KR101914181B1 - Liposome composition containing anti-cancer drug for subcutaneous injection - Google Patents
Liposome composition containing anti-cancer drug for subcutaneous injection Download PDFInfo
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Abstract
The present invention relates to an agent capable of increasing the bioavailability of a water-soluble anticancer agent such as doxorubicin or cisplatin and a patient-customized administration, and a method for producing such a drug. In order to achieve the above object, the present invention is a liposome containing carbonic acid and an anticancer agent, wherein the liposomes are linked by hyaluronic acid to form clusters. The liposome clusters contain an anticancer agent-containing liposome cluster as an active ingredient, The present invention also provides a pharmaceutical composition for subcutaneous injection of an anticancer agent and a method for producing such a pharmaceutical composition.
Description
The present invention relates to pharmaceutical preparations for the effective administration and use of water-soluble anticancer agents such as doxorubicin, cisplatin, etc., and relates to a subcutaneous injection-type preparation capable of patient-customized administration and a method for producing such injection preparations.
Most of the water-soluble anticancer drugs such as doxorubicin and cisplatin are administered by the injection method due to low bioabsorption rate upon oral absorption (Korean Patent Laid-Open Publication No. 2002-0013174). Various studies have been conducted with respect to injection formulations of such anticancer drugs in order to reduce the number of visits to hospitals and to reduce various side effects such as pain.
Recently, liposomes have been extensively studied in which drugs are released and controlled by specific external stimuli (for example, light rays, pH, temperature, ultrasound, etc.) in specific lesions for effective drug delivery by liposomes. In particular, external stimuli-sensitive liposomes using the high intensity focused ultrasound (HIFU) used in non-invasive cancer therapy or near infrared rays used in skin care have a great advantage in that they can be effectively and conveniently applied.
In addition, the development of liposomes that exhibit a contrasting effect or a secondary therapeutic effect other than a simple drug delivery by modifying the surface of the liposome or diversifying the substance to be encapsulated is widely studied.
For example, in U.S. Patent Application Publication No. 2006-0002994, hydrophilic polymers such as polyethylene glycol are introduced on the surface of liposomes to induce ultrasound-induced release. However, in the case of the liposomes prepared by the above-mentioned patent technology, the release of the drug encapsulated in the vicinity of the body temperature of 37 캜 is partially caused, and it is difficult to efficiently control the release of the drug, and various side effects may occur due to the unintended release of the drug.
In US Patent Application Publication No. 2006-0127467, ultrasonic sensitive liposomes were prepared using only non-polar lipids as phospholipids constituting liposomes. However, since the liposome produced by the above-mentioned patent has a disadvantage of releasing the drug depending on only the glass transition temperature (Tg) of the lipid, it is inevitably released by the thermal effect rather than the radiative effect due to the cavitation effect of the ultrasonic wave. It has a disadvantage that it shows limited emission only in ultrasound Hz.
In case of U.S. Patent Application Publication No. 2006-0034771, a liposome capable of an ultrasonic response was prepared by sealing a gas inside the liposome. The prepared liposome can simultaneously display a contrast effect by sealing a gas having a contrast effect. However, the liposomes prepared by the patent were significantly low in the total amount of active substance (1.4 to 1.8%). Therefore, there is a disadvantage that it is not applicable to the encapsulation of a large dose of a drug in a high dose. In addition, the above method has disadvantages in that the physiologically active substance can be denatured by heat, and it is also difficult to produce the physiologically active substance through complicated manufacturing processes such as temperature control and high pressure at the time of sealing. That is, it is not easy to manufacture, and since the unstable gas is sealed, its stability is limited.
Accordingly, a problem to be solved by the present invention is to provide a water-soluble anticancer agent-containing preparation capable of effectively controlling the release of a water-soluble anticancer drug such as doxorubicin and cisplatin, thereby effectively solving the problem of pain and inconvenience due to the necessity of frequent injection, .
In order to solve the above problems, the present invention relates to a liposome containing carbonic acid and a water-soluble anticancer agent, wherein the liposomes are linked by hyaluronic acid to form clusters, And the release of the water-soluble anticancer drug is induced by irradiation.
Preferably, in the present invention, the water-soluble anticancer agent is doxorubicin, cisplatin or a mixture thereof.
Some of the technical ideas used in the present invention are shown in Figs. As shown in FIGS. 1 and 2, when energy such as focused ultrasound or thermal infrared rays is applied to the carbonic acid-containing liposome cluster as an external heat and the temperature of the region where the liposome cluster is administered is increased to 42 ° C or higher, The liposomes are destroyed. Accordingly, the anticancer drug loaded inside is released to the outside and absorbed into cancer cells, thereby achieving the therapeutic purpose. The release of the drug from the liposomes is stopped when the external heat is removed after a certain amount of release. That is, after injecting a certain amount of carbonic acid-containing liposome clusters (diameter of about 5-50 μm) into the dermis of the patient and irradiating the skin with focused ultrasound or thermal infrared rays for a certain period of time, To stop the emission and to be reused repeatedly as needed.
In the present invention, "warm" means a temperature of about 39-50 ° C, and more preferably about 40-43 ° C. For example, it may refer to hyperthermia, one of the therapies for cancer patients in hospitals.
As used herein, the term " about "or" approximately "means having a range of +/- 10%
The liposomes according to the present invention are prepared using phospholipids. For example, one or more of lecithin such as egg yolk lecithin, soybean lecithin and hydrogenated lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and dipalmitoylphosphatidylcholine may be used. As a basic lipid constituting the liposome, the phospholipid is a substance that has been essentially used for the production of a lamellar structure such as a liposome, and it plays an essential role in constituting a double membrane or a multilayer membrane which gives stabilization to particle formation.
The anticancer agent-containing liposome according to the present invention contains lipid containing amino groups and heat-sensitive lipid for the purpose of the present invention. The amine group-containing lipids combine with the hyaluronic acid used in the present invention to form clusters, and the heat-sensitive lipids promote the release of carbon dioxide gas when heated.
As the lipid containing an amine group that can be used in the present invention, a phospholipid having a primary amine can be used, and preferably a saturated lipid having 12 to 20 carbon atoms (e.g., lauric acid, myristic acid, palmitic acid wherein two lipids selected from the group consisting of fatty acid, stearic acid, arachidic acid and / or unsaturated lipids having 16 to 20 carbon atoms (for example, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid) And amino PEG-phosphatidylethanolamine linked at position 2 can be used. More preferably for the purpose of the present invention, lipids containing amine groups that can be used in the present invention include DSPE-PEG amine (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino polyethylene glycol) (E. G., Preferably DSPE-PEG (2000) Amine) may be used.
Preferably, the heat sensitive lipids that can be used in the present invention include 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-stearoyl- sn-glycero-3-phosphocholine, or a mixture thereof. That is, the heat-sensitive lipid according to the present invention preferably has an alkyl chain having 16-18 carbon atoms in the hydrophobic group. When the number of carbon atoms is less than 16, the phase transition temperature may fall below body temperature and the stability of the nanoparticles to be lysed may decrease. When the number of carbon atoms exceeds 18, the size of the liposome particles may increase. This is undesirable due to the nature of water-soluble anticancer drugs.
Preferably, the heat-sensitive lipids (for example, MSPC and / or MPPC) contain 5 to 20% by weight, preferably 5 to 15% by weight, of the total lipids forming the liposome , Even more preferably 5-10 wt%.
In this regard, the lipid containing phosphatidylcholine (PC) in the liposome of the present invention is capable of imparting heat sensitivity to the liposome particles and is preferably contained in an amount of 40-95% by weight based on the total lipid composition.
The liposomes of the present invention may further comprise other neutral lipid (s) within the scope of not impairing the object of the present invention.
Neutral lipids that may be used in the present invention include phosphatidylcholine; Lysophosphatidylcholine; EPC (egg phosphatidylcholine); Palmitoleic acid, oleic acid, linolenic acid, linolenic acid, palmitic acid, stearic acid, and arachidic acid having 12 to 20 carbon atoms and / or unsaturated lipids having 16 to 20 carbon atoms (e.g., palmitoleic acid, arachidonic acid, phosphatidylcholine linked to glycerol (for example, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DOPC (1,2-Dioleoyl-sn- glycero-3-phosphocholine) , Dilinoleoyl phosphatidylcholine (DL), DL (dilauryloyl) phosphatidylcholine (DL), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine 2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-myristoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, DBPC (1,2-diarachidoyl- 3-phosphocholine, DEPC (1,2-dieicosenoyl-sn-glycero-3-phosphocholine), POPC (palmitoyloleoyl phosphatidylcholine), DOPC ioleoyl phosphatidylcholine), DMPC (dimyristoyl phosphatidylcholine)); Phosphatidylethanolamine (PE); Lysophosphatidylethanolamine; Palmitoleic acid, oleic acid, linolenic acid, linolenic acid, palmitic acid, stearic acid, and arachidic acid having 12 to 20 carbon atoms and / or unsaturated lipids having 16 to 20 carbon atoms (e.g., palmitoleic acid, arachidonic acid, phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), dioleyl phosphatidylethanolamine (DOPE), DSPE (distearoyl phophatidylethanolamine), POPE (palmitoyloleoyl phosphatidylethanolamine), DOPE (dioleyl phosphatidylethanolamine)); Polyethylene glycol-phosphatidylethanolamine (PEG-PE); Palmitoleic acid, oleic acid, linolenic acid, linolenic acid, palmitic acid, stearic acid, and arachidic acid having 12 to 20 carbon atoms and / or unsaturated lipids having 16 to 20 carbon atoms (e.g., palmitoleic acid, arachidonic acid) is an alkoxy PEG-PE linked to glycerol (for example, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- [alkoxy (polyethylene glycol)] (DSPE (E.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- [methoxy (polyethylene glycol) 2000] (DSPE-mPEG-2000) DSPE-MPEG-2000 and DSPE-MPEG-5000), MPEG-DPPE (carbonyl methoxypolyethylene glycol-dipalmitoyl phosphatidylethanolamine) (For example, preferably DPPE-MPEG-2000 and DPPE-MPEG-5000), DMPE-MPEG (carbonyl methoxypolyethylene glycol-d imyristoyl phosphatidylethanolamine (for example, preferably DMPE-MPEG-2000 and DMPE-MPEG-5000); or mixtures thereof.
However, for the purpose of the present invention, the drug is also a water-soluble anticancer drug such as doxorubicin, cisplatin, etc., and as a neutral lipid, 1,2-distearoyl-sn-glycero-3- phosphocholine, DSPC, dipalmitoyl phosphatidylcholine (DPPC), DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine), DMPC (dimyristoyl phosphatidylcholine), PLPC (1-palmitoyl-2-linoleoyl-sn 3-phosphocholine, phosphatidylethanolamine (PE), dilauryloyl phosphatidylcholine (EPC), dimyristoyl phosphatidylcholine (DLPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl- myristoyl phosphatidylcholine, SPPC (1-stearoyl-2-palmitoyl phosphatidylcholine), DBPC (1,2-diarachidoyl-snglycero-3-phosphocholine), DEPC (1,2-dieicosenoyl-sn- glycero-3-phosphochohne) palmitoyloleoyl phosphatidylcholine, DSPE (lysophosphatidylcholine), or a mixture thereof, and DPPC, DSPE or It is more preferred to use a mixture of.
The liposomes of the present invention may additionally contain other cationic lipids and / or anionic lipids within the scope not to impair the object of the present invention.
The cationic lipids include dialkyldimethylammonium, dioleoylphosphatidylethanolamine, dioleoyldialkyltrimethylammoniumpropane or dioleoyldialkyldimethylammoniumpropane, but may also include a variety of other well-known in the art Cationic lipids can be used.
Examples of the anionic lipid include dimyristyl glycerophosphate, dipalmitoyl glycerophosphate dimyristyl glycerophosphate, distearoyl glycerophosphate, distearoyl glycerophosphoglycerol, dipalmitoyl glycerophosphoryl glycerol, Di-myristyl glycerophosphorin, dipalmitoyl glycerophosphorus or distearoyl glycerophosphorus, but also various other anionic lipids well known in the art to which the present invention belongs.
For the purpose of the present invention, the lipid containing an amine group in the liposome containing a water-soluble anticancer agent according to the present invention is preferably a DSPE-PEG amine (1,2-distearoyl-sn-glycero-3-phosphoethanolamine- ) And the heat-sensitive lipid is preferably selected from the group consisting of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-stearoyl-2-hydroxy-sn- glycero- 3-phosphocholine, or a mixture thereof.
More preferably for the purposes of the present invention, the neutral lipid that forms the liposome in the liposome containing the water-soluble anticancer agent according to the present invention includes phosphatidylcholine lipid of the same type as DPPC and 1,2-distearoyl-sn- Phosphoethanolamine- [alkoxy (polyethylene glycol)] (DSPE-PEG) (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- [methoxy Polyethylene glycol) 2000 (DSPE-mPEG-2000)] is used as the amino group-containing lipid, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- These lipids serve to donate amine groups necessary for reaction with hyaluronic acid to form clusters, and the PEG polymer plays a role of stabilizing the liposome.
The liposome of the present invention may additionally contain various adjuvants to the extent that the object of the present invention is not impaired. For example, the present invention may also include a polyol as an emulsion stabilizing aid in the production of liposomes, such as propylene glycol, dipropylene glycol, 1,3-butylene glycol, glycerin, methylpropanediol, isoprene Glycol, pentylene glycol, erythritol, xylitol, sorbitol and the like can be used, and as such polyols, glycerin, butylene glycol, propylene glycol and the like are more preferable. The present invention can further utilize ceramides for the production of liposomes. The present invention may further comprise an oligosaccharide in the liposome of the present invention. Such oligosaccharides may be fucoidan, glucosamine, lactose, heparin or the like, and fucoidan is most preferable.
The hyaluronic acid polymer used for clustering the liposomes of the present invention contains a large number of carboxyl groups capable of bonding with the liposomes as the molecular weight increases and is useful for maintaining proper viscosity of the injection. For the purposes of the present invention, the molecular weight of such a hylaronic acid preferably has a weight average molecular weight of about 100,000 to 1,000,000, and more preferably has a weight average molecular weight of about 300,000 to 500,000. When hyaluronic acid having a desirable molecular weight is used, it is possible to prevent the migration of the liposome after injection and to allow the application site to be in the same position for more than three days.
In the liposome cluster containing the water-soluble anticancer agent of the present invention, the liposome preferably has a diameter of about 10-400 nm for the purpose of the present invention, more preferably about 50-300 nm, even more preferably 50-200 nm , Most preferably having a particle diameter of 100-150 nm. When the average particle diameter of the liposome nanoparticles exceeds the above range, the stability of the liposome is lowered or the loading of the water-soluble anticancer agent is decreased.
For the liposome clusters containing the water-soluble anticancer agent of the present invention, the clusters preferably have a diameter of about 1-50 μm for the purposes of the present invention (particularly for the purpose of fixing to the injection site after subcutaneous injection). It is more preferable that the particle diameter of the cluster is 10 to 30 mu m considering the fact that small needles can be used and the pain after injection is small. However, if it is less than 10 μm, the likelihood of particle migration increases.
In the present invention, particle diameters of liposomes and clusters were measured using an electrophoretic light scattering spectrophotometer, and the average particle diameter (D 50 (diameter) of particles having a cumulative volume of 50 vol% ).
The present invention also provides a method for producing a liposome comprising the steps of: (a) preparing a liposome containing a water-soluble anticancer agent and carbonic acid by using lipids containing an amine group and a heat-sensitive lipid; and (b) Wherein the method further comprises the step of forming a liposome cluster through a reaction to form a liposome cluster containing a water-soluble anticancer agent suitable for subcutaneous injection of a water-soluble anticancer agent.
Preferably, the carbonic acid-containing liposome may be formed by enclosing a weak acid having two or more carboxyl groups and ammonium bicarbonate in liposomes. The weak acid can maximize the efficiency of encapsulating the anticancer agent in the range of 50-500 mmol, preferably 200-300 mmol. As the weak acid having two or more carboxyl groups, for example, maleic acid, fumaric acid, succinic acid, tartaric acid, citric acid, or a mixture thereof may be used. More preferably, citric acid is used.
As described above, in the manufacturing method of the present invention, the step of preparing the liposome in the step (a) may further include the use of a neutral lipid, and it is possible to add a cationic lipid and / or anion There is an additional use of gender lipids.
The liposomes of the present invention can be extruded through a filter having a straight passage or a curved passage or can be homogenized using a homogenizer to have a reduced size distribution and the prepared solution is dried by a dehydration method or a freeze drying method .
In the preparation method of the present invention, the liposome loading of the water-soluble anticancer agent can be carried out by various methods commonly known in the field of the present invention. For example, water-soluble anticancer agents can be loaded by conventional hydration methods while producing heat-sensitive liposomes. In addition, an anticancer agent can be enclosed using a pH gradient loading method and a ammonium ion loading method which are generally known in the art (see, for example, Biochimica et Biophysica Acta 1758 (2006) 1633-1640). More specifically, for example, citric acid, which is a preferable weak acid of the present invention, is first sealed and clusters are prepared. When the drug is put into an aqueous solution, osmotic pressure is generated on the surface of the liposome and the drug enters the liposome. A 'remote loading' method can be used in which loading is increased because a large crosslinked body is formed and can not come out again.
In addition, the hydrogencarbonate or the like may be loaded into the liposome by a conventional method in the field of the present invention. For example, a method of hydrating a thermosensitive liposome made up of a saturated aqueous solution of ammonium bicarbonate at a low temperature (about 4 ° C or less) is used to firstly load a saturated solution of carbonic acid, followed by the use of the above- And doxorubicin can be loaded for 10 to 24 hours to complete the second loading.
The production of liposome clusters using hyaluronic acid can be carried out by a conventional method known in the field of the present invention. For the purpose of the present invention, a carbodiimide coupling reaction can be preferably used. For example, the carboxy group is activated by mixing hyaluronic acid and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (or DCC (dicyclohexylcarbodiimide)) and NHS (N-hydroxysuccinimide esters) And the reaction is induced at room temperature for 2 hours or longer to obtain clusters in which hyaluronic acid and liposome are combined.
The pharmaceutical composition according to the present invention may further include pharmaceutically acceptable additives within the range not impairing the object of the present invention, in addition to the aforementioned liposome clusters containing a water-soluble anticancer agent. For example, it may further include a physiological saline solution for injection, an isotonizing agent, a pH adjusting agent, a stabilizer, a surfactant, and the like.
The liposome clusters containing the water-soluble anticancer agent of the present invention are capable of increasing the bioavailability of the water-soluble anticancer agent and capable of patient-customized administration capable of releasing the anticancer agent if necessary. The anticancer agent-containing liposome clusters of the present invention can be locally applied with heat, such as thermal infrared rays, focused ultrasound, etc., because the drug is fixed at the injection site. The liposome clusters of the present invention can also be produced as biodegradable liposomes and can be decomposed without accumulation in the body, and are safe because they are not toxic.
BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments of the invention and, together with the description of the invention, It should not be construed as limited.
Fig. 1 is a conceptual diagram showing some of the technical ideas of the present invention. Carbon dioxide and carbonic acid loaded in liposomes (about 100-400 nm in diameter) are heated above a certain temperature (about 42 ° C), carbon dioxide is vaporized and liposomes are destroyed and the anticancer drug is released. The heat is then removed to stop the emission.
FIG. 2 is a conceptual view showing some of the technical ideas of the present invention. FIG. 2 is a conceptual view showing some of the technical ideas of the present invention. The liposome Fig. After subcutaneous injection, when heated to a temperature of about 42 ° C or higher under a certain degree of heat, the carbonic acid is vaporized to release the anticancer drug.
FIG. 3 is a Cryo-TEM photograph of the carbonic acid liposome of Example 1-3, which is one embodiment of the present invention, in order to examine changes in morphology when heated.
FIG. 4 is a graph showing the results of measurement of gas generation in liposomes under a high temperature condition using an ultrasound imaging apparatus, and a change chart for confirming sensitivity of drug release to external temperature.
Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art to which the present invention belongs.
Example 1: Preparation of carbonic acid-containing liposome
To prepare liposomes, a lipid component, dipalmitoyl phosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- [methoxy (polyethylene glycol) 2000] (DSPE-mPEG-2000), DSPE-mPEG-2000amine (1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N- [amino (polyethylene glycol) -2000] (ammonium salt), and monostearoylphosphatidylcholine (1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine) (MSPC) was used in various ratios of the mass ratios. Each lipid was dissolved in chloroform and then decompressed at 40 ° C using a rotary evaporator. A thin lipid membrane was formed on the wall.
300 mM citric acid and ammonium bicarbonate were gradually added to the lipid membrane to prepare a buffer solution of
Doxorubicin was added to the prepared liposomes in a ratio of 1: 10 (w / w) to the drug (doxorubicin), and was subjected to doxorubicin encapsulation using pH gradient loading method and ammonium ion loading method at 24 ° C for 20 hours. Doxorubicin, which was not encapsulated in the liposomes, was removed using Sephadex G-50 columns. The sealing efficiency was measured using a UV-vis spectrophotometer at a maximum absorption wavelength of 497 nm and calculated by the following equation (1). The sealing efficiency is shown in Table 2 below.
Where C f is the concentration of doxorubicin after dissolution of the liposome in chloroform: methanol (8: 2) (volume ratio) and C i is the concentration of doxorubicin initially added.
Cryo-TEM (Tecnai G2 Spirit, FEI Company, Hillsboro, OR, USA) was used to measure the liposome morphology. After taking 5 μl of the solution, samples were loaded on the carbon film of the copper grid and then rapidly cooled with liquefied nitrogen. This image was recorded with CCD camera (Proscan GmbH, Scheuring, Germany) and analyzed software (Soft Imaging System, GmbH, Munster, Germany) was used.
Comparative Example 1 was prepared in the same manner as in Examples except that lipids of Table 1 were used and that citric acid and ammonium hydrogen carbonate were enclosed.
Table 1 below shows the phospholipid mixing conditions (weight ratio) of Example 1.
Example 2: Clustering by reaction of carbonate-containing liposome with hyaluronic acid polymer
1 ml of a 0.5 wt% hyaluronic acid polymer (weight average MW 350,000) was added to 1 wt% EDC and 1 wt% NHS aqueous solution to induce activation of the carboxyl group. The reaction was induced by slowly stirring the carbonic acid-containing liposome and the hyaluronic acid reaction solution at room temperature for 2 hours or more. After the reaction was completed, the supernatant was discarded by centrifugation at 4000 rpm or higher to remove unreacted materials, and the precipitate was re-dispersed in physiological saline. This operation was repeated three times or more.
Test Example 1: Measurement of particle size and surface charge of the prepared liposome
The particle size and surface charge of the carbonic acid-containing liposome prepared in each Example and Comparative Example 1 were measured using an electrophoresis light scattering spectroscope (ELS-Z, Otuska, Japan). The measurement results are shown in Table 2 below.
Test Example 2: Bubbling of liposome by temperature
Ultrasonography was used to determine the bubble formation of liposomes according to temperature. The ultrasound imaging device used a 7 MHz transducer (TELEMED, Lithuania). A round bottom tube containing 1 ml of liposome was placed in a thermostat and then measured. The ultrasound image was represented by B-mode anatomic images.
Test Example 3: Release characteristics of drug from liposome
The concentration of doxorubicin released at each temperature and time was measured using a UV spectrophotometer to confirm the release characteristics of the drug from the liposome. The release of doxorubicin from liposomes at different temperatures was measured at 2 minutes intervals for Comparative Example 1 and Example 1 at each temperature and then the absorbance of the liposomal solution was measured. The release concentration of doxorubicin over time was measured by a spectrophotometer by taking the liposome solution every 2 minutes after each temperature was kept constant and calculated by the following equation (2).
In the above formula (2), F t is the absorption intensity of the liposome under each condition, F i is the absorption intensity in the range (4) in which no drug is released, F f is the absorption intensity in chloroform: methanol ), Which is the absorbance intensity of doxorubicin measured by treating with liposome.
The physical properties of the liposomes according to the respective embodiments are summarized in Table 2 below.
Concentration (mg / ml) / Encapsulation rate (%)
*; Changes in doxorubicin content in skin after 1 day of mouse skin injection
The physical properties of the carbonic acid-containing liposome prepared in Example 1 were examined. As shown in Table 2, the particle size was not significantly different between 100 and 250 nm, but the mass ratio of DSPE-mPEG-2000 and DSPE-mPEG- The surface charge increased gradually.
In addition, loading rate of anticancer drug was not good because of decreased stability of liposome when MSPC was increased.
As a result of Example 2, in the case of Example 2-5 in which only DSPE-mPEG-2000 was used and DSPE-mPEG-2000amine was not used, there was no increase in the particle size because there was no amine value capable of binding to the carboxy group of the hyaluronic acid polymer. Therefore, it showed a very small size that can be easily transferred from skin tissue to other sites after injection. Clusters near at least 10 microns are required to be fixed after injection to release the drug in near-infrared irradiation.
Cryo-TEM was measured in order to investigate changes in morphology when heat (42 ° C or higher) was applied to the carbonic acid liposome of Example 1-3 according to the present invention. The results are shown in Fig.
As shown in FIG. 3, the carbonic acid liposome of Example 1-3 was seen as a defoamer having a single membrane, and the size of the particles was 100 to 150 nm. This is similar to the previous ELS figures. Before heat treatment, the size was 100 to 150 nm, but after heat treatment, the size was 80 to 100 nm. Before heat treatment, doxorubicin appeared to be encapsulated in liposomes, but it was confirmed that doxorubicin was released to the outside of the liposome after the heat treatment.
FIG. 4 shows the results of measurement of gas evolution in liposomes under a high temperature condition using an ultrasound imaging apparatus, and FIG. 4 shows experimental results for confirming the sensitivity of drug release to external temperature changes .
As shown in FIG. 4, in Comparative Example 1, generation of gas was not observed up to 45 ° C. However, in the case of the carbonic acid liposome of Example 1-3, bubbles were not generated at 37 ° C, but it was confirmed that bubbles were generated at 42 ° C or higher. From this, it was confirmed that ammonium carbonate encapsulated in the liposome generates bubbles.
In order to confirm the sensitivity of the drug release to the external temperature, the release tendency of the drug according to the change of temperature and time was confirmed from the carbonic acid liposomes of Comparative Example 1 and Example 1-3. In the case of the carbonic acid liposome of Example 1-3, the release sensitivity of drug by temperature showed a rapid release of about 80% at 37 - 40 ° C. In addition, Comparative Example 1 showed a release of about 80% or more in the range of 38 - 44 캜. In addition, at 37 캜 or lower, release of the carbonic acid liposome of Comparative Example 1 and Example 1-3 was limited to about 5%. From these results, it was confirmed that the release of the drug is limited, the liposome is stable at a temperature below the body temperature (37 ° C), and the release of the drug from the liposome is rapidly increased when the temperature is higher than the external temperature. The results showed that the ultrasound images showed a relationship with the gas evolution temperature, and that the release of the drug rapidly increased at a temperature of 42 ° C or higher as expected.
On the other hand, to confirm the drug release characteristics with respect to time in the vicinity of the heating temperature, the drug release tendency with time was ascertained from the carbonic acid liposomes of Comparative Example 1 and Example 1-3. In both of the carbonic acid liposomes of Comparative Example 1 and Example 1-3, the release of the drug was not related to the time at the body temperature (37 ° C). On the other hand, in the carbonic acid liposomes of Comparative Example 1 and Example 1-3, drug release was increased by 80% within 10 minutes at 42 ° C and 45 ° C. In particular, the carbonic acid liposome of Example 1-3 emitted about 80% at 1 minute at 42 ° C. In contrast, Comparative Example 1 showed that the drug release rate was slightly slower at 42 ° C than that of the carbonic acid liposome of Example 1-3.
From these results, it can be seen that the release of the drug is minimized at the body temperature (about 37 ° C), so that the liposome is kept relatively stable at the body temperature after injection of the intramuscular injection of the body, Can be predicted.
On the other hand, the carbonic acid liposomes of the examples are expected to effectively utilize such properties in the treatment of solid cancer by maximally increasing the release of the drug within one to two minutes in the range of hyperthermia treatment. That is, when the liposome clusters containing the anticancer agent of the present invention are locally injected into solid tumors and the temperature of the tumor tissues is elevated from the outside, it is possible to minimize the spread of the anticancer agents in the areas other than solid tumors, It is judged that it can be maximized.
Test Example 3: Evaluation of stability of formed clusters
Examples 3-3 and 3-5 were subcutaneously injected into mice, and one day later, the injection site was excised and the content of doxorubicin was measured on the extracted site. The results are shown together in Table 2 above.
As shown in the results of Table 3 (* marked), 0.1 ml of the mouse was subcutaneously injected to the mouse, and the skin was excised 1 day later, and the content of doxorubicin remaining in the skin was measured. Comparing the liposomes of Example 3-5, the liposomes were lost by the body fluid of the skin and doxorubicin liposomes were not observed, but the liposome clusters remained almost the same.
Claims (10)
Wherein the water is irradiated to induce the release of a water-soluble anticancer drug.
The water-soluble anticancer agent is doxorubicin, cisplatin or a mixture thereof,
Wherein the liposome comprises a lipid containing an amine group and a heat sensitive lipid,
The lipid containing the amine group is a phospholipid having a primary amine, wherein two lipids selected from a saturated lipid having 12 to 20 carbon atoms and an unsaturated lipid having 16 to 20 carbon atoms are mixed with glycerol 1 of phosphatidyl ethanolamine and aminopolyethylene glycol (PEG) -phosphatidylethanolamine,
The heat sensitive lipid may be selected from the group consisting of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-stearoyl-2-hydroxy-sn- glycero-3-phosphocholine, Or a mixture thereof. ≪ RTI ID = 0.0 > 11. < / RTI >
(b) forming a liposome cluster by reacting the liposome produced in the step (a) with the hyaluronic acid polymer.
A method for producing a liposome cluster containing a water-soluble anticancer agent suitable for subcutaneous injection of a water-soluble anticancer agent,
The water-soluble anticancer agent is doxorubicin, cisplatin or a mixture thereof,
The lipid containing the amine group is a phospholipid having a primary amine, wherein two lipids selected from a saturated lipid having 12 to 20 carbon atoms and an unsaturated lipid having 16 to 20 carbon atoms are mixed with glycerol 1 of phosphatidyl ethanolamine and aminopolyethylene glycol (PEG) -phosphatidylethanolamine,
The heat sensitive lipid may be selected from the group consisting of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-stearoyl-2-hydroxy-sn- glycero-3-phosphocholine, Or a mixture thereof.
A method for producing a liposome cluster containing a water-soluble anticancer agent suitable for subcutaneous injection of a water-soluble anticancer drug.
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