Combined application of PCNA inhibitor and PARP1 inhibitor

Abstract

The invention discloses a combined application of a PCNA inhibitor and a PARP1 inhibitor in preparation of medicines for treating cancers, which has a synergistic interaction effect and lower toxicity, can be used for treating various cancers with high expression of PCNA and PARP1, especially has a remarkable curative effect on tumors such as liver cancer, colon cancer, triple negative breast cancer and melanoma, and can be used for preparing medicines for treating cancers. The problems of poor treatment effect, narrow indication, easiness in drug resistance, easiness in relapse and the like of independent medication are solved, and a new strategy is provided for tumor treatment.

Description

Technical Field

[0001] The present invention relates to a combined application of a PCNA inhibitor and a PARP1 inhibitor, and in particular to a combined application of a PCNA inhibitor and a PARP1 inhibitor in the preparation of a drug for treating cancer. Background Art

[0002] Proliferating cell nuclear antigen (PCNA) is a highly conserved protein that is closely related to DNA synthesis. PCNA is composed of three subunits, each of which contains two domains. These domains can interact with other proteins and participate in a variety of cell biological processes. Three identical subunits can form a ring structure-a "sliding clamp" that can wrap DNA and slide on the DNA template chain, allowing DNA polymerase to stably bind to the template DNA during DNA replication. In addition, PCNA is also involved in regulating cell cycle progression, DNA damage repair, invasion and metastasis, cell apoptosis and other biological functions. PCNA has been shown to be overexpressed in a variety of tumors such as liver cancer, colorectal cancer, gastric cancer, breast cancer, cervical cancer, prostate cancer, etc., and is involved in regulating the malignant proliferation, invasion and metastasis, and chemotherapy resistance of tumors.

[0003] Poly ADP-ribosyltransferases (PARPs) are sensors of DNA damage response that can recognize and bind to broken DNA chains and then convert them into NAD + It is a substrate that catalyzes the transfer of one or more ADP ribose units to the receptor protein or itself, thereby recruiting other DNA repair proteins to the damage site to jointly repair DNA damage. PARP1 inhibitors bind to the catalytic domain of PARP1, resulting in the inhibition of its enzymatic activity, thereby blocking the DNA base excision repair pathway to repair damaged single-stranded DNA. Unrepaired single-strand DNA damage will be converted into double-stranded DNA breaks through the replication fork. Genes such as BRCA1 / 2 and Rad51 regulate the precise repair of damaged double-stranded DNA through the homologous recombination pathway. When BRCA1 / 2 mutates, the homologous recombination repair pathway is blocked, and the inhibition of PARP1 expression and activity can lead to the blockage of cellular DNA damage repair, the accumulation of DNA fragments and chromosomal instability, and then cell death.

[0004] Although PCNA inhibitors and PARP1 inhibitors have broad application prospects, they still have limitations such as narrow indications and easy development of drug resistance, and cannot fully meet the demand for medication. Summary of the invention

[0005] Purpose of the invention: The present invention aims to provide a synergistic, highly effective and low-toxic drug combination of a PCNA inhibitor and a PARP1 inhibitor.

[0006] Technical solution: The PCNA inhibitor of the present invention is combined with the PARP1 inhibitor for use in the preparation of drugs for treating cancer.

[0007] The present invention found that PARP1 is a direct downstream target of PCNA, and inhibiting the biological expression and function of PARP1 will lead to compensatory activation of the DNA damage repair pathway regulated by PCNA. At the same time, knocking down the expression of PCNA will inhibit the expression of genes such as BRCA1, BRCA2, Rad51, and then block the DNA homologous recombination repair pathway. As important factors in the DNA damage response signal network, PCNA and PARP1 play an important role in regulating DNA replication, DNA damage repair and chromosome stability. Targeting the PCNA / PARP1 signal axis as a treatment strategy has better therapeutic effects and prognosis than a single-target strategy, and is expected to break through the limitations of the targeted single-target treatment strategy, such as strong toxic side effects, narrow indications, and drug resistance. Therefore, jointly inhibiting the expression, function and activity of PCNA and PARP1 may synergistically inhibit the proliferation of malignant tumor cells, and is a new strategy for treating refractory cancers such as liver cancer, colon cancer, breast cancer, melanoma, etc. with high expression of PCNA and PARP1.

[0008] Preferably, the drug is a drug for treating mid- to late-stage solid tumors or hematological tumors with high expression of PCNA and PARP1.

[0009] More preferably, the drug is a drug for treating liver cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, lung adenocarcinoma, gastric cancer, renal cell carcinoma, ovarian cancer, cervical cancer or prostate cancer with high expression of PCNA and PARP1.

[0010] Preferably, the PCNA inhibitor is selected from AOH1160, PCNA-I1, T2AA, AOH1996 or pharmaceutically acceptable salts thereof.

[0011] More preferably, the PCNA inhibitor is AOH1160.

[0012] AOH1160 (N-(2-oxo-2-((2-phenoxyphenyl)amino)ethyl)-1-naphthamide) is a specific inhibitor of PCNA. It interferes with DNA replication, prevents homologous recombination-mediated DNA double-strand damage repair, and causes cell cycle arrest by inhibiting PCNA activity.

[0013]

[0014] Preferably, the PARP1 inhibitor is selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, Pamiparib, Stenoparib, Simmiparib, Saruparib, Senaparib, Veliparib, Venadaparib or pharmaceutically acceptable salts thereof.

[0015] More preferably, the PARP1 inhibitor is Olaparib.

[0016] Olaparib is a small molecule drug targeting the DNA damage response signaling network and has shown good anti-tumor efficacy in clinical practice.

[0017]

[0018] The combined use of other PCNA inhibitors / pharmaceutically acceptable salts thereof and PARP1 inhibitors / pharmaceutically acceptable salts thereof has the same therapeutic effect.

[0019] Preferably, the molar ratio of the PCNA inhibitor to the PARP1 inhibitor is 1:(2-32).

[0020] More preferably, the molar ratio of the PCNA inhibitor to the PARP1 inhibitor is 1:(8-16).

[0021] More preferably, the molar ratio of the PCNA inhibitor to the PARP1 inhibitor is selected from 1:2, 1:4, 1:8, 1:16 or 1:32.

[0022] More preferably, the single dose of the PCNA inhibitor is 0.1-40 mg / kg, and the single dose of the PARP1 inhibitor is 0.1-50 mg / kg.

[0023] More preferably, the single dose of AOH1160 is 0.5-20 mg / kg, and the single dose of Olaparib is 0.5-40 mg / kg.

[0024] Still more preferably, the single dose of AOH1160 is 1-10 mg / kg, and the single dose of Olaparib is 1-20 mg / kg.

[0025] Preferably, the PCNA inhibitor and the PARP1 inhibitor are administered simultaneously or sequentially.

[0026] Further preferably, when the method of simultaneous administration is adopted, two pharmaceutical preparations with single active ingredients can be administered simultaneously, or a compound pharmaceutical preparation containing two active ingredients can be administered simultaneously, such as ordinary tablets, capsules, sustained-release tablets, sustained-release capsules, dispersible tablets, chewable tablets, orally disintegrating tablets, pills, liposomes, solid dispersions, liquid preparations, etc.

[0027] More preferably, when sequential administration is adopted, the PCNA inhibitor can be administered first, or the PARP1 inhibitor can be administered first.

[0028] More preferably, when sequential administration is adopted, the PCNA inhibitor is administered first and then the PARP1 inhibitor is administered for treatment.

[0029] Preferably, the PCNA inhibitor is a sensitizing drug for PARP1 inhibitor.

[0030] More preferably, inhibiting the expression of PCNA can increase the sensitivity of tumors to PARP1.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0032] The targeted combination drug designed by the present invention has a synergistic anti-tumor effect and low toxicity, and can be used to treat various cancers with high expression of PCNA and PARP1, especially liver cancer, colon cancer, triple-negative breast cancer and melanoma. It solves the problems of poor therapeutic effect, narrow indications, easy drug resistance and easy recurrence of single drug use, and provides a new strategy for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A schematic diagram of the synergistic inhibition of malignant proliferation of liver cancer, colon cancer, melanoma and triple-negative breast cancer by AOH1160 and Olaparib in vitro in Example 1; wherein the drug synergy index CI is less than 1.0;

[0034] Figure 2 This is a schematic diagram of the clone formation experiment in Example 1;

[0035] Figure 3 is a schematic diagram of the cell apoptosis experiment in Example 1;

[0036] Figure 4 This is a schematic diagram of the in vivo anti-tumor effect of AOH1160 and / or Olaparib in Example 2;

[0037] Figure 5 The statistical graph of tumor volume and weight in vivo of AOH1160 and / or Olaparib in Example 2;

[0038] Figure 6This is a schematic diagram of the in vivo toxicity evaluation of AOH1160 and / or Olaparib in Example 2;

[0039] Figure 7 Schematic diagram of the anti-tumor effect of PCNA shRNA and / or Olaparib in vivo in Example 3;

[0040] Figure 8 The figure is a statistical graph of tumor volume and weight in vivo in the presence of PCNA shRNA and / or Olaparib in Example 3. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below in conjunction with embodiments.

[0042] Human liver cancer cells HepG2 and human malignant melanoma cells A-375 were cultured with DMEM medium and 10% fetal bovine serum (Bio-Channel) at 5% CO2, 37°C; human colon cancer cells HCT116 were cultured with McCoy's 5A medium and 10% fetal bovine serum at 5% CO2, 37°C; human triple-negative breast cancer cells MDA-MB-231 were cultured with L-15 medium and 10% fetal bovine serum at 5% CO2, 37°C; AOH1160 and Olaparib (MedChemExpress) were dissolved in DMSO. Cell apoptosis detection kit (KeyGEN), crystal violet staining solution (KeyGEN).

[0043] MTT assay for cell proliferation: cells were seeded in a 96-well plate at a density of 3000-5000 cells / well, cultured for 24 hours at 5% CO2 and 37°C, treated with the corresponding drug for 6 days, added with thiazolyl blue dye (MTT) and incubated for 3-4 hours, the culture medium was discarded, DMSO was added to fully dissolve and mix, and cell proliferation was detected. Cell growth inhibition rate = (1-absorbance value of experimental group / absorbance value of control group) × 100%.

[0044] Cloning experiment: 800-1000 cells / well were seeded in a 24-well plate, cultured overnight to allow the cells to adhere to the wall, and then cultured for 14 days after adding the corresponding drugs; fixed with 4% paraformaldehyde for 15 minutes, then stained with 0.1% crystal violet stain for 10 minutes, and observed and photographed with an inverted microscope.

[0045] Cell apoptosis experiment: The cells were seeded in 35 mm culture dishes at a density of 30%-50% per dish, cultured overnight to allow the cells to adhere to the wall, and then treated with corresponding drugs for 6 days; the cells were digested with trypsin and washed twice with PBS; then the cells were stained with propidium iodide and annexin V-FITC. After completion, the cell apoptosis was analyzed using Agilent NovoCyte Quanteon flow cytometer.

[0046] Statistical analysis method: SPSS19.0 software was used for statistical analysis; the measurement data were expressed as x±s, the Student's t test was used for comparison between two groups, the one-way analysis of variance was used for comparison between multiple groups, and the LSD test was used for pairwise comparison between groups. * P<0.05, # P<0.05, + P<0.05 indicates that the difference is statistically significant.

[0047] Example 1: Evaluation of the PCNA inhibitor AOH1160 combined with the PARP1 inhibitor Olaparib in inhibiting the proliferation of human liver cancer cells HepG2, human colon cancer cells HCT116, human malignant melanoma cells A-375 and human breast cancer cells MDA-MB-231 in vitro

[0048] 1. Experimental methods

[0049] (1) MTT assay for cell proliferation

[0050] Four types of tumor cells were treated with DMSO (control), AOH1160, Olaparib, and AOH1160 and Olaparib in combination, wherein the molar ratio of AOH1160 to Olaparib in the combined treatment group was 1:(4-32); after 6 days, cell proliferation was detected using MTT.

[0051] (2) Clone formation assay to detect cell number

[0052] The effect of the combination drug on cell proliferation was detected by cloning assay, and four tumor cells were treated with AOH1160 combined with Olaparib. Among them, the single-drug AOH1160 dose for HepG2 cells was 1.0 μM, the single-drug Olaparib dose was 8.0 μM, the combined dose of AOH1160 was 0.5 μM and 1.0 μM, and the combined dose of Olaparib was 4.0 μM and 8.0 μM. The single-drug AOH1160 dose for HCT116 cells and MDA-MB-231 cells was 0.5 μM, the single-drug Olaparib dose was 4.0 μM, the combined dose of AOH1160 was 0.25 μM and 0.5 μM, and the combined dose of Olaparib was 2.0 μM and 4.0 μM. A375 cells were treated with AOH1160 alone at a dose of 0.5 μM, Olaparib alone at a dose of 8.0 μM, AOH1160 combined at doses of 0.25 μM and 0.5 μM, and Olaparib combined at doses of 4.0 μM and 8.0 μM. After 14 days, the cells were fixed with 4% paraformaldehyde for 15 min, stained with crystal violet for 10 min, and observed and photographed with an inverted fluorescence microscope.

[0053] (3) Cell apoptosis assay

[0054] After HepG2 cells were treated with AOH1160 0.5μM, 1.0μM combined with Olaparib 4.0μM, 8.0μM for 6 days, cell apoptosis was detected by flow cytometry.

[0055] 2. Experimental results

[0056] (1) MTT assay for cell proliferation. The results were used to calculate the synergistic index (CI) using the Chou-Talalay formula, where CI < 1.0 indicated a synergistic effect, CI = 1.0 indicated an additive effect, and CI > 1.0 indicated an antagonistic effect.

[0057] like Figure 1 As shown, when the combined molar ratio of AOH1160 and Olaparib was in the range of 1:(4-32), the CI values ​​in the three tumor cells were all less than 1, indicating that the combination of AOH1160 and Olaparib could synergistically inhibit the malignant proliferation of tumor cells within the measured molar ratio range.

[0058] (2) Number of cell clones

[0059] After AOH1160 and Olaparib were co-treated with a molar ratio of 1:8 in human liver cancer cells HepG2, human colon cancer cells HCT116, and human breast cancer cells MDA-MB-231, and AOH1160 and Olaparib were co-treated with a molar ratio of 1:16 in human malignant melanoma cells A-375, the number of cell clones was significantly less than that of the groups treated with AOH1160 or Olaparib alone ( Figure 2 ), further verifying the synergistic inhibitory effect of AOH11160 combined with Olaparib on tumor cell growth.

[0060] (3) Cell apoptosis

[0061] The analysis of cell apoptosis data showed that compared with the single-drug treatment group, the number of apoptotic cells in the AOH1160 and Olaparib (molar ratio 1:8) combined treatment group increased significantly, indicating that the combination of AOH1160 and Olaparib can synergistically promote HepG2 cell apoptosis ( Figure 3 ).

[0062] The above experimental results show that the combination of AOH1160 and Olaparib has a synergistic anti-tumor effect.

[0063] Example 2: AOH1160 inhibits the growth of human hepatocellular carcinoma cell HepG2 xenograft tumors in vivo by combining and sequentially administering Olaparib

[0064] 1. Experimental methods

[0065] (1) Test drug

[0066] PCNA inhibitor: AOH1160, PARP1 inhibitor: Olaparib.

[0067] Preparation method: AOH1160 is prepared with 10% DMSO, 10% Tween, 40% PEG400 and 40% normal saline; Olaparib is prepared with 10% DMSO, 10% Tween, 30% PEG400 and 50% normal saline.

[0068] (2) Experimental animals

[0069] BALB / cA-nude mice, 7-8 weeks old, female; breeding environment: SPF grade.

[0070] (3) Experimental procedures

[0071] Human liver cancer HepG2 cell line was inoculated into the armpit of nude mice to construct a human liver cancer HepG2 transplant tumor model. When the tumor grew to 50-70 mm 3The animals were randomly divided into HepG2 model group, AOH1160 administration group, Olaparib administration group, AOH1160 / Olaparib combined administration group, and AOH1160 / Olaparib sequential administration group. The AOH1160 administration group was intraperitoneally injected with AOH1160 (20 mg / kg) once a day for 21 days; the Olaparib administration group was intraperitoneally injected with Olaparib (40 mg / kg) once a day for 21 days; the combined administration group was intraperitoneally injected with AOH1160 (10 mg / kg) and Olaparib (20 mg / kg) once a day for 21 days; the sequential administration group was intraperitoneally injected with Olaparib (40 mg / kg) once a day for 10 days, and then intraperitoneally injected with AOH1160 (20 mg / kg) once a day for 11 days (the molar ratio of AOH1160 and Olaparib in the in vivo experiment was 1:2). The volume of transplanted tumors was measured every two days, the mice were weighed, the data were recorded, and the nude mice were killed on day 21. Tumor tissue blocks were surgically removed and weighed, and the tumor inhibition rate (%) was calculated based on the weight and volume of tumor tissue blocks.

[0072] 2. Experimental results

[0073] Compared with the model group, the tumor growth of the AOH1160 and Olaparib groups alone was inhibited to a certain extent. Compared with the single drug treatment group and the model group, the tumor weight and volume of the AOH1160 / Olaparib combined administration group and the sequential administration group were significantly reduced ( Figure 4-5 ).

[0074] The inhibitory effects of the AOH1160 / Olaparib combined administration group and the sequential administration group on human liver cancer cell HepG2 xenograft tumors were stronger than those of the AOH1160 alone group or the Olaparib alone group, and the differences were significant, indicating that AOH1160 and Olaparib can exert better anti-tumor effects in vivo when used in combination or sequentially.

[0075] The preliminary toxicity evaluation results showed that compared with the model group, the body weight of mice in the single-dose group, the combination group, and the sequential administration group did not change significantly, and there was no obvious damage to the heart, liver, spleen, lung, and kidney of the mice ( Figure 6 AOH1160 and Olaparib showed low toxicity whether administered alone, in combination, or sequentially.

[0076] Example 3: Olaparib combined with PCNA shRNA inhibits the growth of human hepatocellular carcinoma cell HepG2 xenograft tumors in vivo

[0077] 1. Experimental methods

[0078] (1) Test drug

[0079] PARP1 inhibitor: Olaparib. PCNA shRNA lentiviral vector (pGV112-shPCNA) and NC shRNA lentiviral vector (pGV112-shNC) were constructed by Shanghai GeneCare Gene Medical Technology Co., Ltd.

[0080] Preparation method: Olaparib is prepared with 10% DMSO, 10% Tween, 30% PEG400 and 50% saline.

[0081] (2) Experimental animals

[0082] BALB / cA-nude mice, 7-8 weeks old, female; breeding environment: SPF grade.

[0083] (3) Experimental procedures

[0084] The HepG2 PCNA shRNA lentiviral stable cell line was constructed, and the HepG2 NC shRNA lentiviral stable cell line was constructed. The HepG2 PCNA shRNA and HepG2 NC shRNA cells in the logarithmic growth phase were inoculated into the armpits of nude mice to construct the corresponding cell xenograft tumor models. When the HepG2 NC shRNA xenograft tumor grew to 60-80 mm 3 The animals were randomly divided into NC shRNA group and NC shRNA + Olaparib administration group. The HepG2 PCNA shRNA xenograft tumor model was randomly divided into PCNA shRNA group and PCNA shRNA + Olaparib administration group. The Olaparib administration group was intraperitoneally injected with Olaparib (40 mg / kg) once a day for 21 days. The volume of the transplanted tumor was measured every two days, the weight of the mice was weighed, the data was recorded, and the nude mice were killed on the 21st day. The tumor tissue blocks were surgically removed and weighed, and the tumor inhibition rate (%) was calculated based on the weight and volume of the tumor tissue blocks.

[0085] 2. Experimental results

[0086] Compared with the NC shRNA group, the tumor growth of the NC shRNA+Olaparib group and the PCNA shRNA group was inhibited to a certain extent. Compared with the NC shRNA+Olaparib group and the PCNA shRNA group, the tumor growth of the PCNAshRNA+Olaparib group was significantly inhibited ( Figure 7-Figure 8 ).

[0087] Compared with the NC shRNA group, inhibition of PCNA expression significantly inhibited the growth of human liver cancer xenografts. Compared with the NC shRNA + Olaparib administration group, inhibition of PCNA expression significantly increased the sensitivity of xenografts to Olaparib, indicating that targeting PCNA and PARP1 can exert a better anti-tumor effect in vivo.

[0088] In summary, the targeted combination drug (combined administration or sequential administration) designed by the present invention can significantly inhibit the growth of tumor cells in vivo and in vitro, and has good prospects for clinical application.

Claims

1. Use of a PCNA inhibitor combined with a PARP1 inhibitor in the preparation of a drug for treating cancer.

2. The use according to claim 1, characterized in that: The medicine is a medicine for treating middle and late stage solid tumors or blood tumors with high expression of PCNA and PARP1.

3. The use according to claim 2, characterized in that: The medicine is a medicine for treating liver cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, lung adenocarcinoma, gastric cancer, renal cell carcinoma, ovarian cancer, cervical cancer or prostate cancer with high expression of PCNA and PARP1.

4. The use according to claim 1, characterized in that: The PCNA inhibitor is selected from AOH1160, PCNA-I1, T2AA, AOH1996 or pharmaceutically acceptable salts thereof.

5. The use according to claim 4, characterized in that: The PCNA inhibitor is AOH1160.

6. The use according to claim 1, characterized in that: The PARP1 inhibitor is selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, Pamiparib, Stenoparib, Simmiparib, Saruparib, Senaparib, Veliparib, Venadaparib or pharmaceutically acceptable salts thereof.

7. The use according to claim 6, characterized in that: The PARP1 inhibitor is Olaparib.

8. The use according to claim 1, characterized in that: The molar ratio of the PCNA inhibitor to the PARP1 inhibitor is 1:(2-32).

9. The use according to claim 1, characterized in that: The PCNA inhibitor and the PARP1 inhibitor are administered simultaneously or sequentially.

10. The use according to claim 1, characterized in that: The PCNA inhibitor is a sensitizing drug for PARP1 inhibitor.

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