Marine symbiotic bacterium-derived anti-tumor pharmaceutical composition

Abstract

The anti-tumor pharmaceutical composition derived from the marine symbiotic bacteria comprises an active component A, an active component B and a medicinal carrier, the active component A is indole alkaloid separated from fermentation liquor of the marine symbiotic bacteria Streptomyces sp.M2021-03, and the molecular formula of the indole alkaloid is H21NO4; the active ingredient B is a polyketone compound separated from a fermentation solution of marine symbiotic bacteria Pseudomonas sp.M2021-07, and the molecular formula of the polyketone compound is C24H30O6; the medicinal carrier is a composite carrier of poloxamer 188 and beta-cyclodextrin in a mass ratio of 1: 2. The active component A (indole alkaloid) blocks mitotic division by inhibiting tumor cell microtubule polymerization, the active component B (polyketone compound) can down-regulate VEGF expression and inhibit angiogenesis, and the inhibition rates of MCF-7 and A549 cells are respectively increased by 40% and 35% compared with those of a single component when the two components are combined.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an anti-tumor pharmaceutical composition from marine symbiotic bacteria. BACKGROUND

[0002] Malignant tumor is one of the major diseases leading to human death worldwide. Although traditional chemotherapeutic drugs are widely used in clinical practice, they have problems such as poor selectivity, strong side effects, and easy drug resistance. In recent years, it has become a research hotspot to explore new anti-tumor active substances from marine organisms and their symbiotic microorganisms. Marine symbiotic bacteria are a kind of microorganisms that form a stable symbiotic relationship with marine animals and plants. Their unique living environment (high pressure, low oxygen, high salt, and oligotrophic) promotes the evolution of special secondary metabolic pathways, which can produce bioactive molecules with novel structures and unique mechanisms of action.

[0003] Studies have shown that secondary metabolites from marine symbiotic bacteria have multiple functions such as anti-tumor, antibacterial, antiviral, and immunomodulatory. For example, indole alkaloids from deep-sea hydrothermal vent symbiotic bacteria have an IC50 value of 0.8 μg / mL against breast cancer cells MCF-7; some polyketide compounds produced by rhizosphere symbiotic bacteria of mangrove can inhibit the proliferation of lung cancer cells A549 by regulating the apoptosis pathway. However, existing researches mainly focus on the isolation and identification of single active ingredients, which have the following technical bottlenecks: poor stability of active ingredients: most of the metabolic products of marine symbiotic bacteria are small molecule compounds, which are easily degraded by light, oxygen, and pH, resulting in reduced drug efficacy in vivo; single target: single component is difficult to block multiple pathways such as proliferation, invasion, and angiogenesis of tumor cells at the same time, which is easy to cause drug resistance; low bioavailability: some active ingredients have poor water solubility, and it is difficult to reach the effective therapeutic window after oral administration; lack of systematic combination research: existing patents mainly focus on the extraction of active ingredients from single strain, and the synergistic mechanism of metabolic products from different strains has not been explored.

[0004] In addition, although clinically commonly used anti-tumor drugs (such as paclitaxel and cisplatin) can inhibit tumor growth in the short term, they are accompanied by severe bone marrow suppression, neurotoxicity, and nephrotoxicity, and there is an urgent need to develop new anti-tumor drug combinations with low toxicity and high efficiency. Based on this, the present application isolates two active ingredients with synergistic effect from specific marine symbiotic bacteria, and optimizes the combination of the active ingredients with a pharmaceutical carrier to form an anti-tumor drug composition with strong stability, multi-target effect, and high bioavailability, thereby solving the above technical problems. SUMMARY

[0005] The present application aims at providing an anti-tumor drug composition from marine symbiotic bacteria to solve the problem of severe bone marrow suppression, neurotoxicity and nephrotoxicity accompanying the short-term inhibition of tumor growth of clinically commonly used anti-tumor drugs such as paclitaxel and cisplatin.

[0006] The above technical objective of the present application is achieved by the following technical scheme:

[0007] The anti-tumor drug composition from marine symbiotic bacteria comprises active ingredient A, active ingredient B and a pharmaceutical carrier, wherein the active ingredient A is an indole alkaloid isolated from the fermentation broth of marine symbiotic bacteria Streptomyces sp. M2021-03, the molecular formula is H 21 NO4; the active ingredient B is a polyketide compound isolated from the fermentation broth of marine symbiotic bacteria Pseudomonas sp. M2021-07, the molecular formula is C 24 H 30 O6; and the pharmaceutical carrier is a composite carrier of poloxamer 188 and β-cyclodextrin with a mass ratio of 1:2.

[0008] Preferably, the mass ratio of the active ingredient A to the active ingredient B is 1:0.8-1.2.

[0009] Preferably, the addition amount of the pharmaceutical carrier is 10%-15% of the total mass.

[0010] Preferably, the IC50 value of the active ingredient A on breast cancer cells MCF-7 is ≤0.7 μg / mL, and the IC50 value of the active ingredient B on lung cancer cells A549 is ≤1.0 μg / mL.

[0011] Preferably, the pharmaceutical composition exerts an anti-tumor effect by inhibiting tumor cell proliferation, invasion and angiogenesis.

[0012] A preparation method of an anti-tumor drug composition from marine symbiotic bacteria, characterized by comprising the following steps:

[0013] S1, marine symbiotic bacteria Streptomyces sp. M2021-03 and Pseudomonas sp. M2021-07 are respectively cultured, and fermentation broth is extracted after fermentation;

[0014] S2, the active ingredient A and the active ingredient B are separated and purified by ethyl acetate extraction and silica gel column chromatography;

[0015] S3, the active ingredient A, the active ingredient B and the pharmaceutical carrier are mixed, high-pressure homogenization treatment is performed to form a nanoemulsion, and the pharmaceutical composition is prepared after freeze-drying.

[0016] Preferably, the fermentation conditions are as follows: Streptomyces sp. M2021-03 is cultured at 28℃ for 7 days, and Pseudomonas sp. M2021-07 is cultured at 37℃ for 48 hours.

[0017] The present application has the following advantages: synergistic effect is significant: the inhibition rate of the combination of active ingredient A (indole alkaloid) and active ingredient B (polyketide) on MCF-7 and A549 cells is increased by 40% and 35% respectively compared with single component; stability and bioavailability are improved: the water solubility of the active ingredients is increased by more than 5 times by the complex carrier (poloxamer 188 + β-cyclodextrin), the active retention rate is more than 95% after the freeze-dried powder is stored at 4℃ for 6 months, and the time to peak blood concentration after intravenous injection is shortened to 1.2 hours (3 hours for traditional preparation); side effects are reduced: animal experiments show that the bone marrow suppression rate (leukopenia) of the present application is reduced by 60% compared with the paclitaxel control group, and no obvious liver and kidney toxicity (normal ALT and Cr indicators) is observed; multi-target mechanism: in addition to directly killing tumor cells, the present application can also inhibit tumor invasion by down-regulating MMP-9 expression, and reduce the activation of tumor-associated fibroblasts (CAFs) by regulating the TGF-β / Smad pathway, thereby blocking the tumor microenvironment support; simple preparation process: the present application uses conventional fermentation technology combined with silica gel column chromatography separation, which does not require complex equipment and is suitable for large-scale production; the preparation of nanoemulsion only requires high-pressure homogenization treatment, which is low in cost and suitable for industrial amplification. DETAILED DESCRIPTION

[0018] An anti-tumor pharmaceutical composition derived from marine symbiotic bacteria, comprising: active ingredient A, active ingredient B and a pharmaceutical carrier, wherein: active ingredient A is an indole alkaloid isolated from the fermentation broth of marine symbiotic bacteria Streptomyces sp. M2021-03, with a molecular formula of H 21 NO4; active ingredient B is a polyketide isolated from the fermentation broth of marine symbiotic bacteria Pseudomonas sp. M2021-07, with a molecular formula of C 24 H 30 O6; and the pharmaceutical carrier is a complex carrier of poloxamer 188 and β-cyclodextrin with a mass ratio of 1:2.

[0019] Preferably, the mass ratio of active ingredient A to active ingredient B is 1:0.8-1.2.

[0020] Preferably, the addition amount of the pharmaceutical carrier is 10%-15% of the total mass.

[0021] Preferably, the IC50 value of the active ingredient A on breast cancer cells MCF-7 is ≤ 0.7 μg / mL, and the IC50 value of the active ingredient B on lung cancer cells A549 is ≤ 1.0 μg / mL.

[0022] Preferably, the pharmaceutical composition exerts an anti-tumor effect by inhibiting tumor cell proliferation, invasion and angiogenesis.

[0023] A preparation method of an anti-tumor pharmaceutical composition derived from marine symbiotic bacteria, characterized in that it comprises the following steps:

[0024] S1, respectively culturing marine symbiotic bacteria Streptomyces sp. M2021-03 and Pseudomonas sp. M2021-07, and extracting the fermentation broth after fermentation;

[0025] S2, purifying the active ingredient A and the active ingredient B by ethyl acetate extraction and silica gel column chromatography;

[0026] S3, mixing the active ingredient A and the active ingredient B with a pharmaceutical carrier, and forming a nanoemulsion by high-pressure homogenization, and then freeze-drying to obtain the pharmaceutical composition.

[0027] Preferably, the fermentation conditions are that Streptomyces sp. M2021-03 is cultured at 28℃ for 7 days, and Pseudomonas sp. M2021-07 is cultured at 37℃ for 48 hours.

[0028] This invention has the following beneficial effects: Significant synergistic effect: Active ingredient A (indole alkaloids) inhibits mitosis by suppressing tumor cell microtubule polymerization, while active ingredient B (polyketide compounds) downregulates VEGF expression and inhibits angiogenesis. The combined use of these two ingredients increases the inhibition rate of MCF-7 and A549 cells by 40% and 35%, respectively, compared to the single-ingredient formulation. Improved stability and bioavailability: The composite carrier (poloxamer 188 + β-cyclodextrin) increases the water solubility of the active ingredients by more than 5 times through inclusion complexation. The lyophilized powder injection retains >95% of its activity after 6 months of storage at 4°C, and the time to peak plasma concentration after intravenous injection is shortened to 1.2 hours (compared to 3 hours for traditional formulations). Reduced toxicity: Animal experiments show that the drug composition of this invention reduces the bone marrow suppression rate (leukopenia) in mice by 60% compared to the paclitaxel control group, and no significant liver or kidney toxicity was observed (ALT and Cr levels were normal). Multi-target mechanism of action: In addition to directly killing tumor cells, it can downregulate MMP-9 expression to inhibit tumor invasion and reduce the activation of tumor-associated fibroblasts (CAFs) by regulating the TGF-β / Smad pathway, thereby blocking tumor microenvironment support. Simple preparation process: It uses conventional fermentation technology combined with silica gel column chromatography, requiring no complex equipment and suitable for large-scale production; the nanoemulsion preparation only requires high-pressure homogenization, which is low-cost and suitable for industrial scale-up.

[0029] Example 1: Isolation and purification of active ingredients

[0030] Strains were cultured: the marine symbiotic bacterium Streptomyces sp. M2021-03 was inoculated into ISP2 medium containing 2% sea salt (4 g / L tryptone, 1 g / L yeast extract, 2 g / L glucose) and cultured at 28°C with shaking for 7 days; Pseudomonas sp. M2021-07 was inoculated into LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) and cultured at 37°C with shaking for 48 hours.

[0031] Fermentation broth extraction: Collect bacterial broth, centrifuge (8000 rpm, 15 min) to obtain supernatant, add an equal volume of ethyl acetate for extraction 3 times, combine organic phases and concentrate to extract; extract is separated by silica gel column chromatography (eluent: petroleum ether-ethyl acetate gradient, volume ratio 10:1→1:1), and purity is detected by HPLC (>98%).

[0032] Structural identification: by nuclear magnetic resonance (NMR) 1 H-NMR, 13 C-NMR and mass spectrometry (MS) confirmed that active ingredient A is an indole alkaloid (named Marineindole A) and active ingredient B is a polyketide compound (named Pseudoployketone B).

[0033] Example 2: Preparation of pharmaceutical composition

[0034] Marineindole A (0.8 g), Pseudoployketone B (0.8 g) were mixed with poloxamer 188 (0.5 g), β-cyclodextrin (1.0 g), and then added with appropriate amount of deionized water, and treated by high pressure homogenizer (pressure 100 MPa) for 10 minutes to form nanoemulsion, and then lyophilized to obtain a light yellow powder of pharmaceutical composition.

[0035] Example 3 (supplement): Verification of anti-tumor activity

[0036] Human breast cancer cells MCF-7 and lung cancer cells A549 in logarithmic growth phase were inoculated in 96-well plates at 1×10 4 cells / well, and then added with different concentrations of active ingredient A, active ingredient B and pharmaceutical composition (A:B = 1:1), respectively, and then cultured for 48 hours, and then the cell survival rate was detected by CCK-8 method.

[0037] The results showed that the IC50 value of the pharmaceutical composition on MCF-7 was 0.35 μg / mL (the IC50 value of active ingredient A alone was 0.6 μg / mL), and the IC50 value on A549 was 0.5 μg / mL (the IC50 value of active ingredient B alone was 0.9 μg / mL); the combination index (CI) was <1 (MCF-7: CI = 0.72; A549: CI = 0.68), indicating that the two had significant synergistic effect.

[0038] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application.

Claims

1. A marine commensal-derived antitumor pharmaceutical composition, characterized by, The mass ratio of the active ingredient A to the active ingredient B is 1:0.8-1.

2. The active ingredient A is an indole alkaloid isolated from the fermentation broth of marine symbiotic bacteria Streptomyces sp. M2021-03, with the molecular formula C 18 H 21 NO4; the active ingredient B is a polyketide compound isolated from the fermentation broth of marine symbiotic bacteria Pseudomonas sp. M2021-07, with the molecular formula C 24 H 30 O6; and the pharmaceutical carrier is a composite carrier of poloxamer 188 and β-cyclodextrin with a mass ratio of 1:

2.

2. The marine commensal-derived antitumor pharmaceutical composition according to claim 1, characterized by: The added amount of the pharmaceutical carrier is 10%-15% of the total mass.

3. The marine commensal-derived antitumor pharmaceutical composition according to claim 1, characterized by: The IC50 value of the active ingredient A on breast cancer cells MCF-7 is ≤0.7 μg / mL, and the IC50 value of the active ingredient B on lung cancer cells A549 is ≤1.0 μg / mL.

4. The marine commensal-derived antitumor pharmaceutical composition according to claim 1, characterized by: The pharmaceutical composition exerts an anti-tumor effect by inhibiting tumor cell proliferation, invasion and angiogenesis.

5. The marine commensal-derived antitumor pharmaceutical composition according to claim 1, characterized by: The method comprises the following steps:

6. A method for preparing an antitumor drug composition derived from marine symbiotic bacteria, characterized in that: S1, culturing marine symbiotic bacteria Streptomyces sp. M2021-03 and Pseudomonas sp. M2021-07 respectively, and extracting the fermentation broth after fermentation; S2, purifying the active ingredient A and the active ingredient B by ethyl acetate extraction and silica gel column chromatography; S3, mixing the active ingredient A, the active ingredient B and the pharmaceutical carrier, and forming a nanoemulsion by high-pressure homogenization, and then freeze-drying to obtain the pharmaceutical composition. The fermentation conditions are as follows: Streptomyces sp. M2021-03 is cultured at 28℃ for 7 days, and Pseudomonas sp. M2021-07 is cultured at 37℃ for 48 hours.

7. The marine commensal-derived antitumor pharmaceutical composition according to claim 6, characterized by: ​