JP7387899B2 - Conjugate consisting of extracellular matrix and anticancer drug and its medical use - Google Patents
Conjugate consisting of extracellular matrix and anticancer drug and its medical use Download PDFInfo
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- JP7387899B2 JP7387899B2 JP2022537781A JP2022537781A JP7387899B2 JP 7387899 B2 JP7387899 B2 JP 7387899B2 JP 2022537781 A JP2022537781 A JP 2022537781A JP 2022537781 A JP2022537781 A JP 2022537781A JP 7387899 B2 JP7387899 B2 JP 7387899B2
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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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
Description
本発明は、エラスチン類似ポリペプチドとインテグリン受容体リガンドとからなる細胞外基質と抗ガン剤接合体及びその医学的用途に関する。 The present invention relates to an extracellular matrix and anticancer drug conjugate comprising an elastin-like polypeptide and an integrin receptor ligand, and medical uses thereof.
癌とは、一般的に人体組織を成している細胞周期に異常が生じて細胞が正常に分化せず、細胞***を続ける疾患であって、開始（ｉｎｉｔｉａｔｉｏｎ）、促進（ｐｒｏｍｏｔｉｏｎ）及び進行（ｐｒｏｇｒｅｓｓｉｏｎ）の３つの段階を経て発生する。癌の原因は、環境や食べ物内に含まれた発ガン物質によって、正常な細胞の遺伝子や癌抑制遺伝子に突然変異が起こり、このような細胞が発ガン物質の継続的な刺激を受けながら非正常に増殖して、癌組織を形成すると知られているが、癌の発生原因については、まだ明確に明らかになっていない。 Cancer is a disease in which there is an abnormality in the cell cycle that generally makes up human tissues, and cells do not differentiate normally and continue to divide. It occurs through three stages: The cause of cancer is that carcinogens contained in the environment or food cause mutations in the genes of normal cells and tumor suppressor genes, and these cells become non-existent while being continuously stimulated by carcinogens. Although it is known to grow normally and form cancerous tissue, the cause of cancer development has not yet been clearly clarified.
癌は、良性腫瘍（ｂｅｎｉｇｎ ｔｕｍｏｒ）と悪性腫瘍（ｍａｌｉｇｎａｎｔ ｔｕｍｏｒ）とに区分されるが、良性腫瘍は、比較的成長速度が遅く、腫瘍の原発生部位から他の組織に移動する転移（ｍｅｔａｓｔａｓｉｓ）が表われないことに対して、悪性腫瘍は、原発部を離れて他の組織に浸潤されて迅速に成長する特徴を有することにより、生命を脅かし、死亡に至る非常に重要な原因となる。 Cancer is classified into benign tumors and malignant tumors. Benign tumors have a relatively slow growth rate and metastasis (metastasis) where the tumor moves from its original site to other tissues. On the other hand, malignant tumors have the characteristic of leaving the primary site and infiltrating other tissues and growing rapidly, which makes them life-threatening and a very important cause of death.
このような癌の治療のためには、手術療法、放射線治療療法及び化学療法などが使われているが、多くの研究にも拘らず、癌患者全体の５０％以上が結局癒されず、死亡すると報告されている。それによる理由は、外科的に切除したとしても、微細に転移した癌細胞を除去することができず、癌が再発するか、多様な抗ガン剤の開発にも拘らず、抗ガン剤を利用した癌治療時に、抗ガン剤に対する癌の細胞死が誘導されないか、初期には反応を示して腫瘍が減るように示すが、治療途中や治療が終わった後、抗ガン剤に対する耐性が生じた癌細胞が急激に増殖するためである。 Surgery, radiation therapy, chemotherapy, etc. are used to treat this type of cancer, but despite much research, more than 50% of all cancer patients end up not being cured and dying. It has been reported. The reason for this is that even if surgically resected, the cancer cells that have metastasized microscopically cannot be removed, and the cancer will recur, or that despite the development of various anticancer drugs, no anticancer drugs can be used. During cancer treatment, cancer cell death is not induced in response to anticancer drugs, or there is an initial response and the tumor appears to be reduced, but resistance to the anticancer drugs develops during or after treatment. This is because cancer cells proliferate rapidly.
化学療法である低分子量抗ガン剤の治療は、多様な類型の癌治療に広く使われてきたが、癌細胞と正常細胞との間の差異点を区別することができない抗ガン剤の非特異性によって、化学療法の臨床的効能は制限的である。 Low-molecular-weight anticancer drug treatment, which is chemotherapy, has been widely used to treat various types of cancer, but the anticancer drugs are non-specific and cannot distinguish between cancer cells and normal cells. Depending on the gender, the clinical efficacy of chemotherapy is limited.
また、抗ガン剤の臨床的効果のために、副作用を誘発させる最大許容量で投与されるが、化学療法の長期的な副作用の１つは、多剤耐性であって、Ｐ糖タンパク質が癌細胞で抗ガン剤をポンピングさせて、多剤耐性を誘導すると知られている。 In addition, for the clinical effectiveness of anticancer drugs, they are administered at the maximum tolerated dose that induces side effects, but one of the long-term side effects of chemotherapy is multidrug resistance, in which P-glycoprotein It is known to induce multidrug resistance by causing cells to pump anticancer drugs.
このような問題点を解決するために、Ｐ糖タンパク質媒介薬物流出メカニズムを克服したり、抗ガン剤を変形させて癌細胞に特異的に薬物を伝達する治療戦略が必要である。 To solve these problems, therapeutic strategies are needed to overcome the P-glycoprotein-mediated drug efflux mechanism or modify anticancer drugs to specifically deliver drugs to cancer cells.
本発明は、腫瘍組織に対する抗ガン剤の接近性を大きく高めて、正常組織に対する副作用を最小化し、癌細胞に対する抗ガン治療の効果を向上させるために、エラスチン類似ポリペプチドとインテグリン受容体リガンドとからなる細胞外基質と抗ガン剤からなる接合体とを抗ガン治療用薬学組成物または薬剤耐性抑制用薬学組成物として提供することである。 The present invention aims to combine elastin-like polypeptides with integrin receptor ligands to greatly increase the accessibility of anticancer drugs to tumor tissues, minimize side effects to normal tissues, and improve the effectiveness of anticancer treatments against cancer cells. An object of the present invention is to provide a conjugate comprising an extracellular matrix consisting of the above and an anticancer drug as a pharmaceutical composition for anticancer treatment or a pharmaceutical composition for suppressing drug resistance.
本発明は、細胞外基質（ＲＥＰ）及び抗ガン剤からなる接合体において、 The present invention provides a conjugate comprising an extracellular matrix (REP) and an anticancer drug,
前記細胞外基質は、エラスチン類似ポリペプチドとインテグリン受容体リガンドとからなり、 The extracellular matrix consists of an elastin-like polypeptide and an integrin receptor ligand,
前記細胞外基質及び抗ガン剤は、リンカーによって互いに連結されることを特徴とする抗ガン治療用細胞外基質及び抗ガン剤接合体を提供する。 The present invention provides an extracellular matrix and anticancer drug conjugate for anticancer treatment, wherein the extracellular matrix and the anticancer drug are connected to each other by a linker.
本発明は、前記細胞外基質及び抗ガン剤接合体を有効成分として含有する癌疾患治療用薬学組成物を提供する。 The present invention provides a pharmaceutical composition for treating cancer diseases containing the extracellular matrix and anticancer drug conjugate as active ingredients.
また、本発明は、前記細胞外基質及び抗ガン剤接合体を有効成分として含有する薬剤耐性抑制用薬学組成物を提供する。 The present invention also provides a pharmaceutical composition for suppressing drug resistance, which contains the extracellular matrix and anticancer drug conjugate as active ingredients.
本発明によれば、エラスチン類似ポリペプチドとインテグリン受容体リガンドとからなる細胞外基質（ＲＥＰ）－ドキソルビシン接合体が、ドキソルビシンまたはＲＥＰ及びドキソルビシン混合物と比較して、非常に向上した抗ガン活性を示し、ドキソルビシン抵抗性を示す癌細胞の薬物抵抗性を抑制させる効果が確認されることによって、前記細胞外基質（ＲＥＰ）－ドキソルビシン接合体は、癌疾患の治療のための効果的な抗ガン治療剤及び抗ガン剤に対する薬物耐性を示す癌細胞の薬剤耐性抑制用薬学組成物として提供されうる。 According to the present invention, an extracellular matrix (REP)-doxorubicin conjugate consisting of an elastin-like polypeptide and an integrin receptor ligand exhibits significantly improved anticancer activity compared to doxorubicin or a mixture of REP and doxorubicin. , it was confirmed that the extracellular matrix (REP)-doxorubicin conjugate has the effect of suppressing drug resistance in cancer cells exhibiting doxorubicin resistance. It can also be provided as a pharmaceutical composition for suppressing drug resistance of cancer cells exhibiting drug resistance to anticancer drugs.
以下、本発明をより詳細に説明する。
本発明は、細胞表面のインテグリン受容体リガンドを通じて癌細胞内に効率的に内在化されて癌細胞の死滅を誘導し、癌細胞のＰ糖タンパク質媒介多剤耐性を克服するために、熱反応性ペプチド配列からなる細胞外基質タンパク質－薬物接合体を確認することによって、本発明を完成した。
The present invention will be explained in more detail below.
The present invention provides a heat-responsive protein that is efficiently internalized into cancer cells through integrin receptor ligands on the cell surface to induce cancer cell death and overcome P-glycoprotein-mediated multidrug resistance in cancer cells. The present invention was completed by identifying an extracellular matrix protein-drug conjugate consisting of a peptide sequence.
本発明は、細胞外基質（ＲＧＤ－ｃｏｎｔａｉｎｉｎｇ Ｅｌａｓｔｉｎ－Ｌｉｋｅ Ｐｏｌｙｐｅｐｔｉｄｅ、ＲＥＰ）及び抗ガン剤からなる接合体において、 The present invention provides a conjugate comprising an extracellular matrix (RGD-containing Elastin-Like Polypeptide, REP) and an anticancer drug,
前記細胞外基質は、エラスチン類似ポリペプチドとインテグリン受容体リガンドとからなるポリペプチドであり、前記細胞外基質及び抗ガン剤は、リンカーによって互いに連結されることを特徴とするガン治療用細胞外基質及び抗ガン剤接合体を提供することができる。 The extracellular matrix for cancer treatment is characterized in that the extracellular matrix is a polypeptide consisting of an elastin-like polypeptide and an integrin receptor ligand, and the extracellular matrix and the anticancer drug are connected to each other by a linker. and an anticancer drug conjugate.
前記細胞外基質は、バリン（ｖａｌｉｎｅ）－グリシン（ｇｌｙｃｉｎｅ）－バリン－プロリン（ｐｒｏｌｉｎｅ）－グリシン（ＶＧＶＰＧ）ポリペプチドであり、前記インテグリン受容体リガンドは、アルギニン（ａｒｇｉｎｉｎｅ）－グリシン－アスパルテート（ａｓｐａｒｔａｔｅ）（ＲＧＤ）である。 The extracellular matrix is a valine-glycine-valine-proline-glycine (VGVPG) polypeptide, and the integrin receptor ligand is an arginine-glycine-aspartate polypeptide. ) (RGD).
より詳細には、前記細胞外基質は、配列番号３で表されるアミノ酸配列である［ＶＧＲＧＤ（ＶＧＶＰＧ）６］ｎで構成され、前記ｎは、配列番号３の反復回数であって、５～２０の整数であることを特徴とする抗ガン治療用細胞外基質及び抗ガン剤接合体である。 More specifically, the extracellular matrix is composed of the amino acid sequence [VGRGD(VGVPG) 6 ] n represented by SEQ ID NO: 3, where n is the number of repeats of SEQ ID NO: 3, ranging from 5 to The present invention is an extracellular matrix and anticancer drug conjugate for anticancer treatment, characterized in that the number is an integer of 20.
より望ましくは、前記細胞外基質は、配列番号２で表されるアミノ酸配列であるＴＧＰＧ［ＶＧＲＧＤ（ＶＧＶＰＧ）６］ｎＷＰＣで構成され、前記ｎは、［ＶＧＲＧＤ（ＶＧＶＰＧ）６］の反復回数であって、５、１０、１２、１５または２０の整数である。 More preferably, the extracellular matrix is composed of TGPG[VGRGD(VGVPG) 6 ] n WPC, which is an amino acid sequence represented by SEQ ID NO: 2, where n is the number of repeats of [VGRGD(VGVPG) 6 ]. and is an integer of 5, 10, 12, 15 or 20.
前記リンカーは、ＯＨ－、ＮＨ２－及びＳＨ－からなる群からそれぞれ異なる２個以上の官能基を有するＣ２～Ｃ５の化合物でもある。 The linker is also a C2-C5 compound having two or more functional groups each different from the group consisting of OH-, NH 2 - and SH-.
より詳細には、前記リンカーは、３－スルホニルプロパンヒドラジド（３－Ｓｕｌｆａｎｙｌｐｒｏｐａｎｅｈｙｄｒａｚｉｄｅ）及び３－メルカプトプロピオン酸（３－Ｍｅｒｃａｐｔｏｐｒｏｐｉｏｎｉｃ ａｃｉｄ）からなる群から選択されるものである。 More particularly, the linker is selected from the group consisting of 3-Sulfonylpropanehydrazide and 3-Mercaptopropionic acid.
前記接合体は、細胞外基質とリンカーのＳＨ－官能基とが結合し、抗ガン剤とリンカーのＯＨ－またはＮＨ２－官能基とが結合して、接合体を成すものである。 The conjugate is one in which the extracellular matrix is bound to the SH-functional group of the linker, and the anticancer agent is bound to the OH- or NH 2 -functional group of the linker.
前記抗ガン剤は、パクリタキセル（ｐａｃｌｉｔａｘｅｌ）、ドキソルビシン（ｄｏｘｏｒｕｂｉｃｉｎ）、シスプラチン（ｃｉｓｐｌａｔｉｎ）、ドセタキセル（ｄｏｃｅｔａｘｅｌ）、タモキシフェン（ｔａｍｏｘｉｆｅｎ）、カンプトテシン（ｃａｍｐｔｏｔｈｅｃｉｎ）、アナストロゾール（ａｎａｓｔｒｏｚｏｌｅ）、カルボプラチン（ｃａｒｂｏｐｌａｔｉｎ）、トポテカン（ｔｏｐｏｔｅｃａｎ）、ベロテカン（ｂｅｌｏｔｅｃａｎ）、イリノテカン（ｉｒｉｎｏｔｅｃａｎ）、グリベック（ｇｌｅｅｖｅｃ）、モノメチルアウリスタチンＥ（ＭＭＡＥ）、メイタンシン（ＤＭ１）、ソラブタンシン（ＤＭ４）、及びビンクリスチン（ｖｉｎｃｒｉｓｔｉｎｅ）からなる群から選択されるものである。 The anticancer drugs include paclitaxel, doxorubicin, cisplatin, docetaxel, tamoxifen, camptothecin, and anastrozole. zole), carboplatin, topotecan ( topotecan, belotecan, irinotecan, gleevec, monomethyl auristatin E (MMAE), maytansine (DM1), soravtansine (DM4), and vincristine. be.
本発明は、前記細胞外基質及び抗ガン剤接合体を有効成分として含有する癌疾患治療用薬学組成物を提供することができる。 The present invention can provide a pharmaceutical composition for treating cancer disease containing the extracellular matrix and anticancer drug conjugate as active ingredients.
前記癌疾患は、肝癌、腎臓癌、肺癌、乳癌、大腸癌、膵臓癌、前立腺癌、脳癌、胃癌、卵巣癌、及び子宮癌からなる群から選択されるものである。 The cancer disease is selected from the group consisting of liver cancer, kidney cancer, lung cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, brain cancer, stomach cancer, ovarian cancer, and uterine cancer.
前記薬学組成物は、腫瘍細胞の細胞死を増加させ、抗ガン剤の薬剤耐性を阻害するものである。 The pharmaceutical composition increases cell death of tumor cells and inhibits drug resistance of anticancer drugs.
本発明は、前記細胞外基質及び抗ガン剤接合体を有効成分として含有する薬剤耐性抑制用薬学組成物を提供することができる。 The present invention can provide a pharmaceutical composition for suppressing drug resistance, which contains the extracellular matrix and anticancer drug conjugate as active ingredients.
前記薬剤耐性は、腫瘍細胞の抗ガン剤耐性である。 The drug resistance is resistance of tumor cells to anticancer drugs.
本発明の一具体例において、前記薬学組成物は、通常の方法によって注射剤、顆粒剤、散剤、錠剤、丸剤、カプセル剤、坐剤、ゲル、懸濁剤、乳剤、点滴剤または液剤からなる群から選択された何れか１つの剤型を使用することができる。 In one embodiment of the invention, the pharmaceutical composition is prepared as an injection, granule, powder, tablet, pill, capsule, suppository, gel, suspension, emulsion, drop, or liquid by conventional methods. Any one dosage form selected from the group consisting of:
本発明の他の具体例において、前記薬学組成物は、薬学組成物の製造に通常使う適切な担体、賦形剤、崩壊剤、甘味剤、被覆剤、膨張剤、滑沢剤、香味剤、抗酸化剤、緩衝液、静菌剤、希釈剤、分散剤、界面活性剤、結合剤、及び潤滑剤からなる群から選択される１つ以上の添加剤をさらに含みうる。 In another embodiment of the invention, the pharmaceutical composition comprises suitable carriers, excipients, disintegrants, sweeteners, coating agents, bulking agents, lubricants, flavoring agents, etc. commonly used in the manufacture of pharmaceutical compositions. It may further include one or more additives selected from the group consisting of antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders, and lubricants.
具体的に、担体、賦形剤及び希釈剤は、ラクトース、デキストロース、スクロース、ソルビトール、マンニトール、キシリトール、エリスリトール、マルチトール、澱粉、アカシアゴム、アルギン酸、ゼラチン、リン酸カルシウム、ケイ酸カルシウム、セルロース、メチルセルロース、非晶質セルロース、ポリビニルピロリドン、水、ヒドロキシ安息香酸メチル、ヒドロキシ安息香酸プロピル、タルク、ステアリン酸マグネシウム及び鉱物油を使用し、経口投与のための固型製剤には、錠剤、丸剤、散剤、顆粒剤、カプセル剤などが含まれ、このような固型製剤は、前記組成物に少なくとも１つ以上の賦形剤、例えば、澱粉、炭酸カルシウム、スクロースまたはラクトース、ゼラチンなどを混ぜて調剤することができる。また、単純な賦形剤以外に、ステアリン酸マグネシウム、タルクのような潤滑剤も使用することができる。経口のための液状製剤としては、懸濁剤、内用液剤、乳剤、シロップ剤などがあり、よく使われる単純希釈剤である水、流動パラフィン以外に、さまざまな賦形剤、例えば、湿潤剤、甘味剤、芳香剤、保存剤などが含まれうる。非経口投与のための製剤には、滅菌された水溶液、非水性溶剤、懸濁剤、乳剤、凍結乾燥製剤、坐剤などが含まれる。非水性溶剤、懸濁剤としては、プロピレングリコール、ポリエチレングリコール、オリーブオイルのような植物性油、オレイン酸エチルのような注射可能なエステルなどが使われる。坐剤の基剤としては、ウイテプゾール（ｗｉｔｅｐｓｏｌ）、マクロゴール、トウイーン（ｔｗｅｅｎ）６１、カカオ脂、ラウリン脂、グリセロゼラチンなどが使われる。 Specifically, carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Using amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, solid formulations for oral administration include tablets, pills, powders, Granules, capsules, etc. are included, and such solid preparations may be prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Can be done. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents are used. , sweeteners, flavoring agents, preservatives, and the like. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, suppositories, and the like. Examples of non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin fat, glycerogelatin, etc. are used.
本発明の一実施例によれば、前記薬学組成物は、静脈内、動脈内、腹腔内、筋肉内、腫瘍内、胸骨内、経皮、鼻側内、吸入、局所、直腸、経口、眼球内または皮内経路を通じて通常の方式で対象体に投与することができる。 According to one embodiment of the invention, the pharmaceutical composition may be intravenous, intraarterial, intraperitoneal, intramuscular, intratumoral, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, ocular. It can be administered to a subject in a conventional manner through intradermal or intradermal routes.
前記細胞外基質及び抗ガン剤接合体の望ましい投与量は、対象体の状態及び体重、疾患の種類及び程度、薬物形態、投与経路及び期間によって変わり、当業者によって適切に選択されうる。本発明の一実施例によれば、これに制限されるものではないが、１日投与量が０．０１～２００ｍｇ／ｋｇ、具体的には、０．１～２００ｍｇ／ｋｇ、より具体的には、０．１～１００ｍｇ／ｋｇである。投与は、一日一回投与することもでき、数回に分けて投与することもでき、これにより、本発明の範囲が制限されるものではない。 The desired dosage of the extracellular matrix and anticancer drug conjugate varies depending on the condition and weight of the subject, the type and severity of the disease, the drug form, the administration route, and the period, and can be appropriately selected by those skilled in the art. According to one embodiment of the invention, the daily dosage is 0.01 to 200 mg/kg, more particularly 0.1 to 200 mg/kg, but not limited thereto. is 0.1 to 100 mg/kg. Administration may be once a day or may be divided into several doses, and the scope of the present invention is not limited thereby.
本発明において、前記「対象体」は、ヒトを含む哺乳動物であるが、これらの例に限定されるものではない。 In the present invention, the "subject" is a mammal including a human, but is not limited to these examples.
以下、本発明の理解を助けるために、実施例を挙げて詳細に説明する。但し、下記の実施例は、本発明の内容を例示するものであり、本発明の範囲が、下記の実施例に限定されるものではない。本発明の実施例は、当業者に本発明をより完全に説明するために提供されるものである。 EXAMPLES Hereinafter, the present invention will be described in detail by way of examples to help understand the present invention. However, the following examples are intended to illustrate the content of the present invention, and the scope of the present invention is not limited to the following examples. These embodiments are provided to fully convey the invention to those skilled in the art.
＜実施例１＞ＲＥＰ（ＲＧＤ－ｃｏｎｔａｉｎｉｎｇ Ｅｌａｓｔｉｎ－Ｌｉｋｅ Ｐｏｌｙｐｅｐｔｉｄｅ）タンパク質の準備
１．ＲＥＰ遺伝子クローニング及び発現
<Example 1> Preparation of REP (RGD-containing Elastin-Like Polypeptide) protein 1. REP gene cloning and expression
単位遺伝子に対するオリゴヌクレオチド（５’－ａａｔｔｃａｔａｔｇｇｇｃｃａｃｇｇｃｇｔｇｇｇｔｃｇｔｇｇｃｇａｔｇｔａｇｇｔｇｔｃｃｃａｇｇｔｇｔｇｇｇｃｇｔａｃｃｇｇｇｃｇｔｔｇｇｔｇｔｔｃｃｔｇｇｔｇｔｃｇｇｃｇｔｇｃｃｇｇｇｃｇｔａｇｇｔｇｔｃｃｃａｇｇｔｇｔｇｇｇｃｇｔｇｃｃｇｇｇｃｔｇｇｃａ－３’）を化学的に合成して、ｐＵＣ１９ベクターのＥｃｏＲＩ及びＨｉｄＩＩＩ部位に挿入した。ＲＥＰ遺伝子の生成のために、ＲＤＬ（ｒｅｃｕｒｓｉｖｅ ｄｉｒｅｃｔｉｏｎａｌ ｌｉｇａｔｉｏｎ）方式で単位遺伝子のオリゴマー化を行った。発現ベクターｐＥＴ－２５ｂ（＋）をＮｄｅＩ及びＨｉｎＤＩＩＩに分解し、ＣＩＰで脱リン酸化させた。 Oligonucleotide for unit gene (5'-aattcatatgggccacggcgtgggtcgtggcgatgtagtgtcccaggtgtgggcgtaccgggcgttggtgttcctggtgtcggcgtgccggcgtaggtgtcccaggtgt gggcgtgccgggctggca-3') was chemically synthesized and inserted into the EcoRI and HidIII sites of the pUC19 vector. To generate the REP gene, unit genes were oligomerized using a recursive directional ligation (RDL) method. Expression vector pET-25b(+) was digested with NdeI and HinDIII and dephosphorylated with CIP.
オリゴヌクレオチド（５’－ｔａｔｇａｃｃｇｇｇｃｃｇｇｇｃｔｇｇｃｃｇｔｇｃｔｇａｔａ－３’）と線形ｐＥＴ－２５ｂ（＋）とを連結して新規な発現ベクターｐＥＴ－２５ｂ（＋）－１を生成した。ＲＥＰ遺伝子をｐＥＴ－２５ｂ（＋）－１プラスミドベクターにクローニングし、熱衝撃方法でＥ．ｃｏｌｉ ＢＬＲ（ＤＥ３）に形質転換させた。 The novel expression vector pET-25b(+)-1 was generated by ligating the oligonucleotide (5'-tatgacgggccgggctggccgtgctgata-3') with linear pET-25b(+). The REP gene was cloned into pET-25b(+)-1 plasmid vector and transformed into E. coli by heat shock method. E. coli BLR (DE3) was transformed.
スターター培養液（２５０ｍＬ ｆｌａｓｋｓ ｃｏｎｔａｉｎｉｎｇ ５０ｍＬ ｏｆ ｍｅｄｉｕｍ ｓｕｐｐｌｅｍｅｎｔｅｄ ｗｉｔｈ １００μｇ／ｍＬ ａｍｐｉｃｉｌｌｉｎ）に－８０℃で保管した形質転換されたＥ．ｃｏｌｉ細胞を接種し、３７℃で一晩撹拌し、培養した。スターター培養液を３，０００ｇで４℃で１５分間遠心分離し、新鮮な培地１０ｍＬで再懸濁させた。発現培養液（４Ｌ ｆｌａｓｋｓ ｃｏｎｔａｉｎｉｎｇ １Ｌ ｏｆ ｍｅｄｉｕｍ ｗｉｔｈ １００μｇ／ｍＬ ａｍｐｉｃｉｌｌｉｎ）に懸濁されたスターター培養液５ｍＬを接種し、３７℃で撹拌して培養した。 Transformed E. E. coli cells were inoculated, stirred and cultured overnight at 37°C. The starter culture was centrifuged at 3,000 g for 15 min at 4°C and resuspended in 10 mL of fresh medium. 5 mL of a starter culture solution suspended in an expression culture solution (4 L flasks containing 1 L of medium with 100 μg/mL ampicillin) was inoculated, and cultured at 37° C. with stirring.
ＯＤ６００が約０．８に到達すれば、ＩＰＴＧ（最終濃度１ｍＭ）を添加して発現を誘導した。誘導３時間後、３，０００ｇで４℃で２０分間遠心分離して細胞を収集した。 When OD600 reached approximately 0.8, expression was induced by adding IPTG (final concentration 1mM). After 3 hours of induction, cells were collected by centrifugation at 3,000g for 20 minutes at 4°C.
２．ＲＥＰタンパク質の精製
収集されたＥ．ｃｏｌｉ細胞を冷たいＰＢＳ緩衝液（ｐＨ７．４）３５ｍＬに再懸濁させ、４℃で音波破壊した。細胞溶解物を１５，０００ｇで４℃で１５分間遠心分離し、溶解されていない細胞の残骸を除去した。細胞溶解物にＮａＣｌ（０．５Ｍ）を添加した後、４２℃に加熱してＲＥＰを凝集させた。
2. Purification of REP protein The collected E. E. coli cells were resuspended in 35 mL of cold PBS buffer (pH 7.4) and sonicated at 4°C. Cell lysates were centrifuged at 15,000 g for 15 min at 4°C to remove unlysed cell debris. After adding NaCl (0.5 M) to the cell lysate, it was heated to 42°C to aggregate REP.
凝集されたタンパク質を１０，０００ｇで４０℃で１５分間遠心分離して溶液から分離した。上澄み液を除去し、ペレットを冷たいＰＢＳ緩衝液に溶解させ、高純度のＲＥＰタンパク質を得るために、追加的にＩＴＣ（ｉｎｖｅｒｓｅ ｔｒａｎｓｉｔｉｏｎ ｃｙｃｌｉｎｇ）を行った。 The aggregated protein was separated from the solution by centrifugation at 10,000 g for 15 minutes at 40°C. The supernatant was removed, the pellet was dissolved in cold PBS buffer, and additional ITC (inverse transition cycling) was performed to obtain high purity REP protein.
＜実施例２＞ＲＥＰタンパク質－薬物接合体の製造
１．ドキソルビシンヒドラゾン（Ｄｏｘｏｒｕｂｉｃｉｎ ｈｙｄｒａｚｏｎｅ）の合成
<Example 2> Production of REP protein-drug conjugate 1. Synthesis of Doxorubicin hydrazone
ドキソルビシン塩酸塩（５８．０ｍｇ、０．１ｍｍｏｌ）を丸底フラスコ内の無水メタノール（１０ｍＬ）に溶解させた。無水メタノール（２ｍＬ）に溶解させた３－（２－ピリジルジチオ）プロパン酸ヒドラジド［３－（２－ｐｙｒｉｄｉｎｙｌｄｉｔｈｉｏ）ｐｒｏｐａｎｏｉｃ ａｃｉｄ ｈｙｄｒａｚｉｄｅ、２７．５ｍｇ、０．１２ｍｍｏｌｅ］をドキソルビシン溶液に添加した。トリフルオロ酢酸（ｔｒｉｆｌｕｏｒｏａｃｅｔｉｃ ａｃｉｄ）３滴を前記反応混合物に添加し、室温で一晩反応物を撹拌した。 Doxorubicin hydrochloride (58.0 mg, 0.1 mmol) was dissolved in anhydrous methanol (10 mL) in a round bottom flask. 3-(2-pyridinyldithio)propanoic acid hydrazide, 27.5 mg, 0.12 mmole, dissolved in anhydrous methanol (2 mL) was added to the doxorubicin solution. Three drops of trifluoroacetic acid were added to the reaction mixture and the reaction was stirred at room temperature overnight.
回転蒸発濃縮機を用いて過量のＭｅＯＨを除去し、無水アセトニトリルで溶媒沈殿させて試料を精製した。 Excess MeOH was removed using a rotary evaporator and the sample was purified by solvent precipitation with anhydrous acetonitrile.
２．ＲＥＰタンパク質－ドキソルビシン接合体の合成及び精製
図４のような構造のＲＥＰタンパク質－ドキソルビシン接合体を製造した。
2. Synthesis and Purification of REP Protein-Doxorubicin Conjugate A REP protein-doxorubicin conjugate having the structure shown in FIG. 4 was produced.
まず、２０ｍＭ ＰＢＳ緩衝液（ｐＨ７．４）にＲＥＰを最終濃度１０ｍｇ／ｍｌになるように溶解させた。１５倍モルの過量のドキソルビシンヒドラゾンをジメチルホルムアミドに溶解させ、ＲＥＰタンパク質溶液を添加した後、室温で一晩反応させた。 First, REP was dissolved in 20 mM PBS buffer (pH 7.4) to a final concentration of 10 mg/ml. A 15-fold molar excess of doxorubicin hydrazone was dissolved in dimethylformamide, and after adding the REP protein solution, the reaction was allowed to proceed overnight at room temperature.
結合後、反応溶液を４２℃に加熱し、３，０００ｒｐｍで３５℃で１０分間遠心分離して、ＲＥＰ－ドキソルビシン接合体を他の反応物から分離し、温度によって可逆的に相変化が表われる特徴を用いてＩＴＣを１回行って、図５のように、ＲＥＰ－ドキソルビシン接合体を精製した。 After binding, the reaction solution was heated to 42°C and centrifuged at 3,000 rpm for 10 min at 35°C to separate the REP-doxorubicin conjugate from other reactants, resulting in a reversible phase change with temperature. A single round of ITC was performed using the features to purify the REP-doxorubicin conjugate as shown in FIG.
また、ＰＢＳで平衡化されたＰＤ－１０カラムを使用して、サイズ排除クロマトグラフィーで追加精製し、該精製されたＲＥＰ－ドキソルビシン接合体の最終濃度を１００μＭで濃縮させ、－８０℃に保管した。 Additionally, it was further purified by size exclusion chromatography using a PD-10 column equilibrated with PBS, and the purified REP-doxorubicin conjugate was concentrated to a final concentration of 100 μM and stored at -80°C. .
３．２’－［３－（２－ピリジルジチオ）プロパンオイル］－パクリタキセル｛２’－［３－（２－ｐｙｒｉｄｉｎｙｌｄｉｔｈｉｏ）ｐｒｏｐａｎｏｙｌ］－Ｐａｃｌｉｔａｘｅｌ｝の合成
パクリタキセル（１．２８ｇ、１．５ｍｍｏｌ）、３－（２－ピリジルジチオ）プロピオン酸［３－（２－ｐｙｒｉｄｉｎｙｌｄｉｔｈｉｏ）ｐｒｏｐａｎｏｉｃ ａｃｉｄ、６４．６ｍｇ、３．０ｍｍｏｌ］及びＤＭＡＰ（ｃａｔ．）をジクロロメタンに溶解させた溶液に１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド［１－ｅｔｈｙｌ－３－（３－ｄｉｍｅｔｈｙｌａｍｉｎｏｐｒｏｐｙｌ）ｃａｒｂｏｄｉｉｍｉｄｅ、４１．６ｍｇ、３．３ｍｍｏｌ］を処理し、前記混合物を室温で１２時間撹拌させた。
3. Synthesis of 2'-[3-(2-pyridinyldithio)propanoyl]-paclitaxel {2'-[3-(2-pyridinyldithio)propanoyl]-Paclitaxel} Paclitaxel (1.28 g, 1.5 mmol), 3 1-Ethyl-3-(3-(2-pyridinyldithio)propanoic acid, 64.6 mg, 3.0 mmol) and DMAP (cat.) were dissolved in dichloromethane. -dimethylaminopropyl)carbodiimide [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 41.6 mg, 3.3 mmol] and the mixture was allowed to stir at room temperature for 12 hours.
有機層を水で洗浄し、無水ＭｇＳＯ４を用いて乾燥させた後、生産物を得るために真空濃縮した。 The organic layer was washed with water, dried using anhydrous MgSO 4 and then concentrated in vacuo to obtain the product.
４．ＲＥＰタンパク質－パクリタキセル接合体の合成及び精製
図４のような構造のＲＥＰタンパク質－パクリタキセル接合体を製造した。
4. Synthesis and Purification of REP Protein-Paclitaxel Conjugate A REP protein-paclitaxel conjugate having the structure shown in FIG. 4 was produced.
まず、最終濃度が１０ｍｇ／ｍｌになるようにＲＥＰ（６０ｍｇ、０．００１ｍｍｏｌ）を１ｍＭ ＥＤＴＡと共に１００ｍＭ ＰＢＳ緩衝液（ｐＨ７．４）に溶解させた。１５倍モル過量の２’－［３－（２－ピリジルジチオ）プロパンオイル］－パクリタキセルをジメチルホルムアミド（２ｍＬ）に溶解させ、ＲＥＰ溶液を添加して室温で一晩反応させた。ＰＢＳ－ＥＤＴＡが含まれた除塩カラムをＰＢＳ－ＥＤＴＡ溶液で平衡化させ、ＲＥＰ－パクリタキセル接合体の反応緩衝液に交換して副産物と過量の未反応試薬とを除去した。 First, REP (60 mg, 0.001 mmol) was dissolved in 100 mM PBS buffer (pH 7.4) with 1 mM EDTA to a final concentration of 10 mg/ml. A 15-fold molar excess of 2'-[3-(2-pyridyldithio)propanoyl]-paclitaxel was dissolved in dimethylformamide (2 mL), REP solution was added, and the mixture was allowed to react overnight at room temperature. A desalting column containing PBS-EDTA was equilibrated with a PBS-EDTA solution and exchanged with a reaction buffer of REP-paclitaxel conjugate to remove byproducts and excess unreacted reagents.
ＲＥＰ－パクリタキセル接合体を１回ＩＴＣ行って、精製した後、ＰＢＳで平衡化されたＰＤ－１０カラムを使用して、サイズ排除クロマトグラフィーで追加精製した。 The REP-paclitaxel conjugate was purified by one round of ITC and further purified by size exclusion chromatography using a PD-10 column equilibrated with PBS.
精製されたＲＥＰ－パクリタキセル接合体を最終１００μＭの濃度で濃縮し、－８０℃に保管した。 The purified REP-paclitaxel conjugate was concentrated to a final concentration of 100 μM and stored at -80°C.
＜実施例３＞ドキソルビシン接合体の比率確認
ＲＥＰのモル吸光係数５，６９０Ｌｍｏｌ－１ｃｍ－１を用いてＲＥＰ濃度を決定し、ドキソルビシンの濃度は、４９５ｎｍでドキソルビシンの吸光度及び吸光係数９，２５０Ｌｍｏｌ－１ｃｍ－１を用いて決定した。
<Example 3> Confirmation of the ratio of doxorubicin conjugate The REP concentration was determined using the molar extinction coefficient of REP of 5,690 Lmol -1 cm -1 , and the concentration of doxorubicin was determined by the absorbance of doxorubicin at 495 nm and the extinction coefficient of 9,250 Lmol - 1 cm −1 .
ＲＥＰ－ドキソルビシン接合体のＲＥＰ濃度を決定するために、２８０ｎｍでのドキソルビシンの吸光度を除き、下記式によって２８０ｎｍの吸光度を用いてＲＥＰ濃度を計算した。 To determine the REP concentration of the REP-doxorubicin conjugate, the absorbance of doxorubicin at 280 nm was removed and the REP concentration was calculated using the absorbance at 280 nm according to the following formula.
［ＲＥＰ］＝［Ａｂｓ２８０－（０．９ｘＡｂｓ４９５）］／５，６９０Ｌｍｏｌ－１ｃｍ－１・ｃｍ [REP] = [Abs 280 - (0.9xAbs 495 )] / 5,690 Lmol -1 cm -1 cm
［ＲＥＰ］は、ＲＥＰ－ドキソルビシン接合体内のＲＥＰの濃度である。 [REP] is the concentration of REP within the REP-doxorubicin conjugate.
ドキソルビシン接合体の比率は、ドキソルビシンの濃度をＲＥＰ濃度で割って計算した。 The proportion of doxorubicin conjugates was calculated by dividing the concentration of doxorubicin by the REP concentration.
＜実施例４＞ＲＥＰ－薬物接合体の分光学的特性の確認
実施例３で合成したＲＥＰ－ドキソルビシン接合体の分光学的特性を確認するために、Ｃａｒｙ １００ ＵＶ－ｖｉｓｉｂｌｅ ｓｐｅｃｔｒｏｐｈｏｔｏｍｅｔｅｒを用いて１５℃で８００ｎｍから２００ｎｍまでＲＥＰ－ドキソルビシン接合体のＵＶ－ｖｉｓスペクトルを確認した。
<Example 4> Confirmation of spectroscopic properties of REP-drug conjugate In order to confirm the spectroscopic properties of the REP-doxorubicin conjugate synthesized in Example 3, it was incubated at 15°C using a Cary 100 UV-visible spectrophotometer. The UV-vis spectrum of the REP-doxorubicin conjugate was confirmed from 800 nm to 200 nm.
その結果、図６のように、ＵＶ－可視スペクトルでＲＥＰは、可視領域で吸収ピークが表われない一方、最大吸収である２８０ｎｍでＵＶ吸収光線が確認された。 As a result, as shown in FIG. 6, in the UV-visible spectrum of REP, no absorption peak appeared in the visible region, but UV absorption light was observed at 280 nm, which is the maximum absorption.
＜実施例５＞ＲＥＰ－薬物接合体の熱的特性の確認
ＲＥＰ－薬物接合体の熱転移特性は、温度勾配が１℃／ｍｉｎであるＣａｒｙ １００ ＵＶ－ｖｉｓｉｂｌｅ ｓｐｅｃｔｒｏｐｈｏｔｏｍｅｔｅｒを用いて温度関数で３５０ｎｍでの接合体溶液の濁度を観察して確認した。転移温度（Ｔｔ）を最大濁度の５０％地点と定義し、ＲＥＰ－薬物転移温度は、下記方程式に適した。
<Example 5> Confirmation of thermal properties of REP-drug conjugate Thermal transition properties of REP-drug conjugate were determined as a function of temperature at 350 nm using a Cary 100 UV-visible spectrophotometer with a temperature gradient of 1°C/min. This was confirmed by observing the turbidity of the conjugate solution. The transition temperature (T t ) was defined as the 50% point of maximum turbidity, and the REP-drug transition temperature was fitted to the equation below.
Ｔｔ＝ａ・Ｌｎ（［ＲＥＰ－Ｄｒｕｇ］）＋ｂ T t =a・Ln([REP-Drug])+b
前記式において、［ＲＥＰ－Ｄｒｕｇ］は、ＲＥＰ－薬物濃度であり、ａは、勾配、ｂは、ＲＥＰ－薬物１μＭのＴｔである。 In the above formula, [REP-Drug] is the REP-Drug concentration, a is the slope, and b is the T t of 1 μM REP-Drug.
その結果、図７のように、ＲＥＰ－ドキソルビシン接合体の逆温度転移が確認された。ＰＢＳ内のタンパク質濃度は、２０μＭ（ドキソルビシン接合体の比率は、０．８６７）であり、冷却速度は、１℃／ｍｉｍであった。 As a result, as shown in FIG. 7, a reverse temperature transition of the REP-doxorubicin conjugate was confirmed. The protein concentration in PBS was 20 μM (doxorubicin conjugate ratio 0.867) and the cooling rate was 1° C./mim.
＜実施例６＞ＲＥＰ－薬物接合体内在化の確認
ＰＣＮＡ－１ヒト膵臓癌細胞を９６ウェルプレート内の１０％ ＦＢＳが含まれたＤＭＥＭ培地にウェル当たり２×１０４細胞密度で分注し、３７℃及び５％ ＣＯ２条件下で２４時間培養した。２４時間付着させた後、培養培地を多様な濃度のＲＥＰ－ドキソルビシンが含まれたＤＭＥＭ（１００μＬ、ｗｉｔｈｏｕｔ ＦＢＳ）培地に交換した。
<Example 6> Confirmation of REP-drug conjugate internalization PCNA-1 human pancreatic cancer cells were dispensed into DMEM medium containing 10% FBS in a 96-well plate at a density of 2 x 104 cells per well. The cells were cultured for 24 hours at 37°C and 5% CO2 . After 24 hours of attachment, the culture medium was replaced with DMEM (100 μL, without FBS) containing various concentrations of REP-doxorubicin.
指定された時間ごとに細胞を冷たいＰＢＳ（ｐＨ７．４）で２回洗浄し、細胞を４’，６－ｄｉａｍｉｄｉｎｅ－２’－ｐｈｅｎｙｌｉｎｄｏｌｅ ｄｉｈｙｄｒｏｃｈｌｏｒｉｄｅで１時間染色した後、ＲＥＰ－ドキソルビシンの内在化をＬｅｉｃａ ＤＭＩ３０００Ｂ ｆｌｕｏｒｅｓｃｅｎｃｅ ｍｉｃｒｏｓｃｏｐｅで確認した。 The cells were washed twice with cold PBS (pH 7.4) at each indicated time, and the cells were stained with 4',6-diamidine-2'-phenylindole dihydrochloride for 1 h before REP-doxorubicin internalization was determined using a Leica probe. Confirmed with a DMI3000B fluorescence microscope.
前記過程で０．５、１、２．５、５及び１０μＭの濃度のＲＥＰ－ドキソルビシン接合体をＰＣＮＡ－１細胞に処理した結果、図８のように、濃度依存的にＲＥＰ－ドキソルビシン接合体の細胞内蓄積が増加したことを確認することができた。 As a result of treating PCNA-1 cells with REP-doxorubicin conjugate at concentrations of 0.5, 1, 2.5, 5, and 10 μM in the above process, as shown in FIG. It was confirmed that intracellular accumulation increased.
また、前記過程でＲＥＰ－ドキソルビシン５μＭをＰＡＮＣ－１細胞に処理し、１及び３時間培養した後、ＰＡＮＣ－１細胞内のＲＥＰ－ドキソルビシン蓄積を確認した結果、図９のように、時間依存的にＰＡＮＣ－１細胞内のＲＥＰ－ドキソルビシン蓄積が増加することが確認された。 In addition, in the above process, PANC-1 cells were treated with 5 μM of REP-doxorubicin, and after culturing for 1 and 3 hours, the accumulation of REP-doxorubicin in PANC-1 cells was confirmed. It was confirmed that REP-doxorubicin accumulation in PANC-1 cells increased.
ＲＥＰ－ドキソルビシン５μＭとＰＡＮＣ－１細胞とを３時間培養し、ＲＥＰ－ドキソルビシンの内在化位置を確認した。 PANC-1 cells were cultured with 5 μM of REP-doxorubicin for 3 hours, and the position of internalization of REP-doxorubicin was confirmed.
その結果、図１０のように、ＲＥＰ－ドキソルビシンは、細胞質（黄色矢印）、核（緑色矢印）及び細胞質と核（青色矢印）いずれもに内在化されることを確認することができた。 As a result, as shown in FIG. 10, it was confirmed that REP-doxorubicin was internalized into the cytoplasm (yellow arrow), the nucleus (green arrow), and both the cytoplasm and nucleus (blue arrow).
ＲＥＰ－ドキソルビシンエンドサイトーシスを測定するために、ＲＥＰ－ドキソルビシン処理前、ＧＲＧＤＮＰ（１００μＭ）、アミロライド塩酸塩（ａｍｉｌｏｒｉｄｅ ｈｙｄｒｏｃｈｌｏｒｉｄｅ、１ｍＭ）、クロルプロマジン（ｃｈｌｏｒｐｒｏｍａｚｉｎｅ、５０μＭ）、ｄｙｎａｓｏｒｅ（８０μＭ）及びゲニステイン（ｇｅｎｉｓｔｅｉｎ、２００μＭ）を３０分間ＰＡＮＣ－１細胞に前処理し、ＰＡＮＣ－１細胞でＲＥＰ－ドキソルビシン接合体の内在化に対するエンドサイトーシスの抑制効果を確認した。 To measure REP-doxorubicin endocytosis, GRGDNP (100 μM), amiloride hydrochloride (1 mM), chlorpromazine (50 μM), dynasore (80 μM) before REP-doxorubicin treatment. and genistein (genistein, 200 μM ) was pretreated on PANC-1 cells for 30 minutes, and the inhibitory effect on endocytosis on internalization of REP-doxorubicin conjugate was confirmed in PANC-1 cells.
その結果、図１１のように、ＲＥＰ－ドキソルビシン接合体は、インテグリン収容体媒介メカニズムを通じて癌細胞に内在化されることを確認することができた。 As a result, as shown in FIG. 11, it was confirmed that the REP-doxorubicin conjugate was internalized into cancer cells through an integrin receptor-mediated mechanism.
一方、対照染色法を用いて細胞内の特定小器官を染色してＲＥＰ－ドキソルビシンの内在化機転、細胞内の移動過程及び作用位置を確認した。 Meanwhile, specific intracellular organelles were stained using a control staining method to confirm the internalization mechanism of REP-doxorubicin, intracellular movement process, and action location.
インテグリン収容体媒介メカニズムを通じてＲＥＰ－ドキソルビシンを捕集した小胞体が腫瘍細胞の内部に移動した後、ＲＥＰ－ドキソルビシンをリソソームに運ぶことを確認するために、ＲＥＰ－ドキソルビシンを処理した細胞にリソゾームのみに特異的に反応するリソトラッカー（ＬｙｓｏＴｒａｃｋｅｒ Ｇｒｅｅｎ）を処理して蛍光顕微鏡で観察した。 To confirm that the endoplasmic reticulum that collected REP-doxorubicin through an integrin receptor-mediated mechanism transports REP-doxorubicin to the lysosomes after its movement into the interior of tumor cells, REP-doxorubicin-treated cells were isolated to lysosomes only. The cells were treated with a specifically reactive LysoTracker (LysoTracker Green) and observed using a fluorescence microscope.
その結果、図１２のように、ＲＥＰ－ドキソルビシンが染色された赤色蛍光と膵臓癌細胞のリソゾームの内部を染色したリソトラッカー（ＬｙｓｏＴｒａｃｋｅｒ Ｇｒｅｅｎ）の緑色蛍光、膵臓癌細胞核を染色した青色蛍光が五番目のイメージでいずれも合わせられて黄色蛍光と確認されることによって、内在化されたＲＥＰ－ドキソルビシンがリソソームの内部に存在することを確認することができた。 As a result, as shown in Figure 12, the red fluorescence that stained REP-doxorubicin, the green fluorescence of LysoTracker Green that stained the inside of the lysosome of pancreatic cancer cells, and the blue fluorescence that stained the nucleus of pancreatic cancer cells were the fifth. By combining all images and confirming yellow fluorescence, it was possible to confirm that internalized REP-doxorubicin was present inside the lysosome.
また、内在化されたＲＥＰ－ドキソルビシンが腫瘍細胞内の特定小器官に存在することを確認するために、ＲＥＰ－ドキソルビシンを処理した腫瘍細胞にミトコンドリアのみに特異的に反応するミトトラッカー（ＭｉｔｏＴｒａｃｋｅｒ Ｇｒｅｅｎ）を処理して蛍光顕微鏡で観察した。 In addition, in order to confirm that internalized REP-doxorubicin exists in specific organelles within tumor cells, tumor cells treated with REP-doxorubicin were treated with MitoTracker Green, which specifically reacts only with mitochondria. were processed and observed using a fluorescence microscope.
その結果、図１３のように、ＲＥＰ－ドキソルビシンが染色された赤色蛍光と膵臓癌細胞の細胞質を染色したミトトラッカー（ＭｉｔｏＴｒａｃｋｅｒ Ｇｒｅｅｎ）の緑色蛍光、膵臓癌細胞核を染色した青色蛍光を確認し、五番目のイメージで赤色蛍光と緑色蛍光とが合わせられて黄色生蛍光と紫色蛍光とで表われることが確認されることによって、内在化されたＲＥＰ－ドキソルビシンは、ミトコンドリアと核内部に存在することを確認することができた。 As a result, as shown in Figure 13, we confirmed the red fluorescence stained with REP-doxorubicin, the green fluorescence of MitoTracker Green that stained the cytoplasm of pancreatic cancer cells, and the blue fluorescence that stained the nucleus of pancreatic cancer cells. By confirming that red fluorescence and green fluorescence are combined to appear as yellow raw fluorescence and purple fluorescence in the second image, it is confirmed that internalized REP-doxorubicin exists inside the mitochondria and nucleus. I was able to confirm.
前記結果から、インテグリン収容体媒介メカニズムを通じてＲＥＰ－ドキソルビシンを捕集した小胞体が膵臓癌細胞の内部に移動した後、リソソームと融合してＲＥＰ－ドキソルビシンをリソソームに運び、リソソームの内部のタンパク質分解酵素は、ＲＥＰ－ドキソルビシンを小分子のペプチド－ドキソルビシンで加水分解してペプチド－ドキソルビシンを細胞質で排出し、該排出された小分子ペプチド－ドキソルビシン接合体が核とミトコンドリアとに浸透して抗ガン効果を表わすことが確認された。 The above results indicate that the endoplasmic reticulum that collects REP-doxorubicin moves into the interior of pancreatic cancer cells through an integrin receptor-mediated mechanism, fuses with lysosomes, transports REP-doxorubicin to lysosomes, and transfers REP-doxorubicin to proteolytic enzymes inside the lysosomes. hydrolyzes REP-doxorubicin with a small molecule peptide-doxorubicin, excretes the peptide-doxorubicin in the cytoplasm, and the excreted small molecule peptide-doxorubicin conjugate penetrates the nucleus and mitochondria and exerts an anticancer effect. It has been confirmed that this is true.
＜実施例７＞ヒト癌細胞に対するＲＥＰ－薬物接合体の細胞毒性の効果確認
Ａ５４９ヒト肺癌細胞、Ｈｅｐ Ｇ２ヒト肝癌細胞、ＨＴ－２９ヒト大腸癌細胞、ＭＤＡ－ＭＢ－２３１ヒト乳癌細胞及びＰＡＮＣ－１ヒト膵臓癌細胞を用いて多様なタイプのヒト癌に対するＲＥＰ－ドキソルビシンの細胞毒性を確認した。
<Example 7> Confirmation of cytotoxic effect of REP-drug conjugate on human cancer cells A549 human lung cancer cells, Hep G2 human liver cancer cells, HT-29 human colon cancer cells, MDA-MB-231 human breast cancer cells, and PANC- The cytotoxicity of REP-doxorubicin against various types of human cancer was confirmed using human pancreatic cancer cells.
癌細胞に対するＩＣ５０値を確認するために、ＰＡＮＣ－１細胞を１０％ ＦＢＳ、２ｍＭ Ｌ－グルタミン（ｇｌｕｔａｍｉｎｅ）、５０μｇ／ｍＬ ストレプトマイシン（ｓｔｒｅｐｔｏｍｙｃｉｎ）及び５０Ｕ／ｍＬ ペニシリン（ｐｅｎｉｃｉｌｌｉｎ）が含まれたＤＭＥＭ培地で培養した。 To confirm the IC50 value against cancer cells, PANC-1 cells were incubated in DMEM medium containing 10% FBS, 2mM L-glutamine, 50μg/mL streptomycin, and 50U/mL penicillin. It was cultured in
細胞を３７℃、９５％相対湿度及び５％ ＣＯ２条件の培養チャンバで培養した。ＰＡＮＣ－１細胞を９６ウェルプレート内の１％ ＦＢＳが含まれた培地にウェル当たり２×１０４細胞を分注し、２４時間培養した。２４時間細胞を付着させた後、培養培地を新鮮なＤＭＥＭ培地（１００μＬ、ｎｏ ＦＢＳ）に取り替えた。 Cells were cultured in a culture chamber at 37°C, 95% relative humidity and 5% CO2 . PANC-1 cells were dispensed at 2×10 4 cells per well into a medium containing 1% FBS in a 96-well plate and cultured for 24 hours. After 24 hours of cell attachment, the culture medium was replaced with fresh DMEM medium (100 μL, no FBS).
ＲＥＰ－ドキソルビシンをドキソルビシンの濃度が０．１、０．２５、０．５、０．７５、１、１．２５、１．５、２．５、５及び１０μＭになるようにウェルに添加するか、パクリタキセルの濃度が０．１、０．２５、０．５、１、２．５、５、７．５、１０、５０及び１００ｎｍになるようにＲＥＰ－パクリタキセルをウェルに添加した。 Add REP-doxorubicin to the wells at concentrations of doxorubicin of 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2.5, 5, and 10 μM. , REP-paclitaxel was added to the wells such that the concentration of paclitaxel was 0.1, 0.25, 0.5, 1, 2.5, 5, 7.5, 10, 50, and 100 nm.
４８時間培養した後、培養培地を除去し、細胞を冷たいＤＭＥＭ培地１００μＬで２回洗浄した。各ウェルにＣＣＫ－８［２－（２－ｍｅｔｈｏｘｙ－４－ｎｉｔｒｏｐｈｅｎｙｌ）－３－（４－ｎｉｔｒｏｐｈｅｎｙｌ）－５－（２，４－ｄｉｓｕｌｆｏｐｈｅｎｙｌ）－２Ｈ－ｔｅｔｒａｚｏｌｉｕｍ］１０μＬを添加し、３７℃、９５％相対湿度及び５％ ＣＯ２条件下で１時間培養した。 After 48 hours of culture, the culture medium was removed and the cells were washed twice with 100 μL of cold DMEM medium. Add 10 μL of CCK-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] to each well, and incubate at 37°C at 95°C. % relative humidity and 5% CO2 for 1 hour.
培養後、４５０ｎｍでｍｉｃｒｏｐｌａｔｅ ｒｅａｄｅｒを用いて吸光度（ＯＤ）を測定した。細胞生存曲線は、ＧｒａｐｈＰａｄ Ｐｒｉｓｍ ８．３．１を利用したｓｉｇｍｏｉｄａｌ ｆｏｕｒ ＰＬ－Ｓｉｇｍｏｉｄａｌ ｆｕｎｃｔｉｏｎに適した。 After culturing, absorbance (OD) was measured at 450 nm using a microplate reader. The cell survival curve was suitable for sigmoidal four PL-Sigmoidal function using GraphPad Prism 8.3.1.
その結果、表１及び表２のように、各癌細胞に対するＲＥＰ－ドキソルビシン接合体（μＭ）とＲＥＰ－パクリタキセル接合体（ｎＭ）とのＩＣ５０値を確認することができた。 As a result, as shown in Tables 1 and 2, it was possible to confirm the IC 50 values of the REP-doxorubicin conjugate (μM) and the REP-paclitaxel conjugate (nM) for each cancer cell.
＜実施例８＞ドキソルビシン、ＲＥＰ及びドキソルビシン混合物及びＲＥＰ－ドキソルビシン接合体の細胞毒性の確認
ドキソルビシン、ＲＥＰ及びドキソルビシン混合物及びＲＥＰ－ドキソルビシン接合体の細胞毒性を比較するために、実施例７のような方法でドキソルビシン、ＲＥＰ及びドキソルビシン混合物及びＲＥＰ－ドキソルビシン接合体をＰＡＮＣ－１細胞で処理してＩＣ５０値を確認した。
<Example 8> Confirmation of cytotoxicity of doxorubicin, REP, doxorubicin mixture, and REP-doxorubicin conjugate In order to compare the cytotoxicity of doxorubicin, REP, doxorubicin mixture, and REP-doxorubicin conjugate, the method as in Example 7 was used. Doxorubicin, REP, doxorubicin mixture, and REP-doxorubicin conjugate were treated with PANC-1 cells to determine IC 50 values.
その結果、図１４のように、ドキソルビシンと比較して、ＲＥＰ及びドキソルビシン混合物の抗ガン効果が４倍増加した一方、ＲＥＰ－ドキソルビシン接合体を２０倍以上増加した抗ガン活性を示すことを確認することができた。 As a result, as shown in FIG. 14, it was confirmed that the anticancer effect of the REP and doxorubicin mixture was increased by 4 times compared to doxorubicin, while the REP-doxorubicin conjugate showed an increased anticancer activity of more than 20 times. I was able to do that.
また、ドキソルビシン敏感性ＭＤＡ－ＭＢ－２３１細胞及びドキソルビシン抵抗性ＭＤＡ－ＭＢ－２３１細胞でドキソルビシン、ＲＥＰ及びドキソルビシン混合物及びＲＥＰ－ドキソルビシン接合体のＩＣ５０（μＭ）値とドキソルビシンの薬物抵抗性指数とを確認した。 In addition, the IC 50 (μM) values of doxorubicin, REP, doxorubicin mixture, and REP-doxorubicin conjugate and the drug resistance index of doxorubicin were determined in doxorubicin-sensitive MDA-MB-231 cells and doxorubicin-resistant MDA-MB-231 cells. confirmed.
その結果、表３のように、ＲＥＰ－ドキソルビシン接合体が処理されたドキソルビシン抵抗性ＭＤＡ－ＭＢ－２３１細胞で薬物抵抗性の克服効果が表われることが確認された。 As a result, as shown in Table 3, it was confirmed that doxorubicin-resistant MDA-MB-231 cells treated with the REP-doxorubicin conjugate exhibited an effect of overcoming drug resistance.
以上、本発明の特定の部分を詳しく記述したところ、当業者にとって、このような具体的な記述は、単に望ましい実施形態に過ぎず、これにより、本発明の範囲が制限されるものではないという点は明白である。したがって、本発明の実質的な範囲は、特許請求の範囲とそれらの等価物とによって定義される。 Although specific parts of the present invention have been described in detail above, those skilled in the art will appreciate that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. The point is clear. Accordingly, the substantial scope of the invention is defined by the claims and their equivalents.
Claims (3)
前記細胞外基質は、エラスチン類似ポリペプチドとインテグリン受容体リガンドとからなり、配列番号３で表されるアミノ酸配列である［ＶＧＲＧＤ（ＶＧＶＰＧ） ６ ］ ｎ (前記ｎは、配列番号３の反復回数であって、５～２０の整数である)で構成されるか、配列番号２で表されるアミノ酸配列であるＴＧＰＧ［ＶＧＲＧＤ（ＶＧＶＰＧ） ６ ］ ｎ ＷＰＣ(前記ｎは、［ＶＧＲＧＤ（ＶＧＶＰＧ） ６ ］の反復回数であって、５、１０、１２、１５または２０の整数である)で構成され、
前記細胞外基質及びドキソルビシンは、リンカーによって互いに連結され、前記リンカーは、ＯＨ－、ＮＨ ２ －及びＳＨ－からなる群からそれぞれ異なる２個の官能基を有するＣ２～Ｃ５の化合物であり、
前記癌疾患は、肝癌、大腸癌、及び膵臓癌からなる群から選択されるいずれかであることを特徴とする、癌疾患治療用薬学組成物。 In a pharmaceutical composition for treating cancer diseases, which contains a conjugate consisting of extracellular matrix (REP) and doxorubicin as an active ingredient,
The extracellular matrix consists of an elastin-like polypeptide and an integrin receptor ligand, and has an amino acid sequence represented by SEQ ID NO: 3 [VGRGD(VGVPG) 6 ] n (n is the number of repeats of SEQ ID NO: 3). TGPG [VGRGD(VGVPG) 6 ] n WPC (the above n is [ VGRGD ( VGVPG ) 6 ] is an integer of 5, 10, 12, 15 or 20),
The extracellular matrix and doxorubicin are connected to each other by a linker, and the linker is a C2 to C5 compound each having two different functional groups from the group consisting of OH-, NH2- and SH-,
A pharmaceutical composition for treating a cancer disease, wherein the cancer disease is one selected from the group consisting of liver cancer, colon cancer, and pancreatic cancer .
The pharmaceutical composition for treating cancer disease according to claim 1 , wherein the pharmaceutical composition increases cell death of tumor cells.
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