Use of cholecalciferol in the preparation of a medicament for the treatment of gastric cancer

Abstract

The application provides an application of doxercalciferol in preparation of a drug for treating gastric cancer. Compared with pancreatic cancer, non-small cell lung cancer, colon adenocarcinoma, cervical cancer and bladder cancer, the inhibitory effect of doxercalciferol on the growth of gastric cancer cells is the strongest.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to the application of ducalciferol in the preparation of drugs for treating gastric cancer. Background Technology

[0002] Gastric cancer is one of the most common malignant tumors worldwide, ranking among the top malignant tumors in both incidence and mortality. Current treatments for gastric cancer mainly include surgical resection, chemotherapy, radiotherapy, and targeted therapy. However, existing treatments suffer from limited efficacy, significant side effects, and a high risk of drug resistance. Therefore, the search for novel, low-toxicity, and highly effective anti-gastric cancer drugs has become a research hotspot in the biomedical field.

[0003] Doxercalciferol (also known as 1α-hydroxyvitamin D2) is an active analog of vitamin D2. Its chemical name is (1α,3β,5Z,7E,22E)-9,10-open-ring ergoster-5,7,10(19),22-tetraen-1,3-diol, and its structural formula is [insert structural formula here]. .

[0004] Ducalciferol is converted into its active metabolite 1α,25-dihydroxyvitamin D2 by hepatic 25-hydroxylase, thereby exerting its physiological role in regulating calcium and phosphorus metabolism. Currently, ducalciferol has been approved by the FDA for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease.

[0005] In recent years, the antitumor activity of vitamin D compounds has attracted increasing attention. Studies have shown that vitamin D and its analogues exert antitumor effects through multiple mechanisms, including inhibiting cell proliferation, inducing cell differentiation and apoptosis, and inhibiting tumor angiogenesis. However, the inhibitory effects of different vitamin D analogues on different types of tumors vary significantly, and some vitamin D analogues have toxic side effects such as hypercalcemia, limiting their clinical application in cancer treatment. Summary of the Invention

[0006] This invention discovers that calciferol can inhibit the growth of various tumor cells, especially exhibiting a stronger inhibitory effect on the growth of gastric cancer cells. Based on this discovery, the technical solution for which protection is sought is proposed in this invention:

[0007] Application of calciferol in the preparation of drugs for treating gastric cancer.

[0008] Calciferol or pharmaceutical compositions containing calciferol are used to treat gastric cancer.

[0009] A decalciferol or pharmaceutical composition containing decalciferol for the treatment of gastric cancer.

[0010] A method for treating gastric cancer involves administering an effective amount of ducalciferol or a pharmaceutical composition containing ducalciferol to patients in need.

[0011] Treating gastric cancer with dulcitol or pharmaceutical compositions containing dulcitol.

[0012] Ducalciferol or pharmaceutical compositions containing ducalciferol are used in the treatment of gastric cancer.

[0013] The gastric cancer is adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or undifferentiated carcinoma. The adenocarcinoma is tubular adenocarcinoma, papillary adenocarcinoma, mucinous adenocarcinoma, or signet ring cell carcinoma.

[0014] The gastric cancer mentioned refers to either early-stage or advanced-stage gastric cancer.

[0015] The gastric cancer mentioned is either intestinal-type gastric cancer or diffuse gastric cancer.

[0016] The gastric cancer is stage 0 (also known as carcinoma in situ), IA, IB, IIA, IIB, IIIA, IIIB, IIIC, or IV. Preferably, it is stage 0, IA, IB, IIA, IIB, IIIA, IIIB, or IIIC.

[0017] The treatment for gastric cancer is neoadjuvant therapy (i.e., treatment with anti-tumor drugs given before surgery) or adjuvant therapy (i.e., treatment with anti-tumor drugs given after surgery).

[0018] The drug or pharmaceutical composition contains calciferol and one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers are various excipients commonly used or known in the pharmaceutical industry, including but not limited to: diluents, binders, antioxidants, pH adjusters, preservatives, lubricants, disintegrants, etc.

[0019] The routes of administration of the drug or drug composition include, but are not limited to: oral; sublingual; sublingual; transdermal; pulmonary; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous; and by implantation of a reservoir or infusion device.

[0020] The dosage form of the drug or pharmaceutical composition may be an oral dosage form, such as tablets, capsules, pills, powders, granules, suspensions, syrups, etc.; or an injectable dosage form, such as an injection solution, powder for injection, etc., administered via intravenous, intraperitoneal, subcutaneous, or intramuscular routes. All dosage forms used are well known to those skilled in the art of pharmaceutical science.

[0021] The dosage of docalciferol in the treatment of gastric cancer is adjusted according to the individual patient's condition (including gastric cancer stage, physical condition, and combination of drugs), and can be used in the range of 2.5 μg / time to 30 μg / time.

[0022] In the treatment of gastric cancer, ducalciferol or pharmaceutical compositions containing ducalciferol can be used in combination with other active substances for treating gastric cancer. These other active substances for treating gastric cancer include, but are not limited to: platinum-based drugs (such as cisplatin, oxaliplatin, etc.), fluorouracil derivatives (such as 5-FU, capecitabine, tegafur, etc.), taxane-based drugs (such as paclitaxel, docetaxel, etc.), irinotecan, and anti-angiogenic targeted therapies (such as ramoximab, apatinib, etc.).

[0023] Terminology definition:

[0024] "And / or" will be considered as a specific disclosure of each of the two specified features or components having or not having the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0025] "Comprising" and "including" have the same meaning and are intended to be open and allow, but do not require, the inclusion of additional elements or steps. When the terms "comprising" or "including" are used herein, the terms "consisting of" and / or "substantially consisting of" are also included and disclosed. Attached Figure Description

[0026] Figure 1 : Inhibition curves of calciferol, vitamin D2 and vitamin D3 on the proliferation of gastric cancer cells NCI-N87.

[0027] Figure 2 : Inhibition curves of proliferation of PANC-1, 5637, A549, Caco-2, and Hela by calciferol, vitamin D2, and vitamin D3.

[0028] Figure 3 Inhibitory curves of calciferol and vitamin D2 on human gastric cancer organoids. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments. It should be noted that the embodiments are not intended to limit the scope of protection of the present invention, and those skilled in the art will understand that any improvements and variations made based on the present invention are within the scope of protection of the present invention.

[0030] All commonly used reagents used in the following examples are commercially available. The biological experiments performed are routine biological experiments in the field and can be conducted according to the instructions in the relevant lab manual or kit.

[0031] Cell lines and sources used in the examples:

[0032]

[0033] Example 1: Inhibitory effect of decalciferol on the proliferation of gastric cancer cells

[0034] NCI-N87 gastric cancer cells in the logarithmic growth phase were harvested at a concentration of 3 × 10⁻⁶ cells / year. 3 Cells were seeded per well in 96-well plates and cultured for 24 hours to allow adherence. Then, dulcitol (Selleck, catalog number: S1467), vitamin D2 (Selleck, catalog number: S4035), and vitamin D3 (Selleck, catalog number: S4063) were added to each well. Eight concentration gradients were prepared for each reagent: 100 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1 μM, and 0.1 μM, with three replicates per concentration. After culturing at 37°C and 5% CO2 for 72 hours, cell viability was determined using the ATP assay (ATP assay kit, Beyotime, catalog number: S0026). The half-maximal inhibitory concentrations (IC50) of dulcitol, vitamin D2, and vitamin D3 against gastric cancer cell lines were calculated. 50 ) DMSO was used as the solvent for the drug, and a negative control was also included.

[0035] Experimental steps for ATP assay of cell viability:

[0036] After obtaining the cell culture plate, equilibrate at room temperature for 10 min, discard the culture supernatant in the cell culture plate, add 50 μL of DPBS to each well, and add 50 μL of ATP standard series solutions (concentrations of 5000, 1500, 500, 150, 50, 15, 5, 0 ng / mL) to the blank area of ​​the cell plate.

[0037] Add 50 μL of CellTiter-Lumi Plus luminescence assay reagent to each sample well and standard solution well.

[0038] Shake at room temperature in the dark for 2 min to promote cell lysis. Incubate at room temperature in the dark (approximately 25°C) for 10 min to allow the luminescence signal to stabilize. Perform detection using the instrument parameters: detection wavelength is the full wavelength range; detection time per well is 1 s. Plot an ATP standard curve based on the detection results, and calculate the ATP content of each sample to determine the relative cell viability. The viability calculation formula is shown below:

[0039] Cell viability (%) = ATP content in the drug treatment group (ng / mL) × 100% / ATP content in the DMSO control group (ng / mL)

[0040] Results: Ducalciferol inhibited the IC50 of NCI-N87 cells. 50 The value is 3.69 μM.

[0041] Example 2: Inhibitory effect of decalciferol on the proliferation of various tumor cells

[0042] Following the method in Example 1, the inhibitory effect of ducalciferol on the proliferation of pancreatic cancer cells (PANC-1), lung cancer cells (A549), colon cancer cells (Caco-2), cervical cancer cells (HeLa), and bladder cancer epithelial cells (5637) was investigated.

[0043] Results: Ducalciferol exhibited varying degrees of inhibitory effects on the proliferation of the aforementioned tumor cells. Specific IC50 values ​​were... 50 The values ​​are shown in Table 1.

[0044] Table 1. IC50 of calciferol, vitamin D2, and vitamin D3 on various tumor cell lines. 50 value

[0045]

[0046] Example 3: Effects of decalciferol on human gastric cancer organoids (3D tumor organoid model)

[0047] Cell plating:

[0048] Organoid preparation: Organoids in the logarithmic growth phase were digested with Tryple, resuspended in DMEM / F12 to form a single-cell suspension, filtered through a 70-micron filter, and the cell filtrate was collected.

[0049] Cell counting: Cell counting is performed using a cell counter to calculate cell density.

[0050] Plating: Resuspend the required number of organoids in Matrigel and mix thoroughly. Add 6 μL of cell suspension to each well of a 96-well plate, with four replicates. Gently shake the culture plate to distribute the cells evenly.

[0051] Gel addition: Place the 96-well plate with cells in a 37°C, 5% CO2 incubator and incubate for 15 minutes each to allow the Matrigel to solidify completely. After solidification, add 200 μL of organoid culture medium to each well.

[0052] Edge wells: Add 200 μL of sterile DPBS to reduce edge effects caused by evaporation.

[0053] Incubation: Place the 96-well plate with cells in a 37°C, 5% CO2 incubator and incubate for 24 hours.

[0054] Chemical treatment:

[0055] Drug dilution: The stock solution was serially diluted with tumor organoid culture medium in sterile centrifuge tubes to prepare working solutions with seven concentration gradients. The stock solution concentration was 10 mM, using DMSO as the solvent. 100 μM: 100-fold dilution of stock solution; 80 μM: 1.25-fold dilution of 100 μM drug; 40 μM: 2-fold dilution of 80 μM drug; 20 μM: 2-fold dilution of 40 μM drug; 10 μM: 10-fold dilution of 100 μM solution; 5 μM: 2-fold dilution of 10 μM drug; 2.5 μM: 2-fold dilution of 5 μM drug.

[0056] Drugs to be tested: Ducalciferol, Vitamin D2

[0057] Control group: DMSO of different concentrations.

[0058] Specific preparation method: Calciferol:

[0059] 100 μM: 9900 μL + 100 μL of drug (stock solution);

[0060] 80 μM: 1600μL+6400μL drug (100 μM);

[0061] 40 μM: 4000μL+4000μL drug (80 μM);

[0062] 20μM: 4000μL+4000μL drug (40 μM);

[0063] 10μM: 5400μL+600μL drug (100 μM);

[0064] 5μM: 2000μL+2000μL drug (10 μM);

[0065] 2.5μM: 2000μL+2000μL drug (5 μM).

[0066] VD2:

[0067] 100 μM: 4950 μL + 50 μL drug (stock solution);

[0068] 80 μM: 600μL+2400μL drug (100 μM);

[0069] 40 μM: 1000μL+1000μL drug (80 μM);

[0070] 20μM: 1000μL+1000μL drug (40 μM);

[0071] 10μM: 1800μL+200μL drug (100 μM);

[0072] 5μM: 1000μL+1000μL drug (10 μM);

[0073] 2.5μM: 1000μL+1000μL drug (5 μM).

[0074] Drug concentrations: 100 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM. 1%, 0.8%, 0.4%, 0.2%, 0.1%, 0.05%, and 0.025% DMSO were used as negative controls.

[0075] Organoid drug sensitivity testing:

[0076] After obtaining the cell culture plate, equilibrate at room temperature for 10 min, discard the supernatant in the cell culture plate, and gently scrape the organoid gel droplet with a 10 μL pipette tip to remove the organoid from the culture plate and expose it from the matrix gel. Add 50 μL of LPBS to each well, and add 50 μL of ATP standard serial solutions (concentrations of 5000, 1500, 500, 150, 50, 15, 5, 0 ng / mL) to the blank area of ​​the cell plate.

[0077] Add 50 μL of CellTiter-Lumi Plus luminescence assay reagent to each sample well and standard solution well.

[0078] Shake at room temperature in the dark for 5 min to promote cell lysis. Incubate at room temperature in the dark (approximately 25°C) for 7 min to allow the luminescence signal to stabilize. Perform detection using the instrument parameters: detection wavelength is the full wavelength range; detection time per well is 1 s. Plot an ATP standard curve based on the detection results, calculate the ATP content of each sample, and thus calculate the relative cell viability. The cell viability calculation formula is shown below:

[0079] Cell viability (%) = ATP content in the drug treatment group (ng / mL) × 100% / ATP content in the DMSO control group (ng / mL)

[0080] Results: To evaluate the direct antitumor activity of dicalciferol against gastric cancer organoids, an in vitro 3D organoid culture model was used to detect the viability of organoids treated with different concentrations of dicalciferol and to calculate their IC50. 50The experimental results showed that ducalciferol's inhibitory effect on the proliferation of gastric cancer organoids was significantly concentration-dependent. Within the concentration range of 2.5 μM to 5 μM, the organoid viability of the ducalciferol-treated group was not significantly decreased compared to the solvent control group (P>0.05), suggesting that the drug had no significant cytotoxic effect within this concentration range. When the concentration was increased to 10 μM, ducalciferol began to significantly inhibit organoid proliferation (P<0.05), and the inhibitory effect was more significant at a concentration of 20 μM. Figure 3 Further calculation of the half-maximal inhibitory concentration (IC50) was performed. 50 ), decalciferol IC 50 The value was 12.92 μM. In comparison, the IC50 of the positive control drug VD2 in the same experimental system was [missing value]. 50 The value was 24.71 μM, which was significantly higher, and the difference between the two was statistically significant (P<0.05). These results indicate that ducalciferol has a significantly better inhibitory effect on the in vitro proliferation of gastric cancer organoids than VD2, suggesting that ducalciferol possesses stronger anti-tumor potential and warrants further development in the field of gastric cancer treatment.

[0081] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of doxorubicin in the preparation of drugs for treating gastric cancer.

2. The application as described in claim 1, wherein the gastric cancer is adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or undifferentiated carcinoma.

3. The application as described in claim 2, wherein the adenocarcinoma is tubular adenocarcinoma, papillary adenocarcinoma, mucinous adenocarcinoma, or signet ring cell carcinoma.

4. The application as described in claim 1, wherein the gastric cancer is early-stage gastric cancer or advanced-stage gastric cancer.

5. The application as described in claim 1, wherein the gastric cancer is intestinal type gastric cancer or diffuse gastric cancer.

6. The application as described in claim 1, wherein the gastric cancer is stage 0, IA, IB, IIA, IIB, IIIA, IIIB, IIIC, or IV.

7. The application as described in any one of claims 1-6, wherein the treatment is neoadjuvant therapy or adjuvant therapy.

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