JP2014084296A - Anticancer agent using expanded carbon fiber and expanded carbon fiber derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a drug delivery system (DDS) for delivering an anticancer agent topically.SOLUTION: An anticancer agent is made by bonding an anticancer component (X) to expanded carbon fibers (ExCFs) represented by the following general formula (1).

Description

  The present invention relates to an anticancer agent using an expanded carbon fiber and an expanded carbon fiber derivative. More specifically, the present invention relates to an anticancer agent in which an anticancer component is bound to expanded carbon fiber, and an expanded carbon fiber derivative useful as an intermediate or precursor of such an anticancer agent.

  Chemotherapy with anticancer drugs is a major strategy to locally shrink primary lesions and suppress distant metastases, and drug efficacy has a major impact on survival. Because the toxicity of anticancer drugs has serious side effects on the kidneys, liver, and other body organs, a more efficient drug delivery system (DDS) that delivers anticancer drugs locally This development is extremely important not only from the viewpoint of reducing side effects, but also from the viewpoint of improving drug efficacy.

  As DDS as an anticancer agent, liposomes, polymer micelles, and the like are used in the clinical stage and have been shown to be effective for cancer treatment. However, new carriers are still being developed with the aim of further improving selectivity and stability, adjusting biocompatibility, and reducing toxicity.

  In particular, low-molecular-weight drugs that are currently commonly used as anticancer drugs are 1) the drug is distributed not only to the target lesion site but also to normal cells. 2) The blood residence time is due to metabolism. Because of the shortcomings such as short and inability to maintain an effective blood concentration, a much larger amount of drug than the actual required amount must be repeatedly administered. There has been a demand for a new DDS carrier of an anticancer drug that can overcome the disadvantages associated with such low-molecular-weight drugs and achieve improved therapeutic efficiency and reduced side effects.

  As candidates for such new DDS carriers, nanocarbon materials such as carbon nanotubes, carbon nanohorns, fullerenes, and graphenes have been attracting attention (see Patent Documents 1 to 4, Non-Patent Documents 1 to 4, etc.). It is considered that nano-sized particles can stay in the body for a long time without being metabolized from the living body. In addition, there is a report that particles having a small particle size are likely to be effectively accumulated in a tumor (EPR (Enhanced Permeability and Retention) effect). Therefore, if a nanocarbon material is used as a carrier for DDS, a large amount of drug is selectively transported to a target lesion site while maintaining the blood circulation time and blood concentration of the drug, and the drug effect is effectively exerted. Therefore, it is expected that treatment efficiency can be improved and side effects can be reduced.

JP 2004-292426 A JP-A-2005-343885 JP 2009-78979 A JP 2009-789993 A

Z. Liu, X. Sun, N. Nakayama-Ratchford, H. Dai, ACS NANO., 2007, 1, 1, 50-56 K. Ajima, M. Yudasaka, T. Murakami, A. Maigne, K. Shiba, S. Iijima, MOLECULAR PHARMACEUTICS., 2005, 2, 6, 475-480 P. Chaudhuri, A. Paraskar, S. Soni, R. A. Mashelkar, S. Swngupta, ACS NANO., 2009, 3, 9, 2505-2514 K. Yang, J. Wan, S. Zhang, Y. Zhang, S. T. Lee, Z. Liu, ACS NANO ,. 2011, 5, 1, 516-522

  However, conventional anticancer agents using nanocarbon materials such as carbon nanotubes as a carrier have room for further improvement in terms of improving drug efficacy and reducing manufacturing costs.

  In view of the above, the object of the present invention is an anticancer agent using a nanocarbon material as a carrier, and has superior medicinal effects than an anticancer agent using a nanocarbon material such as a conventional carbon nanotube as a carrier, It is to provide an anticancer agent that can be produced at a low cost.

  As a result of intensive studies in view of the above problems, the present inventors have focused on expanded carbon fibers as nanocarbon materials that are candidates for new drug carriers. Surprisingly, the anti-cancer component is supported on the expanded carbon fiber, which is surprisingly superior in pharmacological effect and low in cost compared to conventional anti-cancer agents using nanocarbon materials such as carbon nanotubes as drug carriers. The present inventors have found that an anticancer agent having an advantage that it can be produced by, for example, can be obtained, and has reached the present invention.

Therefore, the gist of the present invention is as follows.
[1] An anticancer agent comprising an expanded carbon fiber and an anticancer component bonded thereto.
[2] The anticancer agent of [1] represented by the following general formula (1)
(In the formula (1),
ExCFs represent expanded carbon fibers,
A represents -O- or -NH-,
L represents a single bond or a linker, wherein the linker is a polyethylene chain represented by — (CH ₂ ) _m1 — (where m1 represents an integer of 1 to 22), — (CH ₂ CH ₂ O) _m2 — (where m2 represents an integer from 1 to 50), — (CH ₂ CH (OH)) _m3 — (where m3 represents an integer from 1 to 50) And a sugar chain represented by -MS _m4- (wherein MS each independently represents a divalent monosaccharide component, and m4 is an integer of 1 to 30) Represents), and
X represents an anticancer component,
n represents an integer corresponding to 100 to 5000 μmol per 1 g of the anticancer agent of the formula (1). ).
[3] The anticancer agent of [2], wherein X is represented by the general formula (2)
(In the formula (2),
R ¹ and R ² each independently represent a hydrogen atom or an alkyl group, or R ¹ and R ² are bonded to each other, together with the carbon atom to which R ¹ and R ² are bonded, respectively, unsubstituted or substituted May form a 5- or 6-membered hydrocarbon ring,
R ³ and R ⁴ each independently represent a halogen, a hydroxyl group, or a C1-6 alkoxy group, or R ³ and R ³ are bonded to each other to form a ring structure represented by the formula (3). May be
(In formula (3),
R ¹¹ and R ¹² each independently represent a hydrogen atom, or R ¹¹ and R ¹² are bonded to each other, together with the carbon atom to which R ¹¹ and R ¹² are bonded, an unsubstituted or substituted 3 to 6 member May form a hydrocarbon ring or a heterocyclic ring of
W represents a single bond or —C (═O) —. ). ).
[4] Expanded carbon fiber derivative represented by the general formula (4)
(In the formula (4), ExCFs, n, L, R ¹ and R ² represent the same definitions as the formula (1) of claim 2 and the formula (2) of claim 3).
[5] A preparation comprising the anticancer agent according to any one of [1] to [3] and one or more pharmaceutically acceptable excipients.

  The anticancer agent using the expanded carbon fiber of the present invention is superior in medicinal effect and can be produced at a lower cost than an anticancer agent using a nanocarbon material such as a conventional carbon nanotube as a carrier. .

1A and 1B are SEM images of ExCFs. FIG. 2 is a graph showing the particle size distribution of ExCFs. 3A and 3B are SEM images of CDDP-ExCFs. FIG. 4 is a graph showing the results of XPS analysis of CDDP-ExCFs. FIG. 5 is a graph showing the results of XPS analysis of CDDP-CNTs. FIGS. 6A and 6B are photomicrographs showing the uptake of fluorescently labeled CDDP-ExCFs and fluorescently labeled CDDP-CNTs into metastatic lung lesion cells, respectively. FIG. 7 is a graph showing the relationship between the CDDP concentration of CDDP-ExCFs and CDDP-CNTs and the cell viability. FIGS. 8A to 8F are TUNEL-stained photographs of cells without addition, CNTs, ExCFs, free CDDP, CDDP-CNTs, and CDDP-ExCFs added, respectively. FIG. 9 shows the results of Western blot analysis showing the concentrations of cleaved caspase-3, cleaved PARP-1, and Bax in the CDDP-ExCFs and CDDP-CNTs-added tissues. FIG. 10 is images of lung metastatic tumors at 20 and 40 days after administration in mice in the ExCFs administration group, free CDDP administration group, and CDDP-ExCFs administration group. FIG. 11 is a graph showing the cumulative survival rate of mice in the ExCFs administration group, free CDDP administration group, and CDDP-ExCFs administration group. FIG. 12 is a graph showing changes over time in the tumor volume of mice administered with CDDP-ExCFs and CDDP-CNTs. (A) shows the primary lesion and (b) shows the lung metastatic tumor.

1. Outline The present invention uses an expanded carbon fiber as a DDS carrier, and an anticancer component bonded thereto to form an anticancer agent.

  The expanded carbon fiber is obtained by refining an existing carbon fiber into a nanometer-sized fiber by high-temperature treatment or electrochemical treatment. Various reports have been made on expanded carbon fibers (for example, JP-A-2006-249546, JP-A-2008-163274, JP-A-2008-166661, JP-A-2010-111990, etc.) No investigation has been made on drug carriers such as anticancer agents.

  The inventors of the present invention have focused attention on newly expanded carbon fibers as candidates for drug carriers such as anticancer agents among various nanocarbon materials, and have studied them. As a result, by making the anti-cancer component supported on the expanded carbon fiber and making it an anti-cancer agent, compared to anti-cancer agents using carbon nanotubes that have been regarded as potential drug carrier candidates so far The present inventors have found that an anticancer agent can be efficiently delivered to a target site, and a more remarkable anticancer action and metastasis suppressing action can be obtained. Such a result is extremely unexpected and cannot be predicted from conventional knowledge.

In addition, expanded carbon fibers can be manufactured relatively inexpensively and easily as compared with other nanocarbon materials such as carbon nanotubes and fullerenes. Therefore, by using the expanded carbon fiber as a carrier for an anticancer agent, it is possible to obtain an anticancer agent that is superior in terms of both medicinal effect and production cost as compared with the case of using a carbon nanotube as a carrier. .
Hereinafter, first, the expanded carbon fiber will be described, and then the anticancer agent of the present invention using the expanded carbon fiber will be described.

2. Expanded carbon fiber (1) Features of expanded carbon fiber The expanded carbon fiber used in the anticancer agent of the present invention is not particularly limited, but from the viewpoint of improving hydrophilicity and enhancing the properties as a drug carrier. Preferably has an acidic functional group on the surface, and the amount thereof is preferably large. Here, the acidic functional group refers to a group exhibiting acid properties according to the definition of Lewis, but is usually a group containing an oxygen atom. Examples of the main acidic functional group possessed by the expanded carbon fiber include a carboxyl group and a hydroxyl group, and further, an alkoxy group, an acid anhydride group, and an ether that can be converted into a carboxyl group and a hydroxyl group after hydrolysis or oxidation-reduction. Also included are groups, ketone groups, epoxides and the like. In the present invention, from the viewpoint of imparting hydrophilicity to the expanded carbon fiber, the amount of the acidic functional group of the expanded carbon fiber is usually 1000 mol / g or more, particularly 2000 mol / g or more, more preferably 3000 mol / g or more. It is preferable. Although the upper limit is not particularly limited, it is usually 15000 mol / g or less, particularly 12000 mol / g or less, from the viewpoint of producing expanded carbon fibers and processing thereof.

  Moreover, in the anticancer agent of this invention, it is preferable that an anticancer component couple | bonds with the coupling | bonding group formed by the carboxyl group (-COOH) which exists on the surface of an expanded carbon fiber so that it may mention later. Therefore, as expanded carbon fiber, what contains the carboxyl group (-COOH) of a predetermined ratio or more is preferable. Specifically, the ratio of the carboxyl group of the expanded carbon fiber is usually 1000 mol / g or more, preferably 2000 mol / g or more, more preferably 3000 mol / g or more. The upper limit is not particularly limited, but it is usually 15000 mol / g or less, particularly preferably 12000 mol / g or less, from the viewpoint of the synthesis conditions of the expanded carbon fiber and the reactivity thereof.

  The acidic functional group and carboxyl group of the expanded carbon fiber are, for example, carbon dioxide accompanying a temperature increase under an inert gas as qualitatively analyzed by Fourier Transform Infrared Spectroscopy (FT-IR). Quantitative analysis is possible from the amount generated (derived from carboxyl groups, etc.) and the amount of carbon monoxide generated (derived from hydroxyl groups, etc.). Specifically, for example, the method described in the embodiment can be used.

Although the size of the expanded carbon fiber of this invention is not restrict | limited, For example, it is as follows.
The lower limit of the average diameter of the expanded carbon fiber is usually 50 nm or more, preferably 60 nm or more, more preferably 80 nm or more, from the viewpoint of preventing the carbon fiber itself from becoming an allergen. On the other hand, the upper limit of the average diameter of the expanded carbon fiber is preferably 1000 nm or less, particularly 750 nm or less, and more preferably 500 nm or less, from the viewpoint that it is effectively excluded without being excluded by cells.

  From the viewpoint of preventing the carbon fiber itself from becoming an allergen, the lower limit of the average length of the expanded carbon fiber is usually 50 nm or more, preferably 60 nm or more, and more preferably 80 nm or more. On the other hand, the upper limit of the average length of the expanded carbon fiber is usually 2 μm or less, preferably 1 μm or less, and more preferably 600 nm or less, from the viewpoint that it is effectively excluded without being excluded by cells.

  The average diameter and average length of the expanded carbon fiber can be measured by, for example, a scanning electron microscope (SEM). Specifically, for example, the method described in the embodiment can be used.

(2) Manufacturing method of expanded carbon fiber Such expanded carbon fiber can be manufactured, for example according to the following steps (a) to (c).
(A) Step of preparing an intercalation compound from raw material carbon fiber (b) Step of preparing an expanded carbon fiber by heat-treating the intercalation compound (c) Expansion by performing surface oxidation treatment of the expanded carbon fiber Steps for increasing the total acidic functional groups on the surface of the carbonized carbon fiber and increasing the proportion of carboxyl groups Hereinafter, each step will be described in detail.

(A) Intercalation compound preparation process / raw carbon fiber:
Carbon fibers include pitch-based carbon fibers, PAN-based carbon fibers, rayon-based carbon fibers, vapor-grown carbon fibers, and the like, and any of the raw material carbon fibers used in this step can be used without any particular limitation. However, pitch-based carbon fibers, vapor-grown carbon fibers, and PAN-based carbon fibers are preferred because expanded carbon fibers are easily obtained. Further, among pitch-based carbon fibers, pitch-based carbon fibers containing an optically anisotropic phase, that is, mesophase pitch-based carbon fibers are preferably used because they are particularly easily graphitized and easily obtain expanded carbon fibers. .

  The carbon fiber is obtained by, for example, firing each precursor at a temperature exceeding 800 ° C. after forming each precursor into a fiber shape. As a raw material carbon fiber used for production of the expanded carbon fiber of the present invention, for example, a so-called graphitized fiber that has been heat-treated at about 2900 to 3200 ° C. is suitable.

  The diameter and length of the raw material carbon fiber are not particularly limited, but it is desirable that the diameter and length be small since it is desired to finally obtain an expanded carbon fiber having a large surface area and high light absorption and reactivity. However, if the diameter and length are too small, the handleability may deteriorate and industrial mass production may become difficult. Therefore, it is usually preferable to use carbon fibers having a diameter of 2 μm to 10 μm and a length of 50 μm to 20 cm. More preferably, carbon fibers having a diameter of 5 μm to 15 μm and a length of 1 cm to 15 cm are used.

・ Interlayer compounds:
Carbon fiber has a structure in which multiple graphite structures are overlapped in the length direction, and the crystal structure is highly crystalline and very solid, while the layers are only bonded by van der Waals forces. . Therefore, various low molecular compounds, metal compounds, ions, and the like are easily intercalated between the layers. The resulting compound is an intercalation compound.

As means for obtaining an intercalation compound from raw carbon fibers, there are mainly chemical treatment and electrochemical treatment.
The chemical treatment is a method in which a strong oxidizing agent such as nitric acid or potassium permanganate is allowed to act on carbon fibers in an acid such as sulfuric acid and then treated with excess water. The electrochemical treatment is performed by fixing the raw carbon fibers to the anode in an acid electrolyte and performing electrolysis at a constant current or a constant voltage, and is carried out to introduce compounds and ions between layers. Examples of the acid that is an electrolyte include inorganic acids such as sulfuric acid, concentrated sulfuric acid, nitric acid, concentrated nitric acid, and phosphoric acid, and organic acids such as formic acid and acetic acid. The concentration, voltage, current, treatment time, etc. of the acid electrolyte to be used may be adjusted as appropriate. Specifically, XRD (X-ray Diffraction) or preliminary experiments while confirming the formation ratio of intercalation compounds by weight increase etc. You just have to decide. Usually, a constant current of about 0.1 to 5 A (several A to several tens of A in current density) is energized, and the treatment may be continued until the amount of charge applied to the carbon fiber is 1000 to 10,000 C.

Examples of the chemical treatment include a Hummers-Offman method. In this case, first, for example, after adding concentrated sulfuric acid to a sodium nitrate solution in which carbon fibers are suspended under ice cooling and stirring, potassium permanganate (KMnO ₄ ) is added little by little. The reason why KMnO ₄ is added little by little in the solution is that, since a strong oxidation reaction occurs by addition, it is preferable to carry out the reaction over time. After adding KMnO ₄ and allowing it to stand for a whole day and night, the reaction solution is diluted with water at 70 ° C. to 80 ° C. and left for about 15 minutes. Next, hydrogen peroxide is dropped into the mixed solution until the mixed solution does not foam, to reduce unreacted KMnO _4, and then suction filtration is performed while the mixed solution is warm. Thereby, graphite oxide is obtained as an intercalation compound. The obtained intercalation compound is miniaturized by, for example, heat treatment for 5 seconds to 120 seconds in a temperature range of 400 ° C. to 1000 ° C. Furthermore, you may make it perform refinement | miniaturization processes, such as ultrasonic crushing.

  Intercalation compounds are intercalated with low molecular weight compounds, metal compounds, ions, etc., between graphite layers (stage 1), depending on the degree of intercalation of the compounds, etc. (stage 1) 2) Furthermore, it is classified into various stages such as high-order stage intercalation compounds. As the intercalation compound used in the present invention, a low-order intercalation compound, for example, an intercalation compound of stage 1 or stage 2, is preferred, and an intercalation compound of stage 1 is more preferred. This is because fine expanded carbon fibers are easily obtained in the subsequent steps.

  After the treatment, excess acid and the like adhering to the intercalation compound is removed by washing and further dried. Cleaning may be performed using water or the like. The interlayer distance d (002) plane of the raw carbon fibers is usually 0.3 nm to 0.4 nm. On the other hand, the interlayer distance of the interlayer compound varies depending on the intercalating compound and the like, but extends to approximately 0.65 nm to 1.2 nm. Therefore, the formation of the intercalation compound can be confirmed by measuring the interlaminar distance by X-ray diffraction.

(B) Step for preparing expanded carbon fiber The intercalation compound becomes expanded carbon fiber by heat treatment. The heating temperature may be adjusted as appropriate, but is usually 200 ° C. or higher and 2000 ° C. or lower, more preferably 500 ° C. or higher and 1500 ° C. or lower. The heating time may be relatively short, and is usually about 5 seconds or more and 5 minutes or less. The heating device may be a commonly used one, and for example, an electric furnace or a tubular furnace can be used.

By this heat treatment, the intercalated compound and the like are decomposed and discharged from the interlayer. At that time, the interlayer is expanded and a part of the interlayer bond is broken, and the expanded carbon fiber is expanded from the interlayer compound. Is obtained. This expanded carbon fiber has a larger interlayer distance than that of the raw carbon fiber, and is expanded. For example, the diameter is 2 to 20 times that of the raw carbon fiber, and the bulk density is extremely large as 0.001 to 0.01 g / cm ³ . Formation of the expanded carbon fiber can be confirmed by microscopic observation, bulk density measurement, or the like.

  The above heat treatment increases the interlayer distance in the interlayer compound, and also breaks at the cracked portion, which is shorter than the raw carbon fiber. The diameter and length of the expanded carbon fiber can be measured. For example, the diameter and length of the expanded carbon fiber existing in an arbitrary field of view may be measured by magnifying several tens to several hundreds of times with a scanning electron microscope (SEM).

(C) Surface oxidation treatment step As described above, since the raw carbon fiber is electrolyzed in an acidic solution and heat-treated in air, the expanded carbon fiber includes a carboxyl group on its surface and has an acidic functional group. A group has been introduced. However, the activity of the expanded carbon fiber is not necessarily sufficient, and it is desirable to increase the ratio of carboxyl groups to all acidic functional groups. Therefore, in the present embodiment, the surface oxidation treatment is further performed to increase the acidic functional groups on the surface of the expanded carbon fiber and increase the ratio of carboxyl groups to the total acidic functional groups. It is. By this surface oxidation treatment, in the present embodiment, the ratio of carboxyl groups to all acidic functional groups can be set to 55% or more, for example. For this purpose, it is desirable to first expand the expanded carbon fiber as follows.

・ Powdering:
Preferably, the expanded carbon fiber is pulverized. This is to increase the surface area of the expanded carbon fiber and further increase the amount of carboxyl groups formed on the surface.

  The pulverizing means is not particularly limited, and a conventional method can be used. For example, the expanded carbon fiber may be pulverized by ultrasonic treatment. Ultrasonic treatment can be said to be a suitable pulverization means because the particle size of the obtained expanded carbon fiber can be easily controlled by adjusting the strength and processing time according to the degree of expansion. On the other hand, it is preferable not to use mechanical grinding means such as a ball mill. On the surface of the expanded carbon fiber, many laminated end faces of carbon hexagonal mesh surfaces, so-called edge surfaces, are exposed. This edge surface is very reactive, and in particular, acidic functional groups are often formed on this edge surface. Therefore, when the expanded carbon fiber is pulverized, it is important to preserve the edge surface. However, when the pulverized carbon fiber is pulverized with a ball mill or the like, the edge surface may be damaged.

  The degree of pulverization may be adjusted as appropriate, but it is preferable that the diameter is several nm to several tens of nm, and the maximum length is 10 nm to 2 μm. If the maximum length is 1 μm or less, the surface area is large and the amount of carboxyl groups present on the surface increases, so that highly reactive expanded carbon fibers can be obtained. The size of the pulverized expanded carbon fiber can be measured with a scanning electron micrograph or measured with a zeta electrometer.

・ Surface oxidation treatment:
The means for forming a carboxyl group in the expanded carbon fiber by performing surface oxidation treatment is not particularly limited, and for example, means using acid, ozone or peroxide can be applied. At this time, when an intercalation compound or expanded carbon fiber is obtained from the raw material carbon fiber, various acidic functional groups are formed on the surface because of the heat treatment process, and the carboxyl group can be easily formed by the surface oxidation treatment. Can be increased. Further, as described above, an edge surface is present on the surface of the expanded carbon fiber, and the activity of that portion is high. Therefore, an acidic functional group is easily formed by surface oxidation treatment, and a carboxyl group is easily formed.

  Here, the acidic functional group means a functional group containing an oxygen element. For example, a hydroxyl group (—OH), an aldehyde group (—CHO), a keto group (—C (═O) —), an ether group (—O—). ) And carboxyl groups.

  When the surface of the expanded carbon fiber is oxidized using an acid, the expanded carbon fiber may be heated after being immersed in an acid or an acid aqueous solution. Examples of the acid that can be used include inorganic acids such as nitric acid and sulfuric acid, and organic acids such as formic acid. The heating temperature may be adjusted as appropriate, for example, 60 ° C. or higher and 200 ° C. or lower. The treatment time may be adjusted as appropriate. Specifically, the acidic functional group on the surface is identified and quantified using FT-IR, or the ratio of the carboxyl group to the acidic functional group on the expanded carbon fiber is measured. The time for forming a sufficient amount of carboxyl groups may be used. Usually, it is about 2 hours or more and 24 hours or less.

  When ozone is used, the expanded carbon fiber may be exposed to ozone gas. The concentration of ozone in the ozone gas to be used is, for example, 5% by volume or more and 100% by volume or less. The treatment time may be adjusted as appropriate, and specifically may be determined by a preliminary experiment or the like, but is usually several minutes or more and 5 hours or less.

  When using a peroxide, the peroxide may be dissolved in a solvent and the expanded carbon fiber may be immersed in the solution. Further, the immersion liquid may be heated. Examples of the peroxide include hydrogen peroxide and fuming nitric acid. As the solvent, water or the like can be used. The heating temperature may be adjusted as appropriate, for example, 20 ° C. or more and 50 ° C. or less. By appropriately setting the treatment time, the amount of surface functional groups can be changed. The presence of the surface functional group is specifically confirmed using FT-IR. The amount of the carboxyl group is measured, the ratio of the carboxyl group to the expanded carbon fiber or the total acidic functional group is measured, and the time for which a sufficient amount of the carboxyl group is formed is usually 30 minutes or more, 5 hours or less. When the surface oxidation treatment is performed using an acid or a peroxide, it is preferable to wash the expanded carbon fiber after the treatment. For example, it may be dried after being repeatedly washed with a sufficient amount of water after the treatment.

  The functional group can be introduced to the surface of the expanded carbon fiber by a method other than the above-described method, for example, by an electrochemical treatment in an acidic electrolyte. The expanded carbon fiber is sandwiched between a platinum plate and a platinum mesh, and is used as an anode in an acidic electrolyte. For example, in the case of inorganic acids, nitric acid, sulfuric acid, acetic acid, phosphoric acid, and organic acids are fixed in formic acid or the like. The acidic functional group can be introduced onto the surface of the expanded carbon fiber by performing electrolysis at a current density in the range of 0.1 A / g to 30 A / g under constant current or constant voltage conditions.

・ Expanded carbon fiber with surface oxidation treatment:
By the surface oxidation treatment described above, an acidic functional group is newly formed on the surface of the expanded carbon fiber, particularly the edge surface, and acidic functional groups other than the carboxyl group are oxidized to generate a carboxyl group.

  In order for the expanded carbon fiber to exhibit an excellent effect as a drug carrier, the amount of acidic functional groups, particularly carboxyl groups, present on the surface is very important. In particular, as described above, expanded carbon fibers having a ratio of carboxyl groups in expanded carbon fibers of usually 1000 mol / g or more, particularly 2000 mol / g or more, and more preferably 3000 mol / g or more are very suitable as drug carriers. Excellent drug delivery effect.

  In addition, there is a possibility that an acidic functional group other than the carboxyl group may be involved in the activity. However, as shown in the examples described later, at least the relationship between the activity and the carboxyl group has been confirmed, and surface oxidation treatment is performed. Since the ratio of the carboxyl group in all the acidic functional groups existing on the surface of the expanded carbon fiber is large, the ratio of the carboxyl group in the total acidic functional group is used as a reference in the present invention.

3. Anticancer Agent The anticancer agent of the present invention is obtained by binding an anticancer component to the above-described expanded carbon fiber.

(1) Bonding mode between expanded carbon fiber and anticancer component The binding mode between the expanded carbon fiber and the anticancer component in the anticancer agent of the present invention is not limited, but usually the surface of the expanded carbon fiber. In addition, the anticancer component only needs to be bonded via some linking group. Especially in this invention, it is preferable that the anticancer component has couple | bonded (directly or through a linker) with the coupling group formed by the carboxyl group (-COOH) which exists in the surface of an expanded carbon fiber. Examples of the linking group formed by the carboxyl group include an ester bond (—CO—O—) and a peptide bond (—CO—NH—).
That is, the anticancer agent of the present invention preferably has a structure represented by the following general formula (1).

In the above formula (1), ExCFs represents an expanded carbon fiber.
In the above formula (1), A represents —O— or —NH—.

In the above formula (1), L represents a single bond or a linker. Here, the linker means a divalent group, and examples thereof include a group selected from the following and a group in which two or more of these are combined.
· - _{(CH 2) m1} - polyethylene chain it represented by. Here, m1 represents an integer of 1 to 22.
_{· - (CH 2 CH 2 O} ) m2 - polyethylene glycol chain represented by. Here, m2 represents an integer of 1 to 50.
_{· - (CH 2 CH (OH} )) m3 - polyvinyl alcohol chain represented by. Here, m3 represents an integer of 1 to 50.
-A sugar chain represented by -MS _m4- . Here, each MS independently represents a divalent monosaccharide component, and m4 represents an integer of 1 to 30. Examples of the monosaccharide component include glucose, mannose, galactose, and fructose. These may be D-type or L-type, and may be α-type or β-type. The binding mode of the monosaccharide component is not limited as long as each monosaccharide component allows the glycoside binding mode, but examples include α (1 → 4) bond, α (1 → 6) bond, β (1 → 4). ) Bond, β (1 → 6) bond, and the like.

In said formula (1), X represents an anticancer component. The anticancer component will be described later.
In said formula (1), n is a quantity which prescribes | regulates the introduction number of the anticancer component with respect to an expanded carbon fiber, and represents the integer corresponding to 100-5000 micromol per g.

  In the above formula (1), only the expanded carbon fibers (ExCFs) and the anticancer component-containing group (—CO—A—L—X) bonded thereto are schematically shown. These groups may be bonded to expanded carbon fibers (ExCFs). Examples of other groups include unreacted carboxyl groups (—COOH) and other oxygen-containing functional groups (eg, hydroxyl groups, alkoxy groups, acid anhydride groups, ester groups, epoxide groups, etc.).

(2) Anticancer component The anticancer component refers to a chemical moiety / group that exerts an anticancer drug effect. Specifically, a group derived from a known anticancer agent compound, preferably a monovalent group, and a group holding an active site for exerting an anticancer action can be mentioned. Specifically, groups derived from the following anticancer drug compounds are preferred.

(A) Alkylating agent:
Nitrogen mustards: cyclophosphamide, ifosfamide, melphalan, busulfan, thiotepa, etc.
Nitrosoureas: nimustine, ranimustine, dacarbacin, procarbacin, temozolomide, bendamustine, etc.
Platinum drugs: cisplatin, carboplatin, oxaliplatin, nedaplatin, dahaplatin, etc.

(B) Antimetabolite:
-5-Fluorouracil etc.
(C) Folic acid antimetabolite:
Dihydropteroic acid synthase inhibitor:
-Dihydrofolate reductase (DHFR) inhibitor: methotrexate, trimethoprim, pyrimethamine and the like.

(D) Pyrimidine metabolism inhibitor:
-Thymidylate synthase inhibitors: fluorouracil, flucytosine and the like.
(E) Purine metabolism inhibitor:
-Inosine 5'-phosphate dehydrogenase (IMPDH) inhibitor: 6-mercaptopurine, azathioprine and the like.
-Adenosine deaminase (ADA) inhibitor: Pentostatin and the like.

(F) Nucleotide analogs:
Purine analogs: thioguanine, fludarabine phosphate, cladribine, etc.
Pyrimidine analogs: cytarabine, gemcitabine, etc.
(G) Ribonucleotide reductase inhibitors:
Ribonucleotide reductase inhibitor: hydroxyurea and the like.
(H) Other antimetabolite:
-L-asparaginase and the like.

(I) Topoisomerase inhibitors:
-Camptothecin and its derivatives: irinotecan, nogitane, etc.
Anthracycline drugs: doxorubicin and the like.
-Epipodophyllotoxin drugs: etoposide, etc.
-Quinolone drugs: levofloxacin, ciprofloxacin, etc.

(I) Microtubule polymerization inhibitor:
Vinca alkaloid drugs: vinblastine, vincristine, vindesine, etc.
-Colchicine etc.
(J) Microtubule depolymerization inhibitor:
Taxane type: paclitaxel, docetaxel, etc.

(K) Antitumor antibiotics:
Sarcomacicin, mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin and the like.

(L) Molecular target drug:
Tyrosine kinase inhibitors: imatinib, gefitinib, erlotinib, dasatinib, vandetanib, sunitinib, lapatinib, nilotinib, crizotinib, etc.
-Raf kinase inhibitor: Sorafenib and the like.
-TNF-α inhibitor: etanercept and the like.
Proteasome inhibitor: bortezomib and the like.
Monoclonal antibodies: rituximab, cetuximab, infliximab, baciliximab, tocilizumab, trastuzumab, bevacizumab, omalizumab, mepolizumab, anakinra, gemtuzumab ozogamycin, palivizumab, raribizumab, celtizumab Golimumab, ipilimumab, etc.

  In the present invention, it is possible to use an anticancer component derived from any of the above-mentioned anticancer drug compounds. However, the expanded carbon fiber used as a carrier in the present invention has an advantage that the medicinal effects of the anticancer component can be exhibited more efficiently, although it can be produced relatively inexpensively. In consideration of this point, as an anti-cancer component, targeting performance, although it is cheaper than recent anticancer compounds (for example, molecular targeting drugs) that are excellent in targeting performance but relatively expensive. It can be said that the use of a group derived from a conventional anticancer compound that is inferior in terms of the above results in a more remarkable effect (however, this viewpoint is based on the recent anticancer compound such as a molecular target drug). It never prevents use.)

  Specifically, in the present invention, from the above viewpoint, alkylating drugs, antimetabolites, folic acid antimetabolites, pyrimidine metabolism inhibitors, purine metabolism inhibitors, nucleotide analogs, ribonucleotide reductase inhibitors, or other antimetabolites, etc. It is preferable to use a group derived from as an anticancer component. Among them, it is preferable to use a group derived from an alkylating agent, and it is more preferable to use a group derived from a platinum-based drug.

  Known platinum-based drugs include cisplatin, carboplatin, oxaliplatin, nedaplatin, dahaplatin and the like as described above. Examples of the anticancer component in the anticancer agent of the present invention include groups derived from these known platinum-based drugs and maintaining an anticancer active site.

As the platinum-based drug-derived anticancer component, specifically, a group having a structure represented by the following general formula (2) is preferable.

In the above formula (2), R ¹ and R ² each independently represent a hydrogen atom. Alternatively, R ¹ and R ² may be bonded to each other to form an unsubstituted or substituted 5- or 6-membered hydrocarbon ring together with the carbon atom to which R ¹ and R ² are bonded. Examples of the 5- or 6-membered hydrocarbon ring include a cyclopentane ring and a cyclohexane ring, and a cyclohexane ring is preferable.

In the formula (2), ^{R 3} and ^{R 4} each independently represent a halogen, a hydroxyl group, or C1~6 alkoxy group. Alternatively, R ³ and R ³ may be bonded to each other to form a ring structure represented by the following formula (3).

In the above formula (3), R ¹¹ and R ¹² each independently represent a hydrogen atom. Alternatively, R ¹¹ and R ¹² may be bonded to each other to form an unsubstituted or substituted 3 to 6 membered hydrocarbon ring or heterocyclic ring together with the carbon atom to which R ¹¹ and R ¹² are bonded. The 3- to 6-membered hydrocarbon ring or heterocyclic ring is not limited, but is a cyclopropane ring, cyclobutane ring, cyclopentane ring, cyclohexane ring, azetidine ring, oxetane ring, thietane ring, azolidine ring, oxolane ring. And a thiolane ring. Of these, a cyclobutane ring is preferred.
In said formula (3), W represents a single bond or -C (= O)-.

  The anticancer component may be in the form of a pharmaceutically acceptable salt. As used herein, “pharmaceutically acceptable salt” refers to any acid addition salt or base addition salt that retains the medicinal properties and characteristics of an anticancer component, and is a suitable non-toxic organic or inorganic acid or organic or organic salt. Represents those formed from inorganic bases. Examples of acid addition salts include those derived from inorganic acids (eg hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid) and organic acids (eg p-toluenesulfonic acid, Salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, trifluoroacetic acid, etc.). Examples of the base addition salt include those derived from ammonium hydroxide, potassium hydroxide, sodium hydroxide, quaternary ammonium hydroxide (for example, tetramethylammonium hydroxide). Salt formation by chemical modification of a pharmaceutical compound (ie, drug) is well known to pharmacists as a technique for improving its physical stability, chemical stability, hygroscopicity, fluidity, and solubility. See, for example, Ansel et al., “Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995), pages 196 and 456-457.

(3) Manufacturing method of anticancer agent The manufacturing method of the anticancer agent of this invention is not restrict | limited in particular, It can manufacture by well-known arbitrary methods. Preferably, an expanded carbon fiber having a predetermined amount or more of a carboxyl group on the surface is used as a raw material, and the carboxyl group of the expanded carbon fiber and a functional group (for example, a hydroxyl group or an amino group) of the anticancer component or linker To form a bond (for example, an ester bond or a peptide bond).

  In addition, after binding a part of the anticancer component (for example, a ligand) to the expanded carbon fiber, the remaining portion of the anticancer component (for example, a metal atom) is bonded to the anticancer component. And a prodrug by binding a precursor of an anticancer component to expanded carbon fiber, and then inducing the desired anticancer component in vitro or in vitro to produce an anticancer agent It is also possible to use a method of functioning as

In particular, when producing an anticancer agent having a platinum type anticancer component represented by the above general formula (2), first, an expanded carbon fiber derivative represented by the following general formula (4) is formed. Then, the desired anticancer agent is produced by coordinating the diaminoethyl moiety (—NH—CHR ¹ —CHR ² —NH ₂ ) of the expanded carbon fiber derivative to a platinum atom (Pt). be able to.

In the above formula (4), ExCFs, n, L, R ¹ and R ² represent the same definition as the above formula (1) and formula (2).

  The expanded carbon fiber derivative represented by the above formula (4) is a novel compound, and for example, the production of the anticancer agent of the present invention having an anticancer component represented by the formula (2). Useful as an intermediate or precursor.

(4) Use of anticancer agent The anticancer agent of this invention is effective with respect to various cancers of mammals including a human according to the kind of the anticancer component. Examples of target cancers include pharyngeal cancer, laryngeal cancer, tongue cancer, lung cancer, breast cancer, esophageal cancer, stomach cancer, colon cancer, uterine cancer, ovarian cancer, liver cancer, pancreatic cancer, gallbladder cancer, kidney cancer, prostate Non-epithelial cancer such as cancer, malignant melanoma, thyroid cancer, non-epithelium such as osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, hemangiosarcoma, fibrosarcoma, leukemia, malignant lymphoma, myeloma And so on.

  The anticancer agent of the present invention is usually administered to a patient in a therapeutically effective amount. As used herein, “therapeutically effective amount” means an amount capable of preventing, alleviating or ameliorating symptoms of a disease or extending the survival period of a subject to be treated. The therapeutically effective amount of the anticancer agent of the present invention, that is, the dose can be determined by a method known to those skilled in the art. Such dose may be adjusted for each individual case according to individual requirements (for example, specific compound to be administered, administration route, disease to be treated, patient to be treated). In general, for oral or parenteral administration to an adult weighing about 70 kg, a daily dosage of about 10 mg to about 10,000 mg, preferably about 200 mg to about 1,000 mg, is appropriate, if indicated The upper limit may be exceeded. The daily dose may be administered once or divided into multiple doses, and in the case of parenteral administration, it may be administered by continuous infusion.

  The anticancer agent of the present invention may be administered as it is, but in combination with one or more pharmaceutically acceptable excipients, a preparation (hereinafter sometimes referred to as “the preparation of the present invention”). It can also be used in form. As used herein, “pharmaceutically acceptable” means pharmaceutically acceptable and substantially non-toxic to the patient receiving the compound. Excipients include bases such as lactose, sucrose and corn starch, binders such as gum arabic, corn starch, gelatin, potato starch, alginic acid, corn starch, guar gum, tragacanth gum, gum arabic and other disintegrants, talc, stearic acid, Lubricants such as magnesium stearate, calcium stearate and zinc stearate, as well as pH adjusters, surfactants, dyes, colorants, flavoring agents and the like.

  Formulations of the present invention include formulations suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. The specific dosage form may be appropriately selected according to various conditions such as the type of cancer, lesion site, degree of illness, age of administration subject, dose and number of administration, administration route, etc. Examples include tablets, pills, granules, coatings, capsules, solutions, emulsifiers, suspensions, suppositories, injection solutions, ointments, creams, suppositories and the like.

  The formulation may be provided in unit dosage form for convenience. This can be prepared by any method known in pharmacology. The amount of active ingredient (anticancer agent of the present invention) that can be combined with excipients in the manufacture of a single dosage form varies depending on the patient to be treated and the specific dosage form. The amount of active ingredient that can be combined with excipients in the production of a single dosage form is generally the amount that the active ingredient (the anticancer agent of the present invention) produces a therapeutic effect. In general, the amount of the active ingredient (anticancer agent of the present invention) is usually 1% or more, particularly 5% or more, more preferably 10% or more, and usually 99% or less. Among these, a range of 70% or less, and further 30% or less is preferable.

  The method for preparing the formulation of the present invention comprises the step of mixing the anticancer agent of the present invention with one or more pharmaceutically acceptable excipients. In general, the formulation is prepared by uniformly and intimately mixing the anticancer agent of the present invention with a liquid carrier, a finely divided solid carrier, or both, and then shaping the product as necessary. The

  The formulations of the present invention suitable for oral administration include capsules, cachets, sachets, pills, tablets, lozenges (flavors, usually using sucrose and gum arabic or tragacanth gum), powders, granules, aqueous or non-aqueous In the form of solutions or suspensions in liquids, oil-in-water emulsions, water-in-oil emulsions, elixirs, syrups, lozenges (using inert bases such as gelatin and glycerin, or sucrose and gum arabic), mouthwashes, etc. Thus, there may be mentioned a form containing a predetermined amount of the anticancer agent of the present invention as an active ingredient. The anticancer agent of the present invention may be administered as a bolus, electuary or paste.

  The present invention will be described in more detail with reference to the following examples. However, the present invention is by no means limited to the following examples, and can be implemented with appropriate modifications.

1. Preparation of anticancer agent (1) Example: Preparation of cisplatin-type expanded carbon fibers (CDDP-ExCFs) (a) Preparation of expanded carbon fibers (ExCFs) In a 300 mL beaker, a platinum plate (length) PAN (Polyacrylonitrile) carbon fiber (M60JB manufactured by Toray Industries Inc., fiber diameter 5 μm) trimmed to a length of 5 cm at the lower end of the anode side platinum plate ^It was fixed with ^{(registered trademark)} tape. A silver / silver chloride electrode was attached as a reference electrode beside the anode. In order to sufficiently immerse the carbon fiber, 100 mL of 13 mol / dm ³ nitric acid as an electrolyte was placed in a beaker, and a constant current of 0.5 A was applied until the amount of charge applied to the carbon fiber reached 3600C. The carbon fiber was repeatedly washed with a sufficient amount of water and then air-dried for 24 hours. Thereafter, the carbon fiber was inserted into an electric furnace and subjected to heat treatment at 2600 ° C. for 5 seconds in the air. The treated carbon fiber was pulverized by ultrasonic treatment, dispersed in 13 mol / dm ³ nitric acid (150 mL), and stirred at a temperature of 80 ° C. for 12 hours to oxidize the surface of the expanded carbon fiber. The expanded carbon fibers after the treatment are separated by filtration, thoroughly washed with ultrapure water until the pH of the cleaning solution becomes 7, and then dried at 120 ° C. for 5 hours, whereby expanded carbon fibers (abbreviated as “ExCFs”). You may get it.)

  The morphology of the obtained ExCFs was observed with a scanning electron microscope (SEM, JSM-6701F manufactured by JEOL Ltd.). An example of the obtained SEM image is shown in FIGS. Moreover, the particle size distribution of ExCFs was calculated | required by binarizing the obtained image by the image analysis by WinRoof (made by Mitani Corporation). The results are shown in the graph of FIG. As shown in FIG. 2, the proportion of particles less than 400 nm was 9.17%.

Moreover, by the method of quantifying the amount of carbon dioxide generated while raising the amount of carboxyl groups (—CO ₂ H) of the obtained ExCFs with Fourier Transform Infrared Spectroscopy (FT-IR). When measured, it was about 324 μmol / g.

(B) Introduction of ethylenediamine moiety and platinum into ExCFs

The above ExCFs (1.00 g), p-toluenesulfonic acid (5.70 g, 30.0 mmol), and 2- (2-aminoethyl) ethanol (0.10 mL, 0.99 mmol) were mixed in 100 mL toluene, and the Dean Stark trap was added. And refluxed for 2 days. After removing toluene by decantation, about 50 mL of saturated aqueous sodium hydrogen carbonate solution was added and mixed. The obtained ExCFs was collected by a membrane filter (pore size 10.0 μm), and washed on the filter with toluene, a saturated aqueous sodium hydrogen carbonate solution, water, and methanol, so that an ethylenediamine moiety (on the —CO ₂ H group of ExCFs ( _{-C 2 H 4 -NH-C 2} H 4 -NH 2) was obtained introduced derivative (ExCFs-EDA) 1.00g.

In order to confirm that the ethylenediamine moiety was introduced, each aqueous solution or dispersion (0.01 g / 15 mL) of the original ExCFs and the obtained ExCFs-EDA was prepared, and the pHs were compared. As a result, the original aqueous solution of ExCFs was acidic (pH 6.16), whereas the obtained dispersion of ExCFs-EDA in water was basic (pH 8.48). From this, it was confirmed in the obtained ExCFs-EDA that an ethylenediamine site was introduced into the —CO ₂ H group of ExCFs.

10.0 mg of the ExCFs-EDA was suspended in a 24.1 μmol aqueous solution (10.0 mg / 2.0 mL) of potassium tetrachloroplatinate (II) (K ₂ [PtCl ₄ ], manufactured by Wako Pure Chemical Industries, Ltd.). Reacted. ExCFs after the reaction are washed with toluene, saturated aqueous sodium hydrogen carbonate solution, water and methanol as they are on the membrane filter with a membrane filter (pore size 10.0 μm), and cisplatin type in which platinum atoms are introduced with ethylenediamine as a ligand. 9.42 mg of ExCFs derivative (CDDP-ExCFs) was obtained.

  The obtained CDDP-ExCFs were observed by SEM and analyzed by energy dispersive X-ray spectrometry (EDS). FIG. 3A shows an example of the obtained SEM image, and FIG. 3B shows a corresponding EDS color mapping image. In FIG. 3A, the white particles present on ExCFs represent platinum atoms (Pt). In FIG. 3A, red and yellow dots indicate that the concentration of platinum atoms is high.

The elemental analysis values obtained from the atomic distribution of CDDP-ExCFs calculated by EDS are shown in the following table. It turns out that about 5 mass% of platinum atoms (Pt) are contained. Further, since a considerable amount of nitrogen is also contained, it can be seen that a cisplatin-type anticancer component (CDDP) is formed.

  Moreover, the obtained CDDP-ExCFs was analyzed by X-ray photoelectron spectroscopy (XPS), and the valence of the introduced platinum atom (Pt) was confirmed. The graph of the analysis result by XPS is shown in FIG. As shown in FIG. 4, Pt (II) peaks were observed at 76 and 73 eV, and Pt (0) peaks were observed at 74 and 71 eV. From the above, it can be seen that platinum atoms in CDDP-ExCFs exist in zero and two valences.

(2) Comparative Example: Preparation of cisplatin-type carbon nanotubes (CDDP-CNTs) (a) Preparation of oxidized carbon nanotubes (CNTs) Carbon nanotubes (CNTs) were synthesized by a thermal CVD (thermal chemical vapor deposition) method. That is, by introducing ethanol gas as a raw material of CNTs into a reaction system and causing a chemical reaction on a high-temperature substrate surface, multiwall type (multilayer) CNTs were synthesized. Specifically, by flowing ethanol gas at a flow rate of 130 to 180 sccm into a furnace set with a furnace temperature of 800 ° C. and a furnace pressure of 3.0 to 4.0 × 100 Pa together with argon as a carrier gas. CNTs were formed on the substrate.

  30.0 mg of the obtained multilayer CNTs was added to a 50 mL eggplant flask and suspended in 15 mL of concentrated nitric acid (d = 1.38). The mixture was refluxed at 120 ° C. for 1.5 hours to complete the reaction. Distilled water was added to the reaction mixture, centrifuged (3500 rpm, 30 minutes), and the supernatant was separated by decantation. Distilled water was added again, and the same operation was repeated three times to confirm that the supernatant became neutral, and CNTs were recovered after the oxidation treatment.

The amount of carboxyl groups (—CO ₂ H) contained in the CNTs after the oxidation treatment was measured by a method of quantifying the amount of carbon dioxide generated while raising the temperature with FT-IR, and was about 9.72 μmol / g.

(B) Introduction of ethylenediamine moiety and platinum into CNTs Oxidation treated CNTs 5.0 mg, p-toluenesulfonic acid 1.14 g (6.0 mmol) and 2- (2-aminoethyl) ethanol 0.20 mL (1.97 mmol). And mixed in 200 mL toluene. Refluxed overnight using a Dean-Stark trap. The mixture was allowed to cool to room temperature, and toluene was removed by decantation. Saturated aqueous sodium hydrogen carbonate solution was added to the obtained reaction product, mixed gently, diluted with water, centrifuged (3500 rpm, 30 minutes), and separated by decantation. The same operation was repeated 3 times with distilled water and washed. _3. Derivatives (CNTs-EDA) in which an ethylenediamine moiety (—C ₂ H ₄ —NH—C ₂ H ₄ —NH ₂ ) is introduced into the —CO ₂ H group of CNTs by separating distilled water by decantation. 8 mg was obtained.

In order to confirm that the ethylenediamine site was introduced, each aqueous solution or dispersion (0.01 g / 15 mL) of the original CNTs and the obtained CNTs-EDA was prepared, and the pH was compared. As a result, the original aqueous solution of CNTs was acidic (pH 4.81 to 5.1), whereas the obtained CNTs-EDA dispersion in water was basic (pH 8.1 to 9.1). It was. From this, in the obtained CNTs-EDA, it was confirmed that an ethylenediamine site was introduced into the —CO ₂ H group of CNTs.

The above CNTs-EDA (5.0 mg) was suspended in a 12.0 μmol aqueous solution (5.0 mg / 2.0 mL) of potassium tetrachloroplatinate (II) (K ₂ [PtCl ₄ ], manufactured by Wako Pure Chemical Industries, Ltd.). Reacted. Stirring was stopped, about 70 mL of water was added to the reaction suspension, centrifugation (3500 rpm, 30 minutes) was performed, and the reaction product was separated from water by decantation. About 70 mL of water was added again, and the same operation was repeated three times to wash the CNTs. By removing water by decantation, 4.9 mg of a cisplatin-type CNTs derivative (CDDP-CNTs) into which a platinum atom was introduced with an ethylenediamine moiety as a ligand was obtained.

  The obtained CDDP-CNTs were analyzed by XPS measurement. The graph of the analysis result by XPS is shown in FIG. As shown in FIG. 5, Pt (II) peaks were observed at 76 and 73 eV, and small peaks of Pt (0) were confirmed at 74 and 71 eV. From the above, it can be seen that the platinum atoms in CDDP-CNTs are also present in zero valence and two valences.

2. Evaluation of anticancer agent (1) Preliminary evaluation: cell uptake Prior to evaluation of anticancer agents CDDP-ExCFs and CDDP-CNTs prepared by the above procedure, as a preliminary evaluation, the corresponding carriers ExCFs and CNTs were fluorescent (fluorescein). Labeled to confirm the accumulation effect on the tumor.

(A) Preparation of fluorescently labeled ExCFs and CNTs The fluorescently labeled ExCFs and CNTs were prepared according to the method described in Meng et al., Dyes and Pigments, (2007), 73, 254-260. Specifically, it is as follows.

Preparation of fluorescein-NHS-ester 0.20 g (0.60 mmol) of fluorescein (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.07 g (0.60 mmol) of N-hydroxysuccinimide (NHS, manufactured by Wako Pure Chemical Industries, Ltd.) Was mixed with 5 mL of THF. While stirring the suspension, dicyclohexylcarbodiimide (manufactured by Kishida Chemical Co., Ltd., 0.12 g, 0.60 mmol) was added over 2 days at room temperature. The reaction mixture was filtered, the solvent was removed under reduced pressure, and then treated with column chromatography (silica gel, BW80S manufactured by Fuji Silysia Chemical Ltd.) to give fluorescein-NHS-ester (Rf0.8, acetone, kieselgel TLC) in orange. Obtained as a powder (0.15 g, 58% yield).

-Preparation of fluorescently labeled ExCFs 50 mg of ExCFs (containing about 324 µmol / g -COOH group) prepared in 1 (1) (a) above was added to a 200 mL Erlenmeyer flask, suspended in 100 mL of toluene, and p-toluenesulfonic acid 3 .70 g (19.5 mmol) and 1.0 mL (17 mmol) of ethanolamine were added and sonicated for about 3 minutes. The mixture was refluxed for 24 hours using a Dean-Stark trap. Toluene was decanted, and about 50 mL of a saturated aqueous sodium hydrogen carbonate solution was added and shaken, and dispersed by sonication. ExCFs was collected by filtration with a membrane filter (pore size 10.0 μm), and the amino group was introduced into ExCFs by washing with toluene, saturated aqueous sodium hydrogen carbonate solution, water and methanol as it was on the filter. 0.02 g of this amino group-containing ExCFs was suspended in 5 mL of water, 0.02 g (50 mmol) of the above-mentioned fluorescein-NHS-ester was dissolved in 5 mL of acetonitrile, added to the suspension of ExCFs, and stirred overnight. ExCFs were collected by filtration with a membrane filter (pore size 10.0 μm), and washed with water and methanol on the filter as they were to obtain 0.07 g of fluorescent (fluorescein) -labeled ExCFs.

-Preparation of fluorescently labeled CNTs 13.3 mg of oxidized CNTs prepared in 1 (2) (a) above (approximately 9.72 μmol / g of -COOH group) was suspended in 100 mL of toluene, and 1.88 g of p-toluenesulfonic acid ( 9.88 mmol) and 0.50 mL (8.27 mmol) of ethanolamine were added, and the mixture was refluxed for 24 hours using a Dean-Stark trap. The mixture was allowed to cool to room temperature, and toluene and CNTs were separated by decantation. About 50 mL of saturated aqueous sodium hydrogen carbonate solution was added and mixed to disperse. After centrifugation (3000 rpm, 30 minutes), separation was performed by decantation. The same operation was repeated 3 times with distilled water and washed. Distilled water was separated by decantation to obtain CNTs introduced with amino groups. 10 mg of this amino group-containing CNTs was suspended in 5 mL of water, 10 mg of the above-mentioned fluorescein-NHS-ester was dissolved in 5 mL of acetonitrile, added to the suspension of carbon fiber, and stirred overnight. After centrifugation (3500 rpm, 30 minutes), separation was performed by decantation. The same operation was repeated 6 times with distilled water / methanol and washed. Distilled water was separated by decantation to obtain 10 mg of fluorescent (fluorescein) -labeled CNTs.

(B) Preparation of fluorescently labeled ExCFs and CNTs In order to study the incorporation of the obtained fluorescently labeled ExCFs and CNTs into metastatic lung lesion cells, suspensions of fluorescently labeled CNTs and ExCFs at a concentration of 0.1 mg / ml were used in humans. In addition to the cell culture medium of the osteosarcoma cell line MG63 (obtained from American Type Culture Collection), observation was performed using a microscope system (manufactured by Keyence Corporation, BZ-9000) equipped with a confocal microscope. .

  Examples of the microscopic images obtained are shown in FIGS. 6 (a) and 6 (b). As shown in both images, both fluorescently labeled CNTs and ExCFs were taken up by MG63 cells. Comparing the image of the fluorescently labeled CNTs (FIG. 6A) and the image of the fluorescently labeled ExCFs (FIG. 6B), the fluorescently labeled ExCFs emits stronger fluorescence. Assuming that the fluorescence (fluorescein) introduction rate of the fluorescently labeled CNTs and the fluorescently labeled ExCFs is the same, it is considered that ExCFs is more easily accumulated in cancer cells than CNTs.

(2) In vitro cytotoxicity evaluation The in vitro cytotoxicity of CDDP-CNTs and CDDP-ExCFs was determined by comparing MTT of human osteosarcoma cells (MG63) (3- (4,5-di-methylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) reduction assay was performed.

(A) Materials and methods / materials:
As free cisplatin (hereinafter sometimes referred to as “free CDDP”), Kyowa Kirin Platocin Injection (10 mg per vial) was used.
As the CDDP-ExCFs and CDDP-CNTs, those prepared above were used.
As the non-drug-extruded ExCFs, those prepared in 1 (1) (a) above were used.
As the non-drug-introduced CNTs, self-made multilayer CNTs used as raw materials in the above 1 (2) (a) were oxidized and used.

·Method:
Human osteosarcoma cell line MG63 (obtained from the American Type Culture Collection) was seeded in a 96 well plate at a concentration of 1 × 10 ⁴ cells / well. Free CDDP, CDDP-ExCFs, or CDDP-CNTs was added to each well of cells at a concentration of CDDP equivalent of 1 to 10 μg / ml, respectively, and incubated for 24 hours. Moreover, the cell which added the drug non-introduced ExCFs or drug non-introduced CNTs of the quantity corresponding to the said CDDP-ExCFs or CDDP-CNTs, and the cell (control) which added nothing were incubated for 24 hours, respectively.

(B) Results FIG. 7 shows a graph showing the relationship between the resulting CDDP concentration and cell viability. There was no significant difference in cytotoxicity between the non-added cells and the non-drug-introduced CNTs and non-drug-introduced ExCFs.
The cell (MG63) viability after 24 hours after administration of free CDDP, CDDP-CNTs or CDDP-ExCFs was as shown in the following table.

  From the above results, CDDP-CNTs and CDDP-ExCFs were both more toxic than free CDDP, but CDDP-ExCFs in particular is more toxic than CDDP-CNTs and exhibits excellent anticancer activity. It became clear.

(3) TUNEL immunohistochemical assay In order to visualize apoptosis (cell death) caused by an anticancer agent, immunohistological examination by TUNEL (Terminal deoxynucleotidyltransferase dUTP Nick End Labeling) was performed.

(A) Materials and methods / materials:
As free cisplatin (hereinafter sometimes referred to as “free CDDP”), Kyowa Kirin Platocin Injection (10 mg per vial) was used.
As the CDDP-ExCFs and CDDP-CNTs, those prepared above were used.
As the non-drug-extruded ExCFs, those prepared in 1 (1) (a) above were used.
As the non-drug-introduced CNTs, the oxidation-treated multilayer CNT prepared in the above 1 (2) (a) was used.

·Method:
To human osteosarcoma cell line MG63 (obtained from American Type Culture Collection), free CDDP, CDDP-ExCFs, or CDDP-CNTs was added at a concentration of CDDP equivalent 0-10 μg / mL, respectively. Incubated for 24 hours. Moreover, the cell which added the drug non-introduced ExCFs or drug non-introduced CNTs of the quantity corresponding to the said CDDP-ExCFs or CDDP-CNTs, and the cell (control) which added nothing were incubated for 24 hours, respectively.

  In order to slice a tumor tissue of a mouse and immunostain it, the tumor tissue removed from a living body was made into a paraffin section and then stained with a TUNEL kit. Thereafter, the cells were observed with a BIOREVO microscope system (manufactured by Keyence Corporation, BZ-9000) equipped with a confocal microscope.

(B) Result The obtained micrograph is shown to Fig.8 (a)-(f). In the cells treated with no addition (FIG. 8 (a)), non-drug-introduced CNTs (FIG. 8 (b)) and non-drug-introduced ExCFs (FIG. 8 (c)), almost all TUNEL positivity (fluorescence) was observed. Apoptosis was not confirmed. In cells treated with free CDDP (FIG. 8 (d)), CDDP-CNTs (FIG. 8 (e)) and CDDP-ExCFs (FIG. 8 (f)), TUNEL positivity (fluorescence) was detected. CDDP-CNTs and CDDP-ExCFs both showed stronger fluorescence than free CDDP, but CDDP-ExCFs in particular showed stronger fluorescence than CDDP-CNTs, indicating an excellent anticancer effect. Became.

(4) Western blot analysis Western blot analysis of Bax, PARP-1 and caspase 3 was also performed in order to visualize apoptosis (cell death) caused by anticancer agents. Bax is a protein having an action of promoting apoptosis. PARP-1 is an enzyme having an action of repairing DNA damaged by an anticancer agent or the like. Caspase 3 is an enzyme that has an action of degrading PARP-1 and suppressing DNA repair. When apoptosis is initiated due to DNA damage or the like, induction of Bax and degradation of caspase-3 and PARP-1 are promoted, and DNA repair is inhibited.

(A) Materials and methods / materials:
As free cisplatin (hereinafter sometimes referred to as “free CDDP”), Kyowa Kirin Platocin Injection (10 mg per vial) was used.
As the CDDP-ExCFs and CDDP-CNTs, those prepared above were used.
As the non-drug-extruded ExCFs, those prepared in 1 (1) (a) above were used.
As the non-drug-introduced CNTs, the oxidation-treated multilayer CNT prepared in the above 1 (2) (a) was used.

·Method:
As in 2 (3) (a) above, human osteosarcoma cell line MG63 is obtained under the conditions of each of free CDDP, CDDP-ExCFs, CDDP-CNTs, drug-unintroduced ExCFs, and drug-unintroduced CNTs, or without any addition Incubated with (control). The tumor tissue of the mouse after incubation was taken out and homogenized, and the concentrations of the cleaved caspase 3 (caspase 3 degradation product), cleaved PARP-1 (PARP-1 degradation product), and Bax contained therein were determined. Measured by Western blot analysis.

  Protein concentration was measured by Bradford assay using Bio-Rad protein assay solution. Protein (30 μg) was separated by 12.5% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Santa Cruz Biotechnology). Immunoblotting was performed using a rabbit-derived anti-Bax polyclonal antibody, a rabbit-derived anti-cleaved caspase-3 polyclonal antibody, and a mouse-derived anti-cleaved PARP-1 monoclonal antibody (1: 500; manufactured by Santa Cruz Biotechnology). In addition, a rabbit-derived anti-β-actin antibody (1: 500; manufactured by Sigma) was used as a control, and it was confirmed that each sample was uniformly loaded. This membrane was tested using a goat-derived horseradish peroxidase-conjugated anti-rabbit IgG (1: 1000; manufactured by Santa Cruz Biotechnology) as a probe.

(B) Results The results are shown in FIG. None of the cells treated with additive-free, drug-introduced CNTs and drug-introduced ExCFs showed any induction of Bax, degradation of caspase-3, or degradation of PARP-1, and no apoptosis was confirmed. On the other hand, in cells treated with free CDDP, CDDP-CNTs, and CDDP-ExCFs, induction of Bax, degradation of caspase-3, and degradation of PARP-1 were detected, confirming that apoptosis occurred. It was. Both CDDP-CNTs and CDDP-ExCFs showed stronger Bax induction, caspase-3 degradation, and PARP-1 degradation than free CDDP, especially CDDP-ExCFs being even stronger than CDDP-CNTs. It was revealed that it showed excellent anticancer activity by showing induction of Bax, degradation of caspase-3, and degradation of PARP-1.

(5) In vivo evaluation by measuring lung metastatic lesions
Anti-cancer activity was evaluated by administering free cisplatin, CDDP-ExCFs or CDDP-CNTs to mice transplanted with tumors in the buttock and investigating the size of cancer cells present in the lungs (FIG. 10).

(A) Materials and Methods-Osteosarcoma Cell Line Human osteosarcoma cell line MG63 was obtained from the American Type Culture Collection. In all animals, lung metastases were confirmed by the naked eye and under a microscope within 4 weeks.

-Animals Sterile female BALB / c nude mice (6-8 weeks old) were purchased from CLEA Japan. All animals were kept in a sterile animal facility for at least one week before each experiment. All experiments were conducted according to the animal experiment regulations established by the Oita University Graduate School of Medicine.

Test plan: 1 × 10 ⁶ MG63 cells were implanted subcutaneously into the buttocks of 6-8 week old female nude mice using known techniques for introducing osteosarcoma. All animals developed tumors. Two weeks after transplantation, the animals were divided into 6 groups and subjected to the following treatments.
Preliminary experiments were conducted and the presence of lung metastases was confirmed in 20 mice.

-Evaluation and analysis of results For each group of animals, a slice CT image of the transplanted primary lesion and lung metastatis was taken using a micro CT machine (R_mCT). The CT image was analyzed using I-view-R (Morita Manufacturing Co., Ltd.) as a viewer, the size of the tumor was measured, and the tumor volume was calculated by the formula (π × long axis × short axis × short axis) / 6. .
Analysis of differences between groups was performed by non-repeated measures analysis of variance (ANOVA) and Scheffe test. For each analysis, SPSS ^{(registered trademark)} 18.0 software (manufactured by SPSS Japan) was used.

(B) Results FIG. 10 shows an example of an image of a lung metastasis tumor in a drug-free ExCFs administration group, a free CDDP administration group, and a CDDP-ExCFs administration group, and FIG. 11 is a graph showing the time course of the cumulative survival rate of mice. Respectively. In the image of FIG. 10, white shadows seen in the lung represent cancer cells. In the lungs of mice in the drug-free ExCFs administration group, cancer cells spread to the lungs 20 days after administration, and further to the entire lungs 40 days after administration. Therefore, ExCFs itself has an anticancer effect. You can see almost no. In the mice in the free CDDP administration group, almost no metastasis of cancer cells was confirmed on the 20th day after administration, but on the 40th day after administration, the proliferation of cancer cells was confirmed although it was small compared with the drug-free ExCFs. In the mice in the CDDP-ExCFs administration group, cancer cells hardly proliferated at any time point on the 20th and 40th days after the administration. From this, it can be seen that CDDP-ExCFs has a higher anti-cancer effect than free CDDP.

  The time course of the volume of primary lesions and lung metastatis in mice in all 6 groups is shown in the graphs of FIGS. 12 (a) and 12 (b), respectively. The growth rates of primary lesions and lung metastasis tumors in the drug-free ExCFs administration group and the drug-free CNTs administration group were both comparable to the control group, and no anticancer effect or metastasis suppression effect was observed.

  In contrast, in the free CDDP administration group, the CDDP-ExCFs administration group, and the CDDP-CNTs administration group, the growth rate was suppressed for both primary lesions and lung metastasis tumors, and the anticancer effect and the metastasis suppression effect were exhibited. confirmed. The effect was higher in the CDDP-CNTs administration group than in the free CDDP administration group, which is due to the specific accumulation of nanocarbon materials (ExCFs and CNTs) in cancer cells. It is thought that this is because cancer sites acted efficiently on cancer cells, indicating that these nanocarbon materials are effective as carriers for anticancer agents.

Furthermore, in the CDDP-ExCFs administration group, the primary lesion and lung metastasis suppression effects were more excellent than those in the CDDP-CNTs administration group. Specifically, the volume of the primary lesion in the 45 days elapse after administration, in the CDDP-ExCFs administration group was ^{277.84 ± 41.34mm 3, 426.78.53 ± 33.16mm} 3 of CDDP-CNTs administration group (P <0.01). In addition, the volume of the lung metastasis tumor at 45 days after the administration was 334.94 ± 118.13 mm ³ in the CDDP-ExCFs administration group, and more than 523.61 ± 94.75 mm ³ ) in the CDDP-CNTs administration group. Again, it was significantly reduced (p <0.01). These results indicate that ExCFs is superior to CNTs as a carrier for anticancer agents.

  The anticancer agent of the present invention is useful in the medical field, particularly in the treatment and prevention of various cancers, and its industrial utility value is extremely high.

Claims (5)

  An anticancer agent comprising an expanded carbon fiber combined with an anticancer component. The anticancer agent according to claim 1, which is represented by the following general formula (1):
(In the formula (1),
ExCFs represent expanded carbon fibers,
A represents -O- or -NH-,
L represents a single bond or a linker, wherein the linker is a polyethylene chain represented by — (CH ₂ ) _m1 — (where m1 represents an integer of 1 to 22), — (CH ₂ CH ₂ O) _m2 — (where m2 represents an integer from 1 to 50), — (CH ₂ CH (OH)) _m3 — (where m3 represents an integer from 1 to 50) And a sugar chain represented by -MS _m4- (wherein MS each independently represents a divalent monosaccharide component, and m4 is an integer of 1 to 30) Represents), and
X represents an anticancer component,
n represents an integer corresponding to 100 to 5000 μmol per 1 g of the anticancer agent of the formula (1). ). The anticancer agent according to claim 2, wherein X is represented by the general formula (2).
(In the formula (2),
R ¹ and R ² each independently represent a hydrogen atom or an alkyl group, or R ¹ and R ² are bonded to each other, together with the carbon atom to which R ¹ and R ² are bonded, respectively, unsubstituted or substituted May form a 5- or 6-membered hydrocarbon ring,
R ³ and R ⁴ each independently represent a halogen, a hydroxyl group, or a C1-6 alkoxy group, or R ³ and R ³ are bonded to each other to form a ring structure represented by the formula (3). May be
(In formula (3),
R ¹¹ and R ¹² each independently represent a hydrogen atom, or R ¹¹ and R ¹² are bonded to each other, together with the carbon atom to which R ¹¹ and R ¹² are bonded, an unsubstituted or substituted 3 to 6 member May form a hydrocarbon ring or a heterocyclic ring of
W represents a single bond or —C (═O) —. ). ). Expanded carbon fiber derivative represented by the general formula (4)
(In the formula (4), ExCFs, n, L, R ¹ and R ² represent the same definitions as the formula (1) of claim 2 and the formula (2) of claim 3).   A preparation comprising the anticancer agent according to any one of claims 1 to 3 and one or more pharmaceutically acceptable excipients.