the combination of CDDP + caf and CDDP + CA was calculated as CDDP + cafCA based on the cell viability for each CDDP concentration, as in Example 1. When CI <1.0, a synergistic effect is observed.
In the CDDP + cafCA group, CI <1.0 at all cisplatin concentrations of 0.125 to 2.0 μM, and a synergistic effect was observed on CDDP + caf and CDDP + CA.
3.Isobologram法
Isobologram法によるA375細胞、PANC−1細胞及びTFK−1細胞における相乗効果の解析を行った(図省略)。
実施例1と同様に、CDDP+cafCAの各Effective Doseを示す円形の破線が、それぞれ、CDDP+caf及びCDDP+CAの各Effective Doseを結んだ曲線よりも左下に位置することから、相乗効果が認められた。
CDDP+cafCA群は、50〜90%の全てのEffective Doseで、CI<1.0となり、CDDP+caf及びCDDP+CAに対して、相乗効果が認められた。
3. 3. Isobologram method The synergistic effect on A375 cells, PANC-1 cells and TFK-1 cells by the Isobologram method was analyzed (not shown).
Similar to Example 1, the circular broken line indicating each Effective Dose of CDDP + cafCA is located at the lower left of the curve connecting each Effective Dose of CDDP + caf and CDDP + CA, respectively, so that a synergistic effect was recognized.
In the CDDP + cafCA group, CI <1.0 in all Effective Dose of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.
[軟部肉腫、皮膚癌、膵癌及び胆管癌培養細胞におけるカフェインクエン酸塩のゲムシタビン効果増強の評価]
軟部肉腫、皮膚癌、膵癌及び胆管癌に対するカフェインクエン酸塩の抗癌剤(ゲムシタビン)効果増強を評価した。以下の細胞株及び培地を用いた他は、実施例4と同様の材料及び方法により試験を行った。
[Evaluation of enhanced gemcitabine effect of caffeine citrate in cultured cells of soft tissue sarcoma, skin cancer, pancreatic cancer and cholangiocarcinoma]
The enhancement of the anticancer agent (gemcitabine) effect of caffeine citrate on soft tissue sarcoma, skin cancer, pancreatic cancer and cholangiocarcinoma was evaluated. The test was carried out using the same materials and methods as in Example 4 except that the following cell lines and media were used.
<材料>
・細胞株
軟部肉腫としては、ヒト由来のHT1080細胞(ATCCより入手)を用いた。
皮膚癌としては、ヒト由来のA375細胞(DSファーマバイオメディカル株式会社より入手)を用いた。
膵癌としては、ヒト由来のPANC−1細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
胆管癌としては、ヒト由来のTFK−1細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
・培地
上記の各癌種培養細胞について、HT1080細胞はDMEMを用い、A375細胞、PANC−1細胞及びTFK−1細胞は、いずれもRPMI1640培地を用いた。
<Material>
-Cell line As soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
As the skin cancer, human-derived A375 cells (obtained from DS Pharma Biomedical Co., Ltd.) were used.
As pancreatic cancer, human-derived PANC-1 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As bile duct cancer, human-derived TFK-1 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
-Medium For each of the above-mentioned cultured cells of cancer type, DMEM was used for HT1080 cells, and RPMI1640 medium was used for A375 cells, PANC-1 cells and TFK-1 cells.
<結果>
1.細胞生存率
図52、図53、図54及び図55に、それぞれHT1080細胞、A375細胞、PANC−1細胞及びTFK−1細胞における4つの群(GEM、GEM+caf、GEM+CA、GEM+cafCA)の450 nmの吸光度に基づく細胞生存率を示す。図52、図53、図54及び図55から明らかなように、HT1080細胞(軟部肉腫(線維肉腫))、A375細胞(皮膚癌)、PANC−1細胞(膵癌)及びTFK−1細胞(胆管癌)において、GEM+cafCA群は、全てのゲムシタビン濃度で、他の群と比較して、最も細胞生存率が低かった。
また、分散分析の結果、GEM+cafCA群は、他の全ての群との間で有意差があった(p<0.01)。
よって、カフェインクエン酸塩は、ゲムシタビンと組み合わせて添加することにより、ゲムシタビンの抗癌剤効果を増強することが確認できた。
<Result>
1. 1. Cell viability In FIGS. 52, 53, 54 and 55, the absorbance at 450 nm of four groups (GEM, GEM + caf, GEM + CA, GEM + cafCA) in HT1080 cells, A375 cells, PANC-1 cells and TFK-1 cells, respectively. The cell viability based on is shown. As is clear from FIGS. 52, 53, 54 and 55, HT1080 cells (soft tissue sarcoma (fibrosarcoma)), A375 cells (skin cancer), PANC-1 cells (pancreatic cancer) and TFK-1 cells (bile duct cancer). ), The GEM + cafCA group had the lowest cell viability at all gemcitabine concentrations as compared with the other groups.
In addition, as a result of analysis of variance, the GEM + cafCA group was significantly different from all other groups (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer agent effect of gemcitabine by adding it in combination with gemcitabine.
2.Median Effect法
Median Effect法によるHT1080細胞、A375細胞、PANC−1細胞及びTFK−1細胞における相乗効果の解析を行った(図省略)。相乗効果の指標であるCIは、実施例1と同様に、GEM濃度毎の細胞生存率に基づき、GEM+cafとGEM+CAのコンビネーションをGEM+cafCAとして算出した。CI<1.0のとき、相乗効果が認められる。
GEM+cafCA群は、0.125〜2.0 μMの全てのゲムシタビン濃度で、CI<1.0となり、GEM+caf及びGEM+CAに対して、相乗効果が認められた。
2. 2. Median Effect method The synergistic effect on HT1080 cells, A375 cells, PANC-1 cells and TFK-1 cells was analyzed by the Median Effect method (not shown). As for CI, which is an index of synergistic effect, the combination of GEM + caf and GEM + CA was calculated as GEM + cafCA based on the cell viability for each GEM concentration, as in Example 1. When CI <1.0, a synergistic effect is observed.
In the GEM + cafCA group, CI <1.0 at all gemcitabine concentrations of 0.125 to 2.0 μM, and a synergistic effect was observed for GEM + caf and GEM + CA.
3.Isobologram法
Isobologram法によるHT1080細胞、A375細胞、PANC−1細胞及びTFK−1細胞における相乗効果の解析を行った(図省略)。
実施例1と同様に、GEM+cafCAの各Effective Doseを示す円形の破線が、それぞれ、GEM+caf及びGEM+CAの各Effective Doseを結んだ曲線よりも左下に位置することから、相乗効果が認められた。
GEM+cafCA群は、50〜90%の全てのEffective Doseで、CI<1.0となり、GEM+caf及びGEM+CAに対して、相乗効果が認められた。
3. 3. Isobologram method The synergistic effect of the Isobologram method on HT1080 cells, A375 cells, PANC-1 cells and TFK-1 cells was analyzed (not shown).
Similar to Example 1, the circular broken line indicating each Effective Dose of GEM + cafCA is located at the lower left of the curve connecting each Effective Dose of GEM + caf and GEM + CA, respectively, so that a synergistic effect was recognized.
In the GEM + cafCA group, CI <1.0 in all Effective Dose of 50 to 90%, and a synergistic effect was observed for GEM + caf and GEM + CA.
[癌細胞を担持したマウスにおけるカフェインクエン酸塩の抗癌剤効果増強の評価]
以下の方法により、癌細胞を担持したマウスにおけるカフェインクエン酸塩の抗癌剤効果増強を評価した。さらに、カフェインクエン酸塩及び抗癌剤の濃度を変化させてカフェインクエン酸塩の抗癌剤効果増強を評価した。
[Evaluation of enhancement of anti-cancer drug effect of caffeine citrate in mice carrying cancer cells]
The enhancement of the anticancer drug effect of caffeine citrate in mice carrying cancer cells was evaluated by the following method. Furthermore, the enhancement of the anticancer agent effect of caffeine citrate was evaluated by changing the concentrations of caffeine citrate and the anticancer agent.
<材料>
・マウスは、BALB/c-nu(日本チャールズリバー社より入手)を用いた。
・細胞株
骨肉腫としては、マウス由来のLM8細胞(文部科学省ナショナルバイオリソースプロジェクトを介して、理研BRCより入手)を用いた。
軟部肉腫としては、ヒト由来のHT1080細胞(ATCCより入手)を用いた。
・抗癌剤
シスプラチン(和光純薬工業株式会社より入手)を用いた。
・併用薬
カフェインクエン酸塩(cafCA、クエン酸カフェイン、ノーベルファーマ株式会社より入手)、無水カフェイン(caf、和光純薬工業株式会社より入手)又はクエン酸(CA、和光純薬工業株式会社より入手)を用いた。
・培地
LM8細胞は、RPMI1640培地を用い、HT1080細胞は、DMEMを用いた。
<Material>
-As the mouse, BALB / c-nu (obtained from Charles River Laboratories, Japan) was used.
-As the cell line osteosarcoma, mouse-derived LM8 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As the soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
-The anticancer drug cisplatin (obtained from Wako Pure Chemical Industries, Ltd.) was used.
-Concomitant drug caffeine citrate (cafCA, caffeine citrate, obtained from Nobelpharma Co., Ltd.), anhydrous caffeine (caf, obtained from Wako Pure Chemical Industries, Ltd.) or citric acid (CA, Wako Pure Chemical Industries, Ltd.) Obtained from the company) was used.
-Medium For LM8 cells, RPMI1640 medium was used, and for HT1080 cells, DMEM was used.
<評価方法>
(1)マウス由来のLM8細胞(5×105個)を背部皮下に移植したヌードマウスでの評価系
下記の表2及び図56のスケジュールに従い、癌細胞を担持したマウスにおけるカフェインクエン酸塩の抗癌剤効果増強を評価した。
(2)ヒト由来のHT1080細胞(5×105個)を背部皮下に移植したヌードマウスでの評価系
下記の表2及び図56のスケジュールに従い、癌細胞を担持したマウスにおけるカフェインクエン酸塩の抗癌剤効果増強を評価した。
(3)カフェインクエン酸塩低投与量を特徴とするマウス由来のLM8細胞(5×105個)を背部皮下に移植したヌードマウスでの評価系
下記の表3及び図56のスケジュールに従い、癌細胞を担持したマウスにおけるカフェインクエン酸塩の抗癌剤効果増強を評価した。
(4)抗癌剤低投与量を特徴とするマウス由来のLM8細胞(5×105個)を背部皮下に移植したヌードマウスでの評価系
下記の表4及び図56のスケジュールに従い、癌細胞を担持したマウスにおけるカフェインクエン酸塩の抗癌剤効果増強を評価した。
<Evaluation method>
(1) Evaluation system in nude mice in which mouse-derived LM8 cells (5 × 10 5 ) were transplanted subcutaneously in the back Caffeine citrate in mice carrying cancer cells according to the schedule shown in Table 2 and FIG. 56 below. The enhancement of the anticancer drug effect was evaluated.
(2) according to the schedule in Table 2 and Figure 56 evaluation system of the following human-derived HT1080 cells (5 × 10 5 cells) in nude mice implanted subcutaneously on the back, caffeine citrate in mice carrying cancer cells The enhancement of the anticancer drug effect was evaluated.
(3) Evaluation system in nude mice in which mouse-derived LM8 cells (5 × 10 5 cells) characterized by a low dose of caffeine citrate were transplanted subcutaneously on the back, according to the schedules in Table 3 and FIG. 56 below. The enhancement of the anticancer drug effect of caffeine citrate in mice carrying cancer cells was evaluated.
(4) Evaluation system in nude mice in which LM8 cells (5 × 10 5 ) derived from mice characterized by a low dose of anticancer drug were transplanted subcutaneously on the back, cancer cells were supported according to the schedules in Table 4 and FIG. 56 below. The enhancement of the anticancer drug effect of caffeine citrate was evaluated in the mice.
<評価結果>
マウス由来のLM8細胞を背部皮下に移植したヌードマウスでの評価系の結果を図57及び図58に示す。
ヒト由来のHT1080細胞を背部皮下に移植したヌードマウスでの評価系の結果を図59及び図60に示す。
カフェインクエン酸塩低投与量を特徴とするマウス由来のLM8細胞を背部皮下に移植したヌードマウスでの評価系の結果を図61及び図62に示す。
抗癌剤低投与量を特徴とするマウス由来のLM8細胞を背部皮下に移植したヌードマウスでの評価系の結果を図63及び図64に示す。
図57〜64から明らかなように、CDDP+cafCA群は、他の群と比較して、腫瘍の成長(体積及び重さ)を非常に強く抑制した。加えて、カフェインクエン酸塩及び抗癌剤の濃度を変化させても、CDDP+cafCA群は、他の群と比較して、腫瘍の成長(体積及び重さ)を非常に強く抑制した。
以上の結果により、本発明の抗癌剤(カフェインクエン酸塩)及び治療方法は、In vitro評価系でも抗癌剤増強効果を有することを確認した。
<Evaluation result>
The results of the evaluation system in nude mice in which mouse-derived LM8 cells were transplanted subcutaneously on the back are shown in FIGS. 57 and 58.
The results of the evaluation system in nude mice transplanted with human-derived HT1080 cells subcutaneously on the back are shown in FIGS. 59 and 60.
The results of the evaluation system in nude mice in which LM8 cells derived from mice characterized by a low dose of caffeine citrate were transplanted subcutaneously on the back are shown in FIGS. 61 and 62.
The results of the evaluation system in nude mice in which LM8 cells derived from mice characterized by a low dose of anticancer drug were transplanted subcutaneously on the back are shown in FIGS. 63 and 64.
As is clear from FIGS. 57-64, the CDDP + cafCA group suppressed tumor growth (volume and weight) very strongly as compared with the other groups. In addition, even when the concentrations of caffeine citrate and anticancer agent were changed, the CDDP + cafCA group suppressed tumor growth (volume and weight) very strongly as compared with the other groups.
From the above results, it was confirmed that the anticancer agent (caffeine citrate) and the therapeutic method of the present invention also have an anticancer agent enhancing effect in the in vitro evaluation system.
[安全性の評価]
以下の方法により、本発明の癌治療剤の安全性を評価した。
[Safety evaluation]
The safety of the cancer therapeutic agent of the present invention was evaluated by the following method.
<安全性評価方法>
以下の表5に従い、ヌードマウス(担癌マウスではない)の体重推移を評価した。
外部委託して、前記ヌードマウスを採血、臓器摘出を行い、血液検査及び病理組織学的検査を実施した。
<Safety evaluation method>
According to Table 5 below, the body weight transition of nude mice (not cancer-bearing mice) was evaluated.
The nude mice were outsourced, blood was collected, organs were removed, and blood tests and histopathological tests were performed.
体重推移の評価を図65に示す。各郡では体重増加に有意差がなかった。
血液検査及び病理組織学的検査では、有害事象は認められなかった。
以上により、本発明の癌治療剤及び癌治療方法は安全性に問題がないことを確認した。
The evaluation of the body weight transition is shown in FIG. There was no significant difference in weight gain in each county.
Blood tests and histopathological examination showed no adverse events.
From the above, it was confirmed that the cancer therapeutic agent and the cancer treatment method of the present invention have no problem in safety.
[ADMによる心筋障害の評価]
ADMは心筋障害の有害事象をきたすことがある。そこで、in vitroによるWST−8アッセイにより、本発明のADMを含む癌治療剤による心筋細胞の障害を評価した。本実施例では、心筋細胞の生存細胞数の減少は、ADM+caf、ADM+CA、ADM+cafCAそれぞれが、ADM単独と同等であった。
[Evaluation of myocardial damage by ADM]
ADM can cause adverse events of myocardial damage. Therefore, in vitro WST-8 assay was used to evaluate cardiomyocyte damage caused by the ADM-containing cancer therapeutic agent of the present invention. In this example, the decrease in the number of surviving cardiomyocytes was equivalent to ADM + caf, ADM + CA, and ADM + cafCA, respectively, as compared to ADM alone.
<総論>
以上の結果より、本発明の癌治療剤及び癌治療方法は、以下の有利な効果を有することが明らかになった。
(1)シスプラチンとカフェインクエン酸塩との組合せは、シスプラチン単独、シスプラチンと無水カフェインとの組合せ、シスプラチンとクエン酸との組合せと比較して、有意にシスプラチンの抗癌剤効果をより増強すること。カフェインクエン酸塩の有する抗癌剤増強効果は、無水カフェインの有する抗癌剤増強効果と、クエン酸の有する抗癌剤増強効果との、単なる相加効果ではなく、相乗効果であること。
(2)アドリアマイシンとカフェインクエン酸塩との組合せは、アドリアマイシン単独、アドリアマイシンと無水カフェインとの組合せ、アドリアマイシンとクエン酸との組合せと比較して、有意にアドリアマイシンの抗癌剤効果をより増強すること。カフェインクエン酸塩の有する抗癌剤増強効果は、無水カフェインの有する抗癌剤増強効果と、クエン酸の有する抗癌剤増強効果との、単なる相加効果ではなく、相乗効果であること。
(3)ゲムシタビンとカフェインクエン酸塩との組合せは、ゲムシタビン単独、ゲムシタビンと無水カフェインとの組合せ、ゲムシタビンとクエン酸との組合せと比較して、有意にゲムシタビンの抗癌剤効果をより増強すること。カフェインクエン酸塩の有する抗癌剤増強効果は、無水カフェインの有する抗癌剤増強効果と、クエン酸の有する抗癌剤増強効果との、単なる相加効果ではなく、相乗効果であること。
(4)シスプラチンとカフェインクエン酸塩との組合せは、骨肉腫及び軟部肉腫において、シスプラチン単独、シスプラチンと無水カフェインとの組合せ、シスプラチンとクエン酸との組合せと比較して、有意にシスプラチンの細胞増殖抑制効果をより増強すること。
(5)シスプラチンとカフェインクエン酸塩との組合せは、骨肉腫及び軟部肉腫において、シスプラチン単独、シスプラチンと無水カフェインとの組合せ、シスプラチンとクエン酸との組合せと比較して、有意にシスプラチンのアポトーシス誘導効果をより増強する可能性がある。
(6)安全性に問題がない。
<General>
From the above results, it was clarified that the cancer therapeutic agent and the cancer treatment method of the present invention have the following advantageous effects.
(1) The combination of cisplatin and caffeine citrate significantly enhances the anticancer agent effect of cisplatin as compared with the combination of cisplatin alone, the combination of cisplatin and anhydrous caffeine, and the combination of cisplatin and citric acid. .. The anti-cancer agent enhancing effect of caffeine citrate is not a mere additive effect but a synergistic effect of the anti-cancer agent enhancing effect of anhydrous caffeine and the anti-cancer agent enhancing effect of citric acid.
(2) The combination of adriamycin and caffeine citrate significantly enhances the anticancer drug effect of adriamycin as compared with adriamycin alone, adriamycin and anhydrous caffeine, and adriamycin and citric acid. .. The anti-cancer agent enhancing effect of caffeine citrate is not a mere additive effect but a synergistic effect of the anti-cancer agent enhancing effect of anhydrous caffeine and the anti-cancer agent enhancing effect of citric acid.
(3) The combination of gemcitabine and caffeine citrate significantly enhances the anticancer agent effect of gemcitabine as compared with gemcitabine alone, gemcitabine and anhydrous caffeine, and gemcitabine and citric acid. .. The anti-cancer agent enhancing effect of caffeine citrate is not a mere additive effect but a synergistic effect of the anti-cancer agent enhancing effect of anhydrous caffeine and the anti-cancer agent enhancing effect of citric acid.
(4) The combination of cisplatin and caffeine citrate is significantly more cisplatin in osteosarcoma and soft tissue sarcoma than cisplatin alone, cisplatin plus anhydrous caffeine, or cisplatin plus citric acid. To further enhance the cell growth inhibitory effect.
(5) The combination of cisplatin and caffeine citrate is significantly more cisplatin in osteosarcoma and soft sarcoma than cisplatin alone, cisplatin plus anhydrous caffeine, or cisplatin plus citric acid. It may enhance the effect of inducing apoptosis.
(6) There is no problem with safety.
従来の化学療法よりも強力な治療効果を有する癌治療剤及び癌治療方法を提供する。 Provided are a cancer therapeutic agent and a cancer treatment method having a stronger therapeutic effect than conventional chemotherapy.
Claims (12)
A therapeutic agent for soft tissue sarcoma, which is characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
A gastric cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
A skin cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
A pancreatic cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
A gastric cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with adriamycin, which is an anticancer agent, which is an active ingredient.
A uterine cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with adriamycin, which is an anticancer agent, which is an active ingredient.
A lung cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
A breast cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
A uterine cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
A skin cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
An ovarian cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
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