Use of ovatodiolide in the manufacture of medical composition for improving blood oxygen saturation in a subject

Abstract

A use of a compound in the manufacture of medical composition for improving blood oxygen saturation in a subject, the compound comprises a safe and effective amount of Ovatodiolide, the structural isomers, or the pharmaceutically acceptable salts thereof, the Ovatodiolide includes a chemical structure (I); the said subject is preferably a subject suffering from chronic lung injury; most preferred, the said chronic lung injury includes but is not limited to chronic obstruction pulmonary disease (COPD) or pulmonary fibrosis.

Description

Technical Field

[0001] The present invention provides a use of a compound for preparing a pharmaceutical composition for increasing the blood oxygen concentration of a subject, particularly the use of sylvatol or a pharmaceutically acceptable salt thereof for preparing a pharmaceutical composition for increasing the blood oxygen concentration of a subject. Prior Art

[0002] Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disease characterized by a combination of chronic bronchitis and emphysema, leading to airflow restriction through the airways. COPD is the third leading cause of death worldwide, affecting approximately 10.1% of the population and resulting in over 3 million deaths annually. Its pathological mechanisms involve structural destruction of the small airways, impaired repair of alveolar tissue, and chronic inflammation, leading to bronchitis and emphysema. Emphysema is characterized by alveolar wall destruction and overdistension, resulting in decreased lung elasticity and, consequently, impaired gas exchange. Patients often experience chronic dyspnea, decreased exercise tolerance, increased respiratory effort, and air trapping.

[0003] Long-term exposure to irritants (such as tobacco smoke, air pollution, and occupational dust) triggers the release of inflammatory mediators from alveolar epithelial cells. This, coupled with the continued infiltration of immune cells such as neutrophils, macrophages, lymphocytes, and mast cells, further damages lung tissue structure. Currently, COPD remains incurable. Existing treatment strategies, primarily medications, inhaled bronchodilators, anti-inflammatory drugs, and oxygen therapy, can only alleviate symptoms and slow disease progression. Some patients with severe disease may require pulmonary rehabilitation or surgical procedures (such as lung volume reduction surgery or lung transplantation). However, these treatments remain limited in their ability to repair damaged alveoli and halt disease progression, making COPD treatment a significant global public health challenge.

[0004] Fish needle grass (Anisomeles indica O. Kuntze) is a commonly used herbal remedy in Taiwan. It is also known as guest's grass (from Jiaoling and Meixian, Guangdong), golden sword grass, and herbaceous vine. The Taiwan Ministry of Health and Welfare has included fish needle grass in its list of ingredients suitable for food use, and the entire plant is edible. Fish needle grass is an annual or biennial herb in the Labiatae family. It is primarily distributed in southwestern China, India, the Philippines, Java, and Sumatra in Indonesia. It can be found throughout Taiwan from plains to low-altitude mountainous areas. It is also cultivated sporadically for medicinal purposes in Yuli, Hualien, and Chenggong, Taitung. For traditional folk medicinal purposes, it is collected in summer and autumn. The whole plant or the above-ground parts are harvested, washed, and used fresh or sun-dried. The whole plant has antipyretic, anti-wind, dehumidifying, stomachic, detoxifying, analgesic, and antibacterial properties. It is commonly used by the people to treat colds and fever, abdominal pain and vomiting, food poisoning, cholera, stomachache, gastroenteritis, neurodermatitis, rheumatic bone pain, muscle and bone pain, eczema, swelling, sores, stool poison, and snake bites.

[0005] Our research and development team has been engaged in the breeding of sedge grass (GenBank: GU726292) for a long time and has been conducting a series of studies on the extracts of the whole sedge grass grown on the farm, with a particular focus on the preparation of pure crystalline substances of sedge grass lactone (Ovatodiolide). Specifically, we have carried out extraction, separation, purification, analysis and identification, as well as research on the pharmacological effects of ovatodiolide on anti-inflammatory, antiviral, anti-Helicobacter pylori, anti-cancer, and anti-cancer stem cells. In recent years, our team has completed a test experiment of ovatodiolide against the influenza A and B drug "Tamiflu" (Roche Pharmaceuticals), and found that ovatodiolide extract and ovatodiolide have a good effect in inhibiting influenza virus. At the end of 2019, it was learned that AIDS drugs have shown a positive response to the treatment of patients infected with the new coronavirus; according to literature reports, ovatodiolide can inhibit HIV (Fitoterapia, 2000, 71(5): 574-576.), and subsequent studies have confirmed that ovatodiolide has the effect of inhibiting the new coronavirus. In addition, existing studies have also found that nebulosa lactone can inhibit gastritis caused by Helicobacter pylori in the gastric wall, and can also inhibit the inflammatory response mediated by NF-κB and STAT3.

[0006] Therefore, the present invention infers that nepenthes lactone may also alleviate the symptoms of chronic lung injury caused by chronic inflammation and increase the patient's blood oxygen concentration, and experiments have been conducted to confirm the present invention's inference. Summary of the Invention

[0007] The present invention is based on the discovery that ovatodiolide, its structural isomers, or pharmaceutically acceptable salts thereof can be used to elevate a subject's blood oxygen concentration, thereby improving symptoms of chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and the like. Specifically, the present invention provides a pharmaceutical composition for elevating a subject's blood oxygen concentration, comprising a safe and effective dose of ovatodiolide, its structural isomers, or pharmaceutically acceptable salts thereof, and further comprising a pharmaceutically acceptable carrier.

[0008] The present invention relates to the use of a compound for preparing a pharmaceutical composition for increasing a subject's blood oxygen concentration. The pharmaceutical composition comprises a safe and effective dose of ovatodiolide, a structural isomer thereof, or a pharmaceutically acceptable salt thereof, and further comprises a pharmaceutically acceptable carrier.

[0009] In the present invention, preferably, the system is a subject suffering from chronic lung injury.

[0010] In the present invention, preferably, the chronic lung injury includes but is not limited to chronic obstructive pulmonary disease or pulmonary fibrosis.

[0011] In the present invention, the subject includes but is not limited to mammals or humans.

[0012] In some embodiments, the nebulosa lactone of the present invention is administered at a dosage of about 0.0025-5000 mg / day, for example, about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100, 120, 150, 200, 250, 300, 350, 400, 450, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 mg / day.

[0013] In some embodiments, nebulol is administered in an amount of about 1 ng / kg to about 200 mg / kg, about 1 μg / kg to about 100 mg / kg, or about 1 mg / kg to about 50 mg / kg per unit dose, for example, about 1 μg / kg, about 10 μg / kg, about 25 μg / kg, about 50 μg / kg, about 75 μg / kg, about 100 μg / kg, about 125 μg / kg, about 150 μg / kg, about 175 μg / kg, about 200 μg kg, about 225 μg / kg, about 250 μg kg, about 275 μg kg, about 300 μg / kg, about 325 μg kg, about 350 μg / kg, about 375 μg / kg, about 400 μg / kg, about 425 μg / kg, about 450 μg / kg, about 475 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about 500 μg / kg, about μg / kg, about 525 μg kg, about 550 μg / kg, about 575 μg kg, about 600 μg / kg, about 625 μg / kg, about 650 μg / kg, about 675 μg / kg, about 700 μg / kg, about 725 μg / kg, about 750 μg / kg, about 775 μg / kg, about 800 μg / kg, about 825 μg / kg, about 850 μg / kg, about 875 μg / kg, about 900 μg / kg, about 925 μg / kg, about 950 μg / kg, about 975 μg / kg, about 1 mg / kg, about 4 mg / kg, about 5 mg / kg, about 8 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 The invention relates to an agent that is administered in an amount of about 1 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) unit doses are administered per day.

[0014] In the present invention, the effective dose can be calculated based on the formula provided in the guidelines published by the U.S. FDA (Guidance for Industry: Establishing the Maximum Safe Starting Doses in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, U.S. Department of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research (CDER), July 2005), further taking into account the safety factor to convert equivalent doses between different species.

[0015] The safe and effective dose for mice of the present invention is 1 μg / kg-body weight to 100 μg / kg-body weight; preferably, the safe and effective dose for mice of the present invention is 10 μg / kg-body weight to 80 μg / kg-body weight; more preferably, the safe and effective dose for mice of the present invention is 20 μg / kg-body weight to 60 μg / kg-body weight.

[0016] The safe and effective dose of the present invention in mice, when converted into an equivalent dose for a 60-kg adult, is approximately 0.08 μg / kg-body weight to 8 μg / kg-body weight; preferably, the safe and effective dose of the present invention in mice, when converted into an equivalent dose for a 60-kg adult, is approximately 0.8 μg / kg-body weight to 6.5 μg / kg-body weight; more preferably, the safe and effective dose of the present invention in mice, when converted into an equivalent dose for a 60-kg adult, is approximately 1.3 μg / kg-body weight to 5 μg / kg-body weight.

[0017] In some embodiments, preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0018] In some embodiments, ichthyolide is administered continuously for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, or at least 50 days.

[0019] In some embodiments, nebulol is administered for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) courses of treatment, wherein each course of treatment lasts at least 1 day, 2 days, 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, or at least 50 days; and there is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, two weeks, three weeks, or four weeks between each course of treatment.

[0020] In some embodiments, nephrolactone can be administered orally, buccally, by inhalation spray, sublingually, rectally, transdermally, vaginally, transmucosally, topically, nasally or intestinally; by injection, such as intramuscular injection, subcutaneous injection, intramedullary injection, as well as intrathecally, directly into the brain, in situ, subcutaneously, intraperitoneally, intravenously, intraarticularly, intrasternally, intrahepatically, intralesionally, intracranially, intraperitoneally, nasally, or intraocularly, or other drug delivery methods. Simple diagram description

[0021] FIG1 shows the chemical structural formula of Ovatodiolide (OVA) of the present invention.

[0022] FIG2 is the animal experiment process of the present invention.

[0023] FIG3A shows the results of hematoxylin and eosin (H&E) staining of lung sections of experimental animals of the present invention.

[0024] FIG3B shows the results of the mean linear intercept (MLI) analysis of the alveoli of the experimental animals of the present invention.

[0025] FIG4A shows the analysis results of the lung inspiratory capacity (IC) of the experimental animals of the present invention, with the unit being milliliter (mL).

[0026] FIG4B shows the analysis results of lung tissue elastance (H) of the experimental animals of the present invention, with the unit being cm H2O / mL.

[0027] FIG5 shows the changes in blood oxygen saturation of the experimental animals before and after exercise of the present invention, the unit is percentage (%). Implementation Method

[0028] This specification makes extensive use of many technical and scientific terms commonly used in the field of biotechnology. To ensure a clear and consistent understanding of this specification and the scope of the patent claims, and to provide a clear and consistent understanding of the scope of these terms, the following definitions are provided. Other terms not specifically defined below have the meanings commonly understood by professionals in the relevant field.

[0029] Unless otherwise indicated, the terms "or," "and," and "and" used in this specification refer to "or / and." Furthermore, the terms "include" and "comprising" are not restrictive open-ended conjunctions. The preceding paragraphs are provided for systemic reference only and should not be construed as limiting the subject matter of the invention.

[0030] Unless otherwise specified, "%" used in this specification refers to "weight percent (wt%)." Numerical ranges (e.g., 10%-11% of A) are inclusive of both upper and lower limits (i.e., 10% ≤ A ≤ 11%) unless otherwise specified. Numerical ranges without a defined lower limit (e.g., less than 0.2% of B, or less than 0.2%) may be interpreted as meaning that the lower limit is 0 (i.e., 0% ≤ B ≤ 0.2%). References to "weight percent" of each component may also be expressed as "parts by weight."

[0031] All numerical values ​​disclosed in this specification may have a standard technical measurement error (standard deviation) of ±10%. The word "about" is intended to mean ±10%, ±5%, ±2.5%, or ±1% relative to a given value. In other words, "about 20%" means 20±2%, 20±1%, 20±0.5%, or 20±0.25%.

[0032] Unless otherwise specified, all materials used in this manual are commercially available and easily available.

[0033] As used herein, "enhance," "boost," "enhance," or similar terms refer to increasing or prolonging the intended efficacy or duration of a pharmaceutical composition containing nebulosin. Therefore, when enhancing the therapeutic efficacy of a pharmaceutical composition containing nebulosin, "enhancement" refers to increasing or prolonging the ability of the other therapeutic system to achieve either efficacy or duration.

[0034] As used herein, "treat," "for treatment," and similar terms refer to the administration of an agent to achieve a specific effect. This effect is a therapeutic, partial or complete cure of a disease and / or its symptoms. "Treatment" encompasses any treatment of hepatocellular carcinoma in mammals (particularly humans) and includes: (A) disease inhibition, i.e., arresting its progression; and (B) disease alleviation, i.e., disease regression. In the treatment of tumors (e.g., hepatocellular carcinoma), therapeutic agents can directly reduce the growth and metastasis of tumor cells.

[0035] As used herein, the terms "pharmaceutically acceptable" or "pharmaceutically acceptable" mean that the substance or composition must be compatible with other ingredients of its pharmaceutical formulation and will not aggravate the patient's symptoms.

[0036] The compositions provided herein can be prepared using techniques well known to those skilled in the art by combining the active ingredients or compositions provided herein with at least one pharmaceutically acceptable carrier to form a dosage form suitable for use with the compositions of the present invention. Such dosage forms include, but are not limited to, solutions, emulsions, suspensions, powders, lozenges, tablets, chewing gums, capsules, and other dosage forms similar to or suitable for use with the present invention.

[0037] As used herein, "pharmaceutically acceptable carrier" includes one or more types of ingredients selected from the following: tablets, capsules, granules, syrups, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, injections, and other similar or suitable carrier forms for use in the present invention.

[0038] The composition provided by the present invention may also be appropriately added with one or more dissolution aids, diluents, buffers, colorants, flavoring agents, etc. commonly used in the above-mentioned formulation fields as needed.

[0039] The term "pharmaceutically acceptable excipient" as used herein includes, but is not limited to, at least one of a polymer, a resin, a plasticizer, a filler, a lubricant, a diluent, a binder, a disintegrant, a solvent, a co-solvent, a surfactant, a preservative, a sweetener, a flavoring, a pharmaceutical grade dye or pigment, and a viscosity agent.

[0040] As used herein, a "pharmaceutical composition" refers to a solid or liquid composition in a form, concentration, and purity suitable for administration to a patient and capable of inducing a desired physiological change after administration; the pharmaceutical composition is sterile and / or non-pyrogenic.

[0041] As used herein, "effective amount" refers to the dose necessary to produce or induce a desired biological response and is not intended to be a quantitative measure of therapeutic efficacy. Those skilled in the art will appreciate that the effective amount of a pharmaceutical composition can vary depending on factors such as the desired biological endpoint, the bioactive agent to be delivered, the composition of the encapsulating matrix, and the target tissue.

[0042] 1. Drugs and reagents

[0043] Ovatodiolide (99.7% purity) was provided by National Taitung University. Its chemical structure was confirmed by nuclear magnetic resonance (NMR) and has the chemical structure of formula (I), as shown in Figure 1. The purity of ovatodiolide (>95%) was confirmed by high-performance liquid chromatography (HPLC) (column: RP C18 BDS, 4.6 mm x 150 mm, 4 μm; purchased from Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase consisted of acetonitrile (solvent A) and water containing 0.1% trifluoroacetic acid (solvent B). A linear gradient program was used: 5% trifluoroacetic acid in the first 0 minutes, followed by a linear gradient to 100% trifluoroacetic acid over 40 minutes. The mobile phase flow rate was 1 mL / min, and absorbance was monitored at 220 nm. Dissolve nephrolactone in dimethyl sulfoxide (DMSO) and further dilute in sterile culture medium before use.

[0044] A 1 mM stock solution of nephrolactone was dissolved in DMSO at 15 mg / mL and stored at −20°C. Phosphate-buffered saline (PBS; #P7059), DMSO (#D2650), sulforhodamine B (SRB) reagent (#230162), trypsin / ethylenediaminetetraacetic acid (#T4049) solution, trisaminomethane (Tris) base (#93352), and acetic acid (#695092) were purchased from Sigma Aldrich (St. Louis, MO, USA). Gibco DMEM was purchased from Invitrogen (#11966025, Invitrogen).

[0045] 2. Single-dose oral acute toxicity study of phytosuccinate in rats

[0046] This example tests the single-dose acute oral toxicity safety of the compound of formula (I), ichthyolide, in rats, providing a reference for food safety assessment. This test was conducted in accordance with the Taiwan Ministry of Health and Welfare's Food Safety Assessment - Single-Dose Acute Oral Toxicity Test, the U.S. Environmental Protection Agency (USEPA) (Health Effects Test Guidelines, OPPTS 870.1100, Acute oral toxicity, US EPA 712-C-98-190. In: OPPTS Harmonized Test Guidelines, Series 870.3050, EPA712-C-00-366), and the Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects. No. 420: Acute Oral Toxicity - Fixed Dose Procedure, No. 423: Acute Oral Toxicity - Acute Toxic Class Method, No. 425: Acute Oral Toxicity - Up and Down Method) testing standards. This study investigated the acute toxicity of a single oral dose of the compound (I), ichthyolide, in rats (Sprague-Dawley strain). The compound (I), ichthyolide, was present as slightly yellow crystals with an experimental purity of 99.95%. The compound was prepared in 10% DMSO to a concentration of 0.1 g / mL. Each rat was fed a diet of 10 mL / kg body weight. The rats were orally administered daily based on their body weight, for a final total dose of 1 g / kg body weight. The rats were observed for 14 consecutive days.

[0047] Results showed that after oral administration of the compound (I) - ichthyolide, all rats showed no symptoms of poisoning or mortality. Regarding weekly weight change (g), there were no significant differences in weight gain (WBW) between male and female treated groups compared to the control group. Following the study, changes in blood values, including total white blood cell count (WBC), total red blood cell count (RBC), hematocrit (Hct), mean corpuscular volume (MCV), mean hemoglobin (MCH), mean hemoglobin concentration (MCHC), total platelet count, and WBC count, were not significantly abnormal in both treated and female groups. Serum liver and kidney enzyme levels, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine, were not affected in both male and female treated groups. In terms of absolute organ weight (g) and organ weight percentage, no significant differences were observed between the treated and control groups for the adrenal glands, brain, heart, kidneys, liver, spleen, thymus, testes, or ovaries. Examination of internal organs revealed no gross lesions in the adrenal glands, brain, heart, kidneys, liver, spleen, thymus, testes, or ovaries in the treated groups. Histopathological examinations also revealed no test substance-related pathological changes in the major organs of the group treated with the compound of formula (I) - styracifluolide. Overall, the results indicate that a single oral acute toxicity study of the compound of formula (I) - styracifluolide at 1 g / kg body weight (equivalent to a human dose of approximately 50 mg / kg body weight) did not cause acute poisoning or mortality in rats, and did not induce toxicity-related pathological changes in any major organs.

[0048] 3. 28-day dietary toxicity study of phytosphingolipid lactone in rats

[0049] This example tests the repeated-dose oral toxicity safety of the compound of Formula (I), sylvaline lactone, in rats. This study aims to establish a Material Safety Data Sheet (MSDS) to provide a reference for repeated-dose clinical safety assessments in humans. This study was conducted in accordance with the Taiwan Ministry of Health and Welfare's 28-Day Oral Toxicity Study for Food Safety Assessment (1999) and the 2014 Guidelines for the Safety of Nonclinical Drugs. It also complies with the U.S. Environmental Protection Agency (USEPA) Health Effects Test Guidelines (OPPTS 870.1100, Repeated Dose 28-Day Oral Toxicity Study in Rodents. In: OPPTS Harmonized Test Guidelines, Series 870.3050, EPA712-C-00-366) and the Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects. No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents).

[0050] This study investigated the potential side effects of compound (I) - ichthyolide - on humans for clinical safety assessment. A clinical side effect observation study was conducted on rats after repeated oral administration of compound (I) - ichthyolide for 28 days. Compound (I) - ichthyolide is a slightly yellowish crystal with an experimental purity of 99.95%. The sample was prepared in 5% DMSO. Rats (Sprague-Dawley, SD strain) were divided into four groups: a control group (5% DMSO), a low-dose group (10 mg / kg body weight), a medium-dose group (25 mg / kg body weight), and a high-dose group (50 mg / kg body weight). Each group consisted of 20 rats, half male and half female. Each rat was fed a diet of 10 mL / kg body weight for 28 consecutive days, based on body weight. The test results showed that after oral administration of the compound of formula (I) - nephrolactone to rats for 28 consecutive days, none of the rats developed poisoning symptoms or died due to the test substance.

[0051] After the study, body weight changes, feed consumption, urine and blood levels, serum enzyme levels, and organ weights in male and female rats treated with the compound (I) - sylvatide, compared to the control group, showed slight increases or decreases due to individual differences, but remained within normal values. There was no dose-response correlation between the groups, and these values ​​were not clinically significant and unrelated to the test substance. Examination of all organs in the rats in each group showed no obvious gross pathological changes. Histopathological examinations revealed that the high-dose group did not cause pathological changes related to organ toxicity. The above test results indicate that the compound (I) - ichthyolide - did not cause toxic reactions in any organ of male and female rats after continuous oral administration for 28 days at low doses (10 mg / kg-body weight), medium doses (25 mg / kg-body weight), and high doses (50 mg / kg-body weight). The "no observed adverse effect level" (NOAEL) for the 28-day feeding toxicity study in rats was 50 mg / kg-body weight.

[0052] 4. Analysis of Chromosomal Abnormalities in Mammalian Cell Lines in Vitro by Compound (I) - Isophylline

[0053] This example tests the compound of formula (I), ichthyolide, for chromosomal aberrations in mammalian cell lines in vitro, thereby establishing a Material Safety Data Sheet (MSDS) to provide a reference for safety assessment. Genotoxicity testing was conducted based on the Taiwan Ministry of Health and Welfare's in vitro mammalian cell line chromosomal aberration test for health foods, and in compliance with the U.S. Environmental Protection Agency (USEPA) Health Effects Test Guidelines (OPPTS 870.1100, In vitro mammalian chromosome aberration test, US EPA 712-C-98-190. In: OPPTS Harmonized Test Guidelines, Series 870.3050, EPA 712-C-00-366, 1998) and the Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects. No. 473: In vitro mammalian chromosome aberration test, 1997). This study conducted a chromosomal aberration test on mammalian cells in culture using the compound (I) - ichthyolide. The compound (I) - ichthyolide is a slightly yellowish crystal and was prepared in dimethyl sulfoxide (DMSO). The cytotoxicity test consisted of two parts: one in which CHO-K1 cells were directly exposed to the compound (I) - ichthyolide; the other in which rat liver activated enzyme extract (S9) was used to simulate human metabolism. CHO-K1 cells were treated with a mixture of rat liver activated enzyme extract (S9) and the compound (I) - ichthyolide. In the absence of rat liver activated enzyme extract (S9), a 24-hour cytotoxicity test was conducted at five test doses: 12.5, 15, 17.5, 20, and 25 μM. Results showed that the viability of CHO-K1 cells was approximately 65.6% at a 17.5 μM dose. In addition, a 24-hour cytotoxicity test was conducted in a rat liver activated enzyme extract (+S9) experiment using five test doses of 60, 70, 75, 80, and 90 μM. The results showed that the survival rate of CHO-K1 cells was approximately 60.3% at 75 μM of the compound of formula (I) - ichthyolide, indicating that the compound of formula (I) - ichthyolide is cytotoxic to CHO-K1 cells. This concentration was selected as the maximum dose for the formal test.In a chromosomal aberration assay, compound (I) - yuselaginella lactone was prepared at 12.5, 15, and 17.5 μM (-S9) in each cell culture dish and incubated for 24 hours. Samples were then incubated with S9 at 55, 65, and 75 μM for 3 hours. The cells were then observed for 24 hours to determine if the number and structure of chromosomes in the cells were normal. The results showed that compound (I) - yuselaginella lactone, with or without the S9 mixture metabolic activation system, did not significantly increase the frequency of chromosomal aberrations in CHO-K1 cells compared to the negative control group at 12.5, 15, and 17.5 μM (-S9) and 55, 65, and 75 μM (+S9). There was also no significant change in the location of chromosomal aberrations. These results suggest that compound (I) - yuselaginella lactone, whether in the presence or absence of the S9 mixture, did not exhibit mutagenic effects on the chromosomes of the mammalian CHO-K1 cell line in vitro.

[0054] 5. Micronucleus test of compound (I) - nephrolactone in peripheral blood of mice

[0055] This example tests the genotoxicity of the compound of formula (I), ichthyolide, to establish a Material Safety Data Sheet (MSDS) and provide a reference for safety assessment. The test was conducted in accordance with the mouse peripheral blood micronucleus test (PMM) of the Taiwan Ministry of Health and Welfare's Food Safety Assessment - Genotoxicity Test, and complies with the US Environmental Protection Agency (USEPA) (Mammalian Erythrocyte Micronucleus Test, In: OPPTS Harmonized Test Guidelines, Series 870.5395, EPA 712-C-98-226) and the Organization for Economic Cooperation and Development (OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, No. 474: Mammalian Erythrocyte Micronucleus Test, 1997). This experiment tested the genotoxicity of the compound of formula (I), ichthyolide, on peripheral blood micronucleus in mice (ICR strain). This study primarily evaluated the effect of the compound (Formula (I)) - cyclophosphamide on the incidence of micronuclei in the peripheral blood of rodents in vivo, thereby assessing the extent of direct or indirect damage to erythrocyte chromosomes or mitotic mutations. ICR mice were used as test subjects and were divided into five groups: a negative control, a positive control (60 mg / kg bw per ip), and five male mice (five per group) receiving a low-dose (0.25 g / kg bw), a medium-dose (0.5 g / kg bw), and a high-dose (1 g / kg bw) of the compound (Formula (I)) - cyclophosphamide. Cyclophosphamide was administered via a single gavage tube. The incidence of reticulocytes and the incidence of micronuclei within reticulocytes (‰) were assessed 48 and 72 hours after administration. Results showed no toxicity or body weight differences among the treatment groups 48 and 72 hours after administration of the compound (Formula (I) - cyclophosphamide). Reticulocytes in mouse peripheral blood stained with 0.1% acridine orange stain appeared orange-red under a fluorescence microscope. Yellow-green fluorescent micronuclei, approximately 1 / 20-1 / 5 the size of an erythrocyte, were visible within the reticulocytes. Comparison of the number of reticulocytes and the number of micronuclei within reticulocytes at 48 and 72 hours between the treatment groups with the compound (I) - ichthyolide and the negative control group showed no significant differences. However, the number of reticulocytes in the positive control group was significantly decreased (p < 0.05), and the number of micronuclei within reticulocytes was significantly increased (p < 0.05), compared with the negative control group.Based on the above results, the compound (I) - yuselaginella lactone showed no significant difference in the number of reticulocytes and micronuclei within the peripheral blood of mice compared to the negative control group at all doses, resulting in a negative test result. Therefore, the compound (I) - yuselaginella lactone does not have a chromosomal gene mutation-inducing toxic effect on mouse peripheral blood cells.

[0056] 6. Animal Experimentation

[0057] The animal experiment process of the present invention is shown in FIG2 .

[0058] In specific embodiments of the present invention, animal experiments were performed using 6-week-old male C57BL / 6J Narl mice purchased from the National Laboratory Animal Center. Mice were housed under constant environmental conditions, with room temperature maintained at 20-24°C and relative humidity maintained at 45-65%, with a 12-hour light-dark cycle in a standard animal laboratory, and free access to water and food. All procedures were performed under sterile conditions and were approved by the Laboratory Animal Care and Use Committee of Taipei Medical University.

[0059] Experimental methods:

[0060] A. Induction of emphysema and administration of nebula lactone:

[0061] In this experiment, the induction of emphysema was generally carried out according to the method described in Yeh LY et al. (2022), Front Med (Lausanne), 9:794025.

[0062] Male C57BL / 6JNarl mice were randomly divided into five groups (n = 10 per group), including a normal control group (Control), a pathological control group (PPE), and three experimental groups (i.e., PPE+OVA-1, PPE+OVA-2, and PPE+OVA-3). The mice in the pathological control group and each experimental group were then administered 0.4 IU / mouse of porcine pancreatic elastase (PPE; purchased from Sigma-Aldrich, 39445-21-1; prepared in 50 µL PBS) by intratracheal injection. The same dose of PPE was administered 14 and 28 days after the first PPE injection. The normal control group mice were injected with an equal volume of PBS.

[0063] Then, 35 days after the first PPE injection, the experimental groups were administered 20 μg / kg body weight (PPE + OVA-1), 40 μg / kg body weight (PPE + OVA-2), and 60 μg / kg body weight (PPE + OVA-3) of nebuloside via intratracheal injection. Mice in the pathological and normal control groups were administered an equal volume of PBS. Each group was administered five times weekly for a total of two weeks.

[0064] B. Detection of blood oxygen during exercise:

[0065] On day 48 after the first PPE injection, blood oxygen saturation (SpO2) in each group of mice was measured before and after exercise using a tissue spectrometer (BIOPAC System, Santa Barbara, USA). A non-invasive probe was attached to the tail of each mouse to quantify oxyhemoglobin levels by measuring the absorbance of near-infrared light between 800 and 1000 nm. Deoxyhemoglobin concentrations were determined based on the absorbance of near-infrared light between 600 and 800 nm. Mice then treaded on a treadmill for 5 minutes. SpO2 was then measured again in the tail to determine the difference between pre- and post-exercise levels. Data were analyzed using Biopac Student Lab-BSL software (Upwards Biosystems, Ltd.).

[0066] C. Pulmonary function test:

[0067] On day 49 after the first PPE injection, lung function values ​​of mice in each group were measured using a flexiVent respiratory mechanics system (purchased from SCIREQ, Montreal, Qc, Canada). The system was equipped with an FX1 module and a negative pressure forced expirations (NPFE) device for mice, and was operated by flexiWare v7.2 software.

[0068] At the beginning of the experiment, two consecutive deep inhalations were performed to inflate the lungs to a maximum pressure of 30 cmH₂O to open collapsed lung areas and standardize lung volume. The lungs were allowed to equilibrate at this pressure for 3 seconds, and the volume corrected for gas compression was read as the subject's inspiratory capacity (IC).

[0069] Next, a broadband forced oscillation waveform (Prime-8, P8) with a frequency range of 0.5 to 19.75 Hz was applied to the airway opening of the test animals for 8 seconds to assess the contribution of airway and lung tissue to respiratory responses.

[0070] D. Alveolar Mean Linear Intercept (MLI) analysis:

[0071] On day 49 after the first PPE injection, mice in each group were sacrificed, and their lungs were instilled with 10% formaldehyde via intratracheal instillation. Lung tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Lung H&E images were acquired using Motic Easyscan Pro and Motic DSAssistant software. The mean linear intercept (MLI) was calculated by evaluating the length of the intersecting lines and the number of gridline intercepts in the alveolar space of 10 non-overlapping regions of each lung sample and analyzing them using ImageJ software, as described in Crowley et al. (2019), BMC Pulm Med, 19:206.

[0072] E. Statistical analysis:

[0073] In the examples of the present invention, experimental data were expressed as mean ± standard deviation (SD) and statistically analyzed using Graphpad Prism software version 10. All data were analyzed using one-way analysis of variance (ANOVA) combined with Dunnett's multiple comparisons test to assess differences between each group and the pathological control group. A p < 0.05 result was considered statistically significant.

[0074] Experimental results:

[0075] A. Ichthyolide alleviates lung morphological damage caused by emphysema

[0076] Figure 3 shows lung sections from each group of mice, stained with H&E, and analyzed by MLI. As shown in Figure 3A, compared with the normal control group (Control), the alveoli in the pathological control group (PPE) were significantly dilated, with thinning and even rupture of the alveolar walls, resulting in the disappearance of some alveolar septa. MLI analysis (Figure 3B), showing the mean alveolar spacing, showed a significant increase in alveolar dilation in the pathological control group (PPE) compared with the normal control group (Control). Following administration of different doses of yuselaginella lactone, pathological sections show a significant recovery of alveolar dilation in all experimental groups (PPE+OVA-1; PPE+OVA-2; PPE+OVA-3), with MLI values ​​decreasing. These decreases were most significant in the medium-dose (PPE+OVA-2) and high-dose (PPE+OVA-3) groups, indicating that intratracheal administration of yuselaginella lactone can mitigate lung tissue damage.

[0077] B. Ichthyolide improves lung function and tissue elasticity in mice with emphysema

[0078] Figure 4 shows the lung function test values ​​of each group of mice. As shown in Figure 4A, the inspiratory capacity (IC) of the pathological control group (PPE) was significantly increased compared to the normal control group (Control). This is due to the emphysema-induced alveolar enlargement and reduced lung elasticity after tracheal injection of PPE. Emphysema damages and enlarges the alveolar structure, leading to an increase in lung volume. Simultaneously, due to the loss of elastic fibers, the lungs do not elastically recoil during inspiration, allowing for the inhalation of more air. Following administration of ichthyolide, the IC of mice in all experimental groups (PPE+OVA-1; PPE+OVA-2; PPE+OVA-3) showed a downward trend, with a significant decrease in the medium-dose group (PPE+OVA-2), indicating that ichthyolide can help mitigate the deterioration of lung function.

[0079] Figure 4B shows that compared to the normal control group (Control), the pathological control group (PPE) showed a significant decrease in tissue elastance (H). This was due to the destruction of elastic tissue, changes in alveolar structure, and air trapping during respiration, which prevented normal tissue recovery. In contrast, in the groups injected with hyoscyamine lactone, although lung tissue elasticity in the experimental groups (PPE+OVA-1; PPE+OVA-2; and PPE+OVA-3) did not fully recover to levels comparable to those in the normal control group, there was an upward trend in lung tissue elasticity, with a significant increase in the medium-dose group (PPE+OVA-2).

[0080] C. Ichthyolide improves blood oxygen recovery in mice with emphysema after exercise

[0081] Figure 5 shows the difference in blood oxygen levels before and after exercise in each group of mice. As shown in Figure 5, the pathological control group (PPE) showed a significant increase in blood oxygen difference compared to the normal control group (Control). Because mice experience increased oxygen demand after exercise, and the structural damage to the alveoli in mice with emphysema may affect their oxygen uptake, blood oxygen levels decreased after exercise, indicating low exercise tolerance and impaired pulmonary gas exchange efficiency. In the experimental groups receiving euryselamine (PPE+OVA-1; PPE+OVA-2; PPE+OVA-3), the change in blood oxygen levels before and after exercise was minimal, with significant changes in the high-dose group (PPE+OVA-3). This suggests that euryselamine can stabilize blood oxygen levels in mice with emphysema after exercise, potentially improving ventilation efficiency.

[0082] none

[0083] none

Claims

1. A use of a compound for preparing a pharmaceutical composition for increasing a subject's blood oxygen concentration, wherein: The compound comprises a safe and effective dose of nebulosic acid lactone, its structural isomers or pharmaceutically acceptable salts thereof, wherein the nebulosic acid lactone has a chemical structural formula (I). Formula (I) 2. The use as described in claim 1, wherein: The safe and effective dose is 0.08 μg / kg to 8 μg / kg for a 60 kg adult.

3. The use as described in claim 1, wherein: The safe and effective dose is 0.8 μg / kg to 6.5 μg / kg for a 60 kg adult.

4. The use as described in claim 1, wherein: The safe and effective dose is 1.3 μg / kg to 5 μg / kg for a 60 kg adult.

5. The use as described in claim 1, wherein: The system is an individual suffering from chronic lung injury.

6. The use according to claim 5, wherein: The chronic lung injury includes chronic obstructive pulmonary disease.

7. The use as described in claim 1, wherein: The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

8. The use as described in claim 1, wherein: The individual is a mammal or a human.

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