Use of terpenoids in the treatment of mast cell tumors

Abstract

The present invention provides a method for treating mast cell tumors, comprising administering to a subject an effective amount of a composition, wherein the composition comprises a terpenoid extract derived from Antrodia camphorate or Anisomeles indica.

Description

[Technical Field]

[0001] The present invention relates to herbal terpenoids derived from extracts of Antrodia camphorata and Anisomeles indica, and more particularly to medicinal and edible formulations for the effective treatment of mast cell tumors. [Background technology]

[0002] Mast cell tumors are a type of round cell tumor composed of mast cells that occurs in many non-human animal species. If a mast cell tumor is too large or located to be completely removed surgically, additional treatment such as radiation therapy or chemotherapy may be necessary. Vinblastine and lomustine are common chemotherapeutic agents used to treat mast cell tumors in dogs. Toceranib and masitinib, examples of receptor tyrosine kinase inhibitors, are used to treat mast cell tumors in canines. Both were recently approved by the U.S. Food and Drug Administration as canine-specific anticancer drugs. However, chemotherapeutic techniques can have various side effects depending on the type of drug used. The use of medicinal plant-derived products to manage or inhibit the carcinogenic process offers a safer alternative to using conventional chemicals to treat the disease.

[0003] The medicinal fungus Antrodia camphorata (AC) is a well-known Chinese folk medicine and is known to possess many biological activities. To date, a total of 225 compounds have been isolated, identified, and their structures elucidated, including macromolecules (nucleic acids, proteins, and polysaccharides), small molecules (benzenoids, lignans, benzoquinones, and maleic acid / succinic acid derivatives), terpenoids (lanostan triterpenes, ergostan triterpenes, diterpenes, monoterpenes, and steroids), nucleotides (nucleic acid bases and nucleosides), fatty acids, and fatty acid esters.

[0004] Anisomeles indica, commonly known as "Indian Catmint," is a source of medicinal compounds and possesses a variety of pharmacological effects. Traditionally, this plant has been used as an analgesic, anti-inflammatory agent, and for treating skin conditions. Further research has revealed the presence of various phytochemicals, primarily triterpenes, β-sitosterol, stigmasterol, flavones, apigenin, and ovatodiolide.

[0005] Plant terpenoids have been used in traditional herbal medicine and are abundant in Antrodia camphorate and Anisomeles indica. However, the effectiveness of these terpenoids and their combinations in the treatment of mast cell tumors has not been evaluated. [Brief explanation of the drawing]

[0006] [Figure 1] Figure 1 shows the effects of terpenoids on the cell viability of RBL-2H3 cells and their inhibitory effect on β-hexosaminidase release.

[0007] [Figure 2] Figure 2 shows the effect of terpenoids on the cell viability of RBL-2H3 cells.

[0008] [Figure 3] Figure 3 shows the effects of terpenoids on cell viability and β-hexosaminidase release in RBL-2H3 cells.

[0009] [Figure 4] Figure 4 shows the effects of terpenoids on cell viability and β-hexosaminidase release in RBL-2H3 cells.

[0010] [Figure 5] Figure 5 shows the combined effect of terpenoids on P815 cell viability.

[0011] [Figure 6] Figure 6 shows animal body weight and tumor volume.

[0012] [Figure 7] Figure 7 shows the tumor volume of animals on day 30.

[0013] [Figure 8] Figure 8 shows the tumor weight of animals on day 30.

[0014] [Figure 9] Figure 9 shows the tumor volume of animals on day 30.

[0015] [Figure 10] Figure 10 shows photographs of tumor appearance on day 23.

[0016] [Figure 11] Figure 11 shows H&E histological staining of tumor tissue.

[0017] [Figure 12] Figure 12 shows H&E histological staining of tumor tissues (Mix-HD and AR-DS). Summary of the Invention

[0018] For the convenience of describing the present invention, the central idea expressed in the above Summary of the Invention is illustrated by specific examples. Various items in the embodiments are shown by proportional ratios, scales, variations or substitutions according to the examples, and the entire overview of the above actual elements is not drawn.

[0019] The term "terpenoid" or "terpene" refers to a wide variety of organic compounds, whose basic structure follows general principles: a 2-methylbutane residue, though less precisely, is usually an isoprene unit (C5). n Also known as terpenes, they form the carbon skeleton of terpenes. Approximately 30,000 terpenes are currently known in the literature. Depending on the number of 2-methylbutane (isoprene) subunits, they are classified as hemi-(C5) or mono-(C5). 10 ), sesqui-(C 15 ), G-(C 20 ), Sester-(C 25 ), Tori-(C 30 ) and tetraterpene (C 40 ) can be divided into these categories. [ka]

[0020] In this specification, the terms “subject,” “individual,” “host,” and “patient” are used interchangeably to mean living animals, including humans and non-human animals. A subject may be, for example, an organism having immune cells that can respond to antigenic stimulation and transmit stimuli and inhibitory signals via cell surface receptor binding. A subject may be a mammal, for example, a human or a non-human mammal, such as a dog, cat, pig, cow, sheep, goat, horse, rat, and mouse. The term “subject” does not exclude individuals that are completely normal with respect to disease, or normal in all respects.

[0021] The term “treatment” means therapeutic or preventive measures. Treatment may be administered to subjects with a medical impairment or those likely to eventually suffer impairment, in order to prevent, cure, delay, reduce or improve the severity of one or more symptoms of the impairment or recurrent impairment, or to extend the subject’s survival beyond the survival expected in the absence of such treatment.

[0022] The term "therapeutically effective amount" means an amount of the target compound capable of eliciting a desired response, for example, a biological or medical response of a tissue, system or animal sought by a researcher, veterinarian, physician or other clinician.

[0023] Cell culture

[0024] Mouse mastocytoma cells purchased from the Bioresource Collection and Research Center (Hsinchu City, Taiwan) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, California, USA), 100 U / ml penicillin and 100 μg / ml streptomycin.

[0025] Cell Counting Kit-8 assay for cell viability

[0026] 1×10 per well 5 P815 cells were pretreated with terpenoids for 18 hours. 5 μL of Cell Counting Kit-8 reagent was added to each well, incubated for 4 hours, and then the optical density was measured at 450 nm with a microplate reader (Tecan Sunrise). Using the average optical density (OD, absorbance) of four wells in the indicated group, cell viability was calculated by the following formula: [(OD 実験 -OD ブランク ) / (OD 対照 -OD ブランク )]×100%, wherein OD 実験 is the absorbance of cells treated with 0.1% DMSO or a terpenoid, OD ブランク is the absorbance of wells containing medium only, and OD 対照 is the absorbance of cells treated with 0.1% DMSO only.

[0027] β-hexosaminidase release assay

[0028] RBL-2H3 cells were seeded into a 24-well plate at 0.5×105 Cells were seeded in 0.5 mL wells and treated with the drug every other day for 24 hours. On day 3, 500 μL of anti-DNP-IgE (0.1 μg / mL) was added overnight. On day 4, 160 μL of DNP-BSA (0.1 μg / mL in Tyrode buffer) was added to each well and incubated at 37°C for 1 hour. For each of the 24 wells, 50 μL of cell supernatant and 50 μL of P-NAG were mixed (3 times), and the 96-well plate was incubated at 37°C for 1 hour. 100 μL of stop buffer was added, and absorbance was measured at 405 nm to obtain β-hexosaminidase data. In a 24-well plate, 300 μL of MTT reagent (0.5 mg / mL) was added to each well, and the mixture was reacted at 37°C for 2 hours. The supernatant was removed, and 300 μL of DMSO was added to each well. The absorbance at 570 nm was measured. The data were calculated using Dunnett's test for one-way ANOVA (*p<0.5, **p<0.01, ***p<0.001, ****p<0.0001) and plotted in GraphPad.

[0029] Animal experiments

[0030] All animal experiments were approved by the Laboratory Animal Management and Ethics Committee of National Chung Hsing University, Taiwan. P815 cells were cultured in 200 μL of extracellular matrix gel (1 × 10⁶ cells in 0.2 mL of PBS). 5 The cells were subcutaneously injected into the dorsal region of female BALB / c (6 weeks old) athymic nude mice (body weight, 20±2g). The mice were housed in a pathogen-free environment and randomly divided into two groups (n=5): a vehicle control group (10% DMSO + 90% glyceryl trioctanoate) and a drug group. Mice in both groups were administered daily by forced feeding (gavage feeding) starting on day 6 post-transplantation. Throughout the entire experiment, the mice's food intake and motor activity were carefully monitored, and tumor size was measured every 3 days using electronic calipers, with tumor volume (mm³) being measured. 3 ) is given by the formula: volume = (width 2The calculation was performed according to (×length) / 2. At the end of the experiment (day 20), all mice were euthanized, and the tumors were rapidly collected and weighed.

[0031] Histopathological analysis

[0032] On day 30, mice were sacrificed, the tumors were excised, fixed with 10% formalin and 5% formic acid, and embedded in paraffin. Briefly, tumor sections were stained with hematoxylin-eosin (H&E) and analyzed under a microscope. Tissue sections were degreased with xylene and then treated with ethanol (100%, 95%, 75%, 50%). Tissue was stained with hematoxylin for 1 minute and with eosin for 5 minutes.

[0033] statistical analysis

[0034] All data are presented as mean ± standard deviation. Using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA), one-way ANOVA was performed, followed by Tukey's post-hoc test to analyze statistical differences between groups. A two-tailed t-test was then performed to analyze differences between two independent groups. A p-value < 0.05 was considered statistically significant. [Examples]

[0035] Example 1. Preparation of Antrodia camphorata extract

[0036] 100 grams of Antrodia camphorata fruiting bodies were refluxed with methanol for 6 hours, and the extract was collected and dried to obtain a total of 15 grams of methanol extract of Antrodia camphorata.

[0037] Example 2. Preparation of active ingredients: anthocine K, dehydrosulfurenic acid / sulfurenic acid, pericisponate D, and dehydroebric acid.

[0038] The methanol extract of Antrodia camphorata is further separated by silica column chromatography using n-hexane / ethyl acetate / methanol as the eluent to obtain fractions: [Table 1-1] [Table 1-2]

[0039] Example 3. Preparation of active ingredients: AR100-DS1, AR100-DS4~DS13

[0040] 200 g of ethanol extract of Anisomeles indica was taken and added to a silica-packed chromatography column (10 × 15 cm). Gradient elution was performed using 1200 ml each of the following eluents: n-hexane / ethyl acetate (in ratios of 10 / 1, 5 / 1, 3 / 1, and 1 / 1), hexane / ethyl acetate / methanol (in ratios of 6 / 4 / 1, 3 / 2 / 1), and methanol, yielding 140 g of initial parting liquid.

[0041] The initial 140 g of the separation solution was separated using a silica-packed chromatography column (10 × 15 cm), and gradient elution was performed with 1000 ml each of the following eluents: "dichloromethane," "dichloromethane / methanol (in ratios of 10 / 1, 5 / 1, and 7 / 3)," and "methanol" to obtain a series of separated concentrates. [Table 2-1] [Table 2-2]

[0042] Example 4. Effects of terpenoids on the cell viability of RBL-2H3 cells and inhibitory effect on β-hexosaminidase release.

[0043] As shown in Figure 1, a significant decrease in β-hexosaminidase release was observed in the AR100-DS1 treatment group under safe doses. The results of the triple measurement are shown as the percentage of viable cells. Statistical analysis was performed using one-way ANOVA (Dunnett's multiple comparison test). Results are shown as mean ± SD from at least three independent experiments. Statistical significance is shown (compared to DMSO, ns: no significance, **p<0.01, ***p<0.001, ***p<0.0001).

[0044] Example 5. Effect of terpenoids on the cell viability of RBL-2H3 cells

[0045] As shown in Figure 2, cell viability was measured using the MTT assay. The results of the triple measurement are shown as the percentage of viable cells. Statistical analysis was performed using one-way ANOVA (Dunnett's multiple comparison test). Results are shown as mean ± SD from at least three independent experiments. Statistical significance is shown (compared to DMSO, ns: not significant, **p<0.01, ***p<0.001, ***p<0.0001).

[0046] Example 6. Effects of terpenoids on cell viability and β-hexosaminidase release in RBL-2H3 cells.

[0047] As shown in Figure 3, cell viability was measured using the MTT assay. The results of the triple measurement are shown as the percentage of viable cells. Statistical analysis was performed using one-way ANOVA (Dunnett's multiple comparison test). Results are shown as mean ± SD from at least three independent experiments. Statistical significance is shown (compared to DMSO: ns: no significance, **p<0.01, ***p<0.001, ***p<0.0001).

[0048] Example 7. Effect of terpenoids on β-hexosaminidase release in RBL-2H3 cells.

[0049] As shown in Figure 4, the results of the triple measurement are shown as the percentage of maximum antigen-induced degranulation. Statistical analysis was performed using one-way ANOVA (Dunnett's multiple comparison test). Results are shown as mean ± SD from at least three independent experiments. Statistical significance is shown (compared to DMSO, ns: no significance, **p<0.01, ***p<0.001, ***p<0.0001).

[0050] Example 8. Effect of terpenoids on the cell viability of P815 cells

[0051] Mouse mast cell tumor cells purchased from the Bioresource Collection and Research Center (Hsinchu City, Taiwan) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, California, USA), 100 U / ml penicillin, and 100 μg / ml streptomycin. 1 × 10⁶ cells were cultured per well. 5 P815 cells were pretreated with terpenoids for 18 hours. 5 μL of cell counting kit-8 reagent was added to each well and incubated for 4 hours, after which the optical density was measured at 450 nm using a microplate reader (Tecan Sunrise). Cell viability was calculated using the following formula, based on the average optical density (OD, absorbance) of the four wells of the designated group: [(OD 実験 -OD ブランク ) / (OD 対照 -OD ブランク )] × 100%, here, OD 実験 This is the absorbance of 0.1% DMSO-treated cells or terpenoid-treated cells, OD ブランク This is the absorbance of the well containing only the culture medium. OD 対照 This represents the absorbance of cells treated with 0.1% DMSO alone.

[0052] As shown in Figure 5, the test results indicated that these terpenoids, or combinations of terpenoids, showed significant inhibitory activity against mast cell P815, particularly in the combination groups.

[0053] [Table 3]

[0054] [Table 4]

[0055] Example 10. Effect of terpenoid administration on P815 cell development in nude mice.

[0056] Male BALB / c mice are divided into the following groups: 1. Vehicle (10% DMSO + 90% olive oil) 2. AR100-DS1 (2 mg / kg) 3. AR100-DS1 (7.5 mg / kg) 4. AR100-DS1(2mg / kg)+AR101-DS4(0.1mg / kg) 5. AR100-DS1(7.5mg / kg)+AR101-DS4(1mg / kg) 6. AR101-DS4 (0.06 mg / kg) 7. AR101-DS4 (0.24 mg / kg) 8. AR101-DS2 (1 mg / kg) 9. AR101-DS4 (4 mg / kg)

[0057] Mice were administered 100 μL of the sample intravenously every two days, and the mice were sacrificed on day 30 for histopathological examination.

[0058] As shown in Figure 6A, compared to the volume changes of P815 mast cell tumors in the vehicle group, a significant reduction in tumor growth was observed in the following groups: AR100-DS1 + AR101-DS4 low-dose group, AR100-DS1 + AR101-DS4 high-dose group, AR101-DS2 1 mg / kg group, and AR101-DS2 4 mg / kg group.

[0059] As shown in Figure 6B, this is the average body weight curve over time. No significant changes in animal body weight were observed in any of the groups in this study.

[0060] As shown in Figure 7, compared to the volume change of P815 mast cell tumors in the vehicle group, a significant reduction in tumor growth was observed in the following groups: the low-dose Mix group, the high-dose Mix group, the AR101-DS2 1 mg / kg group, and the AR101-DS2 4 mg / kg group.

[0061] As shown in Figure 8, our test results indicate that Mix HD significantly inhibited the growth of mast cell tumors. Statistical analysis revealed a very low standard deviation for Mix HD, suggesting that terpenoids have strong potential for treating mast cell carcinoma.

[0062] As shown in Figure 9, our test results showed that Mix LD, Mix HD, and AR101-DS2 1 mg / kg significantly inhibited the growth of mast cell tumors. Statistical analysis revealed very low standard deviations for Mix HD and AR101-DS2, suggesting that terpenoids have strong potential for treating mast cell carcinoma.

[0063] As shown in Figure 10, the P815 mast cell tumor was located in the right posterior region near the tail. Compared to tumor volume changes in the vehicle group, a significant reduction in tumor growth was observed in the following groups: Mix LD group, Mix HD group, AR101-DS2 1 mg / kg group, and AR101-DS2 4 mg / kg group.

[0064] As shown in Figure 11, the MIX-HD group, the AR101-DS4 0.06 mg group, and the AR101-DS2 1 mg group all demonstrated the ability to inhibit tumor cell proliferation and induce tumor cell nuclear aggregation. In these circumstances, these processes may have been induced by cellular apoptosis.

[0065] As shown in Figure 12, in the Mix-HD group and the AR101DS2 1 mg / kg group, tumor cell proliferation was inhibited, tumor cell nucleus aggregation occurred, and the rigidity of intercellular connections was lost. Furthermore, the present invention includes the following embodiments. [Aspect 1] A method for treating mast cell tumors, comprising administering an effective amount of a composition to a subject in need thereof, wherein the subject is a non-human animal, and the composition comprises at least one compound and combination thereof selected from the group consisting of the following formulas: [ka] [ka] [Aspect 2] The composition is (a) [ka] and (b) [ka] Or a combination of these, The method according to embodiment 1, including the method described in embodiment 1. [Aspect 3] The composition is [ka] The method according to embodiment 2, including the method described in embodiment 2. [Aspect 4] (a) [ka] and (b)

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Claims

[Claim 1] A pharmaceutical composition for treating mast cell tumors in non-human animals, (a) 【Chemistry 1】 and (b) 【Chemistry 2】 and compounds selected from these combinations A pharmaceutical composition comprising the combination of the above, together with a pharmaceutically acceptable carrier. 【Request Item 2】 【Chemistry 3】 The pharmaceutical composition according to claim 1, comprising a combination of the above.

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