TWI469784B - Therapeutic compositoin for treating cancers - Google Patents

Description

Pharmaceutical composition for treating cancer

The present disclosure is directed to a pharmaceutical composition comprising a novel combination of triterpenoids useful in the treatment of cancer, including cancers that are resistant.

For a long time, in Southeast Asia, edible fungi have been used as nutritional supplements or health foods, among which Ganoderma lucidum is a large-scale, and because of its medical effects, it has been used as a medicinal material for more than a thousand years. . The active ingredients isolated from Ganoderma lucidum generally include triterpenoids, polysaccharides, nucleic acids, polypeptides, and phytosterols. Among these active ingredients, Ganoderma lucidum triterpenoids are most important because of their pharmaceutically active activities, including inhibition of cholesterol synthesis, anticancer, antihypertensive and the like. Ganoderma lucidum triterpenoids include various compounds such as ganoderic acids (GAs), ganodermic acids (GMAs), ganoderic alcohols, ganoderic ketones, and ganoderic aldehydes. In a related study of ganoderic acid, it was found that ganoderic acid has the effect of killing cytotoxicity of cancer cells and inhibiting the amplification thereof. For example, ganoderic acid D (GAD) is known to prevent proliferation of human cervical cancer cells (ie, Hela cells) (Yue et al., Mol Cell Proteomics (2008) 7: 949-961); Acids A and H (GAA and GAH) can inhibit the growth of breast cancer cells or prevent them from invading other tissues (Jiang et al., Int J Mol Med (2008) 21: 577-584); Ganoderma acid X (ganoderic acid X, GAX) inhibits topographic enzyme activity and induces hepatoma cells to enter the apoptotic program (Li et al., Life Sci. (2005) 77, 252-265); Ganoderma lucidum M can effectively inhibit cancer In addition to cell growth, it also inhibits the migration of lung cancer cells (Wang et al., Int Immunopharmacol (2007) 7: 864-870), which is known to induce platelet aggregation (Wang et al., Biochim. Biophys. Acta. 1989) 986, 151-160), inhibition of platelet function (Wang et al., Biochem. J. (1991) 277 (Pt 1), 189-197) and inhibition by thromboxane A2 (Su et al., Biochem. Pharmacol. 1999) 58, 587-595; Su et al., Biochim. Biophys. Acta. (1999b) 1437, 223-234) or prostaglandin E1 (Su et al., Thromb. Res. (1999c) 99, 135-145) in platelets Induced cellular response

The inventors of the present invention have unexpectedly discovered that a novel combination of triterpenoids has the effect of inhibiting specific types of cancer cells, including cancer cells which are resistant and resistant, so that they are not proliferative and, therefore, can be used for treating or preventing cancer. a drug or an adjuvant thereof.

The present disclosure is based, at least in part, on the discovery of a novel combination of triterpenoids having properties that retard or inhibit the growth of cancer cells, which are derived from fruiting bodies or mycelia of Ganoderma lucidum , respectively. In the process of isolation or purification. The above findings suggest that the novel triterpenoid combination in the present disclosure can be used as a cancer, drug or adjuvant that can treat or prevent cancer, including drugs.

Accordingly, a first object of the present disclosure is directed to a pharmaceutical composition that treats or prevents cancer. The pharmaceutical composition comprises a pharmaceutically effective amount of a triterpenoid compound comprising at least ganoderic acid S (GAS), ganoderic acid T (GAT), ganoderic acid Me (GAMe), Ganoderic acid R (GAR) and ganodermic acid S (GMAS); and a pharmaceutically acceptable carrier thereof.

According to a preferred embodiment, the amounts of GMAS, GAMe and GAR, GAT, and GAS are about 0.5-20%, 0.5-20%, 20-75%, and 10-30%, respectively, of the total triterpenoid content. weight ratio). A cancer suitable for treatment or prevention with the pharmaceutical composition may be rectal cancer, liver cancer, breast cancer, lung cancer or blood cancer. In one example, the cancer is lung cancer. In another example, the cancer is a lung cancer that develops resistance to gefitinib.

The novel triterpenoid combination of the present invention comprises from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition, based on the total weight of the pharmaceutical or pharmaceutical composition. In certain embodiments, the total amount of the triterpenoid compound of the present invention is at least about 1% by weight based on the total weight of the pharmaceutical composition. In a particular embodiment, the total amount of the triterpenoid compound of the present invention is at least about 5% by weight based on the total weight of the pharmaceutical composition. In other embodiments, the total amount of the triterpenoid compound of the present invention is at least about 10% by weight based on the total weight of the pharmaceutical composition. In other embodiments, the total amount of the triterpenoid compound of the invention is at least about 25% by weight of the total weight of the pharmaceutical composition.

In certain embodiments, the medicament or medicament of the invention The composition can be used as another auxiliary treatment in addition to other major cancer treatment methods such as surgery, radiation therapy or chemotherapy to improve the therapeutic effect of the cancer.

A second object of the present disclosure is to provide a method of treating cancer. The disclosed method comprises administering to a subject in need of treatment a therapeutically effective amount of a triterpenoid compound comprising GMAS, GAMe and GAR, GAT, and GAS. A cancer suitable for treatment by the method of the invention may be rectal cancer, liver cancer, breast cancer, lung cancer or blood cancer. In one example, the cancer described above is lung cancer. In a preferred embodiment, the cancer is lung cancer that is resistant to gefitinib. The individual can be a mammal, preferably a human.

In certain embodiments, the methods of the invention further comprise additionally administering to the individual, prior to, concurrently with, or after the administration of the pharmaceutical composition of the invention, additional primary cancer treatments such as surgery, radiation therapy, or chemotherapy. Means to improve the therapeutic effect of cancer on the individual.

These and other features of the present disclosure will be more apparent from the following detailed description and appended claims. The above summary and the following detailed description are merely illustrative, and are not intended to limit the scope of the disclosure.

In order to make the description of the present disclosure more detailed and complete, the following description of the embodiments of the present invention and the specific embodiments of the present invention a form. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.

In the following, the term "treatment or treating" includes the prevention (ie, prophylactic), curative or palliative effects that may lead to a desired pharmaceutical and/or physiological effect. Sexual disposal. Preferably, the effect refers to medically partially or completely curing or preventing the growth of cancer cells. In addition, the term "treatment" is used herein to mean the occurrence of one or more symptoms based on partial or complete mitigation, delayed onset, inhibition of progression, reduction in severity, and/or reduction in a particular disease, disorder, and/or medical condition. For the purpose of the test subject (or patient), especially having a medical condition, a symptom of the medical condition, a disease or condition caused by the medical condition, or a condition that would lead to the development of the medical condition. An individual of the prior condition, administering or applying a compound of the present disclosure. An individual who has not developed a significant condition for a particular disease, disorder, and/or medical condition, and/or an individual who has an early onset of the particular disease, abnormality, and/or medical condition may be treated to reduce the occurrence of the particular disease, abnormality, and / or the risk of pathology associated with medical conditions. Herein, the condition, disease, abnormality, and/or medical condition may be carcinoma in situ or metastatic cancer. Reducing one or more signs or clinical indicators means that the treatment is "effective."

"Prophylaxis" here refers to a means of preventing a future event. Based on the prevention of cancer cells in this article or the potential for the transfer of cancer cells due to surgery or diagnostic procedures, therefore, The prophylactic treatment means described herein can be performed prior to, concurrently with, or subsequent to the practice of the procedure or diagnostic procedure.

The term "an effective amount" is intended to achieve the desired effect of reducing the number of cancer cells after a suitable administration period for the purpose of treating cancer.

The words "compound", "composition", "agent" or "medicine or medicament" are used interchangeably herein and refer to when applied to a body (human Or an animal, a compound or composition capable of inducing a desired pharmaceutical and/or physiological response through local and/or systemic action.

The term "administered, administered" or "administration" as used herein refers to the direct administration of the compound or composition, or the administration of a prodrug, derivative, or analog of the active compound. Alternatively, a substantial amount of the active compound can be formed in the subject to be administered.

The words "subject" or "patient" are used interchangeably herein to mean an animal (including a human) that is treatable by the compounds and/or methods. "Individual" or "patient" is used herein to encompass both male and female genders unless otherwise specified. Therefore, "individual" or "patient" includes any mammal, including, but not limited to, human, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc. The compounds benefit from treatment. Animals suitable for treatment with the compounds and/or methods of the invention are preferably human. In general, the words "patient" and "individual" are used interchangeably in this paper. use.

Although numerical ranges and parameters are used to define a broad range of values for the present invention, the relevant values in the specific embodiments are presented as precisely as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the average, depending on the considerations of those of ordinary skill in the art to which the invention pertains. Except for the experimental examples, or unless otherwise explicitly stated, all ranges, quantities, values, and percentages used herein are understood (eg, to describe the amount of material used, the length of time, the temperature, the operating conditions, the quantity ratio, and the like. Are all modified by "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters disclosed in the specification and the appended claims are intended to be At a minimum, these numerical parameters should be understood as the number of significant digits indicated and the values obtained by applying the general carry method.

The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein. In addition, the singular noun used in this specification covers the plural of the noun in the case of no conflict with the context; the plural noun of the noun is also included in the plural noun used.

At least a portion of the present disclosure has been developed based on novel combinations of triterpenoids that are isolated from the body or mycelium of Ganoderma lucidum, and which have the property of combating the proliferation of cancer cells, including drug-resistant cancer cells. Therefore, this novel combination of triterpenoid compounds is potentially useful for treatment Or an agent for preventing cancer or an adjuvant for cancer drugs.

Accordingly, a first aspect of the present disclosure provides a pharmaceutical composition that treats or prevents cancer. The pharmaceutical composition comprises a pharmaceutically effective amount of a triterpenoid compound comprising at least ganoderic acid S (GAS), ganoderic acid T (GAT), ganoderic acid Me (GAMe), Ganoderic acid R (GAR) and ganodermic acid S (GMAS); and a pharmaceutically acceptable carrier thereof.

The triterpenoids of the present disclosure are isolated from Ganoderma genus and can be isolated from fruit bodies or mycelia of Ganoderma lucidum by conventional purification methods, such as GAS, GAT and GAMe. GAR can be isolated or purified using the method disclosed by Xu et al. (App. Microbiol Biotechnol (2010) DOI 10.1007/s00253-010-2576-5); GMAS can be based on Hirotani et al. (Phytochemistry (1987), 26 (10). ), 2797-2803), which is isolated and purified, or in accordance with the method described in Taiwan Patent No. I381844, issued to Chen et al. on January 11, 2013, in an abandoned space bag for culturing Ganoderma lucidum. Ganoderma lucidum mycelium is used as a raw material for separation. Regardless of whether the raw material is from a Ganoderma lucidum fruit body or a mycelium, the separation and purification method generally involves extracting at a temperature higher than room temperature with a solvent (preferably an alcohol solution), and then performing the column layer on the extract. The analysis includes, but is not limited to, high-efficiency liquid chromatography (HPLC) and reverse phase liquid chromatography (Reverse Phase Liquid Chromatograpy), and the like, and steps of concentration, drying, and the like until a dry powder of the active compound of the ganoderma lucidum is obtained.

In certain preferred embodiments, the amount of GMAS is from about 0.5% to about 20% by weight of the total triterpene compound, for example about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14. 14.5, 15.5, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5 or 20% (% by weight), which is calculated as 100% of the total amount of triterpenoids contained in the composition. And got it. In one example, the amount of GMAS is about 1.5% of the total amount of triterpenoids in the composition. In another example, the amount of GMAS is about 2% of the total amount of triterpenoids in the composition. In yet another example, the amount of GMAS is about 8% of the total amount of triterpenoids in the composition.

The amount of the GAMe and GAR is about 0.5-20% by weight of the total triterpenoid content, for example, about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19. 19.5% or 20% by weight, which is calculated as 100% of the total amount of the triterpenoid compound contained in the composition. In one example, the amount of GAMe and GAR is about 4.5% of the total amount of triterpenoids in the composition. In another example, the amount of GAMe and GAR is about 6.5% of the total amount of triterpenoids in the composition. In yet another example, the amount of GAMe and GAR is about 15% of the total amount of triterpenoids in the composition.

The amount of the GAT is about 20-75% by weight of the total triterpenoid content, for example about 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47, 50, 52, 55, 57, 60, 62, 65, 67, 70, 72 or 75% by weight, calculated as the total amount of the triterpenoid compound contained in the composition as 100%. In an example, the GAT The amount is about 65% of the total amount of the triterpenoid compound in the composition. In another example, the amount of GAT is about 70% of the total amount of triterpenoids in the composition. In yet another example, the amount of GAT is about 73% of the total amount of triterpenoids in the composition.

The amount of the GAS is about 10-30% by weight of the total triterpenoid content, for example about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% (% by weight), which is calculated as 100% of the total amount of the triterpenoid compound contained in the composition. In one example, the amount of GAS is about 17% of the total amount of triterpenoids in the composition. In another example, the amount of GAS is about 18% of the total amount of triterpenoids in the composition. In yet another example, the amount of GAS is about 20% of the total amount of triterpenoids in the composition.

The novel triterpenoid combination of the invention (ie, a combination of GMAS, GAMe and GAR, GAT, and GAS) comprises from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition, based on the total weight of the pharmaceutical or pharmaceutical composition. %(weight%). In certain embodiments, the total amount of the triterpenoid compound of the present invention is at least about 1% by weight based on the total weight of the pharmaceutical composition. In a particular embodiment, the total amount of the triterpenoid compound of the present invention is at least about 5% by weight based on the total weight of the pharmaceutical composition. In other embodiments, the total amount of the triterpenoid compound of the present invention is at least about 10% by weight based on the total weight of the pharmaceutical composition. In other embodiments, the total amount of the triterpenoid compound of the invention is at least about 25% by weight of the total weight of the pharmaceutical composition.

Cancers suitable for treatment with the pharmaceutical compositions disclosed herein include, but are not limited to, rectal cancer, liver cancer, breast cancer, lung cancer, or blood cancer. Preferably, the pharmaceutical composition disclosed in the present invention is used to treat the device. Drug-resistant cancers, such as lung cancer that is resistant to gefitinib.

In this context, drug-resistance refers to a specific state of cancer, especially the state of cancer that has developed resistance to a single drug. For example, cancers that have developed resistance include resistance to vinca alkaloids (eg, vinblastine, vincristine, and vinorelvine); Anthracyclines (eg, doxorubicin, daunorubicin, and idarubicin) are resistant; resistant to the microtubule-stable drug, paclitaxel; And drugs that target tyrosine kinase (TK) activity (eg, dasatinib, nilotinib, imatinib, gefitinib) Gefitinib)) is resistant. In a preferred embodiment, a cancer suitable for treatment with a pharmaceutical composition of the invention is lung cancer, which has developed drug-resistant lung cancer against an FDA-approved drug, gefitinib.

In certain embodiments, the pharmaceutical or pharmaceutical compositions disclosed herein can be used as an additional adjunctive therapeutic other than other major cancer treatment modalities such as surgery, radiation therapy, or chemotherapy to improve the The therapeutic effect of cancer.

The above pharmaceuticals or pharmaceutical compositions can be prepared according to well-accepted pharmaceutical processes such as those disclosed in Remington's Pharmaceutical Sciences (17th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa (1985)). . A pharmaceutically acceptable excipient is meant to be compatible with the other ingredients of the pharmaceutical formulation and compatible with the organism.

According to the disclosed embodiments of the present invention, The medicament or pharmaceutical composition disclosed herein, such as a capsule, suspension or syrup for oral administration, is administered by a suitable route of administration or is administered without gastrointestinal route. The mode of administration without gastrointestinal route includes systemic administration such as intramuscular injection, intravenous injection, subcutaneous injection or intraperitoneal injection. Alternatively, it can be applied by means of a transdermal membrane, such as topical skin application or inhalation (eg, intrabronchial, intranasal, intraoral or nasal drops, etc.); or intrarectal administration. Administration may be carried out alone or in combination with conventional pharmaceutically acceptable adjuvants. In a preferred embodiment, the compounds of the invention can be administered to an individual via oral means (e.g., through food).

If administered orally, the novel combination of the triterpenoids of the present invention can be formulated to contain various adjuvants (eg, microcrystalline cellulose, calcium carbonate, dicalcium phosphate, and glycine); various disintegrants (such as starch) , alginic acid and specific citrate); and granule binders (eg, polyvinylpyrrolidone, sucrose, gelatin, and acacia). In addition, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc may also be included. Preferred materials associated therewith include sugars in lactose or milk as well as high molecular weight polyethylene glycols. The above solid dosage forms may also optionally include a coating or shell, such as a casing coating, and a coating to improve the rate of release of any of the pharmaceutically active ingredients. Examples of such coatings are well known to those skilled in the art. In one example, the pharmaceutical composition is formulated as a tablet. In another example, the pharmaceutical composition is a granule formulated to be filled in a soft or hard gelatin capsule or encapsulated in a biodegradable kit. When used in the form of oral suspensions and / or special effects (elixirs), the active ingredients may be combined with various sweeteners or flavors, colorants or dyes. The formulation may, if desired, be added with an emulsifier and/or a suspending agent, and a diluent such as water, alcohol, propylene glycol or glycerin. In certain embodiments, the pharmaceutical compositions of the invention are formulated as liquid dosage forms suitable for oral administration. Such liquid formulations may further include a buffer to maintain the pH. This liquid formulation can also be filled in a soft capsule. The liquid dosage form can be a solution, suspension, emulsion, microemulsion, precipitate or can carry a compound of the invention, a pharmaceutically acceptable derivative, isomer, metabolite, salt or solvate thereof Any liquid medium. The liquid can be designed to improve the solubility of the pharmaceutically acceptable salts of the compounds of the invention to form a drug-containing emulsion or dispersion. When such a dosage form is employed, it is formed by mixing the active compound with at least one pharmaceutically acceptable adjuvant, including, but not limited to, the above-described adjuvants.

If administered in a parenterally manner, the novel combination of the triterpenoids of the present invention can be formulated into a liquid pharmaceutical composition which can be administered by intravenous, intramuscular, subcutaneous or intraperitoneal injection. Sterile solution or suspension. Diluents which can be used in the manufacture of such sterile injectable solutions or suspensions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution, isotonic sodium chloride solution. Fatty acids (e.g., oleic acid) and their glyceride derivatives, or natural pharmaceutically acceptable oils (e.g., olive oil or rapeseed oil) can also be used to make solutions or suspensions for injectable use. Alcohols or carboxymethylcellulose or similar dispersing agents for dilution may also be included in such oily solutions or suspensions. Other commonly used surfactants (e.g., Tweens or Spans series) or emulsifiers, or agents commonly used in the pharmaceutical arts to enhance bioavailability can be used.

The above pharmaceutical or pharmaceutical compositions of the present invention may also be formulated into a variety of dosage forms suitable for topical application to the skin surface. In such embodiments, a variety of known inert carriers suitable for use on the skin surface can be employed to make a suitable dosage form. Such dosage forms include solutions, lotions, creams, gels, ointments, sprays, dermal patches, and the like. Typical inert carriers can be, for example, water, ethanol, polyvinylpyrrolidone, polypropylene glycol, mineral oil, stearic alcohol, and gel-forming materials. The carriers or dosage forms disclosed above are well known to those skilled in the art, and there is no necessary correlation between the choice of suitable dosage form and the efficacy of the pharmaceutical compositions of the present invention.

The above pharmaceuticals or pharmaceutical compositions of the present invention may also be formulated into a variety of dosage forms suitable for mucosal applications, such as buccal and/or sublingual pharmaceutical dosage units, for delivery of drugs through Oral mucosa. A wide variety of biodegradable and pharmaceutically acceptable polymeric adjuvants can be used which provide the pharmaceutical compositions with acceptable adsorption and desired drug release patterns, as well as buccal and/or sublingual drugs. The active ingredient or other ingredients to be administered in the dosage unit are compatible. In general, the above polymeric adjuvants comprise a hydrophilic polymer that adheres to the wetted surface of the oral mucosa. Examples of polymeric adjuvants include, but are not limited to, acrylic acid polymers and copolymers; hydrolyzed polyvinyl alcohol; polyethylene oxides; polyacrylates; Vinyl polymers and copolymers; polyvinylpyrrolidine; dextran; guar gum; Pectins; starch; and cellulosic polymers.

It will be understood that the dosage of the triterpenoid combination of the present invention will vary from individual to individual, not only because of the particular compound or composition employed, the route of administration, the compound (either alone or in combination with one or more drugs) The factors such as the desired response may also be affected by other factors, such as the state or severity of the condition to be treated; the age, sex or weight of the patient, the health of the patient; and the severity of the pathological condition to be treated, Other medical or special dietary content that the patient performs at the same time; and other factors conceivable by those of ordinary skill in the art; and the medical personnel responsible for care may ultimately determine the appropriate dosage based on these factors. The dosage and form of administration can be adjusted to provide a preferred therapeutic response. A therapeutically effective amount also refers to a toxic or detrimental effect of a compound or composition that is less than the therapeutic benefit it brings. In a preferred embodiment, the combination of the triterpenoids of the present invention, when administered, should be administered in an appropriate dosage for a period of time to reduce the number and/or severity of symptoms.

Accordingly, the present disclosure also provides a method of treating cancer in a patient. The disclosed method comprises administering to the patient an effective amount of a pharmaceutical composition of the invention described above, and the pharmaceutical composition is effective to treat cancer in the patient by inhibiting growth and/or replication of cancer cells. Any means capable of efficiently delivering the pharmaceutical composition to a suitable location of action (eg, oral, nasal, pulmonary, percutaneous, or the like, or such as rectal suppository, subcutaneous, intravenous, intramuscular, ophthalmic solution or Ointment or the like) to apply the pharmaceutical composition to a mammal Physically, it is better for humans. Furthermore, the pharmaceutical compositions of the invention may also be administered concurrently with other active ingredients.

Cancers suitable for treatment by the methods of the invention include rectal cancer, liver cancer, breast cancer or lung cancer. Preferably, the compounds of the invention are used to treat cancers that are resistant, including cancers that are resistant to gefitinib.

When the pharmaceutical composition of the present invention is applied to a body, the effective amount to be administered is from about 1 to 100 mg/kg of the individual body weight. The pharmaceutical composition to be administered to the individual per day is used in an amount of about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg body weight; preferably about 30-70 mg/kg body weight. For example, about 30, 40, 50, 60, 70 mg/kg body weight; more preferably about 50 mg/kg body weight. These doses can be administered in a single administration or divided into multiple administrations within one day.

In certain embodiments, the methods of the invention further comprise additionally administering to the individual, prior to, concurrently with, or after the administration of the pharmaceutical composition of the invention, additional primary cancer treatments such as surgery, radiation therapy, or chemotherapy. Means to improve the therapeutic effect of cancer on the individual.

The following examples are presented to illustrate certain aspects of the present disclosure and to assist those skilled in the art to understand and practice the present disclosure. However, the scope of the disclosure is not limited to these embodiments.

Example

Materials and Methods

Cell culture

This study used human breast cancer cell line MDA-MB-231, human liver cell carcinoma strain HepG2 (human hepatocarcinoma cell) Line HepG2), human lung adenocarcinoma cell line A549, human rectal cancer cell line HCT116, human non-small lung cancer cell line PC-9, human non-small lung cancer cell line PC-resistant to gefitinib 9 (PC9-IR), human non-small lung cancer cell line H1650, human myeloid leukocyte cell line K652, human promyelocytic leukocyte cell line HL-60, and mouse lung carcinoma cell line (Lewis lung carcinoma LLC).

MDA-MB-231, HepG2, HCT116, and LLC cells were separately cultured in Dulbecco's modified Eagle's medium (DMEM), PC9, PC9-IR, H1650, K562, and HL-60. Incubate in RPMI medium and add 10% fetal calf serum (FCS), 100 units/ml penicillin, 100 ng/ml streptomycin (Invitrogen, Carlsbad, CA), 2 mM L - Glutamate and non-essential amino acids and sodium pyruvate, and maintained at 37 ° C in a humid environment (5% CO ₂ and 95% air). Cells are usually cultured in a 10 cm culture dish and subcultured as the cells grow to eight minutes. The subculture method was to first aspirate the old culture medium, and then wash the culture cells once with 3 ml of phosphate buffer (PBS), followed by 1 ml of trypsin/EDTA (0.05%/0.025%) at 37 °C. The cells were treated with the solution for 5 minutes, and the cells were detached from the culture plate to be in a suspended state; then, 2 ml of fresh culture solution was added to the suspension cells to neutralize the activity of trypsin. Then, the cells were dispersed to be evenly distributed in the cell suspension, and after a proper ratio of the number of cells was left, the new culture solution was added to an appropriate volume and mixed, and then cultured in a 37 ° C incubator.

Cell viability analysis

Take about 3,000 cells and inoculate them in a flat 96-well plate. In the medium, the culture solution in each culture well was replenished to a volume of 180 L, and then the culture plate was placed in an incubator at 37 ° C overnight. On the next day, a combination of triterpenoids containing different concentrations, gefitinib or imatinib was added to the above culture solution. The final volume in each well was 200 μL, each concentration was triple-coated, placed in a 37 ° C incubator, and cell viability was measured after 48 hours.

Acid phosphatase (ACP) analysis

A large number of living cells can be subject to quality ACP p-nitrophenyl phosphate (p -nitrophenyl phosphate, p -NPP) translate into - nitrophenol (p -nitrophenol, p -NP), p -NP at 405 nm The maximum absorbance at the wavelength, so the absorbance value can be used to quantify the ACP activity in the cell, and then calculate the activity of the cell. To measure cell viability, first aspirate the culture solution, wash once with 200 μL PBS, and add 100 μl of assay buffer (containing 10 mM p- NPP, 0.1 M sodium acetate, 0.1% Triton X-100, and pH approx. 5.5), after reacting at 37 ° C for 30-40 minutes, 10 μl of sodium hydroxide (0.1 N) was added to terminate the reaction, and the absorbance was measured at a wavelength of 405 nm using a SpectraMax M5 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Cell viability, and the cell activity ratio of the untreated drug group was 1, and the cell activity ratio under different conditions was calculated.

Induced carcinoma in situ and in vivo treatment

This test used 6-week-old C57BL/6 and NOD/SCID male rats, kept in a pathogen-free environment, maintained at 22±3°C at room temperature, maintained at 50±20% relative humidity, and maintained 12/12 hours in day and night. Drinking water and food are freely available. Animal feed was used with experimental rodent diet 5001 (purchased from PMI ^® Nutrition International, Inc., MO, USA). For drinking water, boiled tap water is used. All animal experiments were carried out in accordance with the "Guidelines for the Management and Use of Laboratory Animals" published by the Chinese Society of Laboratory Animals.

One day before the experiment, the animals were grouped according to their body weight. After grouping, it was confirmed that there was no statistically significant difference in body weight between the groups, and then the outside of the right thigh was shaved. On the day of the test, the injected tumor cells were subcutaneously injected into the shaved area, and A549 human lung cancer cells or HCT116 human rectal cancer cells (1 × 10 ⁷ cells in 0.1 mL of physiological saline) were injected into NOD/SCID mice, or LLC. Mouse lung cancer cells (1 × 10 ⁶ cells in 0.1 mL of physiological saline) were injected into C57BL/6 mice. Three days after the injection, the pharmaceutical composition of the present invention was administered again, and the day of administration was designated as the first day of the administration test. All dosing solutions were prepared on the same day. At the time of administration, the test animals were fed with a drug-administered suspension by feeding; twice daily, at least three hours apart. The C57BL/6 mice completed the experiment on the 22nd day, and the NOD/SCID mice completed the experiment on the 35th day. After the completion of the experiment, the tumor was removed, the tumor weight was weighed, and the proportion of the pharmaceutical composition of the present invention inhibiting tumor growth was calculated.

Example 1 Inhibition of cancer cell activity in vitro

1.1 pharmaceutical composition

The pharmaceutical compositions of the present disclosure that specifically prevent and/or treat cancer are prepared by mixing the active ingredients into a homogeneous mixture according to the formulation shown in the following table, which may be in accordance with the present disclosure. The method specified is isolated or purified from the fruit body or mycelium of Ganoderma lucidum.

Table 1 Formulation of the pharmaceutical composition of the present disclosure

1.2 Composition 1 of Example 1.1 inhibits the activity of various cancer cells

The efficacy of the pharmaceutical composition 1 disclosed in Example 1.1 for inhibiting the activity of various cancer cell lines, including rectal cancer, liver cancer, breast cancer, lung cancer, and blood cancer, was evaluated by measuring ACP activity in the above manner, and the results are shown in Table 2.

As shown in Table 2, the composition 1 disclosed in Example 1.1 can inhibit the cancer cell line MDA-MB-231, the liver cancer cell line HepG2, the rectal cancer cell line HCT-116, the lung cancer cell line A549, the blood cancer cell line K562 or The activity of cancer cells such as HL-60 is the most sensitive to treatment by blood cancer cell lines, and the least sensitive to breast cancer cell lines.

1.3 Composition 2 of Example 1.1 inhibits the activity of various cancer cells

Similarly, the effect of the pharmaceutical composition 2 of the present disclosure on rectal cancer and lung cancer cell lines was examined using ACP activity as an indicator of cell activity. The results are shown in Table 3.

Summarizing the results of Table 2 and Table 3, the efficacy of Composition 2 of the present invention is at least about 3 times that of Composition 1 in terms of inhibiting the activity of cancer cells, since the amount of GMAS contained in Composition 2 is about Composition 1 The amount of GMAS is 4 times, so it is speculated that the stronger efficacy of Composition 2 (ie, inhibition of cancer cell activity) should be derived from the contribution of GMAS.

1.4 Composition 1 of Example 1.1 inhibits lung cancer cell activity resistant to gefitinib

The effect of the test composition 1 on the lung cancer cell line PC9-IR which had developed resistance to gefitinib was carried out in the manner described in Example 1.1. The concentration of composition 1 or gefitinib inhibiting 50% activity of the cell line, and the cell resistance ratio (RR) of the two cell lines to the drug are summarized in Table 4 below. The cell resistance ratio is a way to determine the susceptibility of a cell strain to a test drug. By dividing the IC ₅₀ concentration of a particular drug against a different cell strain, an RR value can be obtained to represent a cell strain. Sensitivity to the test drug relative to another cell strain.

Both PC-9 or PC9-IR cells showed a significant decrease in cell viability after 72 hours of treatment with gefitinib, and their 50% inhibition of cell viability was 0.0371 μM and 10.6 μM, respectively. Similarly, Composition 1 also inhibited the cell viability of PC-9 or PC9-IR cells, and its concentration which inhibited 50% of cell activity was about 35 μg/mL (35.7 and 34.6 μg/mL, respectively). However, if the cell resistance ratios of composition 1 and gefitinib were compared between the two cell lines, respectively, it was apparent that gefitinib was more effective against the non-resistant PC-9 cell line. In contrast, the cell resistance ratio of the two lung cancer cell lines to the composition 1 was almost the same, indicating that there was no difference in the ability of the composition 1 to kill lung cancer cells with or without drug resistance regardless of whether the cancer cells were resistant or not.

1.5 Composition 1 of Example 1.1 inhibits blood cell cancer activity

The effect of composition 1 on blood cancer cell line HL-60 or K562 was tested in the manner described in Example 1.1. The concentration of composition 1 or imatinib to inhibit 50% activity of the cell line, and the cell resistance ratio (RR) of the two cell lines to the drug are summarized in Table 5 below.

Both HL-60 and K562 cells showed a significant decrease in cell viability after treatment with imatinib for 72 hours, and their 50% inhibition of cell viability was 35 μM and 0.4 μM, respectively. Similarly, Composition 1 also inhibited the cellular activity of HL-60 or K562 cells, and its concentration which inhibited 50% of cellular activity was 25.8 and 30.7 μg/mL, respectively. However, if the cell resistance ratio of the two cell lines to composition 1 and imatinib was compared, it was apparent that imatinib was more effective against the HL-60 cell line. In contrast, the cell resistance ratio of the two blood cancer cell lines to Composition 1 was almost the same, indicating that there was no difference in the ability of Composition 1 to poison both blood cancer cells.

Example 2 Effect of inhibiting cancer cell growth in vivo

2.1 The composition disclosed in Example 1.1 inhibits the growth of cancer cells in vivo

The test animals are induced to produce tumors (e.g., lung cancer and rectal cancer) in the manner disclosed in the "Materials and Methods" section, and then the pharmaceutical composition of the present invention (i.e., the composition disclosed in Example 1.1) is administered to test the present invention. The efficacy of a pharmaceutical composition for inhibiting tumor growth in vivo. The results are shown in Table 6 below.

As shown in Table 6, the two composition formulations disclosed in Example 1.1 (Compositions 1 and 3) were effective in inhibiting the growth of cancer in vivo, and about 80% of lung cancer growth was inhibited by Composition 1, about 50. % of rectal cancer growth can be inhibited by composition 3.

2.2 Triterpenoids inhibit the growth of lung cancer

This embodiment investigates the effect of the total active triterpenoid compound content in the composition on inhibiting tumor growth. This test was carried out using the formulation of Composition 3 of Example 1.1 to increase the total triterpenoid content of the composition without changing the relative proportions of the components (Ganoderma lucidum and/or erythritol) in the formulation. . The results are summarized in Table 7.

This result is consistent with the phenomenon observed in other embodiments of the present invention, that is, the therapeutically effective ingredient in the pharmaceutical composition of the present invention is a triterpenoid. Compared to placebo, it contains only a small amount of triterpenoids The compound of the present invention can produce at least about 50-65% of the growth of cancer by inhibiting the growth of the cancer only in vivo.

Further, since the amount of GMAS contained in the composition 3 was less than 2%, even if the content of the total triterpenoids in the composition was increased, an effect of inhibiting the growth of cancer in an equal ratio was not observed.

In summary, the results of the present invention show that the pharmaceutical compositions of the present disclosure having a combination of novel triterpenoid compounds are potential therapeutic agents for the treatment of cancer, particularly cancers resistant to cancer.

It will be understood that the above-described embodiments and examples are merely illustrative, and that those skilled in the art can align various modifications. The description, examples, and materials set forth above are intended to be illustrative of the present invention and are illustrative of the invention. The present disclosure has been disclosed in the above embodiments, but it is not intended to limit the disclosure, and any person skilled in the art can make various changes and refinements without departing from the spirit and scope of the disclosure. The scope of protection of the disclosure is subject to the definition of the scope of the patent application.

Claims (7)

A pharmaceutical composition for treating cancer comprising a pharmaceutically effective amount of a triterpenoid compound comprising at least ganoderic acid S (GAS), ganoderic acid T (GAT), and ganoderic acid (ganoderic) Acid Me, GAMe), ganoderic acid R (GAR) and ganodermic acid S (GMAS); and a pharmaceutically acceptable carrier thereof. The pharmaceutical composition according to claim 1, wherein the amount of GMAS is from about 1 to 10% by weight based on the total amount of the triterpenoid compound in the composition. The pharmaceutical composition according to claim 1, wherein the amount of GAMe and GAR is about 2-17% by weight based on the total amount of the triterpenoid compound in the composition. The pharmaceutical composition according to claim 1, wherein the amount of GAT is about 65-75% by weight based on the total amount of the triterpenoid compound in the composition. The pharmaceutical composition according to claim 1, wherein the amount of GAS is about 15-20% by weight based on the total amount of the triterpenoid compound in the composition. The pharmaceutical composition according to claim 1, wherein the cancer may be liver cancer, rectal cancer, lung cancer, breast cancer or blood cancer. The pharmaceutical composition according to claim 6, wherein the lung cancer is resistant to gefitinib.