JP2024516326A - Use of polaprezinc in the preparation of a medicament for treating castration-resistant prostate cancer - Google Patents

Abstract

The present invention discloses the use of polaprezinc in preparing a therapeutic drug for castration-resistant prostate cancer, which belongs to the field of biomedical technology. 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 greatly shorten the time from drug discovery to clinical translation, which is of great significance in clinical treatment. [Selected Figure] Figure 4

Description

The present invention belongs to the field of biomedical technology, and specifically relates to the use of polaprezinc in the preparation of a medicament for treating castration-resistant prostate cancer.

Androgen deprivation therapy is the standard treatment for advanced prostate cancer, but 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 castration levels (<50 ng/dL or 1.7 nmol/L); (2) biochemical progression is defined as PSA levels >50% above the nadir on three consecutive weekly measurements 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, symptom progression alone is not considered sufficient to diagnose CRPC.

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 metastatic castration-resistant prostate cancer (mCRPC) patients 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 cure CRPC. Therefore, finding an effective combination treatment strategy has become another research hotspot in CRPC treatment.

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 gene, 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 by 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 and coexist with residual lesions; (3) Tumor cells dynamically express various drug-resistant genes, and during drug treatment, these drug-resistant genes are highly expressed to further reconstruct the drug-resistant expression system and generate drug-resistant cells. In recent years, cellular plasticity has emerged as a target diagnostic evasion model and is a commonality of many cancer drug resistance. 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-persistent cells generated by EPI-001 and Enzalutamide.

EPI-001 (EPI) is an inhibitor of AR and AR-splice variants (AR-Vs) pending clinical development that may be 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.

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.

The technical problem that the present invention aims to solve is to provide an effective CRPC treatment drug that overcomes the drug resistance present in the above-mentioned conventional drugs, significantly improves the therapeutic effect of CRPC by combining Enza and Pola, and exerts an excellent synergistic effect.

The first object of the present invention is to provide a use of polaprezinc in the preparation of a therapeutic agent for castration-resistant prostate cancer.

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

The 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.

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.

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

In one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutical excipient.

In one embodiment of the present invention, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, 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, penetration enhancers, pH value regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, moisturizing agents, absorbents, diluents, flocculants and deflocculants, filter aids and release inhibitors.

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 implant, an embolization agent, a capsule, a tablet, a pill, and an oral liquid.

In one embodiment of the present invention, the pharmaceutical composition may further comprise a pharmaceutical carrier.

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

In one embodiment of the present invention, after extensive 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 used alone and in combination in the treatment of CRPC in animals, and the synergistic effect of the drug combination significantly improves the inhibitory effect of Enza alone on CRPC, and the synergistic effect of the two has been verified in vivo and in vitro.

In one embodiment of the present invention, the present invention creates a drug-resistant cell line capable of recovering 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 in vitro combination of Enza and Pola 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 in animals with Enza and Pola has a more significant anti-CRPC model effect in vivo compared to the single drug group.

The present invention has the following beneficial effects:

The present invention is the first to propose a new strategy based on the preparation of a CRPC therapeutic 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, resulting in very high time and economic costs. The solution of the present invention can realize "new uses for old drugs" and significantly shorten the time from drug discovery to clinical translation.

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. FIG. 2 shows the profile of changes in expression of AR-related genes in DTP and recovery cells, where FIG. 2A shows changes in protein expression detected by WB and FIG. 2B shows changes in transcript expression detected by q-PCR. FIG. 3 shows in vitro efficacy diagrams of EPI, Enza, and Pola in combination in L-DTP cells. FIG. 3A shows bar graphs of relative cell viability in L-DTP-EPI and L-DTP-Enza cells when drugs were combined. FIG. 3B shows bar graphs of CI values in L-DTP-EPI and L-DTP-Enza cells when EPI, Enza, and Pola were combined. FIG. 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; FIG. 4A is a graph showing changes in prostate weight in mice from each group as drug administration progresses; FIG. 4B is a comparative photograph of the prostate glands of mice from each group, which were then excised and photographed; and FIG. 4C is a graph showing changes in body weight in mice from each group as drug administration progresses. FIG. 5 is a graph showing the effects of Enza, Pola and their combination in a C-MYC overexpressing prostate cancer mouse model after continuous administration of Enza led to the development of drug resistance. FIG. 5A is a photograph of HE staining of prostate tissue sections from mice in each group, and FIG. 5B is a photograph of PRDX5 and AR immunohistochemistry of prostate tissue sections from mice in each group, and a bar graph showing the quantification of positive cells.

The present invention is further described below in combination with the drawings and specific examples of 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 technical field.

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

Example 1 Process of Producing DTP in Prostate Cancer LNCaP Cells Using EPI and Enza The prostate cancer L-DTP cell lines resistant to EPI and Enza, L-DTP-EPI and L-DTP-Enza, have restorable properties.

1. Experimental method:
1x106 LNCaP cells were seeded on a 10cm cell culture dish, and after attachment 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, and 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 after 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 third generation (R20) cells after withdrawal were seeded separately in 96-well plates and allowed to adhere overnight. 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. 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 survival rate was counted to draw a survival curve.

その結果は、ＥＰＩ、Ｅｎｚａによって生成されるＬＮＣａＰ－ＤＴＰ細胞の数が非常に少なく、これらの薬剤耐性細胞が全てＥＰＩ、Ｅｎｚａに対して薬剤耐性を有するが、３世代退薬した後に細胞形態が回復でき、薬剤に対する感受性も回復することを示している。 2, 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 withdrawal of DTP-EPI and three generations of DTP-Enza.

The results showed that the number of LNCaP-DTP cells generated by EPI and Enza was very small, and all of these drug-resistant cells were drug-resistant to EPI and Enza, but the cell morphology could be restored after three generations of drug withdrawal, and sensitivity to the drugs was also restored.

Example 2 Establishment of characteristics of expression changes of AR-related genes in DTP and drug withdrawal (recovery cells) After three generations of drug withdrawal of DTP cells, the cell morphology is recovered, and the expression changes of AR-related genes in the cells at this time can also be recovered.

1. Experimental method:
LNCaP cells were seeded on a 10 cm dish, allowed to adhere overnight, and then EPI and Enza were added to treat the cells for 9 days, after which the cells were harvested. During this period, fresh medium containing the drug was replaced every 3 days, and 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 produce L-DTP-EPI and L-DTP-Enza were used as the treatment group. The cells of this treatment group 20 days after withdrawal were used as the recovery group, and the three groups of cells were subjected to cell dissolution, protein extraction and quantification, SDS-PAGE gel electrophoresis, membrane transfer, blocking, primary antibody incubation, secondary antibody incubation, and imaging, after which the expression changes of AR-FL and its related target proteins, and AR-Vs and its related target proteins were observed. 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, and AR-Vs and its related target genes.

2. 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.

The results show that the expression of AR and its associated target proteins PSA, TMPRSS2, AR-Vs and its associated target proteins UBE2C, CDC20 all decreased to different degrees during the DTP stage, but the expression recovered 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 also decreased during the DTP stage, and the expression of R20 recovered to different degrees.

Example 3: Combination use of EPI, Enza and Pola drugs.

Furthermore, we used CCK8 to explain 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.

1. Experimental Methods Drug-resistant L-DTP cells (including L-DTP (EPI) and L-DTP (Enza)) were seeded on a 96-well plate and allowed to adhere. A series of Pola drugs were prepared from high to low concentrations to find the optimal Pola drug concentration. Then, at this concentration, the survival rate of the drug-resistant L-DTP cells was measured by using each of the following combinations: single use (L-DTP (EPI)-Pola), combination [L-DTP (EPI)-combination (EPI + Pola)], and [L-DTP (Enza)-combination (Enza + Pola)]. Finally, the CI value was calculated for L-DTP cells using Calcusyn software.

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 values of L-DTP (EPI) and L-DTP (Enz) drug-resistant cells treated with the combination of EPI and Enza with Pola.

The results show the following. In L-DTP (EPI) cells in which drug resistance was developed by administering EPI continuously 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 administering Enza continuously 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.

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

1. Experimental Method A spontaneous prostate cancer mouse model overexpressing C-MYC (Hi-Myc) was established, and the mice were transformed into mPIN/Cancer mice within 4 months. 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 3 days, with Enza at 10mg/Kg, for a total of 30 days. After that, the necks of several mice were cut off, and the prostate cancer was photographed and weighed. It was found that Enza could significantly alleviate the symptoms, and the prostate weight was reduced to half that of the NC control group. Then, the remaining mice were continuously administered Enza for 30 days using the above method, and it was found that the Enza group had recurrence. 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 combined group, and the corresponding administration treatment was performed. All were intragastric administration once every 3 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.

2. The results are shown in Figures 4 and 5. In Figure 4, Figure 4A is a graph showing the change in prostate weight of mice in each group as drug administration progresses, Figure 4B is a comparative photograph of the prostate gland excised from mice in each group, and Figure 4C is a graph showing the change in body weight of mice in each group as drug administration progresses. In Figure 5, Figure 5A is a photograph of HE staining of prostate tissue sections from mice in each group, and Figure 5B is a photograph of AR and PRDX5 immunohistochemistry of prostate tissue sections from mice in each group, and a bar graph of quantification of positive cells.

The average prostate weight of mice 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 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 induced. At this point, administration was immediately performed separately for each group, explaining the effect of the drug combination.

図４及び表２を組み合わせて分かるように、Ｅｎｚａの単独使用及びＰｏｌａの単独使用に比べてＥｎｚａとＰｏｌａの併用は非常に顕著な効果があり、前立腺の重量は約５３．１４ｍｇに減少することができ、それと同時に、Ｐｏｌａの単独使用の効果はＥｎｚａの単独使用の効果とほぼ同等であり、Ｐｏｌａの単独使用は薬剤耐性のあるＣＲＰＣに対して顕著な効果がないことを示している。 The results of combination and single-agent use by group are shown in Table 2.

As can be seen from the combination of Figure 4 and Table 2, compared with the use of Enza alone and the use of Pola alone, the combination of Enza and Pola has a very significant effect, 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.

The results of HE staining of tissue sections (Figure 5) show 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 to the use of Enza alone and the use of Pola alone, proving that the effect of the combination is significant.

The results of combined and single use by group are shown in Table 3.

Claims (10)

Use of polaprezinc in the preparation of a medicament for treating castration-resistant prostate cancer. The use according to claim 1, characterized in that the use comprises combining polaprezinc with an androgen receptor antagonist to prepare a therapeutic agent for castration-resistant prostate cancer. The composition is a pharmaceutical composition for treating castration-resistant prostate cancer, characterized in that it contains polaprezinc and an androgen receptor antagonist. The pharmaceutical composition according to claim 3, characterized in that the mass ratio of the androgen receptor antagonist to polaprezinc is (1-5):1. The pharmaceutical composition according to claim 3, characterized in that the androgen receptor antagonist comprises one or more of enzalutamide, EPI, abiraterone, and olaparib. The pharmaceutical composition according to claim 3, characterized in that the pharmaceutical composition further comprises a pharmaceutical excipient. The pharmaceutical composition according to claim 6, characterized in that the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, 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, penetration enhancers, pH value adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, moisturizing agents, absorbents, diluents, flocculants and deflocculants, filter aids and release inhibitors. The pharmaceutical composition according to claim 3, characterized in that the pharmaceutical composition may further comprise a pharmaceutical carrier. The pharmaceutical composition according to claim 8, characterized in that the pharmaceutical carrier comprises a microcapsule, a microsphere, a nanoparticle, and a liposome. The pharmaceutical composition according to any one of claims 3 to 9, 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 implant, an embolization agent, a capsule, a tablet, a pill, and an oral liquid.

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