Drug application of combination of IDH1 inhibitor and PARP inhibitor

Abstract

The invention discloses an application of an IDH1 inhibitor combined with a PARP inhibitor in an anti-tumor aspect. The drug combination inhibits DNA damage repair of tumors by inhibiting homologous recombination repair of tumors, increases sensitivity of BRCA1 / 2 wild tumors to PARP inhibitors, solves the drug resistance problem of PARP, also has a synergistic anti-tumor effect, provides a new approach and means for effective treatment of ovarian cancer, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical application of an IDH1 inhibitor combined with a PARP inhibitor, and in particular to an application of an IDH1 inhibitor combined with a PARP inhibitor in the preparation of an anti-tumor drug. Background Art

[0002] Poly (ADP-ribose) polymerase (PARP) is an enzyme that catalyzes the transfer of ADP-ribosyl groups to receptor proteins. It is mostly located in the nucleus of eukaryotic cells and participates in the poly ADP-ribosylation modification of nuclear proteins such as histones, topoisomerases, and DNA polymerases. There are 18 subtypes in the PARP family, of which PARP1 is the most abundantly expressed and most concerned subtype. PARP1 recognizes and binds to DNA break sites through the N-terminus, and plays an important role in base excision repair and single-strand damage repair. As a molecular sensor of DNA damage, inhibition of PARP will directly lead to the accumulation of DNA damage, cell cycle arrest, and apoptosis.

[0003] DNA double-strand break (DSB) is the most serious form of DNA damage, which is mainly repaired through two pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). BRCA1 / 2 plays a vital role in the repair of DNA double-strand breaks mediated by homologous recombination. The deletion or mutation of the BRCA1 / 2 gene can lead to severe damage to HR repair and genomic disorder. Therefore, BRCA1 / 2 is an important tumor suppressor gene. Studies have found that the lack of homologous recombination repair pathways, such as BRCA1 / 2 mutations, makes cells dependent on PARP for repair and sensitive to PARP inhibitors, resulting in a "synthetic lethality" effect.

[0004] The human BRCA1 gene is located on chromosome 17q21, about 81kb, and encodes a BRCA1 protein of about 220kDa, consisting of 1863 amino acid residues. The human BRCA2 gene is located on chromosome 13, long arm region 12, and consists of 27 exons, encoding a BRCA2 protein of 3418 amino acids. BRCA1 and BRCA2 proteins play an important regulatory role in multiple steps of HR repair, and are therefore called key regulatory molecules for HR repair.

[0005] Currently, several PARP inhibitor drugs are on the market or in the clinical trial stage, but most ovarian cancer patients carry wild-type BRCA1 / 2 genes and have poor sensitivity to PARP inhibitor drugs, which limits their widespread clinical application. Summary of the invention

[0006] Purpose of the invention: The present invention aims to provide a pharmaceutical application of an IDH1 inhibitor combined with a PARP inhibitor having a synergistic effect in anti-tumor.

[0007] Technical solution: The IDH1 inhibitor described in the present invention is combined with a PARP inhibitor for use in the preparation of drugs for treating BRCA1 / 2 wild-type tumors.

[0008] The IDH1 inhibitor of the present invention is used in the preparation of a drug for increasing the sensitivity of BRCA1 / 2 wild-type tumors to PARP inhibitors.

[0009] Preferably, the IDH1 inhibitor increases the sensitivity of the tumor to PARP inhibitors by inhibiting DNA damage repair in BRCA1 / 2 wild-type tumors.

[0010] Further preferably, the IDH1 inhibitor inhibits DNA damage repair in BRCA1 / 2 wild-type tumors by inhibiting homologous recombination repair.

[0011] Preferably, the PARP inhibitor is a PARP1 inhibitor.

[0012] Preferably, the drug is a drug that inhibits BRCA1 / 2 wild-type ovarian cancer, breast cancer, prostate cancer, and pancreatic cancer.

[0013] Preferably, the IDH1 inhibitor is selected from Ivosidenib.

[0014] Preferably, the PARP inhibitor is selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Pamiparib, Fluzoparib.

[0015] Preferably, the IDH1 inhibitor is selected from Ivosidenib, and the PARP inhibitor is selected from Olaparib.

[0016] Preferably, the dosage ratio of the IDH1 inhibitor to the PARP inhibitor is 1:5.

[0017] Preferably, the drug is a drug that inhibits the proliferation and metastasis of BRCA1 / 2 wild-type tumors.

[0018] Preferably, the drug is administered in the form of a drug containing a single active ingredient, or a compound drug containing two active ingredients.

[0019] More preferably, when a single active ingredient drug is administered separately, it can be administered simultaneously or sequentially.

[0020] More preferably, when the sequential administration form is adopted, the order of administration is not limited.

[0021] Preferably, the drug is in the form of an oral preparation or an injection preparation.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0023] The drug combination designed by the present invention inhibits tumor DNA damage repair by inhibiting tumor homologous recombination repair, increases the sensitivity of BRCA1 / 2 wild-type tumors to PARP inhibitors, solves the problem of PARP resistance, and also has a synergistic anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The combined use of these drugs has an inhibitory effect on the proliferation of BRCA1 / 2 wild-type ovarian cancer cells;

[0025] Figure 2 The combined inhibitory effect on homologous recombination repair (HR) efficiency in BRCA1 / 2 wild-type ovarian cancer cells;

[0026] Figure 3 It is the accumulation effect of combined use on double-strand DNA break damage in cells;

[0027] Figure 4 The synergistic anti-tumor effect of the combination in nude mice;

[0028] Figure 5 The combined use increases the proportion of cells with DNA damage. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below in conjunction with embodiments.

[0030] Example 1: Evaluation of the inhibitory effect on the proliferation ability of BRCA1 / 2 wild-type ovarian cancer cells

[0031] Experimental design: By adding olaparib and different marketed drugs to BRCA1 / 2 wild-type ovarian cancer cells for combined treatment, the proliferation ability of tumor cells after combined treatment was analyzed by CCK8 to screen for possible combination methods that can sensitize PARP inhibitors.

[0032] Test process:

[0033] 1. Cell treatment

[0034] 10 μM of drugs from the FDA-approved drug library were added to the A2780 cell line, 50 μM of Olaparib was added to the experimental group, and the same volume of DMSO (dimethyl sulfoxide) was added to the control group and treated for 12 hours.

[0035] 2. CCK8 test

[0036] (1) The treated cells were trypsinized, counted, and seeded into 96-well plates with 100 μl of culture medium per well.

[0037] (2) Place the culture plate in a 37°C, 5% CO2 incubator for 24 hours.

[0038] (3) 10 μl of CCK-8 solution was added to each well, and the wells that were added with the corresponding amount of cell culture medium and CCK-8 solution but without cells were used as blank controls.

[0039] (4) The cells were incubated in the cell culture incubator for 0.5 h and the absorbance was measured at 450 nm using an ELISA reader.

[0040] The results showed that the absorbance value of A2780 cells treated with ivosidenib and olaparib in the experimental group was significantly lower than that in the control group, indicating that the combination of ivosidenib and olaparib can effectively inhibit the proliferation of BRCA1 / 2 wild-type ovarian cancer cells ( Figure 1 ).

[0041] Example 2: Evaluation of the inhibitory effect on homologous recombination repair efficiency of BRCA1 / 2 wild-type ovarian cancer cells

[0042] Experimental design: Control reagent, ivosidenib (10 μM), olaparib (50 μM) and the combination of the two (olaparib 50 μM + ivosidenib 10 μM) were added to the cells for 12 hours, and RPA2 and RAD51, that is, DNA damage repair ability, were analyzed by immunofluorescence staining.

[0043] Test process:

[0044] (1) Wash the cells. Discard the culture medium and add room temperature TBS to wash twice, 5 seconds each time.

[0045] (2) Cover with 4% neutral formaldehyde fixative and fix at room temperature for 15 minutes, ensuring that there is enough fixative.

[0046] (3) Remove the fixative and rinse three times with 4°C precooled TBS, 5 minutes each time.

[0047] (4) Treat with ice methanol for 5 to 10 minutes.

[0048] (5) Block with 5% goat serum and incubate at 37°C for 30 minutes.

[0049] (6) Remove the blocking solution, add the primary antibody working solution, and incubate at 4°C overnight.

[0050] (7) Rewarm for 15 minutes and wash with TBST and TBS.

[0051] (8) Add fluorescent secondary antibody working solution and incubate at 37°C for 1 h in dark.

[0052] (9) Add DAPI working solution and incubate at room temperature for 30 minutes in the dark.

[0053] (10) Add anti-fluorescence attenuation mounting medium and observe and collect images under a fluorescence microscope.

[0054] The results showed that compared with the control group (Ctrl), the fluorescence intensity of γH2AX increased in cells treated with ivoanib (Ivo) or olaparib (Ola) alone, indicating increased DNA damage. The fluorescence intensity of RPA2 and RAD51 increased in cells treated with ivoanib or olaparib alone, indicating that the homologous recombination repair process was activated. When ivoanib and olaparib were used in combination (Ola+Ivo), the fluorescence intensity of γH2AX increased further, while the fluorescence intensity of RPA2 and RAD51 decreased, indicating that the combined treatment may have inhibited the efficiency of homologous recombination repair ( Figure 2 ).

[0055] Example 3: Evaluation of the accumulation of DNA double-strand breaks in cells treated with PARP inhibitors and IDH inhibitors

[0056] Experimental design: The cells were treated with control reagent, olaparib 50μM, ivosidenib 10μM and the combination of the two (olaparib 50μM + ivosidenib 10μM) for 12h, and the expression level of γH2AX protein and the shape of the comet assay fluorophore were verified by Western blot experiment using β-actin as the internal reference protein.

[0057] Test process:

[0058] 1. Protein extraction

[0059] (1) Use a cell culture dish of appropriate size for cell plating and prepare to collect samples when the cell density reaches about 90%. Label the culture dishes and centrifuge tubes in advance and pre-cool the centrifuge.

[0060] (2) Pour out all the old culture medium in the well plate or culture dish.

[0061] (3) Wash the plate or dish 2 to 3 times with PBS solution to ensure that there is no residual culture medium. After washing, dry with filter paper to avoid excessive PBS residue affecting the concentration of the lysate.

[0062] (4) Prepare cell lysis buffer: Mix the lysis buffer in appropriate proportions.

[0063] (5) Add an appropriate amount of lysis solution (e.g., 100-200 μl) to each well of the well plate or culture dish. Add dropwise from a high position and try to spread the liquid evenly. Then place the cells on ice for 20 minutes to lyse.

[0064] (6) Use a cell scraper to scrape the cells and collect the cells and lysis solution into a pre-cooled centrifuge tube. After scraping the cells, continue to place them on ice for 20 minutes to ensure that the cells are fully lysed.

[0065] (7) Ultrasonic lysis: Ultrasonicate for 10 seconds, stop for 10 seconds, repeat for 1 minute, and set the power to 30 W. After the ultrasonication is completed, immediately put the centrifuge tube on ice or put it in an ice box to cool down.

[0066] (8) Place the centrifuge tube in a pre-cooled centrifuge and centrifuge at 12,000 rpm for 15 minutes at 4°C. After centrifugation, transfer the supernatant to a new pre-cooled centrifuge tube.

[0067] (9) Protein concentration determination: Take an appropriate amount of protein supernatant to determine the protein concentration.

[0068] 2. Western blot experiment

[0069] (1) Prepare SDS-PAGE separation gel and stacking gel of appropriate concentration as required.

[0070] (2) Loading: Add protein markers and samples into the wells according to the experimental design.

[0071] (3) First, use 80V voltage to transfer the sample to the separation gel, then switch to 120V to continue electrophoresis. After the target protein is dispersed according to the protein marker indication, stop electrophoresis and transfer to the membrane.

[0072] (4) Prepare a tray with sufficient transfer solution, put in the transfer clip with the transparent side facing down, and place the sponge pad, filter paper, and PVDF membrane of appropriate size (activated by soaking in methanol for more than 10 seconds in advance) from bottom to top, and use a small roller to remove bubbles.

[0073] (5) Take out the separation gel and place it flat on the PVDF membrane. Then place the filter paper and sponge pad on it in sequence. After using a roller to remove the bubbles, close the transfer clip and place it in the transfer tank. Pay attention to the placement direction, that is, the transparent side of the transfer clip faces the red side of the transfer tank.

[0074] (6) Place the transfer tank in the electrophoresis apparatus in the corresponding direction, pour in a sufficient amount of transfer solution and place in an ice box for cooling, cover the electrophoresis tank, and soak the entire electrophoresis tank in an ice-water mixture for cooling.

[0075] (7) Start transferring the membrane at a constant voltage of 100 V for 120 minutes. The specific transfer time can be adjusted according to the molecular weight of the protein. The larger the molecular weight, the longer the transfer time.

[0076] (8) After transfer, take out the PVDF membrane from the transfer tank and place it in the prepared 5% skim milk. Place it on a shaker and shake slowly for 1 hour.

[0077] (9) After blocking, wash the milk with PBST, place the target band in the corresponding primary antibody prepared with 5% BSA solution (usually diluted at a ratio of 1:1000), and incubate it on a shaker in a 4°C refrigerator overnight.

[0078] (10) After incubation, remove the strips and wash them four times with PBST, each time for 5 minutes.

[0079] (11) Select a secondary antibody of the corresponding species and dilute it to an appropriate concentration with 5% skim milk. Place the washed PVDF membrane in the prepared secondary antibody and incubate it at room temperature on a shaker for 1 hour.

[0080] (12) After incubation, wash with PBST four times for five minutes each time. After absorbing the attached liquid, add ECL developer prepared by mixing solution A and solution B in a ratio of 1:1 onto the strip, place the strip in the darkroom of the developer for exposure, and use the fully automatic chemiluminescence image analysis system for photographing and analysis.

[0081] 3. Comet Experiment

[0082] (1) Preparation of single cell suspension: Rinse the cells once with cold PBS buffer (without calcium and magnesium), adjust the cell density to 10 after centrifugation. 7 Pieces / ml.

[0083] (2) Drop 80 μl of 0.5% NMA preheated to 56°C onto the frosted surface of a similarly preheated slide, quickly cover with a clean coverslip, and leave at 4°C for 10 minutes to solidify.

[0084] (3) Take 10 μl of PBS containing 1000 cells and 75 μl of 0.5% LMA and mix them at 37°C. Gently remove the cover slip and drop the LMA containing cells onto the first layer of the adhesive plate. Immediately cover it with a clean cover slip and leave it at 4°C for 10 minutes to solidify.

[0085] (4) 85 μl of 0.5% LMA preheated to 37°C was added dropwise onto the solidified LMA layer, and a cover glass was placed on the layer to allow it to solidify.

[0086] (5) Remove the coverslip and immerse the slide in freshly prepared cell lysis buffer for at least 1 hour (make sure that each slide is lysed for the same amount of time). The lysis buffer consists of a detergent and a high salt solution to remove excess cell membranes and histones.

[0087] (6) Remove the slide and rinse it twice with PBS to remove the high concentration of salt on the surface of the slide.

[0088] (7) Place the slide in a horizontal electrophoresis tank and pour in freshly prepared alkaline electrophoresis buffer to cover the surface of the gel by about 0.25 cm. Allow the slide to unwind for 20 minutes to unwind the DNA under alkaline conditions to form single-stranded DNA, which is convenient for electrophoretic migration.

[0089] (8) Perform single cell electrophoresis at 25 V and 300 mA for 20 min.

[0090] (9) After electrophoresis, remove the slide, absorb the electrophoresis buffer with filter paper, and neutralize with Tris-HCl (pH 7.5) for 15 minutes.

[0091] (10) Add 50 μl of staining solution to each slide, cover with a coverslip, and stain for 20 minutes in a dark place.

[0092] (11) Observe and photograph the electrophoresis pattern under a fluorescence microscope. Use comet analysis software to analyze the comet image and the comet DNA ratio (Tail DNA).

[0093] The results showed that in A2780 cells (human ovarian cancer cells) and MDA-MB-231 and MCF-7 cells (human breast cancer cells), the use of Ivo alone had no significant effect on DNA damage, while the use of Olaparib alone was found to have unrepaired DNA breakage damage. When the two were used together, significant DNA damage accumulation was observed in all three cell lines ( Figure 3 A). In addition, in cells with existing DNA damage, the control group (Ctrl) cells were able to repair DNA damage over time, and the expression of γH2AX decreased, while the expression of γH2AX in cells treated with Ivosidenib decreased significantly more slowly, indicating that the repair of cellular DNA damage was inhibited ( Figure 3 B). Similarly, in the comet assay results of MDA-MB-231 cells, the tailing phenomenon of cells using Ivo alone was not obvious, and the cells using Olaparib alone had slight tailing compared with the control group (Ctrl) cells, with moderate DNA damage, while the cells using the combination of the two drugs (Ivo+Ola) showed more obvious tailing and a higher proportion of Tail DNA, indicating a high degree of DNA breakage damage ( Figure 3 C. Figure 3 D).

[0094] Example 4: Nude mouse tumor formation experiment to evaluate the synergistic anti-tumor effect of IDH inhibitors and PARP inhibitors in vivo

[0095] Experimental design: Ovarian cancer cells were inoculated subcutaneously in nude mice. When the tumor grew to a certain size, the mice were randomly divided into 4 groups: control group, IDH inhibitor (ivosidenib) group, PARP inhibitor (olaparib) group and IDH inhibitor (ivosidenib) + PARP inhibitor (olaparib) group. The therapeutic effect was evaluated by immunohistochemical analysis of the proportion of γH2AX-positive cells, i.e. the proportion of cells with DNA damage repair disorders, as well as tumor size and weight.

[0096] Test process:

[0097] 1. Mouse culture

[0098] Six-week-old female BALB / c nude mice were housed in an SPF facility. 7 The cells were suspended in 100 μl PBS and inoculated subcutaneously on the right side of the mouse. 3 The mice were randomly divided into 4 groups: PBS (control), ivosidenib (20 mg / kg), olaparib (100 mg / kg), and a combination of olaparib (100 mg / kg + ivosidenib 20 mg / kg) treatment group. The body weight and tumor volume of nude mice were measured every 3 days. Tumor volume was expressed as width (W) and length (L), i.e., V = (W 2 × L) / 2. When the tumor grew to a certain size, the mice were killed and the transplanted tumor was removed, and the size and weight of the tumor were measured.

[0099] 2. Immunohistochemistry

[0100] (1) Before the experiment, the tumor tissue was sliced.

[0101] (2) Wash the sections three times with PBS, 5 minutes each time.

[0102] (3) Fix the sections with 4% paraformaldehyde at room temperature for 15 to 30 minutes.

[0103] (4) After fixation, wash with PBS three times, 5 minutes each time.

[0104] (5) Use 0.5% Triton X-100 as the permeabilization agent and permeabilize at room temperature for 15 minutes.

[0105] (6) After permeabilization, wash with PBS three times, 5 minutes each time.

[0106] (7) Use 5% BSA (bovine serum albumin) as a blocking agent and block the cells at room temperature for 1 hour.

[0107] (8) Primary antibody incubation: Select a specific antibody against γH2AX and dilute it according to the dilution ratio recommended in the antibody manual. Add the diluted primary antibody dropwise onto the tissue section to ensure that the section is completely covered.

[0108] (9) Place the sections in a humidified chamber and incubate at 4°C overnight.

[0109] (10) Recover the primary antibody and wash the sections three times with PBS, 5 minutes each time.

[0110] (11) Secondary antibody incubation: Select a fluorescently labeled secondary antibody that matches the species of the primary antibody and dilute it according to the dilution ratio recommended in the instructions. Add the diluted secondary antibody dropwise onto the cells and incubate at room temperature for 1 hour in the dark.

[0111] (12) Recover the secondary antibody and wash the sections three times with PBS, 5 minutes each time.

[0112] (13) Use DAPI stain to stain the cell nuclei and incubate at room temperature for 5 to 10 minutes in the dark.

[0113] (14) After staining, wash with PBS three times, 5 minutes each time.

[0114] (15) With the tissue side facing down, mount the slide on a glass slide using an anti-fluorescence fading mountant.

[0115] (16) Observe the sections using a confocal microscope or a fluorescence microscope and collect images.

[0116] The results showed that compared with the control (Ctrl), the tumor volume of the Ivo group and the Olaparib group was reduced, but the tumor was more significantly reduced after the combination (Ivo+Ola); and the tumor weight of the combination group was also significantly smaller than that of the single drug treatment group ( Figure 4 A. Figure 4 B. Figure 4 C). Immunohistochemistry experiments showed that there was no significant γH2AX-positive cell ratio in the Ivo (Ivo)-only treatment group, a small number of γH2AX-positive cells were found in the Olaparib (Ola)-only treatment group, and the γH2AX-positive cell ratio was greatly increased in the Ivo+Ola combination treatment group ( Figure 5 ), the combined administration of IDH inhibitors and PARP inhibitors significantly increased the proportion of DNA damaged cells.

Claims

1. Use of an IDH1 inhibitor combined with a PARP inhibitor in the preparation of a drug for treating BRCA1 / 2 wild-type tumors.

2. Use of an IDH1 inhibitor in the preparation of a drug for increasing the sensitivity of BRCA1 / 2 wild-type tumors to PARP inhibitors.

3. The use according to claim 2, characterized in that: The IDH1 inhibitor increases the sensitivity of the tumor to PARP inhibitors by inhibiting DNA damage repair in BRCA1 / 2 wild-type tumors.

4. The use according to claim 3, characterized in that: The IDH1 inhibitor inhibits DNA damage repair in BRCA1 / 2 wild-type tumors by inhibiting homologous recombination repair.

5. The use according to claim 1 or 2, characterized in that: The drug is a drug for inhibiting BRCA1 / 2 wild-type ovarian cancer, breast cancer, prostate cancer, and pancreatic cancer.

6. The use according to claim 1 or 2, characterized in that: The IDH1 inhibitor is selected from ivosidenib.

7. The use according to claim 1 or 2, characterized in that: The PARP inhibitor is selected from olaparib, rucaparib, niraparib, talazoparib, pamiparib, and fluzoparib.

8. The use according to claim 1 or 2, characterized in that: The IDH1 inhibitor is selected from ivosidenib, and the PARP inhibitor is selected from olaparib.

9. The use according to claim 1 or 2, characterized in that: The dosage ratio of the IDH1 inhibitor to the PARP inhibitor is 1:

5.

10. The use according to claim 1 or 2, characterized in that: The drug is a drug for inhibiting the proliferation and metastasis of BRCA1 / 2 wild-type tumors.

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