Bubble preparation, and tumor suppression and drug delivery system each using bubble preparation

Abstract

[Problem] To provide a bubble preparation capable of significantly suppressing a tumor and efficiently delivering a drug or the like to a site such as a tumor when used together with low-intensity therapeutic ultrasound. [Solution] A bubble preparation which exhibits a tumor suppressing effect when low-intensity therapeutic ultrasound is applied to the bubble preparation, the bubble preparation being characterized by comprising a coating film, which comprises a lipid, and a gas enclosed in the lumen of the coating film, wherein the coating film is an anionic lipid containing at least distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), and the gas contains at least one of perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohexane, or a mixed gas.

Description

Bubble formulations and tumor suppression and drug delivery systems using bubble formulations

[0001] The present invention relates to a bubble formulation suitable for sonoporation systems and a sonoporation system using the bubble formulation.

[0002] Microbubble formulations exist that are specifically designed for both cancer-targeted diagnosis and treatment (Patent Document 1). These formulations utilize microbubbles composed of a lipid shell and a gas core. Specifically, they use shells made from a mixture of distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG). Patent Document 1 shows that a specific molar ratio of these lipids (22-60:30-68:4-12) is important for achieving both bubble stability and lifetime when exposed to ultrasound. This stability allows for long-term imaging of the target area using diagnostic ultrasound at frequencies from 3.0 MHz to 20 MHz.

[0003] On the other hand, systems that utilize ultrasonic frequencies to induce ultrasonic penetration of drugs into tumor cancer cells are known, and in particular, the system described in Patent Document 2 includes a generator for supplying electrical energy at ultrasonic frequencies and at least one ultrasonic probe configured to convert the electrical energy into low-intensity, non-focused pulsed ultrasound defined by operating parameters including ultrasonic frequency and duty cycle. An input device allows an operator to input configuration data including tumor type and drug type. A processor determines the values ​​of the operating parameters based on the input configuration data. The frequency value is determined based at least on the tumor type, and the duty cycle value is determined based at least on the drug type and tumor type. The processor is configured to control the generator and ultrasonic probe to operate according to the determined values.

[0004] Re-table 2016 / 199430WO2019 / 123411

[0005] Patent Document 1 describes an anionic lipid solution containing DSPC:DSPG:DSPE-PEG2000-OMe in a molar ratio of 30:60:10, which is then homogenized while in contact with perfluorocarbon gas, followed by freeze-drying to produce a powder. This powder is then suspended and administered intratumorally to C57BL / 6j mice transplanted with mouse melanoma cells, and the antitumor effect is evaluated by irradiating with ultrasound. The results show that under ultrasound conditions (1 MHz, 4 W / cm²), the antitumor effect was evaluated. 2 Excellent antitumor effects were confirmed under 120 seconds. However, further antitumor effects need to be confirmed in other tumors and deep within the body, and safety evaluations, including long-term side effects and effects on normal tissues, are also required.

[0006] To solve the above problems, we propose the following invention.

[0007] A bubble formulation that exerts a tumor-suppressing effect by applying low-intensity therapeutic ultrasound, wherein the bubble formulation comprises a lipid-based film and a gas sealed within the lumen of the film, the film is an anionic lipid containing at least distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), and the gas contains at least one or a mixture of perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohexane.

[0008] The bubble formulation described above, wherein the coating is an anionic lipid formed from distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG) in a molar ratio of 22-60:30-68:4-12.

[0009] The coating is an anionic lipid formed from distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG) in a molar ratio of 30:60:10, and the gas is perfluoropropane. The bubble preparation described above.

[0010] The low-intensity therapeutic ultrasound has an energy intensity of 0.03 to 50 W / cm 2 of the bubble preparation described above.

[0011] The bubble preparation described above has a diameter of 500 nm to 5 μm.

[0012] The coating is DSPC, DSPG, and a DSPE-PEG derivative, and the DSPE-PEG derivative is DSPE-PEG-OMe, DSPE-PEG-MAL, DSPE-PEG-COOH, DSPE-PEG-NHS, or DSPE-PEG-NH2. The bubble preparation described above.

[0013] A tumor treatment system comprising an ultrasonic probe that generates low-intensity therapeutic ultrasound and a bubble preparation that exhibits a tumor suppression effect by applying the low-intensity therapeutic ultrasound. The bubble preparation consists of a lipid coating and a gas enclosed in the lumen of the coating. The coating is an anionic lipid containing at least distearoyl phosphatidylcholine (DSPC) and distearoyl phosphatidylglycerol (DSPG), and the gas contains at least one of perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohexane or a mixed gas. A tumor treatment system characterized by this.

[0014] Further, the ultrasonic probe is provided with a control unit for generating ultrasound with a low-intensity non-invasive energy intensity. The tumor treatment system described above.

[0015] The tumor treatment system described above is characterized by being used in combination with an anticancer agent.

[0016] The tumor treatment system described above, wherein the anticancer agent is a cytotoxic anticancer agent.

[0017] The tumor treatment system described above, wherein the frequency of the low-intensity therapeutic ultrasound is 1 to 3 MHz.

[0018] The tumor treatment system described above, wherein the peak negative pressure (PNP) at the tumor site is 0.08 to 0.7 MPa.

[0019] The total irradiation energy of the low-intensity therapeutic ultrasound is 4 × 10⁻¹⁰ 0 ~4×10 4 mJ / cm 2 The tumor treatment system described above.

[0020] The tumor treatment system described above, wherein the anticancer agent is gemcitabine, paclitaxel, doxorubicin, irinotecan, or cisplatin.

[0021] The tumor treatment system described above, wherein the tumor is a solid cancer.

[0022] The tumor treatment system described above, wherein the solid tumor is pancreatic cancer, bladder cancer, breast cancer, or ovarian cancer.

[0023] A drug delivery system comprising an ultrasound probe that generates low-intensity therapeutic ultrasound and a bubble formulation that exerts a tumor-suppressing effect by applying low-intensity therapeutic ultrasound, wherein the bubble formulation comprises a lipid coating and a gas sealed in the lumen of the coating, the coating is an anionic lipid containing at least distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), and the gas contains at least one or a mixture of perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohexane.

[0024] Furthermore, the drug delivery system described above comprises a control unit for generating low-intensity, non-invasive ultrasound waves on the ultrasound probe.

[0025] The drug delivery system described above, characterized in that it is used in combination with an anticancer drug.

[0026] The drug delivery system described above, wherein the anticancer agent is a cytotoxic anticancer agent.

[0027] The drug delivery system described above, wherein the anticancer drug is gemcitabine, paclitaxel, doxorubicin, irinotecan, or cisplatin.

[0028] The drug delivery system described above, wherein the frequency of the low-intensity therapeutic ultrasound is 1 to 3 MHz.

[0029] The drug delivery system described above, wherein the peak negative pressure (PNP) at the tumor site is 0.08 to 0.7 MPa.

[0030] The total irradiation energy of the low-intensity therapeutic ultrasound is 4 × 10⁻¹⁰ 0 ~4×10 4 mJ / cm 2 The drug delivery system described above.

[0031] The drug delivery system described above, wherein the tumor is a solid tumor.

[0032] The drug delivery system described above, wherein the solid tumor is pancreatic cancer, bladder cancer, breast cancer, or ovarian cancer.

[0033] The use of the bubble formulation described above for the manufacture of a pharmaceutical product that exerts a tumor-suppressing effect by applying low-intensity therapeutic ultrasound, characterized in that it is used in combination with an anticancer agent.

[0034] By using the bubble formulation described in this disclosure together with low-intensity therapeutic ultrasound, it becomes possible to significantly suppress tumors and efficiently deliver drugs and other substances to tumor sites.

[0035] Figure 1 shows the antitumor effect under the combined use of bubble formulation and low-intensity therapeutic ultrasound. Figure 2 shows the antitumor effect under the combined use of bubble formulation and low-intensity therapeutic ultrasound. Figure 3 shows the antitumor effect under the combined use of bubble formulation and low-intensity therapeutic ultrasound. Figure 4 shows the effect of bubble formulation concentration on the antitumor effect as a change in tumor volume over time. Figure 5 shows the drug concentration in tumor tissue with the combined use of bubble formulation and low-intensity therapeutic ultrasound. Figure 6 shows the antitumor effect of various treatments. Figure 7 shows the tumor volume suppression rate of gemcitabine with the combined use of bubble formulation and low-intensity therapeutic ultrasound. Figure 8 shows the antitumor effect of the combined use of bubble formulation and low-intensity therapeutic ultrasound as a change in tumor volume over time. Figure 9 shows the effect of the combined use of bubble formulation and low-intensity therapeutic ultrasound on the drug concentration in tumor tissue. Figure 10 shows the effect of the combined use of bubble formulation and low-intensity therapeutic ultrasound on the drug concentration in tumor tissue. Figure 11 shows the effect of differences in ultrasound irradiation conditions on the drug concentration in tumor tissue. Figure 12 shows the antitumor effect of the present invention against pancreatic cancer. Figure 13 shows the antitumor effect of the present invention against bladder cancer. Figure 14 shows the antitumor effect of the present invention against breast cancer. Figure 15 shows a list of ultrasound irradiation conditions for the examples. Figure 16 shows a list of anticancer drug combinations and dosages for the examples.

[0036] The present invention will be described below, with illustrative embodiments as examples, along with preferred methods and materials that may be used in carrying out the invention. Unless otherwise specified herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. Any materials and methods equivalent to or similar to those described herein may be used in carrying out the invention. Furthermore, all publications and patents cited herein in connection with the invention described herein constitute part of this specification, for example, as indicating methods, materials, and other things that may be used in the invention. In this specification, antitumor effect or tumor suppression effect includes the effect of reducing tumors.

[0037] <Bubble Formulation> The bubble formulation according to this embodiment consists of a lipid film and a gas sealed in the lumen of the film.

[0038] The coating comprises at least distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), and may further contain 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG). When the coating consists of distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), it is formed in a molar ratio of 9:16 to 3:7, and when it also contains DSPE-PEG, it is an anionic lipid formed in a molar ratio of 22 to 60:30 to 68:4 to 12.

[0039] The gas is a perfluorocarbon gas, specifically containing at least one of perfluoropropane, perfluoroisobutane, and perfluoron-butane, or a mixture thereof. Perfluoropropane is particularly preferred in Japan because it is available for medical use. Other perfluorocarbon gases may also be included, specifically, for example, perfluoromethane, perfluoroethane, perfluoroisooctane, perfluoron-octane, and sulfur hexafluoride. The pressure of the gas being filled is, for example, approximately 0.1 to 1.0 MPa.

[0040] The bubble formulation according to the present invention consists of minute bubbles in which hydrophobic fluorocarbon gas is encapsulated by phospholipids and PEG-lipids, and exists in a liquid as bubble-like bodies with a diameter of 500 nm to 5 μm. The diameter of the bubble formulation is measured, for example, by dynamic light scattering or Coulter counter.

[0041] The coating is formed by combining DSPC, DSPG, and DSPE-PEG in a molar ratio of 22-60:30-68:4-12, which allows it to exhibit sustained resonance and vibration without disintegration upon irradiation with diagnostic ultrasound, thereby enabling long-duration continuous ultrasound imaging.

[0042] Furthermore, in addition to DSPE-PEG, the following phospholipids can be used for DSPC, DSPG, and DSPE-PEG that constitute the coating. Specifically, phosphatidylethanolamines such as dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleyl-phosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), diarachidoyl-phosphatidylethanolamine (DAPE), or dilinoleyl-phosphatidylethanolamine (DLPE); dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidyl ethanolamine (DOPE), dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidyl ethanolamine (DOPE), dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidyl ethanolamine (DOPE), dilauroyl-phosphatidylcholine (DLPC), dilauroyl-phosphatidylcholine (DLPC), dilauroyl-phosphatidylcholine (DLPC), dilauroyl-phosphatidyl ethanolamine (DOPE), dilauroyl-phosphatidyl ethanolamine (DOPE), dilauroyl-phosphatidylcholine (DLPC Phosphatidylcholines such as sphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), diarachidoyl-phosphatidylcholine (DAPC), or dioleyl-phosphatidylcholine (DOPC); phosphatidylserine such as dimyristoyl phosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS), dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylserine (DSPS), and dioleyl phosphatidylserine (DOPS) Tidylserine; Phosphatidic acid derivatives such as dipalmitoylphosphatidic acid (DPPA), dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), diarachidoylphosphatidic acid (DAPA) and its alkali metal salts; dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts, distearoylphosphatidylglycerol (DSPG) and its alkali metal salts Examples include phosphatidylglycerols such as potassium metal salts and dioleyl-phosphatidylglycerol (DOPG); and phosphatidylinositols such as dilauroyl phosphatidylinositol (DLPI), diarachidoyl phosphatidylinositol (DAPI), dimyristoyl phosphatidylinositol (DMPI), dipalmitoyl phosphatidylinositol (DPPI), distearoyl phosphatidylinositol (DSPI), and dioleyl phosphatidylinositol (DOPI).

[0043] In addition, among the DSPC, DSPG, and DSPE-PEG that constitute the coating, other examples besides DSPE-PEG include stearyl PEG, palmitoyl PEG, oleate PEG, myristyl PEG, and lauroyl PEG.

[0044] The molecular weight of PEG can range from 500 to 12000. DSPE-PEG is preferably DSPE-PEG2000, DSPE-PEG3000, or DSPE-PEG5000, and particularly preferably DSPE-PEG2000.

[0045] Furthermore, in addition to DSPC, DSPG, and DSPE-PEG, the coating can also contain the following phospholipids: phosphatidylcholines such as dimyristoyl phosphatidylcholine (DMPC), dilauroyl phosphatidylcholine (DLPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine (DOPC), and dilinoleoyl phosphatidylcholine; phosphatidylglycerols such as dilauroyl phosphatidylglycerol (DLPG), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylglycerol (DOPG), and dilinoleoyl phosphatidylglycerol; and dilauroyl phosphatidylethanolamine (DLPE). Phosphatidylethanolamines such as diphosphophosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), and dilinoleoylphosphatidylethanolamine; and phosphatidylcholines such as dilauroylphosphatidylserine (DLPS), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), and dilinoleoylphosphatidylserine.

[0046] Furthermore, the coating can also contain, in addition to the phospholipids mentioned above, glyceroglycolipids and sphingoglycolipids. Examples of glyceroglycolipids include sulfoxyribosylglycerides, diglycosyldiglycerides, digalactosyldiglycerides, galactosyldiglycerides, and glycosyldiglycerides. Examples of sphingoglycolipids include galactosylcerebrosides, lactosylcerebrosides, and gangliosides.

[0047] The lipid surface of the film can also be modified with ligands targeting target cells, target tissues, or target lesions. Examples of ligands include arginine-glycine-aspartic acid (RGD) sequence peptides and Sigma Protein as ligands for thrombi, and transferrin, folic acid, hyaluronic acid, galactose, or mannose as ligands for cancer cells. Monoclonal antibodies and polyclonal antibodies can also be used as ligands. Preferably, RGD peptides are used, as RGD peptides have the function of specifically binding to cell adhesion factors present on specific cells or thrombi. Therefore, modifying the lipid surface of the film with RGD peptides makes it possible to further facilitate the visualization of thrombi.

[0048] To modify the lipid surface of the coating with ligands, the coating is preferably composed of DSPC, DSPG, and DSPE-PEG derivatives. For example, DSPE-PEG-OMe, DSPE-PEG-Maleimide (MAL), DSPE-PEG-Carboxylic Acid (COOH), DSPE-PEG-N-Hydroxysuccinimide (NHS), or DSPE-PEG-NH2 are preferred.

[0049] The use of DSPE-PEG derivatives in the coating is not essential in the bubble formulation of the present invention.

[0050] Other substances may be added to the coating as needed. For example, the coating may contain sitosterol, cholesterol, dihydrocholesterol, cholesterol esters, phytosterol, stigmasterol, campesterol, cholestanol, lanosterol, 1-O-sterol glucoside, 1-O-sterol maltoside, and mixtures thereof as film stabilizers.

[0051] The bubble formulation for theranostics according to the present invention may also contain or surface-adsorb a drug within its coating. The drug is preferably one that is desired to be maintained at a long-term blood concentration or one that requires targeted administration to specific disease sites or cells. The drug is not particularly limited, but examples include anticancer drugs, antibiotics, anti-asthmatics, antithrombotic drugs, central nervous system drugs, antiparasitic drugs, immunostimulants, peptide drugs, antiviral drugs, and nucleic acid drugs. These drugs can be used together with the bubble formulation for theranostics according to the present invention while bound to a carrier. Examples of carriers include, but are not limited to, LNPs, liposomes, polymer micelles, ADCs, and viral vectors (AAVs).

[0052] In the present invention, the anticancer agent does not necessarily need to be encapsulated within the bubble formulation's coating or adsorbed onto its surface. The tumor-suppressing and drug delivery-promoting effects of the present invention are also exerted when the anticancer agent is administered independently of the bubble formulation and exists in the bloodstream or in the extra-tissue space surrounding the tumor. In other words, the effects of the present invention do not depend on the physical encapsulation of the anticancer agent, but are based on a mechanism in which the permeability of tumor blood vessels and tumor stroma is transiently and reversibly increased by irradiation with low-intensity therapeutic ultrasound, and the bubble formulation, composed of anionic lipids, mechanically responds to this, thereby efficiently transferring the anticancer agent present in the blood vessels into the tumor tissue.

[0053] Anticancer drugs are not limited to a specific category, but examples include doxorubicin, gemcitabine, paclitaxel, cisplatin, mitomycin, bleomycin, 5-fluorouracil, methotrexate, nitrogen mustard, busulfan, oxaliplatin, taxol, and camptothecin. Antibiotics include sulfazene, gentamicin, and streptomycin. Antiasthmatic drugs include theophylline. Antithrombotic drugs include tPA, heparin, low molecular weight heparin, urokinase, thrombomodulin, and streptokinase. Central nervous system drugs include Alzheimer's drugs such as donepezil and memantine. Antiparasitic drugs include meglumine antimonate. Immunostimulants include muramil peptides. Peptide-based drugs include, for example, natural or recombinant α, β, and γ-interferons, interleukins, and superoxide dismutase. Other drugs include, for example, prostaglandins used to treat arteriosclerosis, NF-Kappa B and decoys for arterial occlusion and Vircha's disease, and nucleic acid drugs such as ASO, siRNA, and mRNA.

[0054] Furthermore, bubble formulations for theranostics can contain not only drugs but also genes, either internally or surface-adsorbed. Examples of genes include DNA, RNA, antisense DNA, siRNA, decoys, and therapeutic oligonucleotides.

[0055] <Method for producing the bubble formulation> The bubble formulation for theranostics according to the present invention can be produced as a bubble suspension by stirring a lipid solution with a homogenizer while in contact with perfluorocarbon gas. The production conditions vary depending on the scale and equipment of production, but are 1,000 to 100,000 rpm, 7,000 to 15,000 rpm, and a time of up to 120 minutes.

[0056] Furthermore, the bubble formulation for theranostics according to the present invention can be prepared not only by homogenizer, but also by, for example, shaking. For example, a bubble suspension can be produced by placing a liposome solution in a predetermined ratio into a vial and shaking it with a vial shaker. The bubble formulation prepared in this way will have variations in particle size. In a further embodiment of the present invention, the bubble particle size in such a bubble formulation can be made uniform for use.

[0057] The particle size of the bubble formulation is typically 500 nm to 5 μm in the embodiments described herein, but 1 to 4 μm is preferred. Depending on the composition of the outer lipid shell, when used within the ultrasonic intensity range necessary to induce oscillation, it is preferable to separate and use particles of 1 to 4 μm by fractional centrifugation. In other embodiments, it is also within the scope of the present invention to appropriately select and use particles of 2 to 3 μm or 3 to 5 μm.

[0058] <Lyophilized Powder> The lyophilized powder according to this embodiment is a lyophilized powder obtained by lyophilizing a suspension in which the bubble formulation for theranostics according to the present invention is suspended in a solution of a carbohydrate, which is trehalose or sucrose.

[0059] During freeze-drying, freeze-drying additives may be included for freeze protection and / or dispersion protection. Examples of freeze-drying additives include amino acids such as glycine, sugars such as mannitol, maltose, glucose, lactose, inulin, sucrose, trehalose, or cyclodextrin, polysaccharides such as dextran and chitosan, or polyoxyalkylene glycols such as polyethylene glycol. Buffer components may also be added to maintain optimal pH and osmotic pressure.

[0060] This freeze-dried powder allows for long-term storage of more than three months, and can be restored and used with water for injection and perfluorocarbon gases such as the aforementioned perfluoropropane.

[0061] <Method of Using Bubble Preparation> The bubble preparation according to the present invention is used as follows. That is, a suspension in which the bubble preparation according to the present invention is suspended in water for injection is introduced into the target tissue. The target tissue is not particularly limited, but for example, it is a cancer tissue.

[0062] <Ultrasonic Irradiation> The bubble preparation of the present invention can treat the target tissue by applying low-intensity ultrasonic waves to the target tissue. This frequency is, for example, 0.1 MHz to 7.0 MHz, 0.25 MHz to 2.0 MHz, and preferably 1 to 3 MHz. Under these conditions, cavitation occurs around the target tissue, and when the bubbles vibrate or the bubble preparation is crushed, a jet flow is generated by the release of a large pressure. In addition, in the blood vessels of the target tissue during low-intensity ultrasonic irradiation, an oscillation phenomenon of the bubble preparation occurs, and the permeability is enhanced by a local and reversible transient opening of the vascular endothelium. As a result, the migration of anticancer drugs, which are usually restricted from passing through the blood vessel wall, to the tumor stroma is promoted, and the drug concentration in the tumor increases temporarily and locally without increasing the systemic exposure while minimizing the impact on normal tissues, enhancing the antitumor effect of the anticancer drug. Furthermore, since the temperature of the irradiation site does not rise during low-intensity ultrasonic irradiation, a highly safe and minimally invasive treatment becomes possible. In the present invention, in order to appropriately control the behavior of bubbles and the transient opening of tumor blood vessels, the ultrasonic frequency (MHz), peak negative pressure (PNP Peak Negative Pressure: MPa), and the total irradiation energy (mJ / cm 2 ) are important as the main irradiation conditions. The "total energy irradiated to the tumor" means the amount of energy per unit area imparted to the tumor tissue by ultrasonic irradiation, and is defined based on a plurality of irradiation parameters including the peak negative pressure, irradiation time, duty ratio, and pulse repetition frequency (PRF) in the tumor. The total energy irradiated to the tumor (total irradiation energy) in the present invention is 4×10 0 ~4×10 4 mJ / cm 2It is preferable that the values ​​fall within this range. In particular, peak negative pressure (PNP) is an index that directly reflects the effective sound pressure after being affected by ultrasonic attenuation due to the depth of tumor tissue and individual differences, and can define the degree of mechanical response of the bubble and the degree of increased vascular permeability in tumor tissue with higher reproducibility compared to MI and Intensity, which are device display values. In this invention, it is preferable that the PNP falls within the range of 0.08 to 0.7 MPa. These parameters define the vibration mode of the bubble and the cavitation intensity, and directly affect the effect on the tumor, the degree of increased permeability of tumor blood vessels, and reversibility, so they are appropriately set in order to achieve both tumor treatment effect, drug delivery effect, and safety.

[0063] In the present invention, the effective dosage varies depending on the type of cancer and the patient's biological condition. However, the diameter of the bubbles in the bubble formulation prepared by the method disclosed herein can be appropriately selected within the range of 0.5 to 5 μm depending on the preparation method. In embodiments of the present invention, the preferred bubble diameter is 2 μm or 1 μm. Bubbles of different diameters may be present, or the bubble diameter may be adjusted as appropriate by filtering or other means. The number of bubbles in the bubble formulation can be 1 to 2 × 10⁻¹⁶. 5～12 It can be prepared in the range of / mL. Preferably 1 to 2 × 10 9～11 The value is / mL, and when 2μm bubbles are packed tightly, it is 1 to 2 × 10⁻¹⁶. 11 The effective dose in this disclosure is approximately 1 × 10 / mL, using mice as an example. 3～10 It can be adjusted as needed within the individual range.

[0064] As described above, multiple examples have confirmed that the combination of the bubble formulation according to the present invention and low-intensity therapeutic ultrasound exhibits tumor suppression and drug delivery enhancement effects based on a common mechanism of action, even when the type of anticancer drug, cancer type, tumor model, and administration conditions differ. Therefore, the present invention is not limited to a specific anticancer drug or a specific tumor model, but is a technology that can be widely applied as a pharmaceutical application in combination with anticancer drugs.

[0065] <Preparation of Bubble Formulation> A bubble suspension was prepared by stirring an anionic lipid solution of DSPC, DSPG, and DSPE-PEG2000-OMe in a molar ratio of 22:68:10 using a homogenizer (15000 rpm, 5 minutes) while in contact with perfluoropropane gas (gas flow rate of 12 mL / min).

[0066] Next, a bubble suspension was prepared in the same manner as above, except that the molar ratios of DSPC, DSPG, and DSPE-PEG2000-OMe in the anionic lipid solution were changed as follows.

[0067] DSPC:DSPG:DSPE-PEG2000-OMe = 30:60:10 DSPC:DSPG:DSPE-PEG2000-OMe = 36:54:10 DSPC:DSPG:DSPE-PEG2000-OMe = 45:45:10 DSPC:DSPG:DSPE-PEG2000-OMe = 54:36:10 DSPC:DSPG:DSPE-PEG2000-OMe = 60:30:10 As a comparative example, bubble suspensions were prepared in the same manner as above, except that the molar ratios of DSPC, DSPG, and DSPE-PEG2000-OMe in the anionic lipid solution were changed as follows.

[0068] DSPC:DSPG:DSPE-PEG2000-OMe=0:90:10 DSPC:DSPG:DSPE-PEG2000-OMe=90:0:10

[0069] <Evaluation of antitumor effect against pancreatic cancer> A bubble suspension was prepared by stirring an anionic lipid solution of DSPC:DSPG:DSPE-PEG2000-OMe = 30:60:10 (molar ratio) in a homogenizer (15000 rpm, 5 min) while in contact with perfluoropropane gas (flow rate 12 mL / min). The lipid solution was prepared by mixing 20 mL of the anionic lipid solution liquid in a 50 mL tube. Then, 2 mL of this bubble suspension was dispensed into a 5 mL vial and lyophilized to obtain a lyophilized powder. The vial was refilled with perfluoropropane gas, capped, and the lyophilized formulation was stored at room temperature. 2 mL of ultrapure water (2 mL MilliQ) was added to this lyophilized powder to suspend it, and it was used for the test. The amount of bubble particles varied due to variations in the manufacturing and suspension procedures, but the average number of bubbles was 2.3 × 10⁶. 9 The bubble density was 6.6 μL / mL on average (bubble diameter 1–30 μm), and the bubble volume was 6.6 μL / mL (measurement range: bubble diameter 1–30 μm).

[0070] Next, human-derived pancreatic cancer cells (BxPC-3 cells) 1 × 10 6 Cells / 100μL / mice were transplanted subcutaneously into the posterior dorsal region of BALB / c-nu / nu mice (female, 6 weeks old). Treatment was initiated on the same day in mice in which tumor formation was confirmed 14 days after transplantation. Gemcitabine and nab-paclitaxel (Gem 50mg / kg + NPT 5mg / kg) were used as anticancer drugs. With the treatment initiation day as day 0, 10μL of the bubble formulation, filtered through a 5μm filter, was administered intraveinally in the tail vein four times at 2.5-minute intervals after administration of anticancer drugs on days 0, 4, 8, and 12. Ultrasound was continuously applied percutaneously to the tumor immediately after the first administration of the bubble formulation.

[0071] The experimental groups were: Non-treatment group, gemcitabine and nab-paclitaxel combined treatment group (Gem 50 mg / kg + NPT 5 mg / kg), bubble formulation + ultrasound irradiation group (MB + US), gemcitabine and nab-paclitaxel combined treatment group, and bubble formulation + ultrasound irradiation group (MB + US + Gem 50 mg / kg + NPT 5 mg / kg).

[0072] Treatment was administered a total of four times: on the day the experiment began (day 0), and again at 4, 8, and 12 days later. Tumor diameter was measured until day 24, and tumor volume was confirmed. Tumor volume was calculated as (longest diameter of tumor × shortest diameter of tumor × shortest diameter of tumor) × 0.5 (mm²). 3 The antitumor effect was evaluated using tumor volume as an indicator, calculated using the following formula:

[0073] The ultrasound irradiation conditions were as follows: Equipment: GE C1-6 (GE Healthcare Japan Corporation) Mode: Gen mode Irradiation conditions: Ultrasound frequency: 2.5 [MHz] Tissue surface PNP (peak negative pressure): 0.11 [MPa] Tumor base PNP (peak negative pressure): 0.085 [MPa] Irradiation time: 10 min Total irradiation energy: 18.1 [mJ / cm²] 2 The ultrasonic frequency [MHz] was measured using a hydrophone in water. The tissue surface PNP (peak negative pressure) [MPa] was calculated based on the experimental setup, including whether or not there was attenuation due to the ultrasonic gel between the probe and the tissue, using the measured value obtained using a hydrophone in water. In the embodiments of the present invention, since the tumor is located directly beneath the tissue surface, the tissue surface PNP is almost the same as the tumor surface PNP. The tumor base PNP (peak negative pressure) [MPa] was calculated from the tissue surface PNP by the amount of attenuation due to the tumor, using the average maximum diameter of the tumor. The total irradiation energy was calculated by multiplying the total irradiation energy value per unit time, which was calculated by reflecting the PNP (peak negative pressure) obtained above, the measured duty cycle, pulse repetition frequency (PRF), and, if ultrasonic scanning was performed, the scanning speed, scanning spot size, and scanning range, by the total irradiation time. In the embodiments described later, the ultrasonic irradiation conditions are shown using measured and calculated values, similar to those in this embodiment.

[0074] As shown in Figure 1, the bubble formulation + ultrasound irradiation group (MB + US) demonstrated a superior antitumor effect compared to the gemcitabine and nab-paclitaxel combined administration group, confirming the excellent antitumor effect of the bubble formulation and ultrasound irradiation combination alone as described in this example. Furthermore, it was confirmed that combining it with predetermined amounts of gemcitabine and nab-paclitaxel resulted in even greater antitumor effects.

[0075] <Evaluation of antitumor effect against pancreatic cancer> The bubble formulation was prepared in the same manner as in Example 2. Next, human-derived pancreatic cancer cells (BxPC-3 cells) 1 × 10⁶ 6 Cells / 100μL / mice were transplanted subcutaneously into the posterior dorsal region of BALB / c-nu / nu mice (female, 6 weeks old). Treatment was initiated on the same day in mice in which tumor formation was confirmed 14 days after transplantation. Gemcitabine and nab-paclitaxel (Gem 50mg / kg + NPT 5mg / kg) were used as anticancer drugs. With the treatment initiation day as day 0, 4 days, 8 days, and 12 days later, 40μL of a bubble formulation passed through a 5μm filter was administered intraveinally in the tail vein after administration of anticancer drugs, and immediately afterward, percutaneous ultrasound was irradiated towards the tumor. The experimental groups were: Non-treatment group, gemcitabine and nab-paclitaxel combined treatment group (Gem 50 mg / kg + NPT 5 mg / kg), bubble formulation + ultrasound irradiation group (MB + US), gemcitabine and nab-paclitaxel combined treatment group, and bubble formulation + ultrasound irradiation group (MB + US + Gem 50 mg / kg + NPT 5 mg / kg). A total of four treatments were performed on the day of the experiment (day 0), 4 days later, 8 days later, and 12 days later. Tumor diameter was measured until day 25, and tumor volume was confirmed. Tumor volume was calculated as (longest diameter of tumor × shortest diameter of tumor × shortest diameter of tumor) × 0.5 (mm²). 3 The antitumor effect was evaluated using tumor volume as an indicator, calculated using the following formula. The ultrasound irradiation conditions were as follows: Equipment: GE C1-6 (GE Healthcare Japan Corporation) Mode: Gen mode Irradiation conditions: Ultrasound frequency: 2.5 [MHz] Tissue surface PNP (peak negative pressure): 0.11 [MPa] Tumor base PNP (peak negative pressure): 0.088 [MPa] Irradiation time: 10 min Total irradiation energy: 4.6 [mJ / cm²] 2 ]

[0076] As shown in Figure 3, the bubble formulation + ultrasound irradiation group (MB + US) showed a greater antitumor effect than the gemcitabine and nab-paclitaxel combined administration group, and further antitumor effects were shown when combined with predetermined amounts of gemcitabine and nab-paclitaxel. On the other hand, compared with Figure 1 of Example 2, the difference in the gemcitabine and nab-paclitaxel combined administration group was small, which was thought to be due to the smaller total irradiation energy compared to Example 2.

[0077] <Evaluation of antitumor effect against pancreatic cancer> The bubble formulation was prepared in the same manner as in Example 2. Furthermore, the prepared bubble formulation was prepared in its undiluted form [MB×1], diluted 10-fold with physiological saline [MB×1 / 10], diluted 30-fold [MB×1 / 30], and diluted 60-fold [MB×1 / 60]. Next, human-derived pancreatic cancer cells (BxPC-3 cells) 1×10 6 Cells / 100μL / mice were transplanted subcutaneously into the posterior dorsal region of BALB / c-nu / nu mice (female, 6 weeks old). Treatment was initiated on the same day for mice in which tumor formation was confirmed 14 days after transplantation. Gemcitabine and nab-paclitaxel (Gem 50mg / kg + NPT 5mg / kg) were used as anticancer drugs. With the treatment start day as day 0, 25μL of a bubble formulation passed through a 5μm filter was administered intraveinally in the tail vein after administration of anticancer drugs on days 0, 4, 8, and 12, and immediately afterward, percutaneous ultrasound was irradiated towards the tumor. The experimental groups were: a non-treatment group, a gemcitabine and nab-paclitaxel combined therapy group (Gem 50 mg / kg + NPT 5 mg / kg), a gemcitabine and nab-paclitaxel combined therapy group, and a bubble formulation ([MB×1], [MB×1 / 10], [MB×1 / 30], [MB×1 / 60]) + ultrasound irradiation group ([MB×1] + US + Gem + NPT, [MB×1 / 10] + US + Gem + NPT, [MB×1 / 30] + US + Gem + NPT, [MB×1 / 60] + US + Gem + NPT). A total of four treatments were performed on the day the experiment started (day 0), 4 days later, 8 days later, and 12 days later. Tumor diameter was measured until day 19 to confirm tumor volume. Tumor volume was calculated as (longest diameter of tumor × shortest diameter of tumor × shortest diameter of tumor) × 0.5 (mm) 3The antitumor effect was evaluated using tumor volume as an indicator, calculated using the following formula. The ultrasound irradiation conditions were as follows: Equipment: GE C1-6 (GE Healthcare Japan Corporation) Mode: Gen mode Irradiation conditions: Ultrasound frequency: 2.5 [MHz] Tissue surface PNP: 0.11 [MPa] Tumor base PNP: 0.088 [MPa] Irradiation time: 10 min Total irradiation energy: 4.6 [mJ / cm] 2 ]

[0078] As shown in Figure 4, the gemcitabine and nab-paclitaxel combined therapy group and the bubble formulation + ultrasound irradiation group showed superior antitumor effects at all concentrations ([MB×1] + US + Gem + NPT, [MB×1 / 10] + US + Gem + NPT, [MB×1 / 30] + US + Gem + NPT, [MB×1 / 60] + US + Gem + NPT) compared to the gemcitabine and nab-paclitaxel combined therapy group (Gem 50 mg / kg + NPT 5 mg / kg).

[0079] As shown by the arrows in Figure 4, the number of days until the estimated tumor weight doubled from the treatment start date was calculated from the approximation curves of the estimated tumor weight for each group. In the gemcitabine and nab-paclitaxel combined administration group, it was 8.0 days. In contrast, the gemcitabine and nab-paclitaxel combined administration group and the bubble formulation + ultrasound irradiation group showed an extension of 1.5 days for [MB×1], 2.2 days for [MB×1 / 10], 2.5 days for [MB×1 / 30], and 2.2 days for [MB×1 / 60]. The three groups [MB×1 / 10], [MB×1 / 20], and [MB×1 / 30] showed an extension of 2 days (25%) or more compared to the gemcitabine and nab-paclitaxel combined administration group. From the above, it was shown that the bubble formulation and ultrasound irradiation enhance the tumor-suppressing effect of anticancer drugs over a wide concentration range of 60 times that of the bubble formulation.

[0080] <Evaluation of antitumor effect against pancreatic cancer> The bubble formulation was prepared in the same manner as in Example 2.

[0081] Next, mouse-derived pancreatic cancer cells (PAN02 cells) 1 × 10 6Cells / 100μL / mouse were transplanted intradermally into the posterior dorsal region of C57BL / 6J mice. Treatment was initiated on mice in which tumor formation was confirmed 14 days after transplantation. Gemcitabine and nab-paclitaxel (Gem 50mg / kg + NPT 5mg / kg) were used as anticancer agents. With the treatment initiation day as day 0, 10μL of the bubble formulation, filtered through a 5μm filter, was administered intraveinally in the tail vein four times at 2.5-minute intervals after anticancer drug administration on days 0, 4, 8, and 12. Ultrasound was continuously applied percutaneously to the tumor immediately after the first administration of the bubble formulation.

[0082] The experimental groups were: Non-treatment group, gemcitabine and nab-paclitaxel combined treatment group (Gem 50 mg / kg + NPT 5 mg / kg), bubble formulation + ultrasound irradiation group (MB + US), gemcitabine and nab-paclitaxel combined treatment group, and bubble formulation + ultrasound irradiation group (MB + US + Gem 50 mg / kg + NPT 5 mg / kg).

[0083] The ultrasonic irradiation conditions were as follows: Equipment: ITO US777 (Ito Chotampa Co., Ltd.) Settings: Ultrasonic frequency: 1 [MHz] Intensity: 0.5 W / cm 2 Duty cycle: 5% Irradiation conditions: Tissue surface PNP: 0.13 [MPa] Tumor base PNP: 0.10 [MPa] Irradiation time: 10 min Total irradiation energy: 1.7 × 10⁻¹⁴ 4 [mJ / cm 2 ]

[0084] A total of four treatments were performed: on the day the experiment began (day 0), and again at 4, 8, and 12 days later. Tumor diameter was measured until day 22, and tumor volume was confirmed. Tumor volume was calculated as (longest diameter of tumor × shortest diameter of tumor). 2 ) × 0.5 (mm 3 The antitumor effect was evaluated using tumor volume as an indicator, calculated using the formula shown above. As shown in Figure 2, the antitumor effect of the bubble formulation and ultrasound irradiation was confirmed to be almost equivalent to that of drug administration alone.

[0085] <DDS effect on pancreatic cancer> The bubble formulation was prepared in the same manner as in Example 2.

[0086] Next, mouse-derived pancreatic cancer cells (PAN02 cells) 2 × 10 6 Cells / 100 μL / mouse were transplanted intradermally into the posterior dorsal region of C57BL / 6J mice, and the tumor volume was 300–500 mm². 3 The experiment was conducted once the target level was reached. Gemcitabine (Gem 30 mg / kg) was used as the anticancer drug. After administration of the anticancer drug, 10 μL of the bubble formulation, filtered through a 5 μm filter, was administered via tail vein four times at 2.5-minute intervals. Ultrasound was continuously applied percutaneously to the tumor immediately after the first administration of the bubble formulation. The tumor was collected 4 hours later, and the tissue concentration of gemcitabine was measured.

[0087] The experimental groups were: gemcitabine administration group (Gem 30 mg / kg), gemcitabine (Gem 30 mg / kg) + bubble formulation + ultrasound (tissue surface PNP: 0.11 [MPa]), gemcitabine (Gem 30 mg / kg) + bubble formulation + ultrasound (tissue surface PNP: 0.38 [MPa]), and gemcitabine (Gem 30 mg / kg) + bubble formulation + ultrasound (tissue surface PNP: 0.65 [MPa]).

[0088] The ultrasound irradiation conditions were as follows: Equipment: GE C1-6 (GE Healthcare Japan Corporation) Mode: Gen mode Irradiation conditions:

[0089]

[0090] In this embodiment, as shown in Figure 5, it was confirmed that when the tissue surface PNP was 0.11, drug accumulation in tumor tissue increased. It was demonstrated that by stably and gently vibrating the tissue, drugs from within the blood vessels could be stably delivered to the tissue.

[0091] <DDS effect on pancreatic cancer> The bubble formulation was prepared in the same manner as in Example 2. The creation of tumor-bearing mice and the treatment methods (method of administering the bubble formulation, dose and method of administering gemcitabine, method of irradiation with ultrasound, irradiation conditions and tissue surface PNP: 0.11, 0.38, 0.65 [MPa]) were the same as in Example 6. The experimental groups were: non-treatment group, gemcitabine administration group (Gem 30 mg / kg), gemcitabine (Gem 30 mg / kg) + bubble formulation + ultrasound (tissue surface PNP: 0.11 [MPa]), gemcitabine (Gem 30 mg / kg) + bubble formulation + ultrasound (tissue surface PNP: 0.38 [MPa]), and gemcitabine (Gem 30 mg / kg) + bubble formulation + ultrasound (tissue surface PNP: 0.65 [MPa]). Treatment was administered a total of three times: on the day the experiment began (day 0), 7 days later, and 14 days later. Tumor diameter was measured until day 28, and tumor volume was confirmed. Tumor volume was calculated as (longest diameter of tumor × shortest diameter of tumor × shortest diameter of tumor) × 0.5 (mm²). 3 The antitumor effect was evaluated using tumor volume as an indicator, calculated using the formula shown above. Figure 6 shows the tumor volume on day 28. Figure 7 shows the reduction in tumor volume with gemcitabine monotherapy when bubble and ultrasound are added (1-(tumor volume with MB+US+Gem) / (tumor volume with Gem)). This shows that tumor volume is further suppressed compared to gemcitabine monotherapy in a wide range of tissue surface PNPs from 0.11 to 0.65 [MPa].

[0092] <Antitumor effect against pancreatic cancer> The bubble formulation was prepared in the same manner as in Example 2.

[0093] Next, mouse-derived pancreatic cancer cells (PAN02 cells) 2 × 10 6 Cells / 100μL / mouse were transplanted intradermally into the posterior dorsal region of C57BL / 6J mice, and treatment was initiated on the same day in mice in which tumor formation was confirmed 21 days later. Gemcitabine (Gem 30mg / kg) was used as the anticancer drug. With the treatment start day as day 0, 10μL of the bubble formulation, filtered through a 5μm filter, was administered intraveinally in the tail vein four times at 2.5-minute intervals after anticancer drug administration on days 0, 7, and 14. Ultrasound was continuously applied percutaneously to the tumor immediately after the first administration of the bubble formulation.

[0094] The experimental groups were: a non-treatment group, a gemcitabine administration group (Gem 30 mg / kg), a bubble formulation + ultrasound irradiation group (MB + US), and a gemcitabine and bubble formulation + ultrasound irradiation group (MB + US + Gem 30 mg / kg).

[0095] Treatment was administered a total of three times: on the day the experiment began (day 0), 7 days later, and 14 days later. Tumor diameter was measured until day 28, and tumor volume was confirmed. Tumor volume was calculated as (longest diameter of tumor × shortest diameter of tumor × shortest diameter of tumor) × 0.5 (mm²). 3 The antitumor effect was evaluated using tumor volume as an indicator, calculated using the following formula:

[0096] The ultrasound irradiation conditions were as follows: Equipment: GE C1-6 (GE Healthcare Japan Corporation) Mode: Gen mode Irradiation conditions: Ultrasound frequency: 2.5 [MHz] Tissue surface PNP: 0.11 [MPa] Tumor base PNP: 0.085 [MPa] Irradiation time: 10 min Total irradiation energy: 18.1 [mJ / cm²] 2 ]

[0097] As shown in Figure 8, superior antitumor effects were observed with bubble formulations and ultrasound irradiation alone compared to drug monotherapy.

[0098] <DDS effect on pancreatic cancer> The bubble formulation was prepared in the same manner as in Example 2.

[0099] Next, mouse-derived pancreatic cancer cells (PAN02 cells) 2 × 10 6 Cells / 100 μL / mouse were transplanted intradermally into the posterior dorsal region of C57BL / 6J mice, with a tumor volume of 300-600 mm². 3 Tumor-bearing mice were used in the study. Doxorubicin (Doxil 1 mg / kg) was used as the anticancer agent. After administration of the anticancer agent, 10 μL of the bubble formulation, filtered through a 5 μm filter, was administered intraveinally at 2.5-minute intervals for four times. Ultrasound was continuously applied percutaneously to the tumor immediately after the first administration of the bubble formulation. After 24 hours, the tumor and other organs were collected, and the tissue concentration of doxorubicin was confirmed by fluorescence measurement.

[0100] The experimental groups were a doxorubicin-only group (Doxil 1 mg / kg) and a doxorubicin-bubble formulation + ultrasound irradiation group II (MB + US + Doxil 1 mg / kg).

[0101] Tissue samples were collected 24 hours after administration, and the doxorubicin concentration in 1g of tissue was evaluated.

[0102] The ultrasonic irradiation conditions were as follows: Equipment: ITO US777 (Ito Chotampa Co., Ltd.) Settings: Ultrasonic frequency: 1 [MHz] Intensity: 1.0 W / cm 2 Duty cycle: 5% Irradiation conditions: Tissue surface PNP: 0.18 [MPa] Tumor base PNP: 0.14 [MPa] Irradiation time: 10 min Total irradiation energy: 3.4 × 10⁻¹⁴ 4 [mJ / cm 2 ]

[0103] As shown in Figure 9, it was demonstrated that the tumor concentration of the drug in the tissue was higher with bubble formulation and ultrasound irradiation than with drug administration.

[0104] <Evaluation of DDS for pancreatic cancer> The bubble formulation was prepared in the same manner as in Example 2.

[0105] Next, mouse-derived pancreatic cancer cells (PAN02 cells) 2 × 10 6 Cells / 100 μL / mouse were transplanted intradermally into the posterior dorsal region of C57BL / 6J mice, with a tumor volume of 300-600 mm². 3 Tumor-bearing mice were used. Doxorubicin (Doxil 1 mg / kg) was used as the anticancer agent. After administration of the anticancer agent, 10 μL of the bubble formulation, filtered through a 5 μm filter, was administered intraveinally at 2.5-minute intervals for four times. Ultrasound was continuously applied percutaneously to the tumor immediately after the first administration of the bubble formulation. Tissue samples were collected 24 hours after the start of administration, and the tissue concentration of doxorubicin was measured. The experimental groups were a doxorubicin-only group (Doxil 1 mg / kg) and a doxorubicin + bubble formulation + ultrasound irradiation group (MB + US + Doxil 1 mg / kg).

[0106] The ultrasound irradiation conditions were as follows: Equipment: GE C1-6 (GE Healthcare Japan Corporation) Mode: Gen mode Irradiation conditions: Ultrasound frequency: 2.5 [MHz] Tissue surface PNP (peak negative pressure): 0.11 [MPa] Tumor base PNP (peak negative pressure): 0.085 [MPa] Irradiation time: 10 min Total irradiation energy: 18.1 [mJ / cm²] 2 ]

[0107] As shown in Figure 10, it was confirmed that the accumulation of the drug in tumor tissue increased with the use of bubble formulations and ultrasound irradiation.

[0108] <Evaluation of DDS for pancreatic cancer> The bubble formulation was prepared in the same manner as in Example 2.

[0109] Next, cancer was induced in the pancreas by laparotomy of Wister rats and application of 5 mg / 100 μL DMBA. Gemcitabine (Gem 30 mg / kg) was used as the anticancer drug. After 3 months, 100 μL of the bubble formulation, filtered through a 5 μm filter, was administered via tail vein four or two times at 5-minute intervals to individuals following the administration of the anticancer drug. Ultrasound was continuously applied percutaneously to the tumor immediately after the first administration of the bubble formulation (for 1 minute and 2 minutes). The pancreas was collected 2 hours after ultrasound irradiation, and the intrapancreatic concentration of gemcitabine was measured.

[0110] The experimental groups were: gemcitabine administration group (Gem only 30 mg / kg), gemcitabine + ultrasound irradiation group (1 minute irradiation) (Gem + US (1 minute irradiation) (repeated 4 times)), gemcitabine and bubble formulation + ultrasound irradiation group (1 minute irradiation) (MB + US + Gem 30 mg / kg) (repeated 4 times), gemcitabine and bubble formulation + ultrasound irradiation group (2 minutes irradiation) (MB + US + Gem 30 mg / kg) (repeated 4 times), and gemcitabine and bubble formulation + ultrasound irradiation group (1 minute irradiation) (MB + US + Gem 30 mg / kg) (repeated 2 times).

[0111] The ultrasonic irradiation conditions were as follows: Equipment: MU Rab SCI pr1-6 (MU Laboratories, Inc.) Settings: Ultrasonic frequency: 1.054 [MHz] Intensity: 16 mW / cm2 Duty Cycle: 1% Irradiation Conditions: Tissue surface PNP: 0.22 [MPa] Tumor base PNP: 0.173 [MPa] Irradiation Time: 1 min x 4 x 4 times, 2 min x 4 x 4 times, 1 min x 4 x 2 times Total Irradiation Energy: 3.9 x 10⁻¹⁴ 3 [mJ / cm 2 ]

[0112] As shown in Figure 11, it was confirmed that the accumulation of the drug in tumor tissue increased with the use of bubble formulations and ultrasound irradiation.

[0113] <Tumor-suppressing effect on pancreatic cancer> The bubble formulation was prepared in the same manner as in Example 2.

[0114] Next, mouse-derived pancreatic cancer cells (PAN02 cells) 2 × 10 6 Cells / 100 μL / mouse were transplanted subcutaneously into the posterior dorsal region of C57BL / 6J mice, and tumor formation was confirmed after 21 days. Onivyde (20 mg / kg as irinotecan) was used as the anticancer agent. After administration of the anticancer agent, 10 μL of the bubble formulation, filtered through a 5 μm filter, was administered intravein at 2.5-minute intervals for four times to the tumor-bearing mice. Ultrasound was continuously applied percutaneously to the tumor immediately after the first administration of the bubble formulation.

[0115] The experimental groups were: a non-treatment group, an Onivyde administration group (20 mg / kg of irinotecan), a bubble formulation + ultrasound irradiation group (MB + US), and an Onivyde and bubble formulation + ultrasound irradiation group (MB + US + 20 mg / kg of irinotecan).

[0116] Treatment was administered a total of three times: on the day the experiment began (day 0), at 7 days later, and at 14 days later. Tumor diameter was measured until day 24, and tumor volume was confirmed. Tumor volume was calculated as (longest diameter of tumor × shortest diameter of tumor × shortest diameter of tumor) × 0.5 (mm²). 3 The antitumor effect was evaluated using tumor volume as an indicator, calculated using the following formula:

[0117] The ultrasonic irradiation conditions were as follows: Equipment: ITO US 777 (Ito Chotampa Co., Ltd.) Settings: Ultrasonic frequency: 1 [MHz] Intensity: 0.5 W / cm 2Duty cycle: 5% Irradiation conditions: Tissue surface PNP: 0.13 [MPa] Tumor base PNP: 0.10 [MPa] Irradiation time: 10 min Total irradiation energy: 1.7 × 10⁻¹⁴ 4 [mJ / cm 2 ]

[0118] As shown in Figure 12, the bubble formulation and ultrasound irradiation demonstrated superior antitumor effects compared to drug administration.

[0119] <Tumor-suppressive effect against bladder cancer> The bubble formulation was prepared in the same manner as in Example 2. Next, human-derived bladder cancer cells (UM-UC-3-Luc cells) 1 × 10⁻¹⁶ 6 Cells / mice were transplanted intradermally into the posterior dorsal region of BALB / c-nu / nu mice, and treatment was initiated on the same day in mice in which tumor formation was confirmed 7 days later. Gemcitabine and cisplatin were selected as the anticancer agents. With the treatment initiation day as day 0, 10 μL of the bubble formulation, filtered through a 5 μm filter, was administered intraveinally in the tail vein four times at 2.5-minute intervals on days 0, 4, 8, and 12, after administration of anticancer agents. Ultrasound was continuously applied percutaneously to the tumor immediately after the first administration of the bubble formulation.

[0120] The experimental groups were: a non-treatment group, a gemcitabine and cisplatin combination therapy group (Gem+CDDP: 8 mg / kg of gemcitabine, 0.8 mg / kg of cisplatin), a bubble formulation + ultrasound irradiation group (MB+US), a gemcitabine and cisplatin combination therapy group, and a bubble formulation + ultrasound irradiation group (MB+US+Gem+CDDP: MB+US+gemcitabine, 0.8 mg / kg of cisplatin).

[0121] Treatment was administered a total of four times: on the day the experiment began (day 0), and again at 4, 8, and 12 days later. Tumor diameter was measured up to 24 days later to confirm tumor volume. Tumor volume was calculated as (longest diameter of tumor × shortest diameter of tumor × shortest diameter of tumor) × 0.5 (mm²). 3 The antitumor effect was evaluated using tumor volume as an indicator, calculated using the following formula:

[0122] The ultrasound irradiation conditions were as follows: Equipment: GE C1-6 (GE Healthcare Japan Corporation) Mode: Gen mode Irradiation conditions: Ultrasound frequency: 2.5 [MHz] Tissue surface PNP: 0.11 [MPa] Tumor base PNP: 0.085 [MPa] Irradiation time: 10 min Total irradiation energy: 18.1 [mJ / cm²] 2 As shown in Figure 13, the combination therapy group of gemcitabine and cisplatin, as well as the bubble formulation + ultrasound irradiation group (MB + US + Gem + CDDP), demonstrated superior antitumor efficacy compared to the combination therapy group of gemcitabine and cisplatin (Gem + CDDP).

[0123] <DDS effect on breast cancer> The bubble formulation was prepared in the same manner as in Example 2. Furthermore, the prepared bubble formulation was diluted fourfold with physiological saline and used. Next, mouse-derived breast cancer cells (4T1 cells) 1 × 10⁶ 6 Cells / 100 μL / mouse were transplanted intradermally into the posterior dorsal region of C57BL / 6J mice, and treatment was initiated on the same day in mice in which tumor formation was confirmed 21 days later. Gemcitabine (Gem 50 mg / kg) was used as the anticancer drug. With the treatment start day as day 0, 25 μL of the bubble formulation, filtered through a 5 μm filter, was administered intravein at 2.5-minute intervals four times to tumor-bearing mice on days 0, 7, and 14. Ultrasound was continuously applied percutaneously to the tumor immediately after the first administration of the bubble formulation. After the completion of ultrasound irradiation, the anticancer drug was administered. The experimental groups were: non-treatment group, gemcitabine administration group (Gem 50 mg / kg), bubble formulation + ultrasound irradiation group (MB + US), and gemcitabine and bubble formulation + ultrasound irradiation group (MB + US + Gem 50 mg / kg).

[0124] Treatment was administered a total of three times: on the day the experiment began (day 0), at 4 days later, and at 8 days later. Tumor diameter was measured up to 12 days later, and tumor volume was confirmed. Tumor volume was calculated as (longest diameter of tumor × shortest diameter of tumor × shortest diameter of tumor) × 0.5 (mm²). 3The antitumor effect was evaluated using tumor volume as an indicator, calculated using the following formula. The ultrasound irradiation conditions were as follows: Equipment: GE C1-6 (GE Healthcare Japan Corporation) Mode: Gen mode Irradiation conditions: Ultrasound frequency: 2.5 [MHz] Tissue surface PNP: 0.11 [MPa] Tumor base PNP: 0.085 [MPa] Irradiation time: 10 min Total irradiation energy: 18.1 [mJ / cm] 2 ]

[0125] As shown in Figure 14, a statistically significant difference was observed on day 4, reflecting the effect of the first treatment, when comparing the gemcitabine and bubble formulation + ultrasound irradiation group (Gem + MB + US) with the gemcitabine monotherapy group (Gem). Although no statistically significant difference was observed on day 8, reflecting the effect of the first two treatments, a trend toward the add-on effect of MB + US was observed (p = 0.064).

[0126] Figure 15 shows a list of the target cancers, cancer cells, combination anticancer drugs, and ultrasound irradiation conditions for Examples 1 to 14 described above. Ultrasound frequency: 1-2.5 MHz, PNP at tumor site: 0.085-0.65 MPa, total irradiation energy: 4.6 × 10⁻⁶ 0 ~3.4×10 4 mJ / cm 2 Within this scope, in solid tumors of various sites including pancreatic cancer, bladder cancer, breast cancer, and ovarian cancer, it was shown that a high tumor growth inhibitory effect was obtained by combining bubble formulations, ultrasound, and anticancer drugs with a wide range of anticancer agents, including gemcitabine, gemcitabine + nab-paclitaxel, gemcitabine + cisplatin, doxorubicin, and irinotecan, in multiple cancer models including mouse-derived cancer cells, human-derived cancer cells, and carcinogenesis models, compared to anticancer drugs alone.

[0127] The fact that tumor growth inhibitory effects were obtained over a wide range of 0.085 to 0.65 MPa for PNPs at the tumor site has the advantage of being less affected by variations in ultrasound sound pressure attenuation in the tissue due to variations in tumor depth. Furthermore, the total irradiation energy was 4.6 × 10⁻⁶. 0 ~3.4×10 4 mJ / cm 2A wide range of tumor growth inhibitory effects are obtained, and no animal deaths due to increased total irradiation energy have been observed, offering the advantage of high flexibility in setting irradiation conditions while balancing efficacy and safety. Figure 16 shows a list of target cancers, cancer cells, combination anticancer drugs, and the amount of bubbles administered per dose for Examples 1 to 14. Dosage per body weight: 0.49 to 55.4 × 10⁶ 8 In a wide range of tumors per kg and bubble volumes from 0.14 to 15.9 μL / kg, the combination of bubbles, ultrasound, and anticancer drugs demonstrated a higher tumor growth inhibitory effect compared to anticancer drugs alone, across multiple solid tumor sites, multiple cancer models, and multiple anticancer drugs.

[0128] In this invention, transient and reversible increased permeability of tumor vessels in the presence of bubbles has been confirmed in pancreatic cancer, breast cancer, and bladder cancer models under the same or overlapping irradiation conditions. These cancer types are all solid tumors and share common tumor vascular and microenvironmental characteristics, where material delivery into the tumor is limited due to structurally immature and heterogeneous tumor vessels and high pressure and diffusion resistance resulting from increased stroma. They exhibit similar physical responses to the mechanical effects of ultrasound and bubbles. Therefore, the ultrasound irradiation parameters of this invention are not limited to pancreatic cancer but can be defined as applicable to solid cancers in general, including breast cancer and bladder cancer.

Claims

1. A bubble formulation that exerts a tumor-suppressing effect by applying low-intensity therapeutic ultrasound, wherein the bubble formulation comprises a lipid film and a gas sealed in the lumen of the film, the film is an anionic lipid containing at least distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), and the gas contains at least one or a mixture of perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohexane.

2. The bubble formulation according to claim 1, wherein the coating is an anionic lipid formed from distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG) in a molar ratio of 22-60:30-68:4-12.

3. The bubble formulation according to claim 1, wherein the coating is an anionic lipid formed of distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG) in a molar ratio of 30:60:10, and the gas is perfluoropropane.

4. The low-intensity therapeutic ultrasound is 0.03 to 50 W / cm². 2 The bubble formulation according to claim 1, wherein the energy intensity is [value].

5. The bubble formulation according to claim 1, wherein the diameter is 500 nm to 5 μm.

6. The bubble formulation according to claim 1, wherein the coating is DSPC, DSPG, and a DSPE-PEG derivative, and the DSPE-PEG derivative is DSPE-PEG-OMe, DSPE-PEG-MAL, DSPE-PEG-COOH, DSPE-PEG-NHS, or DSPE-PEG-NH2.

7. A tumor treatment system comprising an ultrasound probe that generates low-intensity therapeutic ultrasound and a bubble formulation that exerts a tumor-suppressing effect by applying low-intensity therapeutic ultrasound, wherein the bubble formulation comprises a lipid coating and a gas sealed in the lumen of the coating, the coating is an anionic lipid containing at least distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), and the gas contains at least one or a mixture of perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohexane.

8. The tumor treatment system according to claim 7, further comprising a control unit for generating low-intensity, non-invasive ultrasound waves on the ultrasound probe.

9. The tumor treatment system according to claim 7 or 8, characterized in that it is used in combination with an anticancer agent.

10. The tumor treatment system according to claim 9, wherein the anticancer agent is a cytotoxic anticancer agent.

11. The tumor treatment system according to claim 10, wherein the anticancer agent is gemcitabine, paclitaxel, doxorubicin, irinotecan, or cisplatin.

12. The tumor treatment system according to any one of claims 9 to 11, wherein the frequency of the low-intensity therapeutic ultrasound is 1 to 3 MHz.

13. The tumor treatment system according to claim 12, wherein the peak negative pressure (PNP) at the tumor site is 0.08 to 0.7 MPa.

14. The total irradiation energy of the low-intensity therapeutic ultrasound is 4 × 10⁻¹⁰ 0 ~4×10 4 mJ / cm 2 The tumor treatment system according to claim 13.

15. The tumor treatment system according to any one of claims 9 to 14, wherein the tumor is a solid tumor.

16. The tumor treatment system according to claim 15, wherein the solid tumor is pancreatic cancer, bladder cancer, or breast cancer.

17. A drug delivery system comprising an ultrasound probe that generates low-intensity therapeutic ultrasound and a bubble formulation that exerts a tumor-suppressing effect by applying low-intensity therapeutic ultrasound, wherein the bubble formulation comprises a lipid coating and a gas sealed in the lumen of the coating, the coating is an anionic lipid containing at least distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), and the gas contains at least one or a mixture of perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohexane.

18. The drug delivery system according to claim 17, further comprising a control unit for generating low-intensity, non-invasive ultrasound waves on the ultrasound probe.

19. The drug delivery system according to claim 17 or 18, characterized in that it is used in combination with an anticancer agent.

20. The drug delivery system according to claim 19, wherein the anticancer agent is a cytotoxic anticancer agent.

21. The drug delivery system according to claim 20, wherein the anticancer agent is gemcitabine, paclitaxel, doxorubicin, irinotecan, or cisplatin.

22. The drug delivery system according to any one of claims 19 to 21, wherein the frequency of the low-intensity therapeutic ultrasound is 1 to 3 MHz.

23. The drug delivery system according to claim 22, wherein the peak negative pressure (PNP) at the tumor site is 0.08 to 0.7 MPa.

24. The total irradiation energy of the low-intensity therapeutic ultrasound is 4 × 10⁻⁶ 0 ~4×10 4 mJ / cm 2 The drug delivery system according to claim 23.

25. The drug delivery system according to any one of claims 19 to 24, wherein the tumor is a solid tumor.

26. The drug delivery system according to claim 25, wherein the solid tumor is pancreatic cancer, bladder cancer, or breast cancer.

27. Use of a bubble formulation according to any one of claims 1 to 6 for the manufacture of a pharmaceutical product that exerts a tumor-suppressing effect by applying low-intensity therapeutic ultrasound, characterized in that it is used in combination with an anticancer agent.

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