KR101971451B1 - Use of 2-hydroxyoleic acid incorporated in lipid vehicle - Google Patents

Abstract

The present invention relates to the use of a lipid vehicle containing 2-hydroxy oleic acid, and more particularly, by inserting 2-hydroxy oleic acid or a pharmaceutically acceptable salt thereof into the lipid membrane of liposomes, such as cancer, obesity, diabetes, etc. It enhances the therapeutic effect of the disease, and provides the effect of using as a drug delivery agent for delivery to the body by including a pharmaceutically active ingredient between the inside and the membrane of the liposome.

Description

２―Use of 2-hydroxyoleic acid incorporated in lipid vehicle}

The present invention relates to the use of lipid vehicles with 2-hydroxyoleic acid.

Because cancer cells are in a state where fatty acid biosynthesis is constantly activated, membrane lipid composition such as fatty acids and phospholipids is different from normal cells, and the change of membrane composition is known to be related to the progression of cancer (Azordegan et al. Mol . Cell Biol. Vol. 374, pp . 223-232, 2013).

Sphingomyelin in cell membrane lipids is biosynthesized with diacylglycerol by delivering a head group of phosphatidylcholine to ceramide, as shown in Equation 1 below, and this synthesis process is sphingomyelin synthase present in intracellular Golgi apparatus or cell membrane. (sphingomyelin synthase, SGMS) is mediated by:

[Equation 1]

Phosphatidylcholine + Ceramide → Sphingomyelin + Diacylglycerol

Cancer cells have less sphingomyelin in cell membranes than normal cells, and activating SGMS to restore sphingomyelin levels in cell membranes to normal levels leads to cell cycle arrest and cell death due to changes in proteins that bind to cell membranes. Therefore, lipid composition control of cancer cell membrane has been suggested as a target of new cancer treatment (Llado et al. Biochim Biophys Acta , vol. 1838 (6), pp. 1619-27, 2014.06).

2-hydroxyoleic acid (or '2OHOA'), a synthetic derivative of oleic acid, is a potent anticancer drug that modulates membrane lipid composition and structure and important membrane protein functions. Reportedly, treatment of 2OHOA (or sodium salt of 2OHOA, NaCHOleate) into cells results in faster insertion of 2OHOA into cancer cell membranes than normal cells, and inserted 2OHOA causes changes in cell membrane lipid composition, membrane fluidity, and structure, Structural changes of activated SGMS promote SM synthesis and increase the level of SM in the cell membrane (Martin et al. Biochim Biophys Acta , vol. 1828 (5), pp. 1405-13, 2013.05). This change in membrane affects the distribution and activity of membrane-bound proteins, resulting in cell cycle arrest or cell death by disturbing cell cycle and apoptosis related signaling systems. Due to this cancer cell-specific induction of apoptosis, 2OHOA is a promising anticancer drug currently designated as a rare drug of glioma (Teres et al. PNAS vol. 109 (22), pp. 8489-8494). , 2012). However, the concentration of 2OHOA suggested in the existing literature, etc., requires a considerably high concentration of 2OHOA at least 100 mM, preferably 200 mM or more, even in experiments using cultured cancer cells.

On the other hand, liposomes are spherical double membrane structure spontaneously formed when dispersing phospholipids such as phosphatidylcholine phospholipids in aqueous solution. Due to the structural properties of liposomes, the aqueous phase is encapsulated with a water-soluble drug, and since a fat-soluble drug can be inserted between phospholipid molecules constituting the membrane, it has been developed as a transporter for various drugs. Incorporating anticancer drugs into nano-sized liposomes overcomes the disadvantages of drug solubility, such as low solubility or chemical instability, and adds enhanced permeablity and retention effects (Maeda). Increasing the concentration of anticancer drugs into the tissue can increase the therapeutic effect while reducing the side effects can be expected.

Since the phosphatidylcholine is the substrate of SGMS, when the substrate and 2OHOA are simultaneously loaded into the liposome-type structure and supplied to cancer cells, the concentration of phosphatidylcholine in the microenvironment is increased and the SM synthesis is further promoted as the 2OHOA is inserted into the cell membrane. It is expected that apoptosis effect can be enhanced, and 2OHOA is a substance that can be easily inserted into the liposome membrane due to its molecular structure. Thus, liposomes based on phosphatidylcholine and 2OHOA as auxiliary components are prepared and used as drug delivery agents. The present invention was completed by developing a 2OHOA insert liposome to be used.

 Azordegan et al. Mol. Cell Biol. Vol. 374, pp. 223-232, (2013)  Llado et al. Biochim Biophys Acta, vol. 1838 (6), pp. 1619-27 (2014.06)  Martin et al. Biochim Biophys Acta, vol. 1828 (5), pp.1405-13 (2013.05)  Teres et al. PNAS vol. 109 (22), pp. 8489-8494 (2012)

It is an object of the present invention to provide a liposome preparation comprising a liposome inserted with 2OHOA.

Another object of the present invention is to provide a drug carrier in which the pharmaceutically active ingredient is inserted or inserted into the liposome into which 2OHOA is inserted.

In order to achieve the above object, the present invention

2-hydroxyoleic acid or a pharmaceutically acceptable salt thereof; And liposomes containing phosphatidylcholine; And

Liposomal formulations comprising a pharmaceutically acceptable carrier are provided.

The invention also

2-hydroxyoleic acid or a pharmaceutically acceptable salt thereof; And liposomes containing phosphatidylcholine; And

Provided is a drug delivery agent comprising a pharmaceutically active ingredient.

The present invention can enhance the therapeutic effect on cancer from a low concentration of 2OHOA by inserting 2-hydroxyoleic acid in liposomes and delivering it to the living body, and the 2OHOA-inserted liposomes contain or insert a pharmaceutically active ingredient to It can be used as a drug carrier that can be delivered into.

Figure 1 illustrates a liposome inserted 2OHOA according to the present invention.
Figure 2 shows the DSC thermogram change of DPPC liposomes by insertion of 2OHOA.
Figure 3 shows the room temperature stability of the liposome inserted 2OHOA.
Figure 4 shows the anticancer activity change of 2OHOA by DMPC addition.
Figure 5 shows the anticancer activity of the liposome inserted 2OHOA.
Figure 6 shows the anticancer activity of the liposome inserted 2OHOA for each concentration of 2OHOA.
Figure 7 compares the cytotoxicity results in normal cells and cancer cells of the liposome inserted 2OHOA.
8 is a result confirming the anticancer activity of MTO mounted on the liposome inserted 2OHOA in non-small cell lung cancer cell line.
9 is a result confirming the anticancer activity of paclitaxel mounted on the liposome inserted 2OHOA in non-small cell lung cancer cell line.
10 is a result confirming the anticancer activity of retinoic acid mounted on the liposome inserted 2OHOA in melanoma B16-F10 culture cells.
11 is a result of confirming the anticancer activity of retinoic acid mounted on the 2OHOA-inserted liposome in the melanoma transplantation animal model, the arrow indicates the time of administration.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention relates to 2-hydroxyoleic acid or a pharmaceutically acceptable salt thereof; And liposomes containing phosphatidylcholine; And

A liposome preparation comprising a pharmaceutically acceptable carrier.

The liposome of the present invention may exhibit a structure in which 2-hydroxyoleic acid (hereinafter referred to as '2OHOA') or a pharmaceutically acceptable salt thereof is inserted into the double membrane of the liposome. More specifically, 2OHOA may be inserted into the double layer of phosphatidylcholine.

According to one embodiment of the present invention, when 2OHOA or a pharmaceutically acceptable salt thereof is inserted between phosphatidylcholine molecules, the intrinsic phase transition temperature of phosphatidylcholine is changed. Therefore, the phase transition temperature of phosphatidylcholine liposomes before and after insertion of 2OHOA or its pharmaceutically acceptable salts was measured by differential scanning calorimetry (DSC) to measure the change in calorie with temperature, and the peak of the phase transition temperature shifted and the calorie peak was measured. It can be seen that the size of is significantly reduced to increase the fluidity of the liposome membrane. That is, 2OHOA or a pharmaceutically acceptable salt thereof is sandwiched between phosphatidylcholine molecules (see FIG. 2). This increase in membrane fluidity may contribute to an increase in the loading efficiency of the mountable fat-soluble or amphiphilic anticancer agent by intercalating into the liposome membrane.

In addition, when 2OHOA or a pharmaceutically acceptable salt thereof is inserted between phosphatidylcholine molecules, the average particle diameter of the phosphatidylcholine liposome may be reduced by 4-5 times to 50 nm to 500 nm, more specifically 50 nm to 200 nm. The particle diameter may be suitable to exert an enhanced permeability & retention (EPR) effect that results in the accumulation of cancer sites (see Table 1).

In addition, when the surface charge measured by zeta potential is inserted between 2OHOA or a pharmaceutically acceptable salt thereof between phosphatidylcholine molecules, the content of 2OHOA or a pharmaceutically acceptable salt thereof increases from neutral to negative so that the phosphatidylcholine liposome is increased to 2OHOA or It can be seen that it is structurally stabilized by the insertion of a pharmaceutically acceptable salt thereof, and since it has a negative charge, it can be seen that 2OHOA or a pharmaceutically acceptable salt thereof is sandwiched between the membranes of liposomes.

Liposomes inserted between the 2OHOA or a pharmaceutically acceptable salt thereof between phosphatidylcholine molecules can be seen to have stability without change in average particle size even when stored at room temperature for one month (see FIG. 3).

In addition, as a result of examining the effect of phosphatidylcholine on the pharmaceutical effect of 2OHOA or its pharmaceutically acceptable salts, an increase in anticancer activity was confirmed (see FIG. 4). This synergistic effect is even more synergistic when 2OHOA or a pharmaceutically acceptable salt thereof is a liposome inserted between phosphatidylcholine molecules. This appears to be due to the insertion of 2OHOA or a pharmaceutically acceptable salt thereof between the phosphatidylcholine molecules, thereby making the entry into the cell membrane easier by liposomes, and the phosphatidylcholine being more useful for sphingomyelin synthase (SGMS). (See Figure 5).

Liposomes in which 2OHOA or a pharmaceutically acceptable salt thereof is inserted between phosphatidylcholine molecules are not toxic to normal cells, but maintain the apoptotic activity of 2OHOA or its pharmaceutically acceptable salt itself against cancer cells (FIG. 7).

As mentioned above, the liposome preparations of the present invention can enhance the therapeutic effect against cancer by enhancing the in vivo incorporation of 2OHOA or a pharmaceutically acceptable salt thereof. Therefore, the liposome preparation of the present invention can be used for the manufacture of anticancer drugs.

Pharmaceutically acceptable salts of 2OHOA include any soluble salt of pharmaceutically acceptable organic or inorganic base. Typical examples of pharmaceutically acceptable inorganic bases are hydroxides, carbonates and hydrogen carbonates of ammonia, calcium, magnesium, sodium and potassium, such as sodium hydroxide, ammonium hydroxide, calcium hydroxide, magnesium carbonate, sodium bicarbonate and potassium hydrogen carbonate. Typical examples of pharmaceutically acceptable organic bases are arginine, betaine, caffeine, choline, N, N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine , Ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, N-methylglucamine, glucamine, glucosamine, histidine, N- (2-hydroxyethyl) piperidine, N- (2-hydroxyethyl ) Pyrrolidine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, theobromine, triethylamine, trimethylamine, tripropylamine and tromethamine.

More specifically, pharmaceutically acceptable salts of 2OHOA are sodium, potassium, ammonium, pyrrolidine, piperidine, N-hydroxyethylpyrrolidone, N-hydroxyethylpiperidine, triethanolamine of 2OHOA. , Diethanolamine, ethylenediamine or diethylamine salt, and the like, but is not limited thereto.

The content of the 2OHOA or a pharmaceutically acceptable salt thereof in the liposome may be 10 to 50 mol%. If it is out of the above range, it may be difficult to expect the anticancer effect of 2OHOA after administration of 2OHOA content is less than or less than the phosphatidylcholine content of the liposomes may be difficult to form a stable liposomes.

The 2-hydroxyoleic acid or a pharmaceutically acceptable salt thereof may be included in a molar ratio of 1: 0.1 to 1 relative to phosphatidylcholine. If it is out of the above range, it may be difficult to form a stable liposome or the anti-cancer effect of 2OHOA due to the low content of 2OHOA.

The phosphatidylcholine is 1,2-di-myristoyl--sn- glycero-3-phosphocholine (1,2-Dimyristoyl-sn-Glycero -3-Phosphocholine, DMPC), 1,2- dipek Sano days - sn - Glycero-3-phosphocholine (1,2-dihexanoyl- sn -glycero-3-phosphocholine, DHPC), 1,2-diheptanoyl- sn -glycero-3-phosphocholine (1,2-diheptanoyl sn -glycero-3-phosphocholine (DHPC), 1,2-dioctanoyl- sn -glycero-3-phosphocholine (1,2-dioctanoyl- sn -glycero-3-phosphocholine), 1,2- Dinonanoyl- sn -glycero-3-phosphocholine (1,2-dinonanoyl- sn -glycero-3-phosphocholine), 1,2-didecanoyl- sn -glycero-3-phosphocholine (1 , 2-didecanoyl- sn -glycero-3-phosphocholine), 1,2-dioundecanoyl- sn -glycero-3-phosphocholine (1,2-diundecanoyl- sn -glycero-3-phosphocholine), 1 , 2-dilauroyl- sn -glycero-3-phosphocholine (1,2-dilauroyl- sn -glycero-3-phosphocholine, DLPC), 1,2-ditridecanoyl-sn-glycero- 3-phosphocholine (1,2-ditridecanoyl- sn -glycero-3 -phosphocholine), 1,2- dipentaerythritol having Noil as -sn- glycero-3-phosphocholine (1,2-dipentadecanoyl- sn -glycero-3-phosphocholine), 3-phosphocholine (1,2 1,2-di-palmitoyl -sn- glycero -dipalmitoyl- sn -glycero-3-phosphocholine (DPPC), 1,2-diheptadecanoyl- sn -glycero-3-phosphocholine (1,2-diheptadecanoyl- sn -glycero-3-phosphocholine), 1 1,2-distearoyl- sn -glycero-3-phosphocholine (1,2-distearoyl- sn -glycero-3-phosphocholine, DSPC), 1,2-dinonadecanoyl-sn-glycero- 3-phosphocholine (1,2-dinonadecanoyl- sn -glycero-3-phosphocholine), 1,2-diarachidyl- sn -glycero-3-phosphocholine (1,2-diarachidoyl- sn -glycero -3-phosphocholine), 1,2-dihenarachidoyl- sn -glycero-3-phosphocholine (1,2-dihenarachidoyl- sn -glycero-3-phosphocholine), 1,2-dibenenoyl- sn-glycero-3-phosphocholine (1,2-dibehenoyl- sn -glycero-3-phosphocholine), 1,2-ditricosanoyl- sn -glycero-3-phosphocholine (1,2- ditricosanoyl- sn -glycero-3-phosphocholine), 1,2-dilignoceroyl- sn -gly Cerro-3-phosphocholine (1,2-dilignoceroyl- sn -glycero-3-phosphocholine), egg phosphatidylcholine, soy phosphatidylcholine, hydrogenated phosphatidylcholine (hydrogenated phosphatidylcholine), 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-Glycero-3-Phosphocholine), 1,2-dimyristooleoyl-sn- Glycero-3-phosphocholine (1,2-dimyristoleoyl- sn -glycero-3-phosphocholine), 1,2-dilinoleyl- sn -glycero-3-phosphocholine (1,2-dilinoleoyl- sn -glycero-3-phosphocholine), 1,2-diecosenoyl- sn -glycero-3-phosphocholine (1,2-dieicosenoyl- sn -glycero-3-phosphocholine), 1,2-dieru Coil-sn-glycero-3-phosphocholine (1,2-dierucoyl- sn -glycero-3-phosphocholine 1,2-didocosahexaenoyl- sn -glycero-3-phosphocholine (1,2 -didocosahexaenoyl- sn -glycero-3-phosphocholine), or 1,2-diecosahexaenoyl- sn -glycero-3-phosphocholine (1,2-dieicosahexaenoyl- sn -glycero-3-phosphocholine) More specifically, phosphatidylcholine may be used as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (1,2-Dimyristoyl-sn-Glycero-3-Phosp). hocholine, DMPC).

The content of phosphatidylcholine in liposomes may be from 50 to 90 mole percent. If it is out of the above range, it may be difficult to form a stable liposome or the anti-cancer effect of 2OHOA due to the low content of 2OHOA.

The lipid vehicle of the above structure may be a liposome prepared by mixing phosphatidylcholine and 2OHOA or a pharmaceutically acceptable salt thereof in a molar ratio of 1: 0.1 to 1. If the molar ratio is outside, it may be difficult to form stable liposomes or the anticancer effect of 2OHOA due to the low content of 2OHOA.

The liposomes may employ well-known liposome manufacturing techniques and are not particularly limited. According to one embodiment of the present invention, phosphatidylcholine and 2OHOA or pharmaceutically acceptable salts thereof are mixed in an organic solvent in a molar ratio, and freeze-dried or vacuum distillation to remove the organic solvent, followed by hydration and sonication. Alternatively, liposomes having a pharmaceutically acceptable salt thereof inserted into the lipid bilayer may be obtained.

The organic solvent may be ethanol, 1-propanol, glycerol, propylene glycol, 1,3-butylene glycol, tertiary butyl alcohol, chloroform or a mixture thereof, but is not limited thereto.

Suitable dosages of the liposome formulations of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to the patient. Can be. Preferably, the subject may be administered to a subject at a dose of 10 to 600 mg / kg / day, more specifically at a dose of 20 to 100 mg / kg / day.

In addition, the liposome preparation of the present invention may have a formulation for intranasal administration, intravenous administration, subcutaneous injection, intrathecal injection, inhalation administration or oral administration, but is not particularly limited thereto.

The invention also

2-hydroxyoleic acid or a pharmaceutically acceptable salt thereof; And liposomes containing phosphatidylcholine; And

It relates to a drug carrier comprising a pharmaceutically active ingredient.

The liposome of the present invention has a structure in which 2OHOA or a pharmaceutically acceptable salt thereof is inserted into the double membrane of the liposome, and the liposome has an aqueous phase portion and a lipid double membrane, so that the aqueous phase contains a water-soluble drug, Since fat-soluble drugs can be inserted between the phospholipid molecules forming the membrane, they can be used as drug carriers.

Thus, the liposomes of the present invention can be delivered to the body by encapsulating the pharmaceutically active ingredient in the inner aqueous phase or inserted into the membrane.

The pharmaceutically active ingredient may be inserted or inserted in the preparation of liposomes. According to one embodiment, the drug is added to the hydration solution in the process of lyophilizing and hydrating a mixture of phosphatidylcholine and 2OHOA or a pharmaceutically acceptable salt thereof, or lyophilizing and hydrating a mixture of phosphatidylcholine and 2OHOA or a pharmaceutically acceptable salt thereof Drugs can be enclosed or inserted by the addition of liposomes to form liposomes.

The pharmaceutical active ingredient may be an anticancer chemotherapeutic agent. Examples of the anticancer chemotherapeutic agents include paclitaxel, docetaxel, cisplatin, carboplatin, oxaliplatin, doxorubicin, daunorubicin, epirubicin Epirubicin, idarubicin, valubicin, mitoxantrone, gefitinib, erlotinib, irinotecan, irinotecan, topotecan, Vinblastine, vincristine, or retinoic acid may be used, but is not particularly limited thereto.

According to one embodiment of the present invention, the anticancer drug mitoxantrone (MTO) is dissolved in an organic solvent together with phosphatidylcholine and 2OHOA or a pharmaceutically acceptable salt thereof, and the MTO is mounted by lyophilization and post-hydration sonication. Liposomes can be prepared. As a result of measuring the loading efficiency of MTO, phosphatidylcholine liposome was confirmed to mount about 3.8% of MTO, but phosphatidylcholine liposome inserted with 2OHOA or a pharmaceutically acceptable salt thereof was able to confirm 100% MTO loading. It was confirmed that the mounting efficiency of the active ingredient is improved. This indicates that the absolute value of the negative charge of the surface charge of the phosphatidylcholine liposome inserted with 2OHOA or its pharmaceutically acceptable salt is greatly reduced by the MTO loading, which is probably due to the electrostatic attraction of MTO and 2OHOA (see Table 2). .

According to another embodiment of the present invention, the DOPC / 2OHOA liposome loaded with paclitaxel, an anticancer agent, exhibits strong anticancer activity against non-small cell lung cancer cell lines, and at a concentration that inhibits the growth of cancer cells by 50%, paclitaxel is 12 nM of MTO. It shows similar anticancer activity at a low concentration of 8 nM compared to (see FIG. 9).

According to another embodiment of the present invention, the DMPC / cholesterol / 2OHOA liposome loaded with retinoic acid, an anticancer agent, was loaded on 90-95% liposomes with retinoic acid, showed strong anticancer activity against malignant melanoma cell line, and did not have 2OHOA. Retinoic acid loaded on liposomes in which 2OHOA was inserted compared to retinoic acid loaded on showed similar anticancer activity at a concentration about 10 times lower (see FIG. 10). The retinoic acid-loaded DMPC / cholesterol / 2OHOA liposomes had similar anticancer effects in the retinoic acid-treated group and 2OHOA-treated group treated with 2OHOA-inserted liposomes in the melanoma mouse model. The retinoic acid treated group mounted on the liposome inserted with 2OHOA showed a remarkably excellent anticancer effect (see FIG. 11). This suggests that the 2OHOA-inserted liposome has an excellent anticancer drug delivery ability, and thus shows an excellent anticancer effect even at a low concentration of anticancer drugs, and also improves the anticancer effect of the same amount of anticancer drugs.

Liposomal formulations or drug delivery agents of the invention may comprise a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers include carriers and vehicles conventionally used in the pharmaceutical arts, and specifically include ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer materials (eg, various Phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, hydrogen carbonate, sodium chloride and zinc salts), colloidal silica , Magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrates, polyethylene glycols, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene glycols or wool, and the like.

In addition, the liposome preparation or drug delivery agent of the present invention may further include a lubricant, a humectant, an emulsifier, a suspension, or a preservative in addition to the above components.

In one embodiment, the liposome preparation or drug delivery agent according to the present invention can be prepared in an aqueous solution for parenteral administration, preferably Hank's solution, Ringer's solution or physically buffered saline. The same buffer solution can be used. Aqueous injection suspensions can be added with a substrate that can increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

Other preferred embodiments of the liposome preparations or drug delivery agents of the invention may be in the form of sterile injectable preparations of sterile injectable aqueous or oily suspensions. Such suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (eg Tween 80) and suspending agents.

In addition, the sterile injectable preparation may be a sterile injectable solution or suspension (eg, a solution in 1,3-butanediol) in a nontoxic parenterally acceptable diluent or solvent. Vehicles and solvents that may be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally employed as a solvent or suspending medium. For this purpose any non-irritating non-volatile oil can be used including synthetic mono or diglycerides.

Suitable dosages of the drug delivery agents of the present invention may be prescribed in various ways by factors such as formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to the patient. Can be.

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

Example 1 Preparation of 2OHOA Inserted Liposomes

After dissolving 16 μmole of phosphatidylcholine and 0-16 μmole of 2OHOA (2-hydroxyoleate type, 2OHOA sodium salt) in tertiary butyl alcohol, the solution was rapidly frozen at minus 80 ° C and freeze-dried in a freeze dryer. After lyophilization for 24 hours, 1 mL of 5% dextrose solution was hydrated to the obtained lipid powder. The temperature of the dispersion during hydration was maintained above the phase transition temperature (DMPC 23 ℃, DPPC 43 ℃, DSPC 56 ℃) of the phosphatidylcholine used. Liposome dispersion spontaneously formed by hydration by stirring at 3,000 rpm for 30 seconds was pulverized and homogenized at 130 ° C. for 30 minutes in a water bath type ultrasonic dispersion machine at 37 ° C. for 30 minutes, and then ultrasonic wave of 250 W intensity for 7 minutes in a water tank type cell dispersing ultrasonic dispersion machine. Treatment gave 2OHOA intercalated liposomes.

Experimental Example 1 Confirmation of 2OHOA Insertion in Liposomes

When 2OHOA is inserted between phosphatidylcholine molecules of the liposome membrane, the phase transition temperature inherent in phosphatidylcholine is changed. Therefore, two liposomes were prepared from the solution of 16 μmole of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) alone or 1.8 μmole of 2OHO in a 9: 1 molar ratio by the method of Example 1. The phase transition temperature of phosphatidylcholine liposomes before and after 2OHOA insertion was measured by differential scanning calorimetry. Approximately 5 mg of the prepared liposome is placed on a standard aluminum pan of a Q2000 differential scanning calorimeter (TA Instruments, USA) and heated at a constant rate from 10 ° C to 70 ° C under a nitrogen atmosphere to measure calorie change with temperature. The graph was obtained.

As shown in Figure 2, the liposome consisting of only DPPC was observed a caloric peak in which DPPC is converted from the gel state to the liquid crystalline (liquid crystalline) at about 41 ℃, but in the case of liposomes prepared with DPPC and 2OHOA mixture of the phase transition temperature The peak shifted to about 39 ° C. and the magnitude of the calorie peak also decreased significantly. These results indicate that 2OHOA molecules are intercalated between DPPC molecules, thereby increasing the fluidity of the liposome membrane by reducing the attraction between DPPC molecules.

Experimental Example 2 Analysis of 2OHOA Encapsulation Efficiency and Physicochemical Properties in Liposomes

DMPC was used as phosphatidylcholine, and 2OHOA-inserted liposomes were prepared at various initial DMPC: 2OHOA ratios, and uninserted 2OHOA was isolated using Amicon Ultra-4 Centrifugal filters (10,000NMWL, Millipore). That is, take 2 mL of the prepared liposome and place it in the upper part of the filter mounting device, and repeat the process of centrifugation at 25 ° C. at 3,000 g for 10 minutes twice, collecting the uninserted 2OHOA solution lowered under the filter, and absorbing at 205 nm with a spectrophotometer. The concentration of uninserted 2OHOA was measured by using a standard calibration curve of 2OHOA prepared in advance, and the concentration of inserted 2OHOA was obtained by subtracting the amount of uninserted 2OHOA from the amount of 2OHOA initially added when preparing liposomes.

The average particle size and particle size distribution of 2OHOA-inserted liposomes were measured by dynamic light scattering method using a fiber-optics particle analyzer (FPAR-1000, Otsuka Electronics, Japan). Samples were diluted 100-fold with filtered 5% dextrose solution before measurement. The surface charge of liposomes was evaluated by diluting each sample with deionized water and measuring by automatic measurement using a Zeta Potentiometer (Zen 600 zetasizer, Malvern, England).

As shown in Table 1, the rate of 2OHOA insertion into liposomes tended to increase as the initial 2OHOA content increased. When 2OHOA was 33% or more of total lipids, more than 95% were inserted into the liposome membrane.

In addition, when 2OHOA was inserted, the average particle diameter of liposomes was reduced by 4-5 times, and thus, the particle size (10 nm-100 nm) suitable for exerting the EPR effect of the cancer site accumulation effect by the nanoparticle loading of the anticancer agent was entered. The measured surface charge increased from neutral to negative 2OHOA content, indicating that the phosphatidylcholine structure liposomes were stabilized by 2OHOA insertion. In addition, it was confirmed once again that negatively charged 2OHOA was intercalated between the membranes and not inside the liposomes.

In addition, as shown in FIG. 3, the prepared 2OHOA-inserted liposomes were stable because there was no change in average particle diameter and the like when stored at room temperature for one month.

Effects of 2OHOA Insertion Efficiency, Average Particle Size and Polydispersity and Surface Charge on Liposomes According to Initial 2OHOA Content in Liposome Preparation Liposomal Composition (μmole / mL) 2OHOA insertion rate
(%) Average particle size
(nm) Polydispersity Zeta potential (mV) DMPC16 - 390 ± 93 0.231 ± 0.033 + 5.9 ± 2.1 DMPC: 2OHOA (9: 1) - - - -26.7 ± 1.7 DMPC: 2OHOA (16: 4) 79.00 81 ± 12 0.293 ± 0.034 -57.3 ± 2.1 DMPC: 2OHOA (16: 5.3) 91.10 90 ± 8 0.327 ± 0.073 -55.9 ± 3.5 DMPC: 2OHOA (16: 8) 95.90 75 ± 0 0.343 ± 0.026 -58.1 ± 1.6 DMPC: 2OHOA (16:16) 98.42 83 ± 14 0.247 ± 0.041 -61.0 ± 4.3

Experimental Example 3 Effect of Concomitant Phosphatidylcholine Solution on the Anticancer Activity of 2OHOA Solution

Although there is phosphatidylcholine that can act as a substrate of SGMS, the hypothesis that SGMS-mediated SM synthesis reaction may be promoted by increasing substrate concentration when phosphatidylcholine is supplied from the outside, thereby increasing apoptosis by SM synthesis. To this end, 0-240 μM DMPC ethanol solution was added together with 120 μM 2OHOA DMSO solution to SCC-1 cells and left for 72 hours in a 5% CO ₂ cell incubator at 37 ° C. for MTT analysis.

For MTT assay, remove the medium from the plate and wash the cells with PBS, then MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrasolium bromide) (0.25 g / 50 mL in PBS) The solution was diluted with DMEM medium so that the solution was 10%, added 0.1 mL each, and incubated for 4 hours. After incubation, walked the medium containing MTT, washed the cells with PBS, added 1 mL of DMSO, and repeatedly inhaled and ejected the DMSO solution with a pipette to allow formazan crystals to dissolve in the cells. Absorbance was measured at. Cell growth rate is expressed in% as compared to the case of treatment with the empty dextrose solution which does not contain 2OHOA.

As shown in FIG. 4, 2OHOA alone inhibited only about 10.8% of cell growth, but when DMPC was added, anticancer activity was increased depending on DMPC dose, and 37.2% was inhibited when 240 μM DMPC supplemented.

These results suggest that supplementation of DMPC promoted SM production by SGMS, resulting in more enhanced anticancer activity of 2OHOA.

Experimental Example 4 Evaluation of Anticancer Activity of Liposomes Inserted with 2OHOA

Head and neck cancer by diluting each of 2OHOA-inserted liposomes prepared by the method of Example 1 (DMPC: 2OHOA = 16: 8) or DMSO (dimethylsulfoxide) solution of free 2OHOA mixed / unmixed with free DMPC with a 5% dextrose solution After being left for 72 hours with the SCC-1 cell line, anticancer activity was measured by MTT assay.

As shown in FIG. 5, 120 μM 2OHOA alone reduced cell growth by 10.8% and 120 μM free 2OHOA + 240 μM free DMPC mixed solution reduced cell growth by 37.2% while containing 2OHOA and DMPC at the same concentration. Liposomes reduced cell growth by about 80%. This means that inserting 2OHOA into liposomes shows synergistic anticancer activity. This is because the inflow of 2OHOA into the cell membrane is made easier by liposomes, and for the same reason, the concentration of DMPC available in the microenvironment around the SGMS increases due to the liposomes.

Figure 6 shows the dose-cell death curves of 2OHOA solution and liposome insertion 2OHOA at various concentrations of 2OHOA. When oleic acid was inserted at the same ratio in liposomes, the anticancer activity was observed because it did not affect cell growth at the same concentration. It confirmed that it is by 2OHOA. In addition, it was confirmed that the growth inhibitory effect of cancer cells was observed at a lower concentration when the 2OHOA administered in the liposome insert type than when administered in the single solution type. That is, 2OHOA inhibited cell growth 4.6 times more effectively in the case of liposome insertion than in the case of single solution at a concentration of 120 μM. The concentration of 2OHOA, which inhibits the growth of cancer cells by 50%, was about 155 μM in the free form but was reduced to 100 μM in the liposome insertion type.

Experimental Example 5 Cytotoxicity Evaluation of 2OHOA Inserted Liposomes in Cultured Cell Lines

Human head and neck cancer UM-SCC-1 cell lines were inoculated at 2,000 per well. DMEM medium containing 10% bovine serum was used as the medium. Inoculated cells were left overnight to allow cells to attach and then 2OHOA solution (40 mM DMSO solution) or 2OHOA insert liposome dispersion was diluted to 5% dextrose to various 2OHOA concentrations and added to each well. After 72 hours of incubation in a 5% CO ₂ cell incubator at 37 ° C., the cell growth rate was expressed in% compared to the case of treating the empty dextrose solution containing no 2OHOA by MTT analysis.

To evaluate the cytotoxicity in normal cells, human lung fibroblast line MRC-5 was inoculated 7,000 per well and tested in the same manner as the cancer cell line using 10% bovine serum-containing DMEM medium as a medium.

As shown in FIG. 7, apoptosis was significantly lower in the MRC-5 cell line, which is normal cells, compared to cancer cells when left with DMPC: 2OHOA liposomes under the same conditions. These results show that cancer cell selective apoptosis activity of free 2OHOA is maintained even when inserted into liposomes.

Example 2 Evaluation of the Use of Liposomes with 2OHOA as an Anticancer Mitoxantrone (MTO) Carrier

Two liposomes were prepared in the same manner as in Example 1, except that 2.5 mg of MTO was dissolved together in 16 μmole of DMPC or 16: 8 μmole mixture solution of DMPC and 2OHOA. MTO not loaded on liposomes was removed by placing 1 mL of the prepared liposomes inside the dialysis membrane with a molecular weight of 10,000 and then dialyzing for 1,000 hours with a 1,000 mL 5% dextrose solution. The obtained MTO-loaded liposome was diluted 100-fold with ethanol, broken, and then absorbed at 660 nm with a spectrophotometer, and compared with a calibration curve of MTO prepared in advance, to determine the efficiency of MTO loading on liposomes.

As shown in Table 2, it was confirmed that only 3.8% of MTO was loaded on liposomes prepared only with DMPC, but 100% was loaded on liposomes into which 2OHOA was inserted. The enhanced MTO loading efficiency in 2OHOA-inserted liposomes is thought to be due to the electrostatic attraction between MTO-2OHOAs, as the absolute value of the negative charge of surface charge of DMPC: 2OHOA liposomes is greatly reduced by MTO loading. .

Evaluation of MTO Loading Efficiency, Average Particle Size, Polydispersity and Surface Charge According to Insertion of 2OHOA in Liposomes Liposomal Composition MTO
Payload efficiency (%) Average particle size
(nm) Polydispersity zeta
electric potential
(mV) DMPC (16μmole) 3.8 83 ± 24 0.255 ± 0.008 +5.4 DMPC: 2OHOA
(16: 8 μmole) 105 56 ± 10 0.322 ± 0.037 -30.7

Example 3 Antitumor Activity Evaluation of 2OHOA Inserted Liposomes with MTO

To evaluate the anticancer activity of 2OHOA-inserted liposomes loaded with MTO, MTO-loaded / 2OHOA-inserted liposomes were prepared in the same manner as in Example 2 except that 2.1 μg of MTO was dissolved together in a 16: 8 μmole mixture solution of DMPC and 2OHOA. Prepared. DMSO solutions of 2OHOA-inserted liposomes and free MTO loaded with MTO were diluted to various MTO concentrations with 5% dextrose solution, respectively, and left for 72 hours with non-small cell lung cancer H460 cell line to measure anticancer activity by MTT assay.

As shown in Figure 8, compared to MTO alone solution, liposome loaded MTO showed much stronger anticancer activity at the same MTO concentration. That is, when the MTO concentration is 75 nM, the MTO alone solution showed 57% cytotoxicity, whereas the liposome loaded MTO showed 95% cytotoxicity.

Example 4 Evaluation of Use of Liposome with 2OHOA as an Anticancer Paclitaxel Transporter

Liposomes were prepared in the same manner as in Example 1, except that 1.8 mg of paclitaxel was dissolved together in a 16: 8 μmole mixture solution of DOPC and 2OHO. Paclitaxel not loaded into liposomes was removed by passing through a 0.8 μm filter mounted in a syringe. Paclitaxel loading concentration on liposomes was determined by HPLC analysis.

As a result, paclitaxel was loaded into 2OHOA insert liposomes at a concentration of 1.5 mg / mL.

DMSO solutions of paclitaxel-loaded 2OHOA-inserted liposomes and free paclitaxel were diluted with various MTO concentrations with 5% dextrose solution, respectively, and left for 72 hours with non-small cell lung cancer H460 cell lines to measure anticancer activity by MTT assay.

As shown in Figure 9, compared to paclitaxel alone solution, liposome loaded paclitaxel showed much stronger anticancer activity at the same paclitaxel concentration. That is, the concentration that inhibits the growth of cancer cells by 50% showed similar anticancer activity at 33% lower concentration of 8 nM for 12 nM liposome loaded MTO for paclitaxel alone solution.

Example 5 Evaluation of the Use of 2OHOA Inserted Liposomes as Anti-cancer All-trans Retinoic Acid Transporters

Two 2OHOA uninserted or inserted liposomes were prepared in the same manner as in Example 1 except that 0.4 mg of retinoic acid was dissolved in a mixture solution of 60 µmole of DMPC and 5 µmole of cholesterol or a mixture solution of 30 µmole of DMPC, 5 µmole of cholesterol and 30 µmole of 2OHOA, respectively. Prepared. Retinoic acid not loaded into liposomes was removed by passing through a 0.8 μm filter mounted in a syringe.

Retinoic acid loading concentration in liposomes at 340 nm was measured by absorbance analysis. Retinoic acid was loaded on 90-95% liposomes.

Retinoic acid loaded 2OHOA-inserted liposomes and 2OHO-non-inserted liposomes were diluted with various retinoic acid concentrations in 5% dextrose solution, respectively, and cultured with malignant melanoma B16-F10 cell line after 72 hours, and anticancer activity was measured by MTT assay. .

As shown in FIG. 10, retinoic acid mounted on 2OHOA-inserted liposomes showed much stronger anticancer activity at the same retinoic acid concentration as compared to 2OHOA-uninserted liposomes. That is, in the case of retinoic acid loaded on 2OHOA-inserted liposomes, the growth of cancer cells was suppressed by about 43% at 10 μM, whereas the retinoic acid loaded on 2OHOA-inserted liposomes inhibited the growth of cancer cells by 45% at 1 μM.

Example 6 Evaluation of Antitumor Activity Improvement Effect in Animal Model of Retinoic Acid (all-trans Retinoic Acid) Mounted in 2OHOA Inserted Liposomes

In order to evaluate the improvement of anticancer activity in the cancer transplantation animal model of retinoic acid loaded on 2OHOA-inserted liposomes, retinoic acid was loaded on liposomes prepared with 60: 5 molar ratio of DMPC and cholesterol. Two 2OHOA-inserted liposomes, 30: 30: 5 molar ratio of DMPC, 2OHOA-inserted liposomes prepared with retinoic acid mounted on liposomes prepared with 2OHOA and cholesterol were prepared.

2 x 10 ⁶ melanoma cell line B16-F10 cell lines derived from mice were transplanted to the dorsal center of Balb / c mice. After the volume of the transplanted cancer cell line reached about 30 mm ³ , mice were treated with 2OHOA solution group (group 1), retinoic acid group loaded on 2OHO-free non-inserted liposomes (group 2), retinoic acid group loaded on 2OHOA-inserted liposomes (group 3). 2OHOA was inserted and randomly divided into 4 groups of empty liposome administration groups without retinoic acid, 6-7 animals per group. The 2OHOA solution was prepared by adding 0.25% hydroxypropylmethylcellulose and 0.2% Tween80 to distilled water to help dissolution. Samples for administration corresponding to each group were intraperitoneally injected three times on a day of 0, 2 and 5 with a retinoic acid concentration of 200 μg / kg of mouse. The shortest diameter (L) and longest diameter (W) of the rock mass were measured at predetermined time intervals, and then the cancer volume was calculated by the formula (L × W ² ) / 2.

As a result, the 2OHOA-inserted liposome-administered group showed significantly greater cancer growth inhibitory effect than the other-administered group (FIG. 11).

Claims (16)

Liposomes prepared from 2-hydroxyoleic acid or a pharmaceutically acceptable salt thereof and phosphatidylcholine; And
Includes a pharmaceutically active ingredient,
Pharmaceutically acceptable salts of 2-hydroxyoleic acid include sodium, potassium, ammonium, pyrrolidine, piperidine, N-hydroxyethylpyrrolidone, N-hydroxyethylpiperi of 2-hydroxyoleic acid. And at least one selected from the group consisting of a dine, triethanolamine, diethanolamine, ethylenediamine and diethylamine salt.
The method of claim 1,
The liposome has a structure in which the 2-hydroxyoleic acid or a pharmaceutically acceptable salt thereof is inserted into the double membrane of the liposome, drug delivery carrier.
delete The method of claim 1,
The drug delivery carrier, wherein the content of 2-hydroxyoleic acid or a pharmaceutically acceptable salt thereof in the liposome is 10 to 50 mol%.
The method of claim 1,
2-hydroxyoleic acid or a pharmaceutically acceptable salt thereof is included in a molar ratio of 1: 0.1 to 1 relative to phosphatidylcholine.
The method of claim 1,
It is 1,2-di-myristoyl-phosphatidylcholine -sn- glycero-3-phosphocholine (1,2-Dimyristoyl-sn-Glycero -3-Phosphocholine, DMPC), 1,2- dipek Sano days - sn - glycero Rho-3-phosphocholine (1,2-dihexanoyl- sn -glycero-3-phosphocholine, DHPC), 1,2-diheptanoyl- sn -glycero-3-phosphocholine (1,2-diheptanoyl- sn -glycero-3-phosphocholine (DHPC), 1,2-dioctanoyl- sn -glycero-3-phosphocholine (1,2-dioctanoyl- sn -glycero-3-phosphocholine), 1,2-dino Nanoyl- sn -glycero-3-phosphocholine (1,2-dinonanoyl- sn -glycero-3-phosphocholine), 1,2- didecanoyl- sn -glycero-3-phosphocholine (1, 2-didecanoyl- sn -glycero-3-phosphocholine), 1,2-dioundecanoyl- sn -glycero-3-phosphocholine (1,2-diundecanoyl- sn -glycero-3-phosphocholine), 1, 2-dilauroyl- sn -glycero-3-phosphocholine (1,2-dilauroyl- sn -glycero-3-phosphocholine, DLPC), 1,2-ditridecanoyl-sn-glycero-3 - phosphocholine (1,2-ditridecanoyl- sn -glycero-3 -phosphocholine), 1,2- di-penta to Kano -sn- glycero-3-phosphocholine (1,2-dipentadecanoyl- sn -glycero-3 -phosphocholine), 1,2- di-palmitoyl -sn- glycero-3-phosphocholine (1, 2- dipalmitoyl- sn -glycero-3-phosphocholine (DPPC), 1,2-diheptadecanoyl- sn -glycero-3-phosphocholine (1,2-diheptadecanoyl- sn -glycero-3-phosphocholine), 1, 2-Distearoyl- sn -glycero-3-phosphocholine (1,2-distearoyl- sn -glycero-3-phosphocholine, DSPC), 1,2-dinonadecanoyl-sn-glycero-3 -Phosphocholine (1,2-dinonadecanoyl- sn -glycero-3-phosphocholine), 1,2-diarachidyl- sn -glycero-3-phosphocholine (1,2-diarachidoyl- sn -glycero- 3-phosphocholine), 1,2-dihenarachidoyl- sn -glycero-3-phosphocholine (1,2-dihenarachidoyl- sn -glycero-3-phosphocholine), 1,2-dibenenoyl-sn 1,2-dibehenoyl- sn -glycero-3-phosphocholine, 1,2-ditricosanoyl- sn -glycero-3-phosphocholine (1,2-ditricosanoyl) sn -glycero-3-phosphocholine), 1,2-dilignoceroyl- sn -glycero- 3-phosphocholine (1,2-dilignoceroyl- sn -glycero-3-phosphocholine), egg phosphatidylcholine, soy phosphatidylcholine, hydrogenated phosphatidylcholine (hydrogenated phosphatidylcholine), 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-Glycero-3-Phosphocholine), 1,2-dimyristooleoyl-sn- Glycero-3-phosphocholine (1,2-dimyristoleoyl- sn -glycero-3-phosphocholine), 1,2-dilinoleyl- sn -glycero-3-phosphocholine (1,2-dilinoleoyl- sn -glycero-3-phosphocholine), 1,2-diecosenoyl- sn -glycero-3-phosphocholine (1,2-dieicosenoyl- sn -glycero-3-phosphocholine), 1,2-dieru Coil-sn-glycero-3-phosphocholine (1,2-dierucoyl- sn -glycero-3-phosphocholine 1,2-didocosahexaenoyl- sn -glycero-3-phosphocholine (1,2 -didocosahexaenoyl- sn -glycero-3-phosphocholine) and 1,2-diecosahexaenoyl- sn -glycero-3-phosphocholine (1,2-dieicosahexaenoyl- sn -glycero-3-phosphocholine) At least one drug delivery agent selected from.
The method of claim 1,
Phosphatidylcholine is a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine, DMPC).
The method of claim 1,
The drug delivery carrier, the content of phosphatidylcholine in liposomes is included in 50 to 90 mol%.
The method of claim 1,
Liposomes are drug carriers having an average particle diameter of 50 nm to 500 nm.
The method of claim 1,
Liposomes are drug carriers having an average particle diameter of 50 nm to 200 nm.
delete delete delete delete The method of claim 1,
The pharmaceutical active ingredient is an anticancer chemotherapeutic agent.
The method of claim 15,
Anticancer chemotherapeutic agents include paclitaxel, docetaxel, cisplatin, carboplatin, oxaliplatin, doxorubicin, daunorubicin, epirubicin and epirubicin. Idarubicin, valubicin, mitoxantrone, gefitinib, erlotinib, irinotecan, topotecan, topotecan, vinblastine ( vinblastine, vincristine and retinoic acid, one or more selected from the group consisting of, a drug carrier.

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