TWI895230B - Use of ovatodiolide in the manufacture of medical composition for improving blood oxygen saturation in a subject - Google Patents

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

Use of ichthyolide for preparing a pharmaceutical composition for increasing a subject's blood oxygen concentration

The present invention provides a compound for use in preparing a pharmaceutical composition for increasing a subject's blood oxygen concentration, particularly a compound of ichthyolide or a pharmaceutically acceptable salt thereof for use in preparing a pharmaceutical composition for increasing a subject's blood oxygen concentration.

Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disease characterized by a combination of chronic bronchitis and emphysema, which restricts airflow 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 pathology involves 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 excessive lung tissue expansion, resulting in decreased elastic recoil and, in turn, impaired gas exchange. Patients often present with chronic dyspnea, decreased exercise tolerance, increased respiratory effort, and air trapping.

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 severely ill patients 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 major global public health challenge.

Fish needle grass ( Anisomeles indica O. Kuntze) is a common folk herb in Taiwan, also known as guest's grass (in 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 found 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 folk medicinal purposes, it is generally 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 public to treat colds, fevers, abdominal pain and vomiting, indigestion, cholera, stomachache, gastroenteritis, neurodermatitis, rheumatism, bone pain, muscle and bone pain, eczema, swelling, ulcers, fecal poisoning, and snake bites.

Our research team has long been engaged in breeding sedge grass (GenBank: GU726292) and conducting a series of studies on extracts from farm-grown sedge grass. We have focused specifically on the preparation of pure crystalline sedge lactone (ovatodiolide). We have conducted extraction, separation, purification, analytical identification, and research into its pharmacological properties, including anti-inflammatory, antiviral, anti-Helicobacter pylori, anti-cancer, and anti-cancer stem cell activities. In recent years, our team has completed a study testing sedge lactone against the influenza A and B drug Tamiflu (Roche), demonstrating that both sedge grass extract and sedge lactone exhibited significant inhibitory effects against influenza viruses. At the end of 2019, it was learned that AIDS drugs had a positive effect on the treatment of patients infected with the new coronavirus. According to literature reports, ichthyolide can inhibit HIV ( Fitoterapia , 2000, 71 (5): 574-576.), and subsequent studies have confirmed that ichthyolide has the effect of inhibiting the new coronavirus. In addition, existing studies have also found that ichthyolide can inhibit gastritis caused by Helicobacter pylori in the gastric wall and can also inhibit inflammatory responses mediated by NF-κB and STAT3.

Therefore, the present invention infers that ichthyolide may also alleviate the symptoms of chronic lung damage caused by chronic inflammation and increase patients' blood oxygen concentration. Experiments have been conducted to confirm the present invention's inference.

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 and improve symptoms of chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and other conditions. Specifically, the present invention provides a pharmaceutical composition for elevating 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.

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.

In the present invention, preferably, the subject is a subject suffering from chronic lung injury.

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

In the present invention, the individual includes but is not limited to mammals or humans.

In some embodiments, the ichthyolide of the present invention is administered in an amount 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.

In some embodiments, ichthyolide 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 460 μg/kg, about 480 μg/kg, about 490 μg/kg, about 500 μg/kg, about 510 μg/kg, about 520 μg/kg, about 530 μg/kg, about 540 μg/kg, about 550 μg/kg, about 560 μg/kg, about 570 μg/kg, about 580 μg/kg, about 590 μg/kg, about 600 μg/kg, about 610 μg/kg, about 620 μg/kg, about 630 μg/kg, about 640 μg/kg, about 650 μg/kg, about 660 μg/kg, about 670 μg/kg, about 680 μg/kg, about 690 μg/kg, about 700 μg/kg, about 710 μg/kg, μg/kg, about 475 μg/kg, about 500 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 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.

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.

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; and even 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.

The safe and effective dose of the present invention for 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 for 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. Even more preferably, the safe and effective dose of the present invention for 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.

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

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.

In some embodiments, ichthyolide is administered for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) courses, wherein each course 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 a delay of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, two weeks, three weeks, or four weeks between each course of treatment.

In some embodiments, ichthyolide can be administered orally, buccally, by inhalation spray, sublingually, rectally, transdermally, vaginally, transmucosally, topically, nasally or enterally; 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.

This specification makes extensive use of many technical and scientific terms commonly used in the field of biotechnology. To provide a clear and consistent understanding of this specification and the scope of the patent application, as well as 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.

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

Unless otherwise specified, "%" used in this specification refers to "weight percent (wt%)." Numerical ranges (e.g., 10%-11% 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% B, or less than 0.2% B) may imply a lower limit of 0 (i.e., 0% ≤ B ≤ 0.2%). The "weight percent" ratio of each component may also be expressed as "parts by weight."

All numerical values disclosed herein may be subject to standard technical measurement error (standard deviation) of ±10%. The term "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%.

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

As used herein, "enhance," "enhance," "potentiate," or similar terms refer to increasing or prolonging the intended efficacy or duration of a pharmaceutical composition containing ichthyolactone. Therefore, when we refer to enhancing the therapeutic efficacy of a pharmaceutical composition containing ichthyolactone, "enhancement" refers to increasing or prolonging the ability of the rest of the therapeutic system to achieve efficacy or duration.

As used herein, the terms "treat," "for treatment," and similar terms refer to the administration of an agent to achieve a specific effect. The 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 suppression, meaning arresting its progression; and (B) disease alleviation, meaning regression of the disease. In the treatment of tumors (e.g., hepatocellular carcinoma), therapeutic agents can directly reduce the growth and metastasis of tumor cells.

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

The compositions provided by the present invention can be prepared using techniques well known to those skilled in the art by combining the active ingredient or composition provided herein with at least one pharmaceutically acceptable carrier to prepare 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, tablets, buccal tablets, troches, chewing gums, capsules, and other dosage forms similar to or suitable for use with the present invention.

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

The compositions provided by the present invention may also contain one or more dissolution aids, diluents, buffers, colorants, flavorings, etc. commonly used in the pharmaceutical preparation field, as needed.

As used herein, "pharmaceutically acceptable excipients" include, but are not limited to, at least one of polymers, resins, plasticizers, fillers, lubricants, diluents, binders, disintegrants, solvents, co-solvents, surfactants, preservatives, sweeteners, flavorings, pharmaceutical-grade dyes or pigments, and viscosity enhancers.

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 the desired physiological changes after administration; the pharmaceutical composition is sterile and/or non-pyrogenic.

As used herein, "effective amount" refers to the amount necessary to produce or induce a desired biological response and is not intended to be a quantitative measure for 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.

1. Drugs and reagents

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) using a RP C18 BDS column (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% A within 0 minutes, then a linear gradient to 100% A over 40 minutes. The mobile phase flow rate was 1 mL/min, and absorbance was monitored at 220 nm. Ichthyolide was dissolved in dimethyl sulfoxide (DMSO) and further diluted in sterile culture medium before use.

A 1 mM stock solution of ichthyolide 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).

2. Single-dose oral acute toxicity study of ichthyolide in rats

This example tests the acute toxicity of the compound of formula (I), ichthyolide, in rats by oral administration at a single dose, providing a reference for food safety assessment. The single-dose acute oral toxicity 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). This study investigated the acute oral toxicity of a single-dose compound (Formula (I)) in rats (Sprague-Dawley strain). Compound (Formula (I)) is a yellowish crystalline solid with an experimental purity of 99.95%. The compound was prepared in 10% DMSO at a concentration of 0.1 g/mL. Each rat received a feed volume of 10 mL/kg body weight. Rats were orally administered daily based on body weight, for a final total dose of 1 g/kg body weight. Observation was continued for 14 days.

Results showed that after oral administration of the compound (Formula (I)) - ichthyolide, all rats showed no signs of poisoning or mortality. Weekly weight change (g) and weight gain in both male and female treated groups were not significantly different from those in the control group. At the end of the experiment, changes in blood values, including total white blood cell count (WBC count), total red blood cell count (RBC count), hematocrit (Hct), mean corpuscular volume (MCV), mean hemoglobin (MCH), mean hemoglobin concentration (MCHC), total platelet count, and white blood cell count, were all normal in both treated and female rats. Serum liver and kidney enzyme levels, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine, were unaffected in both male and female treated rats. Absolute organ weight (g) and organ weight percentages showed no significant differences between the treated and control groups for the adrenal glands, brain, heart, kidneys, liver, spleen, thymus, testes, or ovaries. Internal organ examination revealed no gross lesions in these organs, including the adrenal glands, brain, heart, kidneys, liver, spleen, thymus, testes, or ovaries, in the treated groups. Histopathological examinations revealed no test substance-related pathological changes in any of the major organs of rats treated with the compound (I) - ichthyolide. Overall, oral acute toxicity studies of the compound (I) - ichthyolide at a single dose of 1g/kg body weight (equivalent to approximately 50mg/kg body weight in humans) did not cause acute poisoning or mortality in rats, and no pathological changes in any of the major organs were associated with toxicity.

3. 28-day dietary toxicity test of ichthyolide in rats

This example tests the repeated-dose oral toxicity safety of the compound of formula (I), ichthyolide, in rats. This study aims to establish a Material Safety Data Sheet (MSDS) to provide a reference for clinical safety assessment of repeated administration in humans. The test was conducted in accordance with the Taiwan Ministry of Health and Welfare's 28-Day Oral Toxicity Test for Food Safety Assessment (1999) and the Safety Standard for Non-clinical Drug Tests (2014). 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).

This study investigated the potential toxic effects of compound (I) - ichthyolide - on humans for clinical safety assessment. A 28-day repeated-dose oral toxicity study was conducted in rats. Compound (I) - ichthyolide is a slightly yellowish crystal with an experimental purity of 99.95%. Samples were 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 daily, based on body weight, for 28 consecutive days. The results showed that after 28 consecutive days of oral administration of the compound (I) - ichthyolide, none of the rats showed symptoms of toxicity or died from the test substance.

After the trial, 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) - ichthyolide, compared to the control group, were within normal limits, despite slight increases or decreases due to individual variability. These values also showed no dose-response correlation between groups, were not clinically significant, and were not related to the test substance. Examination of the rats' organs in all groups showed no obvious gross pathological changes. Histopathological examinations revealed that the high-dose group did not cause pathological changes associated with organ toxicity. The above test results show that the compound (I) - ichthyolide - did not cause organ toxicity in male and female rats when continuously fed orally 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.

4. Analysis of Chromosomal Abnormalities in Mammalian Cell Lines in Vitro Using Compound (I) - Ichthyolide

This example tests the compound of formula (I), ichthyolide, for chromosomal abnormalities in mammalian cell lines in vitro, thereby establishing a Material Safety Data Sheet (MSDS) to provide a reference for safety assessment. The genotoxicity test was conducted based on the Taiwan Ministry of Health and Welfare's in vitro mammalian cell line chromosome aberration test for health foods. It also complies 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, EPA712-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 experiment conducted a chromosomal aberration test with mammalian cells in culture using the compound (Formula (I))—Ichthyolide. The compound (Formula (I)) 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 (Formula (I))—Ichthyolide; the other in which rat liver activated enzyme extract (S9) was used to simulate human metabolism. CHO-K1 cells were then exposed to a mixture of rat liver activated enzyme extract (S9) and the compound (Formula (I)—Ichthyolide. In a 24-hour cytotoxicity test without rat liver activated enzyme extract (-S9), five test doses of 12.5, 15, 17.5, 20, and 25 μM were used. The results showed that the 17.5 μM dose of the compound of formula (I) - ichthyolide had an approximately 65.6% survival rate on CHO-K1 cells. 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% when the compound of formula (I) - ichthyolactone was treated with 75 μM, indicating that the compound of formula (I) - ichthyolactone was cytotoxic to CHO-K1 cells. This concentration was selected as the maximum dose for the formal test. For the cell chromosome variation assay, samples of the compound (I) - ichthyolide were prepared at 12.5, 15, and 17.5 μM (-S9) in each cell culture dish and incubated for 24 hours. Cells were also incubated with S9 at 55, 65, and 75 μM for 3 hours. The normality of the chromosome number and structure was observed over a 24-hour period. The results showed that, under the testing conditions of the S9 mixture metabolic activation system, the three dose groups of Formula (I) - ichthyolide, at 12.5, 15, and 17.5 μM (-S9) and 55, 65, and 75 μM (+S9), did not significantly increase the frequency of chromosomal abnormalities in CHO-K1 cells compared to the negative control group, and there was no significant change in the location of chromosomal aberrations. These results, taken together, indicate that Formula (I) - ichthyolide, regardless of whether it contains an S9 mixture or not, has no mutagenic effect on the chromosomes of the mammalian CHO-K1 cell line in vitro.

5. Compound (I) - ichthyolide in peripheral blood micronucleus test in mice

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 product safety assessments. The genotoxicity test was conducted in accordance with the mouse peripheral blood micronucleus test (MME) of the Taiwan Ministry of Health and Welfare's Food Safety Assessment - Genotoxicity Test. The genotoxicity test also adheres to the U.S. 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 study tested the effect of compound (I) - ichthyolide on peripheral blood micronuclei in mice (ICR strain). This study primarily measured the effect of compound (I) - ichthyolide on the incidence of micronuclei in rodents in vivo, thereby assessing the extent of damage caused by direct or indirect genetic mutations in erythrocyte chromosomes or mitosis. The study used ICR mice as test subjects and was divided into five groups: a negative control group, a positive control group (60 mg/kg bw ip), and 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 (I) - ichthyolide. Each group consisted of five male mice. Ichthyolide was administered as a single dose via gastric tube. Reticulocytes and the incidence of micronuclei (‰) within reticulocytes were assessed 48 and 72 hours after administration of the test substance. Results showed no toxic symptoms or weight differences among the treatment groups 48 and 72 hours after administration of the compound (I) - ichthyolide. Reticulocytes in mouse peripheral blood stained with 0.1% acridine orange stain appeared orange-red under a fluorescent 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 of formula (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), while the number of micronuclei within reticulocytes was significantly increased (p < 0.05) compared to the negative control group. In summary, the above results show that the number of reticulocytes and micronuclei within reticulocytes in the peripheral blood of mice treated with compound (I) at all doses was not significantly different from that of the negative control group, resulting in a negative test result. Therefore, compound (I) does not have a chromosomal gene mutation-inducing toxic effect on peripheral erythrocytes in mice.

6. Animal Experiments

The animal experiment process of the present invention is shown in Figure 2.

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

Experimental methods:

A. Induction of emphysema and administration of ichthyolide:

In this experiment, emphysema was induced according to the method described in Yeh L.Y. et al. (2022), Front Med (Lausanne), 9:794025.

Male C57BL/6JNarl mice were randomly divided into five 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). The pathological control group and the experimental groups were then administered 0.4 IU/head of porcine pancreatic elastase (PPE; purchased from Sigma-Aldrich, 39445-21-1; prepared in 50 µL PBS) via intratracheal injection. The same dose of PPE was administered 14 and 28 days after the first PPE injection. The control group mice were injected intratracheally with the same volume of PBS.

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 iridolide via intratracheal injection. Mice in the pathological and normal control groups were administered an equal volume of PBS. Each group received five doses weekly for a total of two weeks.

B. Blood oxygen test during exercise:

On day 48 after the first PPE injection, oxygen saturation (SpO _{2 )} 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. The mice then ran on a treadmill for 5 minutes. Blood oxygen saturation (BOS) was measured again at the tail to determine the difference between pre- and post-exercise levels. The data were analyzed using Biopac Student Lab-BSL software (Upwards Biosystems, Ltd.).

C. Pulmonary function test:

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 operated by flexiWare v7.2 software.

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 the collapsed lung area and standardize lung volume. The lungs were allowed to equilibrate at this pressure for 3 seconds, and the volume corrected for gas compression was recorded as the animal's inspiratory capacity (IC).

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.

D. Alveolar Mean Linear Intercept (MLI) analysis:

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. Mean linear intercept (MLI) was calculated by evaluating the length of intersecting lines within the alveolar space and the number of grid line intercepts in 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.

E. Statistical analysis:

In the present invention, experimental data were expressed as mean ± standard deviation (SD). Statistical analysis was performed 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.

Experimental results:

A. Ichthyolide reduces lung morphological damage caused by emphysema

Figure 3 shows lung sections from each group of mice, stained with H&E, and analyzed using MLI. Figure 3A shows that compared to the control group, the alveoli in the pathological control group (PPE) were significantly dilated, with thinning and even rupture of the alveolar walls, leading to the partial disappearance of 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 to the control group. After administering different doses of ichthyolide, pathological sections revealed that alveolar dilation in mice in each experimental group (PPE+OVA-1; PPE+OVA-2; PPE+OVA-3) recovered significantly, while MLI values decreased. This decrease was most pronounced in the medium-dose (PPE+OVA-2) and high-dose (PPE+OVA-3) groups, demonstrating that intratracheal administration of ichthyolide can alleviate lung tissue damage.

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

Figure 4 shows the lung function test values of mice in each group. Figure 4A shows that 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 alveolar enlargement and reduced elastic recoil capacity of the lungs caused by emphysema in mice after tracheal injection of PPE. Due to the destruction and enlargement of the alveolar structure in emphysema, the lung volume increases. At the same time, due to the loss of elastic fibers, the lungs do not elastically recoil during inhalation, allowing them to inhale more air. After administration of picotolactone, the inspiratory capacity 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 picotolactone helps to alleviate the deterioration of lung function.

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 breathing, which prevented normal tissue recovery. In the groups injected with ichthyolide intratracheally, it was found that although the elasticity of the lung tissue of mice in each experimental group (PPE+OVA-1; PPE+OVA-2; PPE+OVA-3) did not fully recover to that of the normal control group, it did show an upward trend, with a significant increase in the medium-dose group (PPE+OVA-2).

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

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). Exercise increases oxygen demand in mice, and the destruction of alveolar structure in emphysema mice may affect their oxygen uptake capacity. The decrease in blood oxygen levels after exercise indicates low exercise tolerance and impaired gas exchange efficiency in the lungs. In the experimental groups (PPE+OVA-1, PPE+OVA-2, and PPE+OVA-3) given picotolactone, changes in blood oxygen levels before and after exercise were smaller, and the changes were significant in the high-concentration group (PPE+OVA-3). This suggests that picotolactone can help stabilize the blood oxygen levels of mice with emphysema after exercise, helping to improve ventilation efficiency.

without

Figure 1 shows the chemical structure of ovatodiolide (OVA) of the present invention.

Figure 2 shows the animal experiment process of the present invention.

Figure 3A shows the results of hematoxylin and eosin (H&E) staining of lung sections from experimental animals of the present invention.

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

Figure 4A shows the results of the lung inspiratory capacity (IC) analysis of the experimental animals of the present invention, with the unit being milliliters (mL).

FIG4B shows the analysis results of lung tissue elastance (H) of experimental animals in the present invention, with the unit being cm H ₂ O/mL.

Figure 5 shows the changes in blood oxygen saturation before and after exercise in the experimental animals of the present invention, with the unit being percentage (%).

without

Claims (8)

A compound is used to prepare a pharmaceutical composition for increasing a subject's blood oxygen concentration, wherein the compound comprises a safe and effective dose of scutellaria lactone, a structural isomer thereof, or a pharmaceutically acceptable salt thereof, wherein the scutellaria lactone has a chemical structural formula (I). Formula (I) The use as described in claim 1, wherein the safe and effective dose is 0.08 μg/kg to 8 μg/kg for an adult weighing 60 kg. The use as described in claim 1, wherein the safe and effective dose is 0.8 μg/kg to 6.5 μg/kg for an adult weighing 60 kg. The use as described in claim 1, wherein the safe and effective dose is 1.3 μg/kg to 5 μg/kg for an adult weighing 60 kg. The use as described in claim 1, wherein the individual is an individual suffering from chronic lung damage. The use as described in claim 5, wherein the chronic lung injury includes chronic obstructive pulmonary disease. The use as described in claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The use as described in claim 1, wherein the individual is a mammal or a human.