JP2014001204A - Tumor cell-selective anticancer agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anticancer agent containing a compound having novel anticancer action excellent in tumor cell selectivity as an active ingredient, and to provide a drug delivery agent using the compound having novel anticancer action excellent in tumor cell selectivity.SOLUTION: A pharmaceutical composition contains folate-conjugated methyl cyclodextrin as an active ingredient for anticancer action. A pharmaceutical composition contains folate-conjugated methyl cyclodextrin as a drug delivery agent.

Description

  The present invention relates to a new anticancer agent. More specifically, the present invention relates to a novel tumor cell selective anticancer agent.

  Cancer is the leading cause of death in Japan. Although the survival rate has improved for some cancers, there is still insufficient treatment for advanced cancer, and the development of more effective treatments is desired. In addition to anticancer drugs that have a high cell killing effect on cancer cells, it is expected to build a drug delivery system (DDS) that can deliver anticancer drugs to target sites selectively and efficiently. ing. DDS aims to optimize drug treatment by controlling drug pharmacokinetics, and is classified into drug release behavior control, drug absorption promotion, drug targeting to target tissues, and the like. As methods for imparting targeting ability to carriers, ligand modifications such as antibodies, sugar chains, folic acid, and transferrin are known.

  Among them, folic acid is 1) inexpensive, 2) folate receptor (Folate receptor: FR) is overexpressed in various epithelial cancer cells and low in normal cells. It is widely used as a ligand molecule because it is selectively incorporated, 3) it can be administered repeatedly because it has no antigenicity, and 4) it has a relatively small molecular weight that does not affect the intracellular dynamics of the carrier. Yes.

  Cyclodextrins (CyD) are cyclic oligosaccharides that are classified as unimolecular host molecules that incorporate various drugs into their hydrophobic cavities to form inclusion complexes. The supramolecular inclusion properties of CyDs are used in various fields such as food, cosmetics, clinical diagnostics, membrane science, and polymer chemistry. In the pharmacology / pharmaceutics field, the functionality and biocompatibility of CyDs Is used to stabilize pharmaceuticals by complex formation, adjust solubility, improve bioavailability, etc., and is used in pharmaceutical preparations at home and abroad.

  In recent years, various CyDs derivatives with improved functionality and biocompatibility have been developed, and basic research on the application of DDS has been conducted. Some CyDs derivatives include, for example, Methyl-β-CyD (M-β-CyD) and 2,6-Di-O-methyl-α-CyD (DM-α-CyD), Grosse et al. In nude mice transplanted with breast cancer cell MCF7 or human ovarian cancer cell A2780, M-β-CyD was administered intraperitoneally for 2 months (300-800 mg / kg), an anticancer drug, doxorubicin (DOX ) Similar to administration (2 mg / kg), it has been reported to show an effect of suppressing tumor growth compared to control (Non-patent Document 1). Hattori et al. Also prepared folic acid-modified β-CyD, in which folic acid is bound to the primary hydroxyl group of natural β-CyD using two caproic acid molecules as spacers, and is useful as a FR-mediated cancer cell selective DDS carrier. (Non-Patent Document 2).

  Further, the present inventors have made folic acid-modified methylated-β-cyclodextrin (FA-M-β-CyD) obtained by modifying folic acid on methyl-β-cyclodextrin against KB cells (FR highly expressing cells). Concentration-dependent cytotoxicity was observed, but there was little cytotoxicity against A549 cells (FR low-expressing cells), and FA-M-β-CyD cytotoxicity caused by combined use with the anticancer drug DOX It has been reported that sex is significantly enhanced over DOX alone (Non-patent Document 3). Furthermore, the present inventors have reported that when FA-M-β-CyD was directly administered into a tumor of a tumor-bearing mouse model, the growth of the tumor was significantly suppressed (Non-patent Document 4). .

  However, FA-M-β-CyD is required to have a high concentration in order to show cytotoxicity against KB cells (FR highly expressing cells), and the same concentration as anticancer drugs such as DOX. Then, it did not show cytotoxicity against KB cells. Specifically, in order for FA-M-β-CyD to be cytotoxic to KB cells, a high concentration of about 3 orders compared to DOX is required, and it is used as it is as an anticancer agent. There was a problem with that.

Grosse et al., Br. J. Cancer. 78 (1998), 1165-1169. Kenjiro Hattori, Pharmacology, 68 (2008), 398-406. The 131st Annual Meeting of the Pharmaceutical Society of Japan (Shizuoka) March 28-30, 2011, Abstract (29P-0433 “Preparation and physical properties of novel folic acid-modified methylated cyclodextrins as carriers for anticancer drug delivery”) . The 27th Annual Meeting of the Japanese Society for DDS (Tokyo) June 9-10, 2011, abstract (2-B-05 “Evaluation of usefulness of folic acid-modified methylated cyclodextrin as a novel anticancer agent”). K. Motoyama et al., Eur. J. Pharm. Sci., 29 (2006), 111-119. K. Motoyama et al., Biol. Pharm. Bull., 32 (2009), 700-705.

  An object of this invention is to provide the anticancer agent which contains the compound which has the novel anticancer activity excellent in tumor cell selectivity as an active ingredient. Another object of the present invention is to provide a drug delivery agent using a novel compound having an anticancer activity which is selective for tumor cells.

As a result of intensive studies, the inventors have found that folic acid-modified methylated cyclodextrin has an antitumor action, a tumor growth-inhibiting action, or an anticancer action at a concentration that is practically acceptable, that is, as a drug. As a result, the present invention was completed.
That is, based on the following knowledge, the present inventors modified methylation cyclodextrin (methylated CyD) with folic acid, in addition to cancer cell selectivity via folate receptor (FR), methylation. We thought that it was possible to construct new anticancer drugs and DDS carriers with cytotoxic activity via lipid rafts by CyD itself. On the cell membrane, lipid microdomains called lipid rafts, where cholesterol and sphingolipids are localized, are known, and various proteins involved in signal transduction such as receptors are localized. It functions as a scaffold. In recent years, it has been considered that lipid rafts are involved in apoptotic signals because FasL / Fas system apoptosis is mediated by lipid rafts and the apoptosis inducing factor Bad belonging to the Bcl-2 family is localized in rafts. On the other hand, CyD and CyD derivatives solubilize phospholipids and cholesterols, which are the main constituents, from biological membranes such as erythrocyte membranes based on thermodynamic equilibrium under high concentration conditions, depending on the cavity size. , Hemolysis, morphological changes of erythrocytes, and cytotoxicity can be expected. In fact, the present inventors have confirmed that 2,6-Di-O-methyl-α-CyD (DM-α -CyD) acts on the SLR to cause inward-type morphological changes, while Methyl-β-CyD (M-β-CyD) and 2,6-Di-O-methyl-β-CyD ( It was confirmed that DM-β-CyD) acts on the CLR to induce outward projection type morphological changes (Non-Patent Documents 5 and 6).

  Furthermore, the present inventors believe that the in vivo effect is not limited by the insufficient cytotoxic activity of folate-modified methylated cyclodextrin in vitro, but by focusing on the various characteristics of folate-modified methylated cyclodextrin. The present invention was completed by confirming the activity in vivo.

The present invention includes the following.
(1) A pharmaceutical composition which is an anticancer agent containing folic acid-modified methylated cyclodextrin as an active ingredient.
(2) The pharmaceutical composition according to the above (1), which is a pharmaceutical composition for systemic administration.
(3) The pharmaceutical composition according to the above (2), which is an intravenous injection.
(4) The pharmaceutical composition according to any one of (1) to (3), wherein the pharmaceutical composition is used so as to be administered at an interval of at least one week.
(5) The pharmaceutical composition according to any one of (1) to (4) above, wherein the pharmaceutical composition is used in repeated administration of 5 times or less.
(6) The pharmaceutical composition as described in any one of (1) to (5) above, which is used so as to be administered again after confirming an anticancer effect after administration.
(7) The pharmaceutical composition as described in any one of (1) to (3) above, wherein the pharmaceutical composition is used as a single administration.
(8) Any one of the above (1) to (7), wherein 10 to 100 mg / kg body weight of folic acid-modified methylated cyclodextrin is used for human administration A pharmaceutical composition according to 1.
(9) The total dose of repeated administration is used so that folic acid-modified methylated cyclodextrin of 500 mg / kg body weight or less is administered to humans, (1) to (6) above The pharmaceutical composition according to any one of the above.
(10) The pharmaceutical composition according to any one of (1) to (9), wherein the folic acid-modified methylated cyclodextrin is folic acid-modified methyl-β-cyclodextrin.

(11) A pharmaceutical composition which is an anticancer agent, comprising folic acid-modified methylated cyclodextrin as a drug delivery agent.
(12) The pharmaceutical composition according to (11), further comprising a drug having an anticancer effect.
(13) The pharmaceutical composition according to (12), wherein the drug having anticancer activity is doxorubicin.
(14) The pharmaceutical composition according to any one of (11) to (13), which is a pharmaceutical composition for systemic administration.
(15) The pharmaceutical composition according to the above (14), which is an intravenous injection.
(16) The pharmaceutical composition according to any one of (11) to (15), wherein the pharmaceutical composition is used so as to be administered at an interval of at least one week.
(17) The pharmaceutical composition according to any one of (11) to (16) above, wherein the pharmaceutical composition is used in repeated administration of 5 times or less.
(18) The pharmaceutical composition as described in any one of (11) to (17) above, which is used so as to be administered again after confirming an anticancer effect after the administration.
(19) The pharmaceutical composition as described in any one of (11) to (15) above, which is used so as to be administered once.
(20) The pharmaceutical composition according to any one of (11) to (19), wherein the folic acid-modified methylated cyclodextrin is folic acid-modified methyl-β-cyclodextrin.

(21) A method for treating cancer by systemically administering folic acid-modified methylated cyclodextrin.
(22) The method according to (21) above, wherein the systemic administration is intravenous administration.
(23) A method for treating cancer by systemically administering folic acid-modified methylated cyclodextrin at intervals of at least one week or more.
(24) A method for treating cancer by administering folic acid-modified methylated cyclodextrin in five or less repeated doses.
(25) The following steps:
(A) Single systemic administration of folic acid modified methylated cyclodextrin:
(B) a step of confirming the anticancer effect: and (c) a step of systemically administering the folic acid-modified methylated cyclodextrin again only when the anticancer effect is insufficient,
A method for treating cancer by systemically administering a folate-modified methylated cyclodextrin.
(26) A method for treating cancer by a single systemic administration of folic acid-modified methylated cyclodextrin.
(27) A method for treating cancer by administering to a human folic acid-modified methylated cyclodextrin of 10 to 100 mg / kg body weight per time.
(28) A method for treating cancer by administering folate-modified methylated cyclodextrin of 500 mg / kg body weight or less to a human as a total dose of repeated administration.
(29) The pharmaceutical composition according to any one of (21) to (28), wherein the folic acid-modified methylated cyclodextrin is folic acid-modified methyl-β-cyclodextrin.

  The pharmaceutical composition comprising the folic acid-modified methylated cyclodextrin of the present invention as an active ingredient for anticancer action is useful as an anticancer agent at a practical concentration by systemic administration. The pharmaceutical composition containing the folic acid-modified methylated cyclodextrin of the present invention as a drug delivery agent has few or no side effects and is useful as a delivery agent for various drugs.

It is the result of having measured the cytotoxic activity with respect to KB cell and A549 cell of M- (beta) -CyD or FA-M- (beta) -CyD by WST-1 method. It is the result of examining the influence of folic acid (FA) on the cytotoxic activity by FA-M-β-CyD. It is the result of having measured the cytotoxic activity with respect to KB cell of FA-M- (beta) -CyD or DOX by WST-1 method. It is the result of having measured the cytotoxic activity with respect to Colon-26 cell of FA-M- (beta) -CyD by WST-1 method. It is the result of having measured the cytotoxic activity with respect to KB cell and A549 cell of a DOX / FA-M- (beta) -CyD inclusion complex by WST-1 method. It is the result of measuring the antitumor activity of M-β-CyD, FA-M-β-CyD, or DOX using a mouse mouse model for colon cancer colon-26 cell transplantation. It is the result of investigating the effect with respect to the tumor of FA-M-β-CyD using mouse colon cancer Colon-26 cell transplant cancer mouse model. It is the result of having investigated the effect with respect to a mouse | mouth survival rate using the mouse | mouth colorectal cancer Colon-26 cell transplant cancer mouse model of FA-M- (beta) -CyD. It is the result of having investigated the effect with respect to the tumor of FA-M-β-CyD using a human malignant melanoma (Ihara) cell transplanted cancer mouse model. It is the result of having investigated the effect with respect to the tumor of FA-M-β-CyD using a human malignant melanoma (Ihara) cell transplanted cancer mouse model.

Hereinafter, the present invention will be described in detail, but the present invention is not limited to the embodiments described below.
Methylated cyclodextrin is a compound in which the hydroxyl group of cyclodextrin (α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin) is randomly substituted with a methyl group. What is methyl-β-cyclodextrin? , Β-cyclodextrin is a compound in which hydroxyl groups at positions 2, 3 and 6 of 7 glucoses are randomly substituted with methyl groups.

  The folic acid-modified methylated cyclodextrin (FA-M-CyD) of the present invention is a compound in which folic acid is covalently bonded to glucose of cyclodextrin, and the site to which folic acid binds is not particularly limited, The 6-position is preferred, and the 6-position is particularly preferred. The ratio of folic acid binding in the FA-M-CyD of the present invention is not particularly limited as long as it meets the purpose of the present invention (for example, efficient transport to cells having a folate receptor), but the cyclodextrin molecule: Of folic acid molecules, 1: 1 to 1:14 are preferred, 1: 1 to 1: 7 are more preferred, and those in which both molecules are bonded approximately equivalently are particularly preferred. A preferred folic acid modified methylated cyclodextrin in the present invention is folic acid modified methyl-β-cyclodextrin (FA-M-β-CyD).

The FA-M-CyD of the present invention can be synthesized using methods known to those skilled in the art. For example, FA-M-β-CyD or FA-M-α-CyD can be synthesized using M-β-CyD. Alternatively, it can be synthesized by tosylation and amination of the hydroxyl group of M-α-CyD, followed by a condensation reaction to bind folic acid. On the other hand, FA-M-γ-CyD is a naphthalenesulfonylated hydroxyl group of M-γ-CyD. After amination, it can be synthesized by binding folic acid by a condensation reaction.
In the folic acid methylated cyclodextrin of the present invention, M-CyD may be introduced into either α-carboxyl group or γ-carboxyl group of folic acid, and both are included. For example, when M-β-CyD is introduced into folic acid, M-β-CyD is introduced into α-carboxyl group of folic acid (that is, FA-M-β-CyD having γ-carboxyl group) or folic acid Any of those obtained by introducing M-β-CyD into a γ carboxyl group (that is, FA-M-β-CyD having an α carboxyl group) is included in the FA-M-β-CyD of the present invention. Moreover, it is also possible to use both properly in the target cancer type. When synthesizing FA-M-β-CyD, in which M-β-CyD is introduced into the α-carboxyl group of folic acid, it can be synthesized by protecting the γ-carboxyl group of folic acid, binding to folic acid, and removing the protecting group. .

One embodiment of the present invention is a pharmaceutical composition comprising folic acid-modified methylated cyclodextrin as an active ingredient for anticancer activity. The pharmaceutical composition referred to in the present invention means an oral preparation, intravenous injection, intramuscular injection, subcutaneous administration, transdermal absorption agent, nasal administration, intrarectal application, intravaginal application, etc. However, it does not include a form administered directly to the tumor or a form administered intraperitoneally. More preferably, the pharmaceutical composition of the present invention is a pharmaceutical composition for systemic administration and does not include a form for local administration to a tumor. An oral preparation or an intravenous injection is preferable.
The oral preparation as used in the present invention may be in any form and is not particularly limited, and examples thereof include tablets, microcapsules, and syrups.

  The cancer for which the pharmaceutical composition of the present invention can be used is not particularly limited, and can be used for any cancer, but is preferably a cancer that highly expresses a folate receptor. For example, breast cancer, small cell lung cancer, colon cancer, malignant lymphoma, leukemia, testicular cancer, ovarian cancer, pancreatic cancer, lung cancer, pharyngeal cancer, laryngeal cancer, tongue cancer, gingival cancer, esophageal cancer, stomach cancer, bile duct cancer, renal cancer, Examples include bladder cancer, uterine cancer, and prostate cancer.

  The administration period and frequency of the pharmaceutical composition containing the folic acid-modified methylated cyclodextrin of the present invention as an active ingredient for anticancer action are not particularly limited. For example, once daily administration, administration at intervals of 2 or 3 days, administration once a week, administration at intervals of more than one, administration of a single administration Although it can be used, administration at intervals of at least one week is preferable. The pharmaceutical composition of the present invention can exhibit a sufficient anticancer effect even by a single administration. Therefore, the pharmaceutical composition of the present invention can be used for repeated administration of 5 times or less, preferably 2 or 3 times or less. Furthermore, administration of a pharmaceutical composition comprising the folic acid-modified methylated cyclodextrin of the present invention as an active ingredient for anticancer activity not only suppresses the growth of cancer and tumors, but also almost eliminates cancer and tumor masses. You can also expect Therefore, the pharmaceutical composition of the present invention is confirmed to have an anticancer effect after being administered once, and if the disappearance of cancer can be confirmed, the administration can be terminated there, and it is judged that further administration is necessary. In this case, it can be administered again. Any of these various aspects is included in the present invention.

  The dosage of the pharmaceutical composition containing the folic acid-modified methylated cyclodextrin of the present invention as an active ingredient for anticancer activity can be appropriately selected according to the period, number of times, and subject. Although not limited thereto, the dose per administration is, for example, 0.1 to 1000 mg / kg, preferably 1 to 500 mg / kg, more preferably 10 to 100 mg / kg when administered to a human. A sufficient effect at 5 mg / kg has been confirmed in mice, and the estimated effective dose in humans is approximately 60 mg / kg (5 mg / kg x human equivalent constant approximately 12 = 60 mg / kg). In mouse systems, a single dose of FA-M-β-CyD (5 mg / kg) not only suppresses the growth of cancerous tumors, but also shrinks and eventually disappears. ing. Furthermore, in the FA-M-β-CyD (5 mg / kg) administration group, the survival rate of the mice was 100%. From this, a sufficient effect can be expected even with a single dose of the above dose, and even when it is repeatedly administered to humans, the total dose is 1000 mg / kg or less, preferably 500 mg / kg or less, more preferably 100 mg / kg. A sufficient effect can be expected even under kg.

  An aspect of the invention is also a method of treating cancer by systemically administering a folic acid modified methylated cyclodextrin. The form of administering the folic acid-modified methylated cyclodextrin is not particularly limited as long as it is systemic, but for example, oral administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, nasal administration, or intrarectal administration It means administration, etc., and does not include direct administration to tumors or intraperitoneal administration. Preferably, it is oral administration or intravenous administration.

  The cancer to which the method of the present invention is applied is not particularly limited and is as described above. The administration period and frequency of the folic acid-modified methylated cyclodextrin administered in the method of the present invention are not particularly limited and are as described above. The dose of folic acid-modified methylated cyclodextrin in the method of the present invention can be appropriately selected according to the period, number of times, and subject, and is as described above.

  Another embodiment of the present invention is a pharmaceutical composition comprising folate-modified methylated cyclodextrin as a drug delivery agent. Since FA-M-β-CyD of the present invention does not show side effects compared to M-β-CyD, it is useful as a drug delivery agent. The administration period and frequency of the pharmaceutical composition of the present invention as a drug delivery agent are not particularly limited. For example, administration once a day, once every two days, once every three days, once a week, or more than once, only once The form can also be used, and the administration interval can be appropriately selected according to the drug transported by the folic acid-modified methylated cyclodextrin. When the drug to be transported is an anticancer drug, a high anticancer action can be expected due to a synergistic effect with the anticancer action of the folic acid-modified methylated cyclodextrin of the present invention. In such a case, similarly to the above, it can be preferably administered at an interval of at least one week or more, and various administration forms can be used as described above.

  The drug transported in the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include anticancer agents, anti-inflammatory agents, antibiotic agents, molecular imaging agents, and photosensitizers. When transporting cancer drugs, for example, doxorubicin, daunorubicin, bleomycin, imatinib, gefitinib, cyclophosphamide, carmofur, tegafur, fluorouracil, methotrexate, irinotecan, docetaxel, paclitaxel, vinblastine, oxaliplatin, carboplatin, Can do.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to a following example.

Example 1: Preparation of folic acid modified methylated cyclodextrin (FA-M-β-CyD)
FA-M-β-CyD was synthesized according to the report of the inventors R. Onodera et al, J. Incl. Phenom. Macrocycl. Chem. 70, 321-326 (2011). Specifically, as shown in the following synthesis formula, the hydroxyl group of methyl-β-cyclodextrin (M-β-CyD) is tosylated, aminated, and then bound with folic acid (FA) by a condensation reaction to produce FA. -M-β-CyD was prepared. The structure of the obtained FA-M-β-CyD was confirmed by ¹ H-NMR. In addition, the FA substitution degree (DSF) was 1.0 from the study by FAB-MS. Furthermore, investigation by powder X-ray diffraction and suggested scanning calorimetry suggested that FA-M-β-CyD was amorphous.

Example 2: Measurement of cytotoxic activity against KB cells and A549 cells Human oral cancer cell-derived KB cells (FR highly expressing cells, FR (+)) and human lung cancer cell-derived A549 cells (FR low expressing cells, FR (- )), The cytotoxic activity of M-β-CyD, FA-M-β-CyD and DOX was examined by the WST-1 method.
KB cells were seeded in a 96-well plate at 2 × 10 ⁴ cells per well and incubated in RPMI medium (10% FCS) at 37 ° C. for 24 hours. After washing twice with FA-free RPMI medium, serum-free RPMI medium containing M-β-CyD, FA-M-β-CyD or DOX was added and incubated at 37 ° C. for 24 or 48 hours. Subsequently, the wells were washed twice with PBS and 100 μL of HBSS was added. 10 μL of WST-1 reagent (Dojindo Laboratories) was added and incubated at 37 ° C. for 15 minutes. Thereafter, absorbance at 450 nm with respect to a reference wavelength of 620 nm was measured with a plate reader (manufactured by Bio-Rad).

(2-1) Measurement of cytotoxic activity of FA-M-β-CyD Various concentrations (0, 1, 2, 4, 5, 8 and 10 mM) of M-β-CyD or FA-M-β-CyD The cytotoxic activity against KB cells and A549 cells was measured according to the above WST-1 method using a serum-free medium containing The results are shown in FIG.
As already reported by the present inventors, cytotoxicity was shown to be dependent on KB cells (FR highly expressing cells) in a concentration-dependent manner, but almost no cells against A549 cells (FR low expressing cells). It did not show disability. The concentration that shows significant cytotoxic activity against KB cells is as high as the order of mM, and the results show that cytotoxicity is observed at a concentration of 10 mM, to achieve 10 mM in blood concentration in vivo. Was expected to require administration of about 1.76 g / kg.
Further, as reported by the present inventors, the cytotoxic activity by FA-M-β-CyD was significantly suppressed by the addition of folic acid (FA), which is an FR competitive inhibitor (see FIG. 2). .

(2-2) Measurement of cytotoxic activity of low concentration FA-M-β-CyD
Cytotoxic activity against KB cells was measured in the same manner using serum-free medium containing 10 μM FA-M-β-CyD or 10 μM DOX. The results are shown in FIG. DOX showed significant cytotoxic activity, whereas FA-M-β-CyD showed no activity.

Example 3: Measurement of cytotoxic activity against mouse colon cancer Colon-26 cells The cytotoxic activity of FA-M-β-CyD against mouse colon cancer Colon-26 cells (FR highly expressing cells, FR (+)) was measured using WST. -1 method was used.
Colon-26 cells were seeded in a 96-well plate at 2 × 10 ⁴ cells per well and incubated in RPMI medium (10% FCS) at 37 ° C. for 24 hours. After washing twice with FA-free RPMI medium, 0 to 10 mM FA-M-β-CyD-containing serum-free RPMI medium was added and incubated at 37 ° C. for 2 hours. Subsequently, the wells were washed twice with PBS and 100 μL of HBSS was added. 10 μL of WST-1 reagent was added and incubated at 37 ° C. for 30 minutes. Thereafter, the absorbance at 450 nm with respect to a reference wavelength of 620 nm was measured with a plate reader. The results are shown in FIG.
Also in colorectal cancer Colon-26, in order to show cytotoxic activity in vitro, a concentration on the order of mM was necessary.

Example 4: Measurement of cytotoxic activity against KB cells and A549 cells by combined use of FA-M-β-CyD and DOX In the same manner as in Example 2, containing 10 μM FA-M-β-CyD and / or 10 μM DOX The cytotoxic activity of the combined use of FA-M-β-CyD and DOX was measured using a serum-free medium. The results are shown in FIG.
As already reported by the present inventors, 10 μM FA-M-β-CyD alone did not show cytotoxic activity against KB cells, but by adding 10 μM FA-M-β-CyD, The cytotoxic activity of DOX was amplified. On the other hand, it hardly showed cytotoxic activity against A549 cells (FR low expressing cells). The stabilization constant (Kc) of FA-M-β-CyD and DOX was 3.5 × 10 ⁵ / M.

Example 5: Measurement of antitumor activity using a mouse mouse model transplanted with mouse colon cancer Colon-26 cells
Mouse colon cancer Colon-26 cells were cultured in RPMI medium (FA (−) (10% FCS)). 100 μL of Colon-26 cell suspension (2 × 10 ⁵ cells) was inoculated into the hind limbs of BALB / c male mice (4 weeks old). After confirming that the tumor diameter reached 8 mm, the following experiment was conducted.

(5-1) Confirmation of effect on tumor growth After confirming that the tumor diameter of the mouse hindlimb reached 8 mm, 5% mannitol solution, DOX solution (dissolved in 5% mannitol solution), M-β-CyD 100 μL each of the solution (dissolved in 5% mannitol solution) and FA-M-β-CyD solution (dissolved in 5% mannitol solution) were intravenously administered. The dose was 5 mg / kg each.
Changes in tumor volume after administration were observed. The results are shown in FIG. Administration of DOX and M-β-CyD significantly inhibited tumor growth compared to controls. However, a single dose did not completely suppress tumor growth. On the other hand, when FA-M-β-CyD was administered intravenously, no tumor growth was observed despite the single administration. In addition, in mice in the M-β-CyD administration group, it was observed that the pulse increased after administration in all mice (side effects of tachycardia), but such side effects were observed in FA-M-β-CyD. Was not.

(5-2) Confirmation of tumor mass reduction After confirming that the tumor diameter of the mouse hind limb reached 8 mm, 100 μL of the FA-M-β-CyD solution was administered intravenously. The dose was adjusted to 5 mg / kg. Thereafter, the change (reduction) in the tumor volume was confirmed. The results are shown in FIG. Surprisingly, it was confirmed that despite the single intravenous administration of FA-M-β-CyD, the tumor mass shrinks day by day and eventually disappears.

Example 6: Confirmation of effect on mouse survival rate using mouse colon cancer mouse transplanted with colon-26 cells As in Example 5, 5% mannitol solution, DOX (5 mg / kg) solution, and FA -M-β-CyD (5 mg / kg) solution was intravenously administered at 100 μL each, and the dose was 5 mg / kg, and the survival rate of the mice after the administration was observed. The results are shown in FIG. There was no significant difference in survival between control and DOX administration, and all mice died after about 60 days. On the other hand, in the case of administration of FA-M-β-CyD, the death of the mouse was not confirmed even after 120 days of observation, despite the single administration.

Example 7: Measurement of antitumor activity using a mouse model of human malignant melanoma (Ihara) cell transplantation cancer
Malignant melanoma (Ihara) cells were cultured in D-MEM medium (FA (+) (10% FCS)). Nude Rag2 / Jak3 KO male mice (12 weeks old, reared on FA-containing diet) were inoculated with 100 μL of Ihara cell suspension (1 × 10 ⁶ cells) on the left and right backs. After confirming that the tumor diameter reached 5 mm, the following experiment was conducted.

(7-1) Confirmation of effect on tumor growth After confirming that the tumor diameter reached 5 mm, 5% mannitol solution, M-β-CyD solution (dissolved in 5% mannitol solution), and FA-M 100 μL each of -β-CyD solution (dissolved in 5% mannitol solution) was administered intravenously. The dose was 5 mg / kg each.
After administration, body weight and tumor volume were measured every 3 days, and changes were observed. The change in tumor volume and tumor growth rate are shown in FIG. The change in the body weight of the mouse is shown in FIG. It was confirmed that the administration of FA-M-β-CyD significantly inhibited the growth of tumors compared to the administration of M-β-CyD and controls, and human tumor cells also have an antitumor effect. On the other hand, the administration of FA-M-β-CyD did not show any difference in body weight compared to the control, suggesting that there were fewer side effects.

  The above description is merely illustrative of the objects and objects of the present invention and is not intended to limit the scope of the appended claims. Various changes and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings described herein without departing from the scope of the appended claims.

  The pharmaceutical composition of the present invention is useful as an anticancer agent. The pharmaceutical composition of the present invention is also useful as a drug delivery agent.

Claims (20)

  A pharmaceutical composition which is an anticancer agent comprising folic acid-modified methylated cyclodextrin as an active ingredient.   The pharmaceutical composition according to claim 1, which is a pharmaceutical composition for systemic administration.   The pharmaceutical composition according to claim 2, which is an intravenous injection.   The pharmaceutical composition according to any one of claims 1 to 3, which is used so as to be administered at an interval of at least one week.   The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition is used in repeated administration of 5 times or less.   The pharmaceutical composition according to any one of claims 1 to 5, which is used so as to be administered again after confirming an anticancer effect after the administration.   The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is used as a single administration.   The pharmaceutical composition according to any one of claims 1 to 7, characterized in that it is used so that folic acid-modified methylated cyclodextrin of 10 to 100 mg / kg body weight is administered to a human at a time. .   The total dose of repeated administration is used so that folic acid-modified methylated cyclodextrin of 500 mg / kg body weight or less is administered to humans. Pharmaceutical composition.   The pharmaceutical composition according to any one of claims 1 to 9, wherein the folic acid-modified methylated cyclodextrin is folic acid-modified methyl-β-cyclodextrin.   A pharmaceutical composition which is an anticancer agent, comprising folic acid-modified methylated cyclodextrin as a drug delivery agent.   Furthermore, the pharmaceutical composition of Claim 11 containing the chemical | medical agent which has an anticancer action.   The pharmaceutical composition according to claim 12, wherein the drug having anticancer activity is doxorubicin.   The pharmaceutical composition according to any one of claims 11 to 13, which is a pharmaceutical composition for systemic administration.   The pharmaceutical composition according to claim 14, which is an intravenous injection.   The pharmaceutical composition according to any one of claims 11 to 15, which is used so as to be administered at intervals of at least one week or more.   The pharmaceutical composition according to any one of claims 11 to 16, wherein the pharmaceutical composition is used in 5 or less repeated administrations.   The pharmaceutical composition according to any one of claims 11 to 17, wherein the pharmaceutical composition is used so as to be administered again after confirming an anticancer effect after the administration.   The pharmaceutical composition according to any one of claims 11 to 15, which is used so as to be administered in a single dose.   The pharmaceutical composition according to any one of claims 11 to 19, wherein the folic acid-modified methylated cyclodextrin is folic acid-modified methyl-β-cyclodextrin.

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