JP4696259B2 - Novel photosensitive drug and method for producing the same - Google Patents

Description

  The present invention relates to a photosensitive drug capable of selectively killing a target cell by binding to the target cell and receiving light irradiation. Furthermore, the present invention relates to a method for producing the photosensitive drug.

Photodynamic therapy (hereinafter, PDT (P hoto D ynamic T herapy) and hereinafter) includes a photosensitizer PS (photosensitizer), utilizing a photochemical reaction caused by irradiation of ultraviolet laser light or a predetermined wavelength This is a therapeutic method in which active oxygen is generated in a target tissue, and the target tissue is necrotized by the action of the active oxygen, and is applied to the treatment of cancer (for example, see Non-patent Document 1). In the actual treatment of cancer with PDT, a photosensitizing drug is intravenously injected into a patient in advance, the drug is taken into the tumor tissue, and then irradiation with a laser (excimer laser) light having a wavelength that matches the excitation wavelength of the drug is performed. Has been done. The drug taken into the tumor tissue is excited by this laser irradiation, and some excited molecules are in a triplet state. Next, when the excited triplet molecule comes into contact with molecular oxygen dissolved in the tumor tissue, energy is transferred to the molecular oxygen, and the molecular oxygen is activated to become singlet oxygen. Since singlet oxygen is very reactive, bioactive molecules in tumor cells are oxidized and denatured. As a result, apoptosis and the like are induced, and tumor cells are necrotized. The laser used in PDT has a low output of about 1/100 that of a laser scalpel, and the drug accumulates in the tumor tissue so that damage to normal tissue can be minimized. It becomes possible to treat.

Today, the only photosensitizing drugs marketed and used for PDT are polyfimer sodium (trade name Photofrin), but trifluoperazine (hereinafter referred to as TFPZ) and sulfonated aluminum phthalocyanine ( Sulfonated Aluminum Phtalocyanine (hereinafter referred to as SALPC) is also known as a photosensitizer. However, no compound other than those described above can be used as a photosensitive drug.
Roder, B., Nather, D., Proc.SPIE-Int. Soc.Opt. Eng., 1991, 1525, 377-384.

  It is an object of the present invention to provide a new photosensitive drug used for photodynamic therapy and a method for producing the same.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that fluorescent semiconductor quantum dots surface-treated with a polycarboxylic acid compound are excited by ultraviolet rays or laser irradiation of a predetermined wavelength, and thereby become cells. It has been found that it can be used as a photosensitive drug in PDT by exerting an action of necrotizing (killing) and binding the fluorescent semiconductor quantum dot to a substance that can selectively bind to a target cell. The present invention has been completed by making further improvements based on such findings.

That is, the present invention provides the following photosensitive drugs and methods for producing the same:
Item 1. A photosensitizer characterized in that a fluorescent semiconductor quantum dot surface-treated with a polycarboxylic acid compound is bound to a substance that can selectively bind to a target cell.
Item 2. Item 2. The photosensitive agent according to Item 1, wherein the polycarboxylic acid compound is bonded to the surface of the fluorescent semiconductor quantum dot via a sulfur atom.
Item 3. Item 3. The photosensitive drug according to Item 1 or 2, wherein a substance that can selectively bind to a target cell is bonded via a carboxyl group (COOH).
Item 4. Item 4. The photosensitive drug according to any one of Items 1 to 3, wherein the substance that can selectively bind to a target cell is an antibody.
Item 5. Item 5. The photosensitive drug according to any one of Items 1 to 4, wherein the quantum dot has an average particle size of 5 nm or less.
Item 6. Item 6. The photosensitive drug according to any one of Items 1 to 5, wherein the polycarboxylic acid compound is mercaptosuccinic acid.
Item 7. Item 7. The photosensitive drug according to any one of Items 1 to 6, wherein the target cell is at least one selected from the group consisting of leukemia cells, tumor cells, and virus-infected cells.
Item 8. A pharmaceutical composition comprising (a) the photosensitive drug according to any one of Items 1 to 6, and (b) at least one selected from the group consisting of trifluoperazine and sulfonated aluminum phthalocyanine.
Item 9. Mixing a fluorescent semiconductor quantum dot and a polycarboxylic acid compound having two or more COOH groups and a thiol group, binding the compound to the fluorescent semiconductor quantum dot via a sulfur atom, and then targeting via the COOH group A method for producing a photosensitive drug, comprising binding a substance capable of selectively binding to a cell.

  Hereinafter, the present invention will be described in detail.

  The photosensitive drug of the present invention is characterized in that a fluorescent (fluorescent) semiconductor quantum dot surface-treated with a polycarboxylic acid compound is bound to a substance that can selectively bind to a target cell. Is.

  The fluorescent semiconductor quantum dots used in the photosensitive agent of the present invention may be less than the Bohr radius of the exciton, and an example of the average particle diameter is 5 nm or less, preferably about 1 to 5 nm. Bohr radii of CdS, CdSe, and CdTe, which are typical fluorescent semiconductor quantum dots, are 30 nm, 11.2 nm, and 7.8 nm, respectively.

  Examples of the fluorescent semiconductor quantum dots include CdS, CdSe, CdTe, ZnS, ZnSe, ZnTe, etc. as II-VI group semiconductors, and InP, GaP, etc. as III-V groups. Further, those containing a plurality of metal species such as (In, Ga) P, or those containing a plurality of 6B group atoms or 5B group atoms such as Cd (Se, Te) and Ga (As, P) It can also be used. Further, a fluorescent semiconductor quantum dot having a structure in which a certain kind of nanocrystal is used as a core and a semiconductor material of another kind is coated, such as a CdSe / ZnS core / shell nanocrystal, can also be used. Among these fluorescent semiconductor quantum dots, CdSe is particularly preferable because it can be easily applied and can effectively exert the action of killing target cells by ultraviolet irradiation. CdSe exhibits high luminous efficiency, and a fluorescent color from 470 nm to 620 nm can be obtained by controlling the size from 2.3 nm to 5.5 nm.

  A fluorescent semiconductor quantum dot surface-treated with a polycarboxylic acid compound is a compound having a functional group (for example, SH group) capable of binding two or more carboxyl groups and the fluorescent semiconductor quantum dot (ie, a polycarboxylic acid compound); It can be obtained by reacting a fluorescent semiconductor quantum dot with an aqueous solution to bond the compound to the surface of the quantum dot and recovering the quantum dot that has become water-soluble by a COOH group. Examples of the polycarboxylic acid compound include compounds having two or more carboxyl groups (COOH) such as mercaptosuccinic acid and 2,3-dimercaptosuccinic acid.

  Regarding the polycarboxylic acid compound, a compound having one thiol group (SH) and two or more carboxyl groups (COOH) is preferable from the viewpoint of improving water solubility.

  On the other hand, a compound having a plurality of thiol groups, such as 2,3-dimercaptosuccinic acid, may have enhanced binding properties with quantum dots, and is expected to be advantageous.

These compounds can be bonded to a metal constituting the fluorescent semiconductor quantum dot via a thiol group (-SH), and a water-soluble quantum dot having two or more carboxyl groups other than the thiol group can be obtained. . When a quantum dot is treated with a monocarboxylic acid such as HS-CH ₂ COOH, sufficient stability in an aqueous solution cannot be obtained, and the fluorescent semiconductor quantum dot precipitates due to aggregation and can bind to target cells. Bonding (linking) with other substances cannot be performed sufficiently. On the other hand, when the polycarboxylic acid compound of the present invention is used, the aggregation of the fluorescent semiconductor quantum dots does not occur, and is in a dissolved state in an aqueous solution for a long period of 24 to 48 hours or more, and is caused by aggregation. There is virtually no precipitation.

  The treatment for imparting water solubility to the fluorescent semiconductor quantum dots is usually about 0.005 to 0.1 mol, preferably 0.02 to 0, of polycarboxylic acid compound per 1 g of the fluorescent semiconductor quantum dots. Use about 0.03 mol of halogenated hydrocarbons such as chloroform, alcohols such as methanol and ethanol, esters such as ethyl acetate, ethers such as THF and dioxane, presence of organic solvents such as DMF, DMSO, formamide, acetonitrile and acetic acid. The reaction can be advantageously carried out by reacting at a temperature of about 0 ° C. to the boiling point of the solvent for about 10 minutes to 24 hours. After completion of the reaction, a water-soluble fluorescent semiconductor quantum dot surface-treated with these compounds can be obtained by distilling off the solvent.

  For example, when mercaptosuccinic acid is used as the polycarboxylic acid compound, 10 to 150 mg, preferably 30 to 40 mg, of mercaptosuccinic acid may be used per 10 mg of CdSe core nanocrystals.

  If an excessive amount of polycarboxylic acid compound is present, the solution / suspension becomes cloudy. Therefore, it is preferable to remove the excessive amount of polycarboxylic acid compound by Vivaspin ultrafiltration or the like. When a large amount of polycarboxylic acid compound is used, it is difficult to remove excess mercaptosuccinic acid by ultrafiltration or the like, and the solution / suspension tends to be cloudy. When an appropriate amount of the polycarboxylic acid compound is used, it is easy to remove, so that a transparent solution can be obtained.

  The “substance that can selectively bind to the target cell” used in the photosensitizing agent of the present invention is not particularly limited as long as it can selectively bind to the target cell and is pharmaceutically acceptable. In order to easily bond (link) with the fluorescent semiconductor quantum dots, it is desirable to have an amino group.

  Here, the target cell is a cell to be killed by the photosensitive drug of the present invention, and is caused by, for example, expression of a causative gene of a disease such as leukemia cell, tumor cell, virus-infected cell, or expression of a foreign gene. However, the present invention is not limited to these.

  Specific examples of substances that can selectively bind to target cells include antibodies or fragments thereof that selectively bind to target cells, lectins that bind to sugar chains that are specifically present on the cell surface of target cells, and the like. Examples include proteins and peptides.

  Examples of the antibody include IgG, IgA, IgM, IgE and the like. Among these, IgG is preferable. An antibody that selectively binds to a target cell is produced according to a conventionally known method.

In addition, the above-mentioned antibody fragment includes F (ab ′) ₂ obtained by treating the antibody with a protease such as pepsin, and Fab ′ obtained by reducing F (ab ′) ₂ with mercaptan. Examples include Fabs obtained by decomposing antibodies with proteolytic enzymes such as papain, trypsin, chymotrypsin, and plasmin.

  Examples of the lectin include dricos bean lectin (DBA), soybean bean lectin (SBA), wheat germ agglutinin (WGA), and red bean seed lectin (abrin-a). Among these, DBA, SBA, and abrin-a are preferable. A lectin that selectively binds to a target cell is produced by a conventionally known method.

  For example, when the target cell is a leukemia cell, an anti-CD90 antibody or a fragment thereof is exemplified as a specific example of a substance that can selectively bind to the target cell. In addition, when the target cell is a malignant melanoma cell, a specific example of a substance that can selectively bind to the target cell is an anti-GD3 antibody or a fragment thereof.

  The photosensitizing agent of the present invention is not particularly limited in its binding form as long as the fluorescent semiconductor quantum dot and a substance that can selectively bind to the target cell are bound to each other. Examples include those obtained by binding a substance capable of selectively binding to a target cell via a COOH group on the surface of the quantum dot. As described above, 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide, hydrochloride (hereinafter, referred to as “bonding”) can bind a substance that can selectively bind to a target cell via a COOH group on the surface of a fluorescent semiconductor quantum dot. The amino group of the substance that can selectively bind to the target cell after activating the COOH group on the surface of the fluorescent semiconductor quantum dot using a condensing agent in peptide synthesis such as EDC) Dehydration condensation may be performed between the two. As a specific example, a scheme for binding a fluorescent semiconductor quantum dot to an anti-CD90 antibody is shown in FIG.

  The photosensitive drug of the present invention is administered by injection such as intravenous, subcutaneous, intramuscular, or intraperitoneal, and is used as a photosensitizer for photodynamic therapy. As a specific use mode, a method of irradiating a target cell with light having a wavelength that excites a fluorescent semiconductor quantum dot after administering the photosensitive agent and binding the photosensitive agent to the target cell Is mentioned. In this way, the target cells can be killed by exciting the fluorescent semiconductor quantum dots by light irradiation.

  In the photodynamic therapy using the photosensitive drug of the present invention, the irradiated light is not particularly limited as long as it includes a wavelength capable of exciting the fluorescent semiconductor quantum dots of the photosensitive drug, but non-target cells It is desirable that it is not absorbed by the human body (does not adversely affect it). Suitable irradiation light includes ultraviolet rays.

  In performing photodynamic therapy using the photosensitizing agent of the present invention, the amount of photosensitizing agent to be administered to the patient, the time from the administration of the photosensitizing agent to the start of light irradiation, the intensity of the irradiated light, the irradiation time And the like are appropriately set according to the disease to be treated (type of target cell), the type of photosensitive drug to be used, and the like.

  In addition, the photosensitizing agent of the present invention can be further enhanced in the action of killing target cells when used in photodynamic therapy in combination with TFPZ or SALPC. Therefore, as an embodiment of the photosensitizer for photodynamic therapy, a pharmaceutical composition containing (a) the photosensitive drug of the present invention and (b) at least one selected from the group consisting of TFPZ and SALPC is preferable. Used for. In the pharmaceutical composition, the ratio of (a) the photosensitizer of the present invention to the compound (b) is not particularly limited. For example, the ratio of (a) the present invention to 1 part by weight of the compound (b) The ratio in which the photosensitive drug is 10 to 1000 parts by weight is exemplified.

  The photosensitive drug of the present invention is used as a photosensitizer in photodynamic therapy, and can selectively kill target cells such as tumor cells and leukemia cells. The photosensitizing agent of the present invention has an extremely small affinity for a cell because a very small quantum dot in the form of a particle having a diameter of several nanometers is used as a photosensitizing substance, and is effective not only outside the cell but also inside the cell. When exhibiting the above, it has a feature that it can be easily transfected (introduced) into cells.

  As a result of various experiments by the present inventors, the mechanism of action of the photosensitive drug of the present invention was estimated as follows. In the photosensitizing agent of the present invention, a “fluorescent semiconductor quantum dot” exists only on the surface of the target cell by binding a “substance that can selectively bind to the target cell” to the surface of the target cell. When the excitation light is irradiated there, the quantum dots absorb the energy of the light and emit fluorescence. However, a part of the absorbed energy reacts with oxygen existing in the vicinity of the quantum dots to generate oxygen species that are toxic to the living body such as active oxygen and singlet oxygen. These are thought to induce and kill apoptosis in target cells, cells that do not undergo pre-programmed spontaneous cell death, often called apoptosis.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited to these Examples.
Example 1
1. Solubilization of quantum dots and conjugation with antibody CdSe nanocrystals stored under light shielding were dispersed in a chloroform solution, and the concentration was adjusted to 25 mg / ml. As a guideline for concentration adjustment, the maximum absorbance value (hereinafter referred to as OD at MAX) near the absorption edge was set to 0.5. Add 2.5 ml mercaptosuccinic acid (HOOC-CH2-CH (SH) -COOH) solution (30 mg / ml, Aldrich) dissolved in methanol to 12.75 ml of the CdSe nanocrystal solution at room temperature, protected from light And incubated for about 2 hours until the solution became cloudy. Chloroform and methanol used were removed by suction evaporation to obtain dry powder of surface-treated quantum dots in which mercaptosuccinic acid was bonded to the surface of CdSe nanocrystals via sulfur atoms (-S-) derived from mercapto groups. . To the dry powder, 5.5 ml of PBS (-) solution (pH 7.3) was added and dissolved by gently mixing. The lysate was centrifuged (12,000 × g) for 20 minutes at 4 ° C., and an intermediate aqueous layer fraction containing water-soluble CdSe nanocrystals not aggregated was carefully collected. Further, the collected fraction was kept at 4 ° C. overnight and then centrifuged (1,200 × g) at 4 ° C. for 20 minutes to remove insoluble matters. The supernatant containing water-soluble CdSe nanocrystals was concentrated by centrifugal filtration using Vivaspin-20 (fractionated molecular weight: 3,000 MW, manufactured by Sartorius), and after removal of those with a molecular weight of 3,000 or less, Vivaspin-20 (fractionated) The product having a molecular weight of 30,000 or more was removed by centrifugal filtration concentration using a molecular weight of 30,000 MW (manufactured by Sartorius) and purified and concentrated. Finally, water-soluble CdSe nanocrystals were concentrated with Vivaspin-20 and adjusted to the target concentration (OD at MAX = 0.5) in PBS (-) solution (pH 7.3).

  A conjugate of the water-soluble CdSe nanocrystals thus prepared (hereinafter referred to as Qdot) and an anti-CD90 antibody (hereinafter referred to as CD-90) was prepared according to the method shown in FIG. A specific method is as follows. To 1.3 ml Qdot solution dissolved in 150 mM PBS solution (pH 7.3), 150 μl of CD-90 solution (5 mg) prepared in PBS solution (pH 7.3) was added and mixed. Further, 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide, hydrochloride solution prepared to a concentration of 7.2 mg / ml with water was added to the mixed solution, mixed, and reacted at room temperature for 2 hours. Unreacted Qdot and reaction by-product isourea were removed by centrifugal filtration concentration using Vivaspin-6 (limit molecular weight: 3,000 MW, manufactured by Sartorius). The thus-purified “Qdot with CD-90 bound” (hereinafter referred to as QD-CD90) was similarly adjusted with a PBS solution (pH 7.3) to a concentration of 1 mg / ml by centrifugal filtration concentration method. .

2. Reaction of leukemia cells and QD-CD90
Jurkat cells (acute lymphocytic leukemia cells; derived from patients with acute lymphocytic disease) prepared by adjusting QD-CD90 (equivalent to 0.1 mg Qdot) to 1.0 X 10 ⁶ cells / ml with PBS (-) solution (pH 7.3) The stocks were added to 1.2 ml of Tohoku University Institute of Aging Medicine, Medical Cell Resource Center, and allowed to react at room temperature for 30 minutes. Unreacted QD-CD90 was removed by repeating centrifugation (1000Xg / 10min) three times.

3. QD-CD90 binding Jurkat cells QD-CD90 binding Jurkat cells obtained in the ultraviolet radiation above ^{(1.0 X 10 6 cells / ml} ), normal white blood cells isolated from healthy human peripheral blood (1.0 X 10 ⁶ cells / ml ) And a volume ratio of 1: 1, and then the mixture cell solution was irradiated with UV light for 10 minutes (1.4 mW; UV lamp type UV-B 300 nm, UTF-34U Sigma Koki filter) and quenched for 10 minutes three times. Repeated.

In addition, the above mixed cell solution containing TFPZ (final concentration 1 μM, 480 × 10 ⁻⁶ mg / ml) or SALPC (final concentration 1 μM, 560 × 10 ⁻⁶ mg / ml) was subjected to ultraviolet rays under the same conditions as described above. Irradiation was performed.

4). Separation of cells and confirmation by flow cytometry After irradiation with ultraviolet rays, both cells were donated to a Sepharose 6MB column (affinity column chromatography) immobilized with soybean lectin: SBA. The fraction passed without adsorbing on the same column was used as normal lymphocyte cells, and the fraction adsorbed on the same column and eluted with N-acetylgalactosamine was used as QD-CD90-binding Jurkat cells in the experiment. The cells in the eluted fraction were confirmed by flow cytometry using an anti-CD44PE antibody (specifically reacting with normal lymphocyte cells) and an anti-CD90FITC antibody (specifically reacting with Jurkat cells).

5. Effect of UV irradiation on cell viability Cell viability was measured by measuring the amount of ATP in cells using CellTiter Glo (Promega). As a result, the survival rate of UV-treated normal lymphocyte cells was 93% compared to that of untreated cells, whereas the survival rate of UV-treated QD-CD90-binding Jurkat cells was Reduced to 80% compared to untreated. In addition, when treated similarly in the presence of TFPZ and SALPC, the survival rate of QD-CD90-bound Jurkat cells decreased to 64.5% and 57%, respectively, compared to untreated cells. The effect was found to increase significantly (see Table 1).

以上の結果から、QD-CD90は、白血病細胞に対して結合し、紫外線の照射を受けることにより該細胞を選択的に死滅させることが確認され、光線力学療法におけるフォトセンシタイザとして有用であることが確認された。

From the above results, it was confirmed that QD-CD90 binds to leukemia cells and selectively kills the cells by irradiation with ultraviolet rays, and is useful as a photosensitizer in photodynamic therapy. Was confirmed.

It is a figure which shows the scheme which couple | bonds a fluorescent semiconductor quantum dot (CdSe) with an anti-CD90 antibody.

Claims (9)

CdSe surface-treated with a polycarboxylic acid compound is bound to an antibody or a fragment thereof, or a protein or peptide that binds to a sugar chain that is specifically present on the cell surface of a target cell, Photosensitive drug used for necrosis of target cells . The photosensitive drug according to claim 1, wherein the polycarboxylic acid compound is bonded to the surface of CdSe via a sulfur atom. The antibody or fragment thereof, or a protein or peptide that binds to a sugar chain that is specifically present on the cell surface of a target cell, is bound via a carboxyl group (COOH). Photosensitive drug. The photosensitive drug according to any one of claims 1 to 3, wherein the CdSe is bound to an antibody. The photosensitive drug according to any one of claims 1 to 4, wherein the average particle size of CdSe is 5 nm or less. The photosensitive agent according to any one of claims 1 to 5, wherein the polycarboxylic acid compound is mercaptosuccinic acid. The photosensitive drug according to any one of claims 1 to 6, wherein the target cell is at least one selected from the group consisting of leukemia cells, tumor cells, and virus-infected cells. A pharmaceutical composition for treating a tumor , comprising (a) the photosensitive drug according to any one of claims 1 to 6, and (b) at least one selected from the group consisting of trifluoperazine and sulfonated aluminum phthalocyanine. . CdSe and a polycarboxylic acid compound having two or more COOH groups and a thiol group are mixed, and the compound is bonded to CdSe via a sulfur atom, and then the antibody or a fragment thereof, or a target cell via the COOH group A method for producing a photosensitive drug used for necrosis of a target cell, which comprises binding a protein or peptide that binds to a sugar chain that is specifically present on the cell surface.

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