CN111671902B

CN111671902B - Sensitizer drug, drug composition and application

Info

Publication number: CN111671902B
Application number: CN202010198818.4A
Authority: CN
Inventors: 胡海; 罗曼莉; 郑芳
Original assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Current assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2022-08-16
Anticipated expiration: 2040-03-19
Also published as: WO2021184990A1; CN111671902A

Abstract

The sensitizer is a Pin1 inhibitor, and the sensitizer can improve the sensitivity of breast cancer expressing BRCA1 to PARP inhibitors by inhibiting prolyl isomerase Pin1, and is beneficial to improving the clinical effect of tumor targeted therapy. In addition, the medicinal composition of the Pin1 inhibitor and the PARP inhibitor can be used for preparing a therapeutic medicament for targeting HR sensitive tumors, can be used for treating BRCA1 wild type cancers, and has good safety and low toxicity.

Description

Sensitizer drug, drug composition and application

Technical Field

The invention belongs to the technical field of biological medicines, relates to a sensitizer medicine, a medicine composition and application thereof, and particularly relates to a sensitizer medicine for enhancing the sensitivity of a PARP inhibitor for BRCA 1-expressed cancer in radiotherapy and chemotherapy, a medicine composition and application thereof.

Background

The BRCA1 protein is phosphorylated in response to DNA damage, and phosphorylation is essential for the DNA damage repair function of BRCA1 (Science, 1999; 286: 1162-6, Nature, 2000; 404: 201-4). Loss of BRCA1 function is predisposed to breast or ovarian cancer. Furthermore, loss of BRCA1 function predicts a difference in response to a DNA damage repair defect: breast, ovarian, prostate and pancreatic cancer patients with the BRCA1 mutation are sensitive to therapeutic PARP inhibition, which can lead to irreversible DNA damage due to Homologous Recombination (HR) deficiency. PARP inhibitors have become safe and effective drugs for treating BRCA1 mutant breast, ovarian, prostate and pancreatic cancers 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-.

DNA Damage Repair (DDR) requires a perception of the site of DNA damage and a close spatiotemporal fit of repair protein complexes. In eukaryotic cells, these proteins are often activated by the serine/threonine phosphorylation signaling cascade. Phosphorylated serine/threonine residues adjacent to proline are targets for phosphate-specific prolyl isomerization, a post-phosphorylation modification that regulates protein folding, function and stability. 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.). Pin1 binds its phosphorylated target protein through the N-terminal WW domain and then allows its C-terminal PPIase domain to catalyze prolyl bond isomerization in the adjacent peptidyl group.

Generally, Pin1 stabilizes and enhances the function of cyclin-promoting 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-. Pin1 is also expressed in large amounts in breast Cancer, prostate Cancer, pancreatic Cancer, etc. (Nat Rev cancer.2016; 16: 463-78, Cancer Sci.2019; 110: 2442-55.).

All-trans retinoic acid (ATRA), which has few side effects by inducing differentiation, has been clinically used for the treatment of acute promyelocytic leukemia (APML). ATRA was newly found to inhibit the function of Pin1, and PIN1 was a key target for all-trans retinoic acid therapy in acute promyelocytic leukemia and breast cancer (Nat Med.2015; 21: 457-66). Arsenic targets Pin1 and works with tretinoin to inhibit cancer driver pathways and tumor initiating cells (Nature communications.2018; 9: 3069.). In a mouse model, Pin1 overexpression promoted the formation of breast Cancer, while Pin1 inactivation inhibited the occurrence of breast (Embo J, 2004; 23: 3397-. Pin1 also plays an important role in viral, bacterial and parasitic infections and their associated malignancies. Thus, targeting Pin1 represents a new non-toxic strategy that can simultaneously block multiple cancer-driving pathways and eliminate Tumor Initiating Cells (TICs). However, previously discovered inhibitors of Pin1 lack specificity, potency, and/or cell permeability.

Since PARP inhibitor monotherapy is effective in treating BRCA1 mutant breast, ovarian, prostate and pancreatic cancer patients, but is not applicable to BRCA1 wild-type cancer, these tumors cannot be sensitized by combining PARP inhibitors with HR interferents (Proc Natl Acad Sci USA.2016; 113: E4338-47, Cell Rep.2016; 17: 2367-81).

Therefore, there is still a need in the clinic to further study and develop sensitizer drugs or therapies for enhancing the chemoradiotherapy sensitivity of PARP inhibitors of BRCA1 expressing cancers, to increase the sensitivity of PARP inhibitors to BRCA1 expressing tumors, to improve the clinical efficacy of tumor-targeted therapies, and to provide clinicians with better treatment options.

Disclosure of Invention

The invention aims to overcome the technical defects that the existing PARP inhibitor medicine has limited clinical effect on patients with BRCA1 mutant breast cancer, ovarian cancer, prostate cancer and pancreatic cancer and the PARP inhibitor is not suitable for BRCA1 wild type cancer, and provides a sensitizer medicine, a medicine combination and application thereof, which can be used for enhancing the sensitivity of the PARP inhibitor to radiotherapy and chemotherapy of BRCA1 expression cancer.

The technical scheme adopted by the invention is that a sensitizer drug is used for enhancing the sensitivity of PARP inhibitors of BRCA1 expressing cancers in radiotherapy and chemotherapy, and the sensitizer drug comprises a Pin1 inhibitor. The Pin1 inhibitor can be used as a chemo-and radiotherapy sensitizer for enhancing the chemo-and-radiotherapy sensitivity of the PARP inhibitor of the cancer expressed by BRCA1 and enhancing the effect of anti-tumor chemo-radiotherapy.

Preferably, the Pin1 inhibitor is applied to preparing a sensitizer drug for enhancing the sensitivity of the PARP inhibitor of the BRCA1 expressing cancer to radiotherapy and chemotherapy.

Preferably, the Pin1 inhibitor is at least one selected from the group consisting of all-trans retinoic acid (ATRA), ATRA derivatives, Arsenic Trioxide (ATO), Juglone (Juglone), PPIase-Parvulin inhibitors, compound H-371 (derived from the compound on page 53 of patent WO 2019031472), VS10 and API-1.

Preferably, the PARP inhibitor of the present invention is at least one compound selected from the group consisting of Olaparib (Olaparib), Rucaparib, Niraparib (MK-4827), Talazoparib (BMN-673), Veliparib (ABT-888), pamiaparib (BGB-290), INO-1001(3-Aminobenzamide), A-966492, PJ34HCl, UFP-1069, ME-0328, NMS-P118, E7449, Picolinamide, Benzamide, Niraparib (MK-4827) tosynate, NU-1025, Iniparib (BSI-201), AZD-2461, BGP-12.2 HCl, CEP-8983(CK-102), Compound 2X-121, Fuopali (fluoroparib), Compound SC-14, Compound H-340, Compound IBH-1197, and Compound ABiparib 427.

Preferably, according to the 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, which acts as a sensitizer, and its carrier are at a suitable concentration such that a sufficient concentration of the sensitizer is delivered to the tumor, cancer cell or precancerous cell being treated with the PARP inhibitor to increase the sensitivity of the cancer cell to the PARP inhibitor.

The inventors of the present invention surprisingly found that Pin1 inhibition sensitizes breast cells to radiation and PARP inhibition. In particular, the inventors found that Pin1 can stabilize BRCA1 and promote its function in DNA damage repair foci, and that Pin1 inhibition disrupts the stability of BRCA1, disrupts DNA damage repair by HR, resulting in radiosensitivity. In addition, cell viability assays performed by the inventors showed that growth of human breast cancer cell lines MDA-MB 231 and MCF-7 cells was delayed by either Pin1 knockdown or by Olaparib monotherapy, whereas Olaparib treatment in Pin1 knocked down cells resulted in almost complete blockage of cell proliferation.

In addition, effective Pin1 inhibition may bring other benefits, such as disruption of the tumor's cyclinD1 signaling axis and suppression of the dominant mutation p53 found in most triple negative breast cancers, all of which contribute to its efficacy in tumor therapy.

Another object of the present invention is to provide a pharmaceutical composition and its use, the pharmaceutical composition comprising: (1) a sensitizer; (2) PARP inhibitors. Wherein the sensitizer is a Pin1 inhibitor.

Preferably, the Pin1 inhibitor can be at least one selected from the group consisting of all-trans retinoic acid (ATRA), ATRA derivatives, Arsenic Trioxide (ATO), Juglone (Juglone), PPIase-Parvulin inhibitors, compound H-371 (derived from the compound on page 53 of patent WO 2019031472) VS10 and API-1.

The PARP inhibitor may be at least one compound selected from the group consisting of Olaparib (Olaparib), Rucapparib, Nirapparib (MK-4827), Talazolparib (BMN-673), Veliparib (ABT-888), pamiaparib (BGB-290), INO-1001(3-Aminobenzamide), A-966492, PJ34HCl, UFP-1069, ME-0328, NMS-P118, E7449, Picolinamide, Benzamide, Niraparib (MK-4827) tosilate, NU-1025, Iniparib (BSI-201), AZD-2461, BGP-12.2 HCl, CEP-8983(CK-102), Compound 2X-121, Fuopali (fluxaparib), Compound SC-10914, Compound HWH-340, Compound X-1197, sidaramine (IMP-4297), and ABP 76767.

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

Since PARP inhibitor monotherapy is effective in treating patients with BRCA1 mutant breast, ovarian, prostate and pancreatic cancers, it is not applicable to BRCA1 wild-type cancers. The inventors of the present invention have discovered that a Pin1 inhibitor in combination with a PARP inhibitor can be used to treat BRCA1 wild-type (BRCA1-WT) cancer.

According to some embodiments of the invention, the combination of ATRA and Olaparib may 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. These in vitro data indicate that ATRA can act as an HR disrupter through Pin1 knockdown, thus sensitizing BRCA1-WT tumors to PARP inhibition in vivo. Mice bearing MDA-MB 231 xenografts were found to significantly delay tumor growth 5 weeks later by using either single drug ATRA, olaparib or their combined effect. The combination of ATRA with Olaparib effectively reduced the tumor growth proliferation rate and increased the apoptosis rate as assessed by Ki67 staining and TUNEL analysis, respectively.

Therefore, the medicine composition can be used for preparing medicines for treating cancers, has wide targeting property and can be used for targeting 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 tumor, solid tumor, bladder cancer, small cell lung cancer, gastric cancer, transitional cell cancer, cervical cancer, endometrioid cancer, esophageal cancer, squamous cell cancer, glioblastoma, mesothelioma, renal cancer, urethral cancer, uveal melanoma, bile duct cancer, ewing's sarcoma, colorectal cancer.

ATRA or ATRA derivatives may act as potent inhibitors of Pin1, sensitizing tumors to PARP inhibition. In view of the abundant expression of Pin1 in various cancer types, the pharmaceutical compositions of ATRA and Olaparib have broad targeting properties in addition to breast cancer, and can be used to target HR sensitive tumors. On the other hand, the body weight of each group of mice has no significant difference, which indicates that the pharmaceutical composition has no obvious systemic toxicity, further proving the safety of the pharmaceutical composition of the invention.

According to the embodiment of the invention, the effective dose of the PARP inhibitor and the Pin1 inhibitor in the effective components of the pharmaceutical composition is 50 mg-kg of the PARP inhibitor ^-1 d ^-1 The dosage of the Pin1 inhibitor is 1.5 mg-kg ^-1 d ^-1 . Therefore, the medicinal composition/or the medicinal composition of the Pin1 inhibitor and the PARP inhibitor with the effective components in the dosage combination can be used for preparing a therapeutic medicament for targeting HR sensitive tumors.

Preferably, the pharmaceutical composition provided by the invention can be used for preparing a medicament for treating cancer expressing BRCA1, and is used for treating cancer caused by BRCA1 mutation.

Preferably, the pharmaceutical composition provided by the invention can be used for preparing a medicament for treating BRCA1 wild type cancers and treating BRCA1 wild type cancers.

The term "effective dose" as used herein refers to a dose or an effective amount to be administered to a patient. The dose or effective amount to be administered to a patient and the frequency of administration to a subject can be readily determined by one of ordinary skill in the art using known techniques and by observing results obtained under similar conditions. In determining an effective amount or dose, the attending diagnostician will consider a number of factors, including, but not limited to, the potency and duration of action of the compound employed; the nature and severity of the disease to be treated as well as the sex, age, weight, health status and individual responsiveness of the patient to be treated and other relevant conditions.

Preferably, the effective active ingredients in the pharmaceutical composition of the present invention can be mixed with various pharmaceutically acceptable excipients to be made into solid preparation or liquid preparation, such as solid forms of tablets, capsules, granules or powders, or liquid forms of oral liquid, injections, etc. According to the specific embodiment of the present invention, the preferred dosage form is tablet, granule, capsule, but is not limited to the preferred dosage form, and other oral preparations which are conventional in pharmacy are also included. Wherein the tablet, granule and capsule can be common preparation or sustained-release preparation using special technology. When prepared into a solid composition preparation in the form of granules, it may be formulated into a sugar-free form or a sugar-containing form.

The "pharmaceutically acceptable excipient" includes any common excipient that can be used pharmaceutically, such as a disintegrant, a binder, a filler, a lubricant, a matrix material for controlling the release rate of an active ingredient in a sustained-release preparation (i.e., a release rate modifier), etc., so that it has sustained-release or controlled-release activity for sustained-release of a predetermined amount of the active ingredient.

Preferably, the method for preparing the tablet is not particularly limited, and the active ingredient of the pharmaceutical composition of the present invention can be controlled to 100 mesh to 200 mesh using a conventional preparation technique in the art. According to some embodiments of the present invention, the method for preparing the tablet can be performed by granulating by dry rolling, wet method or spray vulcanization, and tabletting, or by directly pulverizing and sieving the pharmaceutical composition and tabletting. According to other embodiments of the present invention, the tablet may be prepared as a single-layer tablet, or as a double-layer tablet, a sustained-release tablet, or the like known in the art. According to a particular embodiment of the invention, the tablet cores may or may not be coated with a film or sugar-coat layer. In addition, a proper amount of flavoring agent can be added in the preparation of the tablet according to the requirement so as to meet the requirements of different tastes.

The pharmaceutical composition of the Pin1 inhibitor and the PARP inhibitor can be used for preparing an antitumor drug for treating targeted HR sensitivity, and can be used for treating BRCA1 wild type cancers.

Compared with the prior art, the invention has the beneficial effects that: the Pin1 inhibitor and the pharmaceutical composition thereof further expand new application and clinical application in clinic. The Pin1 inhibitor can improve the sensitivity of the breast cancer expressing BRCA1 to PARP inhibitors by inhibiting the prolyl isomerase Pin1, and is applied to a sensitizer for enhancing the sensitivity of the PARP inhibitors of the cancer expressing BRCA 1. The medicinal composition of the Pin1 inhibitor and the PARP inhibitor can be used for preparing a tumor treatment medicament with targeted HR sensitivity, can be used for treating BRCA1 wild type cancer, and has good safety and low toxicity.

Drawings

FIG. 1 is a graph showing the results of an in vitro experiment described in example 1 of the present invention demonstrating that Pin1 inhibition sensitizes breast cancer cells to radiation and PARP inhibition; wherein,

a, B, C: inhibition of Pin1 decreased the survival of MCF10A/MCF-7/T47D cells after IR;

d: in BRCA1 mutant (2288delT) SUM149 cells, Pin1 knock-down did not further reduce the efficiency of colony formation after IR post-treatment;

e, F: after Olaparib treatment, the degradation speed of the Pin1 in the cells for knocking down BRCA1 is increased;

g, H, I: after the Pin1 knockout, the wild type BRCA1 breast cancer cells with PARP inhibitor Olaparib have a sharp reduction in colony formation no matter the cells are of the triple negative type or the ER positive type;

j: in BRCA1 mutant (2288delT) SUM149 cells, Pin1 knock-down did not further reduce the efficiency of colony formation after Olaparib treatment;

k, L: pin1 knockdown or Olaparib monotherapy, MDA-MB 231 and MCF-7 growth retardation, resulted in almost complete block of cell proliferation in cells with Pin1 knockdown. When exogenous Pin1 is added to shPin1 MDA-MB 231 and MCF-7 cells, only the wild type can save the synergistic inhibition effect of Pin1 RNAi and Olaparib, and the catalytically inactive Pin1 mutant (S67E) cannot save the synergistic inhibition effect of Pin1 RNAi and Olaparib.

FIG. 2 is a graph showing the results of an in vitro experiment described in example 1 of the present invention demonstrating that Pin1 inhibition sensitizes pancreatic and prostate cancer cells to radiation and PARP inhibition; wherein,

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

FIG. 3 is a graph showing the results of an in vivo experiment described in example 1 of the present invention demonstrating that Pin1 inhibition sensitizes breast cancer cells to radiation and PARP inhibition; wherein,

a, B, C: 2 weeks after tumor cell inoculation, 5 weeks after mouse placebo-controlled or Olaparib treatment, tumor growth was moderately delayed by Pin1 or Olaparib monotherapy, Pin1 was used in combination with Olaparib, tumor growth was delayed, and when exogenous WT Pin1 was added back to shPin1 cells, Olaparib treatment only moderately delayed tumor growth, which was not present in mutant Pin1, and when Pin1 knockout was used in combination with Olaparib, mean tumor volume and weight were significantly reduced;

d, E, F: the Pin1 knockout resulted in a decrease in BRCA1 levels. The Pin1 knockout and Olaparib combination were effective in reducing tumor cell proliferation (assessed by Ki67 staining) and increasing tumor cell apoptosis (detected by TUNEL assay).

FIG. 4 is a graph of the therapeutic effect of ATRA in combination with Olaparib in a triple negative breast cancer PDX that does not contain the BRCA1/2 mutation or a gene change in HR DNA repair in an in vivo assay as described in example 1 of the present invention; wherein, A, B, C: ATRA and Olapaparib combined significantly delayed tumor growth;

d, E: ATRA results in decreased levels of Pin1 and BRCA 1;

f, G: through Ki67 staining and TUNEL analysis and evaluation, the combination of ATRA and Olaparib effectively reduces the growth and proliferation rate of tumors and improves the apoptosis rate;

h: there was no significant difference in body weight of mice in each group, indicating that the combination had no significant systemic toxicity.

Detailed Description

The drawings are only for purposes of illustration and are not to be construed as limiting the invention. For a better understanding of the following embodiments, certain features of the drawings may be omitted, enlarged or reduced, and do not represent the size of an actual product; it will be understood by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted.

Example 1

In vitro and in vivo biological assays

Materials and methods

(1) Medicine

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

(2) Cells and cell culture

ATCC-derived MCF10A cells were cultured in DMEM/F-12 supplemented with 5% horse serum, 20ng/ml Epidermal Growth Factor (EGF), 0.5. mu.g/ml hydrocortisone, 100ng/ml cholera toxin, 10. mu.g/ml insulin and cultured as described in methods.2003; 30: 256-68. ATCC-derived MCF-7, T47D, MDA-MB 231, AU565, HEK293 were cultured in DMEM containing 10% FBS. SUM149 was cultured in Ham's F-12 medium containing 5% bovine serum (FBS), insulin (5. mu.g/mL) and hydrocortisone (2. mu.g/mL).

The radiation dose is chosen according to the experiment and the cells used. MCF10A cells were treated to 5Gy and DNA repair foci were visualized by immunofluorescence assay. Breast cancer cells were assayed for BRCA1 degradation using 10 Gy.

(3) Cell viability and luciferase assay

For cell viability assays, cells were seeded in 96-well plates at a density of 1000(MDA-MB 231), 2000(MCF-7) per well. The following day ATAR or Olaparib was added to the cells. The cell viability was determined by CellTiter-Glo luminescent cell viability assay (Promega) and absorbance was read by Wallac3 plate reader.

(4) Clone formation assay

Cells were seeded at a density of 500(MDA-MB 231), 1000(MCF-7), T47D (1000), 500(MCF10A), SUM149(1000) per well in 6-well plates overnight. Cells were irradiated or treated with olaparib (Olapaparib). The number of surviving clones was checked after 6-7 days.

(5) Plasmid transfection and RNAi experiments

pcDNA3.1 plasmids containing HA-BRCA1, Flag-Pin1, or a fragment containing Myc-tagged BRCA1 were transfected with Lipofectamine 2000 (Invitrogen). The medium was changed to growth medium 8 hours after transfection. Site-directed mutagenesis kit (Agilent) was used to detect point mutations in BRCA1 and Pin1 generated by pcDNA3.1 HA-BRCA1 and Flag-Pin1 plasmids. The successful construction of ShPin1 cells was tested as described in Cell Rep.2015; 11: 111-24. The target sequence for the Pin1 shRNA was 5'-CCACCGTCACACAGTATTTAT-3', targeting the Pin 13 ' UTR. The control shRNA sequence was 5'-UAAGGCUAUGAAGAGAUAC-3'. After lentivirus infection, cells were selected using puromycin.

(6) Immunoblotting

Antibodies include anti-beta-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). Immunoblotting experiments were performed according to standard procedures.

(7) Immunofluorescence detection

MCF10A, MCF-7 cells were irradiated, fixed at the indicated time points, reacted with antibodies against BRCA1 (1: 50, MS110), Pin1 (1: 100), 53BP1 (1: 50, cell signaling) and the corresponding secondary antibodies, and examined with Zeiss Axiovert 200M fluorescence microscope.

(8) Immunohistochemical detection

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

(9) TUNEL assay

Apoptosis was detected by TUNEL method in formalin-fixed and paraffin-embedded sections using the cell death detection kit (Roche). Sections were dewaxed, rehydrated and treated with proteinase K before addition of TUNEL reaction mixture. Staining of the sections was observed by Leica fluorescence microscopy.

(10) In vivo animal experiments

All experiments involving mice were approved by the animal ethics committee of the university of zhongshan and studied according to relevant regulations. Mixing 1.5X 10 ⁶ The shControl or shPin 1-expressing cells with/without exogenous WT Pin1 or mutant Pin1(S67E) MDA-MB 231 were injected into mammary fat pads of 6-week-old BALB/c nude mice (Jackson laboratories). After two weeks, when tumor growth was seen, mice were randomly assigned to the Olaparib-treated group and the control group. Olaparib (50 mg. kg) ^-1 ·d ^-1 ) Or placebo intraperitoneally for 5 weeks. 1.5X 10 for combination therapy of ATRA and Olaparib ⁶ The MDA-MB 231 cells of (A) were injected into fat pads of 6-week-old BALB/c nude mice (Jackson laboratory).

For PDX implantation, three BRCA 1-expressing TNBC specimens were obtained from tumor resection of patients at the grand society commemorative hospital, university of zhongshan, during the year 2017-. Patient informed consent was given as specified by the internal review and ethical committee of the grandson impatient commemorative hospital, details such as cell.2018; 172: 841-56 e 16. 6-week old NSG female mice were anesthetized. Cutting the tumor into 1mm ³ Size, directly embedded in the mammary fat pad to obtain the first generation PDX. Once the first generation PDX reached 1 cm in diameter, it was harvested and cut to 1mm ³ Size, directly embedded in the mammary fat pad to obtainThe second generation of PDX for therapeutic use. PDX from each patient was transplanted into four mice and randomized into four groups. Once tumors were established, mice were randomized into ATRA, Olapaparib, combination and control treatment groups. Use of placebo, olaparide (50mg kg) ^-1 d ^-1 )，ATRA(1.5mg.kg ^- ¹ d ^-1 ) Or olaparide (50mg kg) ^-1 d ^-1 ) And ATRA (1.5 mg.kg) ^-1 d ^-1 ) Intraperitoneal injections were performed for 5 weeks. Tumor size was recorded with calipers using the formula L W ² X 0.5 tumor volume was calculated, where L and W represent length and width, respectively. Mice were sacrificed 5 weeks after treatment. No animals died during the experiment.

Detection of HR DNA repair (AmoyDx) by PDX was done using NGS sequencing, which included 32 genes (AR/ATM/ATR/BARD1/BRAF/BRCA1/BRCA2/BRIP1/CDH1/CDK12/CHEK1/CHEK2/ERBB2/ESR 1/FACCA/FACCL/HDAC 2/HOXB13/KRAS/MRE11/NBN/NRAS/PALB2/PIK3CA/PPP2R2A/PTEN/RAD51B/RAD51C/RAD51D/RAD54L/STK11/TP53) all coding regions and junctions of exons and introns were sequenced by xtNeq 500. The average coverage rate of the sequencing depth target area is more than or equal to 1000 x. The raw data was analyzed by an AmoyDx andsa data analyzer. Genetic variation was screened using MAF < 1% in 1000 genome projects.

(11) Statistical analysis

Data from three laboratory in vitro experiments are presented as mean ± standard deviation. All statistical analyses were performed using the SPSS 16.0 statistical software package. Cell viability, colony formation and tumor volume comparisons at different treatment methods were analyzed using t-test and one-way anova. In all cases, P < 0.05, P < 0.01 and P < 0.001.

(II) results

In vitro experiments were mainly cellular experiments, which demonstrated that Pin1 inhibition sensitizes breast cells to radiation and PARP inhibition. This section induced DNA double strand breaks (DBS) by treatment with Ionizing Radiation (IR) and then examined the effect of Pin1 inhibitors on sensitivity to radiation therapy. The change of cell survival rate of MCF10A/MCF-7/T47D after IR was detected by inhibiting Pin1, the effect of colony formation of wild type BRCA1 breast cancer cells with PARP inhibitor Olaparib was detected by inhibiting Pin1, and the effect of individual and combined use of the Pin1 inhibitor ATRA and the PARP inhibitor Olaparib on the growth of breast/pancreatic/prostate cancer cells was investigated.

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, and that Pin1 inhibition disrupts the stability of BRCA1, disrupts DNA damage repair by HR, leading to radiosensitivity. Inhibition of Pin1 by shRNA decreased cell viability following ionizing radiation (fig. 1A-C). In MCF10A cells (BRCA1 and p53 expression), Pin1 inhibition reduced colony formation at 5Gy IR by about 35% (FIG. 1A), while in MCF-7(BRCA1 and p53 expression) breast cancer cells, colony formation was reduced by about 55% (FIG. 1B). In T47D cells (BRCA1 expression and p53 deletion), colony formation after irradiation was reduced by approximately 74% (fig. 1C). On the other hand, in SUM149 cells, BRCA1 mutant (2288delT), Pin1 knockdown did not further reduce colony formation efficiency after IR treatment (fig. 1D), indicating that the loss of Pin1 and the loss of BRCA1 were equivalent.

Further studies showed that knock-out of Pin1 increased degradation of BRCA1 under IR treatment. Therefore, we analyzed BRCA1 stability and the degradation rate of BRCA1 was faster in Pin1 knockdown cells than control cells after Olaparib treatment (fig. 1E, F), supporting a synergistic lethal effect of PARP inhibition combined with Pin1 depletion. To this end, we investigated the effect of Pin1 depletion on PARP inhibitor sensitivity in wild-type BRCA1 breast cancer cells MDA-MB 231 (triple negative) and MCF-7 and T47D (ER +). We found that wild-type BRCA1 breast cancer cells with the PARP inhibitor Olaparib showed a dramatic decrease in colony formation after Pin1 depletion, whether triple negative or ER positive (fig. 1G-I). On the other hand, in SUM149 cells, the BRCA1 mutant (2288delT), Pin1 knockdown did not further reduce colony formation efficiency after Olaparib treatment (fig. 1J), indicating that the loss of Pin1 and the loss of BRCA1 were equivalent. Similarly, cell viability analysis showed that growth of MDA-MB 231 and MCF-7 cells was delayed by either Pin1 knockdown or by Olaparib monotherapy, whereas Olaparib treatment resulted in almost complete block of cell proliferation in cells with Pin1 knockdown (FIG. 1K, L). In addition, when exogenous Pin1 was added to the shPin1 MDA-MB 231 and MCF-7 cells, only the wild type was able to rescue the synergistic inhibition of Pin1 RNAi and Olaparib, while the catalytically inactive Pin1 mutant (S67E) was unable to rescue the synergistic inhibition of Pin1 RNAi and Olaparib (fig. 1K, L).

It was found that cell growth was delayed in both ATRA and Olaparib, and that the combination of ATRA and Olaparib synergistically inhibited 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 (FIGS. 2A-C). These in vitro data indicate that ATRA can act as an HR disrupter through Pin1 knockdown, thus sensitizing BRCA1-WT tumors to PARP inhibition in vivo.

To measure the effect of Pin1 knock-out on PARP inhibitor response in vivo, MDA-MB 231 cells expressing control or Pin1 shRNA with/without exogenous WT Pin1 or mutant Pin1(S67E) were established in nu/nu mice. Mice were treated with placebo control or Olaparib for 5 weeks 2 weeks after tumor cell inoculation. Single drug treatment with Pin1 or Olaparib moderately delayed graft growth. However, when the Pin1 knockout was used in combination with Olaparib, tumor growth was greatly delayed (fig. 3A, B). While Olapaparib treatment only moderately delayed tumor growth when exogenous WT Pin1 was added back to shPin1 cells, mutant Pin1(S67E) did not (fig. 3A, B). The mean tumor volume of the Pin1 knockout xenografts was 30% less than the control tumors (P < 0.01). When the Pin1 knockout was used in combination with Olaparib, the mean tumor volume and weight were significantly reduced (fig. 3A-C). As expected, Pin1 knock-out resulted in a decrease in BRCA1 levels (fig. 3D, E), which could be restored by addition of exogenous WT Pin1 instead of mutated Pin1 (S67E). The combination of Pin1 knockout and Olaparib was effective in reducing tumor cell proliferation (assessed by Ki67 staining) and increasing tumor cell apoptosis (detected by TUNEL assay) (fig. 3D, F).

The therapeutic effect of ATRA in combination with Olaparib was further tested in PDX of triple negative breast cancer that did not contain BRCA1/2 mutation or core gene changes in HR DNA repair. Three patients' tumors were divided into four groups and treated at a volume of about 100mm3, the combination of ATR and Olaparib significantly prevented tumor growth, whereas monotherapy slowed the growth of these tumors (fig. 4A-C). As expected, ATRA resulted in a decrease in the levels of Pin1 and BRCA1 (fig. 4D, E). The combination of ATRA and Olaparib effectively reduced the proliferation rate of tumor growth and increased the apoptosis rate as assessed by Ki67 and TUNEL staining, respectively (fig. 4F, G). On the other hand, there was no significant difference in body weight of each group of mice, indicating that the combination had no significant systemic toxicity (FIG. 4H).

Example 2

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

Respectively crushing 10g of all-trans retinoic acid and 100 g of olaparib, sieving the crushed materials with a 120-mesh sieve, uniformly mixing the crushed materials with 25 g of croscarmellose sodium, 20 g of mannitol and 15 g of povidone which are sieved with a 80-mesh sieve, adding 30 g of 10% starch slurry to prepare a soft material, sieving the soft material with a 24-mesh sieve to prepare granules, then carrying out ventilation drying at 50 ℃, grading the granules by using the 20-mesh sieve, uniformly mixing the granules with 2 g of colloidal silicon dioxide and 2 g of sodium stearyl fumarate, and tabletting to obtain the pharmaceutical composition tablet.

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.

Claims

1. A pharmaceutical combination comprising a Pin1 inhibitor and a PARP inhibitor, wherein the Pin1 inhibitor is at least one selected from the group consisting of ATRA, Pin1 shRNA, and the PARP inhibitor is Olaparib.

2. The pharmaceutical combination of claim 1, wherein the Pin1 shRNA sequence is 5'-CCACCGTCACACAGTATTTAT-3'.

3. The pharmaceutical combination of claim 1, wherein the Pin1 inhibitor is ATRA and the PARP inhibitor is Olaparib.

4. The pharmaceutical combination of claim 3, wherein the effective amount of ATRA is 1.5 mg-kg ^-1 d ^-1 (ii) a The effective dose of Olaparib is 50mg kg ^-1 d ^-1 。

5. The pharmaceutical combination according to claim 1, for use in the preparation of a medicament for the treatment of a cancer selected from ovarian cancer, peritoneal tumors, 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 carcinoma, cervical cancer, endometrioid cancer, esophageal cancer, squamous cell carcinoma, glioblastoma, mesothelioma, renal cancer, urethral cancer, uveal melanoma, bile duct cancer, ewing's sarcoma, colorectal cancer.

6. A pharmaceutical combination according to any one of claims 1 to 5 for use in the preparation of a medicament for the treatment of an HR-sensitive targeted antineoplastic agent and/or for use in the preparation of a medicament for the treatment of a cancer expressing wild-type BRCA1 and/or for use in the preparation of a medicament for the treatment of wild-type BRCA1 cancer.

Use of a Pin1 inhibitor for the preparation of an agent that enhances the sensitivity of treatment with a PARP inhibitor of BRCA1 expressing cancer, said Pin1 inhibitor being selected from at least one of ATRA, Pin1 shRNA, said PARP inhibitor being Olaparib.

8. The use of claim 7, wherein the Pin1 shRNA sequence is 5'-CCACCGTCACACAGTATTTAT-3'.

9. The use according to claim 7, wherein the Pin1 inhibitor is ATRA and the PARP inhibitor is Olaparib.

10. The use according to claim 7, wherein said BRCA1 expressing cancers include breast, pancreatic, prostate cancer.

Use of an inhibitor of Pin1, the inhibitor of Pin1 being selected from at least one of ATRA, Pin1 shRNA, in the manufacture of a medicament for disrupting the stability of BRCA1 and/or enhancing the sensitivity of BRCA1 to radiotherapy of cancer expressed by tumor cells.

12. The use of claim 11, wherein the Pin1 shRNA sequence is 5'-CCACCGTCACACAGTATTTAT-3'.

13. The use of claim 11, wherein said BRCA 1-expressing cancers include breast, pancreatic, prostate cancer.