Use of stachyose in the preparation of therapeutic drugs for castration-resistant prostate cancer

Abstract

The present invention discloses the use of stachyose in the preparation of a drug for treating castration-resistant prostate cancer, which belongs to the field of biomedicine. The present invention is the first to propose a new strategy for combining stachyose with androgen receptor antagonists to prepare a drug for treating CRPC, and conducts multifaceted and multi-stage validation research. The pharmaceutical composition of the present invention combining stachyose with androgen receptor can be used to treat castration-resistant prostate cancer, which can significantly improve the inhibitory effect of enzalutamide on castration-resistant prostate cancer, and the application of natural compounds to late-stage cancer is of great significance in clinical treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the use of stachyose in the preparation of a therapeutic agent for castration-resistant prostate cancer.

Background Art

[0002] Androgen deprivation therapy is the standard treatment for advanced prostate cancer. However, patients eventually develop castration-resistant prostate cancer (CRPC) after an average of 1 to 3 years of treatment. CRPC refers to prostate cancer in which the disease continues to progress after the first continuous androgen deprivation therapy (ADT). Since 2004, when docetaxel was proven to extend the overall survival of patients with metastatic castration-resistant prostate cancer (mCRPC), drugs for the mCRPC disease stage such as abiraterone acetate, enzalutamide, cabazitaxel, etc. have emerged, changing the treatment situation of these patients, but it is difficult to completely recover CRPC. Therefore, finding other effective treatment targets or combination treatment strategies has become another research hotspot in the treatment of CRPC.

[0003] In recent years, cell plasticity has emerged as a target diagnostic avoidance model and is a commonality of many cancer drug resistances. Blocking new drug resistance pathways can effectively inhibit persister cells. For example, the GPX4 lipid peroxidation pathway is an effective target highly expressed in many persister cell states. So far, it is not known whether there are persister cells in CRPC tumors and whether new effective targets can be found. Therefore, this study focuses on finding an effective combination drug for treating CRPC starting from prostate cancer LNCaP-persister cells generated by EPI-001 and Enzalutamide.

[0004] EPI-001 (EPI) is an inhibitor of arsenic agents (ARs) and AR-splice variants (AR-Vs) that is awaiting clinical development and has potential use in the treatment of CRPC. EPI's target for CRPC is primarily the N-terminal domain (NTD). Enzalutamide (Enza) was the first approved second-generation AR antagonist and has 5-8 times higher affinity for ARs compared to conventional antiandrogens. Based on this, the US FDA approved Enza for CRPC patients in 2012. However, drug resistance to both EPI and Enza typically emerges around 18 months after the onset of CRPC. Therefore, other methods to overcome drug resistance and delay CRPC are urgently needed. [Overview of the project] [Problems that the invention aims to solve]

[0005] The technical problem that this invention aims to solve is to overcome the drug resistance present in the above-mentioned conventional drugs, and to provide an effective CRPC treatment drug that significantly improves the therapeutic effect of CRPC by using Enza and Stac in combination, thereby exhibiting excellent synergistic effects.

[0006] Stachyose (Stac) is a sugar in which two α-galactose molecules are attached to the glucose group of sucrose via 1,6-glucosidic bonds. Its molecular formula is C24H42O21. [Means for solving the problem]

[0007] The first object of the present invention is to provide the use of Stac in the preparation of therapeutic agents for castration-resistant prostate cancer.

[0008] In one embodiment of the present invention, the use includes combining Stac with an androgen receptor antagonist to prepare a therapeutic agent for castration-resistant prostate cancer.

[0009] A second object of the present invention is to provide a pharmaceutical composition for treating castration-resistant prostate cancer, comprising Stac and an androgen receptor antagonist.

[0010] In one embodiment of the present invention, the mass ratio of the androgen receptor antagonist to Stac is (1-8):1. Preferably, the mass ratio of Enza to Stac is 1-3:1.

[0011] In one embodiment of the present invention, the androgen receptor antagonist includes one or more of the following: enzalutamide (Enza), EPI-001 (EPI), abiraterone, and olaparib.

[0012] One embodiment of the present invention further comprises the pharmaceutical composition and the pharmaceutical excipient.

[0013] In one embodiment of the present invention, the pharmaceutical excipients include solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, humectants, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspension aids, coating agents, flavoring agents, anti-adhesion agents, synthesizers, penetration accelerators, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and decoagulants, filtration aids and release inhibitors.

[0014] In one embodiment of the present invention, the dosage form of the formulation includes an injection solution, a lyophilized powder for injection, a sustained-release injection, a liposome injection, a suspension, an implantable agent, an embolizing agent, a capsule, a tablet, a pill, and an oral solution.

[0015] In one embodiment of the present invention, the pharmaceutical composition may further include a pharmaceutical carrier.

[0016] In one embodiment of the present invention, the pharmaceutical carrier includes microcapsules, microspheres, nanoparticles, and liposomes.

[0017] In one embodiment of the present invention, after extensive research and exploration, the present invention discovered a CRPC treatment drug, namely the combination of Enza and Stac. As can be seen from the research results, by creating prostate cancer LNCaP-drug-tolerant persisters (L-DTP) cell lines resistant to EPI and Enza, LNCaP cells acquired reversible drug resistance to EPI and Enza, and the combination of Enza and Stac significantly inhibited cell growth. The effect of the drug combination was verified by CCK8 cell proliferation analysis, and its synergistic effect was determined by the CI value. At the same time, a C-MYC overexpressing prostate cancer mouse model was constructed, and the difference in the effect of Enza and Stac alone versus in combination for the treatment of CRPC in animals was compared. The synergistic effect of the drug combination significantly improved the inhibitory effect of Enza or Stac alone against CRPC, and the synergistic effect of the two drugs has been verified in vivo and in vitro.

[0018] In one embodiment of the present invention, an L-DTP-recoverable drug-resistant cell line was created, and the CI value was calculated using the CCK8 method and Calcusyn software. The results showed that in this cell line, the in vitro combination of Enza and Stac had a synergistic effect against CRPC compared to the use of Enza or Stac alone. By establishing a C-MYC overexpressing prostate cancer mouse model, it was found that in a model in which drug resistance develops after long-term administration of Enza, the group using the combination of Enza and Stac in animals had a more significant anti-CRPC model effect in vivo compared to the monotherapy group. [Effects of the Invention]

[0019] The present invention has the following beneficial effects.

[0020] This invention is of great significance because it is the first to propose a novel strategy for preparing CRPC therapeutics using Stac and for treating CRPC based on the combined use of Enza and Stac, thereby promoting the use of Enza and Stac in the clinical treatment of prostate cancer. Drug research typically takes 8 to 10 years from compound molecule to clinical use and requires a large amount of human and material support, resulting in very high time and economic costs. The solution of this invention enables the reuse of natural compound oligosaccharides, which can significantly shorten the time from drug discovery to clinical application. [Brief explanation of the drawing]

[0021] [Figure 1] Figure 1 shows the formation process of DTP (drug-tolerant persisters) and DTEP (drug-tolerant expanded persisters) cells. [Figure 2] Figure 2 shows the characteristic changes in AR-related protein expression in DTP and DTEP cells. [Figure 3] Figure 3 shows the cyclical effects of DTP and DTEP cells. [Figure 4] Figure 4 shows the in vitro efficacy diagrams of L-DTP cells with the combined use of EPI and Enza with Stac. Figure 4A is a bar graph of the relative viability of L-DTP-EPI and L-DTP-Enza cells with combined use of the drugs, and Figure 4B is a bar graph of the CI values ​​for L-DTP-EPI and L-DTP-Enza cells with the combined use of EPI and Enza with Stac. [Figure 5] Figure 5 shows the effect of Enza, Stac, and their combination on changes in prostate weight after drug resistance developed following continuous administration of Enza in a C-MYC overexpressing prostate cancer mouse model. Figure 5A shows the changes in prostate weight of mice in each group as drug administration progresses. Figure 5B shows comparative photographs of prostates removed from mice in each group. Figure 5C shows the changes in body weight of mice in each group as drug administration progresses. [Figure 6]Figure 6 is a diagram showing the effects of Enza, Stac, and their combination after continuous administration of Enza in a mouse model of C-MYC overexpressing prostate cancer with drug resistance. Figure 6A is a HE staining photograph of prostate tissue sections of mice in each group. Figure 6B is a PRDX5 and AR immunohistochemical photograph of prostate tissue sections of mice in each group. Figure 6C is a bar graph of the quantification of positive cells by immunohistochemistry.

Mode for Carrying Out the Invention

[0022] Hereinafter, the present invention will be further described in combination with the drawings and specific examples of the specification. However, the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and apparatuses used in the present invention are conventional reagents, methods, and apparatuses in the technical field.

[0023] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0024] Example 1 Process of generating DTP / DTEP in prostate cancer LNCaP cells using EPI and Enza and characteristics of this model The prostate cancer L-DTP cell lines L-DTP-EPI and L-DTP-Enza resistant to EPI and Enza can inhibit the expression of AR and its target proteins, and the cell growth inhibition appears as a cell cycle arrest in the G0 / G1 phase.

[0025] 1. Experimental method: 1 × 10⁶ LNCaP cells were seeded in a 10 cm cell culture dish. After adhesion the following day, EPI and Enza were added, respectively, and treated for 9 days. During this period, fresh culture medium containing the drugs was changed every 3 days. For subsequent testing, some cells (i.e., DTP cells) were harvested after 9 days, and the remaining cells were continued to be treated with the drugs. During this period, fresh culture medium containing the drugs was changed every 3 days. For subsequent testing, cells were harvested after 33 days (i.e., DTEP cells). After generating DTP and DTEP cells, the cells were digested and counted, and the percentage of cells in a 1 × 10⁶ cell volume was calculated. The recovered NC, DTP, and DTEP cells were used in the subsequent Western Blot study. These three cell groups underwent cytolysis, protein extraction and quantification, SDS-PAGE gel electrophoresis, membrane transfer, blocking, primary antibody incubation, secondary antibody incubation, and contrast enhancement. Changes in the expression of AR-FL and its associated target proteins, AR-Vs and its associated target proteins, and cell cycle-related proteins were then observed. Flow cytometry: Dead cells were stained with a PI staining cycle kit, and changes in the cell cycle were measured by flow cytometry.

[0026] 2. The results are shown in Figures 1, 2, and 3. Figure 1 is a Giemsa-stained image of DTP and DTEP cells, Figure 2 shows the changes in protein expression of AR & its targets and AR-Vs & its targets in NC, DTP, and DTEP cells, and Figure 3 shows the changes in the expression of cell cycle-related proteins in NC, DTP, and DTEP cells.

[0027] The results showed that LNCaP-DTP cells generated by EPI and Enza accounted for 103.11% of the initial seeded cell volume, while LNCaP-DTEP cells accounted for 110.92%. Both of these drug-resistant cells were resistant to EPI and Enza, becoming spindle-shaped and inhibiting growth under DTP conditions, while DTEP conditions resulted in an increased number of cell clones, resistance to cell growth inhibition, and cell regrowth. Regarding the effects of this cell model on androgen receptor (AR) and its target protein expression, AR protein expression was inhibited under the DTP state, and AR expression was partially restored under DTEP conditions, while the expression of AR targets TMPRSS2 and PSA maintained a consistent trend with AR. Regarding the effects on androgen receptor splice variants (AR-Vs) and their target protein expression, under both the DTP and DTEP states, the expression of AR-Vs and their targets UBE2C and CDC20 proteins both appeared as continuous inhibition. This inhibition was caused by cell cycle arrest. Specifically, P21 (G1 phase marker) increased with DTP, and DTEP recovered to some extent; Cyclin E1 (G1-S phase marker) and CDC6 (G1-S phase marker) decreased with DTP, and DTEP recovered to some extent; and CDC2 (G1-S and G2-M phase marker) decreased with DTP, and DTEP did not recover significantly. Flow cytometry (FACS) analysis revealed that the G0 / G1 phase was arrested with DTP and recovered with DTEP.

[0028] Example 2: In vitro effects of combining EPI and Enza with Stac drugs. Furthermore, using CCK8, we explained the in vitro antitumor effects of Stac drugs on drug-resistant L-DTP (EPI) and L-DTP (Enza) cells, both when used alone and in combination with drug-resistant L-DTP cells.

[0029] 1. Experimental Method Drug-resistant L-DTP cells (including L-DTP(EPI) and L-DTP(Enza)) were seeded in 96-well plates and allowed to adhere. A series of Stac drug concentrations were then prepared from high to low to determine the optimal Stac drug concentration. Subsequently, the viability of drug-resistant L-DTP cells was measured at this concentration for single-drug use (L-DTP(EPI)-Stac), combination use (L-DTP(EPI)-combination(EPI+Stac)), and combination use (L-DTP(Enza)-combination(Enza+Stac)). Finally, CI values ​​were calculated for L-DTP cells using Calcusyn software.

[0030] 2. The results are shown in Figure 4. In Figure 4, Figure 4A is a bar graph of the relative viability of drug-treated cells in L-DTP (EPI) and L-DTP (Enza) drug-resistant cells, and Figure 4B is a bar graph of the CI values ​​in L-DTP (EPI) and L-DTP (Enz) drug-resistant cells, respectively, when EPI and Enza are used in combination with Stac.

[0031] The results are as follows: In L-DTP (EPI) cells that developed drug resistance after continuous administration of EPI for 9 days, no significant inhibitory effect was found with continued administration of EPI. However, a significant inhibitory effect was found when Stac was administered alone, reaching an inhibition rate of 67.48%. Subsequently, when used in combination (EPI + Stac), an inhibition rate of 51.15% was achieved. Similarly, in L-DTP (Enza) cells that developed drug resistance after continuous administration of Enza for 9 days, no significant inhibitory effect was found with continued administration of Enza. However, a significant inhibition rate was found when Stac was administered alone, reaching an inhibition rate of 62.47%. Subsequently, when used in combination (Enza + Stac), an inhibition rate of 51.97% was achieved. When the CI value was calculated, Stac could 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.

[0032] Example 3: Effect of combined use of Enza and Stac in a mouse model of C-MYC overexpressing prostate cancer after continuous administration of Enza has resulted in drug resistance. Furthermore, we described the effect of the combined use of Enza and Stac in mice that relapsed after chemical castration (i.e., continuous administration of Enza) in a mouse model of prostate cancer.

[0033] 1. Experimental Method We constructed a spontaneous prostate cancer mouse model that overexpresses C-MYC (Hi-Myc), and after 4 months, the mice developed mPIN / cancer The mice developed a transition, and were randomly divided into an NC control group (intragastric administration of the solvent) and an Enza administration group. Thereafter, Enza was administered intragastricly once every 3 days at a dose of 10 mg / kg for a total of 30 days. Subsequently, the necks of several mice were amputated, and images of the prostate cancer were taken and their weight measured. It was found that Enza significantly reduced symptoms, and the prostate weight was reduced by half compared to the NC control group. Subsequently, the remaining mice were administered the same method for another 30 days, and recurrence was observed in the Enza group. Thereafter (i.e., when the rats were 6 months old), they were randomly divided into an NC control group (always intragastric administration of the solvent), an Enza monotherapy group, a Stac monotherapy group, and an Enza and Stac combination group. The corresponding treatment was administered intragastricly once every 3 days, with Enza at a dose of 10 mg / kg and Stac at a dose of 80 mg / kg for a total of 30 days. Subsequently, the mice's necks were amputated, and experiments such as photography, weight measurement, and immunohistochemistry were performed on their prostate cancers.

[0034] 2. The results are shown in Figures 5 and 6. In Figure 5, Figure 5A shows the change in prostate weight of mice in each group as drug administration progresses, Figure 5B shows comparative photographs of prostates removed from mice in each group, and Figure 5C shows the change in body weight of mice in each group as drug administration progresses. In Figure 6, Figure 6A shows HE-stained photographs of prostate tissue sections from mice in each group, and Figures 6B and 6C show AR, PRDX5 immunohistochemical photographs, and bar graphs quantifying positive cells of prostate tissue sections from mice in each group.

[0035] The mean prostate weight of mice administered Enza continuously for 30 days was 43.9 mg, compared to 88.7 mg in the NC control group. After 90 days of continuous administration, the mean prostate weight of the Enza group mice was 77.1 mg, compared to 98.2 mg in the NC control group, indicating drug resistance relapse and the onset of CRPC. At this point, the effects of drug combination therapy were explained by immediately administering the drugs separately to each group.

[0036] The results for combination therapy and monotherapy by group are shown in Table 1.

[0037] As can be seen by combining Figure 5 and Table 1, the combination of Enza and Stac has a much more significant effect than the use of Enza alone or Stac alone, and the weight of the prostate can be reduced to approximately 37.068 mg. At the same time, the therapeutic effect of Stac alone is higher than that of Enza alone after drug resistance, indicating that Stac alone has an inhibitory effect on drug-resistant CRPC.

[0038] HE staining results of tissue sections (Figure 6) show that CRPC prostate tumors exhibit significant deformation and fibrosis after combination therapy. Immunohistochemistry shows that the combination of Enza and Stac significantly reduced AR and PRDX5 expression compared to Enza alone and Stac alone. This demonstrates the remarkable effect of the combination therapy.

[0039] The results for combination therapy and monotherapy by group are shown in Table 2.

[0040] TIFF0007869817000002.tif96170

Claims

1. A pharmaceutical composition for treating castration-resistant prostate cancer resistant to EPI or enzalutamide (Enza), comprising only stachyose and EPI or Enza as active ingredients, and not containing any other active ingredients.

2. The pharmaceutical composition according to Claim 1, characterized in that the mass ratio of EPI or Enza to stachyose is (1 to 8):

1.

3. The pharmaceutical composition according to claim 1, further comprising a pharmaceutical excipient.

4. The pharmaceutical composition according to claim 3, characterized in that the pharmaceutical excipient comprises a solvent, propellant, solubilizer, co-solvent, emulsifier, colorant, adhesive, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, flow aid, flavoring agent, preservative, suspension aid, coating agent, flavoring agent, anti-adhesion agent, bonding agent, penetration accelerator, pH adjuster, buffer, plasticizer, surfactant, foaming agent, defoaming agent, thickener, inclusion agent, humectant, absorbent, diluent, flocculant, decoagulant, filtration aid and / or release inhibitor.

5. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition may further contain a pharmaceutical carrier.

6. The pharmaceutical composition according to claim 5, characterized in that the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles, or liposomes.

7. The pharmaceutical composition according to any one of claims 1 to 6, characterized in that the dosage form of the pharmaceutical composition includes an injection solution, a lyophilized powder for injection, a sustained-release injection, a liposome injection, a suspension, an implantable agent, an embolizing agent, a capsule, a tablet, a pill, or an oral solution.

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