KR101002672B1 - PEGylated liposome containing cromolyn and the preparation method thereof - Google Patents

Abstract

The present invention relates to chromoline-containing PEGylated liposomes and a method for preparing the same, and more particularly, to pegylated liposomes containing chromoline and a method for preparing the same. It is about. The chromoline-containing PEGylated liposomes of the present invention have the effect of preventing destruction by phagocytic cells of RES by PEGylation of liposomes, thereby increasing the residence time in the blood to efficiently release the drug at the site of pancreatic cancer. Pancreatic cancer can be prevented. In addition, synergistic effects when administered in combination with gemcitabine may contribute to the prevention of pancreatic cancer.

Chromoline, pegylated liposomes, gemcitabine, pancreatic cancer, pegylated

Description

Pegylated liposome containing cromolyn and the preparation method

The present invention relates to chromoline-containing PEGylated liposomes and a method for preparing the same, and more particularly, to a liposome encapsulated with chromoline having a PEGylated liposome and having a pancreatic cancer prevention and treatment effect.

Pancreatic cancer is one of the most deadly cancers with a five-year survival rate of less than 5% and more than half of the patients diagnosed within six months die. Despite many recent efforts in molecular biology, therapeutic performance has not improved significantly compared to the past. Pancreatic cancer is the most effective treatment for early resection, but only a few patients can be operated on.

As chemotherapy for treating conventional pancreatic cancer, a variety of anticancer agents including gemcitabine have been used, and the present invention requires the combination of anticancer agents and the development of new anticancer agents to enhance anticancer effects.

S100P is a 95-amino acid component of the S100 group, first extracted from the placenta, expressed in more than 90% of pancreatic cancers, and has been reported to be functionally important in pancreatic cancer cell growth and survival, in addition to overexpression in breast and lung cancers. S100P is also known to be resistant to chemotherapy in colorectal cancer cell lines and to reduced survival in lung cancer patients. Taken together, these results suggest that S100P overexpression may increase tumor growth and metastasis and reduce patient survival. Therefore, if the function of S100P is inhibited, it is expected to improve the pancreatic cancer treatment response.

Recently, animal studies by Anderson Cancer Center's team found that a protein called 'S100P' was detected in excess in some cancers, and this protein also plays an important role in the growth and survival of pancreatic cancer cells. The team reported that the protein activates protein receptors on the surface of RAGE cells, which play an important role in Alzheimer's disease, diabetes and cancer.

In particular, Anderson's team has discovered the anticancer activity of cromolyn, which has been widely used as an asthma and allergy drug. In other words, according to the team, chromoline inhibited tumor growth by 70% in rat experiments using human pancreatic cancer cells, especially when combined with chemotherapeutic gemcitabine, compared with 50% of single doses. Up to 85%.

However, since chromoline is used as a coating agent and has a short half-life (1.3 hours), it is essential to study how to effectively deliver it to internal cancer cells. Therefore, the present inventors have focused on a method for increasing the delivery efficiency of chromoline, which is newly known for use in pancreatic cancer.

Liposomes are composed of bilayers of phospholipids, which are basic structures of biological membranes, and point to microvesicles with hydrophilic spaces inside and closed double lipid membranes outside. Liposomes contain water soluble molecules (including DNA) or drugs in the central hydrophilic space, and lipid lipid membranes can attach fat-soluble drugs or bind positively and negatively charged materials. Phospholipids are amphipathic substances, which have a polar molecular group of anionic or amphoteric ions and two nonpolar fat-soluble chains with varying degrees of unsaturation of around 16 hydrocarbons. To form. Therefore, liposomes are widely used in terms of basic science and applied science.

Liposomes interact with proteins and phospholipids in basic science [Lee, J.W. and Kim, H., Arch. Biochem. Biophys., 297: 354 (1992); Hahn, K.H. and Kim, H., J. Biochem., 110: 635 (1991)], Topology and Activity of Proteins in Biofilms, and Fusion of Biofilms (Yun, CH and Kim, H., J. Biochem., 105: 406 (1989); Kim, J. and Kim, H., Biochemistry, 25: 7867 (1986); Kim, J. and Kim, H., Korean Biochem. J., 18: 403 (1985); and It has been used as a model for protein delivery mechanism.

In addition, the field of applied science is used as a model for delivering genetic material to the cells in the cosmetic industry, drug delivery, and in vitro culture, and now it is possible to capture both water-soluble and fat-soluble substances in liposomes, It is easy to target to specific tissues, easy to size and modify, and has little toxicity problem due to the use of phospholipids. It is possible to capture more drugs that can be captured to other drug carriers and to increase the therapeutic effect. As a result, the drug delivery system (DDS) has been attracting attention, particularly as an anticancer drug delivery system.

Therapies that have been conventionally attempted to treat malignancies such as cancer include surgery, radiation therapy or drugs that act directly or indirectly on cancer cells, such as antimetabolites, alkylating agents, mitosis inhibitors. antimitotic drugs and chemotherapy using hormones.

Conventionally, chemotherapy using drugs that directly or indirectly act on cancer cells, such as metabolic antagonists, alkylating agents, mitosis inhibitors, and hormones, is a method for treating cancer. It spreads throughout the body and affects rapidly growing normal cells other than cancer cells, such as bone marrow cells that produce blood, hair follicle cells that grow hair and whiskers rapidly, genital cells that produce eggs and sperm, and gastrointestinal mucosa. Side effects.

In order to overcome the drawbacks of the above anticancer drugs, studies on the liposomes described above as drug carriers for effectively delivering drugs into cells have been in earnest. Hereinafter, a specific example of a conventional drug delivery technique using liposomes will be described.

Korean Patent Laid-Open Publication No. 2005-81889 discloses a method for improving stability in vivo by coating a lipid membrane using scleroglucan, a natural polysaccharide, of liposomes as drug carriers. In addition, the Republic of Korea Patent No. 293572 is a method of producing a polymerized liposome with excellent stability by combining the hydrocarbon chain ends in the molecules constituting the membrane of the liposomes by pH control and in the drug delivery system and precisely does not decompose A drug carrier that can reach a site and slowly administer a drug over a long period of time is described.

Meanwhile, Korean Patent Laid-Open Publication No. 2007-0108135 describes a method for treating cancer in a human body comprising administering pegylated liposome doxorubicin in combination with actinidine 743.

However, there is no disclosure of pegylated liposomes using chromoline, and no direct review of their effects on pancreatic cancer. In addition, there is no disclosure of an optimal method for preparing liposomes for increasing the encapsulation efficiency of chromolines in liposomes and residence time in blood.

Therefore, the present inventors while studying the effective drug delivery method of the chromoline, dipalmitoylphosphatidyl-choline (DPPC), dimylisitoylphosphatidyl-choline (DMPC) and distearoylphosphatidylcholine (distearoylphophatidylcholine, or DSPC) when mixed in the proper ratio, the temperature-sensitive liposomes were produced and found to be released efficiently at tumors with higher temperature conditions (40-41 ℃) than normal tissues. Distearoylphosphoethanolamine-dimethyl polyethylene glycol (distearoylphosphoethanolamine (DSPE-mPEG)) is mixed with and reacted with the liposomes to prepare pegylated liposomes, encapsulated model drug chromoline, and effectively to pancreatic cancer cells. The present invention has been completed by confirming delivery.

It is an object of the present invention to provide chromoline containing PEGylated liposomes.

Another object of the present invention is to provide a method for preparing chromoline-containing PEGylated liposomes.

In order to achieve the object of the present invention, the present invention is composed of DPPC, DMPC, DSPC and DSPE-mPEG, to provide a chromoline-containing PEGylated liposomes containing chromoline.

The present invention also provides a method for preparing the chromoline-containing PEGylated liposome.

The present invention relates to chromoline-containing PEGylated liposomes and a method for preparing the same, and more particularly, to pegylated liposomes containing chromoline and a method for preparing the same. It is about. The chromoline-containing PEGylated liposomes of the present invention have the effect of preventing destruction by phagocytic cells of RES by PEGylation of liposomes, thereby increasing the residence time in the blood to efficiently release the drug at the site of pancreatic cancer. Pancreatic cancer can be prevented. In addition, synergistic effects when administered in combination with gemcitabine may contribute to the prevention of pancreatic cancer.

Hereinafter, the present invention will be described in detail.

As used herein, a "liposome" is a phospholipid bilayer capable of encapsulating an active drug. In addition, the "PEGylated" of the present invention means that the liposome is surface-bound methoxypolyethylene glycol ( mPEG).

The chromoline, chromoline sodium salt and gemcitabine used in the present invention may be commercially available, and each chemical formula is as follows.

[Formula 1]

[Formula 2]

(3)

The chromoline-containing PEGylated liposomes according to the present invention are phospholipids and distearoylphosphoethanolamine-methoxy of dipalmitoylphosphatidylcholine (DPPC), dimyltoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC) It consists of polyethylene glycol (DSPE-mPEG), which is provided in a form in which chromoline is enclosed.

The molar ratio of the components constituting the chromoline-containing PEGylated liposome according to the present invention is preferably DPPC: DMPC: DSPC: DSPE-mPEG = 4: 1: 1: 0.1-0.3.

MPEG constituting the liposome according to the present invention can be used commercially available, the PEG molecular weight is preferably 2,000 to 20,000. In general, when PEGylating liposomes, the effect of preventing destruction by phagocytic cells of RES has the advantage of increasing the residence time in the blood, thereby resulting in an increase in the size of liposomes. In particular, the chromoline-containing PEGylated liposomes of the present invention are preferably administered intravenously. When the molecular weight is 20,000 or more, PEG is more likely to be released into the body through the kidneys without being degraded in the body. Since the long-circulating effect is known to be lowered, it is preferable that the molecular weight of PEG is 2,000-20,000.

The chromoline containing PEGylated liposomes according to the present invention prevent and treat pancreatic cancer.

In addition,

Dissolving DPPC, DMPC, DSPC and DSPE-mPEG in chloroform and concentrating under reduced pressure to prepare a phospholipid film (step 1);

Adding hydrated diethyl ether to the prepared film, stirring, and adding and stirring chromoline to prepare a mixed solution (step 2); And

It provides a method for producing a chromoline-containing PEGylated liposomes comprising the step of sonicating the mixture, concentration under reduced pressure and separating using a column (step 3).

Step 1 is a step of preparing a phospholipid film.

In step 1, DPPC, DMPC, DSPC, and DSPE-mPEG are preferably mixed in a molar ratio of 4: 1: 1: 0.1 to 0.3 and dissolved in chloroform. The components may be dissolved in chloroform and concentrated under reduced pressure using a rotary concentrated evaporator to prepare a phosphorous lipid film.

On the other hand, mPEG of step 1 is preferably a molecular weight of 2,000 to 20,000 PEG.

Step 2 of the present invention is a step of encapsulating chromoline in the prepared phospholipid film. Hydrated diethyl ether was added to the phospholipid film prepared in Step 1, followed by stirring, followed by addition of a chromoline solution dissolved in phosphate buffered saline to prepare a mixed solution.

Step 3 of the present invention is a step of separating the liposomes containing (containing) the chromoline. It is preferable to use a 1 × 12 cm Sephadex G-75 column after sonicating the mixed solution of step 2, concentrating under reduced pressure.

The present invention provides a method for preventing and treating pancreatic cancer in which chromoline-containing PEGylated liposomes are administered in combination with gemcitabine.

In particular, the gemcitabine is preferably used in combination with chromoline in pegylated liposomes, or in the form of pegylated liposomes containing gemcitabine independently.

The present invention also provides an intravenous infusion formulation comprising the chromoline-containing PEGylated liposome as an active ingredient.

The chromoline-containing pegylated liposomes according to the present invention greatly reduce the cell survival rate to 20% or less when used at a concentration of 1000 μM against BxPC-3, a pancreatic cancer cell line expressing S100P. (See FIG. 5B).

In addition, the effect of inhibiting cell proliferation was increased when encapsulated in pegylated liposomes compared to chromoline itself not encapsulated in liposomes (see FIG. 6).

Furthermore, the pegylated liposomes encapsulated with chromoline and gemcitabine were confirmed to have superior cell proliferation inhibitory effect in comparison with each pegylated liposome in the BxPC-3 cell line (see FIG. 7).

Therefore, in the case of chromoline PEGylated liposome of the present invention, the chromoline increases the retention time of chromoline in the blood to maintain the anti-cancer activity of chromoline, and thus can be effectively used for the prevention and treatment of pancreatic cancer. .

The chromoline used in the present invention may be in the form of chromoline itself or a pharmaceutically acceptable salt thereof, and acid salts formed by the pharmaceutically acceptable free acid are useful as salts. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodide. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesul Nate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1- Sulfonates, naphthalene-2-sulfonates or mandelate.

The acid addition salts according to the invention are dissolved in conventional methods, for example, by dissolving chromoline in an excess of aqueous acid solution, and using the water miscible organic solvent, for example methanol, ethanol, acetone or acetonitrile. Can be prepared.

The same amount of chromoline and acid or alcohol in water can be heated and then the mixture is evaporated to dryness or prepared by suction filtration of the precipitated salt.

In addition, bases can be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg, silver nitrate).

The composition may be used as a pharmaceutical composition, and may be various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention may be administered parenterally or orally as desired, and may be administered in one to several times so as to be administered in an amount of 0.1 to 500 mg, preferably 1 to 100 mg per kg of body weight per day. have. The dosage for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples. However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited by the following examples.

Preparation of Reagents, Medium and Cell Lines

Reagents used to prepare pegylated chromoline containing liposomes in this example include DPPC (1,2-dipalmitoyl-rec-glycero-3-phosphocholine), DMPC (1,2-dimyri). Stole-rack-glycero-3-phosphocholine), DL-α-phosphatidylcholine, DSPC (distearoylphosphatidylcholine) were purchased from Sigma Chemical Co .; St. Louis MO, USA, and DSPE- mPEG ₂₀₀₀ (1,2- Distaroyl - sn -glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] is an Avanti Polar Lipids; Alabaster, AL, USA model was purchased from Sigma chromoline sodium salt Chromoline sodium salt was purchased from Sigma as a white powder in a glass vial and stored at room temperature. For the in vitro experiments, a solution was prepared at a concentration of 1 mM in the culture medium used. Lin had the formula is shown in Figure 1, sodium salt.

MTT (3- [4,5-dimethyl thiazol-2-yl] -2,5-diphenyltetrazolium bromide), DMSO (dimethylsulfoxide), chloroform (CDCl3), trypan blue, sepa Dex G-75 columns and the like were purchased from Sigma. In addition, the reagents and solvents used in the experiment were used the first-class and premium reagents.

DMEM (Dulbecco's modified eagle medium), RPMI 1640 medium (RPMI 1640), fetal bovine serum (FBS), trypsin-EDTA, penicillin / streptomycin and the like were purchased from Gibco BRL (Grand Island, NY, USA). BxPC-3 was distributed by the American Type Culture Collection (Manassas, VA, USA) and Panc-1 was distributed by the Korea Cell Line Bank (KCLB, Seoul, Korea).

BxPC-3 cells were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS), and Panc-1 cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% FBS. All cells were maintained in 5% carbon dioxide and 37 ° C. incubator.

Example  One: Chromoline  Containing Pegylated Liposome  Produce

PEGylated chromoline containing liposomes were prepared by reverse-phase-evaporation vesicle (REV).

A mixture was prepared by dissolving DPPC: DMPC: DSPC: DSPE-mPEG ₂₀₀₀ in 1 mL chloroform in a molar ratio of 4: 1: 1: 0.1. In order to remove chloroform from the mixed solution, a phospholipid film was prepared by concentrating under reduced pressure for 10 minutes with a rotary evaporator.

1 mL of hydrated diethyl ether (hydrated diethyl ether) was added to the thin phospholipid film prepared above, followed by stirring. Then, a chromoline solution dissolved in 1 mL of phosphate buffered saline (PBS) was added and stirred to prepare a mixed solution. .

Ultrasonic wave (Sonics & Materials Inc. Vibra CellTM, U.S.A) at 20 kHz intensity was added for 3 minutes while the mixed solution was cold-boiled. After the end of the process ether was added and concentrated under reduced pressure using a rotary concentrated evaporator. Chromoline-sealed liposomes and unsealed liposomes were separated using a 1 × 12 cm Sephadex G-75 column (Sigma, St. Louis MO, USA).

Example  2 ~ 5: PEG  Varying in content Chromoline  Containing Pegylated Liposome  Produce

Instead of mixing the DPPC: DMPC: DSPC: DSPE-mPEG ₂₀₀₀ of Example 1 in a molar ratio of 4: 1: 1: 0.1, DSPE-mPEG ₂₀₀₀ was respectively 0.2 (Example 2) and 0.3 (Example 3). Pegylated chromoline-containing liposomes were prepared in the same manner except mixing at a molar ratio of 0.5 (Example 4) and 1.0 (Example 5).

Experimental Example  One: Chromoline  Containing Pegylated Liposome Encapsulation efficiency  Measure

The calibraion of FIG. 1 shown by measuring the absorbance of chromoline released from the chromoline of chromoline containing pegylated liposomes prepared by varying the PEG content in Examples 1 to 5 using an ultraviolet spectrophotometer. Substituted in the curve was calculated by the following equation.

Encapsulation Efficiency (%) = C _f / C _i x 100

Here, C _f is the concentration of the drug measured after chromoline-encapsulated liposomes are added Triton X-100 (10% v / v) and then cultured at 55 ° C. to completely destroy the structure of liposomes, and C _i Is the concentration of chromoline initially added to enclose chromoline in liposomes. Here, chromoline with an initial concentration of 10 mM was encapsulated.

The measurement results are shown in Table 1.

Encapsulation Efficiency of Chromoline-Containing PEGylated Liposomes According to the Ratio of PEG 10 mM chromoline encapsulation efficiency Chromoline containing PEGylated liposomes = DPPC: DMPC: DSPC: DSPE-mPEG2000 Molar ratio Absorbance (A) Dilution factor Chromoline Conc. (MM) Encapsulation Efficiency (%) 4: 1: 1: 0.1 0.12 100 2.804 28.04 4: 1: 1: 0.2 0.114 100 2.664 26.64 4: 1: 1: 0.3 0.102 100 2.383 23.83 4: 1: 1: 0.5 0.061 100 1.426 14.26 4: 1: 1: 1.0 0.033 100 0.777 7.77

As shown in Table 1, the DSPE-mPEG ₂₀₀₀ has a good sealing efficiency when the molar ratio of 0.1 ~ 0.3, in particular, it was confirmed that the highest sealing efficiency in the case of 0.1.

In order to examine the accuracy of the measured value of the chromoline encapsulation efficiency, the absorbance of the drug according to the concentration of chromoline was measured. The chromoline was adjusted from 0 to 0.25 mM and the absorbance was measured at 295 nm. The measurement results are shown in FIG. 1.

As can be seen in Figure 1, it showed a linearity between the concentration of chromoline and the absorbance. From the above results, it can be seen that the efficiency measurement of the chromoline-containing PEGylated liposome according to the present invention was accurately measured.

Experimental Example 2: Confirmation of S100P Expression in Panc-1 and BxPC-3 Cell Lines

Before examining the anticancer activity of the chromoline-containing PEGylated liposomes according to the present invention by inhibiting S100P against pancreatic cancer cell lines, the expression of S100P of Panc-1 and BxPC-3 cell lines was confirmed.

Panc-1 and BxPC-3 cells, pancreatic cancer cell lines, were washed with PBS for RT-PCR, and total RNA was isolated by treatment with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). After quantifying the isolated RNA, 2D Reaction Mix, sense primer (10μM), antisense primer (10μM), SuperScriptTM III RT / PlatiumTaq Mix (Invitrogen), DEPC water were added to the template RNA, and cDNA synthesis and polymerization chain were added. Amplification by the reaction (PCR) method.

The sequence of Human S100P (Genebank accession no. X65614) primer was prepared as follows.

Forward: 5'-ATGACGGAACTAGAGACAGCCATGGGG-3 '

Reverse: 5'-GGAATCTGTGACATCTCCAGCGCATCA-3 '

And, the sequence of the ß-actin (Genebank accession no. BC016045) primer was prepared as follows. ß-actin was used as a positive control as a house keeping gene.

Forward: 5-ATGATATCGCCGCGCTCGTCGTC-3 '

Reverse: 5'-CGCTCGGCCGTGGTGGTGAA-3 '

After electrophoresis under 100 V using a 1.5% agarose gel, it was stained with ethidium bromide (EtBr) solution to confirm S100P expression by UV-transiluminator.

The result of expression through agarose gel is shown in FIG. 2. As shown in FIG. 2, while S100P expression was hardly expressed in Panc-1 cells, S100P was expressed in BxPC-3 cells.

Experimental Example  3: Chromoline  And Chromoline  Containing Pegylated Liposome  Cytotoxicity Measurement

According to concentration Chromoline  Cytotoxicity

In order to determine the effect on the cell proliferation according to the concentration of chromoline in Panc-1 cells and BxPC-3 cells, it was confirmed using MTT analysis.

Panc-1 cells (2.5 × 10 ³ / well) and BxPC-3 cells (2.5 × 10 ³ / well) were exposed to PBS (control), chromoline, pegylated chromoline containing liposomes for 24 hours, and 20 μL MTT solution (5 mg / mL PBS) was added to the cells and further incubated for 4 hours at 37 ° C., 5% carbon dioxide incubator. The MTT containing medium was aspirated off and the crystal formazan formed by dissolving 100 μL of DMSO. Absorbance was measured at 570 nm using an ELISA Reader (Dynatech MR5000, Dynatech Laboratories, USA).

The survival rate (%) of the cells was calculated by the following formula.

               [OD570 (sample)-OD570 (original)]

Survival Rate (%) = ----------------------------------------- X 100

               [OD570 (control)-OD570 (original)]

The measurement results are shown in Figure 3, on the left shows the effect of chromoline on Panc-1, and on the right shows the effect of chromoline on BxPC-3. As can be seen in Figure 3, IC ₅₀ in Panc-1 cells and 300 μM, the IC ₅₀ in the BxPC-3 cells was confirmed that 100 μM.

According to processing time Chromoline  Cytotoxicity

To determine the effect of chromoline concentration and treatment time on cell proliferation in Panc-1 cells and BxPC-3 cells, it was confirmed using MTT assay. The experimental method was performed in the same manner as the experimental method for examining the effect of chromoline according to the concentration.

The measurement results are shown in Figure 4, on the left shows the effect of chromoline on Panc-1, and on the right shows the effect of chromoline on BxPC-3. As can be seen in Figure 4, after 24 hours, the Panc-1 cells did not change significantly when treated with 0.1 to 100 μM but the cell viability at 1000 μM significantly decreased to 20% or less, while BxPC-3 cells In E. coli it was confirmed that the cell viability at 0.1 to 1000 μM gradually decreased in a concentration-dependent manner.

Chromoline  And Chromoline  contain Pegylated Liposome  Cytotoxicity Comparison

In order to compare the effect on the cell proliferation of chromoline and chromoline containing PEGylated liposomes in Panc-1 cells and BxPC-3 cells, MTT assay was performed in the same manner as the above methods.

The measurement results are shown in FIG. 5. As can be seen in FIG. 5, it was confirmed that chromoline-containing pegylated liposomes in BxPC-3 cells were superior to Panc-1 in inhibiting cell proliferation. In addition, the effect was increased when the chromoline-containing pegylated liposomes were treated than when treated with chromoline alone.

Experimental Example  4: Chromoline  Containing Pegylated Liposomes and Gemcitabine  Containing Pegylated Liposome   Cell proliferation inhibitory effect by simultaneous administration

In order to examine the effects of the combination of chromoline and gemcitabine, pegylated liposomes containing a combination of chromoline and gemcitabine were prepared in Panc-1 cells and BxPC-3 cells, and the cells were subjected to MTT analysis of the liposomes. The effect on proliferation was examined.

Panc-1 cells (2.5 × 10 ³ / well) and BxPC-3 cells (2.5 × 10 ³ / well) were treated with PBS, pegylated liposomes containing chromoline, and pegylated gemcitabine for 24 hours, respectively. Pegylated liposomes containing liposomes and combinations thereof were exposed and 20 μL of MTT solution (5 mg / mL PBS) was added to the cells and incubated for another 4 hours in a 37 ° C., 5% carbon dioxide incubator. The MTT containing medium was aspirated off and 100 μl of DMSO was added to dissolve the crystal formazan formed. Absorbance was measured at 570 nm using an ELISA Reader.

The survival rate (%) of the cells was calculated by the following formula.

                 [OD570 (sample)-OD570 (original)]

Survival Rate (%) = -------------------------------------- X 100

                 [OD570 (control)-OD570 (original)]

The measurement results are shown in FIG. 6. As can be seen in FIG. 6, pegylated liposomes that have been combined with chromoline and gemcitabine together when treated with chromoline-containing pegylated liposomes and gemcitabine-containing pegylated liposomes alone in BxPC-3 Cell proliferation inhibitory effect was much superior. In addition, the effect of the compositions on BxPC-3 was much better than Panc-1.

From this, chromoline-containing pegylated liposomes have an effect of inhibiting cancer proliferation in pancreatic cancer cell lines expressing S100P protein, and in the case of pegylated liposomes sealed after combining chromoline and gemcitabine, Since the synergistic effect of inhibiting growth is significantly superior to the treatment alone, the PEGylated liposomes of the present invention can be used as a useful cancer prevention and treatment composition.

Hereinafter, examples of the pharmaceutical preparation containing the chromoline-containing liposome of the present invention will be described.

Formulation Example 1 Preparation of Pharmaceutical Formulation

1-1. Manufacture of powder

2 g of pegylated liposomes containing chromoline

1 g lactose

After mixing the above components, the airtight cloth was filled to prepare a powder.

1-2. Manufacture of tablets

100 mg pegylated liposome containing chromoline

100 mg corn starch

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

1-3. Preparation of Capsules

100 mg pegylated liposome containing chromoline

100 mg corn starch

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

1-4. Preparation of Injection

10 μg / ml pegylated liposome containing chromoline

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

The chromoline-containing pegylated liposomes prepared in Example 1 were passed through 10 to 20 times through a 0.1-20 μm polycarbonate membrane using a Repex extrusion device (Avestin Inc. Toronto, Canada) to maintain a constant size. It was. The chromoline containing pegylated liposomes are dissolved in a suitable volume of sodium chloride BP for injection, the resulting solution is adjusted to pH 3.5 with dilute hydrochloric acid BP, and the volume is adjusted with sodium chloride BP for injection and sufficiently Mixed. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

1 is a graph measuring absorbance according to the concentration of chromoline.

Figure 2 is an agarose gel picture showing the expression of S100P mRNA in panc-1 and BxPC-3 pancreatic cancer cell lines.

3 is a graph showing cell viability according to the concentration of chromoline in panc-1 (left) and BxPC-3 (right) pancreatic cancer cell lines.

Figure 4 is a graph showing the cell survival rate according to the concentration and the treatment time of chromoline in panc-1 (left) and BxPC-3 (right) pancreatic cancer cell lines.

FIG. 5 is a graph showing the cell survival rate of pegylated liposomes containing chromoline and chromoline in panc-1 and BxPC-3, which are pancreatic cancer cell lines.

FIG. 6 is a comparison of cell viability by chromoline-containing pegylated liposomes, gemcitabine-containing pegylated liposomes, and pegylated liposomes containing chromoline and gemcitabine simultaneously in panc-1 and BxPC-3 pancreatic cancer cell lines. It is shown.

Claims (11)

It consists of phospholipids and distearoylphosphoethanolamine-methoxypolyethylene glycol (DSPE-mPEG) of dipalmitoylphosphatidylcholine (DPPC), dimyltoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC). Cromoline-containing PEGylated liposomes for preventing and treating pancreatic cancer, characterized in that chromoline is encapsulated. The chromoline-containing PEGylated liposome according to claim 1, wherein the mixing ratio of DPPC, DMPC, DSPC and DSPE-mPEG is 4: 1: 1: 0.1-0.3 in molar ratio. The chromoline-containing PEGylated liposome according to claim 1, wherein the mPEG has a molecular weight of 2,000 to 20,000 PEG. delete Dissolving DPPC, DMPC, DSPC and DSPE-mPEG in chloroform and concentrating under reduced pressure to prepare a phospholipid film (step 1); And Adding hydrated diethyl ether to the prepared film, stirring, and adding and stirring chromoline to prepare a mixed solution (step 2); And The method for producing a chromoline-containing PEGylated liposome according to claim 1, comprising the step of sonicating the mixed solution, concentrating under reduced pressure and separating using a column (step 3). The method of claim 5, wherein the mixing ratio of DPPC, DMPC, DSPC, and DSPE-mPEG in step 1 is 4: 1: 1: 0.1-0.3 in molar ratio. The method of claim 5, wherein the mPEG of step 1 is a method for producing chromoline-containing PEGylated liposomes, characterized in that the molecular weight of PEG 2,000 to 20,000. delete delete delete An intravenous injectable formulation comprising the chromoline-containing PEGylated liposome of any one of claims 1 to 3 as an active ingredient.

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