KR101846089B1 - Pegylated liposomal doxorubicin - Google Patents

Abstract

The present invention relates to novel doxorubicin-containing pegylated liposome formulations.
The doxorubicin-containing pegylated liposome preparation of the present invention has a nano size and exhibits a narrow particle size distribution and a high inclusion rate. Therefore, the preparation of the present invention can exhibit the same anticancer efficacy and pharmacokinetics as the conventional liposome-containing pegylated liposome-containing doxorubicin formulation, and can secure a superior quality uniformity and storage stability.

Description

PEGYLATED LIPOSOMAL DOXORUBICIN <br> Patent Searching and Data PEGYLATED LIPOSOMAL DOXORUBICIN WIPO Patent Application WO /

The present invention relates to doxorubicin-containing pegylated liposome formulations.

Doxorubicin (doxorubicin hydrochloride) is one of the most widely used cytotoxic anthracycline antibiotics in cancer chemotherapy and is known to be active against a variety of tumors. The doxorubicin is useful for the treatment of numerous solid tumors and leukemia, and is particularly effective for the treatment of breast cancer associated with multiple therapies. In addition, doxorubicin is a protocol therapy for Kaposi's sarcoma associated with AIDS and shows excellent activity against ovarian cancer, lung cancer, prostate cancer, uterine cancer, head and neck cancer, estrogenic sarcoma and Ewing's sarcoma .

However, doxorubicin has problems indicating cytotoxicity such as cardiac toxicity and bone marrow toxicity, which cause congestive heart failure. Thus, a liposomal doxorubicin preparation in which doxorubicin is encapsulated in liposome is used for effectively reducing the toxicity of doxorubicin and effectively delivering and releasing it to a target organ to be treated.

Liposomes are lipid vesicles composed of a phospholipid bilayer. Liposomes are composed of biomembrane-like constituents and can be utilized as carriers for drugs with nanoparticles, and liposome formulations are characterized by enhanced therapeutic efficacy, reduced side effects, targeted delivery, and delayed release. In particular, when liposomes are used as carriers for anti-tumor drugs, the drug can be targeted and delivered to the tumor area, thereby reducing toxicity and enhancing efficacy. Pegylated liposome formulations in which the surface of the liposome is coated with polyethylene glycol (PEG) polymer are also commonly used to increase the half-life of the liposome preparation and increase the tumor specificity.

Commercially available liposome formulations containing pegylated doxorubicin include Caelyx ( ^R ). United States Patent No. 4,927,571 relates to a process for preparing the above liposome preparation, which comprises dispersing a phospholipid and a cholesterol in an organic solvent, removing the solvent and re-dispersing it in an aqueous solution containing doxorubicin, lactose and a salt, Film Hydration Method).

However, conventional liposome formulations containing doxorubicin do not achieve a high filling rate of 95% or more, and the uniformity of the particle size is lowered. Therefore, in order to enhance the pharmacological effect of the preparation and ensure uniform quality, it is necessary to develop a novel liposome preparation containing doxorubicin, which exhibits a narrow particle size distribution, excellent storage stability, and a high inclusion rate as compared with conventional products.

U.S. Patent No. 4,927,571, "Preparation of injectable Doxorubicin / liposome suspension"

The present inventors have made efforts to develop a liposome preparation in which the cytotoxicity is reduced by improving the quality uniformity of the pegylated liposome preparation containing doxorubicin and increasing the inclusion rate of the drug and reducing the free doxorubicin content.

Accordingly, an object of the present invention is to provide a pegylated liposome preparation containing doxorubicin with improved particle size uniformity and storage stability, and a high encapsulation rate.

In order to achieve the above object,

Liposomes having a lipid bilayer formed, including phospholipids, sterols and PEG-conjugated lipids; And

Wherein the liposome comprises doxorubicin encapsulated within the liposome,

(D50) of 75 to 110 nm and a span value of 0.05 or less. The present invention provides a liposome preparation containing doxorubicin.

The doxorubicin-containing pegylated liposome preparation of the present invention has a nano size and exhibits a narrow particle size distribution and a high inclusion rate. Therefore, the preparation of the present invention exhibits reduced cytotoxicity while exhibiting excellent anticancer efficacy and pharmacokinetics, compared with existing doxorubicin-containing pegylated liposome preparations.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of a liposomal formulation of pegylated, doxorubicin.
Fig. 2 is a graph of particle size distribution of the liposome preparation and the control preparation of Example 1. Fig.
Fig. 3 is a graph comparing the elution characteristics of the liposome preparation and the control preparation of Example 1. Fig.
Fig. 4 is a graph showing the results of single-dose toxicity evaluation of the liposome preparation and the control preparation of Example 1. Fig.
FIG. 5 is a graph comparing the anticancer activity of doxorubicin, the control preparation and the liposome preparation of Example 1, wherein (A) shows the results of the anticancer activity tests on JIMT-4 cells, (B) Results.
6 is a photograph showing comparison of fluorescence emission of doxorubicin, the control preparation and the T47D cell line before / after the liposome preparation treatment of Example 1. Fig.
FIG. 7 shows cytotoxicity test results of flow cytometry of doxorubicin, the control preparation and the liposome preparation of Example 1. FIG.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Doxorubicin  contain Pegylated Liposome  Formulation

The present invention relates to liposomes formed with phospholipids, sterols and PEG-conjugated lipids; And the doxorubicin encapsulated in the liposome, wherein the liposome encapsulating the doxorubicin has an average particle size (D50) of 75 to 110 nm, a span value of 0.05 or less, and an encapsulation rate of 96% or more Wherein the liposomal composition is a liposome.

The pegylated liposome preparation containing doxorubicin of the present invention exhibits a narrow particle size distribution and a high inclusion rate, which is excellent in quality uniformity and storage stability, and has excellent safety due to reduced toxicity by free doxorubicin.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of a liposomal formulation of pegylated, doxorubicin.

Referring to FIG. 1, the liposome preparation containing doxorubicin is coated with a polyethylene glycol (PEG) polymer and has a spherical liposome composed of a lipid bilayer and doxorubicin encapsulated inside the liposome. The PEG coating layer can be formed by the PEG-bonded lipid constituting the lipid bilayer, and exhibits an effect of protecting the liposome preparation from the immune system and increasing the in-vivo circulation time of the preparation.

Doxorubicin is a compound represented by the following general formula (1) and may be used in the form of a water-soluble Doxorubicin hydrochloride.

[Chemical Formula 1]

In the present invention, the phospholipid is a follicular lipid, which is a phosphatidylcholine (PC), phosphatidylserine (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (SM), and the like are not particularly limited.

Specifically, it may be at least one selected from the group consisting of egg yolk lecithin, soybean lecithin, sphingomyelin, hydrogenated soybean phosphatidylcholine, yolk phosphatidylcholine, distearoylphosphatidylcholine, and hydrogenated lecithin, but is not limited thereto. Preferably, it is hydrogenated soybean phosphatidylcholine (HSPC).

The sterol is added in order to control the flowability of the liposome membrane so that the liposome can maintain a more stable structure. The doxorubicin-containing pegylated liposome preparation of the present invention exhibits excellent storage stability at room temperature as well as low temperature, including sterols.

In this case, the storage stability means that the liposome preparation is prevented from being entangled due to the self-adhesion of the liposome during the storage period or the use period of the liposome preparation. The liposome preparation with improved storage stability prevents the liposome particles from becoming larger and maintains a uniform particle size distribution, thereby preventing vascular clogging during intravenous administration to the patient and securing a uniform quality.

As the sterol, at least one selected from the group consisting of cholesterol, cholesterol hexasuccinate, ergosterol, and lanosterol may be used, and cholesterol is preferably used.

The PEG-conjugated lipid means a lipid to which a polyethylene glycol (PEG) polymer, which is a hydrophilic polymer, is bonded at one end. A liposome preparation containing PEG-conjugated lipid as a constituent of the membrane reduces the adsorption between blood proteins and liposomes and thus has an effect of increasing the half-life of the body.

The PEG-conjugated lipids can be used either commercially or directly from phospholipids and PEG.

When PEG-conjugated lipids are used in direct preparation, it is preferred that the PEG used be capped at the end with methoxy, ethoxy or other unreactive groups and the other end with a reactive chemical group. Such PEG may be used by purchasing the PEG having a weight average molecular weight (Mw) of 500 to 20,000 g / mol, or may be used by direct preparation.

The lipid conjugated with the PEG is a follicular lipid, preferably having two hydrocarbon chains, typically an acyl chain and a polar head group. Examples of such lipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidyl acid (PA), phosphatidylinositol (PI), and phosphatidylinositol (PI), which have two hydrocarbon chains, typically about C14 to 22, Myelin (SM), and is preferably phosphatidylethanolamine (PE).

The PEG may be activated at one end by reaction of a suitable activating agent such as cyanuric acid, diimidazole, anhydride, etc., and the activated PEG reacts with the follicular lipid to form a PEG-conjugated lipid. Alternatively, a method such as activating a lipid group instead of activating PEG may be used.

In the present invention, as a PEG-conjugated lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly (ethylene glycol- ), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly (ethylene glycol-5000) (DSPE-mPEG-5000) , And more preferably DSPE-mPEG-2000 having a molecular weight in the range of 2650 to 3050 g / mol is used.

In the pegylated liposome preparation containing doxorubicin of the present invention, the lipid bilayer has a weight ratio of phospholipid, sterol and PEG-lipid lipid of 3 to 9: 1 to 3: 1 to 3 in order to secure stability of the liposome membrane and to achieve uniform particle size distribution , More preferably 3: 1: 1.

Preferably, the doxorubicin is contained in an amount of 8 to 12% by weight based on the total weight of liposomes encapsulating doxorubicin.

The poxylated liposome preparation containing doxorubicin according to the present invention is characterized in that the liposome encapsulated with doxorubicin has an average particle size (D50) of 75 to 110 nm and a span value of 0.05 or less.

In this case, D50 means a particle size corresponding to 50% of the total volume when the particle size of the liposome particle is measured and the volume is accumulated from the small particle.

The span value is a value indicating the degree of particle size distribution of the liposome. The smaller the value, the more uniform and narrow the particle size distribution of the liposome. The span value can be expressed by the following equation (1).

[Equation 1]

Span value = (D90 - D10) / D50

In the above formula (1), D90 and D10 mean particle diameters corresponding to 90% and 10% of the total volume when the particle size of liposome particles is measured and the volume is accumulated from small particles. Since the liposome preparation of the present invention has a uniform particle size distribution with a span value of 0.05 or less, a uniform amount of drug can be enclosed in the liposome, and therefore, it has an advantage that a predictable pharmacokinetic drug behavior is obtained.

The doxorubicin-containing pegylated liposome formulation according to the present invention exhibits a high loading rate of 96% or more. In this case, the inclusion rate represents the content of the drug encapsulated in the liposome with respect to the total drug content. The higher the inclusion rate, the less the ratio of free drug can be evaluated. Since the doxorubicin which is not encapsulated in the liposome exhibits cytotoxicity by acting nonspecifically in the body, a high encapsulation rate is essential for the doxorubicin-containing liposome preparation. The liposome preparation according to the present invention exhibits a high encapsulation rate as described above, and the cytotoxicity is reduced, so that the side effect is small and the target treatment effect is excellent.

In addition, the penetration rate of the doxorubicin-containing pegylated liposome preparation according to the present invention after the treatment with weak ultrasonic waves (20 Hz) is 93% or more. As described above, the liposome preparation of the present invention is excellent in stability, so that a drug encapsulated by a physical impact that may occur during the storage period can be maintained in a sealed state without flowing out of the liposome.

The liposome preparation of pegylated liposomes containing doxorubicin according to the present invention preferably has an osmotic pressure of 360 to 370 mOsmol / kg, more preferably 362 to 365 mOsmol / kg, as measured by a freezing point osmolality meter.

In addition, the liposome preparation of the present invention exhibits a surface potential of approximately -50 to -60 mV and easily binds to the target cell membrane to exhibit a drug effect, and alleviates predation by the reticuloendothelial system (RES). In addition, by having the surface potential as described above, the storage stability can be ensured by the repulsive force between the liposome particles.

The doxorubicin-containing pegylated liposome preparation of the present invention comprises white sugar as a low-temperature protecting material to reduce leakage of the drug encapsulated in the liposome and ensure storage stability. White sugar (sucrose) is a iso-osmotic agent that does not react with liposomes and remains on the inner and outer surfaces of the liposome membrane to strengthen the membrane. The saccharide is preferably contained at a concentration of 5-15% (w / v) of the pegylated liposome preparation containing doxorubicin, more preferably at a concentration of 10%.

METHOD FOR PRODUCING PEPILLARY LIPOSOME PROPERTIES CONTAINING DOXORUBASINE

The pegylated liposome preparation containing doxorubicin according to the present invention can be prepared by a method known in the art. For example, a co-liposome may be prepared, processed to have a uniform particle size of an appropriate size, and then encapsulated with doxorubicin .

Specifically, the liposome preparation of the present invention comprises the steps of S1) adding a phospholipid, sterol and PEG-conjugated lipid to a lower alcohol to prepare a lipid solution, S2) dropping the lipid solution into an inorganic salt aqueous solution to prepare a liposomal dispersion solution , S3) extrusion step, S4) dialysis step, and S5) doxorubicin encapsulation step.

As the lower alcohol which can be used in the step S1, at least one selected from the group consisting of ethanol, isopropyl alcohol and propyl alcohol can be used, and among them, ethanol is preferably used.

The operation temperature of step S1 is preferably from room temperature to 80 DEG C, more preferably from 60 to 80 DEG C, and it is more preferable to proceed under stirring for even dispersion of the lipid component.

Examples of the inorganic salt that can be used in step S2 include an ammonium ion as a cation and include at least one selected from the group consisting of ammonium sulfate, ammonium chloride, ammonium acetate, ammonium formate, and ammonium carbonate, Ammonium sulfate is preferably used. The concentration of the inorganic salt aqueous solution in step S2 is preferably 200 to 300 mM.

The temperature for performing the step S2 is preferably from room temperature to 80 DEG C, more preferably from 60 DEG C to 80 DEG C, and is preferably carried out under stirring so that a liposome of uniform particle size can be produced.

The dropping rate of the lipid solution and the stirring time after dropping are not particularly limited, but the stirring time after dropping is preferably 20 minutes or more, more preferably 30 minutes to 1 hour.

In step S3, the co-liposome dispersion solution prepared in step S2 is passed through an extruder to perform extrusion molding. The extruder may be a LIPEX extruder manufactured by Northern lipids. The filter may be a polycarbonate membrane filter having a pore diameter of 40 to 400 nm, But is preferably from 350 to 850 psi.

The extrusion molding may be repeated several times until the average particle diameter (D90) of the liposome satisfies 150 nm or less. Herein, D90 means that 90% by weight of co-liposome among 100% by weight of co-liposome has a particle diameter of 150 nm or less.

In the present invention, the extrusion molding can be performed by replacing the diameter of the used filter with a smaller one, or by stacking several filters so that the diameter of the filter is reduced according to the advancing direction of the extrusion. At this time, It is preferably 40 to 50 nm. Further, during the extrusion molding, a process of increasing the extrusion pressure may be further performed if necessary.

In step S4, a dialysis process is performed on the co-liposome solution in which the extrusion process is completed. The dialysis process is performed to remove impurities such as inorganic salts remaining in the prepared co-liposome.

As the dialysis solution used in the dialysis process, a buffer solution containing white sugar is used.

White sugar (sucrose) is a iso-osmotic agent that does not react with liposomes and remains on the inner and outer surfaces of the liposome membrane to strengthen the membrane. In particular, the present invention is used as a low-temperature protective material to ensure long-term stability against the distribution period of the liposome preparation. In addition, the white sugar has an effect of reducing drug leakage after the dosing of doxorubicin. In order to ensure the above effect, the concentration of white sugar contained in the aqueous solution is preferably 5 to 15% (w / v), more preferably 10%.

As the buffer, glycine, phosphate, citrate, acetate and histidine are preferable, and histidine is more preferable.

Most preferably, the dialysis solution uses an L-histidine solution containing 10% white sugar.

The dialysis membrane used herein has a molecular weight cut-off (MWCO) of 10,000 to 20,000 daltons, preferably 10,000 to 14,000 daltons, and the material thereof is not particularly limited.

For example, the dialysis membrane may be made of a material such as cellulose, ethylene-vinyl alcohol copolymer, polyacrylonitrile, polymethyl methacrylate, or the like.

Preferably, the dialysis is performed at 0 to 10 ° C., preferably at 3 to 5 ° C., and the dialysis solution is exchanged at intervals of 4 hours while being performed for 12 hours or more. Under such conditions, the purification effect can be obtained without affecting the structure, stability and particle size distribution of the liposome.

In step S5, doxorubicin is encapsulated into the liposome prepared and purified as described above using a pH gradient or chemical gradient.

It is preferable that doxorubicin hydrochloride is used as the doxorubicin to be encapsulated in the co-liposome in the step S5.

The inclusion of doxorubicin can be achieved by dissolving doxorubicin hydrochloride in a dialysis solution of S4 stage, mixing with a liposomal dispersion solution at 55 to 60 DEG C for 0.5 to 5 hours. Thereafter, the pH is adjusted to 6.0 to 7.0 using hydrochloric acid and sodium hydroxide to prepare a liposome preparation in which the liposome is pegylated with doxorubicin.

Preferably, the doxorubicin-containing pegylated liposome preparation prepared through step S5 is stored at a low temperature of 2 to 8 占 폚.

Uses and formulations

The pegylated liposome preparation containing doxorubicin according to the present invention shows an anticancer effect through necrosis and can be used for the prevention or treatment of malignant tumors.

The malignant tumor may be a solid malignant tumor such as adrenocortical cancer, bladder cancer, breast cancer, colorectal cancer, ovarian tumors, connective tissue adenocarcinoma, endocrine tumor, endometrial cancer, epithelial cancer, Ewing sarcoma family tumors , Non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), osteosarcoma, ovarian cancer, pancreatic cancer, ovarian cancer, ovarian cancer, ovarian cancer, (PPNET), peritoneal cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, Kaposi sarcoma, gastric cancer, thymoma, uterine cancer, and Wilms tumor.

The malignant tumors may also be used for the treatment of hematologic malignancies such as leukemia, lymphoma (non-Hodgkin lymphoma), Hodgkin's disease (also referred to as Hodgkin lymphoma), and myeloma such as acute lymphoblastic leukemia (ALL) (CML), chronic leukemia (CML), acute undifferentiated leukemia (AUL), undifferentiated large cell lymphoma (ALCL), leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (AML / TMDS) with mixed lymphoid leukemia (PML), JMML, adult T cell ALL, trilineage myelodysplasia, mixed leukemia (MLL), bone marrow (MDSs), myeloproliferative disorders (MPD), multiple myeloma (MM), and myelogenous sarcoma.

The poxylated liposome preparation containing doxorubicin according to the present invention is usually used as an injection for intravenous administration. It is preferable that doxorubicin hydrochloride is contained in an amount of 1.5 to 2.5 mg per 1 ml of the injection, more preferably 2 mg.

The doxorubicin-containing pegylated liposome preparation according to the present invention may be provided as a preparation to be administered by suspension in an injectable suspension in the final administration.

The injectable suspending agent may include, for example, isotonic agents, thickeners, surfactants, buffers and the like as water-soluble organic carriers.

Examples of the isotonic agent that can be used include water-soluble excipients such as mannitol, sucrose, sorbitol, trehalose, lactose and sodium chloride, or sugars. Examples of the thickening agent include camosodium sodium, carboxymethylcellulose sodium and povidone. Polysorbate 80, polysorbate 20, and the like can be used as the surfactant, and the sorbitan esters can be span 80, span 20, and the like as polyoxyethylene sorbitan. As the buffer, sodium monohydrogenphosphate, anhydrous citric acid, sodium hydroxide, sodium chloride and the like can be used.

The dosage of the pegylated liposome preparation containing doxorubicin according to the present invention can be variously adjusted according to the patient's age, symptoms, dosage form and the like, and is 30.0 to 130.0 mg / day, preferably 38.0 to 95.0 mg / day as doxorubicin hydrochloride &Lt; / RTI &gt;

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the inventions. , And that such changes and modifications are within the scope of the appended claims.

[Example]

Example 1: Preparation of liposome preparation

Glycero-3-phosphoethanolamine-N-methoxy-poly (ethylene glycol-2000) (DSPE-mPEG- 2000) were dispersed in 3.25 ml of ethanol and stirred at 65 캜. The lipid solution was added dropwise to 50 ml of a 250 mM ammonium sulfate solution, mixed and stirred at 65 DEG C for 1 hour to prepare a co-liposome. A 47 mm polycarbonate membrane filter was attached to an extruder (LIPEX extruder, 100 ml, Northern lipids), and the above-mentioned liposome dispersion solution was added and extruded at 500 psi pressure. The extrusion was repeated until the size (D90) of the liposome was 150 nm or less.

The liposome solution whose particle size distribution was adjusted to a desired size by the above extrusion was put into a dialysis membrane having a cut-off molecular weight (MWCO) of 12,000 daltons and loaded on an L-histidine solution containing 10% And dialyzed at 4 &lt; 0 &gt; C for 12 hours.

Next, doxorubicin encapsulation was performed. 100 mg of doxorubicin hydrochloride was dissolved in 5 ml of 10% white sugar-containing L-histidine solution used for dialysis. The liposome dispersion solution purified by dialysis was mixed and allowed to stand at 65 ° C for 1 hour to enclose doxorubicin in the liposome Respectively. The pH of the liposome solution that had been sealed was adjusted to 6.0 to 7.0. The finished liposome preparation was stored at 2-8 ° C in an ampoule.

Experimental Example 1: Analysis of liposome preparation

In order to evaluate the physicochemical properties of the liposome preparations prepared in Example 1, particle size, osmotic pressure and inclusion rate were analyzed. In this experimental example, a Caelyx ^® injection of Janssen, one of the commercially available doxorubicin-containing pegylated liposome preparations, was used.

(1) Particle size analysis

The particle size of liposomes can affect the pharmacokinetics of the body. In other words, if the particle size is too small, the disappearance rate of the body is too fast to exhibit the desired drug efficacy. Conversely, if it is too large, it may affect the capillary blood vessels of the kidney or other organs.

The particle size of Example 1 and the control formulation was analyzed using a particle size analyzer Zetasizer Nano ZS (Malvern), and the results are shown in FIG. As a result of particle size analysis, the average particle size (D50) of the preparation of Example 1 and the control preparation was found to be about 100 nm, and the span value of the preparation of Example 1 was 0.07 and that of Example 1 was 0.03, Of the particles exhibit a narrower particle size distribution. From these results, it can be confirmed that the liposome preparation produced by the production method of the present invention is superior to conventional Celix injection in terms of particle size uniformity.

(2) Osmotic pressure analysis

Osmotic pressure of the liposome preparation of Example 1 was measured using a freezing point osmolality meter (Osmomat 3000, Gonotec), and the average value was 365 mOsmol / kg, which was similar to the control formulation (362 mOsmol / kg).

(3) Measurement of inclusion rate

In order to measure the content of doxorubicin encapsulated in the liposome, HPLC analysis was carried out by the test method described in Korean Pharmacopoeia. The analysis conditions are as follows.

* Column: Stainless steel pipe having an internal diameter of 4.6 mm and a length of 50 mm was charged with 5 탆 octysilyl silica gel

* Mobile phase: 0.02 M ammonium acetate buffer (pH 4.0): 0.02 M ammonium acetate TS solution (50:50, volume ratio)

* Detector: Ultraviolet absorptiometer (measuring wavelength: 254 nm)

* Flow rate: 1.0 ml / min

As a result of the HPLC analysis, the chromatograms of the control preparation and the preparation of Example 1 were confirmed to be similar, and 101.3% and 99.7%, respectively, were found to be suitable for the test standard (90.0-120.0%) compared with the standard product.

On the other hand, supernatant was obtained by centrifugation at 100,000 G for 2 hours using an ultrahigh-speed centrifuge, and the supernatant and the stock solution of the liposome preparation before centrifugation were analyzed using an ultraviolet spectrometer, from which the inclusion rate was calculated. The results are shown in Table 1.

Before centrifugation
Doxorubicin content Supernatant
Doxorubicin content Inclusion rate Example 1 100.2% 4.1% 96.1% Control formulation 100.4% 5.7% 94.7%

As shown in Table 1, the formulation of Example 1 had an incorporation rate of 96.1%, which was higher than that of the control formulation. As the inclusion rate increases, the liposome preparation produced by the present invention can be expected to increase safety in vivo.

(5) Measurement of inclusion rate after ultrasonic treatment

In order to confirm the stability of doxorubicin encapsulated in the liposome, the inclusion rate after treatment with weak ultrasonic waves (20 kHz) was measured using the test method described in the FDA guidance of the US. The results are shown in Table 2.

Incorporation rate before ultrasonic treatment Rate of inclusion after ultrasonic treatment Example 1 96.1% 93.2% Control formulation 94.7% 92.1%

After the ultrasonic treatment, the preparation of Example 1 showed a high packing rate of 93% or more. From this, it can be confirmed that the liposome preparation of the present invention has little leakage of drug encapsulated in ultrasonic treatment, and maintains stability.

Experimental Example 2: Elution test

In order to confirm the in vitro release characteristics of the liposome preparation prepared in Example 1, a dissolution test was conducted using a USP-4 dissolution tester of Sotax. The dissolution test was carried out in such a manner that the liposome preparation was loaded on a phosphate buffer solution of pH 6.5 and the amount of elution was measured at 37 캜 for 4 days.

3 is a graph showing the results of the dissolution test. As shown in FIG. 3, the liposome preparation of Example 1 exhibited a similar elution amount to that of the conventional Celix injection, and it can be judged that the release characteristics in the body are also similar.

Experimental Example 3: Single dose toxicity evaluation

For the safety evaluation of the liposome preparation of the present invention, the following single-dose toxicity evaluation test was performed on the liposome preparation of the control preparation and Example 1. After intravenous injection of 10, 20 and 30 mg / kg of the liposome preparations of Example 1 and the control preparation into the tail vein of the mice (C57BL / 6, 7 wk), the 4-week mortality and general Symptoms were observed. 4 is a graph of the experimental results.

As a result of the experiment, 10 animals were observed in the 30 mg / kg group, 4/10 animals were observed in the 20 mg / kg group, and no mortality was observed in the 10 mg / kg group. The liposome preparation of Example 1 was observed in 10 mg / kg group and 10 and 20 mg / kg treated group did not.

Under this test condition, the approximate lethal dose (ALD) of the control preparation is estimated to be 10 to 20 mg / kg, and the approximate lethal dose of the liposome preparation of Example 1 can be determined to be 20 to 30 mg / kg. From the above results, it can be confirmed that the liposome preparation of the present invention has reduced toxicity and improved stability as compared with the conventional formulation.

Experimental Example 4: Anticancer effect test in breast cancer cell line - Cell proliferation assay

In order to confirm the anticancer efficacy of the pegylated liposome preparation containing doxorubicin, a cell proliferation assay was conducted on doxorubicin not contained in the liposome, the control preparation, and the liposome preparation of Example 1.

   * Cell lines: T47D / GFP and JIMT-1

   * Doxorubicin treatment concentration: 10 ng / ml to 8 μg / ml

Test method: 96 well plate was treated with 4 × 10 ⁴ cells / well of breast cancer cell line, then treated with doxorubicin, the liposome preparation of Example 1, and the control agent, respectively, for 24 hours Then, 10 쨉 l of CCK-8 (cell counting kit-8) solution was treated and incubated for 2 hours. The absorbance of the incubated cell line was confirmed at a wavelength of 450 nm using a spectrophotometer. Fig. 5 (A) shows the results of experiments on JIMT-1 cells, and Fig. 5 (B) shows the results of experiments on T47D cells. In addition, fluorescence of the T47D cell line expressing GFP (green fluorescent protein) was measured using a fluorescence microscope (FIG. 6).

As a result of the measurement of the absorbance, it was confirmed that the liposome preparation of Example 1 and the control preparation inhibited cell growth equally in both cell lines. However, due to the nature of liposome preparations in which toxic liposomes are encapsulated in liposomes and their side effects are reduced, the two liposome formulations have a lower inhibitory effect on cancer cell growth than the same concentration of doxorubicin treatment group.

Experimental Example 5: Anticancer effect test in breast cancer cell line - Cytotoxicity test

In order to confirm the anticancer efficacy of the pegylated liposome preparation containing doxorubicin, the cells were treated with doxorubicin, the control preparation, and the liposome preparation of Example 1 to determine the cell status using flow cytometry (FACS). The results are shown in Fig.

* Cell line: JIMT-1 (radioisotope is not used)

* Doxorubicin treatment concentrations: 100, 600, 1000 ng / ml

* Nuclear staining: Annexin V-FITC apoptosis detection kit-1 (BD, 556547)

The nuclear staining agent contains Annexin V and PI (Propidium iodide) combined with FITC (fluorescein isothiocyanate), and the cause of cell death can be identified by the following principle. In the early stages of apoptosis, the membrane structure is destroyed to expose the PS inside the cell. At this time, phosphatidylserine (PS) and annexin V, which has a high binding power, bind to PS, . On the other hand, PI is a staining substance that binds to the nucleus. When a cell is killed by necrosis and a hole is formed in the cell membrane, PI penetrates and stains the nucleus. Thus, apoptosis can be detected by the reaction of annexin V, and cell necrosis can be detected by the response of PI.

As a result, both the control and the liposome preparation of Example 1 induced cell death by inducing necrosis rather than inducing apoptosis. Table 3 below shows the percentage of cell necrosis in each treatment group. The cell necrosis ratio of the control preparation and the liposome preparation of Example 1 was found to be equivalent, and it was confirmed that the ratio of cell necrosis was increased according to the treatment concentration.

Treatment concentration 100 ng / ml 600 ng / ml 1000 ng / ml Example 1 28.5% 29.6% 32% Control formulation 27.8% 32.7% 33.9%

From the above experimental examples, the doxorubicin-containing liposome preparation according to the present invention exhibits narrow particle size distribution and a high inclusion rate as compared with the conventional preparation, indicating excellent quality uniformity and storage stability. In addition, the doxorubicin-containing liposome preparation according to the present invention has an anticancer effect similar to that of the conventional formulation, but also has cytotoxicity reduced, and thus it is possible to reduce the occurrence of side effects when used as an anticancer agent.

Claims (10)

(DSPE-mPEG-2000), which is formed from hydrogenated soybean phosphatidylcholine, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly Liposomes; And
Wherein the liposome comprises doxorubicin encapsulated within the liposome,
The surface potential of the liposome preparation is -50 to -60 mV,
The weight ratio of the hydrogenated soybean phosphatidylcholine, cholesterol and 1,2-distearoyl-SM-glycero-3-phosphoethanolamine-N-methoxy-poly (ethylene glycol-2000) (DSPE-mPEG-2000) 3 to 9: 1 to 3: 1 to 3,
The liposomes were prepared by mixing the hydrogenated soybean phosphatidylcholine, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy- poly (ethylene glycol- Was dissolved in ethanol, 250 mM ammonium sulfate aqueous solution was added to the mixed solution, and the solution was subjected to compression molding, dialysis, and doxorubicin encapsulation under a pressure of 350 to 850 psi,
The average particle size (D50) of the liposome encapsulating the doxorubicin is 75 to 110 nm, the span value is 0.03 or less,
The incorporation rate of doxorubicin after ultrasonication of the liposome encapsulating the doxorubicin at a frequency of 20 kHz is 93% or more,
The approximate lethal dose of the liposome preparation encapsulating the doxorubicin is greater than 20 mg / kg,
Wherein the doxorubicin comprises 8 to 12 wt% of the total weight of liposomes encapsulating doxorubicin,
The doxorubicin-containing pegylated liposome formulation comprises 10% (w / v) white sugar,
Wherein the liposome preparation is formulated as an injection, and the doxorubicin hydrochloride is contained in an amount of 1.5 to 2.5 mg per 1 mL of the injection. delete delete delete delete delete delete delete delete delete

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