Use of jaceosidin in the preparation of a medicament for increasing blood oxygen concentration in a subject

Abstract

This invention provides the use of a compound in the preparation of a medicament for improving an individual's blood oxygen concentration, the compound comprising a safe and effective dose of strychnine lactone, its structural isomers or pharmaceutically acceptable salts thereof, wherein the strychnine lactone has a chemical structural formula (I); preferably, the individual is an individual suffering from chronic lung injury; more preferably, the chronic lung injury includes, but is not limited to, chronic obstructive pulmonary disease or pulmonary fibrosis.

Description

Technical Field

[0001] This invention provides the use of a compound in the preparation of a medicament for increasing the blood oxygen concentration of a subject, and in particular, the use of a strychnine lactone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for increasing the blood oxygen concentration of a subject. Background Technology

[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 respiratory tract. COPD is the third leading cause of death worldwide, affecting approximately 10.1% of the population and causing more than 3 million deaths annually. Its pathological mechanisms involve the structural destruction of small airways, impaired alveolar tissue repair, and chronic inflammatory responses, resulting in bronchitis and emphysema. Emphysema is characterized by alveolar wall destruction and excessive lung expansion, leading to decreased lung elasticity and impaired gas exchange. Patients often exhibit chronic dyspnea, decreased exercise tolerance, increased respiratory effort, and air trapping.

[0003] Prolonged exposure to irritants (such as tobacco smoke, air pollution, and occupational dust) prompts alveolar epithelial cells to release inflammatory mediators. With the continued infiltration of immune cells such as neutrophils, macrophages, lymphocytes, and mast cells, lung tissue structure is further damaged. Currently, COPD remains incurable; existing treatment strategies can only relieve symptoms and slow disease progression. These mainly include drug therapy, inhaled bronchodilators, anti-inflammatory drugs, and oxygen therapy. Some severely affected patients may require pulmonary rehabilitation or surgery (such as lung volume reduction surgery or lung transplantation). However, these treatments still have limitations in repairing damaged alveoli and halting disease progression, making COPD treatment a significant global public health challenge.

[0004] Fish needle grass (Anisomeles indica O. Kuntze) is a commonly used herb in Taiwan, also known as Kejia Mo Cao (in Jiaoling and Meixian, Guangdong), Jin Jian Cao, and Ben Huo Xiang. Taiwan's health authorities have included fish needle grass in their list of usable food ingredients, and the entire plant is edible. Fish needle grass is an annual or biennial herb belonging to the Lamiaceae family. It is mainly distributed in southwestern China, India, the Philippines, Java and Sumatra in Indonesia, and can be found throughout Taiwan, from plains to low-altitude mountainous areas. There is also sporadic medicinal cultivation in Yuli, Hualien, and Chenggong, Taitung, Taiwan. Traditionally, it is harvested in summer and autumn by pulling up the whole plant or cutting the above-ground parts, washing them, and using them fresh or dried. The whole plant has antipyretic, wind-dispelling, dampness-removing, stomach-strengthening, detoxifying, analgesic, and antibacterial effects. It is commonly used in folk medicine to treat colds and fever, abdominal pain and vomiting, cholera caused by food poisoning, stomach pain, gastroenteritis, neurodermatitis, rheumatic bone pain, muscle and bone pain, eczema, boils, sores, carbuncles, and snake bites.

[0005] Our research team has long been engaged in the breeding of *Needleia serratifolia* (GenBank: GU726292) and has continuously conducted a series of studies on the whole herb extract of *Needleia serratifolia* grown on farms, especially focusing on the preparation of pure crystalline oxytodiolide. Specifically, we have carried out extraction, separation, purification, analysis, identification, and pharmacological studies on its anti-inflammatory, antiviral, anti-Helicobacter pylori, anticancer, and anticancer stem cell effects. In recent years, our team has completed a test experiment comparing oxytodiolide with the influenza A and B drug "oseltamivir" (Roche: Tamiflu), finding that *Needleia serratifolia* extract and oxytodiolide showed good inhibitory effects on influenza virus. At the end of 2019, it was discovered that drugs for treating AIDS showed positive effects in treating patients infected with the novel coronavirus; according to literature reports, oxytodiolide can inhibit HIV (Fitoterapia, 2000, 71(5):574-576.), subsequent studies confirmed that oxytodiolide has an inhibitory effect on the novel coronavirus. In addition, current research has also found that strychnine lactone can inhibit gastritis caused by Helicobacter pylori in the gastric wall, and can also inhibit NF-κB and STAT3-mediated inflammatory responses.

[0006] Therefore, this invention hypothesizes that strychnine lactone may also alleviate symptoms of chronic lung injury caused by chronic inflammation and increase blood oxygen concentration in patients, and puts forward experiments to confirm this hypothesis. Summary of the Invention

[0007] This invention is based on the discovery that ovatodiolide, its structural isomers, or pharmaceutically acceptable salts thereof can be used to increase an individual's blood oxygen concentration and improve symptoms of chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, etc. Specifically, this invention provides a compound for increasing an individual'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] This invention relates to the use of a compound in the preparation of a medicament for increasing an individual'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.

[0009] In this invention, preferably, the individual is an individual suffering from chronic lung injury.

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

[0011] In this invention, the individuals include, but are not limited to, mammals or humans.

[0012] In some embodiments, the strychnine lactone of the present invention is administered at a dose 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, strychnine lactone is administered at unit doses 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, for example at unit doses of 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, or about 325 μg / kg. kg, approximately 350 μg / kg, approximately 375 μg / kg, approximately 400 μg / kg, approximately 425 μg / kg, approximately 450 μg / kg, approximately 475 μg / kg, approximately 500 μg / kg, approximately 525 μg kg, approximately 550 μg / kg, approximately 575 μg kg, approximately 600 μg / kg, approximately 625 μg / kg, approximately 650 μg / kg, approximately 675 μg / kg, approximately 700 μg / kg, approximately 725 μg / kg, approximately 750 μg / kg, approximately 775 μg / kg, approximately 800 μg / kg, approximately 825 μg / kg, approximately 850 μg / kg, approximately 875 μg / kg, approximately 900 μg / kg, approximately 925 μg / kg, approximately 950 μg / kg, approximately 975 μg / kg, approximately 1 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 8 mg / kg, approximately 10 mg / kg Administer in doses of approximately 15 mg / kg, approximately 20 mg / kg, approximately 25 mg / kg, approximately 30 mg / kg, approximately 35 mg / kg, approximately 40 mg / kg, approximately 45 mg / kg, approximately 50 mg / kg, approximately 60 mg / kg, approximately 70 mg / kg, approximately 80 mg / kg, approximately 90 mg / kg, approximately 100 mg / kg, approximately 125 mg / kg, approximately 150 mg / kg, approximately 175 mg / kg, or approximately 200 mg / kg, and administer one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) units daily.

[0014] In this invention, the effective dose can be calculated by further considering the safety factor according to the formula provided in the guidelines published by the US FDA (Guidance for Industry: Establishing the Maximum Safe Starting Doses in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, USDapartment of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research (CDER), July 2005), in order to convert the equivalent dose between different species.

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

[0016] The safe and effective dose for mice of the present invention, when converted to 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 for mice of the present invention, when converted to 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 for mice of the present invention, when converted to 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, the drug preferably also includes a pharmaceutically acceptable carrier.

[0018] In some embodiments, strychnine lactone 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, strychnine lactone is administered in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) treatment cycles, wherein each treatment cycle lasts for 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 the interval between each two treatment cycles is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, two weeks, three weeks, or four weeks.

[0020] In some embodiments, strychnine lactone can be administered orally, orally, via inhalation spray, sublingually, rectally, transdermally, via vaginal mucosa, via transmucosal administration, locally, via the nose or intestine; via injection, such as intramuscular injection, subcutaneous injection, intramedullary injection, as well as intrathecal, direct brain administration, in situ administration, subcutaneous, intraperitoneal, intravenous injection, intra-articular synovial, intrasternal, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal, intraocular injection, or other drug delivery methods. Attached Figure Description

[0021] Figure 1 The chemical structural formula of Ovatodiolide (OVA) of the present invention is shown.

[0022] Figure 2 This is the animal experiment procedure of the present invention.

[0023] Figure 3 A represents the hematoxylin and eosin (H&E) staining results of lung sections from experimental animals used in this invention.

[0024] Figure 3 B represents the mean linear intercept (MLI) analysis results of the alveoli in the experimental animals of this invention.

[0025] Figure 4 A represents the analysis results of the inspiratory capacity (IC) of the lungs of the experimental animals of this invention, in milliliters (mL).

[0026] Figure 4 B represents the tissue elastance (H) analysis results of the experimental animals of this invention, in cm H2O / mL.

[0027] Figure 5 The results of changes in blood oxygen saturation in experimental animals before and after exercise are shown in percentage (%). Detailed Implementation

[0028] This specification extensively uses technical and scientific terms commonly used in the field of biotechnology. In the following description, to provide a clear and consistent understanding of this specification, the scope of the claims, and the scope to which the terms are applied, the following definitions are provided. Other terms not specifically defined below have meanings that are generally understood by those skilled in the art.

[0029] The terms "or," "and," and "and," as used in this specification, unless otherwise stated, refer to "or / and." Furthermore, the terms "comprising" and "including" are not restrictive open-ended conjunctions. The foregoing paragraphs are merely systematic references and should not be construed as limiting the subject of the invention.

[0030] Unless otherwise specified, “%” in this specification refers to “weight percentage (wt%)”; numerical ranges (e.g., 10% to 11% of A) include upper and lower limits unless otherwise specified (i.e., 10% ≤ A ≤ 11%); if no lower limit is defined for a numerical range (e.g., less than 0.2% of B, or B below 0.2%), then the lower limit may be 0 (i.e., 0% ≤ B ≤ 0.2%); the proportional relationship of “weight percentage” of each component can also be replaced by the proportional relationship of “parts by weight”.

[0031] All values ​​disclosed in this specification are subject to a standard technical measurement error (standard deviation) of ±10%. The term "about" is intended to indicate ±10%, ±5%, ±2.5%, or ±1% relative to a given value; that is, "about 20%" represents 20 ±2%, 20 ±1%, 20 ±0.5%, or 20 ±0.25%.

[0032] All materials used in this instruction manual, unless otherwise specified, are commercially available and readily available.

[0033] As used in this instruction manual, the terms "enhance," "improve," "enhance," or similar terms refer to increasing or prolonging the intended efficacy or duration of a drug comprising strychnine. Therefore, "enhance" in the context of enhancing the efficacy of a drug comprising strychnine refers to the ability of a therapeutic system to increase or prolong efficacy or duration.

[0034] As used in this instruction manual, the terms "treatment," "for treatment," and similar expressions refer to the administration of a pharmaceutical agent to achieve a certain effect. This effect is a therapeutic, partial, or complete cure of a disease and / or symptoms of that disease. "Treatment" encompasses any treatment of hepatocellular carcinoma in mammals (particularly humans) and includes: (A) suppressing the disease, i.e., preventing its development; and (B) alleviating the disease, i.e., causing its 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 in this instruction manual, "pharmaceutical acceptable" or "medically acceptable" means that a substance or composition must be compatible with the other components of its pharmaceutical formulation and must not exacerbate the patient's symptoms.

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

[0037] The term "pharmaceutically acceptable carrier" as used in this specification includes one or more of the following types of ingredients: tablets, capsules, granules, syrups, powders, lozenges, capsules, flat capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, injections, and other similar or suitable carrier forms for this invention.

[0038] The compositions provided by this invention may also be appropriately supplemented with one or more commonly used dissolving agents, diluents, buffers, colorants, flavoring agents, etc., in the field of pharmaceutical preparations.

[0039] The “pharmaceutical-acceptable excipients” used in this instruction manual include, but are not limited to, at least one of the following: polymers, resins, plasticizers, fillers, lubricants, diluents, binders, disintegrants, solvents, cosolvents, surfactants, preservatives, sweeteners, flavorings, pharmaceutical-grade dyes or pigments, and viscosity agents.

[0040] The term "medical composition" as used in this instruction manual refers to a solid or liquid composition in a form, concentration and purity suitable for administration to a patient, which, upon administration, can induce the desired physiological changes; the medical composition is sterile and / or non-pyrogenic.

[0041] The term "effective amount" as used in this specification refers to the dose necessary to generate or induce the desired biological response, and is not quantified as required for therapeutic cure. Those skilled in the art will understand that the effective amount of a pharmaceutical composition can vary depending on factors such as the desired biological endpoint, the intended bioactive agent to be delivered, the composition of the encapsulating matrix, and the target tissue.

[0042] Example 1: Drugs and Reagents

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

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

[0045] Example 2: Acute toxicity test of a single oral dose of strychnine lactone in rats.

[0046] This embodiment is a single-dose oral acute toxicity safety test of compound (I) - strychnine lactone in rats, providing a reference for food safety assessment. The experiment was conducted in accordance with the health and safety assessment guidelines of the Taiwan Health Administration – Single-dose oral acute toxicity test, the US 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 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), and other testing standards. This experiment conducted a single-dose oral acute toxicity test on rats (Sprague-Dawley; SD strain) of compound (I) - strychnine lactone. Compound (I) - strychnine lactone is a slightly yellow crystalline substance with a test purity of 99.95%. In the test, it was prepared as a solution with a concentration of 0.1 g / mL in 10% DMSO. Each rat was fed 10 mL / kg body weight orally daily according to its body weight, and the final total dose was 1 g / kg body weight. The rats were observed continuously for 14 days after administration.

[0047] The results showed that after oral administration of compound (I) – strychnine lactone – to rats, none of the rats showed signs of poisoning or died. Regarding weekly weight change (g), there was no significant difference in weekly weight and weight gain between the treated male and female rats and the control group. At the end of the experiment, there were no significant abnormalities in blood values ​​of the treated male and female rats, including: total white blood cell count (WBC count), total red blood cell count (RBC count), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total platelet count, and white blood cell differential. Serum liver and kidney enzyme indicators, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine, were not affected in the treated male and female rats. Regarding the absolute weight (g) and percentage of organ weight, there were no significant differences between the treatment and control groups in organs such as the adrenal glands, brain, heart, kidneys, liver, spleen, thymus, testes, or ovaries. Examination of the internal organs revealed no gross lesions in the treatment group's vital organs, including the adrenal glands, brain, heart, kidneys, liver, spleen, thymus, testes, or ovaries. Histopathological examination showed no histopathological changes related to the test substance in the vital organs treated with compound (I) – strychnine lactone. Overall, the results showed that a single oral dose of compound (I) – strychnine lactone at 1 g / kg body weight (equivalent to approximately 50 mg / kg body weight for human use) did not cause acute poisoning or death in rats in the acute toxicity test, and did not cause any pathological changes related to toxicity in vital organs.

[0048] Example 3: Toxicity test of fish needle lactone on rats after 28 days of feeding

[0049] This embodiment is a repeated-dose oral toxicity safety test of compound (I) – strychnine lactone – in rats, and a material safety data sheet is established based on this to provide a reference for clinical safety assessment of repeated human administration. The test is conducted in accordance with the 28-day feeding toxicity test for health food safety assessment by the health management department of Taiwan, China (1999) and the non-clinical trial safety guidelines for pharmaceuticals (2014), and complies with the testing guidelines of 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 OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents).

[0050] This experiment investigated the potential toxic side effects of compound (I) – strychnine lactone – on humans for clinical safety assessment. A clinical observational study was conducted on the toxicity of compound (I) – strychnine lactone to rats over a 28-day repeated-dose oral administration. Compound (I) – strychnine lactone is a pale yellow crystalline substance with a purity considered to be 99.95%. The sample was prepared with 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 contained 20 rats, half male and half female. Each rat was fed 10 mL / kg body weight orally daily for 28 consecutive days. The results showed that after rats were orally administered compound (I) - strychnine lactone for 28 consecutive days, none of the rats showed any signs of poisoning or died due to the test substance.

[0051] After the experiment, the changes in body weight, feed consumption, urine, blood levels, serum enzyme levels, and organ weight of male and female rats in each treatment group of compound (I) - strychnine lactone, compared with the control group, showed slight increases or decreases due to individual differences, but remained within the normal range. There was no dose-response correlation between groups, no clinical or pathological significance, and it was unrelated to the test substance. Examination of all organs of rats in each group revealed no obvious gross pathological changes. Histopathological examination showed that the high-dose group did not cause any organ toxicity-related pathological changes in any organ. Based on the above test results, compound (I) - strychnine lactone, administered orally to rats for 28 consecutive days at low dose (10 mg / kg-body weight), medium dose (25 mg / kg-body weight), and high dose (50 mg / kg-body weight) did not cause toxic reactions in any organ of male or female rats. The "No observed adverse effect level" (NOAEL) for the 28-day feeding toxicity test in rats was 50 mg / kg-body weight.

[0052] Example 4: Analysis of Chromosomal Abnormalities in In Vitro Mammalian Cell Lines Using Compound (I) - Fish Needle Lactone

[0053] This embodiment describes the analysis of chromosomal aberrations in in vitro mammalian cell lines using compound (I) strychnine lactone, and establishes a Material Safety Data Sheet based on this analysis to provide a reference for safety assessment. The test was conducted according to the in vitro mammalian cell line chromosomal aberration analysis test for health foods issued by the Taiwan Health Administration, and complies with the genotoxicity testing standards of 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, EPA712-C-00-366, 1998) and the OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, No. 473: In vitro mammalian chromosome aberration test, 1997). This experiment conducted a chromosomal aberration test with mammalian cells in culture using compound (I) – strychnine lactone. Compound (I) – strychnine lactone is a pale yellow crystalline substance, prepared with dimethyl sulfoxide (DMSO) for the test. The cytotoxicity test consisted of two parts: one part involved direct treatment of CHO-K1 cells with compound (I) – strychnine lactone, and the other part used rat liver activating enzyme extract (S9) to simulate human metabolism. CHO-K1 cells were treated with a mixture of rat liver activating enzyme extract (S9) and compound (I) – strychnine lactone. In the test without rat liver activating enzyme extract (S9), cytotoxicity was first tested for 24 hours at five test doses of 12.5, 15, 17.5, 20, and 25 μM. The results showed that 17.5 μM of compound (I) – strychnine lactone resulted in a cell survival rate of approximately 65.6% for CHO-K1 cells. In another experiment, a 24-hour cytotoxicity test was conducted using five test doses of 60, 70, 75, 80, and 90 μM in rat liver activating enzyme extract (+S9). The results showed that 75 μM of compound (I) - strychnine lactone resulted in a survival rate of approximately 60.3% for CHO-K1 cells, indicating that compound (I) - strychnine lactone is cytotoxic to CHO-K1 cells. This concentration was selected as the highest dose for the formal test.In the cellular chromosome variation assay, compound (I) – scutellarin – was prepared in 12.5, 15, and 17.5 μM (-S9) solutions in each cell culture dish and co-cultured for 24 hours. Separately, it was co-cultured with S9 at 55, 65, and 75 μM for 3 hours. After 24 hours, the number and structure of cell chromosomes were observed to ensure normality. The results showed that, under the test conditions of the metabolic activation system with or without S9 mixture, the frequency of chromosomal abnormalities in CHO-K1 cells induced by compound (I) – scutellarin – at the three dosage groups (12.5, 15, and 17.5 μM (-S9) and 55, 65, and 75 μM (+S9)) was not significantly increased compared to the negative control group, and there was no significant change in the location of chromosomal abnormalities. In summary, the results indicate that compound (I) – scutellarin – regardless of whether it contains S9 mixture, does not have a mutagenic effect on the chromosomes of the mammalian cell line CHO-K1 in vitro.

[0054] Example 5: Micronucleus test of mouse peripheral blood by compound (I) - strychnine lactone

[0055] This embodiment describes the genotoxicity test of compound (I) – scutellarin – to establish a Material Safety Data Sheet and provide a reference for safety assessment. The test was conducted according to the mouse peripheral blood micronucleus test for health and food safety assessment – ​​genotoxicity testing, as per the guidelines of the US Environmental Protection Agency (USEPA) (Mammalian Erythrocyte Micronucleus Test, In: OPTTS 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 performed the peripheral blood micronucleus test on mouse (ICR strain) using compound (I) – scutellarin. This experiment primarily tested the proportion of micronuclei in peripheral blood of rodents caused by compound (I) cyclophosphamide, thereby assessing the extent of damage caused by gene mutations that directly or indirectly induce erythrocyte chromosome or mitosis. ICR mice were used as the experimental subjects and were divided into five groups: a negative control group, a positive control group (Cyclophosphamide, 60 mg / kg bw), and low-dose (0.25 g / kg bw), medium-dose (0.5 g / kg bw), and high-dose (1 g / kg bw) of compound (I). Five male mice were used in each group. Cyclophosphamide was administered via a gastric tube in a single dose. The incidence of micronuclei (‰) in reticulocytes and reticulocytes in the peripheral blood of the mice was assessed 48 and 72 hours after administration. The results showed that there were no toxic symptoms or differences in body weight among the treatment groups 48 and 72 hours after administration of compound (I) cyclophosphamide. Reticulocytes in peripheral blood of mice appeared orange-red under a fluorescence microscope after staining with 0.1% acridine orange stain. Micronuclei, approximately 1 / 20 to 1 / 5 the size of a red blood cell, exhibited a yellowish-green fluorescence within the reticulocytes. There were no significant differences in the number of reticulocytes and the number of micronuclei within the reticulocytes at 48 and 72 hours between the treatment groups using compound (I) – strychnine lactone – and the negative control group. However, the number of reticulocytes in the positive control group was significantly decreased compared to the negative control group (p<0.05), and the number of micronuclei within the reticulocytes was also significantly increased (p<0.05).Based on the above results, none of the dosage groups of compound (I)-scutellarin showed significant differences in the number of reticulocytes and micronuclei in reticulocytes in the peripheral blood of mice compared with the negative control group, indicating a negative experimental result. Therefore, compound (I)-scutellarin does not have chromosomal gene mutation toxicity to mouse peripheral erythrocytes.

[0056] Example 6 Animal Experiment

[0057] The animal experiment procedure of this invention is as follows: Figure 2 As shown.

[0058] In a specific embodiment of the invention, animal experiments were conducted using 6-week-old male C57BL / 6JNarl mice purchased from an experimental animal center. The mice were housed under constant environmental conditions, with room temperature maintained at 20–24°C and relative humidity at 45–65%, provided with a standard 12-hour light-dark cycle typical of animal laboratories, and had free access to water and food. All procedures were performed under sterile conditions.

[0059] Experimental methods:

[0060] A. The induction of emphysema and the administration of strychnine lactone:

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

[0062] First, male C57BL / 6JNarl mice were randomly divided into 5 groups (n=10 per group), including one normal control group (Control), one pathological control group (PPE), and three experimental groups (i.e., PPE+OVA-1, PPE+OVA-2, and PPE+OVA-3). Then, the pathological control group and each experimental group were administered porcine pancreatic elastase (PPE; purchased from Sigma-Aldrich, 39445-21-1; prepared in PBS, administration volume 50 μL) at a dose of 0.4 IU / mouse via intratracheal injection. The same dose of PPE was administered on days 14 and 28 after the first injection, while the normal control group was administered an equal volume of PBS via intratracheal injection.

[0063] Then, on day 35 following the first PPE injection, mice in the experimental groups were administered strychnine lactone via intratracheal injection at doses of 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), respectively. Mice in the pathological and normal control groups were administered an equal volume of PBS. All groups were administered the medication five times per week for a total of two weeks.

[0064] B. Exercise blood oxygen saturation measurement:

[0065] On day 48 following the first PPE injection, oxygen saturation (SpO2) in each group of mice was measured before and after exercise using a tissue spectrometer (purchased from BIOPAC Systems, Santa Barbara, USA). A non-invasive probe was first attached to the tail of each mouse. The level of oxyhemoglobin was quantified by measuring the absorption rate of near-infrared light at wavelengths between 800 and 1000 nm, while the concentration of deoxyhemoglobin was determined based on the absorption rate of near-infrared light at wavelengths between 600 and 800 nm. The mice then ran on a wheel for 5 minutes, and the oxygen saturation of the tail was measured again to obtain the difference in oxygen saturation before and after exercise. The data were analyzed using Biopac Student Lab-BSL software (Upwards Biosystems, Ltd.).

[0066] C. Lung function testing:

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

[0068] At the start of the experiment, two deep inhalations were performed consecutively, maximally inflating the lungs to a pressure of 30 cmH2O to open the collapsed lung area and standardize lung volume. The lungs were allowed to equalize at this pressure for 3 seconds, and the compressed volume was recorded as the inspiratory capacity (IC) of the test animal.

[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 the airway and lung tissue to the respiratory response.

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

[0071] On day 49 after the first PPE injection, mice in each group were sacrificed, and 10% formaldehyde was injected into the lungs via intratracheal instillation. Lung tissue was then embedded in paraffin and sectioned, and stained with hematoxylin and eosin (H&E). Lung H&E images were acquired using Motic Easyscan Pro and Motic DSAssistant software. Mean Linear Intercept (MLI) was determined by assessing the length of the intersecting lines and the number of grid intercepts in the alveolar space for 10 non-overlapping regions in each lung sample, following the method described in Crowley et al. (2019), BMC Pulm Med, 19:206. ImageJ software was used for analysis to determine the mean alveolar diameter.

[0072] E. Statistical Analysis:

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

[0074] Experimental results:

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

[0076] Figure 3 After displaying lung sections from each group of mice, H&E staining was performed, followed by MLI analysis. Figure 3 As can be seen from Figure A, compared with the normal control group (Control), the alveoli in the pathological control group (PPE) were significantly dilated, and the alveolar walls were thinned or even ruptured, which also resulted in the disappearance of some alveolar septa. Meanwhile, the MLI analysis results ( Figure 3 B) represents the mean alveolar spacing. Compared with the normal control group (Control), the alveolar dilation of the pathological control group (PPE) was significantly increased. After administration of different doses of strychnine lactone, pathological sections showed that the alveolar dilation of mice in each experimental group (PPE+OVA-1; PPE+OVA-2; PPE+OVA-3) was restored, and the MLI values ​​decreased. The decrease was significant in the medium-dose (PPE+OVA-2) and high-dose (PPE+OVA-3) groups, indicating that intratracheal administration of strychnine lactone can alleviate lung tissue damage.

[0077] B. Ficus pluvialis lactone improves lung function and tissue elasticity in mice with emphysema.

[0078] Figure 4 The data displays the lung function test values ​​for each group of mice. Figure 4As shown in Figure A, compared with the normal control group (Control), the inspiratory capacity (IC) of the pathological control group (PPE) was significantly increased. This is because after intratracheal injection of PPE in mice, alveolar enlargement and reduced lung elastic recoil capacity caused by emphysema occur. Emphysema disrupts and enlarges the alveolar structure, leading to increased lung volume. Simultaneously, due to the loss of elastic fibers, the lungs do not recoil during inspiration, preventing the intake of more air. After administration of scutellarin, the inspiratory capacity of mice in all experimental groups (PPE+OVA-1; PPE+OVA-2; PPE+OVA-3) showed a decreasing trend, with a significant decrease in the medium-dose group (PPE+OVA-2), indicating that scutellarin helps alleviate the deterioration of lung function.

[0079] Figure 4 As shown in Figure B, compared with the normal control group (Control), the tissue elasticity (H) of the pathological control group (PPE) was significantly decreased. This was due to the destruction of elastic tissue, changes in alveolar structure, and air trapping during respiration, which prevented normal tissue recovery. In the groups treated with tracheal injection of strychnine lactone, although the elasticity of the lung tissue in each experimental group (PPE+OVA-1; PPE+OVA-2; PPE+OVA-3) did not fully recover to a state comparable to the normal control group, there was an increasing trend in lung tissue elasticity, which was significantly increased in the medium-dose group (PPE+OVA-2).

[0080] C. Ficus pluvialis lactone improves blood oxygen recovery after exercise in mice with emphysema.

[0081] Figure 5 This shows the difference in blood oxygen levels in each group of mice before and after exercise. From... Figure 5 As can be seen, the difference in blood oxygen levels in the pathological control group (PPE) was significantly increased compared to the normal control group (Control). Since mice increase their oxygen demand after exercise, and the alveolar structure damage in emphysematous mice may affect their oxygen uptake capacity, blood oxygen levels decreased after exercise, indicating lower exercise tolerance and impaired pulmonary gas exchange efficiency. The experimental groups administered scutellarin (PPE+OVA-1; PPE+OVA-2; PPE+OVA-3) showed smaller changes in blood oxygen levels before and after exercise, and reached a significant level in the high-concentration group (PPE+OVA-3), indicating that scutellarin can stabilize blood oxygen levels in emphysematous mice after exercise and help improve ventilation efficiency.

Claims

1. Use of a compound in the preparation of a drug to increase an individual's blood oxygen concentration, wherein, The compound comprises a safe and effective dose of strychnine lactone, its structural isomer, or a pharmaceutically acceptable salt thereof, wherein the strychnine lactone has the chemical structural formula (I):

2. The use as described in claim 1, wherein, The safe and effective dosage is 0.08 μg / kg body weight to 8 μg / kg body weight for adults weighing 60 kg.

3. The use as described in claim 1, wherein, The safe and effective dosage is 0.8 μg / kg body weight to 6.5 μg / kg body weight for adults weighing 60 kg.

4. The use as described in claim 1, wherein, The safe and effective dosage is 1.3 μg / kg body weight to 5 μg / kg body weight for adults weighing 60 kg.

5. The use as described in claim 1, wherein, The individual in question is someone suffering from chronic lung injury.

6. The use as described in claim 5, wherein, The chronic lung injury includes chronic obstructive pulmonary disease or pulmonary fibrosis.

7. The use as described in claim 1, wherein, The drug also includes pharmaceutically acceptable carriers.

8. The use as described in claim 1, wherein, The individuals in question are mammals or humans.

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