Composition for preventing or treating brain tumor, comprising liquid plasma

Abstract

A composition comprising a liquid plasma according to the present invention can induce growth inhibition and death of brain tumor cells through the combined effect of radicals, ionic species, and pH effect, and thus can be effectively used for preventing or treating brain tumors. In addition, the present invention is a non-invasive physical and chemical treatment approach with low drug dependence, can exhibit a synergistic effect when used in combination with existing anticancer drugs, and has a high potential for application to the treatment of various nervous system tumors or metastatic brain diseases including glioblastoma and auditory nerve tumors.

Description

Composition for the prevention or treatment of brain tumors containing liquid plasma

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of brain tumors comprising liquid plasma and a method for preparing the same.

[0002] According to data released by the National Cancer Registry, brain and central nervous system cancers rank 3rd in crude incidence among men and 2nd among women in the 0 to 14-year age group, and 3rd overall for both sexes. Brain tumors occur at a rate of approximately 3,000 cases per year in Korea; while benign brain tumors can be cured through curative surgery, malignant brain tumors are treated by combining surgery, radiation therapy, and chemotherapy. Commonly occurring brain tumors include gliomas, meninges, pituitary adenomas, metastatic brain tumors, and acoustic neuromas. Among these, gliomas (glioblastoma) are known as the most common malignant tumor classified as Grade 4 in the WHO brain tumor classification, with an incidence rate of approximately 3 to 4 cases per 100,000 people per year.

[0003] Glioblastoma is a biologically malignant tumor of the nervous system characterized by its occurrence in the central nervous system (CNS). Depending on the location and size of the tumor, glioblastoma can cause a variety of symptoms, which generally include headache, vomiting, visual impairment, drowsiness, and muscle weakness. Symptoms can worsen if the tumor exerts pressure on the central nervous system.

[0004] An acoustic neuroma is a type of schwannoma that is a tumor originating in nerve tissue. Acoustic neuromas primarily originate from the axonal cells of nerves; they grow relatively slowly and may be asymptomatic in the early stages. However, as the tumor grows, it can compress nearby tissues or invade nerves, potentially causing a variety of symptoms. These symptoms may include pain, tingling or numbness, muscle weakness, and paresthesia.

[0005] Meanwhile, treatment methods for brain tumors include surgery, radiation therapy, and chemotherapy, and generally, chemotherapy is used after the cancer has been removed through surgery and radiation therapy. First-line chemotherapy agents for brain tumor treatment include temozolomide, which alkylates or methylates DNA within cells, and second-line agents include Avastin-irinotecan.

[0006] However, due to the drug's refractory and resistance to temozolomide used in the treatment of brain tumors, there is a problem of poor prognosis caused by low cure rates and frequent recurrence and metastasis. Consequently, the 5-year survival rate for malignant brain tumors is only about 20-30%, and many patients die early in the course of tumor treatment, making it necessary to discover new targets to treat this. Accordingly, the inventors confirmed the use of liquid plasma for the treatment of brain tumors and completed the present invention.

[0007] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of brain tumors comprising liquid plasma as an active ingredient.

[0008] Another objective of the present invention is to provide a method for producing liquid plasma for the prevention or treatment of brain tumors.

[0009] Another objective of the present invention is to provide a liquid plasma for the prevention or treatment of brain tumors produced by the above-described manufacturing method.

[0010] To solve the above-mentioned problem, the present invention provides a pharmaceutical composition for the prevention or treatment of brain tumors comprising liquid plasma as an active ingredient, wherein the pH of the liquid plasma is 4.5 to 7.

[0011] According to one embodiment of the present invention, the liquid plasma may be characterized by including nitrogen-based and oxygen-based radicals.

[0012] According to one embodiment of the present invention, the brain tumor may be characterized as being an acoustic neuroma or a glioblastoma.

[0013] According to one embodiment of the present invention, the pharmaceutical composition may be a parenteral formulation and may be selected from intravenous, intra-arterial, intramuscular, subcutaneous, intratumoral or peritumoral injection, subdural, subarachnoid or intraventricular administration, a solution for perfusion or irrigation of the surgical site, a gel, a hydrogel, or a spray-type topical formulation.

[0014] According to one embodiment of the present invention, the pharmaceutical composition may be characterized by detecting PARP cleavage and / or an increase in γ-H2AX signal within 4 to 8 hours after treatment of target cells or target tissues.

[0015] According to one embodiment of the present invention, the pharmaceutical composition may be characterized in that apoptosis is inhibited when combined with a ferroptosis inhibitor, but apoptosis is not inhibited when combined with an iron chelator.

[0016] According to one embodiment of the present invention, the pharmaceutical composition may be characterized by inhibiting apoptosis when neutralizing the pH of the culture medium or microenvironment of the target cell or target tissue.

[0017] In addition, the present invention provides a method for producing a liquid plasma for the prevention or treatment of brain tumors, comprising the following steps: (a) forming a plasma by performing a discharge in water or in an area adjacent to the water surface using an atmospheric pressure plasma generator; and (b) producing a liquid plasma containing nitrogen-based and oxygen-based radicals by dissolving and accumulating reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated by the discharge in water.

[0018] The above manufacturing method may further include the following steps: (c) a step of increasing the concentration of underwater radicals in the liquid plasma.

[0019] In addition, the present invention provides a liquid plasma for the prevention or treatment of brain tumors produced by the manufacturing method described above.

[0020] According to one embodiment of the present invention, the pH of the liquid plasma may be 4.5 to 7.

[0021] The composition containing liquid plasma according to the present invention can effectively induce apoptosis of brain tumor cells, and thus has the effect of promoting the healing of brain tumors.

[0022] Figure 1 shows a bar graph comparing cell viability (%) relative to 0 hours under untreated, 1× PAM, and 2× PAM conditions for acoustic neuroma cell line HEI-193 and glioblastoma cell line U-87 MG.

[0023] Figure 2a shows a line graph comparing cell viability (%) relative to 0 hours over time in acoustic neuroma cells under control, 1× PAW, and 2× PAW conditions.

[0024] Figure 2b shows a line graph representing the change in cell viability (%) from 0 to 24 hours under 2× PAW conditions in acoustic neuroma cells.

[0025] Figure 3 shows a line graph comparing cell survival rates (%) over time (relative to 0 hours) in acoustic neuroma cells treated with a control group (Untreated), 1× PAM, and 2× PAM.

[0026] Figure 4 shows the expression of PARP, p53, p-p53, caspase-3, and H2AX over time after PAM treatment using Western blot.

[0027] Figure 5a shows a bar graph comparing the cell viability (%) relative to 0 hours after treatment with the control group (Untreated, Untreated+NAC) and 2× PAM, and 2× PAM+NAC in acoustic neuroma cells at 0 and 24 hours.

[0028] Figure 5b shows a bar graph comparing the cell viability (%) relative to 0 hours after treatment with the control group (Untreated, Untreated+carboxy-PTIO) and 2× PAM, 2× PAM+carboxy-PTIO at 0 and 24 hours.

[0029] Figure 5c shows a bar graph comparing the cell viability (%) relative to 0 hours after treatment with the control group (Untreated, Untreated+Glutathione) and 2× PAM, 2× PAM+Glutathione, at 0 and 24 hours in acoustic neuroma cells.

[0030] Figure 6a is a bar graph comparing cell viability (%) relative to 0 hours in acoustic neuroma cells with the combined use of z-VAD-fmk, showing the results at 0 and 24 hours for the control group (Untreated), 2× PAM, and 2× PAM+z-VAD-fmk (10, 30, 50 μM) conditions.

[0031] Figure 6b is a bar graph comparing cell viability (%) relative to 0 hours in acoustic neuroma cells with the combination of Necrostatin-1, showing the 0 and 24-hour results for the control group (Untreated), 2× PAM, and 2× PAM+Nec-1 (0.1, 1, 5 μM) conditions.

[0032] Figure 6c is a bar graph comparing cell viability (%) relative to 0 hours in acoustic neuroma cells with the combination of Liproxstatin-1, showing the 0 and 24-hour results for the control group (Untreated), 2× PAM, and 2× PAM+Lip-1 (10, 100, 300 nM) conditions.

[0033] Figure 6d is a bar graph comparing cell viability (%) relative to 0 hours in acoustic neuroma cells with the combination of Ferrostatin-1, showing the 0 and 24-hour results for the control group (Untreated), 2× PAM, and 2× PAM+Fer-1 (0.1, 1, 5 μM) conditions.

[0034] Figure 7 shows a bar graph comparing cell viability (%) relative to 0 hours after 24 hours of treatment with 2× PAM alone and Deferoxamine combined treatment (100, 300, 500, 1000 nM) in acoustic neuroma cells.

[0035] Figure 8a shows a line graph comparing the cell viability (%) over time (relative to 0 hours) in acoustic neuroma cells when 2× PAM-treated medium was neutralized to pH 7.2 with sodium bicarbonate and when it was not neutralized.

[0036] Figure 8b shows a bar graph comparing the change in medium pH according to the liquid plasma concentration.

[0037] Figure 8c shows a line graph comparing cell viability (%) relative to 0 hours over time when acoustic neuroma cells were cultured under pH conditions of 7.4, 5.8, 5.3, and 4.8.

[0038] Figure 9a shows a line graph comparing cell viability (%) relative to 0 hours after treatment with Sham, 1× PAM, and 2× PAM in patient-derived primary acoustic neuroma cells VS-52.

[0039] Figure 9b shows a line graph comparing cell survival rates over time under the same conditions in patient-derived primary acoustic neuroma cells VS-55.

[0040] Figure 9c shows a line graph comparing cell survival rates over time under the same conditions in patient-derived primary acoustic neuroma cells VS-56.

[0041] Figure 9d shows a line graph comparing cell survival rates over time under the same conditions in patient-derived primary acoustic neuroma cells VS-87.

[0042] Figure 10a shows the tumor size (mm²) of the PAW 5× administration group and the control group in a Balb / C nude mouse transplant tumor model. 3 This is a line graph comparing the changes over time of ).

[0043] Figure 10b shows a bar graph comparing tumor weight (mg) in the same model as Figure 10a.

[0044] Figure 10c shows an external photograph of tumor tissue extracted in the same experiment as Figure 10a.

[0045] Figure 10d shows H&E stained pathology images of tumor tissues from the control group and the PAW treatment group in the same experiment as Figure 10a.

[0046] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0047] The present invention provides a pharmaceutical composition for the prevention or treatment of brain tumors comprising liquid plasma as an active ingredient.

[0048] In this specification, "liquid plasma" refers to a liquid in which reactive oxygen species (ROS) and / or reactive nitrogen species (RNS) generated by atmospheric pressure plasma discharge are dissolved or accumulated in water, and specifically includes, but is not limited to, (i) plasma-activated medium (PAM) obtained by exposing cell culture medium to plasma, (ii) plasma-activated water (PAW) obtained by exposing sterile distilled water to plasma, (iii) plasma-activated solution (PAS) based on physiological saline (0.9% NaCl), (iv) plasma-activated solution (PA-PBS) based on phosphate-buffered saline (PBS), (v) plasma-activated solution (PAR or PARL) based on Ringer's solution or lactate Ringer's solution, (vi) plasma-activated solution (PAB) based on buffer solutions such as HEPES, acetate, bicarbonate, etc., (vii) plasma-activated solution based on a balanced salt solution (BSS) or a mixture thereof.

[0049] The above liquid plasma may include nitrogen-based and oxygen-based radicals. Specifically, it may include not only short-lived radicals such as •OH (hydroxyl radical), O2•⁻ (superoxide radical anion), and NO• (nitrogen monooxide radical), but also long-lived species such as H2O2, NO2⁻, NO3⁻, and O₃, and, if necessary, e aq - It may include species such as (hydrated electrons), H• (hydrogen radicals), HO2• (hydroperoxyl radicals). This composition may be controlled according to discharge conditions (e.g., power or power density, discharge time, electrode-water surface distance, type and flow rate of process gas, etc.), but is not limited thereto.

[0050] The pH of the liquid plasma may be 4.5 to 7, and more preferably, may be adjusted to a pH of 4.8 to 6.7. The above pH range is set considering the balance between the persistence of radicals (ROS / RNS) and safety during application, and is suitable for maintaining the stability of long-lived active species (H2O2, NO2⁻, NO3⁻, etc.) formed by plasma treatment while avoiding non-specific acid toxicity that occurs at excessively low pH. If the pH of the aqueous solution exceeds 7, the decomposition of active species is accelerated, which may cause the effective concentration to decrease rapidly; conversely, if the pH drops below 4.5, the cytotoxicity of the acid itself increases, which may lead to non-specific acid damage. In a preferred embodiment of the present invention, plasma irradiation conditions (power, time, electrode-surface distance, process gas composition, flow rate, etc.) may be adjusted so that the pH immediately after preparation falls within the above range, or, if necessary, the pH may be finely adjusted using a buffer or neutralizing agent to a level that does not compromise physiological tolerance.

[0051] The composition of the present invention can be applied to the prevention or treatment of brain tumors, including acoustic neuroma and glioblastoma.

[0052] Vestibular schwannomas are peripheral nerve sheath tumors that primarily occur in the cerebellopontine angle (CPA) and internal auditory canal; although benign, their locational characteristics can cause hearing loss, tinnitus, and balance disorders. The composition of the present invention can be applied in conjunction with surgical resection through methods such as intratumoral or peritumoral local injection, cavity perfusion and irrigation, or stepwise injection via a fine needle. Such local delivery is advantageous for increasing effective exposure at the lesion site without being constrained by the blood-brain barrier. Administration can be performed as a single dose or on a repeated schedule, and the dosage, frequency, and number of doses can be adjusted within medically acceptable ranges depending on the timeline before and after surgery, the extent of the residual lesion, and the auditory nerve preservation strategy. Quality specifications include pH (e.g., 4.5–7), redox potential (ORP), and longevity active species indicators (H₂O₂, NO₂).- , NO3 - Ensure reproducibility between batches by using (etc.).

[0053] Glioblastoma is a malignant glioma characterized by diffuse invasion and rapid proliferation, and carries a high risk of recurrence even after standard therapy (surgery + radiation + chemotherapy). The composition of the present invention can be applied via (i) intraoperative perfusion / irrigation or local application in the form of a gel / hydrogel immediately after resection, (ii) intratumoral or peritumoral injection (repeated catheter-based injection if necessary), or (iii) administration within the central nervous system, such as subdural, subarachnoid, or intraventricular. Such delivery enables local high-concentration exposure, thereby facilitating adjuvant control of invasive residual cells. The administration schedule is established by referring to imaging evaluations and neurological indicators, and the reproducibility of the therapeutic response is enhanced by managing the pH specifications and active tumor indicators of the composition based on batch release standards. It may be used in combination with radiation therapy or chemotherapy if necessary; however, when used in combination, the administration sequence, interval, and local tissue safety are considered during the design process.

[0054] The above pharmaceutical composition may be a parenteral formulation and may be selected from intravenous, intra-arterial, intramuscular, subcutaneous, intratumoral or peritumoral injection, subdural, subarachnoid or intraventricular administration, surgical site perfusion or irrigation solution, gel, hydrogel, or spray-type topical formulation to increase local exposure at the lesion site and avoid the limitations of the blood-brain barrier.

[0055] The liquid formulation is manufactured as a sterile aqueous preparation for injection or perfusion, and, if necessary, is mixed with pharmaceutically acceptable solutions such as physiological saline, PBS, or Ringer's solution to adjust osmotic pressure, ionic strength, and pH (e.g., 4.5–7). For intratumoral and peritumoral injections, small doses can be divided and administered at multiple points using a fine needle or catheter, while subdural, subarachnoid, and intraventricular administration allows for repeated catheter-based infusion via a neurosurgical approach. Immediately after surgery, it can be applied to the surgical cavity as a perfusion and irrigation solution, or uniformly sprayed along the resection margin as a spray formulation to provide local exposure to residual cells.

[0056] Gel or hydrogel formulations can be used to extend residence time at the application site and reduce lavage loss, and viscosity, gelation temperature, and degradation rate are adjusted to suit the surgical procedure and anatomical space. Spray formulations can be designed with spray angles and atomization characteristics to enable uniform application over a wide range of surfaces. For intravenous and intra-arterial administration, dosage and infusion rates are managed considering systemic exposure, and if necessary, selective arterial injection under angiography guidance can be used to target the lesion's supplying vessels.

[0057] To ensure radical preservation and convenience for on-site use, the above pharmaceutical composition may be packaged in sterile single-use containers that are filled and sealed immediately after manufacturing, making them ready-to-use. This packaging design is advantageous for (i) minimizing the loss of active species (ROS / RNS) over time after manufacturing, (ii) blocking microbial contamination and exposure to air (oxygen) and light during the opening and dispensing process, and (iii) suppressing quality variations between batches to enhance the reproducibility of apoptotic efficacy. Additionally, the container may be selected from a material that is chemically compatible with the solution and may be provided in a single-dose volume to prevent reuse after opening. Storage may be carried out under recommended temperature and light-shielding conditions, and the product may be applied immediately after undergoing a simple verification procedure (pH, etc.) prior to use. If necessary, it may include standardized interfaces to enable connection with standard delivery devices such as syringes, catheters, and sprays, and consistency may be managed through basic quality indicators (e.g., pH, appearance, etc.) during batch release.

[0058] The above pharmaceutical composition may result in the detection of PARP cleavage and / or an increase in γ-H2AX signal within 4 to 8 hours after treatment of target cells or target tissues. These changes can be understood as reflecting the progression of apoptosis (detection of cleavage products of poly-ADP-ribose polymerase) and rapid labeling for DNA double-strand damage (phosphorylation of histone H2AX), respectively. The signal can be confirmed by standard methods such as, for example, Western blot, immunofluorescence staining (γ-H2AX foci number / intensity), and flow cytometry, and it is desirable to observe a time-dependent increase compared to a control group (0 hours, untreated, or solvent control).

[0059] The above range of 4 to 8 hours is a representative detection window that may vary depending on the cell line and process conditions, and may be partially advanced or delayed depending on the cell type, treatment concentration, medium conditions, etc. Signal intensity can be quantified by normalizing with an internal loading control (e.g., β-actin) or a nucleoprotein standard, and if necessary, the initiation of apoptosis and damage response following treatment with the composition can be more reliably verified by interpreting it together with auxiliary indicators such as caspase-3 activation and p53 / p-p53 changes.

[0060] When the above pharmaceutical composition is used in combination with a ferroptosis inhibitor (e.g., Liproxstatin-1, Ferrostatin-1), apoptosis may be significantly alleviated (or inhibited) by inhibiting cell membrane lipid peroxidation. On the other hand, when used in combination with an iron chelator (e.g., Deferoxamine), inhibition of apoptosis may not be observed under the same conditions, which suggests that apoptosis caused by the composition may have an atypical ferroptosis character that is not sufficiently inhibited by blocking the typical iron-dependent pathway alone. Evaluation of apoptosis can be performed by conventional viability analysis (MTT, WST-1, CCK-8, etc.) or morphological / molecular marker analysis (PARP cleavage, γ-H2AX, caspase-3 activation, etc.), and the evaluation of the combination effect can be quantified by the degree of recovery compared to single treatment (e.g., increased cell viability, reduced lipid ROS, etc.).

[0061] The above pharmaceutical composition may alleviate or inhibit apoptosis when the pH of the target cell or tissue culture medium or tumor microenvironment is neutralized (e.g., by mixing with sodium bicarbonate or buffer solution). This suggests that the action of active species formed and accumulated by liquid plasma treatment may include pH-dependent factors.

[0062] Whether apoptosis is inhibited can be confirmed by comparing the results before and after neutralization using conventional viability analysis (MTT, WST-1, CCK-8, etc.), morphological observation, or molecular markers (e.g., PARP cleavage, γ-H2AX). The degree of inhibition may vary depending on the timing of neutralization, the type and concentration of the neutralizing agent used, the substrate (physiological saline, PBS, Ringer's solution, culture medium, etc.), and the exposure time, and the present invention is not limited to a specific neutralization method or mechanism.

[0063] The present invention provides a method for producing a liquid plasma for the prevention or treatment of brain tumors, comprising the following steps: (a) forming a plasma by performing a discharge in water or in an area adjacent to the water surface using an atmospheric pressure plasma generator; and (b) producing a liquid plasma containing nitrogen-based and oxygen-based radicals by dissolving and accumulating reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated by the discharge in water.

[0064] In addition, the manufacturing method of the present invention may further include the following step: (c) a step of increasing the concentration of underwater radicals of the liquid plasma.

[0065] Step (a) above is a step of forming plasma by performing a discharge in an underwater or surface-adjacent region using one or more atmospheric pressure plasma generators, such as microwave, radio frequency (RF), dielectric barrier discharge (DBD), atmospheric pressure plasma jet (APPJ), and corona discharge. The process gas may be air, nitrogen, oxygen, argon, helium, or a mixture thereof, and power (or power density), discharge time, electrode-surface distance, and gas flow rate / composition may be set as key control variables.

[0066] In step (b) above, conditions are maintained to dissolve and accumulate reactive oxygen species (ROS) and reactive nitrogen species (RNS) formed by the discharge in water. The liquid substrate may be selected from pharmaceutically acceptable aqueous solutions such as sterile distilled water, physiological saline, PBS, Ringer's solution, balanced salt solution, and cell culture medium, and may be used alone or in combination as needed. Target concentrations of long-life indicator species (H2O2, NO2⁻, NO3⁻, O3, etc.) can be achieved by increasing the discharge intensity and time or by adjusting the gas composition, and excessive heating, acidification, or loss of active species can be prevented by monitoring the temperature, pH, and redox potential (ORP) during the process.

[0067] Step (c) above involves adjusting process variables to increase the underwater radical concentration of the liquid plasma. Specifically, this may include (i) increasing the effective plasma irradiation dose by extending the discharge time or increasing the power (power density), (ii) adjusting the composition or flow rate of the process gas (e.g., changing the air / nitrogen / oxygen / argon / helium or the mixing ratio thereof), (iii) optimizing the electrode-surface distance or discharge location (underwater / adjacent to the surface), (iv) accumulating active species through multi-pass / recirculation exposure, (v) reducing unnecessary late-stage reactions through temperature management and degassing / headspace inert gas purging, and / or (vi) matching the final specification through dilution, concentration, buffering, and neutralization. The execution and intensity of Step (c) may be determined based on real-time / batch-by-batch measurements of quality indicators such as long-life indicator species, pH, and ORP.

[0068] The manufacturing process may be performed under aseptic conditions, or the product may be immediately filled and sealed into sterile single-use containers following sterile filtration after discharge. If necessary, the headspace may be replaced by purging with an inert gas (nitrogen / argon) to minimize post-reaction with oxygen, ozone, etc., and packaging may be performed considering the chemical compatibility and light-blocking properties of the contents and container material. After filling, the integrity of the container-cap seal may be verified, and low-temperature (e.g., 2–8 °C) and light-blocking storage conditions may be specified. If the pH deviates from the target range (e.g., 4.5–7) immediately after manufacturing, it may be intentionally adjusted to operational specifications through buffering, neutralization, or dilution using physiological saline, PBS, Ringer's solution, sodium bicarbonate solution, etc., and reproducibility may be ensured by verifying quality standards, including pH, ORP, and long-lived active species indicators, at batch release.

[0069] The present invention provides a liquid plasma for the prevention or treatment of brain tumors produced by the manufacturing method described above.

[0070] The above liquid plasma is a composition in which reactive oxygen species (ROS) and / or reactive nitrogen species (RNS) generated by atmospheric pressure plasma discharge in a water-in-water or adjacent area are dissolved and accumulated in an aqueous solution, exhibiting activity attributed to nitrogen-based and oxygen-based radicals. The substrate may include sterile distilled water (PAW) and cell culture medium (PAM), as well as pharmaceutically acceptable aqueous solutions such as physiological saline, PBS, and Ringer's solution.

[0071] The pH of the liquid plasma may be 4.5 to 7. This range is an operational specification that is pre-set and adjusted to suit the purpose of application, and is intended to minimize the contribution of non-specific toxicity due to excessive acidity while maintaining the action of plasma-derived active species. If the pH of the stock solution falls outside this range immediately after preparation, it can be adjusted using pharmaceutically acceptable buffering, neutralization, and dilution means, such as physiological saline, PBS, Ringer's solution, or sodium bicarbonate solution.

[0072] pH can be utilized as a quality control standard and, if necessary, managed in parallel with redox potential (ORP) and long-lived active species indicators to ensure consistency in manufacturing processes, packaging, and storage. During the use phase, considering the buffering capacity of the application site and the route of administration (intratumoral injection, surgical irrigation / cleaning, topical application, etc.), procedures for checking the pH immediately before use and making minor adjustments may be included. The above pH range is merely an example, and functionally equivalent variations that provide an equivalent balance of efficacy and safety are also included within the scope of the present invention.

[0073] The present invention will be explained in more detail below through examples, but the following examples are not intended to limit the scope of the invention and should be interpreted as being for the purpose of aiding understanding of the invention.

[0074] Example 1: Brain Tumor Cell Death Effect by Liquid Plasma (PAM) Treatment at Different Concentrations

[0075] Liquid plasma was produced by discharging a plasma torch of a microwave atmospheric pressure plasma generator underwater, in a form in which a large amount of nitrogen-based and oxygen-based radicals were dissolved in water. Liquid plasma was produced at different concentrations depending on the underwater discharge time, and the number of radicals in the water increased proportionally with time.

[0076] After treating acoustic neuroma cell lines (HEI-193) and glioblastoma cell lines (U-87 MG) with the above-mentioned liquid plasma (replaced every 24 hours for a total of 72 hours), changes in cell viability were confirmed.

[0077] Specifically, low tumor cell death effects were observed in both cell lines by treatment with liquid plasma at a concentration of 1× (1h), but cell viability of 50% or less was confirmed by treatment with liquid plasma at a concentration of 2× (2h), confirming that the liquid plasma of the present invention is effective against brain tumor cell death (Fig. 1).

[0078] Example 2: Effect of liquid plasma (PAW) on acoustic neuroma cell death

[0079] Plasma-activated water (PAW) was prepared by directly irradiating sterile distilled water with atmospheric pressure plasma. PAW under 1× and 2× conditions was obtained by adjusting the irradiation time and input power, respectively, and DMEM / F-12 basal medium (plasma-free, w / o plasma) maintained at pH 7.0–7.4 was used for culture.

[0080] The effects of PAW treatment on cell growth inhibition and apoptosis were confirmed using the commercially available vestibular schwannoma cell line HEI-193 (the same cell line was used in all examples below). The control group was cultured without the addition of PAW, while the experimental groups were treated with PAW at concentrations of 1× and 2×. Cell viability was measured at 0, 24, 48, and 72 hours and converted into a percentage (%) relative to the 0-hour control group.

[0081] As a result, the number of cells in the control group increased rapidly over time, proliferating more than 10 times compared to the initial level at 72 hours. On the other hand, the PAW treatment group showed a concentration-dependent growth inhibitory effect; the 1× treatment group had a significantly lower proliferation rate compared to the control group, and the 2× treatment group showed a pattern of almost no increase or a decrease in the number of cells starting from 24 hours (Fig. 2a).

[0082] In addition, detailed measurements of cell viability during the 0–24 hour period in the 2× PAW treatment group showed that cell viability decreased to approximately 60% at 3 hours of treatment and dropped sharply to below 40% after 6 hours. At 24 hours, cell viability was maintained at approximately 20%, confirming that a strong cell death effect occurred within a short period with high-concentration PAW treatment (Fig. 2b).

[0083] Therefore, it has been proven that liquid plasma (PAW) not only inhibits the growth of acoustic neuroma cells but also induces apoptosis within a short period of time when treated at high concentrations.

[0084] Example 3: Tumor cell death effect by PAM treatment independent of culture conditions

[0085] In this embodiment, we investigated whether the cell death effect caused by liquid plasma (PAM, Plasma Activated Medium) is related to starvation. To this end, PAM was prepared by exposing a cell culture medium to plasma, and cells were cultured using the activated medium (PAM) without directly irradiating the cells with plasma. Reactive species generated during PAM preparation include reactive oxygen species (ROS) and reactive nitrogen species (RNS).

[0086] Cells were cultured by dividing them into a control group (untreated with plasma), a 1× PAM treatment group, and a 2× PAM treatment group, and cell viability was measured at 0, 24, 48, and 72 hours after culture. Cell viability was converted into a percentage (%) relative to the 0-hour control group.

[0087] As a result, in the control group, the number of cells gradually increased over time, and in the 1× PAM treatment group, although there was a somewhat inhibitory effect, cell viability was maintained relatively stably. On the other hand, in the 2× PAM treatment group, cell viability decreased rapidly at 24 hours of culture, dropping to below 10%, and low viability was maintained at 48 and 72 hours thereafter (Fig. 3).

[0088] Therefore, it was confirmed that PAM-induced cell death occurs independently of the starvation effect of nutrients in the medium, and that this effect is induced by active species such as ROS and RNS contained within PAM.

[0089] Example 4: Analysis of DNA damage and apoptosis mechanisms induced by PAM treatment

[0090] In this example, to elucidate the mechanism of cell death induced by PAM, cell proteins were extracted and Western blot analysis was performed. The proteins analyzed included PARP, p53, phosphorylated-p53 (p-p53), caspase-3, and H2AX, which are known as indicator proteins of cell death and DNA damage. β-Actin was used as a loading control.

[0091] Experimental results showed that PARP, a protein involved in the intracellular DNA damage repair process, was cleaved by caspase-3 after PAM treatment, with a cleavage band observed between 4 and 8 hours, suggesting that apoptosis was induced. The expression of p53 and p-p53 increased with PAM treatment, demonstrating that they induce DNA damage and stress responses. An activation band for caspase-3 was also confirmed in the 4–8 hour range, confirming the progression of apoptosis.

[0092] In addition, the expression of H2AX increased with PAM treatment, which indicates that when a DNA double-strand break (DSB) occurs, H2AX is phosphorylated and appears in the form of γ-H2AX, meaning that DNA double-strand damage has occurred (Fig. 4).

[0093] Therefore, it was confirmed that PAM-induced apoptosis is accompanied by DNA damage (particularly double-strand breaks), a stress response via the p53 signaling pathway, and caspase-3-mediated apoptosis.

[0094] Example 5: PAM-induced apoptosis is not inhibited by treatment with ROS / RNS scavengers or antioxidants

[0095] In this example, to determine whether PAM-induced apoptosis is dependent on reactive oxygen species (ROS) or reactive nitrogen species (RNS), cell viability was compared after co-treatment with a ROS scavenger, a NO scavenger, and an antioxidant, respectively.

[0096] First, N-acetylcysteine ​​(NAC, a ROS scavenger) was treated simultaneously with PAM. If the apoptotic effect of PAM were ROS-dependent, apoptosis should be inhibited by NAC treatment. However, experimental results showed that the pattern of cell viability reduction was almost identical in both the PAM-alone group and the PAM+NAC-treated group, and no apoptotic inhibitory effect was observed by NAC treatment. This suggests that apoptosis induced by PAM is not merely a ROS-mediated effect (Fig. 5a).

[0097] Next, carboxy-PTIO (NO scavenger) was co-treated with PAM. NO scavengers should exhibit an inhibitory effect in the case of RNS-dependent apoptosis mechanisms. However, cell viability decreased similarly in the PAM alone group and the PAM+PTIO combination group, and although there was a minor statistical difference, no apoptosis inhibitory effect was observed. Therefore, it was confirmed that the effect of PAM is not primarily due to RNS (Fig. 5b).

[0098] Finally, glutathione (GSH, an antioxidant) was co-treated with PAM. Glutathione is a powerful intracellular antioxidant capable of neutralizing ROS / RNS, and if the effect of PAM is based on oxidative stress, a protective effect should be observed. However, the decrease in cell viability was observed identically in the group treated with PAM alone and the group treated with PAM+GSH, indicating that antioxidant treatment also failed to inhibit apoptosis induced by PAM (Fig. 5c).

[0099] From the above results, it was confirmed that PAM-induced apoptosis is not simply caused by the accumulation of ROS or RNS, nor is it inhibited by antioxidants. This suggests that PAM induces apoptosis by activating unique intracellular pathways, including intracellular DNA damage (p53 activation, γ-H2AX increase) and apoptosis signaling (caspase-3 activation, PARP cleavage).

[0100] Example 6: PAM-induced apoptosis is inhibited by a ferroptosis inhibitor

[0101] In this example, to elucidate the mechanism of cell death induced by PAM, cell survival rates were compared after treatment with various cell death inhibitors.

[0102] First, z-VAD-fmk (pan-caspase inhibitor, apoptosis inhibitor) was co-administered to the PAM-treated group. If PAM-induced apoptosis were caspase-dependent apoptosis, apoptosis should be inhibited by z-VAD treatment. However, PAM-induced apoptosis was not significantly inhibited even in the z-VAD-treated group, which implies that PAM does not rely on typical caspase-dependent apoptosis as its primary mechanism (Fig. 6a).

[0103] Next, Necrostatin-1 (Nec-1, a necroptosis inhibitor) was administered in combination with PAM. Nec-1 is a compound that inhibits necroptosis, so if necroptosis is the primary mechanism, the effect of PAM should be inhibited. However, no inhibitory effect on apoptosis was observed in the PAM+Nec-1 treatment group, confirming that apoptosis induced by PAM is unrelated to necroptosis (Fig. 6b).

[0104] On the other hand, when Liproxstatin-1 (Lip-1, a ferroptosis inhibitor and lipid peroxidation inhibitor) and Ferrostatin-1 (Fer-1, a representative ferroptosis inhibitor) were treated in combination with PAM, cell viability was significantly restored. In the group treated with PAM alone, cell viability decreased sharply, whereas in the groups treated with Lip-1 or Fer-1 simultaneously with PAM, cell viability significantly increased. This suggests that apoptosis induced by PAM is directly related to ferroptosis (Figs. 6c, 6d).

[0105] Therefore, it was confirmed that the main mechanism of cell death induced by PAM is ferroptosis, as it is not apoptosis (z-VAD inhibitor independent) or necroptosis (Nec-1 independent) mechanism, but is significantly inhibited by ferroptosis inhibitors (Lip-1, Fer-1).

[0106] Example 7: Apoptosis induced by PAM treatment is independent of iron-dependent mechanism

[0107] In this example, to determine whether cell death caused by PAM corresponds to an iron-dependent ferroptosis mechanism, cell viability was measured after co-treatment with Deferoxamine (DFO), an iron chelator.

[0108] Cells were cultured by dividing them into a group treated with 2× PAM alone and groups treated with 2× PAM in combination with DFO at concentrations of 100 nM, 300 nM, 500 nM, and 1000 nM, respectively. After 24 hours of culture, cell viability was measured, and the cell viability in the PAM alone group and all DFO combined treatment groups decreased to a level close to zero (Fig. 7).

[0109] Increasing the DFO concentration did not show any protective effect against PAM-induced apoptosis, which implies that iron chelation cannot inhibit PAM-induced apoptosis. Therefore, PAM-induced apoptosis has a mechanism different from typical iron-dependent ferroptosis.

[0110] However, considering that the ferroptosis inhibitors (Liproxstatin-1, Ferrostatin-1) in Example 6 inhibited PAM-induced apoptosis, it can be seen that PAM induces non-canonical ferroptosis based on lipid peroxidation rather than iron dependence.

[0111] Example 8: Cell death induced by PAM treatment is closely related to acidification of the medium

[0112] In this example, we investigated how the apoptotic effect of PAM treatment is related to the acidification (pH reduction) of the culture medium.

[0113] First, 2× PAM was prepared and applied to cells, and the pH was adjusted to 7.2 by neutralizing the medium with NaHCO₃. A comparison was made between the group treated with PAM without neutralization and the group treated with PAM without neutralization. As a result, cell viability in the pH-neutralized PAM-treated group recovered to a level similar to that of the untreated control group, suggesting that PAM-induced apoptosis is more significantly influenced by the acidification of the medium than by the simple accumulation of ROS / RNS (Fig. 8a).

[0114] Next, the change in pH of the medium was measured according to the PAM treatment concentration. As the PAM concentration increased, the pH of the medium gradually decreased from 7.4 to 6.7 and 5.9 (Fig. 8b).

[0115] In addition, when cells were cultured under conditions of pH 7.4 (Control), pH 5.8, pH 5.3, and pH 4.8 by artificially adjusting the pH of the medium, cell viability decreased rapidly as the pH decreased, and most cells died, especially under the pH 4.8 condition (Fig. 8c).

[0116] From the above results, it was confirmed that cell death caused by PAM treatment is not explained solely by the accumulation of ROS / RNS, and that acidification of the medium (pH-dependent toxicity) is the main toxic mechanism.

[0117] Example 9: Apoptotic effect of PAM treatment on patient-derived primary tumor cells

[0118] In this example, the effect of PAM was verified not only in commercial cell lines but also in primary vestibular schwannoma cells derived from actual patients.

[0119] Primary tumor cells VS-52, VS-55, VS-56, and VS-87 isolated from patient tissue were cultured into sham-treated (untreated), 1× PAM-treated, and 2× PAM-treated groups. Cell viability for each cell group was measured at 0, 24, 48, and 72 hours and converted into a percentage (%) relative to the 0-hour control group.

[0120] As a result, cell viability was significantly reduced by PAM treatment in all patient-derived tumor cells, and in particular, in the 2× PAM treatment group, cell viability dropped sharply after 24 hours and did not recover even with long-term culture. A more potent apoptotic effect was observed in patient-derived cells than in cell line experiments, which is attributed to the fact that patient-derived cells respond more sensitively to PAM treatment than immortalized cell lines (Figs. 9a-9d).

[0121] Therefore, it was confirmed that the PAM of the present invention consistently induces apoptosis not only at the cell line level but also in actual patient-derived primary tumor cells, which strongly supports the therapeutic applicability of the present invention.

[0122] Example 10: Antitumor effect of PAM treatment in a Balb / C nude mouse transplant tumor model

[0123] In this example, the antitumor effect of PAM (Plasma Activated Medium or Plasma Activated Water) was confirmed in a Balb / C nude mouse transplant tumor model.

[0124] Human acoustic neuroma cells were subcutaneously transplanted into Balb / C nude mice to induce tumor formation, after which they were divided into a control group and a PAW treatment group for comparison. The PAW treatment group was administered five times (5×).

[0125] As a result of the experiment, the tumor size in the control group increased rapidly starting 6 days after transplantation, reaching an average of approximately 180 mm³ at the 15-day mark. In contrast, the increase in tumor size in the PAW-treated group was significantly inhibited, remaining at an average of approximately 50 mm³ even at the 15-day mark. Additionally, tumor weight also significantly decreased in the PAW-treated group compared to the control group (average approximately 100 mg → 20 mg, p<0.01) (Figs. 10a, 10b).

[0126] As a result of dissection, dense cell proliferation was observed in the control group tumor tissue, whereas necrosis and apoptosis were significantly increased in the PAW-treated group tumor tissue (Fig. 10c, Fig. 10d).

[0127] From the above results, liquid plasma exhibits distinct antitumor effects not only in vitro but also in in vivo animal models; in particular, the effects of inhibiting tumor growth and reducing tumor weight upon repeated administration were demonstrated. Therefore, it was confirmed that PAM can be utilized as an anticancer strategy with high potential for actual tumor treatment applications.

[0128] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

[0129] Since the present invention can effectively reduce the survival rate of brain tumor cells using liquid plasma, it can be utilized for the prevention or treatment of brain tumors.

Claims

1. A pharmaceutical composition for the prevention or treatment of brain tumors comprising liquid plasma as an active ingredient, wherein the pH of the liquid plasma is 4.5 to 7.

2. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of brain tumors, wherein the above liquid plasma comprises nitrogen-based and oxygen-based radicals.

3. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of a brain tumor, wherein the brain tumor is an acoustic neuroma or a glioblastoma.

4. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of a brain tumor, wherein the above pharmaceutical composition is a parenteral formulation and is selected from intravenous, intra-arterial, intramuscular, subcutaneous, intratumoral or peritumoral injection, subdural, subarachnoid or intraventricular administration, a solution for perfusion or irrigation of the surgical site, a gel, a hydrogel, or a spray-type topical formulation.

5. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of brain tumors, characterized in that PARP cleavage and / or an increase in γ-H2AX signaling are detected within 4 to 8 hours after treating target cells or target tissues with the above pharmaceutical composition.

6. In Paragraph 1, The above pharmaceutical composition is A pharmaceutical composition for the prevention or treatment of brain tumors, characterized in that apoptosis is inhibited when combined with a ferroptosis inhibitor, but apoptosis is not inhibited when combined with an iron chelator.

7. In Paragraph 1, The above pharmaceutical composition is A pharmaceutical composition for the prevention or treatment of brain tumors, characterized in that apoptosis is inhibited when the pH of the culture medium or microenvironment of the target cell or target tissue is neutralized.

8. A method for preparing liquid plasma for the prevention or treatment of brain tumors comprising the following steps: (a) a step of forming plasma by performing a discharge in an underwater or surface-adjacent area using an atmospheric pressure plasma generator; and (b) A step of dissolving and accumulating reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated by the above discharge in water to produce a liquid plasma containing nitrogen-based and oxygen-based radicals.

9. In Paragraph 8, A method for preparing liquid plasma for the prevention or treatment of brain tumors, comprising the following additional steps: (c) A step of increasing the concentration of underwater radicals in the above liquid plasma.

10. Liquid plasma for the prevention or treatment of brain tumors produced by the manufacturing method according to paragraph 8.

11. In Paragraph 10, Liquid plasma for the prevention or treatment of brain tumors, wherein the pH of the liquid plasma is 4.5 to 7.

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