KR102377334B1 - A Composition for Solubilizing Fingolimod derivative compound and Uses Thereof - Google Patents

Abstract

The present invention relates to a composition for solubilizing a fingolimod derivative compound and a use thereof, and more specifically, to a solubilization composition with improved solubility in water by encapsulating fingolimod, a poorly water-soluble drug, in a specific polymer to form micelles, and a use of the composition for solubilizing the fingolimod derivative compound as a pharmaceutical material. The composition for solubilizing the fingolimod derivative compound according to the present invention can effectively solubilize the fingolimod derivative compound by using a polymeric micelle composed of an association of mPEG-b-PLA. The fingolimod derivative compound is a poorly soluble substance and hardly exists in a water layer, and is solubilized by being encapsulated in micelles. In this way, the fingolimod derivative compound, which is a poorly soluble substance, can be dissolved in the water layer and thus developed as an intravenous injection using water. The intravenous injection can avoid a hepatic first pass effect compared to oral dosage forms, thereby reducing unnecessary drug loss. Compared to oral dosage forms, the intravenous injection can also increase bioavailability, and thus is expected to provide a high effect even with the same amount of drug as an effective administration method. Thus, the development of such formulation enables effective administration of the fingolimod derivative compound.

Description

A Composition for Solubilizing Fingolimod derivative compound and Uses Thereof

The present invention relates to a solubilization composition of a Fingolimod derivative compound and its use, and more particularly, to a water solubility by encapsulating Fingolimod, a poorly water-soluble drug, in a specific polymer to form micelles. It relates to an improved solubilizing composition and the use of the solubilized composition of the fingolimod derivative compound as a pharmaceutical material.

FTY720 (Fingolimod), which is mainly used as a treatment for multiple sclerosis, has recently been studied for its anticancer effect on various cancer cells, and various analogs are being studied. However, analogs such as (R)-FTY720 methyl ether have confirmed anticancer effects on breast cancer cells, but have a disadvantage in that they require many synthesis steps.

In order to overcome the complexity of this synthesis, many analogs including the amide chain of FTY720 have been synthesized. One of the synthesized analogs, N-[4-(2-((4-Methoxybenzyl amino ethyl) phenyl]heptanamide (hereinafter simply referred to as 'MPH')) showed the best anticancer efficacy among them (Molecules. 2018 Oct. 24;23(11):2750., Synthesis of Novel FTY720 Analogs with Anticancer Activity through PP2A Activation).

However, despite these advantages of MPH, there are disadvantages that it is a poorly soluble drug with solubility in water of about 0.37 ± 0.01 mg/mL, and that formulation studies have not been conducted because it is a newly synthesized drug.

A poorly soluble drug means a drug that does not dissolve well in water because it contains a hydrophobic portion in the structure of the compound, and its practicality is often limited due to poor solubility. For example, more than 41% of drugs developed as new drugs are abandoned due to poor solubility, and more than one third of drugs listed in the US Pharmacopeia are classified as poorly soluble drugs.

In order to use such poorly soluble drugs, an additional material to solve the poor solubility must be added, but a number of cases in which use is restricted due to the toxicity of the added material have been reported. For example, in general, emulsification using an emulsifier and collection using liposomes are widely used to solubilize poorly soluble substances, but their use is limited due to the incorporation of foreign substances not derived from the human body and physical instability.

Therefore, in the pharmaceutical application of MPH, which is a poorly soluble material, it is essential to prevent deterioration due to temperature, storage location and period, secure long-term stability, and maintain a homogenized aqueous solution state. It needs to be done without the inclusion of excipients.

However, there has been no research so far to water-soluble MPH, a poorly soluble material, for application as a pharmaceutical or to increase the absorption rate in vivo.

Under this background, the present inventors have conducted various studies to develop a composition capable of improving the solubility of MPH in water, methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer The present invention was completed by identifying that MPH could be effectively solubilized using micelles.

In particular, in the present invention, as a result of applying MPH to various polymers (mPEG- b -PLA, Pluronics® F127 and ^Soluplus® ) that can be used as carriers for poorly soluble drugs, in the case of micelles prepared using Pluronics® F127, the particle size is Since it appears over 1000nm, it is difficult to penetrate into the tumor tissue, and micelles manufactured using Soluplus ^® have a problem in storage stability due to sedimentation over time at refrigerated storage temperature and indoor storage temperature, so it is suitable for application to MPH appeared not to. On the other hand, in the case of micelles prepared using mPEG- b -PLA, the MPH loading efficiency is excellent, and it has a uniform particle size of 25 nm to 35 nm, so it is the first carrier that can be optimized for the solubility and delivery of MPH. identified.

Korean Patent Registration No. 10-1905010

Accordingly, it is an object of the present invention to provide a solubilizing composition of a Fingolimod derivative compound in which the solubility of the Fingolimod derivative compound, which is a poorly soluble material, in water is effectively improved.

Another object of the present invention is to provide a method for preparing a composition for solubilizing a Fingolimod derivative compound in which the solubility of the Fingolimod derivative compound, which is a poorly soluble material, in water is effectively improved.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which can be used as an intravenous preparation by effectively improving the solubility of a poorly soluble material, a Fingolimod derivative compound, in water.

In order to achieve the object of the present invention as described above,

The present invention relates to a methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer micelle encapsulated in a Fingolimod derivative compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof. It provides a solubilization composition of the compound comprising a, fingolimod (Fingolimod) derivatives as an active ingredient.

<Formula 1>

In one embodiment of the present invention, the composition is water-soluble by encapsulating a Fingolimod derivative compound, a poorly water-soluble drug, in a methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer. Solubility may be improved.

In one embodiment of the present invention, the micelles may have a particle size of 25nm to 35nm.

In one embodiment of the present invention, the composition may be stable without deterioration for a long period of time under a temperature condition of 4 ℃ to 37 ℃.

In addition, the present invention comprises the steps of (a) dissolving a Fingolimod derivative compound and a methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer in acetonitrile to prepare a mixture; (b) adding the mixture to a round bottom flask and evaporating the solvent under reduced pressure in a rotary evaporator to obtain a thin film; and (c) hydrating the thin film with distilled water to obtain a micellar solution.

In one embodiment of the present invention, in step (a), the Fingolimod derivative compound and the methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer are 1:20 to It may be mixed in a weight ratio of 1:21.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the solubilized composition of the Fingolimod derivative compound as an active ingredient.

In one embodiment of the present invention, the composition may be a formulation for intravenous injection.

In one embodiment of the present invention, the pharmaceutical composition may further include one or more components selected from the group consisting of amino acids, sugars, lipids, vitamins, electrolytes, pH adjusters, stabilizers, osmotic pressure adjusters and solubilizers. there is.

In one embodiment of the present invention, the cancer is breast cancer, colorectal cancer, uterine cancer, fallopian tube cancer, ovarian cancer, stomach cancer, brain cancer, head and neck cancer, rectal cancer, small intestine cancer, esophageal cancer, lymph gland cancer, gallbladder cancer, lung cancer, non-small cell lung cancer, It may be selected from the group consisting of glioblastoma, skin cancer, kidney cancer, bladder cancer, blood cancer, pancreatic cancer, prostate cancer, thyroid cancer, endocrine adenocarcinoma, oral cancer, liver cancer, and blood cancer.

The composition for solubilizing a fingolimod derivative compound according to the present invention can effectively solubilize the fingolimod derivative compound by using a polymer micelle composed of an association of mPEG- b -PLA. The fingolimod derivative compound is a sparingly soluble substance, hardly present in the aqueous layer, and becomes solubilized by encapsulation in micelles. In this way, the fingolimod derivative compound, which is a poorly soluble substance, can be developed as an intravenous injection using water by allowing it to dissolve in the aqueous layer. Compared to oral dosage forms, intravenous injection can avoid the liver first-pass effect, thereby reducing unnecessary drug loss, and increasing bioavailability compared to oral dosage forms. As a method, the development of such a formulation enables effective administration of the fingolimod derivative compound.

In particular, in the present invention, uniform particles with a size of 25 to 35 nm with excellent tumor penetration ability only in the case of micelles prepared using mPEG- b -PLA among various polymers (mPEG- b -PLA, Pluronics ^® F127 and Soluplus ^® ) It was identified for the first time as a carrier that can be optimized for solubility and delivery of fingolimod as it has a size and excellent storage stability according to temperature.

1 is a schematic diagram showing a method for preparing a nano-micelle preparation containing MPH of the present invention and a schematic diagram after preparation.
2 shows representative particle distribution profiles of MPH-loaded mPEG- b -PLA micelles, MPH-loaded Soluplus ^® micelles and MPH-loaded Pluronics ^® F127 micelles.
3 is a result of evaluating storage stability according to temperature of MPH-loaded mPEG- b -PLA micelles and MPH-loaded Soluplus ^® micelles.
4 is a photograph observing the precipitation of MPH-loaded Soluplus ^® micelles after 5 days at 4 ℃ (a) and 25 ℃ (b) conditions.
Figure 5 shows the in vitro drug release profile of MPH-loaded mPEG- b -PLA micelles (a) and MPH aqueous solution (b).
6 is a graph showing the activity of cancer cells after treatment with MPH-loaded mPEG- b -PLA micelles and MPH aqueous solution for a short period of time (48 hours) in cancer cells (A549, U87). Figure 6 (a) shows the activity of cancer cells according to drug concentration after treating cancer cells (A549) with MPH drug dissolved in organic solvent (DMSO), (b) is MPH-loaded mPEG- b- in cancer cells (A549) Activity of cancer cells according to drug concentration after treatment with PLA solution, (c) is cancer cell activity according to drug concentration after treatment with MPH drug dissolved in organic solvent (DMSO) in cancer cells (U87), (d) is cancer cell (U87) is a graph showing the activity of cancer cells according to drug concentration after treatment with MPH-loaded mPEG- b -PLA solution.
7 shows the activity of cancer cells after treatment with MPH-loaded mPEG- b -PLA micelles and MPH solution (MPH solution dissolved in DMSO, an organic solvent) for a long period (14 days) in cancer cells (A549, U87) by concentration. ((a): A549, (b): U87).

The present invention relates to methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer micelles encapsulated in a Fingolimod derivative compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof. It relates to a solubilization composition of a compound comprising a fingolimod (Fingolimod) derivative, comprising as an active ingredient.

<Formula 1>

It has been reported that the Fingolimod derivative compound has an anticancer effect and the like.

As the Fingolimod derivative compound represented by the above formula, commercially available ones may be used, or may be directly synthesized through a conventionally known method and used.

The Fingolimod derivative compound represented by the above formula may be prepared as a pharmaceutically acceptable salt or solvate according to a method conventional in the art.

As the pharmaceutically acceptable salt, an acid addition salt formed with a pharmaceutically acceptable free acid is useful. Acid addition salts are prepared by conventional methods, for example by dissolving the compound in an aqueous solution of excess acid and precipitating the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equal molar amounts of compound and acid or alcohol in water (eg glycol monomethyl ether) may be heated to dryness followed by evaporation of the mixture, or the precipitated salt may be filtered off with suction.

In this case, organic acids and inorganic acids can be used as free acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. can be used as inorganic acids, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, and citric acid can be used as organic acids. , maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like can be used.

In addition, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

The pharmaceutically acceptable salts of the Fingolimod derivative compound having the structure of the above formula of the present invention include acidic or basic salts that may be present in the compound having the structure of the formula, unless otherwise indicated. do. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of hydroxyl groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen There are salts of phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate), and methods or processes for preparing salts known in the art can be manufactured through

In the present invention, the methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer comprises polyethylene glycol (PEG), which is a hydrophilic material, and polylactic acid, which is a hydrophobic component. It is composed of an association of mPEG- b -PLA, a polymeric polymer of PLA), and serves as a carrier capable of carrying a drug or a carrier capable of delivering a drug.

Although mPEG- b -PLA is a single molecule, when this substance is dissolved in water and exceeds a specific concentration (critical micelle concentration, CMC), spherical micelles are formed. When micelles are formed, a hydrophilic portion (shall) and a hydrophobic portion (core) are formed. The hydrophilic part, the shell, is made of PEG, the hydrophilic part of the polymer, and the hydrophobic part, the nucleus, is made of the hydrophobic part of the polymer, PLA.

In the composition of the present invention, the Fingolimod derivative compound represented by the above formula, which is a poorly soluble substance, hardly exists in the aqueous layer or the hydrophilic part of the PEG-b-PLA polymer micelles, and is distributed in the nucleus, which is the hydrophobic part, within the micelles. Solubilization is performed in such a way that it is encapsulated.

In the present invention, a thin-film hydration method was used as a method for preparing micelles. Briefly, a Fingolimod derivative compound was dissolved in acetonitrile together with a mPEG- b -PLA polymer, transferred to a round-bottomed plastomer, and evaporated at 60°C for 10 minutes using a rotary evaporator to form a thin film. made. Distilled water was added to the thus-prepared film membrane and hydrated for 20 minutes to make a clear micellar solution. After centrifuging the micelle solution, only the supernatant was taken and filtered through a cellulose filter to aseptically remove the precipitated material and remove unhydrated impurities.

The micelles of the present invention prepared by the above method may have an average particle diameter of 25 nm to 35 nm. For reference, when the particle size of micelles is less than 100 nm, phagocytosis and absorption by the reticuloendothelial system (RES) can be avoided, and thus systemic circulation can be prolonged. Therefore, the micelles of the present invention have a sufficiently small particle size to avoid phagocytosis and absorption by the reticuloendothelial system, and thus can stay in the body for a long time, thereby increasing the bioavailability of the drug (fingolimod derivative compound) to be delivered. can

In the present invention, the term “solubilization” refers to a phenomenon in which the solubility of substances that are poorly soluble in water is increased by the presence of substances such as surfactants.

The solubilization composition of the fingolimod derivative compound of the present invention has solubility in water by encapsulating the fingolimod derivative compound, a poorly water-soluble drug, in a methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer. has improved characteristics.

The solubilization composition of the fingolimod derivative compound of the present invention has stable properties without deterioration for a long period of time at a temperature of 4°C to 37°C.

In addition, the present invention provides (a) a Fingolimod derivative compound represented by Formula 1 and a methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer by dissolving it in acetonitrile. preparing a mixture; (b) adding the mixture to a round bottom flask and evaporating the solvent under reduced pressure in a rotary evaporator to obtain a thin film; and (c) hydrating the thin film with distilled water to obtain a micellar solution.

In step (a) of the present invention, the Fingolimod derivative compound and the methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer are mixed in a weight ratio of 1:20 to 1:21 can be

In addition, the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising the solubilized composition of the Fingolimod derivative compound as an active ingredient.

The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. In this case, pharmaceutically acceptable carriers are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose. , polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. may be additionally included in addition to the above components.

Formulations of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained-release formulations of the active compound. It can be, preferably, a solution for intravenous injection is good.

The solubilization composition of a Fingolimod derivative compound, which is an active ingredient of the pharmaceutical composition of the present invention, has stable properties without deterioration for a long period of time at a temperature of 4°C to 37°C. The pharmaceutical composition of the present invention is an intravenous formulation can be usefully used as

When the pharmaceutical composition of the present invention is a formulation for intravenous injection, it may include pharmaceutical ingredients generally included in the formulation for intravenous injection. For example, the intravenous formulation composition may include at least one component selected from the group consisting of amino acids, sugars, lipids, vitamins, electrolytes, pH adjusters, stabilizers, osmotic pressure adjusters, and solubilizers. The specific composition of the amino acid solution for injection, the solution for sugar injection, or the solution for lipid injection is well known in the art.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or locally applied) according to a desired method, and the dosage may vary depending on the condition and weight of the patient, and the disease. Although it varies depending on the degree, drug form, administration route and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, and drug activity of the patient; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient into the body, inactivation rate and excretion rate, disease type, and drugs used in combination.

The pharmaceutical composition of the present invention may be administered to mammals such as human or non-human primates, mice, rats, dogs, cats, horses and cattle, but is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. These Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these Examples.

<Example 1>

Material preparation and cell culture

<1-1> Materials and reagents

Methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG[4k]-b-PLA [2.2k]) was purchased from Advanced Polymer Materials Inc. (Montreal, QC, Canada). Soluplus ^® was purchased from BASF (Ludwigshafen, Rhineland-Palatinate, Germany). Ethanol (EtOH) and acetonitrile (ACN) were manufactured by Fisher Scientific Ltd. (Waltham, MA, USA). Distilled water (DW) was purchased from Tedia (Fairfield, OH, USA). Methanol (MeOH) was purchased from Honeywell Burdick and Jackson (Ulsan, Korea). Pluronics ^® F127, ethyl acetate (EtOAc) and Cremophor EL ^® are sold by Sigma-Aldrich Corp. (St. Louis, MO, USA). All other reagents were of analytical grade or better.

<1-2> Prepare MPH

The MPH used in this experiment was provided by Professor Baek Dong-jae of the Pharmaceutical Synthesis Laboratory, College of Pharmacy, Mokpo University.

<1-3> Cell culture

A549 human non-small cell lung cancer cells used in this experiment were aliquoted from ATCC (American Type Culture Collection, MD, USA), and 90% RPMI-1640 medium (Gibco BRL, Grand Island, NY, USA), 10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY, USA) in a growth medium containing 1% penicillin and streptomycin (Gibco BRL, Grand Island, NY, USA) at 37 °C. , incubated under 5% CO ₂ conditions. In addition, human glioblastoma U-87 cells were also obtained from the American Type Culture Collection (MD, USA), 10% heat-inactivated FBS and antibiotics (100 U/mL of penicillin and 100 In DMEM (Dulbecco's modified Eagle's medium) supplemented with μg/mL of streptomycin), 5% CO ₂ , 95% atmospheric and humid air were maintained at 37°C.

<Example 2>

Preparation of MPH-loaded micelles and characteristics of micelles according to high molecular weight polymers

Polymer micelles loaded with MPH (N-[4-(2-((4-Methoxybenzyl amino ethyl) phenyl]heptanamide) were prepared with various polymers using thin film hydration.

Briefly, various concentrations of MPH and various high molecular polymers (mPEG-b-PLA Soluplus ^® , Plurnonics ^® F127) were dissolved in 1 mL acetonitrile (see Tables 1 to 3 below). The drug-polymer mixture was added to a round bottom flask, and the solvent was evaporated under reduced pressure using a rotary evaporator (EYELA, Bohemia, NY, USA) at 60° C. for 10 minutes to obtain a thin film. After the solvent was completely evaporated, the film was hydrated with 1.4 mL distilled water for 20 minutes to obtain a clear micelle solution. After centrifuging the micelle solution obtained through the above process at 13,000 rpm (Hanil Science lnc., Gimpo, Korea) for 10 minutes, only the supernatant was taken and filtered using a sterile regenerated cellulose filter (0.2 um pore size), and precipitated at the same time Non-hydrated impurities were removed by removing the substances used.

The size and polydispersity index of the nano-solubilized micellar formulation of the present invention was measured with a particle analyzer (Anton Paar, Graz, Austria) instrument. For measurement, the micelle solution was diluted 10-fold and the measurement was performed.

The results are shown in detail in Tables 1 to 3 and FIGS. 1 and 2 below.

Encapsulation efficiency, size, and polydispersity index of micelles with mPEG-b-PLA applied MPH
Initial concentration (mg/mL) Polymer Amount (mg) MPH
final concentration
(mg/mL) EE (%)
(Encapsulation efficiency) Size (nm) Poly dispersity index (PDI) 3 135 1.74 57.97 29.3 0.261 5 135 3.45 69 31.3 0.193 6 90 3.9 65.05 33.1 0.186 6 100 3.11 51.97 33.1 0.243 6 110 4.17 69.58 31.6 0.142 6 125.5 4.67 77.95 29.9 0.196 6 135 4.53 75.52 31.3 0.193 7 135 4.68 66.91 28.6 0.165 8 100 4.33 54.19 30.5 0.167

Encapsulation efficiency, size, and polydispersity index of micelles applied with Soluplus ^® MPH
Initial concentration (mg/mL) Polymer Amount (mg) MPH
final concentration
(mg/mL) EE (%)
(Encapsulation efficiency) Size (nm) Poly dispersity index (PDI) 6 70 4.95 82.5 57.8 0.044 6 125.5 6.07 101 57.7 0.057 6 135 3.34 55.68 69.1 0.049

Encapsulation efficiency, size, and polydispersity index of micelles with Plurnonics ^® F127 applied MPH
Initial concentration (mg/mL) Polymer Amount (mg) MPH
final concentration
(mg/mL) EE (%)
(Encapsulation efficiency) Size (nm) Poly dispersity index (PDI) 6 125.5 4.37 72.88 7196.6 1.531 6 135 3.98 66.39 1221.8 0.417

The size of MPH-containing nano micelles was measured as mPEG-b-PLA 28-33 nm, Soluplus ^® 57-69 nm, and Plurnonics ^® F127 >1000 nm, respectively. Of note, in tumors, micelle-like nano-drug delivery systems accumulate in the tumor mainly through enhanced permeability and retention effects. The micelles with a particle size of 30 nm are generally excellent in penetrating into the tumor tissue, and if the particle size of micelles is smaller than 200 nm, absorption by the reticuloendothelial system (RES) can be avoided, and the time required to circulate throughout the body This could be longer. Considering this relationship between the particle diameter of micelles and the anticancer effect, MPH-encapsulated mPEG-b-PLA micelles and Soluplus ^® micelles were considered as final formulation candidates.

<Example 3>

mPEG-b-PLA, Soluplus ^® Evaluation of storage stability according to temperature of micelles prepared with

In this experiment, the storage safety of micelles prepared by applying mPEG-b-PLA (125.5 mg) and Soluplus ^® (70 mg) to MPH (6 mg), respectively, was evaluated.

In order to measure the storage stability according to the temperature of the nano-solubilized micellar formulation of the present invention, micelles formulated with mPEG-b-PLA, Soluplus ^® in an environment of refrigeration storage temperature of 4°C, indoor storage temperature of 25°C, and body temperature of 37°C was stored for 14 days to measure the size and polydispersity index (PDI) of the micellar formulation.

As a result, as shown in FIG. 3, mPEG-b-PLA micelles were stable at 4°C and 25°C for 14 days, indicating that mPEG-b-PLA micelles can be stored for at least 14 days in a refrigerator or room temperature. suggest In addition, it was stable for 7 days at 37°C measured from the viewpoint of in vivo stability, indicating that micelles can be stable in the body after injection. On the other hand, in the case of Soluplus ^® micelles, the measured size and polydispersity index were confirmed to be stable in all temperature conditions for at least 14 days, but precipitation was observed with the naked eye at 4°C and 25°C after 5 days (see FIG. 4). Soluplus ^® micelles were not considered as the final formulation, because the precipitation of micelles could cause embolism during intravenous injection and serious complications, and mPEG-b-PLA micelles were selected as the final formulation.

<Example 4>

MPH-loaded mPEG- b -Drug release profile of PLA micelles and MPH aqueous solution

In this experiment, the drug release of micelles and MPH aqueous solution (control) prepared by applying mPEG- b -PLA (125.5 mg) to MPH (6 mg) was evaluated. MPH aqueous solution (control) was prepared by dissolving 3 mg of MPH in a mixed solution of acetonitrile: distilled water in a volume ratio of 7:3 (total 1 mL).

After adjusting the MPH concentration of the MPH aqueous solution and the MPH-loaded mPEG- b -PLA micelles to 3 mg/mL, the degree of drug dissolution was measured at the in vitro level. The dissolution test using PBS of pH 7.4 was performed at the same temperature as body temperature at 37°C for 3 hours.

As a result, as shown in FIG. 5 , it was confirmed that 100% of MPH dissolved in the acetonitrile:distilled water mixed solution was released at 90 minutes, and 73% of the MPH was released at 90 minutes and 85% at 180 minutes in micelles.

Through the above results, it was confirmed that the drug release from the MPH-loaded mPEG-b-PLA micelles of the present invention can be continuously released for a longer period of time than in the MPH aqueous solution.

<Example 5>

MPH-loaded mPEG- b -Efficacy evaluation after cancer cell treatment of PLA micelles and MPH solution

In this experiment, drug release of micelles and MPH solutions prepared by applying mPEG- b -PLA (125.5 mg) to MPH (6 mg) was evaluated. MPH solution was prepared by adding 5 mg of MPH to 100 μl of DMSO (dimethyl sulfoxide) and diluting it 1000 times.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was conducted at the in vitro level ( in vitro ) to confirm the kinetics of cancer cells. In 96-wells, 3000 cells each of A549, a non-small cell lung cancer cell, and U87, a human glioblastoma, were laid, and 24 hours later, the MPH solution was diluted twice and the 96-well was drug-treated. The micelles solubilized with mPEG-b-PLA were For treatment with a drug concentration similar to that of the MPH solution, it was diluted 100-fold and diluted two-fold, and the drug was treated in 96-well. After 48 hours of drug treatment, the drug was removed, and the MTT solution was evenly put in each well. After 4 hours, the MTT solution was removed, DMSO was added, the light was blocked with a foil, and the mixture was evenly mixed, followed by measurement with a spectrophotometer at a wavelength of 540 nm. For reference, the IC ₅₀ value is one of the methods for quantifying drug response, and indicates the maximum concentration at the moment when the cell activity is halved when the drug is administered.

As a result, as shown in FIG. 6 and Table 4, the IC ₅₀ values of mPEG-b-PLA micelles containing MPH showed no statistically significant difference in both A549 and U87 compared to the MPH solution. Therefore, it was confirmed that even if MPH was prepared as nano-solubilized micelles, MPH was released without being trapped inside the micelles, and thus the intrinsic anticancer ability of the drug was not deteriorated.

Through the above results, it is confirmed that the MPH-loaded mPEG- b -PLA micelles of the present invention can effectively exhibit anti-cancer effects when applied to actual cancer cells, thereby demonstrating their usefulness as pharmaceuticals.

For reference, DMSO (dimethyl sulfoxide) is an organic solvent that is widely used to dissolve poorly soluble substances in cell experiments, and it is difficult to apply to drugs that are actually applied to the human body because of its strong toxicity. In contrast, the micelles of the present invention contain MPH, a poorly water-soluble drug, in a biodegradable and biocompatible mPEG- b -PLA material so that they can be well soluble in water, and thus have safe properties for the human body.

Therefore, it was objectively confirmed through the above experiment that the MPH-loaded mPEG- b -PLA micelles of the present invention are safe for the human body and have significant anticancer activity.

IC ₅₀ values after treatment of cancer cells (A549, U87) with MPH drugs for a short period of time (48 hours) MPH formulation IC ₅₀ (μM) A549 U87 MPH aqueous solution 26.9 ± 6.64 23.5 ± 9.51 MPH-containing mPEG-b-PLA micelles 22.0 ± 3.09 24.3 ±1.19

In addition, as a cytotoxicity study experiment of the nano-solubilized micelles of the present invention, the clonogenic assay was performed for a longer period (14 days) at the in vitro level ( in vitro ) to confirm the kinetics of cancer cells. Briefly, 200 cells each of A549 and U87 were spread in 6-wells, and after 24 hours, an aqueous solution of MPH dissolved in dimethyl sulfoxide (DMSO) of various concentrations and micelles solubilized with mPEG-b-PLA were treated with drugs. After 14 days of drug treatment, the drug was removed, treated with an aqueous ethanol solution containing crystal violet (0.5%) and left for 30 minutes, washed with clean water, and the number of stained colonies was counted. As in the above MTT assay, MPH aqueous solution and MPH IC50 values of micelles containing mPEG-b-PLA were compared.

As a result, as shown in FIGS. 7 and 5, the IC ₅₀ values of mPEG-b-PLA micelles containing MPH were found to have no statistically significant difference in both A549 and U87 compared with the MPH aqueous solution. Therefore, it was confirmed that even if MPH was prepared as nano-solubilized micelles, MPH was released without being trapped inside the micelles, and thus the intrinsic anticancer ability of the drug was not deteriorated.

IC50 value after treatment of cancer cells (A549, U87) with MPH drug for a long period (14 days) MPH formulation IC ₅₀ (μM) A549 U87 MPH aqueous solution 35.5 ± 6.36 34.0 ± 0.07 MPH-containing mPEG-b-PLA micelles 30.5 ± 0.13 30.4 ± 0.85

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

MPH: N-[4-(2-((4-Methoxybenzyl amino ethyl) phenyl]heptanamide
mPEG-b-PLA: methoxy poly(ethylene glycol)-b-poly(d,l-lactide)

Claims (10)

A composition for solubilizing a Fingolimod derivative compound, comprising:
The composition is a methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer micelle in which a Fingolimod derivative compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof is encapsulated. contains as an active ingredient,
The micelles include a Fingolimod derivative compound and a methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer in a weight ratio of 1:20 to 1:21,
The micelles are characterized in that having a particle size of 25nm to 35nm, fingolimod (Fingolimod) solubilization composition of a derivative compound.
<Formula 1>

According to claim 1,
The composition is characterized in that solubility in water is improved by encapsulating a Fingolimod derivative compound, a poorly water-soluble drug, in a methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer. Ha, fingolimod (Fingolimod) solubilization composition of the derivative compound. delete According to claim 1,
The composition is characterized in that the composition is stable without deterioration for 14 days at a temperature of 4°C to 25°C, a solubilizing composition of a Fingolimod derivative compound. (a) a Fingolimod derivative compound represented by the following Chemical Formula 1 and a methyl-poly(ethylene glycol)-b-poly(d,l-lactide) polymer in a weight ratio of 1:20 to 1:21 in aceto dissolving in nitrile to prepare a mixture;
(b) adding the mixture to a round bottom flask and evaporating the solvent under reduced pressure in a rotary evaporator to obtain a thin film; and
(c) hydrating the thin film with distilled water to obtain a micelle solution, a method for preparing a composition for solubilization of a Fingolimod derivative compound.
<Formula 1>

delete A pharmaceutical composition for preventing or treating non-small cell lung cancer or glioblastoma, comprising the solubilized composition of the Fingolimod derivative compound of claim 1 as an active ingredient. 8. The method of claim 7,
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of non-small cell lung cancer or glioblastoma, characterized in that the formulation for intravenous injection. 9. The method of claim 8,
The pharmaceutical composition is characterized in that it further comprises at least one component selected from the group consisting of amino acids, sugars, lipids, vitamins, electrolytes, pH adjusters, stabilizers, osmotic pressure adjusters, and solubilizers, non-small cell lung cancer or glioma A pharmaceutical composition for preventing or treating blastoma. delete

Images