JP6788278B2 - A pharmaceutical composition comprising a conjugate of a boron-containing compound and a protein. - Google Patents

Description

The present invention relates to pharmaceutical compositions comprising a conjugate of a compound containing boron-10 ( ¹⁰ B) and a tumor-accumulating protein.

Boron Neutron Capture Therapy (BNCT) is a low-energy thermal neutron that is harmless to the human body and is captured by a boron drug, and the reaction between the thermal neutron and ¹⁰ B causes lithium in a microenvironment inside a cell. It is a new cancer treatment method that generates α rays and destroys cancer cells.

Unlike high-energy fast neutrons, low-energy thermal neutrons are harmless to the human body, but the reaction between thermal neutrons and ¹⁰ B produces lithium and helium nuclei (alpha rays), which have an energy of 2.4 MeV. And enough energy to destroy about one cell.

The ideal method of treating cancer is to kill the cancer cells without seriously damaging normal tissue, and selectively deliver boron to the cancer cells for effective therapeutic effect by BNCT. There is a need.

Around the tumor tissue where angioplasty is immature, fine particles such as liposomes tend to accumulate (EPR effect, Enhanced permeation and retention effect). Based on this property, a system for delivering boron to cancer cells using liposomes has been developed. For example, the present inventors have succeeded in developing a system capable of delivering a boron compound at a very high concentration by introducing a boron compound into a lipid bilayer membrane of a liposome (Non-Patent Document 1). In addition, by introducing the boron compound into the liposome membrane into which the boron compound has been introduced, we have succeeded in highly integrating boron (Non-Patent Document 2).

H. Nakamura, Y. Miyajima, T. Takei, S. Kasaoka, K. Maruyama, Chem. Commun. 1910-1911 (2004) H. Koganei, M. Ueno, S. Tachikawa, L. Tasaki, HS Ban, M. Suzuki, K. Shiraishi, K. Kawano, M. Yokoyama, Y. Maitani, K. Ono, H. Nakamura, Bioconjugate Chem. 24, 124-132 (2013)

In this way, the delivery system using liposomes can deliver a large amount of boron to cancer cells, but the selectivity for cancer cells is not very high, and boron is delivered not only to cancer cells but also to the kidneys and spleen. There was a problem of accumulating.

The present invention is to solve the problems of such a conventional boron delivery system and provide a means for selectively delivering a large amount of boron to cancer tissue.

As a result of diligent studies to solve the above problems, the present inventor has a high selectivity in which a boron cluster such as crosododecaborate is bound to maleimide and further, albumin is bound to this compound. It was found to be taken up by tumor tissue. Initially, maleimide was thought to bind only to free cysteine residues in albumin (cysteine residues that do not form disulfide bonds, Cys 34), but it also binds to lysine residues in albumin. We found that we were doing this, and obtained the following ideas from this finding.
1) Since the number of lysine residues present in a protein is much larger than the number of free cysteine residues, it is possible to bind a very large number of maleimide molecules to one protein molecule.
2) Boron clusters can be attached to proteins that do not have free cysteine residues, such as transferrin, via maleimide.
3) A lysine-binding group such as an isothiocyanate group can be used instead of maleimide to bind a boron cluster to a protein.
The present invention has been completed based on the above findings.

That is, the present invention provides the following (1) to (8).
(1) Formula (I): X-LY [In the formula, X represents a group containing ¹⁰ B, L represents a linker, and Y represents a group that binds to a lysine residue. ], Which is a pharmaceutical composition containing a conjugate of a compound represented by lysine residue and a tumor-accumulating protein having a lysine residue, and is a molar ratio of the compound to the protein contained in the composition (the number of moles of the compound). / A pharmaceutical composition characterized in that the number of moles of the protein) is a molar ratio of more than 10.

(2) The pharmaceutical composition according to (1), wherein the tumor-accumulating protein having a lysine residue is transferrin.

(3) The pharmaceutical composition according to (1), wherein the tumor-accumulating protein having a lysine residue is albumin.

(4) The pharmaceutical composition according to any one of (1) to (3), wherein Y in the formula (I) is a maleimidyl group or an isothiocyanate group.

(5) The pharmaceutical composition according to any one of (1) to (4), wherein X in the formula (I) is a group derived from a boron cluster.

(6) The pharmaceutical composition according to any one of (1) to (4), wherein X in the formula (I) is a group derived from crosododecaborate, crosocarborane, or nidcarborane.

(7) Even if L in the formula (I) is an alkylene group (provided that one or more -CH _2- of the alkylene group is substituted with -O-, -S-, -NH-, or -CO- The pharmaceutical composition according to any one of (1) to (6), which is characterized by being (good).

(8) The pharmaceutical composition according to any one of (1) to (7), which is used for boron neutron capture therapy.

This specification includes the contents described in the Japanese patent application, Japanese Patent Application No. 2015-156726 and / or drawings which are the basis of the priority of the present application.

Since the pharmaceutical composition of the present invention is selectively taken up by tumor cells and hardly taken up by normal cells, tumor cells can be selectively destroyed. In addition, since the amount of boron taken up by tumor cells is large, the dose can be reduced.

The figure which shows the synthesis path of MID. The figure which shows the biological distribution of the conjugate of MID and albumin (BSA). The figure which shows the result of the binding experiment of MID and albumin (BSA). The figure which shows the result of the concentration-dependent binding experiment between MID and albumin (BSA). The figure which shows the result of the time-dependent binding experiment between MID and albumin (BSA). Reaction time: 1h (lane1-3), 3h (lane 4), 6h (lane 5), 12h (lane 6), 24h (lane 7), Buffer pH = 7.4. The figure which shows the result of the binding experiment of NCS-dodecaborate sodium salt (compound 11) and transferrin. Buffer (PBS): pH = 7.4 (lane1,2,6), 8.0 (lane3), 9.0 (lane4), 10 (lane5) The figure which shows the result of the concentration-dependent binding experiment of NCS-dodecaborate sodium salt (compound 11) and transferrin. Buffer: pH = 10 (lanes1-5), pH = 7.4 (lanes 6-9) The figure which shows the result of the binding experiment with MID and NCS-dodecaborate sodium salt (Compound 11) to BSA and transferrin. Buffer: pH = 7.4 The figure which shows the result of the binding experiment of MID and albumin (BSA) and transferrin. The figure which shows the result of the pH-dependent binding experiment between MID and albumin (BSA). The figure which shows the result of the incorporation experiment of the boron reagent into mouse colon cancer cells. The figure which shows the result of Western Blotting of the mouse blood treated with MID. The figure which shows the change of the tumor amount in the mouse which injected MID-BSA conjugate.

Hereinafter, the present invention will be described in detail.
The pharmaceutical composition of the present invention has the formula (I): X-LY [in the formula, X represents a group containing ¹⁰ B, L represents a linker, and Y represents a group that binds to a lysine residue. .. ], It is characterized by containing a conjugate of a tumor-accumulating protein having a lysine residue.

In the present invention, the term "conjugate" means a complex formed by binding a compound represented by the formula (I) to a tumor-accumulating protein having a lysine residue.

In the present invention, the "tumor-accumulating protein" means a protein that exhibits a property of selectively collecting in tumor cells or tumor tissues, and includes, for example, a protein that exhibits an EPR effect. In order to obtain the EPR effect, the molecular size of the protein is important. For example, the particle size is preferably 1 to 200 nm, more preferably 4 to 100 nm, and the molecular weight is 40. It is preferably ~ 2000 kDa, more preferably 60-1500 kDa. Therefore, the "tumor-accumulating protein" of the present invention also preferably has such a particle size and / or molecular weight. Specific examples of tumor-accumulating proteins include albumin, transferrin, cytokines, growth factors, immunoglobulins and the like. Examples of albumin include bovine serum albumin (BSA), human serum albumin (HSA), mouse serum albumin, rat serum albumin and the like. When HSA is used as albumin, HSA collected from a patient to which the pharmaceutical composition of the present invention is administered may be used, or other HSA may be used. Examples of transferrin include bovine serum transferrin, human serum transferrin, mouse serum transferrin, rat serum transferrin and the like. The immunoglobulin may be any antibody or antibody fragment. Examples of the antibody fragment include F (ab') ₂ , Fab', Fab, and Fv. The class of immunoglobulin is not particularly limited, and may be any of IgG, IgM, IgA, IgD, and IgE. The immunoglobulin may be of human origin or of non-human animals (eg, mice, rats, rabbits, goats, etc.).

X in the formula (I) may be any group containing ¹⁰ B, and may be a group derived from a compound having one boron atom in the molecule such as BPA, but from a boron cluster. Derived groups are preferred. The boron cluster may be any polyhedral structure that can be used for boron neutron capture therapy, such as crosododecaborate ([B ₁₂ H ₁₂ ] ^2- ), ionic crosocarborane ([CB). ₁₁ H ₁₂ ] ^- ), fat-soluble boron carborane ([C ₂ B ₁₀ H ₁₂ ]), nid carborane ([C ₂ B ₉ H ₁₁ ] ^- ), bisdicarboride metal complex ([(C ₂ B ₉ H ₁₁ ) ₂ M) ]), GB10 ([B ₁₀ H ₁₂ ] ^2- ), etc. The boron atoms contained in the boron cluster may be all ¹⁰ B, but only a part may be ¹⁰ B. In addition, in this specification, the expression "derived group" such as "group derived from a boron cluster" is used, but this removes one hydrogen atom in a boron cluster, for example. Means a group derived by doing so.

Y in formula (I) may be any group as long as it binds to a lysine residue, for example, a maleimidyl group (a group derived by removing a hydrogen atom that binds to a nitrogen atom of maleimide), Isothiocyanate group (-N = C = S), isocyanate group (-N = C = O), aldehyde (-CHO), ketone (-CO-), sulfonyl chloride (-SO ₂ Cl), activated ester (- CO ₂ Su, -CO ₂ Bt, -CO ₂ At, but Su, Bt, At represents the following groups) and the like.

L in the formula (I) may be any as long as it has a function as a linker, but is preferably an alkylene group. However, one or more of the alkylene groups -CH _2- may be substituted with -O-, -S-, -NH-, or -CO-. The number of carbon atoms of the alkylene group is not particularly limited, but is preferably 5 to 20, more preferably 5 to 15. Even when -CH _2- in the alkylene group is replaced with -O-, -S-, or -NH-, it is assumed that these groups have one carbon, and the above-mentioned "number of carbon atoms of the alkylene group" is assumed. Included in. Preferred linkers are -O-CH ₂ -CH ₂ -O-CH ₂ -CH ₂ -NH-CO-CH ₂ -CH ₂ -CH _2- , -S-CH ₂ -CH ₂ -O-CH _{2- CH 2 -NH-CO-CH 2} -CH 2 -CH 2 -, - O-CH 2 -CH 2 -O-CH 2 -CH 2 -, - S-CH 2 -CH 2 -O-CH 2 -CH _2- , -CO-NH-CH ₂ -CH ₂ -CH ₂ -O-CH ₂ -CH ₂ -O-CH ₂ -CH ₂ -O-CH ₂ -CH ₂ -CH ₂ -NH-CO-CH ₂ -CH _2- and so on, but not limited to these.

Specific examples of the compound represented by formula (I), the compound bound to closo dodecaborate a maleimide via a linker described in Example (Compound 1) (M ale i mide- closo- d odecaborate conjugate, Hereinafter, it may be referred to as "MID"), a compound in which an isothiocyanate group is bound to crosododecaborate via a linker (Compound 11) (hereinafter, may be referred to as "NCS-dodecaborate") and the like. be able to.

The molar ratio of the compound represented by the formula (I) to the tumor-accumulating protein having a lysine residue (the number of moles of the compound / the number of moles of the protein) contained in the composition is preferably more than 10. It is more preferably 20 or more, and further preferably 30 or more. By increasing the amount of the compound with respect to the protein in this way, it is considered that the number of molecules of the compound bound to the protein increases, and as a result, the amount of boron incorporated into the tumor tissue increases.

A conjugate of the compound represented by the formula (I) and a tumor-accumulating protein having a lysine residue can be produced by mixing the compound and the protein and reacting the two.

The molar ratio of the protein to the compound at the time of mixing (the number of moles of the compound / the number of moles of the protein) is preferably more than 10, more preferably 20 or more, and more preferably 30 or more, as described above. It is more preferable to have. There is no particular upper limit to this molar ratio, but if the molar ratio of the compound to the protein exceeds a certain value, even if the compound is added, it will no longer bind to the protein, and the added compound can be effectively used. Therefore, it is preferable to set the molar ratio so as not to exceed the value (molar ratio that reaches a plateau).

The pH at the time of reaction may be neutral (for example, 6.4 to 8.4, preferably 6.9 to 7.9) or basic (for example, 9.0 to 11.0, preferably 9.5 to 10.5). Depending on the type of group that binds to the lysine residue, it may not efficiently form a conjugate unless it is basic (for example, if the group that binds to the lysine residue is an isothiocyanate group). In such a case, the pH at the time of reaction is preferably basic.

The reaction temperature is not particularly limited, but is usually 0 to 50 ° C, preferably 10 to 40 ° C.

The reaction time is not particularly limited, but is usually 5 minutes to 24 hours, preferably 1 hour to 12 hours.

The compound represented by the formula (I) can be synthesized according to the method described in Examples, or according to a method obtained by appropriately modifying or modifying those methods with reference to the description thereof. For example, binding a linker to a compound capable of inducing a group containing ¹⁰ B (such as crosododecaborate or BSH) and binding this linker to a compound capable of inducing a group that binds to a lysine residue (such as maleimide or TCDI). Can be synthesized by.

The pharmaceutical composition of the present invention is used for boron neutron capture therapy. That is, the pharmaceutical composition of the present invention is administered to humans or non-human animals (mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.), and then irradiated with low-energy thermal neutrons. Selectively destroys tumor cells. Diseases to be treated include malignant tumors such as brain tumors, malignant melanomas, head and neck cancers, lung cancers, liver cancers, thyroid cancers, skin cancers, bladder cancers, mesenteric tumors, pancreatic cancers, breast cancers, meningiomas and sarcomas. However, it is not limited to these.

When a conjugate of the compound represented by the formula (I) and a tumor-accumulating protein having a lysine residue is used as a pharmaceutical composition, it should be mixed with a pharmaceutically acceptable carrier or diluent according to a known method. Allows this conjugate to be formulated. The dosage form is not particularly limited, and may be an injection, a tablet, a powder, a granule, a capsule, a liquid, a suppository, a sustained-release agent, or the like. The administration method is also not particularly limited, and administration can be performed orally or parenterally (administration by injection into intradermal, intraperitoneal, venous, arterial, or cerebrospinal fluid, infusion, or the like). The dose varies depending on the administration subject, administration method, etc., but for example, when administered as an injection to an adult, one treatment is performed so that the conjugate is 10 to 1000 mg / kg per administration. It can be administered in 1 to several divided doses.

The boron drug used in conventional boron neutron capture therapy has a problem that it is taken up not only by tumor cells but also by normal cells of the kidney and spleen, but the pharmaceutical composition of the present invention is selective for tumor cells. Since it is taken up by normal cells and hardly taken up by normal cells, it has an advantage that tumor cells can be selectively destroyed without seriously damaging normal cells.

In addition, the pharmaceutical composition of the present invention also has an advantage that the dose can be reduced because the amount of boron taken up by the tumor cells is also larger than that of the conventional boron drug.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Example 1] Synthesis of MID (1) Synthesis of compound 3
Compounds known in the literature (Igor B. Sivaev, Nadezhda Yu. Kulikova, Evgeniya A. Nizhnik, Maxim V. Vichuzhanin, Zoya A. Starikova, Andrei A. Semioshkin, and Vladimir I. Bregadze, J. Organomet. Chem. 2008 , 693, 519-525) Using compound 4 (1.3 g, 2.0 mmol) as a starting material, add 1 ml of NaBF ₄ (1.1 g, 10.0 mmol) and 4 M HCl and 70 ml of 1,4-dioxane, and heat. The mixture was heated and refluxed at 100 ° C. for 12 hours. After cooling to room temperature, by-products were filtered off and the filtrate was removed. This precipitate was dissolved in methylene chloride, the precipitate of impurities was removed by filtration through Celite, and then the solid was recrystallized from ethanol and filtered through Kiriyama to obtain Compound 3. Yield 87%
¹ H NMR (400 MHz; CD ₃ Cl): δ 3.72 (m, 6H), 3.30 (m, 18H), 1.53 (m, 16H), 0.99 (m, 24H)

(2) Synthesis of compound 5
Known compounds in the literature (Anna V. Orlova, Nikolay N. Kondakov, Boris G. Kimel, Leonid O. Kononov, Elena G. Kononova, Igor B. Sivaev, and Vladimir I. Bregadze, Appl. Organometal. Chem. 2007 , 21, 98-100) Compound 3 (100.0 mg, 0.2 mmol) was added Bu ₄ NN ₃ (120.7 mg, 0.4 mmol) in methylene chloride solvent and stirred for 24 hours. The reaction mixture was washed 3 times with water, and the aqueous layer was extracted 3 times with methylene chloride. The organic layer was dried over Na ₂ SO ₄ . Then, it was purified by column chromatography to obtain the target compound 5. Developing solvent D: M = 20: 1, R _f = 0.4-0.5. Yield 97%
^{1 1} H NMR (400 MHz; CD ₃ Cl): δ 4.59 (m, 4H), 3.94 (m, 4H), 3.20 (m, 8H), 1.63 (m, 8H), 1.25 (m, 8H), 1.00 ( t, J = 14.8 Hz, 12H)

(3) Synthesis of compound 2
Compound 5 (200.0 mg, 0.27 mmol) was added Ph ₃ P (80.8 mg, 0.31 mmol) in a THF / H ₂ O (10: 1) solvent and stirred for 24 hours. The mixed solvent was removed and washed with 300 ml of an ethyl acetate / diethyl ether mixed solvent to obtain Compound 2. Compound 2 was used for the synthesis of compound MID-TBA (Compound 6) without further purification.

(4) Synthesis of compound MID-TBA (compound 6)
Compound 2 (300.0 mg, 0.42 mmol) was added to 4-Maleimidobutyric Acid (91.0 mg, 0.5 mmol), BOP (221.0 mg, 0.5 mmol) and Et ₃ N (0.088 ml, 0.63 mmol) in a methylene chloride solvent 24. Stirred for hours. Then, 2M HCl solution (10 ml) was added, extracted with methylene chloride, and the organic layer was dried with Na ₂ SO ₄ . The solution was filtered through Celite, the solvent was removed with a rotary evaporator, and the solution was purified by silica gel column chromatography to obtain an oily substance MID-TBA (Compound 6). Developing solvent D: M = 20: 1, R _f = 0.2-0.3. Yield 77%

(5) Synthesis of compound MID-TMA (compound 7)
Compound MID-TBA (Compound 6) (200.0 mg, 0.22 mmol) was dissolved in methanol, 5 equivalents of TMA-Cl (121.8 mg, 1.12 mmol) dissolved in methanol was added thereto, and the mixture was filtered through Kiriyama to form a white solid. The target compound MID-TMA (Compound 7) was obtained. Yield 94%.
MP. 135-136 ^o C; IR (KBr, cm ^-1 ): 3027, 2480, 1708, 1638, 1487, 949, 829; ¹ H NMR (400 MHz; D ₂ O): δ 7.09 (s, 1H) , 6.65 (s, 2H), 3.44 (t, J = 5.2 Hz, 2H), 3.41-3.38 (m, 6H), 3.17 (m, 2H), 3.02 (s, 12H), 2.08 (t, J = 7.2 Hz, 2H), 1.71 (m, 2H); ¹³ C NMR (75 MHz; CD ₃ CN): δ 173.61, 171.57, 134.50, 72.23, 70.51, 67.49, 58.59, 39.37, 37.19, 33.09, 24.54, 23.60, 19.59 , 13.10; ¹¹ B NMR (96.3 MHz; D ₂ O): δ 1.56, -21.09, -22.45, -27.236; HRMS (ESI, negative) m / z calcd. For C ₁₂ H ₂₈ B ₁₂ N ₂ O ₅ Na ₁ [M + Na] ^- : 433.3093, found: 433.3098.

(6) Synthesis of compound MID (compound 1)
Compound MID-TMA (Compound 7) (300.0 mg, 0.54 mmol) was weighed in a 100 ml eggplant flask, 20 ml of distilled water and 25 ml of sodium foam amberlite were added thereto, and the mixture was stirred for 12 hours (KPG-Stirr). did. Sodium amber light was removed by filtration through Kiriyama, and the filtrate was removed with a rotary evaporator and then freeze-dried to obtain the target compound MID (Compound 1). Yield 93%.
¹ H NMR (400 MHz; D ₂ O): δ 6.70 (s, 2H), 3.51-3.38 (m, 8H), 3.20 (m, 2H), 2.16 (t, J = 7.2 Hz, 2H), 1.76 ( m, 2H); ¹³ C NMR (75 MHz; CD ₃ CN): δ 176.26, 173.86, 134.99, 71.50, 69.16, 68.20, 39.78, 33.46, 24.33; ¹¹ B NMR (96.3 MHz; D ₂ O): δ 1.71 , -21.15, -23.00, -27.96; HRMS (ESI, negative) m / z calcd. For C ₁₂ H ₂₈ B ₁₂ N ₂ O ₅ Na ₁ [M + Na] ^- : 433.3093, found: 433.3095.

[Example 2] Biological distribution of MID-BSA conjugate
MID and BSA were mixed in PBS (pH 7.4) to a molar ratio of 10: 1 and reacted at 23 ° C. for 12 hours to form a MID-BSA conjugate. This MID-BSA conjugate was injected into the tail vein of BALB / c mice inoculated with CT26 (mouse colon cancer cell line). The injection volume of MID-BSA conjugate was adjusted to 3 to 30 mg B / kg. Boron concentrations in blood, tumor tissue, liver, kidney, and spleen after injection were measured using ICP-AES.

As shown in FIG. 2, the boron concentration in the tumor tissue reached 60 ppm (injection amount: 30 mg B / kg) or 38 ppm (injection amount: 15 mg B / kg). On the other hand, the boron concentration in the liver, kidney, and spleen was 10 ppm or less. In the boron delivery system using liposomes, boron accumulates not only in the tumor tissue but also in the kidney and spleen (H. Koganei et al., Bioconjugate Chem. 24, 124-132 (2013)). It was surprising that the boron concentration in was low.

[Example 3] Evaluation of binding of MID to BSA Identification of the binding site of albumin other than -SH by blocking -SH of albumin with IAA (2-iodoacetamide) and N-ethymaleimide in advance and then adding MID. I tried. IAA and N-ethymaleimide (10 mM) were added to the BSA solution (100 μM) and stirred for 1 h, then MID (1 mM) was added to proceed the reaction, and SDS-PAGE and Western Blotting were performed. As shown in FIG. 3, it was confirmed by the result of SDS-PAGE that the amount of protein was uniform, and from the result of Western blotting, albumin was also found in the lanes (lane 3 and lane 4) to which the reagent blocking -SH was added. Binding to MID was observed.

Instead of IAA and N-ethylmaleimide, SDS-PAGE and Western Blotting were performed in the same manner as above using N-acetyl succinimide, which is a reagent that selectively blocks lysine residues. As shown in FIG. 3, a weak band was observed when only N-acetyl succinimide was added (lane 5). This band is thought to be due to the binding of MID to albumin-SH. On the other hand, no band was detected when IAA and N-acetyl succinimide were added (lane 6) and when N-ethylmaleimide and N-acetyl succinimide were added (lane 7). From the above, it is considered that MID is bound to albumin at both -SH and lysine residue.

Next, when the concentration dependence of MID-BSA was evaluated, as shown in FIG. 4, the reaction reached a plateau under the reaction condition (lane 4) of BSA: MID = 1:30, and binding even if more MID was added. It was found that the amount did not increase.

After proceeding with the reaction at a reaction ratio of BSA: MID = 1: 100, it was evaluated how long the reaction reached a plateau. As a result, as shown in FIG. 5, it was found that the reaction between BSA and MID proceeded in a time-dependent manner up to a reaction time of 6 hours and then reached a plateau.

[Example 4] Synthesis of NCS-dodecaborate (1) Synthesis of NCS-dodecaborate tetrabutylammonium (TBA) salt (Compound 9)
Compound 8 (400 mg, 0.55 mmol) was added with TCDI (129.9 mg, 0.73 mmol) and Et ₃ N (151.9 μL, 1.10 mmol) in a methylene chloride solvent, and the mixture was stirred overnight. Then, the solvent was removed with a rotary evaporator, and the mixture was washed with 200 mL of a mixed solvent of ethyl acetate: diethyl ether = 1: 1. Impurities were removed by passing the obtained residue through silica gel column chromatography (methylene chloride: methanol = 15: 1) to obtain Compound 9 (319.4 mg, 75%).
^{1 1} H NMR (400 MHz; CD ₃ CN): δ 3.71-3.65 (m, 4H), 3.58-3.54 (m, 4H), 3.10-3.06 (m, 16H), 1.64-1.58 (m, 16H), 1.40 -1.36 (m, 16H), 0.98-0.95 (m, 24H)
MS ESI, negative, m / z calcd. For C ₅ H ₁₉ B ₁₂ NO ₂ S [M / 2] ^2- : 143.5, found: 144.0.

(2) Synthesis of NCS-dodecaborate tetramethylammonium (TMA) salt (Compound 10)
Compound 9 (319.4 mg, 0.41 mmol) was dissolved in methanol, and TMA-Cl (226.7 mg, 2.07 mmol) dissolved in methanol was added dropwise thereto. After stirring for 3 hours, filtration was performed to obtain compound 10 (152 mg, 84%) as a white solid.
¹ H NMR (400 MHz; CD ₃ CN): δ 3.71-3.69 (m, 2H), 3.66-3.64 (m, 2H), 3.54 (m, 4H), 3.10 (s, 24H);
¹³ C NMR (CD ₃ CN): δ 72.56, 68.53, 67.78, 55.35, 45.42
MS ESI, negative, m / z calcd. For C ₅ H ₁₉ B ₁₂ NO ₂ S [M / 2] ^2- : 143.5, found: 143.6.

(3) Synthesis of NCS-dodecaborate sodium salt (Compound 11)

To compound 10 (522 mg, 1.20 mmol), Na + -Amberlite (20 mL) and distilled water (50 mL) were added, and the mixture was stirred overnight. Amberlite was removed by Kiriyama filtration, the solvent was removed by a rotary evaporator, and then lyophilization was performed to obtain Compound 11 (329 mg, 83%).
^{1 1} H NMR (400 MHz; D ₂ O): δ 3.79 (m, 4H), 3.70 (m, 4H)
¹³ C NMR (D ₂ O): δ 71.14, 68.67, 67.66, 45.00
MS ESI, negative, m / z calcd. For C ₅ H ₁₉ B ₁₂ NO ₂ S [M / 2] ^2- : 143.5, found: 143.6.

[Example 5] Evaluation of boron compound binding to human serum transferrin After reacting human serum transferrin (Tf) with NCS-dodecaborate sodium salt (Compound 11), binding evaluation was performed by SDS-PAGE and Western Blotting. The results are shown in FIG. Here, the binding was evaluated by changing the pH of the reaction solution. It was observed that the binding ability to Tf was the highest at pH = 10 (lane 5). In addition, no bond with a boron cluster compound having an amino group at the terminal was observed (lane 6).

Subsequently, the binding evaluation of NCS-dodecaborate sodium salt (Compound 11) to Tf was confirmed in terms of concentration dependence. The reaction was carried out at a concentration ratio of Tf: boron cluster compound = 1: 1, 1:10, 1:50, 1: 100 for a reaction time of 1 hour. The results are shown in FIG. The NCS-dodecaborate sodium salt (Compound 11) bound to Tf in a concentration-dependent manner. It was also found that pH = 10 (Lanes 2-5) binds more efficiently than pH = 7.4 (lanes 6-9).

In addition, a MID binding experiment was performed on Tf. It is known that Tf does not contain free -SH, and compounds containing maleimide usually do not bind to Tf. As shown in FIG. 8, it was found that MID had higher binding ability to BSA at pH 7.4 than NCS-dodecaborate sodium salt (Compound 11) (lanes 2 and 3). Interestingly, it was also found that MID binds to free -SH-free Tf in the same manner as NCS-dodecaborate sodium salt (Compound 11) (lanes 5 and 6).

[Example 6] Evaluation of binding of MID to Tf
MID-Tf conjugates were analyzed by Western Blotting. Tf (0.1 mM) was reacted with MID (1 mM) in PBS buffer (pH 7.4) for 12 hours. For comparison, Na ₂ B ₁₂ H ₁₁ SH (BSH) and Na ₂ B ₁₂ H ₁₂ were also reacted with Tf and analyzed in the same manner (Fig. 9).

As shown in FIG. 9, when Tf was reacted with MID, the binding between the two was confirmed (lane 4). On the other hand, when Tf was reacted with Na ₂ B ₁₂ H ₁₁ SH or Na ₂ B ₁₂ H ₁₂ , no binding was confirmed (lane 5 and lane 6).

[Example 7] Relationship between MID-BSA conjugate and pH
The relationship between BSA-MID binding and pH was investigated by SDS-PAGE and Western Blotting. Under various pH conditions (pH 5-10), BSA (0.1 mM) was treated with MID (1 mM), and the treated mixture was electrophoresed and Western blotting was performed (FIG. 10). Western Blotting was performed using an anti-BSH antibody.

As shown in FIG. 10, under acidic conditions, BSA and MID hardly bound (lane 2 and lane 3).

[Example 8] Evaluation of boron uptake The uptake of boron reagent (MID or MID-BSA conjugate) into mouse colon cancer cells (colon 26 cells) was examined.

The cells were pre-incubated in a culture dish for 24 hours, then treated with a boron reagent (boron concentration was adjusted to 30, 100, or 300 ppm) for 1 hour in the presence or absence of FBS, followed by PBS. Washed with (5 mL x 3). Cells were collected and digested with 2 mL of 60% HClO ₄ /30% H ₂ O ₂ (1: 2) at 70 ° C. for 1 hour. The boron concentration in the solution was measured using ICP-AES. For comparison, the amount of BSH uptake was also examined. The results are shown in FIG. The black bar in the figure shows the boron concentration in the presence of FBS, and the white bar shows the boron concentration in the absence of FBS.

As shown in FIG. 11, the uptake of boron depending on the treatment concentration was observed regardless of which boron reagent was used. In the case of BSH, the boron concentration was lower in the absence of FBS. This suggests that FBS is essential for the uptake of BSH into cells. A phenomenon similar to BSH was observed in the uptake of MID. In contrast, MID-BSA conjugate uptake was suppressed in the presence of FBS and promoted in the absence of FBS. This suggests that intracellular accumulation of MID-BSA conjugates is induced by an albumin-mediated active uptake mechanism.

[Example 9] Western Blotting of mouse blood treated with MID
Mouse blood was incubated in PBS (lane 1) or PBS containing MID (lane 2) for 1 hour. The treated blood was analyzed by Western Blotting using an anti-BSH antibody (Fig. 12).

As shown in FIG. 12, a band binding to the anti-BSH antibody was observed around 60 kDa. This band is believed to be of serum albumin. Therefore, it is considered that MID binds to serum albumin in the circulating blood after injection and exhibits the same action as the MID-BSA conjugate.

[Example 10] Changes in tumor volume in mice injected with MID-BSA conjugate
Binding of MID to BSA was performed in PBS for 12 hours at 37 ° C. Tumor-bearing mice were injected with a PBS solution of MID-BSA from the tail vein and irradiated with thermal neutrons 12 hours after administration in the Kyoto University Research Reactor (KUR). After irradiation, the tumor volume was measured at time intervals. The result is shown in FIG. ○ in the figure shows the case where MID-BSA is not injected and thermal neutrons are irradiated. In the figure, Δ, ●, and ■ indicate the case where 7.5, 15, and 30 mg [ ¹⁰ B] / kg of MID-BSA were injected and irradiated with thermal neutrons, respectively.

As shown in FIG. 13, when MID-BSA was not injected, the amount of tumor increased with the passage of time. In contrast, significant suppression of tumor growth was observed when MID-BSA was infused at volumes of 15 and 30 mg [ ¹⁰ B] / kg. When infused with 30 mg [ ¹⁰ B] / kg of MID-BSA, a temporary increase in tumor size was observed 3 days after irradiation, which is probably acute inflammation caused by a high neutron capture reaction in tumor tissue. It is thought that this is due to. Significant suppression of tumor growth was also observed when infused at a volume of 7.5 mg [ ¹⁰ B] / kg.

The present inventors have previously reported the antitumor effect of boron cluster lipid liposomes in tumor-bearing mice (M. Ueno et al., Bioorg. Med. Chem., 18 (2010) 3059-3065.). In this case, tumor growth was suppressed to some extent for 1 week, but tumor re-growth was observed 1 week after thermal neutron irradiation. These results show that the distribution of boron in tumor tissue is different between liposomes and MID-BSA, where MID-BSA accumulates more aggressively in tumor cells than liposomes, resulting in higher antitumor efficacy in tumor-bearing mice. It is suggested to show. High levels of boron circulate in the blood during irradiation with thermal neutrons, but no serious damage was observed in the area of the right thigh after irradiation. These phenomena were also observed in previous studies of the present inventors of borated liposomes (S. Tachikawa et la., Chem. Commun., 50 (2014) 12325-12328. And H. Koganei et la., Bioconjug. Chem., 24 (2013) 124-132.). The failure is probably dependent on each boron reagent. MID-BSA is thought to not interact with blood vessels and blood contents (eg, red blood cells, white blood cells, and platelets), resulting in minimal damage to blood vessels. Significant boron accumulation was also observed in other organs such as the liver and kidney, but these off-target distributions do not appear to affect toxicity. This is because MID itself is potentially low toxicity (IC ₅₀ > 1 mM) and does not show significant toxicity unless these organs are irradiated with neutrons.

〔experimental method〕
In the above example, SDS-PAGE and Western Blotting were performed as follows.
(1) SDS-PAGE
Compound 11 (0.5 μL, 100 mM) was added to the Tf solution (50 μL, 100 μM), and the mixture was vortexed for 1 hour. After passing the reaction solution through a small molecule removal column (Micro Bio-Spin (registered trademark) 6 Chromatography Columns), 5 × sample buffer (12.5 μL) containing DTT was added, and the mixture was heated at 95 ° C. for 5 minutes. SDS-PAGE (10% acrylamide gel, 200V, 50min) was performed on the obtained sample diluted 30-fold, and CBB staining was performed to visualize the protein band.

(2) Western Blotting
The acrylamide gel obtained by SDS-PAGE was transferred to a membrane filter (300 mA, 45 min), immunoblock was added to the membrane, and the mixture was shaken by a shaker for 1 hour. After washing with TTBS buffer for 10 minutes three times, the primary antibody fixation test using anti-BSH antibody was stirred overnight in an environment of 4 ° C. After washing with TTBS buffer for 10 minutes three times, the secondary antibody fixation test with HRP was stirred in an environment of 4 ° C. for 3 hours. After washing with TTBS buffer for 10 minutes three times, chemiluminescence detection reagent was added and detection measurement was performed with an imager.

All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Since the present invention relates to a composition that can be used as a pharmaceutical product, it can be used in an industrial field such as the pharmaceutical industry.

Claims (6)

Formula (I): X-LY [In the formula, X represents a group containing ¹⁰ B, L is an alkylene group (where one or more -CH _2- of the alkylene group is -O-, -S. -, -NH-, or -CO- may be substituted) , and Y represents a maleimidyl group. ], Which is a pharmaceutical composition containing a conjugate of a compound represented by lysine residue and a tumor-accumulating protein having a lysine residue, and is a molar ratio of the compound to the protein contained in the composition (the number of moles of the compound). / A pharmaceutical composition characterized in that the number of moles of the protein) is a molar ratio of more than 10. The pharmaceutical composition according to claim 1, wherein the tumor-accumulating protein having a lysine residue is transferrin. The pharmaceutical composition according to claim 1, wherein the tumor-accumulating protein having a lysine residue is albumin. The pharmaceutical composition according to any one of claims 1 to 3, wherein X in the formula (I) is a group derived by removing one hydrogen atom in a boron cluster. According to any one of claims 1 to 3, wherein X in the formula (I) is a group derived by removing one hydrogen atom in crosododecaborate, crosocarborane, or nidcarborane. The pharmaceutical composition described. The pharmaceutical composition according to any one of claims 1 to 5 , which is used for boron neutron capture therapy.