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 Antrodia camphorata and Anisomeles indica extracts, particularly to medicinal and dietary formulations for the effective treatment of mast cell tumors. [Background technology]

[0002] Mastocytoma, or mast cell tumor, is a type of round cell tumor composed of mast cells that occurs in many non-human animal species. If a mast cell tumor cannot be completely removed by surgery because of its size or its location, it may require additional treatment such as radiation therapy or chemotherapy. 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 have various side effects depending on the type of drug used. The use of medicinal plant-derived products to manage or block the carcinogenic process offers a safer option than using traditional chemical drugs to treat the disease.

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

[0004] Anisomeles indica, commonly known as "Indian Catmint", is a source of medicamentously active compounds and has various pharmacological effects. The plant has been traditionally used as an analgesic, anti-inflammatory agent, and for skin disorders. Further studies have revealed the presence of various phytochemical constituents, mainly triterpenes, β-sitosterol, stigmasterol, flavones, apigenin, and obatdiolide.

[0005] Plant terpenoids are useful in traditional herbal medicine and are abundant in Antrodia camphorate and Anisomeles indica. However, the efficacy of these terpenoids and their combinations in the treatment of mast cell tumors has not been evaluated. [Brief description of the drawings]

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

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

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

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

[0010] [Diagram 5] FIG. 5 shows the effect of combining terpenoids on P815 cell viability.

[0011] [Figure 6] FIG. 6 shows the animal weights and tumor volumes.

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

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

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

[0015] [Figure 10] FIG. 10 shows a photograph of the tumor appearance on day 23.

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

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

[0018] For the convenience of explaining the present invention, the central ideas expressed in the above summary of the invention are illustrated by specific examples, and various items in the embodiments are shown in proportion, scale, deformation, or substitution for illustrative purposes, and do not depict the entirety of the actual elements described above.

[0019] The term "terpenoid" or "terpene" refers to a large and diverse class of organic compounds whose basic structure follows a general principle: a 2-methylbutane residue, usually, but less precisely, an isoprene unit (C5) n These carbon atoms 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), mono- (C 10 ), Sesqui-(C 15 ), Ji-(C 20 ), Sester-(C 25 ), Tri-(C 30 ) and tetraterpenes (C 40 ) [ka]

[0020] As used herein, the terms "subject", "individual", "host" and "patient" are used interchangeably to mean a living animal, including humans and non-human animals. A subject may be, for example, an organism having immune cells capable of responding to antigenic stimulation and transmitting stimulatory and inhibitory signals via cell surface receptor binding. A subject may be a mammal. For example, a human or non-human mammal, such as a dog, cat, pig, cow, sheep, goat, horse, rat and mouse. The term "subject" does not exclude individuals who are entirely normal with respect to disease, or who are normal in all respects.

[0021] The term "treatment" refers to a therapeutic or prophylactic measure. Treatment may be administered to a subject having a medical disorder or a subject who may ultimately acquire a disorder in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or a recurrent disorder, or to prolong the survival of the subject beyond that which would be expected in the absence of such treatment.

[0022] The term "therapeutically effective amount" refers to an amount of a subject compound that is capable of eliciting a desired response, e.g., a biological or medical response in a tissue, system or animal, as desired by a researcher, veterinarian, physician, or other clinician.

[0023] cell culture

[0024] Mouse mastocytoma cells purchased from Bioresource Collection and Research Center (Hsinchu City, Taiwan) were cultured in Dulbecco's modified Eagle's 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 x 10 per well 5 P815 cells were pretreated with terpenoids for 18 h. 5 μL of Cell Counting Kit-8 reagent was added to each well and incubated for 4 h, then the optical density was measured at 450 nm by a microplate reader (Tecan Sunrise). The average optical density (OD, absorbance) of 4 wells of the indicated group was used to calculate the cell viability with the following formula: [(OD 実験 -OD ブランク ) / (OD 対照 -OD ブランク )] × 100%, where OD 実験 is the absorbance of cells treated with 0.1% DMSO or terpenoids, and OD ブランク is the absorbance of the wells containing medium only. OD 対照 is the absorbance of cells treated with 0.1% DMSO only.

[0027] β-Hexosaminidase release assay

[0028] RBL-2H3 cells were cultured in 24-well plates (0.5 × 105 Cells were seeded in 1000 μL of 0.1 μg / mL per well and drug treatments were added every other day for 24 hours. On the third day, 500 μL of anti-DNP-IgE (0.1 μg / mL) was added overnight. On the fourth day, 160 μL of DNP-BSA (0.1 μg / mL in Tyrode's buffer) was added to each well and incubated at 37°C for 1 hour. For each well of the 24-well plate, 50 μL of cell supernatant and 50 μL of P-NAG were mixed (in triplicate), and the 96-well plate was incubated at 37°C for 1 hour. 100 μL of stop buffer was added, and the absorbance was measured at 405 nm to obtain the data of β-hexosaminidase. In a 24-well plate, 300 μL of MTT reagent (0.5 mg / mL) was added to each well, and the reaction was allowed to proceed at 37°C for 2 hours. The supernatant was removed, and 300 μL of DMSO was added to each well, and the absorbance at 570 nm was measured. The data was calculated by one-way ANOVA with Dunnett's test (*p<0.5, **p<0.01, ***p<0.001, ****p<0.0001) and plotted using Graph pad.

[0029] Animal testing

[0030] All animal experiments were approved by the Laboratory Animal Management and Ethics Committee of National Chung Hsing University, Taiwan. P815 cells (1 × 10 in 0.2 mL of PBS) were cultured in 200 μL of extracellular matrix gel. 5 Cells) were subcutaneously injected into the dorsal region of female BALB / c (6 weeks) athymic nude mice (body weight, 20±2 g). Mice were housed in a pathogen-free environment and randomly divided into two groups (n=5): vehicle control group (10% DMSO+90% glyceryl trioctanoate) and drug group. Mice in both groups were administered daily by gavage feeding starting on day 6 after implantation. During the entire experimental period, the mice were closely observed for food intake and motor activity, and tumor size was measured every 3 days with electronic calipers, and tumor volume (mm 3 ) into the formula: Volume = (Width 2× length) / 2. At the end of the study (day 20), all mice were euthanized and tumors were rapidly harvested and weighed.

[0031] Histopathological analysis

[0032] Mice were sacrificed on day 30, and tumors were excised, fixed in 10% formalin, 5% formic acid, and embedded in paraffin. Briefly, tumor sections were stained with hematoxylin and eosin (H&E) and analyzed by microscopy. Tissue sections were degreased with xylene and then treated with ethanol (100%, 95%, 75%, 50%). Tissues were stained with hematoxylin for 1 min and eosin for 5 min.

[0033] statistical analysis

[0034] All data are presented as mean ± SD. Statistical differences between groups were analyzed by one-way ANOVA followed by Tukey's post-hoc test using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA), and differences between two independent groups were analyzed by unpaired two-tailed t-test. 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 body are refluxed in methanol for 6 hours, and the extract is collected and dried to obtain a total of 15 grams of Antrodia camphorata methanol extract.

[0037] Example 2. Preparation of the active ingredients: anthocin K, dehydrosulfurenic acid / sulfurenic acid, versisponic acid 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 eluent to obtain the following fractions: [Table 1-1] [Table 1-2]

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

[0040] 200 g of the ethanol extract of Anisomeles indica was added to a silica-packed chromatography column (10 x 15 cm) and subjected to gradient elution with 1200 ml of each eluent: n-hexane / ethyl acetate (ratios of 10 / 1, 5 / 1, 3 / 1, 1 / 1), hexane / ethyl acetate / methanol (ratios of 6 / 4 / 1, 3 / 2 / 1), and methanol, to obtain 140 g of initial parting liquid.

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

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

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

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

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

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

[0047] Cell viability was measured using the MTT assay, as shown in Figure 3. Results of triplicate measurements are presented as percentage of viable cells. Statistical analysis was performed using one-way ANOVA (Dunnett's multiple comparison test). Results are presented as mean ± SD from at least three independent experiments. Statistical significance is indicated (ns: not significant, **p<0.01, ***p<0.001, ***p<0.0001 compared to DMSO).

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

[0049] The results of triplicate measurements were expressed as a percentage of maximum antigen-induced degranulation, as shown in Figure 4. Statistical analysis was performed using one-way ANOVA (Dunnett's multiple comparison test). Results are presented as mean values ​​± SD from at least three independent experiments. Statistical significance is indicated (ns: not significant, **p<0.01, ***p<0.001, ***p<0.0001 compared to DMSO).

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

[0051] Mouse mastocytoma cells purchased from Bioresource Collection and Research Center (Hsinchu City, Taiwan) were cultured in Dulbecco's modified Eagle's 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 at 1 × 10 cells per well. 5 P815 cells were pretreated with terpenoids for 18 h. 5 μL of Cell Counting Kit-8 reagent was added to each well and incubated for 4 h, then the optical density was measured at 450 nm by a microplate reader (Tecan Sunrise). The average optical density (OD, absorbance) of 4 wells of the indicated group was used to calculate the cell viability with the following formula: [(OD 実験 -OD ブランク ) / (OD 対照 -OD ブランク )] × 100%, where OD 実験 is the absorbance of 0.1% DMSO-treated or terpenoid-treated cells, and OD ブランク is the absorbance of the wells containing medium only. OD 対照 is the absorbance of cells treated with 0.1% DMSO only.

[0052] As shown in FIG. 5, the test results showed that the effects of these terpenoids or the combination group composed of terpenoids, especially the combination group, showed significant inhibitory ability of mast cell P815.

[0053] [Table 3]

[0054] [Table 4]

[0055] Example 10. Effect of terpenoid administration on the growth of P815 cells in nude mice

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

[0057] Mice were administered 100 μL of sample intravenously every 2 days and sacrificed on day 30 for histopathological examination.

[0058] As shown in Figure 6A, compared with the volume changes of P815 mast cell tumors in the vehicle group, a significant decrease 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, the mean body weight curve over time shows no significant change in the body weight of the animals in all groups in this study.

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

[0061] Our test results showed that Mix HD significantly inhibited the growth of mast cell tumors, as shown in Figure 8. Statistical analysis showed that the standard deviation of Mix HD was very low, which means that terpenoids have strong potential in treating mast cell cancer.

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

[0063] As shown in Figure 10, P815 mastocytoma was located in the right hindquarters close to the tail. Compared with the tumor volume change in the vehicle group, a significant decrease 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, MIX-HD group, AR101-DS4 0.06mg group and AR101-DS2 1mg group showed the ability to inhibit tumor cell proliferation and induce nuclear aggregation of tumor cells, in this situation, these processes may be induced by cell apoptosis.

[0065] As shown in FIG. 12, in the Mix-HD group and the AR101DS2 1 mg / kg group, tumor cell proliferation was inhibited, tumor cell nuclei were aggregated, and the firmness of the junctions between cells was lost.

Claims

1. A pharmaceutical composition for treating mast cell tumors in a non-human animal, comprising a compound selected from the group consisting of the following compounds and combinations thereof, together with a pharmaceutically acceptable carrier: 【Chemical 1】 【Chemistry 2】

2. (a) 【Chemistry 3】 and (b) 【Chemistry 4】 and a compound selected from combinations thereof.

2. The pharmaceutical composition of claim 1, comprising a combination of: 【Request 3】 【Chemical 5】 3. The pharmaceutical composition of claim 2, comprising a combination of:

4. The compound, 【Chemistry 6】 2. The pharmaceutical composition of claim 1, wherein

Images