WO2014178229A1 - Polypeptide having radioactive gallium binding site, and radioactive gallium complex thereof - Google Patents

Abstract

The present invention is a polypeptide, which is an exendin-4 peptide derivative, or a salt thereof. Said polypeptide comprises an amino acid sequence represented by Y-QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ (I). In formula (I), Y- represents an N-terminal α-amino group being attached to a C-terminal carboxyl group of an amino acid sequence represented by any one of B-DLSK (II), H₂N-DLSX (III), B-HGEGTFTSDLSK (IV) or H₂N-HGEGTFTSDLSX (V). In formulas (II) and (IV), B- represents an N-terminal α-amino group modified by a modifying group having a radioactive gallium binding site. In formulas (III) and (V), X represents a lysine residue modified by a modifying group wherein a side chain amino group has a radioactive gallium binding site. The N-terminal α-amino group of the aforementioned B- and the side chain amino group of the lysine residue of the aforementioned X are each a polypeptide or a salt thereof, modified by a modifying group having a radioactive gallium binding site represented by the prescribed general formula.

Description

Polypeptide having radioactive gallium binding site and radioactive gallium complex thereof

The present invention relates to a polypeptide having a radioactive gallium binding site and a radioactive gallium complex thereof.

Insulinoma accounts for about 70% of pancreatic endocrine tumors and overproduces insulin. When performing surgical resection for treatment of insulinoma, it is necessary to identify the tumor site that is localized in the pancreas. The inspection method at that time needs to be performed by inserting a catheter into the pancreas, which is labor intensive and burdens the patient. Therefore, development of a technique capable of non-invasively identifying a localized site of an endocrine tumor is desired.

For example, Patent Document 1 discloses the radioactivity of GLP-1, exendin-3, and exendin-4 in which a labeled molecule into which a radionuclide such as In-111 has been introduced is attached to the C-terminus for noninvasive display of insulinoma. Using labeled peptide derivatives, it has been studied to bind them to glucagon-like peptide 1 receptor (GLP-1R). Insulinomas are known to be tumors in which GLP-1R is overexpressed, and it is expected that insulinomas can be imaged by drugs that bind to GLP-1R.

It is also known that cells expressing GLP-1R secrete insulin. Therefore, it is expected that an agent that binds to GLP-1R can visualize the density of insulin-producing cells in the pancreas in vivo and in vitro. As described in Patent Document 1, imaging of GLP-1 receptor density in the pancreas is particularly important for diabetic patients during and after treatment with pharmaceuticals.

In Patent Documents 2 to 10, a polypeptide derived from exendin-4 is labeled with fluorine-18 or radioactive iodine, whereby non-invasive three-dimensional imaging of pancreatic islets and imaging for quantitative determination of pancreatic islets can be performed. It is being considered.

Special table 2008-511557 gazette International Publication 2010/032509 Pamphlet International Publication 2010/032833 Pamphlet International Publication No. 2011/027584 Pamphlet JP 2011-219442 A International Publication No. 2011/027738 Pamphlet International Publication 2011/040460 Pamphlet International Publication No. 2011/071083 Pamphlet International Publication No. 2012/046845 Pamphlet International publication 2012/108476 pamphlet

By the way, gallium-68 is a positron emitting nuclide having a half-life of 67.7 minutes, and has an appropriate half-life and quantification like fluorine-18. On the other hand, since gallium- ⁶⁸ can be produced by a ⁶⁸ Ge / ⁶⁸ Ga generator, unlike ¹⁸ F-labeled compounds, it can be prepared at the time of use even in a facility that does not have a cyclotron. In addition, SPECT nuclide gallium-67 also exists as radioactive gallium. Since gallium-67 has a long half-life of 3.26 days, basic research on ⁶⁸ Ga-labeled drugs can be facilitated.

However, a radioactive gallium-labeled ligand targeting the glucagon-like peptide 1 receptor (GLP-1R) is not yet known.

The present invention has been made in view of the above circumstances, and provides a radioactive gallium-labeled ligand targeting GLP-1R.

One aspect of the present invention is a polypeptide which is a peptide derivative of exendin-4 or a salt thereof,
The polypeptide consists of an amino acid sequence represented by the following formula (I):
Y-QMEEEAVRLFIEWLKNGGPSSGAPPPS-CONH ₂ (I) (SEQ ID NO: 1)
In the formula (I),
“Y-” indicates that the N-terminal α-amino group is peptide-bonded to the C-terminal carboxyl group of the amino acid sequence represented by the following formulas (II) to (V):
B-DLSK (II) (SEQ ID NO: 2)
H ₂ N-DLSX (III) (SEQ ID NO: 3)
B-HGEGTFTSDLSK (IV) (SEQ ID NO: 4)
H ₂ N-HGEGTFTSDLSX (V) (SEQ ID NO: 5)
In the formulas (II) and (IV), “B-” represents an N-terminal α-amino group modified with a modifying group having the radioactive gallium binding site,
In the formulas (III) and (V), “X” represents a lysine residue in which a side chain amino group is modified with a modifying group having the radioactive gallium binding site,
The N-terminal α-amino group of “B-” and the side chain amino group of the lysine residue of “X” are represented by the following general formula (1):

　（式中、ｍは１～３０の整数である。）で表される基である。〕
で表される放射性ガリウム結合部位を有する修飾基により修飾されている、ポリペプチド又はその塩を提供するものである。 [Wherein n is an integer of 0 or 1, L is an alkyl group having 1 to 15 carbon atoms, or the following general formula (2):

(Wherein m is an integer of 1 to 30). ]
The polypeptide or its salt modified by the modification group which has a radioactive gallium binding site represented by these is provided.

Another aspect of the present invention provides a complex of the above polypeptide and radioactive gallium.

Also, another aspect of the present invention provides a radiopharmaceutical composition containing the above complex.

In addition, another aspect of the present invention provides a method for producing a radioactive gallium complex, comprising a step of reacting the above polypeptide or a salt thereof with radioactive gallium to obtain the above complex.

Further, another aspect of the present invention provides a kit for preparing the above complex comprising the above polypeptide or a salt thereof.

According to the present invention, a radioactive gallium-labeled ligand targeting GLP-1R is provided.

It is a diagram showing the stability of the plasma ^{67 Ga- (Df-Bz-NCS} -Ahx) 9-Ex4 (9-39). ^{67 Ga- (Df-Bz-NCS} -Ahx) 9-Ex4 (9-39) exendin 4 (9-39) in the INS-1 tumor-bearing mice received shows the effect of prior administration. It is a figure which shows the PET / CT image of the INS-1 cancer bearing mouse | mouth using ⁶⁸ Ga-Df-Bz-NCS- (PEG12) 12-Ex4 (9-39). It is a figure which shows the PET / CT image of the INS-1 cancer bearing mouse | mouth using ⁶⁸ Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39).

In the present specification, an asterisk (*) in the structural chemical formula of the substituent (such as the above general formula (1) or general formula (2)) indicates the point of attachment to the rest of the molecule.

[Polypeptide or salt thereof]
The present invention is a polypeptide which is a peptide derivative of exendin-4 or a salt thereof. Exendin-4 is a hormone with a hypoglycemic action contained in saliva secretions of the American poison lizard and is known as a GLP-1R agonist. The polypeptide according to the present invention or a salt thereof is an amino acid sequence of exendin-4 or an amino acid sequence from which 8 amino acids have been deleted from the N-terminus (ie, exendin-4 (9-39): as an antagonist of GLP-1R Which is modified with a N-terminal α-amino group or a modifying group having a radioactive gallium binding site on the amino group of the side chain of lysine at the 9-position. The carboxyl group at the C-terminal is amidated with an amino group from the viewpoint of improving the binding property with GLP-1R.

In the present invention, the “radioactive gallium binding site” refers to a site where deferoxamine is introduced, and this site can be coordinated to a trivalent radioactive gallium cation. Examples of radioactive gallium include Ga-66, Ga-67, and Ga-68. Ga-66 and Ga-68 are preferable, and Ga-68 is more preferable. Ga-66 has a relatively long half-life of 9.5 hours and emits positrons with a particularly large energy of 4.2 MeV. Therefore, Ga-66 is a useful radionuclide in positron emission tomography (PET). Ga-68 is preferred because it has a short half-life of 68 minutes but is sufficient to non-invasively follow many biochemical processes in vivo with PET. On the other hand, Ga-67 (half-life 78 hours) is a useful radionuclide in single photon emission computed tomography (SPECT).

In the present invention, the “modifying group having a radioactive gallium binding site” refers to an N-terminal α-amino group or a side chain of lysine at position 9 and a radioactive gallium binding site in addition to the radioactive gallium binding site. It is preferable to have a linker. This linker may be an alkyl group, a polyethylene glycol linker, or both of them.

That is, the present invention consists of an amino acid sequence represented by the above formula (I). In formula (I), an N-terminal α-amino group (“B-” in formula (II) and formula (IV)) or lysine at position 9 (in formula (III) and formula (V) “ The side chain amino group of X ″) is represented by the above general formula (1). In general formula (1), n is an integer of 0 or 1, but an integer of 1 is preferable.

In the general formula (1), L represents a linker, specifically, an alkyl group having 1 to 15 carbon atoms or a group represented by the general formula (2). When L is an alkyl group having 1 to 15 carbon atoms, it is preferably a linear alkyl group having 1 to 15 carbon atoms, more preferably a linear alkyl group having 1 to 10 carbon atoms, still more preferably Is a 3-7 linear alkyl group. When L is a group represented by the general formula (2), in the general formula (2), m is an integer of 1 to 30, preferably an integer of 5 to 20, and more preferably 10 to 15 Is an integer.

In the polypeptide according to the present invention, from the viewpoint of increasing affinity for GLP-1R, “Y-” in formula (I) is an amino acid sequence represented by formula (II) or formula (V). preferable. That is, a polypeptide represented by the following general formula (3) (SEQ ID NO: 6) or the following general formula (4) (SEQ ID NO: 7) is preferable.

[In the formula (3), n is an integer of 0 or 1, L is an alkyl group having 1 to 15 carbon atoms or the above general formula (2) (wherein m is an integer of 1 to 30). It is group represented by these. ]

[In the formula (4), n is an integer of 0 or 1, and L is an alkyl group having 1 to 15 carbon atoms or the above general formula (2) (wherein m is an integer of 1 to 30). It is group represented by these. ]

As described above, in general formulas (3) and (4), is an integer of 0 or 1, but an integer of 1 is preferable.

In the above formula (I), when “Y-” is the amino acid sequence represented by the formula (II), that is, when the polypeptide according to the present invention is represented by the above general formula (4), the general formula ( In 1) and general formula (4), n is preferably an integer of 1 and L is preferably an alkyl group having 1 to 15 carbon atoms.

In the above formula (I), when “Y-” is an amino acid sequence represented by the formula (V), that is, when the polypeptide according to the present invention is represented by the general formula (3), the general formula (1) ) And general formula (3), n is an integer of 1, and L is a group represented by general formula (2) (wherein m is an integer of 1 to 30). Is preferred.

The polypeptide according to the present invention may form a salt, and such a salt is included in the present invention in a pharmaceutically acceptable salt. “Salts” of the present invention include those derived from inorganic or organic acids, or inorganic or organic bases. Specifically, hydrochloride, hydrobromide, hydroiodic acid, sulfate, nitrate, perchlorate, fumarate, maleate, phosphate, glycolate, lactate, salicylate , Succinate, tartrate, acetate, trifluoroacetate, citrate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, aspartate, glutamate, formate, benzoate , Malonate, naphthalene-2-sulfonate, trifluoroacetate, benzenesulfonate, amine salt and ammonium salt, but are not limited thereto.

Next, a method for producing the polypeptide of the present invention or a salt thereof will be described. The polypeptide of the present invention or a salt thereof can be produced by peptide synthesis according to a conventional method. Examples of the organic chemical peptide synthesis method include a solid phase synthesis method and a liquid phase synthesis method. Peptide synthesis by the solid phase synthesis method is preferred. In the solid-phase synthesis method, the C-terminal of an amino acid or peptide is fixed to a solid-phase carrier via a linker, and the amino acids are sequentially extended to the N-terminal side.

Examples of the peptide synthesis method by the solid phase synthesis method include the Fmoc synthesis method and the Boc synthesis method, and the Fmoc synthesis method is preferable. In the Fmoc synthesis method, an amino acid whose N-terminal α-amino group was protected by Fmoc (9-fluorenylmethyloxycarbonyl group) was used, and the amino group of the amino acid fixed on the solid support and the Fmoc protected group In this method, a peptide bond is formed between a monoamino acid and a carboxylic acid. The peptide is extended by repeating deprotection and washing of Fmoc and addition of an Fmoc-protected monoamino acid after introduction into the solid phase carrier. At this time, for amino acids having a functional group in the side chain, a protecting group is introduced according to the type of the functional group. After extending to the desired length, the protecting group for the side chain functional group is added together with the N-terminal Fmoc. Deprotect to obtain the desired peptide.

Such solid phase peptide synthesis may be performed using an automatic peptide synthesizer. Examples of commercially available automatic peptide synthesizers include 431A (Applied Biosystems) and PSSM-8 (Shimadzu Corporation).

When “Y-” in the formula (I) is a polypeptide having the amino acid sequence of the formula (II) or (IV), the “modifying group having a radioactive gallium binding site” is a peptide bond of Fmoc-protected histidine or aspartic acid. Then, Fmoc may be deprotected and introduced into the N-terminal α-amino group. For example, a linear alkyl carboxylic acid having 1 to 15 carbon atoms having a protected amino group at its end (for example, glycine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-aminopentanoic acid, 6-aminohexanoic acid, 7 -Aminoheptanoic acid, 8-aminooctylic acid, etc.), then the amino group is deprotected and paraisothiocyanate benzyl-desferoxamine (Df-Bz-NCS) is introduced. Thereafter, by deprotecting the functional group on the side chain, a polypeptide in which a modifying group having a radioactive gallium binding site is introduced into the N-terminal α-amino group can be obtained.

On the other hand, when “Y-” in the formula (I) is a polypeptide having the amino acid sequence of the formula (III) or (V), the functional group on the side chain of lysine corresponding to the ninth position of exendin-4 is N-terminal. A protecting group that is deprotected under different conditions from the protecting group for protecting the α-amino group and other side chain functional groups (for example, a trityl group or a derivative thereof, preferably a trityl group, a monomethoxy group) It is preferably protected with a trityl group or a dimethoxytrityl group. After extension by peptide synthesis is completed, only the side chain of lysine that introduces a “modified group having a radioactive gallium binding site” is deprotected, and after introduction of an alkyl linker or polyethylene glycol linker having a protected amino group at the terminal, the linker The terminal amino group of is deprotected and Df-Bz-NCS is introduced. Thereafter, by deprotecting the N-terminal amino group and the side chain functional group, a polypeptide in which a modifying group having a radioactive gallium binding site is introduced into lysine corresponding to the ninth position of exendin-4 is obtained. Can do.

[Radioactive gallium complex]
The polypeptide or salt thereof according to the present invention can form a radioactive gallium complex by reacting with the radioactive gallium. For example, the polypeptide represented by the general formula (3) is reacted with a trivalent radioactive gallium cation to form a radioactive gallium complex (SEQ ID NO: 8) represented by the following general formula (5). Can do.

[In the formula (5), n is an integer of 0 or 1, and L is an alkyl group having 1 to 15 carbon atoms or the above general formula (2) (wherein m is an integer of 1 to 30). Ga ³⁺ is a trivalent radioactive gallium cation. ]

Moreover, the polypeptide represented by the general formula (4) is allowed to form a radioactive gallium complex (SEQ ID NO: 9) represented by the following general formula (6) by acting with a trivalent radioactive gallium cation. Can do.

[In the formula (6), n is an integer of 0 or 1, and L is an alkyl group having 1 to 15 carbon atoms or the above general formula (2) (wherein m is an integer of 1 to 30). Ga ³⁺ is a trivalent radioactive gallium cation. ]

Examples of the trivalent radioactive gallium cation include ⁶⁶ Ga ³⁺ , ⁶⁷ Ga ³⁺ and ⁶⁸ Ga ³⁺ . ^Preferably, a 66 ^{Ga 3+} and ⁶⁸ Ga ^3+, more ^preferably 68 ^{Ga 3+. 66} Ga ³⁺ and ⁶⁸ Ga ³⁺ are suitable for the production of gallium complexes for PET, while ⁶⁷ Ga ³⁺ is suitable for the production of gallium complexes for SPECT.

⁶⁶ Ga ³⁺ uses a cyclotron, ⁶³ Cu (α, n) ⁶⁶ Ga, ⁶⁶ Zn (p, n) ⁶⁶ Ga, ⁶⁸ Zn (p, 3n) ⁶⁶ Ga, ^nat Zn (p, x) ⁶⁶ Ga, etc. It is produced by causing a nuclear reaction of By performing chemical separation from the target, ⁶⁶ Ga ³⁺ suitable for complex production can be obtained. Examples of chemical separation include L.P. C. Brown, Int. J. et al. Appl. Radiat. Isot. 22, 1971, 710-713 can be performed using a solvent-solvent extraction method using isopropyl ether and HCl, in which case ⁶⁶ Ga ³⁺ is separated from the zinc target [ ⁶⁶ Ga] gallium chloride ([ ⁶⁶ Ga] GaCl ₃ ).

⁶⁷ Ga ³⁺ is generated by causing a nuclear reaction such as ⁶⁶ Zn (d, n) ⁶⁷ Ga, ⁶⁸ Zn (p, 2n) ⁶⁷ Ga, ^nat Zn (p, x) ⁶⁷ Ga, etc. using a cyclotron. . When zinc is used as a target, it can be separated from the target by using hydrochloric acid to obtain [ ⁶⁷ Ga] gallium chloride ([ ⁶⁷ Ga] GaCl ₃ ). [ ⁶⁷ Ga] gallium citrate is commercially available from Nippon Mediphysics Corporation as a pharmaceutical product.

⁶⁸ Ga ³⁺ is obtained from a ⁶⁸ Ge / ⁶⁸ Ga generator. Such a generator is, for example, C.I. Loc'h et al, J. MoI. Nucl. Med. 21, 1980, 171-173, and those commercially available from Eckert & Ziegler (Obninsk ⁶⁸ Ge / ⁶⁸ Ga generator). In general, ⁶⁸ Ge is packed in a column made of an organic resin or an inorganic metal oxide such as tin dioxide, aluminum dioxide or titanium dioxide. ⁶⁸ Ga ³⁺ can be obtained, for example, as [ ⁶⁸ Ga] gallium chloride ([ ⁶⁸ Ga] GaCl ₃ ) by eluting from the column using hydrochloric acid as an eluent.

The complex can be formed by bringing the trivalent radioactive gallium cation thus obtained into contact with the polypeptide according to the present invention. Preferably, the trivalent radioactive gallium cation and the polypeptide of the present invention are mixed in a solvent. More preferably, the reaction is carried out under a weakly acidic pH of 4 to 6. As the solvent, for example, a Good's buffer can be used, and a MES buffer is preferably used. At this time, the concentration of MEM is preferably 0.001 to 10 mol / L. In addition, a surfactant such as twin can be added to the solvent, and the concentration can be, for example, 0.01 to 1% by volume.

When a solvent is used, the polypeptide concentration in the complex reaction solution can be, for example, 0.01 to 1000 μmol / L. From the viewpoint of improving the yield, 0.1 to 100 μmol / L is preferable.

The obtained radioactive gallium complex can be purified by high-speed chromatography (HPLC), hydrophobic chromatography, reverse phase chromatography, or the like.

[kit]
The radioactive gallium complex according to the present invention may be prepared using a kit containing the polypeptide according to the present invention or a salt thereof. This kit comprises the polypeptide of the present invention or a salt thereof as it is or in a state dissolved in a solvent. When the polypeptide of the present invention or a salt thereof is provided as it is, the polypeptide of the present invention or a salt thereof can be powdered, for example, a lyophilized powder.

Moreover, the kit according to the present invention may include a container for storing the polypeptide of the present invention. Examples of the shape of the container include a vial and a syringe. The material of the container may be glass or plastic, but when used as a reaction container and charged with a solvent and radioactive gallium to form a complex in the container, radioactive gallium is used. It is preferable to use a material with less adsorption.

Moreover, the kit according to the present invention can include a solvent separately from the polypeptide of the present invention or a salt thereof. As the solvent, those which improve the radiochemical yield are preferable, and examples thereof include Good's buffer (Good's buffer). Among these, MES buffer is preferable.

Moreover, the kit according to the present invention may include an instruction manual describing the method for producing the radioactive gallium complex of the present invention.

[Radiopharmaceutical composition]
In the present invention, the obtained radiogallium complex can be formulated into a form suitable for administration into a living body, and a radiopharmaceutical composition containing the complex as an active ingredient can be obtained. This radiopharmaceutical composition includes pharmacologically acceptable carriers, diluents, emulsions, excipients, bulking agents, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers. Additional components such as a preservative, a preservative, a solubilizer, a preservative, a colorant, and a stabilizer may be included.

The above-mentioned radiopharmaceutical composition can be used for an oral or parenteral administration method, but is preferably used for a parenteral administration method, and is preferably administered intravenously, intraarterially, locally, intraperitoneally. Or, an injection that can be used for administration to the thoracic cavity, subcutaneous administration, intramuscular administration, sublingual administration, transdermal administration, or rectal administration is more preferred. Such an injection can be prepared by dissolving the above-described radioactive gallium complex in water, physiological saline, Ringer's solution, or the like. The concentration of the radioactive gallium complex in the radiopharmaceutical composition may be any concentration that can ensure stability against radiolysis.

The radiopharmaceutical composition of the present invention can be imaged non-invasively by injecting GLP-1R in a living body by administering it to mammals including humans and imaging it with PET or SPECT. Therefore, it is useful for the imaging of insulinoma and the diagnosis, treatment, and prevention of diabetes.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these contents. In the synthesis examples of each polypeptide described in the following examples, each step was repeated a plurality of times as necessary, and an amount necessary for use as an intermediate in other synthesis was ensured.

The following reagents and devices were used in the examples.
The polypeptides to be synthesized and evaluated below were synthesized according to the attached software using an automatic peptide synthesizer (431A, manufactured by Applied Biosystems).
A liquid chromatogram mass spectrometer (LC-MS) consists of a constant speed pump (LC-10AP, manufactured by Shimadzu Corp.), a spectrophotometric detector (SPD-10AP, manufactured by Shimadzu Corp.) and an MS detector (MS-2010, Shimadzu Corp.). (Manufactured by Seisakusho).
Mass spectrometry (MS) was performed using Shimadzu GC-MS-QP Plus.
High-performance liquid chromatography (HPLC) includes a constant speed pump (LC-8A or LC-20A, manufactured by Shimadzu Corporation), a spectrophotometric detector (SPD-20A, manufactured by Shimadzu Corporation), and online NaI (Tl) scintillation detection. A system to which a vessel (NDW-351D, Aloka) was connected was used.
⁶⁸ Ga ^is, 68 ^Ge / 68 Ga- generator ^{(Obninsk 68 Ge / 68 Ga-} Generator, Eckert & Ziegler Co., Ltd.) was used extracted from.
[ ¹²⁵ I] Tyr-GLP-1 (7-36) was purchased from PerkinElmer.
Radioactivity was measured using a Curie meter (IGC-7, manufactured by ALOKA) and an autowell γ counter (Wallac 1480 WIZARD 3, manufactured by PerkinElmer).
Collection of images using a PET / CT apparatus was performed using a GMI FX-3300 Pre-Clinical Imaging System, and 3D-OSEM was used for data analysis.

In addition, regarding the statistical processing of the examples, the data was expressed as an average value ± standard deviation, the significance test was performed using the Tukey-Kramer method, and p <0.05 was regarded as significant. Animal experiments were conducted with the approval of the Kyoto University Animal Experiment Committee.

Abbreviations used in this specification are as follows unless otherwise specified.
Rink Amide MBHA Resin (trade name, manufactured by Merck & Co., Inc.): 4- (2 ′, 4′-dimethoxyphenyl-Fmoc-aminomethyl) -phenoxyacetamide-norleucyl-MBHA
HBTU: 1- [bisdimethylaminomethylene] -1H-benzotriazolium-3-oxide-hexafluorophosphate HOBt: 1-hydroxybenzotriazole DMF: dimethylformamide DCM: dichloromethane Boc-mini-PEG-3TM (trade name) , Manufactured by Peptide International): Boc-11-amino-3,6,9-trioxaundecanoic acid / DCHA
Boc: butoxycarbonyl group DCHA: dicyclohexylamine NMP: N-methylpyrrolidone TFA: trifluoroacetic acid TIS: triisopropylsilane DT: dodecanethiol DIEA: N, N-diisopropylethylamine OBu: tert-butyl ester group Trt: trityl group Pdf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group Mmt: 4-methoxytrityl group Fmoc: 9-fluorenylmethyloxycarbonyl group PEG12: -NH- (C ₂ H ₄ O) ₁₂ -C ₂ H ₄ -C (O)-
Fmoc-Ahx-OH: 6- (Fmoc-amino) hexanoic acid Ahx: 6-aminohexanoyl Df-Bz-NCS: paraisothiocyanate benzyl-desferoxamine (a compound represented by the following formula (7) or a substitution thereof) Base)

PBS (−): phosphate buffered saline without calcium and magnesium% ID / g:% dose / g (% injected dose / g)
OSEM: Ordered subset expectation maximization method

MES: 2-morpholinoethanesulfonic acid monohydrate twin 80 (trade name): polyoxyethylene sorbitan monooleate HEPES: 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid BSA: bovine Serum albumin As the amino acid, L-form was used unless otherwise specified.

Example 1 Synthesis of Df-Bz-NCS- (PEG12) 12-Ex4 The lysine side chain at position 12 of exendin-4 was deferoxamine as a ligand via a polyethylene glycol linker with a repeat number of 12. A modified polypeptide represented by the formula (11) (SEQ ID NO: 10, hereinafter abbreviated as “Df-Bz-NCS- (PEG12) 12-Ex4”) was synthesized by the following method.

1. Synthesis of Protected Peptide Resin A peptide peptide synthesizer (431A) manufactured by Applied Biosystems is used to bind protected amino acids one by one from the carboxyl terminal side according to the attached software (solid phase synthesis method). Synthesis was performed. Rink Amide MBHA Resin (0.25 mmol scale) was used as a starting resin carrier, Fmoc-amino acid derivative was set in the reaction vessel of the peptide synthesizer, and HBTU was used as an activator according to the software attached to the synthesizer. , Dissolved in HOBt and DMF and added to the reaction vessel to react. The obtained resin was gently stirred in piperidine-containing N-methylpyrrolidone to remove the Fmoc group and proceed to condensation of the next amino acid derivative. Among the Fmoc amino acid derivatives used, amino acids having functional groups in the side chains are Asp (OBu), Ser (OBu), Lys (Boc), Lys (Mmt), Gln (Trt), Glu (OBu), and Trp (respectively). Boc), Arg (Pbf), Asn (Trt) were used. Amino acids were sequentially extended according to the sequence to obtain a protected peptide resin (SEQ ID NO: 11) represented by the following formula (12).

Boc-HGEGFTSDLSK (Mmt) QMEEEAVRLFIEWLKNGGPSSGAPPPS-Rink Amide MBHA (12)

From the protected peptide resin represented by the above formula (12), treatment with 1.5 vol% TFA / 5 vol% TIS / 93.5 vol% DCM protects the amino group of the lysine side chain at position 12 of exendin-4 After removing the Mmt group, Fmoc-PEG12-OH (Fmoc-N-amido-dPEG12-acid, manufactured by QUANTA Bio-design) was introduced by the HATU-HOAt method, and the protected peptide resin represented by the following formula (13) (SEQ ID NO: 12) was obtained.

Boc-HGEGFTSDLSK (Fmoc-PEG12-) QMEEEAVRLFIEWLKNGGPSSGAPPPS-Rink Amide MBHA (13)

After removing the Fmoc group of (Fmoc-PEG12-) introduced into the amino group of the lysine side chain from the protected peptide resin represented by the above formula (13) using 20% by volume piperidine / NMP, Df-Bz -Protected peptide resin (SEQ ID NO: 13) represented by the following formula (14) was obtained by introduction through a condensation reaction using NCS (p-SCN-Bn-Deferoxamine, manufactured by Macrocyclics) and DIEA as a base.

Boc-HGEGFTSDLSK (Df-Bz-NCS-PEG12-) QMEEEAVRLFIEWLKNGGPSSGAPPPS-Rink Amide MBHA (14)

2. Deprotection and Cleavage from Resin The obtained protected peptide resin was prepared by standard deprotection conditions using TFA [TFA / TIS / water / DT: 95 / 2.5 / 2.5 / 2.5 (v / v) Then, the mixture was treated at room temperature for 2 hours to simultaneously perform deprotection and cleaving of the peptide from the resin. After the carrier resin was filtered off from the reaction solution, TFA was distilled off, ether was added to the residue, and the precipitated crude product was collected by filtration.

3. Isolation and purification of target peptide The obtained sparsely produced peptide was subjected to a water-acetonitrile elution system (0.1% by volume) containing 0.1% by volume TFA using an HPLC preparative apparatus (reverse phase column (ODS), 30 × 250 mm). Volume% TFA-containing water: 0.1 volume% TFA-containing acetonitrile (volume ratio) = gradient from 70:30 (0.01 minutes) to 10:90 (20 minutes)), flow rate 3.5 mL / min, elution time 9 .2 minutes), and acetonitrile was distilled off, followed by lyophilization powder to obtain the peptide represented by the above formula (11) as a TFA salt. In the above formulas (12) to (14), the side chain protecting group was omitted.

(Example 2) < ⁶⁸ > Ga-Df-Bz-NCS- (PEG12) 12-Ex4 labeling synthesis study Df-Bz-NCS- (PEG12) 12-Ex4 synthesized in Example 1 was converted to 0.01 mmol / L MES. buffer (pH 5.5) solution obtained by dissolving in (5μmol / L) 20μL to ⁶⁸ Ge ^{/ 68} Ga- extracted ⁶⁸ Ga solution from generator (1.2 mol / L sodium acetate buffer, 200 [mu] L) was added 20 [mu] L. The mixture was allowed to stand at room temperature for 30 minutes. Purification was performed using an HPLC analyzer (LC-20A, manufactured by Shimadzu Corporation) using a reverse phase column (COSMOSIL 5C ₁₈ -AR-II (10 × 250 mm), manufactured by Nacalai Tesque). The mobile phase was gradient from 0.1 volume% TFA-containing water: 0.1 volume% TFA-containing acetonitrile (volume ratio) = 70: 30 (0.01 minutes) to 10:90 (20 minutes) with a flow rate of 3.5 mL. / Minute (elution time 9.2 minutes).

Example 3 Labeled synthesis of ⁶⁷ Ga-Df-Bz-NCS- (PEG12) 12-Ex4 Df-Bz-NCS- (PEG12) 12-Ex4 (100 μmol / L) in 30 μL of 0.01 mol / MES buffer (0.1 vol% twin 80) was dissolved, and a solution of [ ⁶⁷ Ga] gallium chloride (4.77 MBq, 1 μL) was added. The mixture was allowed to stand at room temperature for 5 minutes, and production of the target product was confirmed by LC-MS. Purification was performed using a HPLC analyzer (LC-20A, manufactured by Shimadzu Corporation) and a reverse phase column (COSMOSIL 5C ₁₈ -AR-II, 10 × 250 mm, manufactured by Nacalai Tesque). The mobile phase is gradient from 0.1 volume% TFA-containing water: 0.1 volume% TFA-containing acetonitrile (volume ratio) = 70: 30 (0.01 minutes) to 10:90 (20 minutes). It was 5 mL / min (elution time 9.2 minutes).

(Example 4) Synthesis of Ga-Df-Bz-NCS- (PEG12) 12-Ex4 (unlabeled) [ ⁶⁷ Ga] The same as Example 3 except that non-radioactive gallium chloride was used instead of gallium chloride. Thus, non-radioactive Ga-Df-Bz-NCS- (PEG12) 12-Ex4 was synthesized. The desired product was identified by LC / MS and purified by reverse phase HPLC.

Example 5 Synthesis of (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) The N-terminus of exendin-4 (9-39) was defenoxamined as a ligand via an n-pentyl linker. A modified polypeptide represented by the formula (21) (SEQ ID NO: 14, hereinafter abbreviated as “(Df-Bz-NCS-Ahx) 9-Ex4 (9-39)”) was synthesized by the following method.

1. Synthesis of protected peptide resin Using Rink Amide MBHA Resin (0.25 mmol scale) as a starting resin carrier, Fmoc-amino acid derivative is set in the reaction vessel of the peptide synthesizer and activated according to the software attached to the synthesizer The agents HBTU and HOBt were dissolved in DMF and added to the reactor for reaction. The resulting resin was gently stirred in piperidine-containing N-methylpyrrolidone to remove the Fmoc group and proceed to condensation of the next amino acid derivative. Among the Fmoc amino acid derivatives used, amino acids having a functional group in the side chain are Asp (OBu), Ser (OBu), Lys (Boc), Gln (Trt), Glu (OBu), Trp (Boc), Arg ( Pbf) and Asn (Trt) were used. Amino acids were sequentially extended according to the sequence to obtain a protected peptide resin (SEQ ID NO: 15) represented by the following formula (22).

Fmoc-DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-Rink Amide MBHA (22)

Fmoc-Ahx-OH was removed from the protected peptide resin represented by the above formula (22) by the HBTU-HOBt method by removing the Fmoc group to obtain a protected peptide resin (SEQ ID NO: 16) represented by the following formula (23).

Fmoc-Ahx-DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-Rink Amide MBHA (23)

After removing the Fmoc group from the protected peptide resin represented by the formula (23), the protected peptide represented by the following formula (24) is reacted with Df-Bz-NCS (p-SCN-Bn-Deferoxamine, manufactured by Macrocyclics) and DIEA. A resin (SEQ ID NO: 17) was obtained.

Df-Ahx-DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-Rink Amide MBHA (24)

2. Deprotection and excision from resin The obtained protected peptide resin was subjected to standard deprotection conditions using TFA [TFA / TIS / water / DT: 95 / 2.5 / 2.5 / 2.5 (v / v) ] At room temperature for 2 hours to simultaneously perform deprotection and release of the peptide from the resin. After the carrier resin was filtered off from the reaction solution, TFA was distilled off, and ether was added to the residue to precipitate a crude product precipitate.

3. Isolation and purification of target peptide The crude peptide thus obtained was purified using an HPLC preparative apparatus (reverse phase column (ODS, 30 × 250 mm), water-acetonitrile system containing 0.1 vol% TFA (0.1 vol%). TFA-containing water: 0.1% by volume TFA-containing acetonitrile (volume ratio) = gradient from 70:30 (0.01 minutes) to 10:90 (20 minutes), flow rate 3.5 mL / min, elution time 9.2 In the above formulas (22) to (24), the target peptide represented by the formula (21) was obtained as a TFA salt after the acetonitrile was distilled off and lyophilized powder. The side chain protecting group was omitted.

(Example 6) ⁶⁸ Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) labeling synthesis study (Df-Bz-NCS-Ahx) 9-Ex4 (9-) synthesized in Example 5 39) was used as a labeling precursor, and labeling conditions with ⁶⁸ Ga were examined. MES buffer (pH 5.5) of (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) synthesized in Example 5 at various concentrations (shown in the cell of “peptide concentration” in Table 1 below) in dissolved solution (0.5-50μmol / L) 20μL to ⁶⁸ Ge ^{/ 68} Ga- extracted ⁶⁸ Ga solution from generator (1.2 mol / L sodium acetate buffer, 200 [mu] L) was added 20 [mu] L. The mixture was allowed to stand at room temperature for 30 minutes. Purification was performed using an HPLC analyzer (LC-20A, manufactured by Shimadzu Corporation) using a reverse phase column (COSMOSIL 5C ₁₈ -AR-II (10 × 250 mm), manufactured by Nacalai Tesque). The mobile phase was gradient from 0.1 volume% TFA-containing water: 0.1 volume% TFA-containing acetonitrile (volume ratio) = 70: 30 (0.01 minutes) to 10:90 (20 minutes) with a flow rate of 3.5 mL. / Minute (elution time 9.2 minutes).

The examination results are shown in Table 1. The concentration of MES buffer and the radiochemical yield by adding Twin 80 were examined (Table 1, Experiment No. 1-10). As a result, when the (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) concentration was 50 μmol / L, high radiochemical yields were achieved under all conditions, and the MES buffer concentration was high. In the case of the condition (1.0 mol / L), a tendency to decrease the radiochemical yield was shown. As a result of examining the MES buffer concentration conditions, there was a tendency that the radiochemical yield increased with decreasing MES concentration (Table 1, Experiment Nos. 5, 6, and 8). Moreover, as a result of examining the radiochemical yield due to the addition of Twin 80 as a solubilizer, both (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) concentration was 50 μmol / L. High and no significant difference was seen (Table 1, Experiment Nos. 1-4). (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) concentration 5μmol / L shows significant improvement in radiochemical yield from 5.7% to 88.8% with or without twin 80 added (Table 1, Experiment Nos. 6 and 7). As a further purpose of improving the specific activity, were subjected to labeling study at (Df-Bz-NCS-Ahx ) 9-Ex4 (9-39) concentration of 0.5 [mu] mol / L ^condition, 68 Ga-labeled body It was obtained only in a low yield (Table 1, Experiment Nos. 9 and 10). From these results, it was revealed that the optimal Ga labeling condition was a condition in which 0.1% by volume of Twin 80 was added to 0.01 mol / L MES buffer (pH 5.5).

Example 7 Labeled synthesis of ⁶⁷ Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) (5 μmol / L) It was dissolved in 300 μL of 0.1 mol / MES buffer (0.1 vol% twin 80), and a solution of [ ⁶⁷ Ga] gallium chloride (14.8 MBq, 4 μL) was added. The mixture was allowed to stand at room temperature for 5 minutes, and production of the target product was confirmed by LC-MS. Purification was performed using a HPLC analyzer (LC-20A, manufactured by Shimadzu Corporation) and a reverse phase column (COSMOSIL 5C ₁₈ -AR-II, 10 × 250 mm, manufactured by Nacalai Tesque). The mobile phase is gradient from 0.1 volume% TFA-containing water: 0.1 volume% TFA-containing acetonitrile (volume ratio) = 70: 30 (0.01 minutes) to 10:90 (20 minutes). It was 5 mL / min (elution time 9.2 minutes).

(Example 8) Synthesis of Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) (unlabeled) Except that [ ⁶⁷ Ga] gallium chloride was used instead of non-radioactive gallium chloride In the same manner as in Example 7, non-radioactive Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) was synthesized. The desired product was identified by LC / MS and purified by reverse phase HPLC. The HPLC conditions were the same as in Example 7.

(Example 9) Evaluation of affinity for GLP-1R Ga-Df-Bz-NCS- (PEG12) 12-Ex4 and Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) The affinity for GLP-1R was evaluated by binding inhibition experiments using G ^125-1 receptor membrane protein and [ ¹²⁵ I] Tyr-GLP-1 (7-36) as a competitive ligand.
Binding Buffer (50 mmol / L HEPES, 5 mmol / L magnesium chloride and 0.2 vol% BSA in water, pH 7.4) 155 μL, Ga-Df-Bz-NCS- (PEG12) 12-Ex4 synthesized in Example 4, Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39), GLP-1 (7-36) amide (Glucagon-like Peptide 1 (Human, 7-36 Amide, peptide research) synthesized in Example 8 ), Exendin 4 amide (Exendin 4, manufactured by WAKO) or exendin 4 (9-39) amide (Exendin Fragment 9-39, manufactured by Sigma-Aldrich Co. LLC.) (0, 10 ⁻¹¹ , 10 ⁻¹⁰⁾ , ^{10 -9, 10} -8, 10 ^{7, 10 -6, 10 -5 mol} / L) 20μL, [125 I] Tyr-GLP-1 (7-36) (5nmol / L) 20μL, Membrane Preparation Recombinant Human GLP-1 (Millipore Corp., 200 units 1 mL) 5 μL was mixed and shaken for 2 hours at room temperature.The shaken liquid was subjected to B / F separation using glass fiber filter paper, and the filter paper was washed with Buffer (25 mmol / L HEPES, 0.5 mol / L sodium chloride). Then, the radioactivity remaining on the filter paper was measured using an autowell γ counter, and the obtained measurement result was GraphPad Prism version 5.03 ( Using GraphPad Software) Analysis, and to calculate the IC50.

The results are shown in Table 2. As a result, Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) is 9.32 nmol / L, which is more than the parent compound exendin 4 (9-39) amide (30.24 nmol / L). It was confirmed to have a high affinity. Ga-Df-Bz-NCS- (PEG12) 12-Ex4 is inferior in affinity to exendin 4 amide (6.09 nmol / L) which is a parent compound, but exendin 4 (9-39) amide (30. 24 nmol / L) was confirmed to have a higher affinity.

(Example 10) Evaluation of stability in plasma In order to confirm the stability of ⁶⁷ Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) in vivo, stability in plasma in vitro Sex was evaluated. Plasma was collected from BALB / c nu / nu mice (male, 4 weeks) and shaken at 37 ° C. for 10 minutes. ⁶⁷ Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) (370 kBq / 10 μL) synthesized by the method shown in Example 7 was mixed with plasma (200 μL), and 30,60 at 37 ° C. , 120, 240 minutes. Methanol (100 μL) was added to the shaking solution to aggregate plasma protein components, and centrifuged at 4 ° C. at 10,000 × g for 5 minutes to obtain a supernatant. The supernatant was filtered using a Milex filter-GV (13 mm), and using a Radio-HPLC analyzer (LC-20A, manufactured by Shimadzu Corporation), a reverse phase column (COSMOSIL 5C ₁₈ -AR-II, 10 × 250 mm) , Manufactured by Nacalai Tesque). The mobile phase is gradient from 0.1 volume% TFA-containing water: 0.1 volume% TFA-containing acetonitrile (volume ratio) = 70: 30 (0.01 minutes) to 10:90 (20 minutes). The ratio of unchanged substance was calculated from the analysis at 5 mL / min.

The results are shown in FIG. As a result, it was confirmed that 78% of the unchanged product was present even after 240 minutes, and 94% of the unchanged product was present even after 60 minutes almost at the same time as the half life of ⁶⁸ Ga.

(Example 11) Pharmacokinetic evaluation using normal mice ⁶⁷ Ga-Df-Bz-NCS- (PEG12) 12-Ex4 synthesized by the method shown in Example 3, ⁶⁷ Ga- synthesized by the method shown in Example 7 As a basic evaluation of (Df-Bz-NCS-Ahx) 9-Ex4 (9-39), pharmacokinetic evaluation was performed using normal mice (ddY mice, 6 weeks old, male). From the viewpoint of ease of handling, in this study, ⁶⁷ Ga was used instead of ⁶⁸ Ga. ⁶⁷ Ga-Df-Bz-NCS- (PEG12) 12-Ex4 or ⁶⁷ Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) (18.5-37.0 kBq / 100 μL under anesthesia ) Was administered from the mouse tail vein. After administration, each organ (pancreas, blood, heart, lung, stomach, small intestine, large intestine, liver, spleen, kidney) was removed (n = 5) at 5, 15, 30, 60, and 120 minutes. The weight and radioactivity of each organ were measured, and the accumulation amount (% ID / g) was calculated from the radioactivity per unit weight. The results for ⁶⁷ Ga-Df-Bz-NCS- (PEG12) 12-Ex4 are shown in Table 3, and the results for ⁶⁷ Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) are shown in Table 4. .

(Example 12) Evaluation of pharmacokinetics using INS-1 tumor-bearing mice Tumor transplanted animals (INS-1 tumor-bearing mice) were prepared by the following method. BALB / c nu / nu mice (female, 4 weeks old) were purchased from Japan SLC. The animals were reared under a 12-hour / 12-hour day / night cycle condition, and feed and water were freely given. INS-1 cells (provided by Kyoto University Graduate School of Medicine, Department of Diabetes Nutrition) were suspended in PBS (−) and implanted subcutaneously into the right lower limb (2.5 × 10 ⁶ -5.0 × 10 ⁶ cells / 100 μL PBS (-) / Animal). Tumor volume, based on the (length) × ^(width) 2/2 was measured, using a mouse became 100 mm ³ or more in the evaluation.
⁶⁷ Ga-Df-Bz-NCS- (PEG12) 12-Ex4 or ⁶⁷ Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) without anaesthesia to the INS-1 tumor-bearing mice prepared above. ) (18.5-37.0 kBq / 100 μL) was administered from the tail vein of the mouse. After administration, it is 15, 30, 60, 120 minutes for ⁶⁷ Ga-Df-Bz-NCS- (PEG12) 12-Ex4, and about ⁶⁷ Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) Was sacrificed at 5, 15, 30, 60, 120 minutes, and each organ (pancreas, blood, heart, lung, stomach, small intestine, large intestine, liver, spleen, kidney, tumor, muscle) was removed (n = 5). ). The weight and radioactivity of each organ were measured, the radioactivity after attenuation correction was obtained from the measured value, and the accumulation amount (% ID / g) was calculated from the radioactivity per unit weight.

The results for ⁶⁷ Ga-Df-Bz-NCS- (PEG12) 12-Ex4 are shown in Table 5. ^Further, Table 6 shows the results of 67 Ga- (Df-Bz-NCS -Ahx) 9-Ex4 (9-39). As a result, ⁶⁷ Ga-Df-Bz-NCS- (PEG12) 12-Ex4 was confirmed to have high accumulation in the tumor at 26.2% ID / g 30 minutes after administration (Table 5). Further, as for the adjacent organ ratio of the pancreas important for imaging, a high adjacent organ ratio of a tumor / pancreas ratio of 1.71, a tumor / blood ratio of 6.75, and a tumor / liver ratio of 17.30 can be obtained 30 minutes after administration. (Table 5). In addition, ⁶⁷ Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) confirmed 13.1% ID / g and high accumulation in the tumor 30 minutes after administration (Table 6). ). In addition, as for the adjacent organ ratio of the pancreas important for imaging, a high adjacent organ ratio such as a tumor / pancreas ratio of 2.85, a tumor / blood ratio of 2.67, and a tumor / liver ratio of 1.63 can be obtained 30 minutes after administration. (Table 6).

(Example 13) Inhibition evaluation using INS-1 tumor-bearing mice The accumulation of ⁶⁷ Ga-Df-Bz-NCS- (PEG12) 12-Ex4 in Example 12 in a tumor is a GLP-1R-specific accumulation. In order to examine the fact, the change in the accumulation amount by pre-administration of Ex4 (9-39) was examined. Exendin 4 (9-39) (Glucagon-like Peptide 1 (Human, 7-36 Amide, Peptide Laboratories) (20 μg / 100 μL) was administered under anesthesia, and after 30 minutes, ⁶⁷ Ga- (Df-Bz- NCS-Ahx) 9-Ex4 (9-39) (18.5-37.0 kBq / 100 μL) was administered from the tail vein, 30 minutes after the administration, each organ was removed, and the weight and radiation of each organ The amount of accumulation (% ID / g) was calculated from the radioactivity per unit weight.

The results are shown in FIG. In the figure, “(−) Ex (9-39)” is a result of a mouse not pre-administered exendin 4 (9-39), and “(+) Ex (9-39)” is exendin 4 (9-39) results for mice pre-administered with 77.6% tumor accumulation (p <0.001) and 65.8% pancreas accumulation (p <0.001). ) And a significant decrease.

(Example 14) PET / CT imaging of INS-1 tumor-bearing mouse ⁶⁸ Ga-Df-Bz-NCS- (PEG12) 12-Ex4 (18.5 MBq / 50 μL) synthesized under the conditions shown in Example 2 An INS-1 tumor-bearing mouse prepared by the method shown in Example 12 was intravenously injected without anesthesia, and anesthetized with isoflurane (2.0%) from 5 minutes after administration. After 20 minutes from administration, the PET / CT apparatus (FX -300, manufactured by Gamma Medica Co., Ltd.) for 10 minutes. Thereafter, CT imaging (60 kV, 310 μA) was performed. Image reconstruction was performed using 3D-OSEM.

The results are shown in FIG. 3A shows a coronal plane image, FIG. 3B shows a sagittal plane image, and FIG. 3C shows a cross-sectional image. As a result, the tumor transplanted to the right leg was depicted.

(Example 15) PET / CT imaging of INS-1 tumor-bearing mouse 0.01 mol / L MES buffer (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) synthesized in Example 5 ( pH 5.5) to a solution (5μmol / L) 200μL, after adding Tween 80 to so as to contain 0.1 ^vol%, 68 ^Ge / 68 Ga- extracted from the generator ^[68 Ga] gallium chloride solution (209MBq (5 .65 mCi), 200 μL) was added and allowed to stand at room temperature for 5 minutes. Purification was performed as described in Example 6. The obtained ⁶⁸ Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) (18.5 MBq / 50 μL) was transferred to an INS-1 cancer-bearing mouse prepared by the method shown in Example 12. Intravenous injection was performed without anesthesia, and anesthesia with isoflurane (2.0%) was aspirated from 5 minutes after administration, and PET imaging was performed for 10 minutes using a PET / CT apparatus (FX-3300, manufactured by Gamma Medica) from 20 minutes after administration. Thereafter, CT imaging (60 kV, 310 μA) was performed. Image reconstruction was performed using 3D-OSEM.

The results are shown in FIG. As a result, the tumor transplanted to the right leg was depicted in the cross-sectional image.

As mentioned above, although embodiment and the Example of this invention were described, these are the illustrations of this invention and various structures other than the above are also employable.

Claims (8)

A polypeptide which is a peptide derivative of exendin-4 or a salt thereof,
The polypeptide consists of an amino acid sequence represented by the following formula (I):
Y-QMEEEAVRLFIEWLKNGGPSSGAPPPS-CONH ₂ (I) (SEQ ID NO: 1)
In the formula (I),
“Y-” indicates that the N-terminal α-amino group is peptide-bonded to the C-terminal carboxyl group of the amino acid sequence represented by the following formulas (II) to (V):
B-DLSK (II) (SEQ ID NO: 2)
H ₂ N-DLSX (III) (SEQ ID NO: 3)
B-HGEGTFTSDLSK (IV) (SEQ ID NO: 4)
H ₂ N-HGEGTFTSDLSX (V) (SEQ ID NO: 5)
In the formulas (II) and (IV), “B-” represents an N-terminal α-amino group modified with a modifying group having the radioactive gallium binding site,
In the formulas (III) and (V), “X” represents a lysine residue in which a side chain amino group is modified with a modifying group having the radioactive gallium binding site,
The N-terminal α-amino group of “B-” and the side chain amino group of the lysine residue of “X” are represented by the following general formula (1):

[Wherein n is an integer of 0 or 1, L is an alkyl group having 1 to 15 carbon atoms, or the following general formula (2):

(Wherein m is an integer of 1 to 30). ]
A polypeptide or a salt thereof, which is modified with a modifying group having a radioactive gallium binding site represented by the formula: In the formula (I), “Y-” indicates that the N-terminal α-amino group is peptide-bonded to the C-terminal carboxyl group of the amino acid sequence represented by the formula (II). 2. The polypeptide or a salt thereof according to claim 1, wherein in general formula (1), n is an integer of 1 and L is a linear or branched alkyl group having 1 to 15 carbon atoms. In the formula (I), “Y-” represents that the N-terminal α-amino group is peptide-bonded to the C-terminal carboxyl group of the amino acid sequence represented by the formula (V), The polypeptide or its salt of Claim 1 whose n is an integer of 1 in General formula (1), and L is group represented by the said General formula (2). A complex of the polypeptide according to any one of claims 1 to 3 and radioactive gallium. A radiopharmaceutical composition comprising the complex according to claim 4. The radiopharmaceutical composition according to claim 5, which is used for imaging of insulinoma. A method for producing a radioactive gallium complex, comprising the step of reacting the polypeptide according to any one of claims 1 to 3 or a salt thereof with radioactive gallium to obtain the complex according to claim 4. A kit for preparing the complex according to claim 4, comprising the polypeptide according to any one of claims 1 to 3 or a salt thereof.

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