JP2005047821A - Diagnostic or therapeutic medicine using cyclopentadienyl carbonyl group-containing radiactive compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a radioactive metal-labeled compound, such as a radioactive metal-labeled antibody, which little accumulates the radioactive metal in kidney and can quickly be excreted into urine, to provide an image-diagnostic or therapeutic medicine composition containing the labeled compound as an active ingredient, and to provide an image-diagnostic or therapeutic method. <P>SOLUTION: This diagnostic or therapeutic agent labeled with a radioactive metal represented by formula 1: M-A-B-C-D-E-F [M is a radioactive metal selected from Tc-99m, Re-186 and Re-188; A is a group derived from cyclopentadienyl tricarbonyl or cyclopentadienyl tricarbonyl group; B and C are each an arbitrary amino acid residue; D is spacer 1; E is spacer 2 (when D is directly bound to F, the spacer 2 may not exist); F is an antibody, a protein such as an antibody fragment, or a physiologically active substance such as a peptide]. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００２２】
上記スキーム１及びスキーム２は、本願化合物であるＭ−Ａ−Ｂ−Ｃ−Ｄ−Ｅ−Ｆ（式１）の合成ルートの一例を示している。スキーム１には、式１のＭ−Ａ−Ｂ−Ｃ−Ｄ部分の合成ルートが、スキーム２には、Ｅ−Ｆ部分の合成ルート及びＭ−Ａ−Ｂ−Ｃ−Ｄ部分とＥ−Ｆ部分を結合して、本願化合物であるＭ−Ａ−Ｂ−Ｃ−Ｄ−Ｅ−Ｆ（式１）を合成するルートが示されている。
【００２３】
スキーム１のＮ^α−（ｔ−ｂｕｔｏｘｙｃａｒｂｏｎｙｌ）−ｇｌｙｃｉｎｅ（１）は、本願化合物である式１のＢの部分に対応するグリシンのアミノ基に保護基としてｔ−ブトキシカルボニル基（ｔ−Ｂｏｃ）が結合したものである。この化合物（１）とＮ−ヒドロキシスクシンイミド（ＮＨＳ）を反応させて、Ｎ−Ｓｕｃｃｉｎｉｍｉｄｙｌ−Ｎ^α−（ｔ−ｂｕｔｏｘｙｃａｒｂｏｎｙｌ）−ｇｌｙｃｉｎｅ（２）を得ることができる。
Ｎ^ε−Ｍａｌｅｏｙｌ−Ｌ−ｌｙｓｉｎｅ・ＨＣｌ（３）は、式１のＤ−Ｃ部分に対応する化合物であり、リジン（Ｃ）の末端のアミノ基とマレオイル基（Ｄ）とが結合した化合物である。
Ｎ，Ｎ−ジイソプロピルエチルアミン（ＤＩＥＡ）により、上記化合物（２）と（３）を結合させ、Ｎ^α−（ｔ−ｂｕｔｏｘｙｃａｒｂｏｎｙｌ）−ｇｌｙｃｙｌ−Ｎ^ε−ｍａｌｅｏｙｌ−Ｌ−ｌｙｓｉｎｅ（４）を得ることができる。次に、この化合物（４）をアニソールに溶かし、トリフルオロ酢酸（ＴＦＡ）を加えてグリシンの保護基であるｔ−Ｂｏｃをはずして、Ｇｌｙｃｙｌ−Ｎ^ε−Ｍａｌｅｏｙｌ−Ｌ−ｌｙｓｉｎｅ・ＴＦＡ（５）を得る。この化合物（５）は、本願化合物である式１のＤ−Ｃ−Ｂ部分に対応する化合物であり、ここに、Ｄはマレオイル基、Ｃはリジン残基、Ｂはグリシンである。
【００２４】
次に、スキーム１の［^１８６Ｒｅ］Ｔｒｉｃａｒｂｏｎｙｌ（ｃａｒｂｏｘｙｃｙｃｌｏｐｅｎｔａｄｉｅｎｙｌ）ｒｈｅｎｉｕｍ（ＣｐＴＲ−ＣＯＯＨ）（６）とＮ−ヒドロキシスクシンイミド（ＮＨＳ）を反応させて、Ｎ−Ｈｙｄｒｏｘｙｓｕｃｃｉｎｉｍｉｄｙｌ ｔｒｉｃａｒｂｏｎｙｌ（ｃａｒｂｏｘｙｃｙｃｌｏｐｅｎｔａｄｉｅｎｙｌ）ｒｈｅｎｉｕｍ（［^１８６Ｒｅ］ＣｐＴＲ−ＣＯＯＳｕ）（７）を得ることができる。化合物（７）は、本願化合物である式１のＭ−Ａ部分に対応する化合物であり、ここに、Ｍはレニウム、Ａは−ＣＯＯＳｕ基（Ｓｕはスクシンイミジル基）を有するシクロペンタジエニル基を表す。
この化合物［^１８６Ｒｅ］ＣｐＴＲ−ＣＯＯＳｕ（７）を、上記化合物Ｇｌｙｃｙｌ−Ｎ^ε−Ｍａｌｅｏｙｌ−Ｌ−ｌｙｓｉｎｅ・ＴＦＡ（５）と反応させて、［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＯＨ（８）を得ることができる。この化合物（８）は、本願化合物である式１のＤ−Ｃ−Ｂ−Ａ−Ｍ部分に対応する化合物である。
【００２５】
スキーム２のＦａｂ−ＮＨ_２は、本願化合物である式１のＦ部分に相当する化合物である。このＦａｂ−ＮＨ_２を式１のＥ部分に相当する２−イミノチオラン（２−ＩＴ）と反応させ、Ｆａｂ−ＮＨ−Ｃ（＝ＮＨ）−ＣＨ_２−ＣＨ_２−ＣＨ_２−ＳＨを得ることができる。すなわち、Ｆａｂ−ＮＨ−Ｃ（＝ＮＨ）−ＣＨ_２−ＣＨ_２−ＣＨ_２−ＳＨは、式１のＦ−Ｅ部分に相当する化合物である。
最後に、式１のＦ−Ｅ部分に相当するＦａｂ−ＮＨ−Ｃ（＝ＮＨ）−ＣＨ_２−ＣＨ_２−ＣＨ_２−ＳＨと、式１のＤ−Ｃ−Ｂ−Ａ−Ｍ部分に対応する化合物［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＯＨ（８）を反応させ、Ｍ−Ａ−Ｂ−Ｃ−Ｄ−Ｅ−Ｆ（式１）で表される本願化合物を得ることができる。
【００２６】
スキーム２では、Ｆ−Ｅ部分を合成し、スキーム１で合成したＤ−Ｃ−Ｂ−Ａ−Ｍ部分と結合させて本願の式１の化合物を得るルートを例示したが、ＤがＦと結合し得る官能基を有するときには、スキーム１で合成したＤ−Ｃ−Ｂ−Ａ−Ｍ部分と直接Ｆ部分を結合させることも可能である。この場合は、式１でＥが存在しない化合物、Ｍ−Ａ−Ｂ−Ｃ−Ｄ−Ｆが本願化合物として得られることになる。
【００２７】
本発明の化合物を有効成分とする放射性診断剤または放射性治療薬等の医薬組成物は、本願化合物が含有する生理活性物質の性質に基づき、腫瘍、炎症、感染症、心循環器疾患、脳・中枢系疾患等の各種疾患および臓器・組織の画像診断、あるいは治療に用いられる。本化合物は、腎臓に蓄積することなく速やかに体外に排泄されるので、腹部のバックグラウンドとなる放射能が少ないため、画像診断においては腹部の病巣部位の検出・診断が容易となる。また、放射性治療の場合には、腎臓への被曝を低減できる。また、被曝の低減が可能な分、投与量を増加させ、標的臓器へ放射能を大量に集め、治療効果を高めることも可能になる。
【００２８】
本発明の化合物を、放射性診断剤または放射性治療剤として供する場合は、上述した方法によって調製される放射性金属標識物を更にＨＰＬＣ法により精製し不純物および未反応物を取り除いた後に使用しても良い。
【００２９】
本発明の化合物は、薬学的に許容される添加物と混合することにより、水溶性の放射性診断剤または放射性治療剤に調製することができる。かかる添加物としては、薬学的に許容されるアスコルビン酸、ｐ−アミノ安息香酸等の安定化剤、水性緩衝液等のｐＨ調節剤、Ｄ−マンニトール等の賦形剤、および放射化学的純度を改良するのに役立つクエン酸、酒石酸、マロン酸、グルコン酸ナトリウム、グルコヘプトン酸ナトリウム、等があげられる。またこれらの添加物を加えた凍結乾燥品、凍結溶液の形態でも提供が可能である。
【００３０】
本発明の化合物を含有してなる放射性診断剤および放射性治療剤は、静脈内投与等の非経口投与、あるいは経口投与により投与でき、その投与量は患者の年齢、体重、適当な放射線イメージング装置、および対象疾患の状態などの諸条件を考慮し、イメージングおよび治療が可能と考えられる放射能および投与量が決定される。
ヒトを対象とする場合、Ｔｃ−９９ｍ標識体を用いた診断剤の投与量は、Ｔｃ−９９ｍの放射能量として３７ＭＢｑ〜１１１ＭＢｑである。Ｒｅ−１８６またはＲｅ−１８８標識体を用いた治療剤の場合は、放射能量として３７ＭＢｑ〜１８５００ＭＢｑの範囲であり、好ましくは３７０ＭＢｑ〜７４００ＭＢｑである。
【００３１】
【実施例１】
以下に説明するすべての実施例では、日本原子力研究所より供給された^１８６ＲｅＯ_４ ⁻溶液を０．０１Ｎ ＮａＯＨにて中和して用いた。Ｎａ［^１２５Ｉ］ＩはＩＣＮ ｂｉｏｍｅｄｉｃａｌｓ， Ｉｎｃより購入した。逆相ＨＰＬＣは、Ｃｏｓｍｏｓｉｌ ５Ｃ_１８−ＡＲ−３００（４．６ ｘ １５０ｍｍ， Ｎａｃａｌａｉ Ｔｅｓｑｕｅ）を用い、０．１％トリフルオロ酢酸（ｔｒｉｆｌｕｏｒｏａｃｅｔｉｃ ａｃｉｄ（ＴＦＡ））を含有する水（Ａ）と０．１％ ＴＦＡを含有するアセトニトリル（Ｂ）をＡ：Ｂ ＝ ８０：２０からＡ：Ｂ ＝ ２０：８０へ３０分間で変換するｇｒａｄｉｅｎｔ法（ｓｏｌｖｅｎｔ ｓｙｓｔｅｍ １）により、流速１．０ｍＬ／ｍｉｎで溶出した。分子篩ＨＰＬＣは５ Ｄｉｏｌ−３００（７．５ ｘ ６００ｍｍ， Ｎａｃａｌａｉ Ｔｅｓｑｕｅ）を用い、溶出溶媒として０．１Ｍリン酸緩衝液 ｐＨ ６．８（ｓｏｌｖｅｎｔ ｓｙｓｔｅｍ ２）により溶出した。溶出液はフラクションコレクター（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｎｃｅｓ， ＲｅｄｉＦｒａｃ）により０．５分間隔で分取後、Ａｕｔｏ ｗｅｌｌ γｓｙｓｔｅｍ（ＡＲＣ−３８０Ｍ， Ａｌｏｋａ）で放射能を測定した。薄層クロマトグラフィー（ＴＬＣ）は、シリカゲルプレート（Ｓｉｌｉｃａ ｇｅｌ ６０Ｆ_２５４， Ｍｅｒｋ）を使用し、クロロホルム（ｓｏｌｖｅｎｔ ｓｙｓｔｅｍ ３）又は生理食塩水（ｓｏｌｖｅｎｔ ｓｙｓｔｅｍ ４）を展開溶媒とした。ＳｅｐＰａｋ ｐｌｕｓ（Ｃ１８ ｓｈｏｒｔ ｂｏｄｙ；３６０ｍｇ／ｃａｒｔｒｉｄｇｅ， Ｗａｔｅｒｓ）はエタノール６ｍＬを流した後、Ｈ_２Ｏ ６ｍＬを流して前処理した後に使用した。サンプルを添加後Ｈ_２Ｏ ５ｍＬ、次いでエタノール２〜３ｍＬで溶出した。最初の１００μＬは廃棄した。トリカルボニル（シクロペンタジエニルカーボネート）レニウム（Ｔｒｉｃａｒｂｏｎｙｌ（ｃｙｃｌｏｐｅｎｔａｄｉｅｎｙｌｃａｒｂｏｎａｔｅ）ｒｈｅｎｉｕｍ（［^１８６Ｒｅ］ＣｐＴＲ−ＣＯＯＨ））、及びトリカルボニル［（シクロペンタジエニルカルボニルアミノ）アセテート］レニウム（Ｔｒｉｃａｒｂｏｎｙｌ［（ｃｙｃｌｏｐｅｎｔａｄｉｅｎｙｌｃａｒｂｏｎｙｌ ａｍｉｎｏ）ａｃｅｔａｔｅ］ｒｈｅｎｉｕｍ（ＣｐＴＲ−Ｇｌｙ））は、以前本願発明者らが報告した方法に基づき合成した（非特許文献２５および２６参照。）。その他の試薬は、全て特級のものをそのまま使用した。
【００３２】
ＣｐＴＲ − ＣＯＯＨ （６）の合成：
（１）［^１８６Ｒｅ］Ｔｒｉｃａｒｂｏｎｙｌ（ｍｅｔｈｏｘｙｃａｒｂｏｎｙｌｃｙｃｌｏｐｅｎｔａｄｉｅｎｙｌ）ｒｈｅｎｉｕｍ（ＣｐＴＲ−ＣＯＯＭｅ）の合成
本化合物は以前本発明者らが報告した方法（非特許文献２５参照）を少し変更して合成した。
ポータブルリアクター（０．８×８．５ ｃｍ；耐圧ガラス工業）に１，１’−ビス（メトキシカルボニル）フェロセン（１２ｍｇ， ４０μｍｏｌ）、ヘキサカルボニルクロム（１９ｍｇ， ８６．３μｍｏｌ）、塩化スズ（ＩＩ）無水物（３７ｍｇ， １９５．１μｍｏｌ）を加えた。［^１８６Ｒｅ］ＲｅＯ_４ ⁻水溶液は、真空下で溶媒を留去させた後、乾燥メタノール（５００μＬ）に抽出し、前記ポータブルリアクターに加えた。容器を密閉後、撹拌しながら２００℃の油浴中で６０分反応させた。氷上で１０分以上冷却し室温に戻した後、反応溶媒を減圧下で留去した。残渣をクロロホルムに溶かし、クロロホルムを溶出溶媒とするシリカゲルクロマトグラフィーで精製することにより、［^１８６Ｒｅ］ＣｐＴＲ−ＣＯＯＭｅを得た。放射化学的収率（６３％）、放射化学的純度（９５％以上）はそれぞれＴＬＣ（ｓｏｌｖｅｎｔ ｓｙｓｔｅｍ ３）により求めた。
【００３３】
（２）［^１８６Ｒｅ］Ｔｒｉｃａｒｂｏｎｙｌ（ｃａｒｂｏｘｙｃｙｃｌｏｐｅｎｔａｄｉｅｎｙｌ）ｒｈｅｎｉｕｍ（ＣｐＴＲ−ＣＯＯＨ）（６）の合成
次いで、［^１８６Ｒｅ］ＣｐＴＲ−ＣＯＯＭｅをジオキサン（２００μＬ）に溶かし、２Ｎ ＮａＯＨ水溶液（６００μＬ）を加えた。室温で１０分間撹拌した後、ｃｏｎｃ． ＨＣｌで反応液を酸性にして、活性化したＳｅｐＰａｋカラムを用いて精製し、［^１８６Ｒｅ］ＣｐＴＲ−ＣＯＯＨ（６）を得た。放射化学的収率（９５．２％）、放射化学的純度（９５％以上）はそれぞれＴＬＣ（ｓｏｌｖｅｎｔ
ｓｙｓｔｅｍ ３）により求めた。
【００３４】
【実施例２】
Ｎ ^α −（ｔ−ｂｕｔｏｘｙｃａｒｂｏｎｙｌ）−ｇｌｙｃｙｌ−Ｎ ^ε −ｍａｌｅｏｙｌ−Ｌ−ｌｙｓｉｎｅ （４）の合成：
（１）Ｎ−Ｓｕｃｃｉｎｉｍｉｄｙｌ−Ｎ^α−（ｔ−ｂｕｔｏｘｙｃａｒｂｏｎｙｌ）−ｇｌｙｃｉｎｅ（２）の合成
Ｎ^α−（ｔ−ｂｕｔｏｘｙｃａｒｂｏｎｙｌ）−ｇｌｙｃｉｎｅ（１）（０．８ｇ，４．５７ｍｍｏｌ）とＮ−ｈｙｄｒｏｘｙｓｕｃｃｉｎｉｍｉｄｅ（ＮＨＳ， ０．５５ｇ， ４．７８ｍｍｏｌ）をＮ，Ｎ’−ジメチルホルムアミド（ＤＭＦ，２０ｍＬ）に溶かして氷冷し、０℃で１，３−ジシクロヘキシルカルボジイミド（ＤＣＣ， １．０３ｇ， ５．０４ｍｍｏｌ）を加え、０〜５℃で４時間、その後室温で１６時間撹拌した。析出した沈殿物をろ過し、ろ液を減圧下で留去した。
イソプロピルアルコールから再結晶し、白色結晶の上記化合物（２）を１．０３ｇ（収率８３．６％）得た。
^１Ｈ−ＮＭＲ（ＣＤＣｌ_３）：δ １．４４（９Ｈ， ｓ， Ｂｏｃ）， ２．８０（４Ｈ， ｄ， ｓｕｃｃｉｎｉｍｉｄｅ）， ４．２０（２Ｈ， ｄ， ＣＨ_２）
ＦＡＢ−ＭＳ：理論値（Ｃ_１１Ｈ_１７Ｎ_２Ｏ_６ （Ｍ＋Ｈ）^＋）：ｍ／ｚ ２７３、測定値：２７３
【００３５】
（２）Ｎ^α−（ｔ−ｂｕｔｏｘｙｃａｒｂｏｎｙｌ）−ｇｌｙｃｙｌ−Ｎ^ε−ｍａｌｅｏｙｌ−Ｌ−ｌｙｓｉｎｅ（４）の合成
Ｗａｋｉｓａｋａらの方法（非特許文献２７参照）に従って合成したＮ^ε−Ｍａｌｅｏｙｌ−Ｌ−ｌｙｓｉｎｅ・ＨＣｌ（３）（２００ｍｇ， ０．７６ｍｍｏｌ）をＤＭＦ（５ｍＬ）に溶かし、Ｎ，Ｎ’−ジイソプロピルエチルアミン（ＤＩＥＡ， １３２．９μＬ， ０．７８ｍｍｏｌ）を加えた。ＤＭＦ（５ｍＬ）に溶かした化合物（２）（１７２．７ｍｇ， ０．６３ｍｍｏｌ）を滴下し、常温で一晩撹拌した。反応液を減圧留去した後、残渣を酢酸エチルに溶かし、ｐＨ ５〜６に希釈した硫酸で３回洗浄した。無水硫酸カルシウムで乾燥し、ろ過したろ液を減圧下で留去して、無色油状の上記化合物（４）を１５５．７ｍｇ（収率６４．０％）得た。
^１Ｈ−ＮＭＲ（ＣＤＣｌ_３）：δ １．３０−１．９１（６Ｈ， ｍ， （ＣＨ_２）_３）， １．４１（９Ｈ， ｓ， Ｂｏｃ）， ４．５２（１Ｈ， ｓ， ＣＨ）， ５．６０（１Ｈ， ｓ， ＮＨ）， ６．６７（２Ｈ， ｓ， ｍａｌｅｉｍｉｄｅ）， ７．０８（１Ｈ， ｓ， ＮＨ）
ＦＡＢ−ＭＳ：理論値（Ｃ_１７Ｈ_２６Ｎ_３Ｏ_７ （Ｍ＋Ｈ）^＋）ｍ／ｚ ３８４、測定値：３８４
【００３６】
【実施例３】
［ ^１８６ Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＯＨ （８）の合成：
（１）Ｇｌｙｃｙｌ−Ｎ^ε−Ｍａｌｅｏｙｌ−Ｌ−ｌｙｓｉｎｅ・ＴＦＡ（５）の合成
実施例２で得られた化合物（４）（１９１．５ｍｇ， ０．５０ｍｍｏｌ）をアニソール（５０μＬ）に溶かし、トリフルオロ酢酸（ＴＦＡ，９５０μＬ）を少しずつ加え、そのまま室温で撹拌した。１時間後、Ｎ_２ガスを用いてＴＦＡを留去し、エーテルを加えた。析出した結晶をろ取し、白色結晶の化合物Ｇｌｙｃｙｌ−Ｎ^ε−Ｍａｌｅｏｙｌ−Ｌ−ｌｙｓｉｎｅ・ＴＦＡ（５）を１１２．６ｍｇ（収率６６．２％）得た。
^１Ｈ−ＮＭＲ（ＣＤ_３ＯＤ）：δ １．３３−１．９５（６Ｈ， ｍ，（ＣＨ_２）_３）， ４．４２（１Ｈ， ｓ， ＣＨ）， ６．６８（２Ｈ， ｓ， ｍａｌｅｉｍｉｄｅ）
ＦＡＢ−ＭＳ：理論値（Ｃ_１２Ｈ_１８Ｎ_３Ｏ_５ （Ｍ＋Ｈ）^＋）ｍ／ｚ ２８４、測定値：２８４
【００３７】
（２）Ｎ−Ｈｙｄｒｏｘｙｓｕｃｃｉｎｉｍｉｄｙｌ ｔｒｉｃａｒｂｏｎｙｌ（ｃａｒｂｏｘｙｃｙｃｌｏｐｅｎｔａｄｉｅｎｙｌ）ｒｈｅｎｉｕｍ（［^１８６Ｒｅ］ＣｐＴＲ−ＣＯＯＳｕ）（７）の合成
［^１８６Ｒｅ］ＣｐＴＲ−ＣＯＯＨ（６）をジクロロメタン（２００μＬ）に溶解し、ＮＨＳ（２．０ｍｇ， １７．３μｍｏｌ）、ＤＣＣ（２．０ｍｇ， ９．７μｍｏｌ）を加え、室温で５分間撹拌した。Ｎ_２ガスにて溶媒を除去した後、アセトニトリルに溶解し、ＲＰ−ＨＰＬＣにて精製した。放射化学的収率（７６％）、放射化学的純度（９５％以上）はそれぞれクロロホルムを展開溶媒とするＴＬＣ（ｓｏｌｖｅｎｔ ｓｙｓｔｅｍ ３）により求めた。
【００３８】
（３）［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＯＨ（８）の合成
Ｇｌｙｃｙｌ− Ｎ^ε−ｍａｌｅｏｙｌ−Ｌ−ｌｙｓｉｎｅ・ＴＦＡ（５）（１２．５ｍｇ， ３６．７μｍｏｌ）をＮ，Ｎ’−ジメチルホルムアミド（ＤＭＦ； ８０μＬ）に溶解し、Ｎ，Ｎ’−ジイソプロピルエチルアミン（ＤＩＥＡ； ６．３μＬ， ３６．７μｍｏｌ）を少しずつ加えた後、ＤＭＦ（９０μＬ）に溶解した［^１８６Ｒｅ］ＣｐＴＲ−ＣＯＯＳｕ（７）を少しずつ加えた。室温で１時間程撹拌した後、逆相ＨＰＬＣ（ＲＰ−ＨＰＬＣ）にて精製した。ＲＰ−ＨＰＬＣはＣｏｓｍｏｓｉｌ ５Ｃ_１８−ＡＲ−３００（４．６ ｘ １５０ｍｍ， Ｎａｃａｌａｉ Ｔｅｓｑｕｅ）を用い、０．１％ ｔｒｉｆｌｕｏｒｏａｃｅｔｉｃ ａｃｉｄ（ＴＦＡ）を含有する水（Ａ）と０．１％ ＴＦＡ を含有するアセトニトリル（Ｂ）をＡ：Ｂ ＝ ８０：２０からＡ：Ｂ ＝ ２０：８０へ３０分間で変換するｇｒａｄｉｅｎｔ法により、流速１．０ｍＬ／ｍｉｎで溶出した。放射化学的収率（４９．６％）、放射化学的純度（９１％以上）はそれぞれクロロホルムを展開溶媒とするＴＬＣ（ｓｏｌｖｅｎｔ ｓｙｓｔｅｍ ３）により求めた。
【００３９】
【実施例４】
［ ^１８６ Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＩＴ−Ｆａｂ の合成：（１）Ｆａｂフラグメントのチオール化（２−イミノチオラン（２−ＩＴ）接合Ｆａｂの合成）
２ｍＭのエチレンジアミン四酢酸（ＥＤＴＡ）を含む、０．１６Ｍホウ酸緩衝液（ｐＨ８．５）を用いてＦａｂを調整し（１．０ｍｇ／ｍＬ， ２００μＬ）、同じ緩衝液に溶解した２−イミノチオラン（２−ＩＴ；２．５ｍｇ／ｍＬ， ７．４μＬ）を加え、３０分間静かに撹拌した。２ｍＭのＥＤＴＡを含む０．１Ｍリン酸緩衝液（ｐＨ ６．０）で平衡化したＳｅｐｈａｄｅｘ Ｇ−５０を用いたスピンカラム法により、過剰の２−ＩＴを除去した。１分子あたりに導入されたチオールの数は２，２’−ジピリジルジスルフィド（ＤＰＳ）を用いて測定した。その結果、２−ＩＴによりチオール化した抗体Ｆａｂフラグメントは抗体一分子当たり３．０個のチオール基を有していた。
【００４０】
（２）［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＩＴ−Ｆａｂの合成
［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＯＨ（８）に上記のチオール化したＦａｂ（１ｍｇ／ｍＬ， １００μＬ）を加え、９０分間静かに撹拌した。次いで０．１Ｍリン酸緩衝液（ｐＨ ６．０）に溶かしたヨードアセトアミド（１４．８μＬ， １０．０ｍｇ／ｍＬ）を加え、３０分間撹拌した。０．５Ｍの酢酸緩衝液（ｐＨ ６．０）で平衡化したＳｅｐｈａｄｅｘ Ｇ−５０を用いるスピンカラム法により精製した。
上記（１）で得られたチオール化抗体Ｆａｂフラグメントに、［^１８６Ｒｅ］ＣｐＴＲ−ＧＫ（Ｍ）−ＯＨ（８）を混ぜ合わせることにより、［^１８６Ｒｅ］ＣｐＴＲ−ＧＫ（Ｍ）−ＩＴ−Ｆａｂを放射化学的収率３０％、放射化学的純度９０％以上で得た。
【００４１】
【実施例５】
［ ^１８６ Ｒｅ］ＣｐＴＲ−Ｆａｂ の合成：
実施例３−（２）で得られた［^１８６Ｒｅ］ＣｐＴＲ−ＣＯＯＳｕ（７）を１０μＬのＤＭＦに溶解し、Ｆａｂ溶液１００μＬ（１ｍｇ／ｍＬ， ０．１６Ｍホウ酸緩衝液ｐＨ ９．５）にゆっくり加えた。１時間後、０．５Ｍの酢酸緩衝液（ｐＨ ６．０）で平衡化したＳｅｐｈａｄｅｘ Ｇ−５０を用いるスピンカラム法により精製した。放射化学的収率、放射化学的純度はそれぞれ生理食塩水を展開溶媒とするＴＬＣにより求めた。その結果、［^１８６Ｒｅ］ＣｐＴＲ−Ｆａｂは、［^１８６Ｒｅ］ＣｐＴＲ−ＣＯＯＳｕ（７）と抗体Ｆａｂフラグメントを混ぜ合わせることにより、放射化学的収率３０％、放射化学的純度９５％以上で得られた。
【００４２】
【実施例６】
［ ^１２５ Ｉ］ＨＭＬ−ＩＴ−Ｆａｂ の合成：
［^１２５Ｉ］ＨＭＬ−ＩＴ−Ｆａｂは藤岡らの方法（非特許文献１８参照）に従い合成した。
ＨＭＬ（ε−Ｎ−マレオイル−Ｌ−リジン）の有機スズ前駆体を１％の酢酸を含むメタノールに溶解し、［^１２５Ｉ］ＮａＩ及び酸化剤であるＮ−クロルコハク酸を加えて、［^１２５Ｉ］ＨＭＬを合成した。この［^１２５Ｉ］ＨＭＬに、実施例５と同様の方法でチオール化したＦａｂを加え、スピンカラム法により精製して［^１２５Ｉ］ＨＭＬ−ＩＴ−Ｆａｂを得た。
【００４３】
【実施例７】
マウス体内分布試験
実施例４で得られた［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＩＴ−Ｆａｂと実施例６で得られた［^１２５Ｉ］ＨＭＬ−ＩＴ−Ｆａｂの混液、あるいは実施例５で得られた［^１８８Ｒｅ］ＣｐＴＲ−Ｆａｂをそれぞれ生理食塩水に溶解し、一群３−４匹の６週齡ｄｄＹ系雄性マウスに一匹あたり１１．１ｋＢｑ（１００μｌ）になるように尾静脈から投与した。投与１０，３０分，１，３，６時間後に屠殺して臓器を摘出し、重量と放射能を測定した。また、投与後６時間までに排泄された糞便と尿を採取し、放射能を測定した。更に投与後６時間までに排泄された尿を０．４５μｍのフィルターでろ過した後、分子篩ＨＰＬＣにより分析した。また低分子画分の分析は尿を１０−ｋＤａの限外濾過膜で濾過し、濾液を逆相ＨＰＬＣで分析して尿中に排泄された放射能の化学形を検討した。この時、逆相ＨＰＬＣは、Ｃｏｓｍｏｓｉｌ ５Ｃ_１８−ＡＲ−３００（４．６ ｘ １５０ｍｍ， Ｎａｃａｌａｉ Ｔｅｓｑｕｅ）を用い、０．１％トリフルオロ酢酸（ＴＦＡ）を含有する水（Ａ）と０．１％ ＴＦＡを含有するアセトニトリル（Ｂ）をＡ：Ｂ ＝ ８０：２０からＡ：Ｂ ＝ ２０：８０へ３０分間で変換するｇｒａｄｉｅｎｔ法により、流速１．０ｍＬ／ｍｉｎで溶出した。分子篩ＨＰＬＣは５ Ｄｉｏｌ−３００（７．５ ｘ ６００ｍｍ， Ｎａｃａｌａｉ Ｔｅｓｑｕｅ）を用い、溶出溶媒として０．１Ｍリン酸緩衝液ｐＨ ６．８により溶出した。溶出液はフラクションコレクター（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｎｃｅｓ， ＲｅｄｉＦｒａｃ）により０．５分間隔で分取後、Ａｕｔｏ ｗｅｌｌ γｓｙｓｔｅｍ（ＡＲＣ−３８０Ｍ， Ａｌｏｋａ）で放射能を測定した。
【００４４】
正常マウスにおける［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＩＴ−Ｆａｂ、［^１８６Ｒｅ］ＣｐＴＲ−Ｆａｂ、［^１２５Ｉ］ＨＭＬ−ＩＴ−Ｆａｂの体内放射能分布を検討した結果を表１−３に示す。表中の放射能量の値は、平均値（標準偏差）を、％ＩＤ／ｇ（臓器重量（ｇ）当りの全投与放射能に対する百分率）で示した。いずれのＲＩ標識抗体フラグメントの血液からの消失速度は同程度であった。また、いずれのＲＩ標識抗体フラグメントの肝臓や腸間への放射能集積に相違は観られなかった。一方、［^１８６Ｒｅ］ＣｐＴＲ−Ｆａｂと［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＩＴ−Ｆａｂとの腎臓への放射能集積には投与早期から大きな相違が観察された。
［^１８６Ｒｅ］ＣｐＴＲ−Ｆａｂは投与後３０分を最大値とする腎臓への集積を示したが、［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＩＴ−Ｆａｂは［^１２５Ｉ］ＨＭＬ−ＩＴ−Ｆａｂと同様に投与早期から腎臓の放射能を大きく低減した。
【００４５】
【表１】

【表２】

【表３】

【００４６】
【実施例８】
腎臓の被曝線量の推定比較
実施例７で示した通り、［^１８６Ｒｅ］ＣｐＴＲ−Ｆａｂをマウスに投与した際、腎臓への放射能集積とそれに続く消失が観察された。一方、［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＩＴ−Ｆａｂは［^１２５Ｉ］ＨＭＬ−ＩＴ−Ｆａｂと同様にマウスに投与後、早期から腎臓への放射能集積を低減した（図１）。図１の四角印は、［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＩＴ−Ｆａｂ投与群、三角印は［^１２５Ｉ］ＨＭＬ−ＩＴ−Ｆａｂ投与群、丸印は［^１８６Ｒｅ］ＣｐＴＲ−Ｆａｂ投与群を示す。
それぞれの標識抗体の腎集積面積の比較から、［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＩＴ−Ｆａｂは腎臓への被曝線量を［^１８６Ｒｅ］ＣｐＴＲ−Ｆａｂの１／３以下に低減することが示された。
【００４７】
【実施例９】
尿中放射能の分析
［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＩＴ−Ｆａｂをマウス尾静脈に投与し、６時間後までに尿中に排泄された放射能量の分析を行った（図２）。分子篩ＨＰＬＣで分析したところ、５０％以上の放射能が低分子画分に溶出された（図２（Ａ））。さらに、分子量１万カットの限外濾過膜により溶出した低分子画分をＲＰ−ＨＰＬＣによって分析したところ、６５％以上の放射能がＣｐＴＲ−Ｇｌｙに一致する保持時間に溶出された（図２（Ｂ））。
【図面の簡単な説明】
【図１】図１は、［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＩＴ−Ｆａｂ、［^１２５Ｉ］ＨＭＬ−ＩＴ−Ｆａｂ、［^１８６Ｒｅ］ＣｐＴＲ−Ｆａｂ投与後のマウスの放射能量の生体内分布を示した図である。％ＩＤ／ｇは、臓器重量（ｇ）当りの全投与放射能に対する百分率を平均値で示した値である。
【図２】図２は、［^１８６Ｒｅ］ＣｐＴＲ−Ｇｌｙ−Ｌｙｓ（Ｍａｌｅ）−ＩＴ−Ｆａｂ投与後６時間後までに、マウスの尿中に排泄された放射能量を示した図である。（Ａ）は分子篩ＨＰＬＣで分析した結果であり、（Ｂ）は逆相ＨＰＬＣ（ＲＰ−ＨＰＬＣ）で分析した結果である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a radioactive metal-labeled compound that makes it possible to reduce radioactivity retention in the kidney when diagnosing or treating a diseased part of a living body, and a drug containing the compound as an active ingredient.
[0002]
[Prior art]
A compound in which a physiologically active substance such as a protein such as a monoclonal antibody or a polyclonal antibody or a peptide such as a peptide is labeled with a radioactive metal and a pharmaceutical composition thereof are widely used as a radiopharmaceutical for diagnostic imaging or treatment of a diseased site in a living body. (For example, refer nonpatent literature 1-5.). Known radioactive metals include technetium-99m, indium-111 and gallium-67 for diagnostic purposes, and yttrium-90, rhenium-186 or rhenium-188 for therapeutic purposes. When a radioactive metal element is labeled on an antibody or the like, a metal chelating agent is usually used. Known metal chelating agents include EDTA, DTPA, diaminodithio compounds, cyclam, and DOTA. These chelating agents may be preliminarily bound to an antibody or the like and then labeled with a radioactive metal, or may be labeled with an antibody or the like after forming a radioactive metal chelate (for example, Non-patent Documents 6-10). reference.).
[0003]
However, since the compound often accumulates in the kidney, it is known to cause exposure in the kidney or kidney damage (see Non-Patent Documents 11 and 12). When an antibody is fragmented into Fab, single chain Fv, or the like, distribution to a target site such as a disease site is promoted, and thus Fab and Fv are often used. On the other hand, since Fab and Fv are more prominently accumulated in the kidney, there is concern about kidney exposure and the risk of kidney damage, and dosage adjustment is necessary (see Non-Patent Documents 13-17).
[0004]
In addition to the above-mentioned radiometals, radiohalogens for labeling, such as fluorine-18, iodine-123, and iodine-131, are also known. These radioactive halogen elements are also widely used as radiodiagnostic drugs or radiotherapeutic drugs by labeling them with antibodies and peptides in the same manner as the above-mentioned radiometal elements.
[0005]
Recently, a compound in which radioactive iodine is labeled on hippuric acid and further bound to the Fab fragment of the antibody via ε-N-maleyl-L-lysine (HML) has been reported (see Non-Patent Documents 18 and 19). Compared with a compound in which radioactive tyrosine residues of conventional antibodies are labeled with radioactive iodine, the compound has a low level of renal radioactivity and rapid urinary excretion. This excretion mechanism is based on the experimental results in rats, and the glycine-lysine bond in the compound is selectively cleaved by the action of the brush border membrane enzyme present in the renal cortex, and the metabolite iodohippuric acid is found in the urine. It was found that it was due to rapid excretion.
[0006]
Although radioactive iodine is widely used for both diagnosis and treatment, it is difficult to prepare in the hospital and cannot be used in an emergency. Therefore, it is expected to use an antibody stably labeled with a radioactive metal that can be adjusted only by mixing the radioactive metal and the ligand-bound antibody when necessary. However, there are no reports of compounds that have achieved sufficiently low renal radioactivity accumulation and rapid urinary excretion as indicated by radioiodine-labeled antibodies in radiometal-labeled antibodies and the like (see Non-Patent Documents 20-24). .
[Non-Patent Document 1]
Behr ™, 8 others, “Comparison of full-length and fragmented anti-CEA monoclonal antibody labeled with technetium-99m for immunoscintigraphy in colorectal cancer ( (Comparison of complete versus fragmented-technium-99m-labeled anti-CEA monoclonal antigens for immunoscinctography in collective cancer), J N. 430-441
[Non-Patent Document 2]
Maloney DG, 6 others, “Phase I clinical trial with increased use of single agent infusion of chimeric anti-CD20 monoclonal antibody (IDEC-D2B8) in patients with recurrent B-cell lymphoma (Phase I clinical) triusing escalating single-dose infusion of chimeric anti-CD20 monoclonal antibodies (IDEC-C2B8) in party with recurrent B-cell, 94), 84. 2457-2466
[Non-Patent Document 3]
Maloney DG, Grillo-Lopez AJ, White CA, et al., “IDEC-C2B8 (rituximab) anti-CD20 monoclonal antibody for patients with relapsed low-grade non-Hodgkin lymphoma Treatment (IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapeutic in patients with relaxed low-grade non-Hodgkin's lymphoma), Vol. 90, 1997. 2188-2195
[Non-Patent Document 4]
Wiseman GA, Leigh B, Erwin WD, et al., “Rituximab—Radiation dosimetry resurgence results for Zevalin radioimmunotherapy of refractory non-Hodgkin lymphoma. radioimmunotherapy of rituximab-refractory non-Hodgkin lymphoma) ", Cancer, 2002, Vol. 94, p. 1349-1357
[Non-Patent Document 5]
Wittig TE, Gordon LI, Cabanillas F, et al., “Yttrium in patients with relapsed or refractory low grade thyroid follicular adenocarcinoma or modified B-cell non-Hodgkin lymphoma. ibritumomab tiuxetan were labeled with 90 radioimmunotherapy and randomized controlled trials of rituximab immunotherapy (randomized controlled trial of yttrium-90-labeled ibritumomab tiuxetan radioimmunotherapy versus rituximab immunotherapy for patients with relapsed or refractory low-grade, follicular, or transformed B-cell non-Hodgkin's lymphoma), J Clin Oncol, 2002, Vol. 20, pp. 2453-2463.
[Non-Patent Document 6]
Ono M, Arano Y, Mukai T, et al., “Plasma protein binding of the hydrazinonicotinamide-derived polypeptides and peptides labeled with technetium-99m (Plasma protein binding of 99m) Tc-labeled hydrazino nicotinamide derivivated polypeptides and peptides), Nucl Med Biol, 2001, Vol. 28, p. 155-164
[Non-Patent Document 7]
Ono M, et al., "99m-Tc-HYNIC-derivatized ternary ligand ((99m) Tc for low protein binding in vivo of a polypeptide labeled with 99m-Tc" -HYNIC-derivatized tertiary ligands for (99m) Tc-labeled polypeptides with low in vivo protein binding), Nucl Med Biol, 2001, 28 p. 215-224
[Non-Patent Document 8]
Akizawa H, Arano Y, Mifune M, et al., “Significance of Indium-111-DTPA-chelates in renal radioactivity levels of Indium-111-DTPA-conjugated peptides. of (111) In-DTPA chelate in renal radioactivity levels of (111) In-DTPA-conjugated peptides), Nucl Med Biol, 2001, Vol. 28, p. 459-468
[Non-patent document 9]
Mukai T, et al., 8 "Synthesis and evaluation of a monoactive DOTA derivative for the residual level based on indium-111 to measure protein pharmacokinetics." indium-111-based reseduralizing label to estimate protein pharmaceuticals), J Pharm Pharmacol, 2002, Vol. 54, p. 1073-1081
[Non-Patent Document 10]
Arano Y, et al., 8 “Reassessment of diethylenetriaminepentaacetic acid (DTPA) as a chelating agent for indium-111 labeling of polypeptides using newly synthesized monoreactive DTPA derivatives. -Pentaacetic acid (DTPA) as a chelating agent for indium-111 labeling of polypeptids using a newly synthesized monoactive DTPA deliberate 39, 19). 3451-3460
[Non-Patent Document 11]
Otte A, 7 others, “Yttrium-90 DOTATOC: first clinical results (Yttrium-90 DOTATOC)”, Eur J Nucl Med, 1999, Vol. 26, p. 1439-1447
[Non-Patent Document 12]
Cybulla M, 3 others, “End-stage renal disease after treatment 90Y-DOTATOC”, Eur J Nucl Med, 2001, Vol. 28. p. 1552-1554
[Non-Patent Document 13]
Baum RP, et al., “Initial clinical results with technetium-99m-labeled LL2 monoclonal antibody in technetium-99m-labeled LL2 monoclonal antibody fragment in radioimmunodetection of B-cell lymphoma. radioimmunodetection of B-cell lymphomas) ", Cancer, 1994, Vol. 73, p. 896-899
[Non-Patent Document 14]
Buzis WC, et al., 4 "Dosimetric evaluation of immunoscintigraphical indigenous monoclonality of 111-labeled monoclonal antibody indium-111 labeled monoclonal antibody fragment. participants with ovarian cancer) ", J Nucl Med, 1992, vol. 33, p. 1113-1120
[Non-Patent Document 15]
DePalatis LR, 3 others, “Lu-177 alpha- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraacetic acid-CC49 Fab related to radioimmunoconjugate injection Lysine Reduces Radioactivity Accumulation in Lysine (Lycine redes renal accumulation of radioactivity associative with injection of the [177Lu] alpha- [2- (4-aminophenyl) etyl, 4] -1,4,7,10-tetraacetic acid-CC49 Fab radioimmunoconjugate) ", Cancer Res, 199 Year, Vol. 55, p. 5288-5295
[Non-Patent Document 16]
Ulteme ME, Bridger GJ, Abrams MJ, et al., “Tumor imaging with 99-technium-99m-labeled hydrazinonicotinamide-Fab ′ junction. -Labeled hydrazinonictinamide-Fab 'conjugates) ", J Nucl Med, 1997, Vol. 38, p. 133-138
[Non-Patent Document 17]
Wu C, et al., “Bio-distribution and catabolism of Ga-67-labeled anti-Tac dsFv fragment”, Bioconjug 97 Chem Chem, 19 Year, Volume 8, p. 365-369
[Non-Patent Document 18]
Renal metabolism of 3'-iodohypril N (epsilon) -maleoil-L-lysine (HML) conjugate-Fab fragment (Renal metabolism of 3'-iodopuripurpur (3) -iodohippurpur epsilon) -maleyl-L-lysine (HML) -conjugated Fab fragments), Bioconjug Chem, 2001, Vol. 12, p. 178-185
[Non-Patent Document 19]
Arano Y, Fujioka Y, Akizawa H, et al., “Chemical design of radioactive antibody for low kidney radioactivity levels (Chemical design of radioactive antibody). real radioactivity levels), Cancer Res, 1999, 59, p. 128-134
[Non-Patent Document 20]
Akizawa H, Arano Y, “Changes in pharmacokinetics through the metabolizable binding of radiolabeled antibodies. Altering pharmacokinetics of pharmacokinetics of monoclonal antibodies. by the interpolation of metabolizable links. Metabolizable linkers and pharmacokinetics of monoclonal antigens), Q J Nucl Med, 2002, 46 p. 206-223
[Non-patent document 21]
Arano Y, “Delivery of diagnostic agents for gamma-imaging”, Adv Drug Deliv Rev, 1999, 37, p. 103-120
[Non-Patent Document 22]
Li L, et al., “Reduction of kidney absorption of radioactive metal-labeled peptide conjugated to recombinant antibody fragment by site-specific conjugation of DOTA-peptide to Cys-diabody (Reduction of kidney uptake in) ”radio-labeled peptide linked conjugated to recombinant anti-fragmentation. Site-specific conjugation of DOTA-peptides to a Cys. 985-995
[Non-Patent Document 23]
Verbeek K, 3 others, “Preparation and preliminary evaluation of 99m Tc-EC-For-MLFK”, Nucl Med Biol, 200 Volume 29, p. 585-592
[Non-patent Document 24]
Kim MK, Jeong HJ, Kao CH, et al. “Improved renal clearance and tumor targeting of 99m Tc-labeled anti-Tac monoclonal antibody Fab by chemical modification ( Improved renal clearance and tumor targeting of 99m Tc-labeled anti-Tac monoclonal antibody Fab by chemical modification, Vol. 29, Nucl Med Biol. 29. 139-146
[Non-Patent Document 25]
Waferity (Uehara T), 8 others, “In vivo recognition of cyclopentadienyltrivinylbonrhenium (CpTR) 3rd year, CpTR), 200th year” (CpTR) Volume, p. 327-334
[Non-Patent Document 26]
Spradau TW, 4 others, “Synthesis and biological evaluation of Tc-99m cyclopentadienyltricarbonylrhenium labeled octreotide, Tc-99m-cyclopentadientylethylenetylenedientylethylenetylene Med Biol, 1999, 26, p. 1-7
[Non-Patent Document 27]
Wakisaka K., 8 others, “L-lysine as a metabolizable bond that stabilizes plasma and liberates m-iodohippuric acid after lysosomal proteolysis and a novel radioactivity for protein radiopharmaceuticals iodination reagent (a novel radioiodination reagent for protein radiopharmaceuticals with L-lysine as a plasma-stable metabolizable linkage to liberate m-iodohippuric acid after lysosomal proteolysis) ", J Med Chem, 1997 years, Vol. 40, p. 2643-2652
[0007]
[Problems to be solved by the invention]
In view of the state of the prior art, the present invention provides a radioactive metal-labeled compound such as an antibody that has little radioactive accumulation in the kidney and can be rapidly excreted in urine, and diagnostic imaging using the labeled compound as an active ingredient The purpose of the present invention is to provide a pharmaceutical composition for use or treatment, and a diagnostic imaging method or therapeutic method.
[0008]
[Means for Solving the Problems]
That is, the present invention provides the following formula 1
M-A-B-C-D-E-F (Formula 1)
(In Formula 1,
M: Technetium-99m (Tc-99m), rhenium-186 (Re-186)
Or a radioactive metal selected from rhenium-188 (Re-188)
A: a group derived from a cyclopentadienyl tricarbonyl group or a cyclopentadienyl tricarbonyl group
B: Any amino acid residue
C: Any amino acid residue
D: Spacer 1
E: Spacer 2 (however, if D and F can be directly bonded, spacer 2 may not be present)
F: Proteins such as antibodies and antibody fragments, or physiologically active substances such as peptides
Indicate)
The present invention relates to a diagnostic agent and a therapeutic agent labeled with a radioactive metal represented by
In the present invention, technetium-99m, indium-111, gallium-67, etc. can be used as radioactive metals for diagnostic agents, and yttrium-90, rhenium-186, rhenium-188, etc. can be used for treatment.
[0009]
The present invention also provides:
M-A-B-C-D (Formula 2)
(In Formula 2,
M: Technetium-99m (Tc-99m), rhenium-186 (Re-186)
Or a radioactive metal selected from rhenium-188 (Re-188)
A: cyclopentadienyl tricarbonyl group or cyclopentadienyl tricarbonyl group optionally having a carbonyl group
B: Any amino acid residue
C: any amino acid residue,
D: Spacer 1
Indicate)
And a process for producing the same are also provided.
The radioactive metal-labeled cyclopentadienyl tricarbonyl compound represented by the above formula 2 is MA-COOR (M represents the radioactive metal according to claim 11, and A represents a cyclopentadienyl tricarbonyl group. R represents an alkyl group or N-hydroxysuccinimidyl group) and H-B-C-D (B, C and D are the same as above, and H represents a hydrogen atom). Can be manufactured. Here, the alkyl group means an alkyl group having 1 to 6 carbon atoms.
[0010]
【The invention's effect】
The present invention provides a radiodiagnostic agent and a radiotherapeutic agent comprising as an active ingredient a radio-labeled compound that is metabolized in vivo without being accumulated in the kidney and the resulting radiometabolite is rapidly excreted in the urine. Became possible.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below.
A feature of the present invention is that compound A labeled with a radioactive metal is bound to a physiologically active substance such as an antibody, antibody fragment or peptide via an amino acid sequence BC that is selectively cleaved in the kidney. The compound is selectively enzymatically degraded, and the metabolite (MAB) is rapidly excreted in the urine without accumulating in the kidney.
It has already been known that the amino acid sequence glycine-lysine is cleaved by a brush border membrane enzyme present in vivo, particularly in the renal cortex (see Non-Patent Document 18). In fact, a compound in which radioactive iodine is labeled to hippuric acid and further bound to the Fab fragment of the antibody via ε-N-maleoyl-L-lysine (HML) is labeled with the radioactive tyrosine residue of the conventional antibody. Compared with the compound, the level of radioactivity in the kidney was low, and rapid urinary excretion was shown. This excretion mechanism is based on the experimental results in rats, and the glycine-lysine bond in the compound is selectively cleaved by the action of the brush border membrane enzyme present in the renal cortex, and the metabolite iodohippuric acid is found in the urine. It was found that it was due to rapid excretion.
[0012]
However, no effective means for excretion from the kidney has been reported with radioactive metal-labeled physiologically active substances (see Non-Patent Documents 20-24). Enzymes are generally known to strictly identify the chemical structure of a compound that is to be a substrate. Therefore, when the glycine-lysine sequence is simply bound to the radioactive metal label A, it is predicted whether or not the chemical structure including the structure (chemical form) before and after the glycine-lysine bond can serve as a substrate for the brush border membrane enzyme. Is not easy. Moreover, even if it has undergone enzyme metabolism, it has been difficult to design a chemical structure (chemical form) so that the metabolite A-B is rapidly excreted in the urine.
[0013]
The compound of the present invention uses a cyclopentadienyl tricarbonyl group (A) as a radioactive metal chelate moiety, and the amino acid sequence BC represented by glycyllysine and a maleoyl group represented by this cyclopentadienyl metal (MA). Is bound and labeled with a physiologically active substance (F) such as an antibody via BCD. In addition, the compound of the present invention binds and labels a physiologically active substance (F) such as an antibody via B-C-D-E in which spacer 1 (D) is further bound to spacer 2 (E). You can also. Cyclopentadienyl tricarbonyl metal is a complex structure that easily and stably holds a metal, and has recently been widely used as a useful compound for radioactive metal labeling. The present inventors have recognized that the cyclopentadienyl tricarbonyl metal derived from glycyl group is recognized as hippuric acid and promotes excretion due to its similarity to hippuric acid (non-patented). Reference 25). Therefore, various compounds were synthesized by binding cyclopentadienyltricarbonyl metal derived from glycyl group to an antibody as a physiologically active substance via lysine and a spacer, and attempted to be administered to a living body. As a result, the inventors succeeded in finding a chemical structure M-A-B-C-D-E-F of Formula 1 including a BC bond selectively cleaved by a renal brush border membrane enzyme in a living body. In particular, the amino acid sequence B—C derivatized cyclopentadienyl tricarbonyl metal represented by glycyllysine is a substrate for brush border membrane enzymes even in the presence of the bond of cyclopentadienyl tricarbonyl metal, and lysine and glycine. It has been shown to undergo selective cleavage between. Furthermore, it was found that MA-B, which is a metabolite generated by cutting between B-C, is rapidly excreted in urine without accumulating in the kidney.
[0014]
M-A is a cyclopentadienyl tricarbonyl metal labeled with a radioactive metal. Radioactive metal M is technetium-99m (^99mTc), Indium-111 (¹¹¹In) and gallium-67 (⁶⁷Ga), etc., and for treatment, yttrium-90 (⁹⁰Y), rhenium-186 (¹⁸⁶Re) or rhenium-188 (¹⁸⁸Re) and the like. In addition, a bonding group such as a carbonyl group can be introduced into A.
[0015]
B and C each represent an arbitrary amino acid residue. Examples of the amino acid residue of B include a glycine residue, and examples of the amino acid residue of C include an amino acid residue selected from lysine, phenylalanine, or tyrosine. Of these, a preferred BC combination is glycine-lysine.
[0016]
D is a spacer 1 selected from a maleoyl group or a group derived from a carboxylic acid active ester, and forms a covalent bond with the side chain amino group of amino acid residue C and also forms a covalent bond with E or direct F It is a compound having a functional group. Examples of the carboxylic acid active ester include hydroxysuccinimide and tetrafluorophenol. The spacer 1 of D is preferably a maleoyl group.
[0017]
E is a spacer 2 typified by iminothiol derived from 2-iminothiolane, and forms a covalent bond with D by previously derivatizing a physiologically active substance such as an antibody with E. When the physiologically active substance has a functional group capable of directly binding to D, E is not essential.
[0018]
F is a physiologically active substance composed of a protein such as an antibody (Mab) or antibody fragment F (ab) '2, or a peptide.
[0019]
Specifically, the following compounds can be exemplified as preferred embodiments of the compound represented by Formula 1 of the present invention.
Cyclopentadienyltricarbonylrhenium (¹⁸⁶Re) -Glycine-Lysine-ε-N-Maleoyl-2-iminothiolane-Mab
Cyclopentadienyltricarbonylrhenium (¹⁸⁸Re) -Glycine-Lysine-ε-N-Maleoyl-2-iminothiolane-Mab
Cyclopentadienyltricarbonyltechnetium (^99mTc) -Glycine-Lysine-ε-N-Maleoyl-2-iminothiolane-Mab
Cyclopentadienyltricarbonylrhenium (¹⁸⁶Re) -Glycine-Lysine-ε-N-Maleoyl-2-iminothiolane-Fab '
Cyclopentadienyltricarbonylrhenium (¹⁸⁶Re) -Glycine-Lysine-ε-N-Maleoyl-2-iminothiolane-peptide
[0020]
Hereinafter, synthesis examples of the compound of the present invention will be described with reference to Schemes 1 and 2.
[0021]

[0022]
The above-mentioned scheme 1 and scheme 2 show an example of a synthetic route of MA-B-C-D-F (formula 1) which is the present compound. Scheme 1 shows the synthetic route for the M-A-B-C-D moiety of Formula 1 and Scheme 2 shows the synthetic route for the E-F moiety and the M-A-B-C-D moiety and E-F. A route for synthesizing M-A-B-C-D-E-F (formula 1), which is a compound of the present invention, is shown.
[0023]
N in Scheme 1^α-(T-butoxycarbonyl) -glycine (1) is a compound in which a t-butoxycarbonyl group (t-Boc) is bonded as a protecting group to the amino group of glycine corresponding to the B portion of Formula 1 which is the present compound. . By reacting this compound (1) with N-hydroxysuccinimide (NHS), N-Succinimidyl-N^α-(T-butoxycarbonyl) -glycine (2) can be obtained.
N^ε-Maleoyl-L-lysine.HCl (3) is a compound corresponding to the DC part of Formula 1, and is a compound in which the amino group at the terminal of lysine (C) and the maleoyl group (D) are bonded.
The above compounds (2) and (3) are combined with N, N-diisopropylethylamine (DIEA), and N^α-(T-butyoxycarbonyl) -glycyl-N^ε-Maleyl-L-lysine (4) can be obtained. Next, this compound (4) is dissolved in anisole, trifluoroacetic acid (TFA) is added to remove t-Boc, which is a protecting group for glycine, and Glycyl-N^ε-Obtain Maleoyl-L-lysine TFA (5). This compound (5) is a compound corresponding to the DCB part of Formula 1 which is the present compound, wherein D is a maleoyl group, C is a lysine residue, and B is glycine.
[0024]
Next, [¹⁸⁶[Re] Tricarbonyl (carboxycyclopentadienyl) rhenium (CpTR-COOH) (6) and N-hydroxysuccinimide (NHS) to react with N-hydroxysuccinimidyl tricarbonyl (carbonoxy)¹⁸⁶Re] CpTR-COOSu) (7). The compound (7) is a compound corresponding to the MA part of the formula 1 which is the present compound, wherein M is rhenium, and A is a cyclopentadienyl group having a —COOSu group (Su is a succinimidyl group). To express.
This compound [¹⁸⁶Re] CpTR-COOSu (7) is converted into the above compound Glycyl-N^ε-Reaction with Maleoyl-L-lysine TFA (5)¹⁸⁶Re] CpTR-Gly-Lys (Male) -OH (8) can be obtained. This compound (8) is a compound corresponding to the DCBAM portion of Formula 1 which is the present compound.
[0025]
Fab-NH in Scheme 2₂Is a compound corresponding to the F moiety of Formula 1 which is the present compound. This Fab-NH₂Is reacted with 2-iminothiolane (2-IT) corresponding to the E moiety of formula 1 and Fab-NH-C (= NH) -CH₂-CH₂-CH₂-SH can be obtained. That is, Fab-NH-C (= NH) -CH₂-CH₂-CH₂-SH is a compound corresponding to the FE moiety of Formula 1.
Finally, Fab-NH-C (= NH) -CH corresponding to the FE moiety of Formula 1₂-CH₂-CH₂-SH and the compound corresponding to the D-C-B-A-M moiety of formula 1 [¹⁸⁶Re] CpTR-Gly-Lys (Male) -OH (8) can be reacted to obtain the compound of the present invention represented by M-A-B-C-D-F (Formula 1).
[0026]
Scheme 2 illustrates the route of synthesizing the FE moiety and linking it to the DCBAM moiety synthesized in Scheme 1 to obtain the compound of Formula 1 of the present application. When it has a functional group that can be used, it is also possible to directly bond the F portion to the DCBAM portion synthesized in Scheme 1. In this case, a compound in which E is not present in Formula 1, M-A-B-C-D-F, is obtained as the present compound.
[0027]
A pharmaceutical composition such as a radiodiagnostic agent or a radiotherapeutic agent comprising the compound of the present invention as an active ingredient is based on the nature of the physiologically active substance contained in the compound of the present application, such as tumor, inflammation, infection, cardiovascular disease, brain / It is used for diagnostic imaging or treatment of various diseases such as central diseases and organs / tissues. Since this compound is rapidly excreted outside the body without accumulating in the kidney, the radioactivity that forms the background of the abdomen is small, and therefore, the detection and diagnosis of the lesion site in the abdomen is facilitated in image diagnosis. Further, in the case of radiotherapy, exposure to the kidney can be reduced. In addition, since the dose can be reduced, the dose can be increased, and a large amount of radioactivity can be collected in the target organ to enhance the therapeutic effect.
[0028]
When the compound of the present invention is used as a radiodiagnostic agent or radiotherapeutic agent, the radioactive metal label prepared by the above-mentioned method may be used after further purification by HPLC method to remove impurities and unreacted substances. .
[0029]
The compound of the present invention can be prepared into a water-soluble radiodiagnostic agent or radiotherapeutic agent by mixing with a pharmaceutically acceptable additive. Such additives include pharmaceutically acceptable stabilizers such as ascorbic acid and p-aminobenzoic acid, pH regulators such as aqueous buffers, excipients such as D-mannitol, and radiochemical purity. Citric acid, tartaric acid, malonic acid, sodium gluconate, sodium glucoheptonate, etc. useful for improvement. Moreover, it can be provided in the form of a freeze-dried product or a frozen solution to which these additives are added.
[0030]
A radiodiagnostic agent and a radiotherapeutic agent comprising the compound of the present invention can be administered by parenteral administration such as intravenous administration or oral administration, and the dosage is determined by the patient's age, weight, appropriate radiographic imaging device, Taking into account various conditions such as the condition of the target disease and the radioactivity and dose that can be imaged and treated are determined.
When targeting humans, the dosage of the diagnostic agent using the Tc-99m label is 37 MBq to 111 MBq as the amount of radioactivity of Tc-99m. In the case of a therapeutic agent using a Re-186 or Re-188 label, the radioactivity is in the range of 37 MBq to 18500 MBq, preferably 370 MBq to 7400 MBq.
[0031]
[Example 1]
All examples described below were supplied by the Japan Atomic Energy Research Institute.¹⁸⁶ReO₄ ⁻The solution was used after neutralization with 0.01N NaOH. Na [¹²⁵I] I was purchased from ICN biomedicals, Inc. Reversed phase HPLC was performed using Cosmosil 5C.₁₈-Using AR-300 (4.6 x 150 mm, Nacalai Tesque), water (A) containing 0.1% trifluoroacetic acid (TFA) and acetonitrile containing 0.1% TFA (B ) Was eluted at a flow rate of 1.0 mL / min by the gradient method (solvent system 1) for converting A: B = 80: 20 to A: B = 20: 80 in 30 minutes. For molecular sieve HPLC, 5 Diol-300 (7.5 × 600 mm, Nacalai Tesque) was used, and elution was carried out with 0.1 M phosphate buffer pH 6.8 (solvent system 2) as an elution solvent. The eluate was fractionated at 0.5 minute intervals using a fraction collector (Amersham Biosciences, RediFrac), and the radioactivity was measured using an Autowell γ system (ARC-380M, Aloka). Thin layer chromatography (TLC) was performed on silica gel plates (Silica gel 60F).₂₅₄, Merck), and chloroform (solvent system 3) or physiological saline (solvent system 4) as a developing solvent. SepPak plus (C18 short body; 360 mg / cartridge, Waters)₂Used after pretreatment with flowing 6 mL of O. H after adding sample₂Eluted with 5 mL O and then 2-3 mL ethanol. The first 100 μL was discarded. Tricarbonyl (cyclopentadienyl carbonate) rhenium (Tricarbonyl (cyclopentadienyl carbonate) rhenium ([¹⁸⁶Re] CpTR-COOH)), and tricarbonyl [(cyclopentadienylcarbonylamino) acetate] rhenium (Tricarbonyl [(cyclopentadienylcarbonylamino) acetate] rhenium (CpTR-Gly)), a method previously reported by the present inventors. (See Non-Patent Documents 25 and 26). All other reagents were used as they were.
[0032]
CpTR − COOH Synthesis of (6):
(1) [¹⁸⁶[Re] Tricarbonyl (methycarbonylcyclopentadienyl) rhenium (CpTR-COOMe)
This compound was synthesized by slightly modifying the method previously reported by the present inventors (see Non-Patent Document 25).
1,1′-bis (methoxycarbonyl) ferrocene (12 mg, 40 μmol), hexacarbonylchromium (19 mg, 86.3 μmol), tin (II) chloride in a portable reactor (0.8 × 8.5 cm; pressure glass industry) Anhydride (37 mg, 195.1 μmol) was added. [¹⁸⁶Re] ReO₄ ⁻The aqueous solution was distilled off under vacuum, extracted into dry methanol (500 μL), and added to the portable reactor. After sealing the container, the mixture was reacted for 60 minutes in an oil bath at 200 ° C. with stirring. After cooling on ice for 10 minutes or more and returning to room temperature, the reaction solvent was distilled off under reduced pressure. The residue was dissolved in chloroform and purified by silica gel chromatography using chloroform as an elution solvent.¹⁸⁶Re] CpTR-COOMe was obtained. The radiochemical yield (63%) and radiochemical purity (95% or more) were determined by TLC (solvent system 3).
[0033]
(2) [¹⁸⁶[Re] Tricarbonyl (carbocycle diadenyl) rhenium (CpTR-COOH) (6)
Then, [¹⁸⁶Re] CpTR-COOMe was dissolved in dioxane (200 μL) and 2N NaOH aqueous solution (600 μL) was added. After stirring at room temperature for 10 minutes, conc. Acidify the reaction with HCl and purify using an activated SepPak column, [¹⁸⁶Re] CpTR-COOH (6) was obtained. The radiochemical yield (95.2%) and the radiochemical purity (95% or more) are TLC (solvent).
It was determined by system 3).
[0034]
[Example 2]
N ^α -(T-butyoxycarbonyl) -glycyl-N ^ε -Maleyl-L-lysine Synthesis of (4):
(1) N-Succinimidyl-N^αSynthesis of-(t-butoxycarbonyl) -glycine (2)
N^α-(T-butyoxycarbonyl) -glycine (1) (0.8 g, 4.57 mmol) and N-hydroxysuccinimide (NHS, 0.55 g, 4.78 mmol) were dissolved in N, N'-dimethylformamide (DMF, 20 mL). The mixture was ice-cooled, 1,3-dicyclohexylcarbodiimide (DCC, 1.03 g, 5.04 mmol) was added at 0 ° C., and the mixture was stirred at 0 to 5 ° C. for 4 hours and then at room temperature for 16 hours. The deposited precipitate was filtered, and the filtrate was distilled off under reduced pressure.
Recrystallization from isopropyl alcohol gave 1.03 g (yield: 83.6%) of the compound (2) as white crystals.
¹H-NMR (CDCl₃): Δ 1.44 (9H, s, Boc), 2.80 (4H, d, succinimide), 4.20 (2H, d, CH₂)
FAB-MS: Theoretical value (C₁₁H₁₇N₂O₆  (M + H)⁺): M / z 273, measured value: 273
[0035]
(2) N^α-(T-butoxycarbonyl) -glycyl-N^ε-Synthesis of maleyl-L-lysine (4)
N synthesized according to the method of Wakisaka et al. (See Non-Patent Document 27)^ε-Maleoyl-L-lysine · HCl (3) (200 mg, 0.76 mmol) was dissolved in DMF (5 mL), and N, N′-diisopropylethylamine (DIEA, 132.9 μL, 0.78 mmol) was added. Compound (2) (172.7 mg, 0.63 mmol) dissolved in DMF (5 mL) was added dropwise and stirred overnight at room temperature. After the reaction solution was distilled off under reduced pressure, the residue was dissolved in ethyl acetate and washed three times with sulfuric acid diluted to pH 5-6. The filtrate was dried over anhydrous calcium sulfate and filtered, and the filtrate was distilled off under reduced pressure to obtain 155.7 mg (yield 64.0%) of the above compound (4) as a colorless oil.
¹H-NMR (CDCl₃): Δ 1.30-1.91 (6H, m, (CH₂)₃), 1.41 (9H, s, Boc), 4.52 (1H, s, CH), 5.60 (1H, s, NH), 6.67 (2H, s, maleimide), 7.08 ( 1H, s, NH)
FAB-MS: Theoretical value (C₁₇H₂₆N₃O₇  (M + H)⁺) M / z 384, measured value: 384
[0036]
[Example 3]
[ ¹⁸⁶ Re] CpTR-Gly-Lys (Male) -OH Synthesis of (8):
(1) Glycyl-N^ε-Synthesis of Maleoyl-L-lysine TFA (5)
The compound (4) obtained in Example 2 (191.5 mg, 0.50 mmol) was dissolved in anisole (50 μL), trifluoroacetic acid (TFA, 950 μL) was added little by little, and the mixture was stirred as it was at room temperature. 1 hour later, N₂TFA was distilled off using gas and ether was added. The precipitated crystals were collected by filtration to give a white crystalline compound Glycyl-N.^ε-Maleoyl-L-lysine.TFA (5) 112.6 mg (yield 66.2%) was obtained.
¹H-NMR (CD₃OD): δ 1.33-1.95 (6H, m, (CH₂)₃), 4.42 (1H, s, CH), 6.68 (2H, s, maleimide)
FAB-MS: Theoretical value (C₁₂H₁₈N₃O₅  (M + H)⁺) M / z 284, measured value: 284
[0037]
(2) N-Hydroxysuccinimidyl tricarbonyl (carboxycyclopentadienyl) rhenium ([¹⁸⁶Re] CpTR-COOSu) (7)
[¹⁸⁶Re] CpTR-COOH (6) was dissolved in dichloromethane (200 μL), NHS (2.0 mg, 17.3 μmol) and DCC (2.0 mg, 9.7 μmol) were added, and the mixture was stirred at room temperature for 5 minutes. N₂After removing the solvent with gas, it was dissolved in acetonitrile and purified by RP-HPLC. The radiochemical yield (76%) and the radiochemical purity (95% or more) were each determined by TLC (solvent system 3) using chloroform as a developing solvent.
[0038]
(3) [¹⁸⁶Synthesis of Re] CpTR-Gly-Lys (Male) -OH (8)
Glycyl-N^ε-Maleyl-L-lysine · TFA (5) (12.5 mg, 36.7 μmol) was dissolved in N, N′-dimethylformamide (DMF; 80 μL), and N, N′-diisopropylethylamine (DIEA; 6.3 μL) was dissolved. , 36.7 μmol) was added little by little and then dissolved in DMF (90 μL) [¹⁸⁶Re] CpTR-COOSu (7) was added in small portions. After stirring at room temperature for about 1 hour, the mixture was purified by reverse phase HPLC (RP-HPLC). RP-HPLC is Cosmosil 5C₁₈-Using AR-300 (4.6 x 150 mm, Nacalai Tesque), water (A) containing 0.1% trifluoroacetic acid (TFA) and acetonitrile (B) containing 0.1% TFA were A: B = Elution was performed at a flow rate of 1.0 mL / min by the gradient method in which 80:20 was converted to A: B = 20:80 in 30 minutes. The radiochemical yield (49.6%) and the radiochemical purity (91% or more) were determined by TLC (solvent system 3) using chloroform as a developing solvent.
[0039]
[Example 4]
[ ¹⁸⁶ Re] CpTR-Gly-Lys (Male) -IT-Fab Synthesis of:(1) Thiolation of Fab fragment (synthesis of 2-iminothiolane (2-IT) -conjugated Fab)
Fab was prepared using a 0.16M borate buffer solution (pH 8.5) containing 2 mM ethylenediaminetetraacetic acid (EDTA) (1.0 mg / mL, 200 μL), and 2-iminothiolane dissolved in the same buffer solution ( 2-IT; 2.5 mg / mL, 7.4 μL) was added and gently stirred for 30 minutes. Excess 2-IT was removed by a spin column method using Sephadex G-50 equilibrated with 0.1 M phosphate buffer (pH 6.0) containing 2 mM EDTA. The number of thiols introduced per molecule was measured using 2,2'-dipyridyl disulfide (DPS). As a result, the antibody Fab fragment thiolated with 2-IT had 3.0 thiol groups per antibody molecule.
[0040]
(2) [¹⁸⁶[Re] CpTR-Gly-Lys (Male) -IT-Fab Synthesis
[¹⁸⁶The above thiolated Fab (1 mg / mL, 100 μL) was added to [Re] CpTR-Gly-Lys (Male) -OH (8) and gently stirred for 90 minutes. Subsequently, iodoacetamide (14.8 μL, 10.0 mg / mL) dissolved in 0.1 M phosphate buffer (pH 6.0) was added and stirred for 30 minutes. Purification was carried out by spin column method using Sephadex G-50 equilibrated with 0.5 M acetate buffer (pH 6.0).
To the thiolated antibody Fab fragment obtained in (1) above, [¹⁸⁶[Re] CpTR-GK (M) -OH (8)¹⁸⁶Re] CpTR-GK (M) -IT-Fab was obtained with a radiochemical yield of 30% and a radiochemical purity of 90% or higher.
[0041]
[Example 5]
[ ¹⁸⁶ Re] CpTR-Fab Synthesis of:
Obtained in Example 3- (2) [¹⁸⁶Re] CpTR-COOSu (7) was dissolved in 10 μL of DMF and slowly added to 100 μL of Fab solution (1 mg / mL, 0.16 M borate buffer pH 9.5). After 1 hour, purification was performed by a spin column method using Sephadex G-50 equilibrated with 0.5 M acetate buffer (pH 6.0). The radiochemical yield and radiochemical purity were each determined by TLC using physiological saline as a developing solvent. as a result,[¹⁸⁶[Re] CpTR-Fab is [¹⁸⁶Re] CpTR-COOSu (7) and antibody Fab fragment were combined to give a radiochemical yield of 30% and a radiochemical purity of 95% or more.
[0042]
[Example 6]
[ ¹²⁵ I] HML-IT-Fab Synthesis of:
[¹²⁵I] HML-IT-Fab was synthesized according to the method of Fujioka et al. (See Non-Patent Document 18).
An organotin precursor of HML (ε-N-maleyl-L-lysine) is dissolved in methanol containing 1% acetic acid, [¹²⁵I] NaI and the oxidizing agent N-chlorosuccinic acid were added,¹²⁵I] HML was synthesized. this[¹²⁵I] A thiolated Fab was added to HML in the same manner as in Example 5, and purified by spin column method [¹²⁵I] HML-IT-Fab was obtained.
[0043]
[Example 7]
Mouse biodistribution test
Obtained in Example 4 [¹⁸⁶Re] CpTR-Gly-Lys (Male) -IT-Fab and obtained in Example 6 [¹²⁵I] HML-IT-Fab mixed solution or obtained in Example 5 [¹⁸⁸Re] CpTR-Fab was each dissolved in physiological saline, and administered to the group of 3-4 6-week-old ddY male mice via the tail vein at 11.1 kBq (100 μl) per mouse. At 10, 30 minutes after administration, 1, 3, and 6 hours after sacrifice, the organs were removed and the weight and radioactivity were measured. In addition, feces and urine excreted by 6 hours after administration were collected and the radioactivity was measured. Further, urine excreted by 6 hours after administration was filtered through a 0.45 μm filter, and then analyzed by molecular sieve HPLC. The low molecular fraction was analyzed by filtering urine with a 10-kDa ultrafiltration membrane and analyzing the filtrate by reverse phase HPLC to examine the chemical form of radioactivity excreted in urine. At this time, reverse phase HPLC was performed using Cosmosil 5C.₁₈-Using AR-300 (4.6 x 150 mm, Nacalai Tesque), water (A) containing 0.1% trifluoroacetic acid (TFA) and acetonitrile (B) containing 0.1% TFA A: Elution was performed at a flow rate of 1.0 mL / min by the gradient method in which B = 80: 20 to A: B = 20: 80 in 30 minutes. Molecular sieve HPLC was performed using 5 Diol-300 (7.5 × 600 mm, Nacalai Tesque) and eluting with 0.1M phosphate buffer pH 6.8 as an elution solvent. The eluate was fractionated at 0.5 minute intervals using a fraction collector (Amersham Biosciences, RediFrac), and the radioactivity was measured using an Autowell γ system (ARC-380M, Aloka).
[0044]
In normal mice [¹⁸⁶Re] CpTR-Gly-Lys (Male) -IT-Fab, [¹⁸⁶Re] CpTR-Fab, [¹²⁵I] The results of examining the in vivo radioactivity distribution of HML-IT-Fab are shown in Table 1-3. The value of the radioactivity in the table is the average value (standard deviation) expressed as% ID / g (percentage of total administered radioactivity per organ weight (g)). The rate of disappearance of any RI-labeled antibody fragment from blood was similar. In addition, no difference was observed in the radioactivity accumulation of any RI-labeled antibody fragment in the liver or intestine. on the other hand,[¹⁸⁶[Re] CpTR-Fab and [¹⁸⁶A significant difference was observed in the accumulation of radioactivity in the kidney with [Re] CpTR-Gly-Lys (Male) -IT-Fab from the early stage of administration.
[¹⁸⁶Re] CpTR-Fab showed accumulation in the kidney with a maximum value of 30 minutes after administration.¹⁸⁶[Re] CpTR-Gly-Lys (Male) -IT-Fab is [¹²⁵I] Similar to HML-IT-Fab, the radioactivity of the kidney was greatly reduced from the early stage of administration.
[0045]
[Table 1]

[Table 2]

[Table 3]

[0046]
[Example 8]
Estimated comparison of kidney dose
As shown in Example 7, [¹⁸⁶When Re] CpTR-Fab was administered to mice, radioactive accumulation in the kidney and subsequent loss was observed. on the other hand,[¹⁸⁶[Re] CpTR-Gly-Lys (Male) -IT-Fab is [¹²⁵I] After administration to mice in the same manner as HML-IT-Fab, the accumulation of radioactivity in the kidney was reduced early (FIG. 1). The square mark in FIG.¹⁸⁶[Re] CpTR-Gly-Lys (Male) -IT-Fab administration group, triangle mark is [¹²⁵I] HML-IT-Fab administration group, circles are [¹⁸⁶The Re] CpTR-Fab administration group is shown.
From the comparison of the area of renal accumulation of each labeled antibody,¹⁸⁶[Re] CpTR-Gly-Lys (Male) -IT-Fab¹⁸⁶It was shown to reduce to 1/3 or less of [Re] CpTR-Fab.
[0047]
[Example 9]
Analysis of urinary radioactivity
[¹⁸⁶Re] CpTR-Gly-Lys (Male) -IT-Fab was administered to the tail vein of mice, and the amount of radioactivity excreted in urine by 6 hours was analyzed (FIG. 2). When analyzed by molecular sieve HPLC, 50% or more of the radioactivity was eluted in the low molecular fraction (FIG. 2 (A)). Furthermore, when the low molecular fraction eluted by the ultrafiltration membrane having a molecular weight of 10,000 cut was analyzed by RP-HPLC, 65% or more of the radioactivity was eluted at a retention time corresponding to CpTR-Gly (FIG. 2 ( B)).
[Brief description of the drawings]
FIG. 1 is a diagram of [¹⁸⁶Re] CpTR-Gly-Lys (Male) -IT-Fab, [¹²⁵I] HML-IT-Fab, [¹⁸⁶It is the figure which showed the biodistribution of the radioactivity amount of the mouse | mouth after administration of [Re] CpTR-Fab. % ID / g is a value indicating the average value of the percentage of the total administered radioactivity per organ weight (g).
FIG.¹⁸⁶It is the figure which showed the amount of radioactivity excreted in the urine of a mouse | mouth by 6 hours after administration of [Re] CpTR-Gly-Lys (Male) -IT-Fab. (A) is the result analyzed by molecular sieve HPLC, and (B) is the result analyzed by reverse phase HPLC (RP-HPLC).

Claims (17)

M-A-B-C-D-E-F (Formula 1)
(In Formula 1,
M: Technetium-99m (Tc-99m), rhenium-186 (Re-186)
Or a radiometal A selected from rhenium-188 (Re-188): a group B derived from a cyclopentadienyl tricarbonyl group or a cyclopentadienyl tricarbonyl group: any amino acid residue C: any amino acid residue Group D: Spacer 1
E: Spacer 2 (however, if D and F can be directly bonded, spacer 2 may not be present)
F: protein, such as an antibody or antibody fragment, or physiologically active substance such as a peptide)
A diagnostic and therapeutic drug comprising a radiometal-labeled cyclopentadienyl tricarbonyl compound represented by the formula: The drug of claim 1, wherein BC is an amino acid sequence that is selectively cleaved by a kidney enzyme. The agent according to claim 1, wherein the group derived from the cyclopentadienyl tricarbonyl group of A is a cyclopentadienyl tricarbonyl group having a carbonyl group. The drug according to claim 1, wherein the amino acid residue of B is a glycine residue. The drug according to claim 1, wherein the amino acid residue of C is an amino acid residue selected from lysine, phenylalanine or tyrosine. The drug of claim 1, wherein B-C is glycyl-lysine. D is a spacer that forms a covalent bond with amino acid residue C and has a functional group that can bind to spacer E, or a spacer that can directly bind to bioactive substance F having a functional group that can bind to D. Item 1. Drug of item 1. The drug according to claim 7, wherein D is a spacer selected from a group derived from a maleoyl group or a carboxylic acid active ester. The agent of claim 1, wherein E is a spacer having two functional groups covalently bound to D and F. 10. The agent of claim 9, wherein E is iminothiol or E is not present as a spacer. The drug according to claim 1, wherein F is a physiologically active substance selected from a peptide such as a monoclonal antibody, a polyclonal antibody, albumin, or other plasma protein, or a peptide such as somatostatin, chemotactic factor, laminin and the like. M-A-B-C-D (Formula 2)
(In Formula 2,
M: Radiometal selected from technetium-99m (Tc-99m), rhenium-186 (Re-186) or rhenium-188 (Re-188) A: cyclopentadienyl tricarbonyl group or cyclopentadienyl tricarbonyl Group B derived from a group: any amino acid residue C: any amino acid residue,
D: Spacer 1
Indicate)
A cyclopentadienyl tricarbonyl compound labeled with a radioactive metal. 13. The compound according to claim 12, wherein the group derived from the cyclopentadienyl tricarbonyl group of A is a cyclopentadienyl tricarbonyl group having a carbonyl group. The compound of claim 12, wherein the amino acid residue of B is a glycine residue. The compound of claim 12, wherein the amino acid residue of C is an amino acid residue selected from lysine, phenylalanine or tyrosine. 13. The compound of claim 12, wherein D is a spacer selected from a maleoyl group or a group derived from a carboxylic acid active ester. A process for producing a radiometal-labeled cyclopentadienyl tricarbonyl compound represented by formula 2 of claim 12,
M-A-COOR (M represents a radioactive metal according to claim 12, A represents a cyclopentadienyltricarbonyl group, R represents an alkyl group or an N-hydroxysuccinimidyl group), and H -B-C-D (B, C and D are as defined in claim 12, H represents a hydrogen atom),
M-A-B-C-D (Formula 2)
(M, A, B, C and D are the same as in claim 12).

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