JP6839447B2 - Peptides with accumulation specific to biliary tract cancer and their use - Google Patents

Description

The present invention relates to peptides having a specific accumulation in biliary tract cancer and their use.
The present application claims priority based on Japanese Patent Application No. 2015-196227 filed in Japan on October 1, 2015, the contents of which are incorporated herein by reference.

Biliary tract cancer is a cancer consisting of bile duct cancer, gallbladder cancer, and duodenal papilla cancer. The number of deaths from bile duct cancer and gallbladder cancer in Japan is about 8,900 for men and about 9,300 for women in 2013. In 2010, the prevalence of bile duct cancer and gallbladder cancer was approximately 11,300 males and 11,300 females, accounting for 2% and 3% of the total cancer prevalence, respectively. In addition, among patients with cholangiocarcinoma and gallbladder cancer, surgery is applicable to about 20% of the total, and the survival rate 5 years after the surgery is about 30%.
Biliary tract cancer has few subjective symptoms at an early stage, and may have progressed to some extent when symptoms such as jaundice, white stool, jaundice urine, and abdominal pain appear, and early detection is important in treatment. .. The biliary tract cancer in the present specification includes adenocarcinoma derived from bile duct epithelium, adenosquamous carcinoma, and intrahepatic cholangiocarcinoma histopathologically.

Biliary tract cancer (particularly intrahepatic or extrahepatic bile duct epithelial-derived cancer) can be examined and diagnosed by blood biochemical examination, abdominal ultrasonography, abdominal contrast CT examination, endoscopic retrograde pancreatobiliary angiography (ERCP:). Examples thereof include endoscopic retrograde cholangiopancreatography) and FDG-PET (Fluorodeoxy glucose-Positron Emission Tomography) examination.

By the way, in the trend in the medical field using peptides as biomaterials, cell membrane penetrating (cell absorptive) peptides such as Tat, penetratin, and polyargine have been attracting attention.
However, since these peptides are widely and non-selectively absorbed without distinction between normal cells or normal tissues and tumor cells or tumor tissues, the treatment of malignant tumors requiring targeted drug transport is DDS (Drag Delivery). Application to System) tools is difficult to use in that it causes serious side effects. In particular, cell membrane penetrating (cell-absorbing) peptides such as Tat, which are widely used in experimental systems worldwide, are known to have the property of causing accumulation in the liver (see, for example, Non-Patent Document 1).
In contrast, cyclic RGD is the only peptide that has been medicinalized. Cyclic RGD is the alpha _v beta ₃ integrin has been reported to be highly expressed in endothelial cells of the neovasculature or in existing vessels (and some tumor cells) has been targeted, the point of action on the vascular hyperpermeability Since it has, it is applied as an imaging agent or a DDS agent not only alone but also in combination with other pharmaceuticals (see, for example, Patent Document 1).

Japanese Patent No. 5721140

Vives E., et al., A Truncated HIV-1 Tat Protein Basic Domain Rapidly Translocates through the Plasma Membrane and Accumulates in the Cell Nucleus, J. Biol. Chem., 272, 16010-16017, 1997.

In the above-mentioned examination and diagnosis method for biliary tract cancer (particularly cancer derived from intrahepatic or extrahepatic bile duct epithelium), the criterion for the examination result is the determination of abnormal shadow. There is a limit to the accuracy of shadow determination, including the extent of lesions.
Further, since the cyclic RGD described in Patent Document 1 is not a peptide that targets tumor cells and tumor tissues themselves, it is novel in that it has the ability to directly capture cancer, and is used in anticancer medical technology. There was still room for improvement in terms of aspects.

The present invention has been made in view of the above circumstances, and provides a novel peptide having a specific accumulation that acts directly on biliary tract cancer cells and tissues.

That is, the present invention includes the following aspects.
[1] A peptide according to any one of the following (a) to (d) and having a cyclic structure .
(A) Ri Do an amino acid sequence comprising the sequence represented by SEQ ID NO: 3, and a peptide from L- amino ing,
(B) Among the sequences represented by SEQ ID NO: 3, the amino acid sequence comprises a sequence in which the fourth arginine residue from the N-terminal is replaced with an isoleucine residue, an alanine residue, or a lysine residue, and is composed of L. -Peptide consisting of amino acids and having biliary tract cancer-specific accumulation ,
(C) A peptide consisting of an amino acid sequence including the sequence represented by SEQ ID NO: 7 and consisting of a D-amino acid.
(D) Among the sequences represented by SEQ ID NO: 7, the amino acid sequence comprises a sequence in which the fourth arginine residue from the C-terminal is replaced with an isoleucine residue, an alanine residue, or a lysine residue, and is composed of D. -Peptide consisting of amino acids and having biliary tract cancer-specific accumulation
[2] A peptide consisting of an amino acid sequence containing the sequence represented by any of SEQ ID NOs: 3, 5 and 6 and consisting of L-amino acids, or
The peptide according to [1], which comprises an amino acid sequence including the sequence represented by SEQ ID NO: 7 and is a peptide composed of a D-amino acid.
[3] A peptide consisting of an amino acid sequence including the sequence represented by SEQ ID NO: 3 and consisting of an L-amino acid, or
The peptide according to [1] or [2], which comprises an amino acid sequence including the sequence represented by SEQ ID NO: 7 and is a peptide composed of a D-amino acid.
[ 4 ] A nucleic acid comprising the peptide according to any one of [1] to [3].
[ 5 ] A vector comprising the nucleic acid according to [4].
[ 6 ] A pharmaceutical composition comprising the peptide according to any one of [1] to [3] and one or more selected from the group consisting of a labeling substance, a modifying substance and a physiologically active substance. Things .
[ 7 ] The pharmaceutical composition according to [6 ], wherein the labeling substance is a stable isotope, a radioisotope or a fluorescent substance.
[ 8 ] The pharmaceutical composition according to [6 ] or [ 7 ], wherein the modifying substance is a sugar chain or polyethylene glycol.
[ 9 ] The pharmaceutical composition according to any one of [6] to [8] , which is for treatment or diagnosis of biliary tract cancer.

According to the present invention, it is possible to provide a novel peptide having an accumulation specific to biliary tract cancer. In addition, biliary tract cancer can be detected easily, with high sensitivity, and selectively.

It is a fluorescence micrograph of various biliary tract cancer cells to which various peptides were added in Test Example 1. 3 is a fluorescence micrograph of M213 cells supplemented with Peptide3 and chlorpromazine in Test Example 2. 3 is a fluorescence micrograph of M213 cells supplemented with Peptide3 and Dynasaw in Test Example 2. 3 is a fluorescence micrograph of M213 cells supplemented with Peptide3 and EIPA in Test Example 2. 3 is a fluorescence micrograph of M214 cells to which Peptide3 (L form) in Test Example 3 was added and cultured at different serum concentrations. 3 is a fluorescence micrograph of M214 cells to which Peptide4 (D form) in Test Example 3 was added and cultured at different serum concentrations. 3 is a fluorescence micrograph of various biliary tract cancer cells to which Peptide 3 was added in Test Example 4. It is a fluorescence micrograph of various hepatocellular carcinoma cells to which Peptide3 was added in Test Example 5. 3 is a fluorescence micrograph of various cancer cells to which Peptide 3 was added in Test Example 6. 3 is a fluorescence micrograph of cells derived from normal tissues to which Peptide 3 was added in Test Example 7. 3 is a fluorescence micrograph of cholangiocarcinoma cells and normal cells derived from the bile duct co-cultured with Peptide 3 in Test Example 8. It is a fluorescence micrograph of bright field and dark field of various tissues of a mouse in which M214 cells in Test Example 9 were transplanted and Peptide3 (L form) was intraperitoneally administered. It is a fluorescence micrograph of bright field and dark field of various tissues of a mouse in which a liver transplanted with M156 cells in Test Example 10 was transplanted and Peptide3 (L form) was intraperitoneally administered. It is a fluorescence micrograph of various cells to which Peptide4 (D form) was added in Test Example 11. It is a fluorescence micrograph of bright field and dark field of various tissues of a mouse in which TKKK cells in Test Example 12 were transplanted and Peptide 3 (L body) or Peptide 4 (D body) was intraperitoneally administered. It is a graph which shows the fluorescence intensity in various tissues of the mouse which transplanted the TKKK cell in Test Example 12 and intraperitoneally administered Peptide3 (L body). It is a graph which shows the fluorescence intensity in various tissues of the mouse which transplanted the TKKK cell in Test Example 12 and intraperitoneally administered Peptide4 (D body). It is a graph which shows the fluorescence intensity in various tissues of the mouse which transplanted the TKKK cell in Test Example 12 and intraperitoneally administered Peptide3 (L form) or Peptide4 (D form). It is a fluorescence micrograph of bright field and dark field of various tissues of a mouse in which M156 cells in Test Example 13 were transplanted and Peptide4 (D body) was intraperitoneally administered. 3 is a fluorescence micrograph of bile duct cancer cells contained in ascites of a bile duct cancer patient to which Peptide 3 was added in Test Example 14.

[Peptide with accumulation specific to biliary tract cancer]
In one embodiment, the present invention provides the following peptides (a) or (b).
(A) A peptide consisting of an amino acid sequence containing the sequence represented by SEQ ID NO: 1.
(B) A peptide consisting of an amino acid sequence containing a sequence having 60% or more identity with the sequence represented by SEQ ID NO: 1 and having an accumulation specific to biliary tract cancer.

The peptide of this embodiment is a novel peptide having an accumulation specific to biliary tract cancer.

The present inventors have found a novel peptide having an accumulation specific to biliary tract cancer by the in-vitro virus (IVV) method, and have completed the present invention.
In the IVV method, puromycin, a type of antibiotic, is bound to the 3'end of mRNA via a PEG (polyethylene glycol) spacer, and a cell-free translation reaction is performed using this as a template to combine puromycin with the protein. A simple mRNA-protein linking molecule IVV covalently linked via is constructed. The present inventors have constructed an IVV library by independently producing IVV. After in vitro fishing of IVV containing a protein that binds to bait from this IVV library, the mRNA linked to it is reverse-transcribed, amplified by PCR, and the base sequence is decoded. Allows you to identify interacting proteins in very small amounts (more than a thousand times more sensitive than mass spectrometry).

The peptide of this embodiment includes the peptide of (a) below.
(A) A peptide consisting of an amino acid sequence containing the sequence represented by SEQ ID NO: 1.

The amino acid sequence represented by SEQ ID NO: 1 in (a) above is the sequence represented by the following amino acid sequence.
LVXGARLVVR (SEQ ID NO: 1)
[In the amino acid sequence represented by SEQ ID NO: 1 above, X is an isoleucine residue (I), an alanine residue (A), an arginine residue (R), a lysine residue (K) or a histidine residue (H). ). ]

The peptide (a) above has a specific accumulation property for biliary tract cancer. Further, the peptide of the present embodiment has an accumulation specific to biliary tract cancer even if it is a peptide consisting only of the amino acid sequence represented by SEQ ID NO: 1.

As used herein, the term "bile duct cancer" means a cancer consisting of bile duct cancer, gallbladder cancer and duodenal papilla cancer and bile duct cancer. In addition, cholangiocarcinoma means a malignant tumor that develops from the epithelium of the bile duct, and depending on the site of the bile duct that has developed, extrahepatic cholangiocarcinoma, hilar region cholangiocarcinoma and distal cholangiocarcinoma, and the liver It can be divided into internal bile duct cancer (bile duct cell cancer). Gallbladder cancer means a malignant tumor that begins in the gallbladder and cystic duct. The duodenal papilla is composed of a papillary bile duct, a papillary pancreatic duct, a common duct, and a large duodenal papilla, and duodenal papilla cancer means cancer that has developed at the above-mentioned site. In the histopathological classification, it refers to adenocarcinoma, adenosquamous carcinoma, and intrahepatic bile duct cell carcinoma.

As used herein, the term "biliary tract cancer-specific accumulation" means the property of being highly absorbed and accumulated in biliary tract cancer cells as compared with tumor cells of normal tissues in vivo and other strains. .. As shown in Examples described later, it is presumed that the peptide of this embodiment is absorbed into biliary tract cancer cells by clathrin-dependent endocytosis due to dynamin.

In the amino acid sequence represented by SEQ ID NO: 1, X is preferably an amino acid residue having hydrophilicity, and is an arginine residue (R), a lysine residue (K) or a histidine residue (H). Is more preferable, and from the viewpoint of toxicity, it is even more preferable that the arginine residue (R) is used.

Among the amino acid sequences represented by SEQ ID NO: 1 in (a) above, more specifically, the amino acid sequence represented by SEQ ID NO: 2 below can be mentioned.
LVRGARLVVR (SEQ ID NO: 2)

The peptide of the present embodiment includes the peptide of the following (b) as a peptide functionally equivalent to the peptide of the above (a).
(B) A peptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having an accumulation specific to biliary tract cancer.

In order to be functionally equivalent to the peptide of (a) above, it has 60% or more identity. The identity is preferably 70% or more, more preferably 80% or more, further preferably 85% or more, particularly preferably 90% or more, and most preferably 95% or more.
Furthermore, the peptide (b) has a specific accumulation property for biliary tract cancer.

Here, the sequence identity of the target amino acid sequence with respect to the reference amino acid sequence can be obtained, for example, as follows. First, the reference amino acid sequence and the target amino acid sequence are aligned. Here, each amino acid sequence may include a gap so as to maximize the sequence identity. Subsequently, the number of matching amino acids in the reference amino acid sequence and the target amino acid sequence can be calculated, and the sequence identity can be determined according to the following formula (1).
"Sequence identity (%)" = [Number of matched amino acids] / [Total number of amino acids in the target amino acid sequence] x 100 (1)

The peptide of (a) or (b) above preferably has a cyclic structure. The cyclic structure facilitates absorption only into biliary tract cancer cells. Further, the peptide of (a) or (b) may be composed of an L-amino acid, a D-amino acid, or a combination thereof, and is preferably a Retro-Inverso type substituted with a D-amino acid. ..
The Retro-Inverso type makes it less susceptible to intracellular enzymatic degradation. The half-life of the peptide (a) or (b) composed of only L-amino acids is 3 to 6 hours, whereas the half-life of the Retro-Inverso type is extended to 8 to 12 hours. Can be done.
The L-amino acid is a naturally occurring amino acid, and the D-amino acid is the one in which the chirality of the L-amino acid residue is inverted. It may also be chemically modified to increase its specific accumulation in biliary tract cancer or to optimize other physical characteristics.

The peptide of (a) or (b) is preferably further provided with a cysteine residue at the N-terminal and C-terminal. Specific examples thereof include the amino acid sequence represented by SEQ ID NO: 3 below.
CLVRGARLVVRC (SEQ ID NO: 3)
By providing cysteine residues at the N-terminal and C-terminal, the peptide of the present embodiment can take a cyclic form by utilizing the disulfide bond between thiol groups possessed by the cysteine residue.

[Nucleic acid encoding peptide]
In one embodiment, the present invention provides nucleic acids encoding the peptides described above.

According to the nucleic acid of the present embodiment, a peptide having an accumulation specific to biliary tract cancer can be obtained.

Examples of the nucleic acid encoding the above peptide include a nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO: 4, or 80% or more, for example 85% or more, for example 90% or more, eg, 90% or more of the nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO: 4. Examples thereof include nucleic acids including any combination of base sequences encoding each amino acid which is a constituent component of a peptide having 95% or more identity and specific accumulation in biliary tract cancer. The base sequence shown in SEQ ID NO: 4 is the base sequence of the nucleic acid encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 3 above.

Here, the sequence identity of the control base sequence with respect to the reference base sequence can be determined, for example, as follows. First, the reference base sequence and the target base sequence are aligned. Here, each base sequence may include a gap so as to maximize the sequence identity. Subsequently, the number of matching bases in the reference base sequence and the target base sequence can be calculated, and the sequence identity can be determined according to the following formula (2).
"Sequence identity (%)" = [Number of matched bases] / [Total number of bases in the target base sequence] x 100 (2)

[Vector containing nucleic acid encoding peptide]
In one embodiment, the invention provides a vector containing the nucleic acids described above.

According to the vector of the present embodiment, a peptide having an accumulation specific to biliary tract cancer can be obtained.

The vector of this embodiment is preferably an expression vector. The expression vector is not particularly limited, and for example, a plasmid derived from Escherichia coli such as pBR322, pBR325, pUC12 and pUC13; a plasmid derived from bacillus such as pUB110, pTP5 and pC194; a plasmid derived from yeast such as pSH19 and pSH15; Bacteriophages; viruses such as adenovirus, adeno-associated virus, lentivirus, vaccinia virus, baculovirus, retrovirus, hepatitis virus; and vectors modified thereto can be used.

In the above-mentioned expression vector, the promoter for expressing the above-mentioned peptide is not particularly limited, and for example, animal cells such as EF1α promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-tk promoter and the like can be used. A promoter for expression as a host, a promoter for expression using a plant cell as a host such as a 35S promoter of Califlower Mosaic Virus (CaMV), a REF (rubber election promoter) promoter, a polyhedrin promoter, an insect cell such as a p10 promoter, etc. A promoter for expression as a host can be used. These promoters can be appropriately selected depending on the host expressing the above-mentioned peptides.

The expression vector described above may further have a multicloning site, enhancer, splicing signal, polyA addition signal, selectable marker, origin of replication and the like.

[Career]
In one embodiment, the invention provides a carrier comprising the peptides described above.

According to the carrier of the present embodiment, the target substance can be easily and efficiently transported to biliary tract cancer.

The carrier of the present embodiment preferably further comprises a labeling substance or a modifying substance. In addition, the carrier of the present embodiment may include both a labeling substance and a modifying substance. The labeling substance or modifying substance may be physically or chemically bound to the above-mentioned peptide, either directly or via a linker. Specifically, it may be a coordination bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any known bond, linker, and bond method can be adopted.

Examples of the labeling substance include stable isotopes, radioactive isotopes, fluorescent substances, positron emission tomography (PET) nuclei, single photon emission computed tomography (SPECT) nuclei, and SPECT nuclei. Examples thereof include magnetic resonance imaging (MRI) contrasting agents, computed tomography (CT) contrasting agents, and magnetic materials. Of these, stable isotopes, radioactive isotopes or fluorescent substances are preferable. By providing the above-mentioned labeling substance, it is possible to easily and highly sensitively confirm whether or not the target substance has been transported to biliary tract cancer.

Examples of stable isotopes include ¹³ C, ¹⁵ N, ² H, ¹⁷ O, and ¹⁸ O. Radioisotopes include, for example, ³ H, ¹⁴ C, ¹³ N, ³² P, ³³ P, and ³⁵ S. When the labeling substance is a stable isotope or a radioisotope, the above-mentioned peptide may be prepared by using a stable isotope-labeled amino acid or a radioisotope-labeled amino acid. Amino acids labeled with stable isotopes or radioactive isotopes include 20 types of amino acids (alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine). , Valine, tryptophan, cysteine, aspartic acid, glutamine), and is not particularly limited as long as it is an amino acid contained in the above-mentioned peptide. Further, the amino acid may be L-form or D-form, and can be appropriately selected as needed.

The above-mentioned peptides labeled with stable isotopes or radioisotopes are prepared by expressing the above-mentioned vectors containing nucleic acids encoding the above-mentioned peptides in a system in which stable isotope-labeled amino acids or radioisotope-labeled amino acids are present. can do. Examples of the system in which the stable isotope-labeled amino acid or the radioisotopically-labeled amino acid is present include a cell-free peptide synthesis system and a live cell peptide synthesis system in which the stable isotope-labeled amino acid or the radioisotope-labeled amino acid is present. .. That is, in a cell-free peptide synthesis system, a peptide may be synthesized using a stable isotope-labeled amino acid or a radioactive isotope-labeled amino acid in addition to a stable isotope-unlabeled amino acid or a radioactive isotope-unlabeled amino acid as a material, or a live cell peptide synthesis system. In the above-mentioned, by culturing the cells transformed with the above-mentioned vector containing the above-mentioned peptide-encoding nucleic acid in the presence of a stable isotope-labeled amino acid or a radioisotope-labeled amino acid, the above-mentioned above-mentioned peptide-encoding nucleic acid is contained. The above-mentioned peptides labeled with stable isotopes or radioactive isotopes can be prepared from the vector.

Stable isotope-labeled or radioisotopically-labeled expression of the above-mentioned peptide using a cell-free peptide synthesis system can be expressed by the above-mentioned vector containing a nucleic acid encoding the above-mentioned peptide or the above-mentioned stable isotope-labeled amino acid or radioisotope. In addition to labeled amino acids, stable isotope-unlabeled amino acids or radioactive isotope-unlabeled amino acids necessary for the synthesis of the above-mentioned peptides labeled with stable isotopes or radioactive isotopes, cell extracts for cell-free peptide synthesis, It can be carried out using an energy source (high-energy phosphate bond-containing substance such as ATP, GTP, creatin phosphate, etc.) or the like. The reaction conditions such as temperature and time can be appropriately selected and carried out. For example, the temperature is 20 to 40 ° C., preferably 23 to 37 ° C., and the reaction time is 1 to 24 hours, preferably 1 to 24 hours. 10 to 20 hours.

In the present specification, the term "cell extract for cell-free peptide synthesis" refers to a translation system involved in protein synthesis such as ribosomes and tRNA, or a plant cell or animal cell containing components necessary for the transcription system and translation system. It means fungal cells and extracts from bacterial cells. Specific examples thereof include cell extracts of Escherichia coli, wheat germ, rabbit reticulocyte, mouse L-cell, Ehrlich ascites cancer cell, HeLa cell, CHO cell, budding yeast and the like. Preparation of such cell extracts can be described, for example, in Pratt, J. et al. M. Et al., Translation and translation-a practical application (1984), pp. According to the method described in 179-209, the above cells are disrupted using a French press, glass beads, an ultrasonic disruptor or the like, and a buffer solution containing several kinds of salts for solubilizing protein components and ribosomes is prepared. In addition, it can be homogenized and centrifuged to precipitate insoluble components.

In addition, the expression of the above-mentioned peptides labeled with stable isotopes or radioisotopes using a cell-free peptide synthesis system is described, for example, in Premium Expression Kit (manufactured by Cellfree Science) equipped with a wheat germ extract, Escherichia coli extraction. RTS 100 with liquid, E.I. Commercially available kits such as Escherichia coli HY Kit (manufactured by Roche Applied Science) and Cell-free-kun Quick (manufactured by Taiyo Nippon Sanso Co., Ltd.) may be used as appropriate. When the expressed stable isotope-labeled or radioisotope-labeled above-mentioned peptide is insoluble, it may be appropriately solubilized with a protein denaturing agent such as guanidine hydrochloride or urea. The above-mentioned peptides labeled with stable isotopes or radioisotopes are further prepared by fractionation treatment by fractionation centrifugation, sucrose density gradient centrifugation, etc., or purification treatment using affinity column, ion exchange chromatography, etc. You can also do it.

Expression of the above-mentioned stable isotope-labeled or radioisotopically-labeled peptide using a live cell peptide synthesis system introduces the above-mentioned vector containing a nucleic acid encoding the above-mentioned peptide into a living cell and nourishes the living cell. And antibiotics, as well as stable isotope-labeled amino acids or radioisotopic-labeled amino acids, stable isotope-labeled peptides or radioactive isotope-labeled peptides necessary for the synthesis of the above stable isotope-labeled amino acids or radioisotope-non-labeled amino acids. It can be carried out by culturing in a culture solution containing a labeled amino acid or the like. Here, the living cell is not particularly limited as long as it is a living cell capable of expressing the above-mentioned vector containing the nucleic acid encoding the above-mentioned peptide, for example, a mammalian cell line such as Chinese hamster ovary (CHO) cell, or a mammalian cell line. Living cells such as Escherichia coli, yeast cells, insect cells, and plant cells can be mentioned, and Escherichia coli is preferable from the viewpoint of convenience and cost effectiveness. The expression of the above-mentioned vector containing the nucleic acid encoding the above-mentioned peptide is incorporated into an expression vector designed to be expressed in each living cell by gene recombination technology, and the expression vector is introduced into the living cell. It can be carried out. In addition, the above-mentioned vector containing the nucleic acid encoding the above-mentioned peptide can be introduced into living cells by a method suitable for the living cells to be used, for example, electroporation method, heat shock method, calcium phosphate method, lipofection. Method, DEAE dextran method, microinjection method, particle gun method, method using virus, FuGENE (registered trademark) 6 Transfection Reagent (manufactured by Roche), Lipofectamine 2000 Reagent (manufactured by Invitrogen), Lipofectamine LTX Reagent A method using a commercially available transfection reagent such as Lipofectamine 3000 Reagent (manufactured by Invitrogen) and the like can be mentioned.

The above-mentioned stable isotope-labeled or radioisotope-labeled peptide expressed by the live cell peptide synthesis system disrupts or extracts living cells containing the above-mentioned stable isotope-labeled or radioisotope-labeled peptide. Can be prepared by. Examples of the crushing treatment include a freeze-thaw method, a French press, glass beads, a homogenizer, a physical crushing treatment using an ultrasonic crushing device, and the like. Further, as the extraction treatment, for example, an extraction treatment using a protein denaturing agent such as guanidine hydrochloride or urea can be mentioned. The above-mentioned peptides labeled with stable isotopes or radioisotopes are further subjected to fractionation treatment by fractionation centrifugation, sucrose density gradient centrifugation, etc., purification treatment using affinity column, ion exchange chromatography, etc. It can also be prepared.

Examples of the fluorescent substance include known quantum dots, indocyanine green, 5-aminolevulinic acid (5-ALA; metabolite protoporphyrin IX (PP IX)), near-infrared fluorescent dye (for example, Cy5.5, Cy7, AlexaFluoro, etc.). ), Other known fluorescent dyes (for example, GFP, FITC (Fluorescein), TAMRA, etc.), etc. The above-mentioned fluorescent substance-labeled peptide contains the above-mentioned vector containing a fluorescent substance and a nucleic acid encoding the above-mentioned peptide. May be prepared by the above-mentioned cell-free peptide synthesis system or live cell peptide synthesis system without using stable isotope-labeled amino acids or radioactive isotope-labeled amino acids.

The nuclides for PET and SPECT are preferably, for example, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁷ Cu, ⁶⁴ Cu, ⁴⁸ V. , Tc-99m, ²⁴¹ Am, ⁵⁵ Co, ⁵⁷ Co, ¹⁵³ Gd, ¹¹¹ In, ¹³³ Ba, ⁸² Rb, ¹³⁹ Ce, Te-123m, ¹³⁷ Cs, ⁸⁶ Y, ⁹⁰ Y, ^185/187 Re, ^186/188 Examples thereof include Re, ¹²⁵ I, or a complex thereof, or a combination thereof. The above-mentioned peptide labeled with a PET nuclide or a SPECT nuclide may be prepared by preparing the above-mentioned vector containing the nucleic acid encoding the above-mentioned peptide by the above-mentioned cell-free peptide synthesis system or live cell peptide synthesis system.
Examples of the MRI contrast agent, CT contrast agent and magnetic material include gadolinium, Gd-DTPA, Gd-DTPA-BMA, Gd-HP-DO3A, iodine, iron, iron oxide, chromium, manganese, or a complex thereof, or a chelate thereof. Examples include a complex. The above-mentioned peptide labeled with an MRI contrast agent, a CT contrast agent or a magnetic substance can be physically or chemically obtained by directly or via a linker between the above-mentioned peptide and the MRI contrast agent, the CT contrast agent or the magnetic substance. It may be prepared by combining them. Specifically, it may be a coordination bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any known bond, linker, and bond method can be adopted.

Examples of the modifying substance include sugar chains and polyethylene glycol (PEG). By providing the above-mentioned modifying substance, the target substance can be easily and efficiently absorbed into biliary tract cancer cells. The above-mentioned peptide modified with the modifying substance may be prepared by physically or chemically binding the modifying substance and the above-mentioned peptide directly or via a linker. Specifically, it may be a coordination bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any known bond, linker, and bond method can be adopted.

In the carrier of the present embodiment, the target substance can be appropriately selected depending on the intended use. For example, when used for imaging of biliary tract cancer, the above-mentioned labeling substance is provided as the target substance as described later. Moreover, when it is used for the treatment or diagnostic use of biliary tract cancer, a physiologically active substance can be provided as a target substance as described later. The target substance may be physically or chemically bound to the above-mentioned peptide, either directly or via a linker. Specifically, it may be a coordination bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any known bond, linker, and bond method can be adopted.

Further, in the carrier of the present embodiment, when the target substance is a protein, a fusion protein containing the target substance and the above-mentioned peptide can be produced, for example, by the following method. First, a host is transformed with an expression vector containing a nucleic acid encoding a fusion protein. Subsequently, the host is cultured to express the fusion protein. Conditions such as the composition of the medium, the temperature and time of culturing, and the addition of the inducer can be determined by those skilled in the art according to known methods so that the transformant grows and the fusion protein is efficiently produced. Further, for example, when an antibiotic resistance gene is incorporated into an expression vector as a selection marker, a transformant can be selected by adding an antibiotic to the medium. Subsequently, the fusion protein expressed by the host is purified by an appropriate method to obtain the fusion protein.

The host is not particularly limited as long as it is a living cell capable of expressing an expression vector containing a nucleic acid encoding a fusion protein, and is not particularly limited as long as it is a mammalian cell line such as a Chinese hamster ovary (CHO) cell or a virus (for example, an adeno). Viruses, adeno-related viruses, lentiviruses, vaccinia viruses, baculoviruses, retroviruses, hepatitis viruses and other viruses), bacteria (eg, Escherichia coli, etc.) and other microorganisms, yeast cells, insect cells, plant cells and other living cells Can be mentioned.
Furthermore, in the carrier of the present embodiment, an expression vector containing a nucleic acid encoding the above-mentioned fusion protein may be directly introduced into a biliary tract cancer cell or tissue and expressed.

[Pharmaceutical composition]
In one embodiment, the present invention provides a pharmaceutical composition comprising the above-mentioned carriers and bioactive substances.

According to the pharmaceutical composition of the present embodiment, biliary tract cancer can be selectively treated.

In the present specification, the "physiologically active substance" is not particularly limited as long as it is effective for the treatment of biliary tract cancer, for example, drugs such as anticancer agents, nucleic acids, antibodies and antibodies that specifically bind to biliary tract cancer. Fragments, aptamers and the like can be mentioned.
As the "physiologically active substance", a molecular-targeted drug having a cytotoxic activity selective for biliary tract cancer is preferable, but cytotoxic, which is used as a conventional anticancer agent because it is selectively accumulated for biliary tract cancer by the above-mentioned carriers. It may be a medicine.
In addition, the physiologically active substance may be physically or chemically bound to the above-mentioned carriers directly or via a linker. Specifically, it may be a coordination bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any known bond, linker, and bond method can be adopted. Further, in the pharmaceutical composition of the present embodiment, the above-mentioned carrier may contain the above-mentioned labeling substance or modifying substance.

Nucleic acids include, for example, siRNA, miRNA, antisense, or artificial nucleic acids that compensate for their functions.

The antibody can be produced, for example, by immunizing a rodent animal such as a mouse with a peptide derived from biliary tract cancer as an antigen. It can also be prepared, for example, by screening a phage library. Examples of the antibody fragment include Fv, Fab, scFv and the like.

Aptamers are substances that have a specific binding ability to biliary tract cancer. Examples of the aptamer include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers having a specific binding ability to biliary tract cancer can be selected by, for example, the systematic evolution of ligand by explicit enrichment (SELEX) method or the like. In addition, peptide aptamers having a specific binding ability to biliary tract cancer can be selected by, for example, the Two-hybrid method using yeast.

The pharmaceutical composition of the present embodiment can be used for biliary tract cancer diagnosis, biliary tract cancer therapeutic effect diagnosis, pathological analysis, biliary tract cancer treatment, or diagnosis of diseases associated with biliary tract cancer, pathological condition analysis, treatment, therapeutic effect diagnosis. .. As a diagnostic method, for example, PET, SPECT, CT, MRI, an endoscope, a fluorescence detector and the like can be used.

<Dose>
The pharmaceutical composition of the present embodiment takes into consideration the age, sex, body weight, symptoms, treatment method, administration method, treatment time, etc. of the test animal (various mammals including humans or non-human animals, preferably humans). It is adjusted as appropriate.
For example, when the pharmaceutical composition of the present embodiment is injected intravenously (Intravenous: iv) by an injection, 5 mg or more per 1 kg of body weight per 1 kg body weight of a test animal (preferably human). It is preferable to administer the amount of peptide, more preferably the amount of peptide of 5 mg or more and 15 mg or less, and particularly preferably the amount of peptide of 5 mg or more and 10 mg or less.

As for the number of administrations, it is preferable to administer once to several times per week on average.
Examples of the administration form include methods known to those skilled in the art, such as intraarterial injection, intravenous injection, subcutaneous injection, intranasal, intraperitoneal, transbronchial, intramuscular, percutaneous, or oral. Therefore, intravenous injection or intraperitoneal administration is preferable.

<Composition component>
The pharmaceutical composition of this embodiment comprises a therapeutically effective amount of the carrier and bioactive agent described above, as well as a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers or diluents are excipients, diluters, bulking agents, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners, flavors. Examples include agents, solubilizers, additives and the like. By using one or more of these carriers, pharmaceutical compositions in the form of injections, liquids, capsules, suspensions, emulsions, syrups and the like can be prepared.
A colloidal dispersion system can also be used as the carrier. The colloidal dispersion system is expected to have an effect of enhancing the in vivo stability of the peptide and an effect of enhancing the transferability of the peptide to a specific organ, tissue, or cell. Examples of the colloidal dispersion system include polyethylene glycol, polymer composites, polymer aggregates, nanocapsules, microspheres, beads, oil-in-water emulsifiers, micelles, mixed micelles, and lipids including liposomes. Liposomal or artificial membrane vesicles, which have the effect of efficiently transporting peptides to the organs, tissues, or cells of the body, are preferred.

Examples of formulation in the pharmaceutical composition of the present embodiment include those used orally as sugar-coated tablets, capsules, elixirs, and microcapsules as needed.
Alternatively, aseptic solutions with water or other pharmaceutically acceptable liquids, or those used parenterally in the form of injections of suspensions can be mentioned. Furthermore, pharmacologically acceptable carriers or diluents, specifically sterile water or saline, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, excipients, vehicles, etc. Examples thereof include those formulated by appropriately combining with an antiseptic, a binder and the like and mixing in a generally accepted unit dose form required for pharmaceutical practice.

Additives that can be mixed with tablets and capsules include, for example, binders such as gelatin, corn starch, traganth gum, gum arabic, excipients such as crystalline cellulose, swelling such as corn starch, gelatin and alginic acid. Agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavors such as peppermint, reddish oil or cherry are used. When the dispensing unit form is a capsule, the above-mentioned material can further contain a liquid carrier such as fat or oil.

Aseptic compositions for injection can be formulated according to routine formulation practices using vehicles such as distilled water for injection.
Aqueous solutions for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizers such as. It may be used in combination with alcohol, specifically ethanol, polyalcohol such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-50.

Examples of the oily liquid include sesame oil and soybean oil, and benzyl benzoate and benzyl alcohol may be used in combination as solubilizing agents. In addition, a buffer (for example, phosphate buffer, sodium acetate buffer, etc.), a soothing agent (for example, procaine hydrochloride, etc.), a stabilizer (for example, benzyl alcohol, phenol, etc.), an antioxidant, etc. are blended. You may. The prepared injection solution is usually filled in a suitable ampoule.

In the case of an injection, it can also be prepared as an aqueous or non-aqueous diluent, suspension, or emulsion as described above. Such sterilization of the injection can be performed by filtration sterilization with a filter, blending of a bactericidal agent, or the like. The injectable preparation can be produced as a form of preparation for errands. That is, a sterile solid composition can be prepared by a freeze-drying method or the like, and the composition can be dissolved in distilled water for injection or another solvent before use.

<Treatment method>
One aspect of the invention provides a pharmaceutical composition comprising the aforementioned carriers and bioactive substances for the treatment of biliary tract cancer.
Also, one aspect of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned carrier and bioactive agent, as well as a pharmaceutically acceptable carrier or diluent.
In addition, one aspect of the present invention provides a therapeutic agent for biliary tract cancer, which comprises the pharmaceutical composition.
Also, one aspect of the invention provides the use of the carriers and bioactive substances described above for the manufacture of therapeutic agents for biliary tract cancer.
In addition, one aspect of the present invention provides a method for treating biliary tract cancer, which comprises administering an effective amount of the above-mentioned carrier and bioactive substance to a patient in need of treatment.

[Methods for imaging biliary tract cancer]
In one embodiment, the present invention provides a method for imaging biliary tract cancer, using the carriers described above.

According to the method of the present embodiment, biliary tract cancer can be detected easily, with high sensitivity and selectively.

In the method of this embodiment, the carrier described above preferably comprises a labeling substance. Further, it may be provided with a modifying substance. Examples of the labeling substance and the modifying substance include the same substances as those described above.

For example, when the above-mentioned carrier containing the labeling substance is added to biliary tract cancer cells, the amount of the above-mentioned carrier containing the labeling substance added is preferably 1 μM or more and 4 μM or less in the culture medium. In addition, it is possible to evaluate whether or not it is accumulated in biliary tract cancer cells 30 minutes or more and 3 hours or less after the addition.

Further, for example, when the above-mentioned carrier having a fluorescent substance as a labeling substance is injected intravenously (Intravenous: iv) by an injection, the body weight of 1 kg per administration is given to the test animal (preferably human). It is preferable to administer an amount of 5 mg or more of the peptide, more preferably an amount of 5 mg or more and 15 mg or less of the peptide, and particularly preferably an amount of 5 mg or more and 10 mg or less of the peptide.
Further, for example, when the above-mentioned carrier having a stable isotope, a nuclide for PET or a nuclide for SPECT as a labeling substance is injected intravenously (Intravenous: iv) by an injection, the stable isotope to be used and the nuclide for PET are used. Alternatively, the dose may be determined from the radiation dose according to the type of SPECT nuclide.

In the method of the present embodiment, for example, PET, SPECT, CT, MRI, an endoscope, a fluorescence detector and the like can be used as the method for detecting the above-mentioned carrier including the labeling substance.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1] Synthesis of peptide Protein-RNA chimeric random type having a 12-amino acid residue peptide as a phenotype and an mRNA coding sequence as a corresponding genotype via puromycin, which was originally prepared. Using a peptide library (in-vivro library; IVVL), each peptide (Peptide 1 to 4) shown in Table 1 below was separated and identified according to a known IVV (in-vitro virus) method. In addition, each of the identified peptides derived from IVVL was synthesized under the FITC (Fluorescein isothiocyanate) label and subjected to hydrochloride treatment. In addition, r9 (9-residue continuous D-arginine) is a non-selective membrane-permeable peptide currently widely used.
All of these were obtained by consignment synthesis to Sigma-Aldrich Japan (Genosis Division).

The cell lines and origins of the various cells used in Test Examples 1 to 13 below are as shown in Table 2 below. These are those that the inventor has subcultured and maintained in the laboratory.

For bile duct cancer cells, MMNK-1 cells, liver cancer cells, HeLa cells, U2OS cells, A172 cells, A549 cells, MCF7 cells, GCIY cells, Lovo cells, BxPC3 cells, KPK cells, PC-3 cells, NHDF cells, 10 The cells were cultured using RPMI1640 medium containing% FBS (RPMI1640 medium). Hepatocyte cells were cultured in hepatocyte medium containing 5% FBS (Hepatocyte medium).
HPNE cells were cultured using a CS-C medium kit containing 5% FBS. NuLi-1 cells were cultured in BEGM medium (Bronchial Epithelial Growth Medium, Serum-free). Kidney cells were cultured in a growth medium for normal human renal epithelial cells (RenaLife Comp Kit). TIME cells were cultured using an EBM-2-MV Bullet kit (Endothelial Cell Basal Medium-2 Bullet kit).

[Test Example 1] Confirmation test of accumulation of peptide in biliary tract cancer cells In M156 cells, M213 cells, M214 cells and KKU-100 cells, 4 μM of Peptide 1, 2 and 3 prepared in Example 1, respectively, in a medium. It was added so as to be. The cells were cultured at 37 ° C. for 60 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope. Before microscopic examination, the culture supernatant to which the peptide was added is removed, washed 3 times with 1 × PBS (-), then trypsinized to remove adherent cells, and immediately transferred to a new 96-well plate to prepare a new culture solution. After resuspension, microscopic examination was performed. The results are shown in FIG.

From FIG. 1, fluorescence was detected in all cells of Peptide 1, 2 and 3. In particular, fluorescence was strongly detected in cells to which Peptide3 was added. From the above, it was clarified that Peptide3 has high permeability into bile duct cancer cells.

[Test Example 2] Confirmation test of accumulation of peptide in biliary tract cancer cells using inhibitor Peptide3 or r9 prepared in Example 1 was added to M213 cells so as to be 4 μM in a medium, and at 37 ° C. The cells were cultured for 60 minutes. As an inhibitor for investigating the inhibition of peptides into bile duct cancer cells, 0, 5, 20, 50, 100 μM chlorpromazine or 0, 2, 10, 50, 100 μM 5- (0, 2, 10, 50, 100 μM 5- (20 minutes after the start of culture). N-Ethyl-N-isopropyl) amiloride (EIPA) was added after 30 minutes with 0, 10, 20, 40 and 80 μM dynaso, respectively. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIGS. 2A-2C.
Chlorpromazine is an inhibitor that inhibits clathrin-dependent endocytosis. Dynasaw is also an inhibitor that inhibits clathrin-dependent endocytosis due to dynamin. In addition, EIPA is an inhibitor that inhibits macropinocytosis (pinocytosis).

From FIGS. 2A to 2C, the fluorescence in the cells to which r9 was added decreased as the amount of EIPA added increased, whereas the fluorescence in the cells to which Peptide3 was added decreased as the amount of Dynasaw added increased. , It became clear that it will decrease. From this, it was inferred that the uptake of Peptide3 into cholangiocarcinoma cells was due to clathrin-dependent endocytosis due to dynamin.

[Test Example 3] Confirmation test of peptide accumulation in biliary tract cancer cells in media having different serum concentrations Peptide3 or Peptide4 prepared in Example 1 was added to M214 cells in a medium containing 0%, 10%, and 50% FBS. Was added to 4 μM and cultured at 37 ° C. for 60 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIGS. 3A and 3B.
Note that Peptide3 and Peptide4 are peptides having the same amino acid sequence, Peptide3 is composed of only L-form amino acids, and Peptide4 is composed of only D-form amino acids. That is, Peptide3 and Peptide4 are in a mirror isomer relationship.

From FIGS. 3A and 3B, it was confirmed that the cells to which Peptide4 was added showed stronger fluorescence even after 120 minutes than the cells to which Peptide3 was added. In addition, even in an environment with a high serum concentration of 50% FBS, albumin contained in the serum inhibits the uptake into bile duct cancer cells, but the cells to which Peptide4 is added are cholangiocarcinoma cells. It became clear that the concentration of serum was high. Medium containing 50% FBS is close to human blood concentration. Therefore, it was speculated that Peptide3 and Peptide4 can be accumulated in cholangiocarcinoma even when added into the human body by, for example, intravenous injection.

[Test Example 4] Confirmation test of accumulation of peptides in various biliary tract cancer cells Peptide3 (4 μM) prepared in Example 1 and Hoechst (Dojindo Co., Ltd.), which is a nuclear stain, were applied to various biliary tract cancer cells shown in Table 1 above. ) Was added, and the cells were cultured at 37 ° C. for 120 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG.

From FIG. 4, fluorescence of Peptide3 was detected in M156 cells, M213 cells, M214 cells, KKU-100 cells and TKKK cells.

[Test Example 5] Confirmation test of accumulation of peptide in various bile cell cancer cells Peptide3 (4 μM) prepared in Example 1 and Hoechst (nuclear stain agent) were used in various bile cell cancer cells shown in Table 1 above. (Manufactured by Dojindo Co., Ltd.) was added, and the cells were cultured at 37 ° C. for 120 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG.

From FIG. 5, almost no fluorescence was detected in PLC / PRF / 5 cells and HepG2 cells.

[Test Example 6] Confirmation Test of Accumulation of Peptide in Cancer Cells Derived from Other Various Tissues Peptide3 (4 μM) prepared in Example 1 and M156 cells derived from other various tissues shown in Table 1 above were used. Hoechst (manufactured by Dojindo Co., Ltd.), which is a nuclear stain, was added and cultured at 37 ° C. for 120 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG.

From FIG. 6, almost no fluorescence was detected in GCIY cells and PC-3 cells.

[Test Example 7] Confirmation test of accumulation of peptides in normal cells derived from various tissues Peptide3 (4 μM) prepared in Example 1 and a nuclear stain were applied to normal cells derived from various tissues shown in Table 1 above. Hoechst (manufactured by Dojindo Co., Ltd.) was added, and the cells were cultured at 37 ° C. for 120 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG.

From FIG. 7, no fluorescence was detected in any of the cells.

From Test Examples 4 to 7, it was confirmed that the peptide of the present invention has a specific accumulation property for cholangiocarcinoma cells.

[Test Example 8] Confirmation test of accumulation of peptides in co-cultured cholangiocarcinoma cells and normal cells derived from bile ducts Peptide3 (4 μM) prepared in Example 1 and Peptide3 (4 μM) prepared in Example 1 were applied to co-cultured M156 cells and MMNK-1 cells. A medium containing Hoechst (manufactured by Dojindo Co., Ltd.), which is a nuclear stain, was added and cultured at 37 ° C. for 60 minutes. At the same time, Peptide3 (4 μM) prepared in Example 1 and Hoechst (manufactured by Dojindo Co., Ltd.), which is a nuclear stain, were added to the co-cultured M156 cells and MMNK-1 cells, and the cells were cultured at 37 ° C. for 30 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG.

From FIG. 8, it was clarified that Peptide3 was not taken up by MMNK-1 cells, but was taken up only by M156 cells. From this, it was clarified that the peptide of the present invention has a specific accumulation only in cholangiocarcinoma cells, unlike the conventional cell-permeable peptide such as Tat.

[Test Example 9] Evaluation test of peptide accumulation in various tissues in human cholangiocarcinoma cell transplanted mouse NOD-SCID mouse (purchased from Charles River Japan, Inc.) in which M214 cells were intraperitoneally (ip) injected 6 weeks (Aged female mouse) was prepared as a human cholangiocarcinoma cell transplantation model (human leukemia-xenograft model). Subsequently, 150 μg of Peptide3 prepared in Example 1 was intravenously (iv) injected into 20 g of mouse body weight. The abdomen was opened 90 minutes after administration, and the tumor lesions and the distribution of peptides were observed under a fluorescent stereomicroscope. The results are shown in FIG. In FIG. 9, gal means gallbladder, live means liver, st means stomach, sp means spleen, br means brain, hr means heart, kd means kidney, lu means lung, and tumor means malignant tumor. Further, in FIG. 9, "Bright Field" is an image taken in a bright field, and "FITC" is an image taken in a dark field. The upper image is an image of the mouse when the abdomen is opened, the lower left image is an image of various excised tissues, and the lower right image is an image of sliced various excised tissues. ..

From FIG. 9, it was confirmed that Peptide3 was accumulated only in the gallbladder and malignant tumors.

[Test Example 10] Evaluation test of peptide accumulation in various tissues in mice transplanted with liver transplanted with human cholangiocarcinoma cells 6 weeks after purchasing liver transplanted with M156 cells from NOD-SCID mice (Japan Charles River Co., Ltd.) It was transplanted to an old female mouse). Subsequently, 150 μg of Peptide3 prepared in Example 1 was intravenously (iv) injected into 20 g of mouse body weight. The abdomen was opened 90 minutes after administration, and the tumor lesions and the distribution of peptides were observed under a fluorescent stereomicroscope. The results are shown in FIG. In FIG. 10, gal means gallbladder, live means liver, st means stomach, sp means spleen, br means brain, hr means heart, kd means kidney, lu means lung, and tumor means malignant tumor. Further, in FIG. 10, "Bright Field" is an image taken in a bright field, and "FITC" is an image taken in a dark field. The upper image is an image of the mouse when the abdomen is opened, the lower left image is an image of various excised tissues, and the lower right image is an image of sliced various excised tissues. ..

From FIG. 10, it was confirmed that Peptide3 was accumulated only in the gallbladder and malignant tumors.

[Test Example 11] Confirmation test of peptide accumulation in various cells Peptide4 (4 μM) prepared in Example 1 and a nuclear stain were applied to various biliary tract cancer cells, MMNK-1 cells and Hepatocytes shown in Table 1 above. Hoechst (manufactured by Dojindo Co., Ltd.) was added and cultured at 37 ° C. for 120 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG.

From FIG. 11, fluorescence was detected only in cholangiocarcinoma cells. Further, when compared with the bile duct cancer cells to which Peptide3 was added, stronger fluorescence was detected in the bile duct cancer cells to which Peptide4 was added. Further, fluorescence was detected in SSP25 cells, OCUG-1 cells, TFK-1 cells and HuCCT1 cells in which fluorescence was not detected in Peptide3. From the above, it was confirmed that Peptide4, which is a D-form, has higher accumulation in bile duct cancer cells.

[Test Example 12] Evaluation test of peptide accumulation in various tissues in human cholangiocarcinoma cell transplanted mice NOD-SCID in which TKKK cells were injected intraperitoneally (ip) and a liver transplanted with TKKK cells was further transplanted. A mouse (a 6-week-old female mouse purchased from Charles River Japan) was prepared as a human cholangiocarcinoma cell transplantation model (human leukemia-xenograft model). Subsequently, 150 μg of Peptide3 or Peptide4 prepared in Example 1 was injected intravenously (iv) with respect to 20 g of mouse body weight. The abdomen was opened 90 minutes after administration, and the tumor lesions and the distribution of peptides were observed under a fluorescent stereomicroscope. The results are shown in FIG. In FIG. 12, gal means gallbladder, live means liver, st means stomach, sp means spleen, br means brain, hr means heart, kd means kidney, lu means lung, and tumor means malignant tumor. Further, in FIG. 12, "Bright Field" is an image taken in a bright field, and "FITC" is an image taken in a dark field. Further, the upper image is an image of the tissue removed from the mouse to which Peptide3 was added, and the lower image is an image of the tissue removed from the mouse to which Peptide4 was added. The image on the left is an image of various extracted tissues, and the image on the right is an image of slices of various extracted tissues.

From FIG. 12, it was confirmed that Peptide3 and Peptide4 were accumulated only in malignant tumors. In addition, stronger fluorescence was detected in Peptide 4 than in Peptide 3.

In addition, FIGS. 13A and 13B are graphs showing the ratio of fluorescence intensity detected in various tissues when the fluorescence detected in each tissue is quantified in Test Example 12 and the fluorescence detected in the pancreas is set to 1. Is. Further, FIG. 14 is a graph showing the ratio of the fluorescence intensity detected in the malignant tumor when the fluorescence detected in each tissue is quantified and the fluorescence detected in each tissue is set to 1.
From FIGS. 13A and 13B, in the mice administered with Peptide3, the fluorescence detected in the malignant tumor was about 8.6 times the fluorescence detected in the liver, whereas in the mice administered with Peptide4, the fluorescence detected in the malignant tumor was malignant. The fluorescence detected was about 45.8 times the fluorescence detected in the liver.
Further, from FIG. 14, when the fluorescence in any tissue was set to 1, it was detected in the malignant tumor in the mouse to which Peptide4 was administered rather than the fluorescence detected in the malignant tumor in the mouse to which Peptide3 was administered. Fluorescence was a higher value.

From the above, it was confirmed that Peptide4, which is a D-form, has higher accumulation in bile duct cancer cells.

[Test Example 13] Evaluation test of peptide accumulation in various tissues in mice transplanted with liver transplanted with human cholangiocarcinoma cells 6 weeks after purchasing liver transplanted with M156 cells from NOD-SCID mice (Japan Charles River Co., Ltd.) It was transplanted to an old female mouse). Subsequently, 150 μg of Peptide4 prepared in Example 1 was intravenously (iv) injected into 20 g of mouse body weight. The abdomen was opened 90 minutes after administration, and the tumor lesions and the distribution of peptides were observed under a fluorescent stereomicroscope. The results are shown in FIG. In FIG. 15, gal means gallbladder, live means liver, and tumor means malignant tumor. Further, in FIG. 15, "Bright Field" is an image taken in a bright field, and "FITC" is an image taken in a dark field. The upper image is an image of the mouse when the abdomen is opened, the lower left image is an image of the removed liver and malignant tumor, and the lower right image is a slice of the removed liver and malignant tumor. It is an image of a thing.

From FIG. 15, it was confirmed that Peptide4 was accumulated only in the gallbladder and malignant tumors.

[Test Example 14] Evaluation test of peptide accumulation in cholangiocarcinoma cells contained in ascites of cholangiocarcinoma patients Peptide3 (4 μM) and a nuclear stain prepared in Example 1 were used in the ascites of 3 cholangiocarcinoma patients. Hoechst (manufactured by Dojindo Co., Ltd.) was added, and the cells were cultured at 37 ° C. for 120 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG. In FIG. 16, “Phase + Hoechst” indicates a bright field including a nuclear-stained image.

From FIG. 16, it was confirmed that Peptide3 has accumulation not only in cultured cells but also in bile duct cancer cells derived from bile duct cancer patients.

According to the present invention, it is possible to provide a novel peptide having an accumulation specific to biliary tract cancer. In addition, biliary tract cancer can be detected easily, with high sensitivity, and selectively.

Claims (9)

A peptide according to any one of the following (a) to (d) and having a cyclic structure .
(A) Ri Do an amino acid sequence comprising the sequence represented by SEQ ID NO: 3, and a peptide from L- amino ing,
(B) Among the sequences represented by SEQ ID NO: 3, the amino acid sequence comprises a sequence in which the fourth arginine residue from the N-terminal is replaced with an isoleucine residue, an alanine residue, or a lysine residue, and is composed of L. -Peptide consisting of amino acids and having biliary tract cancer-specific accumulation ,
(C) A peptide consisting of an amino acid sequence including the sequence represented by SEQ ID NO: 7 and consisting of a D-amino acid.
(D) Among the sequences represented by SEQ ID NO: 7, the amino acid sequence comprises a sequence in which the fourth arginine residue from the C-terminal is replaced with an isoleucine residue, an alanine residue, or a lysine residue, and is composed of D. -Peptide consisting of amino acids and having biliary tract cancer-specific accumulation A peptide consisting of an amino acid sequence containing the sequence represented by any of SEQ ID NOs: 3, 5 and 6 and consisting of L-amino acids, or
The peptide according to claim 1, which is a peptide consisting of an amino acid sequence including the sequence represented by SEQ ID NO: 7 and also consisting of a D-amino acid. A peptide consisting of an amino acid sequence including the sequence represented by SEQ ID NO: 3 and consisting of an L-amino acid, or
The peptide according to claim 1 or 2, which is a peptide consisting of an amino acid sequence including the sequence represented by SEQ ID NO: 7 and also consisting of a D-amino acid. A nucleic acid comprising the peptide according to any one of claims 1 to 3. A vector comprising the nucleic acid according to claim 4. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 3 and one or more selected from the group consisting of a labeling substance, a modifying substance and a physiologically active substance. The pharmaceutical composition according to claim 6, wherein the labeling substance is a stable isotope, a radioactive isotope, or a fluorescent substance. The pharmaceutical composition according to claim 6 or 7, wherein the modifying substance is a sugar chain or polyethylene glycol. The pharmaceutical composition according to any one of claims 6 to 8, which is for treatment or diagnosis of biliary tract cancer.