JP2016069520A - Improved type folic acid modified cyclodextrin derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a folic acid cyclodextrin compound whose solubility is high and whose tumor cells accumulation ability is superior by transformation of folic acid modified cyclodextrin having a problem that preparation of a water solution as a vein injection is difficult because its solubility to water is low.SOLUTION: A compound has hydrophilic substituents such as a polyethylene glycol group and hydroxypropyl group introduced to folic acid modified cyclodextrin. The transformed folic acid modified cyclodextrin has superior tumor accumulation ability and is available as a drug delivery system applicable as various formulations including a vein injection.SELECTED DRAWING: None

Description

  The present invention relates to a folic acid-modified cyclodextrin having high water solubility.

  The present inventors have heretofore found that folate-modified cyclodextrin is molecularly recognized by the folate receptor protein on the surface of the tumor cell and binds to the tumor cell through a strong interaction with the antigen-antibody reaction level (Patent Document 1). . Among the folic acid cyclodextrin compounds reported so far, heptakis-6-amino-β-cyclodextrin (exemplary compound 1 in Patent Document 1) (hereinafter referred to as “ND1”) is a typical compound. This is a structure in which 7 folic acids are bonded to the 6-position carbon atom via a linker composed of an aminocaproic acid dimer. ND1 shows accumulation in cancer cells by the action of folic acid, and is expected to be applied to cancer treatment and cancer diagnosis as a DDS carrier capable of delivering drugs to tumor cells. The present inventors have also confirmed that folic acid-modified cyclodextrin (hereinafter referred to as “ND201”) in which the ND1 linker is replaced with an aminocaproic acid monomer also shows drug delivery to tumor cells.

  While such a folic acid-modified cyclodextrin is expected to have applicability in its ability to accumulate tumors, it is difficult to prepare an aqueous solution as an intravenous injection because of its low solubility in water in practical use. There was a problem of being. For example, ND1 and ND201 have a low solubility in pure water of about 1 mg / mL, and as such, it was difficult to use at high concentrations.

  Until now, there has been no report on a compound in which PEG is chemically bonded to cyclodextrin. As an example of using cyclodextrin and PEG in combination, there is only a report by Davis et al. (See Non-Patent Document 1 and Non-Patent Document 2). Davis et al. Use nanoparticles in which a cyclodextrin polymer is encapsulated with adamantane-conjugated PEG in clinical experiments for gene delivery. Here, PEG is conjugated mainly for the purpose of increasing cell affinity.

  On the other hand, hydroxypropylation has been frequently used as a method for solubilizing cyclodextrins. In particular, many products have been put to practical use in solubilization of β-cyclodextrin (Non-patent Document 3).

International Publication No. WO2009 / 041666

M.M. E. Davis et al. , Nature 08956 (2010) M.M. E. Davis, Molecular Pharmaceuticals, 6, 659-668 (2009) Chemical Reviews, 98, 1471-2076 (1998).

  In order to improve the solubility of the folic acid-modified cyclodextrin, the present inventors tried to increase the solubility in a basic aqueous solution by formulating ammonium salt formation or Na salt formation. For example, after dissolving in a pH 8.0 alkaline aqueous solution, a buffer solution of pH 7.4 was added to improve the solubility. However, since this method requires preparation at the time of use, it was considered that more practical and simple means were necessary.

  Therefore, as a result of studying various other means for increasing the solubility of the compound, the present inventors have found that a hydrophilic substituent (a polyethylene glycol group (hereinafter referred to as “PEG group”)) and a hydroxypropyl group (hereinafter, referred to as “polypropylene group”). By introducing the “HP group”), etc.), the solubility of the compound itself was improved, and a folic acid-modified cyclodextrin more suitable for practical use was successfully obtained. Furthermore, the present inventors have confirmed whether such a hydrophilic group-introduced folic acid-modified cyclodextrin can maintain the ability to accumulate tumor cells. In comparison, it was found that it has a better ability to accumulate tumor cells, and the present invention was completed.

  Therefore, the present invention relates to a folic acid modified cyclodextrin having no hydrophilic group introduced, and more specifically to the invention described below. Note that throughout the present specification, unless otherwise defined, or unless otherwise specified, each symbol included in the general formula (I) is represented by the following general formula (I ) Has the same definition as in the description.

  Cyclodextrin compound represented by the following general formula (I)

[Wherein m represents an integer of 6 to 8,
m R ¹ 's each independently represent a hydrogen atom, a hydroxyl group, -NH-Y-R ² group, -NHCONH-Y-R ² group, -NHCO-Y-R ² group, -NH-Y-NHCO. -Y-NH-Y-R ² group, -NH-Y-NHCO-Y-R ² group, -NHCO-Y-NHCO-Y-NH-R ² group, -NHCO-Y-NHCO-Y-NHCO- Y—R ² group, —NHCONH—Y—NHCO—Y—NHCO—Y—R ² group, —Z group, —NH—Y—R ² —Z group, —NHCONH—Y—R ² —Z group, — ^NHCO-Y-R 2 -Z group, -NH-Y-NHCO-Y -NH-Y-R 2 -Z group, -NH-Y-NHCO-Y -R 2 -Z group, -NHCO-Y-NHCO ^-Y-NH-R 2 -Z group, and, -NHCO-Y-NHCO-Y -NHCO-Y-R 2 -Z group, -NHCONH Y-NHCO-Y-NHCO- Y-R 2 -Z group, represents a group selected from the group consisting of,
Here, at least two of the m R ¹ groups are —Z group, —NH—Y—R ² —Z group, —NHCONH—Y—R ² —Z group, —NHCO—Y—R ² —. Z group, -NH-Y-NHCO-Y -NH-Y-R 2 -Z group, -NH-Y-NHCO-Y -R 2 -Z group, -NHCO-Y-NHCO-Y -NH-R 2 -Z group, and a group selected from the group consisting of -NHCO-Y-NHCO-Y- NHCO-Y-R 2 -Z group,
m X ¹ , m X ² , and m X ³ are each independently a hydrogen atom, a hydroxyl group, —R ² , —NH—Y—R ² group, —NHCONH—Y—R ^2. Groups, —NHCO—Y—R ² groups, —NH—Y—NHCO—Y—NH—Y—R ² groups, —NH—Y—CONH—Y—R ² groups, and —NH—Y—NHCO— Represents a group selected from the group consisting of YR ² groups;
Here, each Y independently represents a group selected from an alkylene group, a phenylene group, and a -O- group,
Here, each R ² independently represents a polyethylene glycol group or a hydroxypropyl group,
Here, the Z group represents a group represented by the following formula (II)

In the formula, n represents an integer of 0 to 3.]

  In the present specification, “cyclodextrin” (sometimes abbreviated as “CyD”) means a substance in which 6 to 8 D-glucoses are cyclically bonded by α1,4 bonds. Six, seven, and eight D-glucoses are cyclically linked by α1,4 bonds, and the clodextrins are respectively α-cyclodextrin (α-CyD), β-cyclodextrin (β-CyD), And γ-cyclodextrin (γ-CyD).

  In the present specification, “folic acid-modified cyclodextrin” or “Fol-cyclodextrin” means that two or more primary hydroxyl groups at the 6-position of glucopyranose constituting cyclodextrin have the same or different folic acid. It means a cyclodextrin compound substituted with a substituent. For example, the folic acid-modified cyclodextrin in the present specification includes ND1 (per-Fol-cap2-β-cyclodextrin: Exemplified Compound 1 described in WO2009 / 041666) represented by the following structural formula and ND201.

  In the present specification, the hydrophilic group-introduced folic acid-modified cyclodextrin means a compound in which the above-mentioned folic acid is modified and the crodextrin is further modified with a hydrophilic group. The hydrophilic group may be introduced into the primary hydroxyl group at the 6-position of glucopyranose constituting cyclodextrin together with folic acid or separately from folic acid, or at the 2nd or 3rd position of glucopyranose constituting cyclodextrin. It may be bonded to a secondary hydroxyl group. Alternatively, the hydrophilic group may be introduced by replacing the hydrogen atom at the 3-position of glucopyranose constituting the cyclodextrin.

  In the present specification, the alkylene group means a linear or branched divalent saturated hydrocarbon group having 1 to 10 carbon atoms (C1-10 alkylene group), such as a methylene group or an ethylene group. N-propylene group, i-propylene group, n-butylene group, sec-butylene group, t-butylene group, isobutylene group, n-pentylene group, isopentylene group, 2,3-dimethylpropylene group, n-hexylene group, 2-methylpentylene group, 2,2-dimethylbutylene group, 2,3-dimethylbutylene group, n-heptylene group, 2-methylhexylene group, 3-methylhexylene group, 2,2-dimethylpentylene group 2,3-dimethylpentylene group, 2,4-dimethylpentylene group, 2,5-dimethylpentylene group, 2,2,3-trimethylbutylene group, 2,2,4-triene Tylbutylene group, 2,2,5-trimethylbutylene group, 2,3,4-trimethylbutylene group, 2,3,5-trimethylbutylene group, 2,4,4-trimethylbutylene group, n-octylene group, n- Nonylene group, n-decalene group, etc. are mentioned, Preferably it is C1-6 alkylene group, More preferably, a methylene group, ethylene group, n-propylene group, i-propylene group, n-butylene group, sec -A butylene group, a t-butylene group, an isobutylene group, a pentylene group, an isopentylene group, or a 2,3-dimethylpropylene group. More preferably, it is a linear C1-5 alkyl group.

  In this specification, the “phenylene group” is a 1,4-phenylene group, a 1,3-phenylene group, or a 1,4-phenylene group.

In the present specification, the hydrophilic group means a polyethylene glycol group and a hydroxypropyl group. In the general formula (I), the hydrophilic group is represented by R ² . In the compound of the present invention, the hydrophilic group may be a position (X ¹ , X ² and X ³ in the general formula (I)) different from the folic acid group (Z in the general formula (I), the same applies hereinafter). , The same position as the folic acid group (R ¹ in the general formula (I)) may be used. When the hydrophilic group is present at the same position as the folic acid group, the folic acid group may be bonded to the cyclodextrin skeleton via the hydrophilic group, or a part of R ¹ in the general formula (I) (for example, 1-2) May be hydrophilic groups and the rest may be folic acid groups. In the latter case, since the number of folic acids bound to cyclodextrin is reduced, there is a concern that the nanocluster effect may be reduced accordingly. This decrease in the number of folic acid is considered to have no effect on the tumor accumulation ability of folate-modified cyclodextrins. In addition, the presence of a hydrophilic group on the same side as the folic acid group may affect the behavior of folic acid and its inclusion, but the hydrophilic group is sufficiently large and flexible from the whole molecule. Therefore, it is considered that there is no influence.

In the present specification, the “polyethylene glycol group” means a group derived from an ethylene glycol polymer (HO— (CH ₂ —CH ₂ —O) _n —H). Specifically, the “polyethylene glycol group” is not particularly limited as long as it is a group having a — (CH ₂ —CH ₂ —O) _n — structure, for example, — (CH ₂ —CH ₂ — _O) n -H group, and _- including (Cl to 6) alkyl group _{- (CH 2 -CH 2 -O)} n. Here, the (C1-6) alkyl group means a linear or branched monovalent saturated hydrocarbon group having 1 to 6 carbon atoms, such as a methyl group, an ethyl group, or an n-propyl group. I-propyl group, n-butyl group, sec-butyl group, t-butyl group, isobutyl group, n-pentyl group, isopentyl group, 2,3-dimethylpropyl group, n-hexyl group, 2-methylpentyl group 2,2-dimethylbutyl group, or 2,3-dimethylbutyl group. The degree of polymerization of polyethylene glycol is not particularly limited as long as the object of the present invention can be achieved. For example, it can be 20 to 80, preferably 30 to 60, and more preferably 40. ~ 50.

In the present specification, the “hydroxypropyl group” means a group represented by — (CH ₂ —CH (CH ₃ ) —O) _t —H. The degree of polymerization of hydroxypropyl can be, for example, 1 to 20, preferably 2 to 10, more preferably 3 to 8, and most preferably 4 to 6.

R ¹ in the compound represented by the general formula (I) is preferably a hydrogen atom, a hydroxyl group, —NHCO—Y—NHCO—Y—NHCO—Y—R ² group, —NHCONH—Y—NHCO—Y—NHCO. —YR ² group and a group selected from the group consisting of —Z group, and more preferably —NHCO— (CH ₂ ) ₅ —NHCO— (CH ₂ ) ₅ —NHCO—CH ₂ — ( _{CH 2 CH 2 O) p -CH} 3 _{group, -NHCONH- (CH 2) 5 -NHCO-} (CH 2) 5 -NHCO-CH 2 - (CH 2 CH 2 O) p -CH 3 group, or -Z It is a group.

  In the group represented by the formula (II), n is an integer of 0 to 3, preferably 1 or 2.

X ¹ in the compound represented by the general formula (I) is preferably a hydrogen atom, —NH—Y—R ² group, or —NH—Y—CONH—Y—R ² group, more preferably A hydrogen atom, —NH— (CH ₂ ) ₅ —CONH— (CH ₂ ) ₃ — (CH ₂ CH ₂ O) _p —CH ₃ , or —NH— (CH ₂ ) ₃ — (CH ₂ CH ₂ O) _p— CH ₃ group.

X ² in the compound represented by the general formula (I) is preferably a hydrogen atom or a hydroxyl group.

  In the general formula (I), Y is preferably an alkylene group, more preferably a methylene group, a propylene group, or a pentylene group.

  Particularly preferably, the hydrophilic group-introduced folic acid-modified cyclodextrin compound of the present invention is the following compound.

PND1-1 (PEG-per-Fol-cap2-β-cyclodextrin): In the compound represented by the general formula (I), m is an integer of 6 to 8, and R ¹ is a —Z group (n = 2), and one X ¹ is —NH— (CH ₂ ) ₅ —CONH— (CH ₂ ) ₃ — (CH ₂ CH ₂ O) _p —CH ₃ group (the average value of p is 46 to 47), (m−1) X ¹ are hydrogen atoms, one X ² is a hydrogen atom, (m−1) X ² are hydroxyl groups, and X ³ is a hydroxyl group. A compound.

PND1-3: In the compound represented by the general formula (I), m is an integer of 6-8, ^{R 1} is a -Z groups (n = 2), is one of ^{X 1,} - NH— (CH ₂ ) ₃ — (CH ₂ CH ₂ O) _p —CH ₃ group (average value of p is 46 to 47), (m−1) X ¹ are hydrogen atoms, 1 A compound in which X ² is a hydrogen atom, (m−1) pieces of X ² are hydroxyl groups, and X ³ is a hydroxyl group.

PND1-7: In the compound represented by the general formula (I), m is an integer of 6 to 8, (m−r) R ¹ is a —Z group (n = 2), and r R ¹ groups are —NHCO— (CH ₂ ) ₅ —NHCO— (CH ₂ ) ₅ —NHCO—CH ₂ — (CH ₂ CH ₂ O) _p —CH ₃ groups (average value of p is 46 to 47) A compound in which X ¹ is a hydrogen atom, X ² is a hydroxyl group, and X ³ is a hydroxyl group.

PND1-8: In the compound represented by the general formula (I), m is an integer of 6 to 8, (m−r) R ¹ is a —Z group (n = 2), and r R ¹ groups are —NHCONH— (CH ₂ ) ₅ —NHCO— (CH ₂ ) ₅ —NHCO—CH ₂ — (CH ₂ CH ₂ O) _p —CH ₃ (average value of p is 46 to 47) A compound in which X ¹ is a hydrogen atom, X ² is a hydroxyl group, and X ³ is a hydroxyl group.

ND201-HP: In the compound represented by the general formula (I), m is an integer of 6 to 8, s R ¹ is a -Folate group (n = 1), and the remaining R ¹ is A hydroxyl group, X ¹ is a hydrogen atom, and t X ² and / or X ³ are —O (CH ₂ CH (CH ₃ ) 0) _q —H groups (q is an integer up to 6) (t = 1-16), and the remaining X ² and / or X ³ is a hydroxyl group. Note that in this specification, ND201-tHP is bonded to t hydroxypropyl (HP) groups (—O (CH ₂ CH (CH ₃ ) 0) _q —H groups (q is an integer up to 6)). It means ND201.

  In addition to the above compounds, the compounds of the present invention include pharmacologically acceptable esters of these compounds. Here, the “pharmacologically acceptable ester” is a compound containing a group that is metabolized in vivo to give the compound of the present invention, and is an ester that can be administered into the body as a medicine. . In the present specification, the ester includes not only an ester-bonded compound but also an amide-bonded compound. Esters may be degraded by in vivo esterases to give active compounds. For example, as the ester, substituted or unsubstituted lower alkyl ester, lower alkenyl ester, lower alkylamino lower alkyl ester, acylamino lower alkyl ester, acyloxy lower alkyl ester, aryl ester, aryl lower alkyl ester, amide, lower Examples thereof include alkylamides and hydroxide amides. The ester is preferably a propionic acid ester or an acyl ester.

  The present invention also relates to a pharmaceutical composition containing a compound represented by formula (I) as an active ingredient. The type of pharmaceutical composition is not particularly limited, and dosage forms include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, An injection agent etc. are mentioned. These preparations can be prepared according to a conventional method. In the case of a liquid preparation, it may be dissolved or suspended in water or other appropriate solvent at the time of use. Tablets and granules may be coated by a known method. In the case of injection, it is prepared by dissolving the compound of the present invention in water, but it may be dissolved in physiological saline or glucose solution as necessary, and a buffer or preservative may be added. Good. It is provided in any dosage form for oral or parenteral administration. For example, a pharmaceutical composition for oral administration in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions or liquids, for intravenous administration, for intramuscular administration, Alternatively, it can be prepared as a pharmaceutical composition for parenteral administration in the form of injections, drops, transdermal absorbents, transmucosal absorbents, nasal drops, inhalants, suppositories, etc. for subcutaneous administration. Injections, infusions, and the like can be prepared as powdered dosage forms such as freeze-dried forms, and can be used by dissolving in an appropriate aqueous medium such as physiological saline at the time of use.

  Since the folic acid-modified cyclodextrin of the present invention is excellent in solubility while maintaining the directivity to cancer cells, it can be formulated into a desired dosage form. The folic acid-modified cyclodextrin of the present invention is useful as a DDS for the purpose of targeting cancer because of its superior ability to accumulate tumor cells. For example, the folic acid-modified cyclodextrin of the present invention can provide an anticancer agent with fewer side effects by inclusion or binding of a substance having anticancer activity. The folic acid-modified cyclodextrin of the present invention can provide a clearer diagnostic image of cancer by inclusion or binding of a substance having a labeling action. In addition, the hydrophilic group-introduced folic acid-modified cyclodextrin of the present invention can be prepared as a relatively high concentration folic acid-modified cyclodextrin aqueous solution by dramatically increasing the solubility. As a result, it can be administered as an injection into the bloodstream of mammals and used as a cancer therapeutic or cancer diagnostic agent, as well as in non-clinical and clinical trials for cancer therapeutic agents and cancer diagnostic agents. Can be used.

  In addition, since the hydrophilic group-introduced folic acid-modified cyclodextrin of the present invention shows excellent tumor accumulation ability, the inclusion of an anticancer agent enables the inclusion of the anticancer agent to the target cancer. Can be transported. In addition, by inclusion of a contrast agent, it can be efficiently transported to the cancer that is the target of the included contrast agent. Furthermore, the hydrophilic group-introduced folic acid-modified cyclodextrin of the present invention has high water solubility, and since it becomes a polymer, it enhances retention in the bloodstream, avoids RES (phagocytic cells), and is directly taken into cancer cells The effect can be exerted by active targeting by being activated and passive targeting by the EPR effect.

Doxorubicin (DOX) -encapsulated ND1 (per-Fol-cap2-β-CyD) and doxorubicin (DOX) -encapsulated PND1-1 (PEG-per-Fol-cap2-β-CyD) against KB (FR (+)) cells It is a graph showing anti-tumor activity. The vertical axis indicates the cell viability (%) after 24 hours of drug treatment, the horizontal axis indicates the type of the treated drug, and in order from the left, control (control), doxorubicin alone (DOX alone), doxorubicin / per- Fol-cap2-β-cyclodextrin (ND1) (molar ratio 1: 1) (DOX / cap2 (1: 1)), doxorubicin / PEG-per-Fol-cap2-β-cyclodextrin (PND1-1) (mol) Ratio 1: 1) (DOX / PEG-cap2 (1: 1)), per-Fol-cap2-β-cyclodextrin (ND1) (cap2 (10 μM)), and PEG-per-Fol-cap2-β- Cyclodextrin (PND1-1) (PEG-cap2 (10 μM)) is shown. Values are mean ± S. E. Indicates. The influence of PEG-per-Fol-cap-β-cyclodextrin (PND1-1 and PND1-3) on the antitumor activity of vinblastine is shown. The vertical axis represents the cell viability (%). In the above figure, the horizontal axis indicates the sample used, and FA (−) RPMI medium (control), VLB (10 μM), VLB / PND1-1 (1: 1) (VBL / ND1-PEG1 (1: 1) in this order. )), VBL / PND1-1 (1: 2) (VBL / PEG1 (1: 2)), ND1-PND1-1 (10 μM) (ND1-PEG1 (10 μM), PND1-1 (20 μM) (ND1-PEG1) In the figure below, the horizontal axis indicates the sample used, and FA (−) RPMI medium (control), VLB (10 μM), VLB / PND1-3 (1: 1) (VBL / ND1-PEG3 (1: 1)), VBL / PND1-3 (1: 2) (VBL / ND1-PEG3 (1: 2)), PND1-3 (10 μM), PND1-3 (20 μm) M) (ND1-PEG1 (20 μM)). The result of the uptake | capture evaluation of PND1-1 (FIG. 3A) and PND1-3 (FIG. 3B) in KB (FR +) and CHO (FR-) cell is shown. The fluorescence intensity of FITC of the cell by flow cytometry is shown. FIG. 3C shows the integrated amount (vertical axis) of the fluorescence intensity of cell FITC by flow cytometry. It is a graph which shows the result of having measured the influence of folic acid on the intracellular uptake of PND1-1 by KB (FR (+)) cell and CHO (FR (−)) cell. The bar graph in the lower graph shows the average fluorescence intensity of FITC-labeled PND1-1 (10 mM), FITC-labeled PND1-1 (10 mM) + folic acid (4 mM). It is a graph showing the influence of PND1-1 and PND1-3 on tumor weight and body weight in intravenous administration of doxorubicin (5 mg / kg) in vivo. The vertical axis represents (A) tumor volume (mm ³ ) and (B) body weight (g), respectively. The horizontal axis shows the elapsed time (days) from doxorubicin administration. Symbols in the figure are: white circle: control group (control), white diamond: doxorubicin single administration group (5 mg / kg) (DOX alone (5 mg / kg)), white triangle: doxorubicin / PND1-1 composite Body administration group (DOX / PND1-1), black triangle: doxorubicin / PND1-3 administration group (DOX / PND1-3), respectively. The influence of PND1-1 and PND1-3 on the survival rate of tumor mice is shown. Symbols in the figure are: white circle: control group (5% Mannitol), white diamond: doxorubicin single administration group (5 mg / kg), white triangle: doxorubicin / PND1-1 = 1: 1 complex administration group, Open square: shows doxorubicin / PND1-3 = 1: 1 complex administration group. n = 3 to 7, and the value represents an average value. It is a graph which shows the result of having investigated the influence of the ND201 derivative on the antitumor activity of doxorubicin (DOX) using (A) colon-26 cell and (B). A: MS spectrum of ND200-6HP which is an intermediate, and B: HPLC chromatogram of ND201-6HP obtained by leaf oxidation of ND200-6HP. Analytical HPLC conditions, column: SEC type (volume exclusion column), JAIGEL GS-310 (trade name, Nippon Analytical Industry), two φ21.5 mm × 500 mm, mobile phase: 1/15 M ammonium carbonate aqueous solution (pH 8.2) ) / Methanol = 100/5, flow rate: 5.0 ml / min, detection: UV258 nm The result of having evaluated the solubility of ND201 and ND201-6HP (ND201-HP) is shown. The vertical axis represents permeability, and the horizontal axis represents the amount (mL) of Meyron intravenous injection (pH 7.9). It is the photograph which compared doxorubicin distribution in the cell after adding doxorubicin, a doxorubicin / ND201 complex, and a doxorubicin / ND201-6HP complex to a KB cell. The illustration on the left is a schematic diagram of the experiment. The right photograph is a photograph of DIC (differential interference contrast: showing cells), TRITC label (showing ND201 or ND201-6HP), and their superposition (Merge). The comparison of the fluorescence intensity in doxorubicin, doxorubicin / ND201, and doxorubicin / ND201-6HP complex incorporated in KB cells is shown. The vertical axis represents the fluorescence intensity. It is the graph which compared the uptake | capture of ND201 and ND201-6HP by KB cell in the presence / absence of folic acid. The vertical axis represents the average fluorescence intensity (represents the amount of ND201 and ND201-6HP incorporated), and the horizontal axis represents the sample used. The sample shows ND201 in the absence of folic acid, ND201 in the presence of folic acid, ND201-6HP in the absence of folic acid, and ND201-6HP in the presence of folic acid in order from the left. It is a graph which shows the antitumor activity with respect to KB cell of doxorubicin independent, doxorubicin / ND201 complex, and doxorubicin / ND201-6HP complex. The vertical axis shows the cell viability (%) with the control as 100%, and the horizontal axis shows the sample used. The result of having evaluated the anti-tumor effect in vivo of ND201 and ND201-6HP complex by the tumor volume is shown. The vertical axis represents tumor volume (mm ³ ), and the horizontal axis represents the number of days after administration.

  The folic acid-modified cyclodextrin used as a raw material for the hydrophilic group-introduced folic acid-modified cyclodextrin of the present invention includes International Patent Publications WO 2004/085487 and WO 2009/041666, and Japanese Patent Application Publication No. 2008-285567, No. According to the method described in 2009-61429 and 2011-190341, it is compoundable. For example, the ND1 compound can be synthesized by the method described in Example 1 described in WO2009 / 041666.

  In one aspect, the invention provides a compound of the formula

  It is related with the manufacturing method of the compound represented by general formula (I) characterized by introduce | transducing a polyethyleneglycol group or a hydroxypropyl group into the 2-position, 3-position, or 6-position hydroxyl group of the compound represented by this.

(Introduction of a hydrophilic group at the 2nd or 3rd position of cyclodextrin)
Introduction of a hydrophilic group (polyethylene glycol group or hydroxypropyl group, the same shall apply hereinafter) to the folic acid-modified cyclodextrin at position 2 (X ³ ) or position 3 (X ¹ or X ² ) Fukudome et al., Tetrahedron Lett. , 47, 6599-6602 (2006), by modifying the secondary position of the folate-modified cyclodextrin.

  Specifically, the secondary hydroxyl group at the 2-position of cyclodextrin is monotosylated by the action of tosylimidazole, and then the C2-C3-position epoxy ring is formed with a base catalyst. Then, (1) the amino group of aminocaproic acid is reacted with the cyclodextrin at the 3rd or 2nd position and the remaining carboxylic acid group is reacted with the hydrophilic group amine, or (2) the hydrophilic group amine is reacted directly. Hydrophilic groups can be attached. The product can be purified by conventional HPLC column purification methods to give the compounds of the invention.

(Introduction of hydrophilic group to cyclodextrin position 6)
The introduction of a hydrophilic group at position 6 (R ¹ ) of folic acid-modified cyclodextrin is an intermediate in the process of synthesis of folic acid-modified cyclodextrin, and peraminocaproyl-β-cyclodextrin (hereinafter abbreviated as “ND100”). Prior to modification with folic acid, it is reacted with a hydrophilic group —COOH to introduce a hydrophilic group into part of the amino group (1-2 in one molecule of cyclodextrin), and then introduce folic acid into the remaining R ¹ Can also be synthesized. Many reactive hydrophilic group-COOH compounds are commercially available (for example, NOF Corporation, Tokyo).

  In another aspect, the present invention relates to a method for treating cancer comprising administering to a patient in need thereof an effective amount of the compound represented by the general formula (I) encapsulated with a substance having anticancer activity. Or it relates to a preventive method. Or this invention relates to the compound represented by the said general formula (I) which included the substance which has an anticancer effect | action for using for the treatment or prevention of cancer. In still another embodiment, the present invention relates to a diagnosis of cancer comprising administering to a patient in need thereof an effective amount of the compound represented by the general formula (I) encapsulated with a substance having the above-mentioned action. The present invention relates to a detection or labeling method. Alternatively, the present invention relates to a compound represented by the above general formula (I) in which a substance having a labeling action for use in diagnosis, detection or labeling of cancer is included.

  In the present specification, the term “cancer” includes epithelial malignant tumors, hematopoietic malignant tumors derived from the spinal cord, and in particular, ovarian cancer (such as non-mucinous ovarian cancer), uterine cancer, endometrial cancer. Breast cancer, breast cancer, prostate cancer, testicular cancer (such as testicular choriocarcinoma), brain cancer (such as ependymoma), throat cancer, lung cancer, lung adenocarcinoma, kidney cancer (such as renal cell cancer), liver cancer, colon cancer (such as Colon cancer, etc.), pleural mesothelioma, sarcoma, chronic and acute myeloid leukemia, and various metastatic cancers such as lung metastatic cancer.

  The substance having a labeling action that can be included in the compound represented by the general formula (I) is not particularly limited as long as it is a substance that can be included in cyclodextrin and can be used as a label. [3-18F] -α-methyltyrosine (18F-FMT) (for positron emission tomography (PET)); fluorescent dye compound such as fluorescein (for fluorescence / endoscopy); gadolinium such as gadolinium chelate aromatic compound (Gd) compound (for nuclear magnetic resonance imaging (MRI)); iodine compound such as barium compound, iodine, or 1,3,5-triiodobenzene (for X-ray computed tomography (X-ray CT)) Can be mentioned.

  The pharmaceutical composition of the present invention can be formulated by a conventional method using a normal pharmaceutically acceptable carrier. When preparing a solid preparation for oral administration, add excipients to the active ingredient and, if necessary, binders, disintegrants, lubricants, etc., and then add solvents, granules, powders, capsules, etc. by conventional methods. And When preparing an injection, a pH adjuster, a buffer, a stabilizer, a solubilizing agent, etc. may be added to the main drug as necessary to obtain a subcutaneous or intravenous injection by a conventional method.

  The pharmaceutical composition of the present invention can be used in oral dosage forms or parenteral dosage forms such as injections and drops. When this compound is administered to mammals or the like, it may be administered orally as tablets, powders, granules, syrups, etc., or may be administered parenterally as injections or drops. The dose can be appropriately set depending on the degree of symptoms, age, body weight, sex, administration route, administration form, reactivity to drugs, disease type, etc. For example, it is usually 50 to 500 mg per day per day for an adult. Administer in several divided doses.

  Hereinafter, examples will be shown to describe the present invention in more detail, but the present invention is not limited to these examples. It should be noted that all documents cited throughout this application are incorporated herein by reference in their entirety.

(Example 1) Synthesis of PND1-1 PND1-1 was synthesized by the route of ND1 → ND1-COOH → ND1-CONH-PEG.
Specifically, 100.5 mg of ND1 synthesized according to the method described in International Patent Publication WO2009 / 041666 was dissolved in 4 ml of DMSO, and 4.2 mg of 1- (p-toluenesulfonyl) imidazole and 4 mg of cesium carbonate were added to DMSO. A solution dissolved in 2 ml was added and reacted at room temperature for 40 hours. Next, to this reaction mixture, 6 mg of 6-amino-n-caproic acid (amino cap) and 58.8 mg of 1,4-diazabicyclo [2,2,2] octane (DABCO) were added and reacted for 24 hours. . This reaction solution was equipped with a SEC type column (JAIGEL GS-310 φ21.5 mm × 500 mm) in a preparative HPLC apparatus, and water was flowed as an eluent at a flow rate of 10.0 ml / min and eluted at 230 nm with UV detection. In the preparative HPLC chromatogram, the single sharp peak component (area ratio 35%) having a retention time of 6.05 min that flowed out first was collected and freeze-dried to obtain 40.0 mg.

  40.0 mg of the obtained product was dissolved in 4.0 ml of DMSO, 47.2 mg of N, N′-dicyclohexylcarbodiimide (DCC), 48.9 mg of N-imidosuccinic acid (HONSu) and PEG amine (NOF, (SUNBRIGHT MEPA-20H MW2000) was added to 38.7 mg and reacted at room temperature for 24 hours. A SEC column (JAIGEL GS-310 φ21.5 mm × 500 mm) was used for the HPLC apparatus, and the eluent was water: methanol 100: 5, and a peak of 12.7 min (21%) was collected, freeze-dried, and PND1- 1 was obtained. The yield was 65.9 mg (47% yield). Purity confirmation by analytical HPLC is 1 mg of 1 ml of 2.7 M ammonia solution 25 μl, SEC column (JAIGEL GS-310 7.6 mm × 300 mm) eluent 1/15 M PBS buffer solution, flow rate 1.0 ml / min, detection wavelength At 230 nm, a peak with an area ratio of 79% was observed at a retention time of 5.16 min. A peak area ratio of 21%, which is considered to be folic acid or its related substance, was observed at a retention time of 9.3 min. According to the measurement of PND1-1 by MALDI-TOF MS (Bruker Daltonics fexAnalysis), it has a wide peak at 4000 to 6000 centering on m / z 5000 and shows a sharp mountain distribution that seems to be a PEG group-introduced product. A main peak group was seen. Apart from this, a weak second peak group distributed at 6000 to 8000 centered on 7000 was also slightly observed. In this second peak group, a sharp mountain distribution indicating the introduction of PEG groups was not observed. The water solubility of PND1-1 was 10 mg / ml, which was more than 10 times that of ND1.

(Example 2) Synthesis of PND1-3 PND1-3 was synthesized by a route of ND1 → [ND1-epoxy] → ND1-PEG. Specifically, 21.4 mg of ND1 synthesized according to the method described in International Patent Publication WO2009 / 041666 is dissolved in 1 ml of DMSO, and 1.4 mg of 1- (p-toluenesulfonyl) imidazole and 0.9 mg of cesium carbonate are added. And allowed to react at room temperature for 19 hours. Next, 15 mg of 1,4-diazabicyclo [2,2,2] octane (DABCO) and 80 mg of PEG amine (NOF, MW2000) were added to the reaction mixture and reacted at room temperature for 24 hours. Using two SEC columns JAIGEL GS-310 φ21.5 mm × 500 mm, a peak of 13.2 min (49%) at a detection wavelength of 230 nm was fractionated with eluent water, and freeze-dried to obtain PND1-3. The yield was 4.6 mg. Measurement of this substance by MALDI-TOF MS (Bruker Daltonics fex Analysis) has a broad peak at 4000 to 6000 centering on m / z 5000, and a main peak group showing a sharp mountain distribution that seems to be a PEG group-introduced product. It was seen. The water solubility of PND1-3 was 10 mg / ml, which was more than 10 times that of ND1.

(Example 3) In vitro cell growth inhibitory action of doxorubicin-encapsulated PND1-1 KB cells (5 × 10 ⁴ cells / well) were cultured in a medium not containing folic acid (RPM11640SigmaAldrich) at 37 ° C. for 24 hours. After washing the cells once with PBS, 10 μM doxorubicin (DOX), 10 μM doxorubicin / per-Fol-cap2-β-cyclodextrin (ND1) (molar ratio 1: 1), doxorubicin / PEG-per-Fol-cap2-β -Cyclodextrin (PND1-1) (molar ratio 1: 1) 10 μM, per-Fol-cap2-β-cyclodextrin (ND1) 10 μM, and PEG-per-Fol-cap2-β-cyclodextrin (PND1-1) ) It was cultured at 37 ° C. for 24 hours in 150 μL of medium containing 10 μM. n = 8-11. After washing once with PBS, 100 μL of fresh HBSS and 10 μL of WST-1 reagent were added to each well and incubated at 37 ° C. for 30 minutes. The cell viability (%) was calculated by measuring with a plate reader (S: 450 nm, R: 620 nm) and calculating that only cells having metabolic activity showed fluorescence when the cell growth reagent WST-1 was used.

  The results are shown in FIG. In vitro, doxorubicin-encapsulated PND1-1 showed cell growth inhibitory effects comparable to doxorubicin alone and doxorubicin-encapsulated ND1.

Example 4 In Vitro Cell Growth Inhibitory Effect of Vinblastine Encapsulation PND1-1 and PND1-3 Inhibiting the Antitumor Activity of Vinblastine PEG-per-Fol-cap-β-cyclodextrin (PND1-1 and PND1-3) The influence of was investigated. 5 × 10 ⁴ KB cells were prepared in a 96-well plate. Incubate at 37 ° C. for 24 hours and wash once with PBS (150 μL. Then add 150 μL of each sample. Incubate for 24 hours at 37 ° C. Wash with PBS (150 μL). Add WST-1 (10 μL). HBSS (100 μL) was added and measured with a plate reader (S: 450 nm, R: 620 nm) As a control, FA (−) RPMI medium without folic acid, VLB alone (10 μM), VLB / PND1-1 (1: 1), VBL / PND1-1 (1: 2), PND1-1 (10 μM), and PND1-1 (20 μM) were used.

  The results are shown in FIG. The antitumor activity of vinblastine and PND1-1 complex in KB cells was not 1: 1, but was exhibited by 1: 2. The antitumor activity of vinblastine and PND1-3 complex was significantly observed at 1: 2.

(Example 5) Evaluation of intracellular uptake of PND1-1 and PND1-3 by KB cells (FR (+)) and CHO cells (FR (-)) PND1-1 KB (FR +) and CHO (FR-) cells Internal uptake evaluation was performed. KB cells or CHO cells were prepared to 4 × 10 ⁵ cells / 35 mm dish. After incubation (37 ° C., 24 h), various samples (1 mL) were added once by washing with a medium (1 mL) × 1. Then, after incubation (37 ° C., 1 h), the cells were lysed with PBS (1 mL) × 1 times and PBS (1 mL) and FACS-treated (FL1). After incubation for 1 hour at 37 ° C. by flow cytometry, the FITC fluorescence intensity of the cells was determined. As various samples, control: FA (−) medium, FITC-labeled PND1-1 (10 μM), and FITC-labeled PND1-3 (10 μM) were used.

Next, uptake of PND1-3 into KB (FR +) and CHO (FR-) cells was evaluated. In the experimental operation, KB cells or CHO cells were prepared to 4 × 10 ⁵ cells / 35 mm dish. After incubation (37 ° C., 24 h), various samples (1 mL) were added once by washing with a medium (1 mL) × 1. Then, after incubation (37 ° C., 1 h), the cells were lysed with PBS (1 mL) × 1 times and PBS (1 mL) and FACS-treated (FL1). After incubation for 1 hour at 37 ° C. by flow cytometry, the FITC fluorescence intensity of the cells was determined. As various samples, control: FA (−) medium, FITC-labeled PND1-1 (10 μM), and FITC-labeled PND1-3 (10 μM) were used. The concentration of PND1-1 and PND1-3 is 10 μM.

  The results are shown in FIG. The lower bar graph shows the result of calculating the integrated value of FITC fluorescence intensity of cells after 1 hour incubation at 37 ° C. using flow cytometry. According to this, the uptake of PND1-3 was significantly increased 2-fold compared with PND1-1. The uptake was low in CHO cells with low FR.

(Example 6) Evaluation of influence of folic acid on intracellular uptake of PND1-1 and PND1-3 by KB cells (FR (+)) and CHO cells (FR (-)) (1) Influence of folic acid on intracellular uptake Effect Prepare KB cells and CHO cells at 4 × 10 ⁵ cells / 35 mm dish, and after incubation (37 ° C., 23 h), wash with medium (1 mL) × 1 times and add folic acid (4 mM: 1 mL) Incubation (37 ° C., 1 h, folic acid added group only). Next, various samples (1 mL) were added, incubated (37 ° C., 1 h), washed with PBS (1 mL) × 1 time, cells were lysed with PBS (1 mL), and FACS-treated (FL1). Samples were control: FA (−) medium, FITC labeled PND1-1 (10 mM), FITC labeled PND1-1 (10 mM) + folic acid (4 mM), FITC labeled PND1-3 (10 mM), and RITC labeled ND1-3 (10 mM) + folic acid (4 mM) was used. The FICT fluorescence intensity of the cells was measured by a flow cytometer after incubation at 37 ° C. for 1 hour. The concentration of PND1-1 is 10 mM, and the concentration of folic acid is 4 mM.

(2) Effect of folic acid on intracellular uptake KB cells and CHO cells were prepared to 4 × 10 ⁵ cells / 35 mm dish, incubated (37 ° C., 23 h), washed with medium (1 mL) × 1 time, and folic acid ( 4 mM: 1 mL) was added and pre-incubation (37 ° C., 1 h, folic acid added group only). Next, various samples (1 mL) were added, incubated (37 ° C., 1 h), washed with PBS (1 mL) × 1 time, cells were lysed with PBS (1 mL), and FACS-treated (FL1). Samples were control: FA (−) medium, FITC labeled PND1-1 (10 mM), FITC labeled PND1-1 (10 mM) + folic acid (4 mM), FITC labeled PND1-3 (10 mM), and RITC labeled ND1-3 (10 mM) + folic acid (4 mM). The FICT fluorescence intensity of the cells was measured by a flow cytometer after incubation at 37 ° C. for 1 hour. The concentration of PND1-1 is 10 mM, and the concentration of folic acid is 4 mM.

  The results are shown in FIG. The effect of adding folic acid is summarized in the bar graph at the bottom of FIG. No inhibitory effect due to the addition of folic acid (4 mM) was observed in either KB or CHO cells.

(Example 7) In vivo antitumor action of doxorubicin-encapsulated PND1-1 and PND1-3 (1) Influence of PND1-1 and PND1-3 on the antitumor effect of doxorubicin (in vivo)
BALB / c mice (×, 4 weeks old) were inoculated with Colon-26 cells (2 × 10 ⁵ cells / 100 mL) on the left hind limb. After confirming that the tumor diameter was 8 mm, doxorubicin / PND1-1, doxorubicin / PND1-3 (1: 1, 100 μL) was intravenously administered. Tumor volume and body weight were measured over time for about 30 days.

(2) Effect of PND1-1 and PND1-3 on survival rate of tumor mice The following samples (100 μL) were intravenously administered to tumor-bearing mice (BALB / c male, Colon-26 cells). 5% Mannitol (control), doxorubicin alone (5 mg / kg), doxorubicin / PND1-1 = 1: 1 complex, doxorubicin / PND1-3 = 1: 1 complex. n was set to 3-7.

  The results are shown in FIGS. From FIG. 5, DOX and PND1-1 and PND1-3 completely suppressed tumor growth over 30 days. It is estimated that there is no weight change and no side effects. The survival rate in FIG. 6 was 100% or more for PND1-3 over 100 days. Even PND1-1 was 100% for 80 days or more. Good anti-tumor activity was seen.

Synthesis PND1-4 (Example 8) PND1-4 was synthesized in _{ND1 → ND1-NH 2 → ND1} -NHCO-PEG path. Specifically, ND1100.5 mg synthesized according to the method described in International Patent Publication WO2009 / 041666 is dissolved in 4 ml of DMSO, 4.2 mg of 1- (p-toluenesulfonyl) imidazole and 2 mg of cesium carbonate are added, and For 40 hours. Next, 3.0 mg of hexamethylene diamine and 58.8 mg of 1,4-diazabicyclo [2,2,2] octane (DABCO) were added to this reaction mixture, and reacted for 24 hours. This reaction solution was fractionated on a SEC column JAIGELGS-310φ21.5 mm × 500 mm using water as an eluent at a flow rate of 10.0 ml / min and a detection of 230 nm. In the HPLC chromatogram, the first sharp peak component (area ratio 35%) that flowed out first was collected and freeze-dried to obtain 38.0 mg of ND1 hexamethylene derivative.

  PEG2000OSuME-020AS (NOF) 2.77 mg was reacted with 1 mg of DMSO solution overnight at room temperature for 8 mg of ND1 hexamethylene derivative. The product was purified by HPLC as it was. Using a SEC column JAIGEL φ21.5 mm × 500 mm, a peak retention time of 12.5 min was collected at a detection wavelength of 230 mm, and lyophilized to obtain 2.4 mg (30%) of PND1-4. The HPLC purity was 98%.

Example 9 Synthesis of PND1-2 PND1-2 was synthesized by the route of ND100 (Cap2) + PEG-OSu → ND100-NHCO-PEG → ND1-PEG (primary position). Specifically, ND1 precursor 6-heptakis (5-aminocaprylamidecaprylamide) -β-cyclodextrin synthesized according to the method described in International Patent Publication WO2009 / 041666 was charged with NMM 2.6 mg and PEGOSu 7.4 mg overnight at room temperature. After the reaction, 51.9 mg of folic acid and 33.3 mg of DMT-MM were reacted at room temperature for 45 hours. The product was purified by HPLC as it was. The first peak was collected and lyophilized to obtain 20.0 mg of PND1-2.

(Example 10) Synthesis of PND1-7 PND1-7 was synthesized by the route of ND100 → ND100-NHCO-PEG (STEP1) and ND100-NHCO-PEG → PND1-7 (STEP2).

(Step 1) 200.0 mg of 6-heptakis (5-aminocaprylamidecaprylamide) -β-cyclodextrin (ND100) synthesized according to the method described in International Patent Publication WO2009 / 041666 is dissolved in 4 ml of methanol, and PEG-COOH ( NOF: ME-020-AC (MW2000)) 118.3 mg, DMT-MM 21.3 mg and N-methylmorpholine 100 μl were added and stirred at room temperature for 16 hours, followed by heating at 35 ° C. for 23 hours. Add 4 ml of 1/15 M ammonium carbonate aqueous solution to 8 ml of this reaction solution, and use HPLC equipment to add 1/15 M ammonium carbonate aqueous solution (pH 8.2) to two SEC columns (JAIGELGS-310φ21.5 mm × 500 mm). The eluent was collected at a flow rate of 5.0 ml / min and a detection UV of 286 nm. In the HPLC chromatogram, the components of the single sharp peak A flowing out first and the single sharp peak B flowing out second were separated. Each was freeze-dried to obtain two white powders A (23.5 mg) and B (85.9 mg). In the measurement of this product by MALDI-TOFMS (Bruker Daltonicsfex Analysis), A showed a broad peak presumed to be ND100 in which the PEG group was 2-substituted at 6600 to 8000 centering on m / z 7220. Further, a trace amount of a broad peak seen as PEG-COOH was observed near m / z 2000. In B, a broad peak presumed to be ND100 in which 1 to PEG group was substituted at 4100 to 5900 around m / z 4966 was observed. Further, a trace amount of a broad peak seen as PEG-COOH was observed near m / z 2000. Similarly, a trace amount of a broad peak presumed to be ND100 in which the PEG group was substituted by 2 near m / z 7000 was confirmed. When the impurities were ignored, the yield of ND100 with 2-substituted PEG groups was 4.0%, and the yield of ND100 with 1-substituted PEG groups was 24%.

(Step 2) ND10080.8 mg with 1 substituted PEG group and 189.3 mg of folic acid were dissolved in 40 ml of DMSO, 137.8 mg of DMT-MM and 50 ml of methanol were added and reacted at 35 ° C. for 48 hours. It was confirmed under the following conditions by analytical HPLC with a SEC column that the resulting compound was Exemplified Compound ND1-PEG # 7 (PND1-7). A peak different from that of the starting material ND100-NHCO-PEG was observed at around 9.0 minutes. The progress of the reaction was confirmed under the following conditions using an analytical HPLC equipped with a SEC column, and it was confirmed that the progress of the reaction was completed in 48 hours.

Analytical HPLC condition column: SEC type (volume exclusion column)
JAIGEL GS-310-A (trade name, manufactured by Nippon Analytical Industry)
φ7mm × 500mm
Mobile phase: 1/15 M aqueous ammonium carbonate solution (pH 8.2) / methanol = 100/5
Flow rate: 1.0 ml / min
Detection: UV258nm

  The reaction mixture formed a precipitate in acetone. The precipitate was separated by filtration and then added with a 1/15 M aqueous ammonium carbonate solution adjusted to pH 8.2 to make 50 ml, and fractionated using a preparative HPLC equipped with a SEC column under the following conditions. In the HPLC chromatogram, the component of the single sharp peak that flowed out first (retention time around 26 minutes) was collected and lyophilized to obtain 49.9 mg (42% yield) of PND1-7. The HPLC purity of the fraction was 97%. In addition, it was confirmed that free folic acid was present at a peak area ratio of 4%. This indicates the presence of 1.4 molecules of folic acid per molecule of PND1-7.

  Measurement of this product by MALDI-TOF MS (Bruker Daltonics fex Analysis) showed a broad peak presumed to be PEG-free ND1 at 4500-6000 centering on m / z 5000. Further, there was a broad peak at 6000 to 8000 centering on m / z 7200, and a peak showing a mountain-like distribution that seems to have one PEG group was observed. The peak of two or more PEG groups and the peak of the raw material ND100 were not observed.

Preparative HPLC condition column: SEC type (volume exclusion column)
JAIGEL GS-310 (trade name, Japan Analytical Industry)
Two mobile phases of φ21.5 mm × 500 mm: 1/15 M ammonium carbonate aqueous solution (pH 8.2) / methanol = 100/5
Flow rate: 5.0 ml / min
Detection: UV258nm

(Example 11) Continuous simple synthesis of PND1-7 20.3 mg of 6-heptakis (5-aminocaprylamidecaprylamide) -β-cyclodextrin (ND100) synthesized according to the method described in International Patent Publication WO2009 / 041666 was added to 5 ml. Dissolved in methanol, 17 mg of PEG-COOH (NOF), 10.0 mg of DMT-MM and 100 μl of N-methylmorpholine were added, and the mixture was heated at 35 ° C. for 23 hours. Subsequently, 167 mg of folic acid, 202.5 mg of DMT-MM, and 3 ml of methanol were added and reacted at 35 ° C. for 94 hours.

  The purification operation is as follows. This reaction solution was made up to 50 ml by adding 1/15 M ammonium carbonate aqueous solution, and SEC column JAIGEL GS-310 φ21.5 mm × 500 mm was used as two eluents with ammonium carbonate aqueous solution (pH 8.0) at a flow rate of 7.0 ml / min. And fractionated at detection UV 286 nm. In the HPLC chromatogram, the component of the single sharp peak flowing out first was collected and lyophilized to obtain 16.4 mg (yield 30%) of PEGylated ND1 (PND1-7). The HPLC purity of the fraction was 78%. In addition, it was confirmed that free folic acid was present at a peak area ratio of 18%. Measurement of this product by MALDI-TOF MS (Bruker Daltonics fex Analysis) showed a broad peak presumed to be PEG-free ND1 at 4500-6000 centering on m / z 5000. Further, there was a broad peak at 6000 to 8000 centering on m / z 7200, and a peak showing a mountain-like distribution that seems to have one PEG group was observed. The peak of two or more PEG groups and the peak of the raw material ND100 were not observed. As a result of observing the same product by proton NMR, it was confirmed that 9 molecules of free folic acid were present.

  Free folic acid that could not be removed by preparative HPLC purification caused the product to form a precipitate in a mixture of water and acetone. The precipitate was dissolved again in water and adjusted to pH 5. The precipitate formed there was removed by Celite filtration, and the solution was freeze-dried to obtain a product. In the analytical HPLC chromatogram, the residual free folic acid was determined to be 3% or less in area ratio. The area ratio of the product ND1-PEG7 was 95%.

(Example 12) In vitro evaluation of PND1-7 (1) Effect of ND201 derivative on antitumor activity of DOX (Colon 26)
Colon-26 cells were seeded at 5 × 10 ⁴ cells / well in a 96-well plate using RPMI 1640 (Fol-free) medium containing 10% (v / v) FBS. After incubation (24 h, 37 ° C.), the plate was washed with 150 mL of PBS. Next, 150 μL of serum-free RPMI 1640 (Fol-free) medium containing various samples was added. Samples were serum-free RPMI 1640 (Fol-free) medium (Control), doxorubicin alone (10 mM) (DOX alone), DOX / PND1-7 (molar ratio 1: 1) (10 mM), DOX / 8HP-ND201 (molar ratio) 1: 1) (10 mM). Next, it was incubated (24 h, 37 ° C.) and washed with 150 mL of PBS. HBSS (100 mL) and WST-1 solution (10 mL) were added, followed by incubation (30 min, 37 ° C.). Absorbance measurement with a microplate reader (S: 450 nm, R: 620 nm)

(2) Effect of ND201 derivative on antitumor activity of DOX (KB cell, see FIG. 8B)
KB cells were seeded at 5 × 10 ⁴ cells / well in a 96-well plate using RPMI 1640 (Fol-free) medium containing 10% (v / v) FBS. After culturing (24 hours, 37 ° C.), the plate was washed with 150 μL of PBS. 150 μL of sample-containing serum-free RPMI 1640 (Fol-free) medium was added. Samples were serum-free RPMI 1640 (Fol-free) medium (Control), doxorubicin alone (10 mM) (DOXalone, DOX / PND1-7 (molar ratio 1: 1) (10 mM), DOX / 8HP-ND201 (molar ratio 1: 1) (10 mM) After culturing (24 h, 37 ° C.), washing with 150 mL of PBS, adding 100 mL of HBSS and 10 μL of WST-1 solution, incubating (30 min, 37 ° C.), and then using a microplate reader The absorbance was measured (S: 450 nm, R: 620 nm).

  The results are shown in FIG. In the in vitro experiment, in the Colon-26 cells, the cell killing effect was observed in any of the complexes of ND201-8HP and PND1-7. The same was true for KB cells.

Example 13 Synthesis of PND1-8 PND1-8 was synthesized by the route of ND100 → ND100-NHCONH-PEG → PND1-8. Specifically, 50.6 mg of PEG-COOH (NOF Corporation SUNBRIGHT ME-020AC) in a nitrogen stream is made into 5 ml of DMSO solution, 50 μl of DPPA and 15.2 mg of DABCO are added, and the oil bath is heated at 125 ° C. Heated for hours. After cooling to room temperature, 77.6 mg of ND1 precursor 6-heptakis (5-aminocaprylamidecaprylamide) -β-cyclodextrin (ND100) synthesized according to the method described in International Patent Publication WO2009 / 041666 is dissolved in 3 ml of DMSO. And added. Further, 30 mg of DABCO was added and reacted at room temperature for 45 hours. Next, 233 mg of folic acid, equimolar amounts of DMT-MM and 58.8 mg of 1,4-diazabicyclo [2,2,2] octane (DABCO) were added to the reaction mixture and reacted for 45 hours.

  This reaction solution was fractionated into a SEC column JAIGEL GS-310 φ21.5 mm × 500 mm with an ammonium carbonate aqueous solution (pH 8.0) as an eluent at a flow rate of 7.0 ml / min and detection of 286 nm. In the HPLC chromatogram, the first sharp peak component (area ratio 34%) that flowed out first was collected and freeze-dried to obtain 30 mg of PEGylated ND1 (PND1-8) (yield 32%). The HPLC purity was 80%.

  The product was measured by MALDI-TOF MS (Bruker Daltonics fex Analysis), with a broad peak at 4500-6500 centering on m / z 5323, showing a single distribution indicating a single PEG group. The peak of was seen. The peak of 3081 of ND100 of the raw material and PEG body 2290 was also seen.

  In PND1-8, PEG-COOH is changed to PEG-NCO with a DPPA reagent to increase the reactivity, and after a 1: 1 molar ratio reaction is performed, the PEG-introduced bond is a urethane bond. Folic acid was introduced into the remaining R2 position through an amide bond. Compared with PND1-7, the synthesis was completed in a short time and with good yield.

  Here, in the synthesis of PND1-7 and PND1-8, the number of folic acids introduced is inevitably 6 instead of 7. There is a concern that the nanocluster effect may be reduced by that amount, but empirically, there is an example where 6 associations showed the same association with the antibody, and it is considered that there is no influence. Moreover, since PEG-NCO used in the synthesis of PND1-8 has good reactivity with the secondary hydroxyl group, it can be introduced into the secondary position.

Example 14 Synthesis of ND201-6HP After dissolving 300 mg of ND200 (Per (HCl · aminoCap) -β-CD) in 5.0 ml of 0.37 M NaOH aqueous solution at room temperature, 67.5 μl while cooling in an ice bath. Of propylene oxide was slowly added dropwise over 20 minutes. Thereafter, the mixture was returned to room temperature and stirred for 20 hours. The completion of the reaction was confirmed by TLC, and disappearance of the raw material spots was confirmed. The reaction was terminated by neutralization with 0.5M HCl. Dialyzed to remove salt. The dialysate was changed once. After lyophilization, HPD ND200 was obtained in a yield of 100.3 mg.

  Next, 701.0 mg of folic acid was added to 40 ml of DMSO and dissolved by heating to 70 ° C. 100.3 mg of HP ND200 obtained earlier was dissolved in 12.9 ml of DMSO at room temperature. To this solution, 174.6 μl of N-methylmorpholine and 439.4 mg of DMT-MM were dissolved in a mixed solution of 100 μl of methanol and 2 ml of DMSO. The same amount of DMT-MM solution was added 5 times over 11 days, and the progress of the reaction was followed by HPLC. After 11 days, acetone was reprecipitated and suction filtered. The sample was dialyzed using 1M ammonia. After dialysis was repeated twice, the solution was concentrated, dissolved in ammonia carbonate solution, and fractionated by HPLC to obtain ND201-6HP. The yield was 59.9 mg. The product was confirmed by TLC (developing solution: butanol, ethanol, water, ammonia, coloration: iodine) with an Rf value of 0.67. HP ND200 was confirmed by MS spectrum, and the structure was also confirmed by HP ND201 HPLC chromatogram.

  FIG. 8 shows the MS spectrum data used to confirm the structure of HP-converted ND200 and the HPLC data of HP-ND201. From the MS spectrum, 1-9 HP groups were introduced. From the HPLC data, the production of a leaf-oxidized product was confirmed at a retention time consistent with ND201.

(Example 15) Evaluation of solubility of ND201-6HP 4.0 mg of ND201 or ND201-6HP was dissolved in 1.5 ml of Meyron's intravenous solution (pH 7.9) and treated with ultrasound at room temperature for 30 minutes. The transmittance at 660 nm was measured. Solvents were sequentially added thereto, and ultrasonic treatment and transmittance measurement were repeated. The maximum dissolution concentration was observed for ND201-6HP with a transmittance of 57.9%, whereas ND201 had a transmittance of 28% under 9 ml of solvent. The transmittance was converted to absorbance, and the ratio of dissolved concentration was determined from the comparison.

  The results are shown in FIG. From the comparison of 660 nm transmittance% in 9 ml of intravenous injection of Meyron (pH 7.9), A = −log T (−log 0.579 / −log 0.28) = 0.5527 / 0.2380 = 2.32 Sex ratio was sought. The solubility of ND201-6HP increased by 2.32 times as compared with ND201 due to HP conversion.

(Example 16) Evaluation of intracellular uptake of ND201-6HP by KB cells (FR (+)) Comparison of distribution amount of doxorubicin in doxorubicin, doxorubicin / ND201 complex and doxorubicin / ND201-6HP complex in KB cells did. KB cells (5 × 10 ⁴ cells / well) were cultured in a folic acid-free medium at 37 ° C. for 24 hours. The cells were washed once with a folic acid-free medium and then cultured at 37 ° C. for 24 hours in 1 ml of each medium containing doxorubicin, doxorubicin / ND201, and doxorubicin / ND201-6HP. After washing without folic acid, it was scanned with a fluorescence microscope. The concentration of doxorubicin, ND201 and ND201-6HP is 10 μM.

  The results are shown in FIGS. ND201-6HP had almost the same uptake into KB cells in vitro as ND201. As a result, it was shown that the amount of ND201-6HP incorporated into the cells of doxorubicin was similar to that of ND201.

(Example 17) Evaluation of the influence of folic acid on intracellular uptake of ND201-6HP by KB cells (FR (+)) TRITC-labeled ND201 and TRITC-labeled ND201-6HP folic acid into cells The effect of uptake was examined. The fluorescence intensity of TRITC was measured after incubation at 37 ° C. for 1 hour with a flow cytometer. The concentration of ND201 and ND201-6HP is 10 μM. The concentration of folic acid in the ND201 and ND201-HP systems is 10 μM.

  The results are shown in FIG. TRITC labeled ND201-6HP showed up to 4 times the uptake of KB cells compared to TRITC labeled ND201. However, uptake was greatly reduced by the addition of folic acid (10 mM). This is thought to be an inhibitory effect of folic acid on this system.

(Example 18) In vitro cell growth inhibitory action of doxorubicin-encapsulated ND201-6HP shows the antitumor activity of doxorubicin, doxorubicin / ND201 complex and doxorubicin / ND201-6HP complex in KB cells treated for 24 hours. The concentration of doxorubicin, ND201, and ND201-6HP is 10 μM.

  The results are shown in FIG. The doxorubicin-encapsulated ND201-6HP was further improved in the KB tumor cell growth inhibitory effect than ND201.

(Example 19) In vivo antitumor effect of doxorubicin-encapsulated ND201-6HP in the left hind limb of BALB / c male mice (4 weeks old, 20 g) 10% (v / v) FBS-containing RPMI 1640 (Fol) -Free) Colon-26 cell suspension cultured in medium was seeded at 2 × 10 ⁵ cells / 100 μL. About 10 days later, mice whose tumor diameter reached 8 mm were used for in vivo experiments. 100 μL of mannitol solution (control), doxorubicin solution (DOX 5 mg / kg), DOX / ND201, DOX / ND201-6HP complex solution (DOX / ND201-HP) were administered into the tail vein of cancer-bearing mice. Doxorubicin was used in an amount corresponding to 5 mg / kg. Tumor volume and body weight were followed over time for 31 days after administration. Survival was monitored.

The results are shown in FIG. Doxorubicin-encapsulated ND201-6HP showed a tumor growth inhibitory effect about 3 times that of ND201 over 30 days.

Claims (1)

Cyclodextrin compound represented by the following general formula (I)
[Wherein m represents an integer of 6 to 8,
m R ¹ 's each independently represent a hydrogen atom, a hydroxyl group, -NH-Y-R ² group, -NHCONH-Y-R ² group, -NHCO-Y-R ² group, -NH-Y-NHCO. -Y-NH-Y-R ² group, -NH-Y-NHCO-Y-R ² group, -NHCO-Y-NHCO-Y-NH-R ² group, -NHCO-Y-NHCO-Y-NHCO- Y—R ² group, —NHCONH—Y—NHCO—Y—NHCO—Y—R ² group, —Z group, —NH—Y—R ² —Z group, —NHCONH—Y—R ² —Z group, — ^NHCO-Y-R 2 -Z group, -NH-Y-NHCO-Y -NH-Y-R 2 -Z group, -NH-Y-NHCO-Y -R 2 -Z group, -NHCO-Y-NHCO ^-Y-NH-R 2 -Z group, and, -NHCO-Y-NHCO-Y -NHCO-Y-R 2 -Z group, -NHCONH Y-NHCO-Y-NHCO- Y-R 2 -Z group, represents a group selected from the group consisting of,
Here, at least two of the m R ¹ groups are —Z group, —NH—Y—R ² —Z group, —NHCONH—Y—R ² —Z group, —NHCO—Y—R ² —. Z group, -NH-Y-NHCO-Y -NH-Y-R 2 -Z group, -NH-Y-NHCO-Y -R 2 -Z group, -NHCO-Y-NHCO-Y -NH-R 2 -Z group, and a group selected from the group consisting of -NHCO-Y-NHCO-Y- NHCO-Y-R 2 -Z group,
m X ¹ , m X ² , and m X ³ are each independently a hydrogen atom, a hydroxyl group, —R ² , —NH—Y—R ² group, —NHCONH—Y—R ^2. Groups, —NHCO—Y—R ² groups, —NH—Y—NHCO—Y—NH—Y—R ² groups, —NH—Y—CONH—Y—R ² groups, and —NH—Y—NHCO— Represents a group selected from the group consisting of YR ² groups;
Here, each Y independently represents a group selected from an alkylene group, a phenylene group, and a -O- group,
Here, each R ² independently represents a polyethylene glycol group or a hydroxypropyl group,
Here, the Z group represents a group represented by the following formula (II)
In the formula, n represents an integer of 0 to 3.]