Fluorescence enhancer

Abstract

The present invention provides: a protoporphyrin IX fluorescence enhancer comprising a nucleic acid having a G4 structure, the nucleic acid having the G4 structure being a nucleic acid that is bound to or complexed with a substance having cell membrane permeability; a method for producing a fluorine-containing G4 compound, in which an amidide derivative compound containing a fluorine-containing group and a nucleic acid having a guanine quadruplex structure are bound by a phosphoramidite method to synthesize a fluorine-containing G4 compound in which the nucleic acid having the guanine quadruplex structure and the fluorine-containing group are linked via a linking group; a medicinal composition having the fluorescence enhancer as an active ingredient; a cancer cell visualization kit containing the fluorescence enhancer and 5-aminolevulinic acid; and a cancer cell visualization method in which the fluorescence enhancer and 5-aminolevulinic acid are introduced into an in-vitro cancer cell.

Description

Fluorescence enhancer

[0001] The present invention relates to a fluorescence enhancement technique for protoporphyrin IX (PpIX), which is useful for photodynamic diagnosis (PDD) using 5-aminolevulinic acid (5-ALA). This application claims priority to Japanese Patent Application No. 2023-216872, filed on December 22, 2023, the contents of which are incorporated herein by reference.

[0002] In diagnostic imaging, including endoscopic examinations, there is a strong demand in clinical settings for technology that can easily identify even the smallest lesions without overlooking them. Furthermore, in surgical procedures, being able to visualize cancer sites can help prevent the risk of leaving cancer behind. For this reason, research into photodynamic diagnosis, which uses light to distinguish between tumors and normal tissue, is being widely conducted.

[0003] A typical photosensitizer used in photodynamic diagnosis is 5-ALA. 5-ALA is a natural amino acid naturally present in the living organisms of animals and plants. Administered to the body, 5-ALA is taken up into cells, metabolized in the cytoplasm, and then metabolized to PpIX in mitochondria. In normal cells, ferrous iron is coordinated to PpIX by an iron-adding enzyme (ferrochelatase, FECH), ultimately resulting in the biosynthesis of heme, which is necessary for maintaining vital activities. On the other hand, in cancer cells, energy production by oxidative phosphorylation in mitochondria is reduced, anaerobic glycolysis in the cytoplasm is significantly increased (Warburg effect), and the activity of FECH, which converts PpIX to heme, is lower than in normal cells. This reduces the heme synthesis reaction, inhibits heme biosynthesis, and results in excessive accumulation of PpIX. PpIX has the optical property of emitting red fluorescence (600-740 nm) when excited with blue visible light (375-445 nm). Therefore, when irradiated with blue visible light, cancer cells emit red fluorescence due to the accumulation of PpIX, whereas normal cells do not emit red fluorescence because PpIX has been converted to heme. Thus, blue visible light makes it possible to visually distinguish and recognize cancer cells from normal cells (Non-Patent Document 1). Clinically, photodynamic diagnosis is performed by administering 5-ALA to cancer patients to excessively accumulate PpIX in cancer cells, and then irradiating the biological tissue with blue visible light to visualize only cancerous lesions, causing them to glow red.

[0004] When irradiated with excitation light, PpIX undergoes photooxidation to convert to photoprotoporphyrin (PPp), resulting in a decrease in fluorescence intensity and bleaching (Non-Patent Document 2). Because PpIX bleaching can result in false positives, conventional photodynamic diagnosis recommends minimizing observation under blue light sources to avoid bleaching. Specifically, cancer lesions are observed, resected, and stopped bleeding under a white light source. Lesions emitting red fluorescence from PpIX are quickly confirmed under a blue light source, and then resected and stopped bleeding under a white light source. However, because white light also contains wavelengths that excite red fluorescence, rapid operation is required even under a white light source. Thus, photodynamic diagnosis using PpIX requires large doses of 5-ALA in addition to the short observation time available in the same area. However, high doses can cause side effects such as photosensitivity, liver dysfunction, hypotension, nausea, and vomiting.

[0005] On the other hand, one type of higher-order structure formed by guanine-rich nucleic acids is the guanine quadruplex (G-quadruplex, G4) structure. The G4 structure is a stable higher-order structure formed by stacking two or more planar structures called G-quartets, which are formed from four guanine molecules through Hoogsteen hydrogen bonding between guanines, through π-π stacking. G4 structures in RNA are involved in RNA splicing, RNA transport, mRNA translation, and the like (Non-Patent Document 3).

[0006] Furthermore, compounds having a polyfluoro structure are known to be stable and low in toxicity in vivo, and to have excellent uptake into cells and escape from endosomes (Non-Patent Document 4). Taking advantage of these properties, studies are being conducted to introduce a polyfluoro structure into oligonucleotides or peptide nucleic acids as a moiety capable of penetrating cell membranes.

[0007] Ishizuka et al., International Immunopharmacology, 2011, vol.11(3), p.358-365.Juzenas et al., Journal of Photochemistry and Photobiology B: Biology, 2001, vol.61(1-2), p.78-86.Rhodes et al., Nucleic Acids Research, 2015, vol.43(18), p.8627-8637.Zhang et al., MRS Communications, 2018, vol.8, p.303-313.Ferreira et al., ChemistryOpen, 2012, vol.1, p.106-114.

[0008] An object of the present invention is to provide a fluorescence enhancer for enhancing fluorescence emitted from intracellular protoporphyrin IX, and a kit for visualizing cancer cells, which comprises the fluorescence enhancer.

[0009] The present inventors discovered that by coexisting PpIX with a nucleic acid having a G4 structure bound or complexed with a substance having cell membrane permeability, the intensity of red fluorescence emitted from PpIX upon irradiation with blue visible light can be significantly enhanced, and thus completed the present invention.

[0010] That is, the present invention includes the following aspects. [1] A fluorescence enhancer for protoporphyrin IX, comprising a nucleic acid having a G4 structure, wherein the nucleic acid having a G4 structure is bound to or complexed with a substance having cell membrane permeability. [2] The fluorescence enhancer of [1], wherein the nucleic acid having a G4 structure bound to the substance having cell membrane permeability is a fluorine-containing G4 compound in which a nucleic acid having a G-quadruplex structure and a fluorine-containing group are linked via a linking group, and the fluorine-containing group is a perfluoroalkyl group having 1 to 10 carbon atoms and no ether-bonding oxygen atom between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms and 1 to 5 ether-bonding oxygen atoms between carbon atoms. [3] The fluorescence enhancer of [1], wherein the fluorine-containing group is a group represented by the following general formula (F2) or (F3):

[0011]

[0012] [In the formula, R Ｆ３ is a perfluoroalkylene group having q3 carbon atoms; R Ｆ４ is a perfluoroalkyl group having q4 carbon atoms; n1, q3, and q4 are natural numbers such that n1 + q3 + q4 is 3 or more and 10 or less; R Ｆ５ is a perfluoroalkylene group having q5 carbon atoms, and R Ｆ６ is a perfluoroalkyl group having q6 carbon atoms; and n2, q5, and q6 are natural numbers such that 2×n2+q5+q6 is 4 or more and 10 or less.] [4] The fluorescence enhancer according to [2] or [3], wherein the linking group is an alkylene group, an alkenylene group, —C(═O)—, —NH—, —O—, —S—, —C(═O)—O—, —O-C(═O)—, —C(═O)NH—, —NH-C(═O)—, —NH-C(═O)—O—, —O-C(═O)-NH—, —O-P(═O)(OH)—O—, a polyethylene glycol group, or a group consisting of a combination thereof. [5] The fluorescence enhancer according to [2] or [3], wherein the fluorine-containing G4 compound is a group represented by the following general formula (1):

[0013]

[0014] [In formula (1), G ０ is a nucleic acid that forms a G-quadruplex structure and may be modified with a substance other than nucleic acids; R ＦＥ is a perfluoroalkyl group having 1 to 10 carbon atoms and no ether-bonding oxygen atoms between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonding oxygen atoms between carbon atoms; Z １ [6] The fluorescence enhancer according to any one of the above [2] to [4], wherein the fluorine-containing G4 compound is a compound represented by the following general formula (2):

[0015]

[0016] [In formula (2), G ０ is a nucleic acid that forms a G-quadruplex structure and may be modified with a substance other than nucleic acids; R ＦＥis a perfluoroalkyl group having 1 to 10 carbon atoms and no ether-bonding oxygen atoms between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonding oxygen atoms between carbon atoms; Z ２ [7] The fluorescence enhancer of [1] above, wherein the nucleic acid having a G4 structure is a nucleic acid complexed with a lipid nanoparticle. [8] The fluorescence enhancer of [2] above, wherein the nucleic acid having a G4 structure is a compound represented by the following general formula (G1):

[0017]

[0018] [In formula (G1), G ｓ is a base sequence in which s consecutive Gs (guanines); s represents an integer of 2 to 5; N represents A (adenine), C (cytosine), G (guanine), T (thymine), or U (uracil); N ｒ１ is a base sequence in which r1 consecutive Ns are present; N ｒ２is a base sequence in which r2 consecutive Ns are present; r1 and r2 each represent an integer between 0 and 7, satisfying 1≦r1+r2≦7; and when 2≦r1+r2≦7, the multiple Ns may be the same or different bases.] [9] A method for producing a fluorine-containing G4 compound, comprising bonding an amidite derivative compound containing a fluorine-containing group with a nucleic acid that forms a G-quadruplex structure by a phosphoramidite method, to synthesize a fluorine-containing G4 compound in which the nucleic acid that forms a G-quadruplex structure and the fluorine-containing group are linked via a linking group.

[10] A method for producing a fluorine-containing G4 compound according to [9], wherein the fluorine-containing group is a perfluoroalkyl group having 1 to 10 carbon atoms and no ether-bonding oxygen atom between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonding oxygen atoms between carbon atoms.

[11] A pharmaceutical composition containing, as an active ingredient, the fluorescence enhancer of any one of [1] to [8].

[12] A kit for visualizing cancer cells, containing the fluorescence enhancer of any one of [1] to [8] and 5-aminolevulinic acid.

[13] A method for visualizing cancer cells, comprising introducing the fluorescence enhancer of any one of [1] to [8] and 5-aminolevulinic acid into cancer cells ex vivo.

[0019] The fluorescence enhancer according to the present invention has a nucleic acid that takes a G4 structure, thereby reducing the bleaching of PpIX. Furthermore, since the fluorescence enhancer is bound or complexed with a substance that is cell membrane permeable, it also has excellent cell membrane permeability. Therefore, by coexisting the fluorescence enhancer with PpIX, the intensity of red fluorescence emitted from PpIX upon irradiation with blue visible light can be enhanced. In particular, the cancer cell visualization kit and cancer cell visualization method according to the present invention enable cancer cells in which PpIX, a metabolic product of 5-aminolevulinic acid, has accumulated to be visualized with high sensitivity using blue visible light.

[0020] In Example 1, the synthesized phosphoramidite derivative compound (EEA-CH ２ -amidite) １1 is a H-NMR spectrum of the phosphoramidite derivative compound (EEA-CH) synthesized in Example 1. ２ -amidite) １９ 1 is a F-NMR spectrum of the phosphoramidite derivative compound (EEA-CH) synthesized in Example 1. ２ -amidite) ３１ 1 is a P-NMR spectrum of the phosphoramidite derivative compound (EEA-CH) synthesized in Example 1. ２ 1 shows the results of HR-Mass analysis of the fluorine-containing oligomer RNA (EEA-(UAGGGU) -amidite) synthesized in Example 1. ４ 1 shows the results of MALDI-TOF MS analysis of PpIX (RNA). ４ FIG. 1 shows the results of measuring the fluorescence emission spectrum of a sample containing RNA and a control sample containing only PpIX. ４ The sample in Test 1 to which RNA, RNase A, and PpIX were added, the sample in Test 2 to which only PpIX was added, and EEA-(UAGGGU) ４ This figure shows the results of examining the intensity of red fluorescence emitted from PpIX after 1 hour or 2 hours of incubation for the sample in Test Group 3 to which RNA and PpIX were added. ４ The samples were prepared by mixing RNA and PpIX at a molar ratio of 1:0, 1:1, 1:2, or 1:5. １ 1H-NMR spectrum. In Example 2, 5-ALA and EEA-(UAGGGU) ４ Breast cancer cells with added RNA (Test Group 1), breast cancer cells with added 5-ALA alone (Test Group 2), and 5-ALA and EEA-(UAGGGU) ４ The bright field image (top row) and red fluorescent image (bottom row) of breast cancer cells (test group 3) to which no RNA was added. In Example 3, 5-ALA and EEA-(UAGGGU) were administered to mice bearing tumor tissue derived from the breast cancer cell line 4T1. ４1 shows red fluorescence images of each tissue after administration of RNA (upper row) or 5-ALA alone (lower row). ２ -amidite) １ 1H-NMR spectrum of the phosphoramidite derivative compound (EEEA-CH ２ -amidite) １９ 1 is a F-NMR spectrum of the phosphoramidite derivative compound (EEEA-CH) synthesized in Example 4. ２ -amidite) ３１ 1 is a P-NMR spectrum of the phosphoramidite derivative compound (EEEA-CH ２ 1 shows the results of HR-Mass analysis of the fluorine-containing oligomer RNA (EEEA-(UAGGGU) -amidite) synthesized in Example 4. ４ 1 shows the results of MALDI-TOF MS analysis of the phosphoramidite derivative compound (BEA-CH) synthesized in Example 4. ２ -amidite) １ 1H-NMR spectrum of the phosphoramidite derivative compound (BEA-CH) synthesized in Example 4. ２ -amidite) １９ 1 is a F-NMR spectrum of the phosphoramidite derivative compound (BEA-CH) synthesized in Example 4. ２ -amidite) ３１ 1 is a P-NMR spectrum of the phosphoramidite derivative compound (BEA-CH) synthesized in Example 4. ２ 1 shows the results of HR-Mass analysis of the fluorine-containing oligomer RNA (BEA-(UAGGGU) -amidite) synthesized in Example 4. ４ 1 shows the results of MALDI-TOF MS analysis of the phosphoramidite derivative compound (C5PE-CH ２ -amidite) １ 1H-NMR spectrum of the phosphoramidite derivative compound (C5PE-CH２ -amidite) １９ 1 is a F-NMR spectrum of the phosphoramidite derivative compound (C5PE-CH ２ -amidite) ３１ 1 is a P-NMR spectrum of the phosphoramidite derivative compound (C5PE-CH ２ 1 shows the results of HR-Mass analysis of the synthesized fluorine-containing oligomer RNA (C5PE-(UAGGGU) -amidite). ４ In Example 4, PpIX was treated with EEEA-(UAGGGU) as an oligomer RNA. ４ RNA, BEA-(UAGGGU) ４ RNA, or C5PE-(UAGGGU) ４ 1 shows the results of measuring the fluorescence emission spectrum of a sample containing added RNA and a control sample containing only PpIX. ４ RNA or (UAGGGU) ４ FIG. 1 shows the results of measuring the fluorescence emission spectrum of a sample containing RNA and a control sample containing only PpIX. ４ Breast cancer cells with added RNA (Test Group 1), 5-ALA and (UAGGGU) ４ 1 shows bright-field images (top row) and red fluorescent images (bottom row) of breast cancer cells to which RNA was added (Test Group 2) and breast cancer cells to which only 5-ALA was added (Test Group 3). ４ 1 shows the results of MALDI-TOF MS analysis of EEEA-L-(UAGGGU) in the presence of RNase in Example 6. ４ RNA and (UAGGGU) ４ Figure 29A shows the results of examining the effect of RNA on PpIX fluorescence. ４The sample in Test 1 to which RNA, RNase A, and PpIX were added, and EEEA-L-(UAGGGU) ４ The results are shown for the sample in Test Group 2, to which RNA and PpIX were added, and the sample in Test Group 3, to which only PpIX was added. ４ A sample from Test 1 to which RNA, RNase A, and PpIX were added, a sample from Test 2 to which only PpIX was added, and (UAGGGU) ４ The results of the sample in Test Group 3, to which RNA, RNase A, and PpIX were added, are shown. ４ 1 shows bright-field images (top row) and red fluorescent images (bottom row) of breast cancer cells to which RNA was added (Test Group 1) and breast cancer cells to which only 5-ALA was added (Test Group 2). ４ A sample containing RNA-containing LNP ("RNA in LNP") and a sample containing PpIX containing L-(UAGGGU) ４ This figure shows the results of measuring the fluorescence emission spectrum of a sample to which RNA was added ("RNA") and a control sample containing only PpIX ("control"). ４1 shows bright-field images (upper row) and red fluorescent images (lower row) of breast cancer cells to which RNA-containing LNP was added (Test Group 1) and breast cancer cells to which only 5-ALA was added (Test Group 2). This figure shows the results of MALDI-TOF MS analysis of synthesized Cy5-modified oligomeric RNA in Reference Example 1. This figure shows the results of fluorescence emission spectrum measurement of a sample in which Cy5-modified oligomeric RNA was added to PpIX and a control sample containing only PpIX in Reference Example 1. This figure shows the results of fluorescence emission spectrum measurement of a sample in which L-biotin-modified oligomeric RNA was added to PpIX and a control sample containing only PpIX in Reference Example 2. This figure shows the results of examining the fluorescence intensity of red fluorescence emitted from PpIX 1 to 5 hours after incubation for the sample in Test Area 1, to which only L-biotin-modified oligomeric RNA and PpIX were added, the sample in Test Area 2, to which L-biotin-modified oligomeric RNA, PpIX, and RNase A were added, and the sample in Test Area 3, to which only PpIX was added, in Reference Example 2.

[0021] In the present invention and the present specification, the term "nucleic acid" refers to a molecule in which nucleotides are linked by phosphodiester bonds. Such nucleotides include not only natural nucleotides (naturally occurring nucleotides, D-nucleotides) such as deoxyribonucleotides and ribonucleotides, but also artificial nucleotides that are modified from natural nucleotides and can form phosphodiester bonds with natural nucleotides. Artificial nucleotides include natural nucleotides in which the side chains etc. of the ribose backbone have been modified with a functional group such as an amino group, nucleotides in which the hydroxyl group at the 2' position of the ribose backbone has been substituted with a methoxy group, a fluoro group, a methoxyethyl group etc., phosphorothioate nucleotides (nucleotides in which the oxygen atom of the phosphate group has been substituted with a sulfur atom), morpholino nucleotides (nucleotides in which ribose or deoxyribose has been substituted with a morpholine ring), BNA (Bridged Nucleic Acid), HNA (Hexitol Nucleic Acid), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), TNA (Threeose Nucleic Acid), GNA (Glycerol Nucleic Acid), CeNA (Cyclohexenyl Nucleic Acid), SNA (Serinol Nucleic Acid), etc. Artificial nucleotides also include L-type nucleotides and nucleotides in which the side chains etc. have been modified. "Nucleic acid" includes molecules such as DNA and RNA in which only one or more naturally occurring nucleotides are linked by phosphodiester bonds, molecules in which one or more naturally occurring nucleotides are linked by phosphodiester bonds with one or more artificial nucleotides, and molecules in which only one or more artificial nucleotides are linked by phosphodiester bonds.

[0022] In the present invention and the present specification, "C ｐ１－ｐ２ " (p1 and p2 are positive integers satisfying p1<p2) means that the group has p1 to p2 carbon atoms.

[0023] In the present invention and the present specification, "C １－１０ The "alkyl group" is an alkyl group having 1 to 10 carbon atoms, and may be a straight chain or a branched chain. ２－１０The "alkyl group" is an alkyl group having 2 to 10 carbon atoms, and may be a straight chain or a branched chain. １－１０ Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, and a decyl group.

[0024] In the present invention and the present specification, "C １－６ The "alkyl group" is an alkyl group having 1 to 6 carbon atoms, and may be a straight chain or a branched chain. １－６ Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, and a hexyl group.

[0025] In the present invention and this specification, an "alkylene group" is a divalent group obtained by removing two hydrogen atoms from a saturated hydrocarbon, and may be straight-chain or branched. Examples of the alkylene group include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, an octamethylene group, a nonamethylene group, a methylmethylene group, an ethylmethylene group, a methylethylene group, a methylpropylene group, an ethylethylene group, a dimethylmethylene group, a 1,2-dimethylethylene group, a 1,1-dimethylethylene group, a 1-ethylpropylene group, a 2-ethylpropylene group, and a 1,2-dimethylpropylene group. Examples of the alkyl group include a methyl group, a 2,2-dimethylpropylene group, a 1-propylpropylene group, a 2-propylpropylene group, a 1-methyl-1-ethylpropylene group, a 1-methyl-2-ethylpropylene group, a 1-ethyl-2-methylpropylene group, a 2-methyl-2-ethylpropylene group, a 1-methylbutylene group, a 2-methylbutylene group, a 3-methylbutylene group, a 2-ethylbutylene group, a 1-methylpentylene group, a 2-ethylpentylene group, and a 1-methylhexylene group.

[0026] In the present invention and this specification, an "alkenylene group" is a divalent group obtained by removing two hydrogen atoms from a hydrocarbon having at least one double bond, and may be linear or branched. Examples of the alkenylene group include ethenyl, propenyl, butenyl, butadienyl, pentenyl, pentadienyl, hexenyl, hexadienyl, hexatrienyl, heptenyl, heptadienyl, heptatrienyl, octenyl, octadienyl, and octatrienyl groups.

[0027] In the present invention and the present specification, "C １－1０ A "perfluoroalkyl group" is a group in which all hydrogen atoms of an alkyl group having 1 to 10 carbon atoms are substituted with fluorine atoms, and is a completely halogenated C １－１０ Also called alkyl group. １－１０ Examples of perfluoroalkyl groups include perfluoromethyl group, perfluoroethyl group, perfluoropropyl group, perfluoroisopropyl group, perfluorobutyl group, perfluoroisobutyl group, perfluorosec-butyl group, perfluorotert-butyl group, perfluoropentyl group, perfluoroisopentyl group, perfluoroneopentyl group, perfluorotert-pentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, and perfluorodecyl group.

[0028] In the present invention and the present specification, the term "ether-bonded oxygen atom" refers to an oxygen atom that connects carbon atoms, and does not include oxygen atoms in which oxygen atoms are connected in series. The number of ether-bonded oxygen atoms that an alkyl group having Nc carbon atoms (Nc is an integer of 2 or more) can have is up to Nc-1. ２－１０ The term "alkyl group" refers to a C ２－１０ It is a group having at least one oxygen atom by an ether bond between carbon atoms of the alkyl group. Hereinafter, an "alkyl group having an oxygen atom by an ether bond between carbon atoms" may be referred to as an "ether bond-containing alkyl group."

[0029] In the present invention and the present specification, "a carbon atom having an ether-bonding oxygen atom between carbon atoms, ２－１０ The "perfluoroalkyl group" is C ２－１０ Ether bond-containing C having at least one ether bond oxygen atom between the carbon atoms of the alkyl group ２－１０ It is a group in which all hydrogen atoms of an alkyl group have been substituted with fluorine atoms. Hereinafter, a "perfluoroalkyl group having an ether-bonded oxygen atom between carbon atoms" may be referred to as an "ether bond-containing perfluoroalkyl group."

[0030] The fluorescence enhancer according to the present invention enhances the intensity of red fluorescence emitted from PpIX upon irradiation with blue visible light, and comprises a nucleic acid having a G4 structure. The nucleic acid having a G4 structure stabilizes the structure of PpIX by interacting with PpIX in the G4 structure. Due to its structure-stabilizing effect on PpIX, the nucleic acid having a G4 structure can enhance the intensity of red fluorescence emitted from PpIX.

[0031] In one embodiment, the fluorescence enhancer according to the present invention preferably comprises a nucleic acid having a G4 structure with improved cell membrane permeability. By improving cell membrane permeability, the fluorescence enhancer can further enhance the detection sensitivity of intracellular PpIX. In the present invention and this specification, "cell membrane permeability" includes both the property of a substance directly passing through the cell membrane and the property of a substance being taken up into the cell membrane via membrane transport such as endocytosis.

[0032] Examples of nucleic acids having a G4 structure with improved cell membrane permeability include nucleic acids in which a nucleic acid having a G4 structure is bound to a substance having cell membrane permeability. Examples of cell membrane permeable groups include hydrophobic groups such as fluorine-containing groups. Examples of substances having cell membrane permeability include fluorine-containing group-containing compounds and cell membrane penetrating peptides (CPPs). Examples of CPPs include peptides derived from the TAT protein of the HIV virus, peptides derived from Antennapedia, and polyarginine peptides (WO 2008 / 089491).

[0033] Examples of nucleic acids having a G4 structure with improved cell membrane permeability include nucleic acids in which the G4 structure is complexed with a substance having cell membrane permeability. For example, the cell membrane permeability of the nucleic acid having a G4 structure can be improved by complexing the nucleic acid having a G4 structure with a drug delivery system (DDS) carrier having cell membrane permeability. The DDS carrier may be a nanoparticle or a microparticle. In addition, the cell membrane permeability of the nucleic acid having a G4 structure can be improved by complexing CPP with the nucleic acid having a G4 structure. Examples of nanoparticles include liposomes, polymeric micelles, dendrimers, lipid nanoparticles (LNPs), and exosomes, with LNPs being preferred.

[0034] <Fluorine-Containing G4 Compound> Examples of substances having cell membrane permeability include fluorine-containing group-containing compounds. A nucleic acid in which a nucleic acid having a G4 structure is bound to a fluorine-containing group-containing compound can be used as the fluorescence enhancer of the present invention. It is particularly preferable that the fluorescence enhancer of the present invention comprises a fluorine-containing G4 compound in which a nucleic acid having a G4 structure and a fluorine-containing group are linked via a linking group. The fluorine-containing G4 compound stabilizes the structure of PpIX by interacting with PpIX in the G4 structure. Furthermore, the fluorine-containing G4 compound has excellent cell membrane permeability due to the fluorine-containing group. Due to the PpIX structure stabilization effect and cell membrane permeability, the fluorine-containing G4 compound can particularly enhance the fluorescence intensity of red fluorescence emitted from PpIX in cells. Hereinafter, the fluorine-containing G4 compound used as the fluorescence enhancer of the present invention may be referred to as the "fluorine-containing G4 compound of the present invention."

[0035] The nucleic acid having the G4 structure contained in the fluorine-containing G4 compound according to the present invention is a 5'-(N ｒ１ G ｓ N ｒ2 )-3' (general formula (G1)) (G4 consensus sequence) is one or more, preferably 2 to 5, more preferably 3 to 5, even more preferably 3 to 4, and particularly preferably 4. In the G4 consensus sequence, s represents an integer of 2 or more and 5 or less, and "Gｓ " represents a "base sequence in which s Gs (guanines) are consecutive." In the G4 consensus sequence, N represents "A (adenine) or C (cytosine) or G (guanine) or T (thymine) or U (uracil)," and r1 and r2 each represent an integer between 0 and 7, satisfying 1≦r1+r2≦7. "N ｒ１ "teeth," ｒ１ A base sequence in which N is consecutive is called "N ｒ２ "teeth," ｒ２ Each of the above means a "base sequence in which N's are consecutive." When 2≦r1+r2≦7, the multiple N's may be the same type of base or different types of bases. In the G4 consensus sequence contained in the fluorine-containing G4 compound according to the present invention, the multiple N's are preferably each independently A, T or U. Examples of the G4 consensus sequence include 5'-(UAGGGU)-3' (formula (G1-1)), 5'-(GGGUA)-3' (formula (G1-2)), 5'-(GGGTT)-3' (formula (G1-3)), 5'-(UUAGGG)-3' (formula (G1-4)), 5'-(UAGGGGU)-3' (formula (G1-5)), 5'-(UAGGU)-3' (formula (G1-6)), and the like. The G4 structure in the fluorine-containing G4 compound according to the present invention may be of a parallel type, an antiparallel type, or a hybrid type. When the nucleic acid having a G4 structure contained in the fluorine-containing G4 compound according to the present invention has two or more G4 consensus sequences, it is preferable that these G4 consensus sequences are all the same G4 consensus sequence, but as long as it can form a G4 structure, the nucleic acid may be composed of two or more types of G4 consensus sequences. For example, the two or more G4 consensus sequences contained in the nucleic acid having a G4 structure contained in the fluorine-containing G4 compound according to the present invention may be N ｒ１ and N ｒ2 may be different from each other, and s may be different from each other.

[0036] The nucleic acid having a G4 structure contained in the fluorine-containing G4 compound according to the present invention may be a nucleic acid having one or more G4 consensus sequences so as to be able to form a G4 structure. For example, it may be a single-stranded nucleic acid in which 2 to 5, preferably 4, G4 consensus sequences are present in series, either directly or via a linker sequence, or a branched nucleic acid. For example, a nucleic acid in which a single-stranded nucleic acid containing one G4 consensus sequence is bound to the end of each arm of a four-armed molecule, a nucleic acid in which single-stranded nucleic acids containing two G4 consensus sequences are bound to the end of each arm of a two-armed molecule, or a nucleic acid in which a single-stranded nucleic acid containing one G4 consensus sequence is bound to the end of one arm of a two-armed molecule and single-stranded nucleic acids containing three G4 consensus sequences are bound to the end of the other arm can also be used as the nucleic acid having a G4 structure contained in the fluorine-containing G4 compound according to the present invention. An example of a nucleic acid in which a single-stranded nucleic acid containing one G4 consensus sequence is bound to the tip of each arm of a four-arm molecule is the nucleic acid described in Non-Patent Document 5, in which a single-stranded nucleic acid containing one G4 consensus sequence is bound to the four arms of Trebler Phosphoramidite.

[0037] The nucleic acid having a G4 structure contained in the fluorine-containing G4 compound according to the present invention may be composed of two or more nucleic acid molecules. For example, it may be a nucleic acid having a G4 structure formed by polymerization of two single-stranded nucleic acids in which two consensus sequences exist in series directly or via a linker sequence. Alternatively, it may be a nucleic acid having a G4 structure formed by aggregation of four single-stranded nucleic acids each containing one G4 consensus sequence.

[0038] The nucleic acid having a G4 structure contained in the fluorine-containing G4 compound according to the present invention may be DNA, RNA, or a nucleic acid containing both deoxyribonucleotides and ribonucleotides. It may also be a nucleic acid consisting of only natural nucleotides, a nucleic acid containing natural nucleotides and one or more artificial nucleotides, or a nucleic acid consisting of only one or more artificial nucleotides. The nucleic acid having a G4 structure used in the present invention is preferably an enzyme-resistant nucleic acid, more preferably an L-nucleic acid, in order to prevent degradation within cells.

[0039] The nucleic acid having a G4 structure contained in the fluorine-containing G4 compound according to the present invention may be a nucleic acid consisting of only a G4 structure, or may be a nucleic acid having a region consisting of another base sequence in addition to the G4 structure. The other base sequence is not particularly limited as long as it is a sequence that does not inhibit the interaction between the fluorine-containing G4 compound according to the present invention and PpIX. Examples of the other base sequence include functional nucleic acids such as nucleic acid aptamers and nucleic acid probes, and may also be a nucleic acid chain that serves as a linker.

[0040] The nucleic acid having a G4 structure contained in the fluorine-containing G4 compound according to the present invention may be modified with a substance that does not contribute to the formation of the G4 structure, as long as the G4 structure is not impaired. The substance that modifies the nucleic acid having a G4 structure may be a nucleic acid or a substance other than nucleic acid. The modifying substance other than nucleic acid is not particularly limited, and examples thereof include amino acids, peptides, proteins, monosaccharides, oligosaccharides, sugar chains, lipids, and low-molecular-weight compounds. The modifying substance may be a chimeric molecule consisting of a combination of two or more of these substances, or may be a chimeric molecule of these substances and a nucleic acid. Examples of the chimeric molecule include glycoproteins and lipoproteins.

[0041] The modifying substance for modifying a nucleic acid having a G4 structure may have a specific function or may be a substance without a particular function. Examples of substances having a specific function include molecules that specifically or selectively bind to a specific substance, such as antibodies, sugar-binding molecules, hormone-binding molecules, transporter molecules, ligands (agonists, antagonists), cell surface antigen-binding molecules, etc. In addition, substances that have a specific function but do not specifically bind to a specific molecule, such as cell membrane-permeable peptides and liposomes, and labeling substances such as fluorescent substances, are also used as modifying substances for modifying a nucleic acid having a G4 structure. Since this can facilitate the purification and detection of the fluorine-containing G4 compound according to the present invention, it is also preferable that the nucleic acid having a G4 structure be modified with biotin, a tag peptide (His tag, Myc tag, Flag tag, etc.), a fluorescent substance, etc.

[0042] The nucleic acid having a G4 structure and the substance that modifies it may be bonded directly or via a linker, which may be a single-stranded nucleic acid chain, a double-stranded nucleic acid chain, a peptide linker, or an alkylene glycol chain such as a polyethylene glycol chain.

[0043] The fluorine-containing group contained in the fluorine-containing G4 compound according to the present invention is a C １－１０ Perfluoroalkyl group or C having 1 to 5 ether-bonded oxygen atoms between carbon atoms ２－１０ Hereinafter, "a C alkyl group having no ether-bonding oxygen atom between carbon atoms" will be referred to as a "C alkyl group having no ether-bonding oxygen atom between carbon atoms." １－１０ Perfluoroalkyl group or C having 1 to 5 ether-bonded oxygen atoms between carbon atoms ２－１０ perfluoroalkyl group" by R ＦＥ That's what they say.

[0044] R ＦＥ C does not have an ether-bonding oxygen atom between carbon atoms １－１０ In the case of a perfluoroalkyl group, the group may be linear or branched. １－１０ The perfluoroalkyl group exhibits sufficient cell membrane permeability and also suppresses toxicity to cells. ２－１０ Perfluoroalkyl groups are preferred, and linear C ３－１０ Perfluoroalkyl groups are more preferred, and linear C ４－１０ Perfluoroalkyl groups are more preferred.

[0045] R ＦＥ is an ether bond oxygen-containing C ２－１０ In the case of a perfluoroalkyl group, the group is preferably, for example, a group represented by any one of the following general formulae (F1) to (F4).

[0046]

[0047] In general formula (F1), R Ｆ１is a perfluoroalkylene group having q1 carbon atoms, and R Ｆ２ is a perfluoroalkyl group having q2 carbon atoms. q1 and q2 are natural numbers such that the sum of both is 2 or more and 10 or less. In general formula (F2), R Ｆ３ is a perfluoroalkylene group having q3 carbon atoms, and R Ｆ４ is a perfluoroalkyl group having q4 carbon atoms. n1, q3, and q4 are natural numbers such that n1 + q3 + q4 is 3 or more and 10 or less. In general formula (F3), R Ｆ５ is a perfluoroalkylene group having q5 carbon atoms, and R Ｆ６ is a perfluoroalkyl group having q6 carbon atoms. n2, q5, and q6 are natural numbers such that 2×n2+q5+q6 is 4 or more and 10 or less. In general formula (F4), R Ｆ７ is a perfluoroalkylene group having q7 carbon atoms, and R Ｆ８ is a perfluoroalkyl group having q8 carbon atoms. n3, q7, and q8 are natural numbers such that 3×n3+q7+q8 is 5 or more and 10 or less.

[0048] R in general formula (F1) Ｆ１ , R in general formula (F2) Ｆ３ , R in general formula (F3) Ｆ５ and R in general formula (F4) Ｆ７ R may be a linear perfluoroalkylene group or a branched perfluoroalkylene group. Ｆ１ , R Ｆ３ , R Ｆ５ , and R Ｆ７ As for C １－３ A perfluoroalkylene group is preferred, a methylene group, an ethylene group, a methylmethylene group, an ethylmethylene group, a dimethylmethylene group, or a group in which all hydrogen atoms of a methylethylene group have been substituted with fluorine atoms is more preferred, a methylene group, a methylmethylene group, an ethylmethylene group, or a dimethylmethylene group in which all hydrogen atoms have been substituted with fluorine atoms is even more preferred, and a methylene group or a methylmethylene group in which all hydrogen atoms have been substituted with fluorine atoms is even more preferred.

[0049] R in general formula (F1) Ｆ２ , R in general formula (F2)Ｆ４ , R in general formula (F3) Ｆ６ and R in general formula (F4) Ｆ８ R may be a linear perfluoroalkyl group or a branched perfluoroalkyl group. Ｆ２ , R Ｆ４ , R Ｆ６ , and R Ｆ８ As the C １－５ Perfluoroalkyl groups are preferred, and linear C １－５ Perfluoroalkyl groups are more preferred, and linear C ２－４ Perfluoroalkyl groups are more preferred.

[0050] The fluorine-containing group contained in the fluorine-containing G4 compound according to the present invention can be, for example, an ether bond-containing perfluoroalkyl group contained in the fluorine compounds described in JP-A-2006-321797 and JP-A-2011-1377 together with their synthesis methods.

[0051] In the fluorine-containing G4 compound according to the present invention, the linking group linking the nucleic acid having a G4 structure to the fluorine-containing group is not particularly limited as long as it is a group capable of linking the nucleic acid having a G4 structure to the fluorine-containing group, and any divalent or higher organic group can be used. Examples of the divalent or higher organic group include an alkylene group, an alkenylene group, -C(=O)-, -NH-, -O-, -S-, -C(=O)-O-, -O-C(=O)-, -C(=O)NH-, -NH-C(=O)-, -NH-C(=O)-O-, -O-C(=O)-NH-, -O-P(=O)(OH)-O-, a polyethylene glycol (PEG) group (-(C ２ H ４ O)n- (n is a natural number), etc. Furthermore, groups in which two or more hydrogen atoms have been removed from a ring such as a pyrrole ring, a pyrazole ring, an imidazole ring, a triazole ring, a pyridine ring, a pyrimidine ring, a pyrazine ring, an oxazole ring, a thiazole ring, a furan ring, a thiophene ring, or a benzene ring can also be used as the divalent or higher linking group.

[0052] When the linking group is an alkylene group, the alkylene group is １－１０An alkylene group (an alkylene group having 1 to 10 carbon atoms) is preferred, and may be a straight chain or a branched chain. When the linking group is an alkenylene group, the alkenylene group is preferably C ２－１０ An alkylene group (an alkenylene group having 2 to 10 carbon atoms) is preferred, and may be linear or branched. By further removing a hydrogen atom from the alkylene group or alkenylene group, a trivalent or higher valent linking group can be obtained.

[0053] In the fluorine-containing G4 compound according to the present invention, the linking group linking the nucleic acid having a G4 structure and the fluorine-containing group may be a divalent or higher organic group obtained by appropriately combining the divalent or higher organic groups listed above.Furthermore, a linking group obtained by appropriately combining two or more of these linking groups to form a trivalent or higher linking group as a whole can also be used as the linking group between the nucleic acid having a G4 structure and the fluorine-containing group.When the linking group is a trivalent or higher organic group, the linking group can be linked to other substances in addition to the nucleic acid having a G4 structure and the fluorine-containing group.Examples of such other substances include the modifying substances that modify the nucleic acid having a G4 structure.

[0054] In the fluorine-containing G4 compound according to the present invention, the linking group linking the nucleic acid having a G4 structure to the fluorine-containing group is preferably an alkylene group, an alkenylene group, -C(=O)-, -NH-, -O-, -S-, -C(=O)-O-, -O-C(=O)-, -C(=O)NH-, -NH-C(=O)-, -NH-C(=O)-O-, -O-C(=O)-NH-, -O-P(=O)(OH)-O-, a PEG group, or a group consisting of a combination thereof. １－１０ Alkylene group -O-P(=O)(OH)-O-, -C ２－１０ Alkenylene group -O-P(=O)(OH)-O-, -C １－１０ Alkylene group -CO-, -C １－１０ Alkylene group -CO-O-, -C ２－１０ Alkenylene group -CO-, -C ２－１０ Alkenylene group -CO-O-, -C １－１０ Alkylene group -NH-, -C ２－１０ An alkenylene group -NH- is more preferred, and -C １－１０ Alkylene group -O-P(=O)(OH)-O-, -C２－１０ More preferred is the alkenylene group -O-P(=O)(OH)-O-.

[0055] The fluorine-containing G4 compound according to the present invention may be a compound in which a fluorine-containing group is linked via a linking group to the 5'-end side of a nucleic acid having a G4 structure, or a compound in which a fluorine-containing group is linked via a linking group to the 3'-end side of a nucleic acid having a G4 structure.

[0056] When the nucleic acid having a G4 structure in the fluorine-containing G4 compound according to the present invention is a single-stranded nucleic acid, a compound in which a fluorine-containing group is linked to the 5'-end or 3'-end of the single-stranded nucleic acid via a divalent linking group, and various modifying substances are linked to the remaining end via a divalent linking group can be used as the fluorine-containing G4 compound according to the present invention. Furthermore, when the nucleic acid having a G4 structure in the fluorine-containing G4 compound according to the present invention has a branched structure containing two or more single-stranded nucleic acids, a compound in which a fluorine-containing group is linked to either the 5'-end or 3'-end of the nucleic acid via a divalent linking group, and various modifying substances are linked to either the other 5'-end or 3'-end of the nucleic acid to which the fluorine-containing group is not linked via a divalent linking group can be used as the fluorine-containing G4 compound according to the present invention. Furthermore, the fluorine-containing G4 compound according to the present invention may also be a compound in which various modifying substances are linked to either the 5'-end or 3'-end of the nucleic acid having a G4 structure via a divalent linking group, and a fluorine-containing group is further linked to the modifying substance via a divalent linking group.

[0057] Examples of the fluorine-containing G4 compound according to the present invention include compounds represented by the following general formula (1): ０ is a nucleic acid having a G4 structure, and may be modified with a substance other than nucleic acid. １ is a linking group. ＦＥ is a C having no ether-bonding oxygen atom between carbon atoms. １－１０ Perfluoroalkyl group or C having 1 to 5 ether-bonded oxygen atoms between carbon atoms ２－１０ A nucleic acid having a G4 structure (which may be modified with a substance other than nucleic acid), a linking group, and R ＦＥThe above-mentioned materials can be used.

[0058]

[0059] The compound represented by general formula (1) is preferably, for example, a compound represented by the following general formulas (1-1) to (1-5). ０ and G ’０ is a nucleic acid having a G4 structure, which may be modified with a substance other than nucleic acid. １１ , Z １１ , Z １３ , and Z １４ are each independently a divalent linking group, Z １５ is a trivalent linking group. The divalent linking group and the trivalent linking group can be the organic groups listed above as the linking group. １１ , L １２ , and L １３ are each independently a modifying substance. As the modifying substance, the substances listed above as modifying substances for nucleic acids having a G4 structure can be used.

[0060]

[0061] The fluorine-containing G4 compound according to the present invention is preferably a compound represented by the following general formula (2), since it can be easily synthesized by utilizing the phosphoramidite method (solid phase synthesis method) which is widely used in nucleic acid synthesis. ０ and R ＦＥ is the same as in the general formula (1). ２ is a linking group, specifically, Z １ The same groups as those shown below can be used.

[0062]

[0063] The compound represented by the general formula (2) can be, for example, a fluorine-containing group (R ＦＥ The compound can be synthesized by combining an amidite derivative compound containing R with a nucleic acid having a G4 structure by the phosphoramidite method. ＦＥThe fluorine-containing G4 compound according to the present invention is synthesized by condensing (coupling) the amidite group in an amidite derivative compound containing the following with the hydroxyl group at the 5'-position of a nucleic acid having a G4 structure. The coupling reaction can be carried out in the same manner as in a general nucleic acid synthesis reaction using the phosphoramidite method, and can also be synthesized using a commonly used automatic nucleic acid synthesizer.

[0064] The R ＦＥ The amidite derivative compound containing R ＦＥ It can be synthesized by introducing a phosphoryl amidite group into the hydroxyl group of a compound having a hydroxyl group. ＦＥ An example of the compound having a hydroxyl group is a compound represented by the following general formula (3): ＦＥ and Z ２ is the same as in the general formula (2). ２ As for C １－６ An alkylene group is preferred, and a methylene group, an ethylene group, or a propylene group is more preferred. The hydroxyl group in general formula (3) may be protected with a 4,4'-dimethoxytriphenylmethyl group (DMTr) or the like.

[0065]

[0066] The method for introducing a phosphorylating amidite group into a compound represented by general formula (3) is not particularly limited as long as it allows the introduction of a phosphorylating amidite group, and various synthetic methods can be used. Commercially available phosphoramidite reagents such as 2-cyanoethyldiisopropylchlorophosphoramidite (CAS RN. 89992-70-1) and 2-cyanoethyltetraisopropylphosphoramidite (CAS RN. 102691-36-1) can also be used. For example, the hydroxyl group of a compound represented by general formula (3) can be phosphoramiditeized with 2-cyanoethyldiisopropylchlorophosphoramidite in the presence of N,N-diisopropylethylamine using acetonitrile as a solvent.

[0067] The synthesized fluorine-containing G4 compound such as the compound represented by general formula (2) can be isolated and purified by various methods such as ion chromatography, gel filtration chromatography, reverse phase chromatography, and normal phase chromatography.

[0068] <G4 nucleic acid-containing LNP> In one embodiment, the fluorescence enhancer according to the present invention preferably comprises a nucleic acid in which a nucleic acid having a G4 structure is complexed with an LNP. The nucleic acid having a G4 structure complexed with an LNP is taken up into the cell membrane by membrane transport by the LNP, and then released from the LNP, thereby enhancing the fluorescence intensity of the red fluorescence emitted from PpIX in the cell. Hereinafter, the complex of a nucleic acid having a G4 structure and an LNP used as a fluorescence enhancer according to the present invention may be referred to as the "G4 nucleic acid-containing LNP according to the present invention."

[0069] The LNP used in the G4 nucleic acid-containing LNP according to the present invention is not particularly limited. LNP is basically composed of four types of lipid components: cationic lipid, phospholipid, cholesterol, and PEG lipid (polyethylene glycol-modified lipid). As these lipids, known lipids used as constituent lipids of LNP in gene therapy, gene modification technology, etc., or modified versions thereof can be used as appropriate. For example, the LNPs described in U.S. Patent No. 11,191,849 and U.S. Patent No. 11,684,577 can be used, and LNPs modified as appropriate from these can also be used.

[0070] The nucleic acid having G4 structure used in the LNP containing G4 nucleic acid of the present invention can be the same nucleic acid as that having G4 structure used in the fluorine-containing G4 compound of the present invention.The complex between the nucleic acid having G4 structure and LNP can be produced by encapsulating the nucleic acid having G4 structure in LNP.For example, by micelleizing the constituent lipid of LNP in a solution containing the nucleic acid having G4 structure, the LNP containing the nucleic acid having G4 structure, that is, the LNP containing the nucleic acid having G4 structure can be obtained.

[0071] The fluorine-containing G4 compound according to the present invention and the G4 nucleic acid-containing LNP according to the present invention can be used as is or mixed with a pharmacologically acceptable carrier or the like to form a pharmaceutical composition. That is, the fluorescence enhancer according to the present invention, which includes the fluorine-containing G4 compound according to the present invention and the G4 nucleic acid-containing LNP according to the present invention, can be used as an active ingredient of a pharmaceutical composition. As the pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as formulation materials are used, and are incorporated as excipients, lubricants, binders, disintegrants in solid formulations; solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. in liquid formulations. In addition, formulation additives such as preservatives, antioxidants, colorants, and sweeteners can also be used as needed. These carriers and formulation additives can be appropriately selected from those commonly used in pharmaceutical formulations.

[0072] A pharmaceutical composition containing the fluorine-containing G4 compound of the present invention or the G4 nucleic acid-containing LNP of the present invention can be produced by a method commonly used in the pharmaceutical technology field, for example, a method described in the Japanese Pharmacopoeia, etc. The content of the fluorine-containing G4 compound of the present invention in the pharmaceutical composition varies depending on the administration route, dosage form, intended dose, etc., but is, for example, about 0.1 to 100% by mass.

[0073] The dosage of the fluorine-containing G4 compound according to the present invention and the G4 nucleic acid-containing LNP according to the present invention will vary depending on the subject, route of administration, tissue to be administered, etc., but for example, when administered orally or parenterally to an adult patient, the dosage of the fluorine-containing G4 compound according to the present invention per dose is usually about 0.01 to 250 mg / kg body weight, preferably 0.01 to 200 mg / kg body weight, more preferably 0.01 to 100 mg / kg body weight, even more preferably 0.1 to 50 mg / kg body weight, and still more preferably 1.0 to 40 mg / kg body weight.

[0074] The fluorine-containing G4 compound according to the present invention and the G4 nucleic acid-containing LNP according to the present invention serve as fluorescence enhancers that enhance the intensity of red fluorescence emitted from PpIX upon irradiation with blue visible light, and are therefore particularly suitable as pharmaceutical compositions to be used in combination with 5-ALA in photodynamic diagnosis for visualizing cancer cells using 5-ALA. By using the fluorine-containing G4 compound according to the present invention or the G4 nucleic acid-containing LNP according to the present invention in combination, the intensity of the red fluorescence derived from PpIX can be improved, thereby reducing the amount of 5-ALA required for visualization of cancer cells and suppressing side effects caused by excessive administration of 5-ALA. Furthermore, the fluorine-containing G4 compound according to the present invention and the G4 nucleic acid-containing LNP according to the present invention can also suppress bleaching of PpIX, allowing for more sufficient time for observation of cancer lesions under a blue light source than when 5-ALA is administered alone, thereby improving the accuracy of identifying cancer lesions.

[0075] For example, when using 5-ALA to visualize cancer cells during surgical treatment, 5-ALA is generally orally administered 2 to 4 hours before the induction of anesthesia during surgery. The fluorine-containing G4 compound and the G4 nucleic acid-containing LNP according to the present invention may be orally administered simultaneously with 5-ALA, or may be orally administered independently of 5-ALA at any time after administration of 5-ALA and before the induction of anesthesia. Furthermore, they may be applied or sprayed onto tumor tissue 10 to 30 minutes before irradiation with blue visible light during surgery.

[0076] By combining the fluorine-containing G4 compound according to the present invention or the G4 nucleic acid-containing LNP according to the present invention with 5-ALA to prepare a kit, visualization of cancer cells using 5-ALA can be more easily performed. In a kit for visualization of cancer cells containing a fluorescence enhancer consisting of the fluorine-containing G4 compound according to the present invention, the 5-ALA contained in the kit may be a pharmacologically acceptable salt. Examples of such salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, and sulfate; organic acid salts such as formate, acetate, propionate, toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, and malate; alkali metal salts such as lithium salt, sodium salt, and potassium salt; alkaline earth metal salts such as magnesium and calcium salt; and metal salts such as aluminum and zinc; ammonium salts such as ammonium salt, alkylammonium salts such as tetramethylammonium salt; and organic amine salts such as triethylamine salt, piperidine salt, morpholine salt, and toluidine salt.

[0077] The 5-ALA contained in the cancer cell visualization kit together with the fluorine-containing G4 compound of the present invention may be an ALA derivative. The ALA derivative is not particularly limited as long as it is a compound that can be metabolized in the body to cause PpIX to accumulate in cancer cells. Examples of the ALA derivative include alkyl esters of 5-ALA, such as methyl ester, ethyl ester, propyl ester, butyl ester, and pentyl ester.

[0078] The fluorine-containing G4 compound according to the present invention and the G4 nucleic acid-containing LNP according to the present invention are useful not only for visualizing cancer cells in the body of an animal, but also for visualizing cancer cells ex vivo. Because the fluorine-containing G4 compound according to the present invention has excellent cell membrane permeability, it can be introduced into cancer cells simply by adding it directly to a culture medium for cancer cells together with 5-aminolevulinic acid.

[0079] Cancer cells visualized by the fluorine-containing G4 compound according to the present invention or the G4 nucleic acid-containing LNP according to the present invention and 5-ALA are not particularly limited, as long as they are cancer cells in which PpIX accumulates upon introduction of 5-ALA. The fluorine-containing G4 compound according to the present invention and the G4 nucleic acid-containing LNP according to the present invention can improve the visualization of various cancer cells. Examples of such cancer cells include brain tumors (malignant gliomas), head and neck cancer, adrenocortical cancer, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, endometrial cancer, esophageal cancer, Ewing's tumor, gallbladder cancer, Hodgkin's disease, hypopharyngeal cancer, laryngeal cancer, lip and oral cavity cancer, liver cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, melanoma, mesothelioma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, testicular cancer, and thyroid cancer.

[0080] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0081] Example 1 A phosphoramidite containing a fluorine-containing group was coupled with a single-stranded RNA having a G4 structure to synthesize a fluorine-containing G4 compound in which the single-stranded RNA having a G4 structure was modified with a fluorine-containing group. The effect of this compound on the intensity of red fluorescence emitted from PpIX upon irradiation with blue visible light was investigated.

[0082] (1) EEA-CH ２ Synthesis of EEA-CH ２ OH (compound (1)) and 2-cyanoethyldiisopropylchlorophosphoramidite (compound (2)) were coupled to form phosphoramidite (EEA-CH ２ -amidite) was synthesized.

[0083]

[0084] EEA-CH ２OH (276 mg, 0.83 mM) was reacted with 2-cyanoethyldiisopropylchlorophosphoramidite (compound (2): 0.18 mL, 0.87 mM, 1.05 equivalents) in the presence of N,N-diisopropylethylamine (DIPEA: 0.57 mL, 3.32 mM, 4 equivalents) using acetonitrile as a solvent at room temperature for 3 hours to obtain a phosphoramidite derivative compound (400 mg, yield 90% by mass).

[0085] The synthesized phosphoramidite derivative compounds １ H. １９ F. ３１ The P-NMR spectrum charts are shown in Figures 1 to 3, respectively. The HR-Mass results of the phosphoramidite derivative compound are shown in Figure 4. As a result, a peak was detected that matched the expected molecular weight of the amidite derivative compound (536.9862). These results indicate that the synthesized phosphoramidite derivative compound is the target EEA-CH ２ -Amidite was identified.

[0086] (2)EEA-(UAGGGU) ４ Synthesis of RNA D-type single-stranded RNA with G4 structure (UAGGGU) ４ ) and EEA-CH ２ The fluorine-containing G4 compound (EEA-(UAGGGU)) was then bonded to the fluorine-containing G4 compound (EEA-(UAGGGU)) by the phosphoramidite method. ４ RNA) was synthesized.

[0087]

[0088] First, a fluorine-containing oligomer RNA (EEA-(UAGGGU)) was synthesized using an automatic DNA / RNA synthesizer. ４) was synthesized. Next, AMA (28% aqueous ammonia:methylamine = 1:1 (volume ratio)) was added to the CPG (Controlled Pore Glass) carrier in the automated synthesizer, and the synthesized fluorine-containing oligomeric RNA was excised. The AMA solution in which the fluorine-containing oligomeric RNA was dissolved was incubated at 65°C for 10 minutes to deprotect the nucleic acid bases. Next, dimethyl sulfoxide (DMSO), triethylamine (TEA), and triethylamine trihydrofluoride (TEA·3HF) were added to the fluorine-containing oligomeric RNA, and the mixture was incubated at 65°C for 2.5 hours in a dry bath incubator to deprotect the tert-butyldimethylsilyl group (TBDS) of the fluorine-containing oligomeric RNA. Thereafter, the solvent was removed, and the product was dissolved in sterile water and then simply purified using a Glen-Pak cartridge to obtain a crude product. The crude product was treated by reverse phase HPLC (High Performance Liquid Chromatography) to obtain the target fluorine-containing oligomer RNA (EEA-(UAGGGU) ４ RNA was purified.

[0089] The obtained fluorine-containing oligomeric RNA was analyzed by MALDI-TOF MS (Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry). The analysis results are shown in FIG.

[0090] (3) Measurement of the fluorescence emission spectrum of PpIX. KCl (final concentration 100 mM) and K-PO ４ The reaction buffer was prepared by adding fluorine-containing oligomer RNA (EEA-(UAGGGU)) (final concentration: 20 mM). ４RNA) was added to the mixture to adjust the total sample volume to 200 μL. A control sample without added RNA was also prepared. After annealing, PpIX dissolved in DMSO (final concentration 1 μM) was added to each sample, and the mixture was incubated at room temperature in a dark place for 2 hours. After incubation, the sample was placed in a fluorometer cell, and the fluorescence emission spectrum was measured using a spectrofluorometer.

[0091] The results of the fluorescence emission spectrum measurement are shown in Figure 6. EEA-(UAGGGU) ４ In the sample to which RNA was added (in the figure, "F-modified"), a prominent peak derived from PpIX was detected at a wavelength of around 630 nm compared to the control sample to which RNA was not added (in the figure, "control"). ４ It was found that RNA enhanced the fluorescence intensity derived from PpIX.

[0092] (4) EEA-(UAGGGU) in the presence of RNase ４ Effect of RNA on PpIX fluorescence The reaction buffer used in (3) above was added with fluorine-containing oligomer RNA (EEA-(UAGGGU)) at a final concentration of 10 μM. ４ Two samples (Test Groups 1 and 3) containing the reaction buffer (RNA) and one sample (Test Group 2) containing only the reaction buffer were prepared. After annealing, PpIX (final concentration 1 μM) dissolved in DMSO was added to each sample. RNase A was added only to the sample in Test Group 1. The samples were then incubated at 37°C for 2 hours in a dry bath incubator.

[0093] The fluorescence intensity of each sample was measured 1 and 2 hours after the start of incubation using an in vivo imaging system (Lumazone, Shoshin EM). A 405 nm / 625 nm filter was used as the excitation / emission filter. Fluorescence intensity was quantified using imaging software (Intelligent Imaging Innovations). The measurement results are shown in Figure 7. In the sample from Test 1 (labeled "1"), which contained the RNA-cleaving enzyme RNase A, fluorescence fading was measured 1 hour after the start of incubation, and fluorescence was almost undetectable after 2 hours. In the sample from Test 3 (labeled "3"), which did not contain RNase A, strong fluorescence was observed 1 hour after the start of incubation and continued to be measured even after 2 hours. In the sample from Test 2 (labeled "2"), which contained only PpIX, fluorescence was almost undetectable both 1 and 2 hours after the start of incubation. The fluorescence fading of the sample in Test Area 1 was EEA-(UAGGGU) ４ This indicates that the RNA was cleaved by RNase A, destabilizing the G4 structure, which led to the degradation of PpIX and a decrease in fluorescence intensity. ４ It was found that RNA binds to PpIX and enhances the intensity of red fluorescence derived from PpIX. The test conditions and the results of fluorescence intensity are shown in Table 1.

[0094]

[0095] (5)EEA-(UAGGGU) ４ Structural analysis of binding between RNA and PpIX: The reaction buffer used in (3) above was diluted with fluorine-containing oligomer RNA (EEA-(UAGGGU)) at a final concentration of 0.8 mM. ４ Four samples containing 1000kJ / ml of RNA were prepared. After annealing, PpIX dissolved in DMSO was added to each sample to a final concentration of 0, 0.8, 1.6, or 4.0 mM. The samples were then left to stand overnight at 4°C. ２ After adding O, the mixture was placed in a tube for NMR measurement and analyzed using a superconducting nuclear magnetic resonance apparatus (NMR). １H-NMR spectra were measured at room temperature.

[0096] For each sample １ The results of H-NMR signal measurement are shown in Figure 8. Of the four spectra in the figure, "1:0" represents the result of a sample without added PpIX (PpIX concentration: 0 mM), "1:1" represents the result of a sample with a PpIX concentration of 0.8 mM, "1:2" represents the result of a sample with a PpIX concentration of 1.6 mM, and "1:5" represents the result of a sample with a PpIX concentration of 4.0 mM. As shown in Figure 8, the peaks of the uracil (U) bases H6 and H1' decreased with increasing PpIX concentration. These results indicated that PpIX binds to the U base position at the 5' end of the G4 structure.

[0097] Based on the obtained structural analysis results, EEA-(UAGGGU) was analyzed using Discovery Studio 4.5 (BIOVIA). ４ Molecular modeling simulation of the binding between RNA and PpIX revealed that PpIX binds to the 5' end of the G4 structure.

[0098] Furthermore, EEA-(UAGGGU) ４ Molecular dynamics simulations were performed on the binding of RNA to PpIX, revealing that the potential energy of 5'-end binding is significantly lower than that of side binding, indicating stabilization.

[0099] [Example 2] Breast cancer cell line 4T1 (obtained from JCRB cell bank) was treated with 5-ALA and EEA-(UAGGGU) synthesized in Example 1. ４ RNA was introduced, and the fluorescence intensity of each cell was examined.

[0100] Breast cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM) at 37°C in a humidified atmosphere (5% CO by volume). ２ ) were cultured in three 35 mm glass-bottom dishes. ４Cells were seeded at a cell count per dish and cultured for 24 hours to allow adhesion and proliferation (preculture). Next, 5 mM 5-ALA was added to the culture medium of two of the three dishes (Test Group 1 and Test Group 2), and nothing was added to the remaining one (Test Group 3). After 21 hours of culture, the culture medium was removed using an aspirator. Next, 1 μM EEA-(UAGGGU)4RNA was added to the culture medium of one of the three dishes (Test Group 1), and nothing was added to the remaining two dishes (Test Group 2 and Test Group 3). After another 24 hours of culture, fluorescent images of the cells in each dish were obtained using a confocal microscope set to a laser excitation wavelength of 405 nm and a detector fluorescence wavelength of 580 nm to 680 nm. Microscopic images of each cell are shown in Figure 9. In Figure 9, the upper row is a bright-field image, and the lower row is a fluorescent image.

[0101] As shown in Figure 9, in the cells of Test Group 2 to which only 5-ALA was added, almost no red fluorescence derived from PpIX was observed, as in the cells of Test Group 3 to which 5-ALA was not added. ４ In the cells of Test Group 1, which were added together with RNA, strong red fluorescence derived from PpIX was observed. ４ It was confirmed that RNA enhances the red fluorescence derived from PpIX accumulated in cancer cells.

[0102] [Example 3] Using a mouse cancer model, tumors in the animal body were treated with 5-ALA and EEA-(UAGGGU) synthesized in Example 1. ４ Visualization was performed using RNA.

[0103] Nude mice (6-week-old, female) were inoculated with breast cancer cell line 4T1 (8 × 10 ５ After one week from the transplantation, the formation of a subcutaneous tumor was confirmed, and 5-ALA (5 mg / mouse) was administered. ４ RNA (0.3 mg / mouse) was administered. 5-ALA and EEA-(UAGGGU) ４All RNAs were dissolved in saline and injected subcutaneously. Control mice were administered 5-ALA alone. The mice were dissected, and the red fluorescence intensity in each tissue was measured using an in vivo imaging system (Lumazone, Shoshin EM Co., Ltd.) in the same manner as in Example 1.

[0104] Transmitted light images and red fluorescent images of each tissue are shown in Figure 10. The mice administered 5-ALA with EEA-(UAGGGU) showed significantly higher fibroblast growth than the mice administered 5-ALA alone. ４ The intensity of red fluorescence emitted from the tumor tissue was clearly enhanced in mice administered with RNA. ４ Although the amount of RNA administered was as very small as 0.3 mg / mouse, an effect of enhancing the intensity of red fluorescence was observed, demonstrating that the fluorescence enhancer comprising the fluorine-containing G4 compound according to the present invention is highly effective.

[0105] Example 4 A phosphoramidite containing three types of fluorine-containing groups was coupled with a D-single-stranded RNA having a G4 structure to synthesize a fluorine-containing G4 compound in which the single-stranded RNA having a G4 structure was modified with a fluorine-containing group.

[0106] (1) EEEA-CH ２ Synthesis of EEEA-CH ２ OH (compound (3)) and 2-cyanoethyldiisopropylchlorophosphoramidite (compound (2)) were coupled to form phosphoramidite (EEEA-CH ２ -amidite) was synthesized.

[0107]

[0108] EEEA-CH ２ OH (250 mg, 0.65 mM) was reacted with 2-cyanoethyldiisopropylchlorophosphoramidite (compound (2): 0.19 mL, 0.49 mM, 1.1 equivalents) in the presence of DIPEA (0.232 mL, 1.34 mM, 3 equivalents) using acetonitrile as a solvent at room temperature for 3 hours to obtain a phosphoramidite derivative compound (105 mg, yield 80% by mass).

[0109] The synthesized phosphoramidite derivative compounds １ H. １９ F. ３１ The P-NMR spectrum charts are shown in Figures 11 to 13, respectively. The HR-Mass results of the phosphoramidite derivative compound are shown in Figure 14. As a result, a peak was detected that matched the expected molecular weight (648.09) of the amidite derivative compound. These results indicate that the synthesized phosphoramidite derivative compound is the target EEEA-CH ２ -Amidite was identified.

[0110] (2)EEEA-(UAGGGU) ４ Synthesis of RNA D-type single-stranded RNA with G4 structure (UAGGGU) ４ ) and EEEA-CH ２ The fluorine-containing G4 compound (EEEA-(UAGGGU)) was then bonded to the fluorine-containing G4 compound (EEEA-(UAGGGU)) by the phosphoramidite method. ４ RNA) was synthesized.

[0111]

[0112] First, a fluorine-containing oligomer RNA (EEEA-(UAGGGU)) was synthesized using an automatic DNA / RNA synthesizer. ４ ) was synthesized. Next, AMA (28% aqueous ammonia:methylamine = 1:1 (volume ratio)) was added to the CPG carrier in the automated synthesizer, and the synthesized fluorine-containing oligomeric RNA was excised. The AMA solution in which the fluorine-containing oligomeric RNA was dissolved was incubated at 65°C for 10 minutes to deprotect the nucleic acid bases. Next, DMSO, TEA, and TEA-3HF were added to the fluorine-containing oligomeric RNA, and the mixture was incubated at 65°C for 2.5 hours in a dry bath incubator to deprotect the TBDS group of the oligomeric RNA. Thereafter, the solvent was removed, and the product was dissolved in sterile water, followed by simple purification using a Glen-Pak cartridge to obtain a crude product. The crude product was treated by reverse-phase HPLC to isolate the desired fluorine-containing oligomeric RNA (EEEA-(UAGGGU) ４ RNA was purified.

[0113] The obtained fluorine-containing oligomeric RNA was analyzed by MALDI-TOF MS, and the analysis results are shown in FIG.

[0114] (3) BEA-CH ２ Synthesis of BEA-CH by the following synthesis reaction ２ OH (compound (4)) and 2-cyanoethyldiisopropylchlorophosphoramidite (compound (2)) were coupled to form phosphoramidite (BEA-CH ２ -amidite) was synthesized.

[0115]

[0116] BEA-CH ２ OH (300 mg, 0.69 mM) was reacted with 2-cyanoethyldiisopropylchlorophosphoramidite (compound (2): 0.22 mL, 0.66 mM, 1.1 equivalents) in the presence of DIPEA (0.3 mL, 1.80 mM, 3 equivalents) using acetonitrile as a solvent at room temperature for 3 hours to obtain a phosphoramidite derivative compound (110 mg, yield 30% by mass).

[0117] The synthesized phosphoramidite derivative compounds １ H. １９ F. ３１ The P-NMR spectrum charts are shown in Figures 16 to 18, respectively. The HR-Mass results of the phosphoramidite derivative compound are shown in Figure 19. As a result, a peak was detected that matched the molecular weight (632.0921) of the expected amidite derivative compound. From these results, it was confirmed that the synthesized phosphoramidite derivative compound was the target BEA-CH ２ -Amidite was identified.

[0118] (4) BEA-(UAGGGU) ４ Synthesis of RNA D-type single-stranded RNA with G4 structure (UAGGGU) ４ ) and BEA-CH ２ The fluorine-containing G4 compound (BEA-(UAGGGU)) was then bonded to the fluorine-containing G4 compound (BEA-(UAGGGU)) by the phosphoramidite method. ４ RNA) was synthesized.

[0119]

[0120] First, a fluorine-containing oligomer RNA (BEA-(UAGGGU)) was synthesized using an automatic DNA / RNA synthesizer. ４ Next, in the same manner as in (2) above, the fluorine-containing oligomer RNA synthesized from the automatic synthesizer was cut out, and the nucleic acid bases and the TBDS group were deprotected. After that, simple purification using a Glen-Pak cartridge and purification treatment using reversed-phase HPLC were carried out to obtain the desired fluorine-containing oligomer RNA (BEA-(UAGGGU) ４ RNA was purified.

[0121] The obtained fluorine-containing oligomeric RNA was analyzed by MALDI-TOF MS, and the analysis results are shown in FIG.

[0122] (5) C5PE-CH ２ Synthesis of C5PE-CH ２ OH (compound (5)) and 2-cyanoethyldiisopropylchlorophosphoramidite (compound (2)) were coupled to obtain phosphoramidite (C5PE-CH ２ -amidite) was synthesized.

[0123]

[0124] C5PE-CH ２ OH (250 mg, 0.68 mM) was reacted with 2-cyanoethyldiisopropylchlorophosphoramidite (compound (2): 0.21 mL, 0.649 mM, 1.1 equivalents) in the presence of DIPEA (0.3 mL, 1.77 mM, 3 equivalents) using acetonitrile as a solvent at room temperature for 3 hours to obtain a phosphoramidite derivative compound (100 mg, yield 30% by mass).

[0125] The synthesized phosphoramidite derivative compounds １ H. １９ F. ３１The P-NMR spectrum charts are shown in Figures 21 to 23, respectively. The HR-Mass results of the phosphoramidite derivative compound are shown in Figure 24. As a result, a peak was detected that matched the molecular weight (563.5883) of the expected amidite derivative compound. From these results, it was confirmed that the synthesized phosphoramidite derivative compound was the target C5PE-CH ２ -Amidite was identified.

[0126] (6) C5PE-(UAGGGU) ４ Synthesis of RNA D-type single-stranded RNA with G4 structure (UAGGGU) ４ ) and C5PE-CH ２ The fluorine-containing G4 compound (C5PE-(UAGGGU)) was obtained by combining the fluorine-containing G4 compound (C5PE-(UAGGGU)) with the fluorine-containing G4 compound (C5PE-(UAGGGU)) by the phosphoramidite method. ４ RNA) was synthesized.

[0127]

[0128] First, a fluorine-containing oligomer RNA (C5PE-(UAGGGU)) was synthesized using an automatic DNA / RNA synthesizer. ４ Next, in the same manner as in (2) above, the fluorine-containing oligomer RNA synthesized from the automatic synthesizer was cut out, and the nucleic acid bases and the TBDS group were deprotected. After that, simple purification using a Glen-Pak cartridge and purification treatment using reversed-phase HPLC were carried out to obtain the desired fluorine-containing oligomer RNA (C5PE-(UAGGGU) ４ RNA was purified.

[0129] The obtained fluorine-containing oligomeric RNA was analyzed by MALDI-TOF MS, and the analysis results are shown in FIG.

[0130] (7) Measurement of the fluorescence emission spectrum of PpIX - 1 Three types of synthesized fluorine-containing oligomeric RNA (EEEA-(UAGGGU) ４ RNA, BEA-(UAGGGU) ４ RNA, C5PE-(UAGGGU) ４ The fluorescence emission spectrum of PpIX was measured using the RNA. ４The reaction buffer was prepared by adding fluorine-containing oligomer RNA (EEEA-(UAGGGU)) (final concentration: 20 mM). ４ RNA, BEA-(UAGGGU) ４ RNA, or C5PE-(UAGGGU) ４ RNA) was added to the mixture to adjust the total sample volume to 200 μL. A control sample without added RNA was also prepared. After annealing, PpIX dissolved in DMSO (final concentration 1 μM) was added to each sample, and the mixture was incubated at room temperature in a dark place for 2 hours. After incubation, the sample was placed in a fluorometer cell, and the fluorescence emission spectrum was measured using a spectrofluorometer.

[0131] The results of fluorescence emission spectrum measurement are shown in Figure 26A. Regardless of the type of fluorine-containing oligomeric RNA, a prominent peak derived from PpIX was detected at a wavelength of around 630 nm in the samples to which fluorine-containing oligomeric RNA was added, compared to the control sample to which no RNA was added. From these results, it is clear that EEEA-(UAGGGU) ４ RNA, BEA-(UAGGGU) ４ RNA, and C5PE-(UAGGGU) ４ All RNAs were EEA-(UAGGGU) ４ As with RNA, it was found that the fluorescence intensity derived from PpIX was enhanced.

[0132] (8) Measurement of the fluorescence emission spectrum of PpIX-2 C5PE-(UAGGGU) as oligomeric RNA ４ RNA or (UAGGGU) ４ The fluorescence emission spectrum of PpIX was measured in the same manner as in (7) above, except that RNA was used.

[0133] The results of the fluorescence emission spectrum measurement are shown in Figure 26B. ４ RNA) and oligomeric RNA ((UAGGGU) ４In the sample to which fluorine-containing oligomer RNA (C5PE-(UAGGGU) was added), a prominent peak derived from PpIX was detected at a wavelength of around 630 nm compared to the control sample to which no RNA was added. ４ RNA) and oligomeric RNA ((UAGGGU) ４ When compared with the sample to which fluorine-containing oligomer RNA (C5PE-(UAGGGU) ４ A more pronounced peak was detected in the sample to which RNA (RNA) was added. These results demonstrate that oligomeric RNA, regardless of whether it is modified with a fluorine-containing group, enhances the fluorescence intensity derived from PpIX, and that the introduction of a fluorine-containing group further enhances the effect of oligomeric RNA in enhancing the fluorescence intensity derived from PpIX.

[0134] [Example 5] 5-ALA and C5PE-(UAGGGU) synthesized in Example 4 were added to breast cancer cell line 4T1 (obtained from JCRB cell bank). ４ RNA was introduced, and the fluorescence intensity of each cell was examined.

[0135] Three 35mm glass bottom dishes, 5x10 ４ The cells were seeded at 100 cells / dish and cultured for 24 hours to allow the cells to adhere and proliferate (preculture). Next, 500 μM 5-ALA was added to the culture medium of three dishes, and the cells were cultured for 21 hours. After removing the culture medium with an aspirator, 1 μM C5PE-(UAGGGU) was added to the culture medium of one of the three dishes. ４ RNA was added (Test Group 1), and 1 μM (UAGGGU) was added to one culture medium. ４ RNA was added to one dish (Test 2), and nothing was added to the remaining dish (Test 3). After culturing for another 24 hours, fluorescent images of the cells in each dish were taken using an all-in-one fluorescent microscope set to a filter excitation wavelength of 395-415 nm and a detector fluorescence wavelength of 625-655 nm. Microscopic images of each cell are shown in Figure 27. In Figure 27, the top row is a bright-field image, and the bottom row is a fluorescent image.

[0136] As shown in Figure 27, in the cells of Test Group 3 to which only 5-ALA was added, almost no red fluorescence derived from PpIX was observed. ４ RNA or (UAGGGU) ４ In the cells of Test Group 1 and Test Group 2, which were added together with RNA, strong red fluorescence derived from PpIX was observed. When comparing the cells of Test Group 1 and Test Group 2, stronger red fluorescence derived from PpIX was observed in the cells of Test Group 1. From these results, it was confirmed that C5PE-(UAGGGU) ４ RNA and (UAGGGU) ４ It was found that RNA enhances the red fluorescence derived from PpIX accumulated in cancer cells, and that the enhancement of PpIX-derived red fluorescence by oligomeric RNA is further enhanced by introducing a fluorine-containing group into the oligomeric RNA.

[0137] Example 6: A phosphoramidite containing three types of fluorine-containing groups was coupled with an L-single-stranded RNA having a G4 structure to synthesize a fluorine-containing G4 compound in which the single-stranded RNA having a G4 structure was modified with a fluorine-containing group. The effect of the obtained fluorine-containing G4 compound on PpIX fluorescence in the presence of RNAase was investigated.

[0138] (1) L-type single-stranded RNA (L-(UAGGGU)) with a G4 structure ４ Synthesis of L-type oligomer RNA (L-(UAGGGU) ４ Next, in the same manner as in Example 4 (2), the fluorine-containing oligomeric RNA synthesized from the automatic synthesizer was excised, and the nucleic acid bases and the TBDS group were deprotected. After that, simple purification using a Glen-Pak cartridge and purification treatment using reversed-phase HPLC were carried out to obtain the desired L-type oligomeric RNA (L-(UAGGGU) ４ RNA was purified.

[0139] The obtained L-type oligomeric RNA was analyzed by MALDI-TOF MS, and the analysis results are shown in FIG.

[0140] (2)EEEA-L-(UAGGGU) ４Synthesis of RNA L-type oligomer RNA (L-(UAGGGU) ４ RNA) and EEEA-CH synthesized in Example 4(1). ２ The fluorine-containing G4 compound, fluorine-containing oligomer RNA (EEEA-L-(UAGGGU) ４ Specifically, L-(UAGGGU) was synthesized as an oligomeric RNA. ４ Purified EEEA-L-(UAGGGU) was prepared in the same manner as in Example 4(2), except that RNA was used. ４ RNA was obtained.

[0141] (3) EEEA-L-(UAGGGU) in the presence of RNase ４ Effect of RNA on PpIX fluorescence. Autoclaved ultrapure water was diluted with KCl (final concentration 100 mM) and K-PO ４ The reaction buffer was prepared by adding fluorine-containing oligomer RNA (EEEA-L-(UAGGGU)) (final concentration: 20 mM) to the reaction buffer. ４ Two samples (Test Groups 1 and 2) containing the reaction buffer (RNA) and one sample (Test Group 3) containing only the reaction buffer were prepared. After annealing, PpIX (final concentration 1 μM) dissolved in DMSO was added to each sample. RNase A was added only to the sample in Test Group 1. The samples were then incubated at 37°C for 2 hours in a dry bath incubator.

[0142] One and two hours after the start of incubation, the fluorescence intensity of each sample was measured using an in vivo imaging system (Lumazone, manufactured by Shoshin EM). A 405 nm / 625 nm filter was used as the excitation / emission filter. Fluorescence intensity was quantified using imaging software (manufactured by Intelligent Imaging Innovations). The measurement results are shown in Figure 29A. In the sample of test group 3 to which only PpIX was added (labeled "3" in the figure), almost no fluorescence was measurable either one hour or two hours after the start of incubation. On the other hand, in the case of EEEA-L-(UAGGGU)４ In the test groups 1 and 2 to which RNA was added, strong fluorescence was observed 1 hour after the start of incubation, regardless of whether RNase A was added or not, and fluorescence was still measured even after 2 hours. From these results, it was concluded that EEEA-L-(UAGGGU) ４ It was found that RNA binds to PpIX, enhancing the intensity of the red fluorescence derived from PpIX, and is resistant to RNase A, so that PpIX is not degraded even in the presence of RNase A, and the fluorescence intensity does not change. The conditions and fluorescence intensity results for each test group are shown in Table 2.

[0143]

[0144] (4) EEEA-L-(UAGGGU) in the presence of RNase ４ RNA and (UAGGGU) ４ Effect of RNA on PpIX fluorescence The reaction buffer used in (3) above was diluted with L-type fluorine-containing oligomer RNA (EEEA-L-(UAGGGU)) at a final concentration of 10 μM. ４ One sample (Test Group 1) contained 10 μM of D-type oligomer RNA ((UAGGGU) ４ One sample (Test Group 3) containing the reaction buffer (RNA) and one sample (Test Group 2) containing only the reaction buffer were prepared. After annealing, each sample was added with PpIX (final concentration 1 μM) dissolved in DMSO. RNase A was added only to the samples in Test Groups 1 and 3. The samples were then incubated at 37°C for 2 hours in a dry bath incubator.

[0145] The fluorescence intensity of each sample was measured 1 and 2 hours after the start of incubation in the same manner as in (3) above. The measurement results are shown in Figure 29B. In the sample of Test Group 2 to which only PpIX was added (labeled "2" in the figure), almost no fluorescence was measurable either 1 or 2 hours after the start of incubation. On the other hand, in the case of EEEA-L-(UAGGGU) ４ In Test Group 1, where RNA and RNase A were added, strong fluorescence was observed one hour after the start of incubation, and fluorescence was still measured even after two hours. (UAGGGU)４ In test group 3, where RNA and RNase A were added, fluorescence fading was measured one hour after the start of incubation, and almost no fluorescence was measured after two hours. The fluorescence fading of the sample in test group 3 was (UAGGGU) ４ This indicates that the RNA was cleaved by RNase A, destabilizing the G4 structure, which led to the degradation of PpIX and a decrease in fluorescence intensity. ４ It was found that RNA binds to PpIX and enhances the intensity of red fluorescence derived from PpIX. The test conditions and the results of fluorescence intensity are shown in Table 3.

[0146]

[0147] [Example 7] Breast cancer cell line 4T1 (obtained from JCRB cell bank) was treated with 5-ALA and EEEA-L-(UAGGGU) synthesized in Example 6. ４ RNA was introduced, and the fluorescence intensity of each cell was examined.

[0148] Two 35mm glass bottom dishes, 5x10 ４ The cells were seeded at 100 cells / dish and cultured for 24 hours to allow the cells to adhere and proliferate (preculture). Then, 500 μM 5-ALA was added to the culture medium of two dishes, and the cells were cultured for 21 hours. After removing the culture medium with an aspirator, 1 μM EEEA-L-(UAGGGU) was added to the culture medium of one of the two dishes. ４ RNA was added to one dish (Test 1), and nothing was added to the remaining dish (Test 2). After culturing for another 24 hours, fluorescent images of the cells in each dish were taken using an all-in-one fluorescent microscope set to a filter excitation wavelength of 395-415 nm and a detector fluorescence wavelength of 625-655 nm. Microscopic images of each cell are shown in Figure 30. In Figure 30, the upper row is a bright-field image, and the lower row is a fluorescent image.

[0149] As shown in Figure 30, in the cells of Test Group 2 to which only 5-ALA was added, almost no red fluorescence derived from PpIX was observed. ４In the cells of Test Group 1, which were added together with RNA, strong red fluorescence derived from PpIX was observed. ４ It was found that RNA enhanced the red fluorescence derived from PpIX accumulated in cancer cells.

[0150] [Example 8] L-(UAGGGU) synthesized in Example 6 ４ RNA was encapsulated in LNP and complexed with LNP. ４ The effect of RNA-containing LNPs on PpIX fluorescence was examined.

[0151] (1) L-(UAGGGU) ４ Preparation of RNA-containing LNPs L-type oligomeric RNA (L-(UAGGGU) ４ RNA) was dissolved in 50 mM sodium acetate buffer adjusted to pH 5.0. Ionized lipid, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and PEG-DMG (polyethylene glycol-modified dimyristoylglycerol) were added to ethanol at a molar ratio of 50 / 10 / 38.5 / 1.5 and dissolved. An aqueous solution of L-oligomeric RNA and a lipid ethanol solution were mixed so that the amount (mass ratio) of L-oligomeric RNA relative to the total lipid amount was 0.1 (wt / wt)%. The resulting mixed solution was dialyzed overnight against phosphate buffer (PBS) at pH 7.4 using a dialysis tube (MWCO (molecular weight cutoff): 100 kD). The dialyzed LNP solution was sterilized by filtration using a 0.22 μm membrane filter, and L-(UAGGGU) ４ A solution containing RNA-containing LNPs was prepared.

[0152] (2) Measurement of the fluorescence emission spectrum of PpIX. KCl (final concentration 100 mM) and K-PO ４ The reaction buffer was prepared by adding L-(UAGGGU) (final concentration: 20 mM) to the reaction buffer. ４ RNA-containing LNP (converted to oligomeric RNA concentration) or L-type oligomeric RNA (L-(UAGGGU) ４RNA) was added to the mixture to adjust the total sample volume to 200 μL. A control sample without added RNA was also prepared. After annealing, PpIX dissolved in DMSO (final concentration 1 μM) was added to each sample, and the mixture was incubated at room temperature in a dark place for 2 hours. After incubation, the sample was placed in a fluorometer cell, and the fluorescence emission spectrum was measured using a spectrofluorometer.

[0153] The results of the fluorescence emission spectrum measurement are shown in Figure 31. L-(UAGGGU) ４ The sample containing RNA-containing LNP and the L-type oligomeric RNA (L-(UAGGGU) ４ In the sample to which L-(UAGGGU) was added, a prominent peak derived from PpIX was detected at a wavelength of around 630 nm compared to the control sample to which no RNA was added. ４ The sample containing RNA-containing LNP and the L-type oligomeric RNA (L-(UAGGGU) ４ When comparing the samples to which L-(UAGGGU) ４ A more pronounced peak was detected in the sample containing RNA-containing LNP. These results demonstrate that the fluorescence intensity derived from PpIX can be enhanced by encapsulating oligomeric RNA in LNPs and forming a complex between the two, compared to the case of oligomeric RNA alone.

[0154] (3) Detection of PpIX in breast cancer cells. Three 35 mm glass-bottom dishes were filled with 5 × 10 ４ The cells were seeded at a cell count of 100 / dish and cultured for 24 hours to allow the cells to adhere and proliferate (preculture). Next, 500 μM 5-ALA was added to two of the culture media in the three dishes (Tests 1 and 2), and nothing was added to the remaining culture medium (Test 3). After 21 hours of culture, the culture medium was removed using an aspirator. Next, 1 μM L-(UAGGGU) was added to the culture medium in one of the three dishes. ４ RNA-containing LNP (converted to oligomeric RNA concentration) was added (Test Group 1), and 1 μM L-(UAGGGU) ４RNA was added to one dish (Test 2), and nothing was added to the remaining dish (Test 3). After culturing for another 24 hours, fluorescent images of the cells in each dish were taken using an all-in-one fluorescent microscope set to a filter excitation wavelength of 395-415 nm and a detector fluorescence wavelength of 625-655 nm. Microscopic images of each cell are shown in Figure 32. In Figure 32, the upper row is a bright-field image, and the lower row is a fluorescent image.

[0155] As shown in Figure 32, in the cells of Test Group 3 to which only 5-ALA was added, almost no red fluorescence derived from PpIX was observed. ４ RNA-containing LNP or L-(UAGGGU) ４ In the cells of Test Group 1 and Test Group 2, which were added together with RNA, strong red fluorescence derived from PpIX was observed. When comparing the cells of Test Group 1 and Test Group 2, stronger red fluorescence derived from PpIX was observed in the cells of Test Group 1. From these results, it was confirmed that L-(UAGGGU) ４ RNA-containing LNP and L-(UAGGGU) ４ It was found that RNA enhances the red fluorescence derived from PpIX accumulated in cancer cells, and that the enhancement of PpIX-derived red fluorescence by oligomeric RNA is further enhanced by complexing the oligomeric RNA with LNP.

[0156] [Reference Example 1] Single-stranded RNA having a G4 structure ((UAGGGU) ４ ) was labeled with the fluorescent substance Cy5, and the effect on the fluorescence of PpIX was examined.

[0157] First, EEA-CH ２ Cy5-CH instead of amidite ２ Cy5-modified oligomeric RNA was synthesized and purified using the phosphoramidite method and simple purification by reversed-phase HPLC in the same manner as in Example 1, except that a Cy5-amidite was used.

[0158]

[0159] The obtained Cy5-modified oligomeric RNA was analyzed by MALDI-TOF MS, and the analysis results are shown in Figure 33.

[0160] The obtained Cy5-modified oligomeric RNA was annealed in the same manner as in Example 1, and then incubated with PpIX at room temperature in a dark place for 2 hours. After incubation, the sample was placed in a fluorometer cell and the fluorescence emission spectrum was measured using a spectrofluorometer.

[0161] The results of fluorescence emission spectrum measurement are shown in Figure 34. In the sample to which Cy5-modified oligomer RNA was added (in the figure, "Cy5-RNA"), a prominent peak derived from PpIX was detected at a wavelength of around 630 nm, compared to the control sample to which no RNA was added (in the figure, "Control"). In addition, a peak derived from Cy5 was also detected at a wavelength of around 670 nm. From these results, it can be seen that Cy5-modified oligomer RNA is capable of activating EEA-(UAGGGU) ４ As with RNA, it was found that the fluorescence intensity derived from PpIX was enhanced, and in addition, Cy5 also emitted light due to fluorescence resonance energy transfer (FRET).

[0162] [Reference Example 2] L-type single-stranded RNA ((UAGGGU) ４ ) was synthesized and labeled with biotin to examine its effect on the fluorescence of PpIX.

[0163] First, the use of L-type oligonucleotides instead of D-type oligonucleotides and the use of EEA-CH ２ Biotin-CH instead of amidite ２ Except for using a phosphoramidite, L-biotin-modified oligomeric RNA was synthesized and purified in the same manner as in Example 1 using the phosphoramidite method and simple purification by reversed-phase HPLC.

[0164]

[0165] The obtained L-biotin-modified oligomeric RNA was annealed in the same manner as in Example 1, and then incubated with PpIX at room temperature in a dark place for 2 hours. After incubation, the sample was placed in a fluorometer cell and its fluorescence emission spectrum was measured using a spectrofluorometer.

[0166] The results of fluorescence emission spectrum measurement are shown in Figure 35. A significant PpIX-derived peak was detected at a wavelength of around 630 nm in the sample containing L-biotin-modified oligomeric RNA (labeled "L-biotin RNA" in the figure), compared with the control sample containing no RNA (labeled "Control" in the figure). These results demonstrate that L-biotin-modified oligomeric RNA enhances the fluorescence intensity derived from PpIX.

[0167] The obtained L-biotin-modified oligomeric RNA was annealed and then incubated with PpIX at room temperature in the dark for 2 hours in the same manner as in Example 1, except that the incubation time after the addition of RNase A was set to 1 to 5 hours. RNase A was then added and further incubation was performed, and the fluorescence intensity of each sample was measured over time. The fluorescence intensity was quantified using imaging software (Intelligent Imaging Innovations).

[0168] The measurement results are shown in Figure 36. Test group 1 (labeled "1" in the figure) is a sample to which only L-biotin-modified oligomeric RNA and PpIX were added, without the addition of the RNA-cleaving enzyme RNase A. Test group 2 (labeled "2" in the figure) is a sample to which L-biotin-modified oligomeric RNA, PpIX, and RNase A were added, and test group 3 (labeled "3" in the figure) is a sample to which only PpIX was added, without the addition of L-biotin-modified oligomeric RNA and RNase A. Fluorescence was observed in both test group 1 and test group 2 samples from 1 hour to 5 hours after the start of incubation. In the test group 3 sample, fluorescence was almost undetectable after 1 hour of incubation. These results demonstrate that L-biotin-modified oligomeric RNA binds to PpIX and enhances the intensity of the red fluorescence derived from PpIX. Furthermore, it was revealed that L-type biotin-modified oligomeric RNA is not degraded by RNase A.

[0169] The present invention provides a fluorescence enhancer for PpIX, which comprises a fluorine-containing G4 compound obtained by modifying a nucleic acid having a G4 structure with a fluorine-containing group, and a method for visualizing cancer cells using the same. The fluorescence enhancer according to the present invention has excellent PpIX structural stabilization ability and cell membrane permeability, and is therefore particularly useful for visualizing cancer cells in an animal body using 5-ALA, and is expected to be used in the fields of medicine and biotechnology.

Claims

1. A fluorescence enhancer for protoporphyrin IX, which consists of a nucleic acid having a G4 structure and is bound or complexed with a substance that is cell membrane permeable.

2. The fluorescence enhancer according to claim 1, which comprises a fluorine-containing G4 compound in which a nucleic acid having a G-quadruplex structure and a fluorine-containing group are linked via a linking group, and the fluorine-containing group is a perfluoroalkyl group having 1 to 10 carbon atoms and no ether-bonding oxygen atoms between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonding oxygen atoms between carbon atoms.

3. The fluorine-containing group is represented by the following general formula (F2) or (F3): [In the formula, R Ｆ３ is a perfluoroalkylene group having q3 carbon atoms; R Ｆ４ is a perfluoroalkyl group having q4 carbon atoms; n1, q3, and q4 are natural numbers such that n1+q3+q4 is 3 or more and 10 or less; R Ｆ５ is a perfluoroalkylene group having q5 carbon atoms, R Ｆ６ n2, q5 and q6 are natural numbers such that 2×n2+q5+q6 is 4 or more and 10 or less.

4. The fluorescence enhancer according to claim 2, wherein the linking group is an alkylene group, an alkenylene group, -C(=O)-, -NH-, -O-, -S-, -C(=O)-O-, -O-C(=O)-, -C(=O)NH-, -NH-C(=O)-, -NH-C(=O)-O-, -O-C(=O)-NH-, -O-P(=O)(OH)-O-, a polyethylene glycol group, or a group consisting of a combination thereof.

5. The fluorine-containing G4 compound is represented by the following general formula (1): [In formula (1), G ０ is a nucleic acid that has a G-quadruplex structure and may be modified with a substance other than nucleic acid; R ＦＥ is a perfluoroalkyl group having 1 to 10 carbon atoms that does not have an ether-bonding oxygen atom between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms that has 1 to 5 ether-bonding oxygen atoms between carbon atoms; Z １ The fluorescence enhancer according to claim 2 , which is a compound represented by the following formula:

6. The fluorine-containing G4 compound is represented by the following general formula (2): [In formula (2), G ０ is a nucleic acid that has a G-quadruplex structure and may be modified with a substance other than nucleic acid; R ＦＥ is a perfluoroalkyl group having 1 to 10 carbon atoms that does not have an ether-bonding oxygen atom between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms that has 1 to 5 ether-bonding oxygen atoms between carbon atoms; Z ２ The fluorescence enhancer according to claim 2 , which is a compound represented by the following formula:

7. The fluorescence enhancer according to claim 1, wherein the nucleic acid having the G4 structure is a nucleic acid complexed with a lipid nanoparticle.

8. The nucleic acid having the G4 structure is represented by the following general formula (G1): [In formula (G1), G ｓ is a base sequence having s consecutive Gs (guanines); s represents an integer between 2 and 5; N represents A (adenine), C (cytosine), G (guanine), T (thymine), or U (uracil); N ｒ１ is a base sequence in which r1 consecutive N's are present; N ｒ２ is a base sequence having r2 consecutive N's; r1 and r2 each represent an integer of 0 to 7 satisfying 1≦r1+r2≦7; when 2≦r1+r2≦7, the multiple N's may be the same or different bases.

9. A method for producing a fluorine-containing G4 compound, comprising binding an amidite derivative compound containing a fluorine-containing group to a nucleic acid that forms a G-quadruplex structure by a phosphoramidite method to synthesize a fluorine-containing G4 compound in which the nucleic acid that forms a G-quadruplex structure and the fluorine-containing group are linked via a linking group.

10. A method for producing a fluorine-containing G4 compound according to claim 9, wherein the fluorine-containing group is a perfluoroalkyl group having 1 to 10 carbon atoms and no ether-bonding oxygen atoms between carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms and having 1 to 5 ether-bonding oxygen atoms between carbon atoms.

11. A pharmaceutical composition comprising the fluorescence enhancer according to any one of claims 1 to 8 as an active ingredient.

12. A kit for visualizing cancer cells, comprising the fluorescence enhancer according to any one of claims 1 to 8 and 5-aminolevulinic acid.

13. A method for visualizing cancer cells, comprising introducing the fluorescence enhancer according to any one of claims 1 to 8 and 5-aminolevulinic acid into cancer cells outside of a vitro system.

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