Application of Stachyose in Preparation of Drug for Treating Castration-Resistant Prostate Cancer

Abstract

Disclosed is an application of Stachyose in preparation of a drug for treating castration-resistant prostate cancer, belonging to the technical field of biological medicine. The disclosure proposes a new strategy of using Stachyose in combination with an androgen receptor antagonist to prepare a drug for treating CRPC for the first time, and conducts multi-angle and multi-level verification research. The drug composition of the Stachyose in combination with the androgen receptor in the disclosure can be used for treating castration-resistant prostate cancer, and significantly improves the effect of Enzalutamide on inhibiting castration-resistant prostate cancer. The natural compound is applied to the advanced stage of cancer, and has important clinical therapeutic significance.

Claims

1. An method of use of Stachyose in preparation of a drug for treating castration-resistant prostate cancer, comprising: using an effective amount of Stachyose as an active ingredient of the drug.

2. The method according to claim 1, comprising using the Stachyose alone or in combination with an androgen receptor antagonist to prepare a drug for treating castration-resistant prostate cancer.

3. A method for treating castration-resistant prostate cancer, comprising: administering an effective amount of Stachyose to a castration-resistant prostate cancer patient in need.

4. A drug composition for treating castration-resistant prostate cancer, wherein the composition comprises Stachyose and an androgen receptor antagonist.

5. The drug composition according to claim 4, wherein a mass ratio of the androgen receptor antagonist to the Stachyose is (1-8):1.

6. The drug composition according to claim 4, wherein the androgen receptor antagonist comprises any one or more of the following: Enzalutamide, EPI, Abiraterone, and Olaparib.

7. The drug composition according to claim 4, wherein the drug composition also comprises pharmaceutical excipients.

8. The drug composition according to claim 7, wherein the pharmaceutical excipients comprise a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, an adhesive, a disintegrant, a filler, a lubricant, a wetter, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending aid, a coating material, an aromatic agent, an anti-binding agent, an integration agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foamer, a defoamer, a thickener, an inclusion agent, a humectant, an absorbent, a diluent, a flocculant, a deflocculant, a filter aid, and a release retardant.

9. The drug composition according to claim 4, wherein the drug composition also comprise drug carriers.

10. The drug composition according to claim 9, wherein the drug carriers comprise microcapsules, microspheres, nanoparticles and liposomes.

11. The drug composition according to claim 4, wherein the dosage forms of the drug composition comprise injections, lyophilized powder for injection, controlled-release injections, liposome injections, suspensions, implants, emboli, capsules, tablets, pills, and oral liquids.

Description

BRIEF DESCRIPTION OF FIGURES

[0022] FIG. 1 shows a forming process of DTP (drug-tolerant persisters) and DTEP (drug-tolerant expanded persisters) cells in Example 1.

[0023] FIG. 2 shows characteristics of expression changes of AR-related proteins in the DTP and DTEP cells in Example 1.

[0024] FIG. 3 shows cycle effects of the DTP and DTEP cells in Example 1.

[0025] FIG. 4A shows a bar graph of a relative survival rate of drug combination cells on L-DTP-EPI and L-DTP-Enza cells in Example 2. FIG. 4B shows a bar graph of a CI value of EPI and Enza in combination with Stac respectively on the L-DTP-EPI and L-DTP-Enza cells.

[0026] FIG. 5A shows a graph of changes in prostate weight of mice in an Enza single-drug group 1 (10 mg/Kg), an Enza single-drug group 2 (90 mg/Kg), a Stac single-drug group 1 (80 mg/Kg), a Stac single-drug group 2 (90 mg/Kg) and an Enza and Stac combination group (10 mg/Kg+80 mg/Kg) with administration in Example 3. FIG. 5B shows photographs of stripped prostates of mice in the Enza single-drug group 1 (10 mg/Kg), the Stac single-drug group 1 (80 mg/Kg) and the Enza and Stac combination group (10 mg/Kg+80 mg/Kg) in Example 3. FIG. 5C shows a graph of changes in body weight of mice in the Enza single-drug group 1 (10 mg/Kg), the Stac single-drug group 1 (80 mg/Kg) and the Enza and Stac combination group (10 mg/Kg+80 mg/Kg) with administration in Example 3.

[0027] FIG. 6A shows HE staining graphs of prostate tissue slices of mice in the Enza single-drug group 1 (10 mg/Kg), the Stac single-drug group 1 (80 mg/Kg) and the Enza and Stac combination group (10 mg/Kg+80 mg/Kg) in Example 3. FIG. 6B shows PRDX5 and AR immunohistochemical graphs of the prostate tissue slices of mice in the Enza single-drug group 1 (10 mg/Kg), the Stac single-drug group 1 (80 mg/Kg) and the Enza and Stac combination group (10 mg/Kg+80 mg/Kg) in Example 3. FIG. 6C shows a quantitative bar graph of immunohistochemical positive cells of mice in the Enza single-drug group 1 (10 mg/Kg), the Stac single-drug group 1 (80 mg/Kg) and the Enza and Stac combination group (10 mg/Kg+80 mg/Kg) in Example 3.

DETAILED DESCRIPTION

[0028] The disclosure is further described below with reference to the accompanying drawings of the specification and specific examples, but the examples do not limit the disclosure in any form. Unless otherwise specified, the reagents, methods and devices used in the disclosure are conventional reagents, methods and devices in the technical field.

[0029] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1: Process of Using EPI and Enza to Produce DTP/DTEP in Prostate Cancer LNCaP Cells and Characteristics of this Model

[0030] EPI and Enza-tolerant prostate cancer L-DTP cell strains L-DTP-EPI and L-DTP-Enza can inhibit the protein expression of AR and targets thereof, and the cell growth inhibition manifests as cycle arrest in a G0/G1 phase.

[0031] 1. Experimental Method:

[0032] 1×10.sup.6 LNCaP cells were inoculated in a 10 cm cell culture dish, and after adherence on the second day, EPI and Enza were respectively added for treatment for 9 days. In the period, for replacement, a fresh drug-containing medium was used every three days for culture, some cells (DTP cells) were collected for subsequent experiments after 9 days, and the remaining cells continued to be treated with drugs. In the period, for replacement, a fresh drug-containing medium was used every three days for culture, and DTEP cells were collected after 33 days for subsequent experiments. After the DTP and DTEP cells were produced, the cells were digested and counted to calculate the percentage of the cells in 1×10.sup.6 cells. The collected NC, DTP and DTEP cells were used for subsequent Western Blot experiments: the three groups of cells were subjected to cell lysis, protein extraction and quantification, SDS-PAGE gel electrophoresis, membrane transfer, blocking, primary antibody incubation, secondary antibody incubation and development, then, the expression changes of AR-FL and its related target proteins and AR-Vs and its related target proteins were observed, and the expression changes of cell cycle-related proteins were observed. Flow cytometry: dead cells were stained by a PI-stained cycle kit, and cell cycle changes were determined by a flow cytometry.

[0033] 2. The results were shown in FIG. 1, FIG. 2 and FIG. 3. FIG. 1 shows a Giemsa staining graph of DTP and DTEP cells. FIG. 2 shows protein expression changes of AR & its targets and AR-Vs & its targets in NC, DTP and DTEP cells. FIG. 3 shows expression changes of cell cycle-related proteins in NC, DTP and DTEP cells.

[0034] The results showed that the number of the LNCaP-DTP cells produced by EPI and Enza accounted for 103.11% of the initial seed cells, and the LNCaP-DTEP cells accounted for 110.92%. These drug-tolerant cells were tolerant to EPI and Enza, and the cells became spindle-shaped and the growth was inhibited in DTP. In DTEP, cell clones were increased, cell growth inhibition was resisted, and cells were re-proliferated. The effects of this cell model on the protein expressions of an androgen receptor (AR) and its targets: the protein expression of AR was inhibited in a DTP state, and the expression of AR was recovered to a certain extent in DTEP; and the expression of targets TMPRSS2 and PSA of AR kept the same trend as AR. Protein expression effects of androgen receptor splice variants (AR-Vs) and their targets: in DTP and DTEP states, the protein expressions of AR-Vs and their targets UBE2C and CDC20 were continuously inhibited. This inhibition was caused by cell cycle arrest and was specifically manifested as follows: P21 (marker in G1 phase) was up-regulated in DTP, and recovered to a certain extent in DTEP; Cyclin El (marker in G1-S phase) and CDC6 (marker in G1-S phase) were down-regulated in DTP, and recovered to a certain extent in DTEP; and CDC2 (marker in G1-S and G2-M phases) was down-regulated in DTP, but not significantly recovered in DTEP. Through flow cytometry (FACS) analysis, it was found that the G0/G1 phase was arrested in DTP and recovered in DTEP.

Example 2: In-Vitro Effects of EPI and Enza in Combination with Stac Respectively

[0035] Furthermore, CCK8 was used alone or in combination in drug-tolerant L-DTP cells to demonstrate an anti-tumor effect of Stac in drug-tolerant L-DTP (EPI) and L-DTP (Enza) cells in vitro.

[0036] 1. Experimental Method:

[0037] Drug-tolerant cells L-DTP (including L-DTP (EPI) and L-DTP (Enza)) were inoculated in a 96-well plate, and after adherence, a series of Stac drugs with concentrations from high to low were prepared to find an optimal Stac drug concentration. Then, this concentration was used for measuring the survival rates of single (L-DTP (EPI)-Stac) and combined [L-DTP (EPI)-combination (EPI+Stac)] and [L-DTP (Enza)-combination (Enza+Stac)] in drug-tolerant cells L-DTP respectively. Finally, Calcusyn software was used for calculating a CI value in L-DTP cells.

[0038] 2. The results were shown in FIG. 4A and FIG. 4B. FIG. 4A and FIG. 4B show in-vitro pharmacodynamic graphs of EPI and Enza in combination with Stac respectively in L-DTP cells, and FIG. 4A shows a bar graph of a relative survival rate of drug combination cells on L-DTP (EPI) and L-DTP (Enza) drug-tolerant cells; and FIG. 4B shows a bar graph of a CI value of EPI and Enza in combination with Stac respectively on the L-DTP (EPI) and L-DTP (Enz) drug-tolerant cells.

[0039] The results showed that in the drug-tolerant L-DTP (EPI) cells produced after continuous administration of EPI for 9 days, and if EPI continued to be added, there was no obvious inhibiting effect; if Stac was added alone, a significant inhibiting effect was achieved, and an inhibition rate was 67.48%; and when EPI and Stac were combined, an inhibition rate was 51.15%. Similarly, in the drug-tolerant L-DTP (Enza) cells produced after continuous administration of Enza for 9 days, if Enza continued to be added, there was no obvious inhibiting effect; if Stac was added alone, a significant inhibiting effect was achieved, and an inhibition rate was 62.47%; and when Enza and Stac were combined, an inhibition rate was 51.97%. By calculating the CI value, it was found that Stac can achieve a high synergistic effect of 0.49 in L-DTP-EPI cells and a high synergistic effect of 0.54 in L-DTP (Enza) cells.

Example 3: Drug Combination Effects of Enza and Stac Combination Scheme on C-MYC-Over-Expressed Prostate Cancer Mouse Model after Generation of Tolerance by Continuous Administration of Enza

[0040] Further, the effect of Enza and Stac combination in mice relapsing after chemical castration (continuous administration of Enza) was illustrated in a prostate cancer mouse model.

[0041] 1. Experimental Method:

[0042] A C-MYC (Hi-Myc)-over-expressed spontaneous prostate cancer mouse model was constructed, and at 4 months, the mice developed mPIN/Cancer transition. At the moment, the mice were randomly divided into an NC control group (intragastrical administration with solvents) and an Enza administration group. Then, intragastrical administration was performed every three days, and Enza administration was performed at 10 mg/Kg for a total of 30 days. Then, the necks of some mice were severed, and the prostate cancers of the mice were taken, photographed and weighed. It was found that Enza can significantly reduce the symptoms, and the prostate weight was reduced by half compared with that in the NC control group. Then, according to the above method, the remaining mice were administered for 30 days, and it was found that some mice in the Enza group relapsed. Then, mice (mice aged 6 months) were randomly divided into: an NC control group (intragastrical administration with solvents all the time), an Enza single-drug group 1, an Enza single-drug group 2, a Stac single-drug group 1, a Stac single-drug group 2 and an Enza and Stac combination group, the corresponding administration treatment was performed, and all mice were intragastrically administered once every three days. The dosage of Enza in the Enza single-drug group 1 was kept at 10 mg/Kg each time, the dosage of Enza in the Enza single-drug group 2 was kept at 90 mg/Kg each time, the dosage of Stac in the Stac single-drug group 1 was kept at 80 mg/Kg each time, the dosage of Stac in the Stac single-drug group 2 was kept at 90 mg/Kg each time, the dosages of Enza and Stac in the Enza and Stac combination group were kept at 10 mg/Kg and 80 mg/Kg each time, and the administration was performed for a total of 30 days. Then, the necks of the mice were severed, and the prostate cancers of the mice were taken, photographed, weighed and subjected to experiments including immunohistochemistry.

[0043] 2. The results were shown in FIG. 5A to FIG. 5C and FIG. 6A to FIG. 6C. FIG. 5A to FIG. 5C are graphs showing drug combination effects of Enza, Stac and drug combination on changes in prostate weight of the C-MYC-over-expressed prostate cancer mouse model after generation of tolerance by continuous administration of Enza, and FIG. 5A shows a graph of changes in prostate weight of each group of mice with administration; FIG. 5B shows comparison photographs of stripped prostates of each group of mice; and FIG. 5C shows a graph of changes in weight of each group of mice with administration. FIG. 6A to FIG. 6C are graphs showing drug combination effects of Enza, Stac and drug combination scheme on the C-MYC-over-expressed prostate cancer mouse model after generation of tolerance by continuous administration of Enza, and FIG. 6A shows HE staining graphs of prostate tissue slices of each group of mice; and FIG. 6B and FIG. 6C respectively show AR and PRDX5 immunohistochemical graphs of the prostate tissue slices and a quantitative bar graph of positive cells of each group of mice.

[0044] An average of the prostate weights of the mice after continuous administration of Enza for 30 days was 43.9 mg, and at the moment, an average in the NC control group was 88.7 mg. The administration was continued, after 90 days, it was found that an average of the prostate weights of the mice in the Enza group was changed to 77.1 mg, and at the moment, an average in the NC control group was 98.2 mg, indicating that drug tolerance relapse was caused, resulting in CRPC. At the moment, the grouped administration was performed immediately to illustrate the drug combination effects.

[0045] The results of grouped combination and single use were shown in Table 1:

TABLE-US-00001 TABLE 1 Administration effects of different administrations (30 d of administration) after drug tolerance relapse Average of prostate Administration weights of mice NC control group 102.01 mg  Continued Enza single-drug group 1 54.42 mg (10 mg/Kg) after drug tolerance relapse Continued Enza single-drug group 2 53.83 mg (90 mg/Kg) after drug tolerance relapse Stac single-drug group 1 (80 mg/Kg 50.86 mg Stac) after drug tolerance relapse Stac single-drug group 2 (90 mg/Kg 47.50 mg Stac) after drug tolerance relapse Enza and Stac combination group 37.07 mg (10 mg/Kg Enza + 80 mg/Kg Stac) after drug tolerance relapse

[0046] It can be seen from FIG. 5A to FIG. 5C and Table 1 that the combination of Enza and Stac has a very significant effect compared with the single use of Enza and the single use of Stac, and the prostate weight can be reduced to about 37.068 mg. At the same time, it can be seen that the treatment effect of the single use of Stac was better than that of the single use of Enza after drug tolerance, indicating that the single use of Stac has an inhibiting effect on drug-tolerant CRPC.

[0047] It can be seen from the HE staining results of the tissue slices (FIG. 6A to FIG. 6C) that the prostate tumor of CRPC has obvious regression and fibrosis after drug combination, and it can be seen from immunohistochemistry that the combination of Enza and Stac significantly reduces the expressions of AR and PRDX5 compared with the single use of Enza and the single use of Stac, thereby proving that the combination effects were significant.

[0048] The results of grouped combination and single use were shown in Table 2:

TABLE-US-00002 TABLE 2 Administration effects of different administrations (30 d of administration) after drug tolerance relapse AR PRDX5 Administration expression/% expression/% NC control group 93.7 47.0 Continued Enza single-drug group 1 89.4 78.4 (10 mg/Kg) after drug tolerance relapse Continued Enza single-drug group 2 82.6 80.0 (90 mg/Kg) after drug tolerance relapse Stac single-drug group 1 (80 mg/Kg 61.5 68.2 Stac) after drug tolerance relapse Stac single-drug group 2 (90 mg/Kg 57.4 60.9 Stac) after drug tolerance relapse Enza and Stac combination group 46.3 39.2 (10 mg/Kg Enza + 80 mg/Kg Stac) after drug tolerance relapse