阅读说明：本技术 藤黄绿脓菌素和自噬抑制剂的药物组合物及用途 (Pharmaceutical composition of pyoluteorin and autophagy inhibitor and application ) 是由 钱忠明 包勇 于 2020-06-11 设计创作，主要内容包括：本发明属于生物医药领域,涉及用于治疗肿瘤等疾病的药物组合物,具体涉及藤黄绿脓菌素和自噬抑制剂的药物组合。本发明的一种药物组合包括藤黄绿脓菌素和自噬抑制剂。经实验,结果表明,该药物组合通过联合用药可以显著抑制癌细胞的生长,并且具有明显的协同作用,相对于藤黄绿脓菌素单用具有更强的抑制肿瘤的效果。本发明为临床的肿瘤治疗提供新的治疗干预方案,具有良好的临床应用前景。(The invention belongs to the field of biological medicines, relates to a pharmaceutical composition for treating diseases such as tumors and the like, and particularly relates to a pharmaceutical composition of gamboge pyocyancin and an autophagy inhibitor. The drug combination comprises pyoluteorin and an autophagy inhibitor. The experiment shows that the medicine composition can obviously inhibit the growth of cancer cells through combined medication, has obvious synergistic effect and has stronger tumor inhibiting effect compared with the single use of the pyoluteorin. The invention provides a new therapeutic intervention scheme for clinical tumor treatment and has good clinical application prospect.)

1. A pharmaceutical composition comprising pyoluteorin and an autophagy inhibitor;

the structural formula of the pyoluteorin is shown as the following formula,

2. the pharmaceutical composition of claim 1, wherein the autophagy inhibitor is selected from the group consisting of one or more of phosphatidylinositol 3-kinase inhibitor, chloroquine, Thapsigargin, bafilomycin a1, desipramine, trichostatin, and phenylacetylene sulfonamide.

3. The pharmaceutical composition of claim 2, wherein the phosphatidylinositol 3-kinase inhibitor is selected from the group consisting of 3-MA, Wortmannin, and LY 294002.

4. The pharmaceutical composition according to claim 2, wherein the phosphatidylinositol 3-kinase inhibitor is preferably chloroquine or 3-MA.

5. Use of a pharmaceutical composition according to any one of claims 1 to 4 for the preparation of an anti-tumor medicament.

6. The use according to claim 5, wherein the antitumor agent is an agent for the prophylaxis and/or treatment of a tumor disease.

7. The use according to claim 5, wherein the anti-neoplastic drug is a tumor cell proliferation inhibitor.

8. The use of claim 7, wherein the tumor cell is a pancreatic cancer cell, a breast cancer cell, a ductal breast cancer cell, a liver cancer cell, an esophageal cancer cell, a colorectal cancer cell, or a prostate cancer cell.

9. The use of claim 8, wherein the tumor cells are pancreatic ductal adenocarcinoma cells, pancreatic cancer cells, breast cancer cells, or breast ductal carcinoma cells.

10. The use according to claim 8, wherein the cell is a cell with low p-JNK pathway activity.

Technical Field

The invention belongs to the field of biological medicines, relates to a pharmaceutical composition for treating diseases such as tumors and the like, and particularly relates to a combined drug of Pyoluteorin (pyroluteorin) and an autophagy inhibitor, and a medicinal application of the pharmaceutical composition.

Background

The prior art discloses that tumors are characterized by unlimited proliferation of cells and are a great threat to human health worldwide today. For example, pancreatic cancer is one of the most aggressive human cancers in the world. Despite significant progress over the past decades, the treatment of pancreatic cancer has not changed much, and overall survival rates for 5 years are still less than 6%. The first-line treatment regimen for pancreatic cancer is still dominated by the chemotherapeutic gemcitabine. However, gemcitabine tends to cause drug resistance and side effects, and thus the development of new more effective anti-pancreatic cancer drugs is imminent.

Recent studies have found that immunity plays an important role in the development of tumors. Autophagy is an evolutionarily conserved physiological process that maintains homeostasis under external stress and maintains the balance of the body's energy supply by degrading misfolded proteins and damaged organelles.

Pyoluteorin (pyroluteorin) is a natural secondary bacterial metabolite isolated from pseudomonas fluorescens, and the current research on this compound is mainly focused on its antibacterial and antifungal activity. However, its antitumor activity has been rarely reported. Recent research shows that the gamboge pyocin can induce the apoptosis of triple negative breast cancer cells, and indicates that the gamboge pyocin has an important function in treating tumors. Therefore, the research on the action mechanism of the gamboge pyocyancin in the tumor and the construction of a novel pharmaceutical composition of the gamboge pyocyancin have important significance for clinical popularization in the aspect of anti-tumor of the gamboge pyocyancin.

Disclosure of Invention

The invention aims to provide a pharmaceutical composition for treating diseases such as tumors and the like based on the current situation of the prior art, and particularly relates to a combined composition of pyocin (pyroluteorin) and an autophagy inhibitor and a novel medicinal application thereof.

The invention is based on the following research results:

in the research process, the gamboge pyocin is found to be capable of inducing apoptosis of triple-negative breast cancer cells, the research of the application finds that the gamboge pyocin can obviously induce apoptosis in pancreatic cancer cells, and simultaneously, the gamboge pyocin can also induce autophagy of pancreatic cancer cells, and the specific mechanism of the induced autophagy of the cells is to promote phosphorylation of Bcl-2 by activating a p-JNK pathway, so that the Bcl-2 is separated from Beclin1 protein, and the Beclin1 protein is promoted to be combined to a Vps34 complex, and finally the activity of Vps34 and the occurrence of autophagy are promoted. The pyoluteorin-induced autophagy of cells can obviously resist the apoptosis effect induced by the pyoluteorin-induced autophagy of cells. After the gamboge pyocyancin is combined with the autophagy inhibitor, the cell autophagy induced by the gamboge pyocyancin is obviously inhibited, and the cell apoptosis induced by the gamboge pyocyancin is further enhanced. The killing effect on pancreatic cancer cells is obviously increased.

The invention provides a pharmaceutical composition, which comprises pyoluteorin and an autophagy inhibitor.

The embodiment of the invention shows that the pharmaceutical composition can obviously inhibit the growth of pancreatic cancer cells by adopting combined medication and has obvious synergistic action. The pyoluteorin is used as a drug combination of an apoptosis inducer and an autophagy inhibitor 3-MA, and can be applied to the treatment of tumors, particularly pancreatic cancer.

In the combination of gamboge pyocin and autophagy inhibition of the invention, the gamboge pyocin is gamboge pyocin and derivatives thereof with similar structure and similar function.

The structural formula of the pyoluteorin in the combined medicine is shown in figure 1.

The autophagy inhibitor in the combined medicine comprises one or more of phosphatidylinositol 3-kinase (PI3K) inhibitor, chloroquine, Thapsigargin (TG, Thapsigargin), bafilomycin A1, Desipramine (DCMI), Trichostatin (TSA) and phenylacetylene sulfonamide.

Inhibitors of phosphatidylinositol 3-kinase (PI3K) include: 3-MA, Wortmannin (Wortmannin), LY294002(CAS:154447-36-6), and the like. Chloroquine and 3-MA are preferred.

3-methyladenine (3-MA) is an inhibitor of phosphatidylinositol 3-kinase (PI 3K). PI3K plays an important role in many biological processes, including controlling the activation of mTOR, a key regulator of autophagy. 3-MA inhibits autophagy by inhibiting class III PI3K from blocking autophagosome formation, and is also an early inhibitor of autophagy.

Chloroquine can increase the pH of lysosome, thereby inhibiting the fusion of autophagosome and lysosome and the degradation of lysosomal protein, and inhibiting autophagy in the later stage of autophagy.

The pharmaceutical composition also comprises pharmaceutically acceptable carriers and excipients. The carrier may be any conventional carrier and excipient in the pharmaceutical art. The choice of a particular carrier and excipient will depend on the mode of administration or disease type and state used to treat a particular patient. The preparation of suitable pharmaceutical compositions for a particular mode of administration is well within the knowledge of those skilled in the pharmaceutical art. For example, carriers, excipients, diluents, fillers, solvents, supporting agents, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, and the like, which are conventional in the pharmaceutical field, may be included as the pharmaceutically acceptable carrier or excipient. If necessary, flavoring agent, antiseptic, sweetener, etc. can also be included.

The invention also provides the application of the pharmaceutical composition in preparing antitumor drugs.

The anti-tumor drug can be a drug for preventing and/or treating tumor diseases. For example, the anti-tumor drug is a tumor cell proliferation inhibitor.

The tumor cell can be a malignant tumor cell, namely a cancer cell, and is preferably a pancreatic cancer cell, a breast ductal carcinoma cell, a liver cancer cell, an esophageal cancer cell, a colorectal cancer cell or a prostate cancer cell.

The cancer cells are pancreatic ductal adenocarcinoma cells, pancreatic cancer cells, breast cancer cells or breast ductal carcinoma cells.

The cells are those with low p-JNK pathway activity.

The experimental result shows that compared with the single use of the gamboge pyocin, the combination of the gamboge pyocin and the autophagy inhibitor CQ or 3-MA remarkably increases the inhibition effect of the gamboge pyocin on the tumor; the combination of the pyoluteorin and the autophagy inhibitor 3-MA can obviously inhibit the generation and development of tumors in a mouse body; pyoluteorin has synergistic effect in combination with autophagy inhibitor.

The invention has the advantages that:

the gamboge pyocyanin and the autophagy inhibitor are proved to be respectively used for clinical treatment, and the combined medication can obviously improve the anti-tumor capability of the medicament to generate synergistic effect and improve the clinical treatment effect, and has high safety, so the invention provides a new and more effective treatment intervention scheme for clinically treating tumor diseases.

Drawings

FIG. 1 is a structural formula diagram of pyoluteorin;

FIG. 2 is a western blot result graph of protein expression of LC3 in pancreatic cancer cell line BxPC 3;

FIG. 3 is a photograph of immunofluorescence confocal imaging in pancreatic cancer cell line BxPC 3;

FIG. 4 is an image obtained by transmission electron microscopy in pancreatic cancer cell line BxPC 3;

FIG. 5 is a diagram of relative cell viability of pancreatic cancer cell lines BxPC3 and Capan-1 after treatment, panel A with pyoluteorin and 3-MA and panel B with pyoluteorin and CQ;

FIG. 6 is a tumor growth curve of PDX model of pancreatic cancer.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. The examples, in which specific conditions are not specified, were carried out according to the conventional experimental conditions in molecular biology and immunology. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1 Guttin pyocyancin induces apoptosis

In this example, a western blot experiment is performed to verify that pyoluteorin can induce autophagy of cells. The specific experimental steps are as follows:

digesting the pancreatic cancer cells BxPC3 with logarithmic growth, inoculating the pancreatic cancer cells into a 6-well plate, treating the cells for 24 hours by using pyocins (0,0.5,1 and 2 mu M) with different concentrations, then collecting and cracking the cells, taking 20 mu g of protein to perform a western blot experiment, and detecting the expression change condition of an apoptosis index Caspase 3;

example 2 Guttin pyocyancin induces autophagy

In the embodiment, the gamboge pyocyanin can induce autophagy of cells through western blot experiments, immunofluorescence experiments and transmission electron microscope experiments. The specific experimental steps are as follows:

logarithmically growing pancreatic cancer cells BxPC3 were digested, seeded in 6-well plates, treated with pyoluteorin, and subjected to concentration gradient and time gradient experiments:

concentration gradient experiment: treating cells for 24 hours by using pyoluteorin (0,0.5,1 and 2 mu M) with different concentrations, then collecting and cracking the cells, taking 20 mu g of protein to perform a western blot experiment, and detecting the expression change condition of autophagy index LC 3;

time gradient experiment: treating cells with pyoluteorin (1 μ M) for 8 hours, 16 hours and 24 hours, then collecting and cracking the cells, taking 20 μ g of protein to perform a western blot experiment, and detecting the expression change condition of autophagy index LC 3; the result of Western Blot detection is shown in FIG. 2.

Immunofluorescence assay:

BxPC3 cells in exponential growth phase were taken, transiently transfected with GFP-LC3 and cultured for 24 hours. After trypsinization, centrifuging at 1500rpm for 5min, discarding supernatant, uniformly beating with corresponding culture medium to obtain cell suspension, counting with a counter, diluting into cell suspension with concentration of 5.6 × 104 cells/mL, inoculating into a confocal dish, placing in a cell culture box at 200 μ L per well for 24 h. Pyoluteorin (1 μ M) was then added, and an equal amount of DMSO was added as a control, followed by incubation for 24 hours. The supernatant was removed and the cells were washed once with PBS, then fixed with 100. mu.L/well of 4% paraformaldehyde for 15min at room temperature, washed 3 times with PBS, incubated with 10. mu.g/mL Hoechst in PBS for 5min at room temperature, and imaged on Opera. In imaging, the objective lens selects a 40X water lens, 9 pictures are randomly and automatically acquired in each hole, and 488 excitation detection GFP protein and 425 excitation Hoechst detection cell nucleus are respectively used for excitation detection, and the result is shown in figure 3.

Transmission electron microscope experiment:

BxPC3 cells were harvested in logarithmic growth phase, pyoluteorin (1. mu.M) was added to the control, and an equal amount of DMSO was added to the control, followed by 24 hours of culture. Adherent cells were then scraped off, the cells were transferred along with the medium to a 15ml centrifuge tube, centrifuged at 1500rpm for 5min, and the supernatant was discarded. The cells were resuspended in 1ml PBS, added to a 1.5ml EP tube, centrifuged at 1500rpm for 5min, the supernatant discarded, and then freshly prepared 2.5% glutaraldehyde fixative was injected and tested on the machine after sectioning, the results are shown in FIG. 4.

Example 3 combination of pyoluteorin and autophagy inhibitor to inhibit tumor cell activity

In the embodiment, the gamboge pyocin is combined with CQ or 3-MA respectively, and the CCK8 method is used for verifying that the combination of the gamboge pyocin and the autophagy inhibitor can obviously increase the apoptosis promoting function of the gamboge pyocin. The experimental procedure was as follows:

the pyoluteorin and CQ are used together:

collecting exponential growth pancreatic cancer cell lines BxPC3 and Capan-1, centrifuging at 1500rpm for 5min, discarding supernatant, re-suspending with corresponding culture medium to obtain cell suspension, counting with cell counter, and diluting to concentration of 3.0 x 10⁴Cell suspensions of individual cells/ml were seeded in 96-well plates at 40. mu.L per well, leaving one well without cells as background control. Cells were cultured in FBS medium supplemented with 10% carbon dioxide at 37 ℃ in 5% carbon dioxide. Cells were placed in an incubator overnight and were treated with 10 μ M DMSO as a blank by adding either pyoluteorin (1 μ M), CQ (5 μ M) or both (pyoluteorin 1 μ M, CQ 5 μ M). After 48 hours of incubation in an incubator, the culture was aspirated, 50. mu.l of fresh culture containing 5. mu.L of CCK-8 reagent was added to each well, and after 1-2 hours of incubation in an incubator, the absorbance was measured at a wavelength of 450nm using Envision with a reference wavelength of 650 nm. After subtracting the background value of wells of no species from all the well readings, the proliferation inhibition rate IR (%) of the test compound to the cells was calculated as (OD value to control well-OD value administration well)/OD value to control well x 100%. The experiment was repeated 3 times, and the average of 3 experiments was calculated as the final indicator of inhibitory ability. The cell activity values obtained by the assay are shown in FIG. 5A.

The pyoluteorin and 3-MA are used together:

collecting exponential growth pancreatic cancer cell lines BxPC3 and Capan-1, centrifuging at 1500rpm for 5min, discarding supernatant, re-suspending with corresponding culture medium to obtain cell suspension, counting with cell counter, and diluting to concentration of 3.0 x 10⁴Cell suspensions of individual cells/ml were seeded in 96-well plates at 40. mu.L per well, leaving one well without cells as background control. Cells were cultured in FBS medium supplemented with 10% carbon dioxide at 37 ℃ in 5% carbon dioxide. Cells were placed in an incubator overnight and either pyoluteorin (1 μ M), 3-MA (5mM) or a combination of both (pyoluteorin 1 μ M, 3-MA 5mM) was added, respectively, with 10 μ M DMSO treatment as a blank. After 48 hours of incubation in an incubator, the culture was aspirated, 50. mu.l of fresh culture containing 5. mu.L of CCK-8 reagent was added to each well, and after 1-2 hours of incubation in an incubator, the absorbance was measured at a wavelength of 450nm using Envision with a reference wavelength of 650 nm. After subtracting the background value of wells of no species from all the well readings, the proliferation inhibition rate IR (%) of the test compound to the cells was calculated as (OD value to control well-OD value administration well)/OD value to control well x 100%. The experiment was repeated 3 times, and the average of 3 experiments was calculated as the final indicator of inhibitory ability. The cell activity values obtained by the assay are shown in FIG. 5B.

According to fig. 5, it is shown that the combination of gamboge pyocin and autophagy inhibitor CQ or 3-MA significantly increases the tumor-inhibiting effect of gamboge pyocin compared to the single use of gamboge pyocin.

Example 4 combination of pyoluteorin and autophagy inhibitor 3-MA in a mouse model to inhibit tumor growth

Human pancreatic cancer cell line BxPC3 cells according to the size of 5 x 10⁶The cells/mouse were subcutaneously inoculated to the right axilla of 5 to 6 weeks old nude mice, respectively, to form transplantable tumors, and then used after passage in nude mice. Collecting tumor tissue in vigorous growth stage, and cutting into 1.5-2.0 mm under aseptic condition³Left and right, inoculated subcutaneously in the right axilla of nude mice. Measuring the diameter of the transplanted tumor by using a vernier caliper when the tumor volume reaches 100mm³On the left and right, 4 groups were randomly divided, and 5 to 6 nude mice were each group. The first group was a negative control group, and equal amounts of solvent were given; the second group isInjecting 10mg/kg of pyoluteorin into tail vein every 2 days; the third group was 3-MA (25mg/kg) administered in tail vein every two days; the fourth group was a combination of these two drugs, i.e., 10mg/kg pyoluteorin and 25mg/kg 3-MA per two days of tail vein injection. Tumor length (B, tumor diameter) width (a, tumor transverse diameter) was measured every 3 days, and from this tumor volume V ═ a × B was calculated²/2。

The relative tumor proliferation rate is calculated as follows: RTV is Vt/V0. Where Vt is the tumor volume at a certain time and V0 is the tumor volume at the beginning of the administration. The relative tumor proliferation rate calculation formula is as follows: T/C (%) ═ tumor proliferation rate relative to treatment group/tumor proliferation rate relative to solvent control group x 100%.

The formula for the percentage of tumor inhibition: percent tumor inhibition is (tumor weight of control group-tumor weight of administration group)/tumor weight of control group x 100%.

The evaluation standard of the curative effect is as follows: T/C (%) >60 was considered ineffective; T/C (%). ltoreq.60 and tumor volumes were considered effective with a statistical treatment P <0.05 compared to the solvent control group. Statistical analysis of the metrology data was performed using a two-sided T-test. The growth curve is shown in FIG. 6. As can be seen, the combination of pyoluteorin and autophagy inhibitor 3-MA can obviously inhibit the generation and development of tumors in mice.

Example 5 combination of pyoluteorin and autophagy inhibitor has synergistic effect

The formula Q ═ E (A + B)/(E (A)) + E (B) -E (A) x E (B)) is calculated by using a standard synergy index, the (E (A + B) is the inhibition rate of the combination of two medicines, the E (A) is the inhibition rate of the single medicine of A, and the E (A) is the inhibition rate of the single medicine of B), the synergy effect statistics of pyoluteorin and CQ is carried out, and the synergy effect is defined when the Q value is more than 1.15. In pancreatic cancer cell line BxPC3, E (pyoluteorin) ═ 0.23, E (CQ) ═ 0.16, E (pyoluteorin + CQ) ═ 0.84, Q ═ 1.57, showed synergistic effects.

The above description is only for the specific embodiments of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present disclosure should be covered within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.