JP2019043892A - Oligopeptide derivatives and pharmaceuticals using the same - Google Patents
Oligopeptide derivatives and pharmaceuticals using the same Download PDFInfo
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- JP2019043892A JP2019043892A JP2017168845A JP2017168845A JP2019043892A JP 2019043892 A JP2019043892 A JP 2019043892A JP 2017168845 A JP2017168845 A JP 2017168845A JP 2017168845 A JP2017168845 A JP 2017168845A JP 2019043892 A JP2019043892 A JP 2019043892A
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- Prior art keywords
- oligopeptide
- radioactive
- cyclic
- glycine
- arginine
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Abstract
Description
本発明は、癌細胞やそれにより亢進された組織のリガンドに結合し放射線診療に用いるオリゴペプチド誘導体、及びそれを用いた医薬に関するものである。 TECHNICAL FIELD The present invention relates to an oligopeptide derivative used for radiological treatment by binding to a ligand of a cancer cell or a tissue promoted thereby, and a medicine using the same.
放射性同位元素である放射性核種（ＲＩ）を有する放射性医薬品は、治療や診断のような診療に用いられている。このような放射性核種として例えば、治療用に89Ｓｒ、90Ｙ、135Ｉ、131Ｉ、125Ｉ、153Ｓｍ、186/188Ｒｅ、211Ａｔ、223Ｒａなどが知られ、体内診断用に18Ｆ、67/68Ｇａ、99ｍＴｃ、111Ｉｎ、123Ｉ、133Ｘｅなどが知られ、体外診断用に3Ｈ、125Ｉなどが知られている。 Radiopharmaceuticals having a radioisotope, a radionuclide (RI), are used in medical care such as treatment and diagnosis. For example, 89 Sr, 90 Y, 135 I, 131 I, 125 I, 153 Sm, 186/188 Re, 211 At, 223 Ra, etc. are known as such radionuclides for therapeutic use, and 18 F for in vivo diagnosis. , 67/68 Ga, 99m Tc, 111 In, 123 I, 133 Xe, etc. are known, and 3 H, 125 I etc. are known for in vitro diagnosis.
放射性核種内用療法（Radionuclide Therapy）は、放射性核種内用療法用薬剤を静注や経口で投与し、癌原発巣や転移巣のような病巣部位にこの放射性薬剤を集積させ、放射性薬剤から放出されるα線又はβ線のような放射線により、病巣部位の癌細胞を破壊するというものである。 Radionuclide internal therapy (Radionuclide Therapy) consists of intravenous or oral administration of a radionuclide therapeutic drug, which accumulates the radiopharmaceutical at focal sites such as primary or metastatic cancer, and releases the radiopharmaceutical from the radiopharmaceutical Radiation such as alpha rays or beta rays destroys cancer cells at the lesion site.
日本で保険適応となっている放射性核種内用療法用薬剤として、β線を放出するヨウ化ナトリウム(核種：131Ｉ)、塩化ストロンチウム(同89Ｓｒ)及び放射標識抗CD20抗体(同90Ｙ)や、α線を放出する塩化ラジウム(同223Ｒａ)などがある。 Sodium iodide (Nuclide: 131 I), Strontium chloride ( 89 Sr), and radio-labeled anti-CD20 antibody ( 90 Y) that emit β-rays as medicines for radionuclide internal medicine that are covered by insurance in Japan And radium chloride (the same 223 Ra) that emits alpha rays.
これらの薬剤を用いた放射性核種内用療法は、例えば甲状腺機能亢進症や甲状腺癌において甲状腺がヨウ素を取り込む性質を利用したり、骨転移などの骨代謝の亢進した病態の領域にラジウムが集積する性質を利用したりして、放射性核種のヨウ化ナトリウムや塩化ラジウムの薬剤を投与し、甲状腺癌等の病巣部位や骨転移領域で、α線やβ線を放出させて、癌細胞を破壊して死滅させ、抗腫瘍効果を発揮するというものである。 For radionuclide internal therapy using these agents, for example, the thyroid uptake property of iodine in hyperthyroidism and thyroid cancer is used, and radium accumulates in the area of pathological conditions in which bone metabolism such as bone metastasis is enhanced. Taking advantage of the properties, the drug of radionuclide sodium iodide or radium chloride is administered, and alpha rays and beta rays are emitted at focal sites such as thyroid cancer and bone metastasis areas to destroy cancer cells. To kill and to exert an antitumor effect.
β線を放出する放射性核種内用療法用薬剤は病巣部位の癌細胞に集積したとしても、β線は飛程が０．５ｍｍ程度と比較的長いため癌細胞や近隣の正常細胞まで到達し、癌細胞の破壊のみならず正常細胞までも損傷してしまう。一方、α線を放出する放射性核種内用療法用薬剤は病巣部位の癌細胞に集積すると、α線は、β線に比べ約４００倍もの高い線エネルギー付与(LET)（α線:80keV/μm、β線:0.2 keV/μm）を有し、強い細胞傷害性を持ち飛程が極めて短い。そのため、α線はその癌細胞の標的部位にしか到達せず癌細胞を破壊するが、近隣の正常細胞を損傷しない。 Even if the radionuclide internal therapy drug that emits beta radiation accumulates in the cancer cells at the focal site, the beta radiation has a relatively long range of about 0.5 mm and reaches the cancer cells and nearby normal cells, Not only destruction of cancer cells but also normal cells are damaged. On the other hand, when the radionuclide internal therapy drug that emits alpha rays accumulates in the cancer cells at the lesion site, the alpha rays have a linear energy transfer (LET) about 400 times higher than beta rays (alpha rays: 80 keV / μm) , Β rays: 0.2 keV / μm), having strong cytotoxicity and extremely short range. Therefore, alpha rays only reach the target site of the cancer cells and destroy the cancer cells, but do not damage neighboring normal cells.
このようなα線を放出する放射性核種として211Ａｔも知られている。211Ａｔは安定同位体が存在しないため、詳しい物理的性質が未だに不明なハロゲン元素である。Ａｔは、ＩやＢｒなど他のハロゲンと類似した化学的性質・化学反応性を持つ。そのため、既知のハロゲン誘導体での合成法を応用してＡｔ標識して、治療用に放射性核種として211Ａｔを導入し得る。211Ａｔは半減期が７．２時間で短く安全性が高い。このような211Ａｔを各種病巣部位などの標的部位に効率良く集積できれば、放射性核種内用療法において高い治療効果が期待できる。 211 At is also known as a radionuclide that emits such alpha rays. 211 At is a halogen element whose detailed physical properties are still unknown because stable isotope does not exist. At has similar chemical properties and chemical reactivity to other halogens such as I and Br. Therefore, synthetic methods with known halogen derivatives can be applied to At labeling to introduce 211 At as a radionuclide for treatment. 211 At has a short half life of 7.2 hours and high safety. If such 211 At can be efficiently accumulated at target sites such as various lesion sites, a high therapeutic effect can be expected in radionuclide internal therapy.
ところで、細胞質膜を貫通する受容体であって細胞接着に関与する接着因子であるインテグリンは、１８種類のαサブユニット、８種類のβサブユニットが確認されている（非特許文献１）。そのうち、αｖβ３インテグリンは、血管新生のマーカーであり、また様々な癌細胞に過剰発現している（非特許文献２）。腫瘍による血管新生の亢進の際の血管の内皮細胞にαｖβ３インテグリンが発現したり、腫瘍の癌原発巣の癌細胞にαｖβ３インテグリンが発現したりしている。アルギニン−グリシン−アスパラギン酸(RGD)配列を含むペプチドはαｖβ３インテグリンに対して高親和性を示す（非特許文献３） By the way, 18 types of α subunits and 8 types of β subunits have been confirmed as integrins that are receptors that penetrate the plasma membrane and are involved in cell adhesion (Non-Patent Document 1). Among them, α v β 3 integrin is a marker of angiogenesis and is overexpressed in various cancer cells (Non-patent Document 2). Or expression of α v β 3 integrin in endothelial cells of the blood vessel at the time of the enhancement of angiogenesis, the α v β 3 integrin on the cancer cells of the proto-primary tumor of the tumor is or expressed by the tumor. A peptide containing an arginine-glycine-aspartic acid (RGD) sequence exhibits high affinity for α v β 3 integrin (Non-patent Document 3)
特許文献１に、環状ＲＧＤモチーフおよびスペーサー配列により共有結合で連結された２つのエキスタチンＣ末端部分を含有する、αｖβ３インテグリンに対する選択的アフィニティーを示す、αｖβ３インテグリンのアンタゴニスト化合物が記載されており、放射性同位元素を錯化することができるキレート化剤が例示されている。
特許文献２に、α３β１−インテグリン、α５β１−インテグリン、αｖβ３−インテグリンおよびαｖβ５−インテグリン、および他のインテグリンのうちの少なくとも一つを阻害すること、およびヒトまたは動物の対象者におけるｂＦＧＦ産生を阻害すること、のうちの少なくとも一方を行うためのものであって、対象者に有効量のＲＧシステイン酸ペプチドまたはその誘導体を投与する工程を含む方法が記載されている。特許文献２には、ＲＧシステイン酸ペプチドまたはその誘導体は放射性標識されており、かつ腫瘍を検出するために用いられる方法が例示されている。
αｖβ３インテグリンに対して高親和性を示し、各種腫瘍の癌原発巣の放射線診断や放射性核種内用療法に用いることができるアルギニン−グリシン−アスパラギン酸配列を含むペプチドが求められている。 There is a need for a peptide having an arginine-glycine-aspartate sequence that exhibits high affinity to α v β 3 integrin and can be used for radiodiagnosis of cancer primary lesions of various tumors and internal radionuclide therapy.
本発明は前記の課題を解決するためになされたもので、αｖβ３インテグリンに対して高親和性を示し各種腫瘍の癌原発巣の診療に汎用性があり、臓器よりも腫瘍への高い集積性を示し、簡素かつ迅速に製造でき、安全で、放射線診断や放射性核種内用療法に用いることができるもので医薬として利用できる、放射標識されたアルギニン−グリシン−アスパラギン酸配列を含むオリゴペプチド誘導体、及びそれを用いた医薬を提供することを目的とする。 The present invention was made to solve the above-mentioned problems, and has high affinity to α v β 3 integrin, which is versatile for medical treatment of cancer primary lesions of various tumors, and is higher for tumors than organs. An oligopeptide comprising a radiolabeled arginine-glycine-aspartic acid sequence that exhibits accumulation, can be simply and rapidly manufactured, is safe, can be used for radiodiagnosis and radionuclide internal therapy, and can be used as a medicine It aims at providing a derivative and a medicine using the same.
前記の目的を達成するためになされた本発明のオリゴペプチド誘導体は、環状オリゴペプチド配列からなりアルギニン−グリシン−アスパラギン酸配列を有しつつ、放射性同位体で標識されているというものである。 The oligopeptide derivative of the present invention made to achieve the above-mentioned purpose is that it is labeled with a radioactive isotope while being composed of a cyclic oligopeptide sequence and having an arginine-glycine-aspartic acid sequence.
このオリゴペプチド誘導体は、インテグリンに、結合し、配位し、及び／又は親和することができるものである。 The oligopeptide derivative is capable of binding, coordinating and / or affinity to an integrin.
このオリゴペプチド誘導体は、前記放射性同位体が、放射性ヨウ素、放射性アスタチン、放射性ストロンチウム、放射性イットリウム、放射性ラジウム、放射性ビスマス、放射性アクチニウム、放射性ルテチウム、放射性レニウム、及び放射性銅から選ばれる何れかの放射性核種を含んでいることが好ましい。 This oligopeptide derivative is any radioactive nuclide selected from the group consisting of radioactive iodine, radioactive astatine, radioactive strontium, radioactive yttrium, radioactive radium, radioactive bismuth, radioactive actinium, radioactive lutetium, radioactive rhenium, and radioactive copper Is preferably contained.
このオリゴペプチド誘導体は、記放射性同位体がα線を放出するものである。 This oligopeptide derivative is one in which the radioactive isotope emits alpha rays.
このオリゴペプチド誘導体は、前記放射性同位体が、例えば211Ａｔを含んでいるというものである。 The oligopeptide derivative is such that the radioactive isotope contains, for example, 211 At.
このオリゴペプチド誘導体は、前記環状オリゴペプチド配列のアミノ酸残基の芳香環基に直接、若しくは前記環状オリゴペプチド配列へスペーサー基を介して又は介さずに結合した芳香環基に、前記放射性同位体が結合していることが好ましい。 In this oligopeptide derivative, the radioisotope is attached to an aromatic ring group linked directly to an aromatic ring group of an amino acid residue of the cyclic oligopeptide sequence or to the cyclic oligopeptide sequence via or without a spacer group. It is preferred that they be linked.
このオリゴペプチド誘導体は、前記環状オリゴペプチド配列が、アルギニン−グリシン−アスパラギン酸−Ｄ−アミノ酸配列を有していると一層好ましい。 In this oligopeptide derivative, it is more preferable that the cyclic oligopeptide sequence has an arginine-glycine-aspartic acid-D-amino acid sequence.
このオリゴペプチド誘導体は、前記環状オリゴペプチド配列中の前記Ｄ−アミノ酸が、Ｄ−フェニルアラニン、又はＤ−チロシンであると、なお一層好ましい。 In this oligopeptide derivative, it is even more preferable that the D-amino acid in the cyclic oligopeptide sequence is D-phenylalanine or D-tyrosine.
このオリゴペプチド誘導体は、例えば環状（アルギニン−グリシン−アスパラギン酸−フェニルアラニン−リジン）オリゴペプチド誘導体、環状（アルギニン−グリシン−アスパラギン酸−チロシン−リジン）オリゴペプチド誘導体、環状（アルギニン−グリシン−アスパラギン酸−フェニルアラニン−バリン）オリゴペプチド誘導体、又は環状（アルギニン−グリシン−アスパラギン酸−チロシン−バリン）オリゴペプチド誘導体である。 This oligopeptide derivative is, for example, cyclic (arginine-glycine-aspartate-phenylalanine-lysine) oligopeptide derivative, cyclic (arginine-glycine-aspartate-tyrosine-lysine) oligopeptide derivative, cyclic (arginine-glycine-aspartic acid- Phenylalanine-valine) oligopeptide derivatives or cyclic (arginine-glycine-aspartate-tyrosine-valine) oligopeptide derivatives.
このオリゴペプチド誘導体は、環状（アルギニン−グリシン−アスパラギン酸−Ｄ−フェニルアラニン−リジン）オリゴペプチド誘導体、環状（アルギニン−グリシン−アスパラギン酸−Ｄ−チロシン−リジン）オリゴペプチド誘導体、環状（アルギニン−グリシン−アスパラギン酸−Ｄ−フェニルアラニン−バリン）オリゴペプチド誘導体、又は環状（アルギニン−グリシン−アスパラギン酸−Ｄ−チロシン−バリン）オリゴペプチド誘導体であると、一層好ましい。 This oligopeptide derivative is a cyclic (arginine-glycine-aspartate-D-phenylalanine-lysine) oligopeptide derivative, a cyclic (arginine-glycine-aspartic acid-D-tyrosine-lysine) oligopeptide derivative, a cyclic (arginine-glycine- More preferably, they are aspartic acid-D-phenylalanine-valine) oligopeptide derivatives or cyclic (arginine-glycine-aspartic acid-D-tyrosine-valine) oligopeptide derivatives.
前記の目的を達成するためになされた本発明の医薬は、前記のオリゴペプチド誘導体を含有するというものである。 The medicament of the present invention made to achieve the above-mentioned purpose is one containing the above-mentioned oligopeptide derivative.
本発明のオリゴペプチド誘導体は、簡素な構造で、選択的にインテグリンとりわけαｖβ３インテグリンに対して高親和性を示して結合、配位し及び／又は親和でき、従来のオリゴペプチド誘導体やその放射性同位体標識化合物に比べ、臓器よりも腫瘍への高い集積性を示す腫瘍特異性に優れるものである。 The oligopeptide derivative of the present invention has a simple structure and can selectively bind, coordinate, and / or have high affinity for integrins, particularly α v β 3 integrin, and can be a conventional oligopeptide derivative or the like Compared to radioactive isotope labeled compounds, it is superior in tumor specificity which shows higher accumulation in tumors than organs.
このオリゴペプチド誘導体は、簡易且つ迅速に簡便に高純度で製造できるため、半減期が比較的長い放射性同位体でも数日程度と比較的短い放射性同位体でも含有させることができ、安全性が高く、品質に優れている。さらにこのオリゴペプチド誘導体は、医薬として用いることができる。とりわけ半減期が７．２時間と短い211Ａｔを放射性同位体として用いる場合であっても、簡便な合成法で投与の少し前に調製できるので、放射能を十分に活用して放射線治療効果を発揮できる。 Since this oligopeptide derivative can be easily and rapidly produced in high purity simply and easily, it can be contained as a radioactive isotope having a relatively long half life or a relatively short isotope date of about several days, and the safety is high. Is excellent in quality. Furthermore, this oligopeptide derivative can be used as a medicine. In particular, even if 211 At, which has a short half-life of 7.2 hours, is used as a radioactive isotope, it can be prepared a short time before administration by a simple synthesis method. It can be demonstrated.
このオリゴペプチド誘導体は、腫瘍組織、例えば腫瘍の癌原発巣の癌細胞、転移しつつある微小癌転移組織又は転移してしまった転移組織、腫瘍による新生血管への高い集積性に起因して、放射性同位体による高い腫瘍組織選択的撮像能や癌細胞やそれに起因する殺傷能を有する。 This oligopeptide derivative is due to the high accumulation of tumor tissue, for example, cancer cells in the primary tumor of a tumor, metastatic micrometastatic tissue or metastasized metastatic tissue, and neovascularization by the tumor. It has high tumor tissue selective imaging ability by radioactive isotopes, and cancer cells and killing ability due to it.
特に放射性同位体が、α線を放出する211Ａｔであると癌細胞のみを破壊し正常細胞を損傷しない。 In particular, when the radioactive isotope is 211 At that emits an alpha ray, only cancer cells are destroyed and normal cells are not damaged.
このオリゴペプチド誘導体を含有する医薬は、経口投与や静注投与又は腫瘍組織への直接投与により、癌細胞やそれによる新生血管の内皮細胞のインテグリンとりわけαｖβ３インテグリンにオリゴペプチド誘導体を集積させて、腫瘍組織の選択的な放射線診断に利用して正確で確実な画像診断や、腫瘍組織へ有効成分を集積させる放射性核種内用療法に利用して確実で安全な癌細胞の破壊やアポトーシスを誘導することができる。 A drug containing this oligopeptide derivative is allowed to accumulate the oligopeptide derivative on integrins, particularly α v β 3 integrin of cancer cells and their neovascular endothelial cells by oral administration, intravenous administration or direct administration to tumor tissue. To use it for selective radiodiagnosis of tumor tissue for accurate and reliable image diagnosis and radionuclide internal therapy for accumulating active ingredients in tumor tissue for reliable and safe destruction of cancer cells and apoptosis. It can be induced.
また、このオリゴペプチド誘導体を含有する医薬は、腫瘍組織への集積性が高いので、放射線診断にも用いることができ、安全で確実に腫瘍組織部位、腫瘍組織によって亢進した血管部位、微小癌転移乃至癌転移した部位、郭清すべき腋窩リンパ節部位などを、特定したり、腫瘍の進行状況を診断したりするのに、用いることができる。 In addition, since the drug containing this oligopeptide derivative has high accumulation in tumor tissue, it can also be used for radiodiagnosis, and it is safe and reliable for tumor tissue site, blood vessel site enhanced by tumor tissue, micro cancer metastasis The site of cancer metastasis, the axillary lymph node site to be dissected, etc. can be identified or used to diagnose the progress of the tumor.
以下、本発明を実施するための形態を詳細に説明するが、本発明の範囲はこれらの形態に限定されるものではない。 Hereinafter, although the form for implementing this invention is demonstrated in detail, the scope of the present invention is not limited to these forms.
本発明のオリゴペプチド誘導体は、アミノ酸配列が環状オリゴペプチド配列のみからなりアルギニン−グリシン−アスパラギン酸配列を有しつつ、放射性同位体で標識されているものである。 The oligopeptide derivative of the present invention is one in which the amino acid sequence consists only of a cyclic oligopeptide sequence and is labeled with a radioactive isotope while having an arginine-glycine-aspartic acid sequence.
オリゴペプチド誘導体は、アルギニン−グリシン−アスパラギン酸配列を有していると、インテグリン、そのファミリーの中でも特にαｖβ３インテグリンを認識してそこへ結合し、配位し、及び／又は親和することができるものである。 The oligopeptide derivative, having an arginine-glycine-aspartic acid sequence, recognizes, binds to, coordinates with, and / or affinity to an integrin, particularly an α v β 3 integrin among the family. It is possible to
この放射性同位体が、放射性ヨウ素（135Ｉ、131Ｉ、125Ｉ）、放射性アスタチン（211Ａｔ）、放射性ストロンチウム（89Ｓｒ）、放射性イットリウム（90Ｙ）、放射性ラジウム（223Ｒａ）、放射性ビスマス（212/213Ｂｉ）、放射性アクチニウム（225Ａｃ）、放射性ルテチウム（177Ｌｕ）、放射性レニウム（186/188Ｒｅ）、及び放射性銅（64/67Ｃｕ）から選ばれる何れかの放射性核種及びそれらが崩壊した放射性核種又はそれの安定同位体核種を含んでいてもよい。 The radioactive isotopes are radioactive iodine ( 135 I, 131 I, 125 I), radioactive astatine ( 211 At), radioactive strontium ( 89 Sr), radioactive yttrium ( 90 Y), radioactive radium ( 223 Ra), radioactive bismuth ( 212/213 Bi), radioactive actinium ( 225 Ac), radioactive lutetium ( 177 Lu), radioactive rhenium ( 186/188 Re), and any radioactive nuclide selected from radioactive copper ( 64/67 Cu) and their decay Or a stable isotope or radionuclide thereof.
中でも、Ｘ線を放射する125Ｉ、α線を放射する211Ａｔが好ましい。とりわけ、この放射性同位体が211Ａｔであると、放射性核種内用療法用薬剤として用いられるオリゴペプチド誘導体が病巣部位の癌細胞やそれにより亢進した血管内皮細胞のαｖβ３インテグリンに結合等をして短時間で集積し、α線が、死滅させるべき癌細胞等の組織のみに到達して損傷を惹き起こして破壊及びアポトーシスを誘導するが近隣の正常細胞を損傷しない。 Among them, 125 I emitting X-rays and 211 At emitting α-rays are preferable. In particular, when the radioactive isotope is 211 At, an oligopeptide derivative used as a medicine for internal radionuclide therapy binds to α v β 3 integrin of cancer cells in the lesion site or vascular endothelial cells enhanced thereby. It accumulates in a short time, and alpha rays reach only tissues such as cancer cells to be killed, causing damage and inducing destruction and apoptosis but not neighboring normal cells.
オリゴペプチド誘導体は、アルギニン−グリシン−アスパラギン酸配列を含む環状オリゴペプチド配列に直接、若しくはスペーサー基を介して又は介さずに結合した芳香環基に、前記放射性同位体が結合していてもよい。その芳香環基はフェニル基のような炭化水素芳香環であっても複素芳香環であってもよい。オリゴペプチド誘導体は、環状オリゴペプチド配列中にフェニルアラニン残基やチロシン残基がある場合にその芳香環に前記放射性同位体が直接結合していてもよい。スペーサー基を介して結合した芳香環基は、例えば前記環状オリゴペプチド配列中にリジン残基がある場合にその末端の遊離ε−アミノ基に結合するもので前記放射性同位体が結合しているベンゾイル基、ベンジルオキシカルボニル基が挙げられる。 The radioactive peptide may be bound to an aromatic ring group linked directly or with or without a spacer group to a cyclic oligopeptide sequence containing an arginine-glycine-aspartic acid sequence. The aromatic ring group may be a hydrocarbon aromatic ring such as a phenyl group or a heteroaromatic ring. In the oligopeptide derivative, when there is a phenylalanine residue or a tyrosine residue in the cyclic oligopeptide sequence, the radioactive isotope may be directly bound to the aromatic ring. An aromatic ring group linked via a spacer group is, for example, a benzoyl group linked to a free ε-amino group at its end when a lysine residue is present in the cyclic oligopeptide sequence, and in which the radioactive isotope is linked. And benzyloxycarbonyl group.
オリゴペプチド誘導体は、例えば環状ペンタオリゴペプチド配列からなるもので、官能基が非アミノ酸で保護されているものであってもよいが、環状のオリゴペプチド配列に別なアミノ酸が結合していないものである。この環状のペンタオリゴペプチド配列は、天然型Ｌアミノ酸から構成されていてもよく、一つ又は複数が非天然型Ｄアミノ酸で置換されて構成されていてもよい。 The oligopeptide derivative is composed of, for example, a cyclic pentaoligopeptide sequence, and the functional group may be protected with a non-amino acid, but another amino acid is not bound to the cyclic oligopeptide sequence. is there. This cyclic pentaoligopeptide sequence may be composed of naturally occurring L amino acids, or may be composed of one or more being substituted with non-naturally occurring D amino acids.
オリゴペプチド誘導体中、環状オリゴペプチド配列は、例えばアルギニン−グリシン−アスパラギン酸−Ｄ−アミノ酸配列を有している。中でも、オリゴペプチド誘導体中、環状オリゴペプチド配列は、環状（アルギニン−グリシン−アスパラギン酸−任意のアミノ酸−リジン）又は環状（アルギニン−グリシン−アスパラギン酸−任意のアミノ酸−バリン）が好ましく、環状（アルギニン−グリシン−アスパラギン酸−Ｄ−アミノ酸−リジン）又は環状（アルギニン−グリシン−アスパラギン酸−Ｄ−アミノ酸−バリン）であると一層好ましい。環状ペンタペプチド配列中の任意のアミノ酸はチロシン又はフェニルアラニンであることが好ましく、中でもＤ−チロシン又はＤ−フェニルアラニンであると一層好ましい。 In the oligopeptide derivative, the cyclic oligopeptide sequence has, for example, an arginine-glycine-aspartic acid-D-amino acid sequence. Among the oligopeptide derivatives, cyclic oligopeptide sequences are preferably cyclic (arginine-glycine-aspartic acid-any amino acid-lysine) or cyclic (arginine-glycine-aspartic acid-any amino acid-valine), cyclic (arginine-arginine) It is more preferable that -glycine-aspartate-D-amino acid-lysine or cyclic (arginine-glycine-aspartate-D-amino acid-valine) is used. Preferably, any amino acid in the cyclic pentapeptide sequence is tyrosine or phenylalanine, more preferably D-tyrosine or D-phenylalanine.
オリゴペプチド誘導体は、放射性同位体で標識された環状（アルギニン−グリシン−アスパラギン酸−Ｄ−アミノ酸−リジン）オリゴペプチド誘導体、又は放射性同位体で標識された環状（アルギニン−グリシン−アスパラギン酸−Ｄ−アミノ酸−バリン）であると、αＶβ３インテグリンへの親和性が高くなる。 The oligopeptide derivative is a cyclic (arginine-glycine-aspartic acid-D-amino acid-lysine) oligopeptide derivative labeled with a radioactive isotope, or a cyclic (arginine-glycine-aspartic acid-D-labeled with a radioactive isotope) The amino acid-valine) has a high affinity for α v β 3 integrin.
オリゴペプチド誘導体は、より具体的には、下記化学式(Ｉ)
化学式(Ｉ)で表わされるオリゴペプチド誘導体の合成法は、例えば非放射性ヨウ素で置換したフェニルアラニン残基を有する環状（アルギニン−グリシン−アスパラギン酸−ヨウ素置換フェニルアラニン−リジン）オリゴペプチドをペプチド固相合成で作製し、次いで非放射性ヨウ素−放射性ヨウ素置換反応、例えばヨウ素基を有機スズで置換しクロラミンＴやＮＣＳのような酸化剤の存在下で放射性ヨウ素イオン(125Ｉ−)又は放射性アスタチンイオン(211Ａｔ−)で置換するというものである。 The synthesis method of the oligopeptide derivative represented by the chemical formula (I) is, for example, a solid phase synthesis of a cyclic (arginine-glycine-aspartic acid-iodine substituted phenylalanine-lysine) oligopeptide having a nonradioactive iodine substituted phenylalanine residue by peptide solid phase synthesis A nonradioactive iodine-radioactive iodine substitution reaction, such as substituting the iodine group with an organotin, in the presence of an oxidizing agent such as chloramine T or NCS, for example, radioactive iodine ion ( 125 I − ) or radioactive astatine ion ( 211 At -) is that substituting.
また、別なオリゴペプチド誘導体は、具体的には、環状（アルギニン−グリシン−アスパラギン酸−チロシン−リジン）オリゴペプチド誘導体、とりわけ下記化学式(II)
化学式(II)で表わされるオリゴペプチド誘導体の合成法は、環状（アルギニン−グリシン−アスパラギン酸−Ｄ−チロシン−リジン）オリゴペプチドをペプチド固相合成で作製し、次いでクロラミンＴ存在下で放射性ヨウ素イオン(125Ｉ−)をチロシン残基の水酸基の隣に導入するというものである。 The synthetic method of the oligopeptide derivative represented by the chemical formula (II) is to prepare cyclic (arginine-glycine-aspartic acid-D-tyrosine-lysine) oligopeptide by peptide solid phase synthesis, and then radioiodine ion in the presence of chloramine T ( 125 I − ) is to be introduced next to the hydroxyl group of a tyrosine residue.
さらに、別なオリゴペプチド誘導体は、具体的には、下記化学式(III)
化学式(III)で表わされるオリゴペプチド誘導体の合成法は、例えば、Ｎ-スクシンイミジル ３-(トリ-n-ブチルスズ)ベンゾエート(N-Succinimidyl 3-(tri-n-butylstannyl)benzoate：ATE)にt-ブチルハイドロパーオキサイド(tert-Butyl hydroperoxide)存在下で放射性ヨウ素イオン(125Ｉ−)を反応させてＮ-スクシンイミジル ３-125ヨードベンゾエート(N-Succinimidyl 3-125iodobenzoate)とし、未置換の環状（アルギニン−グリシン−アスパラギン酸−フェニルアラニン−リジン）オリゴペプチドに反応させて、トリエチルアミン存在下で、放射性ヨウ素標識ベンンゾアミドとして保護されている環状（アルギニン−グリシン−アスパラギン酸−フェニルアラニン−リジン）オリゴペプチド化合物を得るというものである。なお、環状（アルギニン−グリシン−アスパラギン酸−チロシン−リジン）からも同様にして放射性ヨウ素(125Ｉ)置換体を得ることができる。 The synthesis method of the oligopeptide derivative represented by the chemical formula (III) is, for example, t- to N-succinimidyl 3- (tri-n-butyltin) benzoate (N-Succinimidyl 3- (tri-n-butylstannyl) benzoate: ATE) butyl hydroperoxide (tert-butyl hydroperoxide) radioactive iodide ions in the presence of (125 I -) and was reacted N- succinimidyl 3- 125 iodobenzoate (N-succinimidyl 3- 125 iodobenzoate) , unsubstituted cyclic (arginine -Glycine-aspartate-phenylalanine-lysine) oligopeptide is reacted to obtain a cyclic (arginine-glycine-aspartate-phenylalanine-lysine) oligopeptide compound protected as radioiodine-labeled bennzamide in the presence of triethylamine It is a thing. Radioactive iodine ( 125I ) substitution can be obtained from cyclic (arginine-glycine-aspartic acid-tyrosine-lysine) in the same manner.
本発明の医薬は、前記オリゴペプチド誘導体を有効成分として含有するというものである。 The medicament of the present invention contains the oligopeptide derivative as an active ingredient.
この医薬を用いて、上皮成長因子受容体（ＥＧＦＲ）イメージング試験を行うことができる。 This drug can be used to conduct epidermal growth factor receptor (EGFR) imaging studies.
この医薬は、前記オリゴペプチド誘導体をαｖβ３インテグリンに結合し、配位し、及び／又は親和する有効成分として含有し、必要に応じ、非毒性で不活性の薬学的に許容しうる賦形剤、例えば固体状、半固体状もしくは液状の希釈剤、分散剤、充填剤及び担体と混合することにより、製剤化されている。さらに安定剤、保存剤、ｐＨ調整剤、結合剤、崩壊剤、界面活性剤、滑沢剤、流動性促進剤、矯味剤、着色剤、香料防腐剤、媒質、生理食塩水、別な薬効を有する薬剤が添加剤として含まれていてもよい。 This drug contains the oligopeptide derivative as an active ingredient that binds to, coordinates with, and / or interacts with the α v β 3 integrin and, if necessary, a non-toxic inactive pharmaceutically acceptable agent. It is formulated by mixing with excipients, for example, solid, semisolid or liquid diluents, dispersants, fillers and carriers. Stabilizers, preservatives, pH adjusters, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, flavor preservatives, medium, saline, and other medicinal effects The medicine which it has may be included as an additive.
この医薬の剤形は、例えばエリキシル剤、カプセル剤、顆粒剤、丸剤、軟膏、懸濁剤、液剤、腸溶剤、乳剤、硬膏剤、坐剤、散剤、錠剤、シロップ剤、注射剤、トローチ剤、軟膏剤、ハップ剤、リニメント剤、リモナーデ剤、ローション剤が挙げられる。液状媒体に溶解させてもよく懸濁させてもよく、固体状媒体に分散させたものであってもよい。 The dosage form of this medicine is, for example, elixir, capsule, granule, pill, ointment, suspension, solution, enteric agent, emulsion, plaster, suppository, powder, tablet, syrup, injection, troche Agents, ointments, haptics, liniments, limonade agents, lotions. It may be dissolved or suspended in a liquid medium, or may be dispersed in a solid medium.
この医薬は、経口で投与してもよいが、吸収・分布・代謝の観点から静脈注射・点滴で投与することが好ましく、腫瘍組織近傍に直接注入してもよい。 Although this medicine may be administered orally, it is preferable to administer by intravenous injection / instillation from the viewpoint of absorption, distribution and metabolism, and may be directly injected in the vicinity of tumor tissue.
この医薬中、前記オリゴペプチド誘導体は、トレーサ量という薬理作用を示さないごく微量の化学量で標識し、精製後、投与するというものである。 In this medicine, the oligopeptide derivative is labeled with a very small amount of stoichiometry which does not show the pharmacological effect of a tracer amount, and is administered after purification.
この医薬の投与量、用量は、前記オリゴペプチド誘導体である有効成分の有効性、投与の形態・経路、癌の進行ステージ、患者の体型・体重・年齢、併用する他の疾患の治療薬の種類や量に応じ、適宜選択される。その投与は、１〜３０日間隔又は１〜６月間隔で間欠的に投与される。 The dosage and dose of this medicine depend on the efficacy of the active ingredient that is the oligopeptide derivative, the form and route of administration, the stage of cancer progression, the patient's body type, body weight and age, and other therapeutic agents used in combination. It is selected appropriately according to the amount. The administration is administered intermittently at intervals of 1 to 30 days or at intervals of 1 to 6 months.
この医薬は、以下のように使用されて、効能を発揮する。例えば211Ａｔを有するオリゴペプチド誘導体を含有した医薬を、腫瘍を治療すべき患者に投与する。医薬に含有されているオリゴペプチド誘導体が、経口投与又は血中投与により血液等を介して流動し又は腫瘍部位近傍に投与されて、腫瘍部位に到達する。オリゴペプチド誘導体は、環状オリゴペプチド配列の存在によって、腫瘍の癌原発巣の癌細胞に発現し又は腫瘍による血管新生の亢進の際の血管の内皮細胞に発現しているαｖβ３インテグリンにおけるアルギニン−グリシン−アスパラギン酸配列に対する受容体部位に結合し、配位し、及び／又は親和する。その結果、このオリゴペプチド誘導体が、癌細胞や、腫瘍による血管新生の亢進の際の血管内皮細胞に専ら集積して留まる。その際、オリゴペプチド誘導体の放射性同位体211Ａｔがα線を放出する。α線は飛程距離が短く、結合等をした腫瘍組織の癌細胞等を選択的に放射能で破壊してアポトーシスを誘導し死滅させる。しかしα線はその近傍の正常細胞にまで到達できないので正常組織を損傷しない。 This medicine is used as follows to exert its effect. For example, a medicament containing an oligopeptide derivative having 211 At is administered to a patient whose tumor is to be treated. The oligopeptide derivative contained in the drug is flowed through blood or the like by oral administration or blood administration or is administered near the tumor site to reach the tumor site. The oligopeptide derivative is expressed in the α v β 3 integrin expressed in the cancer cells of the primary tumor of the tumor or in the endothelial cells of blood vessels in promoting tumor angiogenesis by the presence of the cyclic oligopeptide sequence. -Bind to, coordinate with, and / or affinity receptor sites for the glycine-aspartate sequence. As a result, this oligopeptide derivative is accumulated exclusively in cancer cells and vascular endothelial cells at the time of tumor-induced angiogenesis. At that time, the radioactive isotope 211 At of the oligopeptide derivative emits alpha rays. The alpha ray has a short range distance, and selectively destroys cancer cells and the like of the tumor tissue which has been bound or the like by radioactivity to induce apoptosis and to be killed. However, alpha rays do not damage normal tissues because they can not reach normal cells in the vicinity.
この医薬を放射線治療に用いる場合を例に説明したが、放射線診断に用いる場合でも、同様に集積するので、放射線診断の際にも癌細胞や、腫瘍による血管新生の亢進の際の血管内皮細胞を検知することができるが、その近傍の正常組織を損傷しない。 Although the case where this medicine is used for radiation therapy has been described as an example, since it is accumulated similarly even when used for radiodiagnosis, cancer cells and vascular endothelial cells at the time of promotion of angiogenesis by tumors are also accumulated at the time of radiodiagnosis But can not damage normal tissue in the vicinity.
以下に、本発明のオリゴペプチド誘導体を合成し、医薬としての有用性について、実証した例を示す。 The following is an example of synthesizing the oligopeptide derivative of the present invention and demonstrating its utility as a pharmaceutical.
用いた試薬、及び機器は以下の通りである。
（試薬・機器）
ペプチド固相合成に必要な試薬である2-クロロトリチル クロライド レジン(2-chlorotrityl chloride resin)、Fmoc-Arg(Pbf)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Gly-OH、Fmoc-D-Tyr(tBu)-OH、Fmoc-D-Phe-OHは渡辺化学工業株式会社(東京, 日本)、Fmoc-Lys(Boc)-OH、ヒト精製αvβ3インテグリン、薄層クロマトグラフィー (TLC Silica gel 60F254)はMerck (Darmstadt, ドイツ)、1-ヒドロキシベンゾトリアゾールハイドレート)1-hydroxybenzotriazolehydrate (HOBt)、1,3-diisopropylcarbodiimide (DIPCDI)は国産化学株式会社(東京, 日本)、N,N-ジメチルホルムアミド(N,N-dimethylformamide)(DMF)は和光純薬工業株式会社(大阪, 日本)、[125I]NaIは、Perkin Elmer (Waltham, MA, 米国)より購入し、その他試薬はナカライテスク(京都, 日本)より購入し、試薬はすべて特級試薬を用いた。211At-は国立大学法人大阪大学より供給を受けた。U87MG (ヒトグリオーマ細胞株)はDSファーマバイオメディカル株式会社(大阪, 日本)より購入した。逆相HPLCについて、Comosil 5C18-AR-II (4.6 × 150 mm)、Comosil 5C18-AR-II (10 × 150 mm)カラムはナカライテスク株式会社(京都, 日本)、送液ユニットLC-20AD及びLC-20AR、カラムオーブンCTO-10A及びCTO-20A、吸光度検出器のSPD-10A及びSPD-20Aは株式会社島津製作所(京都, 日本) の製品を用い、エレクトロンスプレーイオン化質量測定装置(ESI-MS)のJMS-T100TDは日本電子株式会社(JEOL) (東京, 日本)、放射線測定装置オートウェルガンマシステムのAccuFLEX gamma ARC-7010は株式会社日立ヘルスケア・マニュファクチャリング(柏, 日本)、及びWallac Wizard 1470 Gamma Counter はPerkin Elmer (Waltham, MA, 米国)を用いた。
The reagents and instruments used are as follows.
(Reagents and equipment)
Peptide solid phase is a reagent necessary for the synthesis of 2-chlorotrityl chloride resin (2-chlorotrityl chloride resin), Fmoc-Arg (Pbf) -OH, Fmoc-Asp (O t Bu) -OH, Fmoc-Gly-OH, Fmoc-D-Tyr (t Bu ) -OH, Fmoc-D-Phe-OH Watanabe chemical industry Co., Ltd. (Tokyo, Japan), Fmoc-Lys (Boc) -OH, human purified α v β 3 integrin, a thin layer Chromatography (TLC Silica gel 60F 254 ) is Merck (Darmstadt, Germany), 1-hydroxybenzotriazole hydrate) 1-hydroxybenzotriazole hydrate (HOBt), 1,3-diisopropylcarbodiimide (DIPCDI) is a domestic chemical corporation (Tokyo, Japan) N, N-dimethylformamide (DMF) is purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), [ 125 I] NaI is purchased from Perkin Elmer (Waltham, MA, USA), Other reagents were purchased from Nacalai Tesque (Kyoto, Japan), and all reagents used were special grade reagents. 211 At - was supplied by the national university corporation Osaka University. U87MG (human glioma cell line) was purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). About reverse phase HPLC, Comosil 5C 18 -AR-II (4.6 x 150 mm), Comosil 5C 18 -AR-II (10 x 150 mm) column is Nacalai Tesque, Inc. (Kyoto, Japan), liquid transfer unit LC-20 AD And LC-20AR, column ovens CTO-10A and CTO-20A, and SPD-10A and SPD-20A of absorbance detectors manufactured by Shimadzu Corporation (Kyoto, Japan) using an electron spray ionization mass spectrometer (ESI- MS) JMS-T100TD from Nippon Denshi Co., Ltd. (JEOL) (Tokyo, Japan), AccuFLEX gamma ARC-7010 for radiation measurement system autowell gamma system from Hitachi Healthcare Manufacturing Co., Ltd. (柏, Japan), and The Wallac Wizard 1470 Gamma Counter used Perkin Elmer (Waltham, MA, USA).
（実施例１：化学式(Ｉ)タイプのオリゴペプチド誘導体）
前記化学式(Ｉ)で表わされるオリゴペプチド誘導体は、下記化学反応式[１]〜[４]に示す方法で合成した。また得られたオリゴペプチド誘導体は、有効性試験に用いた。その合成の詳細は以下の通りである。
Example 1: Oligopeptide Derivative of the Formula (I) Type
The oligopeptide derivative represented by the above chemical formula (I) was synthesized by the method shown in the following chemical reaction formulas [1] to [4]. Moreover, the oligopeptide derivative obtained was used for the efficacy test. The details of the synthesis are as follows.
Fmoc-4-ヨード-D-フェニルアラニン（Fmoc-4-iodo-D-phenylalanine）(1)の合成
既存の方法に従い、化学反応式[１]に示すように、Ｄ−フェニルアラニンにI2及びNaIO3を反応させヨード-D-フェニルアラニン(D-Phe(4-I)-OH)とした後、Fmoc-OSuをK2CO3存在下で反応させることにより、Fmoc-4-ヨード-D-フェニルアラニン(1)(Fmoc-D-Phe(4-I)-OH)を得た。その理化学分析結果は、DART-MS (m/z calcd for ([M+H]+): 514.04 found: 514.04)であり、化合物(1)の構造を支持する。
Synthesis of Fmoc-4-iodo-D-phenylalanine (Fmoc-4-iodo-D-phenylalanine) (1) According to the existing method, as shown in the chemical reaction formula [1], I 2 and NaIO 3 for D-phenylalanine To give iodo-D-phenylalanine (D-Phe (4-I) -OH) and then reacting Fmoc-OSu in the presence of K 2 CO 3 to give Fmoc-4-iodo-D-phenylalanine ( 1) (Fmoc-D-Phe (4-I) -OH) was obtained. The physicochemical analysis result is DART-MS (m / z calcd for ([M + H] + ): 514.04 found: 514.04), which supports the structure of compound (1).
c[Arg(Pbf)-Gly-Asp(OtBu)-4-iodo-D-Phe-Lys(Boc)] (2)の合成
化合物１を用いて、化学反応式[２]に示すように、通常のFmoc固相合成法に従いレジン結合ペプチドとFmocアミノ酸とをN,N-ジイソプロピルカルボジイミドとHOBt存在下、N,N-ジメチルホルムアミド(DMF)中で攪拌することにより結合させ、ピペリジンによりFmoc基を脱保護する操作を繰り返すことによりペプチド鎖を伸長させて、同式に示す固相支持体樹脂（黒丸）に結合した鎖状のオリゴペプチドとし、次いで30%ヘキサフルオロイソプロパノール(HFIP)で固相支持体樹脂から切り出してH2N-Asp(OtBu)-D-Phe-Lys(Boc)-Arg(Pbf)-Gly-OHとし、ジフェニルリン酸アジド(DPPA)及びNaHCO3存在下、c[Arg(Pbf)-Gly-Asp(OtBu)-4-iodo-D-Phe-Lys(Boc)] (2)を合成した。逆相HPLCによる精製を行い、白色粉末の化合物(2)(63.8 mg, 37.4 %)を得た。逆相HPLCにはCosmosil 5C18-AR-II (10 × 150 mm)カラムを使用し、移動相は0.1 %テトラフルオロ酢酸(TFA)を含有する水:メタノール(20:80)から、10分間で(10:90)に変換するグラジエント法にてUV = 220 nm、流速4.0 mL/minの条件で行った。その理化学分析結果は、ESI-MS (m/z calcd for ([M+H]+): 1138.41 found: 1138.66)であり、化合物(2)の構造を支持する。
c with Compound 1 [Arg (Pbf) -Gly-Asp (O t Bu) -4-iodo-D-Phe-Lys (Boc)] (2), as shown in reaction formula [2] The resin-bound peptide and Fmoc amino acid are combined by stirring in N, N-dimethylformamide (DMF) in the presence of N, N-diisopropylcarbodiimide and HOBt according to a conventional Fmoc solid phase synthesis method, and the Fmoc group is obtained by piperidine The peptide chain is extended by repeating the procedure of deprotecting it to form a linear oligopeptide bound to the solid support resin (black circle) shown by the same formula, followed by solid phase with 30% hexafluoroisopropanol (HFIP) Cleavage from the support resin to H 2 N-Asp (O t Bu) -D-Phe-Lys (Boc) -Arg (Pbf) -Gly-OH in the presence of diphenyl phosphate azide (DPPA) and NaHCO 3 c was synthesized [Arg (Pbf) -Gly-Asp (O t Bu) -4-iodo-D-Phe-Lys (Boc)] (2). Purification by reverse phase HPLC gave the compound (2) (63.8 mg, 37.4%) as a white powder. Reverse phase HPLC using a Cosmosil 5C 18 -AR-II (10 x 150 mm) column, mobile phase from water: methanol (20:80) containing 0.1% tetrafluoroacetic acid (TFA) over 10 minutes It was carried out under the conditions of UV = 220 nm and a flow rate of 4.0 mL / min by the gradient method of conversion to (10:90). The physicochemical analysis result is ESI-MS (m / z calcd for ([M + H] + ): 1138.41 found: 1138.66), which supports the structure of compound (2).
I-c(Arg-Gly-Asp-D-Phe-Lys) (3)の合成
化学反応式[４]に示すように、化合物(2)(10mg: 8.79μmol)をTFA:水:トリイソプロピルシラン((i-Pr)3SiH)(38:1:1, 500 μL)の混合溶液に溶解し、室温にて2時間撹拌を行った。溶媒を窒素ガスにより留去し、残留物を逆相HPLCにて精製し、白色粉末の化合物I-c(Arg-Gly-Asp-D-Phe-Lys) (3) (6.1 mg, 95.2%)を得た。逆相HPLCにはCosmosil 5C18-AR-II (10 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(70:30)から、10分間で(50:50)に変換するグラジエント法にてUV = 220 nm、流速4.0 mL/minの条件で行った。その理化学分析結果は、ESI-MS (m/z calcd for ([M+H]+): 730.21 found: 730.18)であり、化合物(3)の構造を支持する。
Synthesis of Ic (Arg-Gly-Asp-D-Phe-Lys) (3) As shown in the chemical reaction formula [4], compound (2) (10 mg: 8.79 μmol) was dissolved in TFA: water: triisopropylsilane ((( It was dissolved in a mixed solution of i-Pr) 3 SiH) (38: 1: 1, 500 μL) and stirred at room temperature for 2 hours. The solvent was distilled off with nitrogen gas, and the residue was purified by reverse phase HPLC to obtain Compound Ic (Arg-Gly-Asp-D-Phe-Lys) (3) (6.1 mg, 95.2%) as a white powder. The Reverse phase HPLC uses a Cosmosil 5C 18 -AR-II (10 x 150 mm) column and the mobile phase is from 50: 50 in 10 minutes from water: methanol (70:30) containing 0.1% TFA. It carried out on the conditions of UV = 220 nm and the flow rate of 4.0 mL / min by the gradient method converted to (iii). Its physicochemical analysis result is ESI-MS (m / z calcd for ([M + H] + ): 730.21 found: 730.18), which supports the structure of compound (3).
Sn(nBu)3-c[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(Boc)] (4)の合成
化学反応式[３]に示すように、化合物(2) (5.6mg 4.9μmol)を、無水1,4-ジオキサン (400μL)に溶解し、ビス(トリブチルスズ([(nBu)3Sn]2) (5.96μL: 2.4当量)、及びビス(トリフェニルホスフィン)パラジウム(II) ジクロリド(PdCl2[P(C6H5)3]2)(517.26μL: 0.15当量)を加え、窒素雰囲気下、60℃に加熱しながら2時間撹拌後、逆相HPLCによる精製を行い、白色粉末のSn(nBu)3-c[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(Boc)] (4) (512μg, 8.00%)を得た。逆相HPLCにはCosmosil 5C18-AR-II (10 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(5:95)から、10分間で(2.5:97.5)に変換するグラジエント法にてUV = 220 nm、流速4.0 mL/minの条件で行った。その理化学分析結果は、ESI-MS (m/z calcd for ([M+H]+): 1302.62 found: 1302.62)であり、化合物(4)の構造を支持する。
Sn (nBu) 3 -c, as shown in [Arg (Pbf) -Gly-Asp (O t Bu) -D-Phe-Lys (Boc)] Synthesis reaction formula (4) [3], compound (2 ) (5.6 mg 4.9 μmol) is dissolved in
Sn(nBu)3-c(Arg-Gly-Asp-D-Phe-Lys) (5)の合成
化学反応式[４]に示すように、化合物(3)(2.0 mg 2.74 μmol)を、無水メタノール(300 μL)に溶解し、N,N-ジイソプロピルメチルアミン(DIEA, 2.5当量)、及びビス(トリブチルスズ) ([(nBu)3Sn]2)(5.82 μL, 4.0当量)を加え、窒素ガスを吹きかけた。その後、トリス(ジベンジリデンアセトン)ジパラジウム(0)(Pd2(dba)3)(384.6 μg, 0.15 当量)を加え、窒素雰囲気下、70℃に加熱しながら12時間撹拌後、逆相HPLCによる精製を行い、白色粉末のSn(nBu)3-c(Arg-Gly-Asp-D-Phe-Lys) (5) (839.7μg, 34.3%)を得た。逆相HPLCにはCosmosil 5C18-AR-II (10 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(30:70)から、10分間で(25:75)に変換するグラジエント法にてUV = 220 nm、流速4.0 mL/minの条件で行った。その理化学分析結果は、ESI-MS (m/z calcd for ([M+H]+): 894.42 found: 894.44)であり、化合物(5)の構造を支持する。
Synthesis of Sn (nBu) 3 -c (Arg-Gly-Asp-D-Phe-Lys) (5) As shown in the chemical reaction formula [4], Compound (3) (2.0 mg 2.74 μmol) was dissolved in anhydrous methanol Dissolve in (300 μL), add N, N-diisopropylmethylamine (DIEA, 2.5 equivalents), and bis (tributyltin) ([(nBu) 3 Sn] 2 ) (5.82 μL, 4.0 equivalents), and add nitrogen gas. I sprayed it. Thereafter, tris (dibenzylideneacetone) dipalladium (0) (Pd 2 (dba) 3 ) (384.6 μg, 0.15 equivalent) is added, and after stirring for 12 hours while heating to 70 ° C. under nitrogen atmosphere, reversed phase HPLC Purification was performed to obtain Sn (nBu) 3 -c (Arg-Gly-Asp-D-Phe-Lys) (5) (839.7 μg, 34.3%) as white powder. Reversed phase HPLC uses a Cosmosil 5C 18 -AR-II (10 x 150 mm) column and the mobile phase is from water: methanol (30:70) containing 0.1% TFA for 10 minutes (25:75) It carried out on the conditions of UV = 220 nm and the flow rate of 4.0 mL / min by the gradient method converted to (iii). The physicochemical analysis result is ESI-MS (m / z calcd for ([M + H] + ): 894.42 found: 894.44), which supports the structure of compound (5).
125I-c[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(Boc)] (6)の作製
化学反応式[３]に示すように、化合物(4) (30μg, 23.0nmol)をエタノール(79 μL)に溶解し、クロラミンT/エタノール溶液(1 mg/mL, 15 μL)、1%酢酸/エタノール溶液(21 μL)、及び[125I]NaI (3.7 MBq, 3 μL)を加えて室温にて10分間放置し、逆相HPLCにて精製することで125I-c[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(Boc)] (6) (放射化学的収率: 82.9%、放射化学的純度: 96.6%)を得た。逆相HPLCにはCosmosil 5C18-AR-II (4.6 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(20:80)から20分間で(0:100)に変換するグラジエント法にて、流速1.0 mL/minの条件で行った。
125 Ic as shown in [Arg (Pbf) -Gly-Asp (O t Bu) -D-Phe-Lys (Boc)] Preparation chemical equation (6) [3], compound (4) (30 [mu] g, 23.0 nmol) is dissolved in ethanol (79 μL), chloramine T / ethanol solution (1 mg / mL, 15 μL), 1% acetic acid / ethanol solution (21 μL), and [ 125 I] NaI (3.7 MBq, 3 μL) ) was added and left at room temperature for 10 minutes, 125 Ic [Arg (Pbf) -Gly-Asp (O t Bu) -D-Phe-Lys (Boc)] by purified by reverse phase HPLC (6) (Radiochemical yield: 82.9%, radiochemical purity: 96.6%) was obtained. Reversed phase HPLC uses a Cosmosil 5C 18 -AR-II (4.6 x 150 mm) column and the mobile phase is 0.1% TFA in water: methanol (20:80) over 20 minutes (0: 100) The reaction was carried out under the condition of a flow rate of 1.0 mL / min by the gradient method of converting into
125I-c(Arg-Gly-Asp-D-Phe-Lys) (7)の作製（化合物(6)使用）
化学反応式[３]に示すように、化合物(6)をTFA:水:トリイソプロピルシラン(38:1:1, 80 μL)の混合溶液に溶解し、室温にて90分間撹拌を行った。溶媒を窒素ガスにより留去し、残留物を逆相HPLCにて精製し、125I-c(Arg-Gly-Asp-D-Phe-Lys) (7) (放射化学的収率: 75.1%、放射化学的純度: 97.0%。以下、125I-c(RGDfK)と略記することがある)を得た。逆相HPLCにはCosmosil 5C18-AR-II (4.6 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(70:30)から20分間で(30:70)に変換するグラジエント法にて、流速1.0 mL/minの条件で行った。この化合物(7)と、放射性同位体非標識でヨウ素置換体である化合物(3)であるI-c(Arg-Gly-Asp-D-Phe-Lys) (3)との逆相HPLCによる分析と比較したところ、同じリテンションタイムに夫々単一の溶出ピークが検出され、化合物(7)であることが確認された。
Preparation of 125 Ic (Arg-Gly-Asp-D-Phe-Lys) (7) (using Compound (6))
As shown in the chemical reaction formula [3], the compound (6) was dissolved in a mixed solution of TFA: water: triisopropylsilane (38: 1: 1, 80 μL) and stirred at room temperature for 90 minutes. The solvent is distilled off with nitrogen gas, the residue is purified by reverse phase HPLC, and 125 Ic (Arg-Gly-Asp-D-Phe-Lys) (7) (radiochemical yield: 75.1%, radiochemistry Target purity: 97.0% (hereinafter sometimes abbreviated as 125 Ic (RGDfK)). Reverse phase HPLC using a Cosmosil 5C 18 -AR-II (4.6 x 150 mm) column, mobile phase from water: methanol (70:30) containing 0.1% TFA (30:70) for 20 minutes The reaction was carried out under the condition of a flow rate of 1.0 mL / min by the gradient method of converting into Reversed-phase HPLC analysis and comparison of this compound (7) with the radioactive isotope unlabeled and iodine-substituted compound (3) Ic (Arg-Gly-Asp-D-Phe-Lys) (3) As a result, each single elution peak was detected at the same retention time, and it was confirmed to be compound (7).
125I-c(Arg-Gly-Asp-D-Phe-Lys) (7)の作製 (化合物(5)使用)
化学反応式[４]に示すように、化合物(5) (30μg, 33.6 nmol)をアセトニトリル(5 μL)に溶解し、1%酢酸/アセトニトリル溶液(10 μL)、[125I]NaI (3.7 MBq, 3 μL)、及びN-クロロスクシンイミド(NCS)/アセトニトリル溶液(1 mg/mL, 15 μL)を加えて80℃に加熱しながら15分間反応させたのち、亜硫酸水素ナトリウム(NaHSO3)水溶液(1 mg/mL, 15 μL)で反応を停止し、逆相HPLCにて精製することで化合物(7) (放射化学的収率: 83.1%、放射化学的純度: 96.1%)を得た。逆相HPLCにはCosmosil 5C18-AR-II (4.6 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(70:30)から20分間で(30:70)に変換するグラジエント法にて、流速1.0 mL/minの条件で行った。前記と同様に、この化合物(7)と、化合物(3)であるI-c(Arg-Gly-Asp-D-Phe-Lys)との逆相HPLCによる分析と比較したところ、同じリテンションタイムに夫々単一の溶出ピークが検出され、化合物(7)であることが確認された。
Preparation of 125 Ic (Arg-Gly-Asp-D-Phe-Lys) (7) (Use Compound (5))
As shown in the chemical reaction formula [4], the compound (5) (30 μg, 33.6 nmol) is dissolved in acetonitrile (5 μL), 1% acetic acid / acetonitrile solution (10 μL), [ 125 I] NaI (3.7 MBq , 3 μL), and N-chlorosuccinimide (NCS) / acetonitrile solution (1 mg / mL, 15 μL) and react for 15 minutes while heating to 80 ° C., and then an aqueous solution of sodium bisulfite (NaHSO 3 ) (NaHSO 3 ) The reaction was quenched with 1 mg / mL, 15 μL) and purified by reverse phase HPLC to obtain a compound (7) (radiochemical yield: 83.1%, radiochemical purity: 96.1%). Reverse phase HPLC using a Cosmosil 5C 18 -AR-II (4.6 x 150 mm) column, mobile phase from water: methanol (70:30) containing 0.1% TFA (30:70) for 20 minutes The reaction was carried out under the condition of a flow rate of 1.0 mL / min by the gradient method of converting into Similar to the above, when the compound (7) and the compound (3) Ic (Arg-Gly-Asp-D-Phe-Lys) are analyzed by reversed-phase HPLC, the single retention time is identical to the single retention time. One elution peak was detected and confirmed to be compound (7).
211At-c[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(Boc)] (8)及び211At-c(Arg-Gly-Asp-D-Phe-Lys) (9)の作製
化学反応式[３]に示すように、化合物(4) (30μg, 23.0 nmol)をアセトニトリル(5 μL)に溶解し、1%酢酸/アセトニトリル溶液(10 μL)、[211At]At-/H2O (3.7 MBq, 3 μL)、及びN-クロロスクシンイミド (NCS)/アセトニトリル溶液(1 mg/mL, 15 μL)を加えて80℃に加熱しながら15分間反応させたのち、亜硫酸水素ナトリウム(NaHSO3)水溶液(1 mg/mL, 15 μL)で反応を停止することで211At-c[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(Boc)] (8) (放射化学的収率: 91.2%)を得た。その後、溶媒を窒素ガスにより留去し、残留物をTFA:水:トリイソプロピルシラン(38:1:1, 80 μL)の混合溶液に溶解し、室温にて10分間振とうした。溶媒を窒素ガスにより留去後、残留物を逆相HPLCにて精製し、211At-c(Arg-Gly-Asp-D-Phe-Lys) (9) (放射化学的収率: 33.3%、放射化学的純度: 97.7%。以下、211At-c(RGDfK)と略記することがある)を得た。逆相HPLCにはCosmosil 5C18-AR-II (4.6 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(70:30)にて、流速1.0 mL/minの条件で行った。この化合物(9)と、放射性同位体非標識でヨウ素置換体である化合物(3)であるI-c(Arg-Gly-Asp-D-Phe-Lys)との逆相HPLCによる分析と比較したところ、リテンションタイムが近いところに、夫々単一の溶出ピークが検出され、化合物(9)であることが確認された。
211 At-c [Arg (Pbf ) -Gly-Asp (O t Bu) -D-Phe-Lys (Boc)] (8) and 211 At-c (Arg-Gly -Asp-D-Phe-Lys) ( Preparation of 9) As shown in the chemical reaction formula [3], compound (4) (30 μg, 23.0 nmol) is dissolved in acetonitrile (5 μL), 1% acetic acid / acetonitrile solution (10 μL), [ 211 At] After adding At − / H 2 O (3.7 MBq, 3 μL) and N-chlorosuccinimide (NCS) / acetonitrile solution (1 mg / mL, 15 μL) and reacting for 15 minutes while heating to 80 ° C. sodium bisulfite (NaHSO 3) solution (1 mg / mL, 15 μL ) by stopping the reaction at 211 at-c [Arg (Pbf ) -Gly-Asp (O t Bu) -D-Phe-Lys (Boc ] (8) (radiochemical yield: 91.2%) was obtained. After that, the solvent was distilled off with nitrogen gas, the residue was dissolved in a mixed solution of TFA: water: triisopropylsilane (38: 1: 1, 80 μL), and shaken at room temperature for 10 minutes. After distilling off the solvent with nitrogen gas, the residue is purified by reverse phase HPLC, and 211 At-c (Arg-Gly-Asp-D-Phe-Lys) (9) (radiochemical yield: 33.3%, Radiochemical purity: 97.7% (hereinafter sometimes abbreviated as 211 At-c (RGDfK)). Reversed phase HPLC uses a Cosmosil 5C 18 -AR-II (4.6 x 150 mm) column, the mobile phase is water: methanol (70:30) containing 0.1% TFA, and the flow rate is 1.0 mL / min. It went on condition. Comparing this compound (9) with the radioactive isotope unlabeled and iodine-substituted compound (3) Ic (Arg-Gly-Asp-D-Phe-Lys) by reversed-phase HPLC, A single elution peak was detected near the retention time, and it was confirmed that this was a compound (9).
211At-c(Arg-Gly-Asp-D-Phe-Lys) (9)の作製 (化合物5使用)
化学反応式[４]に示すように、化合物(5) (30μg, 33.6 nmol)をアセトニトリル(5 μL)に溶解し、1%酢酸/アセトニトリル溶液(10 μL)、[211I]At-/H2O (7.4 MBq, 100 μL)、及びN-chlorosuccinimide (NCS)/アセトニトリル溶液(1 mg/mL, 15 μL)を加えて80℃に加熱しながら15分間反応させたのち、亜硫酸水素ナトリウム(NaHSO3)水溶液(1 mg/mL, 15 μL)で反応を停止し、逆相HPLCにて精製することで211At-c(Arg-Gly-Asp-D-Phe-Lys) (9) (放射化学的収率: 50.1%、放射化学的純度: 96.9%)を得た。逆相HPLCにはCosmosil 5C18-AR-II (4.6 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(70:30)にて、流速1.0 mL/minの条件で行った。前記と同様に、この化合物(9)と、前記の化合物(3)であるI-c(Arg-Gly-Asp-D-Phe-Lys) (3)との逆相HPLCによる分析と比較したところ、リテンションタイムが近いところに、夫々単一の溶出ピークが検出され、化合物(9)であることが確認された。
Preparation of 211 At-c (Arg-Gly-Asp-D-Phe-Lys) (9) (using Compound 5)
Compound (5) (30 μg, 33.6 nmol) is dissolved in acetonitrile (5 μL) as shown in the chemical reaction formula [4], 1% acetic acid / acetonitrile solution (10 μL), [ 211 I] At − / H After adding 2 O (7.4 MBq, 100 μL) and N-chlorosuccinamide (NCS) / acetonitrile solution (1 mg / mL, 15 μL) and reacting for 15 minutes while heating to 80 ° C., sodium bisulfite (NaHSO 4) 3 ) The reaction is quenched with an aqueous solution (1 mg / mL, 15 μL) and purified by reverse phase HPLC to give 211 At-c (Arg-Gly-Asp-D-Phe-Lys) (9) (radiochemistry) Target yield: 50.1%, radiochemical purity: 96.9%). Reversed phase HPLC uses a Cosmosil 5C 18 -AR-II (4.6 x 150 mm) column, the mobile phase is water: methanol (70:30) containing 0.1% TFA, and the flow rate is 1.0 mL / min. It went on condition. Similar to the above, when compared with the analysis by reversed phase HPLC of this compound (9) and Ic (Arg-Gly-Asp-D-Phe-Lys) (3) which is the above-mentioned compound (3), retention At times where the times were close, single elution peaks were detected, respectively, and it was confirmed that this was compound (9).
（比較例１-1：化学式(Ｉ)タイプの放射性同位体非標識のオリゴペプチド誘導体）
化学式(Ｉ)タイプに対する放射性同位体非標識のオリゴペプチド誘導体である下記化学式
なお、実施例１で合成した211Ａｔで標識した誘導体（化合物(9)）と比較例１-1における非標識のＩで置換した誘導体（化合物(3)）との逆相HPLCによる分析にはCosmosil 5C18-AR-II (4.6 × 150 mm)カラムを使用し、標識した誘導体に対し放射性強度と非標識の誘導体に対し220nm紫外線とで検出し、移動相は0.1%のTFAを含有する水:メタノール(70:30)、流速1.0 mL/minの条件で行ったところ、211Ａｔで標識した誘導体の方がやや遅いリテンションタイムに、夫々単一の溶出ピークが現れ、高い純度であることが示された。
Comparative Example 1-1: Radioisotope-Unlabeled Oligopeptide Derivative of the Formula (I) Type
A radioisotopic unlabeled oligopeptide derivative for the type of formula (I) type
For analysis of the 211 At-labeled derivative (compound (9)) synthesized in Example 1 and the unlabeled I-substituted derivative (compound (3)) in Comparative Example 1-1 by reverse-phase HPLC Using a Cosmosil 5C 18 -AR-II (4.6 x 150 mm) column, the mobile phase is detected with radioactive intensity for labeled derivatives and 220 nm UV for unlabeled derivatives, the mobile phase is water containing 0.1% TFA : When conducted under conditions of methanol (70:30) and a flow rate of 1.0 mL / min, a single elution peak appears at a slightly slower retention time for the derivative labeled with 211 At, and the purity is high Indicated.
（比較例１-2：化学式(Ｉ)タイプのハロゲン非置換のオリゴペプチド誘導体）
化学式(Ｉ)タイプに対するハロゲン非置換のオリゴペプチド誘導体であるc(Arg-Gly-Asp-D-Phe-Lys) (10) (以下、c(RGDfK)と略記することがある)は、実施例１での原料としてヨウ素非置換のＤ−フェニルアラニンを用いたこと以外は、実施例１の方法に準じ、以下のようにして合成した。
The halogen-unsubstituted oligopeptide derivative c (Arg-Gly-Asp-D-Phe-Lys) (10) (hereinafter sometimes abbreviated as c (RGDfK)) to the chemical formula (I) type is an example It synthesize | combined as follows according to the method of Example 1 except having used iodine non-substituted D-phenylalanine as a raw material in 1.
（実施例２：化学式(II)タイプのオリゴペプチド誘導体）
前記化学式(II)で表わされるオリゴペプチド誘導体は、実施例１での原料としてヨウ素置換のＤ−フェニルアラニンに代えてＤ−チロシンを用いたいこと以外は、実施例１の方法に準じて後述の化合物(13)を合成し、下記化学式
The oligopeptide derivative represented by the above chemical formula (II) is a compound described later according to the method of Example 1 except that it is desired to use D-tyrosine instead of iodine-substituted D-phenylalanine as a raw material in Example 1 (13) is synthesized, the following chemical formula
（比較例２-1：化学式(II)タイプの非放射性のハロゲン置換のオリゴペプチド誘導体）
化学式(II)タイプに対する非放射性のハロゲン置換のオリゴペプチド誘導体である下記化学式
A nonradioactive halogen-substituted oligopeptide derivative to the formula (II) type:
（比較例２-2：化学式(II)タイプのハロゲン非置換のオリゴペプチド誘導体）
化学式(II)タイプに対するハロゲン非置換のオリゴペプチド誘導体である下記化学式
The following chemical formula, which is a non-halogenated oligopeptide derivative to the chemical formula (II) type:
（実施例３：化学式(III)タイプのオリゴペプチド誘導体）
前記化学式(III)で表わされるオリゴペプチド誘導体は、比較例１-2で得た化合物(10)(c(RGDfK))を用い、下記化学反応式[５]
ATE (0.5 mg, 0.98 μmol)をエタノール(55 μL)に溶解し、クロラミンT/エタノール溶液(4 mg/mL, 15 μL)、1%酢酸/エタノール溶液(21 μL)、及び[125I]NaI (3.7 MBq, 24 μL)を加えて室温にて10分間放置し、溶媒を、窒素ガスにより約50 μL程度に濃縮後、水(50 μL)を加え、逆相HPLCを用いて精製し、[125I]SIBを得た。逆相HPLCにはCosmosil 5C18-AR-II (4.6 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(50:50)から20分間で(30:70)に変換するグラジエント法にて、流速1.0 mL/minの条件で行った。[125I]SIBを含む溶液を、50℃に加熱しながら、窒素ガスにて溶媒を完全に留去し、DMF (50 μL)に溶解後、c(RGDfK) (50 μg, 68 nmol) に加え、TEA (0.5 μL)を加えて室温にて40分インキュベートを行った後に逆相HPLCにより精製を行い、化合物(14) (放射化学的収率: 30%、放射化学的純度: 99%)を得た。逆相HPLCにはCosmosil 5C18-AR-II (4.6 × 150 mm)カラムを使用し、移動相は0.1%のTFAを含有する水:メタノール(60:40)から20分間で(10:90)に変換するグラジエント法にて、流速1.0 mL/minの条件で行った。
なお、実施例３で合成した標識した誘導体(化合物(14))と後述する比較例３で合成した非標識の誘導体(化合物(15))との逆相HPLCによる分析にはCosmosil 5C18-AR-II (4.6 × 150 mm)カラムを使用し、標識した誘導体に対し放射性強度と非標識の誘導体に対し220nm紫外線とで検出し、移動相は0.1%のTFAを含有する水と0.1%のTFAを含有するメタノールとの60:40〜10:90での0〜20minのグラジエントにて、流速1.0 mL/minの条件で行ったところ、同じリテンションタイムに夫々単一の溶出ピークが現れ、標識／非標識以外は同一であることが示された。
Example 3 Oligopeptide Derivative of the Formula (III) Type
The oligopeptide derivative represented by the above chemical formula (III) is a compound represented by the following chemical reaction formula [5], using the compound (10) (c (RGDfK)) obtained in Comparative Example 1-2.
ATE (0.5 mg, 0.98 μmol) is dissolved in ethanol (55 μL), chloramine T / ethanol solution (4 mg / mL, 15 μL), 1% acetic acid / ethanol solution (21 μL), and [ 125 I] NaI Add (3.7 MBq, 24 μL) and leave at room temperature for 10 minutes. Concentrate the solvent to about 50 μL with nitrogen gas, add water (50 μL), and purify using reverse phase HPLC [ < 125 > I] SIB was obtained. Reverse phase HPLC using a Cosmosil 5C 18 -AR-II (4.6 x 150 mm) column, mobile phase from water: methanol (50:50) containing 0.1% TFA (30:70) for 20 minutes The reaction was carried out under the condition of a flow rate of 1.0 mL / min by the gradient method of converting into The solution containing [ 125 I] SIB was completely distilled off with nitrogen gas while heating to 50 ° C., dissolved in DMF (50 μL), and then dissolved in c (RGDfK) (50 μg, 68 nmol) In addition, TEA (0.5 μL) was added and incubation was carried out at room temperature for 40 minutes, followed by purification by reverse phase HPLC to obtain compound (14) (radiochemical yield: 30%, radiochemical purity: 99%) I got Reverse phase HPLC using a Cosmosil 5C 18 -AR-II (4.6 x 150 mm) column, mobile phase from water: methanol (60:40) containing 0.1% TFA for 20 minutes (10:90) The reaction was carried out under the condition of a flow rate of 1.0 mL / min by the gradient method of converting into
In addition, Cosmosil 5C 18 -AR is used for analysis of the labeled derivative (compound (14)) synthesized in Example 3 and the unlabeled derivative (compound (15)) synthesized in Comparative Example 3 described later by reverse phase HPLC. Using a -II (4.6 x 150 mm) column and detecting radioactive intensity for labeled derivatives and 220 nm UV for unlabeled derivatives, the mobile phase is water containing 0.1% TFA and 0.1% TFA And a gradient of 0 to 20 min at 60: 40 to 10: 90 with methanol containing a water at a flow rate of 1.0 mL / min, each single elution peak appears at the same retention time, and It was shown to be identical except for unlabeled.
（比較例３：化学式(III)タイプの放射性同位体非標識のオリゴペプチド誘導体）
化学式(III)タイプに対する放射性同位体非標識のオリゴペプチド誘導体である下記化学式
ESI-MS (m/z calcd for ([M+H]+): 834 found: 834)であり、化合物(15)の構造を支持する。
Comparative Example 3 Radioisotope-Unlabeled Oligopeptide Derivative of the Formula (III) Type
The following chemical formula is a radioisotopic unlabeled oligopeptide derivative to the chemical formula (III) type
ESI-MS (m / z calcd for ([M + H] + ): 834 found: 834) and supports the structure of compound (15).
以下に、本発明を適用するオリゴペプチド誘導体及び本発明を適用外のオリゴペプチド誘導体を評価した方法と、その結果を示す。 Below, the method to which the oligopeptide derivative which applied this invention and the oligopeptide derivative which does not apply this invention were evaluated, and the result are shown.
（評価試験１：αvβ3 インテグリンに対するin vitroでの結合アッセイ(Binding Assay)）
αvβ3インテグリンとの親和性の評価は過去に報告された方法（Dijkgraaf Iら，“Improved targeting of the αvβ3 integrin by multimerisation of RGD peptides.” EJNMMI.， 2007年, 第34巻第2号，p.267-273）をわずかに変更し、以下のように行った。ヒト精製αvβ3インテグリン(200 μg/mL)を600 ng/mLとなるようにcoating buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2 0.5 mM MgCl2, 1 mM MnCl2, pH 7.4)に溶解し、96穴イムノロックウェルモジュールプレート (Thermo, Waltham, MA, 米国)に100 μLずつ加え、4℃で一晩放置させた。その後、ウェルをbinding buffer [0.1% 牛血清アルブミン(BSA) / coating buffer, 200 μL]で2度洗浄した後blocking buffer (1% BSA / coating buffer, 200 μL)を加え、2時間室温にて放置後、binding buffer (200 μL)で2度洗浄した。次に放射性リガンドとして125I-c(RGDyK)、非放射性リガンドとしてI-c(RGDfK)、c(RGDfK)を用い、また、放射性リガンドとして125I-c(RGDfK)、211At-c(RGDfK)、非放射性リガンドとしてc(RGDfK)を用い、ウェルに放射性リガンド(37 kBq, 95 μL)、及び非放射性リガンド(2 nM - 2 mM, 5 μL)を加え、37℃にて1時間振とうを行った後、ウェルをbinding buffer (200 μL, 300 μL, 350 μL、合計3度)を用いて洗浄し、ウェルの放射能を測定した。
(Evaluation test 1: In vitro binding assay for α v β 3 integrin (Binding Assay))
Evaluation of affinity for α v β 3 integrin has been reported in the past (Dijkgraaf I et al., “Improved targeting of the αv β3 integral by multimerisation of RGD peptides.” EJNMMI., 2007, Vol. 34, No. 2, We changed p. 267-273) slightly and carried out as follows. Coating buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl 2 0.5 mM MgCl 2 , 1 mM MnCl 2 , pH so that human purified α v β 3 integrin (200 μg / mL) is 600 ng / mL Then, 100 μL of each was added to a 96-well immuno lock well module plate (Thermo, Waltham, Mass., USA) and allowed to stand overnight at 4 ° C. Thereafter, the wells are washed twice with binding buffer [0.1% bovine serum albumin (BSA) / coating buffer, 200 μL], blocking buffer (1% BSA / coating buffer, 200 μL) is added, and left at room temperature for 2 hours. Then, it was washed twice with binding buffer (200 μL). Next, 125 Ic (RGDyK) as the radioligand, Ic (RGDfK) and c (RGDfK) as the nonradioactive ligand, and 125 Ic (RGDfK) and 211 At-c (RGDfK) as the radioligand as the nonradioactive ligand c (RGDfK), radioligand (37 kBq, 95 μL) and non-radioactive ligand (2 nM-2 mM, 5 μL) are added to the wells and shaken at 37 ° C for 1 hour, then the wells Were washed with binding buffer (200 μL, 300 μL, 350 μL, 3 times in total) to measure the radioactivity of the wells.
なお、この評価試験１中、アスタチンに安定同位体が存在しないため、非放射性リガンドを固定して、放射性リガンドを変化させて評価したものである。その結果を、下記表１に示す。
表１から明らかな通り、125I-c(RGDyK)、125I-c(RGDfK)、211At-c(RGDfK)のαｖβ３インテグリンへの親和性を調べたところ、高い親和性を有するという結果が得られた。 As is clear from Table 1, when the affinity of 125 Ic (RGDyK), 125 Ic (RGDfK), and 211 At-c (RGDfK) to α v β 3 integrin was examined, the result of having high affinity was obtained. It was done.
また、この評価試験１と同様にして、安定同位体が存在している核種の場合、放射性リガンドを固定して、コールド体を比較して評価した。その結果を、下記表２に示す。
（評価試験２：担癌モデルマウスにおける体内放射能分布）
U87MGヒトグリオーマ細胞をEMEMメディウムで5% CO2、37℃条件下において培養し、細胞数5×106をヌードマウス[4 weeks, BALB/c Slc-nu/nu, female (15-19 g; Japan SLC)]の背面皮下へ移植した。28日後、125I-c(RGDfK)、及び211At-c(RGDfK)の混合液(37 kBq, 74 kBq/100 μL)を担癌モデルマウスに尾静脈投与した。投与1、4時間後にマウスを屠殺し、各臓器を摘出、臓器の重量と放射能を測定した。211Atの放射能は70-120 keVのエネルギーレンジにて測定を行い、125Iは211Atが減衰した後に1-80 keVのエネルギーレンジで測定した。
(Evaluation test 2: Radioactivity distribution in tumor-bearing model mice)
U87MG human glioma cells are cultured in EMEM medium at 5% CO 2 at 37 ° C., and the cell number is 5 × 10 6 in nude mice [4 weeks, BALB / c Slc-nu / nu, female (15-19 g; Japan SLC)] was implanted subcutaneously on the back of the patient. Twenty-eight days later, a mixed solution (37 kBq, 74 kBq / 100 μL) of 125 Ic (RGDfK) and 211 At-c (RGDfK) was administered to the tumor-bearing model mice via the tail vein. One and four hours after administration, the mice were sacrificed, each organ was removed, and the weight and radioactivity of the organ were measured. 211 radioactivity At performs measurements at the energy range of 70-120 keV, 125 I is 211 At was measured with an energy range of 1-80 keV after attenuation.
その結果を図１に示す。図１から明らかな通り、125I-c(RGDfK)、及び211At-c(RGDfK)の両化合物は腫瘍組織に高く集積していた。また、125I-c(RGDfK)、及び211At-c(RGDfK)の両化合物が類似の体内分布を示すこと、ヨウ素アニオン、アスタチンアニオンの集積臓器として知られている胃への放射能集積が低いことから、生体内で顕著な脱ヨウ素化、脱アスタチン化は起こっていないことが分かった。 The results are shown in FIG. As is clear from FIG. 1, both 125 Ic (RGDfK) and 211 At-c (RGDfK) were highly accumulated in tumor tissue. Further, 125 Ic (RGDfK), and 211 both compounds of At-c (RGDfK) indicate the biodistribution similar, it iodine anion, is radioactive accumulation in the stomach known as integrated organ A statin anion lower From the above, it was found that significant deiodination and deastatination did not occur in vivo.
また、125I-c(RGDyK)と125I-c(RGDfK)、125I-c(RGDyK)、及び125IB-c(RGDfK)については、単独液としたこと以外は同様にして体内放射能分布を測定した。その結果を、夫々図２〜図４に示す。図２から明らかな通り、125I-c(RGDyK)、125I-c(RGDfK)及び125I-c(RGDyK)の三化合物は腫瘍組織に高く集積していた。また、125I-c(RGDyK)、125I-c(RGDfK)及び125I-c(RGDyK)の三化合物が類似の体内分布を示すこと、ヨウ素アニオンの集積臓器として知られている胃への放射能集積が低いことから、生体内で顕著な脱ヨウ素化は起こっていないことが分かった。また、図４から明らかな通り、125IB-c(RGDfK)は腫瘍組織にも腫瘍組織へ幾分低い集積性を示したが、腸への高い集積性(小腸；投与後1時間に54.9%ID/g)を示した。 The radioactivity distribution in the body was similarly measured except that 125 Ic (RGDyK) and 125 Ic (RGDfK), 125 Ic (RGDyK), and 125 IB-c (RGDfK) were used alone. The results are shown in FIGS. 2 to 4 respectively. As apparent from FIG. 2, three compounds of 125 Ic (RGDyK), 125 Ic (RGDfK) and 125 Ic (RGDyK) were highly accumulated in the tumor tissue. Further, 125 Ic (RGDyK), 125 Ic (RGDfK) and three compounds of 125 Ic (RGDyK) indicate the biodistribution similar, that radioactive accumulation in the stomach known as integrated organ iodine anion is low From the results, it was found that significant deiodination did not occur in vivo. Also, as is clear from FIG. 4, 125 IB-c (RGDfK) showed somewhat lower accumulation in tumor tissues as well as in tumor tissues, but high accumulation in the intestine (small intestine; 54.9% one hour after administration ID / g) is shown.
（評価試験３：非放射性リガンドによる阻害試験）
上記と同様に担癌モデルマウスを作製し、c(RGDfK) (0.2 mg/mouse)を125I-c(RGDfK)、及び211At-c(RGDfK)の混合液(37 kBq, 74 kBq/100 μL)と同時に尾静脈投与した。投与1時間後にマウスを屠殺し、各臓器を摘出、臓器の重量と放射能を測定した。211Atの放射能は70-120 keVのエネルギーレンジにて測定を行い、125Iは211Atが減衰した後に1-80 keVのエネルギーレンジで測定した。
(Evaluation test 3: inhibition test by non-radioactive ligand)
Tumor-bearing model mice are prepared in the same manner as above, and a mixture of c (RGDfK) (0.2 mg / mouse), 125 Ic (RGDfK), and 211 At-c (RGDfK) (37 kBq, 74 kBq / 100 μL) At the same time, tail vein was administered. One hour after administration, the mice were sacrificed, each organ was removed, and the weight and radioactivity of the organ were measured. 211 radioactivity At performs measurements at the energy range of 70-120 keV, 125 I is 211 At was measured with an energy range of 1-80 keV after attenuation.
その結果を、図５に示す。図５から明らかな通り、c(RGDfK)同時投与によるブロッキング実験により腫瘍への集積が著しく低下したことより、125I-c(RGDfK)、及び211At-c(RGDfK)の腫瘍への集積は、αｖβ３インテグリンを介していることが分かった。 The results are shown in FIG. Figure 5 is clear from, than the accumulation in the tumor was significantly reduced by c (RGDfK) blocking experiments by simultaneous administration, 125 Ic (RGDfK), and accumulation in tumors of 211 At-c (RGDfK) has, alpha It turned out that v beta 3 integrin is mediated.
これらの結果から、I-c(RGDfK)はαVβ3インテグリンに対し、c(RGDfK)と同程度の高い結合親和性を示すことが分かった。また、125I-c(RGDfK)の体内放射能分布実験では125I-c(RGDyK)と類似した分布傾向が観察された。さらに211At-c(RGDfK)は125I-c(RGDfK)と類似した体内動態を示した。125I-c(RGDfK)では、投与早期に腎臓への放射能集積(投与後1時間に6.7%ID/g)が観察されたが、滞留せずにクリアランスされ、腫瘍への放射能集積(投与後1時間に4.1%ID/g)は125IB-c(RGDfK)に比べ高く、125I-c(RGDyK)と類似した体内分布傾向が観察された。 From these results, it was found that Ic (RGDfK) exhibits as high binding affinity as that of c (RGDfK) for α v β 3 integrin. Further, in the body radioactivity distribution experiment 125 Ic (RGDfK) it was observed distribution tendency similar to 125 Ic (RGDyK). Furthermore, 211 At-c (RGDfK) showed similar pharmacokinetics to 125 Ic (RGDfK). In 125 Ic (RGDfK), radioactive accumulation in the kidney (6.7% ID / g one hour after administration) was observed early in administration, but it was cleared without retention, and radioactive accumulation in tumor (post-administration) 4.1% ID / g in 1 hour) is higher than the 125 IB-c (RGDfK), biodistribution tendency similar to 125 Ic (RGDyK) was observed.
なお、本発明のオリゴペプチド誘導体自身が医薬を兼ねる例として記載したが、患者に投与する際には、錠剤や注射剤の医薬製剤である医薬として調製して用いることが可能である。 In addition, although the oligopeptide derivative of this invention itself was described as an example which served as a pharmaceutical, when administering to a patient, it is possible to prepare and use as a pharmaceutical which is a pharmaceutical formulation of a tablet or an injection.
本発明のオリゴペプチド誘導体は、癌原発巣や転移組織の癌細胞を特異的に攻撃して破壊する医薬品として有用であり、産業上の利用可能性がある。 INDUSTRIAL APPLICABILITY The oligopeptide derivative of the present invention is useful as a medicine that specifically attacks and destroys cancer cells in a primary tumor or a metastatic tissue, and has industrial applicability.
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| CN110256532A (en) * | 2019-07-03 | 2019-09-20 | 湖北强耀生物科技有限公司 | A kind of RGD cyclic peptide synthetic method |
| CN110379468A (en) * | 2019-07-17 | 2019-10-25 | 成都火石创造科技有限公司 | A kind of improved chemical molecular formula cutting method |
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| CN110256532A (en) * | 2019-07-03 | 2019-09-20 | 湖北强耀生物科技有限公司 | A kind of RGD cyclic peptide synthetic method |
| CN110256532B (en) * | 2019-07-03 | 2023-04-14 | 湖北强耀生物科技有限公司 | RGD cyclopeptide synthesis method |
| CN110379468A (en) * | 2019-07-17 | 2019-10-25 | 成都火石创造科技有限公司 | A kind of improved chemical molecular formula cutting method |
| CN110379468B (en) * | 2019-07-17 | 2022-08-23 | 成都火石创造科技有限公司 | Improved chemical molecular formula segmentation method |
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