JP2009137884A - Pharmaceutical composition for ced method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for the CED (convection-enhanced delivery) method, exhibiting its own high medicinal activity with broad distribution over the brain tissue in brain treatment by the CED method, and applicable to various medicaments including hydrophobic medicaments. <P>SOLUTION: The pharmaceutical composition for the CED method is such as to include medicament-encapsulated polymer micelles as the active ingredient in an aqueous medium, wherein the medicament-encapsulated polymer micelles are such that a medicament is encapsulated in polymer micelles formed of a block copolymer having a hydrophilic region and a hydrophobic region, wherein the hydrophilic region and the hydrophobic region are disposed outside and inside respectively. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a pharmaceutical composition used in the CED method.

  In recent years, in addition to surgical therapy and radiation therapy for malignant gliomas (glioblastoma, anaplastic astrocytoma, etc.), various therapies such as chemotherapy, immunotherapy, gene therapy, and molecular targeted therapy have been tried. However, the improvement of treatment results has not progressed much compared to the 1980s. In particular, glioblastoma, which accounts for 32% of gliomas, has a very poor 5-year survival rate of about 7%. The oral alkylating agent listed in the drug price list in 2006 is the only anti-cancer drug that has been confirmed by phase III trials to prolong survival in patients with primary glioblastoma. The median survival time is only 14.6 months, which is limited to an extended effect of about 2 months compared to treatment with radiation therapy alone. Furthermore, recurrent malignant gliomas are now recognized as difficult to treat.

For example, in the treatment of malignant glioma, when a systemic administration of a therapeutic drug is performed intravenously, there is a problem of permeability through the blood brain barrier (BBB) of the therapeutic drug. . That is, since the tight junction of the vascular endothelial cell layer is made very dense at the blood-brain barrier, hydrophilic drugs hardly permeate. Furthermore, the hydrophobic drug that can normally permeate the cell membrane is also discharged to the blood side by the action of the P-glycoprotein that is expressed in large numbers in the BBB. For example, even if the BBB permeability of a therapeutic drug is obtained, in order to ensure an effective drug concentration for central nervous system tumors, the drug dose is increased because it causes a number of side effects to the whole body. It is limited and the therapeutic effect cannot be so high.
As described above, because of the inherent problem of BBB penetration, it can be said that chemotherapy for brain tumors is more difficult than for other solid cancers. In fact, among all solid cancers, brain tumors, together with pancreatic cancer, belong to the most difficult class of anticancer drugs. The CED (Convection-Enhanced Delivery) method is attracting attention as a new administration method designed to overcome the difficulty of anticancer drug treatment for this malignant glioma.

  Unlike conventional local intracerebral administration using diffusion, the CED) method uses a microinfusion pump and a catheter, continuously infuses a drug at a very low rate using a drug as a positive pressure, and bulk flows through the cell gap. It is a local administration method in the new brain that causes convection in the flow and provides a wide and high concentration drug distribution in the brain tissue gap without mechanical damage to the brain structure. According to the CED method, since the dosage is an order of magnitude smaller than that of systemic administration, systemic toxicity can be suppressed to a level with no problem. The distribution of drugs in the brain can be controlled by the volume and rate of injection, and systemic complications due to drugs are said to be negligible.

  In continuous injection by the CED method, it is necessary to keep a very low flow rate of 1 to 5 μL / min stably for a long time. There are a wide variety of drugs that can be administered by the CED method, and various drugs have been tried in rat brain tumor transplantation models, and their effectiveness has been reported. So-called conventional low-molecular-weight anticancer drugs, boronated drugs for neutron capture therapy, suicide genes for gene therapy, proteins and peptides that cause cell death, or cytokines for immunostimulation, It is roughly divided into immunotoxins. Among these, those using low-molecular-weight anticancer agents have been used for a long time in the field of treatment for humans, and are considered to be most suitable for practical use. However, when a low molecular weight anticancer drug is administered by the CED method, it is necessary to administer the drug by binding or encapsulating the drug carrier, not by administering the drug alone. This is because a low-molecular-weight anticancer agent moves from the cancer tissue at the administration site into the blood, and thus is discharged at a high rate from the cancer tissue and cannot spread widely throughout the cancer tissue. By binding or encapsulating the drug carrier, the rate of draining the cancer tissue to the blood can be greatly reduced, so that the anticancer drug can be widely distributed throughout the cancer tissue.

  The CED method (Convection-Enhanced Delivery), which is a direct brain tumor injection method, is ideal as a selective chemotherapy for cancer. However, even if a solution of a low molecular weight anticancer agent is administered by the CED method in practice, the solution cannot be exerted because it cannot be spread and distributed throughout the cancer tissue. This is because a low molecular weight anticancer agent migrates from cancer tissue into the blood and is discharged from the cancer tissue at a high rate and is not widely distributed in the cancer tissue.

In order to solve the above problems, a low molecular weight anticancer agent is enclosed in a drug carrier system, the rate of transfer from cancer tissue to blood is reduced, and it is widely distributed in cancer tissue by convection generated by CED method. It is effective to make it. As this carrier, liposome has been studied conventionally. However, there are two problems with liposomes for use as carriers.
(1) Difficult to apply to hydrophobic drugs. When a highly hydrophobic drug is encapsulated in liposomes, it is not encapsulated in the inner aqueous phase, but is encapsulated in a bilayer membrane composed of an outer phospholipid. Since this bilayer is only as thick as two lipid molecules, its drug content is low. In addition, when the drug content is increased, the properties of the bilayer are greatly changed by the influence of the physicochemical properties of the drug molecules, so that it cannot function as a drug carrier.
(2) Since the drug release is controlled only by one phospholipid bilayer, it is either slightly faster or slower than the desired release behavior.

J. Andrew MacKaya, Dennis F. Deenb and Francis C. Szoka, Jr., Brain Research Volume 1035, Issue 2, 28 February 2005, Pages 139-153 M. Yokoyama, T. Okano, Y. Sakurai, S. Fukushima, K. Okamoto, and K. Kataoka, Selective delivery of adriamycin to a solid tumor using a polymeric micelle carrier system, J. Drug Targeting, 7 (3), 171186 (1999) Kumi Kawano, Masato Watanabe, Tatsuhiro Yamamoto, Masayuki Yokoyama, Praneet Opanasopit, Teruo Okano, and Yoshie Maitani, Enhanced antitumor effect of camptothecin loaded in long-circulating polymeric micelles, J. Controlled Release, 112, 329 332 (2006)

  The object of the present invention is a brain treatment method by the CED method, which can be widely distributed in brain tissue and exhibit high drug activity, and can be applied to many kinds of drugs including hydrophobic drugs. It is to provide a composition.

  As a result of diligent research, the inventors of the present application have formed a drug inside a polymer micelle formed from a block copolymer having a hydrophilic region and a hydrophobic region, wherein the hydrophilic region is located outside and the hydrophobic region located inside. By using a drug-encapsulated polymer micelle encapsulated as a drug carrier, it was found that when a drug is administered to the brain by the CED method, it can be widely distributed in brain tissue and exhibit high drug activity, Further, the inventors have conceived that a large amount of drug can be encapsulated by using this polymer micelle as compared with liposome even when the drug is hydrophobic.

  That is, the present invention is formed from a block copolymer having a hydrophilic region and a hydrophobic region, wherein the drug is enclosed in a polymer micelle in which the hydrophilic region is arranged outside and the hydrophobic region is arranged inside. Provided is a pharmaceutical composition for CED method containing molecular micelle as an active ingredient in an aqueous medium.

  When administered to the brain by the CED method, the pharmaceutical composition of the present invention can be widely distributed in brain tissue and exhibit high drug activity, and even a hydrophobic drug can be administered in large quantities be able to. Therefore, when the pharmaceutical composition of the present invention containing an anticancer agent as a drug is directly administered to a brain cancer tumor by the CED method, the anticancer agent is widely distributed in cancer tissues and exhibits high anticancer activity, and has an excellent cancer therapeutic effect. It can be demonstrated.

  The polymeric micelle contained in the pharmaceutical composition of the present invention is a polymeric micelle formed from a block copolymer having a hydrophilic region and a hydrophobic region, wherein the hydrophilic region is disposed outside and the hydrophobic region is disposed inside. Yes, the drug is sealed inside. Such drug-encapsulated polymer micelles themselves and methods for producing the same have been studied and publicly known by the present inventors (for example, Non-Patent Document 2 and Non-Patent Document 3).

  In the polymer micelle, the “hydrophilic region” and the “hydrophobic region” are so hydrophilic that both micelles are formed with the hydrophilic region outside and the hydrophobic region inside in water. It means each different area.

  Preferable examples of the structure constituting the hydrophilic region include, but are not limited to, polyethylene glycol, poly (vinyl alcohol), poly (vinyl pyrrolidone), and the like. The end of the hydrophilic region (on the opposite side of the hydrophobic region, that is, the end of the hydrophilic region of the block copolymer) is hydrogen, lower (1 to 6 carbon atoms, the same applies hereinafter) alkyl group, lower hydroxyalkyl group, lower An alkylamino group and the like are preferable, but not limited thereto. The molecular weight of the hydrophilic region is preferably about 2000 to 20,000, and more preferably about 4000 to 14000.

  Preferred examples of the structure constituting the hydrophobic region include a hydrophobic group in part or all of free carboxyl groups such as poly (aspartic acid), poly (glutamic acid), poly (acrylic acid) and poly (methacrylic acid). However, it is not limited to these. Aspartic acid and glutamic acid that can be used in the hydrophobic region have two carboxyl groups in one unit, and either carboxyl group may be used for peptide bonding of the main chain. Thus, it may be a copolymer obtained by subjecting different carboxyl groups to main chain peptide bonds. Examples of the hydrophobic group include, but are not limited to, groups containing an aromatic ring such as a benzyl group, a phenyl group, and a naphthyl group. Alternatively, as specifically shown in the Examples, when the drug encapsulated in the micelle is a hydrophobic drug containing an aromatic ring such as adriamycin, the drug is used as the carboxyl group as the hydrophobic group. It may be covalently bound to. In this case, when the drug has an amino group such as adriamycin, the amino group and the carboxyl group can be easily amide-bonded using a coupling reagent such as carbodiimide. When a hydrophobic drug is covalently bonded to the hydrophobic region, the polymer micelle can be administered to the tissue and after disintegration, the drug covalently bonded to the hydrophobic region can also exert drug activity. The ratio of the carboxyl group in the hydrophobic region to which the hydrophobic group is bonded is usually about 20 mol% to 80 mol%, preferably about 30 mol% to 70 mol%. The end of the hydrophobic region (opposite to the hydrophilic region, ie, the end of the hydrophobic region of the block copolymer) is hydrogen, lower alkyl group, hydroxyl group, lower alkyloxy, phenyl lower alkyl, phenyl lower alkyloxy, lower alkyl Phenyl, lower alkylphenyloxy, lower alkoxycarbonyl, phenyl-lower alkoxycarbonyl, lower alkylaminocarbonyl, phenyl-lower alkylaminocarbonyl groups and the like are preferred, but not limited thereto. In addition, the number of free and carboxyl groups to which the hydrophobic group is bound contained in the hydrophobic region (the polymerization unit is one in the polymerized state such as aspartic acid, glutamic acid, (meth) acrylic acid described above). In the case of having a free carboxyl group, the degree of polymerization of the unit is usually 10 to 50, preferably about 20 to 30.

The hydrophilic region and the hydrophobic region may be directly bonded or may be bonded via a linker. As such a linker, —O—, —NH—, —OCO—, —OCONH—, —NHCO—, —NHCONH—, —COO—, —CONH—, — (CH ₂ ) _n — (n is 1 to 1) 6), -R ^1- (CH ₂ ) _n- (n is an integer of 1-6, R ¹ is -O-, -NH-, -OCO-, -OCONH-, -NHCO-, -NHCONH- , —COO—, —CONH—), — (CH ₂ ) _n —R ² — (n is an integer of 1 to 6, R ² is —O—, —NH—, —OCO—, —OCONH—, —NHCO -, -NHCONH-, -COO-, -CONH-), -R ^1- (CH ₂ ) _n -R ^2- (n is an integer of 1 to 6, R1 and R ² are independently of each other -O-,- NH-, -OCO-, -OCONH-, -NHCO-, -NHCONH-, -COO-, -CONH-), but not limited thereto.

  The particle size (diameter) of the polymer micelle is preferably 10 nm to 100 nm. The first reason that the polymer micelle particle size is preferably within this range is to suppress uptake into glial cells in the brain. In the case of intravenous administration, suppressing the uptake of macrophage-shaped Kupffer cells in the liver by reducing the particle size of the drug carrier to about 200 nm or less was a necessary requirement for achieving cancer targeting (particles It is thought that the larger the diameter, the larger the surface area that interacts with the cells, and the more easily phagocytosed). Although not yet elucidated at this time, avoiding phagocytosis of glial cells that perform macrophage-like functions in the brain is considered to be an important matter for reaching cancer cells efficiently. Moreover, it is because the behavior which spreads by diffusion after the end of administration or is discharged from the treatment site depends on the particle size. It can be said that the particle size in the above range is preferable to achieve optimization of the reach of the anticancer agent and the duration of the anticancer activity.

  The particle size of the polymer micelle can be adjusted by changing the molecular weight of the block copolymer constituting the polymer micelle. In order to achieve a particle size of 10 nm to 100 nm, the molecular weight of the block copolymer is usually about 1,000 to 40,000, preferably about 3,000 to 20,000. Since the particle size of the polymer micelle can be increased by increasing the molecular weight of the block copolymer, the particle size of the polymer micelle can be easily adjusted. At the time of continuous administration by the CED method, the injected drug solution spreads by convection, so the spreading method does not depend on the size of the polymer micelle, but it is also important that the particle size can be controlled at 100 nm or less. Conceivable. The reason for this is that the behavior of spreading or evacuating from the treatment site after administration is dependent on the particle size. Therefore, it is considered that it is a great advantage that the particle size can be changed in the range of 10 nm to 100 nm in order to achieve the optimization of the anticancer drug reach and the anticancer activity duration.

  The drug encapsulated in the polymer micelle is not limited as long as it is desired to be administered by the CED method. For example, anticancer drugs, boronated drugs for neutron capture therapy, suicide genes used for gene therapy And proteins and peptides that cause cell death, cytokines and immunotoxins for the purpose of immunostimulation, and the like. Among these, preferable examples include anticancer agents administered for the treatment of brain cancer tumors. In particular, a hydrophobic low molecular weight anticancer agent, which is difficult with known liposomes, is a preferred example. Specific examples of such low molecular weight anticancer agents include, but are not limited to, adriamycin, camptothecin, daunorubicin, vinplastin, vincristine, SN-38, BCNU, ACNU, temozolomide, paclitaxel, taxotere and the like. is not.

  The drug-encapsulated polymer micelle used in the present invention can be prepared by a known method described in, for example, Non-Patent Document 2, Non-Patent Document 3, and the like. That is, polymer micelles containing a drug inside can be obtained by forming polymer micelles in an aqueous medium containing a block copolymer and a drug. If the drug is hydrophobic, a solution of the drug in an organic solvent such as dimethylformamide can be added to the aqueous block copolymer solution. The polymer micelle can be formed by simply stirring this aqueous solution, or can be formed by a solvent evaporation method or the like in which water is gradually evaporated to increase the block copolymer concentration to form a polymer micelle. The concentration of the block copolymer in the solution when forming the polymer micelle is usually about 0.05% to 30% by weight, preferably about 0.5% to 10% by weight, and the concentration of the drug is usually 0.01%. It is about 20% by weight to 20% by weight, preferably about 0.05% to 10% by weight. It is preferable to remove the drug and other low molecular weight components not encapsulated in the polymer micelle by dialysis. The formation of polymer micelles can be performed at room temperature.

  The polymer micelle described above is contained in a suspended (suspended) state in an aqueous medium. The “aqueous medium” is a medium containing water as a main component, and physiological saline or phosphate buffered saline is usually used. The concentration of the polymer micelle in the pharmaceutical composition is set so that the drug concentration administered by the CED method becomes a predetermined concentration. The drug concentration is appropriately set according to the nature of the drug, the condition of the patient, the dose, etc., for example, in the case of an anticancer drug, usually about 0.1 mg / mL to 10 mg / mL, especially 1 mg / mL to 4 mg / mL Degree.

  Administration of the pharmaceutical composition of the present invention by the CED method can be carried out in the same manner as in the past. That is, a catheter having a CED injection needle loaded at the tip is connected to a known CED apparatus (including a microinjection pump and a syringe such as a Hamilton syringe (trade name)), and a hole opened in the skull is formed. In the case of humans, the pharmaceutical composition is usually injected at a flow rate of about 1 to 5 μL / min. The infusion time is appropriately set according to the medicine to be infused, the type of disease, the patient's condition, and the like.

  Advantages of using drug-encapsulated polymer micelles as pharmaceutical compositions administered by the CED method include (1) small particle size, (2) slow transition from cancer tissue to blood flow, and (3) appropriate drug release Speed can be mentioned. The polymer micelle sufficiently satisfies the above three requirements and becomes an ideal drug carrier system for the CED method. Hereinafter, these will be described.

(1) Small particle size When the polymer micelle has a small particle size of 10 nm to 100 nm, it is possible to suppress uptake into glial cells in the brain. In the case of intravenous administration, suppressing the uptake of macrophage-shaped Kupffer cells in the liver by reducing the particle size of the drug carrier to about 200 nm or less was a necessary requirement for achieving cancer targeting (particles It is thought that the larger the diameter, the larger the surface area that interacts with the cells, and the more easily phagocytosed). Although not yet elucidated at this time, avoiding phagocytosis of glial cells that perform macrophage-like functions in the brain is thought to be an important matter for reaching cancer cells efficiently. Moreover, it is because the behavior which spreads by diffusion after the end of administration or is discharged from the treatment site depends on the particle size. Optimization of anticancer drug reach and anticancer activity duration can be achieved.

(2) Slow transition from cancer tissue to blood flow There are roughly two mechanisms for transition from tissue to blood flow. In the case of hydrophobic low-molecular-weight drugs, phospholipids of vascular endothelial cells that make up blood vessels It directly permeates the cell membrane, which is a molecular membrane. In this cell membrane permeation mechanism, the migration rate is large. In addition, the transition mechanism is a mechanism that permeates intercellular junctions and intracellular channels of vascular endothelial cells. In this transition mechanism, the transition speed is lower than in the former case. Since the polymer micelle is discharged into the bloodstream only by the latter mechanism, its speed is small. Therefore, the CED method is suitable for obtaining a distribution that is small in the bloodstream and spreads throughout the cancer tissue.

(3) Appropriate drug release rate In the CED method, continuous administration is carried out over several hours to several tens of hours in human clinical practice. Therefore, it is considered that the release of the drug from the drug carrier needs to have a half-life adjusted to several hours to several tens of hours, but it is generally difficult to control within this range. When an anticancer drug is chemically bonded to an antibody or a synthetic polymer, it is necessary to set the chemical bond to a special one. In the case of liposomes, drug release is controlled only by a phospholipid bilayer, and therefore, it tends to be either slightly faster or slower than the desired release behavior. For example, in a liposome preparation approved in Japan as a targeting preparation for the anticancer drug adriamycin in Japan in 2006, the release of the drug is a factor that suppresses the higher expression of anticancer activity. (Adriamycin is originally a drug that easily permeates phospholipid bilayer membranes. If it is encapsulated in liposomes as it is, drug release is too fast. However, there is a fact that the release has almost disappeared and the anticancer activity has been lowered.

  On the other hand, polymer micelles have the advantage that drug release can be controlled by the micelle inner core (not a single membrane) having various physical properties such as liquid, solid, and crystal. In addition, various physical properties can be obtained by relaxation phenomena due to complex polymer chain entanglement (a wide range of physical properties can be achieved by controlling the molecular structure only from very hard materials to elastic materials such as rubber). In fact, it has already been demonstrated that drug release from polymeric micelles can be controlled over a very wide range of half-lives from minutes to tens of hours. That is, more specifically, the half-life of drug release can be controlled by the chemical structure of the hydrophobic polymer chain, the chain length of the polymer chain constituting the polymer, and the drug micelle content, If a hydrophobic polymer chain with high crystallinity and solid properties is used, the half-life can be shortened. Conversely, if a hydrophobic polymer chain with low crystallinity and strong liquid properties is used, the half-life is reduced. Can be lengthened. The chain length of the polymer chain constituting the polymer and the content of the drug in micelles can be considered both with and without affecting the half-life.

  Hereinafter, the present invention will be described more specifically based on examples. In addition, this invention is not limited to the Example demonstrated below.

Preparation of Adriamycin-Encapsulated Polymer Micelle-Containing Pharmaceutical Composition Polymer micelles encapsulating the anticancer drug adriamycin (also known as doxorubicin) were prepared by the method described in Non-Patent Document 2. The polymer micelle was formed from a PEG-P (Asp (ADR)) block copolymer shown in the following scheme 1, and the anticancer drug adriamycin was encapsulated in the formed polymer micelle. A block copolymer in which the molecular weight of the polyethylene glycol chain used is 12,000, the number of aspartic acid chain units (x + y in Scheme 1) is 22, and the number of aspartic acid residues that bind adriamycin is 59 mol%. Was used. This block copolymer PEG-P (Asp (ADR)) was synthesized as follows. 400 mg of PEG-P (Asp) was dissolved in 44 mL of N, N-dimethylformamide, 283 mg of adriamycin hydrochloride was added, and 64 mg of triethylamine was further added. To this was added 189 mg of water-soluble carbodiimide hydrochloride, and the mixture was stirred at room temperature for 24 hours. When the reaction was confirmed by HPLC, the binding rate of adriamycin was 59 mol%. This solution was dialyzed 6% with 3% acetic acid methanol solution using a dialysis membrane, and then 5 times with water to remove adriamycin and other low molecular reagents that were not chemically bound, and PEG-P (Asp (ADR)) An aqueous solution is obtained.

  Adriamycin was physically encapsulated using an aqueous PEG-P (Asp (ADR)) solution as follows. The aqueous PEG-P (Asp (ADR)) solution was concentrated by ultrafiltration and dialyzed twice with DMF. A solution prepared by dissolving 131 mg of adriamycin hydrochloride and 30 mg of triethylamine in 54 mL of DMF was added to about 10 mL of this polymer solution, and the mixture was stirred for 1.5 hours at room temperature. This solution was dialyzed 3 times against water for injection to remove adriamycin and other low molecular weight reagents. The obtained micelle solution was concentrated by ultrafiltration and prepared with saline so that the concentration of encapsulated adriamycin was 2.0 mg / mL to obtain a 0.9 wt% saline solution containing adriamycin encapsulated polymer micelle. The weight ratio of physisorbed adriamycin and polymer moiety in the polymer micelle was 2.0: 13.0.

Next, administration of adriamycin-encapsulated polymeric micelle-containing pharmaceutical composition by the CED method and its toxicity and therapeutic effect will be described.
The pharmaceutical composition prepared in Example 1 and containing adriamycin-encapsulated polymer micelles in physiological saline was used in the following animal experiments.

(1) Analysis of drug distribution to brain tumor by CED method Fischer 344 rat was used as a host of rat brain tumor model. A 9L rat glioma established cell line (source: National Cancer Center) was used as a tumor cell to be transplanted, and an Eagle's minimum essential medium containing 10% fetal calf serum was used as a medium. The number of transplanted cells was 5 × 10 ⁵ . The CED method was performed with an injection system using a step-design injection needle with a backflow prevention function reported by Krauze et al. In 2005 (reference: Krauze MT, Saito R, Noble C, Tamas M, Bringas J, Park JW). , Berger MS, Bankiewicz K., Reflux-free cannula for convection-enhanced high-speed delivery of therapeutic agents. J Neurosurg. 103 (5): 923-9 (2005)). On day 7 after tumor implantation, the injection rate started from 0.2 μL / min and gradually increased from 0.5 μL / min to 0.8 μL / min. Injection was performed for 15 minutes, 10 minutes, and 15 minutes for a total of 40 minutes, respectively, and the injection volume was 20 μL in total. The amount of drug injection was monitored by measuring the distance of drug movement in the drug line connected to the injection needle. The brain distribution of the drug was analyzed by observing the tomographic slice of the brain by observing fluorescence derived from adriamycin.

  As a result, when the anticancer drug adriamycin was administered alone, the drug distribution was limited to the insertion site of the injection needle and did not spread over a wide range from the administration point. In contrast, polymer micelles encapsulating adriamycin provided a good drug distribution that surrounded not only the inside of the tumor but also the surrounding area of the transplanted tumor. Malignant glioma has the property of infiltrating the surrounding normal brain tissue, and local recurrence from the infiltrated tumor cells becomes a problem in the treatment. The drug distribution of micelles was the result of predicting high anticancer activity in the therapy targeting infiltrated tumor cells.

  Next, drug distribution in the brain of normal rats was analyzed. The drug concentration was 2 mg / ml in terms of adriamycin, and 5 normal Sprague-Dawley rats were used. Rats were euthanized immediately after drug injection, and cerebrum was collected and frozen. Frozen sections were prepared with a thickness of 25 μm, drug distribution was observed, photographed, and captured with a fluorescence microscope, and the area of drug distribution observed on each pathological tissue section was measured using image analysis software (NIH image) . The drug distribution volume Vd (volume of distribution) was calculated by multiplying this by the slice thickness of the frozen section. Furthermore, polymer micelles that do not physically adsorb adriamycin (polymer micelles consisting only of PEG-P (Asp (ADR)) block copolymer in Scheme 1) and commercially available liposome preparations that are put into practical use as adriamycin-encapsulated preparations FIG. 1 shows the result of comparison with the same analysis.

  Compared to the administration of adriamycin alone, the drug distribution volume is more than 3 times by micelle encapsulation. In addition, the drug distribution volume was almost the same as doxil of the liposome preparation. Furthermore, there is no significant difference compared to polymeric micelle carriers that do not physically adsorb adriamycin, which means that physically encapsulated adriamycin is reliably distributed in brain tissue by polymeric micelles. Means that. In FIG. 1, error bars indicate 1 SD (standard deviation) around the average value.

(2) Evaluation of toxicity to brain tissue by CED injection Normal Sprague-Dawley rats were used for evaluation of drug distribution in the brain of normal rats. Rats were euthanized 3 weeks after drug injection, and brain tissues were paraffin-wrapped. A pathological tissue section was prepared after embedding (slice thickness: 5 μm). During the period from drug injection to euthanasia, rat skin condition, body weight, and the presence or absence of neurological deficits were observed. The histopathological sections were stained with hematoxylin and eosin, observed with a microscope, and histopathological analysis was performed. The amount of drug injected was 20 μL as in the CED method described above. None of the rats showed obvious neurological loss symptoms or weight loss during the observation period, but on the histopathological section, necrotic changes consistent with the drug distribution occurred in a dose-dependent manner. This was mild compared to adriamycin alone. When adriamycin encapsulated in liposomes was administered, the necrotic change was slight compared with the previous two, and this was thought to be a result of the long drug retention of liposomes.

(3) Evaluation of in vivo anticancer activity by CED method A rat 9L glioma model was used as in (1) above. Comparison was made between four groups: a control group (injected with physiological saline), an adriamycin single administration group, a commercially available adriamycin-encapsulated liposome preparation administration group, and an adriamycin micelle administration group. The drug injection was performed on the seventh day after tumor implantation, and the drug concentration was 0.2 mg / ml in terms of adriamycin for each group. Rats that became unable to eat or move with increasing brain tumors were euthanized and the observation was terminated. The longest observation period was 90 days, and rats that survived to this point were euthanized after the end of the observation period. The cerebrums of euthanized rats were collected, embedded in paraffin, and histopathologically analyzed by hematoxylin and eosin staining. The results of the survival test were displayed as a Kaplan-Meier curve and analyzed by the log-rank test (Fig. 2). Adriamycin alone administration and adriamycin encapsulated doxyl of other liposome preparations did not significantly increase the survival time compared to the control. In contrast, a significant survival-prolonging effect was obtained only with adriamycin-encapsulated polymer micelles. All rats that survived for 50 days or longer survived until 90 days.

Preparation of a camptothecin-encapsulated polymeric micelle-containing pharmaceutical composition will be described.
A block copolymer (PEG-P (Asp (Bzl)) having a structure represented by the following formula was produced by the method described in Non-Patent Document 3. Specifically, it was produced as follows: Polyethylene glycol-b -Dissolve 200 mg of polyaspartic acid block copolymer (PEG-P (Asp), polyethylene glycol chain molecular weight 5,000 and polyaspartic acid chain unit number 25) in 2 mL of N, N-dimethylformamide, and leave the aspartic acid residue. 2.0 times molar equivalent of 1,8-diazabicyclo [5,4,0] 7-undecene (DBU) and benzyl bromide with respect to the group were added and reacted at 50 ° C. for 15 hours.
The molecular weight of the polyethylene glycol chain in the produced block copolymer (PEG-P (Asp (Bzl)) was 5,000, the number of units of the polyaspartic acid moiety was 25, and the benzyl esterification rate of the aspartic acid residue was 70%.

  As described in Non-Patent Document 3, 40% by weight of camptothecin was encapsulated in polymer micelles by a solvent evaporation method with respect to this block copolymer. Specifically, this was performed as follows. A solution prepared by dissolving 5.0 mg of the block copolymer prepared above and 2.0 mg of camptothecin in 2.0 mL of chloroform was stirred in a nitrogen stream to evaporate the chloroform. Distilled water was added thereto, and ultrasonic waves were irradiated for 2 minutes with a probe type ultrasonic irradiation device. The obtained micelle solution was concentrated to a required concentration by an ultrafiltration device.

Next, the administration of the camptothecin-encapsulated polymeric micelle-containing pharmaceutical composition by the CED method and its toxicity and therapeutic effect will be described.
The pharmaceutical composition prepared in Example 3 and containing camptothecin-encapsulated polymer micelles in physiological saline was subjected to animal experiments. That is, the survival test by the CED method was performed on the rats transplanted with 9L cells in the same manner as in Example 2. The results are shown in FIG.

  As shown in FIG. 3, the drug was administered by CED method on the seventh day after cancer transplantation. The controls were camptothecin solution (dissolved in organic solvent dimethyl sulfoxide (DMSO) because it is not required in water), solvent DMSO, and block copolymer that forms polymer micelles (block copolymer without encapsulating camptothecin) PEG-P (Asp (Bzl)) In both of these controls, the previous rat died by day 21, but in contrast, camptothecin-encapsulated polymer micelles showed a significant survival-prolonging effect. It was.

The adriamycin alone, a commercially available adriamycin-encapsulated liposome preparation, adriamycin-encapsulated polymer micelles (present invention) and polymer micelles without encapsulated drugs are directly administered into the rat brain by the CED method. FIG. It is a figure which shows the anticancer activity at the time of administering an adriamycin enclosure polymer micelle directly in a rat brain by CED method compared with the anticancer activity in a control group. It is a figure which shows the anticancer activity at the time of administering a camptothecin inclusion polymeric micelle directly in a rat brain by CED method compared with the anticancer activity in a control group.

Claims (3)

  Effective drug-encapsulated polymer micelles, which are formed from block copolymers with hydrophilic and hydrophobic regions, and encapsulate drugs inside polymer micelles with the hydrophilic regions on the outside and the hydrophobic regions on the inside A pharmaceutical composition for the CED method, which is contained in an aqueous medium as a component.   The medicament according to claim 1, wherein the drug is an anticancer drug, and the CED method is effective for treating a cancer tumor in the brain.   The medicament according to claim 1 or 2, wherein the polymer micelle has a particle size of 10 nm to 100 nm.