Sonosensitizer composition comprising phthalocyanine compound with paramagnetic ions bonded thereto, and use thereof

Abstract

The present invention relates to: a sonosensitizer composition that does not exhibit phototoxicity, the sonosensitizer composition comprising a phthalocyanine compound with paramagnetic ions bonded thereto, or a pharmaceutically acceptable salt thereof; and a use thereof. According to an aspect, a sonosensitizer composition comprising a phthalocyanine compound with paramagnetic ions of copper (II) or nickel (II) bonded thereto or a pharmaceutically acceptable salt thereof can effectively kill cancer cells, by exhibiting excellent ROS generation activity when exposed to ultrasound, without exhibiting phototoxicity, which is one of the side effects of conventional photosensitizers. In addition, the sonosensitizer composition has sonodynamic properties, improved tumor specificity, and excellent biocompatibility, and thus can be effectively used as an anticancer agent. Meanwhile, the present invention was achieved with the support of the following in-hospital research project under the supervision of Seoul National University Bundang Hospital. [In-Hospital Research Project Supporting Present Invention] [MSRI Project Number] 13-2021-0003 [Specialized Research Management Agency] Seoul National University Bundang Hospital [Project Name] Development of tumor-targeting high-sensitivity ultrasound-responsive agent using focused ultrasound equipment and research on immunomodulatory mechanism [Contribution Rate] 50% [Main Organization] Seoul National University Bundang Hospital [Research Period] 01 March 2021 - 31 August 2024

Description

Ultrasonic sensitizer composition comprising a phthalocyanine compound bonded with a paramagnetic ion and use thereof

[0001] The present invention relates to an ultrasound sensitizer composition comprising a phthalocyanine compound or a pharmaceutically acceptable salt thereof bound to a paramagnetic ion that does not exhibit phototoxicity, and a use thereof.

[0002] Cancer is a group of abnormal cells that develops due to the disruption of the balance between cell division and death, resulting in continuous division and proliferation. Massive investments are being made worldwide in the development of treatments to cure cancer. Currently, clinically available cancer treatments include chemotherapy, radiation, targeted therapy, and surgical resection. However, these methods have limitations, making the development of cancer treatment technologies that minimize side effects crucial.

[0003] Meanwhile, reactive oxygen species (ROS) are byproducts of cellular metabolic reactions in living organisms and are known to be involved in cell signaling and homeostasis. With the discovery that ROS can kill cancer cells, new anticancer therapies that control intracellular ROS are emerging.

[0004] Among these new therapies, photodynamic therapy (PDT) is known as a minimally invasive treatment method that utilizes exogenous ROS generated by photosensitizers under light irradiation. For example, PDT involves intravenously administering a photosensitizer to a subject and then irradiating it with appropriate light. The excited photosensitizer then activates oxygen molecules, converting them into singlet oxygen or generating new radicals, selectively attacking and destroying cancer cells or various tumor tissues. Despite its low systemic toxicity and ability to selectively destroy tumors, PDT has the disadvantage of being limited to treating superficial or localized tumors due to the limited depth of light penetration into tissue. Furthermore, because the photosensitizers currently available in PDT are metabolized slowly by normal cells, patients must be protected from light for 30 days after treatment to prevent photosensitivity dermatitis.

[0005] On the other hand, unlike PDT, which uses light as an energy source, sonodynamic therapy (SDT) is known to induce cancer cell death through ROS generated from the ultrasound sensitizer and sonocavitation by irradiating ultrasound (US) with an ultrasound sensitizer together. Furthermore, because SDT can focus ultrasound on a specific tumor site, it can selectively destroy cancer cells while minimizing damage to surrounding normal cells. In addition, it can penetrate deeper into tumor tissue, which is advantageous for treating deep-seated tumors. Therefore, it is receiving great attention as a potential alternative to PDT in cancer treatment.

[0006] Because the therapeutic efficacy of SDT largely depends on the effectiveness of the ultrasound sensitizer, the development of safe and effective ultrasound sensitizers is crucial for the success of SDT. However, most commonly used ultrasound sensitizers are photosensitizer-derived. This has hindered their clinical application due to the potential for phototoxicity, a side effect of conventional photosensitizers. This phenomenon, characterized by the generation of reactive oxygen species (ROS) by light exposure, can also damage normal cells.

[0007] Therefore, there is a need for the development of novel ultrasound sensitizers that are non-phototoxic and have improved ultrasound dynamic properties, enhanced tumor specificity, and superior biocompatibility compared to commercially available products.

[0008] One aspect provides an ultrasound sensitizer composition comprising a phthalocyanine compound or a pharmaceutically acceptable salt thereof to which a paramagnetic ion, such as copper(II) or nickel(II), is bound.

[0009] Another aspect provides a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a phthalocyanine compound or a pharmaceutically acceptable salt thereof, to which a paramagnetic ion, such as copper(II) or nickel(II), is bound.

[0010] Another aspect provides a method of treating cancer comprising administering the pharmaceutical composition to a subject other than a human.

[0011] One aspect provides an ultrasound sensitizer composition comprising a phthalocyanine compound or a pharmaceutically acceptable salt thereof bound to a paramagnetic ion, such as copper(II) or nickel(II).

[0012] Hereinafter, the present invention will be described in more detail.

[0013] Unless otherwise defined, all technical terms used in this specification have the same meaning as commonly understood by those skilled in the art. Furthermore, numerical values ​​described herein are assumed to include the meaning of "about," even if not explicitly stated.

[0014] The term "include" in this specification is used to indicate that other components may be added and / or interposed, rather than to the exclusion of other components, unless specifically stated otherwise.

[0015] As used herein, the term “combination thereof” means a mixture or combination of one or more of the described components.

[0016] As used herein, the term "sonodynamic therapy (SDT)" refers to a treatment method that includes treating a subject with a pathological condition (e.g., cancer) with an ultrasound sensitizer and irradiating light to activate the ultrasound sensitizer and obtain a therapeutic effect.

[0017] As used herein, the term "sonosensitizer" comprehensively refers to a substance that, when exposed to an appropriate frequency, absorbs vibrational energy and then generates reactive oxygen species, which can damage or destroy cells.

[0018] In this specification, the term "phototoxicity" refers to a phenomenon in which normal cells other than the targeted abnormal cells (e.g., cancer cells) are damaged due to the generation of reactive oxygen species by light encountered in daily life.

[0019] The present inventors selected phthalocyanine compounds as sonosensitizers to develop a novel sonosensitizer that is free of phototoxicity and exhibits improved sonodynamic properties (excellent sonosensitization, high molecular extinction co-efficiency in the far infrared and visible ranges, and low dark toxicity) compared to commercially available products, while also exhibiting excellent chemical stability and biocompatibility. In addition, a sonosensitizer composition comprising a phthalocyanine compound or a pharmaceutically acceptable salt thereof, which is based on a complex in which a paramagnetic ion is bound to the macrocyclic structure of phthalocyanine, was synthesized. Specifically, the paramagnetic ion may be Cu(II) or Ni(II), and the paramagnetic ion can generate a phthalocyanine compound with reduced cross-talk between systems, and can attenuate or completely suppress the phototoxic activity of the phthalocyanine-based sonosensitizer due to the heavy metal ion effect based on metal-ligand charge transfer (MLCT).

[0020] The term "pharmaceutically acceptable salt" as used herein means any organic or inorganic addition salt of a compound represented by Chemical Formula 1, etc., which has an effective effect at a concentration that is relatively non-toxic and harmless to the patient, and in which side effects due to this salt do not diminish the beneficial effects of the compound represented by Chemical Formula 1, etc. These salts may use inorganic acids and organic acids as the free acid.

[0021] The above ultrasound sensitizer may include a phthalocyanine compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0022] [Chemical Formula 1]

[0023]

[0024] In the above chemical formula 1, M is Cu(Ⅱ) or Ni(Ⅱ),

[0025] R may be one selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, sulfonic acid, amidosulfuric acid, phosphoric acid, nitric acid, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, tartaric acid, citric acid, oxalic acid, maleic acid, maleic acid, ethanesulfonic acid, salicylic acid, benzoic acid, paratoluenesulfonic acid, and methanesulfonic acid. Specifically, R may be a sulfonic acid, and when the R group is selected as a sulfonic acid, the inherent aggregation tendency of the phthalocyanine compound can be suppressed, thereby improving the therapeutic efficiency of PDT or SDT, and thus can be used as an effective ultrasound sensitizer.

[0026] In addition, the phthalocyanine compound or a pharmaceutically acceptable salt thereof may be a compound represented by the following chemical formula 2 or 3.

[0027] [Chemical Formula 2]

[0028]

[0029] [Chemical Formula 3]

[0030]

[0031] As used herein, the “ultrasonic sensitizer comprising a phthalocyanine compound or a pharmaceutically acceptable salt thereof” refers to a phthalocyanine compound that is modified to form Cu among the phthalocyanine compound and paramagnetic ions. 2+ or Ni 2+ Cu containing 2+ or Ni 2+- may refer to a complex sulfonated phthalocyanine, for example, CuPc-SO3 or NiPc-SO3. In the present specification, "ultrasound sensitizer comprising a phthalocyanine compound or a pharmaceutically acceptable salt thereof" may be used interchangeably with a phthalocyanine complex, a sensitizer compound, a phthalocyanine ultrasonic sensitizer, a phthalocyanine-based ultrasonic sensitizer, or a phthalocyanine compound.

[0032] The above phthalocyanine compound or a pharmaceutically acceptable salt thereof may be characterized by not generating reactive oxygen species (ROS) even when irradiated with light of, for example, 300 to 700 nm, 350 to 700 nm, 400 to 700 nm, 450 to 700 nm, 500 to 700 nm, 550 to 700 nm, or 600 to 700 nm, so that phototoxicity may be significantly suppressed or not appear.

[0033] The frequency and intensity of ultrasound that can be used for the phthalocyanine compound or the pharmaceutically acceptable salt thereof can be selected based on the need to induce activation of the phthalocyanine compound at the target site. Specifically, the ultrasound frequency that can be used for the phthalocyanine ultrasound sensitizer compound can be, for example, 0.01 to 10.0 MHz, 0.1 to 9 MHz, 0.1 to 8 MHz, 0.2 to 10 MHz, 0.2 to 8 MHz, 0.5 to 7 MHz, 0.5 to 5 MHz, 0.5 to 4 MHz, 0.5 to 3 MHz, 0.5 to 2 MHz, or 0.5 to 1.5 MHz, which are typically used in medical ultrasound, and the phthalocyanine compound or the pharmaceutically acceptable salt thereof can exhibit ultrasound sensitizing activity for ultrasound of the above wavelength.

[0034] The above phthalocyanine compound or pharmaceutically acceptable salt thereof has, for example, an emission intensity of 0.01 to 10.0 W / cm 2 , 0.01 to 8 W / cm 2 , 0.01 to 7 W / cm 2 , 0.01 to 5 W / cm 2 , 0.1 to 10.0 W / cm 2 , 0.1 to 8 W / cm 2 , 0.1 to 7 W / cm 2 , 0.1 to 5 W / cm 2 , 0.1 to 4 W / cm 2 , or 0.2 to 3.5 W / cm 2 Ultrasound can be applied at an intensity of , and when ultrasound is applied at this intensity, ROS can be generated. The cancer treatment effect by ultrasound will depend on the selected intensity, that is, for low ultrasound intensities, the treatment time will be long, and for higher ultrasound intensities, the treatment time will be shorter. Ultrasound can be applied continuously or in a pulsed manner and can be delivered as a focused or columnar beam.

[0035] Any source capable of producing acoustic energy (e.g., ultrasound) may be used in the methods described herein. The source must be capable of directing the energy to a target site, and may include, for example, a probe or a device capable of directing the energy to the target tissue.

[0036] The ultrasound sensitizer comprising the above phthalocyanine compound or a pharmaceutically acceptable salt thereof may further comprise microbubbles. The microbubbles may be selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine (DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and It may include at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[succinyl(polyethylene glycol)-2000] (DSPE-PEG2k-NHS).

[0037] Additionally, the microbubbles may include, for example, DSPC and DSPE-PEG2k-NHS in a molar ratio of, for example, 0.1:1 to 10:1, 1:1 to 10:1, 2:1 to 10:1, 3:1 to 10:1, 4:1 to 10:1, 5:1 to 10:1, 6:1 to 10:1, 7:1 to 10:1, 8:1 to 10:1 or 9:1.

[0038] In one embodiment, the gas filled inside the microbubbles may be a known gas, and examples thereof include, but are not limited to, carbon dioxide, helium, nitrogen, argon, sulfur hexafluoride, and perfluorinated gases. Specifically, the gas may be a fluoride gas containing fluorine gas, and examples thereof include one of perfluoropropane (C3F8), sulfur hexafluoride (SF6), perfluoropentane, decafluorobutane, and perfluorohexane.

[0039] In one embodiment, when microbubbles are destroyed by ultrasound, the gas filled inside the microbubbles provides a pressure wave simultaneously with the destruction of the microbubble structure, thereby allowing the ultrasound sensitizer loaded on the surface or inside the microbubble or processed together with the microbubble to be forcibly injected into the tissue of the target cancer cell, thereby improving the delivery efficacy of the ultrasound sensitizer.

[0040] In one embodiment, microbubbles can be moved to a target site by irradiating ultrasound with a specific output (i.e., intensity, time, duty cycle parameters, etc.), wherein the specific output parameter value of the ultrasound used to move the microbubbles is, for example, 1 to 3 W / cm in intensity. 2 , the time is 1 to 2 minutes, and the duty cycle may preferably be 10 to 60%, but is not limited thereto.

[0041] In addition, in one embodiment, the microbubbles can be destroyed at the target site using ultrasound of a specific output and the therapeutic drug can be released, wherein the specific output parameter value of the ultrasound used to destroy the microbubbles is, for example, 1 to 3 W / cm in intensity. 2 , the time is 1 to 2 minutes, and the duty cycle may preferably be 10 to 60%, but is not limited thereto.

[0042] The above phthalocyanine compound or a pharmaceutically acceptable salt thereof can generate ROS when ultrasonic waves are irradiated with the above intensity for, for example, 30 to 300 seconds, 30 to 240 seconds, 30 to 180 seconds, 45 to 120 seconds, 45 to 100 seconds, 50 to 90 seconds, 50 to 80 seconds, 55 to 70 seconds, or 55 to 65 seconds.

[0043] The above phthalocyanine compound or a pharmaceutically acceptable salt thereof may be characterized in that it is irradiated with ultrasound, for example, 4 to 48 hours, 4 to 30 hours, 4 to 26 hours, or 4 to 25 hours after administration of the composition, and when irradiated within the above time, the ability to generate ROS may be increased.

[0044] The above ultrasound sensitizer, when administered to a living body, exhibits selective toxicity toward cancer cells by specifically binding to specific target cells (e.g., cancer cells) and then generating a toxic substance that can kill cancer cells when activated by ultrasound.

[0045] Mechanistically, the ultrasound sensitizer of the present invention selectively accumulates in tumor tissues, and then is activated by ultrasound to combine with oxygen and form highly chemically reactive singlet oxygen ( 1 This is due to a mechanism that directly damages cancer cells by generating O2: singlet oxygen, and not only has an indirect effect of killing cancer cells by damaging micro-blood vessels around the tumor and preventing the supply of nutrients to cancer tissues, but also the ultrasound sensitizer composition can selectively accumulate in cancer tissues due to oxidative stress, and the ultrasound sensitizer composition can generate active oxygen to treat cancer or suppress cancer metastasis.

[0046] Another aspect provides a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a phthalocyanine compound or a pharmaceutically acceptable salt thereof, to which a paramagnetic ion, such as copper(II) or nickel(II), is bound.

[0047] The above phthalocyanine compound or a pharmaceutically acceptable salt thereof may be a compound represented by the following chemical formula 1;

[0048] [Chemical Formula 1]

[0049]

[0050] In the above chemical formula 1, M is Cu(Ⅱ) or Ni(Ⅱ),

[0051] R may be one selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, sulfonic acid, amidosulfuric acid, phosphoric acid, nitric acid, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, tartaric acid, citric acid, oxalic acid, maleic acid, maleic acid, ethanesulfonic acid, salicylic acid, benzoic acid, paratoluenesulfonic acid, and methanesulfonic acid. Specifically, R may be a sulfonic acid, and when the R group is selected as a sulfonic acid, the inherent aggregation tendency of the phthalocyanine compound can be suppressed, thereby improving the therapeutic efficiency of PDT or SDT, and thus can be used as an effective ultrasound sensitizer.

[0052] In addition, the phthalocyanine compound or a pharmaceutically acceptable salt thereof may be a compound represented by the following chemical formula 2 or 3.

[0053] [Chemical Formula 2]

[0054]

[0055] [Chemical Formula 3]

[0056]

[0057] In the present specification, when a composition including an ultrasonic sensitizer including the phthalocyanine compound is used as a pharmaceutical composition, the composition may include the effective ingredient in an amount of 0.0001 to 50 wt% based on the total weight of the composition.

[0058] The pharmaceutical composition may further comprise microbubbles. The microbubbles may be selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine (DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and It may include at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[succinyl(polyethylene glycol)-2000] (DSPE-PEG2k-NHS).

[0059] Additionally, the microbubbles may include, for example, DSPC and DSPE-PEG2k-NHS in a molar ratio of 0.1:1 to 10:1.

[0060] The above pharmaceutical composition may contain, in addition to the above effective ingredient, one or more effective ingredients having the same or similar function.

[0061] The term "cancer" as used in this specification means or describes a physiological condition in mammals that is typically characterized by uncontrolled cell growth and may include, without limitation, malignant tumors that grow rapidly while infiltrating surrounding tissues and spread or metastasize throughout the body, thereby threatening life. The above cancers are lung cancer, laryngeal cancer, stomach cancer, colon / rectum cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine sarcoma, small intestine cancer, uterine cancer, cervical cancer, urethral cancer, prostate cancer, melanoma, bone cancer, kidney cancer, skin cancer, bone cancer, muscle cancer, fatty cancer, fibrous cell carcinoma, leukemia, lymphoma, multiple myeloma, tumors arising from nerve tissue, triple negative breast cancer, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity / paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer. There may be one or more types selected from the military.

[0062] The pharmaceutical composition may further comprise a pharmaceutical additive selected from the group consisting of a pharmaceutically acceptable carrier, a diluent, a binder, a disintegrant, a lubricant, and any combination thereof. The pharmaceutically acceptable carrier included in the pharmaceutical composition is one commonly used in formulation, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0063] The pharmaceutical composition may contain about 0.1 to 500 mg of an ultrasound sensitizer including a phthalocyanine compound as an active ingredient as a free base per unit dosage form, and may contain the ultrasound sensitizer in a proportion of 0.5 to 90 wt%, 0.5 to 80 wt%, 0.5 to 70 wt%, 0.5 to 60 wt%, or 0.5 to 50 wt%, specifically 1 to 40 wt%, based on the total weight of the formulation. The compound may be included in the pharmaceutical composition in an amount sufficient to achieve its efficacy or activity. Those skilled in the art can select and implement the quantitative upper or lower limit of the compound included in the composition within an appropriate range.

[0064] The composition for use in the present invention may be used in the form of an aqueous suspension in water or a saline solution, for example, phosphate-buffered saline.

[0065] The pharmaceutical composition may be for oral or parenteral use, and the pharmaceutical composition may be administered orally or parenterally. It may be formulated as an oral or parenteral dosage form. The parenteral dosage form may be an injection or a topical application. The topical application may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. In the case of parenteral administration, it may be administered by, for example, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. In addition, the composition may be administered by any device that allows the active substance to travel to target cells.

[0066] Another aspect provides a method of treating cancer comprising administering the pharmaceutical composition to a subject other than a human.

[0067] The above method of treating cancer may be a method in which cancer cells are killed by reactive oxygen species generated in response to administration of an ultrasound sensitizer including a phthalocyanine compound included as an active ingredient in the above pharmaceutical composition.

[0068] The above composition may be administered in combination with drugs such as steroids, anti-inflammatory agents, and antibiotics to effectively prevent or treat cancer or secondary diseases arising therefrom in a subject. The combination administration may be administered sequentially, simultaneously, or separately to the subject.

[0069] The above treatment method involves administering a pharmaceutical composition in a therapeutically effective dosage. An ultrasound sensitizer containing a phthalocyanine compound within the pharmaceutical composition is then distributed to a desired part of the body or target. Once administered to the body, the target is exposed to ultrasound at a frequency and intensity that achieves the desired therapeutic effect.

[0070] The above administration may be 0.001 μg / mL to 1,000 mg / mL of the compound per subject per day, for example, 0.01 μg / mL to 500 mg / mL, or 0.1 μg / mL to 150 mg / mL. The administration may be administered once a day or divided into several times. The dosage of the pharmaceutical composition may vary depending on the patient's condition and weight, the severity of the disease, the drug form, the administration route, and the duration, but may be appropriately selected by those skilled in the art.

[0071] An ultrasound sensitizer composition comprising a phthalocyanine compound or a pharmaceutically acceptable salt thereof, to which a paramagnetic ion, such as copper(II) or nickel(II), is bound according to an aspect, exhibits excellent ROS generation activity upon exposure to ultrasound, thereby effectively killing cancer cells, without causing phototoxicity, which is one of the side effects of conventional photosensitizers. In addition, the ultrasound sensitizer composition has sonodynamic properties, enhanced tumor specificity, and excellent biocompatibility, and thus can be effectively utilized as an anticancer agent.

[0072] Figure 1 is a diagram showing the chemical structures of CuPc-SO3 and NiPc-SO3.

[0073] Figure 2 is a diagram showing the photophysical properties of CuPc-SO3, NiPc-SO3, and ZnPc in DMF solution. λ abs is the maximum absorption wavelength (nm), ε is the molecular extinction coefficient (ХΧ10 5 M -1 cm -1 ), λ em is the maximum emission wavelength (nm), f is the fluorescence quantum yield. △ is the singlet oxygen quantum yield.

[0074] Figure 3 is a diagram showing the results of confirming the HPLC chromatogram of CuPc-SO3 (Figure 3A) and the HPLC chromatogram of NiPc-SO3 (Figure 3B), and the ESI-MS spectrum of CuPc-SO3 (Figure 3C) and the ESI-MS spectrum of NiPc-SO3 (Figure 3D), respectively.

[0075] Figure 4A shows the absorption spectra of 4.0 μM CuPc-SO3, NiPc-SO3, and ZnPc in DMF solution, and Figure 4B shows the fluorescence spectra of 4.0 μM CuPc-SO3, NiPc-SO3, and ZnPc in DMF solution (Figure 4B). The experiment was performed under excitation at 610 nm (slit = 2.5 / 2.5).

[0076] Figure 5 is a graph confirming the results of photosensitivity ROS generation by CuPc-SO3, NiPc-SO3, and ZnPc. The absorption spectrum changes over time in dichlorodihydrofluorescein diacetate (DCFH-DA) solutions containing 1 μM CuPc-SO3, NiPc-SO3, or ZnPc are shown in Figure 5A for the measurement result for the CuPc-SO3 solution, Figure 5B for the measurement result for the NiPc-SO3 solution, and Figure 5C for the measurement result for the NiPc-SO3 solution. Light irradiation was performed at 660 nm (slit width = 15-1.5, Xe-lamp). In addition, the result of plotting the change in absorption intensity at 523 nm in Figures 5A to 5C is shown in Figure 5D. Here, the ROS generation rate was represented by the slope of each item shown in the graph.

[0077] Figure 6 shows the photosensitive singlet oxygen ( 1As a road to confirm the production of O2), the absorption spectrum changes over time in 9,10-diphenylanthracene (DPA) solutions containing ZnPc, CuPc-SO3 or NiPc-SO3 are shown in Fig. 6A for the ZnPc solution, Fig. 6B for the CuPc-SO3 solution and Fig. 6C for the NiPc-SO3 solution. The light irradiation was performed at 660 nm (slit width = 15-1.5, Xe-lamp). In addition, the results of plotting the change in absorption intensity at 383 nm in Figs. 6A to 6C are shown in Fig. 6D.

[0078] Figure 7 shows the results of measuring the generation of reactive oxygen species (ROS) caused by ultrasonic stimulation of CuPc-SO3, NiPc-SO3, and ZnPc, under ultrasonic (0.2 W / cm) 2 , 20% duty cycle, 1 MHz). The absorption spectrum changes over time in dichlorodihydrofluorescein diacetate (DCFH-DA) solutions containing 1 μM CuPc-SO3, NiPc-SO3, or ZnPc exposed to a 100 MHz (1 MHz) irradiation source are shown in Fig. 7A for the CuPc-SO3 solution, Fig. 7B for the NiPc-SO3 solution, and Fig. 7C for the ZnPc solution. In addition, the change in absorption intensity at 524 nm in Figs. 7A to 7C is plotted in Fig. 7D. Here, the rate of ROS production is represented by the slope of each item shown in the graph. Also shown in Fig. 7E are the results of measuring the relative fluorescence intensity for CuPc-SO3 and ZnPc in DCFH-DA with or without microbubble treatment, and the solution was exposed to various ultrasonic environments (US - means no ultrasonic irradiation, US ++ means 1.5 W / cm 2 , 1 MHz with 30% duty cycle, US +++ is 3 W / cm 2, 50% duty cycle, 1 MHz conditions). Figure 7F is a diagram showing the results of measuring the relative fluorescence intensity using DCFH-DA under ultrasonic irradiation conditions that treated microbubbles. The conditions of the ultrasonic treatment are US -, no ultrasonic irradiation, and US +, 1.5 W / cm. 2 , 1 minute at 20% duty cycle, US++ is 1.5 W / cm 2 , 30% duty cycle, 1 minute, US +++ is 3 W / cm 2 , the condition is 1 minute with 50% duty cycle.

[0079] Figure 8 is a diagram showing the results of measuring the generation of reactive oxygen species (ROS) caused by ultrasound sensitization in a control solution that does not contain an ultrasound sensitizer, and ultrasound (0.2 W / cm 2 The results show the time-dependent absorption spectrum changes of DCF solutions containing 1 μM control solution exposed to a 20% duty cycle, 1 MHz.

[0080] Figure 9 shows the photoreactive cytotoxicity of CuPc-SO3, NiPc-SO3, and ZnPc. The results of cell viability analysis for the group without light irradiation (Figure 9A) and the group treated with various concentrations (0.1 to 50 μM) of CuPc-SO3, NiPc-SO3, or ZnPc and DMSO as a control for 24 hours were confirmed. The results are as follows: 660 nm light irradiation (100 mW / cm 2 for 5 minutes; 30 J / cm 2 ) is a diagram showing the results of confirming the cell survival rate for a group (Fig. 9B).

[0081] Figure 10 is a diagram showing the results of apoptosis analysis of CuPc-SO3, NiPc-SO3, and ZnPc in 4T1 cancer cells, confirming the ultrasonic dynamic cytotoxicity of CuPc-SO3, NiPc-SO3, and ZnPc. Conditions of ultrasound treatment are US -, no ultrasound irradiation, and US +, 1.5 W / cm2 , 1 minute at 20% duty cycle, US++ is 1.5 W / cm 2 , the condition is 1 minute with 30% duty cycle.

[0082] The present invention will be described in more detail below through experimental examples and examples. However, these experimental examples and examples are intended to exemplify the present invention and the scope of the present invention is not limited to these experimental examples and examples.

[0083] Example 1. Synthesis of CuPc-SO3 and NiPc-SO3

[0084] The complete synthetic process of the two compounds (CuPc-SO3 and NiPc-SO3) is briefly shown in the following reaction schematic diagram 1, and the specific synthetic process of CuPc-SO3 and NiPc-SO3 is as follows.

[0085] [Reaction schematic 1]

[0086]

[0087] First, the synthesis of CuPc-SO3 as its salt form, CuPc-SO3Na, was carried out as follows. A mixture of 434 mg of 4-sulfo-phthalic acid trisodium salt (1.39 mmol), 4.2 mg of 0.0361 mmol of ammonium molybdate, 41.6 mg of 0.78 mmol of ammonium chloride, 500 mg of 8.3 mmol of urea, and 43.8 mg of 0.26 mmol of CuCl22H2O was dissolved in nitrobenzene (5 mL) and heated at 180°C for 3 days. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed with methanol, and dried. The crude product was then dissolved in 11 mL of 1 N hydrochloric acid saturated with sodium chloride. The solution was heated at 80°C for 1 hour, cooled to room temperature, and then filtered. The resulting solid was added to 7 mL of 0.1 N sodium hydroxide, heated at 80°C for 1 hour, and then cooled to room temperature. The sodium salt of the compound slowly precipitated from the solution. The formed precipitate was filtered and dried in a vacuum oven to obtain CuPc-SO3 in an 85% yield.

[0088] With respect to NiPc-SO3, a complex containing Ni(II) was prepared by a synthetic method similar to that of CuPc-SO3 described above, and the reaction yield was confirmed to be 80%.

[0089] Example 2. Experimental information and experimental materials

[0090] UV-Vis and fluorescence spectra were recorded using a Jasco V-750 spectrophotometer and a Shimadzu RF-5301PC spectrofluorometer, respectively. MALDI-TOF / TOF-MS spectra were acquired using a Shimadzu LCMS-2020 and a Bruker Ultraflextreme (Korea Basic Science Institute), respectively. Ultrasonic experiments were performed using an Intelect Mobile Ultrasound (DJO, LLC). Reversed-phase HPLC analysis was performed on a Dr. Maish GmbH ReproSil 100 C 18, 5 μm (250 X 20 mm) column using a Waters HPLC system (YL9100). Zinc phthalocyanine (ZnPc) was purchased from Aldrich.

[0091] Example 3. Spectroscopic data measurement

[0092] For UV / Vis and fluorescence spectra measurements, dimethyl sulfoxide (DMSO) stock solutions of CuPc-SO3 and NiPc-SO3 were diluted with aqueous buffer, Hanks' Balanced Salt Solution (HBSS, 5% DMSO), or DMF to prepare 4 μM solutions. For fluorescence spectra measurements, samples were excited at 610 nm using excitation and emission slit widths of 2.5 nm.

[0093] Example 4. Preparation of microbubbles

[0094] All phospholipid compounds were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and Yoon et al. (Theranostics 2014; 4(11):1133-1144) and Kim et al. (Pharmaceuticals 2021; 14(1), 6). 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[succinyl(polyethylene glycol)-2000] (DSPE-PEG2k-NHS) were dissolved in chloroform at a molar ratio of 9:1. The chloroform was then evaporated, and the phospholipid film was hydrated in PBS (lipid 1 mg / mL) and dispersed in a constant temperature ultrasonic bath. Microbubbles (MB) were generated by injecting sulfur hexafluoride (SF6) gas into the headspace of a lipid suspension vial using Vialmix◎ (Definity, North Billerica, MA, USA) for 45 s. The number of microbubbles was calculated by converting them to volume by counting them under an optical microscope.

[0095] Example 5. In vitro ROS analysis

[0096] 2',7'-Dichlorofluorescein diacetate reagent (DCFDA, also called DCFH-DA) is a fluorescent dye that measures hydroxyl, peroxyl and other reactive oxygen species (ROS), and DCFDA is known to be a nonfluorescent compound that is oxidized by ROS to 2',7'-dichlorofluorescein (DCF) and emits fluorescence. 10 μM ZnPc or 10 μM CuPc-SO3Na was added to HBSS solution together with DCFDA (25 μM) and 100 mL per sample, i.e. approximately 2 Х 10 9 Ultrasound (US) was applied with or without adding microbubbles under the following conditions (1.0 MHz, 1.5 W / cm 2 , 1 minute at 20% or 30% duty cycle or 3 W / cm 2, 1 min at 50% duty cycle). After an additional 30 min or 3 h of reaction, the fluorescence values ​​of the supernatant were quantitatively measured at Ex / Em = 485 / 535 nm using a fluorometer (SpectraMax iD3 multimode plate reader (Molecular Devices Corp., Sunnyvale, CA, USA)).

[0097] Example 6. In vitro cytotoxicity assay

[0098] To determine cytotoxicity, the Cellomax Cell Viability Kit (Precaregene, Hanam, Gyeonggi-do) was used according to the manufacturer's instructions in an environment with three compounds. The 4T1 mouse breast cancer cell line was purchased from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a humidified CO2 incubator. 4T1 cells were seeded at 1.0 × 10 per well in a 96-well plate. 4 Each cell was inoculated. Twenty-four hours after inoculation, the culture medium was replaced with fresh medium (80 μL) containing 0, 0.1, 1, 5, 10, 30, and 50 μM of ZnPc, CuPc-SO3Na, or NiPc-SO3Na. The cells were cultured for an additional 24 h at 37°C and then washed twice with PBS. Then, the cells were illuminated with a 660 nm LED lamp (DavinchK, Seoul, Korea) at 100 mW / cm 2 30 J / cm for 5 minutes 2The cells were investigated. Cell viability was evaluated by the CCK-8 assay based on the absorbance at a wavelength of 450 nm using a microplate reader (Bio-TekELx800, USA). For the MTT method, 10 μL of 12 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Thermo Fisher Scientific) was added to each well and incubated at 37°C for 4 hours. As a negative control (cell-free blank), 10 μL of MTT solution was added to 100 μL of medium alone. 100 μL of SDS-HCl solution was added to the above solution and incubated for an additional 4 hours. The absorbance at 570 nm was measured using a SpectraMax iD3 multimode plate reader, and the percentage of viable cells was calculated according to the following formula: Cell viability (%) = (OD 처리 - OD blank ) / (OD 대조군 - OD blank ) Х 100.)

[0099] Cytotoxicity by SDT was assessed by live / dead cell staining. Live cells were distinguished by the presence of ubiquitous intracellular esterase activity, were cell-permeabilized by calcein AM, and stained green (excitation / emission = ~495 nm / ~515 nm). In contrast, EthD-1 entered damaged cell membranes and stained nucleic acids with red fluorescence (excitation / emission = ~495 nm / ~635 nm). 4T1 cells were treated with 10 μM ZnPc or 10 μM CuPc-SO3Na and 100 μL of microbubbles per sample, resulting in approximately 2 х 10 9 was added with microbubbles and then ultrasonically (1.0 MHz, 1.5 W / cm 2, 20% or 30% duty cycle, 1 min) were investigated. After 24 h, live / dead cell staining was performed according to the manufacturer's manual, and the stained cells were observed for fluorescence expression using a Carl Zeiss confocal laser scanning microscope (CLSM; Oberkochen, Baden-Wurttemberg, Germany).

[0100] Example 7. Detection of apoptosis

[0101] Apoptosis was quantified by fluorescence measurement using an Annexin V-APC apoptosis detection kit (Biolegend Inc., 8999 BioLegend Way, San Diego, CA, USA) that combines propidium iodide (PI) and fluorophore allophycocyanin (APC). Cells were washed with cold BioLegend cell staining buffer and then incubated in annexin V binding buffer at 0.25–1.0 χ 10 7 Cells were resuspended at a concentration of 10 cells / mL. 5 μL of APC-Annexin V and 10 μL of PI solution were added to 100 μL of cell suspension in a 5-mL test tube and incubated for 15 minutes at room temperature in the dark. 400 μL of Annexin V binding buffer was added to each tube and analyzed by flow cytometry (BD FACSAria III; BD Life Sciences, San Jose, CA, USA). The data collected by flow cytometry were analyzed according to the following criteria: viable (Annexin V-negative, PI-negative), early apoptotic (Annexin V-positive, PI-negative), late apoptotic (Annexin V-positive, PI-positive), and necrotic (Annexin V-negative, PI-positive) cell populations.

[0102] Example 8. Fluorescence quantum yield ( f) Calculation

[0103] The fluorescence quantum yield was determined by comparing the fluorescence intensity of the sample (S: sensitizer) with the fluorescence intensity of the fluorescence standard (R: reference) using the following mathematical equation:

[0104] [Mathematical Formula 1]

[0105]

[0106] In the above mathematical formula 1 is the quantum yield, A is the integrated area under the corrected fluorescence spectrum, OD is the optical density, and n is the refractive index. Also, the subscripts S and R represent the sample and reference, respectively, and the fluorescence quantum yield ( ) was used as a reference for the calculation.

[0107] Example 9. Singlet oxygen quantum yield ( ) calculate

[0108] Singlet oxygen quantum yield ( ) was calculated according to the literature of N Adarsh ​​et al (Organic Letters. 12, 5720-5723 (2010). The relative quantum yield was calculated as 0.52, which is the value of ZnPc as a reference in DMF (W Spiller et al, Journal of Porphyrins and Phthalocyanines, vol. 2, no. 2, pp. 145-158, 1998). Air-saturated DMF was obtained by bubbling air for 15 min. The absorbance of 1,3-diphenylisobenzofuran (DPBF) was adjusted to about 1.0 in air-saturated dichloromethane. After adding the sample sensitizer to the cuvette and adjusting the absorbance of the sensitizer to about 0.1, some measurements were performed in the dark, and the cuvette was exposed to monochromatic light at the peak absorption wavelength for 3 min. After irradiation, the absorbance was measured several times. After that, for each photosensitizer The slope of the DPBF maximum absorbance versus time plot at 414 nm was calculated. The singlet oxygen quantum yield was calculated according to the following mathematical equation (2).

[0109] [Equation 2]

[0110]

[0111] In the above mathematical expression 2, m is the slope of the difference in absorbance change of DPBF at 414 nm according to the irradiation time, and F is the absorption correction coefficient, F = 1 - 10 -OD (OD at the investigation wavelength) and PF is the absorbed flux (μEinstein dm -3 s -1 )am.

[0112] Experimental Example 1. Synthesis and Characterization of CuPc-SO3 and NiPc-SO3

[0113] In order to synthesize an ultrasound sensitizer to solve the problems of existing ultrasound sensitizers, phthalocyanine (Pc) was selected as an ultrasound sensitizer due to its excellent ultrasound sensitization, high molecular extinction co-efficiency in the far infrared and visible range, low dark toxicity, excellent chemical stability, and excellent biocompatibility. In addition, sulfonation of phthalocyanine suppresses its inherent aggregation tendency and improves the therapeutic efficiency of PDT or SDT, making it more suitable for clinical application, making it effectively usable as an ultrasound sensitizer. The macrocyclic structure of phthalocyanine is Zn 2+ , Cu 2+ or Ni 2+ It can form a complex containing a wide range of metal ions including Cu. Among the metal ions, Cu 2+ or Ni 2+ Paramagnetic ions such as Cu can produce phthalocyanine compounds with reduced cross-talk effects between systems, and the paramagnetic ions can weaken or completely inhibit the phototoxic activity of phthalocyanine compounds due to the heavy metal ion effect based on metal-ligand charge transfer (MLCT). Therefore, in order to synthesize an ultrasonic sensitizer based on phthalocyanine without phototoxicity, a paramagnetic ion such as Cu 2+ or Ni 2+ - Complex sulfonated phthalocyanines (CuPc-SO3 and NiPc-SO3) were synthesized by the method of Experimental Example 1, and their chemical structure is shown in Figure 1. In addition, Zn was used as a closed-shell diamagnetic ion. 2+ Zn containing 2+As a complex phthalocyanine sensitizer, ZnPc, which is currently in clinical use, was set as a reference sensitizer. In order to analyze the characteristics of the synthesized sulfonated phthalocyanine ultrasonic sensitizer, an experiment was performed to confirm the photophysical properties of CuPc-SO3, NiPc-SO3, and ZnPc in DMF solution, and the results are shown in Fig. 2. The HPLC chromatograms of CuPc-SO3 and NiPc-SO3 were confirmed and shown in Figs. 3A and 3B. In addition, the absorption spectrum and fluorescence spectrum of 4.0 μM CuPc-SO3, NiPc-SO3, and ZnPc in DMF solution were confirmed and shown in Figs. 4A and 4B, respectively.

[0114] As confirmed in Figures 3C and 3D, the ESI-MS results of CuPc-SO3 are C 32 H 12 CuN8Na4O 12 S4[MH] - The silver was 982.83, the found value was 981.1, and the ESI-MS result of NiPc-SO3 was C 32 H 12 N8Na4NiO 12 S4[MH] - The value of CuPc-SO3 was 976.9, and the found value was 977.84. In addition, the HPLC retention time of CuPc-SO3 was 3.139 minutes, and the HPLC retention time of NiPc-SO3 was 3.317 minutes. It was confirmed that the characteristics confirmed in Figures 3A to 3D are completely consistent with the expected structures of CuPc-SO3 and NiPc-SO3 compounds.

[0115] As confirmed in Fig. 2 and Fig. 4A, a distinct absorption peak attributed to the phthalocyanine core was clearly observed in the synthesized compound. The molecular extinction coefficient (ε) for CuPc-SO3 was 2.65 Х 10 5 M -1 cm -1 (λ abs= 675 nm), the molecular extinction coefficient (ε) for NiPc-SO3 is 2.67 Х 10 5 M -1 cm -1 (λ abs = 669 nm), and for ZnPc it was 2.62 Х 10 5 M -1 cm -1 (λ abs = 669 nm) was confirmed (where λ abs is the maximum absorption wavelength (nm), ε is the molecular extinction coefficient (Х10 5 M -1 cm -1 ), λ em is the maximum emission wavelength (nm). Nevertheless, as confirmed in Fig. 2 and Fig. 4B, when irradiated at 610 nm, which is the excited state, the emission bands of CuPc-SO3 and NiPc-SO3 were completely suppressed, which is thought to be caused by the MLCT-based heavy metal ion effect. In contrast, under the same experimental conditions, it was confirmed that ZnPc exhibited significant fluorescence emission centered around 677 nm ( f = 0.17; see equation 1).

[0116] Experimental Example 2. Photosensitization properties of CuPc-SO3 and NiPc-SO3

[0117] Because phototoxicity related to ROS generation occurs when irradiated with light, the photoresponsive properties of Pc-SO3 were analyzed before applying them to in vitro SDT studies. The ROS production capacity of CuPc-SO3, NiPc-SO3, and ZnPc during light irradiation was analyzed in aqueous media using a commercial ROS probe, 2',7'-dichlorodihydrofluorescein diacetate. The ROS production capacity of 1 μM CuPc-SO3, NiPc-SO3, or ZnPc is shown in Figures 5A, 5B, and 5C, respectively, and the ROS production rate is shown in Figure 5D as a result of plotting the change in absorption intensity at 523 nm.

[0118] As confirmed in Fig. 5A, when the CuPc-SO3 solution containing DCF was irradiated with a 660 nm Xe-lamp, the fluorescence intensity of DCF showed almost no change. Also, as confirmed in Fig. 5B, the fluorescence intensity of NiPc-SO3 showed almost no change. In contrast, in the case of ZnPc in Fig. 5C, a significant spectral change was observed, confirming that a significant amount of ROS was generated. As confirmed in Fig. 5D, the ROS generation rates of the CuPc-SO3 and NiPc-SO3 solutions were about 6.8 and 15.3, respectively, which were slower than the generation rate of ZnPc, which was 109.9.

[0119] Also, photosensitive singlet oxygen by ZnPc, CuPc-SO3 and NiPc-SO3 1An experiment was conducted to confirm the production of O2. The absorption spectrum changes over time in 9,10-diphenylanthracene (DPA) solutions containing ZnPc, CuPc-SO3 or NiPc-SO3 were measured and shown in FIGS. 6A to 6C, respectively. In addition, the results of plotting the changes in absorption intensity at 383 nm in FIGS. 6A to 6C are shown in FIG. 6D. As confirmed in FIGS. 6A to 6D and in Equation 2 of Example 9, the singlet oxygen QY ( ) were confirmed to be 0.07 and 0.02, respectively, when irradiated with light at 660 nm. These singlet oxygen QY( ) was found to be significantly smaller than the QY value of ZnPc (0.56 in DMF), a sensitizer being clinically tested. Therefore, it was confirmed that the ultrasonic sensitizer containing the CuPc-SO3 and NiPc-SO3 compounds synthesized in Experimental Example 1 can be effectively utilized as an ultrasonic sensitizer that can prevent phototoxicity by inhibiting the generation of reactive oxygen species that cause phototoxicity.

[0120] Experimental Example 3. Ultrasonic sensitivity characteristics of CuPc-SO3 and NiPc-SO3

[0121] To confirm whether the compound can be effectively used as an ultrasound sensitizer, an experiment was conducted to confirm the ultrasound sensitizing properties of the compound. An experiment was conducted to determine whether CuPc-SO3 induces the generation of ROS that damages tumor cells by ultrasound irradiation, and the results of the ROS production levels of CuPc-SO3, NiPc-SO3, and ZnPc under ultrasound exposure are shown in Fig. 7. The measurement of the level of ROS production was measured using the principle that fluorescence is emitted when DCF reacts with ROS. Specifically, the measurement results for the CuPc-SO3 solution are shown in Fig. 7A, the measurement results for the NiPc-SO3 solution are shown in Fig. 7B, and the measurement results for the ZnPc solution are shown in Fig. 7C, and the ROS measurement results for the control solution that did not contain the ultrasound sensitizer are shown in Fig. 8. In addition, the results of plotting the change in absorption intensity at 524 nm in Figs. 7A to 7C are shown in Fig. 7D.

[0122] As confirmed in Figures 7A and 7C, the ROS production levels of CuPc-SO3 solution and ZnPc were increased by ultrasound (0.2 W / cm 2 , 20% duty cycle, 1 MHz) was confirmed to increase as the exposure time increased up to 40 minutes. In contrast, as can be seen in Fig. 8, almost no spectral change was observed in the control group under the same experimental conditions, confirming that ROS production was hardly observed. As confirmed in Fig. 7B, it was confirmed that DCF emission was somewhat observed in the NiPc-SO3 solution as the ultrasound exposure time increased. As confirmed in Fig. 7D, the ROS production rate of CuPc-SO3 was 60.3, the ROS production rate of NiPc-SO3 was 14.1, and the ROS production rate of ZnPc was 89.2.

[0123] An experiment was conducted to determine whether adding microbubbles to a synthesized ultrasound sensitizer compound with suppressed phototoxicity could enhance the sonoluminescence effect and thereby increase ultrasonic cavitation, thereby enhancing the SDT therapeutic effect. The relative fluorescence intensities of the solutions exposed to various ultrasound environments after adding CuPc-SO3 or ZnPc to DCFH-DA with or without microbubbles are measured in Fig. 7E. In addition, the relative fluorescence intensities of CuPc-SO3 or ZnPc under ultrasound irradiation conditions with microbubbles are shown in Fig. 7F.

[0124] As confirmed in Figs. 7E and 7F, when ultrasonic cavitation was induced by adding microbubbles, the production of ROS in CuPc-SO3 and ZnPc was significantly increased, and it was confirmed that ROS production also increased as the ultrasonic exposure energy increased (from US+ to US++, US+++). In particular, when microbubbles were treated together with CuPc-SO3, ROS production was confirmed to be approximately 2.4 times higher than that of ZnPc under ultrasonic exposure. Therefore, the synthesized CuPc-SO3 compound can be utilized as a more effective ultrasonic sensitizer for cancer treatment using SDT, and it can be expected that the efficiency of tumor treatment can be increased as an effective ultrasonic sensitizer when microbubbles are administered together as an adjuvant.

[0125] Experimental Example 4. In vitro phototoxicity and anticancer activity of CuPc-SO3 and NiPc-SO3, which are ultrasound sensitizers, as anticancer agents.

[0126] In order to confirm whether the generation of ROS confirmed in the above experimental example affects tumor cell death, photoreactivity and ultrasound sensitivity by CuPc-SO3, NiPc-SO3 and ZnPc, an experiment was performed, and the photoreactive cytotoxicity of CuPc-SO3, NiPc-SO3 and ZnPc was confirmed as a result shown in Fig. 9. Specifically, the cell viability of the mouse breast cancer cell line 4T1 treated with various concentrations (0.1 to 50 μM) of CuPc-SO3, NiPc-SO3 or ZnPc and DMSO as a control group for 24 hours is confirmed as a result of the group without light irradiation in Fig. 9A, and 660 nm light irradiation (100 mW / cm 2 for 5 minutes; 30 J / cm 2 ) is shown in Fig. 9B. In addition, as a method for confirming the sonodynamic cytotoxicity of CuPc-SO3, NiPc-SO3, and ZnPc, the results of apoptosis analysis of CuPc-SO3, NiPc-SO3, and ZnPc in 4T1 cancer cells are shown in Fig. 10.

[0127] As confirmed in Figures 9A and 9B, ZnPc was irradiated with light (660 nm laser light, 100 mW / cm 2 When the induced photoreactive cytotoxicity was investigated for 10 minutes, it was significantly increased in a dose-dependent manner compared to when not exposed to light, but in the presence of various concentrations of CuPc-SO3 and 0.1 to 10 mM. It was confirmed that no difference in cytotoxicity was observed regardless of the presence or absence of light irradiation when NiPc-SO3 was treated.

[0128] To confirm the activity of sonodynamic therapy, an ultrasound sensitization experiment was conducted, and apoptosis was analyzed according to the presence or absence of ultrasound irradiation in cancer cells treated with an ultrasound sensitizer compound together with microbubbles. As confirmed in Fig. 10, compared to the control group that was not irradiated with ultrasound (US -), the group treated with the ZnPc solution in the ultrasound irradiated group showed an approximately 1.6-fold increase in the number of apoptotic cells in the control group under the condition of US++. On the other hand, the group treated with the CuPc-SO3 solution in the group that was exposed to ultrasound showed a significant increase in the number of early apoptotic cells by approximately 2.8-fold under the condition of US++ compared to the group that was not exposed to ultrasound. In addition, it was confirmed that the number of apoptotic cells increased as the ultrasound exposure energy increased from US+ to US++, confirming that the anticancer activity significantly increased in a ultrasound energy-dependent manner.

[0129] In summary, we confirmed that the CuPc-SO3 ultrasound sensitizer compound, which does not exhibit photoreactivity and thus solves the phototoxicity problem, can significantly enhance apoptosis in 4T1 cancer cells compared to ZnPc, which is currently in clinical use, when exposed to ultrasound at an effective intensity. Therefore, we confirmed that the CuPc-SO3 and NiPc-SO3 compounds with reduced phototoxicity can play the role of ultrasound sensitizers that can be utilized for effective anticancer treatment when administered together with microbubbles as an adjuvant while preventing phototoxicity when utilized in SDT therapy.

Claims

1. A sonosensitizer composition comprising a phthalocyanine compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof; [Chemical Formula 1] In the above chemical formula 1, M is Cu(Ⅱ) or Ni(Ⅱ), R is one selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, sulfonic acid, amidosulfuric acid, phosphoric acid, nitric acid, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, tartaric acid, citric acid, oxalic acid, maleic acid, maleic acid, ethanesulfonic acid, salicylic acid, benzoic acid, paratoluenesulfonic acid, and methanesulfonic acid.

2. An ultrasound sensitizer composition according to claim 1, wherein the phthalocyanine compound or a pharmaceutically acceptable salt thereof is a compound represented by the following chemical formula 2 or 3. [Chemical Formula 2] [Chemical Formula 3] 3. An ultrasonic sensitizer composition according to claim 1, characterized in that the compound does not generate reactive oxygen species (ROS) even when irradiated with light of 300 to 700 nm.

4. An ultrasonic sensitizer composition according to claim 1, wherein the compound exhibits ultrasonic sensitizing activity for ultrasonic waves having a wavelength of 0.1 to 10 MHz.

5. In claim 1, the compound has a luminescence intensity of 0.01 to 10.0 W / cm 2 An ultrasound sensitizer composition that generates ROS when irradiated with ultrasound of an intensity of .

6. An ultrasonic sensitizer composition according to claim 1, wherein the composition further comprises microbubbles.

7. In claim 6, the microbubbles are selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine (DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and An ultrasound sensitizer composition comprising at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[succinyl(polyethylene glycol)-2000] (DSPE-PEG2k-NHS).

8. An ultrasound sensitizer composition according to claim 7, wherein the microbubbles comprise DSPC and DSPE-PEG2k-NHS in a molar ratio of 0.1:1 to 10:

1.

9. A pharmaceutical composition for preventing or treating cancer, comprising a phthalocyanine compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient; [Chemical Formula 1] In the above chemical formula 1, M is Cu(Ⅱ) or Ni(Ⅱ), R is one selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, sulfonic acid, amidosulfuric acid, phosphoric acid, nitric acid, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, tartaric acid, citric acid, oxalic acid, maleic acid, maleic acid, ethanesulfonic acid, salicylic acid, benzoic acid, paratoluenesulfonic acid, and methanesulfonic acid.

10. In claim 9, the cancer is lung cancer, laryngeal cancer, stomach cancer, colon / rectum cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine sarcoma, small intestine cancer, uterine cancer, cervical cancer, urethral cancer, prostate cancer, melanoma, bone cancer, kidney cancer, skin cancer, bone cancer, muscle cancer, fatty cancer, fibrous cell carcinoma, leukemia, lymphoma, multiple myeloma, tumor arising in nerve tissue, triple negative breast cancer, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity / paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, A pharmaceutical composition comprising at least one selected from the group consisting of thymic cancer, mediastinal tumor, and esophageal cancer.

11. A pharmaceutical composition according to claim 9, further comprising a pharmaceutical additive selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, and any combination thereof.

12. A method for treating cancer, comprising administering to a subject other than a human a pharmaceutical composition according to any one of claims 9 to 11.

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