Pharmaceutical composition for treating osteosarcoma

Abstract

The invention belongs to the technical field of new drugs. The invention provides a pharmaceutical composition for treating osteosarcoma. The pharmaceutical composition provided by the invention comprises gossypol and honokiol. The invention develops a novel pharmaceutical composition for treating osteosarcoma, which can effectively inhibit the growth of osteosarcoma cells and has better safety.

Description

Technical Field

[0001] The invention belongs to the technical field of new drugs, and specifically relates to a pharmaceutical composition for treating osteosarcoma. Background Art

[0002] Osteosarcoma is a common malignant bone tumor that mainly occurs in adolescents and young adults. Traditional treatment methods include surgical resection of the tumor and preoperative or postoperative chemotherapy. However, single treatment methods often have limited effects. The resistance of osteosarcoma patients to chemotherapeutic drugs is a problem that seriously affects the treatment effect, and the incidence of resistance can be as high as 30% to 50%. Combination therapy can overcome drug resistance to a certain extent and improve the treatment effect and prognosis of patients. Therefore, drug synergistic treatment regimens have become the focus of research. At present, the commonly used chemotherapy regimens for osteosarcoma patients mainly include combinations formed by cisplatin (P), doxorubicin (A), high-dose methotrexate (M), ifosfamide (I) and etoposide (E), such as MAP, IFO / MAP, API and other regimens. However, these drugs often cause significant side effects, with severe gastrointestinal reactions, hematological toxicity, hepatorenal toxicity and alopecia being the most common.

[0003] At present, the number of achievements in the research and development of pharmaceutical compositions for the treatment of osteosarcoma is limited. For example, Chinese patent ZL 201810309050.6 discloses a pharmaceutical composition with synergistic anti-osteosarcoma efficacy, which comprises apatinib, a VEGFR2 selective inhibitor, and GSK-J4, a histone demethylase inhibitor; another example is Chinese patent ZL202210570031.5, which provides a pharmaceutical composition for the treatment of osteosarcoma, which comprises procollagen C-terminal protease enhancer 1 and cisplatin.

[0004] However, people have not yet figured out the rules for developing a drug composition for treating osteosarcoma, which leads to greater challenges in further developing related drug combinations. In addition, most of the drugs currently used to treat osteosarcoma are traditional chemotherapy drugs with high toxicity. Therefore, how to develop a drug composition based on a substance with lower toxicity to treat osteosarcoma is a technical problem that needs to be solved urgently in this field. Summary of the invention

[0005] In view of the defects of the prior art, the present invention aims to provide a pharmaceutical composition for treating osteosarcoma, which uses substances with lower toxicity as components of the composition and has a synergistic effect on inhibiting the growth of osteosarcoma cells.

[0006] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0007] A pharmaceutical composition for treating osteosarcoma, comprising gossypol and honokiol.

[0008] Preferably, the molar ratio of gossypol to honokiol is 1:1.

[0009] A pharmaceutical preparation for treating osteosarcoma, comprising the aforementioned pharmaceutical composition.

[0010] Furthermore, the pharmaceutical preparation also includes pharmaceutically acceptable excipients.

[0011] The present invention also provides the use of the above-mentioned pharmaceutical composition in preparing a drug for inhibiting the proliferation of osteosarcoma cells.

[0012] The pharmaceutical composition of the present invention is a plant extract drug. Plant extract drugs have the following significant advantages over traditional chemotherapy drugs: plant extracts generally have lower toxicity than chemotherapy drugs. This allows patients to tolerate better during treatment and reduces treatment interruptions or dose adjustments due to side effects. Plant extracts generally contain a variety of active ingredients that can fight tumor cells through multi-target mechanisms of action (such as anti-oxidation, anti-inflammatory, apoptosis induction, angiogenesis inhibition, etc.). This multi-target action helps reduce the occurrence of drug resistance and may improve the therapeutic effect. In addition, plant extracts are generally widely available and have relatively low production costs, so they can provide more economical treatment options and reduce the financial pressure on patients.

[0013] As shown in the embodiments of the present invention, the present invention finds that gossypol is an efficient osteosarcoma therapeutic drug, and honokiol can enhance the sensitivity of osteosarcoma cells to gossypol, and the two together exert a synergistic killing effect on osteosarcoma cells. Interestingly, honokiol does not produce a significant killing effect on osteosarcoma cells at the concentration that exerts this enhanced effect. This result suggests that the strengthening effect of honokiol on gossypol is not dependent on the toxic effect of honokiol itself.

[0014] Beneficial effects of the present invention:

[0015] The present invention develops a new drug composition for treating osteosarcoma, which can effectively inhibit the growth of osteosarcoma cells and has good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result diagram of the effect of the combination of gossypol and cyclophosphamide on the activity of SJSA-1 cells;

[0017] Figure 2 This is the result diagram of the effect of the combination of gossypol and ifosfamide on the activity of SJSA-1 cells;

[0018] Figure 3 This is the result diagram of the effect of the combination of gossypol and doxorubicin on the activity of SJSA-1 cells;

[0019] Figure 4 This is the result diagram of the effect of the combination of gossypol and etoposide on the activity of SJSA-1 cells;

[0020] Figure 5 This is the result diagram of the effect of the combination of gossypol and gemcitabine on the activity of SJSA-1 cells;

[0021] Figure 6 This is the result diagram of the effect of the combination of gossypol and bleomycin on the activity of SJSA-1 cells;

[0022] Figure 7 This is the result diagram of the effect of the combination of gossypol and vincristine on the activity of SJSA-1 cells;

[0023] Figure 8 This is the result diagram of the effect of the combination of gossypol and carnosol on the activity of SJSA-1 cells;

[0024] Fig. 9 This is the result diagram of the effect of the combination of gossypol and ginkgo biloba on the activity of SJSA-1 cells;

[0025] Fig.10 This is the result diagram of the effect of the combination of gossypol and honokiol on the activity of SJSA-1 cells;

[0026] Fig.11 This is the result diagram of the effect of the combination of gossypol and xanthohumol on the activity of SJSA-1 cells;

[0027] Fig.12 is the CI value of the two-drug synergy coefficient of the eleven drug combinations;

[0028] Fig.13 The figure shows the experimental results of treating K7M2 osteosarcoma cell line with different concentrations of gossypol and honokiol alone or in combination;

[0029] Fig.14 The figure shows the experimental results of treating HOS osteosarcoma cell line with different concentrations of gossypol and honokiol alone or in combination;

[0030] Fig.15 The figure shows the experimental results of treating MG63 osteosarcoma cell line with different concentrations of gossypol and honokiol alone or in combination;

[0031] Fig.16 The figure shows the experimental results of treating Saos-2 osteosarcoma cell line with different concentrations of gossypol and honokiol alone or in combination;

[0032] Fig.17 This is a graph showing the safety experimental results of gossypol combined with magnolol in the treatment of osteosarcoma. DETAILED DESCRIPTION

[0033] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technical personnel in this field based on the above invention content still fall within the scope of protection of the present invention.

[0034] Example 1

[0035] 1.1 Main Reagents

[0036] Osteosarcoma cell lines (HOS, K7M2, MG63) and mouse calvarial osteoblast precursors (MC3T3-E1) were purchased from Wuhan Pronocell Life Science Co., Ltd.; human osteosarcoma cell lines (Saos-2 and SJSA-1) were purchased from the cell bank of the Chinese Academy of Medical Sciences; fetal bovine serum was from Shanghai ExCellBio; dimethyl sulfoxide (DMSO) was purchased from Shanghai Aladdin Biotechnology Co., Ltd.; DMEM medium and 0.25% trypsin were purchased from Gibco, USA; CCK-8 kit was purchased from Apexbio, USA; Gossypol (Cat. No.: R01 3819, purity>95%, DMSO as solvent), Honokiol (Honokiol, Catalog No.: R011878, purity 98%, DMSO as solvent), Methotrexate (Methotrexate, Catalog No.: R000499, purity 99%, DMSO as solvent), Doxorubicin (Doxorubicin, Catalog No.: R017518, purity 98%, DMSO as solvent), Gemcitabine (Gemcitabine, Catalog No.: R002322, purity 98%, DMSO as solvent), Vincristine (Vincristin e, product number: R055634, purity 98%, PBS as solvent), carnosol (Carnosol, product number: R022244, purity 98%, DMSO as solvent), xanthohumol (Xanthohumol, product number: R025341, purity 99%, DMSO as solvent) and etoposide (Etoposide, product number: R003588, purity 98%, DMSO as solvent) were purchased from Shanghai Luoen Biotechnology Co., Ltd., Cis-platinum (Cis-platinum, product number: D109812, purity Pt, 65%, ddH2O as solvent) were purchased from Shanghai Luoen Biotechnology Co., Ltd. As solvent) was purchased from Shanghai Aladdin Biotechnology Co., Ltd., Ifosfamide (Cat. No. S46617, purity 98%, DMSO as solvent), Cyclophosphamide (Cat. No. S30563, purity 97%, DMSO as solvent), Bleomycin (Cat. No. B73357, purity ≥ 90%, PBS as solvent) and Ginkgetin (Cat. No. B21584, purity HPLC ≥ 98%, DMSO as solvent) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0037] 1.2 Cell culture and grouping

[0038] In the preliminary evaluation experiment of the synergistic killing effect of the combined scheme, the inventor used human osteosarcoma cell SJSA-1 as an in vitro model. The cell experiment was divided into an experimental group (drug group, cotton pol group, and cotton pol + drug combination group) and a blank control group (containing an equal volume of solvent as the experimental group). The drugs in the drug group include commonly used chemotherapy drugs for the treatment of osteosarcoma and compounds with good activity reported in current basic research. High-glucose DMEM containing 10% fetal bovine serum was used as a complete culture medium, and the mother liquor was gradiently diluted with the complete culture medium to obtain a drug culture medium containing different target concentrations. Cultured in an incubator with 5% CO2 and 37°C.

[0039] In the universal evaluation experiment of the synergistic killing effect of the combination scheme, we used human osteosarcoma cells (HOS, MG63 and Saos-2) and mouse osteosarcoma cells (K7M2) as in vitro models. The cell experiment was divided into experimental groups (gossypol group, magnolol group, and gossypol + magnolol combined group) and blank control group (containing the same volume of solvent as the experimental group). The mother liquor was gradiently diluted with complete culture medium to obtain a medium containing drugs at different target concentrations. High-glucose DMEM containing 10% fetal bovine serum was used as the complete culture medium, and the mother liquor was gradiently diluted with complete culture medium to obtain a medium containing drugs at different target concentrations. Cultured in an incubator with 5% CO2 and 37°C.

[0040] In the safety evaluation of the combination regimen, the cell experiment was divided into an experimental group (cottonpol + honokiol combined group, methotrexate group, doxorubicin group, etoposide group, cisplatin group and ifosfamide group) and a blank control (NC) group. Since DMSO can affect the activity of platinum compounds, except for cisplatin, which uses ddH2O as a solvent, the remaining drugs were prepared with DMSO as a solution to prepare a mother solution with a concentration of 1mM. To ensure the activity of the drugs, all drugs were prepared immediately, and the process from preparation to use did not exceed 1 hour. High-glucose DMEM containing 10% fetal bovine serum was used as a complete culture medium, and the mother solution was gradiently diluted with the complete culture medium to obtain a drug culture medium containing different target concentrations. Cultured in an incubator with 5% CO2 and 37°C. 1.3CCK-8 method was used to detect the effect of cottonpol and the drug of interest alone or in combination on the activity of SJSA-1 cells.

[0041] SJSA-1 cells grown to 80% to 90% confluence were digested with 0.05% trypsin and plated at 5 × 10 3100 μL / well was inoculated in a 96-well plate, and 0, 0.1, 0.4, 0.7, 1, 4, 7, 10, 40, 70, 100 uM of cottonyphenol and 0, 0.1, 0.4, 0.7, 1, 4, 7, 10, 40, 70, 100 uM of the drug of interest were added and cultured for 24 hours. In order to determine the effect of the combination of cottonyphenol and the drug of interest on osteosarcoma cells, 0, 0.1, 0.4, 0.7, 1, 4, 7, 10, 40, 70, 100 uM cottonyphenol was mixed with the drug of interest at a concentration ratio of 1:1 and then treated with SJSA-1 cells for 24 hours. After 24 hours of treatment, the old culture medium was discarded and fresh complete culture medium containing 10% CCK-8 was added for 2 hours. The absorbance at 450nm (A) was detected by a microplate reader. The absorbance at 650nm was used as the reference wavelength. The cell inhibition rate was calculated after deducting the absorbance of the well without CCK-8. Cell inhibition rate = 1-[(A 实验孔 -A 空白孔) / (A 对照孔 -A 空白孔) ], and the synergy coefficient CI value of the two drugs was calculated using Comosyn software.

[0042] 1.4 CCK-8 assay to detect the effects of gossypol and honokiol alone or in combination on the activity of HOS, Saos-2, MG63 and K7M2 cells

[0043] The osteosarcoma cells grown to 80% to 90% confluence were digested with 0.05% trypsin and plated at 5×10 3 pcs / well / 100 μL were inoculated in a 96-well plate, and 0, 0.1, 0.4, 0.7, 1, 4, 7, 10, 40, 70, 100 uM of gossypol and 0, 0.1, 0.4, 0.7, 1, 4, 7, 10, 40, 70, 100 uM of honokiol were added and cultured for 24 hours. In order to determine the effect of the combination of gossypol and honokiol on osteosarcoma cells, 0, 0.1, 0.4, 0.7, 1, 4, 7, 10, 40, 70, 100 uM of gossypol and honokiol were mixed at a concentration ratio of 1:1 and then treated with osteosarcoma cells for 24 hours. After 24 hours of treatment, the old culture medium was discarded and fresh culture medium containing 10% CCK-8 was added for 2 hours. The absorbance at 450nm (A) was detected by a microplate reader. The absorbance at 650nm was used as the reference wavelength. The cell inhibition rate was calculated after deducting the absorbance of the well without CCK-8. Cell inhibition rate = 1-[(A 实验孔 -A 空白孔) / (A 对照孔 -A 空白孔) ], and the synergy coefficient CI value of the two drugs was calculated using Comosyn software.

[0044] 1.5 CCK-8 method to detect the effect of the combination of gossypol and honokiol and first-line clinical drugs on the activity of MC3T3T-E1 cells

[0045] MC3T3T-E1 cells grown to 80% to 90% confluence were digested with 0.05% trypsin and plated at 5 × 10 3 Each well was inoculated with 100 μL of each cell per well in a 96-well plate, and 0, 0.1, 0.4, 0.7, 1, 4, 7, 10, 40, 70, 100 uM of methotrexate, doxorubicin, etoposide, cisplatin and ifosfamide were added, respectively. The gossypol and honokiol combination group was added with 0, 0.1, 0.4, 0.7, 1, 4, 7, 10, 40, 70, 100 uM of gossypol and 0, 0.1, 0.4, 0.7, 1, 4, 7, 10, 40, 70, 100 uM of honokiol. After 24 hours of treatment in each group, the old culture medium was discarded, and fresh culture medium containing 10% CCK-8 was added for 2 hours. The absorbance at 450 nm (A) was detected using an ELISA reader, and the absorbance at 650 nm was used as the reference wavelength. The cell inhibition rate was calculated after deducting the absorbance of the well without CCK-8. Cell inhibition rate = {1-[(A 实验孔 -A 空白孔) / (A 对照孔 -A 空白孔) ]}×100%, the inhibition rate of each group was fitted and the half inhibition concentration (IC50) was calculated.

[0046] In the present invention, we used different concentrations of gossypol and drugs of interest (including methotrexate, doxorubicin, gemcitabine, vincristine, etoposide, ifosfamide, cyclophosphamide, bleomycin, ginkgetin, carnosol, xanthohumol, and honokiol) to treat human osteosarcoma cells SJSA-1 alone or in combination, and cells treated with an equal volume of solvent served as a blank control group.

[0047] After 24 hours of treatment, discard the old culture medium and wash twice with PBS to eliminate the influence of the absorbance of the drug itself on the experimental results. Add fresh culture medium containing 10% CCK-8 and continue to culture in the cell culture incubator for 2 hours. Use an enzyme marker to detect the absorbance of each group of culture medium at 450nm, and the absorbance at 650nm is used as the reference wavelength. The group without CCK-8 was used as a blank control group to eliminate the influence of culture medium, cells and well plate itself on absorbance. Each group of data was repeated 3 times independently. Calculate the average inhibition rate of each group, and use Comosyn software to calculate the synergistic coefficient CI value of the two drugs.

[0048] The experimental results showed that honokiol had no significant inhibitory effect on SJSA-1 cells in the concentration range of 0.1 to 100 μM, but it could synergistically exert a higher osteosarcoma cell toxicity than gossypol alone (molar concentration ratio = 1:1), with a half inhibitory concentration (IC50) in the range of 1 to 10 μM, and a CI value of 0.55, which was the combination with the strongest synergistic effect among the combinations of gossypol and drugs of interest ( Figures 1 to 12 ).

[0049] In the present invention, we use different concentrations of gossypol and honokiol to treat 4 osteosarcoma cell lines (HOS, MG63 and Saos-2 are human osteosarcoma cells, and K7M2 is mouse osteosarcoma cells) alone or in combination, and the cells treated with equal volume solvents are blank control groups. After 24 hours of treatment, the old culture medium was discarded and washed twice with PBS to eliminate the influence of the absorbance of the drug itself on the experimental results. Fresh culture medium containing 10% CCK-8 was added and cultured in a cell culture incubator for 2 hours. The absorbance of each group of culture medium at 450nm was detected by an ELISA instrument, and the absorbance at 650nm was used as the reference wavelength. The group without CCK-8 was used as a blank control group to exclude the influence of culture medium, cells and well plate itself on absorbance. Each group of data was repeated 3 times independently. The average inhibition rate of each group was calculated, and the synergistic coefficient CI value of the two drugs was calculated using Comosyn software. The experimental results showed that honokiol had no significant inhibitory effect on the four osteosarcoma cells in the concentration range of 0.1 to 100 μM, but it could synergize with gossypol (molar concentration ratio = 1:1) to exert higher osteosarcoma cell toxicity, with a half inhibitory concentration (IC50) in the range of 1 to 10 μM and a CI value in the range of 0.1 to 0.7. Among them, the strongest synergy was in the MG63 cell line, with a CI value of 0.21, showing a strong synergistic effect ( Figure 13 to Figure 16 ).

[0050] In addition, in order to test the safety of gossypol combined with honokiol in the treatment of osteosarcoma, we used mouse calvarial osteoblast precursors (MC3T3-E1) as a normal cell model and used CCK-8 to explore the effects of current clinical first-line osteosarcoma treatment drugs and the gossypol & honokiol combination regimen on the activity of MC3T3-E1 cells. Fig.17 As shown, the effect of gossypol + honokiol group (molar concentration ratio = 1:1) on the activity of MC3T3-E1 cells was significantly less than that of the first-line osteosarcoma treatment drugs in the concentration range of 0.1 to 100 μM. This result suggests that the combination of gossypol & honokiol shows better safety than other single drugs (cisplatin, doxorubicin, methotrexate, ifosfamide, etoposide) at the same concentration.

Claims

1. A pharmaceutical composition for treating osteosarcoma, characterized in that: The composition includes gossypol and honokiol.

2. A pharmaceutical composition for treating osteosarcoma according to claim 1, characterized in that: The molar ratio of the cottonpol to the honokiol is 1:

1.

3. A pharmaceutical preparation for treating osteosarcoma, characterized in that: The pharmaceutical preparation comprises the pharmaceutical composition according to claim 1 or 2.

4. A pharmaceutical preparation for treating osteosarcoma according to claim 3, characterized in that: The pharmaceutical preparation also includes pharmaceutically acceptable excipients.

5. Use of the pharmaceutical composition according to claim 1 or 2 for preparing a drug for inhibiting the proliferation of osteosarcoma cells.

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