KR102764982B1 - Composition for penetration of blood-brain barrier comprising sonosensitive liposomes as an effective ingredients - Google Patents

Abstract

The present invention relates to an ultrasound-responsive liposome for blood-brain barrier penetration, wherein the ultrasound-responsive liposome not only has excellent drug encapsulation efficiency and drug release effect by ultrasound, but also can effectively penetrate the blood-brain barrier when stimulated by ultrasound. In particular, the ultrasound-responsive liposome according to the present invention can circulate in the body for a long time, so that the blood-brain barrier penetration efficiency is even better, and has high affinity for brain tumor cells, so that the delivery effect to the tumor site is excellent. Therefore, the ultrasound-responsive liposome according to the present invention can be utilized as a drug delivery vehicle for delivering a treatment agent for brain diseases to the brain. In particular, the present inventors have discovered optimal cavitation conditions capable of stably opening the blood-brain barrier in order to further improve the drug delivery effect of the ultrasound-responsive liposome, and thus, when the ultrasound-responsive liposome according to the present invention for blood-brain barrier penetration is combined with the above cavitation conditions, it is expected that excellent therapeutic effects will be obtained for various brain diseases.

Description

{Composition for penetration of blood-brain barrier comprising sonosensitive liposomes as an effective ingredient}

The present invention relates to a composition for penetrating the blood-brain barrier, including an ultrasound-responsive liposome.

As the incidence of brain tumors, brain infections caused by bacteria or viruses, and neurological diseases is increasing, the demand for technology that can accurately deliver drugs to the brain is increasing. In particular, the most commonly performed treatment for brain tumors is surgical treatment that involves cutting the skull and removing the tumor area.

However, craniotomy not only places a burden on the patient, but there is also a concern about side effects due to damage to nerve cells during surgery. However, surgical treatment is still used because, even when drugs targeting the brain are used, drug delivery is hindered by the blood-brain barrier (BBB) that protects the brain. Therefore, anticancer drugs used to treat brain diseases such as brain tumors are primarily limited to agents that can penetrate the blood-brain barrier, and this also makes it difficult to achieve a complete therapeutic effect because it is difficult to accurately deliver drugs to the diseased area. Therefore, the development of a technology to accurately deliver drugs to brain tissue is urgent.

Recently, research on the technology of delivering drugs to the brain using ultrasound is being conducted worldwide. Cavitation caused by ultrasound and ultrasound contrast agents, microbubbles, can temporarily open the blood-brain barrier and is being applied as a drug delivery method for treating brain diseases. To this end, after injecting microbubbles, ultrasound is focused three-dimensionally and the ultrasound is precisely emitted to the brain tumor area. The microbubbles located near the blood-brain barrier locally open the blood-brain barrier, and the drug is delivered to the brain tissue and induces a treatment mechanism for brain diseases. However, the currently used anticancer drugs are small molecule chemotherapeutic agents, which are rapidly excreted from the body when injected into the body and rapidly absorbed into normal tissue, resulting in a limitation in the delivery effect to the brain.

Therefore, if a drug delivery system with high blood-brain barrier penetration efficiency and long-term blood circulation is developed and applied together with ultrasound and microbubbles, drugs can be delivered to the brain more effectively.

Korean Patent Publication No. 1180558

As a result of exemplary research to solve the above problems, the inventors of the present invention have completed an ultrasound-responsive liposome (IMP302) that can not only effectively pass through the blood-brain barrier but also circulate in the body for a long time, thereby further enhancing the blood-brain barrier penetration efficiency and having a high affinity for brain tumor cells, thereby maximizing the drug delivery effect to the tumor site.

Accordingly, the purpose of the present invention is to provide a composition for penetrating the blood-brain barrier, comprising the ultrasound-responsive liposome as an active ingredient.

Another object of the present invention is to provide a drug delivery vehicle for penetrating the blood-brain barrier, comprising the ultrasound-responsive liposome.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating brain diseases, comprising the ultrasound-responsive liposome.

Another object of the present invention is to provide a method for preparing the ultrasound-responsive liposome for penetration through the blood-brain barrier.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

The present invention provides a blood-brain barrier penetration composition comprising ultrasound-responsive liposomes containing DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE-mPEG2000 (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), cholesterol, and lyso-PC (lysophosphatidylcholine) as an active ingredient. The composition comprises a pharmaceutical composition.

In one embodiment of the present invention, the DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lyso-PC may be included in a molar ratio (mole%) of 1 to 50 : 1 to 10 : 5 to 80 : 0.1 to 50 : 0.1 to 20, but is not limited thereto.

In another embodiment of the present invention, the DSPC is present in an amount of 0.1 to 50% by dry weight relative to the total liposome.

The above DSPE-mPEG2000 is 3 to 50% by dry weight relative to the total liposome.

The above DOPE is 1 to 80% by dry weight relative to the total liposome,

The cholesterol is present in an amount of 0.05 to 40% by dry weight relative to the total liposome, and

The above lyso-PC may be included in an amount of 0.5 to 10% by dry weight relative to the total liposome, but is not limited thereto.

In another embodiment of the present invention, the ultrasound-responsive liposome may further include, but is not limited to, one or more selected from the group consisting of sphingolipids and polysorbates.

In addition, the present invention provides a composition for penetrating the blood-brain barrier, comprising an ultrasound-responsive liposome containing DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine) and a sphingolipid as an active ingredient.

In another embodiment of the present invention, the DOPE may be included in a molar ratio (%) of 5 to 80 with respect to the entire liposome, and the sphingolipid may be included in a molar ratio (%) of 5 to 80 with respect to the entire liposome, but is not limited thereto.

In another embodiment of the present invention, the ultrasound-responsive liposome may further comprise, but is not limited to, DSPE-mPEG2000.

In another embodiment of the present invention, the DSPE-mPEG2000 may be included in a molar ratio (%) of 1 to 20 relative to the entire liposome, but is not limited thereto.

In another embodiment of the present invention, the ultrasound-responsive liposome may satisfy one or more characteristics selected from the group consisting of, but not limited to:

(a) particle size of 100 to 200 nm; and

(b) The ratio of the drug loaded into the liposome to the total drug added is 50-100%.

In another embodiment of the present invention, the ultrasound-responsive liposomes can, but are not limited to, cross the blood-brain barrier when exposed to ultrasound.

In another embodiment of the present invention, the composition may be, but is not limited to, for delivering a drug into the brain.

In another embodiment of the present invention, the drug may be, but is not limited to, a treatment for a brain disease.

In another embodiment of the present invention, the brain disease may be at least one selected from the group consisting of, but is not limited to, brain tumor, brain infection caused by bacteria or viruses, Parkinson's disease, encephalitis, stroke, apoplexy, Alzheimer's disease, Lou Gehrig's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, epilepsy, thrombosis, embolism, cerebral infarction, cerebral apoplexy, small infarction, and cerebral circulation metabolic disorder.

In another embodiment of the present invention, the brain disease treatment agent is vincristine, vinblastine, vinflunine, vindesine, vinorelbine, temozolomide, carmustine, lomustine, cabazitacel, docetaxel, larotaxel, ortataxel, paclitaxel, tesetaxel, isabepilone, lomustine, procarbazine, rituximab, tocilizumab, temozolomide, carboplatin, erlotinib, irinotecan, enzastaurin, vorinostat, doxorubicin, cisplatin, Gleevec, 5-fluorouracil, tamoxifen, topotecan, belotecan, imatinib, floxuridine, gemcitabine, leuprolide, flutamide, zoledronate, methotrexate, camptothecin, hydroxyurea, The composition may be at least one selected from the group consisting of, but is not limited to, streptozocin, valrubicin, retinoic acid, mechlorethamine, chlorambucil, busulfan, doxifluridine, mitomycin, prednisone, afinitor, mitoxantrone, levodopa, carbidopa, entacapone, tolcapone, dopamine agonists, donepezil, galantamine, rivastigmine, memantine, anticholinergics, and amantadine.

In another embodiment of the present invention, the ultrasound-responsive liposomes may be hydrated with, but are not limited to, ammonium sulfate, ammonium citrate, or TEA-SOS.

In another embodiment of the present invention, the composition may be administered sequentially or simultaneously with the sonication, but is not limited thereto.

In another embodiment of the present invention, the ultrasound may satisfy one or more characteristics selected from the group consisting of, but not limited to:

(a) the frequency of the ultrasound is 20 kHz to 3 MHz; and

(b) Duty cycle is 0.5 to 20%.

In another embodiment of the present invention, the sonication may be performed sequentially or simultaneously with the administration of microbubbles, but is not limited thereto.

In addition, the present invention provides a drug delivery vehicle for penetrating the blood-brain barrier, comprising ultrasound-responsive liposomes containing DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE-mPEG2000 (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), cholesterol, and lyso-PC (lysophosphatidylcholine) as an active ingredient.

In addition, the present invention provides a drug delivery vehicle for penetrating the blood-brain barrier, comprising an ultrasound-responsive liposome containing DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine) and a sphingolipid as an active ingredient.

In addition, the present invention provides a method for preparing the blood-brain barrier penetration composition, comprising the following steps:

(S1) a step of dissolving at least one selected from the group consisting of DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lyso-PC in a first organic solvent;

(S2) a step of evaporating the organic solvent to prepare a liposome thin film; and

(S3) A step of hydrating the above liposome film with an aqueous solution.

In one embodiment of the present invention, the first organic solvent may be at least one selected from the group consisting of dimethylacetamide, dimethylformamide, dimethyl sulfoxide, chloroform, methanol, ethanol, and ether, but is not limited thereto.

In another embodiment of the present invention, the first organic solvent in step (S1) may be one to which polysorbate has been added, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating brain diseases, which comprises the ultrasound-responsive liposome as an active ingredient. Preferably, the ultrasound-responsive liposome is loaded with a drug.

In addition, the present invention provides a method for preventing or treating brain diseases, comprising a step of administering an effective amount of the ultrasound-responsive liposome to a subject in need thereof. Preferably, the method may further comprise a step of treating with ultrasound and/or a step of administering microbubbles.

In addition, the present invention provides a use of the ultrasound-responsive liposome for preventing or treating brain diseases.

The present invention also provides a use of the composition for the manufacture of a medicament for treating brain diseases.

In addition, the present invention provides a method for delivering a drug to the brain, comprising the step of administering an effective amount of the ultrasound-responsive liposome loaded with a drug to a subject in need thereof. Preferably, the method may further comprise the step of treating with ultrasound and/or the step of administering microbubbles.

The present invention also provides a use of the ultrasound-responsive liposome for drug delivery to the brain.

The present invention also provides the use of the ultrasound-responsive liposomes for preparing brain-targeting drug delivery vehicles.

The present invention relates to an ultrasound-responsive liposome for blood-brain barrier penetration, wherein the ultrasound-responsive liposome not only has excellent drug encapsulation efficiency and drug release effect by ultrasound, but also can effectively penetrate the blood-brain barrier when stimulated by ultrasound. In particular, the ultrasound-responsive liposome according to the present invention can circulate in the body for a long time, so that the blood-brain barrier penetration efficiency is even better, and has high affinity for brain tumor cells, so that the delivery effect to the tumor site is excellent. Therefore, the ultrasound-responsive liposome according to the present invention can be utilized as a drug delivery vehicle for delivering a treatment agent for brain diseases to the brain. In particular, the present inventors have discovered optimal cavitation conditions capable of stably opening the blood-brain barrier in order to further improve the drug delivery effect of the ultrasound-responsive liposome, and thus, when the ultrasound-responsive liposome according to the present invention for blood-brain barrier penetration is combined with the above cavitation conditions, it is expected that excellent therapeutic effects will be obtained for various brain diseases.

Figure 1 shows the results of quantitative analysis using FACS of the degree of penetration into brain tumor cells of ultrasound-responsive liposomes, Doxil liposomes, and Marquibo liposomes for blood-brain barrier penetration according to the present invention.
Figure 2a shows the results of observing the brain tumor cell penetration effect of ultrasound-responsive liposomes and Doxil liposomes for blood-brain barrier penetration according to the present invention using a confocal fluorescence microscope (red: liposomes, blue: nuclei, magnification: 10X).
Figure 2b shows another result of observing the brain tumor cell penetration effect of ultrasound-responsive liposomes and Doxil liposomes for blood-brain barrier penetration according to the present invention using a confocal fluorescence microscope (red: liposomes, blue: nuclei, magnification: 10X).
Figure 3 shows the results of MTT analysis to confirm the cancer cell killing effect of the blood-brain barrier-penetrating ultrasound-responsive liposomes, Doxil liposomes, and free doxorubicin (freeDOX) according to the present invention on brain tumor cells, with or without sonication.
Figure 4a shows an image of the penetration of Evans-blue (EB) dye into brain tissue through the blood-brain barrier according to the number of microbubbles, and the results of a quantitative analysis of the penetration efficiency.
Figure 4b shows the results of H&E staining to confirm the stability of brain tissue according to the number of microbubbles.
Figure 5a shows an image of brain tissue penetration by Evans-blue dye through the blood-brain barrier under ultrasound conditions.
Figure 5b shows the results of quantitative analysis of the penetration efficiency of Evans-blue dye through the blood-brain barrier according to ultrasound conditions.
Figure 5c shows the results of H&E staining to confirm the stability of brain tissue according to ultrasound conditions.
Figure 6 shows the results of confirming the brain tissue delivery effect and major organ distribution pattern of ultrasound-responsive liposomes and Marquibo liposomes for blood-brain barrier penetration according to the present invention through blood-brain barrier penetration in mice.
Figure 7 shows the results of confirming the distribution patterns in major organs under ultrasonic conditions of liposomes without added sphingomyelin (IMP302-004) and liposomes containing sphingomyelin (IMP302-005).
Figure 8 shows the results of comparing the blood-brain barrier penetration effects of ultrasound-responsive liposomes (IMP302-004) and Doxil liposomes according to the present invention.

The present invention relates to a composition for penetrating the blood-brain barrier, and has been completed by developing an ultrasound-responsive liposome that not only has excellent drug encapsulation efficiency and an ultrasound-induced drug release effect, but can also effectively penetrate the blood-brain barrier when stimulated by ultrasound.

Specifically, in one embodiment of the present invention, the physical properties of liposomes were compared according to the types of hydration solutions (ammonium sulfate, ammonium citrate, and TEA-SOS), and as a result, all three liposomes showed a particle size of about 200 nm and had excellent drug loading rates. In particular, it was confirmed that loading the drug at 60° C. for 2 hours using ammonium sulfate or ammonium citrate as a hydration solution was the most optimized condition for drug loading into liposomes (Example 1).

In another embodiment of the present invention, as a result of comparing the physical properties and characteristics of liposomes according to the type of lipid constituting the liposome, it was confirmed that liposomes prepared with at least one selected from the group consisting of (i) DOPE; and (ii) DSPC, DSPE-mPEG2000, cholesterol, lyso-PC, and sphingomyelin were the most excellent in terms of drug loading rate and ultrasound sensitivity (Example 2).

In another embodiment of the present invention, when the ultrasound-responsive liposome according to the present invention was cultured together with a brain tumor cell line, it was confirmed that the ultrasound-responsive liposome could effectively penetrate into the brain tumor cells (Examples 3 and 4).

In another embodiment of the present invention, when the ultrasound-responsive liposome loaded with an anticancer agent was cultured together with a brain tumor cell line and treated with ultrasound, it was confirmed that the ultrasound-responsive liposome had an excellent anticancer effect on brain tumor cells (Example 5).

In another embodiment of the present invention, in order to further enhance the drug delivery effect into the brain of the ultrasound-responsive liposome of the present invention, conditions for sonication and microbubble cavitation that can stably open the blood-brain barrier were discovered (Examples 6 and 7).

In another embodiment of the present invention, when the blood-brain barrier was opened by treating a mouse model with ultrasound and microbubbles, the ultrasound-responsive liposomes according to the present invention were administered, and it was confirmed that the liposomes effectively penetrated into the brain tissue (Examples 8 and 9).

That is, since it has been confirmed that the ultrasound-responsive liposome for blood-brain barrier penetration according to the present invention can effectively pass through the blood-brain barrier and accurately deliver the encapsulated drug to the brain, it is expected to be utilized as a drug delivery vehicle for the treatment of various diseases.

Hereinafter, the present invention will be described in detail.

The present invention provides a blood-brain barrier penetrable composition and/or drug delivery vehicle, comprising as an active ingredient an ultrasound-responsive liposome, wherein the ultrasound-responsive liposome comprises at least one selected from the group consisting of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE-mPEG2000 (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), cholesterol, lyso-PC (lysophosphatidylcholine), and sphingolipid. The composition includes a pharmaceutical composition.

Preferably, the present invention comprises:

i) a composition for penetrating the blood-brain barrier, comprising as an active ingredient an ultrasound-responsive liposome comprising DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE-mPEG2000 (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), cholesterol, and lyso-PC (lysophosphatidylcholine);

ii) a composition for penetrating the blood-brain barrier, comprising as an active ingredient an ultrasound-sensitive liposome comprising DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine) and a sphingolipid; and/or

iii) Provided is a composition for penetrating the blood-brain barrier, comprising as an active ingredient an ultrasound-responsive liposome containing DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), a sphingolipid, and DSPE-mPEG2000.

In one embodiment of the present invention, the ultrasound-responsive liposome may further comprise polysorbate.

In the present invention, “DOPE” is an abbreviation for 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, and the lipid is known to form heteroliposomes with DOTAP, which is used as a drug delivery vehicle.

In the present invention, “DSPC” is an abbreviation for 1,2-Distearoyl-sn-glycero-3-phosphocholine, which refers to a phospholipid composed of two stearic acids attached to a phosphatidylcholine head group.

In the present invention, “DSPE-mPEG2000” is an abbreviation for 1,2-Distearoyl-sn-Glycero-3-Phosphoethanol amine-N-methoxy-poly(ethylene glycol2000) (1,2-Distearoyl-sn-Glycero-3-Phosphoethanol amine with conjugated methoxyl poly(ethylene glycol2000)), and refers to a PEGylated derivative of 1,2-distearoyl-sn-glycero-3-PE (DSPE).

In the present invention, "cholesterol" refers to a type of sterol (a modified steroid) and a lipid found in the cell membrane of all animal cells. The cholesterol according to the present invention includes a derivative of cholesterol. The derivative of cholesterol may be, for example, sitosterol, ergosterol, stigmasterol, 4,22-stigmasteradien-3-one, stigmasterol acetate, lanosterol, cycloartenol, or a combination thereof. Cholesterol is located in the lipid bilayer, and the permeability can be lowered or increased by controlling the amount thereof, and can be used regardless of the ratio in the liposome.

In the present invention, “Lyso-PC” is an abbreviation for lysophosphatidylcholine, a phospholipid derived from phosphocholine whose head group is choline, and is a representative term for lipids consisting of one acyl chain, and various types of lyso-PC are known.

In the present invention, the lyso-PC may be represented by the following chemical formula 1 or 2, but is not limited thereto. Preferably, the lyso-PC may be 1-LPC (lysophosphatidylcholine) or 2-LPC represented by the following chemical formula 1 or 2, but is not limited thereto.

[Chemical Formula 1]

[Chemical formula 2]

(In the above chemical formula 1 or 2, R is a C ₆ to C ₂₆ alkyl group, a C ₆ to C ₂₆ alkenyl group, a C ₆ to C ₂₆ alkynyl group, a substituted or unsubstituted C ₆ to C ₂₆ cycloalkyl, a substituted or unsubstituted C ₆ to C ₂₆ aryl group, a substituted or unsubstituted C ₇ to C ₂₆ arylalkyl group, or H.)

In the present invention, the lyso-PC is lyso-PC (6:0), lyso-PC (7:0), lyso-PC (8:0), lyso-PC (9:0), lyso-PC (10:0), lyso-PC (11:0), lyso-PC (12:0), lyso-PC (13:0), lyso-PC (14:0), lyso-PC(15:0), lyso-PC(16:0), 2-lyso-PC(16:0), lyso-PC(17:0), lyso-PC(17:1), lyso-PC(18:0), lyso-PC(18:1), lyso-PC(18:2), 2-lyso-PC(18:0), 2-lyso-PC(18:1), lyso-PC(19:0), It may be at least one selected from the group consisting of lyso-PC(20:1), lyso-PC(20:4), lyso-PC(22:0), lyso-PC(22:5), lyso-PC(24:0), lyso-PC(26:0) and lyso-PC(20:5), but is not limited thereto.

Additionally, preferably, the lyso-PC may be MSPC (1-Stearoyl-2-lyso-sn-glycero-3-phosphocholine), but is not limited thereto.

In the present invention, “sphingolipid” means a lipid comprising a skeleton of sphingoid bases to which a fatty acid can be attached via an amide bond and a head group of primary hydroxyl. Sphingolipids are found in all animals, plants, fungi, and some prokaryotes or viruses, and are known to play an important role in maintaining the structure and function of cell membranes and transmitting intercellular signals. In the present invention, the sphingolipid is not limited to a specific type, but may preferably be at least one selected from the group consisting of sphingosine, ceramide, sphingomyelin, cerebroside, and ganglioside.

Preferably, the sphingolipid according to the present invention is sphingomyelin. In the present invention, “sphingomyelin” refers to a sphingophospholipid composed of phosphocholine and ceramide or phosphoethanolamine head groups. Natural sphingomyelin is found in the membranes of animal cells, particularly in the membranous sheath surrounding the axons of nerve cells.

In the present invention, “polysorbate” refers to a nonionic co-surfactant derived from ethoxylated sorbitan esterified with fatty acid. Polysorbate is generally known to act as an emulsifying agent and solubilizer for liposomes. In the case of the ultrasound-responsive liposome according to the present invention, the blood-brain barrier penetration efficiency can be improved by additionally including polysorbate. In the present invention, the polysorbate may be selected from polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80, but is not limited thereto. Preferably, the polysorbate is polysorbate 80.

In one embodiment of the present invention, the DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lyso-PC may be included in a molar ratio (mole %) of 1 to 50 : 1 to 10 : 5 to 80 : 0.1 to 50 : 0.1 to 20, but is not limited thereto. The present inventors have confirmed through specific experiments that liposomes satisfying the above composition ratios have excellent drug loading rate, ultrasound sensitivity, and blood-brain barrier permeability.

More specifically, the DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lyso-PC are in a molar ratio of 1~50 : 1~10 : 5~80 : 0.1~50 : 0.1~20, a molar ratio of 1~50 : 1~10 : 5~68 : 0.1~50 : 0.1~20, a molar ratio of 1~50 : 10~15 : 5~80 : 0.1~50 : 0.1~20, a molar ratio of 1~50 : 1~20 : 5~70 : 0.1~35 : 0.1~15, a molar ratio of 1~50 : 1~10 : 5~70 : 0.1~50 : 0.1~20, a molar ratio of 1~40 : 1~9 : 35~80 : 0.1~40 : 1~10 mole ratio (mole%), 1~40 : 1~10 : 20~70 : 0.1~50 : 1~10 mole ratio (mole%), 1~40 : 1~10 : 20~68 : 0.1~50 : 1~8 mole ratio (mole%), 1~30 : 1~10 : 10~80 : 0.1~50 : 0.1~20 mole ratio (mole%), 1~30 : 1~9 : 5~70 : 0.1~40 : 0.1~20 mole ratio (mole%), 1~30 : 1~9 : 5~68 : 0.1~40 : 0.1~20 mole ratio (mole%), 1~30 : 1~10 : 20~80 : 0.1~50 : 2~10 mole ratio (mole%), 1~30 : 1~20 : 20~68 : 0.1~40 : 1~11 mole ratio (mole%), 1~20 : 1~10 : 15~80 : 1~48 : 1~12 mole ratio (mole%), 10~40 : 3~7 : 40~68 : 0.1~45 : 0.1~11 mole ratio (mole%), 0.1~20 : 1~8 : 63~67 : 0.1~32 : 3~11 mole ratio (mole%), 5~15 : 1~7 : 60~67 : 10~32 : 1~11 mole ratio (mole%), 8~12 : 3~6 : 50~66 : It may be included in a molar ratio (mole%) of 10~32 : 5~11, or a molar ratio (mole%) of 0.1~20 : 1~8 : 53~57 : 0.1~30 : 5~10, but is not limited thereto.

In addition, in the present invention, the DSPC is present in an amount of 1 to 50 molar ratio (mole%), 1 to 40 molar ratio (mole%), 1 to 30 molar ratio (mole%), 1 to 25 molar ratio (mole%), 1 to 20 molar ratio (mole%), 1 to 15 molar ratio (mole%), 1 to 7 molar ratio (mole%), 5 to 50 molar ratio (mole%), 5 to 40 molar ratio (mole%), 5 to 35 molar ratio (mole%), 5 to 30 molar ratio (mole%), 5 to 25 molar ratio (mole%), 5 to 20 molar ratio (mole%), 5 to 15 molar ratio (mole%), 7 to 12 molar ratio (mole%), 25 to 35 molar ratio (mole%), 27 to 32 molar ratio (mole%), or 35 to May be included in a molar ratio of 45 mole%, but is not limited thereto.

In addition, in the present invention, the DSPE-mPEG2000 may be included in a molar ratio (mole%) of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 3 to 8, 3 to 7, or 4 to 6, molar ratio (mole%) relative to the total liposome, but is not limited thereto.

In addition, in the present invention, the DOPE is present in an amount of 5 to 80 molar ratio (mole%), 5 to 75 molar ratio (mole%), 5 to 70 molar ratio (mole%), 5 to 68 molar ratio (mole%), 10 to 75 molar ratio (mole%), 10 to 70 molar ratio (mole%), 10 to 68 molar ratio (mole%), 20 to 75 molar ratio (mole%), 20 to 70 molar ratio (mole%), 20 to 68 molar ratio (mole%), 30 to 75 molar ratio (mole%), 30 to 70 molar ratio (mole%), 30 to 68 molar ratio (mole%), 40 to 80 molar ratio (mole%), 40 to 75 molar ratio (mole%), 40 to 70 molar ratio (mole%), 40 to 68 The mole ratio may be comprised of, but is not limited to, 45 to 80 mole %, 45 to 70 mole %, 50 to 80 mole %, 50 to 70 mole %, or 50 to 68 mole %.

In addition, in the present invention, the cholesterol is present in an amount of 0.1 to 50 molar ratio (mole%), 0.1 to 45 molar ratio (mole%), 1 to 45 molar ratio (mole%), 5 to 45 molar ratio (mole%), 5 to 40 molar ratio (mole%), 5 to 35 molar ratio (mole%), 5 to 30 molar ratio (mole%), 5 to 25 molar ratio (mole%), 5 to 20 molar ratio (mole%), 5 to 15 molar ratio (mole%), 10 to 40 molar ratio (mole%), 10 to 35 molar ratio (mole%), 10 to 30 molar ratio (mole%), 10 to 25 molar ratio (mole%), 10 to 20 molar ratio (mole%), 20 to 40 molar ratio (mole%), 20 to 35 molar ratio relative to the total liposome. (mole%), 25 to 40 molar ratio (mole%), 25 to 35 molar ratio (mole%), 27 to 32 molar ratio (mole%), 30 to 40 molar ratio (mole%), 5 to 10 molar ratio (mole%), or 40 to 50 molar ratio (mole%), but is not limited thereto.

In addition, in the present invention, the lyso-PC is present in an amount of 0.1 to 20 molar ratio (mole%), 0.1 to 15 molar ratio (mole%), 0.1 to 10 molar ratio (mole%), 1 to 20 molar ratio (mole%), 1 to 10 molar ratio (mole%), 1 to 9 molar ratio (mole%), 1 to 8 molar ratio (mole%), 1 to 7 molar ratio (mole%), 1 to 6 molar ratio (mole%), 3 to 20 molar ratio (mole%), 3 to 15 molar ratio (mole%), 3 to 10 molar ratio (mole%), 3 to 7 molar ratio (mole%), 2 to 10 molar ratio (mole%), 3 to 7 molar ratio (mole%), 2 to 6 molar ratio (mole%), 5 to 20 molar ratio (mole%), 5 to The molar ratio may be comprised of, but is not limited to, 15 mole %, 5 to 10 mole %, 7 to 20 mole %, 7 to 15 mole %, 7 to 12 mole %, 8 to 12 mole %, 9 to 11 mole %, 4 to 6 mole %, or 6 to 8 mole %.

In another embodiment of the present invention, the DSPC is present in an amount of 0.1 to 50% by dry weight relative to the total liposome.

The above DSPE-mPEG2000 is 3 to 50% by dry weight relative to the total liposome.

The above DOPE is 1 to 80% by dry weight relative to the total liposome,

The cholesterol is present in an amount of 0.05 to 40% by dry weight relative to the total liposome, and

The above lyso-PC may be included in an amount of 0.5 to 10% by dry weight relative to the total liposome, but is not limited thereto.

In the present invention, the DSPC may be included in an amount of 0.1 to 50 dry weight %, 0.1 to 45 dry weight %, 0.1 to 40 dry weight %, 1 to 40 dry weight %, 1 to 30 dry weight %, 1 to 20 dry weight %, 1 to 15 dry weight %, 1 to 13 dry weight %, 3 to 20 dry weight %, 5 to 20 dry weight %, 7 to 20 dry weight %, 5 to 15 dry weight %, 7 to 13 dry weight %, or 9 to 11 dry weight % relative to the total liposome, but is not limited thereto.

In the present invention, the DSPE-mPEG2000 may be included in an amount of 3 to 50 dry weight %, 5 to 40 dry weight %, 10 to 30 dry weight %, 10 to 25 dry weight %, 10 to 20 dry weight %, 15 to 30 dry weight %, 15 to 25 dry weight %, 15 to 20 dry weight %, 16 to 19 dry weight %, or 17 to 19 dry weight % relative to the total liposome, but is not limited thereto.

In the present invention, the DOPE is present in an amount of 1 to 80 dry weight %, 5 to 80 dry weight %, 10 to 80 dry weight %, 15 to 80 dry weight %, 20 to 80 dry weight %, 25 to 80 dry weight %, 30 to 80 dry weight %, 30 to 75 dry weight %, 30 to 70 dry weight %, 30 to 65 dry weight %, 30 to 60 dry weight %, 30 to 55 dry weight %, 30 to 50 dry weight %, 40 to 80 dry weight %, 40 to 70 dry weight %, 50 to 80 dry weight %, 50 to 70 dry weight %, 55 to 65 dry weight %, 60 to 65 dry weight %, 60 to 63 dry weight %, or 60 to 62 dry weight % relative to the total liposome. May include, but are not limited to:

In the present invention, the cholesterol is present in an amount of 0.05 to 40 dry weight%, 1 to 40 dry weight%, 1 to 30 dry weight%, 1 to 20 dry weight%, 1 to 15 dry weight%, 1 to 13 dry weight%, 1 to 10 dry weight%, 3 to 20 dry weight%, 3 to 15 dry weight%, 3 to 13 dry weight%, 3 to 10 dry weight%, 5 to 20 dry weight%, 5 to 15 dry weight%, 5 to 13 dry weight%, 5 to 10 dry weight%, 6 to 8 dry weight%, 10 to 40 dry weight%, 10 to 35 dry weight%, 10 to 30 dry weight%, 10 to 25 dry weight%, 10 to 20 dry weight%, 15 to 40 dry weight%, 15 may be included in an amount of, but is not limited to, from 15 to 25% by dry weight, from 15 to 22% by dry weight, or from 20 to 30% by dry weight.

In the present invention, the lyso-PC is present in an amount of 0.5 to 10 dry weight%, 0.5 to 8 dry weight%, 0.5 to 7 dry weight%, 0.5 to 6 dry weight%, 0.5 to 5 dry weight%, 0.5 to 4 dry weight%, 1 to 8 dry weight%, 1 to 7 dry weight%, 1 to 6 dry weight%, 1 to 4 dry weight%, 2 to 8 dry weight%, 2 to 7.5 dry weight%, 2 to 6 dry weight%, 2 to 5 dry weight%, 2 to 4 dry weight%, 2.5 to 7.5 dry weight%, 2.5 to 7 dry weight%, 2.5 to 6.5 dry weight%, 3 to 8 dry weight%, 3 to 7 dry weight%, 3 to 6 dry weight%, 3 to 5 dry weight%, 3 to It can be comprised in an amount of, but is not limited to, 4% by dry weight, 5 to 10% by dry weight, 5 to 9% by dry weight, 5 to 8% by dry weight, 5 to 7% by dry weight, 5 to 6% by dry weight, or 5 to 5.5% by dry weight.

The ultrasound-responsive liposome according to the present invention may be characterized in that it contains DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine) and sphingolipid as active ingredients, and does not contain DSPC, cholesterol, and Lyso-PC.

Preferably, the ultrasound-responsive liposome according to the present invention may be characterized in that it contains DOPE, DSPE-mPEG2000, and sphingolipid as active ingredients, and does not contain DSPC, cholesterol, and Lyso-PC.

In one embodiment of the present invention, the DOPE is present in a molar ratio (mole%) of 5 to 80, 5 to 75, 5 to 70, 5 to 60, 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 33, 5 to 20, 5 to 10, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 20 to 80, 20 to 70, molar ratio (mole%), relative to the total liposome. It may be included in a molar ratio of 20 to 60 mole %, 20 to 50 mole %, 30 to 80 mole %, 30 to 70 mole %, 30 to 60 mole %, 35 to 55 mole %, or 40 to 55 mole %, but is not limited thereto.

In another embodiment of the present invention, the sphingolipid is present in a molar ratio of 5 to 80 mole%, 5 to 75 mole%, 5 to 70 mole%, 5 to 60 mole%, 5 to 50 mole%, 5 to 45 mole%, 5 to 40 mole%, 5 to 35 mole%, 5 to 20 mole%, 5 to 10 mole%, 10 to 80 mole%, 10 to 70 mole%, 10 to 60 mole%, 10 to 50 mole%, 20 to 80 mole%, 20 to 70 mole%, 20 to 60 mole% relative to the total liposome. (mole%), 20 to 50 molar ratio (mole%), 30 to 80 molar ratio (mole%), 30 to 70 molar ratio (mole%), 30 to 60 molar ratio (mole%), 35 to 55 molar ratio (mole%), or 40 to 55 molar ratio (mole%), but is not limited thereto.

In another embodiment of the present invention, the DSPE-mPEG2000 may be included in a molar ratio of 1 to 20 (mole%), 1 to 18 (mole%), 1 to 16 (mole%), 1 to 14 (mole%), 1 to 12 (mole%), 5 to 20 (mole%), 6 to 20 (mole%), 7 to 20 (mole%), 8 to 20 (mole%), 9 to 20 (mole%), 7 to 15 (mole%), 7 to 13 (mole%), 5 to 15 (mole%), 5 to 13 (mole%), or 8 to 12 (mole%), but is not limited thereto.

In another embodiment of the present invention, the DOPE, sphingolipid, and DSPE-mPEG2000 may be included in a molar ratio (mole%) of 20-60 : 20-60 : 1-20, a molar ratio (mole%) of 20-70 : 20-70 : 5-15, a molar ratio (mole%) of 30-60 : 20-70 : 5-20, a molar ratio (mole%) of 35-50 : 35-50 : 8-15, a molar ratio (mole%) of 25-60 : 25-60 : 8-15, or a molar ratio (mole%) of 40-55 : 40-55 : 8-15, but is not limited thereto.

In addition, in the present invention, the DOPE is included in an amount of 1 to 80% by dry weight relative to the entire liposome, the sphingolipid is included in an amount of 1 to 80% by dry weight relative to the entire liposome, and the DSPE-mPEG2000 may not be included or may be included in an amount of 3 to 50% by dry weight relative to the entire liposome, but is not limited thereto.

The liposome of the present invention is formed by amphiphilic compounds including phospholipids. These amphiphilic compounds are typically arranged at the interface between an aqueous medium and an essentially water-insoluble organic solvent, thereby stabilizing emulsified solvent microdroplets. The amphiphilic compounds include compounds having a molecule having a hydrophilic polar head moiety (e.g., a polar or ionic group) capable of reacting with an aqueous medium and a hydrophobic organic tail moiety (e.g., a hydrocarbon chain) capable of reacting with, for example, an organic solvent. Amphiphilic compounds are compounds that can stabilize mixtures of substances that cannot normally be mixed otherwise, such as a mixture of two immiscible liquids (e.g., water and oil), a mixture of a liquid and a gas (e.g., gas microbubbles in water), or a mixture of a liquid and insoluble particles (e.g., metal nanoparticles in water).

In addition, the ultrasound-responsive liposome according to the present invention may be hydrated with ammonium sulfate, ammonium citrate, or TEA-SOS. That is, the inner buffer of the liposome may be ammonium sulfate, ammonium citrate, or TEA-SOS. Preferably, the ultrasound-responsive liposome according to the present invention is hydrated with ammonium sulfate or ammonium citrate.

The ultrasound-responsive liposome according to the present invention is characterized by being able to penetrate (i.e., pass through or migrate) the blood-brain barrier. That is, the ultrasound-responsive liposome can penetrate the blood-brain barrier and migrate into brain tissue. In particular, the ultrasound-responsive liposome according to the present invention can penetrate the blood-brain barrier while maintaining its physical properties and characteristics. Therefore, the ultrasound-responsive liposome according to the present invention can be used as a carrier for delivering a drug into the brain, i.e., into brain tissue. That is, the blood-brain barrier-penetrating composition according to the present invention can be for delivering a drug loaded in the ultrasound-responsive liposome into brain tissue.

In the present invention, the “blood-brain barrier (BBB)” is a vascular barrier that separates the brain and blood, and refers to a barrier that protects the brain by blocking foreign substances such as pigments, drugs, and toxins from entering brain tissue. The blood-brain barrier is distributed throughout the cerebral blood vessels surrounding brain cells, and brain capillary endothelial cells are tightly bound to the blood-brain barrier, and the surrounding area is tightly surrounded by glial cells, preventing drugs or metabolites from entering the interior through a tight structure. Most of the substances constituting the blood-brain barrier are composed of phospholipids.

The liposome according to the present invention is characterized in that it passes through the blood-brain barrier when exposed to ultrasound.

The term “ultrasound” used in the present invention generally refers to sound waves exceeding the frequency of 16 Hz to 20 kHz, which is the sound wave frequency that can be heard by human ears, and high-intensity focused ultrasound introduces focused ultrasound that provides continuous, high-intensity ultrasonic energy to a focus, and can produce instantaneous thermal effects (65-100℃), cavitation effects, mechanical effects, and sonochemical effects depending on the energy and frequency. Ultrasound is not harmful when passing through human tissue, but high-intensity ultrasound that forms a focus generates sufficient energy to cause coagulation necrosis and thermocautery effects regardless of the type of tissue.

In the present invention, the liposome is an ultrasound-responsive liposome. The ultrasound-responsive liposome may mean that the permeability of the liposome increases when exposed to ultrasound. Therefore, when the liposome is exposed to ultrasound, the drug contained in the liposome may be released. Alternatively, the ultrasound-responsive liposome may mean that the blood-brain barrier permeability of the liposome increases when exposed to ultrasound. Therefore, when the liposome is exposed to ultrasound, the liposome may pass through the blood-brain barrier and be delivered to the brain, and similarly, the drug loaded in the liposome may be delivered to the brain by ultrasound.

In the present invention, the ultrasound refers to a sound wave having a frequency higher than the audible frequency range of 16 Hz to 20 kHz. The ultrasound may be, but is not limited to, high intensity focused ultrasound (HIFU), high intensity unfocused ultrasound, or a combination of the two. HIFU refers to ultrasound that focuses high intensity ultrasound energy at one place to create a concentrated focus. Depending on which image is viewed to perform the HIFU treatment, there are ultrasound-guided high intensity focused ultrasound (Ultrasound-guided HIFU) and MRI-guided HIFU. The frequency of the ultrasound is, for example, 1 kHz to 100 kHz, 1 kHz to 90 kHz, 1 kHz to 80 kHz, 1 kHz to 70 kHz, 1 kHz to 60 kHz, 1 kHz to 50 kHz, 1 kHz to 40 kHz, 1 kHz to 30 kHz, 1 kHz to 20 kHz, 1 kHz to 10 kHz, 20 kHz to 3.0 MHz, 40 kHz to 2.0 MHz, 60 kHz to 2.0 MHz, 80 kHz to 2.0 MHz, 100 kHz to 2.0 MHz, 150 kHz to 2.0 MHz, 200 kHz to 2.0 MHz, 250 kHz to 2.0 MHz, 300 kHz to 2.0 MHz, 350 kHz to 2.0 MHz, 400 kHz to 2.0 MHz, 450 kHz to 2.0 MHz, 500 kHz to 2.0 MHz, 550 kHz to 2.0 MHz, 600 kHz to 2.0 MHz, 650 kHz to 2.0 MHz, 700 kHz to 2.0 MHz, 750 kHz to 2.0 MHz, 800 kHz to 2.0 MHz, 850 kHz to 2.0 MHz, 900 kHz to 2.0 MHz, 950 kHz to 2.0 MHz, 600 kHz to 1.5 MHz, 650 kHz to 1.5 MHz, 700 kHz to 1.5 MHz, 750 kHz to 1.5 MHz, 800 kHz to 1.5 MHz, 850 kHz to 1.5 MHz, 900 kHz to 1.5 MHz, 950 kHz to 1.5 MHz, 1 MHz to 1.5 MHz, 600 kHz to 1.3 MHz, 650 kHz to 1.3 MHz, 700 kHz to 1.3 MHz, 750 kHz to 1.3 MHz, 800 kHz to 1.3 MHz, 850 kHz to 1.3 MHz, 900 kHz to 1.3 MHz, 950 kHz to 1.3 MHz, 600 kHz to 1.1 MHz, 650 kHz to 1.1 MHz, 700 kHz to 1.1 MHz, 750 kHz to 1.1 MHz, 800 kHz to 1.1 MHz, It may be, but is not limited to, 850 kHz to 1.1 MHz, 900 kHz to 1.1 MHz, 950 kHz to 1.1 MHz, 600 kHz to 1 MHz, 650 kHz to 1 MHz, 700 kHz to 1 MHz, 750 kHz to 1 MHz, 800 kHz to 1 MHz, 850 kHz to 1 MHz, 900 kHz to 1 MHz, or 950 kHz to 1 MHz.

The above liposome may have secured stability. The stability means that the drug encapsulated in the liposome in the blood is not released when not exposed to ultrasound. The inventors of the present invention confirmed that when the release rate of the drug encapsulated in the liposome according to the ultrasound-responsive liposome according to the present invention was measured every 20 minutes for 60 minutes at room temperature (20-25° C.) under vortexing conditions, the drug release rate was at most 30%. That is, since the liposome according to the present invention maintains a stable structure in an environment without ultrasound, the drug is hardly released.

The particle size of the liposome according to the present invention can be, for example, 50 nm to 500 nm, 50 nm to 400 nm, 50 nm to 300 nm, 50 nm to 200 nm, 60 nm to 200 nm, 70 nm to 200 nm, 80 nm to 200 nm, 90 nm to 200 nm, 100 nm to 200 nm, 110 nm to 190 nm, 120 nm to 180 nm, 130 nm to 170 nm, 140 nm to 170 nm, 140 nm to 160 nm, or about 150 nm. According to a specific embodiment, it can be 100 nm to 200 nm. The particle size distribution of liposomes can be measured using a Zetasizer Nano ZS (Malvern) by Dynamic Light Scattering (DLS) analysis after diluting the liposomes 10-fold. However, the method for measuring the particle size distribution of liposomes is not limited thereto, and can be measured according to other methods known in the art and converted to an equivalent level of value.

The ultrasound-responsive liposome according to the present invention can carry a drug. The term “carrying” may be used interchangeably with the term “encapsulation” or “loading.”

The term “drug” as used herein refers to any compound having a desired biological activity. The desired biological activity includes an activity useful for diagnosing, curing, alleviating, treating, or preventing a disease in humans or other animals.

In the present invention, the mixing ratio of the drug and the ultrasound-responsive liposome may be preferably 1:2 to 1:50 by mass ratio (w/w %), more preferably 1:2 to 1:40, 1:2 to 1:35, 1:2 to 1:30, 1:2 to 1:25, 1:10 to 1:50, 1:10 to 1:40, 1:10 to 1:35, 1:10 to 1:30, 1:10 to 1:35, 1:2 to 1:20, 1:2 to 1:18, 1:2 to 1:16, 1:2 to 1:14, 1:2 to 1:12, 1:2 to 1: The mass ratio (w/w %) may be from 10.5, 1:2.5 to 1:10.5, 1:2.5 to 1:5, 1:2 to 1:4, 1:2.5 to 1:4.5, 1:2.5 to 1:4, 1:2.5 to 1:3, 1:1:5 to 1:10.5, 1:5.5 to 1:10.5, 1:6 to 1:10.5, 1:6.5 to 1:10.5, 1:7 to 1:10.5, 1:7 to 1:10.5, 1:7 to 1:10, 1:7 to 1:9, or about 1:8, but is not limited thereto as long as the drug can be efficiently encapsulated into the liposome.

When the drug is treated into the liposome according to the present invention in order to load the drug into the liposome, the ratio of the drug loaded into the liposome relative to the total drug added may be 50 to 100%. That is, the liposome according to the present invention may have a drug loading rate of 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 76% to 100%, 77% to 100%, 80% to 100%, 85% to 100%, 87% to 100%, 88% to 100%, 90% to 100%, or 95% to 100%, but is not limited thereto.

The above encapsulation rate may refer to the loading rate for the amount of drug added, but is not limited thereto. The method for measuring the drug encapsulation rate of liposomes can be calculated by treating the drug to liposomes, separating the unencapsulated drug using the size exclusive chromatography (SEC) method, and measuring the absorbance of the encapsulated drug and the unencapsulated drug. However, the method for measuring the drug encapsulation rate is not limited thereto, and can be measured according to other methods known in the art, and can be converted to a value at an equivalent level.

In the present invention, the drug-encapsulated ultrasound-responsive liposome may have a drug release rate of, but is not limited to, 10% to 100%, 50% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, or 75% to 100%. The drug release rate may refer to a release rate with respect to the amount of drug added, but is not limited thereto. Here, the release rate measurement method may be quantified by treating the liposome with ultrasound, separating the drug released from the liposome and the liposome from which the drug was released by the SEC method, and measuring the absorbance of the drug. However, the drug release rate calculation method is not limited thereto, and may be measured by another method known in the art, and may be converted to a value at an equivalent level.

In the present invention, the drug-encapsulated ultrasound-responsive liposome may have a drug release rate of 0.1% to 100%, 0.1% to 80%, 0.1% to 60%, 0.1% to 40%, 0.1% to 30%, 0.1% to 20%, 0.1% to 10%, or 30% or less in the blood in a non-ultrasonic state, but is not limited thereto.

The composition according to the present invention can be used for the purpose of delivering a drug into the brain by penetrating the blood-brain barrier. That is, the present invention provides a drug delivery vehicle for penetrating the blood-brain barrier, which comprises the ultrasound-responsive liposome according to the present invention as an active ingredient.

In the present invention, the “drug” may be included without limitation as long as it is a drug that acts on the brain. That is, the drug may be included without limitation in specific type or component as long as it is a drug that is intended to be delivered to the brain. Preferably, the drug may be a treatment for brain disease.

In the present invention, “brain disease” means a pathological state in which the function or structure of brain tissue is reversibly/irreversibly deteriorated or lost due to damage to brain tissue, cerebral blood vessels, and brain nerves. Preferably, the brain disease may be selected from the group consisting of brain tumor, bacterial or viral brain infection, Parkinson’s disease, encephalitis, stroke, apoplexy, Alzheimer’s disease, Lou Gehrig’s disease, Huntington’s disease, Pick’s disease, Creutzfeldt-Jakob disease, epilepsy, thrombosis, embolism, cerebral infarction, stroke, small infarction, and cerebral circulation metabolic disorder, but is not limited thereto, and all diseases related to the brain are included.

Most preferably, the brain disease may be a brain tumor (brain cancer). Preferably, the brain tumor may be selected from, but is not limited to, astrocytoma, glioma, brainstem glioma, pituitary adenoma, glioblastoma, oligodendroglioma, glioblastoma multiforme, oligodendroglioma, oligodendroglioma, ependymoma, medulloblastoma, hemangioblastoma, meningioma, pituitary adenoma, craniopharyngioma, and choroid papilloma.

The term “cancer” or “tumor” used in the present invention is a general term for a disease caused by cells that have aggressive characteristics in which cells divide and grow while ignoring normal growth limits, invasive characteristics in which cells infiltrate surrounding tissues, and metastatic characteristics in which cells spread to other parts of the body.

“Drugs for treating brain diseases” include vincristine, vinblastine, vinflunine, vindesine, vinorelbine, temozolomide, carmustine, lomustine, cabazitacel, docetaxel, larotaxel, ortataxel, paclitaxel, tesetaxel, isabepilone, lomustine, procarbazine, rituximab, tocilizumab, temozolomide, carboplatin, erlotinib, irinotecan, enzastaurin, vorinostat, doxorubicin, cisplatin, Gleevec, 5-fluorouracil, tamoxifen, topotecan, belotecan, imatinib, floxuridine, gemcitabine, leuprolide, flutamide, zoledronate, methotrexate, camptothecin, hydroxyurea, streptozocin, It may be at least one selected from, but is not limited to, valrubicin, retinoic acid, mechlorethamine, chlorambucil, busulfan, doxifluridine, mitomycin, prednisone, afinitor, mitoxantrone, levodopa, carbidopa, entacapone, tolcapone, dopamine agonists, donepezil, galantamine, rivastigmine, memantine, anticholinergics, and amantadine.

In addition, the present invention provides a pharmaceutical composition for preventing or treating brain diseases, which comprises the ultrasound-responsive liposome according to the present invention as an active ingredient. Preferably, the ultrasound-responsive liposome is loaded with a brain disease treatment agent.

In the present invention, “prevention” means any act of suppressing or delaying the onset of a brain disease by administering a composition according to the present invention.

In the present invention, “treatment” means any act in which the symptoms of a brain disease are improved or beneficially changed by administering a composition according to the present invention.

The term “pharmaceutical composition” in the present invention means a composition manufactured for the purpose of preventing or treating brain diseases, and each may be formulated and used in various forms according to conventional methods. For example, it may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and may be formulated and used in the form of skin external preparations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas, suppositories, and sterile injection solutions.

In addition to the ultrasound-responsive liposome according to the present invention, the composition may further include at least one selected from the group consisting of an anticancer agent, an imaging contrast agent, an antibiotic, an anti-inflammatory agent, a protein, a cytokine, a peptide, and an antibody.

In addition, the pharmaceutical composition according to the present invention may further include appropriate carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. At this time, carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. When formulating, the composition is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants and surfactants commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can be used, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can be used, such as witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosa administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, etc.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by considering all of the above factors, and this can be easily determined by those skilled in the art. Administration may be administered once a day or divided into several times.

In the present invention, “administration” means providing a predetermined composition of the present invention to a subject by any appropriate method.

In the present invention, “subject” means a subject in need of treatment of a disease and to which the composition of the present invention can be administered, and more specifically, means a mammal such as a human or non-human primate, mouse, dog, cat, horse, or cow.

The composition according to the present invention can be administered sequentially or simultaneously with the sonication. Preferably, the composition can be administered immediately after the sonication. In particular, the ultrasound can be applied to the brain. The present inventors have confirmed safe ultrasound conditions that can open the blood-brain barrier without causing damage to brain tissue, and when the pharmaceutical composition is administered while emitting ultrasound under the above conditions, the drug delivery efficiency of the liposome according to the present invention to the brain can be further enhanced.

Preferably, the frequency of the ultrasound may be, but is not limited to, 20 kHz to 3 MHz, 20 kHz to 2 MHz, 20 kHz to 1.5 MHz, or 20 kHz to 1 MHz.

Preferably, the intensity of the ultrasound may be, but is not limited to, 0.1 to 5 W, 0.1 to 4 W, 0.1 to 3 W, 0.1 to 2 W, 0.1 to 1.5 W, 0.5 to 3 W, 0.5 to 2 W, or 0.7 to 1.5 W.

Additionally, the duty cycles of the ultrasound may be, but are not limited to, 0.5 to 20%, 0.5 to 15%, 0.5 to 12%, 0.5 to 10%, 0.5 to 7%, 0.7 to 10%, 0.8 to 10%, 0.9 to 10%, 1 to 10%, 1 to 9%, 1 to 8%, 1 to 7%, 1 to 6%, 1 to 5%, 1 to 4%, 1 to 3%, 2 to 10%, 3 to 10%, or 4 to 10%.

Additionally, the ultrasound may be emitted for, but is not limited to, 10 to 300 seconds, 10 to 250 seconds, 10 to 200 seconds, 10 to 150 seconds, 10 to 120 seconds, 10 to 100 seconds, 10 to 90 seconds, 10 to 80 seconds, 10 to 70 seconds, 20 to 100 seconds, 30 to 100 seconds, 40 to 100 seconds, 50 to 100 seconds, 50 to 90 seconds, 50 to 80 seconds, or 50 to 70 seconds.

In addition, the sonication may be performed sequentially or simultaneously with the administration of microbubbles. Preferably, the sonication may be performed immediately after the administration of microbubbles. In the present invention, microbubbles are bubbles having an average size of 1 to 10 μm that act as an ultrasound contrast agent, and cause cavitation together with ultrasound to temporarily open the blood-brain barrier.

Preferably, the microbubbles are present in an amount of 10 to 1×10 ¹⁰ pieces/g, 10 to 1×10 ⁹ pieces/g, 10 to 1×10 ⁸ pieces/g, 10 to 1×10 ⁷ pieces/g, 10 to 1×10 ⁶ pieces/g, 10 to 1×10 ⁵ pieces/g, 10×10 ² to 1×10 ⁸ pieces/g, 10×10 ³ to 1×10 ⁸ pieces/g, 10×10 ⁴ to 1×10 ⁸ pieces/g, 1×10 ⁶ to 9×10 ⁶ pieces/g, 1×10 ⁶ to 8×10 ⁶ pieces/g, 1×10 ⁶ to 7×10 ⁶ pieces/g, 1×10 ⁶ to 6×10 ⁶ pieces/g, based on the total weight of the object. 2×10 ⁶ to 7×10 ⁶ units/g, or 4×10 ⁶ to 7×10 ⁶ units/g can be injected, but is not limited thereto.

In addition, the present invention provides a method for preparing the ultrasound-responsive liposome, comprising the following steps:

(S1) a step of dissolving at least one selected from the group consisting of DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lyso-PC in a first organic solvent;

(S2) a step of evaporating the organic solvent to prepare a liposome thin film; and

(S3) A step of hydrating the above liposome film with an aqueous solution.

In the present invention, the first organic solvent may be at least one selected from the group consisting of dimethylacetamide, dimethylformamide, dimethyl sulfoxide, chloroform, methanol, ethanol, and ether, but is not limited thereto.

In the present invention, the aqueous solution may be, but is not limited to, ammonium sulfate, ammonium citrate, or TEA-SOS, and may be appropriately selected depending on the type of drug loaded into the liposome. Most preferably, the aqueous solution is ammonium sulfate.

In the present invention, the first organic solvent in step (S1) may be one to which polysorbate has been added, but is not limited thereto.

The above manufacturing method may additionally include a step of extruding the hydrated liposome through an extruder after the step (S3). The temperature for extruding the liposome may be adjusted in a variety of ways from room temperature to the phase transition temperature range of each substance, and the number of extrusions may be repeated an appropriate number of times to uniformly size the liposome.

In addition, the present invention provides a method for loading a brain disease treatment agent into an ultrasound-responsive liposome for penetrating the blood-brain barrier, the method comprising a step of stirring the brain disease treatment agent and the ultrasound-responsive liposome.

Preferably, the stirring step can be performed at 25 to 70 °C, 30 to 70 °C, 40 to 70 °C, 50 to 70 °C, 55 to 70 °C, 50 to 65 °C, or 55 to 65 °C.

Additionally, the stirring step can be performed for 30 minutes to 5 hours, 30 minutes to 4 hours, 30 minutes to 3 hours, 1 hour to 5 hours, 1 hour to 4 hours, or 1 hour to 3 hours.

The terms used in the present invention are selected from the most widely used general terms possible while considering the functions of the present invention, but they may vary depending on the intention of engineers working in the field, precedents, the emergence of new technologies, etc. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meanings thereof will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined based on the meanings of the terms and the overall contents of the present invention, rather than simply the names of the terms.

Throughout the specification of the present invention, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components can be included, unless otherwise specifically stated. The terms "about," "substantially," and the like, used throughout the specification of the present invention, are used in a meaning at or close to the numerical value when manufacturing and material tolerances inherent in the stated meaning are presented, and are used to prevent unscrupulous infringers from unfairly utilizing the disclosure in which exact or absolute values are mentioned to aid the understanding of the present invention.

Throughout the specification of the present invention, the term "a combination of these" included in the expressions in the Makushi format means a mixture or combination of one or more selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

[Example]

The materials used in the manufacture of ultrasound-responsive liposomes for blood-brain barrier penetration according to the present invention are as described in the table below.

Example 1. Preparation of liposomes and encapsulation of drugs for ultrasound-responsive brain tumor treatment

Ultrasound-responsive liposomes (IMP302) optimized for brain tumor treatment were prepared. First, liposomes having the lipid compositions shown in Table 1 below were prepared and tested to evaluate the solvent affecting drug loading efficiency.

Composition and composition ratio of liposomes for ultrasound-responsive brain tumor treatment (molar ratio, %) DSPC DSPE-PEG Cholesterol DOPE MSPC Molar ratio (%) 10 5 30 65 5 Sheep (mg) 2.99 5.31 4.39 18.3 0.98

1-1. Preparation and hydration of lipid thin films

The total amount of lipid was fixed at 32 mg per sample, and after dissolving everything in 2 mL of chloroform, a lipid thin film was prepared by completely evaporating the chloroform using a rotary evaporator. Thereafter, each lipid thin film was hydrated using 250 mM ammonium sulfate, ammonium citrate, or TEA-SOS as an interior buffer. Hydration was achieved by stirring the lipid thin film and each solution at 200 rpm at 50 °C, and hydration was achieved at a lipid concentration of 32 mg/mL.

1-2. Liposome size control using size extrusion

The liposomes manufactured through the above Example 1-1 have a multi-lamellar structure and a polydisperse size distribution. In order to transform the liposomes into a uni-lamellar structure and to adjust the size to 100 to 200 nm, an extruder equipped with a polycarbonate filter (size extruder mini; Avanti) was used. The size extrusion was controlled using a polycarbonate filter whose pore size was 200 nm. The extrusion was performed back and forth 10 to 20 times with a syringe to obtain liposomes in a monodisperse state.

1-3. Exchanging the exterior buffer

To induce remote loading, the exterior buffer of the manufactured liposomes was exchanged with deionized water (DW) using a PD-10 column. The exchange was performed by loading 2 mL of liposome solution onto the PD-10 column and then eluting with 4 mL of DW, thereby obtaining liposomes in which the exterior buffer was exchanged with deionized water.

1-4. Encapsulation of Vincristin sulfate

To encapsulate vincristine sulfate into the obtained liposomes, vincristine sulfate and lipid were mixed in deionized water at a ratio of 1:20 (w/w%), and stirred at 150 rpm for 2 hours at a temperature of 37 °C or 60 °C. Thereafter, unencapsulated vincristine sulfate was removed using a PD-10 column in the same manner as described above. The loading efficiency of vincristine in the liposomes was quantitatively analyzed by measuring the absorbance at 294 nm using UV-vis.

1-5. Characterization of ultrasound-responsive liposomes for blood-brain barrier penetration and confirmation of optimal manufacturing method

According to the above examples, ultrasound-responsive liposomes for blood-brain barrier penetration were prepared by the lipid thin film hydration method. In order to confirm the characteristics of liposomes according to the type of hydration solution used in liposome preparation, the liposome size distribution and the encapsulation rate of vincristine sulfate according to the type of hydration solution were compared. The results are shown in Table 2 below.

Size distribution and encapsulation rate of vincristine sulfate according to interior buffer used in the preparation of ultrasound-responsive liposomes for blood-brain barrier penetration Interior buffer Size distribution (d.nm) Loading efficiency (w/w %, 60℃) Ammonium sulfate (250 mM) 159.1 ± 69.2 4.6 Ammonium citrate (250 mM) 140.2 ± 38.9 4.9 TEA-SOS (250mM) 220.3 ± 75.2 4.2

As shown in Table 2, the size distribution of the ultrasound-responsive liposomes for blood-brain barrier penetration prepared using each internal buffer all showed a size of approximately 200 nm. In addition, the encapsulation rate (w/w %) of vincristine sulfate into each liposome was found to be at a level equivalent to that of Marqibo, a commercialized liposome preparation.

In addition, in order to determine the optimal conditions for loading vincristine sulfate into the ultrasound-responsive liposomes for blood-brain barrier penetration prepared with ammonium citrate, the loading efficiency according to temperature and time during the stirring step of vincristine sulfate and liposomes was compared and evaluated using UV-vis. The loading effect for each temperature or time condition is as follows.

Comparative evaluation of loading efficiency of vincristine sulfate on ultrasound-responsive liposomes for blood-brain barrier penetration as a function of time and temperature Experimental conditions Size distribution (d.nm) Loading efficiency (w/w %) Temperature (37 ℃, 2h) 128.7 ± 40.1 3.5 Temperature (60 ℃, 2h) 132.5 ± 35.9 4.7 Loading time (60 ℃, 24h) 135.5 ± 42.4 5.1

The loading efficiency of vincristine sulfate according to temperature was 3.5% at 37℃ and 4.7% at 60℃, respectively. In other words, loading vincristine at a temperature of 60℃ was found to be more effective in terms of loading efficiency. In addition, when the loading time conditions of 2 hours and 24 hours were compared, similar loading efficiencies were shown for both 2 hours and 24 hours, and the size distribution of liposomes was found to remain stable without change during the loading process of vincristine sulfate.

Through the above examples, it was confirmed that the ultrasound-responsive liposomes for blood-brain barrier penetration, finally loaded with vincristine sulfate, were most effective for loading vincristine when prepared using ammonium citrate (pH3.2) or ammonium sulfate (pH6.4) as an internal buffer, and that loading the drug into the liposomes at 60°C for 2 hours was the most optimized condition for more effective loading.

Example 2. Comparison of the characteristics of ultrasound-responsive liposomes for blood-brain barrier penetration according to lipid composition

In order to confirm the optimal composition of ultrasound-responsive liposomes for brain tumor treatment, the physical properties and characteristics according to the lipid types constituting the liposomes were compared and evaluated. The composition of each liposome manufactured for the comparative experiment is as shown in Table 4 below.

Composition and composition ratio (molar ratio, %) of ultrasound-responsive liposomes for blood-brain barrier penetration Geological composition No. Lipid composition and molar ratio (%) DSPC DSPE-mPEG2k cholesterol DOPE MSPC sphingomyelin Polysorbate 80 004 10 5 30 65 5 - - 004P 10 5 30 65 5 Addition 005 - 10 - 45 - 45 - 006 - - - 50 - 50 - 007 - - - 10 - 90 - 008 - - - 90 - 10 - 009 - - 10 45 - 45 - 010 - - - 60 - 40 - 011 - - - 30 - 70 - 012 - - - 20 - 80 - 013 - - - 70 - 30 - 013P 69.95 29.95 adding *Polysorbate 80 was prepared by adding 0.1% (v/v) to the solvent.

Each liposome was prepared according to the preparation method of Example 1, but for a clear comparison, doxorubicin-HCl, which has a higher loading efficiency for liposomes than vincristine, was used. The 004P and 013P formulations containing polysorbate 80 were prepared by adding 0.1% (v/v) of polysorbate 80 to the solvent.

2-1. Analysis of liposome encapsulation and encapsulation rate of doxorubicin-HCl

Encapsulation of doxorubicin-HCl was accomplished by mixing doxorubicin-HCl and liposomes in a weight ratio of 1:8 and stirring at 150 rpm for 2 hours at 37°C. Unloaded doxorubicin-HCl was removed using a PD-10 column. Doxorubicin-HCl loaded into liposomes was quantified by analyzing the absorbance at 475 nm using UV-vis.

Through basic property analysis, candidate compositions of ultrasound-responsive liposomes for brain tumor treatment were initially screened, and drug release tests by ultrasound were performed to derive the optimal composition. The component size distribution and doxorubicin-HCl encapsulation effect of ultrasound-responsive liposomes for blood-brain barrier penetration are shown in Table 5 below.

Liposome composition ratio by composition; and comparative evaluation results of physical properties and doxorubicin-HCL loading efficiency of each liposome Geology Composition number 004 004P 005 006 007 008 009 010 011 012 013 013P Marqibo DOPE 65 65 45 50 10 90 45 40 30 20 70 70 sphingomyelin 45 50 90 10 45 60 70 80 30 30 60 DSPE-mPEG2k 5 5 10 Choles
terol 30 30 10 40 DSPC 10 10 MSPC 5 5 Polysorbate 80 0.1%
(v/v) 0.1%
(v/v) Experimental Results Size (d.nm) 123.9 128.2 135.2 143.6 127.8 X
(particle instability) 128.2 139.1 134.2 122.5 123.5 146.2 141.3 PDI 0.062 0.075 0.055 0.079 0.089 0.092 0.096 0.112 0.091 0.095 0.097 0.148 Entrapment Efficiency of doxorubicin-HCl (%) 98.27 95.3 97.47 94.99 19.55 96.32 93.02 94.42 94.19 51.32 94.1 19.06

Each liposome manufactured with the composition according to Table 5 showed an overall size distribution of 100 to 150 nm, a polydisperse index of 0.1, and a uniform size distribution. In addition, the loading efficiency of doxorubicin-HCl also showed an excellent loading rate in which more than 90% of the added doxorubicin dose was loaded into the liposomes. As an exception, only composition 008 showed an unstable particle state during the manufacturing stage and aggregated within a short period of time, and the aggregation tended to become more severe over time. However, the other compositions did not show particle aggregation or drug leakage over time. In order to select the most optimized composition among the above liposomes, compositions 004, 005, and 013, which are expected to have the highest drug encapsulation efficiency and high DOPE content and thus excellent ultrasound sensitivity and long circulation, were selected as candidate compositions.

2-2. Analysis of drug release effect by ultrasound

The ultrasound response test of liposomes was performed using compositions 004, 005, and 013 selected as candidate compositions. The analysis of the drug release rate by ultrasound was performed using an ultrasonicator. The drug-encapsulated liposomes were placed in the ultrasonicator, and then treated with ultrasound at a frequency of 24 kHz, an amplitude of 20% (92 W/cm ² ) for 60 seconds. The drug released from the liposomes was separated using a PD-10 column and quantitatively analyzed by measuring the absorbance at 295 nm with UV-vis. As experimental controls, composition 011 containing relatively less ultrasound-sensitive phospholipids and liposomes loaded with vincristine sulfate or doxorubicin-HCl, which are commercial products, were used.

The loading rate of vincristine sulfate or doxorubicin-HCl for each liposome and the drug release rate by ultrasound are shown in Table 6 below.

Results of drug loading rate and ultrasound sensitivity evaluation according to liposome composition Liposome 004 005 011 013 Doxil Loading efficiency
(%) Doxorubicin 98.49 96.48 94.75 51.80 93.64 Vincristine 73.31 68.74 61.16 81.16 58.73 Release (%) Doxorubicin 50.73 42.32 8.78 72.22 19.90 Vincristine 66.48 63.48 66.87 54.46 56.72

First, when comparing the loading efficiency, in the case of doxorubicin-HCl, compositions 004, 005, 011, and Doxil showed high loading efficiency with more than 90% of the added drug being loaded. Only in the case of composition 013, aggregation of liposomes occurred during the loading process of doxorubicin-HCl, resulting in a somewhat low loading efficiency of 51.8%. However, in the case of vincristine sulfate, all compositions showed high loading efficiencies of 70 to 80%.

The drug release rate by ultrasound response was evaluated by quantitative analysis of the drug released from each liposome in response to ultrasound stimulation. In the case of liposomes containing doxorubicin-HCl, the drug release rate by ultrasound response was higher in the order of the composition ratio of DOPE, confirming that the composition ratio of DOPE was proportional to the ultrasound response of liposomes. In the case of ultrasound-responsive liposomes containing vincristine sulfate, most liposomes showed similar levels of ultrasound response under the same ultrasound conditions.

Therefore, based on the above experimental results, liposomes of compositions 004 and 013, which were most efficiently loaded with vincristine, a drug for treating brain tumors, and showed the best ultrasound response when loaded with doxorubicin, were judged to be the optimal liposome compositions with the best properties, drug loading efficiency, and ultrasound response. In particular, although composition 013 had a somewhat low loading rate for doxorubicin, it was included in the optimal liposome composition because it showed a high loading rate for vincristine at a level similar to that of other compositions.

2-3. Confirmation of the effect of polysorbate 80 on the ultrasonic sensitivity of liposomes

Additionally, in order to evaluate the effect of polysorbate 80 on ultrasound-responsive liposomes for blood-brain barrier penetration, polysorbate 80 was added to liposomes of compositions 004 and 013, which had the highest release amounts of vincristine sulfate and doxorubicin-HCl, and then the physical properties and ultrasound sensitivity of the liposomes were evaluated. In the present invention, polysorbate 80 is added as a component to increase the BBB permeability of the liposome preparation. Liposomes were prepared by containing 0.1% (v/v) of polysorbate 80 in the solvent, and all other processes were performed in the same manner. The physical properties of liposomes containing polysorbate 80 and the ultrasound-responsive release rate of doxorubicin-HCl are as shown in Table 7 below.

004P 013P Size (d.nm) 128.2 146.2 Loading efficiency (%) 95.3 94.1 Release (%) 73.9 65.9

As can be seen in Table 7, when polysorbate 80 was added to the 004 or 013 liposome composition, the particle size distributions were 128.2 and 146.2 nm, respectively, and the drug release rates by ultrasound response were 73.9% and 65.9%, respectively. Polysorbate 80 was added to increase the BBB permeability of the liposome, and the experimental results show that polysorbate 80 does not affect the physical properties of the liposome before promoting the blood-brain barrier permeation of the ultrasound-responsive liposome.

Example 3. Evaluation of brain tumor cell line penetration ability of ultrasound-responsive liposomes for blood-brain barrier penetration through FACS analysis

In order to evaluate the brain tumor cell line penetration ability according to the composition of liposomes, an experiment was conducted using the brain tumor cell line U87MG. In order to quantitatively analyze the effect of each liposome on the penetration of the cell line, FACS analysis was performed, and the ultrasound-responsive liposomes for blood-brain barrier penetration used in the experiment were labeled with a fluorescent dye, DiI, to detect the red wavelength. DiI dye exhibits fluorescence at an excitation/emission wavelength of 549/565 nm, which is a red wavelength.

The experiment was performed as follows. Before liposome treatment, U87MG cell line was cultured in a cell culture medium without serum for 1 hour to perform starvation. After starvation, each liposome solution was treated at a concentration of 400 μg/ml, and after incubation with liposomes for 2 or 4 hours, FACS analysis was performed. The number of cells used was 3×10 ⁵ . The results of quantitative analysis of the brain tumor cell penetration ability of liposomes are shown in Fig. 1. Among the ultrasound-responsive liposomes for penetrating the blood-brain barrier according to the present invention, liposomes having compositions 004 and 013 and Doxil showed high endocytosis efficiency, as most of the liposomes penetrated into the cells within 2 hours after being treated to U87MG cells. The penetration efficiencies for U87MG were found to be high in the order of Doxil, 004, 013, and 011 liposomes.

In addition, when brain tumor cells and liposomes were incubated for 4 hours, liposomes of compositions 004, 013, and Doxil showed cell penetration rates that increased over time and showed penetration efficiency close to 100%, and liposomes of composition 011 also showed cellular endocytosis efficiency that continuously increased over time. In particular, the brain tumor cell line penetration effect of the ultrasound-responsive liposomes according to the present invention was shown to be about twice as high as that of Marqibo, a commercial liposome used to load vincristine drugs.

The above results demonstrate that the ultrasound-responsive liposome for blood-brain barrier penetration according to the present invention can effectively penetrate brain tumor cells and deliver drugs.

Example 4. Verification of cell penetration ability of ultrasound-responsive liposomes for blood-brain barrier penetration using confocal fluorescence microscopy

To select the optimal liposome composition for brain tumor treatment, liposomes of various compositions were treated to U87MG cell line, a brain tumor cell line, and the cellular penetration effect of each liposome over time was evaluated using confocal fluorescence microscopy. To confirm the intracellular location of each liposome by fluorescence, liposomes were labeled with DiI fluorescent dye and detected by emitting red wavelength. U87MG cells were seeded in cell culture chambers, incubated overnight, and starved for 1 h in serum-free medium before each treatment with liposomes. After starvation, each liposome was treated at a concentration of 400 μg/ml, and cellular uptake was induced by incubating for 2 or 4 h, and then the particles were washed. Afterwards, cells were fixed with 4% paraformaldehyde for confocal microscopy, and then microscopic photography was performed. In order to analyze the degree of penetration and the penetration mechanism into brain tumor cells according to the liposome composition, imaging was performed using a confocal fluorescence microscope. For fluorescence photography, liposomes labeled with DiI were photographed at a red wavelength (ex/em: 549/565 nm), and the nuclei of cells were stained with DAPI to appear blue (ex/em: 358/461 nm). The morphology of cells was confirmed by optical photography with DIC. The results of photographing the results of penetration into U87MG cells over time of the 004 and 013 composition liposomes of the present invention, and the Doxil composition liposomes are shown in Figs. 2a and 2b.

As a result of photographing the time-dependent penetration effect of each liposome particle into brain tumor cells using a confocal fluorescence microscope, it was found that both 004 and 013 composition liposomes according to the present invention effectively penetrated into U87MG cells within 2 hours, which was consistent with the results of the FACS analysis of Example 3. All liposomes of each composition were accurately located in the cytoplasm of U87MG cells and exhibited dot-shaped fluorescence, indicating that the liposomes were endocytosed while maintaining the particle shape. The morphology of U87MG cells was compared between the groups treated with the liposomes according to the present invention or the Doxil composition liposomes and the untreated control group, and no particular changes were found. These results suggest that the cytotoxicity of the ultrasound-responsive liposomes themselves according to the present invention is very low.

Example 5. Verification of brain tumor cell killing effect of ultrasound-responsive liposomes for blood-brain barrier penetration

In order to confirm the brain tumor therapeutic effect of the ultrasound-responsive liposome according to the present invention, the anticancer effect was verified using brain tumor cells, U87MG cells. U87MG cells were seeded at 5× ¹⁰⁴ cells per well in a 96-well plate and cultured for 24 hours. Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% antibiotics was used as the cell culture medium. For the efficacy evaluation, liposomes with a composition of 004 loaded with doxorubicin, liposomes with a composition of Doxil, and free doxorubicin were added to the cells at various concentrations (0, 1.25, 2.5, 5, 10 μg/mL). In addition, in order to compare and analyze the degree of cancer cell death according to the presence or absence of ultrasound irradiation, liposomes irradiated with ultrasound and liposomes not treated with ultrasound were treated to each designated well. After drug treatment, the cells were incubated for 4 hours, and after removing all added drugs, they were washed three times with phosphate buffered saline (0.01 M, pH 7.4), and cultured for 72 hours with the cell culture medium to compare the degree of cancer cell proliferation. The cancer cell killing effect was quantitatively analyzed by measuring the absorbance at a wavelength of 570 nm using an MTT assay.

The results of confirming the cancer cell killing effect of each liposome are shown in Fig. 3. According to the present invention In the case of ultrasound-responsive liposomes for penetrating the blood-brain barrier, the cancer cell killing effect was significantly increased after ultrasound treatment compared to before ultrasound treatment. On the other hand, in the case of Doxil, which is not ultrasound-sensitive, there was no difference in the cancer cell killing effect before and after ultrasound treatment. In particular, the 004 composition liposome according to the present invention was found to have an anticancer effect about twice as high as that of Doxil liposome. The above results show that the ultrasound liposome for penetrating the blood-brain barrier according to the present invention has an excellent anticancer effect on brain tumor cells.

Example 6. Exploration of microbubble conditions for opening the blood-brain barrier and comparative analysis of Evans blue penetration efficiency according to the conditions

To derive the optimal ultrasound conditions for opening the blood-brain barrier (BBB), the degree of BBB opening was compared and analyzed using Evans blue. Evans blue is a very small dye with a molecular weight of 961 Da and is widely used to analyze the degree of BBB opening. The experiment was performed with normal BALB/c nude mice. After intravenous injection of microbubbles, ultrasound was emitted using VIFU2000, a high-intensity focused ultrasound device. After temporary opening of the BBB through cavitation of ultrasound and microbubbles, Evans blue (3%, w/w) was immediately intravenously injected into the body, and the brain was removed 24 hours later. Before brain removal, blood was drained through normal saline perfusion to remove variables that could be caused by blood. Brain tissue was immersed in formamide and incubated overnight at 70°C to extract Evans blue within the brain tissue. The degree of blood-brain barrier opening was quantitatively analyzed by measuring the absorbance at wavelengths of 670 nm and 740 nm using UV-vis.

Factors that can affect the cavitation phenomenon of ultrasound and microbubbles, which is the mechanism for opening the blood-brain barrier, include ultrasound conditions such as ultrasound frequency, intensity, duty cycle, and ultrasound emission time, and the number of microbubbles also has an effect. Therefore, in order to confirm the ultrasound conditions and the number of microbubbles that can open the blood-brain barrier, the degree of opening of the blood-brain barrier according to the number of microbubbles was first quantitatively analyzed. Table 8 below shows the design of a comparative experiment for the degree of opening of the blood-brain barrier according to the number of microbubbles.

Experiment to determine microbubble conditions for opening the blood-brain barrier Experiment No. Ultrasound Microbubble
(MB: SonoVue) DC, PRF Time
/spot Power(w) 1 Freq. 1 MHz
DC : 5%
PRF : 1 Hz 60s 1 2×10 ⁶ /g dog 2 5×10 ⁶ /g each 3 10×10 ⁶ /g pieces

The results of the analysis of the penetration efficiency of Evans blue accumulated in the brain through the blood-brain barrier under each open condition of Table 8 above are shown in Fig. 4a. As can be seen in the image and graph of Fig. 4a, as the number of microbubbles increased, the degree of opening of the blood-brain barrier increased, which improved the penetration efficiency of Evans blue. The results indicate that as the number of microbubbles increased, the number of microbubbles that resonate with ultrasound increased, which resulted in cavitation, which increased the penetration efficiency of the dye into the brain tissue. In addition, as a result of performing H&E staining on the brain tissue penetrated by Evans blue, it was confirmed that there was no toxicity due to Evans blue penetration into the brain tissue (Fig. 4b).

Example 7. Exploration of ultrasound conditions for opening the blood-brain barrier and comparative analysis of Evans blue penetration efficiency according to the conditions

Continuing from Example 6, in order to confirm the ultrasonic conditions for opening the blood-brain barrier, the penetration efficiency of Evans blue according to the ultrasonic conditions (intensity, frequency, and duty cycle) and ultrasonic treatment time was quantitatively analyzed. Table 9 below shows the comparative experimental design of the degree of blood-brain barrier opening according to the ultrasonic conditions. The number of injected microbubbles was standardized to 5×10 ⁶ /g.

Experimental confirmation of ultrasound conditions for opening the blood-brain barrier Experiment No. Power (W) PRF (Hz) Duty cycle (%) Times (s) 1 1 1 1 60 2 120 3 180 4 5 60 5 10 6 2 1 7 3

After intravenous administration of microbubbles, ultrasound treatment was performed immediately, and after completion of the ultrasound treatment, 3% (w/w) Evans blue was administered intravenously. After 24 hours, the brain was removed from which blood was completely removed through saline perfusion. Quantitative analysis of Evans blue was performed in the same manner as in Example 5.

The results of the analysis of the penetration effect of Evans blue according to the ultrasound conditions are shown in Fig. 5a. As shown in the figure, as the intensity and emission time of the ultrasound increased, the overall blood-brain barrier opening effect increased, and the penetration effect of Evans blue also increased proportionally. Even in the experimental group treated with the lowest intensity ultrasound (Experimental Group 1), Evans blue effectively penetrated into the tissue at the site exposed to ultrasound, and continuously increased as the exposure time increased. In addition, the penetration effect of Evans blue increased as the intensity and duty cycle of the ultrasound increased.

Quantitative analysis results showed that increasing the ultrasound intensity was a more important factor in enhancing the penetration effect of Evans blue than increasing the ultrasound emission time. In addition, increasing the duty cycle showed a similar pattern of increasing Evans blue penetration efficiency as increasing the ultrasound exposure time (Fig. 5b).

However, since some individuals in the above experimental groups died, H&E staining was additionally performed to evaluate the brain tissue stability under each ultrasound condition to determine whether the brain tissue penetration of Evans blue was caused by the safe temporary opening of the blood-brain barrier or by damage to the brain tissue. As a result, as shown in Fig. 5c, in the experimental groups exposed to high-intensity ultrasound (experimental groups 6 and 7), bleeding was observed in a significantly smaller area compared to the total brain area and the ultrasound-exposed area, whereas no bleeding occurred in the experimental groups exposed to low-intensity ultrasound (experimental groups 1 to 5). Based on the above results, it was confirmed that the safe ultrasound conditions that do not induce brain tissue damage are the ultrasound conditions of experimental groups 1, 2, and 6.

Example 8. Analysis of blood-brain barrier penetration efficiency of ultrasound-responsive liposomes for blood-brain barrier penetration

In order to analyze the blood-brain barrier penetration efficiency of the ultrasound-responsive liposome according to the present invention, the liposomes were labeled with a near-infrared (NIR) wavelength DiD fluorescent dye, and fluorescence analysis was performed using an in vivo Imaging System (IVIS). In order to open the blood-brain barrier, ultrasound was emitted for 60 seconds under the ultrasound conditions derived from Example 7, which are 1 W, 1%-duty cycle, and 1 Hz-PRF. The opening of the blood-brain barrier was performed in the same manner as described in Examples 6 and 7, and each liposome was intravenously injected immediately after the end of the ultrasound emission. Twenty-four hours after the liposome injection, each organ was extracted and fluorescence photography was performed using IVIS. By analyzing the fluorescence images and quantitatively analyzing the fluorescence intensity for each organ, the most optimal liposome composition for blood-brain barrier penetration was searched.

Among the liposomes used in the experiment, except for the 004 composition liposomes, the remaining liposomes contain sphingomyelin, a representative type of sphingolipid. Sphingomyelin is known to be able to penetrate the blood-brain barrier, and is known to be capable of long-term circulation in the bloodstream and have a long half-life. Therefore, in order to find liposomes with the optimal composition that can effectively pass through the blood-brain barrier and accumulate in the brain, the blood-brain barrier penetration efficiency of liposomes according to the composition ratio of sphingomyelin was compared and analyzed. The composition of each liposome used in the experiment is as shown in Table 10 below.

Composition and ratio (molar ratio, %) of liposomes used in blood-brain barrier penetration experiments Lipid 004 005 006 007 011 Marqibo DOPE 65 45 50 10 30 - sphingomyelin - 45 50 90 70 60 DSPE-mPEG2k 5 10 - - - - Cholesterol 30 - - - - 40 DSPC 10 - - - - - lyso-PC 5 - - - - -

Liposomes manufactured with the composition according to Table 10 were each injected into normal mice, and ultrasound was emitted under the ultrasound conditions of 1 W, 1% DC, and 60 s, which were confirmed to be safe conditions for brain tissue in Example 7. The results of blood-brain barrier penetration efficiency and organ distribution for each liposome are shown in Figure 6.

As a result of analyzing the distribution pattern by organ, the liposomes used in this experiment showed a common tendency to accumulate the most in the liver, and were also accumulated to a significant level in the spleen. The above results are thought to be because the liver recognizes liposomes as foreign substances and excretes them from the body, as the liposomes have a size of about 100 to 200 nm. In addition, it has been reported in previous studies that DOPE tends to accumulate in the spleen.

First, when observing the brain of the experimental group that did not emit ultrasound, liposomes of all compositions were not detected. In other words, it was shown that the penetration of liposomes into the brain tissue was inhibited by the blood-brain barrier. This is because the blood-brain barrier generally actively permeates only substances at the level of small molecules, so it is thought that liposomes with a large size of about 100 to 200 nm could not pass through the blood-brain barrier despite the permeation function of sphingomyelin.

On the other hand, when the opening of the blood-brain barrier was induced using ultrasound and microbubbles, liposomes were detected in the area exposed to ultrasound regardless of the composition of the liposomes. The increase in delivery efficiency into brain tissue according to the increase in the content of sphingomyelin was not particularly observed. Compared with the blood-brain barrier penetration effect of sphingomyelin known previously, the liposomes according to the present invention were about 2 to 3 times larger in size. Liposomes having a size of about 20 to 50 nm have the possibility of penetrating the blood-brain barrier, but liposomes measuring up to 100 nm do not easily penetrate the blood-brain barrier. To overcome this, when the opening of the blood-brain barrier is induced, it is judged that the opened path of the blood-brain barrier should show a gap of 100 nm or more.

In addition, in order to confirm the effect of sphingomyelin on the blood-brain barrier penetration effect of liposomes, liposomes of composition 004 without added sphingomyelin and liposomes of composition 005 containing other sphingomyelins were compared and analyzed, and it was confirmed that they penetrated the blood-brain barrier at a similar level (Fig. 7).

In addition, a comparative experiment of liposomes of compositions 004 and 005 was conducted by setting the duty cycle to 1 or 5% among the ultrasonic conditions. Other conditions were set to the safe ultrasonic conditions confirmed in Example 7. As a result, as can be confirmed in Fig. 7, it was found that a 5% duty cycle improved the permeation efficiency of both liposomes more than a 1% duty cycle. In addition, as the duration time increased, the fluorescence intensity increased, indicating that the blood-brain barrier permeation effect of the liposomes was improved. The above results show that the blood-brain barrier permeation rate of liposomes can be further increased as the duty cycle degree and ultrasonic duration increase.

Example 9. Comparison of blood-brain barrier penetration effects of ultrasound-responsive liposomes and Doxil liposomes for blood-brain barrier penetration

In order to compare the brain delivery effects of ultrasound-responsive liposomes for blood-brain barrier penetration according to the present invention and non-responsive liposomes, Doxil, liposomes having a composition of 004 and liposomes having a composition of Doxil were prepared and a comparative experiment was conducted. The experimental method was performed in the same manner as in the above example, and the degree of brain tissue delivery was quantitatively analyzed by measuring fluorescence intensity with IVIS.

The experimental results are shown in Fig. 8. Both the ultrasound-responsive liposome (004 composition liposome) according to the present invention and the non-responsive liposome, Doxil, detected fluorescence at the site where ultrasound was emitted, confirming that both liposomes penetrated the blood-brain barrier due to the cavitation of ultrasound and microbubbles. However, in the case of the ultrasound-responsive liposome according to the present invention, the delivery effect to the brain was shown to be improved by nearly twice compared to the Doxil liposome. The above results show that the ultrasound-responsive liposome for penetrating the blood-brain barrier according to the present invention not only has excellent ultrasound sensitivity, but also has a higher penetration effect into brain tissue, so that it can be delivered to brain tissue more effectively.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (26)

An ultrasound-responsive liposome comprising, as an active ingredient, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and lysophosphatidylcholine (lyso-PC),
A composition for penetrating the blood-brain barrier, characterized in that the DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lyso-PC are contained in a molar ratio (mole %) of 8 to 12: 3 to 6: 50 to 66: 10 to 32: 5 to 11. delete In the first paragraph,
The above DSPC is 0.1 to 50% by dry weight relative to the total liposome,
The above DSPE-mPEG2000 is 3 to 50% by dry weight relative to the total liposome.
The above DOPE is 1 to 80% by dry weight relative to the total liposome,
The cholesterol is present in an amount of 0.05 to 40% by dry weight relative to the total liposome, and
A composition for penetrating the blood-brain barrier, characterized in that the lyso-PC is contained in an amount of 0.5 to 10% by dry weight relative to the total liposome.
In the first paragraph,
A composition for penetrating the blood-brain barrier, characterized in that the ultrasound-responsive liposome further comprises at least one selected from the group consisting of sphingolipids and polysorbates.
delete delete delete delete delete In the first paragraph,
A composition for penetrating the blood-brain barrier, characterized in that the ultrasound-responsive liposome passes through the blood-brain barrier when exposed to ultrasound.
delete delete delete delete In the first paragraph,
A composition for penetrating the blood-brain barrier, characterized in that the ultrasound-responsive liposome is hydrated with ammonium sulfate, ammonium citrate, or triethylammonium sucrose octasulfate (TEA-SOS). In the first paragraph,
A composition for penetrating the blood-brain barrier, characterized in that the composition is administered sequentially or simultaneously with sonication.
In Article 16,
The composition for penetrating the blood-brain barrier is characterized in that the ultrasound satisfies one or more characteristics selected from the group consisting of:
(a) the frequency of the ultrasound is 20 kHz to 3 MHz; and
(b) Duty cycle is 0.5 to 20%.
In Article 16,
A composition for penetrating the blood-brain barrier, characterized in that the sonication is performed sequentially or simultaneously with the administration of microbubbles.
An ultrasound-responsive liposome comprising, as an active ingredient, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and lysophosphatidylcholine (lyso-PC),
A drug delivery vehicle for penetrating the blood-brain barrier, characterized in that the DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lyso-PC are contained in a molar ratio (mole%) of 8 to 12: 3 to 6: 50 to 66: 10 to 32: 5 to 11. A method for producing a composition for penetrating the blood-brain barrier of claim 1, comprising the following steps:
(S1) A step of dissolving DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lyso-PC in a first organic solvent at a molar ratio (mole%) of 8 to 12: 3 to 6: 50 to 66: 10 to 32: 5 to 11;
(S2) a step of evaporating the organic solvent to prepare a liposome thin film; and
(S3) A step of hydrating the above liposome film with an aqueous solution. In Article 20,
A manufacturing method, characterized in that the first organic solvent is at least one selected from the group consisting of dimethylacetamide, dimethylformamide, dimethyl sulfoxide, chloroform, methanol, ethanol, and ether.
In Article 20,
A manufacturing method, characterized in that the first organic solvent of the above step (S1) has polysorbate added thereto. In Article 19,
The above ultrasound-responsive liposome is a drug delivery vehicle for penetrating the blood-brain barrier, which satisfies one or more characteristics selected from the group consisting of:
(a) particle size of 100 to 200 nm; and
(b) The ratio of the drug loaded into the liposome to the total drug added is 50-100%. In Article 19,
The above drug delivery vehicle is a drug delivery vehicle for penetrating the blood-brain barrier for delivering drugs into the brain. In Article 19,
The above drug is a treatment for brain disease,
A drug delivery system for penetrating the blood-brain barrier, characterized in that the brain disease is at least one selected from the group consisting of brain tumor, bacterial or viral brain infection, Parkinson's disease, encephalitis, stroke, apoplexy, Alzheimer's disease, Lou Gehrig's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, epilepsy, thrombosis, embolism, cerebral infarction, cerebral apoplexy, small infarction, and cerebral circulation metabolic disorder. In Article 25,
The above brain disease treatment agents include vincristine, vinblastine, vinflunine, vindesine, vinorelbine, temozolomide, carmustine, lomustine, cabazitacel, docetaxel, larotaxel, ortataxel, paclitaxel, tesetaxel, isabepilone, lomustine, procarbazine, rituximab, tocilizumab, temozolomide, carboplatin, erlotinib, irinotecan, enzastaurin, vorinostat, doxorubicin, cisplatin, 5-fluorouracil, tamoxifen, topotecan, belotecan, imatinib, floxuridine, gemcitabine, leuprolide, flutamide, zoledronate, methotrexate, camptothecin, hydroxyurea, streptozocin, valrubicin, A drug delivery system for penetrating the blood-brain barrier, characterized in that at least one selected from the group consisting of retinoic acid, mechlorethamine, chlorambucil, busulphan, doxifluridine, mitomycin, prednisone, afinitor, mitoxantrone, levodopa, carbidopa, entacapone, tolcapone, dopamine agonists, donepezil, galantamine, rivastigmine, memantine, anticholinergics, and amantadine.