WO2017170564A1 - Nanoparticulate formulation for cancer disease treatment - Google Patents

Abstract

[Problem] To provide a novel DDS having SN-38 as the medicinal component. [Solution] An excellent SN-38 ester derivative with cancer cell proliferation inhibitory activity is provided which is effective in nanodrug production.

Description

Nanoparticulate preparation for cancer disease treatment

The present invention relates to a drug used for the treatment of cancer diseases, particularly a compound in which a water-soluble and hardly absorbable drug 10-hydroxy 7-ethylcamptothecin (SN-38) and a fat-soluble organic acid are bound, The present invention relates to a nanoparticle, a method for producing a nanoparticulate preparation for treating cancer diseases by nanoparticulating the compound, and a nanoparticulate preparation for treating cancer diseases.

An anticancer drug, camptothecin, and its specific derivatives are known. Many of camptothecin and its derivatives are extremely insoluble in water, which limits the clinical use of this drug. Water-soluble camptothecin derivatives include 9-dimethylaminomethyl-10-hydroxycamptothecin (Topotecan), 7-[(4-methylpiperazino) methyl] -10,11-ethylenedioxycamptothecin, 7-[(4- Methylpiperazino) methyl] -10,11-methylenedioxycamptothecin, and 7-ethyl-10- [4- (1-piperidino) -1-piperidino] carbonyloxycamptothecin (CPT-11). Among these, CPT-11 (Irinotecan / Camptosar, registered trademark) was approved for use in humans by the US Food and Drug Administration in June 1996.

It was found in Patent Document 1 by Miyasaka et al. In 1984 that the active body of CPT-11 is 10-hydroxy-7-ethylcamptothecin (SN-38). However, SN-38 is not directly administered to human cancer patients because of its poor water solubility. On the other hand, in recent years, it has been reported that in human cancer patients, SN-38 is further metabolized to form inactivated glucuronide species. Although NK012, which is a preparation obtained by polymerizing SN-38 into a polymer micelle, has been developed, there are various problems with micellized nanoparticles, and their practical application is questioned.

In addition, although SN-38 liposome preparation (LE-SN-38) has been clinically developed, it cannot pass the primary tumor reaction endpoint. Therefore, a new drug development method using SN-38 as a medicinal ingredient and using a new DDS (Drug Delivery System) is required.

Conventionally, nanoparticles of poorly soluble drugs are produced by mechanically pulverizing coarse particles with a ball mill or the like. However, it is difficult to reduce the particle size to the nanometer scale, and the variation in the particle size is large.

On the other hand, when a drug is administered to the human body, a poorly soluble drug is often used as an aqueous suspension drug in which the drug is dispersed in water.

Aqueous suspension drugs have the property that nanoparticulate drug components dispersed in water stay in the body, and are therefore effective for exerting their effects locally or over a long period of time. Depending on the use, it is used as an oral preparation, an external preparation for skin, an external preparation for nasal cavity, an external preparation for ophthalmology and the like.

An aqueous suspension drug is usually produced by dispersing a drug pulverized into nanoparticles into water using a surfactant or the like. However, the particle size of the drug obtained by pulverization is relatively large, and the variation in particle size is also large, so that a large particle component settles due to long-term storage and rapid changes in the environment such as temperature. There's a problem. In addition, due to the variation in particle size, the quality of the aqueous suspension drug is likely to vary, and even if the drug is manufactured by the same manufacturing method, the retention time is too short to be effective. There is also a problem that side effects may occur due to the time being too long. Therefore, a method for obtaining drug nanoparticles having a small particle size and a narrow particle size distribution has been demanded.

On the other hand, Patent Document 2 discloses a conjugate-based antifungal or antibacterial prodrug formed by coupling an antifungal or antibacterial agent with at least one linker and / or carrier. Yes. Here, since a fatty acid, a surfactant, a polymer, or the like is used as the carrier, there are problems such as safety and practical application.

Patent Document 3 discloses a cholesterol-modified cancer therapeutic compound, a bile acid-modified cancer therapeutic compound, a bile acid derivatized modified cancer therapeutic compound, an emulsion, a microemulsion, a micelle preparation containing these compounds, these And a method for administration of the compounds and preparations and a method for treating cancer using these compounds and preparations.

However, since surfactants are used, micellar nanoparticles have various problems and their practical application is questioned.

US4473692 Japanese Patent Application 2014-517203 WO2005 / 118612

There was an urgent need for the development of a new DDS with SN-38 as a medicinal ingredient that can solve one or more of the disadvantages or problems of the prior art described above.

In view of the above situation, the present inventors have intensively studied the development of a new DDS having SN-38 as a medicinal ingredient. As a result, the present inventors have surprisingly found that SN-38 ester derivatives in which a specific fat-soluble organic acid is introduced into the 10-position hydroxy group of SN-38 are effective for producing nanopharmaceuticals and have excellent cancer. It was found to show cell growth inhibitory activity. The present inventor has completed the present invention based on this finding.

That is, the present invention solves the above problems by providing the inventions described in [1] to [24] below.

[1]
Formula (1):

(In the formula, X is a hydrogen atom, a hydroxy group, an optionally substituted alkyl group, an optionally substituted alkoxy group or an optionally substituted acyloxy group, and the 5-position and 6-position of the cholesterol skeleton) The carbon-carbon bond between is a single bond or a double bond).
[2]
The compound according to [1], wherein X is a hydrogen atom, and the carbon-carbon bond between the 5th and 6th positions of the cholesterol skeleton is a single bond.
[3]
X is a hydroxy group, an optionally substituted alkyl group, an optionally substituted alkoxy group or an optionally substituted acyloxy group, and a carbon atom between the 5-position and 6-position of the cholesterol skeleton— The compound according to [1], wherein the carbon bond is a double bond.
[4]
The compound according to [3], wherein X is a hydroxy group.
[5]
The compound according to [3], wherein X is an acyloxy group.
[6]
The compound according to [5], wherein the acyloxy group is an alkylcarbonyloxy group.
[7]
The alkylcarbonyloxy group is an acetyloxy group, [6]
Compound described in 1.
[8]
Formula (2):

(Wherein R is an optionally substituted alkyl group).
[9]
The compound according to [8], wherein R is a C ₁ to C ₂₀ alkyl group.
[10]
The compound according to [8], wherein R is a C ₁ -C ₆ alkyl group.
[11]
The compound according to [8], wherein R is a methyl group.
[12]
Formula (3):

(Wherein n represents an integer of 1 to 20).
[13]

A compound selected from the group consisting of:
[14]
[1] Nanoparticles comprising the compound according to any one of [13] to nanoparticles.
[15]
The nanoparticle according to [14], having an average particle size of 10 to 200 nm.
[16]
[14] or [15], which is obtained by injecting a solution in which the compound according to any one of [1] to [13] is dissolved in water into a water-miscible organic solvent. The manufacturing method of the nanoparticle of description.
[17]
[1] A pharmaceutical composition comprising the compound according to any one of [13].
[18]
[14] A pharmaceutical composition comprising the nanoparticle according to [15].
[19]
[1] A therapeutic agent for cancer diseases comprising the compound according to any one of [13].
[20]
[14] or a therapeutic agent for cancer diseases comprising the nanoparticle according to [15].
[21]
The therapeutic agent for cancer disease according to [19] or [20], wherein the cancer disease is a solid tumor.
[22]
[21] The solid tumor is esophageal cancer, stomach cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, laryngeal cancer, lung cancer, prostate cancer, bladder cancer, breast cancer, uterine cancer or ovarian cancer. Therapeutic agent for cancer diseases.
[23]
[1] The nanoparticulate preparation for cancer disease treatment according to any one of [13].
[24]
[14] or an injection containing the nanoparticle according to [15].

Unless otherwise stated, the terms used in this specification and claims have the meanings described below.

The “alkyl group” means a saturated aliphatic hydrocarbon group, for example, a linear or branched alkyl group having 1 to 20 carbon atoms, for example, a methyl group, an ethyl group, a propyl group, an isopropyl, unless otherwise specified. Group, butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentyl group, hexyl group and the like C ₁ -C ₆ alkyl group, heptyl group, 1-methylhexyl group, 5-methylhexyl group, 1, 1-dimethylpentyl group, 2,2-dimethylpentyl group, 4,4-dimethylpentyl group, 1-ethylpentyl group, 2-ethylpentyl group, 1,1,3-trimethylbutyl group, 1,2,2- Trimethylbutyl, 1,3,3-trimethylbutyl, 2,2,3-trimethylbutyl, 2,3,3-trimethylbutyl, 1-propylbutyl, 1,1,2, -Tetramethylpropyl group, octyl group, 1-methylheptyl group, 3-methylheptyl group, 6-methylheptyl group, 2-ethylhexyl group, 5,5-dimethylhexyl group, 2,4,4-trimethylpentyl group, 1-ethyl-1-methylpentyl group, nonyl group, 1-methyloctyl group, 2-methyloctyl group, 3-methyloctyl group, 7-methyloctyl group, 1-ethylheptyl group, 1,1-dimethylheptyl group 6,6-dimethylheptyl group, decyl group, 1-methylnonyl group, 2-methylnonyl group, 6-methylnonyl group, 1-ethyloctyl group, 1-propylheptyl group, n-nonyl group, n-decyl group, etc. C ₁ -C ₆ alkyl groups are preferred.

The term “alkoxy group” refers to an (alkyl) -O— group having 1 to 20 carbon atoms in which the alkyl portion has the above-mentioned meaning, for example, methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, sec -C ₁ -C ₆ alkoxy groups such as butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy, hexyloxy, isohexyloxy, etc. It is not a thing.

Examples of the “acyloxy group” include alkylcarbonyloxy group, alkoxycarbonyloxy group, carbamoyloxy group, alkylcarbamoyloxy group, alkylsulfinyloxy group, alkylsulfonyloxy group and the like.

“Alkylcarbonyloxy group” means an (alkyl) -C (═O) —O— group having 1 to 20 carbon atoms in which the alkyl portion has the above-mentioned meaning, for example, an acetoxy group or a propionyloxy group _{A 1} to C ₆ alkylcarbonyloxy group may be mentioned, but is not limited thereto.

“Alkoxycarbonyloxy group” refers to an (alkyl) -O—C (═O) —O— group having 1 to 20 carbon atoms in which the alkoxy moiety has the above meaning, such as a methoxycarbonyloxy group, ethoxy carbonyloxy group, propoxycarbonyl group, C _{1 ~} C ₆ alkoxy such as butoxycarbonyl group - is carbonyl group) can be mentioned, but not limited thereto.

“Alkylcarbamoyloxy group” refers to an (alkyl) -NH—C (═O) —O— group having 1 to 20 carbon atoms in which the alkyl moiety has the above meaning, for example, methylcarbamoyloxy group, ethyl A C ₁ -C ₆ alkylcarbamoyloxy group such as a carbamoyloxy group), but is not limited thereto.

“Alkylsulfinyloxy group” means an (alkyl) -SO—O— group having 1 to 20 carbon atoms in which the alkyl moiety has the above-mentioned meaning, for example, methylsulfinyloxy group, ethylsulfinyloxy group, propylsulfinyl group Examples thereof include, but are not limited to, C ₁ -C ₆ alkylsulfinyloxy groups such as oxy group and isopropylsulfinyloxy group.

“Alkylsulfonyloxy group” means an (alkyl) -SO ₂ —O— group having 1 to 20 carbon atoms in which the alkyl moiety has the above-mentioned meaning, for example, methylsulfonyloxy group, ethylsulfonyloxy group, propyl Examples thereof include, but are not limited to, a C ₁ -C ₆ alkylsulfonyloxy group such as a sulfonyloxy group and an isopropylsulfonyloxy group.

“The alkyl group which may be substituted, the alkoxy group which may be substituted or the acyloxy group which may be substituted” may be substituted or unsubstituted. When substituted, the substituent may be substituted at any substitutable position, and preferably the substituent is a halogen, a hydroxy group, a cyano group, a nitro group, or the like.

The anti-cancer active ingredient SN-38 is chemically bonded with a fat-soluble organic acid (hydrophobic molecule) to improve the cell membrane affinity and permeability of the ingredient, and to efficiently form nanoparticles in the reprecipitation method. By urging, the function of the drug for treating cancer containing such a medicinal ingredient can be remarkably improved.

As the fat-soluble organic acid, any substance known to those skilled in the art can be used. For example, cholesterols such as cholesterol, various derivatives such as hydrogenated dihydrocholesterol (for example, colanic acid or cholenoic acid), hinokitiol Organic acid derivatives (for example, hinokitiol glutaric acid), esters or salts with various fatty acids, and hydrophobic amino acids and hydrophobic peptides.

A typical example of the fat-soluble organic acid is an organic acid represented by the formula (4) or the formula (5).

In the formula (4), X is a hydrogen atom, a hydroxy group, an optionally substituted alkyl group, an optionally substituted alkoxy group or an optionally substituted acyloxy group, the 5-position of the cholesterol skeleton and The carbon-carbon bond between the 6 positions is a single bond or a double bond. In the formula (5), n represents an integer of 1 to 20, preferably an integer of 1 to 6, and more preferably 3.

A suitable representative example of the fat-soluble organic acid is a compound in which X is a hydrogen atom, and the carbon-carbon bond between the 5-position and the 6-position of the cholesterol skeleton is a single bond.

As a suitable representative example of the fat-soluble organic acid, X is a hydroxy group, an optionally substituted alkyl group, an optionally substituted alkoxy group or an optionally substituted acyloxy group, and a cholesterol skeleton. Examples of the carbon-carbon bond between the 5-position and 6-position of the compound include a double bond. X is preferably a hydroxy group or an acyloxy group (eg, alkylcarbonyloxy), more preferably an acetyloxy group.

Examples of the fat-soluble organic acid include the following, but are not limited thereto.

Specific examples of the compound of the formula (1) of the present invention include the following, but are not limited thereto.

An example of the photograph image (SEM image) image | photographed with the scanning electron microscope of the SN-38 hinokitiol derivative nanoparticle of this invention is shown. An example of the photograph image (SEM image) image | photographed with the scanning electron microscope of the SN-38 colanic acid derivative nanoparticle of this invention is shown. An example of the photograph image (SEM image) image | photographed with the scanning electron microscope of the surface modified SN-38 colanic acid derivative nanoparticle by the albumin of this invention is shown. The result of the in-vitro anticancer activity test of SN-38 derivative nanoparticles of the present invention and irinotecan is shown. The result of the in-vivo anticancer activity test of SN-38 colanic acid derivative nanoparticles surface-modified with albumin and irinotecan is shown.

Synthesis of compounds of formula (1), formula (2) and formula (3) of the present invention The compounds of formula (1), formula (2) and formula (3) of the present invention are prepared by methods known to those skilled in the art. Can be manufactured. For example, SN-38 ester is produced by dehydrating condensation of SN-38 or a derivative thereof and a fat-soluble organic acid such as colanic acid, cholenic acid, hinokitiol or a derivative thereof in the presence of a base and a solvent. Can do.

Examples of bases that can be generally used in the production of SN-38 esters include organic bases such as aliphatic tertiary amines, aromatic tertiary amines, nitrogen-containing heterocyclic compounds, and inorganic bases such as NaOH. . Here, as the aliphatic tertiary amine, trialkylamines substituted with an aliphatic chain having 1 to 6 carbon atoms (for example, trimethylamine, triethylamine, tripropylamine, tributylamine, N, N-diisopropylethylamine, N, N— Dimethylpropylamine and the like, and the aromatic tertiary amine is an aniline in which a fatty acid chain having 1 to 6 carbon atoms is substituted on the nitrogen atom (for example, N, N-dimethylaniline, N, N-diethylaniline, etc.) ) Are nitrogen-containing heterocyclic compounds such as pyridines (specifically, pyridine, N, N-dimethylaminopyridine (DMAP), N, N-diethylaminopyridine, etc.), diazines (specifically, 1,2-diazine, 1,3-diazine, 1,4-diazine, etc.), triazines (specifically, 1,3,4-triazine, etc.) Can be exemplified methyl imidazole, hexamethylenetetramine, and 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) nitrogen-containing heterocyclic compounds such as, respectively. The amount of the base used may be 1.0 to 6.0 mol, preferably 1.0 to 3.0 mol, per 1 mol of SN-38.

Commonly used solvents for the preparation of SN-38 esters include suitable solvents such as methylene chloride, ethylene dichloride (EDC), tetrahydrofuran, ethyl acetate, dimethylformamide (DMF) or dimethoxyethane (DME). .

The reaction temperature for producing the SN-38 ester can be arbitrarily selected within the temperature range below the boiling point of the solvent, but a range of 0 to 60 ° C. can be exemplified as a preferable reaction temperature. More preferably, it is 20 to 30 ° C. Although the reaction time of the reaction is not unambiguous because it is affected by the reaction conditions (for example, the type and amount of the compound and solvent used, reaction temperature, etc.), it is usually 1 to 24 hours.

Method for Producing Nanoparticles of Compound of Formula (1) of the Present Invention In producing nanoparticles of the compound of formula (1) of the present invention, the compound of formula (1) is first dissolved in an organic solvent which is a good solvent. Is done. The organic solvent can be appropriately selected as long as it can dissolve the compound of the formula (1) and is miscible with water. Specific examples of the organic solvent include, but are not limited to, acetone, tetrahydrofuran, dioxane, acetonitrile, methanol, ethanol, propanol, N-methylpyrrolidone, dimethyl sulfoxide, and the like. Or can be used in combination. As the organic solvent, acetone, tetrahydrofuran, ethanol and dimethyl sulfoxide are particularly preferable from the viewpoints of solubility and safety.

Since the concentration of the compound of formula (1) of the present invention in the organic solvent is a large factor that affects the particle size of the produced nanoparticles, the concentration is appropriately changed according to the desired particle size. As will be described later, when obtaining nanoparticles having an average particle size of 10 to 200 nm, the dissolution concentration is usually adjusted to about 0.1 to 15% by mass, but nanoparticles having an average particle size of 10 to 200 nm are used. Is prepared using a solution of around 0.5% by mass.

When an organic solvent solution of a compound of formula (1) (hereinafter simply referred to as an organic solvent solution) is injected into water which is a poor solvent, nanoparticles of the compound of formula (1) in a state dispersed in water are generated. . In this invention, when the compound of Formula (1) has two or more fat-soluble organic acids, the solubility to the water of the compound of Formula (1) becomes lower than original SN-38. As a result, the compound of formula (1) crystallizes more rapidly in water than the original SN-38, resulting in nanoparticles with smaller particle size and particle size distribution.

The amount of water into which the organic solvent solution is injected is usually 10 times or more based on the volume of the compound of the formula (1) of the present invention. There is no particular upper limit, but if there is too much water, the concentration of the nanoparticles of the compound of formula (1) will become thin and it will be necessary to concentrate, so it is usually 100 times or less.

When the temperature of the water into which the organic solvent solution is injected is changed, the particle size of the produced nanoparticles also changes, so the formula obtained by keeping the water constant at a specific temperature during the injection of the organic solvent solution The particle size of the nanoparticles of the compound (1) can be controlled. For example, when water at a low temperature is used, the particle size of the nanoparticles of the compound of formula (1) tends to increase. Usually, the temperature is controlled in the range of −20 ° C. to 60 ° C. according to the desired particle size.

It is preferable that the injection of the organic solvent solution is performed in a short time in agitated water in order to prevent variation in particle size. By stirring for a while after injection, an aqueous dispersion in which the crystallized or nanoparticulated compound of formula (1) is uniformly dispersed in water can be obtained. The obtained nanoparticles of the compound of the formula (1) can be used as an aqueous suspension medicine while being dispersed in water, and can be obtained as a fine particle powder by performing a solid-liquid separation operation such as filtration. It can also be separated.

When used as an aqueous suspension drug, it can be used as is, depending on the application and the type of good solvent used, but usually an organic solvent added as a good solvent improves the safety of the drug. Removed. There is no restriction | limiting in the removal method of an organic solvent, It can remove from an aqueous suspension by a well-known method. Usually, the organic solvent is easily removed by distilling off under reduced pressure (or normal pressure), but can also be removed using dialysis.

By performing the above operations, nanoparticles of the compound of formula (1) having a very small particle size and a narrow particle size distribution can be obtained, such as the temperature of water, the type of good solvent, the amount added, etc. It is possible to produce nanoparticles of the compound of the formula (1) having a large particle size while maintaining a narrow particle size distribution by controlling the average particle size by changing the production conditions. Nanoparticles of the compound of formula (1) having a diameter of about 10 nm to 200 nm can be produced.

The compound of the formula (1) is hydrolyzed in the living body to the original form of the drug SN-38 by the change of pH when passing through the living body and the action of an enzyme such as esterase. Therefore, since it functions as SN-38 itself in a living body, it is possible to ensure the drug efficacy and safety of the nanoparticles of the compound of formula (1) of the present invention.

Although the present invention has been described with reference to the most practical and preferred embodiments at the present time, the invention is limited to the embodiments disclosed herein. However, the present invention can be modified as appropriate without departing from the scope or spirit of the invention that can be read from the claims and the entire specification, and the nanoparticle of the compound of the formula (1) accompanied by such a change and the production method thereof are also described in the present invention. It should be understood as being included in the technical scope of the invention. In addition, the manufacturing method of the nanoparticle of the compound of Formula (2) and Formula (3) of this invention is based on said manufacturing method of the compound nanoparticle of Formula (1) of this invention.

In order to use the EPR effect (enhanced permeability and retention effect), the size of the nanoparticles needs to be 10 nm or more in order to suppress renal excretion. In addition, since it is not captured by macrophages such as spleen and Kupffer cells of the liver, it must be 200 nm or less (Nanomedicine for cancer targeting, www.dojindo.co.jp/letterj/151/review/01 .html (Review, Professor Yoshiki Katayama)). Accordingly, the average particle size of the nanoparticles of the present invention is preferably 10 to 200 nm, and more preferably 10 to 100 nm.

Furthermore, the present invention relates to various therapeutic pharmaceutical compositions containing the above-mentioned nanoparticulate preparation for cancer treatment. Such pharmaceutical compositions include any pharmaceutical formulation known to those skilled in the art, depending on various conditions such as the subject of administration, administration route, application, form, and type and amount of the drug included. Various other buffers, adjuvants, additives, and the like that are allowed can be appropriately contained.

The tumor to be treated with the nanoparticulate preparation for cancer disease treatment of the present invention is not particularly limited but is a solid tumor, specifically, esophageal cancer, stomach cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer. Liver cancer, laryngeal cancer, lung cancer, prostate cancer, bladder cancer, breast cancer, uterine cancer or ovarian cancer. Target sites include tumor cells, tissues, organs or organs and their interiors.

The nanoparticulate preparation for cancer disease treatment of the present invention is prepared, for example, in a parenteral, oral, transdermal, nasal or pulmonary administration form.

Compositions of the invention containing pharmaceutically acceptable excipients can be prepared by any of the well-known dispensing techniques including mixing excipients with drugs or therapeutic agents.

As parenteral, for example, it can be formulated as a sterile aqueous solution for injection administration.

The pharmaceutical composition according to the present invention comprises, for example, neoplasms derived from epithelial cells (epithelial carcinoma) such as brain cancer, bone cancer, basal cell carcinoma, adenocarcinoma, esophageal cancer, small intestine cancer and gastric cancer, colon cancer, liver cancer, bladder Benign or malignant including cancers such as cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancer, prostate cancer, renal carcinoma and other known cancers that affect epithelial cells throughout the body Useful for prevention, amelioration and / or treatment of tumor / neoplasm. Cancers for which the compositions of the invention are considered particularly useful are gastrointestinal cancers, particularly colorectal cancer, lung cancer, especially small cell lung cancer, cervical cancer and pancreatic cancer.

The individual dose at which the composition according to the present invention is administered to obtain a therapeutic effect will of course depend on the individual administration environment including, for example, age, weight, patient status, route of administration and the like. Individual dosing schedules can be tailored to a particular subject based on their individual needs and the professional judgment of the person who supervises the administration of the composition.

The dosage range employed will depend on the route of administration, the age, weight and condition of the patient being treated. For example, the daily dose of the composition of the present invention is generally 1 to 1000 mg / m ² body surface area, preferably 10 to 500 mg / m ² body for parenteral administration, specifically bolus or intravenous administration by infusion. Surface area. The dose may be administered at one time, or may be divided into several portions at various time intervals. A specific example of a suitable schedule for parenteral administration is 6 weeks of administering 125 mg / m ² body surface area over 90 minutes by intravenous infusion on the first day of each week from week 1 to week 4. Dosing schedule. In another treatment regimen, parenteral administration of 350 mg / m ² body surface area is given intravenously over 90 minutes every 3 weeks.

Hereinafter, the present invention will be specifically described with reference to examples, but the technical scope of the present invention is not limited to these descriptions.

SN-38 hinokitiol derivative ((S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ', 4': 6,7] Synthesis of indolizino [1,2-b] quinolin-9-yl (5-isopropyl-7-oxocyclohepta-1,3,5-trien-1-yl) glutarate

SN-38 glutaric acid ester was synthesized based on the description in known literature (L.LMao at al., Chemical Communications 2012, 48, 395-397).

SN-38 glutarate (50 mg, 0.0987 mmol) and hinokitiol (16.2 mg, 0.0987 mmol) were added to an eggplant-shaped flask and dissolved in 1.0 ml of dehydrated CH ₂ Cl ₂ . To this solution, EDC (37.8 mg, 0.197 mmol) and DMAP (1.2 mg, 0.00987 mmol) were added and stirred for 3 hours. Saturated aqueous ammonium chloride solution was added to the reaction solution to stop the reaction, and the mixture was transferred to a separatory funnel. After extraction with chloroform, the chloroform layer was washed with water and a saturated aqueous sodium chloride solution. The chloroform layer was dehydrated with anhydrous magnesium sulfate, filtered with absorbent cotton, and then the solvent was distilled off under reduced pressure. And it refine | purified by the silica gel column chromatography which made the developing solvent chloroform / methanol = 100/1-30/1. The color of the purified crystals was pale yellow, and SN-38 hinokitiol derivative (56.3 mg, 0.0863 mmol) was obtained in a yield of 87%.

¹ HNMR (CDCl ₃ , 400 MHz): d 1.05 (3H, t, J = 7.2 Hz), 1.25, (6H, d, J = 6.8 Hz) 1.40 (3H, t, J = 7.2 Hz), 1.54-1.84 (2H, m), 2.25-2.32 (2H, m), 2.78-2.95 (4H, m), 3.16 (2H, q, J = 7.6 Hz), 3.70 (1H, s), 5.26 (2H, s), 5.31 (1H, d, J = 16 Hz), 5.76 (1H, d, J = 16 Hz), 6.97-7.19 (4H, m), 7.58 (1H, dd, J = 9.2, 2.4 Hz), 7.64 (1H , s), 7.87 (1H, d, J = 2.4 Hz), 8.23 (1H, d, J = 9.2 Hz).
HR-MS (ESI-TOF): m / z calcd.for C ₃₇ H ₃₇ N ₂ O ₉ ([M + H] ⁺ ) 653.2494, found 653.2488.

SN-38 colanic acid derivative ((S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7 Indolizino [1,2-b] quinolin-9-yl (R) -4-((5S, 8R, 9S, 10S, 13R, 14S, 17R) -10,13-dimethylhexadecahydro-1H-cyclopenta [ a) Synthesis of phenanthrene-17-yl) pentanoate)

SN-38 (80.6 mg, 0.205 mmol) and colanic acid (77.4 ng, 0.215 mmol) were added to a two-necked eggplant-shaped flask and dissolved in 10 ml of dehydrated CH ₂ Cl ₂ . To this solution, EDC (84.9 mg, 0.443 mmol) and DMAP (5.5 mg, 0.0450 mmol) were added and stirred for 24 hours. The reaction solution was transferred to a separatory funnel, chloroform was added, and the mixture was washed with a saturated aqueous ammonium chloride solution, water, and an aqueous sodium chloride solution. The chloroform layer was dehydrated with anhydrous magnesium sulfate, filtered with absorbent cotton, and then the solvent was distilled off under reduced pressure. And it refine | purified by the silica gel column chromatography which made the developing solvent chloroform / methanol = 100/1. The color of the purified crystals was pale yellow, and SN-38 colanic acid derivative (149 mg, 0.203 mmol) was obtained in a yield of 99%.

¹ HNMR (CDCl ₃ , 400 MHz): d 0.69 (3H, s), 0.92-2.01 (48H, m), 2.57-2.61 (1H, m), 2.67-2.71 (1H, m), 3.14 (2H, q , J = 7.6 Hz), 3.94 (1H, s), 5.24 (2H, s), 5.29 (1H, d, J = 16 Hz), 5.74 (1H, d, J = 16 Hz), 7.53 (1H, dd , J = 9.2, 2.4 Hz), 7.64 (1H, s), 7.79 (1H, d, J = 2.4 Hz), 8.21 (1H, d, J = 9.2 Hz).
HR-MS (ESI-TOF): m / z calcd.for C ₄₆ H ₅₉ N ₂ O ₆ ([M + H] ⁺ ) 735.4368, found 735.4358.

SN-38 cholenoic acid derivative ((S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6,7 Indolizino [1,2-b] quinolin-9-yl (R) -4-((3S, 8S, 9S, 10R, 13R, 14S, 17R) -3-hydroxy-10,13-dimethyl-2,3 , 4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenanthren-17-yl) pentanoate)

TBS-cholenic acid was synthesized based on the description in known literature (T. A. Spencer at al., Journal of Medicinal Chemistry 2001, 44, 886-897.).

SN-38 (50 mg, 0.127 mmol) and TBS-cholenic acid (62.3 mg, 0.127 mmol) were added to the eggplant-shaped flask and dissolved in 1.3 ml of dehydrated CH ₂ Cl ₂ . To this solution, EDC (48.7 mg, 0.254 mmol) and DMAP (1.6 mg, 0.0127 mmol) were added and stirred for 3 hours. Saturated aqueous ammonium chloride solution was added to the reaction solution to stop the reaction, and the mixture was transferred to a separatory funnel. After extraction with chloroform, the chloroform layer was washed with water and a saturated aqueous sodium chloride solution. The chloroform layer was dehydrated with anhydrous magnesium sulfate, filtered with absorbent cotton, and then the solvent was distilled off under reduced pressure. And it refine | purified by the silica gel column chromatography which made the developing solvent chloroform / methanol = 100/1. The color of the purified crystals was pale yellow, and SN-38 cholate TBS protector (38.4 mg, 0.0445 mmol) was obtained in a yield of 35%.

SN-38 cholate TBS protector (38.4 mg, 0.0445 mmol) was added to an eggplant-shaped flask and dissolved in CH ₂ Cl ₂ / MeCN / H ₂ O = 1.5 mL / 1 mL / 0.5 mL. LiBF ₄ (20.9 mg , 0.222 mmol) and stirred at 80 ° C. for 5 hours. Saturated aqueous sodium hydrogen carbonate solution was added to this solution to stop the reaction, and the solution was transferred to a separatory funnel. After extraction with chloroform, the chloroform layer was washed with water and a saturated aqueous sodium chloride solution. The chloroform layer was dehydrated with anhydrous magnesium sulfate, filtered with absorbent cotton, and then the solvent was distilled off under reduced pressure. And it refine | purified by the silica gel column chromatography which made the developing solvent chloroform / methanol = 100/1. The color of the purified crystals was pale yellow, and SN-38 cholate derivative (27.0 mg, 0.0360 mmol) was obtained in a yield of 81%.

¹ HNMR (THF-d ₈ , 400 MHz): d 0.76 (3H, s), 0.93-1.64 (29H, m), 1.80-2.21 (9H, m), 2.57-2.61 (1H, m), 2.67-2.71 (1H, m), 3.19-3.32 (4H, m), 5.26-5.30 (4H, m), 5.51-5.56 (2H, m), 7.49 (1H, s), 7.55 (1H, dd, J = 9.2, 2.4 Hz), 7.91 (1H, d, J = 2.4 Hz), 8.15 (1H, d, J = 9.2 Hz).
HR-MS (ESI-TOF): m / z calcd.for C ₄₆ H ₅₇ N ₂ O ₇ ([M + H] ⁺ ) 749.4160, found 749.4144.

SN-38 acetylcholenic acid derivative ((S) -4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ′, 4 ′: 6, 7] Indolizino [1,2-b] quinolin-9-yl (R) -4-((3S, 8S, 9S, 10R, 13R, 14S, 17R) -3-acetoxy-10,13-dimethyl-2, Synthesis of 3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenanthren-17-yl) pentanoate)

Acetylcholenic acid was synthesized based on the description in known literature (Y.YShen at al., Journal of Fluorine Chemistry 2002, 113, 13-15.).

SN-38 (84.7 mg, 0.216 mmol) and acetylcholenoic acid (108 mg, 0.259 mmol) were added to the eggplant-shaped flask, and dehydrated CH ₂ Cl ₂ was dissolved in 2.2 ml. To this solution, EDC (82.8 mg, 0.254 mmol) and DMAP (2.6 mg, 0.0216 mmol) were added and stirred for 18 hours. Saturated aqueous ammonium chloride solution was added to this solution to stop the reaction, and the solution was transferred to a separatory funnel. After extraction with chloroform, the chloroform layer was washed with water and a saturated aqueous sodium chloride solution. The chloroform layer was dehydrated with anhydrous magnesium sulfate, filtered with absorbent cotton, and then the solvent was distilled off under reduced pressure. And it refine | purified by the silica gel column chromatography which made the developing solvent chloroform / methanol = 100/1. The color of the purified crystals was light yellow, and SN-38 acetylcholenic acid derivative (136 mg, 0.0445 mmol) was obtained in a yield of 80%.

¹ HNMR (CDCl ₃ , 400 MHz): d 0.73 (3H, s), 0.92-1.64 (30H, m), 1.82-2.01 (11H, m), 2.32-2.34 (2H, m), 2.56-2.62 (1H , m), 2.68-2.76 (1H, m), 3.15 (2H, q, J = 7.6 Hz), 3.70 (1H, s), 4.59-4.65 (1H, m), 5.28 (2H, s), 5.32 ( 1H, d, J = 16 Hz), 5.38 (1H, d, J = 4.8 Hz), 5.76 (1H, d, J = 16 Hz), 7.55 (1H, dd, J = 9.2, 2.4 Hz), 7.64 ( 1H, s), 7.82 (1H, d, J = 2.4 Hz), 8.24 (1H, d, J = 9.2 Hz).
HR-MS (ESI-TOF): m / z calcd.for C ₄₈ H ₅₉ N ₂ O ₈ ([M + H] ⁺ ) 791.4266, found 791.4260.

Method for producing nanoparticles
100 μL of a SN-38 derivative THF solution prepared to 10 mM was injected into 10 ml of water at room temperature using a syringe to obtain an aqueous dispersion of SN-38 derivative nanoparticles. A dialysis membrane (fractionated molecular weight: 12,000-14,000) filled with an aqueous dispersion of nanoparticles (10 mL) is placed in a beaker containing 100 mL of distilled water and distilled twice every 5 hours. The water in the nanoparticle dispersion was removed by exchanging water. Then, the final concentration became 0.1 mM by adjusting the volume of the nanoparticle dispersion in the dialysis membrane to 10 mL.

The particle size was as follows.
SN-38 hinokitiol derivative 200 nm
SN-38 colanic acid derivative 50 nm
SN-38 cholenoic acid derivative 50 nm
SN-38 acetylcholenic acid derivative 50 nm

Test example 1

in vitro activity test

Human hepatoma cell line HepG2 was seeded in a 96-well plate at 2 × 10 ⁴ cells / well. On the next day, the irinotecan and SN-38 derivative nanoparticle dispersion was added to the HepG2 cell culture so as to be 0.01 to 10 μM. Thereafter, the cells were cultured for 48 hours, and cell viability was measured by a colorimetric method using Cell Counting Kit-8 (manufactured by DOJINDO) and a microplate reader (manufactured by BIO-RAD). As a result, irinotecan showed almost no anticancer activity under these conditions, whereas various SN-38 samples showed remarkable anticancer activity (FIG. 3).

Test example 2

Surface modification with albumin and in vivo anticancer activity test using mouse (1) Surface modification with albumin 200 μL of SN-38 cholanic acid derivative prepared in 4.2 mM was added to bovine serum albumin (BSA) solution (10 mg / mL) was added to 1 mL of water using a microsyringe to obtain a 0.7 mM nanoparticle dispersion. THF was removed by dialysis. The surface modification with albumin contributes to the improvement of the dispersion stability of the nanoparticles. In addition to albumin, such an effect includes effective proteins such as globulin, hemoglobin, fibrinogen, antithrombin, transferrin, ceruloplasmin, chylomicron, lipaprotein, glycoprotein and the like.
(2) In vivo anticancer activity test A suspension of 4T1 cells (derived from mouse breast cancer) prepared at 1x10 ⁷ cells / mL in the back of two nude mice (BALB / c, male, 5 weeks old, n = 5). Tumors were formed by subcutaneous injection of 100 μL, and administration of anticancer agents was started after the tumors grew to 3-5 mm. As an anticancer agent, SN-38 colanic acid derivative nanoparticle dispersion liquid surface-modified with BSA and irinotecan aqueous solution as a comparison target were administered at 100 μL per time. The SN-38 colanic acid derivative nanoparticle dispersion and the irinotecan solution were prepared in physiological saline so that the amounts of SN-38 were 10 mg and 1 mg, respectively, with respect to 1 kg of mouse body weight. Administration was performed 5 times at a frequency of once every two days, and the change in tumor volume over time was evaluated. As a result, it was clarified that the SN-38 colanic acid derivative nanoparticle dispersion (1 mg / kg) showed the same level of activity as compared with the case where 10 times the amount of irinotecan solution (10 mg / kg) was administered. It was.

Nanoparticles of the invention according to the present application show remarkable anticancer activity, and unlike micelles containing surfactant as a constituent requirement, they do not make surfactant a constituent requirement, so practical use and safety are expected. .

Claims (24)

1. Formula (1):
   

   

   (In the formula, X is a hydrogen atom, a hydroxy group, an optionally substituted alkyl group, an optionally substituted alkoxy group or an optionally substituted acyloxy group, and the 5-position and 6-position of the cholesterol skeleton) The carbon-carbon bond between is a single bond or a double bond).
2. The compound according to claim 1, wherein X is a hydrogen atom, and the carbon-carbon bond between the 5th and 6th positions of the cholesterol skeleton is a single bond.
3. X is a hydroxy group, an optionally substituted alkyl group, an optionally substituted alkoxy group or an optionally substituted acyloxy group, and a carbon atom between the 5-position and 6-position of the cholesterol skeleton— The compound according to claim 1, wherein the carbon bond is a double bond.
4. The compound according to claim 3, wherein the X is a hydroxy group.
5. The compound according to claim 3, wherein X is an acyloxy group.
6. The compound according to claim 5, wherein the acyloxy group is an alkylcarbonyloxy group.
7. The compound according to claim 6, wherein the alkylcarbonyloxy group is an acetyloxy group.
8. Formula (2):
   

   

   (Wherein R is an optionally substituted alkyl group).
9. The compound according to claim 8, wherein R is a C ₁ -C ₂₀ alkyl group.
10. The compound according to claim 8, wherein R is a C ₁ -C ₆ alkyl group.
11. The compound according to claim 8, wherein R is a methyl group.
12. Formula (3):
    

    

    (Wherein n represents an integer of 1 to 20).
13. 

    A compound selected from the group consisting of:
14. A nanoparticle obtained by forming the compound according to any one of claims 1 to 13 into a nanoparticle.
15. The nanoparticles according to claim 14, wherein the average particle diameter is 10 to 200 nm.
16. The nanoparticle according to claim 14 or 15, wherein the nanoparticle according to claim 14 or 15 is obtained by injecting, into water, a solution in which the compound according to any one of claims 1 to 13 is dissolved in a water-miscible organic solvent. Manufacturing method.
17. A pharmaceutical composition comprising the compound according to any one of claims 1 to 13.
18. A pharmaceutical composition comprising the nanoparticles according to claim 14 or 15.
19. A therapeutic agent for cancer diseases comprising the compound according to any one of claims 1 to 13.
20. A therapeutic agent for cancer diseases containing the nanoparticles according to claim 14 or 15.
21. The cancer disease therapeutic agent according to claim 19 or 20, wherein the cancer disease is a solid tumor.
22. The solid tumor is esophageal cancer, stomach cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, laryngeal cancer, lung cancer, prostate cancer, bladder cancer, breast cancer, uterine cancer or ovarian cancer. Therapeutic agent for cancer diseases.
23. The nanoparticulate preparation for cancer disease treatment according to any one of claims 1 to 13.
24. An injection containing the nanoparticle according to claim 14 or 15.
    
    
    
    

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