Application of composition containing istuss in preparation of anti-prostatic cancer drugs

Abstract

The invention relates to the technical field of prostatic cancer treatment, in particular to application of an istuss-containing composition in preparation of an anti-prostatic cancer drug. The invention discloses a composition which can enable an androgen receptor to be re-expressed and is combined with a medicine for targeting the androgen receptor to inhibit prostatic cancer, and the composition contains islas. Due to the re-expression of the androgen receptor, the tumor is re-sensitive to an endocrine therapeutic drug which targets the androgen receptor and has low toxicity. Therefore, the purpose of inhibiting the prostatic cancer, especially castration resisting the growth of the prostatic cancer, can be achieved through combined use of endocrine therapeutic drugs with relatively low toxicity. Meanwhile, the anti-PSMA antibody provided by the invention can specifically recognize PSMA and has good binding affinity. When the anti-PSMA antibody is combined with eiss for use, the lethality of the anti-PSMA antibody to prostatic cancer is remarkably increased, and the anti-PSMA antibody has a synergistic effect and can be effectively used for targeted therapy of prostatic cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of prostate cancer treatment, and in particular to an application of an ispins-containing composition in the preparation of an anti-prostate cancer drug. Background Art

[0002] Prostate cancer refers to an epithelial malignant tumor that occurs in the prostate. In 2004, WHO's "Pathology and Genetics of Tumors of the Urinary System and Male Reproductive Organs" listed the pathological types of prostate cancer as adenocarcinoma (acinar carcinoma), ductal adenocarcinoma, urothelial carcinoma, squamous cell carcinoma, and adenosquamous carcinoma. Among them, prostate adenocarcinoma accounts for more than 95%, so what we usually call prostate cancer refers to prostate adenocarcinoma. In 2012, the incidence of prostate cancer in my country's tumor registration areas was 9.92 / 100,000, ranking sixth in the incidence of male malignant tumors. The age of onset is at a low level before the age of 55, and gradually increases after the age of 55. The incidence rate increases with age, and the peak age is 70 to 80 years old. Patients with familial hereditary prostate cancer develop at a slightly earlier age, and patients aged ≤55 years account for 43%.

[0003] At present, androgen deprivation therapy (ADT) is regarded as the standard treatment for prostate cancer. This therapy reduces the level of androgen in the body or inhibits androgen receptors (AR) through surgery or the use of hormone receptor inhibitors, thereby slowing the progression of prostate cancer. However, most patients will develop drug resistance within 2 to 3 years after receiving androgen deprivation therapy, and the disease will evolve into castration-resistant prostate cancer (CRPC). Castration-resistant prostate cancer is a special type of prostate cancer, which is characterized by no longer being sensitive to conventional androgen deprivation therapy and can continue to develop even when receiving ADT treatment at low levels of blood testosterone. CRPC is usually accompanied by a high risk of metastasis, and is prone to metastasis to bones and lymph nodes. CRPC with metastasis is usually accompanied by a lower survival period. In addition, some patients with early prostate cancer will choose radical surgery or radical radiotherapy, and some low-risk and elderly patients can also choose active monitoring according to specific circumstances, and wait for further treatment after the disease progresses. However, it cannot fundamentally cure prostate cancer, and the above treatment methods are also relatively harmful to the subjects.

[0004] Therefore, there is a need to provide more effective therapeutic agents for prostate cancer to achieve targeted elimination of prostate cancer cells. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides an application of an ispins-containing composition in the preparation of an anti-prostate cancer drug. The present invention provides a method for targeted inhibition of kinesin superfamily member 11 (KIF11) to re-express or sensitize androgen receptors in prostate cancer, re-sensitize tumors to therapeutic drugs targeting androgen receptors with less toxicity, and then use anti-PSMA antibodies in combination to achieve the purpose of inhibiting the growth of prostate cancer, especially CRPC.

[0006] On the one hand, in order to achieve the above-mentioned purpose of the invention, the present invention provides the use of Ispins in preparing a combined anti-prostate cancer drug.

[0007] Among them, Ispinesib (ISP), also known as SB-715992, CK-0238273, is the first small molecule inhibitor of KIF11 and is an effective, specific, allosteric inhibitor. Ispinesib changes the binding of KIF11 to microtubules and inhibits the movement of KIF11 by preventing the release of ADP without changing the release of KIF11-ADP complex in microtubules. Ispinesib inhibits the proliferation of multiple cancer cells, induces apoptosis, and causes tumor regression in vivo. When Ispinesib is used in combination with anti-PSMA antibodies, it has stronger killing activity.

[0008] Unexpectedly, the inventors of the present invention have found that Ispins can sensitize or re-express androgen receptors, thereby resensitizing tumors to endocrine therapy targeting androgen receptors with less toxicity. In this way, the purpose of inhibiting CRPC growth can be achieved by combining endocrine therapy drugs targeting androgen receptors.

[0009] In the above-mentioned use of the present invention, the Ispins used are substances having the following structural formula:

[0010]

[0011] The molecular formula of Ispins is C 30 H 33 ClN4O2, CAS: 336113-53-2, the English name is (R)-N-(3-aminopropyl)-N-(1-(3-benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylpropyl)-4-methylbenzamide.

[0012] Preferably, in the above-mentioned use of the present invention, Ispins is used for preparing a combined anti-CRPC drug.

[0013] Preferably, in the above-mentioned use of the present invention, Ispins is used in combination with a drug targeting androgen receptor.

[0014] For example, such drugs targeting androgen receptors can be endocrine therapy drugs for prostate cancer, including but not limited to:

[0015] 1. The first generation of anti-androgen drugs: Flutamide, Bicalutamide, Nilutamide;

[0016] 2. Second-generation anti-androgen drugs: Apalutamide, Enzalutamide, Darolutamide.

[0017] On the other hand, in order to achieve the purpose of the present invention, the present invention also provides a composition that can activate or sensitize androgen receptors and is combined with a drug targeting androgen receptors to inhibit prostate cancer, wherein the composition contains Ispins.

[0018] Preferably, the prostate cancer targeted by the composition of the present invention is castration-resistant prostate cancer.

[0019] In the above-mentioned composition of the present invention, the Ispins used are substances having the following structural formula:

[0020]

[0021] The above-mentioned composition of the present invention also includes at least one pharmaceutically acceptable excipient, which is selected from the group consisting of but not limited to the following substances: excipients, disintegrants, carriers, sustained-release agents, adhesives, fillers, packaging capsules, various processing aids, etc.

[0022] Preferably, the composition of the present invention is in the form of an oral preparation, such as various oral tablets and capsules.

[0023] The present invention found that Ispins can re-express or sensitize androgen receptors, thereby re-sensitizing tumors to endocrine therapeutic drugs that target androgen receptors and have lower toxicity. In this way, the purpose of inhibiting the growth of prostate cancer, especially CRPC, can be achieved by combining the use of endocrine therapeutic drugs with lower toxicity.

[0024] Preferably, in the above-mentioned use of the present invention, Ispins is used in combination with an anti-PSMA antibody.

[0025] On the other hand, in order to achieve the purpose of the present invention, the present invention also provides a composition that can activate or sensitize androgen receptor and inhibit prostate cancer in combination with anti-PSMA antibodies, wherein the composition contains Ispins.

[0026] Preferably, the prostate cancer targeted by the composition of the present invention is castration-resistant prostate cancer.

[0027] In the above-mentioned composition of the present invention, the Ispins used are substances having the following structural formula:

[0028]

[0029] The above-mentioned composition of the present invention also includes at least one pharmaceutically acceptable excipient, which is selected from the group consisting of but not limited to the following substances: excipients, disintegrants, carriers, sustained-release agents, adhesives, fillers, packaging capsules, various processing aids, etc.

[0030] Preferably, the composition of the present invention is in the form of an oral preparation, such as various oral tablets and capsules.

[0031] On the other hand, one of the purposes of the present invention is to provide an anti-prostate cancer drug comprising the following components: 2-10 nM Ispins, 5-10 ng / mL anti-PSMA antibody.

[0032] Preferably, the anti-prostate cancer drug comprises the following components: 2 nM Ispins, 5 ng / mL anti-PSMA antibody.

[0033] Preferably, the anti-prostate cancer drug comprises the following components: 5 nM Ispins, 8 ng / mL anti-PSMA antibody.

[0034] Preferably, the anti-prostate cancer drug comprises the following components: 10 nM Ispins, 10 ng / mL anti-PSMA antibody.

[0035] Preferably, the heavy chain variable region sequence of the PSMA antibody is shown as SEQ ID NO.1, and the light chain variable region sequence is shown as SEQ ID NO.2.

[0036] Further preferably, the PSMA antibody heavy chain variable region includes CDR1, CDR2 and CDR3, whose sequences correspond to SEQ ID NOs. 3-5, respectively, and the PSMA antibody light chain variable region includes CDR4, CDR5 and CDR6, whose sequences correspond to SEQ ID NOs. 6-8, respectively.

[0037] On the other hand, the present invention also provides a method for preparing an anti-prostate cancer drug, characterized in that the method comprises the following steps:

[0038] 1) Weigh the Ispins by weight;

[0039] 2) preparing anti-PSMA antibodies;

[0040] 3) The Ispins prepared in step 1) and the anti-PSMA antibody prepared in step 2) are prepared in physiological saline according to the dosage ratio.

[0041] Preferably, the anti-PSMA antibody in step 2) is prepared by using hybridoma technology to immunize BALB / c mice with PSMA protein as an antigen, then isolate mouse spleen cells and fuse them with SP2 / 0 myeloma cells to screen for anti-PSMA antibodies.

[0042] The advantages of the present invention are as follows: The present invention found that Ispins can re-express or sensitize androgen receptors, thereby re-sensitizing tumors to endocrine therapeutic drugs that target androgen receptors and have less toxicity. In this way, the purpose of inhibiting the growth of prostate cancer, especially CRPC, can be achieved by combining with less toxic antibody drugs.

[0043] In addition, the anti-PSMA antibody provided by the present invention can specifically recognize PSMA and has good binding affinity. When used in combination with Ispins, its lethality to prostate cancer is significantly increased, and there is a synergistic effect, and it can be effectively used for targeted treatment of prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a scatter plot of correlation analysis of transcription levels of KIF11 and AR in prostate cancer tissues;

[0045] Figure 2 is a scatter plot of correlation analysis of transcription levels of KIF11 and KLK3 in prostate cancer tissues;

[0046] Figure 3 is a scatter plot of correlation analysis of transcription levels of KIF11 and TMPRSS2 in prostate cancer tissues;

[0047] Figure 4 is a series of box plots of the relative expression of KIF11 in androgen-sensitive prostate cancer and androgen-resistant prostate cancer gene datasets;

[0048] Figure 5 is a comprehensive box plot of the relative expression of KIF11 in androgen-sensitive prostate cancer or androgen-sensitive prostate cancer;

[0049] Figure 6 Scatter plot showing the correlation between transcriptional expression levels of KIF11 and tumor proliferation pathway markers in prostate cancer tissues;

[0050] Figure 7is the immunoblot of androgen receptor in the treated and control groups of PC3 cells and C4-2B cells;

[0051] Figure 8 is the relative expression bar graph of androgen receptor, prostate-specific antigen and transmembrane serine protease 2 transcription levels in the PC3 cell and C4-2B cell treated and control groups;

[0052] Fig. 9 is the cell proliferation curve of PC3 cells treated with enzalutamide or ispins;

[0053] Fig.10 is the cell proliferation curve of PC3 cells after treatment with apalutamide or ispins;

[0054] Fig.11 is the cell proliferation curve of C4-2B cells after being treated with enzalutamide or ispins;

[0055] Fig.12 is the cell proliferation curve of C4-2B cells after being treated with apalutamide or ispins;

[0056] Fig.13 are EdU and Hoechst staining images of PC3 cells treated with enzalutamide or ispins (left) and quantification bar graphs of EdU ratios in each group (right);

[0057] Fig.14 are EdU and Hoechst staining images of PC3 cells after treatment with apalutamide or ispins (left) and quantification bar graphs of EdU ratios in each group (right);

[0058] Fig.15 are EdU and Hoechst staining images of C4-2B cells treated with enzalutamide or ispins (left) and quantification bar graphs of EdU ratios in each group (right);

[0059] Fig.16 The EdU and Hoechst staining images of C4-2B cells after treatment with apalutamide or ispins (left) and the EdU ratio quantification bar graph of each group (right);

[0060] Fig.17 The 40X and 400X images of clones in each group after PC3 cells were treated with enzalutamide or ispins (left) and the quantitative bar graph of the diameter of clones in each group (right);

[0061] Fig.18 The 40X and 400X images of clones in each group after PC3 cells were treated with apalutamide or ispins (left) and the quantitative bar graph of the diameter of clones in each group (right);

[0062] Fig.19 The 40X and 400X microscope images of each group of clones after C4-2B cells were treated with enzalutamide or ispins (left) and the quantitative bar graph of the diameter of each group of clones (right);

[0063] Fig. 20 The 40X and 400X microscope images of each group of clones after C4-2B cells were treated with apalutamide or ispins (left) and the quantitative bar graph of the diameter of each group of clones (right);

[0064] Fig.21 is the PC3 tumor volume growth curve;

[0065] Fig. 22 is a bar graph of tumor weight in each group;

[0066] Fig.23 It is the curve graph of the percentage change of weight of nude mice in each group;

[0067] Fig.24 This is a real picture of the complete tumor of nude mice in each group;

[0068] Fig.25 is a bar graph of AR, PSA, and TMPRSS2 transcription levels in nude mouse xenograft tumors;

[0069] Fig.26 is the immunoblot of AR and PSA in nude mouse xenograft tumors;

[0070] Fig. 27 It is the drug's specific killing effect on prostate cancer cells;

[0071] Fig.28 It is a curve chart of drug effect on prostate tumor volume growth. DETAILED DESCRIPTION

[0072] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0073] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0074] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0075] Example 1

[0076] Correlation between androgen signaling pathway-related markers and KIF11 expression: GEPIA online data analysis of differential gene expression in prostate cancer showed that KIF11 was positively correlated with AR in transcriptome expression. The prostate cancer dataset showed that KIF11 was negatively correlated with kallikrein 3 (KLK3) downstream of AR in the androgen signaling pathway in transcriptome expression; similarly, KIF11 was negatively correlated with transmembrane serine protease 2 (TMPRSS2) downstream of AR in the androgen signaling pathway; please refer to Figure 1-Figure 3 , Figure 1-Figure 3 The scatter plots of the correlation analysis between the transcription levels of KIF11 and AR in prostate cancer tissue, the scatter plots of the correlation analysis between the transcription levels of KIF11 and KLK3 in prostate cancer tissue, and the scatter plots of the correlation analysis between the transcription levels of KIF11 and TMPRSS2 in prostate cancer tissue are shown respectively.

[0077] Example 2

[0078] The results of the differential gene expression analysis of KIF11 in different types of prostate cancer showed that the expression level of KIF11 in androgen-resistant prostate cancer (G2) was higher than that in androgen-sensitive prostate cancer (G1). Figure 4-Figure 5 , Figure 4 is a series of box plots of the relative expression of KIF11 for the androgen-sensitive prostate cancer gene expression dataset and the androgen-resistant prostate cancer gene expression dataset. Figure 5 is a comprehensive box plot of the relative expression of KIF11 in androgen-sensitive prostate cancer or androgen-insensitive prostate cancer.

[0079] Example 3

[0080] The analysis of KIF11 in prostate cancer tissues showed a positive correlation with tumor proliferation and related indicators, which was statistically significant. The Spearman correlation coefficient between the two was 0.73, indicating a strong correlation between the two. Figure 6 , Figure 6 Scatter plot showing the correlation between transcriptional expression levels of KIF11 and tumor proliferation pathway markers in prostate cancer tissues.

[0081] Example 4

[0082] Application of the drug Ispins, which targets and inhibits KIF11, to induce androgen receptor expression in androgen-independent prostate cancer cells PC3:

[0083] Using cell culture medium as solvent, 10^-9M IspIn culture medium was prepared, and androgen-independent prostate cancer cell lines PC3 and C4-2B were incubated with it for 48 hours. Western Blot and real-time fluorescence quantitative polymerase chain reaction (RT-PCR) were performed on the two incubated cells. It was detected that the protein level and messenger RNA (mRNA) level of androgen receptor in PC3 cells treated with IspIn were increased compared with the control group, and the androgen receptor downstream genes prostate specific antigen (PSA) and transmembrane serine proteinase 2 (TMPRSS2) were also increased; MDA-MB-453 was used as a positive control in the immunoblot; C4-2B cells themselves have androgen receptors, and there was no obvious change after IspIn treatment; please refer to Figure 7-Figure 8 ,in Figure 7 is the immunoblot of androgen receptor in the treated and control groups of PC3 cells and C4-2B cells; Figure 8 It is a relative expression bar graph of the transcriptional levels of androgen receptor, prostate specific antigen and transmembrane serine protease 2 in the PC3 cell and C4-2B cell treatment group and the control group. The cells used in the present invention include but are not limited to PC3, C4-2B, DU145, Vcap, 22Rv1.

[0084] Example 5

[0085] Prostate cancer cells treated with IspIns are sensitive to growth inhibition by Enzalutamide (ENZA) or Apalutamide (APA):

[0086] Prostate cancer cells PC3 and C4-2B were divided into 4 groups, namely control group, ISP group, single-drug group and combination group. The control group was treated with drug solvent DMSO, the ISP group was treated with ispins (concentration 10^-9M), the single-drug group was treated with enzalutamide (concentration 10^-5M) or apalutamide (concentration 10^-5M), and the combination group was treated with the same concentration of ispins + enzalutamide or ispins + apalutamide. The CCK8 method was used to detect the absorbance values ​​at 450 nm at 0 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours, and a cell proliferation curve was prepared based on the absorbance values. The results showed that the absorbance value of the combined group of PC3 cells and C4-2B cells was lower than that of other groups, indicating that ispins combined with enzalutamide or apalutamide can significantly inhibit the growth of castration-resistant prostate cancer cells. Please refer to Figure 9-12 ,in, Fig. 9 is the cell proliferation curve of PC3 cells treated with enzalutamide or ispins; Fig.10 is the cell proliferation curve of PC3 cells after treatment with apalutamide or ispins; Fig.11is the cell proliferation curve of C4-2B cells after being treated with enzalutamide or ispins; Fig.12 It is a cell proliferation curve of C4-2B cells after being treated with apalutamide or ispins.

[0087] Example 6

[0088] EdU proliferation experiment: The prostate cancer cells PC3 and C4-2B were divided into 4 groups, namely the control group, ISP group, single-drug group and combination group. The control group was treated with the drug solvent DMSO, the ISP group was treated with ispins (concentration 10^-9M), the single-drug group was treated with enzalutamide (concentration 10^-5M) or apalutamide (concentration 10^-5M), and the combination group was treated with the same concentration of ispins + enzalutamide or ispins + apalutamide. The EdU staining and Hoechst staining of each group of cells were detected according to the instructions of the EdU kit, and quantified according to the EdU ratio value. The results showed that the EdU staining and quantification ratio values ​​of the combined group of PC3 cells and C4-2B cells were lower than those of other groups, indicating that ispins combined with enzalutamide or apalutamide can significantly inhibit the proliferation of castration-resistant prostate cancer cells. Please refer to Figure 13-Figure 16 ,in, Fig.13 are EdU and Hoechst staining images of PC3 cells treated with enzalutamide or ispins (left) and quantification bar graphs of EdU ratios in each group (right); Fig.14 are EdU and Hoechst staining images of PC3 cells after treatment with apalutamide or ispins (left) and quantification bar graphs of EdU ratios in each group (right); Fig.15 are EdU and Hoechst staining images of C4-2B cells treated with enzalutamide or ispins (left) and quantification bar graphs of EdU ratios in each group (right); Fig.16 The EdU and Hoechst staining images of C4-2B cells after treatment with apalutamide or ispins (left) and the EdU ratio quantification bar graph of each group (right).

[0089] Example 7

[0090] Soft agar clone formation experiment: The prostate cancer cells PC3 and C4-2B were divided into 4 groups, namely the control group, ISP group, single-drug group and combination group. The control group was treated with drug solvent, the ISP group was treated with ispins (concentration 10^-9M), the single-drug group was treated with enzalutamide (concentration 10^-5M) or apalutamide (concentration 10^-5M), and the combination group was treated with the same concentration of ispins + enzalutamide or ispins + apalutamide. The cells in each group were cultured in soft agar for 15 days, and the diameters were measured and quantitatively compared after clone formation. The results showed that the number of clones formed and the diameter of the clones in the combination group of PC3 cells and C4-2B cells were lower than those in other groups, indicating that ispins combined with enzalutamide or apalutamide can significantly inhibit the growth and proliferation of castration-resistant prostate cancer cells. Please refer to Figure 17-Figure 20 ,in, Fig.17 The 40X and 400X images of clones in each group after PC3 cells were treated with enzalutamide or ispins (left) and the quantitative bar graph of the diameter of clones in each group (right); Fig.18 The 40X and 400X images of clones in each group after PC3 cells were treated with apalutamide or ispins (left) and the quantitative bar graph of the diameter of clones in each group (right); Fig.19 The 40X and 400X microscope images of each group of clones after C4-2B cells were treated with enzalutamide or ispins (left) and the quantitative bar graph of the diameter of each group of clones (right); Fig. 20 The 40X and 400X microscope images of each group of clones after C4-2B cells were treated with apalutamide or ispins (left) and the quantitative bar graph of the diameter of each group of clones (right).

[0091] Example 8

[0092] Animal tumor formation experiment: 4-week-old male Balbc / nude mice (Vitamin Liva) were raised in an SPF environment for one week, and each nude mouse was subcutaneously injected with 200ul cell suspension containing about 5×10^7 PC3 cells. 3Drug treatment was started at about 100 mg / kg. Ispins was intraperitoneally injected at a concentration of 1 mg / kg, once every 4 days; apalutamide and enzalutamide were gavaged at a concentration of 10 mg / kg, once a day. The long and short diameters of the tumor were measured every 3 days, and the volume formula was (long diameter × short diameter^2) / 2. The nude mice were divided into 6 groups with 5 mice in each group, namely the control group, ispins group, enzalutamide group, apalutamide group, ispins + enzalutamide group, and ispins + apalutamide group. The nude mice were killed by cervical dislocation on the 18th day after treatment, and the complete tumor was removed. The experimental results showed that after the prostate cancer tumors in nude mice were treated with combined drugs (ISP+ENZA or ISP+APA), the growth rate was significantly inhibited, the tumor weight was reduced, and there was no statistically significant change in the weight of the nude mice in each drug group. Then, tissue immunoblotting and qPCR experiments were performed on nude mouse tumors to detect the protein expression of AR and PSA in tumors in vivo, and the transcription levels of AR, PSA, and TMPRSS2. The results showed that Ispins induction could induce the expression of AR, PSA, and TMPRSS2. Figure 21-26 ,in, Fig.21 is the PC3 tumor volume growth curve; Fig. 22 is a bar graph of tumor weight in each group; Fig.23 It is the curve graph of the percentage change of weight of nude mice in each group; Fig.24 This is a real picture of the complete tumor of nude mice in each group; Fig.25 is a bar graph of the transcription levels of AR, PSA, and TMPRSS2 in transplanted tumors; Fig.26 The figure is the immunoblot of AR and PSA in the transplanted tumor.

[0093] Example 9

[0094] An anti-prostate cancer drug comprises the following components: 2nM Ispins and 5ng / mL anti-PSMA antibody.

[0095] Among them, anti-PSMA antibodies were screened by using hybridoma technology to immunize BALB / c mice with PSMA protein as antigen, then separating mouse spleen cells and fusing them with SP2 / 0 myeloma cells. The binding of candidate anti-PSMA antibodies to endogenous human PSMA expressed on human prostate cancer cells was determined by flow cytometry. The results showed that the EC50 value of anti-PSMA antibody binding to endogenous PSMA was 1.95nM, confirming that it has strong and specific binding to endogenous PSMA.

[0096] The isolated antibodies were sequenced and identified, and the heavy chain variable region sequence of the PSMA antibody of the present invention is shown in SEQ ID NO. 1, and the light chain variable region sequence is shown in SEQ ID NO. 2. The PSMA antibody heavy chain variable region includes CDR1, CDR2 and CDR3, and their sequences correspond to SEQ ID NO. 3-5, respectively, and the PSMA antibody light chain variable region includes CDR4, CDR5 and CDR6, and their sequences correspond to SEQ ID NO. 6-8, respectively.

[0097] The preparation method of the anti-prostate cancer drug comprises the following steps:

[0098] 1) Weigh the Ispins by weight;

[0099] 2) preparing anti-PSMA antibodies;

[0100] 3) The Ispins prepared in step 1) and the anti-PSMA antibody prepared in step 2) are prepared in physiological saline according to the dosage ratio.

[0101] Example 10

[0102] An anti-prostate cancer drug comprises the following components: 5 nM Ispins, 8 ng / mL anti-PSMA antibody. The preparation method is the same as that of Example 9.

[0103] Embodiment 11

[0104] An anti-prostate cancer drug comprises the following components: 10 nM Ispins, 10 ng / mL anti-PSMA antibody. The preparation method is the same as that of Example 9.

[0105] Comparative Example 1

[0106] An anti-prostate cancer drug comprises 10 nM Ispence, which is dissolved in physiological saline for later use.

[0107] Comparative Example 2

[0108] An anti-prostate cancer drug comprises 10 ng / mL anti-PSMA antibody, which is dissolved in physiological saline for later use.

[0109] Verification Example 1

[0110] Anti-prostate cancer drugs have specific killing effects on prostate cancer cells: 2×10 5 C4-2B tumor cells were added to the cell culture dish and incubated at 37℃5%CO2 for 2h. After they adhered to the wall, 100μL of drug was added for 6h. A blank group was set up, that is, an equal amount of cell culture medium was added. After the treatment, the cell killing rate was analyzed using the instructions of the LDH detection kit. The results are shown in Fig. 27As shown: the anti-prostate cancer drugs described in Examples 9-11 of the present invention have significant killing effects on C4-2B tumor cells, while the killing effects of Comparative Examples 1-2 on C4-2B tumor cells are limited, further confirming that the combination of Ispins and anti-PSMA antibodies has a synergistic killing effect on prostate cancer.

[0111] Verification Example 2

[0112] Animal tumor formation experiment: 4-week-old male Balbc / nude mice (Vitamin Liva) were kept in an SPF environment for one week, and each nude mouse was subcutaneously injected with a 5×10 7 200 μL cell suspension of C4-2B cells was used. When the tumor volume was 200 mm 3 Drug treatment was started at about 3:00 p.m. The tumor model mice were equally divided into a blank group, a single drug group 1, a single drug group 2 and a combination drug group, with 5 tumor model mice in each group. Among them, the combination drug group used the drug described in Example 11 for intraperitoneal injection at a concentration of 1 mg / kg, once every 4 days; the single drug group 1 used the drug described in Comparative Example 1 for intraperitoneal injection at a concentration of 1 mg / kg, once every 4 days; the single drug group 2 used the drug described in Comparative Example 2 for intraperitoneal injection at a concentration of 1 mg / kg, once every 4 days; the blank group was treated with an equal amount of normal saline injection. The long and short diameters of the tumor were measured every 3 days, and the volume formula is (long diameter × short diameter ^ 2) / 2. The nude mice were killed by cervical dislocation on the 18th day after treatment, and the complete tumor was removed. The experimental results are as follows Fig.28 It shows that after the prostate cancer tumors in nude mice received the combined drug group, the growth rate was significantly inhibited and the tumor weight was reduced; while the inhibition of the tumor was weaker under the single drug group treatment.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

[0114] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. Use of Ispins in the preparation of combined anti-prostate cancer drugs.

2. The use according to claim 1, characterized in that The Ispins is used for preparing a combined anti-CRPC drug.

3. The use according to claim 2, characterized in that The ispins can activate the re-expression of androgen receptor or sensitize androgen receptor.

4. The use according to claim 2, characterized in that The Ispins are combined with a drug that targets the androgen receptor.

5. The use according to claim 1, characterized in that The Ispins described is a substance having the following structural formula:

6. A composition capable of activating the re-expression of androgen receptor or sensitizing androgen receptor and combining with a drug targeting androgen receptor to inhibit prostate cancer, characterized in that: The composition contains Ispins.

7. The composition according to claim 6, characterized in that The prostate cancer is castration-resistant prostate cancer.

8. The composition according to claim 7, characterized in that The Ispins are Substances with the following structural formula:

9. The composition according to claim 8, characterized in that The composition also includes at least one pharmaceutically acceptable excipient.

10. The use according to claim 2, characterized in that The ISPINS is used in combination with an anti-PSMA antibody, wherein the PSMA antibody heavy chain variable region includes CDR1, CDR2 and CDR3, whose sequences correspond to SEQ ID NOs. 3-5, respectively, and the PSMA antibody light chain variable region includes CDR4, CDR5 and CDR6, whose sequences correspond to SEQ ID NOs. 6-8, respectively.

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