JP6177044B2 - Novel sugar-linked photosensitizers for photodynamic therapy - Google Patents

Description

  The present invention relates to a chlorin derivative having excellent antitumor activity and a metal complex thereof, and a pharmaceutical composition containing the chlorin derivative and a metal complex thereof.

  Photodynamic therapy (PDT) is a method in which a photosensitizer is administered into a blood vessel or tissue of a target disease patient, accumulated in cancer or tumor tissue, and then irradiated with light of a specific wavelength. This is a treatment method that specifically causes cancer cells to die. PDT is a minimally invasive cancer treatment method that can selectively and specifically kill only tumor cells by photochemical reaction.

  In PDT, a photosensitizer is excited by light of a specific wavelength, and singlet oxygen is generated from triplet oxygen by direct or indirect energy transfer from the activated photosensitizer, and this singlet oxygen produces a tumor. It is thought that cells are destroyed.

  Porphyrin derivatives are well known as photosensitive substances used in PDT, and polyhematoporphyrin ether and talaporphyrin sodium have already been put into practical use. However, since hematoporphyrin and its dimer have a maximum absorption wavelength of 630 nm and a molar extinction coefficient as low as 3000, the therapeutic effect of PDT is limited to surface cancer. There is also the problem of side effects. Therefore, various porphyrin derivatives have been studied for the purpose of improving these points.

  Since PDT generally damages not only cancer and tumor tissues but also normal tissues, it is required to use as little photosensitizer as possible. In order to minimize damage to normal tissues around cancer and tumor tissues, it is preferable that the photosensitizer is selectively and specifically accumulated in cancer and tumor tissues compared to normal tissues. Furthermore, in order to reduce side effects, it is required that the cytotoxicity is low under conditions where no light is irradiated. Recently, it was discovered that by attaching a sugar to the porphyrin ring, it is possible to reduce cytotoxicity under dark conditions while maintaining strong cytotoxicity under light irradiation. Using sugar-linked porphyrin derivatives as drugs for PDT It has been proposed.

  In Patent Document 1 and Non-Patent Document 1, S-glucosylated or S-galactosylated 5,10,15,20-tetrakis (tetrafluorophenyl) chlorin is prepared, and these have phototoxicity. It has been reported that S-glucosylated compounds showed stronger phototoxicity than S-galactosylated compounds.

Further, in Non-Patent Document 2, 5,10,15,20-tetrakis [4- (β-D-glucopyranosylthio) -2,3,5,6-tetrafluorophenyl described in the above document is used. ] -2,3- [Methano (N-methyl) iminomethano] chlorine (hereinafter abbreviated as H ₂ TFPC-SGlc) compared with PDT in clinical use in Japan for Talaporfin As a result, H ₂ TFPC-SGlc is about 30 times more cytotoxic to cancer cells in vitro, and it has been reported that tumor growth was significantly suppressed even in vivo compared to talaporfin.

  Patent Document 2 describes a porphyrin derivative in which a monosaccharide selected from galactose, mannose, xylose, and glucose is bound by S-glycosylation, and the porphyrin derivative has a photocytotoxic effect, It is described for use in mechanical therapy. Patent Document 2 also describes a chlorin derivative. However, only glucose is bonded, and a phenyl group substituted with fluorine is not described.

International Publication No. 2008/102669 Special Table 2005-500335

S. Hirohara et al., Journal of Photochemistry and Photobiology B: Biology, 97, 22-33 (2009) M. Tanaka et al., Anticancer Research, 31, 763-770 (2011)

However, there is a demand for a photosensitive substance having a higher tumoricidal cell effect than H ₂ TFPC-SGlc reported in Patent Document 1 and Non-Patent Documents 1 and 2.

  Therefore, an object of the present invention is to provide a chlorin derivative having excellent antitumor activity and a metal complex thereof.

  Macrophages that infiltrate the stroma of cancerous tissue are called tumor-associated macrophages (TAM). TAM is associated with (1) cancer cell proliferation, promotion of invasion, (2) promotion of tumor angiogenesis, (3) tumor immunosuppression, and (4) promotion of metastasis. Therefore, it is considered that TAM can be targeted to suppress tumors.

Since TAM expresses the mannose receptor, the present inventors used the compound 5,10,15,20-tetrakis [4 in which the glucose of H ₂ TFPC-SGlc was replaced with mannose in order to target this TAM. -(α-D-Mannopyranosylthio) -2,3,5,6-tetrafluorophenyl] -2,3- [methano (N-methyl) iminomethano] chlorine (hereinafter abbreviated as H ₂ TFPC-SMan) In some cases, various tests were conducted. Then, H ₂ TFPC-SMan are, surprisingly, even for normal cancer cells other than TAM represent similar cell killing effect as H ₂ TFPC-SGlc, thereon, in vivo than H ₂ TFPC-SGlc The knowledge that it shows a significantly high tumor reduction effect was acquired.

  The present invention has been completed based on these findings, and provides the following chlorin derivatives or metal complexes thereof, pharmaceutical compositions and the like.

(I) Chlorine derivative or its metal complex
(I-1) General formula (I):

[Wherein, R ^{1 to} R ²⁰ are the same or different and represent F-, mannose-S-, mannose-DS- or mannose-ODS-, and at least one of these is mannose-S-, mannose -DS- or mannose-ODS- is shown. Here, D represents a hydrocarbon group.
R ²¹ and R ²² are the same or different and each represents a hydrogen atom, alkyl, or E— (CH ₂ ) _n —. Alternatively, R ²¹ and R ²² may be bonded to each other to form a ring. Here, E represents aryl, amino, alkylamino, imino or cycloalkyl, and n represents an integer of 0 to 3. Or a metal complex thereof.
(I-2) The chlorin derivative or the metal complex thereof according to (I-1), wherein R ^{5 to} R ²⁰ all represent F-.
(I-3) The chlorin derivative according to (I-1) or (I-2), wherein R ^{1 to} R ⁴ are the same or different and each represents mannose-S-, mannose-DS-, or mannose-ODS- Or a metal complex thereof.
(I-4) The metal complex is represented by the general formula (II):

[Wherein M represents a metal atom. R ^{1 to} R ²² are the same as described above. ] The chlorin derivative or its metal complex as described in (I-1).
(I-5) The chlorin derivative or metal complex thereof according to (I-4), wherein M represents a transition metal atom.
(I-6) The chlorin derivative or a metal complex thereof according to (I-4) or (I-5), wherein M represents Pt, Pd, Zn, or Au.
(I-7) The chlorin derivative or metal complex according to any one of (I-1) to (I-6), wherein the mannose is D (+)-mannose.

(II) Pharmaceutical composition
(II-1) A pharmaceutical composition comprising the chlorin derivative or a metal complex thereof according to any one of (I-1) to (I-7).
(II-2) The pharmaceutical composition according to (II-1), which is used for photodynamic treatment of a tumor or solid cancer.
(II-3) The pharmaceutical composition according to (II-1), which is used for photodynamic treatment of skin diseases.

  The chlorin derivative bound to mannose of the present invention has an antitumor activity superior to that of the previously reported chlorin derivative bound to glucose. In addition, since the chlorin derivative of the present invention has an optimal wavelength of red visible light 650-660 nm, which is a longer wavelength region than porphyrin compounds, it is expected to be applied to photodynamic treatment of cancer cells in deeper areas. The

₂ is a diagram showing an NMR spectrum of H ₂ TFPC-SMan in Synthesis Example 1. FIG. (a) ¹ H NMR spectra in CD ₃ OD (CD ₃ OD = 3.34 ppm), (b) ¹⁹ F NMR spectra in CD ₃ OD (CF ₃ COOH = -76.05 ppm) 6 is a graph showing caspase 3/7 activity (% control) after PDT in Test Example 2. * p <0.05 10 is a graph showing a tumor volume ratio after PDT in Test Example 3. * p <0.05 In Test Example 4 is a graph showing a ratio of from above F4 / 80 staining of cell number / mm ^2, CD206 staining of cell number ^{/ mm 2, CD206 / F4 /} 80. *** p <0.001 for control and chlorin, ### P <0.001 for control, chlorin and GC It is a photograph which shows uptake | capture of the mannose connection chlorin into a cancer cell by observation with a confocal laser microscope.

  Hereinafter, the present invention will be described in detail.

Chlorine derivative or metal complex thereof The chlorin derivative of the present invention is represented by the following general formula (I).

[Wherein, R ^{1 to} R ²⁰ are the same or different and represent F-, mannose-S-, mannose-DS- or mannose-ODS-, and at least one of these is mannose-S-, mannose -DS- or mannose-ODS- is shown. Here, D represents a hydrocarbon group.
R ²¹ and R ²² are the same or different and each represents a hydrogen atom, alkyl, or E— (CH ₂ ) _n —. Alternatively, R ²¹ and R ²² may be bonded to each other to form a ring. Here, E represents aryl, amino, alkylamino, imino or cycloalkyl, and n represents an integer of 0 to 3. ]

  The hydrocarbon group may be either linear or branched, and is preferably a hydrocarbon group having 1 to 6 carbon atoms. Examples of the hydrocarbon group include a methylene group, ethylene group, propylene group, α-butylene group, β-butylene group, cyclohexylene, o-phenylene group, m-phenylene group, p-phenylene group, and the like.

  In the chlorin derivative of the present invention, mannose may be bonded to any of the o-position, m-position and p-position of the phenyl group, and two or more mannoses may be bonded to each phenyl group.

R ^{5 to} R ²⁰ are preferably all F-. R ^{1 to} R ⁴ are preferably mannose-S-, mannose-DS- or mannose-ODS-, more preferably mannose-S-.

In the chlorin derivative of the present invention, preferably, R ^{5 to} R ²⁰ all represent F- and at least one of R ^{1 to} R ⁴ represents mannose-S-, mannose-DS- or mannose-ODS-. In this case, R ^{1 to} R ⁴ are more preferably all mannose-S-, mannose-DS- or mannose-ODS-, and more preferably all mannose-S-.

  The mannose may be either D (+)-mannose or L (−)-mannose, but is preferably D (+)-mannose. Mannose may be either α-type or β-type.

  Alkyl may be linear or branched, and is preferably alkyl having 1 to 6 carbon atoms. Examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl and the like. Alkyl includes the alkyl portion of alkylamino.

  Alkylamino may be mono-substituted amino or di-substituted amino by alkyl.

  Aryl means a monocyclic or polycyclic group consisting of a 5- or 6-membered aromatic hydrocarbon ring, and specific examples include phenyl and naphthyl.

  Cycloalkyl is preferably cycloalkyl having 3 to 7 carbon atoms. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.

R ²¹ and R ²² may combine with each other to form a ring, and examples of the ring formed include a 3- to 7-membered saturated or unsaturated hydrocarbon ring or a heterocycle. Examples of the group in which R ²¹ and R ²² are bonded include —CH ₂ —N (R) —CH ₂ — (wherein R is a hydrogen atom, methyl, ethyl or the like).

  Examples of the chlorin derivative of the present invention include 5,10,15,20-tetrakis [4- (α-D-mannopyranosylthio) -2,3,5,6-tetrafluorophenyl] chlorin, 10,15,20-tetrakis [4- (α-D-mannopyranosylthio) -2,3,5,6-tetrafluorophenyl] -2,3- [methano (N-methyl) iminomethano] chlorine, etc. Is mentioned.

  The metal complex of the chlorin derivative of the present invention is preferably a metal complex represented by the following general formula (II).

[Wherein M represents a metal atom. R ^{1 to} R ²² are the same as described above. ]

  Examples of M include transition metals, Group II, Group III, and Group IV metals, preferably transition metals, and more preferably Pt, Pd, Zn, and Au.

  The chlorin derivative and its metal complex of the present invention can be chemically synthesized by referring to a known method (for example, International Publication No. 2008/102669).

  The chlorin derivative of the present invention has an antitumor activity superior to the previously reported chlorin derivative bound with glucose. In addition, the chlorin derivative of the present invention or a metal complex thereof can be expected to be excellent in cancer cell accumulation and selectivity and to suppress TAM present in cancer tissue stroma.

Pharmaceutical Composition The pharmaceutical composition of the present invention is characterized by containing the above chlorin derivative or a metal complex thereof.

  Since the chlorin derivative of the present invention has an antitumor activity superior to the previously reported chlorin derivative bound to glucose, the pharmaceutical composition of the present invention is suitable for photodynamic treatment of tumors or solid cancers. Useful.

  The types of cancer applicable to the photodynamic therapy in the present invention are cancers classified as malignant tumors. Specific cancer types include esophageal cancer, lung cancer, stomach cancer, cervical cancer, uterine cancer, uterine cancer, skin cancer, prostate cancer, colon cancer, brain tumor, bladder cancer, liver cancer, pancreatic cancer, bile duct cancer, Examples include kidney cancer, head and neck cancer, breast cancer, and ovarian cancer. Skin cancer includes skin metastasis of visceral cancer in addition to primary (squamous cell carcinoma, basal cell carcinoma, epidermal appendage cancer).

  Further, since the chlorin derivative of the present invention and its metal complex have affinity for benign tumors in addition to malignant tumors, the pharmaceutical composition of the present invention is suitable for skin psoriasis, sun keratosis, etc. It is also useful for photodynamic treatment of disease.

  Furthermore, by utilizing the tumor accumulation property of the chlorin derivative of the present invention, it can be used for diagnosis of cancer by combined use with PET or the like.

  The chlorin derivative of the present invention has an optimal wavelength of red visible light 650-660 nm, which is a longer wavelength region than porphyrin compounds, and thus is suitable not only for surface cancer but also for tumors or solid cancers in regions deeper than the epidermis. Application can be expected.

  The pharmaceutical composition of the present invention can be administered by catheter, intravenous or intramuscular injection, and can be administered by parenteral route. In addition, it can be administered through an endoscopic spray tube or percutaneously, and can be directly injected into a tumor tissue deep in the body.

  The pharmaceutical composition of the present invention is administered to mammals including humans. In addition, the pharmaceutical composition of the present invention can optionally contain a biologically acceptable carrier, excipient, etc. depending on the form of use. In addition, the pharmaceutical composition of the present invention can be produced according to conventional means.

  When the pharmaceutical composition of the present invention is prepared as an injectable preparation, it can be formulated into a sterile aqueous solution or dispersion containing a chlorin derivative or a metal complex thereof, or a sterile lyophilizing agent. Examples of the liquid carrier include water, physiological saline, ethanol, hydrous ethanol, glycerol, propylene glycol, vegetable oil and the like.

  In the pharmaceutical composition of the present invention, diluents such as sucrose, lactose, dicalcium phosphate, carboxymethylcellulose; lubricants such as magnesium stearate, calcium stearate, talc; starch, molasses, polyvinylpyrrolidone, glucose, cellulose, derivatives thereof, etc. Of binders.

  The dosage of the pharmaceutical composition of the present invention can be appropriately determined finally based on the judgment of a doctor in consideration of the type of dosage form, administration method, patient age and weight, patient symptom, and the like.

  The photodynamic therapy in the present invention is performed by irradiating a site to be treated with a light beam containing an absorption band of a chlorin derivative or a metal complex thereof after administering the pharmaceutical composition of the present invention. Specifically, the tumoricidal cell effect can be exerted by generating singlet oxygen by irradiation with light of 500 nm or more, but it is preferable to irradiate with light having a high proportion of light having the maximum absorption wavelength.

The amount of light irradiation in the photodynamic treatment in the present invention is not particularly limited as long as a therapeutic effect is obtained, and is, for example, 1 to 100 J / cm ² .

  Examples of the light source include a laser, a halogen lamp, and an LED. The laser is not particularly limited as long as a light beam having a wavelength necessary for excitation can be obtained, and examples thereof include a dye laser, an argon laser, and a semiconductor laser.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited by an Example.

Synthesis Example 1 Mannose-linked chlorin In the (3) S-glucosylation step of Example 1 of Patent Document 1, instead of 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosylthioacetyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosylacetylthioacetyl synthesized based on J. Defaye et al., Carbohydrate Research, 130, 317-321 (1984) 5,10,15,20-tetrakis [4- (α-D-mannopyranosylthio) -2,3, where the sugar is mannose, in the same manner as in Example 1 of Patent Document 1. , 5,6-tetrafluorophenyl] -2,3- [methano (N-methyl) iminomethano] chlorin (H ₂ TFPC-SMan (M-chlorin)) was obtained (yield 49.9%). MALDI-TOF-MASS (Bruker): m / z for C ₇₁ H ₆₁ F ₁₆ N ₅ O ₂₀ S ₄ ([M] ⁺ ) Calcd 1735.25, Found 1735.23. Anal (Perkin-Elmer 240C). Found (Calcd for C ₇₁ H ₆₁ F ₁₆ N ₅ O ₂₀ S ₄ · 5H ₂ O): C, 46.90 (46.69); H, 3.66 (3.92); N, 3.91. NMR spectrum (JEOL JNM-EX400): Figure 1 a) ¹ H-NMR, (b) ¹⁹ F-NMR

Further, 5,10,15,20-tetrakis (pentafluorophenyl-2,3- [methano (N-methyl) iminomethano] chlorine (hereinafter abbreviated as H ₂ TFPC (chlorin)), which is a comparative reference substance, Based on the process described in (2) Production of H ₂ TFPC (conversion from porphyrin derivative to chlorin derivative) in Example 1 of Patent Document 1, H ₂ TFPC-SGlc (G-chlorin) Synthesis was performed based on the steps described in Example 3 (3) Production of H ₂ TFPC-SAcGlc (S-glucosylation) and (4) Production of H ₂ TFPC-SGlc (Removal of acetyl protecting group).

Test example 1
Cell lines (gastric cancer (MKN28, MKN45), colon cancer (HT29, HCT116, CT26)) were seeded at 5 × 10 ³ cells / well in a 96-well plate. As the medium, RPMI1640 + 10% FBS, McCOY's 5A + 10% FBS, and l-DMEM + 10% FBS were used. It was then incubated at 37 ℃ 5% CO ₂ 24 hours.

A drug solution (H ₂ TFPC-SMan, H ₂ TFPC-SGlc, H ₂ TFPC) adjusted to seven concentrations and a medium were mixed and administered to the plate. Then, incubated for 4 hours at 37 ℃ 5% CO _2.

The medium was removed, washed once with PBS, and PBS was added again. A 660 nm red LED was irradiated at 22 J / cm ² . Thereafter, PBS was removed, the medium was returned to the culture, and incubated at 37 ° C. with 5% CO ₂ for 24 hours. Using the WST-8 assay kit (Dojindo, Kumamoto, Japan), 50% cell growth inhibitory concentration was calculated and the effect of PDT was determined.

  The results are shown in Table 1.

  From the results in Table 1, it can be seen that M-chlorin exhibits a cell killing effect equivalent to that of G-chlorin and a very strong cell killing effect compared to chlorin.

Test example 2
CT26 cells were seeded in a 96-well plate at 5 × 10 ³ cells / well, cultured in 1-DMEM + 10% FBS medium, and chlorin, G-chlorin or M-chlorin was administered to each well in an amount of 0.1 μM. 4 hours later, 660 nm red light LED was irradiated at 22 J / cm ² and immediately after irradiation (0 hours), 1, 2, 4, and 8 hours later, Caspace 3/7 was added to the Caspase-Glo 3/7 Assay kit (Promega ).

  The results are shown in FIG. FIG. 2 shows that M-chlorin and G-chlorin strongly induced apoptosis by PDT compared to chlorin.

Test example 3
1 × 10 ⁶ mouse colon cancer cell line CT26 was subcutaneously injected into BALB / c mice (7 mice for each group, 4 weeks old female). One week later, when a subcutaneous tumor having a diameter of about 5 mm was formed, BALB / c mice were intraperitoneally administered chlorin, G-chlorin or M-chlorin (6.25 μmol / kg each). 24 hours after administration, 22 J / cm ² of 660 nm red LED was irradiated. Seven days later, chlorin, G-chlorin or M-chlorin (each 6.25 μmol / kg) was intraperitoneally administered again. 24 hours after administration, 22 J / cm ² of 660 nm red LED was irradiated. Thereafter, the tumor size was measured over time, and the tumor volume was calculated by major axis × minor axis × minor axis.

  The results are shown in FIG. FIG. 3 shows that in the allograft model, M-chlorin has a significant tumor reduction effect compared to G-chlorin, chlorin and control.

Test example 4
A subcutaneous tumor of mouse colon cancer cell line CT26 was prepared in BALB / c mice, and PDT was performed in the same manner as in Test Example 3 above. Three days after the operation, the subcutaneous tumor was removed, fixed with formalin, anti-F4 / 80 monoclonal antibody (ab6640, Abcam) as a macrophage marker, and anti-CD206 monoclonal antibody (MCA2235T, Bio-Rad) as a TAM marker. ), And the number of macrophages and TMA present in the stroma of each tumor was counted and quantified.

  The results are shown in FIG. From FIG. 4, F4 / 80 and CD206 showed a significant decrease in the number of cells in G-chlorin and M-chlorin compared to control and chlorin, but there was a significant difference between G-chlorin and M-chlorin. However, when compared with the ratio of CD206 / F4 / 80, M-chlorin is clearly different from the others, indicating that it specifically acts on M2 macrophages (TAM).

Test Example 5
CT26 cells were cultured on a preparation, 2 μM M-chlorin was administered into the culture, and washed after 4 or 24 hours. After washing, place a cover glass, embed the surroundings, excite with a confocal laser microscope (Nikon confocal laser microscope C1) with 543 nm laser light irradiation, and in the cells in the range of 605-675 nm The light emitted from chlorin was observed.

  The results are shown in FIG. From FIG. 5, uptake of M-chlorin into CT26 was confirmed. It was revealed that M-chlorin does not adhere to the surface of cancer cells, but passes through the cell membrane of cancer cells and is taken into the cytoplasm and exerts a cytocidal effect by PDT upon irradiation with light.

Claims (10)

Formula (I):

[Wherein, R ^{1 to} R ²⁰ are the same or different and represent F-, mannose-S-, mannose-DS- or mannose-ODS-, and at least one of these is mannose-S-, mannose -DS- or mannose-ODS- is shown. Here, D represents a hydrocarbon group.
R ²¹ and R ²² are the same or different and each represents a hydrogen atom, alkyl, or E— (CH ₂ ) _n —. Alternatively, R ²¹ and R ²² may be bonded to each other to form a ring. Here, E represents aryl, amino, alkylamino, imino or cycloalkyl, and n represents an integer of 0 to 3. Or a metal complex thereof. The chlorin derivative or metal complex thereof according to claim 1, wherein R ^{5 to} R ²⁰ all represent F-. The chlorin derivative or metal complex thereof according to claim 1 or 2, wherein R ^{1 to} R ⁴ are the same or different and each represents mannose-S-, mannose-DS-, or mannose-ODS-. The metal complex has the general formula (II):

[Wherein M represents a metal atom. R ^{1 to} R ²² are the same as described above. The chlorin derivative of Claim 1 represented by these, or its metal complex.   The chlorin derivative or metal complex thereof according to claim 4, wherein M represents a transition metal atom.   The chlorin derivative or metal complex thereof according to claim 4 or 5, wherein M represents Pt, Pd, Zn or Au.   The chlorin derivative or metal complex according to any one of claims 1 to 6, wherein the mannose is D (+)-mannose.   The pharmaceutical composition containing the chlorin derivative or its metal complex as described in any one of Claims 1-7.   The pharmaceutical composition according to claim 8, which is used for photodynamic treatment of a tumor or a solid cancer.   The pharmaceutical composition according to claim 8, which is used for photodynamic treatment of skin diseases.

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