JP5263805B2 - Polymer complexes stabilized by amphiphilic polymer ligands and test and pharmaceutical compositions - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a polymer complex as a novel biomaterial that can quickly, easily and stably coordinate to a metal atom even under a mild condition, and a pharmaceutical composition utilizing the same. <P>SOLUTION: Disclosed is a polymer complex comprised of at least one metal atom and an amphipathic block copolymer comprising a hydrophobic ligand and a hydrophilic polymer, more specifically, a polymer complex comprised of at least one species of metal atoms and an amphipathic block copolymer comprising a hydrophilic polymer and a hydrophobic ligand consisting of a peptide or a depsipeptide having a helical structure comprising an amino acid having a heterocycle whose side chain can coordinate to a metal atom. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

The present invention relates to a polymer complex using an amphiphilic ligand and a composition thereof. More specifically, the present invention relates to a polymer complex composed of a polymer ligand made amphiphilic by a block copolymer of a hydrophobic ligand and a hydrophilic polymer, and a metal atom coordinated thereto, and a composition thereof. .
Since the polymer complex of the present invention can easily and stably coordinate metal atoms under mild conditions even in an aqueous environment, as a biomaterial or pharmaceutical composition, more specifically, a radiopharmaceutical, Paramagnetic agents and fluorescent agents are useful for diagnosis and treatment of various abnormal tissues, particularly for diagnosis and treatment of certain types of cancer.

(1) Metallic proteins in terms of metal atoms and polymer complexes:
Proteins derived from living organisms that bind metal atoms relatively tightly are called metal proteins, and function as an enzyme or electron carrier that transports and stores metal ions and has an active center due to metal atoms. (For example, Non-Patent Document 1). Amino acid side chains and main chains constituting proteins are mainly used for the binding sites of metal atoms. In particular, histidine having a heterocyclic imidazole group is excellent as a ligand for a nitrogen atom, and is considered to have been used for many metalloproteins in the course of chemical evolution of metalloenzymes (for example, Non-Patent Document 2). .

An overview of metal proteins that have metal atoms bonded by histidine side chains.
(a) Fe protein
Heme protein in which Fe ions are bound as heme (Fe-porphyrin complex), which is a low-molecular metal complex, depending on the form of Fe atom binding, non-heme in which Fe ions are bound to protein without heme Proteins are classified as iron-sulfur proteins, in which Fe ions are bound to proteins as clusters with sulfur atoms. In cytochrome c of the heme protein, the nitrogen atom of the imidazole group of histidine is coordinated to the Fe ion in heme (for example, Non-Patent Document 3). Coordination of histidine side chains to the two Fe ions of methane monooxygenase (for example, Non-Patent Document 4) for non-heme proteins, and [4Fe4S] cluster of hydrogenase (for example, Non-Patent Document 5) for iron-sulfur proteins It can be seen.

(b) Cu protein Cu proteins called Type I, Type II, and Type III, which have been spectroscopically classified before the crystal structure is elucidated, all have histidine side chain coordination. In plastocyanin classified into Type I (for example, Non-Patent Document 6), two nitrogen atoms of imidazole groups are coordinated. In addition, galactose oxidase (for example, Non-Patent Document 7) can be cited as Type II, and hemocyanin having two Cu ions as Type III (for example, Non-Patent Document 8). In addition, ascorbate oxidase (for example, Non-Patent Document 9) and nitrite reductase (for example, Non-Patent Document 10), which are multi-Cu proteins, also show coordination of histidine side chains.

(c) Zn protein In carboxypeptidase A (for example, Non-Patent Document 11), carbonic acid dehydrogenase (for example, Non-Patent Document 12), and alcohol dehydrogenase (for example, Non-Patent Document 13), the Zn ion at the enzyme active center The reactivity is controlled by coordination of 1 to 3 imidazole groups. Unlike enzymes, histidine side chain coordination is also found in proteins called zinc fingers (for example, Non-Patent Document 14) that bind to DNA and perform gene expression and control.

(d) Other metal proteins In urease having a plurality of Ni ions (for example, Non-Patent Document 15) and nitrogenase having a cluster composed of Fe-Mo-S (for example, Non-Patent Document 16), a metal atom Coordination bond of nitrogen atom of imidazole group is known.

  In the above example where a nitrogen atom is coordinated to a metal atom, a common structural feature for strongly holding the metal atom can be considered. (A) A strong coordination bond between a metal atom and an imidazole ring (heterocyclic structure). (b) Chelate effect by protein polypeptide chain as a polymer ligand. It is considered to be characterized by four points: (c) protein amphiphile structure as a polymer ligand, and (d) highly molecularly designed polymer ligand (coordination bond, chelate effect, amphiphile structure). It is done.

(a) and (b) are widely used as a basic concept of coordination chemistry to stabilize not only metal proteins but also low-molecular metal complexes. The relatively strong coordination bond to the metal atom by the imidazole ring in (a) is not only the σ bond that σ-donates the lone pair of nitrogen atom to the metal atom, but also the d orbital of the metal atom and the p orbital of the heterocyclic ring. This is because there is a π bond. There are two types of π bonds: π-back donation of electrons from the filled d orbitals to the empty π ^* orbitals of the heterocyclic ring, and π from the heterocyclic π orbitals to the empty d orbitals of the metal atoms. There is a donation. A heterocyclic structure such as an imidazole ring can cope with these two types of π bonds depending on the oxidation state of the metal atom, so that it is widely suitable for coordination bonds regardless of the type of metal atom. . As a result, it is considered that a heterocyclic structure such as an imidazole ring has been found in many metalloprotein ligands in the course of chemical evolution.

  The chelating effect (b) can be explained mainly by the entropy effect (Non-patent Document 17). That is, the stability of a metal atom in a protein is derived from a chelate structure in which a ligand is linked by a polypeptide chain. In addition, the enthalpy effect derived from the difference in ligand coordination energy, such as the difference between water molecules and imidazole rings, contributes to the retention of metal atoms in proteins as part of the chelate effect.

  The protein amphiphile structure in (c) is a hydrophilic environment because the protein outer shell dissolves in an aqueous environment, and the active center is in a hydrophobic environment to strengthen the coordination bond between the metal atom and the nitrogen atom. This can be understood (Non-patent Document 18). The reason why the hydrophobic environment occurred in the process of chemical evolution is that it prevents water molecules from entering the metal protein, and the water molecules that occupy most of the aqueous solution (approximately 50 mol / L) coordinate to the metal atoms. It can be considered that this is to prevent the separation of the metal atom from the active center.

  When the metal protein is considered as a polymer complex as described above, it can be understood that the polypeptide chain stabilizes the metal atom as an amphiphilic ligand. However, there have been no examples of utilizing the metal atom retention mechanism by metalloproteins as biomaterials or pharmaceutical compositions.

(2) Drugs in terms of metal atoms and polymer complexes:
Conventionally, various ligands that form complexes are used when using radiation generated from metal atoms as a pharmaceutical (disruption of abnormal tissue) or when binding to other pharmaceuticals as a tracer (detection of abnormal tissue). I came. These ligands are generally called “chelating agents” even when they do not use the chelating effect in a strict sense, and are important development fields of radiopharmaceuticals.

A radiopharmaceutical is a drug aimed at “detection” and “destruction” of an abnormal tissue in a living body by radiation. Research on this pharmaceutical includes (a) production of radioisotopes, (b) development of polymer probes (eg, monoclonal antibodies) used for tissue detection, and (c) combination with polymer probes used for tissue detection. Three important points are the development of “chelating agents” to be used. For example, when a metal element is used for the radioisotope, the monoclonal antibody and the metal atom are not directly complexed, but the metal atom is complexed to the chelating agent bound to the monoclonal antibody. This method is widely used as radiation therapy or radiation diagnosis in which abnormal tissue is destroyed by radiation or an image of the abnormal tissue is created (for example, Patent Documents 1 to 5). In addition, diagnostic imaging of abnormal tissues can be performed in the same manner using a polymer complex of paramagnetic metal ions, “chelators” and antibodies instead of radioisotopes (MRI method). This is also actively studied as a radiopharmaceutical in a broad sense (for example, Patent Documents 6 to 9).

  Conventionally, “chelating agents” that have been used in clinical and research as radiopharmaceuticals are EDTA type ligands (for example, DTPA (= diethylenetriaminepentaacetic acid). Multidentate coordination, such as ligands (eg, DOTA (= 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)) that forms chelate compounds by coordination with metal ions It is a child (for example, Patent Documents 10 to 12). These “chelating agents” are hydrophilic ligands and are metal amphiphilic polymer complexes in that they stabilize metal atoms using only the chelating effect of multidentate ligands. It is very different from protein.

(3) Disadvantages of “chelating agents” as pharmaceutical compositions:
Of the conventional “chelating agents”, DTPA, which is widely used, is characterized in that there are many examples of use. However, because it is a hydrophilic low molecular weight ligand consisting of carboxylic acid and tertiary amine, (a) it is not a ligand tailored to the type of metal atom and oxidation state as seen in metal proteins. There are three problems: (b) exchange reaction with a more stable metal ion, and (c) bond formation with a polymer probe using DTPA anhydride. (a) and (b) can be problematic because metal ions dissociate from the “chelating agent” and accumulate in specific tissues (although it is not DTPA, it is a research example of ⁶⁴ Cu and a macrocyclic chelate ligand. Patent Document 20). (c) has the disadvantages that the reactivity of the DTPA anhydride used to generate the monoclonal antibody-chelating agent bond is low, and the pH must be reacted for a long time in an alkaline condition of 8.5 to 9.0. .

  In addition, relatively new “chelators”, macrocyclic chelate ligands (eg, DOTA) are characterized by high stability of the complex. However, when (a) metal ions are introduced, complex formation does not occur at 4 ° C to room temperature at all, or it takes a very long time. (B) It is necessary to apply heat at 50 to 80 ° C for a short time. There are two points as problems (for example, Non-Patent Document 21). This is because when a metal ion is introduced into a monoclonal antibody-chelating agent-complex used for detection or destruction of abnormal tissue, there is a concern that the protein structure is denatured due to high temperature and the antibody titer is lowered.

  In order to solve the problems of these “chelating agents”, a macrocyclic chelating ligand with improved DOTA has been studied (for example, Non-Patent Document 22). However, the basic skeleton and coordinating atoms are the same, and in terms of stabilizing metal atoms using only the chelate effect of multidentate ligands, it can be expected that conventional “chelating agents” will not be greatly improved. Conceivable.

(3) Advantages of metalloproteins as polymer complexes:
Metallic proteins do not require high-temperature treatment in a reconstitution reaction in which metal atoms are introduced into polypeptide chains that do not have metal atoms (apoproteins). In this reconstitution reaction, metal atoms are often introduced very stably (generation of holoprotein) in a very short time even at room temperature. Thus, from the viewpoint of the polymer complex, the metal protein has extremely attractive characteristics. However, there has been no example in which these properties are used as biopolymer materials or pharmaceutical compositions.

(4) Advantages of metalloproteins as polymer complexes:
When an amphiphilic polymer compound is dispersed in water, a plurality of polymer chains associate to form a micelle structure having a hydrophobic inner core and a hydrophilic outer shell. In general, polymer micelles have a critical micelle concentration of 1 / 1,000-1 / 10,000 of low molecular micelles represented by soap. In addition, the particle size of the polymer micelle is in the range of 10-100 nm and easily leaks from the blood vessel wall of cancer tissue or inflammatory tissue (EPR effect; for example, Non-Patent Document 23). As a result, it is known that effective drugs encapsulated in micelles can be accumulated in these abnormal tissues and the drug can be exposed to the abnormal tissues over a long period of time.

Examples of amphiphilic polymers that form polymer micelles include polylactic acid-block-polyethylene glycol block copolymers (for example, Non-Patent Document 24), poly (γ-benzyl glutamic acid) -block-polyethylene glycol (for example, , Patent Document 13), and poly (β-benzylaspartic acid) -block-polyethylene glycol (for example, Patent Document 14; Non-Patent Document 25) is well known. Furthermore, it is also known that an anticancer agent is carried or encapsulated in a hydrophobic inner core (Patent Documents 15 and 16), and exhibits tissue selectivity to cancer tissue and anticancer activity (for example, Non-Patent Document 26; Patent Documents 17 and 18). The disadvantage of these amphiphilic polymer compounds is that the hydrophobic sequence is functioned by polymer polymerization reaction (polylactic acid is ring-opening polymerization of lactide, poly (βbenzylaspartic acid) is NCA polymerization method). Sex is imparted by random chemical modification. Therefore, it has been impossible to precisely design the metal atom retention mechanism with an arbitrary molecular structure.
No. 6-47560 No. 6-51720 JP 6-76440 7-62032 JP-A-8-325297 US Patent 5750660 US Pat. US Patent 5,316,757 US Patent 5362476 US Patent 5342606 US Patent 5428139 US Patent 5,316,757 JP-A-5-955 Japanese Patent Laid-Open No. 6-206832 JP-A-11-335267 JP-A-6-107565 JP-A-6-206815 JP-A-5-117385 Harry B. Gray, Proceedings of National Academy of Science of the United States of America, 2003, vol. 100, pp. 3563-3568. Fujio Egami, Journal of Biochemistry, 1975, vol. 77, pp. 1165-1169. Ashida Tamaichi et al., Journal of Biochemistry, 1973, vol. 73, pp. 463-465. Amy C. Rosenzweig et al., Nature, 1993, vol. 366, pp. 537-543. Anne Volbeda et al., Nature, 1995, vol. 373, pp. 580-587 Thomas P. Garrett et al., Journal of Biological Chemistry, 1984, vol. 259, pp. 2822-2825. Nobutoshi Ito et al., Nature, 1991, vol. 350, pp. 87-90. Wil P. Gaykema et al., Journal of Molecular Biology, 1986, vol. 187, pp. 255-275. Albrecht Messerschmidt et al., Journal of Molecular Biology, 1989, vol. 206, pp. 513-529. Jean LeGall et al., Science, 1991, vol. 253, pp. 438-442. DC Rees et al., Journal of Molecular Biology, 1982, vol. 160, pp. 475-498. Proceedings of National Academy of Science of the United States of America, 1975, vol. 72, pp. 51-55. JP Samama et al., European Jounal of J Biochemistry, 1977, vol. 81, pp. 403-409. NP Pavletich et al., Science, vol. 252, pp. 809-817. E. Jabri et al., Science, 1995, vol. 268, pp. 998-1004. Georgiadis et al., Science, 1992, vol. 257, pp. 1653-1659. Inorganic Chemistry: Principles of Structure and Reactivity, Fourth Edition; JE Huheey, EA Keiter, RL Keiter Eds .; Harper Collins College Publishers: New York, 1993; pp. 522-531. Hiroyuki Oku, Doctral Thesis, Osaka University, 1995, pp. 1-4. Yasushi Arano et al., Journal of Medicinal Chemistry, 1996, vol. 39, pp. 3451-3460. Laura A. Bass et al., Bioconjugate Chemistry, 2000, vol. 11, pp 527-532. JB Stimmel et al., Bioconjugate Chemistry, 1995, vol. 6, pp. 219-225. David A. Keire et al., Inorganic Chemistry, 2001, vol. 40, pp. 4310-4318. Maeda Hiroshi et al., Journal of Controlled Release, 2000, vol. 65, pp. 271-284. Yukio Nagasaki et al., Macromolecules, 1998, vol. 31, pp. 1473-1479 Kwon GS et al., Pharmaceutical Research, 1995, vol. 12, pp. 192-195. Yokoyama M. et al., Journal of Drug Targeting, 1999, vol. 7, pp. 171-186

  An object of the present invention is to provide a polymer complex as a novel biomaterial capable of rapidly and simply and stably coordinating metal atoms even under mild conditions, and a pharmaceutical composition using the same. is there.

  In order to achieve the above object, the present inventors paid attention to the structural features of metalloproteins. That is, attention was paid to a feature that has not been noticed in the past, (c) amphiphilic structure of a protein as a polymer ligand. In addition to this, two features that are also used in low molecular weight ligands: (a) relatively strong coordination bonds between metal atoms and heterocyclic structures (pyridine ring and imidazole ring) and (b) polypeptide chain The chelating ligand according to We planned the chemical synthesis of novel polymer complexes with ligands that combine these three properties.

So far, the present inventors have a β- (3-pyridyl) -L-alanine (Pal) residue as a metal atom binding site, and the sequence of -Leu-Leu-Aib- for Helix induction. The introduced peptide Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-OEt (3) (Boc-SEQ ID NO: 11-OEt) has been studied. This compound has excellent solubility in organic solvents and forms a 1: 1 chelate complex with CoCl _{2 in} CH ₃ CN (eg, Ryo Nakai et al., Peptide Science 2002, The Japanese Peptide Society, 2003, pp. 313-316.).
Therefore, the chelating amphiphilic polymer ligand Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-, in which polyethylene glycol ₄₀₀₀ (PEG ₄₀₀₀ ) is bound to the C-terminus of peptide 3. Aib-Phe-PEG ₄₀₀₀ (chelate type, 2) (Boc-SEQ ID NO: 12-PEG ₄₀₀₀ ) was synthesized.

Peptide 2 was synthesized by the liquid phase method. Peptide 2 with the above sequence attached on PEG ₄₀₀₀ is activated in order from the N-terminus by activating the carboxy terminus of each fragment Boc-Leu-Leu-Aib-OH or Boc-Pal-OH with N-hydroxysuccinimide (HOSu) as a HOSu ester. The reaction was performed after isolation. Boc-Pal-OSu was used in the reaction as it was without isolation. As a result, chelate-type amphiphilic polymer ligand 2 (0.74 g, 0.13 mmol) was obtained using PEG (4.0 g, 1.0 mmol) as a starting material. The product at each reaction step was followed by mass spectrometry (MALDI-TOF-MS). As a result, a change in molecular weight corresponding to the elongation of the fragment or amino acid was detected, and the production of the target product was confirmed (FIG. 1).

The resulting chelate-type amphiphilic polymer ligand 2 formed micelles in an aqueous solution (FIG. 2, FIG. 3, critical micelle concentration). This ligand was found to be able to stably coordinate various metal ions at the hydrophobic part to form a polymer complex (FIGS. 4 to 6, complex formation constant with CoCl ₂ , K = [ CoCl ₂ -2] / [CoCl ₂ ] [2] = 1.2 x 10 ⁵ M ^-1 ). Subsequently, the dynamics in the mouse body by a polymer complex using ¹¹¹ InCl ₃ as a radioactive metal ion was examined (Tables 1 and 2). As a result, it was found that a large amount of polymer complex was excreted within 24 hours in other tissues although it accumulated a little in the kidney.

In order to confirm accumulation in the tumor tissue, mice were examined by gamma rays ( ¹¹¹ complex with ¹¹¹ InCl ₃ , ¹¹¹ In-2) and fluorescence (polymer ligand with carboxyfluorescein, CF-2). The kinetics in the body was examined (FIG. 9, Table 3). As a result, the tumor tissue was successfully detected by a method using γ-rays and fluorescence images.
As described above, the present invention has been completed as a result of intensive studies on various amphiphilic polymer ligands and complexes from synthesis to pharmacokinetics.

（５）側鎖に金属原子に配位可能な複素環を有するアミノ酸を含むヘリックス構造を有するデプシペプチドまたはペプチドが下記一般式(A)、（B）または(C)で表される、請求項３または４に記載の高分子錯体。
R₁−Xaa−R₄ (A)
R₁−Xaa−R₂−Yaa−R₄ (B)
R₁−Xaa−R₂−Yaa−R₃−Zaa−R₄ (C)
（式中、XaaおよびYaaおよびZaaは側鎖に金属原子に配位可能な複素環を有するアミノ酸を表し、R₁はXaaに結合した、アミノ酸、ペプチド、ヒドロキシカルボン酸、もしくはデプシペプチド、または水素原子もしくはアミノ基の保護基を表し、R₂およびR₃はXaaおよびYaaに結合したアミノ酸、ペプチド、ヒドロキシカルボン酸もしくはデプシペプチドを表し、R₄はXaaまたはYaaまたはZaaに結合した、アミノ酸、ペプチド、ヒドロキシカルボン酸もしくはデプシペプチド、またはXaaまたはYaaまたはZaaが直接親水性ポリマーに結合した単結合を表す。）
（６）側鎖に金属原子に配位可能な複素環を有するアミノ酸が、下記一般式(IV)〜（VIII)の何れかのアミノ酸である、（３）〜（５）の何れかの高分子錯体。

（Pal はβ-(3-pyridyl)-L-alanine残基を表し; His は L-histidine残基を表し; X₁とX₂はアルキル基、シリル基、アミド基、ウレタン基、エーテル基、もしくはエステル基を表す。）
（７）一般式(A)が下記一般式（a）であり、一般式（B）が下記一般式（b）であり、一般式（C）が下記一般式（c）である、（５）の高分子錯体。
Y₁-Leu-Leu-Aib-Xaa-Leu-Leu-Aib-Phe-（a）（配列番号１）
Y₁-Leu-Leu-Aib-Xaa-Leu-Leu-Aib-Yaa-Leu-Leu-Aib-Phe-（b）（配列番号２）
Y₁-Leu-Leu-Aib-Xaa-Leu-Leu-Aib-Yaa-Leu-Leu-Aib-Zaa-Leu-Leu-Aib-Phe-（c）（配列番号３）
（Aib は aminoisobutylic acid 残基を表し、Y₁はアミノ酸、ペプチド、ヒドロキシカルボン酸、もしくはデプシペプチドまたはアミノ基の保護基を表す。）
（８）親水性ポリマーがポリアルキレングリコールである、（１）〜（７）の何れかの高分子錯体。
（９）金属原子がアルカリ土類金属、遷移金属、後遷移金属、ランタニド、アクチニドから選択される、（１）〜（８）の何れかの高分子錯体。
（１０）金属原子が放射性核種、または常磁性イオン、蛍光性イオン、もしくはこれらの金属クラスターを形成する金属原子である、（９）の高分子錯体。
（１１）金属原子が、⁷Be、⁴⁷Ca、⁴⁶Sc、⁴⁷Sc、⁴⁸V、⁵¹Cr、⁵²Mn、⁵⁴Mn、⁵²Fe、⁵⁹Fe、⁵⁵Co、⁵⁶Co、⁵⁷Co、⁵⁸Co、⁶³Ni、⁶⁰Cu、⁶¹Cu、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶²Zn、⁶⁵Zn、⁶⁶Ga、⁶⁷Ga、⁶⁸Ga、⁸⁹Sr、⁸⁶Y、⁸⁷Y、⁸⁸Y、⁹⁰Y、⁸⁸Zr、⁸⁹Zr、⁹⁵Zr、^95mTc、⁹⁶Tc、^99mTc、⁹⁹Mo、⁹⁷Ru、¹⁰³Ru、⁹⁹Rh、^101mRh、¹⁰⁵Rh、¹⁸⁶Rh、¹⁰⁵Cd、¹⁰⁷Cd、^107mAg、¹¹¹Ag、¹¹¹In、^114mIn、^117mSn、¹³¹Ba、¹³⁹Ce、¹⁴¹Ce、¹⁴⁵Eu、¹⁴⁷Eu、¹⁴⁶Gd、¹⁴⁹Gd、¹⁴⁹Pm、¹⁵³Tb、¹⁵⁵Tb、¹⁶⁶Ho、¹⁶⁷Tm、¹⁶⁹Yb、¹⁷¹Lu、¹⁷²Lu、¹⁷⁷Lu、^177mLu、¹⁷⁵Hf、¹⁷⁸W、¹⁸³Re、¹⁸⁵Re、¹⁸⁶Re、¹⁸⁷Re、¹⁸⁸Re、¹⁹¹Pt、¹⁹⁰Ir、¹⁹²Ir、¹⁸⁸Pt、¹⁹¹Pt、^193mPt、^195mPt、¹⁹²Ir、¹⁹⁸Au、¹⁹⁹Au、²⁰¹Tl、²⁰³Hgからなる群より選ばれる1種または複数の放射性核種である、（１）〜（９）の何れかの高分子錯体。
（１２）金属原子が低分子金属錯体を形成した金属原子である、（１）〜（９）の何れかの高分子錯体。
（１３）低分子金属錯体が、ポルフィリン配位子、クロリン配位子、シッフ塩基配位子、チオラート配位子、チオエーテル配位子、イミダゾール配位子、フェノラート配位子からなる群より選ばれる1種または複数の配位子を有する低分子錯体である、（１２）の高分
子錯体。
（１４）（１）〜（１３）の何れかの高分子錯体を含む医薬組成物。
（１５）がんの治療または診断のための、（１４）の医薬組成物。 That is, the present invention is as follows.
(1) A polymer complex comprising an amphiphilic block copolymer comprising a hydrophobic ligand and a hydrophilic polymer and one or more metal atoms.
(2) The polymer complex of (1), wherein the amphiphilic block copolymer is represented by the following general formula (I), (II) or (II ′):
Hydrophilic polymer-block-hydrophobic ligand (I)
Hydrophobic ligand-block-hydrophilic polymer (II)
Hydrophobic ligand-block-hydrophilic polymer-block-hydrophobic ligand (II ')
(3) The polymer complex according to (1) or (2), wherein the hydrophobic ligand is a depsipeptide or peptide having a helix structure including an amino acid having a heterocycle capable of coordinating to a metal atom in a side chain. (4) a depsipeptide or peptide having a helix structure containing an amino acid having a heterocycle capable of coordinating to a metal atom in a side chain, an amino acid having a heterocycle capable of coordinating to a metal atom in a side chain, leucine, phenylalanine, (3) The polymer complex comprising at least one selected from γ-protected glutamic acid, ε-protected lysine, methionine, hydroxycarboxylic acid and α-methylamino acid represented by the following general formula (III).

(5) A depsipeptide or peptide having a helix structure containing an amino acid having a heterocycle capable of coordinating to a metal atom in a side chain is represented by the following general formula (A), (B) or (C): Or 5. the polymer complex according to 4;
R ₁ −Xaa−R ₄ (A)
R ₁ −Xaa−R ₂ −Yaa−R ₄ (B)
R ₁ −Xaa−R ₂ −Yaa−R ₃ −Zaa−R ₄ (C)
(In the formula, Xaa, Yaa and Zaa represent an amino acid having a heterocyclic ring capable of coordinating to a metal atom in the side chain, and R ₁ is an amino acid, peptide, hydroxycarboxylic acid, depsipeptide, or hydrogen atom bonded to Xaa. Alternatively, it represents an amino-protecting group, R ₂ and R ₃ represent an amino acid, peptide, hydroxycarboxylic acid or depsipeptide bound to Xaa and Yaa, and R ₄ represents an amino acid, peptide, hydroxy bound to Xaa or Yaa or Zaa Carboxylic acid or depsipeptide, or Xaa or Yaa or Zaa represents a single bond directly bonded to a hydrophilic polymer.)
(6) The high amino acid according to any one of (3) to (5), wherein the amino acid having a heterocyclic ring that can be coordinated to a metal atom in the side chain is any one of the following general formulas (IV) to (VIII): Molecular complex.

(Pal represents a β- (3-pyridyl) -L-alanine residue; His represents an L-histidine residue; X ₁ and X ₂ are alkyl groups, silyl groups, amide groups, urethane groups, ether groups, Or represents an ester group.)
(7) General formula (A) is the following general formula (a), General formula (B) is the following general formula (b), General formula (C) is the following general formula (c), (5 ) Polymer complex.
Y ₁ -Leu-Leu-Aib-Xaa-Leu-Leu-Aib-Phe- (a) (SEQ ID NO: 1)
Y ₁ -Leu-Leu-Aib-Xaa-Leu-Leu-Aib-Yaa-Leu-Leu-Aib-Phe- (b) (SEQ ID NO: 2)
Y ₁ -Leu-Leu-Aib-Xaa-Leu-Leu-Aib-Yaa-Leu-Leu-Aib-Zaa-Leu-Leu-Aib-Phe- (c) (SEQ ID NO: 3)
(Aib represents an aminoisobutylic acid residue, and Y ₁ represents an amino acid, peptide, hydroxycarboxylic acid, depsipeptide or amino protecting group.)
(8) The polymer complex according to any one of (1) to (7), wherein the hydrophilic polymer is polyalkylene glycol.
(9) The polymer complex according to any one of (1) to (8), wherein the metal atom is selected from alkaline earth metals, transition metals, post-transition metals, lanthanides, and actinides.
(10) The polymer complex according to (9), wherein the metal atom is a radionuclide, or a paramagnetic ion, a fluorescent ion, or a metal atom that forms a metal cluster thereof.
(11) The metal atom is ⁷ Be, ⁴⁷ Ca, ⁴⁶ Sc, ⁴⁷ Sc, ⁴⁸ V, ⁵¹ Cr, ⁵² Mn, ⁵⁴ Mn, ⁵² Fe, ⁵⁹ Fe, ⁵⁵ Co, ⁵⁶ Co, ⁵⁷ Co, ⁵⁸ Co, ⁶³ Ni, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶² Zn, ⁶⁵ Zn, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁹ Sr, ⁸⁶ Y, ⁸⁷ Y, ⁸⁸ Y, ⁹⁰ Y, ⁸⁸ Zr, ⁸⁹ Zr, ⁹⁵ Zr, ^95m Tc, ⁹⁶ Tc, ^99m Tc, ⁹⁹ Mo, ⁹⁷ Ru, ¹⁰³ Ru, ⁹⁹ Rh, ^{101 m} Rh, ¹⁰⁵ Rh, ¹⁸⁶ Rh, ¹⁰⁵ Cd, ¹⁰⁷ Cd, ^{107 m} Ag, ¹¹¹ Ag, ¹¹¹ In , ^114m In, ^117m Sn, ¹³¹ Ba, ¹³⁹ Ce, ¹⁴¹ Ce, ¹⁴⁵ Eu, ¹⁴⁷ Eu, ¹⁴⁶ Gd, ¹⁴⁹ Gd, ¹⁴⁹ Pm, ¹⁵³ Tb, ¹⁵⁵ Tb, ¹⁶⁶ Ho, ¹⁶⁷ Tm, ¹⁶⁹ Yb, ¹⁷¹ Lu, ¹⁷² Lu, ¹⁷⁷ Lu, ^177m Lu, ¹⁷⁵ Hf, ¹⁷⁸ W, ¹⁸³ Re, ¹⁸⁵ Re, ¹⁸⁶ Re, ¹⁸⁷ Re, ¹⁸⁸ Re, ¹⁹¹ Pt, ¹⁹⁰ Ir, ¹⁹² Ir, ¹⁸⁸ Pt, ¹⁹¹ Pt, ^{193 m} Pt, ^{195 m} Pt, The polymer complex according to any one of (1) to (9), which is one or more radionuclides selected from the group consisting of ¹⁹² Ir, ¹⁹⁸ Au, ¹⁹⁹ Au, ²⁰¹ Tl, and ²⁰³ Hg.
(12) The polymer complex according to any one of (1) to (9), wherein the metal atom is a metal atom forming a low molecular metal complex.
(13) The low molecular metal complex is selected from the group consisting of a porphyrin ligand, a chlorin ligand, a Schiff base ligand, a thiolate ligand, a thioether ligand, an imidazole ligand, and a phenolate ligand. The polymer complex according to (12), which is a low-molecular complex having one or more ligands.
(14) A pharmaceutical composition comprising the polymer complex according to any one of (1) to (13).
(15) The pharmaceutical composition of (14) for cancer treatment or diagnosis.

Since the polymer complex of the present invention can easily and stably coordinate metal atoms under mild conditions even in an aqueous environment, as a biomaterial or pharmaceutical composition, more specifically, a radiopharmaceutical, Paramagnetic agents and fluorescent agents are useful for diagnosis and treatment of various abnormal tissues, particularly for diagnosis and treatment of certain types of cancer.

The present invention provides a polymer complex comprising an amphiphilic block copolymer comprising a hydrophobic ligand and a hydrophilic polymer and one or more metal atoms.
Examples of the amphiphilic block copolymer include those represented by the following general formula (I) or (II) or (II ′).
Hydrophilic polymer-block-hydrophobic ligand (I)
Hydrophobic ligand-block-hydrophilic polymer (II)
Hydrophobic ligand-block-hydrophilic polymer-block-hydrophobic ligand (II ')

  The hydrophobic ligand is preferably a hydrophobic depsipeptide or a hydrophobic peptide having a helix structure containing an amino acid having a heterocyclic ring that can be coordinated to a metal atom in the side chain. Here, examples of the amino acid having a heterocyclic ring include an amino acid having a heterocyclic ring containing a nitrogen atom and an amino acid having a heterocyclic ring containing a sulfur atom. Examples of amino acids having a heterocyclic ring containing a sulfur atom include cysteine and its derivatives.

ここで、Pal はβ-(3-pyridyl)-L-alanine残基を表し; His は L-histidine残基を表し、X₁とX₂はアルキル基、シリル基、アミド基、ウレタン基、エーテル基、もしくはエステル基を表す。 Examples of amino acids having a heterocyclic ring containing a nitrogen atom include amino acids of the following general formulas (IV) to (VIII).

Where Pal represents a β- (3-pyridyl) -L-alanine residue; His represents an L-histidine residue, and X ₁ and X ₂ are alkyl groups, silyl groups, amide groups, urethane groups, ethers Represents a group or an ester group.

Aはアミノ酸側鎖を表し、疎水性の置換基が好ましい。 Examples of the hydrophobic peptide having a helical structure include peptides composed of an amino acid having a heterocyclic ring that can be coordinated to a metal atom in the side chain and other hydrophobic amino acids.
Other hydrophobic amino acids include leucine, phenylalanine, γ (carboxyl group) -protected glutamic acid, ε (amino group) -protected lysine, methionine, and the group consisting of α-methyl amino acids represented by the following general formula (III) One or more selected are preferred. In addition, when using an unnatural amino acid as another hydrophobic amino acid, it is preferable to use an amino acid having no branch at the β-position carbon atom in order to stabilize the helix.

A represents an amino acid side chain, and is preferably a hydrophobic substituent.

Examples of the α-methylamino acid represented by the general formula (III) include 2-aminoisobutylic acid (Aib), α-methylphenylalanine, α-methylvaline and the like.
In fact, many peptides of 2-aminoisobutylic acid and α-methylvaline are synthesized as amino acid residues that stably generate a helix. In addition, α-methylphenylalanine has been clinically applied as a radiopharmaceutical by introducing a fluorine atom, and it is easily expected to stabilize the helix.

As other amino acids, amino acids having a hydrophobic protecting group introduced in the side chain can also be used. Examples having such protecting ^{groups, Arg (N G -nitro),} Arg (N G - (4-methoxy-2,3,6-trimethylbenzenesulfonyl)), Arg (N G, N G -bis-benzyloxycarbonyl) , Arg ( ^NG- tosyl), Lys (N-ε-acetyl), Lys (N-ε-tert-butoxucarbonyl), Trp (N-in-tert-butoxycarbonyl), Trp (N-in-formyl), Ser (O-benzyl), Ser (O-tert-butyl), Ser (O-methyl), Ser (O-ethyl), Gln (γ-4.4'-dimethoxybenzhydryl), Gln (γ-trityl), Gln (γ- xanthyl), Glu (γ-trityl), Glu (γ-tert-butyl ester), Glu (γ-benzyl ester), Glu (γ-cyclohexyl ester), Cys (S-acetamidomethyl), Cys (Sp-methoxybenzyl), Cys (Sp-methylbenzyl), Cys (S-trityl), Tyr (O-methyl), Tyr (O-ethyl), Tyr (O-benzyl), Tyr (O-2,6-dichlorobenzyl), Tyr (O- tert-butyl), Tyr (O-2-bromobenzyl), Asp (β-cyclohexyl ester), Asp (β-benzyl ester), Thr (O-benzyl), Thr (O-tert-butyl), Thr (O- methyl), Thr (O-ethyl), Asn (γ-trityl), Asn (γ-xanthyl), Asn (β-tert-butyl ester), His (N-im-benzyloxymethyl), His (N-im
-dinitrophenyl), His (N-im-tosyl), His (N-im-triyl), His (N-im-tert-butoxycarbonyl), His (N-im-methyl), His (N-im-ethyl) Can also be used.

On the other hand, examples of the hydrophobic depsipeptide having a helical structure include depsipeptides composed of an amino acid having a heterocycle capable of coordinating to a metal atom in the side chain, hydroxycarboxylic acid, and other amino acids.
Here, as the hydroxycarboxylic acid, lactic acid, leucine acid, malic acid and the like can be used.

The helix sequence can be designed with reference to the table of TE Creighton, PROTEINS-Structures and Properties-Second Edition, WH Freeman and Company, 1993, pp. 256. You can refer to the table of pp. 154.
The length of the hydrophobic depsipeptide or hydrophobic peptide having a helical structure containing an amino acid having a heterocyclic ring that can be coordinated to a metal atom in the side chain is preferably 5 to 20.

As a depsipeptide or peptide having a helix structure containing an amino acid having a heterocycle capable of coordinating to a metal atom in the side chain, one having a sequence represented by the following general formula (A), (B) or (C) Can be mentioned.
R ₁ −Xaa−R ₄ (A)
R ₁ −Xaa−R ₂ −Yaa−R ₄ (B)
R ₁ −Xaa−R ₂ −Yaa−R ₃ −Zaa−R ₄ (C)
In these formulas, Xaa, Yaa and Zaa represent an amino acid having a heterocyclic ring capable of coordinating to a metal atom in the side chain as described above.
R ₁ represents an amino acid, peptide, hydroxycarboxylic acid, or depsipeptide, or a hydrogen atom or an amino-protecting group (acetyl group, alkoxycarbonyl group, etc.) bonded to Xaa.
R ₂ and R ₃ represent an amino acid or peptide bonded to Xaa, Yaa and Zaa, or a hydroxycarboxylic acid or depsipeptide. When R ₂ and R ₃ are peptides or depsipeptides, it is preferably a peptide (depsipeptide) consisting of 3 or 2 amino acids (a total of 3 or 2 amino acids and a hydroxycarboxylic acid).
R ₄ represents an amino acid, peptide, hydroxycarboxylic acid or depsipeptide bonded to Xaa or Yaa or Zaa. In the amphiphilic block copolymer, a hydrophilic polymer is bonded via the carboxy terminus of R ₄ . On the other hand, R ₄ may be a single bond (a state in which Xaa (formula IV), Yaa (formula V), or Zaa (formula VI) is bonded directly to the hydrophilic polymer).

Specific examples of the sequences of the general formulas (A), (B), and (C) include the sequences of the following general formulas (a), (b), and (c), respectively.
Y ₁ -Leu-Leu-Aib-Xaa-Leu-Leu-Aib-Phe- (a) (SEQ ID NO: 1)
Y ₁ -Leu-Leu-Aib-Xaa-Leu-Leu-Aib-Yaa-Leu-Leu-Aib-Phe- (b) (SEQ ID NO: 2)
Y ₁ -Leu-Leu-Aib-Xaa-Leu-Leu-Aib-Yaa-Leu-Leu-Aib-Zaa-Leu-Leu-Aib-Phe- (c) (SEQ ID NO: 3)
Here, Aib represents an aminoisobutylic acid residue, and Y ₁ represents an amino acid, peptide, hydroxycarboxylic acid, depsipeptide or amino group protecting group bonded to the amino group of Leu.

The following sequences can also be exemplified.
(d) Boc-Leu-Leu-Aib-His-Leu-Leu-Aib-Phe- (Boc-SEQ ID NO: 4)
(e) Boc-Leu-Leu-Aib-His-Leu-Leu-Aib-His-Leu-Leu-Aib-Phe- (Boc-SEQ ID NO: 5)
(f) Boc-Leu-Leu-Aib-His-Leu-Leu-Aib-His-Leu-Leu-Aib-His-Leu-Leu-Aib-Phe- (Boc-SEQ ID NO: 6)
(g) Boc-Leu-Leu-Ala-His-Phe-Leu-Phe-Lac-Leu-Phe- (Boc-SEQ ID NO: 7)
(Lac = lactic acid residue)
(h) Boc-Phe-Leu-Phe-Lac-His-Leu-Leu-Ala-His-Phe-Leu-Phe-Lac-Leu-Phe- (Boc-SEQ ID NO: 8)
(i) Boc-Leu-Leu-Aib-Cys-Leu-Leu-Aib-His-Leu-Leu-Aib-Phe- (Boc-SEQ ID NO: 9)
(j) Boc-Leu-Leu-Aib-His-Leu-Leu-Aib-Cys-Leu-Leu-Aib-Phe- (Boc-SEQ ID NO: 10)

In these sequences, His may have a protecting group such as His (N-im-benzyloxymethyl) and Cys may have Cys (S-acetamidomethyl) and Cys (S-methyl).

The amino acids constituting the peptide or depsipeptide as described above may be either L-form or D-form, but the helix is preferably formed from only one conformation amino acid. When the helix is composed of an L-form amino acid and an α-methyl amino acid corresponding to the L-form amino acid, a right-handed helix is formed, and the helix is α-form corresponding to the D-form amino acid and the D-form amino acid. When composed of methyl amino acids, a left-handed helix is formed.
Such sequences can be synthesized according to ordinary peptide synthesis methods.
The hydrophobic ligand may be labeled with a fluorescent substance. Examples of the fluorescent substance include rhodamine and fluorescein, which can be introduced into the amino group at the amino terminal of the hydrophobic ligand.

A polyalkylene glycol is preferable as the hydrophilic polymer for forming the block copolymer with the hydrophobic ligand as described above. Examples of the polyalkylene glycol include polyethylene glycol, polypropylene glycol, polybutylene glycol and the like, and polyethylene glycol is preferable.
The molecular weight of the hydrophilic polymer is preferably 100 to 20000, more preferably 500 to 10,000.

  The method for binding the hydrophobic ligand and the hydrophilic polymer is not particularly limited. For example, the hydrophilic polymer terminal is modified with NHS (N-hydroxysuccinimide), maleimide, methoxy, etc. The method of making it react with the carboxyl group of the carboxy terminal of is mentioned. Monoethylene PEG, multi-arm PEG, or the like can be used as the polyethylene glycol having the terminal modified.

Examples of the metal atom coordinated to the hydrophobic ligand include alkaline earth metals, transition metals, post-transition metals, lanthanides, and actinides. Classification of these metal elements James H. Huheey, are described in Inorganic Chemistry 4 ^th Ed..

  The metal atom may be a radionuclide, a paramagnetic ion, a fluorescent ion, or a metal atom that forms a metal cluster thereof. Paramagnetic ions include metal ions having unpaired electrons. Preferred examples of the fluorescent ion include rare earth element ions and metal complexes thereof.

Radionuclides include ⁷ Be, ⁴⁷ Ca, ⁴⁶ Sc, ⁴⁷ Sc, ⁴⁸ V, ⁵¹ Cr, ⁵² Mn, ⁵⁴ Mn, ⁵² Fe, ⁵⁹ Fe, ⁵⁵ Co, ⁵⁶ Co, ⁵⁷ Co, ⁵⁸ Co, ⁶³ Ni, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶² Zn, ⁶⁵ Zn, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁹ Sr, ⁸⁶ Y, ⁸⁷ Y, ⁸⁸ Y, ⁹⁰ Y, ⁸⁸ Zr, ⁸⁹ Zr ⁹⁵ Zr, ^{95 m} Tc, ⁹⁶ Tc, ^{99 m} Tc, ⁹⁹ Mo, ⁹⁷ Ru, ¹⁰³ Ru, ⁹⁹ Rh, ¹⁰¹ Rh, ¹⁰⁵ Rh, ¹⁸⁶ Rh, ¹⁰⁵ Cd, ¹⁰⁷ Cd, ^{107 m} Ag, ¹¹¹ Ag, ¹¹¹ In, ^{114 m} In, ^117m Sn, ¹³¹ Ba, ¹³⁹ Ce, ¹⁴¹ Ce, ¹⁴⁵ Eu, ¹⁴⁷ Eu, ¹⁴⁶ Gd, ¹⁴⁹ Gd, ¹⁴⁹ Pm, ¹⁵³ Tb, ¹⁵⁵ Tb, ¹⁶⁶ Ho, ¹⁶⁷ Tm, ¹⁶⁹ Yb, ¹⁷¹ Lu, ¹⁷² Lu, ¹⁷⁷ Lu, ^177m Lu, ¹⁷⁵ Hf, ¹⁷⁸ W, ¹⁸³ Re, ¹⁸⁵ Re, ¹⁸⁶ Re, ¹⁸⁷ Re, ¹⁸⁸ Re, ¹⁹¹ Pt, ¹⁹⁰ Ir, ¹⁹² Ir, ¹⁸⁸ Pt, ¹⁹¹ Pt, ^{193 m} Pt, ^{195 m} Pt, ¹⁹² Ir ¹⁹⁸ Au, ¹⁹⁹ Au, ²⁰¹ Tl, ²⁰³ Hg and the like.

The metal atom may already form a low molecular metal complex. For example, metal complexes are formed with ligands such as porphyrin ligand, chlorin ligand, Schiff base ligand, thiolate ligand, thioether ligand, imidazole ligand, phenolate ligand, etc. A metal atom is mentioned.

Examples of the method of forming a polymer complex of an amphiphilic block copolymer and a metal atom include a method of mixing the copolymer and the metal atom in an aqueous solution, and a method of mixing in an organic solvent such as acetonitrile. It is done.
In the case of using an organic solvent, it is preferable to remove the organic solvent by drying after forming a polymer complex.

The present invention provides a composition comprising the above polymer complex. In addition to the above-described polymer complex, the composition may contain components that are acceptable in the preparation process, such as a buffer, a lyophilization aid, a stabilization aid, a solubilization aid, and an antibacterial agent.
Examples of the buffer include phosphate, citrate, sulfosalicylate, acetate, and the like.
Examples of the lyophilization aid include mannitol, lactose, sorbitol, dextran, Ficoll, and polyvinylpyrrolidine (PVP).
Examples of stabilizing aids include ascorbic acid, cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite, gentisic acid, inositol and the like.
Examples of solubilizers include ethanol, glycerin, polyoxyethylene sorbitan monooleate, sorbitan monooleate, polysorbates, poly (oxyethylene) poly (oxypropylene) poly (oxyethylene) block copolymers (Pluronics), and lecithin. Is mentioned.
Antibacterial agents include benzyl alcohol, benzalkonium chloride, chlorobutanol, methyl paraben, propyl paraben, butyl paraben and the like.

  In a composition containing a polymer complex, it is not necessary for all amphiphilic ligands to coordinate with metal atoms, and ligands without coordinated metal atoms coexist. Also good.

The composition of the present invention can be used as a pharmaceutical composition.
For example, when a radionuclide as described above is used as a coordination metal atom, it can be used for treatment or diagnosis of cancer. That is, the polymer complex of the present invention in which micelles are formed leaks from cancer tissue in the living body, and can be “detected” and “destructed” by radiation emitted from the radionuclide. Tumor tissue can be detected by a SPECT camera (γ-ray) or a PET camera (positron).

On the other hand, the compound which has pharmacological activity can also be included in a micelle, and it can also be set as a pharmaceutical composition. In particular, since the polymer complex of the present invention is likely to accumulate in cancer tissue, by incorporating an anticancer agent in the micelle, the drug accumulates in the cancer tissue and can be exposed to the cancer tissue for a long period of time. . Examples of the anticancer agent include adreamycin and doxorubicin.
In addition, by using a polymer complex with fluorescent labeling, tumor tissue can be clearly detected with fluorescence.
Such a pharmaceutical composition can be developed by referring to the inventors' related research and known methods. (For example, Tomohiro Suda et al., Peptide Science 2005, The
Japanese Peptide Society, 2006, pp. 495-498; Aya Inoue et al., Peptide Science 2006, The Japanese Peptide Society, 2006, pp. 54-55; Kataoka Kazunori et al., Modern Medicine, Latest Medical Company, 2006, vol 61, pp. 1092-1101; Hiroshi Maeda, Latest Medicine, Latest Medicine, 2006, vol 6
1, pp. 1138-1148.)

Hereinafter, Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (1) (Boc-SEQ ID NO: 13-PEG ₄₀₀₀ ), which is an amphiphilic ligand, is an embodiment of the present invention. ) And Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (2) (Boc-SEQ ID NO: 12-PEG ₄₀₀₀ ) 1 and 2. These mass spectrometry data are shown in Example 3. Details of the method for synthesizing the polymer complex using these as ligands are shown in Examples 4 and 5, respectively. The pharmacokinetics when the obtained polymer complex is administered to mice as a physiological saline solution are shown in Example 6, and the pharmacokinetics when administered to cancer-bearing mice are shown in Example 7. In addition, common procedures in chemical synthesis are shown as synthesis procedures 1 to 4. However, the following specific examples do not limit the present invention, and it is needless to say that the protective group and the condensing agent can be appropriately changed, for example, by replacing them with other conventional ones.

  Also, for example, in the synthesis of amphiphilic ligands using other peptide sequences or depsipeptide sequences, other amino acids or hydroxycarboxylic acids can be used in the same manner or in a known manner. (For example, Hiroyuki Oku et al., Journal of Organometallic Chemistry, 2007, vol. 692, pp. 79-87.).

In the following examples, the following abbreviations were used.

(Amino acid derivative)
Boc-Leu-OH = N-α-t-butoxycarbonyl-L-leucine
Boc-Phe-OH = N-α-t-butoxycarbonyl-L-phenylalanine
Boc-Pal-OH = N-α-t-butoxycarbonyl-β- (3-pyridyl) -L-alanine
HCl ・ H-Aib-OEt = 2-aminoisobutyric acid ethyl ester hydrochloride

(Main chain protecting group of amino acid)
Boc = tert-butoxycarbonyl (t-Bu-O-CO-)
OEt = ethyl ester (-O-CH ₂ -CH ₃ )

(Main chain protecting group of amino acid)
OSu = succinimide ester

(Hydrophilic polymer)
PEG ₄₀₀₀ = Polyethylene glycol ₄₀₀₀ (molecular weight 4000)

(Peptide synthesis reagents and related compounds)
DCC = N, N'-dichlorohexylcarbodiimide
DCUrea = dicyclohexylurea
HOSu = N-hydroxysuccinimide
HOBt = 1-hydroxybenzotriazole
TFA = trifluoroacetic acid
(Boc) ₂ O = di-t-butyl carbonate
NMM = N-methylmorpholine
EDC.HCl = 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride
DMAP = N, N'-dimethylaminopyridine
CFDA = Carboxyfluorescein diacetate
CF = Carboxyfluorescein

(solvent)
THF = tetrahydrofuran
CHCl ₃ = chloroform
CDCl ₃ = deuterated chloroform
AcOEt = ethyl acetate
DMSO-d ₆ = deuterated dimethyl sulfoxide
MeOH = methanol

[Synthesis Procedure 1: Synthesis of Boc-L-amino acid]
L-amino acid or side chain protected L-amino acid (1.0 mol) was dissolved in 4M NaOH (250 mL, 1.0 mol) and dissolved in a minimum amount of dioxane while slowly cooling with ice-MeOH (Boc) ₂ O (240.0 g, 1.1 mol) was added slowly over 30 minutes. The mixture was stirred for 1 hour in an ice bath and 1.5 hours at room temperature. The precipitated NaHCO ₃ is filtered off, adjusted to pH 3.0 and extracted with AcOEt. The extraction solution was washed with a 10% aqueous citric acid solution and then dried over Na ₂ SO ₄ . After the desiccant was filtered off, the filtrate was concentrated under reduced pressure, and hexane was added to the residue for crystallization. Thereafter, recrystallization was performed with AcOEt-hexane to obtain Boc-L-amino acid.

[Synthesis Procedure 2: Deprotection Reaction of Amino Group Terminal, Synthesis of De-Boc Compound]
The peptide compound in which the amino group was protected with N-α-t-butoxycarbonyl was placed in a 300 mL eggplant flask and dissolved in TFA (or 4M HCl in dioxane) in a fume hood. The lid was immediately covered with a calcium chloride tube to prevent moisture from entering. After confirming the completion of the reaction by TLC, it is concentrated and repeatedly added Et ₂ O until concentrated until the TFA odor (or hydrochloric acid odor) disappears, and concentrated to finally obtain a white powder of TFA salt (or hydrochloride). The yield is almost quantitative.

[Synthesis Procedure 3: Condensation Reaction Using Condensing Agent]
Peptide compound (2.1 mmol) in which amino group is protected with N-α-t-butoxycarbonyl and carboxyl end is deprotected is placed in a 300 mL Erlenmeyer flask, dissolved in distilled CHCl ₃ and condensed with HOBt (0.28 g, 2.1 mmol). Agent EDC · HCl (0.40 g, 2.1 mmol) (or DCC (0.43 g, 2.1 mmol)) was added and stirred. Next, the amino group is deprotected in the above synthesis procedure 2 in a 300 ml eggplant flask, and the peptide compound (0.7 mmol) of the TFA salt is added to neutralize the TFA salt with NMM. Neutralization can be confirmed at approximately equimolar (0.075 mL, 0.70 mmol), but may be slightly higher in the case of a salt with poor crystallinity. The two solutions are mixed and stirred, and then immediately cooled with ice to start the reaction. Slowly return to room temperature and stir overnight. This mixture is concentrated by an evaporator and dissolved in AcOEt, and then 10% aqueous citric acid solution, distilled water, saturated aqueous NaHCO ₃ solution, distilled water, and saturated brine are used in this order (excluding DCUrea insoluble in AcOEt when DCC is used). Wash, dry over Na ₂ SO ₄ and concentrate to obtain an oily or colorless powdered condensation product. This is purified by silica gel chromatography (distilled CH ₃ Cl-hexane or AcOEt-benzene) or gel filtration chromatography (Pharmacia LH20, DMF or MeOH). In the case of a colorless powder, it may be purified by recrystallization using a solvent system of AcOEt-distilled Et ₂ O or distilled CH ₃ Cl-hexane. Yields can be obtained in the range of approximately 70-90%.

[Synthesis Procedure 4: Condensation Reaction Using Active Ester]
Peptide compound (3.0 mmol) in which amino group is protected with N-α-t-butoxycarbonyl (Boc-) and carboxyl terminal is active esterified (-OSu) is placed in a 300 mL Erlenmeyer flask and stirred in distilled THF . Next, the peptide compound (1.0 mmol) of the TFA salt (or HCl salt) whose amino group has been deprotected in the above synthesis procedure 2 is placed in a 300 ml eggplant flask, and the TFA salt (or HCl salt) is neutralized with NMM. Neutralization can be confirmed with almost equimolar (0.1 mL, 1.0 mmol) NMM, but it may be slightly higher in the case of a salt with poor crystallinity. The reaction is started at room temperature while mixing and stirring the two solutions. Continue stirring for approximately 2-3 days. This mixture can be processed in the same manner as in synthesis procedure 3 to give the product. Yields can be obtained in the range of approximately 70-90%.

[Example 1]
(1) Synthesis of Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (1) (Boc-SEQ ID 13-PEG ₄₀₀₀ ) (1a: Synthesis of Boc-Phe-OSu)
Boc-Phe-OH (6.3 g, 25 mmol) and HOSu (3.5 g, 30 mmol) were subjected to a condensation reaction in the same manner as in Synthesis Procedure 3 using DCC (6.2 g, 30 mmol). However, HOSu does not require a neutralization reaction with NMM because the -OH group, not the amino group, undergoes dehydration condensation. After the reaction, colorless Boc-Phe-OSu was obtained. Yield 8.7 g (96% yield). ¹ H-NMR (CDCl ₃ , 300 MHz): 7.63 (d, 1H, Boc-Phe NH); 7.24 (5H, Phe, C ₆ H _5- ); 4.54 (1H , Phe αCH ₂ ); 3.14, 2.91 (2H, Phe βCH ₂ ); 2.81 (s, 4H, -OSu); 1.40 (s, 9H, Boc t-Bu).

(1b: Synthesis of Boc-Phe-PEG ₄₀₀₀ )
Boc-Phe-OSu (4.4 g, 12 mmol) and PEG ₄₀₀₀ (16 g, 4.0 mmol) were subjected to a condensation reaction in the presence of DMAP (0.050 g, 0.40 mmol) in THF by the method of synthesis procedure 3. The reaction was carried out over 2 days. ^The amount of Boc-Phe introduced into PEG ₄₀₀₀ was confirmed by ¹ H-NMR. Two equivalents can be introduced per molecule of PEG ₄₀₀₀ . When the amount introduced was insufficient, Boc-Phe-OSu and DMAP were added again to carry out the reaction. Yield, 15.3 g (84% yield). ¹ H-NMR (DMSO-d ₆ , 300 MHz): 7.23 (10H, Phe C ₆ H _5- ); 4.12 (2H, Phe αCH); 3.4-3.6 (360H , PEG ₄₀₀₀ -CH _2- ); 2.85, 2.98 (4H, Phe βCH ₂ ); 1.31 (s, 18H, Boc t-Bu).

(1c: Synthesis of Boc-Leu-Aib-OEt)
Boc-Leu-OH.H ₂ O (44.9 g, 180 mmol), HCl.H-Aib-OEt (25.1 g, 150 mmol), NMM (16.5 mL, 150 mmol), DCC (37.1 g, 180 mmol) And a condensation reaction was performed by the method of Synthesis Procedure 3 to obtain a colorless product. Yield, 27.2 g (52% yield). ¹ H-NMR (CDCl ₃ , 300 MHz): 6.74 (s, 1H, Aib NH); 4.92 (d, 1H, Leu NH); 4.18 (t, 2H, OEt -CH _2- ); 4.15 (1H, Leu αCH); 1.71 (1H, Leu γCH; 2H, Leu βCH ₂ ); 1.54 (s, 6H, Aib βCH ₃ ); 1.45 (9H, Boc t-Bu); 1.26 (q, 3H, OEt -CH ₃ ) .0.93 (6H, Leu δCH ₃ ).

(1d: Synthesis of Boc-Leu-Leu-Aib-OEt)
Boc-Leu-OH.H ₂ O (13.0 g, 52.2 mmol), HCl.H-Leu-Aib-OEt (12.2 g, 43.5 mmol, synthesized from Boc-Leu-Aib-OEt by the method of synthesis procedure 2), NMM (4.8 mL, 44 mmol), DCC (10.8 g, 52.2
Using (mmol), a condensation reaction was performed by the method of Synthesis Procedure 3 to obtain a colorless product. Yield, 14.9 g (75% yield). ¹ H-NMR (CDCl ₃ , 300 MHz): 6.78 (s, 1H, Aib ³ NH); 6.51 (d, 1H, Leu ² NH); 4.92 (d, 1H , Leu ¹ NH); 4.92 (d, 1H, Leu NH); 4.38 (1H, Leu ² αCH); 4.16 (t,
2H, OEt -CH _2- ); 4.08 (1H, Leu ¹ αCH); 1.70 (2H, Leu ^1,2 γCH; 4H, Leu ^1,2 βCH ₂ )); 1.55 (s, 6H, Aib ³ βCH ₃ ) 1.44 (s, 9H, Boc t-Bu); 1.24 (q, 3H, OEt -CH ₃ ) .0.91
(6H, Leu δCH ₃ ).

(1e: Synthesis of Boc-Leu-Leu-Aib-OH)
Boc-Leu-Leu-Aib-OEt (14.8 g, 32.3 mmol) was placed in a 500 mL eggplant flask, and methanol was added to dissolve it. 1M NaOHaq. (32.3 mL, 32.3 mmol) was added and stirred. Stirring was stopped in 4 hours, followed by concentration under reduced pressure. Distilled water was added and the system was acidified with 4M hydrochloric acid, followed by extraction with EtOAc. The EtOAc solution was washed successively with distilled water and saturated brine. After drying over Na ₂ SO ₄ and concentrating under reduced pressure, hexane was added for crystallization. Recrystallization from AcOEt-hexane gave a colorless product. Yield, 10.0 g (72% yield). ¹ H-NMR (CDCl ₃ , 300 MHz): 7.33 (d, 1H, Leu ² NH);
7.24 (s, 1H, Aib ³ NH); 5.24 (d, 1H, Leu ¹ NH); 4.59 (1H, Leu ² αCH); 4.17 (1H, Leu ¹ αCH); 1.60 (2H, Leu ^1,2 γCH; 4H, Leu ^1,2 βCH ₂ ); 1.56 (s, 6H, Aib ³ βCH ₃ );
1.48 (s, 9H, Boc t-Bu); 0.92 (12H, Leu δCH ₃ ).

(1f: Synthesis of Boc-Leu-Leu-Aib-OSu)
Boc-Leu-Leu-Aib-OEt (14.8 g, 32.3 mmol) was placed in a 500 mL eggplant flask, and methanol was added to dissolve it. 1M NaOHaq. (32.3 mL, 32.3 mmol) was added and stirred. Stirring was stopped in 4 hours, followed by concentration under reduced pressure. Distilled water was added and the system was acidified with 4M hydrochloric acid, followed by extraction with EtOAc. The EtOAc solution was washed successively with distilled water and saturated brine. After drying over Na ₂ SO ₄ and concentrating under reduced pressure, hexane was added for crystallization. Recrystallization from AcOEt-hexane gave a colorless product. Yield, 10.0 g (72% yield). ¹ H-NMR (CDCl ₃ , 500 MHz): 6.96 (s, 1H, Aib ³ NH);
6.52 (d, 1H, Leu ² NH); 4.86 (d, 1H, Leu ¹ NH); 4.41 (1H, Leu ² αCH); 4.07 (1H, Leu ¹ αCH); 1.66 (2H, Leu ^1,2 γCH; 4H, Leu ^1,2 βCH ₂ ); 1.25 (s, 6H, Aib ³ βCH ₃ );
1.39 (s, 9H, Boc t-Bu); 0.92 (12H, Leu δCH ₃ ).

(1 g: Synthesis of Boc-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (Boc-SEQ ID No. 14-PEG ₄₀₀₀ ) )
Boc-Leu-Leu-Aib-OSu (1.1 g, 2.0 mmol), HCl.H-Phe-PEG ₄₀₀₀ (4.2 g, 1.0 mmol; produced from Boc-Phe-PEG ₄₀₀₀ by the method of synthesis procedure 2, HCl. H-Phe- mole ratio was confirmed to be about 1 equivalent to PEG ₄₀₀₀ by ¹ H-NMR), and NMM (0.29 mL, 0.66 mmol) was used for the condensation reaction by the method of Synthesis Procedure 4. Went. The reaction was allowed to proceed over 2 days. A colorless product was obtained. The introduction amount of Boc-Leu-Leu-Aib- was clarified by ¹ H-NMR to be about 1 equivalent with respect to PEG ₄₀₀₀ . Yield, 3.2 g (70% yield). Mp = 41-43 ° C. ¹ H-NMR (DMSO-d ₆ , 500 MHz): 7.16-7.91 (4H, amide NH; 4H, Phe C ₆ H _5- ) ; 6.88 (d, 1H, Leu ¹ NH); 3.7-4.5 (4H, αCH); 3.4-3.6 (360H, PEG ₄₀₀₀ -CH _2- ); 1.2-1.3 (9H, Boc t-Bu); 0.92 (12H , Leu δCH ₃ ).

(1h: Synthesis of Boc-Pal-OSu)
Boc-Pal-OH (1.0 g, 3.8 mmol) and HOSu (0.51 g, 4.5 mmol) are condensed using DCC (0.93 g, 4.5 mmol) in the same manner as in Synthesis Procedure 3 and Boc-Phe-OSu. Went. After the reaction, the colorless product was directly used for the next reaction. The yield is almost quantitative.

(1i: Synthesis of Boc-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (Boc-SEQ ID NO: 15-PEG ₄₀₀₀ ) )
Boc-Pal-OSu (0.36 g, 1.0 mmol), HCl.H-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (3.0 g, 0.66 mmol; Boc-Leu-Leu-Aib-Phe- Using PEG ₄₀₀₀ ) and NMM (0.070 mL, 0.66 mmol), a condensation reaction was performed by the method of Synthesis Procedure 4. The reaction was allowed to proceed for 3 days. A colorless product was obtained. The amount of Boc-Pal- introduced relative to PEG ₄₀₀₀ was observed to be about 0.7 equivalent by ¹ H-NMR. This is thought to be T ₂ broadening due to an apparent increase in molecular weight accompanying aggregation in solution. Subsequent compounds similarly have a broad ¹ H-NMR signal. Therefore, the integration ratio is described as 1.0 equivalent to PEG ₄₀₀₀ . Yield, 2.4 g (78% yield). Mp = 44-46 ° C. ¹ H-NMR (DMSO-d ₆ , 500 MHz): 7.17-8.42 (7H, amide NH; 5H, Phe C ₆ H _5- ) 7.03 (1H, Leu NH); 3.6-4.6 (7H, αCH); 3.4-3.6 (360H, PEG ₄₀₀₀ -CH _2- ); 1.2-1.3 (9H, Boc t-Bu); 0.81, 0.88 (12H, Leu δCH ₃ ).

(1j: Synthesis of Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (Boc-SEQ No. 13-PEG ₄₀₀₀ ) (1))
Boc-Leu-Leu-Aib-OSu (0.51 g, 0.98 mmol), 2HCl.H-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (2.3 g, 0.49 mmol; Boc-Pal- A condensation reaction was performed by the method of Synthesis Procedure 4 using Leu-Leu-Aib-Phe-PEG ₄₀₀₀ ) and NMM (0.050 mL, 0.98 mmol). The reaction was allowed to proceed over 2 days. A colorless product was obtained. Yield, 2.0 g (82% yield). Mp = 47-48 ° C. ¹ H-NMR (DMSO-d ₆ , 500 MHz): 7.17-8.42 (7H, amide NH; 5H, Phe C ₆ H _5- ) 7.03 (1H, Leu NH); 3.6-4.6 (8H, αCH); 3.4-3.6 (360H, PEG ₄₀₀₀ -CH _2- ); 1.2-1.3 (9H, Boc t-Bu);
0.78, 0.81 (12H, Leu δCH ₃ ).

[Example 2]
(2) Synthesis of Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (2) (Boc-SEQ ID NO: 12-PEG ₄₀₀₀ ) (2a: Boc -Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (Boc-SEQ No. 16-PEG ₄₀₀₀ ) synthesis)
Boc-Pal-OSu (0.28 g, 0.76 mmol), 2HCl.H-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (1.89 g, 0.38 mmol), NMM (0.080 mL, 0.76 mmol) ) Was used for the condensation reaction by the method of Synthesis Procedure 4. The reaction was allowed to proceed over 2 days to give a colorless product. Yield, 1.8 g (92% yield). Mp = 45-47 ° C. ¹ H-NMR (DMSO-d ₆ , 500 MHz): 7.12-8.41 (9H, amide NH; 5H, Phe C ₆ H _5- ) 3.6-4.6 (7H, αCH); 3.4-3.6 (360H, PEG ₄₀₀₀ -CH _2- ); 1.2-1.3 (9H, Boc t-Bu);
0.8-0.9 (24H, Leu δCH ₃ ).

(2b: Synthesis of Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (2) (Boc-SEQ ID 12-PEG ₄₀₀₀ ) )
Boc-Leu-Leu-Aib-OSu (0.26 g, 0.25 mmol), 2HCl.H-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (1.3 g, 0.25 mmol), NMM (0.080 mL, 0.75 mmol) And the condensation reaction was carried out by the method of Synthesis Procedure 4. The reaction was allowed to proceed over 2 days. A colorless product was obtained. Yield, 0.74 g (54% yield). Mp = 46-49 ° C. ¹ H-NMR (DMSO-d ₆ , 500 MHz): 7.12-8.43 (12H, amide NH; 5H, Phe C ₆ H _5- ) 3.6-4.6 (7H, αCH); 3.4-3.6 (360H, PEG ₄₀₀₀ -CH _2- ); 1.2-1.3 (9H, Boc t-Bu);
0.8-0.9 (36H, Leu δCH ₃ ).

なお、Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe（Boc-配列番号１２）とPEG₄₀₀₀の比を２：１とすることで以下のような共重合体を得ることもできる。

また、モノメトキシ化したPEG₄₀₀₀を用いた場合は、Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe（Boc-配列番号１２）とPEG₄₀₀₀の比を１：１である以下のような共重合体を得ることもできる。 The expected structure of Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (2) (Boc-SEQ ID 12-PEG ₄₀₀₀ ) is shown below. .

By setting the ratio of Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe (Boc-SEQ ID NO: 12) to PEG ₄₀₀₀ to 2: 1, Can also be obtained.

When monomethoxylated PEG ₄₀₀₀ is used, Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe (Boc-SEQ ID NO: 12) and PEG ₄₀₀₀ The following copolymer having a ratio of 1: 1 can also be obtained.

[Example 3]
(3) Mass Spectrometry by MALDI-TOF Method MALDI-TOF spectra were measured for the polymer ligands obtained in Examples 1 and 2. From the spectrum (FIG. 1), the intermediate and final product obtained at each step of the synthesis can be confirmed by molecular weight. As shown in FIG. 1, the spectrum was observed as a molecular weight corresponding to an association of two molecules. Also, the mass change between each spectrum corresponded to the expected fragment or amino acid extension. Therefore, the production of the target product was confirmed by mass spectrometry.

[Example 4]
(4) Measurement of critical micelle concentration (cmc) Polymeric ligand obtained in Example 2 , Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe- For PEG ₄₀₀₀ (Boc-SEQ ID NO: 12-PEG ₄₀₀₀ ) (2) , cmc was measured (Y. Nagasaki et al., Macromolecules, 1998, vol. 31, pp. 1473-1479.). The JASCO FP6200 fluorescence spectrophotometer was used for the fluorescence spectrum.
As a hydrophobic probe, a change in the fluorescence spectrum of pyrene was used. 10 mg of 1 was dissolved in 10 mL of saturated pyrene aqueous solution (˜0.14 mg / mL) and allowed to stand at 20 ° C. for 1 day. The spectrum was measured at 20 ° C., and the fluorescence intensity at 390 nm was recorded when the excitation light was scanned from 300 nm to 360 nm (FIG. 2). The intensity ratio at 337 nm and 334 nm obtained from the spectrum was plotted against the concentration of 1 (FIG. 3), and the concentration that greatly changed the graph was cmc. As a result, the critical micelle concentration of 1 was found to be 0.060 mg / mL.

[Example 5]
(5) Synthesis of polymer complex by CoCl ₂ Polymer ligand obtained in Example 2 Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG _The change in visible absorption spectrum when ₄₀₀₀ (Boc-SEQ ID NO: 12-PEG ₄₀₀₀ ) (2) was titrated into an aqueous solution of CoCl ₂ (0.1 mM) was measured. From FIG. 4, it was found that in the range where the concentration of 2 (calculated assuming the average molecular weight of 2 as 5505) is large (0.1 to 0.4 mM), the baseline of 400-800 nm increases as the concentration of 2 increases. This is interpreted as an increase in apparent absorbance as 2 increases in concentration, as 2 forms larger aggregates and scatters incident light.

From Fig. 5, in the range where the concentration of 2 (calculated assuming the average molecular weight of 2 as 5505) is small (0 to 0.1 mM), the increase in baseline from 400 to 800 nm is almost negligible as the concentration of 2 increases. It was. In addition, the dd absorption of Co (II) ions changed while showing the isoabsorption points at 547, 575, 606, 654, and 675 nm as the concentration of 2 (calculated assuming the average molecular weight of 2 as 5505) increased. Thus it was found that the 2 and CoCl ₂ is one type of complex formed. Furthermore, the change in the absorption maximum at 590 nm was plotted as shown in FIG. 6, and the binding constant of the complex formation reaction was calculated by the nonlinear least square method. Assuming K = [CoCl ₂ -2] / [CoCl ₂ ] [2] for the complexation equilibrium reaction, results of Δabs∞ = 0.0086 and K = 1.2 × 10 ⁵ M ⁻¹ were obtained.

The polymer complex obtained here is a crosslinked type [(CoCl ₂ ) ₂ (Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Aib- Phe- (SEQ ID NO: 12) PEG ₄₀₀₀ ) ₂ ], or the mononuclear form shown in FIG. 10D [CoCl ₂ (Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib -Phe- (SEQ ID NO: 12) PEG ₄₀₀₀ )].
In addition, the structure of the polymer complex consisting of Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (Boc-SEQ ID NO: 13-PEG ₄₀₀₀ ) (1) and CoCl ₂ is also crosslinked (see FIG. 10A) and a mononuclear type (FIG. 10B) were considered to exist.

[Example 6]
(6) Synthesis of Polymer Complex ( ¹¹¹ In-1) with ¹¹¹ InCl ₃ Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ obtained in Example 1 (Boc-SEQ ID NO: 13-PEG ₄₀₀₀ ) A complex formation reaction with ¹¹¹ InCl ₃ was performed using (1).

(6a: Complex formation using CH ₃ CN)
1 (410 μg) was dissolved in 200 μL of CH ₃ CN in an Eppendorf tube. 20 μL of ¹¹¹ InCl ₃ solution (manufactured by Nippon Mediphysics, ˜20 MBq / mL) was added thereto and stirred well for 15 minutes. When including a pharmaceutical compound such as an anticancer agent, it was added at the same time. A vortex mixer and an ultrasonic washer were used for stirring. When applying ultrasonic waves, attention was paid to the temperature rise of the solution. Thereafter, the solvent was distilled off with a rotary evaporator and nitrogen gas. Even after 90 minutes, the residue was in the form of a highly viscous oil, so physiological saline was added to the oil obtained as it was and redissolved, and the mixture was stirred well for 15 minutes. At the time of redissolving, the concentration was adjusted appropriately for examining pharmacokinetics or pharmacological activity. The resulting solution was passed through a sterile filter. Radiochemical purity was tested from the distribution of ¹¹¹ In by thin layer chromatography (Developed with Gelmal Sciences ITLC-SG, silica gel glass fiber plate, physiological saline). The ¹¹¹ In that forms a complex has a radioactivity at the origin, and the ¹¹¹ In that does not form a complex has a radioactivity at the deployment tip. The purity of the obtained polymer complex solution of ¹¹¹ In-1 was 99.5%.

(6b: Complex formation using physiological saline)
1 (320 μg) was dissolved in 1600 μL of physiological saline in a glass test tube. To this was added 15 μL of ¹¹¹ InCl ₃ solution (manufactured by Nippon Mediphysics, ˜20 MBq / mL), and the mixture was well stirred for 15 minutes using an ultrasonic cleaner. When encapsulating a pharmaceutical compound such as an anticancer agent, it is added simultaneously but requires longer stirring. At the time of stirring, it was necessary to pay attention to the temperature rise of the solution. After stirring for 15 minutes, thin layer chromatography (ITLC-SG manufactured by Gelmal Sciences, silica gel glass fiber plate, developed with physiological saline) was performed, and the radiochemical purity was 99.0%.

[Example 7]
(7) Synthesis of polymer complex ( ¹¹¹ In-2) by ¹¹¹ InCl ₃ Polymer ligand Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu- obtained in Example 2 Leu-Aib-Phe-PEG ₄₀₀₀ (Boc-SEQ ID NO: 12-PEG ₄₀₀₀ ) (2) was used to perform a complex formation reaction with ¹¹¹ InCl ₃ .

(7a: Complex formation using CH ₃ CN)
2 (410 μg) was dissolved in 200 μL of CH ₃ CN in an Eppendorf tube. 20 μL of ¹¹¹ InCl ₃ solution (manufactured by Nippon Mediphysics, ˜20 MBq / mL) was added thereto and stirred well for 10 minutes. When including a pharmaceutical compound such as an anticancer agent, it was added at the same time. A vortex mixer or an ultrasonic cleaner may be used for stirring. When applying ultrasonic waves, attention was paid to the temperature rise of the solution. Thereafter, the solvent was distilled off with a rotary evaporator or nitrogen gas. The residue became a colorless solid after 15-30 minutes. The obtained solid was re-dissolved with physiological saline and stirred well for 10 minutes. At the time of redissolving, the concentration was adjusted appropriately for examining pharmacokinetics or pharmacological activity. The resulting solution was passed through a sterile filter. Radiochemical purity was assayed by thin layer chromatography (ITLC-SG manufactured by Gelmal Sciences, silica gel glass fiber plate, developed with physiological saline). The radiochemical purity of the ¹¹¹ In-2 polymer complex obtained was 98.5-99.8% in all cases. This method using CH ₃ CN has a slightly larger number of operations, but it has been found that ¹¹¹ In is rapidly incorporated into the polymer ligand 2 with high efficiency.

(7b: Complex formation using physiological saline)
2 (420 μg) was dissolved in 2100 μL of physiological saline in a glass test tube. 20 μL of ¹¹¹ InCl ₃ solution (manufactured by Nippon Mediphysics, ˜20 MBq / mL) was added thereto, and stirred well for 15 minutes using an ultrasonic cleaner. When encapsulating a pharmaceutical compound such as an anticancer agent, it is added simultaneously but requires longer stirring. At the time of stirring, it was necessary to pay attention to the temperature rise of the solution. After stirring for 15 minutes, the radiochemical purity was assayed by thin layer chromatography (ITLC-SG manufactured by Gelmal Sciences, developed with silica gel glass fiber plate, physiological saline) and found to be 98.0%. When the concentration of saline is low (200 μL) and the concentration is high, ¹¹¹ In coordination is slow (about 50% in the purity test). became. Although the method using physiological saline is simple, it was found that complex formation takes a little time depending on the concentration, and it is necessary to confirm the complex formation by a purity test.

[Example 8]
(8) the polymer complex ^{(111 In-1, 111 In} -2) of the gel permeation chromatography polymer complex ^{(111 In-1, 111 In} -2) relative molecular mass and state of complexation of the gel permeation chromatography Analyzed by graphy (GPC). The apparatus was an LC-10 inert HPLC system manufactured by Shimadzu Corporation, the GPC column was TOSOH SW-3000, and the eluent was phosphate buffer (pH 7.0) at 0.2 mL / min. A UV detector (220 nm) and a γ-ray detector were used to detect the sample. The sample concentration was 1 μg / mL to eliminate the effect of micelle bodies. At 10 μg / mL or more, only a large peak derived from the aggregates at the elution position of 2.0 to 2.5 mL and a wide elution curve from 2.5 to 5.0 mL appeared, and information on the molecular weight of the monomer was not given.

Using a UV detector (220 nm) to detect the sample, ¹¹¹ In-1 has three peaks corresponding to molecular weights of 5000 (monomer), 10100 (dimer), and 15100 (trimer). It was seen (Fig. 7a). ¹¹¹ In-2 showed a sharp peak at a molecular weight of 5500 (FIG. 7b).

When a γ-ray detector was used for detection of the sample, ¹¹¹ In-1 prepared in Example 6a showed a wide elution peak derived from the 1 to 3 mer and a large tailing. When the GPC measurement was performed again using the fraction showing the maximum peak (no. 24), the same broad elution peak was shown, but no tailing was observed (FIG. 8a). ¹¹¹ In-2 showed a sharp elution peak derived from the monomer, and tailing did not occur (FIG. 8b). Similarly, even when the GPC chromatogram of the fraction showing the maximum peak (no. 23) was measured, a sharp elution peak derived from the monomer was shown.

When considered as a polymer complex, ¹¹¹ In-1 was not separated by silica gel chromatography, but GPC was found to be coordinated in such a way that ¹¹¹ In was separated. 1 is coordinated in such a way that ¹¹¹ In is easily dislodged in the GPC column (for example, a slightly weak coordination bond between one Pal side chain and a PEG chain on the hydrophobic helix) and can be purified by GPC. I found it suitable. 2 was coordinated in such a way that ¹¹¹ In would not come off in the GPC column (eg, chelate coordination by two Pal side chains on a hydrophobic helix), and GPC purification was not necessarily required .

[Example 9]
(9) Detection of tumor tissue with polymer ligand (2) According to synthesis procedure 2, the Boc group of polymer ligand (2) was deprotected to generate an amino group. Carboxyfluorescein diacetate active ester (CFDA-OSu) was coupled to this amino group by synthesis procedure 4. When DMAP was added in a catalytic amount (0.2 equivalent to 2), the reaction proceeded efficiently. CF-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe (SEQ ID NO: 12) -PEG ₄₀₀₀ (3) obtained after purification was added to a physiological saline solution (approximately 1 mg / 1 mL) ) Was administered (100 μL / animal) to nude mice transplanted with fiber cancer cells at the base of the hind limbs. Two hours later, the mouse was dissected and each tissue was observed with a fluorescence imager (Maestro manufactured by CRi). As a result, not only tumor tissue but also metastatic cancer to the liver could be detected (FIG. 9). Thus, it was found that the polymer ligand (2) has a property of accumulating in the tumor tissue.

[Example 10]
(10) using a polymer complex ^{(111 In-1, 111 In} -2) polymer complex created in pharmacokinetic examples 6 and 7 of ^{(111 In-1, 111 In} -2), weight 28~30g The biodistribution behavior of male ddy mice (weight 28-30 g) was examined. 20μg / 100μL per animal was administered from the tail vein, and after sacrifice and dissection after 1, 4 and 24 hours, each organ was weighed, and the radioactivity was quantified with a γ-counter (% dose / g). ) Was calculated. Tables 1 and 2 show the distribution of ¹¹¹ In-1 and ¹¹¹ In-2 in the body. Under this condition, accumulation in the kidney is frequently observed, but when a higher concentration of the polymer complex is administered, accumulation in the liver is gradually increased. In the case of ¹¹¹ In-2, the accumulation in the kidney (~ 20% dose / g) peaks after 4 to 24 hours, and the accumulation in the liver is small but gradually increases after 24 and 48 hours (2.6 to 4.0%) dose / g). Along with this, the blood concentration decreased rapidly (10.0% dose / g 1 hour after administration to 1.0% dose / g 24 hours later). In addition, accumulation in bone (6.7-9.3% dose / g) is considered to originate from ¹¹¹ In ions liberated by decomposition of the polymer complex. There was no characteristic accumulation for other tissues. ¹¹¹ In-1 showed the same tendency.

[Example 11]
(11) Accumulation of polymer complex ( ^111In -2) in tumor tissue Using the polymer complex ( ^111In -2) prepared in Examples 6 and 7, fiber cancer cells were transplanted to the base of the hind limb. Biodistribution behavior in SCID mice was investigated. 40 μg / 100 μL (40 g / mouse) per animal was administered from the tail vein, and after 4 hours after sacrificed dissection, each organ was weighed and the radioactivity was quantified with a γ-counter. g) was calculated. Table 3 shows the distribution in the body. Under these conditions, accumulation in the kidney (24% dose / g) was often observed, but accumulation in the tumor tissue was 5.8% dose / g. Thus, it was found that the polymer complex ( ^111In -2) has a property of accumulating in the tumor tissue.

FIG. 1 shows Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (Boc-SEQ ID NO: 13-PEG ₄₀₀₀ ) (1) prepared in Examples 1 and 2 and its synthesis intermediate. Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (Boc-SEQ ID NO: 12-PEG ₄₀₀₀ ) (2) and its synthetic intermediates, It is the spectrum measured by MALDI-TOF mass spectrometry. FIG. 2 shows Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (Boc-SEQ ID NO: 12-PEG ₄₀₀₀ ) (2 ) prepared in Example 2. ) Are excitation fluorescence spectra of a saturated pyrene aqueous solution at each concentration. Excitation wavelength = 300 to 360 nm; Detection wavelength = 390 nm; Slit width = 5 nm; Measurement temperature 20 ° C. FIG. 3 shows the Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ prepared in Example 2 (Boc-SEQ ID NO: 12-PEG ₄₀₀₀ ) (2 ), The intensity ratio (I ₃₃₇ / I ₃₃₄ ) at 337 nm and 334 nm is plotted against the concentration of 2 for the excitation fluorescence spectrum (FIG. 2) of the saturated pyrene aqueous solution at each concentration. The concentration that greatly changed the graph was defined as the critical micelle concentration (cmc). FIG. 4 shows the Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ prepared in Example 2 (Boc-SEQ ID NO: 12-PEG ₄₀₀₀ ) (2 ) Is a diagram showing an apparent spectral change when it was dropped into an aqueous solution of CoCl ₂ (0.1 mM). As the concentration of 2 (calculated with an average molecular weight of 2 as 5505) increased (0.1, 0.2, 0.3, 0.4 mM), the baseline at 400-800 nm increased. This represents an increase in apparent absorbance as 2 increases in concentration, as 2 forms larger aggregates and scatters incident light. FIG. 5 shows the Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ prepared in Example 2 (Boc-SEQ ID NO: 12-PEG ₄₀₀₀ ) (2 Is a diagram showing a change in spectrum when the solution is dropped into an aqueous solution of CoCl ₂ (0.1 mM). The dd absorption of Co (II) ions changed with increasing isotopes at 547, 575, 606, 654, and 675 nm as the concentration of 2 (calculated assuming the average molecular weight of 2 as 5505) increased. Strong absorption maxima were observed at 590 and 660 nm. FIG. 6 shows Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (Boc-SEQ ID NO: 12-PEG ₄₀₀₀ ) (2 ) prepared in Example 2. Is a graph plotting the change in absorbance at 590 nm with respect to the apparent change in spectrum when it was dropped into an aqueous solution of CoCl ₂ (0.1 mM). When the concentration of 2 was 0.1 mM or less, as shown in FIG. 5, it was considered that scattering due to aggregation was almost negligible, and the binding constant was calculated by the nonlinear least square method. By using K = [CoCl ₂ -2] / [CoCl ₂ ] [2] in the calculation formula, the results of Δabs∞ = 0.86 and K = 1.2 × 10 ⁵ M ⁻¹ were obtained. FIGS. 7a and 7b show gel permeation chromatograms detected by ultraviolet light (220 nm) of a polymer complex ( ¹¹¹ In-1, ¹¹¹ In-2), which is an embodiment of the present invention. (GPC device, Shimadzu LC-10 inert HPLC system; GPC column, TOSOH SW-3000; eluent, phosphate buffer (pH 7.0) 0.2 mL / min; UV detector, 220 nm) ¹¹¹ In− Three peaks with molecular weight considered to be equivalent to 5000 (monomer), 10100 (dimer), and 15100 (trimer) were observed for 1 (FIG. 7a). ¹¹¹ In-2 showed a sharp peak at a molecular weight of 5500 (FIG. 7b). FIGS. 8a and 8b show gel permeation chromatograms detected by γ-rays of polymer complexes ( ¹¹¹ In-1 and ¹¹¹ In-2), respectively, which are one embodiment of the present invention. (GPC device, Shimadzu LC-10 inert HPLC system; GPC column, TOSOH SW-3000; eluent, phosphate buffer (pH 7.0) 0.2 mL / min; γ-ray counter sampled every 0.2 mL) Chromatogram was prepared by quantification.) When a γ-ray detector was used to detect the sample, ¹¹¹ In-1 showed a wide elution peak derived from 1 to 3 mer and large tailing (Fig. 8a-as prepared) . Among them, when GPC measurement was performed again using fraction No. 23 showing the maximum peak, the same broad elution peak was shown but no tailing was observed (FIG. 8a-fraction). ¹¹¹ In-2 showed a sharp elution peak derived from the monomer, and tailing did not occur (FIG. 8b-as prepared). The same result was obtained when GPC measurement was performed again using fraction no. 24 showing the maximum peak (FIG. 8b-fraction). FIG. 9 shows the Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ prepared in Example 2 (Boc-SEQ ID NO: 12-PEG ₄₀₀₀ ) (2 CF-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe- (SEQ ID NO: 12) PEG ₄₀₀₀ (3) was administered to cancer-bearing nude mice (approximately A fluorescent image of metastatic cancer to the liver detected at 100 μg / animal) is shown (photograph). (At 2 hours after administration, each organ was removed and observed with a fluorescence imager (Maestro manufactured by CRi). The schematic diagram of the complex formation structure of the polymer complex of this invention. FIGS. 10A and 10B show Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (Boc-SEQ ID NO: 13-PEG ₄₀₀₀ ), respectively. The cross-linked or mononuclear structure of a polymer complex comprising (1) and CoCl ₂ is shown. FIGS. 10C and 10D show Boc-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Pal-Leu-Leu-Aib-Phe-PEG ₄₀₀₀ (Boc-SEQ ID 12-PEG ₄₀₀₀ ) (2) and CoCl, respectively. The cross-linked or mononuclear structure of the polymer complex consisting of ₂ .

Claims (6)

¹¹¹ In and amphiphilic block copolymer composed of hydrophobic ligand and polyalkylene glycol
A polymer complex comprising
A depsipeptide having a helix structure, wherein the hydrophobic ligand is represented by the following general formula (a), (b) or (c) and includes an amino acid having a heterocycle capable of coordinating to a metal atom in a side chain, A polymer complex that is a peptide.
Y ₁ -Leu-Leu-Aib-Xaa-Leu-Leu-Aib-Phe- (a) (SEQ ID NO: 1)
Y ₁ -Leu-Leu-Aib-Xaa-Leu-Leu-Aib-Yaa-Leu-Leu-Aib-Phe- (b) (SEQ ID NO: 2)
Y ₁ -Leu-Leu-Aib-Xaa-Leu-Leu-Aib-Yaa-Leu-Leu-Aib-Zaa-Leu-Leu-Aib-Phe- (c) (SEQ ID NO: 3)
(Wherein, Xaa, Yaa and Zaa is lower following general formula any of (IV) ~ (VI), represents an amino acid having a coordinatable heterocycles metal atom in the side chain, Aib is Aminoisobutylic acid residues Y ₁ represents an amino acid, peptide, hydroxycarboxylic acid, depsipeptide or amino protecting group.)

(X ₁ and X ₂ represent hydrogen, an alkyl group, a silyl group, an amide group, a urethane group, an ether group, or an ester group.) The polymer complex according to claim 1, wherein the amphiphilic block copolymer is represented by the following general formula (I), (II), or (II ').
Polyalkylene glycol-block-hydrophobic ligand (I)
Hydrophobic ligand-block-polyalkylene glycol (II)
Hydrophobic ligand-block-polyalkylene glycol-block-hydrophobic ligand (II ') The polymer complex according to claim 1 or 2, wherein the metal atom is a metal atom forming a low molecular metal complex. The low molecular metal complex is selected from the group consisting of porphyrin ligand, chlorin ligand, Schiff base ligand, thiolate ligand, thioether ligand, imidazole ligand, phenolate ligand or The polymer complex according to claim 3, which is a low-molecular complex having a plurality of ligands. The pharmaceutical composition containing the polymer complex of any one of Claims 1-4. The pharmaceutical composition according to claim 5 for treating or diagnosing cancer.

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