Use of polaprezinc in the preparation of a medicament for treating castration-resistant prostate cancer

Abstract

The present invention discloses the use of polaprezinc in the preparation of a therapeutic drug for castration-resistant prostate cancer, which belongs to the field of biomedicine. The present invention is the first to propose a new strategy for combining Pola with an androgen receptor antagonist to prepare a therapeutic drug for CRPC, and conducts multifaceted and multi-stage validation research. The pharmaceutical composition of the present invention combining polaprezinc and 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, realize a new use of old drugs, and significantly shorten the time from drug discovery to clinical translation, which is of great significance in clinical treatment.

Description

[Technical field]

[0001] The present invention relates to the field of biomedical technology, specifically to the use of polaprezinc in the preparation of a medicament for the treatment of castration-resistant prostate cancer. [Background technology]

[0002] Although androgen deprivation therapy is the standard treatment for advanced prostate cancer, patients eventually develop castration-resistant prostate cancer (CRPC) after an average of 1-3 years of treatment. According to the 2014 CUA Manual, CRPC refers to prostate cancer in which the disease continues to progress after the first continuous androgen deprivation therapy (ADT). The following conditions must be met simultaneously: (1) serum testosterone is maintained at castrate levels (<50 ng / dL or 1.7 nmol / L) and (2) biochemical progression is defined as PSA levels >50% above the nadir on three consecutive weekly scans with an absolute increase of >2 ng / ml, or radiographic progression is defined as two or more new lesions on bone scintigraphy or enlarging soft tissue lesions as assessed by RECIST. Currently, symptomatic progression alone is not considered sufficient to diagnose CRPC.

[0003] Previously, CRPC patients had no effective treatment and could only receive some palliative treatments. Since docetaxel was proven to be able to extend the overall survival of patients with metastatic castration-resistant prostate cancer (mCRPC) in 2004, drugs for the mCRPC disease stage, such as abiraterone acetate, enzalutamide, and cabazitaxel, have emerged, which have changed the treatment situation for these patients, but it is difficult to completely reverse CRPC. Therefore, finding an effective combination treatment strategy has become another research hotspot in CRPC treatment.

[0004] Recently, many scholars have proposed a new tumor cell drug resistance mechanism - persister cells, namely tumor cell plasticity, minimal residual disease, or drug-tolerant persisters (DTP). The characteristic of this mechanism is that in a drug-resistant state, tumor cells do not depend on the drug target pathway, but survive through other pathways, but no mutations occur in the target genes, and drug sensitivity is restored after a certain period of withdrawal. Currently, three hypotheses have been proposed to create this mechanism: (1) a small number of drug-resistant cells originally exist and increase after drug treatment through Darwinian evolution; (2) a small number of drug-resistant cells are epigenetically modified by a small portion of cancer cells to generate drug-resistant cells, which coexist with residual lesions; and (3) tumor cells dynamically express various drug-resistant genes, and during drug treatment, these drug-resistant genes are highly expressed, which further reconstructs the drug-resistant expression system and generates drug-resistant cells. In recent years, cell plasticity has emerged as a target evasion model and is a commonality of drug resistance in many cancers. Blocking new drug resistance pathways can effectively inhibit persister cells, for example, the GPX4 lipid peroxidation pathway is an effective target that is 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 effective combination drugs to treat CRPC, starting from prostate cancer LNCaP-persister cells generated by EPI-001 and Enzalutamide.

[0005] EPI-001 (EPI) is an inhibitor of AR and AR-splice variants (AR-Vs) awaiting clinical development and potentially used to treat CRPC. The target of EPI for CRPC is mainly the N-terminal domain (NTD). Enzalutamide (Enza) is the first approved second-generation AR antagonist, and has 5-8 times higher affinity for AR than conventional antiandrogens. In 2012, the US FDA approved Enza for CRPC patients based on this. However, drug resistance to the treatment of CRPC, whether EPI or Enza, usually appears around 18 months. Therefore, other methods to overcome drug resistance and delay CRPC are urgently needed.

[0006] Polaprezinc (Pola) is a chelated form of zinc and L-carnosine. It is the first zinc-related drug approved in Japan and is in clinical use to treat gastric ulcers. The results indicate that Pola may be effective in treating pressure ulcers. A 2013 study showed that Pola, when used in combination, may be effective in treating small intestinal mucosal damage caused by long-term use of aspirin. Summary of the Invention [Problem to be solved by the invention]

[0007] The technical problem to be solved by the present invention is to provide an effective therapeutic agent for CRPC that overcomes the drug resistance present in the above-mentioned conventional drugs, significantly improves the therapeutic effect of CRPC by using Enza and Pola in combination, and exerts an excellent synergistic effect. [Means for solving the problem]

[0008] A first object of the present invention is to provide a use of polaprezinc in the preparation of a medicament for treating castration-resistant prostate cancer.

[0009] In one embodiment of the invention, the use comprises combining polaprezinc with an androgen receptor antagonist to prepare a medicament for treating castration-resistant prostate cancer.

[0010] A second object of the present invention is to provide a pharmaceutical composition for treating castration-resistant prostate cancer, comprising polaprezinc and an androgen receptor antagonist.

[0011] In one embodiment of the present invention, the mass ratio of the androgen receptor antagonist to polaprezinc is (1-5): 1. Preferably, the mass ratio of Enza to Pola is 1-2:1.

[0012] In one embodiment of the invention, the androgen receptor antagonist comprises one or more of enzalutamide (Enza), EPI-001 (EPI), abiraterone, and olaparib.

[0013] In one embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutical excipient.

[0014] In one embodiment of the present invention, the pharmaceutical excipients include solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, taste masking agents, preservatives, suspension aids, coating materials, flavorings, anti-adhesion agents, integrating agents, permeation enhancers, pH value adjusters, buffers, plasticizers, surfactants, foaming agents, anti-foaming agents, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculating and deflocculating agents, filter aids and release inhibitors.

[0015] In one embodiment of the present invention, the dosage forms of the formulation include injection solutions, lyophilized powders for injection, sustained release injections, liposome injections, suspensions, implants, embolic agents, capsules, tablets, pills, and oral liquids.

[0016] In one embodiment of the invention, the pharmaceutical composition may further comprise a pharmaceutical carrier.

[0017] In one embodiment of the invention, the pharmaceutical carrier comprises a microcapsule, a microsphere, a nanoparticle, or a liposome.

[0018] In one embodiment of the present invention, after a large amount of research and exploration, the present invention has found a CRPC treatment drug, namely, the combination of Enza and Pola. As can be seen from the research results, by creating a prostate cancer LNCaP-drug-tolerant persisters (L-DTP) cell line resistant to EPI and Enza, LNCaP cells acquire reversible drug resistance to EPI and Enza, and the combination of Enza and Pola can significantly inhibit cell growth, and the drug combination effect is verified by CCK8 cell proliferation analysis, and the synergistic effect is determined by the CI value. At the same time, a C-MYC overexpressing prostate cancer mouse model is constructed to compare the difference in the effect of Enza and Pola alone and in combination in the treatment of CRPC in animals, and the synergistic effect of the combination of drugs significantly improves the inhibitory effect of Enza alone against CRPC, and the synergistic effect of the two has been verified in vivo and in vitro.

[0019] In one embodiment of the present invention, the present invention creates a drug-resistant cell line that can recover L-DTP, and calculates the CI value using the CCK8 method and Calcusyn software. As a result, it was shown that in this cell line, the combination of Enza and Pola in vitro has a synergistic effect against CRPC compared to the single use of Enza or Pola. By establishing a C-MYC overexpressing prostate cancer mouse model, it was found that in a model in which drug resistance develops due to long-term administration of Enza, the combination group of Enza and Pola in animals has a more significant anti-CRPC model effect in vivo than the single drug group. Effect of the Invention

[0020] The present invention has the following beneficial effects:

[0021] This invention is the first to propose a new strategy based on the preparation of a CRPC drug using Pola and the combined use of Enza and Pola, and describes its mechanism of action, which promotes the use of Enza and Pola in the clinical treatment of prostate cancer, and is of great significance. Drug research takes an average of 8 to 10 years from compound molecules to clinical use, and requires a lot of human and material support, which results in very high time and economic costs. The solution of the present invention can realize "new uses of old drugs" and significantly shorten the time from drug discovery to clinical translation. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 is an explanatory diagram of the recoverable characteristics of DTP (drug-tolerant persisters) after drug withdrawal. FIG. 1A shows the process of cell recovery after DTP and drug withdrawal, and FIG. 1B shows the recovery characteristics of sensitivity to the corresponding drugs after three generations of DTP-EPI and three generations of DTP-Enza withdrawal. [Diagram 2] Figure 2 shows the characteristics of expression changes of AR-related genes in DTP and withdrawal (Recovery Cells), where Figure 2A shows the changes in protein expression detected by WB, and Figure 2B shows the changes in transcript expression detected by q-PCR. [Diagram 3] Figure 3 shows the in vitro efficacy of EPI, Enza, and Pola in combination in L-DTP cells. Figure 3A shows a bar graph of the relative cell viability in L-DTP-EPI and L-DTP-Enza cells when drugs were combined. Figure 3B shows a bar graph of the CI values ​​in L-DTP-EPI and L-DTP-Enza cells when EPI, Enza, and Pola were combined. [Figure 4]Figure 4 is a graph showing the effect of Enza, Pola, and their combination on changes in prostate weight in a C-MYC-overexpressing prostate cancer mouse model after continuous administration of Enza led to the development of drug resistance. Figure 4A is a graph showing changes in prostate weight in mice in each group as drug administration progressed. Figure 4B is a comparative photograph of the prostate glands removed from mice in each group, and Figure 4C is a graph showing changes in body weight in mice in each group as drug administration progressed. [Diagram 5] Figure 5 shows the effects of Enza, Pola and their combination in a mouse model of C-MYC-overexpressing prostate cancer after continuous administration of Enza had developed drug resistance. Figure 5A shows HE-stained photographs of prostate tissue sections from mice in each group, and Figure 5B shows PRDX5 and AR immunohistochemical photographs of prostate tissue sections from mice in each group, as well as bar graphs quantification of positive cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention will be further described below in combination with the drawings and specific examples in the specification, but the examples are not intended to limit the present invention in any manner. Unless otherwise specified, the reagents, methods and devices used in the present invention are conventional reagents, methods and devices in the art.

[0024] Unless otherwise stated, all reagents and materials used in the following examples are commercially available.

[0025] Example 1: Process for producing DTP in prostate cancer LNCaP cells using EPI, Enza Prostate cancer L-DTP cell lines resistant to EPI and Enza, L-DTP-EPI and L-DTP-Enza, have reversible properties.

[0026] 1. Experimental method: 1×106 LNCaP cells were seeded on a 10cm cell culture dish, and after adhesion the next day, EPI and Enza were added and treated for 9 days. During this period, fresh medium containing the drug was replaced every 3 days, and the drug was withdrawn after 9 days. The medium was replaced with fresh medium and the cells were cultured normally in an incubator. They were passaged on the 6th, 12th, and 17th days, respectively, and photos of the cell morphology of DTP (9th day), R5 (5th day of withdrawal), R10, and R20 were taken with an inverted microscope. After generating DTP and cloned DTP cells, the cells were digested and counted, and the percentage of DTP cells in the total L-parent cells was calculated. L-parent cells and the three generations of cells after withdrawal (R20) were seeded in 96-well plates, respectively, and allowed to adhere overnight. After that, a series of EPI and Enza were prepared from high to low concentrations, and the control group was added to the wells. Three duplicate wells were set up for each concentration, and after 48 hours of incubation in a cell incubator, CCK8 was added. After 4 hours, the cell OD value at 450 nm wavelength was detected using a full-wavelength multi-function microplate reader, and the viability was counted to draw a survival curve.

[0027] 2, and the results are shown in Figure 1 and Table 1. In Figure 1, Figure 1A shows the process of cell recovery after DTP and withdrawal, and Figure 1B shows the recovery characteristics of the sensitivity to the corresponding drugs after three generations of DTP-EPI and three generations of DTP-Enza withdrawal. TIFF0007660714000001.tif88170The results show that the number of LNCaP-DTP cells generated by EPI and Enza is very small, and all of these drug-resistant cells have drug resistance to EPI and Enza, but the cell morphology can be restored after three generations of drug withdrawal, and the sensitivity to the drugs is also restored.

[0028] Example 2 Establishment of characteristics of expression changes of AR-related genes in DTP and withdrawal (Recovery Cell) cells The cell morphology of DTP cells recovered after three generations of drug withdrawal, and the changes in the expression of AR-related genes in the cells at this time could also be restored.

[0029] 1. Experimental method: LNCaP cells were seeded in 10 cm dishes and allowed to adhere overnight. After treatment with EPI and Enza for 9 days, the cells were harvested. During this period, fresh medium containing the drugs was replaced every 3 days. The remaining DTP cells were harvested in the same manner 20 days after withdrawal. LNCaP cells treated with DMSO for 9 days were used as the NC control group, and drug-resistant cells treated with EPI and Enza for 9 days to generate L-DTP-EPI and L-DTP-Enza were used as the treatment group. The cells in this treatment group 20 days after withdrawal were used as the recovery group. 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 imaging to observe the expression changes of AR-FL and its associated target proteins, and AR-Vs and its associated target proteins. At the same time, q-RTPCR measurements were performed on the three groups of cells to detect the expression changes of AR-FL and its related target genes, AR-Vs and its related target genes.

[0030] 2, and the results are shown in Figure 2. In Figure 2, Figure 2A shows the changes in protein expression detected by WB, and Figure 2B shows the changes in transcript expression detected by q-RTPCR.

[0031] The results showed that the expression of AR and its associated target proteins PSA, TMPRSS2, AR-Vs and its associated target proteins UBE2C, CDC20 were all decreased to different degrees during the DTP stage, but the expression was restored after withdrawal R20; similarly, the expression of AR-Vs and its associated target genes, AR-Vs and its associated target genes, and growth markers: AKT1, C-MYC were also decreased during the DTP stage, and the expression was restored to different degrees during R20.

[0032] Example 3 Combination of EPI, Enza and Pola drugs.

[0033] Furthermore, we used CCK8 to describe the in vitro antitumor effects of Pola drugs in drug-resistant L-DTP(EPI) and L-DTP(Enza) cells, either alone or in combination with other drugs.

[0034] 1. Experimental method Drug-resistant cells L-DTP (including L-DTP(EPI) and L-DTP(Enza)) were seeded on a 96-well plate, and after adhesion, a series of Pola drugs from high to low concentrations were prepared to find the optimal Pola drug concentration, and then the viability of the drug-resistant cells L-DTP was measured at this concentration by using alone (L-DTP(EPI)-Pola), combination [L-DTP(EPI)-combination(EPI+Pola)], [L-DTP(Enza)-combination(Enza+Pola)]. Finally, the CI value was calculated for L-DTP cells using Calcusyn software.

[0035] 2. The results are shown in Figure 3. In Figure 3, Figure 3A is a bar graph of the relative survival rate of L-DTP (EPI) and L-DTP (Enza) drug-resistant cells treated with the combination of drugs, and Figure 3B is a bar graph of the CI value of L-DTP (EPI) and L-DTP (Enz) drug-resistant cells treated with the combination of EPI and Enza with Pola, respectively.

[0036] The results show the following. In L-DTP (EPI) cells in which drug resistance was developed by continuous administration of EPI for 9 days, no significant inhibitory effect was found even with continued administration of EPI, and no significant inhibitory rate was found even with administration of Pola alone, but in the case of combined use (EPI + Pola), a 55.74% inhibitory rate could be reached. Similarly, in L-DTP (Enza) cells in which drug resistance was developed by continuous administration of Enza for 9 days, no significant inhibitory effect was found even with continued administration of Enza, and no significant inhibitory rate was found even with administration of Pola alone, but in the case of combined use (Enza + Pola), a 60.765% inhibitory rate could be reached. When the CI value was calculated, Pola could reach a high synergistic effect of 0.525 in L-DTP-EPI cells and a high synergistic effect of 0.695 in L-DTP (Enza) cells.

[0037] Example 4: Effect of combined use of Enza and Pola after continuous administration of Enza in a mouse model of C-MYC-overexpressing prostate cancer and development of drug resistance Furthermore, in a mouse model of prostate cancer, we described the effect of combining Enza with Pola on mice that relapsed after chemical castration (i.e., continuous administration of Enza).

[0038] 1. Experimental method We established a spontaneous prostate cancer mouse model overexpressing C-MYC (Hi-Myc), and within 4 months, the mice were mPIN / Cancer At this time, the mice were randomly divided into an NC control group (intragastric administration of solvent) and an Enza administration group, and then intragastric administration was performed once every three days, with Enza at 10mg / Kg, for a total of 30 days. After that, several mice were decapitated, and the prostate cancer was photographed and weighed. It was found that Enza could significantly alleviate the symptoms, and the prostate weight was reduced by half compared to the NC control group. Then, the remaining mice were continuously administered Enza for 30 days using the above method, and it was found that there was a recurrence in the Enza group. After that (i.e., when the rats were 6 months old), the mice were randomly divided into an NC control group (always intragastric administration of solvent), an Enza single agent group, a Pola single agent group, and an Enza and Pola combination group, and the corresponding administration treatment was performed. All were intragastric administration once every three days, with Enza at 10mg / Kg and Pola at 20mg / Kg each time, for a total of 30 days. The mice were then decapitated and the prostate cancer was photographed, weighed, and subjected to immunohistochemistry and other experiments.

[0039] 2, the results are shown in Figure 4 and Figure 5. In Figure 4, Figure 4A is a graph showing the change in the weight of the prostate of each group of mice with the progress of drug administration, Figure 4B is a comparative photograph of the prostate of each group of mice taken by excising it, and Figure 4C is a graph showing the change in the weight of each group of mice with the progress of drug administration. In Figure 5, Figure 5A is a HE stained photograph of the prostate tissue section of each group of mice, and Figure 5B is a bar graph showing the AR, PRDX5 immunohistochemistry photograph of the prostate tissue section of each group of mice and the quantification of positive cells.

[0040] The average prostate weight of mice that were administered Enza for 30 consecutive days was 41.2 mg, while the average weight of the NC control group was 85.3 mg. After 60 consecutive days of administration, the average prostate weight of the mice in the Enza group was 78.4 mg, while the average weight of the NC control group was 95.6 mg, indicating that drug-resistant recurrence had occurred and CRPC had been caused. At this point, the drugs were immediately administered separately to explain the effect of the combination therapy.

[0041] The results of combination and single-agent use by group are shown in Table 2. TIFF0007660714000002.tif71170As can be seen from the combination of Figure 4 and Table 2, the combination of Enza and Pola has a very significant effect compared with the use of Enza alone and the use of Pola alone, and the prostate weight can be reduced to about 53.14 mg. At the same time, the effect of using Pola alone is almost the same as that of using Enza alone, indicating that the use of Pola alone has no significant effect on drug-resistant CRPC.

[0042] HE staining of tissue sections (Figure 5) showed that after combination treatment, CRPC prostate tumors showed significant deformation and fibrosis. Immunohistochemistry showed that the combination of Enza and Pola significantly reduced the expression of AR and PRDX5 compared with Enza alone and Pola alone, proving the significant effect of the combination.

[0043] The results of combination and single-agent use by group are shown in Table 3. TIFF0007660714000003.tif96170

Claims

1. Use of polaprezinc in the preparation of a medicament for treating castration-resistant prostate cancer, the use comprising combining polaprezinc with an androgen receptor antagonist to prepare a medicament for treating castration-resistant prostate cancer.

2. A pharmaceutical composition for treating castration-resistant prostate cancer, comprising polaprezinc and an androgen receptor antagonist.

3. The pharmaceutical composition according to claim 2, characterized in that the mass ratio of the androgen receptor antagonist to polaprezinc is (1-5):

1.

4. The pharmaceutical composition according to claim 2, characterized in that the androgen receptor antagonist comprises one or more of enzalutamide and EPI.

5. The pharmaceutical composition of claim 2, further comprising a pharmaceutical excipient.

6. The pharmaceutical composition according to claim 5, characterized in that the pharmaceutical excipient comprises a solvent, a propellant, a solubilizer, a co-solvent, an emulsifier, a colorant, an adhesive, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a flow aid, a taste masking agent, a preservative, a suspension aid, a coating material, a flavoring agent, an anti-adhesion agent, a integrating agent, a penetration enhancer, a pH value adjuster, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickener, an inclusion agent, a moisturizing agent, an absorbent, a diluent, a flocculating agent, a deflocculating agent, a filter aid and / or a release inhibitor.

7. 3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition further comprises a pharmaceutical carrier.

8. 8. The pharmaceutical composition of claim 7, wherein the pharmaceutical carrier comprises a microcapsule, a microsphere, a nanoparticle or a liposome.

9. The pharmaceutical composition according to any one of claims 2 to 8, wherein 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 implant, an embolization agent, a capsule, a tablet, a pill, or an oral liquid.

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