WO2021184990A1 - Sensitizer drug, drug combination and use - Google Patents

Abstract

A sensitizer drug, a drug combination and use thereof. The sensitizer is a Pin1 inhibitor, which increases the sensitivity of BRCA1-expressing cancers to PARP inhibitors by inhibiting prolyl isomerase Pin1, contributing to improve clinical effects of targeted tumor therapy. The drug combination of a Pin1 inhibitor and a PARP inhibitor can be used for preparing a therapeutic drug targeting HR-sensitive tumors, can be used for treating BRCA1 wild-type cancers, and has good safety and low toxicity.

Description

A sensitizer drug and drug combination and application Technical field

The invention belongs to the technical field of biomedicine, and relates to a sensitizer drug and drug combination and application thereof, in particular to a sensitizer drug and drug combination and application for enhancing the sensitivity of BRCA1 expression cancer PARP inhibitor to radiotherapy and chemotherapy. .

Background technique

PARP, poly (ADP-ribose) polymerase, is an enzyme closely related to DNA repair under stress conditions. When DNA damage breaks, it will activate PARP. As a molecular sensor of DNA damage, it has the function of recognizing and binding to the DNA break position, and then activates and catalyzes the poly-ADP ribosylation of the receptor protein and participates in the DNA repair process. PARP is very important for cell stability and survival. When PARP is inhibited, the repair process mediated by it will also be hindered. For example: PARP inhibitor binds to the catalytic site of PARP1 or PARP2, causing the PARP protein to fail to fall off the DNA damage site and be bound to the DNA. PRAP will cause DNA replication forks to stall and DNA replication cannot proceed smoothly during DNA replication. When PARP is inhibited and the repair process mediated by it is blocked, cells usually trigger a repair method called homologous recombination (HR) repair to repair the error. However, when the HR repair function is also abnormal, other DNA repair methods used by cells usually introduce large-scale genome reorganization, leading to cell death, that is, synthetic lethality. This is also the reason why the prior art PARP inhibitors are effectively used in HR-sensitive cancers.

BRCA1 protein is an important protein in the process of DNA damage repair. It will be phosphorylated in response to DNA damage, and phosphorylation is necessary for the DNA damage repair function of BRCA1 (Science, 1999; 286: 1162-6, Nature, 2000; 404: 201-4). The loss of BRCA1 function indicates a difference in response to DNA damage repair defects. For example, BRCA1 plays an important role in the repair of homologous recombination. When the function of this protein is damaged, it will cause HR repair dysfunction. This is also one of the reasons why breast, ovarian, prostate and pancreatic cancer patients with BRCA1 mutations are sensitive to therapeutic PARP inhibition; because of their homologous recombination (HR) defects, PARP inhibition can lead to insurmountable DNA damage. PARP inhibitors have become safe and effective drugs for the treatment of BRCA1 mutant breast cancer, ovarian cancer, prostate cancer and pancreatic cancer with HR deficiency (N Engl J Med, 2009; 361:123-34, N Engl J Med, 2019; 381 : 317-27, N Engl J Med, 2015; 373: 1697-708).

However, PARP inhibitor therapy also has drawbacks. Although PARP inhibitor monotherapy can effectively treat BRCA1 mutant breast, ovarian, prostate and pancreatic cancer patients, it is not suitable for BRCA1 wild-type cancer. Moreover, existing research shows that it is difficult to sensitize these tumors by combining PARP inhibitors with HR interfering agents (Proe Natl Acad Sci USA. 2016; 113: E4338-47, Cell Rep. 2016; 17: 2367-81) .

Therefore, further research and development of sensitizer drugs or therapies for enhancing the sensitivity of BRCA1-expressing cancers to PARP inhibitors to increase the sensitivity of BRCA1-expressing cancers to PARP inhibitors and improve tumor targeting are still needed in the clinic. The clinical effect of treatment and provide clinicians with better treatment options.

Summary of the invention

The present invention aims to overcome the limited clinical effects of existing PARP inhibitor drugs for BRCA1 mutant breast cancer, ovarian cancer, prostate cancer, and pancreatic cancer patients, and the technical defects that PARP inhibitors are not applicable to BRCA1 wild-type cancers, and provide one A sensitizer drug and drug combination and application, which can be used to enhance the sensitivity of BRCA1-expressing cancer with PARP inhibitors to radiotherapy and chemotherapy.

The technical scheme adopted by the present invention is a sensitizer drug for enhancing the radiochemotherapy sensitivity of BRCA1 expressing cancer PARP inhibitors, and the sensitizer drug includes a Pin1 inhibitor. The Pin1 inhibitor of the present invention can be used as a chemotherapy and radiotherapy sensitizer, used to enhance the sensitivity of BRCA1 expression cancer PARP inhibitor to radiotherapy and chemotherapy, and to enhance the effect of anti-tumor chemotherapy and radiotherapy.

Prolyl cis-trans isomerase Pin1 can specifically recognize the pSer/Thr-Pro domain of the protein, and plays an important role in cell cycle, transcription regulation, cell proliferation and differentiation, such as: DNA damage repair (DDR) needs Sense DNA damage sites and need to repair protein complexes in close coordination in space and time. In eukaryotic cells, these proteins are often activated by the serine/threonine phosphorylation signal cascade; and the phosphorylated serine/ Threonine residues are targets of phosphate-specific prolyl isomerization, and Pin1 is closely related to this process. Pin1 binds its phosphorylated target protein through the N-terminal WW domain, and then makes its C-terminal PPIase domain catalyze the prolyl bond isomerization of adjacent peptide groups. The two domains of Pin1 cooperate closely to catalyze the cis-trans isomerization of substrate proteins. This conformational change can significantly affect the folding, function and stability of many substrate proteins of Pin1. This is a post-phosphorylation modification.

Existing studies have found that Pin1 is closely related to the development of cancer. For example, this post-phosphorylation modification of signal transduction molecules by prolyl isomerase Pin1 can coordinate mitosis and be abnormally activated in cancer (Nat Cell Biol , 2005; 7: 435-41, Nat Rev Cancer, 2007; 7: 381-8.); Generally, Pin1 stabilizes and enhances the function of pro-cyclin proteins, such as cyclin D1 (Proc Natl Acad Sci USA, 2002 ;99: 1335-40, Oncol Rep, 2006; 16: 491-6.), c-Jun (Embo J, 2001; 20: 3459-72), Raf-1 (Mol Cell, 2005; 17: 215-24 ), NF-k B (Mol Cell, 2003; 12: 1413-26), beta-Catenin (Nat Cell Biol, 2001; 3: 793-801) and Topo II (Mol Cell, 2007; 26: 287-300) . Pin1 is also highly expressed in breast cancer, prostate cancer, pancreatic cancer, etc. (Nat Rev Cancer. 2016; 16: 463-78, Cancer Sci. 2019; 110: 2442-55.); Pin1 is in viral, bacterial and parasitic infections It also plays an important role in malignant tumors. Pin1 is generally overexpressed in tumors and is related to poor clinical prognosis.

Such results show that Pin1 may be an effective target for the diagnosis or treatment of cancer. Existing studies have also carried out studies on the effect of inhibiting Pin1 on the occurrence and development of cancer. For example, existing studies have found that ATRA can inhibit the function of Pin1, and PIN1 is the treatment of acute promyelocytic leukemia and breast cancer with all-trans retinoic acid. Key targets (Nat Med. 2015; 21: 457-66); arsenic targets Pin1 and cooperates with retinoic acid to inhibit cancer-driving pathways and tumor-initiating cells (Nature communications.2018; 9:3069.); in In mouse models, overexpression of Pin1 promotes the formation of breast cancer, while inactivation of Pin1 inhibits breast cancer (Embo J, 2004; 23: 3397-407) or prostate cancer (Clin Cancer Res, 2005; 11: 7523-31) Therefore, targeting Pin1 represents a new non-toxic strategy that can simultaneously block multiple cancer-driving pathways and eliminate tumor-initiating cells (TICs). However, the Pin1 inhibitory treatment technology still has some shortcomings. For example, the previously discovered Pin1 inhibitor lacks specificity, efficacy and/or cell permeability, and the therapeutic effect is limited.

The present invention creatively discovered that Pin1 inhibitor can enhance the sensitivity of BRCA1 expressing cancer to PARP inhibitor treatment, and the combined application of the two is significantly improved compared to Pin1 inhibition or PARP inhibition alone. Moreover, it can be seen from the results of the examples that the inhibition of Pin1 affects the stability of the key protein BRCA1 for HR repair, and can enhance the sensitivity of BRCA1-expressing cancers to PARP inhibitors. Moreover, although the prior art each discloses the application of Pin1 inhibition or PARP inhibition in cancer, the application of Pin1 inhibition alone to cancers that normally express BRCA1 is taken as an example, because this type of cancer is not affected by PARP inhibitors. PARP inhibition will not occur, and the DNA repair process can still proceed normally. Therefore, it is often more difficult to find the effect of Pin1 inhibitors on BRCA1, a key protein in HR repair that is activated when PARP is inhibited, and its use as a PARP inhibitor in the treatment. The role of sensitizers.

Preferably, the Pin1 inhibitor of the present invention is used to prepare a sensitizer drug for enhancing the sensitivity of BRCA1 expression cancer PARP inhibitor to radiotherapy and chemotherapy.

Preferably, the Pin1 inhibitor of the present invention is selected from all-trans retinoic acid (ATRA), ATRA derivatives, arsenic trioxide (ATO), juglone, PPIase-Parvulin inhibitor, compound H-371 (derived from Patent WO2019031472 on page 53 of the compound), at least one of VS10, API-1, Pin1 shRNA, and the target sequence of the Pin1 shRNA is 5'-CCACCGTCACACAGTATTTAT-3'.

Preferably, the PARP inhibitor of the present invention is selected from Olaparib (Olaparib), Rucaparib, Niraparib (MK-4827), Talazoparib (BMN-673), Veliparib (ABT-888), pamiparib (BGB-290 ), INO-1001(3-Aminobenzamide), A-966492, PJ34 HCl, UFP-1069, ME-0328, NMS-P118, E7449, Picolinamide, Benzamide, Niraparib(MK-4827) tosylate, NU-1025, Iniparib( BSI-201), AZD-2461, BGP-12·2HCl, compound CEP-8983 (CK-102), compound 2X-121, Fuzuopali (fluzoparil), compound SC-10914, compound HWH-340, compound IDX -1197, at least one of simmiparib, compound IMP-4297, and compound ABT-767.

Preferably, according to an embodiment of the present invention, the effective dose of the Pin1 inhibitor as a sensitizer is 1.5 mg·kg ^-1 d ^-1 .

Preferably, the Pin1 inhibitor of the present invention, as a sensitizer, and its carrier have a suitable concentration so that a sufficient concentration of the sensitizer can be delivered to the tumor, cancer cell, or precancerous cell treated with the PARP inhibitor To increase the sensitivity of cancer cells to PARP inhibitors.

Preferably, it also includes a pharmaceutically acceptable carrier for transporting the sensitizer to the tumor, cancer cell, or precancerous cell where the PARP inhibitor acts.

The inventors of the present invention unexpectedly discovered that Pin1 inhibition sensitizes breast cells to radiation and PARP inhibition. Specifically, the inventors found that Pin1 can stabilize BRCA1 and promote its function in DNA damage repair foci, Pin1 inhibition will destroy the stability of BRCA1, and damage DNA damage repair through HR, resulting in sensitivity to radiation. In addition, the cell viability analysis experiment conducted by the inventors showed that through Pin1 knockdown or Olaparib monotherapy, the growth of human breast cancer cell lines MDA-MB 231 and MCF-7 cells was delayed, while in Pin1 knockdown cells, Olaparib The treatment resulted in almost complete blockage of cell proliferation.

In addition, effective Pin1 inhibition may bring other benefits, such as destroying the cyclinD1 signal axis of tumors and inhibiting the dominant mutation p53 found in most triple-negative breast cancers, all of which contribute to its use in tumor therapy The effect of.

Preferably, the present invention also provides the use of Pin1 inhibitors in the preparation of drugs for enhancing the sensitivity of PARP inhibitors in the treatment of cancers, and the cancers are cancers expressing BRCA1. The Pin1 inhibitor is selected from at least one of all-trans retinoic acid, all-trans retinoic acid derivatives, arsenic trioxide, juglone, PPIase-Parvulin inhibitor, compound H-371, VS10, API-1, Pin1 shRNA, The target sequence of the Pin1 shRNA is 5'-CCACCGTCACACAGTATTTAT-3'.

Another object of the present invention is to provide a drug combination and its application, the drug combination comprising: (1) a sensitizer; (2) a PARP inhibitor. Wherein the sensitizer is a Pin1 inhibitor.

Preferably, the drug combination is in the form of a drug composition or a drug combination product.

Preferably, the Pin1 inhibitor can be selected from all-trans retinoic acid (ATRA), ATRA derivatives, arsenic trioxide (ATO), juglone, PPIase-Parvulin inhibitor, compound H-371 (derived from patent Compound on page 53 of WO2019031472) at least one of VS10, API-1, Pin1 shRNA, and the target sequence of Pin1 shRNA is 5′-CCACCGTCACACAGTATTTAT-3′. Among them, all-trans retinoic acid (ATRA) as a Pin1 inhibitor has also been used clinically to treat acute promyelocytic leukemia (APML). It can bind to Pin1 to inhibit its activity and cause Pin1 to degrade with almost no side effects. An ideal Pin1 inhibitor.

The PARP inhibitor can be selected from Olaparib (Olaparib), Rucaparib, Niraparib (MK-4827), Talazoparib (BMN-673), Veliparib (ABT-888), pamiparib (BGB-290), INO-1001 ( 3-Aminobenzamide), A-966492, PJ34 HCl, UFP-1069, ME-0328, NMS-P118, E7449, Picolinamide, Benzamide, Niraparib (MK-4827) tosylate, NU-1025, Iniparib (BSI-201), AZD -2461, BGP-12·2HCl, compound CEP-8983 (CK-102), compound 2X-121, Fuzuopali (fluzoparil), compound SC-10914, compound HWH-340, compound IDX-1197, simmiparib At least one of minopyrim), compound IMP-4297, and compound ABT-767.

Preferably, the drug combination of the present invention is used for the treatment of cancer.

Because PARP inhibitor monotherapy can effectively treat BRCA1 mutant breast cancer, ovarian cancer, prostate cancer and pancreatic cancer patients, it is not suitable for BRCA1 wild-type cancer. The inventors of the present invention found that Pin1 inhibitors and PARP inhibitors can be used to treat BRCA1 wild-type (BRCA1-WT) cancers.

According to some embodiments of the present invention, the combination of ATRA and Olaparib can synergistically inhibit cell growth, not only in MDA-MB 231 and MCF-7 cells, but also in BRCA1/2-WT pancreatic cancer cells PANC-1 and prostate cancer cells VCaP is also like this. These in vitro data indicate that ATRA can act as a HR disruptor through Pin1 knockout, thus making BRCA1-WT tumors sensitive to PARP inhibition in vivo. By using a single drug ATRA, Olapaparib or their combination, mice carrying MDA-MB 231 xenografts found that the combination of ATRA and Olapaparib significantly delayed tumor growth after 5 weeks. As assessed by Ki67 staining and TUNEL analysis, the combination of ATRA and Olaparib effectively reduced the tumor growth and proliferation rate and increased the apoptosis rate.

Therefore, the drug combination of the present invention can be used to prepare a drug for the treatment of cancer, which has a wide range of targeting properties and can be used to target HR-sensitive tumors.

Preferably, the cancer of the present invention is selected from the group including but not limited to: ovarian cancer, peritoneal tumor, fallopian tube cancer, breast cancer, pancreatic cancer, prostate cancer, non-small cell lung cancer, head and neck tumors, solid tumors, bladder cancer, Small cell lung cancer, gastric cancer, transitional cell cancer, cervical cancer, endometrioid cancer, esophageal cancer, squamous cell carcinoma, glioblastoma, mesothelioma, kidney cancer, urethral cancer, uveal melanoma, bile duct Cancer, Ewing's sarcoma, colorectal cancer.

ATRA or ATRA derivatives can be used as effective Pin1 inhibitors to make tumors sensitive to PARP inhibition. In view of the abundant expression of Pin1 in a variety of cancer types, in addition to breast cancer, the pharmaceutical composition of ATRA and Olaparib also has a wide range of targeting properties and can be used to target HR-sensitive tumors. On the other hand, there is no significant difference in the body weight of each group of mice, indicating that the pharmaceutical composition has no obvious systemic toxicity, which further proves the safety of the pharmaceutical combination of the present invention.

According to an embodiment of the present invention, the effective doses of the active ingredients PARP inhibitor and Pin1 inhibitor in the pharmaceutical combination are respectively 50 mg·kg ^-1 d ^-1 of the PARP inhibitor, and the Pin1 inhibitor The dose is 1.5 mg·kg ^-1 d ^-1 . Therefore, the active ingredient is a drug combination/or a pharmaceutical composition of a Pin1 inhibitor and a PARP inhibitor in the above-mentioned dose combination, which can be used to prepare a therapeutic drug targeting HR-sensitive tumors. Preferably, the PARP inhibitor is olaparib.

Preferably, the drug combination of the present invention can be used to prepare a drug for treating cancers expressing BRCA1 for the treatment of cancers caused by BRCA1 mutations.

Preferably, the drug combination of the present invention can be used to prepare a drug for treating BRCA1 wild-type cancer, for the treatment of BRCA1 wild-type cancer.

Preferably, it also includes a pharmaceutically acceptable carrier for transporting the sensitizer to the tumor, cancer cell, or precancerous cell where the PARP inhibitor acts.

Preferably, the sensitizer is Pin1 inhibitor all-trans retinoic acid, and the effective dose is 1.5 mg·kg ^-1 d ^-1 ; the PARP inhibitor is olaparib, and the effective dose is 50 mg·kg ^-1 d ^-1 .

Preferably, the present invention also provides the use of the above-mentioned drug combination in the preparation of drugs for treating HR-sensitive cancers, drugs for treating cancers expressing wild-type BRCA1, and/or drugs for treating BRCA1 wild-type cancers

The term "effective dose" as used herein refers to the dose or effective amount to be administered to the patient. The dosage or effective amount to be administered to the patient and the frequency of administration to the subject are easily determined by those of ordinary skill in the art by using known techniques and by observing the results obtained under similar conditions. When determining the effective amount or effective dose, the attending diagnostician will consider many factors, including but not limited to, the potency and duration of the compound used; the nature and severity of the disease to be treated and the gender of the patient to be treated , Age, weight, health status and individual responsiveness and other related conditions.

Preferably, the effective active ingredient in the pharmaceutical composition of the present invention can be mixed with a variety of pharmaceutically acceptable excipients to form a solid preparation or a liquid preparation form, such as a solid form such as a tablet, capsule, granule or powder. , Or oral liquid, injection and other liquid forms. According to specific embodiments of the present invention, the preferred dosage forms are tablets, granules, and capsules, but they are not limited to the preferred dosage forms, and also include other conventional oral preparations in pharmacy. Among them, tablets, granules, and capsules can be ordinary preparations, or they can be slow and controlled release preparations using special technology. When prepared into a solid composition preparation in the form of granules, it can be made into a sugar-free type or a sugar-containing type.

"Pharmaceutically acceptable excipients" include any common excipients that can be used pharmaceutically, such as disintegrants, binders, fillers, lubricants, slow-release formulations used to control the release rate of active ingredients The matrix material (i.e., the release rate adjustment), etc., make it have slow-release or controlled-release activity, so that it can continuously release a predetermined amount of active ingredient.

Preferably, the method for preparing the tablet is not particularly limited, and conventional preparation techniques in the field can be used to control the particle size of the active ingredient of the pharmaceutical composition of the present invention to 100 mesh to 200 mesh. According to some embodiments of the present invention, the method for preparing tablets may adopt granulating techniques such as dry rolling, wet or vulcanization spraying, and then compressing tablets, or directly crushing the pharmaceutical composition through a sieve and then compressing the tablets. According to other embodiments of the present invention, it can be prepared as a single-layer tablet, or as a double-layer tablet, a sustained-release tablet, and other tablet types known in the art. According to specific embodiments of the present invention, the outside of the tablet core may be coated with a film coating layer or a sugar coating layer or not coated with a film coating layer or a sugar coating layer. In addition, appropriate flavoring agents can also be added in the preparation of tablets to meet the needs of different tastes.

The pharmaceutical composition of Pin1 inhibitor and PARP inhibitor of the present invention can be used to prepare anti-tumor drugs that are sensitive to targeted HR, and can be used to treat BRCA1 wild-type cancers.

Compared with the prior art, the beneficial effects of the present invention are: the Pin1 inhibitor and the drug combination thereof of the present invention further expand new uses and clinical applications in clinical practice. Pin1 inhibitors can increase the sensitivity of BRCA1-expressing breast cancers to PARP inhibitors by inhibiting prolyl isomerase Pin1, and the application of sensitizers for enhancing the sensitivity of BRCA1-expressing cancers to PARP inhibitors. This new application study No report has been seen before. The drug combination of Pin1 inhibitor and PARP inhibitor of the present invention can be used to prepare targeted HR-sensitive tumor therapeutic drugs, which can be used to treat BRCA1 wild-type cancers, and has good safety and low toxicity.

Description of the drawings

Figure 1 is a graph showing the experimental results of the in vitro experiment in Example 1 of the present invention verifying that Pin1 inhibition makes breast cancer cells sensitive to radiation and PARP inhibition; in which,

A, B, C: Inhibition of Pin1 will reduce the survival rate of MCF10A/MCF-7/T47D cells after IR;

D: In BRCA1 mutant (2288delT) SUM149 cells, Pin1 knockdown did not further reduce the efficiency of clone formation after IR post-treatment;

E, F: After Olaparib treatment, the degradation rate of BRCA1 in Pin1 knockdown cells increased;

G, H, I: After Pin1 is knocked out, wild-type BRCA1 breast cancer cells with PARP inhibitor Olaparib, whether triple-negative or ER-positive, the colony formation is drastically reduced;

J: In BRCA1 mutant (2288delT) SUM149 cells, Pin1 knockdown did not further reduce the clone formation efficiency after Olaparib treatment;

K, L: Pin1 knockdown or Olaparib monotherapy, MDA-MB 231 and MCF-7 growth delay, Olaparib treatment in Pin1 knockdown cells almost completely blocked cell proliferation. When exogenous Pin1 is added to shPin1 MDA-MB 231 and MCF-7 cells, only the wild type can rescue the synergistic inhibition of Pin1 RNAi and Olaparib, while the non-catalytically active Pin1 mutant (S67E) cannot rescue the Pin1 RNAi and Olaparib Synergistic inhibition.

Fig. 2 is a graph showing the experimental results of in vitro experiments verifying that Pin1 inhibition makes pancreatic cancer cells and prostate cancer cells sensitive to radiation and PARP inhibition according to Example 1 of the present invention; wherein,

A, B, C: ATRA or Olaparib can delay cell growth, and the combination of ATRA and Olaparib can synergistically inhibit cell growth, not only in breast cancer MDA-MB 231(A), but also in BRCA1/2-WT pancreatic cancer cells PANC-1 (B) neutralizes prostate cancer cell VCaP (C).

Figure 3 is a diagram showing the experimental results of the in vivo experiment in Example 1 of the present invention verifying that Pin1 inhibition makes breast cancer cells sensitive to radiation and PARP inhibition; in which,

A, B, C: 2 weeks after tumor cell inoculation, mice are treated with placebo or Olaparib for 5 weeks. Pin1 or Olaparib monotherapy can moderately delay tumor growth. The combination of Pin1 and Olaparib can delay tumor growth and remove the foreign source. When WT Pin1 is added back to shPin1 cells, Olapaparib treatment only moderately delays tumor growth. Mutant Pin1 has no such effect. When Pin1 knockout is used in combination with Olaparib, the average tumor volume and weight are significantly reduced;

D, E, F: Pin1 knockout leads to a decrease in BRCA1 levels. The combination of Pin1 knockout and Olaparib can effectively reduce tumor cell proliferation (assessed by Ki67 staining) and increase tumor cell apoptosis (detected by TUNEL analysis).

Figure 4 is a diagram of the therapeutic effect of the combination of ATRA and Olaparib in the triple-negative breast cancer PDX that does not contain BRCA1/2 mutations or genetic changes in HR DNA repair in the in vivo experiment described in Example 1 of the present invention; wherein,

A, B, C: The combination of ATRA and Olapaparib significantly delayed tumor growth;

D, E: ATRA causes the level of Pin1 and BRCA1 to decrease;

F, G: As assessed by Ki67 staining and TUNEL analysis, the combination of ATRA and Olaparib effectively reduced the tumor growth and proliferation rate and increased the apoptosis rate;

H: There is no significant difference in the body weight of each group of mice, indicating that the combination has no obvious systemic toxicity.

Detailed ways

It should be pointed out that the following detailed descriptions are all illustrative and are intended to provide further explanations for the application. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

It should be noted that the terms used here are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and/or "including" are used in this specification, they indicate There are features, steps, operations, devices, components, and/or combinations thereof.

The present invention will now be further explained in conjunction with specific examples. The following embodiments are only for explaining the present invention, but do not constitute a limitation to the present invention. The test samples and test procedures used in the following examples include the following content (if the specific conditions of the experiment are not indicated in the examples, usually follow the conventional conditions or the conditions recommended by the reagent company; the reagents used in the following examples , Consumables, etc., if there is no special instructions, they can be obtained from commercial channels).

Example 1

In vitro and in vivo biological testing

(1) Materials and methods

(1) Drugs

ATRA (Sigma) is dissolved in 150 mM DMSO and diluted in 1:1 PBS and 1M NaOH working solution. 200 ml is administered intraperitoneally at a dose of 1.5 mg/kg, once a day. The preparation of Olaparib (Selleckchem) was carried out according to the literature Cancer Discov. 2012; 2:1048-63.

(2) Cells and cell culture

MCF10A cells derived from ATCC were cultured in DMEM/F-12, added with 5% horse serum, 20ng/ml epidermal growth factor (EGF), 0.5μg/ml hydrocortisone, 100ng/ml cholera toxin, 10μg/ml insulin, Cultivation is described in the literature Methods. 2003; 30: 256-68. MCF-7, T47D, MDA-MB 231, AU565, and HEK293 derived from ATCC were cultured in DMEM containing 10% FBS. SUM149 was cultured in Ham's F-12 medium containing 5% bovine serum (FBS), insulin (5μg/mL) and hydrocortisone (2μg/mL).

The radiation dose is selected according to the experiment and the cells used. MCF10A cells were treated with 5Gy, and the DNA repair foci were observed by immunofluorescence assay. Breast cancer cells were tested for BRCA1 degradation with 10Gy.

(3) Cell viability and luciferase determination

For cell viability determination, cells were seeded in 96-well plates at a density of 1000 (MDA-MB 231) per well and 2000 (MCF-7) per well. ATAR or Olaparib was added to the cells the next day. CellTiter-Glo Luminescent Cell Viability Assay (Promega) determines cell viability, and Wallac 3 plate reader reads the absorbance value.

(4) Clone formation assay

Cells were seeded in a 6-well plate overnight at a density of 500 (MDA-MB 231), 1000 (MCF-7), T47D (1000), 500 (MCF10A), and SUM149 (1000) per well. Olapaparib (Olapaparib) irradiates or treats cells. The number of surviving clones was measured after 6-7 days.

(5) Plasmid transfection and RNAi experiment

The pcDNA3.1 plasmid containing HA-BRCA1, Flag-Pin1 or the Myc tag BRCA1 fragment was transfected with Lipofectamine 2000 (Invitrogen). The medium was changed to growth medium 8 hours after transfection. Site-directed mutagenesis kit (Agilent) detects the point mutations of BRCA1 and Pin1 generated by pcDNA3.1 HA-BRCA1 and Flag-Pin1 plasmids. The successful detection of ShPin1 cell construction is described in the literature Cell Rep. 2015; 11: 111-24. The target sequence of Pin1 shRNA is 5'-CCACCGTCACACAGTATTTAT-3', which targets Pin1 3'UTR. The control shRNA sequence is 5'-UAAGGCUAUGAAGAGAUAC-3'. After lentivirus infection, puromycin was used to select cells.

(6) Western blot

Antibodies include anti-β-actin (1∶10,000, sigma), HA tag (1∶2000, Cell signaling), Flag tag (1∶2000, Cell signaling), Myc tag (1∶1000, 9E10, Santa Cruz) And BRCA1 antibody (1:500, MS110, Calbiochem), mouse antibody Pin1 (1:3000) (e.g. Cancer Res. 2014; 74: 3603-16). Perform western blotting experiments according to standard procedures.

(7) Immunofluorescence detection

Irradiate MCF10A, MCF-7 cells, fix them at the specified time point, and react with antibodies against BRCA1 (1:50, MS110), Pin1 (1:100), 53BP1 (1:50, cell signaling) and corresponding secondary antibodies. Check with Zeiss Axiovert 200M fluorescence microscope.

(8) Immunohistochemical detection

The formalin-fixed and paraffin-embedded tumors were stained with Pin1 (1:200), Ki67 (BD biosciences), and BRCA1 (1:100MS110) according to standard protocols. The DAB solution (Vector Laboratories) mixture was immunolabeled, and then counterstained with hematoxylin.

(9) TUNEL determination

Cell death detection kit (Roche) was used to detect apoptosis in formalin-fixed and paraffin-embedded sections by TUNEL method. The sections were deparaffinized, rehydrated and treated with proteinase K, and then added to the TUNEL reaction mixture. Observe the staining of the sections with a Leica fluorescence microscope.

(10) In vivo animal experiment

All experiments involving mice were approved by the Animal Ethics Committee of Sun Yat-Sen University and conducted research in accordance with relevant regulations. 1.5×10 ⁶ MDA-MB 231 cells expressing shControl or shPin1 with or without exogenous WT Pin1 or mutant Pin1 (S67E) were injected into the mammary fat pad of 6-week-old BALB/c nude mice (Jackson Laboratories). Two weeks later, when tumor growth was visible, the mice were randomly divided into an Olaparib treatment group and a control group. Olaparib (50mg·kg ^-1 ·d ^-1 ) or placebo was injected intraperitoneally for 5 weeks. For the combination therapy of ATRA and Olaparib, 1.5×10 ⁶ MDA-MB 231 cells were injected into the fat pad of 6-week-old BALB/c nude mice (Jackson Laboratories).

Regarding PDX implantation, three BRCA1-expressing TNBC specimens were collected during tumor resection from patients at Sun Yat-sen Memorial Hospital of Sun Yat-sen University between 2017 and 2018. The patient’s informed consent was given in accordance with the internal review and ethics committee of Sun Yat-sen Memorial Hospital. The details are described in the document Cell.2018;172:841-56e16. 6-week-old female NSG mice were anesthetized. The tumor was cut into a size of 1mm ³ and directly embedded in the breast fat pad to obtain the first-generation PDX. Once the first-generation PDX reaches 1 cm in diameter, it is harvested, cut into 1mm ³ size, and directly embedded in the breast fat pad to obtain the second-generation PDX for treatment. The PDX of each patient was transplanted into four mice and randomly divided into four groups. Once tumors formed, the mice were randomly divided into ATRA, Olapaparib, combination and control treatment groups. Use placebo, olaparib (50mg·kg ^-1 d ^-1 ), ATRA (1.5mg.kg ^-1 d ^-1 ) or olaparib (50mg·kg ^-1 d ^-1 ) and ATRA (1.5 mg.kg ^-1 d ^-1 ) was injected intraperitoneally for 5 weeks. Use a caliper to record the tumor size, and use the formula L×W ² ×0.5 to calculate the tumor volume, where L and W represent length and width, respectively. The mice were sacrificed after 5 weeks of treatment. No animals died during the experiment.

The HR DNA repair (AmoyDx) detection of PDX was completed by NGS sequencing, including 32 genes (AR/ATM/ATR/BARD1/BRAF/BRCA1/BRCA2/BRIP1/CDH1/CDK12/CHEK1/CHEK2/ERBB2/ESR1/FANCA/ FANCL/HDAC2/HOXB13/KRAS/MRE11/NBN/NRAS/PALB2/PIK3CA/PPP2R2A/PTEN/RAD51B/RAD51C/RAD51D/RAD54L/STK11/TP53) through NextSeq500 all coding regions and junction regions of exons and introns Perform sequencing. The average coverage rate of the sequencing depth target area is ≥1000×. The raw data is analyzed by AmoyDxANDAS data analyzer. In the 1000 genome project, use MAF<1% to screen out genetic variants.

(11) Statistical analysis

The in vitro experimental data of the three laboratories are expressed in terms of mean±standard deviation. All statistical analysis was performed using SPSS 16.0 statistical software package. The cell viability, colony formation and tumor volume under different treatment methods were compared by t-test and one-way analysis of variance. In all cases, *P<0.05, **P<0.01 and ***P<0.001.

(2) Results

In vitro experiments are mainly cell experiments, through cell experiments to verify that Pin1 inhibition makes breast cells sensitive to radiation and PARP inhibition. This part uses ionizing radiation (IR) treatment to induce DNA double-strand breaks (DBS) and then detects the effect of Pin1 inhibitors on radiotherapy sensitivity. By inhibiting Pin1 to detect the changes in the survival rate of MCF10A/MCF-7/T47D cells after IR, by inhibiting Pin1, to detect the colony formation of wild-type BRCA1 breast cancer cells with the PARP inhibitor Olaparib, and to study the Pin1 inhibitors ATRA and PARP The effect of the inhibitor Olaparib alone and in combination on the growth of breast cancer/pancreatic cancer/prostate cancer cells.

The inventors of the present invention found that phosphate-specific prolyl isomerase Pin1 can stabilize BRCA1 and promote its function in DNA damage repair foci. Inhibition of Pin1 will destroy the stability of BRCA1 and damage DNA damage repair through HR, leading to Sensitive to radiation. Inhibition of Pin1 by shRNA will reduce the cell survival rate after ionizing radiation (Figure 1A-C). In MCF10A cells (BRCA1 and p53 expression), Pin1 inhibition reduced colony formation under 5Gy IR by about 35% (Figure 1A), while in MCF-7 (BRCA1 and p53 expression) breast cancer cells, colony formation was reduced Approximately 55% (Figure 1B). In T47D cells (BRCA1 expression and p53 deletion), colony formation after irradiation was reduced by approximately 74% (Figure 1C). On the other hand, in SUM149 cells, the BRCA1 mutant (2288delT), Pin1 knockdown did not further reduce the colony formation efficiency after IR treatment (Figure 1D), indicating that the loss of Pin1 and the loss of BRCA1 are equivalent.

Further studies showed that the knockout of Pin1 increased the degradation of BRCA1 under IR treatment. Therefore, we analyzed the stability of BRCA1. After Olaparib treatment, the degradation rate of BRCA1 in Pin1 knockdown cells was faster than that in control cells (Figure 1E, F), which supports the synergistic lethal effect of PARP inhibition combined with Pin1 depletion . For this reason, we studied the effect of Pin1 depletion on the sensitivity of PARP inhibitors in wild-type BRCA1 breast cancer cells MDA-MB 231 (triple negative) and MCF-7 and T47D (ER+). We found that after Pin1 was depleted, wild-type BRCA1 breast cancer cells with PARP inhibitor Olaparib, whether triple-negative or ER-positive, showed a sharp decrease in colony formation (Figure 1G-I). On the other hand, in SUM149 cells, BRCA1 mutant (2288delT), Pin1 knockdown did not further reduce the colony formation efficiency after Olaparib treatment (Figure 1J), indicating that the loss of Pin1 and the loss of BRCA1 are equivalent. Similarly, cell viability analysis showed that the growth of MDA-MB 231 and MCF-7 cells was delayed by Pin1 knockdown or Olaparib monotherapy, while Olaparib treatment in Pin1 knockdown cells almost completely blocked cell proliferation (Figure 1K , L). In addition, when exogenous Pin1 was added to shPin1MDA-MB 231 and MCF-7 cells, only the wild type could rescue the synergistic inhibition of Pin1 RNAi and Olaparib, while the non-catalytically active Pin1 mutant (S67E) could not rescue Pin1 RNAi and Olaparib. Synergistic inhibition of Olaparib (Figure 1K, L).

It was found that both ATRA and Olaparib can delay cell growth, and the combination of ATRA and Olaparib can synergistically inhibit cell growth, not only in MDA-MB 231 and MCF-7 cells, but also in BRCA1/2-WT pancreatic cancer cells PANC- This is also true in 1 and prostate cancer cells VCaP (Figure 2A-C). These in vitro data indicate that ATRA can act as a HR disruptor through Pin1 knockout, thus making BRCA1-WT tumors sensitive to PARP inhibition in vivo.

In order to measure the effect of Pin1 knockout on the response to PARP inhibitors in vivo, MDA-MB 231 cells expressing control or Pin1 shRNA with or without exogenous WT Pin1 or mutant Pin1 (S67E) were established in nu/nu mice . Two weeks after tumor cell inoculation, mice were treated with placebo or Olaparib for 5 weeks. Pin1 or Olaparib monotherapy can moderately delay the growth of the graft. However, when Pin1 knockout was used in combination with Olaparib, tumor growth was greatly delayed (Figure 3A, B). When exogenous WT Pin1 was added back to shPin1 cells, Olapaparib treatment only moderately delayed tumor growth, while mutant Pin1 (S67E) did not have this effect (Figure 3A, B). The average tumor volume of Pin1 knockout xenografts was 30% smaller than the control tumor (P<0.01). When Pin1 knockout was used in combination with Olaparib, the average tumor volume and weight were significantly reduced (Figure 3A-C). As expected, Pin1 knockout resulted in a decrease in BRCA1 levels (Figure 3D, E), which can be restored by adding exogenous WT Pin1 instead of mutated Pin1 (S67E). The combination of Pin1 knockout and Olaparib can effectively reduce tumor cell proliferation (assessed by Ki67 staining) and increase tumor cell apoptosis (detected by TUNEL analysis) (Figure 3D, F).

In the PDX of triple-negative breast cancer that does not contain BRCA1/2 mutations or core gene changes in HR DNA repair, the therapeutic effect of the combination of ATRA and Olaparib was further tested. Divide the tumors of three patients into four groups and ^{treat them when the volume reaches about 100mm 3.} The combination of ATR and Olaparib can significantly prevent the growth of tumors, while monotherapy can only slow down the growth of these tumors (Figure 4A-C) . As expected, ATRA caused a decrease in Pin1 and BRCA1 levels (Figure 4D, E). As assessed by Ki67 and TUNEL staining, the combination of ATRA and Olaparib effectively reduced the proliferation rate of tumor growth and increased the rate of apoptosis (Figure 4F, G). On the other hand, there was no significant difference in the body weight of each group of mice, indicating that the combination had no obvious systemic toxicity (Figure 4H).

Example 2

Pharmaceutical composition containing 10 mg of all-trans retinoic acid and 100 mg of olaparib

After 10 grams of all-trans retinoic acid and 100 grams of olaparib were crushed through a 120-mesh sieve, they were combined with 25 grams of croscarmellose sodium, 20 grams of mannitol, and 15 grams of poly Mix the vitamin ketone evenly, add 30 grams of 10% starch slurry to make soft material, pass through a 24-mesh sieve to granulate, then ventilate and dry at 50 degrees Celsius, and use a 20-mesh sieve to sizing, then mix with 2 grams of colloidal silicon dioxide and 2 Grams of sodium stearyl fumarate are mixed uniformly and compressed to obtain a pharmaceutical composition tablet.

Obviously, the above-mentioned embodiments of the present invention are merely examples to clearly illustrate the technical solutions of the present invention, and are not intended to limit the specific implementation manners of the present invention. Any modification, equivalent replacement and improvement made within the spirit and principle of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims (16)

1. A sensitizer drug, which is characterized in that it is used to enhance the radiochemotherapy sensitivity of a PARP inhibitor for BRCA1 expressing cancer, and the sensitizer drug includes a Pin1 inhibitor.
2. The sensitizer drug according to claim 1, wherein the Pin1 inhibitor is selected from the group consisting of all-trans retinoic acid, all-trans retinoic acid derivatives, arsenic trioxide, juglone, PPIase-Parvulin inhibitor, compound H At least one of -371, VS10, API-1, Pin1 shRNA, the target sequence of Pin1 shRNA is 5'-CCACCGTCACACAGTATTTAT-3'.
3. The sensitizer drug of claim 1, wherein the Pin1 inhibitor is all-trans retinoic acid, and the effective dose of the sensitizer drug is 1.5 mg·kg ^-1 d ^-1 .
4. The sensitizer drug of claim 1, wherein a specific concentration of the sensitizer drug and its carrier are used for delivery to tumors, cancer cells, or precancerous cells treated with PARP inhibitors to increase cancer The sensitivity of cells to PARP inhibitors.
5. The sensitizer drug according to claim 1, further comprising a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is used to transport the sensitizer to the tumor, cancer cell, or PARP inhibitor acting as the sensitizer. Precancerous cells.
6. The sensitizer drug according to claim 1, characterized in that it is used to prepare a drug for enhancing the sensitivity of BRCA1-expressing cancer with PARP inhibitors to radiotherapy and chemotherapy.
7. Use of a Pin1 inhibitor in the preparation of a drug that enhances the sensitivity of a PARP inhibitor to treat cancer, the cancer being BRCA1 expressing cancer.
8. The use according to claim 7, wherein the Pin1 inhibitor is selected from the group consisting of all-trans retinoic acid, all-trans retinoic acid derivatives, arsenic trioxide, juglone, PPIase-Parvulin inhibitor, compound H-371, At least one of VS10, API-1, and Pin1 shRNA, the target sequence of the Pin1 shRNA is 5'-CCACCGTCACACAGTATTTAT-3'.
9. A drug combination characterized by comprising: (1) a sensitizer; (2) a PARP inhibitor.
10. The pharmaceutical combination according to claim 9, wherein the sensitizer is a Pin1 inhibitor,

    The Pin1 inhibitor is selected from at least one of all-trans retinoic acid, all-trans retinoic acid derivatives, arsenic trioxide, juglone, PPIase-Parvulin inhibitor, compound H-371, VS10, API-1, Pin1 shRNA , The target sequence of the Pin1 shRNA is 5'-CCACCGTCACACAGTATTTAT-3';

    The PARP inhibitor is selected from olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, pamiparib, 3-Aminobenzamide, A-966492, PJ34 HCl, UFP-1069, ME-0328, NMS-P118, E7449, Picolinamide, Benzamide , Niraparib tosylate, NU-1025, Iniparib, AZD-2461, BGP-12·2HCl, compound CEP-8983, compound 2X-121, compound SC-10914, compound HWH-340, compound IDX-1197, simmiparib, compound IMP- 4297. At least one of compound ABT-767.
11. The drug combination according to claim 9 or 10, characterized in that it is used to prepare a medicine for treating cancer.
12. The drug combination according to claim 11, wherein the cancer is selected from the group consisting of ovarian cancer, peritoneal tumor, fallopian tube cancer, breast cancer, pancreatic cancer, prostate cancer, non-small cell lung cancer, head and neck tumors, solid tumors , Bladder cancer, small cell lung cancer, gastric cancer, transitional cell cancer, cervical cancer, endometrioid cancer, esophageal cancer, squamous cell carcinoma, glioblastoma, mesothelioma, kidney cancer, urethral cancer, uvea Melanoma, cholangiocarcinoma, Ewing's sarcoma, colorectal cancer.
13. The drug combination according to any one of claims 9-12, characterized in that it is used to prepare an anti-tumor drug for the treatment of targeted HR sensitivity and/or a drug for the treatment of cancer expressing wild-type BRCA1 and/ Or it can be used to prepare drugs for treating BRCA1 wild-type cancer.
14. The pharmaceutical combination according to claim 9, further comprising a pharmaceutically acceptable carrier for transporting the sensitizer to the tumor, cancer cell or precancerous cell.
15. The pharmaceutical combination according to claim 9, wherein the sensitizer is Pin1 inhibitor all-trans retinoic acid, and the effective dose is 1.5 mg·kg ^-1 d ^-1 ; the PARP inhibitor is olaparib, which is effective The dose is 50 mg·kg ^-1 d ^-1 .
16. The use of the drug combination according to any one of claims 9-15 in the preparation of drugs for treating HR-sensitive cancers, drugs for treating cancers expressing wild-type BRCA1, and/or drugs for treating BRCA1 wild-type cancers.

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