Application of chrysophanin in prevention and treatment of brain glioma

Abstract

The application discloses application of a golden orchid glycoside in preparation of a drug product for preventing and treating brain glioma, and in particular, application of the golden orchid glycoside in inhibiting growth of brain glioma. It is found through a cell activity experiment that the golden orchid glycoside can significantly inhibit proliferation of brain glioma cells; the golden orchid glycoside can inhibit growth of intracranial tumors of mice, which indicates that the golden orchid glycoside is expected to be further researched and developed as an anti-brain glioma drug, and further expands the effect of research and development progress of the anti-brain glioma drug.

Description

Technical Field

[0001] This invention relates to the field of glioma drug development technology, and in particular to the application of Anoectochilus roxburghii glycoside in the preparation of drugs for the prevention and treatment of glioma. Background Technology

[0002] Gliomas account for approximately 75% of primary malignant brain tumors in adults and a high percentage (40%-50%) of all types of intracranial tumors. The annual incidence of gliomas is 5-8 per 100,000, and their 5-year mortality rate is second only to pancreatic and lung cancer among all cancers, seriously threatening people's health. Among the various subtypes of gliomas, glioblastoma is the most malignant. Glioblastoma (GBM) accounts for approximately 57.3% of all gliomas and 48.3% of all malignant brain tumors. Currently, surgery combined with radiotherapy and chemotherapy is the most common clinical treatment strategy. However, tumor cell resistance to treatment allows some residual lesions to regrow, resulting in unsatisfactory outcomes. Newly diagnosed GBM patients typically survive less than 15 months, while recurrent GBM patients survive less than 6 months, indicating a very poor prognosis. Current immunotherapies such as vaccination, checkpoint blockade, and chimeric antigen receptor T-cell therapy have not improved median overall survival in GBM patients, failing to provide a survival benefit.

[0003] While the term "glioma" is not explicitly recorded in ancient Chinese literature, similar descriptions exist for conditions such as headache, true headache, syncope, and epilepsy. Glioma is attributed to congenital deficiencies, excessive sexual activity, and fright damaging the kidneys, leading to kidney deficiency and malnourishment of the brain. This allows pathogenic factors to invade, impairing the brain's vital energy and hindering the distribution of body fluids. Furthermore, the accumulation of blood stasis and phlegm creates toxins that accumulate in the brain, eventually developing into a glioma. In Traditional Chinese Medicine (TCM), symptoms such as "dementia," "mental retardation," "forgetfulness," and "depression" are often attributed to liver stagnation affecting the spleen, stomach weakness leading to phlegm production, accumulation in the chest, and obstruction of the heart, resulting in mental confusion (from *Bianzheng Qiwen*). TCM plays a crucial role in the prevention and treatment of gliomas and related complications. Its advantages lie primarily in alleviating adverse reactions to radiotherapy and chemotherapy, enhancing their efficacy, improving clinical symptoms, enhancing quality of life, reducing recurrence rates, and prolonging patient survival.

[0004] Golden Thread Orchid (Anoectochilus roxburghii) is a traditional medicinal herb from southern Fujian. The entire plant is used medicinally; it is neutral in nature and sweet in taste, clearing heat, cooling the blood, dispelling wind, and promoting diuresis. Clinically, it is often used for the prevention and treatment of hyperglycemia, hyperlipidemia, and hypertension, and as an adjunct therapy in tumor surgery and radiotherapy / chemotherapy. It is a medicinal and edible plant with high economic value. Currently, there are no reports of its components being used in the treatment of gliomas. Summary of the Invention

[0005] In one aspect, this disclosure provides the use of roximate in the preparation of a medicament for the prevention or treatment of gliomas in subjects.

[0006] In some implementations, the subject is a mammal, such as a human or a mouse.

[0007] On the other hand, this disclosure provides the application of roximate in inhibiting the growth of glioma cells in vitro.

[0008] On the other hand, this disclosure provides a pharmaceutical composition for the prevention or treatment of gliomas in subjects, comprising anoectochilin and pharmaceutically acceptable excipients.

[0009] In some embodiments, the active ingredient in the pharmaceutical composition is 1-99% by weight of anoectochilin.

[0010] In some embodiments, the pharmaceutical composition is a tablet, injection, capsule, granule, powder, syrup, solution, suspension, or aerosol.

[0011] On the other hand, this disclosure provides the use of the above-described pharmaceutical composition in the preparation of a medicament for inhibiting the growth of glioma tumors.

[0012] On the other hand, this disclosure provides a method for preventing or treating gliomas, comprising administering a therapeutically effective amount of anoectochilin or the above-described pharmaceutical composition to a subject in need.

[0013] In some implementations, the subject is a mammal, such as a human or a mouse. Attached Figure Description

[0014] Figure 1 This is a diagram showing the experimental results of a study on the inhibitory effect of Anoectochilin on the growth of glioma cells.

[0015] Figure 2 Figure 1 shows the experimental results of a study on the inhibitory effect of roxithromycin on intracranial tumors in mice. (A) Fluorescence imaging results; (B) Fluorescence intensity changes over time; (C) Comparison of fluorescence intensity among groups.

[0016] Figure 3 The results show that Anoectochilin has no hepatotoxicity, nephrotoxicity, or organ toxicity in mice. (A) Changes in mouse body weight over time; (B) AST results; (C) ALT results; (D) UREA results; (E) UA results; (F) CREA results; (G) Staining results of heart, liver, spleen, lung, and kidney. Detailed Implementation

[0017] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this disclosure. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0018] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this disclosure. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof, and also cover situations consisting of such features, etc.

[0019] "Treatment" refers to the administration of treatment to a subject to obtain a beneficial or desired clinical outcome. As used herein, "treatment" encompasses a variety of treatments, including administration of any possible medicine to the subject, surgery, radiation, etc. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: relief of one or more symptoms, reduction of disease severity, prevention or delay of disease spread, delay of disease recurrence, delay or slowing of disease progression, improvement of disease condition, inhibition of disease or disease progression, inhibition of its development, and remission (whether partial or complete remission). The methods provided herein cover any one or more of these therapeutic aspects. According to the above, "treatment" does not require the complete removal or complete remission of all symptoms of a disease or condition. "Treatment" refers to the administration of treatment to a subject to prevent the occurrence of a disease or its symptoms, or to reduce the likelihood of the occurrence of a disease or its symptoms.

[0020] The term "therapeutic effective amount" refers to an amount of active compound sufficient to elicit a biological or medical response in a subject as desired by a clinician. The "therapeutic effective amount" of this invention can be determined by those skilled in the art based on factors such as route of administration, subject weight, age, and medical condition. For example, a typical daily dose range can be from 0.01 μg to 100 mg or more of the active ingredient per kg of body weight.

[0021] When referring to pharmaceutical compositions, the term "pharmaceuticalally acceptable excipient" refers to solid or liquid diluents, fillers, antioxidants, stabilizers, and other substances that can be safely administered to humans and / or animals without excessive adverse side effects, while also being suitable for maintaining the activity of the drug or active agent contained therein. Depending on the route of administration, a variety of excipients well known in the art can be used, including, but not limited to, sugars, starches, cellulose and their derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifiers, isotonic saline, and / or pyrogen-free water. The pharmaceutical compositions provided herein can be formulated into clinically acceptable dosage forms such as powders and injections. The pharmaceutical compositions of the present invention can be administered to subjects via any suitable route, such as oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, inhalation, or transdermal administration.

[0022] "Subject" refers to an animal, such as a mammal, including (but not limited to) humans, rodents, apes, felines, canines, equines, bovines, suidae, sheep, goats, laboratory mammals, livestock mammals, locusts mammals, and pet mammals. Subjects can be male or female and can be of any age, including infants, young children, adolescents, adults, and the elderly. In some instances, a subject refers to an individual who requires treatment for a disease or condition. In some instances, a subject receiving treatment can be a patient who suffers from or is at risk of developing a condition associated with the treatment. In specific instances, a subject is a human being, such as a human patient. This term is generally used interchangeably with "patient," "subject of examination," "subject of treatment," etc. As described in the background section, glioma is a serious intracranial tumor, and the development of chemical monomers capable of crossing the blood-brain barrier and possessing good inhibitory activity for the treatment of glioma is of great significance. This disclosure provides the use of anoectochilus roxburghii glycoside as an active ingredient in the preparation of drugs for the prevention and treatment of glioma.

[0023] This disclosure demonstrates, through in vitro cell experiments, that roximately quinquefolioside inhibits the proliferation of glioma cells. Different concentrations of roximatefolioside were used to treat glioma cell lines, confirming that the inhibitory effect of roximatefolioside on glioma cells is concentration- and time-dependent. Roximatefolioside at concentrations above 0.25 mM exhibits inhibitory effects on glioma cells. Using GL261-based orthotopic glioma mice as a model, roximatefolioside was administered via gavage at low, medium, and high doses of 15 mg / kg, 30 mg / kg, and 60 mg / kg for 21 consecutive days. The growth of intracranial gliomas in the roximatefolioside-treated mice was significantly inhibited.

[0024] This invention provides a pharmaceutical composition comprising roximate. Preferably, roximate, as an active ingredient, comprises 1-99% by weight of the pharmaceutical composition (or formulation). Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. It may also further comprise conventional excipients such as flavoring agents. Preferably, the pharmaceutical composition may be in the form of tablets, injections, capsules, granules, powders, syrups, solutions, suspensions, or aerosols, and may be present in a suitable solid or liquid carrier or diluent and in a suitable sterile apparatus for injection or infusion, prepared according to conventional pharmaceutical preparation methods. For example, the pharmaceutical composition of this invention can be administered orally to human patients in the form of tablets or capsules.

[0025] Compared with existing technologies, the beneficial effects of this invention are as follows: Gliomas, as primary brain tumors with extremely poor prognosis, are difficult to treat effectively with traditional chemotherapy drugs due to the presence of the blood-brain barrier. This disclosure, through cell activity experiments, found that *Anoectochilus roxburghii* glycosides can significantly inhibit the proliferation of glioma cells; and in in vivo mouse glioma treatment experiments, it was found that *Anoectochilus roxburghii* glycosides can inhibit the growth of intracranial tumors in mice. This suggests that *Anoectochilus roxburghii* glycosides hold promise for further development as an anti-glioma drug, expanding the progress in the research and development of anti-glioma drugs. Further research results show that in a mouse orthotopic glioma xenograft model, *Anoectochilus roxburghii* glycosides can effectively reduce tumor volume and inhibit tumor growth in mice.

[0026] To enable those skilled in the art to better understand the technical solutions of this disclosure, the technical solutions of this invention will be described in detail below with reference to relevant experimental data.

[0027] Example 1: Study on the inhibitory effect of Anoectochilus roxburghii glycoside on glioma cells

[0028] This embodiment uses the GL261 glioma cell line as a research model to study the effect of roxithromycin on the growth of glioma cells. Cells were cultured for 24 hours in media containing 8 mM, 4 mM, 2 mM, 1 mM, 0.5 mM, and 0.25 mM roxithromycin, respectively. Figure 1 It can be observed that as the concentration of roxithromycin increases, the inhibitory effect on glioma cell growth becomes stronger (P<0.001).

[0029] Example 2: Study on the inhibitory effect of Anoectochilin on intracranial tumors in mice

[0030] Modeling: GL261-luc cells in logarithmic growth phase were digested with trypsin and placed in centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 min, the supernatant culture medium was discarded, and the cells were resuspended in PBS and kept on ice. C57BL / 6 mice were captured, weighed, and injected intraperitoneally with 1.25% aphthylamine. Hair removal cream was used to remove hair from the scalp. The mice were anesthetized and fixed on a stereotaxic apparatus. The scalp was disinfected by wiping with 75% alcohol. A longitudinal incision was made along the midline of the scalp with a scalp, and the periosteum was dissected with forceps to expose the sagittal cruciate ridges of the skull. The mouse head was leveled, and the cross-section of the sagittal ridges was... Move the needle 2mm to the right and 0.5mm down to locate the position, and slowly drill a small hole using a skull drill. Use a micro-injection needle to draw 3μL of GL261-luc cell suspension, insert the needle vertically downwards and slowly into the brain 3mm, then withdraw the needle upwards 0.5mm to leave enough space for the cell suspension. Then slowly inject the cell suspension into the mouse brain. After injection, leave the needle in the brain for 5 minutes, then slowly move the needle to remove it so that the GL261-luc cells can adhere to the brain tissue. Suture the wound, apply antibiotics to prevent brain infection, place the mouse on a constant temperature electric blanket, and feed the mouse normally after it has started to move normally.

[0031] Grouping: One week after modeling, the patients were divided into groups: sham surgery group (no intervention after surgery), model group (administered via gavage with the same dose of physiological saline), temozolomide (TMZ) group (administered via gavage at 30 mg / kg TMZ), high-dose anoectochilin group ("High-dose group", administered via gavage at 60 mg / kg anoectochilin), medium-dose anoectochilin group ("Medium-dose group", administered via gavage at 30 mg / kg anoectochilin), and low-dose anoectochilin group ("Low-dose group", administered via gavage at 15 mg / kg anoectochilin). The administration was once daily for 21 days.

[0032] Tumor fluorescence imaging in mice was monitored on days 14 and 21 during the intervention process. D-fluorescein potassium working solution (15 mg / mL) was prepared using sterile DPBS, filtered through a 0.2 μm filter for sterilization, and injected intraperitoneally at a concentration of 10 μL / g body weight. The fluorescence signal reached its strongest stable plateau 10-20 minutes after injection. Mice were then anesthetized and placed in a small animal in vivo imaging system for imaging analysis using an IVIS system. The survival time of the mice was recorded.

[0033] Figure 2 The results showed that on day 21 of administration, compared to the model group, the positive control drug TMZ reduced the fluorescence intensity of mouse tumors, and the roxithromycin-treated group reduced the fluorescence intensity of mouse tumors in a dose-dependent manner. Figure 2 A and Figure 2C), and the medium and high doses of roxithromycin showed significant differences compared to the model group (*P<0.05), indicating that both roxithromycin and the positive control drug TMZ could inhibit the growth of mouse gliomas. In particular, the high-dose group essentially eliminated tumor cells. Changes in fluorescence intensity over time also reflected these effects (C). Figure 2 B).

[0034] Example 3: Anoectochilin showed no hepatotoxicity, nephrotoxicity, or organ toxicity in mice.

[0035] After the experiment, mouse serum was collected for biochemical analysis. ALT and AST levels were used to determine hepatotoxicity, and UREA, UA, and CREA levels were used to determine nephrotoxicity. Heart, liver, spleen, lungs, and kidneys were collected from mice for HE staining to determine the drug's toxicity to these organs.

[0036] Figure 3 Experimental results showed that roximate did not affect the body weight changes in mice. Figure 3 A) showed no hepatotoxicity or nephrotoxicity in mice. Figure 3 B-3F) is also non-toxic to the heart, liver, spleen, lungs, and kidneys of mice. Figure 3 G).

[0037] The above descriptions are merely some specific embodiments of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. The use of Anoectochilus roxburghii glycoside in the preparation of a drug for the prevention or treatment of glioma in subjects.

2. The application as described in claim 1, wherein the subject is a mammal.

3. The application as described in claim 2, wherein the mammal is a human or a mouse.

4. Application of Anoectochilin in inhibiting the growth of glioma cells in vitro.

5. Use of a pharmaceutical composition in the preparation of a medicament for inhibiting the growth of glioma tumors, wherein the pharmaceutical composition comprises anoectochilin and pharmaceutically acceptable excipients.

6. The application as described in claim 5, wherein the active ingredient in the pharmaceutical composition is 1-99% by weight of the total pharmaceutical composition.

7. The application as described in claim 6, wherein the pharmaceutical composition is a tablet, injection, capsule, granule, powder, syrup, solution, suspension, or aerosol.

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