Use of alpha-mangostin in preparation of anti-tumor immunological drugs

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the novel use of α-trachytin in the preparation of antitumor immunotherapeutic drugs. Pharmacological experiments show that α-trachytin significantly inhibits tumor growth in osteosarcoma-bearing mice, and its activity is dose-dependent. α-trachytin upregulates tumor immune markers CD8, CD56, F4 / 80, and PD-L1. Furthermore, sequential co-administration of α-trachytin with anti-PD-1 antibodies enhances the sensitivity of immunotherapies to anti-PD-1 antibodies, demonstrating its promising application prospects in the treatment of osteosarcoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the novel use of α-dextrin in the preparation of antitumor immunotherapeutic drugs. Background Technology

[0002] Osteosarcoma (OS) is a malignant bone tumor that commonly affects children and adolescents. Conventional treatment involves radical resection and chemotherapy, but some patients experience recurrence or lung metastasis, leading to a significant decrease in postoperative survival. Significant breakthroughs have been achieved in tumor immunotherapy, with immune checkpoint inhibitors, especially PD-1 / PD-L1 antibodies, showing good efficacy in treating various solid tumors. However, research on soft tissue tumors such as osteosarcoma is still in its early stages. Therefore, developing novel drugs for the treatment of osteosarcoma is of paramount importance.

[0003] Mangosteen (Garcinia mangostana L.) is an evergreen tree belonging to the genus Garcinia in the family Clusiaceae. Commonly known as mangosteen, it is often called the "Queen of Fruits." The mangosteen peel contains flavonoids, xanthones, anthocyanins, proanthocyanidins, and other active ingredients, and is commonly used as a traditional medicine in some Southeast Asian regions to treat diarrhea, dysentery, and infections. α-mangostin, a xanthone compound extracted from the mangosteen peel, is abundant and possesses various biological activities. Studies have shown that α-mangostin has potential anti-tumor effects, such as inhibiting cell proliferation, inducing tumor cell cycle arrest, inducing apoptosis, and autophagy; α-mangostin has shown inhibitory activity against various cancer types, including gastric cancer, breast cancer, liver cancer, and glioblastoma. However, research on its effects on osteosarcoma is limited.

[0004] α-Typocytosine, with the structural formula shown in Formula I below.

[0005]

[0006] Alpha-dextrin has a structural core similar to that of tacrine and possesses a unique tricyclic aromatic system (C6-C3-C6). A search revealed no reports of alpha-dextrin being used to treat osteosarcoma by activating tumor immunity. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention provides a new use of α-dextrin in the preparation of immunotherapeutic drugs for antitumor (especially osteosarcoma).

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0009] This invention provides the use of α-dextrin in the preparation of antitumor immunotherapeutic drugs.

[0010] Alternatively, in the above-described uses, the drug is used to treat osteosarcoma.

[0011] Alternatively, in the above-mentioned uses, α-dextrin can be used as the sole active ingredient in the preparation of antitumor immunotherapeutic drugs.

[0012] Preferably, the α-dextrin induces upregulation of tumor immune markers CD8, CD56, F4 / 80 and PD-L1, and the α-dextrin activates tumor immune activity.

[0013] Alternatively, in the above-described uses, the drug comprises the active ingredient α-dextrin and an immune checkpoint inhibitor.

[0014] Preferably, the active ingredient contained in the drug consists of α-dextrin and an immune checkpoint inhibitor.

[0015] More preferably, the mass ratio of α-dextrin to the immune checkpoint inhibitor is 5:1 to 15:1.

[0016] More preferably, the mass ratio of the α-dextrin to the immune checkpoint inhibitor is 10:1.

[0017] Preferably, α-dextrin enhances the sensitivity of immune checkpoint inhibitors.

[0018] In addition, preferably, the combination of α-dextrin and immune checkpoint inhibitors has a significant synergistic effect.

[0019] Alternatively, in the above-described uses, the active ingredient of the drug may be in the same pharmaceutical preparation or in different pharmaceutical preparations, and the active ingredient may be applied simultaneously or sequentially.

[0020] Alternatively, in the above-described uses, the α-retropine may be administered before or after the administration of the immune checkpoint inhibitor.

[0021] Preferably, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[0022] More preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody, such as nivolumab or pembrolizumab, or an anti-PD-L1 antibody, such as atezolizumab or duvastatin.

[0023] Alternatively, in the above-described uses, the drug comprises an active ingredient and one or more pharmaceutically acceptable excipients.

[0024] Alternatively, in the above-described uses, the drug may be an oral or non-oral formulation.

[0025] Preferably, the oral preparation is selected from one or more of tablets, capsules, pellets, granules, powders, oral films, or oral liquids.

[0026] Preferably, the non-oral preparation may be selected from one or more of injections, ointments, creams, or suppositories.

[0027] The pharmaceutically acceptable excipients described in this invention include conventional pharmaceutical solvents (such as water, ethanol, propylene glycol, and injectable oils), diluents (such as starch, powdered sugar, dextrin, lactose, pregelatinized starch, microcrystalline fibers, inorganic calcium salts (such as calcium sulfate, calcium hydrogen phosphate, and pharmaceutical-grade calcium carbonate), mannitol, vegetable oils, and polyethylene glycol), binders (such as water, ethanol, starch paste, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, and hydroxypropyl methylcellulose), and disintegrants (such as dry starch, sodium carboxymethyl starch, and low-substituted hydroxypropyl cellulose). The ingredients include: cellulose, croscarmellose, croscarmellose sodium carboxymethyl cellulose, etc.; lubricants (such as magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol, magnesium lauryl sulfate, etc.); absorption enhancers (such as surfactants, Azone, EDTA, salicylic acid, amino acid ethylamine derivatives, acetoacetates, β-dicarboxylic acid esters, aromatic acidic compounds, aliphatic acids, etc.); preservatives (such as benzoic acid, hydroxypropyl butyl ester, hydroxypropyl methyl ester, phenol, m-cresol, etc.); and flavoring agents (such as sucrose, stevioside, etc.).

[0028] When used to treat osteosarcoma, the drug of the present invention is administered to humans or other mammals. For this purpose, the mass of the drug of the present invention ingested or administered, calculated as α-dextrin, based on an adult body weight of 60-70 kg, is typically 120-700 mg per person per day, more preferably 300-400 mg per person per day.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] Pharmacological studies showed that α-dextrin significantly inhibited tumor growth in osteosarcoma-bearing mice in a dose-dependent manner. α-dextrin also upregulated tumor immunomodulatory markers CD8, CD56, F4 / 80, and PD-L1. Furthermore, sequential co-administration of α-dextrin with anti-PD-1 antibodies enhanced the sensitivity of the immunomodulatory drugs to the anti-PD-1 antibodies, demonstrating its promising potential for anti-osteosarcoma applications. Attached Figure Description

[0031] The present invention will be further described below with reference to the accompanying drawings.

[0032] Figure 1 The study illustrates the inhibition of tumor growth in mice bearing K7M2 osteosarcoma by α-retropine. Figure a shows the tumor volume growth curve; figure b shows a gross photograph of the dissected tumor tissue (scale bar = 5 cm); figure c shows the tumor weight; and figure d shows the immunohistochemical staining of the tumor tissue (scale bar = 100 μm). Each group consisted of 10 animals (n = 10), with three replicates per group, and the average values ​​were taken. Data are expressed as mean ± standard mean error (Means ± SD). **: The treatment group was compared to the control group; P < 0.01.

[0033] Figure 2 The study illustrates the inhibition of tumor volume growth in mice bearing K7M2 osteosarcoma by α-dextrin combined with anti-PD1. Figure a shows the tumor volume growth curve; figure b shows a gross photograph of the tumor tissue after dissection (scale bar = 5 cm); and figure c shows the tumor weight. Each group consisted of n = 10 animals, with three replicates per group, and the average values ​​were taken. Data are expressed as mean ± standard mean error (Means ± SD). *: Treatment group vs. control group, P < 0.05; **: Treatment group vs. control group, P < 0.01.

[0034] Figure 3 The side effects analysis of α-dextrin combined with anti-PD1 administration is shown. Figure a is a line graph of mouse body weight during the administration period; Figure b is the statistical analysis of serum biochemical indicators; Figure c is the statistical analysis of relative weight of organs (heart, liver, spleen, lung, and kidney); Figure d is the HE staining of organs (scale bar = 50 μm). The number of experimental animals in each group was n = 10. Detailed Implementation

[0035] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products. The purchase details of some raw materials and reagents are as follows:

[0037] α-Diocylin was purchased from Shaanxi Baoji Chenguang Biotechnology Co., Ltd., and its structure was determined by UV, MS, NMR and other methods. Its purity was above 98%.

[0038] K7M2 mouse osteosarcoma cells were purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0039] PRMI 1640 medium was purchased from Thermo Fisher Scientific, USA. 0.25% trypsin digestion solution (containing EDTA) and phosphate-buffered saline (PBS) were purchased from Beijing Zhongke Maichen Technology Co., Ltd. Sodium chloride injection was purchased from Regen Biotech Co., Ltd. All other reagents were of analytical grade and purchased from a Beijing chemical plant.

[0040] Example 1 α-Dextrin inhibits tumor growth in osteosarcoma-bearing mice.

[0041] 1.1 Test animals: BALb / c mice, 6-8 weeks old, male, Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0042] 1.2 Test Drug:

[0043] Low-dose α-dextrin (α-MG) solution (50 mg / kg): Weigh 10 mg α-MG, add 2 mL of physiological saline, mix well by sonication before use, and administer 0.2 mL / 20 g per mouse by gavage.

[0044] High-dose α-MG solution (100mg / kg): Weigh 20mg α-MG, add 2mL of physiological saline, mix well by sonication before use, and administer 0.2mL / 20g to each mouse by gavage.

[0045] 1.3K7M2 mouse osteosarcoma transplantation model:

[0046] (1) K7M2 cells in the logarithmic growth phase were digested with trypsin, centrifuged, and the supernatant of the culture medium was removed. An appropriate amount of PBS solution was added, and the cells were counted using a cell counting chamber. The cell density was adjusted to 2 × 10⁻⁶ cells / year. 7 Cells / mL. Place the cell suspension on ice and use a 1mL syringe to draw 200μL of the suspension and subcutaneously inoculate it into the right back of mice (cell count: 4 × 10⁶ / mL). 6 (each / each).

[0047] (2) Observe the tumor growth. On the 7th day after tumor bearing, the tumor in the mouse reached 100 mm. 3 The above indicates successful tumor bearing. Mice that successfully bore tumors were randomly divided into three groups: a control group, a low-dose group (50 mg / kg), and a high-dose group (100 mg / kg).

[0048] (3) Prepare the appropriate dosage of α-dextrin with sodium chloride solution and dissolve it using ultrasound. Administer by gavage once daily for 2 weeks.

[0049] (4) Record the mouse weight daily during the administration period; measure the tumor's long diameter (a) and short diameter (b) with calipers every 2 days, and calculate the tumor volume (mm). 3 ) = 1 / 2 × a × b 2A tumor growth curve was plotted. The tumor volume is approximately 2000 mm². 3 He was executed.

[0050] (5) Twenty-four hours after the last administration, mice were euthanized by cervical dislocation, the tumor was removed, photographed, and weighed. A portion of the tumor tissue was fixed in 4% paraformaldehyde and subjected to immunohistochemical staining. The specific steps are as follows:

[0051] 1) Dehydrate, embed in paraffin, and section each fixed tumor group, with each section being 4-5 μm thick.

[0052] 2) Dewax the paraffin sections with xylene, dehydrate them with graded ethanol, and wash them three times with PBS.

[0053] 3) Place in citrate buffer for antigen retrieval and incubate at 95°C for 10 min.

[0054] 4) Discard the citrate buffer and add 3% H2O2. Incubate for 30 min.

[0055] 5) Discard H2O2, wash 3 times with PBS, and block with 10% goat serum at room temperature for 30 min.

[0056] 6) Discard the blocking solution, wash 3 times with PBS, add the corresponding 1:100 antibody dilution solution, and incubate overnight at 4°C.

[0057] 7) Wash the sections three times with PBS, add goat anti-rabbit IgG dilution solution and incubate at room temperature for 1 hour.

[0058] 8) Wash the slices three times with PBS, add diaminobenzidine (DAB) chromogenic solution to the slices, and incubate at room temperature for 10 min.

[0059] 9) Wash three times with PBS, then dehydrate with graded ethanol, clear with xylene, mount with neutral resin, observe protein expression under a microscope, and take pictures with a digital pathology slide scanner.

[0060] 1.4 Test Results

[0061] To verify the tumor-suppressive effect of α-dextrin on osteosarcoma-bearing mice in vivo, a K7M2 osteosarcoma xenograft mouse model was established. Figure 1 a and Figure 1As shown in b, the rate of tumor growth gradually slowed down with increasing administration days after gavage administration of different doses of α-dextrin (50 and 100 mg / kg), exhibiting a certain dose-dependent effect. In particular, the high dose of α-dextrin (100 mg / kg) showed a significant inhibitory effect on tumor growth after the sixth day of gavage, and thereafter maintained a slow growth rate. After the last administration, compared with the control group of K7M2 xenograft mice, the tumor volume of mice in each dose group was significantly reduced (P<0.01).

[0062] Subsequently, by weighing the tumors in each group, it was found that α-dextrin could dose-dependently regulate the tumor weight in tumor-bearing mice. Figure 1 c, P<0.01). These results suggest that α-retropine can inhibit the growth of K7M2 osteosarcoma in vivo.

[0063] Immunohistochemical results showed that the T-cell marker CD8, NK-cell marker CD56, and macrophage marker F4 / 80 were almost not expressed in K7M2 osteosarcoma tissue, suggesting poor immune cell infiltration in this tumor. This situation was improved after administration of α-dextrin. With increasing dosage, the number of brownish-yellow CD8-positive granules in the cytoplasm increased significantly. The positive expression areas of CD56 and F4 / 80 were also significantly upregulated. More importantly, the expression of PD-L1 on the surface of tumor cells was significantly upregulated after administration of α-dextrin. These results suggest that α-dextrin can increase the number of tumor-infiltrating lymphocytes and activate anti-tumor immune activity.

[0064] The results of this embodiment are explained as follows:

[0065] 1) α-Dextrin can effectively inhibit the growth of osteosarcoma;

[0066] 2) α-Dextrin can significantly upregulate immune cell-related indicators in osteosarcoma.

[0067] Example 2 α-Dextrin combined with anti-PD1 inhibits tumor growth in osteosarcoma-bearing mice.

[0068] 2.1 Test animals: BALb / c mice, 6-8 weeks old, male, Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0069] 2.2 Test Drug:

[0070] α-Daucusin (α-MG) solution (50mg / kg): Weigh 10mg α-MG, add 2mL of physiological saline, mix well by sonication before use, and administer 0.2mL / 20g to each mouse by gavage.

[0071] Anti-PD1 (mouse IgG2a, catalog number BE0146, BioXCell BE0146: InVivoMAb anti-mousePD-1(CD279)) and isotype control IgG2a (catalog number BE0189) were purchased from BioXCell, USA, at a concentration of 10 mg / mL. Therefore, 10 μL per mouse was administered using a microsyringe.

[0072] 2.3K7M2 mouse osteosarcoma transplantation model:

[0073] The treatment model was essentially the same as the 1.3K7M2 mouse osteosarcoma transplantation model. Successfully tumor-bearing BALb / c mice were randomly divided into four groups: saline + IgG2a, saline + anti-PD1, α-dextrin + IgG2a, and α-dextrin + anti-PD1. α-dextrin 50 mg / kg was administered by gavage once daily for two consecutive days; subsequently, each mouse was intraperitoneally injected with 100 μg of anti-PD1 or IgG2a once daily for two consecutive days. Each treatment cycle lasted four days, for a total of four cycles and 16 days.

[0074] 2.4 Test Results

[0075] Given that α-dextrin can upregulate PD-L1 expression in tumors, α-dextrin was further combined with anti-PD-1 in an alternating manner. First, α-dextrin was administered orally for 2 days to activate the anti-tumor immune microenvironment, followed by intraperitoneal injection of anti-PD-1 solution for 2 days, completing one round of treatment. A total of 4 rounds of treatment were administered, lasting 16 days. The tumor volume growth curve of K7M2 was recorded every 2 days. Figure 2 a) The study found that compared to the control group, the α-dextrin combined with anti-PD1 group showed a significant slowdown in tumor volume growth rate, and the tumor size on the last day was significantly different from the control group (P<0.01), suggesting that α-dextrin can enhance the sensitivity of this type of immunotherapy. Furthermore, intraperitoneal injection of anti-PD1 alone or oral administration of α-dextrin alone also reduced the tumor volume growth rate, and the tumor size on the last day was statistically significant compared to the control group (P<0.05). In summary, tumor growth curves and gross tumor tissue photographs (…) Figure 2 b) and tumor weight statistics ( Figure 2 c) Both suggest that the effects of using immunotherapy drugs or α-dextrin alone are similar, but the combined use of the two has a more significant effect on inhibiting osteosarcoma growth.

[0076] Finally, a line graph showing the body weight of K7M2 tumor-bearing mice during the drug administration period was plotted. Figure 3a) Studies have confirmed that, compared with the control group, there were no statistically significant changes in body weight in mice across the treatment groups, including the anti-PD1 monotherapy group, the α-dextrin monotherapy group, and the α-dextrin combined with anti-PD1 group (P>0.05). Furthermore, serum biochemical parameters (ALT, AST, Urea, CK, and CK-MB) were not significantly different in each group. Figure 3 b) Relative organ weight ( Figure 3 c) and the morphology of the heart, liver, spleen, lungs, and kidneys ( Figure 3 d) No significant difference or obvious change suggests that the toxic side effects of α-dextrin combined with the immunomodulatory drug anti-PD1 in osteosarcoma-bearing mice are negligible.

[0077] In summary, this invention provides a novel use of α-dextrin in the preparation of immunotherapeutic drugs for antitumor (especially osteosarcoma), thereby offering new options for the clinical treatment of osteosarcoma.

[0078] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of a combination of α-mangostin and an anti-PD-1 antibody for the manufacture of a medicament for the treatment of osteosarcoma, characterized in that: The mass ratio of the alpha-mangostin to the anti-PD-1 antibody is 10:

1.

2. Use according to claim 1, characterized in that: The medicine comprises a combination of active ingredients alpha-mangostin and an anti-PD-1 antibody and one or more pharmaceutically acceptable adjuvants.

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