CN110420330B

CN110420330B - Pharmaceutical application of PI3K and MTH1 targeted drug composition

Info

Publication number: CN110420330B
Application number: CN201910653369.5A
Authority: CN
Inventors: 林凡; 周婷婷; 陈真
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2019-07-19
Filing date: 2019-07-19
Publication date: 2020-05-29
Anticipated expiration: 2039-07-19
Also published as: CN110420330A

Abstract

A PI3K and MTH1 targeted drug composition and its application are provided. The small molecule targeted pharmaceutical composition and the pharmaceutical preparation provided by the invention comprise a first preparation formed by a PI3K inhibitor or a pharmaceutically acceptable carrier thereof and a second preparation formed by an MTH1 inhibitor or a pharmaceutically acceptable carrier thereof, and are reasonable in compatibility, so that the drug sensitivity of the PI3K inhibitor can be improved by the combined use of the two preparations, the effect of the MTH1 small molecule inhibitor on the anti-tumor aspect can be exerted, and the treatment effect of the PI3K inhibitor is improved. Experiments prove that the combined administration of the PI3K inhibitor and the MTH1 inhibitor remarkably enhances the activity inhibition of tumor cells compared with the single administration, reduces the single administration dosage, can reduce the drug toxicity, is expected to become a new treatment scheme aiming at tumors with undesirable treatment effect of the PI3K inhibitor, and has wide application prospect.

Description

Pharmaceutical application of PI3K and MTH1 targeted drug composition

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of a combined medicine of two PI3K small-molecule inhibitors BKM120 and GDC0941 and two MTH1 small-molecule inhibitors TH588 and TH287 in inhibiting growth of tumor cells, in particular to application in preparing a medicine for inhibiting growth of brain glioma and lung cancer cells.

Background

With the rapid development of human socioeconomic, the incidence of malignant tumor is increasing, which seriously threatens human life and health and causes heavy burden to society and families, therefore, it is of great significance to explore the pathogenesis of malignant tumor and find effective therapeutic targets and therapeutic drugs. According to the cancer statistical report of 2018, the estimated new disease rate of national malignant tumors is 380.4 ten thousand (211.4 ten thousand for men and 169.0 ten thousand for women), more than 1 thousand people are diagnosed as cancer every day on average, and 7 people are diagnosed as cancer every minute. According to the disease cases, the lung cancer is the first disease in the whole country, and the lung cancer is about 78.1 ten thousand per year, and then the lung cancer, the colorectal cancer, the liver cancer and the breast cancer are sequentially developed. The world health organization published 2004 that the incidence rates of the new common lung cancer types are adenocarcinoma of the lung, squamous carcinoma, small cell lung cancer and large cell lung cancer from high to low, and the incidence rates of the different lung cancer types are 31.5%, 29.4%, 17.8% and 9.2% in turn. The lung cancer is a malignant tumor with high morbidity and high mortality, and the early diagnosis and treatment have important meanings for prolonging the life cycle of patients and improving the life quality of the patients.

Brain glioma is the most common primary intracranial tumor, and according to the data of the american brain tumor registry (CBTRUS), 47% of malignant central nervous system tumors are Glioblastoma (GBM), one of the worst prognosis tumors among all tumors, and have the characteristics of strong invasiveness and rapid progression. The current standard treatment for glioblastoma is maximal surgical resection combined with radiotherapy and chemotherapy. The only chemotherapeutic drug Temozolomide (TMZ) that has been clinically proven to penetrate the blood-brain barrier and be effective against glioblastoma also only extends median survival in patients from 12 months to 14.1 months. Observing the Genome map of the glioblastoma multiforme drawn by TCGA (the Cancer Genome atlas), 88% of the glioblastoma multiforme shows that gene mutation of RTK/RAS/PI3K signal pathway, including amplification and mutation of various RTK (receptor Tyrosine kinase), activation mutation of PI3K (coding gene is PI3 KCA) for phosphorylation of PIP2 to PIP3, or inactivation mutation of Cancer suppressor PTEN for reversing the reaction by dephosphorylation, and inactivation mutation for negative regulation of RAS signal pathway NF1 all result in abnormal up-regulation of PI3K-AKT-mTOR pathway to promote tumor cell growth and proliferation.

BKM120 is a class i PI3K small molecule inhibitor, IC50 acting on p110 α/β/δ/γ is 52nM/166nM/116nM/262nM, respectively, and has a large number of clinical studies in tumors such as PI3K-AKT-mTOR signaling pathway activated, which can reduce phosphorylation of downstream effector AKT, thereby inhibiting tumor cell proliferation, BKM120 as a PI3K inhibitor has important clinical significance in tumors such as PI3K signaling pathway activated mutated breast cancer, non-small cell lung cancer, ovarian cancer, and the like, thus, gliomas as a PI3K high frequency rate tumor, BKM120 as a potential study drug has significant clinical significance in using BKM120 as a potential study drug in the invention, we screened out different gliomas cell lines with different concentrations of BKM120, found that BKM120 can inhibit activation of AKT signaling pathway at 1 μ M concentration, and found that BKM120 can inhibit most gliomas cell lines but inhibit proliferation of gliomas cell lines at a PI3 activation pathway 120U K, but the cd 120 cell lines at a tumor cell line activating p 120, cd 120 c 300, cd 120 c 3 c p-c 3 c p-c 120 c p 120 c 3 c 120 c p 120 c 3 c p 120 c p1 c p 120 c p1 c 3 c p1 c p 120 c p1 c p2 c p1 c p2 c p3 c p2 c p3 c p1 c p2 c p 102 c p1 c p2 c.

TH588 is a potent and selective inhibitor of MTH1 with an IC50 of 5nM, and in vitro studies TH588 induces DNA damage in tumor cells and induces an ATM-p53 mediated death response and DNA repair; in vivo studies found TH588 significantly inhibited tumor growth in SW480, MCF7 and xenograft tumor mice bearing the patient BRAF V600E mutation. MTH1 (MutT homolog 1) acts as a pyrophosphatase that efficiently cleans up oxidized nucleotides in the pool of nucleotides, thereby protecting rapidly proliferating cancer cells from lethal damage from high levels of ROS, but normal cells can survive the repair of DNA oxidative damage through other alternative pathways in the absence of MTH1 enzyme. The significant difference in the necessity of MTH1 between normal and cancer cells made it possible to target MTH1 for this therapy to treat cancer, and we found by big data analysis that the expression level of MTH1 protein in brain glioma cells was significantly higher than that of normal cells.

According to the invention, 601 small molecule targeted drugs in a drug library are combined by 10 mu M concentration and 1 mu M concentration of BKM120 to screen out an MTH1 inhibitor TH588, the obvious synergistic inhibition effect of the 1 mu M concentration of BKM120 and the 10 mu M concentration of TH588 in U251 is found, and the concentration gradient search is carried out to find that the 1 mu M concentration of BKM120 and the 2 mu M concentration of TH588 in various brain glioma cell systems including U251 have stronger synergistic inhibition effect, the U251 cell growth inhibition effect is stronger than that of single drugs including BKM120 and TH588, the drug concentration is lower than that of the single drugs including BKM120 and TH588, the problem of drug resistance of the U251 cell to the single drug of BKM120 is solved, and the cytotoxicity of the single drug with high concentration is reduced. TH287 is another potent selective MTH1 inhibitor with an IC50 of 0.8nM, and we also showed significant synergistic inhibition using BKM120 in combination with TH287 in the U251 cell line. In the research of a synergistic inhibition mechanism, the PI3K inhibitor and the MTH1 inhibitor can inhibit the growth of tumor cells by inducing the apoptosis of the tumor cells and DNA damage.

Disclosure of Invention

The technical problem to be solved is as follows: the invention provides a PI3K and MTH1 targeted drug composition and application thereof. The pharmaceutical composition is a PI3K inhibitor and an MTH1 inhibitor. The scheme of the invention can specifically inhibit the growth of brain glioma and lung cancer cells, and can improve the curative effect of the PI3K inhibitor in inhibiting the growth of tumor cells.

The technical scheme is as follows: a PI3K and MTH1 targeted drug composition contains PI3K inhibitor and MTH1 inhibitor.

Preferably, the PI3K inhibitor is BKM120 or GDC-0941.

Preferably, the concentration of the BKM120 is 0 to 4 μ M, and the concentration of the GDC0941 is 0 to 100 μ M;

preferably, the MTH1 inhibitor is TH588 or TH 287.

Preferably, the TH588 concentration is 0 to 10. mu.M, and the TH287 concentration is 0 to 4. mu.M.

The PI3K and MTH1 targeted drug composition is applied to the preparation of drugs for inhibiting the growth of tumor cells.

The cancer is brain glioma or lung cancer.

Has the advantages that: the invention provides a PI3K and MTH1 small molecule inhibitor composition and a pharmaceutical preparation, wherein the PI3K small molecule inhibitor is BKM120 and GDC 0941; the MTH1 small molecule inhibitor is TH588 and TH 287; the pharmaceutical formulation comprises BKM120 at a concentration of 0 to 4 μ Μ or GDC0941 at a concentration of 0 to 100 μ Μ; TH588 at a concentration of 0 to 10. mu.M or TH287 at a concentration of 0 to 4. mu.M; the two can improve the drug sensitivity of the PI3K inhibitor and play the role of the MTH1 inhibitor in resisting tumors, thereby synergistically enhancing the treatment effect of the PI3K inhibitor. Experiments prove that in U251 cells, the combination of the BKM120 and TH588 can inhibit cell proliferation by inducing apoptosis and promoting DNA damage, and the synergistic inhibition effect of the BKM120 and the TH588 in the U251 cells is still remarkable no matter the BKM120 is replaced by GDC-0941 or the TH588 is replaced by TH 287. Meanwhile, the synergistic inhibition of BKM120 and TH588 in lung cancer cells PC9 and H460 still exists. Indicating that inhibitors of MTH1 enhance the therapeutic efficacy of PI3K inhibitors in tumor cells. The small molecule inhibitor composition and the pharmaceutical preparation can be applied to preparing antitumor drugs and have wide application prospects.

Drawings

FIG. 1 is a graph of the effect of different concentrations of BKM120 on the signaling pathways of U251, U87, T98G cells; after GBM cell lines were treated with different concentrations of BKM120 for 24h, the intracellular levels of phospho-AKT (Ser473, Thr 308), phospho-P70S6K (Thr 389), and phospho-S6 (Ser 235/236) decreased in concentration gradient, indicating that the AKT/mTOR signaling pathway was inhibited. Under the same concentration, the sensitive cell lines U87 and T98G have obvious inhibition compared with the signal channel of the drug-resistant cell line.

FIG. 2 is a graph of the effect of different concentrations of BKM120 on long-term growth of various glioma cell lines; in addition to the drug resistant cell lines U251 and SNB19, BKM120 at a concentration of 1 μ M produced a significant inhibition of the long-term growth of most brain glioma cells.

Figure 3 is a graph of the combined effect of BKM120 and TH588 at different concentrations in various brain glioma cells; TH588 and BKM120, in combination at different concentrations, showed synergistic inhibition in the SNB19, LN18, LN229, a172, U251, U118MG cell lines, in addition to the T98G cell line.

FIG. 4 is a graph of the effect of different concentrations of BKM120 and TH588 single drugs, and different combinations of the two drugs, on long-term cell growth in various glioma cell lines; the combination of the BKM120 and TH588 in SNB19, LN229, U251 and LN18 cell lines has a remarkably stronger inhibition effect on the long-term growth of cells than that of a single drug.

FIG. 5 is a graph of the effect of BKM120 and TH588 single and combined drugs on apoptosis; after U251 cells were treated with 1 μ M BKM120, 2 μ M TH588 alone or in combination for 72 hours, the level of apoptosis was increased in the two-drug combination compared to the single-drug treatment group.

FIG. 6 is a diagram of comet assay testing the effect of BKM120 and TH588 single drug and combined drug on cellular DNA damage; after the U251 cells are treated by 1 mu M BKM120 and 2 mu M TH588 alone or in combination for 24 hours, the trailing length of the U251 cells accounts for 9 percent of the length of the cells when the two medicines are combined, and compared with the single medicine group, the DNA damage is obviously strengthened.

FIG. 7 is a graph of flow cytometry to detect the effect of single and combined BKM120 and TH588 drugs on cellular DNA damage; after the U251 cells are treated by 1 mu M BKM120 and 2 mu M TH588 alone or in combination for 24 hours, when the two medicines act in combination, the number of the U251 cells with DNA damage also accounts for 52 percent, and the DNA damage degree of the U251 cells is stronger than that of the single medicine.

FIG. 8 is a graph of the combined effect of BKM120 and TH588 in the U251 cell line; treating U251 cells for 72h under different concentrations of BKM120 and TH588, and calculating synergy index of the two drugs by Compuyn software, wherein the horizontal line in the figure indicates that CI =1, and the two drugs have synergy when CI < 1; BKM120 and TH588 have a synergistic inhibitory effect in the U251 cell line.

FIG. 9 is a graph showing the combined effect of GDC-0941 and TH588 in U251 cell line; treating U251 cells for 72h under different concentration combinations of Pan-PI3K inhibitors GDC0941 and TH588, and calculating synergy index of the two drugs by using Compuyn software, wherein the horizontal line in the figure represents CI =1, and the two drugs have synergy when CI < 1; GDC0941 and TH588 had synergistic inhibition in the U251 cell line.

FIG. 10 is a graph of the combined effect of BKM120 and TH287 on the U251 cell line; the combination of different concentrations of the BKM120 and the MTH1 inhibitor TH287 is used to treat U251 cells for 72h, and the compactyn software is used to calculate the synergy index of the two drugs, wherein the horizontal line in the figure indicates that CI =1, and the two drugs have synergy when CI < 1; BKM120 and TH287 have synergistic inhibitory effects in the U251 cell line.

FIG. 11 is a graph of the combined effect of BKM120 and TH588 in a H460 cell line; treating H460 cells for 72H under different concentrations of BKM120 and TH588, and calculating synergy index of the two drugs by using Compuyn software, wherein the horizontal line in the figure indicates that CI =1, and the two drugs have synergy when CI < 1; BKM120 and TH588 have synergistic inhibitory effects in the lung cancer H460 cell line.

FIG. 12 is a graph of the combined effect of BKM120 and TH588 in PC9 cell line. Treating H460 cells for 72H under different concentrations of BKM120 and TH588, and calculating synergy index of the two drugs by using Compuyn software, wherein the horizontal line in the figure indicates that CI =1, and the two drugs have synergy when CI < 1; BKM120 and TH588 have synergistic inhibitory effects in the lung cancer PC9 cell line.

Detailed Description

The following examples are presented to enable one of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.

The invention aims to search a medicament aiming at PI3K inhibitor-resistant tumor cells, improve the curative effect of a PI3K inhibitor in treating tumors and improve the survival time of tumor patients. Research shows that different medicines can achieve the effect of multi-target multi-inhibition when used together, and the combined medication becomes a trend in the treatment of malignant tumors. Different medicines are combined for use, a certain synergistic effect can be generated, so that the dosage of single medicine is reduced, the accumulation of medicine toxicity in vivo is reduced, the superposition of adverse reactions is avoided, and the curative effect of the medicine is improved.

The U251 cell line is selected at the beginning of the research because the incidence rate of PI3K activated mutation in brain glioma is high, and the BKM120 serving as a PI3K inhibitor has a large amount of clinical researches in tumors of PI3K signal channel activation mutation, such as breast cancer, non-small cell lung cancer, ovarian cancer and the like, and can be applied to treatment of PI3K activated mutation tumors in the future. It is expected that BKM120 could inhibit the growth of all PI 3K-activated mutant glioma cells, but experiments demonstrated that in PI 3K-activated mutant glioma cells U251, BKM120 at a concentration of 1 μ M was effective in inhibiting activation of the AKT signaling pathway, but not in inhibiting cell proliferation, and therefore it was hypothesized that U251 cell proliferation was associated with an unknown signaling pathway in addition to being dependent on PI3K signaling pathway activation. Thus, we used a drug library containing 601 small molecule targeted drugs targeting 178 targets, and used each drug in the library at a concentration of 10 μ M in combination with a concentration of 1 μ M BKM120 to find an MTH1 inhibitor TH588 that is synthetic lethal to BKM120 in the U251 cell line. Through the research of concentration gradient, the combination of 1 mu M concentration of BKM120 and 2 mu M concentration of TH588 can inhibit the proliferation of most brain glioma cells, wherein the cell line comprises U251 cell line in which the growth of the BKM120 cannot be inhibited by single medicine. And the mechanism research shows that the combination of the BKM120 and the TH588 inhibits the proliferation of the U251 cells by inducing apoptosis and promoting DNA damage. Whether BKM120 was replaced with GDC-0941 or TH588 was replaced with TH287, the synergistic inhibitory effect of both drugs in U251 cells was still significant. Meanwhile, the synergistic inhibition of BKM120 and TH588 in lung cancer cells PC9 and H460 still exists. Indicating that inhibitors of MTH1 enhance the therapeutic efficacy of PI3K inhibitors in tumor cells. The small molecule inhibitor composition and the pharmaceutical preparation can be applied to preparing antitumor drugs and have wide application prospects.

The invention provides an application of a PI3K inhibitor and MTH1 inhibitor composition and a pharmaceutical preparation in preparing antitumor drugs.

The BKM120 is a PI3K small molecule inhibitor, acts on IC50 of p110 α/β/delta/gamma, is 52nM/166nM/116nM/262nM respectively, and can reduce phosphorylation of downstream effector AKT in tumors activated by PI3K-AKT-mTOR signaling pathway so as to inhibit tumor cell proliferation, the BKM120 is used as a PI3K inhibitor and has a great deal of clinical research in tumors of breast cancer, non-small cell lung cancer, ovarian cancer and the like activated by PI3K signaling pathway, therefore, the brain glioma is used as a tumor with high frequency rate of PI3K and has important clinical significance when the BKM120 is used as a potential research drug, the GDC-0941 is another effective PI3K α/delta inhibitor, the IC50 is 3 nM in a cell-free test and has moderate selectivity on p110 β (11 times) and p110 gamma (25 times), and the obvious synergistic effect of inhibiting TH when the GDC-0941 and the screened TH are combined in U251 cells.

TH588 is a potent and selective inhibitor of MTH1 with an IC50 of 5nM, and in vitro studies TH588 induces DNA damage in tumor cells and induces an ATM-p53 mediated death response and DNA repair; in vivo studies found TH588 significantly inhibited tumor growth in SW480, MCF7 and xenograft tumor mice bearing the patient BRAFV600E mutation. TH287 is another potent selective MTH1 inhibitor with an IC50 of 0.8nM, and we also showed significant synergistic inhibition using BKM120 in combination with TH287 in the U251 cell line.

The source of BKM120, GDC-0941, and TH588, TH287 is not particularly limited and may be prepared using any source known to those skilled in the art, such as commercially available sources, or using conventional techniques known in the art.

Further, the first formulation of the PI3K inhibitor and a pharmaceutically acceptable carrier, and the second formulation of the MTH1 inhibitor and a pharmaceutically acceptable carrier.

Further, the concentration of the BKM120 is 0 to 4 mu M, and the concentration of the GDC0941 is 0 to 100 mu M; TH588 concentration is 0 to 10. mu.M, TH287 concentration is 0 to 4. mu.M.

The invention also provides the pharmaceutical composition and pharmaceutically acceptable auxiliary materials.

Preferably, the dosage form of the pharmaceutical preparation is injection, tablet, capsule, granule, suspension, emulsion, solution, sol, freeze-dried powder injection, mucilage, aerosol, microcapsule, microsphere, liposome, micelle, sustained release preparation or controlled release preparation.

The invention also provides the application of the medicine and the combined medicine in preparing anti-cancer medicines.

Preferably, the cancer is primarily brain glioma and lung cancer.

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings of the present specification, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Example 1

Western blot is adopted to detect the change of signal paths of U251, T98G and U87 glioma cell lines under the single-drug treatment of BKM120 with different concentrations, and the specific implementation scheme is as follows:

1. the cell lines U251, T98G and U87 were 6 x 10 one day in advance⁵The cells were plated in 6cm dishes, 7 dishes for each cell.

2. Single drug BKM120 was prepared at concentrations of 0.1. mu.M, 1. mu.M, 2. mu.M, 4. mu.M, 8. mu.M, and 10. mu.M.

3. After the cells are well paved, 5mL of the medicine in the step 2 is added into each dish 24h, and DMSO with the concentration of 2 per mill is added into a control group for treatment for 24 h.

4. After 24h of drug treatment, the cells were washed twice with PBS, drained, 200. mu.L of cell lysate was added to each dish, the lysed cells were collected into 1.5mL EP tubes with a scraper, lysed for half an hour at 4 ℃, centrifuged at 13000rpm for 15min, and the supernatants were stored at-80 ℃.

5. The protein content of each sample was measured by BCA method.

6. Preparing 10% polyacrylamide gel for protein separation, loading 20ug of protein samples into each hole, performing 100V constant voltage electrophoresis until bromophenol blue disappears, performing 250mA wet rotation for 2h, sealing 3% BSA for 3 hours, performing primary antibody incubation at 4 ℃ for 6h, performing 1 xTBST membrane washing for 3 times, performing each time for 10min, performing secondary antibody incubation at room temperature for 1h, performing 1 xTBST membrane washing for 5 times, and performing ECL color development exposure for 6min each time.

As shown in fig. 1, when the BKM120 concentration was 1 μ M, the p-AKT (Ser473) expression was significantly reduced in U251, U87, T98G, indicating that PI3K activity was effectively inhibited; moreover, the 1 mu M concentration of the BKM120 can effectively inhibit the expression of p-s6, and an important inhibition effect on cell growth should be generated.

Example 2

A clone formation experiment is adopted to detect the long-term growth condition of various brain glioma cell lines under the action of a BKM120 single drug, and the specific embodiment is as follows:

1. the 8 glioma cell lines U251, LN18, SNB19, U118, LN229, U87, a172, T98G were seeded in 12-well plates in an amount of 800 cells per well.

2. BKM120 was prepared at 500nM, 750nM and 1 μ M concentrations and a set of blanks was set at 2% DMSO.

3. After 48 hours, after the cells adhere to the wall in the step 1, 2mL of the medicine prepared in the step 2 is added into each hole, the medicine is changed once in 3 days, and after the continuous culture is carried out for 14 days, the medicine is removed for 3 days.

4. Removing the culture medium after removing the drug for 3 days, washing with PBS 2 times, washing with deionized water 1 time, fixing with 4% paraformaldehyde for 15min, washing with deionized water 1 time, dyeing with 1 × Giemsa dye solution in dark place for 30min, washing with deionized water 3 times, and taking pictures for storage.

As shown in fig. 2, a1 μ M concentration of BKM120 produced significant inhibition of long-term growth of most glioma cells, except for the U251 and SNB19 cell lines.

Example 3

The cell proliferation conditions of the BKM120 and the TH588 under single medicine and two-medicine combination are detected by Alamar blue, and the SI value is calculated, wherein the specific embodiment is as follows:

1. u251, SNB19 were seeded into 96-well plates per 1000 cells the day before; LN229, a72, LN18, U118MG, T98G were plated in 96-well plates at 2000 cells per well, 3 replicates per cell per concentration, and controls were set.

2. Preparing single medicines of BKM120100nM, BKM1201 mu M, TH 58810 mu M, TH 5882 and 5882 mu M; double-drug BKM120100nM + TH 58810 μ M, BKM120100nM + TH 5882 μ M, BKM1201 μ M + TH 58810 μ M, BKM1201 μ M + TH 5882 μ M.

3. And (2) absorbing the old culture medium after the cells adhere to the wall in the step 1, adding 100 mu L of mixed solution of the complete culture medium and 10 mu L of LAlamar Blue into each hole, incubating for 4 hours in a cell culture box with 37 ℃ and 5% carbon dioxide, detecting absorbance by using a chemiluminescence instrument under the conditions of excitation light wavelength 534nm and emission light wavelength 584nm, and recording data of each hole as Day0 data.

4. Cells which were tested for Day0 data were aspirated away from the Alamar Blue containing mixture, added to the drug formulated in step 2 on each well, and monitored for cell growth on the third Day by Alamar Blue as described in step 2 and recorded as Day3 data.

5. The cell survival rate under the single action of TH588 is recorded as Rc; the cell survival rate under the action of a single drug of the BKM120 is recorded as Cd; the cell survival rate without the action of the drug is recorded as Cc; the cell survival rate under the condition of combining the two medicines is recorded as Rd; ECE = (Rc/Cc) × (Cd/Cc); OCE = Rd/Cc; SI = ECE-OCE; when the SI is more than 0, the combination of the two medicines has synergistic effect; when the SI is less than 0, the combination of the two medicines has antagonism. Based on the data recorded by Day3, the SI value for each cell was calculated and plotted as shown in FIG. 3.

As shown in FIG. 3, when TH588 concentration was 10. mu.M and BKM120 concentration was 1. mu.M, both drugs showed synergistic inhibition in SNB19, LN229, A172 and LN18 cell lines; when the concentration of TH588 is 2 muM and the concentration of BKM120 is 1 muM, the two medicines have synergistic inhibition effect in SNB19, LN229, A172, U251 and U118MG cell lines; TH588 concentration was 2. mu.M, BKM120 concentration was 100nM and TH588 concentration was 10. mu.M, with BKM120 concentration of 100nM and no synergistic inhibition in T98G. Therefore, 1. mu.M BKM120 and 2. mu.M TH588 are the optimal synergistic inhibitory concentrations for the glioma cell line.

Example 4

A clone formation experiment is adopted to detect the long-term growth conditions of various brain glioma cells under the conditions of single drug BKM120, single drug TH588 and the combination of BKM120 and TH588, and the specific embodiment is as follows:

1. the U251, SNB19 cell lines were seeded one day in 12-well plates per 1000 cells, and a172, U118MG, LN18, LN229, and T98G in 12-well plates per 2000 cells.

2. Preparing single-drug BKM120100nM, single-drug BKM1201 μ M, single-drug TH 5882 μ M, two-drug BKM1201 μ M + TH 5882 μ M; two drugs of BKM120100nM + TH 5882 μ M.

3. And (3) after the cells in the step (1) are completely attached to the wall, adding the medicine in the step (2) into the attached cells, changing the liquid every three days, adding the medicine for culturing for 14 days, and dyeing after removing the medicine for 3 days.

As shown in fig. 4, the combination of BKM120 and TH588 in SNB19, LN229, U251 cell lines had significantly greater inhibition of long-term cell growth than the inhibition of the two drugs alone.

Example 5

And detecting the apoptosis condition of the U251 cells under the single-drug action and the double-drug action of the BKM120 and the TH588 by using Annexin V-FITC. The specific embodiment is as follows:

1. u251 cells were arranged at 4X 10⁴Each hole is planted in a 6-hole plate, and pairs are arrangedAccording to the group, a BKM120 single medicine group, a TH588 single medicine group and a BKM120+ TH588 two medicine combination group; each group is provided with 3 multiple holes; and the flow cytometer is reserved in advance.

2. Single drug formulations, BKM1201 μ M, TH 5882 μ M and BKM1201 μ M + TH 5882 μ M were prepared.

3. And (3) adding the culture medium in the step (2) after the cells adhere to the wall, and culturing for 3 days.

4. The 6cm dish of medium was completely transferred to a 15mL centrifuge tube, and the cells were washed twice with PBS and transferred to a 15mL centrifuge tube.

5. Adding 300 mu L of pancreatin, shaking uniformly, quickly absorbing 200u of pancreatin (ensuring that cells can be covered by pancreatin and avoiding pancreatin pairs containing EDTA) and stopping with 1mL of complete culture medium, transferring to a 15mL centrifuge tube, washing once with 1mL of complete culture medium, transferring to 1500rpm in the centrifuge tube, centrifuging for 5min, absorbing and discarding supernatant, washing cells with 1mL of precooled PBS, centrifuging for 5min at 300g and 4 ℃ for 5min, and repeating once; removing PBS by suction, adding 100. mu.L of 1XBinding buffer to resuspend cells, adding 2.5. mu.L of annexin V-FITC and 5. mu.L of PI stabilizing Solution, and gently mixing; incubating at room temperature in dark for 15min, and mixing uniformly every 5 min; adding 400 mu L of 1 × Binding Buffer, mixing uniformly, placing on ice, and waiting for flow detection (completed within 1 hour) to analyze and count the test data.

As shown in figure 5, the U251 apoptosis rate under the action of single drug BKM1201 μ M is only 6.5%, the U251 apoptosis rate under the action of single drug TH 5882 μ M is 9.5%, but the U251 apoptosis rate under the combined action of two drugs reaches 18%, the apoptosis quantity caused by the combined action of two drugs is obviously higher than that caused by single drug,p<0.05 difference was statistically significant.

Example 6

A comet assay is adopted to detect the DNA damage condition of U251 cells under the combined action of a single BKM120 drug, a single TH588 drug and two drugs. The specific embodiment is as follows:

1. u251 cell line was performed at 1.5X 10⁵Each well is planted in a 6-well plate, and a BKM120 single medicine group, a TH588 single medicine group, a two-medicine combination group, a positive control group and a negative control group are arranged in the well;

2. preparing 1 mu M of single drug of BKM120, 2 mu M of single drug of TH588 and 1201 mu M + of BKMTH 5882 μ M double drug, 100 μ MH₂O₂。

3. After the U251 cells in the step 1 are completely attached to the wall, the culture medium is changed into the medicine (except for positive control) in the step 2 for treatment for 24 hours;

4. after 24h of drug treatment, the cells were washed once with 1 XPBS and counted after trypsinization. The counted cells were washed once more with PBS and then resuspended with PBS to a concentration of 1X 106 cells/mL.

5. Tabletting: dropping 100 μ L of normal melting point agarose dissolved in 0.75% PBS on a frosted glass slide, covering with a cover glass to avoid air bubbles, freezing at 4 deg.C for 10min, and carefully removing the cover glass after solidification to obtain the first layer of glue (lower layer of glue)

6. Mixing 10 μ L cell suspension with 90 μ L low melting point agarose (mixed well with PBS 0.75% at 40-42 deg.C), dripping on the first layer of gel, covering with cover glass, standing in refrigerator at 4 deg.C for 30min, solidifying, and removing the cover glass carefully to obtain the second layer of gel (upper layer of gel)

7. Cell lysis slide was placed in freshly prepared pre-cooled cell lysate (10mM Tris pH =10,2.5M Nacl, 0.1M EDTA2Na, 10% DMSO, 1% Tritox-100) protected from light and lysed overnight at 4 ℃.

8. Washed three times with enzyme reaction buffer (40mM HEPES pH =8.0, 0.1M Kcl, 0.5mM EDTA2Na, bsa0.2 mg/mL);

9. electrophoresis: the slide was placed in a horizontal electrophoresis tank and allowed to stand in a precooled alkaline electrophoresis buffer (10M NaOH (pH =13), 0.5M EDTA2Na) for 20min, 25V for electrophoresis, 300mA for current, 30min for electrophoresis.

10. Neutralizing and dyeing, namely soaking the slide in a neutralizing solution (0.4M Tris-HCl) after electrophoresis is finished, and dyeing the slide in a dark place for 15min at the concentration of 2.5ug/mLDAPI 20 mu L;

11. photographs were taken under a fluorescent microscope (DAPI using uv excitation);

12. the ratio of the length of the tail of the cells in each group to the total length of the cells was calculated by Casp software, and the higher the ratio, the greater the DNA damage.

As shown in fig. 6, when 1 μ M BKM120 was used alone, the U251 cell tail length accounted for approximately 4% of the cell length; 2 mu M TH588 single drug actionWhen used, the tail length of the U251 cell accounts for about 5% of the length of the cell; however, when the two medicines are combined, the ratio of the tail length of the U251 cell to the cell length reaches 9 percent, and compared with the single medicine group, the DNA damage is obviously strengthened.p<0.05, the difference was statistically significant.

Example 7

And detecting gamma H2AX positive cells by using a flow cytometry technology, so as to detect the DNA damage condition of the U251 cells. The specific embodiment is as follows:

1. u251 cells were plated at 1.2X 10⁴Each well was seeded in 6-well plates; a BKM120 single medicine group, a TH588 single medicine group, a two-medicine combination group and a negative control group are arranged; (reservation flow cytometer in advance);

2. preparing single-drug BKM1201 μ M, single-drug TH 5882 μ M, double-drug BKM1201 μ M + TH 5882 μ M;

3. after the cells adhere to the wall in the step 1, adding the medicine in the step 2, wherein the medicine is 3 mL/hole, and treating for 48 hours;

4. after the drug treatment is finished, digesting and centrifuging the cells, removing supernatant, and resuspending 1mL of PBS;

5. 3mL of 90% analytical alcohol (precooled at-20 ℃) was placed in a 15mL centrifuge tube, and 1mL of PBS heavily-spun cells were added while vortexing (gentle shaking instrument), and fixed overnight at-20 ℃;

6. 1500rpm, 8min, remove supernatant, 1mL FASC spin again and transfer to 1.5mL EP tube, 5000rpm 2min, remove supernatant, 500 μ L FACS spin again, 5000rpm 2min, remove supernatant;

7. preparing an antibody: 1:50 (5 samples with 5. mu.L antibody, 250. mu.L FACS, mix, 50. mu.L for one sample);

8. adding the prepared antibody, incubating at room temperature in dark for 1h, and flicking once in 20 min;

9. adding 1mL FACS for re-rotation, centrifuging, and rotating at 5000rpm for 2 min;

10. removing supernatant, adding 500 μ L FACS,5000rpm for 2 min;

11. add 300. mu.L FACS to re-spin and detect by flow cytometry.

As shown in fig. 7, when 1 μ M BKM120 was used alone, the number of U251 cells with DNA damage accounted for 40% of the total; when 2 mu MTH588 single drug acts, DNA damage occursThe number of U251 cells of (a) also accounted for about 40% of the total; however, when the two drugs are combined, the number of U251 cells with DNA damage also accounts for 52 percent of the total number, which indicates that the DNA damage degree of the U251 cells is stronger when the two drugs are combined.p<0.05, the difference was statistically significant.

Example 8

Concentrations of BKM120 were combined with concentrations of TH588 and observed for synergistic inhibition in the U251 cell line. The specific embodiment is as follows:

1. the U251 cells were seeded in 96-well plates at 1500 cells per well;

2. setting concentration gradients, wherein six concentration gradients of each drug are combined in pairs to prepare 36 groups of drug-containing complete culture media with final concentration;

BKM120 ( 0μM、0.5μM、0.75μM、1μM、2μM、4μM)

TH588 ( 0μM、1μM、1.5μM、2μM、4μM、8μM)

3. discarding the old culture medium containing the medicine after the cells are plated for 24h, and replacing the fresh culture medium containing the medicine with different concentration combinations in the step 2;

4. adding drug for 72h, discarding the old culture medium containing the drug, adding a mixed solution containing 100 μ L of complete culture medium and 10 μ L of AlamarBlue to each well, setting a negative control, incubating in a cell incubator containing 5% carbon dioxide at 37 ℃ for 4h, detecting absorbance by using a chemiluminescence apparatus under the conditions of excitation light wavelength of 534nm and emission light wavelength of 584nm, and recording data of each well.

5. The two-drug synergy index was calculated using the Compuyn software and then analyzed for mapping.

The synergy index of the two drugs was calculated using the Compusyn software as shown in fig. 8, where the horizontal line indicates CI =1 and where CI >1 the two drugs are antagonistic; when CI =1, the two medicines have the addition function; when CI is less than 1, the two medicines have synergistic effect; the results in the figure show that BKM120 and TH588 have a synergistic inhibitory effect in the U251 cell line.

Example 9

Multiple concentrations of GDC0941 were combined with multiple concentrations of TH588 and observed for synergistic inhibition in the U251 cell line. The specific embodiment is as follows:

1. the U251 cells were seeded in 96-well plates at 1500 cells per well;

GDC0941 ( 0μM、1μM、2μM、10μM、20μM、100μM)

TH588 ( 0μM、2μM、4μM、5μM、6μM、8μM)

The synergy index of the two drugs was calculated using the Compusyn software as shown in fig. 9, where the horizontal line indicates CI =1 and where CI >1 the two drugs are antagonistic; when CI =1, the two medicines have the addition function; when CI is less than 1, the two medicines have synergistic effect; the results show that GDC0941 and TH588 have a synergistic inhibitory effect in the U251 cell line.

Example 10

Multiple concentrations of BKM120 were combined with multiple concentrations of TH287 and observed to determine whether they had a synergistic inhibitory effect in the U251 cell line. The specific embodiment is as follows:

1. the U251 cells were seeded in 96-well plates at 1500 cells per well;

2. setting concentration gradients, BKM 1205 concentration gradients and TH 2876 concentration gradients, and combining two by two to prepare 30 groups of drug-containing complete culture media with final concentration;

BKM120 ( 0μM、0.5μM、1μM、2μM、4μM、)

TH287 ( 0μM、0.25μM、0.5μM、1μM、2μM、4μM)

The synergy index of the two drugs was calculated as shown in figure 10 using the compactyn software, with the horizontal line indicating CI =1, and when CI >1 the two drugs were antagonistic; when CI =1, the two medicines have the addition function; when CI is less than 1, the two medicines have synergistic effect; the results in the figure show that BKM120 and TH287 have a synergistic inhibitory effect in the U251 cell line.

Example 11

Concentrations of BKM120 were combined with concentrations of TH588 and observed for synergistic inhibition in the H460 cell line. The specific embodiment is as follows:

1. h460 cells were seeded in 96-well plates at 2000 cells per well;

2. setting concentration gradients, namely, BKM 1206 concentration gradients and TH 5885 concentration gradients, and combining two of the two gradients to prepare 30 groups of drug-containing complete culture media with final concentration;

BKM120 ( 0μM、0.2μM、0.4μM、0.6μM、0.8μM、1μM)

TH588( 0μM、2μM、3μM、4μM、6μM)

3. and (3) after the cells are plated for 24h, discarding the old culture medium containing the medicine, and replacing the fresh culture medium containing the medicine and with different concentration combinations in the step 2.

The synergy index of the two drugs was calculated using the Compusyn software as shown in fig. 11, where the horizontal line indicates CI =1 and where CI >1 the two drugs are antagonistic; when CI =1, the two medicines have the addition function; when CI is less than 1, the two medicines have synergistic effect; the results in the figure show that BKM120 and TH588 have a synergistic inhibitory effect in the H460 cell line.

Example 12

Concentrations of BKM120 were combined with concentrations of TH588 and observed for synergistic inhibition in the PC9 cell line. The specific embodiment is as follows:

1. PC9 cells were seeded in 96-well plates at 2000 cells per well;

2. setting concentration gradients, BKM 1205 concentration gradients and TH 5886 concentration gradients, and combining two by two to prepare 30 groups of drug-containing complete culture media with final concentration;

BKM120 ( 0μM、0.5μM、0.75μM、1μM、2μM)

TH588( 0μM、2μM、3μM、4μM、8μM、10μM)

The synergy index of the two drugs was calculated using the Compusyn software as shown in fig. 12, where the horizontal line indicates CI =1 and where CI >1 the two drugs are antagonistic; when CI =1, the two medicines have the addition function; when CI is less than 1, the two medicines have synergistic effect; the results in the figure show that BKM120 and TH588 have a synergistic inhibitory effect in the PC9 cell line.

Claims

1. The application of the targeted medicine composition containing 1 mu M of BKM120 and 2 mu M of TH588 in preparing the medicine for inhibiting the U251 glioma cell line.