WO2014178229A1 - 放射性ガリウム結合部位を有するポリペプチド、及び、その放射性ガリウム錯体 - Google Patents

放射性ガリウム結合部位を有するポリペプチド、及び、その放射性ガリウム錯体 Download PDF

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WO2014178229A1
WO2014178229A1 PCT/JP2014/056553 JP2014056553W WO2014178229A1 WO 2014178229 A1 WO2014178229 A1 WO 2014178229A1 JP 2014056553 W JP2014056553 W JP 2014056553W WO 2014178229 A1 WO2014178229 A1 WO 2014178229A1
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group
polypeptide
formula
radioactive gallium
terminal
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PCT/JP2014/056553
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English (en)
French (fr)
Japanese (ja)
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英郎 佐治
暢也 稲垣
木村　寛之
健太郎 豊田
洋和 松田
博樹 松本
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国立大学法人京都大学
日本メジフィジックス株式会社
アークレイ株式会社
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Priority to JP2015514773A priority Critical patent/JPWO2014178229A1/ja
Publication of WO2014178229A1 publication Critical patent/WO2014178229A1/ja

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0478—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group complexes from non-cyclic ligands, e.g. EDTA, MAG3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides

Definitions

  • 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.
  • 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.
  • 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.
  • labeled peptide derivatives it has been studied to bind them to glucagon-like peptide 1 receptor (GLP-1R).
  • GLP-1R glucagon-like peptide 1 receptor
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • GLP-1R glucagon-like peptide 1 receptor
  • the present invention has been made in view of the above circumstances, and provides a radioactive gallium-labeled ligand targeting GLP-1R.
  • 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)
  • Y- indicates that the N-terminal ⁇ -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)
  • B- represents an N-terminal ⁇ -amino group modified with a modifying group having the
  • 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.
  • Another aspect of the present invention provides a radiopharmaceutical composition containing the above complex.
  • 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.
  • kits for preparing the above complex comprising the above polypeptide or a salt thereof.
  • a radioactive gallium-labeled ligand targeting GLP-1R is provided.
  • an asterisk (*) in the structural chemical formula of the substituent indicates the point of attachment to the rest of the molecule.
  • 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 ⁇ -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.
  • 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.
  • radioactive gallium examples 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).
  • PET positron emission tomography
  • 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.
  • Ga-67 half-life 78 hours
  • SPECT single photon emission computed tomography
  • the “modifying group having a radioactive gallium binding site” refers to an N-terminal ⁇ -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.
  • the present invention consists of an amino acid sequence represented by the above formula (I).
  • an N-terminal ⁇ -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).
  • n is an integer of 0 or 1, but an integer of 1 is preferable.
  • L represents a linker, specifically, an alkyl group having 1 to 15 carbon atoms or a group represented by the general formula (2).
  • 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.
  • m is an integer of 1 to 30, preferably an integer of 5 to 20, and more preferably 10 to 15 Is an integer.
  • 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.
  • 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. ]
  • 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. ]
  • n is preferably an integer of 1 and L is preferably an alkyl group having 1 to 15 carbon atoms.
  • 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 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.
  • the polypeptide of the present invention or a salt thereof can be produced by peptide synthesis according to a conventional method.
  • 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.
  • 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.
  • the Fmoc synthesis method an amino acid whose N-terminal ⁇ -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
  • 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.
  • 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.
  • automatic peptide synthesizers include 431A (Applied Biosystems) and PSSM-8 (Shimadzu Corporation).
  • 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 ⁇ -amino group.
  • 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.
  • the amino group is deprotected and paraisothiocyanate benzyl-desferoxamine (Df-Bz-NCS) is introduced.
  • Df-Bz-NCS paraisothiocyanate benzyl-desferoxamine
  • 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 ⁇ -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.
  • the polypeptide or salt thereof according to the present invention can form a radioactive gallium complex by reacting with the radioactive gallium.
  • 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).
  • SEQ ID NO: 8 represented by the following general formula (5).
  • 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).
  • Ga 3+ is a trivalent radioactive gallium cation.
  • 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.
  • 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).
  • 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+ .
  • 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 ( ⁇ , 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.
  • 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. .
  • a nuclear reaction such as 66 Zn (d, n) 67 Ga, 68 Zn (p, 2n) 67 Ga, nat Zn (p, x) 67 Ga, etc.
  • zinc 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.
  • 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).
  • 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.
  • 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.
  • a solvent for example, a Good's buffer can be used, and a MES buffer is preferably used.
  • the concentration of MEM is preferably 0.001 to 10 mol / L.
  • a surfactant such as twin can be added to the solvent, and the concentration can be, for example, 0.01 to 1% by volume.
  • the polypeptide concentration in the complex reaction solution can be, for example, 0.01 to 1000 ⁇ mol / L. From the viewpoint of improving the yield, 0.1 to 100 ⁇ mol / L is preferable.
  • the obtained radioactive gallium complex can be purified by high-speed chromatography (HPLC), hydrophobic chromatography, reverse phase chromatography, or the like.
  • 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.
  • the polypeptide of the present invention or a salt thereof can be powdered, for example, a lyophilized powder.
  • the kit according to the present invention may include a container for storing the polypeptide of the present invention.
  • a container for storing the polypeptide of the present invention examples 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.
  • the kit according to the present invention can include a solvent separately from the polypeptide of the present invention or a salt thereof.
  • a solvent those which improve the radiochemical yield are preferable, and examples thereof include Good's buffer (Good's buffer).
  • MES buffer is preferable.
  • kit according to the present invention may include an instruction manual describing the method for producing the radioactive gallium complex of 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.
  • 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.
  • each step was repeated a plurality of times as necessary, and an amount necessary for use as an intermediate in other synthesis was ensured.
  • LC-MS liquid chromatogram mass spectrometer
  • High-performance liquid chromatography 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.
  • LC-8A or LC-20A constant speed pump
  • SPD-20A spectrophotometric detector
  • Tl NaI 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 ⁇ 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.
  • 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:
  • 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
  • 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.
  • 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.
  • 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).
  • Df-Bz -Protected peptide resin 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.
  • 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.
  • 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 (5 ⁇ mol / L) 20 ⁇ L 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.
  • buffer (pH 5.5) solution obtained by dissolving in (5 ⁇ mol / L) 20 ⁇ L 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.
  • Example 3 Labeled synthesis of 67 Ga-Df-Bz-NCS- (PEG12) 12-Ex4 Df-Bz-NCS- (PEG12) 12-Ex4 (100 ⁇ mol / L) in 30 ⁇ L 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 ⁇ L) 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.
  • 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.
  • 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-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).
  • the protected peptide represented by the following formula (24) is reacted with Df-Bz-NCS (p-SCN-Bn-Deferoxamine, manufactured by Macrocyclics) and DIEA.
  • Df-Bz-NCS p-SCN-Bn-Deferoxamine, manufactured by Macrocyclics
  • DIEA DIEA
  • 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 (0.5-50 ⁇ mol / L) 20 ⁇ L 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).
  • Example 7 Labeled synthesis of 67 Ga- (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) (Df-Bz-NCS-Ahx) 9-Ex4 (9-39) (5 ⁇ mol / L) It was dissolved in 300 ⁇ L of 0.1 mol / MES buffer (0.1 vol% twin 80), and a solution of [ 67 Ga] gallium chloride (14.8 MBq, 4 ⁇ L) 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.
  • 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.
  • Binding Buffer 50 mmol / L HEPES, 5 mmol / L magnesium chloride and 0.2 vol% BSA in water, pH 7.4 155 ⁇ L, 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.
  • 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
  • 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 ⁇ L) synthesized by the method shown in Example 7 was mixed with plasma (200 ⁇ L), and 30,60 at 37 ° C. , 120, 240 minutes.
  • Methanol 100 ⁇ L 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 ratio of unchanged substance was calculated from the analysis at 5 mL / min.
  • 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.
  • 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 ⁇ L 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.
  • (18.5-37.0 kBq / 100 ⁇ L) was administered from the tail vein of the mouse.
  • 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).
  • 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 (Glucagon-like Peptide 1 (Human, 7-36 Amide, Peptide Laboratories) (20 ⁇ g / 100 ⁇ L) was administered under anesthesia, and after 30 minutes, 67 Ga- (Df-Bz- NCS-Ahx) 9-Ex4 (9-39) (18.5-37.0 kBq / 100 ⁇ L) 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.
  • Example 14 PET / CT imaging of INS-1 tumor-bearing mouse 68 Ga-Df-Bz-NCS- (PEG12) 12-Ex4 (18.5 MBq / 50 ⁇ L) 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.
  • the PET / CT apparatus FX -300, manufactured by Gamma Medica Co., Ltd.
  • CT imaging 60 kV, 310 ⁇ A
  • Image reconstruction was performed using 3D-OSEM.
  • FIG. 3A shows a coronal plane image
  • FIG. 3B shows a sagittal plane image
  • FIG. 3C shows a cross-sectional image.
  • 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 (5 ⁇ mol / L) 200 ⁇ L, 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 ⁇ L) 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 ⁇ L) 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 ⁇ A) was performed. Image reconstruction was performed using 3D-OSEM.

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JP2011507863A (ja) * 2007-12-19 2011-03-10 イミューノメディクス、インコーポレイテッド タンパク質、ペプチド、および他の分子のｆ−１８標識のための改良された方法および組成物
WO2012046845A1 (ja) * 2010-10-08 2012-04-12 国立大学法人京都大学 ペプチド誘導体及びその使用
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JP2018517761A (ja) * 2015-03-10 2018-07-05 セラグノスティクス リミテッドＴｈｅｒａｇｎｏｓｔｉｃｓ Ｌｉｍｉｔｅｄ 放射性核種錯体を調製するための方法およびキット
JP2021130675A (ja) * 2015-03-10 2021-09-09 セラグノスティクス リミテッドTheragnostics Limited 放射性核種錯体を調製するための方法およびキット
JP7109627B2 (ja) 2015-03-10 2022-07-29 セラグノスティクス リミテッド 放射性核種錯体を調製するための方法およびキット
JP2022153493A (ja) * 2015-03-10 2022-10-12 セラグノスティクス リミテッド 放射性核種錯体を調製するための方法およびキット
US11826436B2 (en) 2015-03-10 2023-11-28 Theragnostics Limited Methods and kits for preparing radionuclide complexes
JP7412487B2 (ja) 2015-03-10 2024-01-12 セラグノスティクス リミテッド 放射性核種錯体を調製するための方法およびキット

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