Treatment of tumors with TTfields and Aurora kinase inhibitors
阅读说明：本技术 用TTFields和Aurora激酶抑制剂治疗肿瘤 (Treatment of tumors with TTfields and Aurora kinase inhibitors ) 是由 莫舍·吉拉迪 戴玛·克雷斯 阿晴·谭米 罗莎·S·舒奈德曼 于 2019-04-08 设计创作，主要内容包括：可以通过将Aurora激酶抑制剂(例如MLN8237或另一种Aurora A激酶抑制剂、AZD1152或另一种Aurora B激酶抑制剂)施用于癌细胞,并且将频率为100至300 kHz(例如200 kHz)的交变电场施加于癌细胞,来降低癌细胞的生存力。此外,可以通过将Aurora激酶抑制剂施用于受试者,并且将频率为100至300 kHz(例如200 kHz)的交变电场施加于受试者的靶区域(例如,脑),来治疗受试者中的癌症(例如,成胶质细胞瘤)。(The viability of cancer cells can be reduced by applying an Aurora kinase inhibitor (e.g., MLN8237 or another Aurora a kinase inhibitor, AZD1152, or another Aurora B kinase inhibitor) to the cancer cells and applying an alternating electric field having a frequency of 100 to 300 kHz (e.g., 200 kHz) to the cancer cells. In addition, a cancer (e.g., glioblastoma) in a subject may be treated by administering an Aurora kinase inhibitor to the subject and applying an alternating electric field having a frequency of 100 to 300 kHz (e.g., 200 kHz) to a target region (e.g., brain) of the subject.)
1. Use of a therapeutic agent comprising an Aurora kinase inhibitor in the manufacture of a medicament for therapeutically reducing the viability of a cancer cell, wherein the therapeutic agent is administered to a cancer cell and an alternating electric field having a frequency of 100 kHz to 300 kHz is applied to the cancer cell.
2. The use of claim 1, wherein the Aurora kinase inhibitor comprises an Aurora A kinase inhibitor.
3. The use of claim 1, wherein the Aurora kinase inhibitor comprises an Aurora B kinase inhibitor.
4. The use of claim 1, wherein the Aurora kinase inhibitor comprises MLN 8237.
5. The use of claim 1, wherein the Aurora kinase inhibitor comprises AZD 1152.
6. The use of claim 1, wherein the Aurora kinase inhibitor is selected from the group consisting of AZD1152, alisertib (MLN8237), darussertib (PHA-739358), AT9283, PF-03814735 and AMG 900.
7. The use of claim 1, wherein the alternating electric field is applied at least partially simultaneously with the administration of the therapeutic agent.
8. The use of claim 1, wherein the alternating electric field is applied for a duration of at least 72 hours.
9. The use of claim 1, wherein the frequency of the alternating electric field is from 180 kHz to 220 kHz, from 190 kHz to 210 kHz, from 195 kHz to 205 kHz, or about 200 kHz.
10. The use of claim 1, wherein the Aurora kinase inhibitor is administered to the cancer cells at a therapeutically effective concentration, and wherein the alternating electric field has a field strength of at least 1V/cm in at least some of the cancer cells.
11. The use of claim 10, wherein the therapeutically effective concentration of the Aurora kinase inhibitor is reduced by at least 50% relative to the dose of the Aurora kinase inhibitor known to be therapeutically effective in the absence of an alternating electric field.
12. The use of claim 10, wherein the therapeutically effective concentration of the Aurora kinase inhibitor is from about 12.5 nM to about 100 nM.
13. The use of claim 10, wherein the therapeutically effective concentration of the Aurora kinase inhibitor is from 25 nM to 75 nM.
14. Use of a therapeutic agent comprising an Aurora kinase inhibitor in the manufacture of a medicament for the therapeutic treatment of cancer in a subject, wherein the therapeutic agent is administered to a target region of a subject at a therapeutically effective concentration, and an alternating electric field having a frequency of 100 kHz to 300 kHz is applied to the target region of the subject.
15. The use of claim 14, wherein the Aurora kinase inhibitor comprises an Aurora a kinase inhibitor.
16. The use of claim 14, wherein the Aurora kinase inhibitor comprises an Aurora B kinase inhibitor.
17. The use of claim 14, wherein the Aurora kinase inhibitor comprises MLN 8237.
18. The use of claim 14, wherein the Aurora kinase inhibitor comprises AZD 1152.
19. The use of claim 14, wherein the cancer comprises glioblastoma.
20. The use of claim 14, wherein the Aurora kinase inhibitor is selected from the group consisting of AZD1152, alisertib (MLN8237), darussertib (PHA-739358), AT9283, PF-03814735 and AMG 900.
21. The use of claim 14, wherein the alternating electric field is applied at least in part after administration of the therapeutic agent and prior to elimination or depletion of the Aurora kinase inhibitor from the body of the subject.
22. The use of claim 14, wherein the alternating electric field is applied for a duration of at least 72 hours.
23. The use of claim 14, wherein the frequency of the alternating electric field is from 180 kHz to 220 kHz, from 190 kHz to 210 kHz, from 195 kHz to 205 kHz, or about 200 kHz.
24. The use of claim 14, wherein the alternating electric field has a field strength of at least 1V/cm in at least some target regions of the subject.
25. The use of claim 14, wherein the therapeutically effective concentration of the Aurora kinase inhibitor is reduced by at least 50% relative to the dose of the Aurora kinase inhibitor known to be therapeutically effective in the absence of an alternating electric field.
26. The use of claim 14, wherein the therapeutically effective concentration of the Aurora kinase inhibitor is from about 12.5 nM to about 100 nM.
27. The use of claim 14, wherein the therapeutically effective concentration of the Aurora kinase inhibitor is from 25 nM to 75 nM.
28. A therapeutic agent comprising an Aurora kinase inhibitor for use in therapeutically reducing the viability of a cancer cell, wherein the therapeutic agent is administered to a cancer cell and an alternating electric field having a frequency of 100 kHz to 300 kHz is applied to the cancer cell.
29. The therapeutic agent of claim 28, wherein the Aurora kinase inhibitor comprises an Aurora a kinase inhibitor.
30. The therapeutic agent of claim 28, wherein the Aurora kinase inhibitor comprises an Aurora B kinase inhibitor.
31. The therapeutic agent of claim 28, wherein the Aurora kinase inhibitor comprises MLN 8237.
32. The therapeutic agent of claim 28, wherein the Aurora kinase inhibitor comprises AZD 1152.
33. The therapeutic agent of claim 28, wherein the Aurora kinase inhibitor is selected from the group consisting of AZD1152, alisertib (MLN8237), darussertib (PHA-739358), AT9283, PF-03814735, and AMG 900.
34. The therapeutic agent of claim 28, wherein the alternating electric field is applied at least partially concurrently with the administration of the therapeutic agent.
35. The therapeutic agent of claim 28, wherein the alternating electric field is applied for a duration of at least 72 hours.
36. The therapeutic agent of claim 28, wherein the frequency of the alternating electric field is 180 kHz to 220 kHz, 190 kHz to 210 kHz, 195 kHz to 205 kHz, or about 200 kHz.
37. The therapeutic agent of claim 28, wherein the Aurora kinase inhibitor is administered to the cancer cells at a therapeutically effective concentration, and wherein the alternating electric field has a field strength of at least 1V/cm in at least some of the cancer cells.
38. The therapeutic agent of claim 37, wherein the therapeutically effective concentration of the Aurora kinase inhibitor is reduced by at least 50% relative to a dose of the Aurora kinase inhibitor known to be therapeutically effective in the absence of an alternating electric field.
39. The therapeutic agent of claim 37, wherein the therapeutically effective concentration of the Aurora kinase inhibitor is from about 12.5 nM to about 100 nM.
40. The therapeutic agent of claim 37, wherein the therapeutically effective concentration of the Aurora kinase inhibitor is from 25 nM to 75 nM.
41. A therapeutic agent comprising an Aurora kinase inhibitor for use in the therapeutic treatment of cancer in a subject, wherein the therapeutic agent is administered to a target region of a subject at a therapeutically effective concentration and an alternating electric field having a frequency of 100 kHz to 300 kHz is applied to the target region of the subject.
42. The therapeutic agent of claim 41, wherein the Aurora kinase inhibitor comprises an Aurora A kinase inhibitor.
43. The therapeutic agent of claim 41, wherein the Aurora kinase inhibitor comprises an Aurora B kinase inhibitor.
44. The therapeutic agent of claim 41, wherein the Aurora kinase inhibitor comprises MLN 8237.
45. The therapeutic agent of claim 41, wherein the Aurora kinase inhibitor comprises AZD 1152.
46. The therapeutic agent of claim 41, wherein the cancer comprises glioblastoma.
47. The therapeutic agent of claim 41, wherein the Aurora kinase inhibitor is selected from the group consisting of AZD1152, alisertib (MLN8237), darussertib (PHA-739358), AT9283, PF-03814735, and AMG 900.
48. The therapeutic agent of claim 41, wherein the alternating electric field is applied at least partially after administration of the therapeutic agent and prior to elimination or depletion of the Aurora kinase inhibitor from the body of the subject.
49. The therapeutic agent of claim 41, wherein the alternating electric field is applied for a duration of at least 72 hours.
50. The therapeutic agent of claim 41, wherein the frequency of the alternating electric field is 180 kHz to 220 kHz, 190 kHz to 210 kHz, 195 kHz to 205 kHz, or about 200 kHz.
51. The therapeutic agent of claim 41, wherein the alternating electric field has a field strength of at least 1V/cm in at least some target regions of the subject.
52. The therapeutic agent of claim 41, wherein the therapeutically effective concentration of the Aurora kinase inhibitor is reduced by at least 50% relative to a dose of Aurora kinase inhibitor known to be therapeutically effective in the absence of an alternating electric field.
53. The therapeutic agent of claim 41, wherein the therapeutically effective concentration of the Aurora kinase inhibitor is from about 12.5 nM to about 100 nM.
54. The therapeutic agent of claim 41, wherein the therapeutically effective concentration of the Aurora kinase inhibitor is from 25 nM to 75 nM.
Background
Tumor treatment fields (TTfields) are created by applying a low intensity, medium frequency, alternating electric fieldAnd an effective antitumor therapeutic pattern delivered. To date, TTFields therapy has received FDA approval for the treatment of glioblastoma multiforme brain tumors. In one example, TTFields therapy uses a wearable and portable device (optube)®) To be delivered. The delivery system may include four adhesive, non-invasive, insulated "transducer arrays", an electric field generator, a rechargeable battery, and a carrying case. The transducer array may be applied to the skin near the tumor and connected to the field generators.
TTfields may use, for example, Inovitro in a preclinical settingTMThe TTFields bench system was applied in vitro. InovitroTMIncluding TTFields generators and substrates, each containing 8 ceramic dishes. Cells were plated on 22 mm circular coverslips placed inside each dish. TTFields are applied using two pairs of orthogonal transducer arrays in each dish, insulated by a high dielectric constant ceramic. The orientation of the TTFields in each dish was switched by 90 ° every 1 second, thus covering most of the axis of cell division.
SUMMARY
Certain types of cancer (e.g., glioblastoma) can be improved or treated with TTfields and Aurora kinase inhibitors (e.g., balacet (AZD1152), alisertib (MLN8237), darussotat (PHA-739358), AT9283, PF-03814735, and AMG 900). See, e.g., Bavetsias et al, Aurora Kinase Inhibitors: Current Status and Outlook, Front Oncol. 2015; 5: 278. All cited references and publications are incorporated herein in their entirety.
One aspect of the invention relates to the use of a therapeutic agent comprising an Aurora kinase inhibitor in the manufacture of a medicament for the therapeutic reduction of the viability of cancer cells, wherein the therapeutic agent is administered to cancer cells and an alternating electric field having a frequency of 100 kHz to 300 kHz is applied to cancer cells.
In one embodiment, the Aurora kinase inhibitor comprises an Aurora a kinase inhibitor.
In one embodiment, the Aurora kinase inhibitor comprises an Aurora B kinase inhibitor.
In one embodiment, the Aurora kinase inhibitor comprises MLN 8237.
In one embodiment, the Aurora kinase inhibitor comprises AZD 1152.
In one embodiment, the Aurora kinase inhibitor is selected from AZD1152, alisertib (MLN8237), darussertib (PHA-739358), AT9283, PF-03814735 and AMG 900.
In one embodiment, the alternating electric field is applied at least partially concurrently with the administration of the therapeutic agent.
In one embodiment, the alternating electric field is applied for a duration of at least 72 hours.
In one embodiment, the frequency of the alternating electric field is 180 kHz to 220 kHz, 190 kHz to 210 kHz, 195 kHz to 205 kHz, or about 200 kHz.
In one embodiment, the Aurora kinase inhibitor is administered to the cancer cells at a therapeutically effective concentration, and wherein the alternating electric field has a field strength of at least 1V/cm in at least some of the cancer cells.
In one embodiment, the therapeutically effective concentration of the Aurora kinase inhibitor is reduced by at least 50% relative to a dose of the Aurora kinase inhibitor known to be therapeutically effective in the absence of an alternating electric field.
In one embodiment, the therapeutically effective concentration of the Aurora kinase inhibitor is from about 12.5 nM to about 100 nM.
In one embodiment, the therapeutically effective concentration of the Aurora kinase inhibitor is from 25 nM to 75 nM.
Another aspect of the invention relates to the use of a therapeutic agent comprising an Aurora kinase inhibitor in the manufacture of a medicament for the therapeutic treatment of cancer in a subject, wherein the therapeutic agent is administered to a target region of the subject at a therapeutically effective concentration and an alternating electric field having a frequency of 100 kHz to 300 kHz is applied to the target region of the subject.
In one embodiment, the Aurora kinase inhibitor comprises an Aurora a kinase inhibitor.
In one embodiment, the Aurora kinase inhibitor comprises an Aurora B kinase inhibitor.
In one embodiment, the Aurora kinase inhibitor comprises MLN 8237.
In one embodiment, the Aurora kinase inhibitor comprises AZD 1152.
In one embodiment, the cancer comprises a glioblastoma.
In one embodiment, the Aurora kinase inhibitor is selected from AZD1152, alisertib (MLN8237), darussertib (PHA-739358), AT9283, PF-03814735 and AMG 900.
In one embodiment, the alternating electric field is applied at least partially after administration of the therapeutic agent and before the Aurora kinase inhibitor is eliminated or depleted from the body of the subject.
In one embodiment, the alternating electric field is applied for a duration of at least 72 hours.
In one embodiment, the frequency of the alternating electric field is 180 kHz to 220 kHz, 190 kHz to 210 kHz, 195 kHz to 205 kHz, or about 200 kHz.
In one embodiment, the alternating electric field has a field strength of at least 1V/cm in at least some target regions of the subject.
In one embodiment, the therapeutically effective concentration of the Aurora kinase inhibitor is reduced by at least 50% relative to a dose of the Aurora kinase inhibitor known to be therapeutically effective in the absence of an alternating electric field.
In one embodiment, the therapeutically effective concentration of the Aurora kinase inhibitor is from about 12.5 nM to about 100 nM.
In one embodiment, the therapeutically effective concentration of the Aurora kinase inhibitor is from 25 nM to 75 nM.
Yet another aspect of the invention relates to a therapeutic agent comprising an Aurora kinase inhibitor for use in therapeutically reducing the viability of a cancer cell, wherein the therapeutic agent is administered to a cancer cell and an alternating electric field having a frequency of 100 kHz to 300 kHz is applied to the cancer cell.
In one embodiment, the Aurora kinase inhibitor comprises an Aurora a kinase inhibitor.
In one embodiment, the Aurora kinase inhibitor comprises an Aurora B kinase inhibitor.
In one embodiment, the Aurora kinase inhibitor comprises MLN 8237.
In one embodiment, the Aurora kinase inhibitor comprises AZD 1152.
In one embodiment, the Aurora kinase inhibitor is selected from AZD1152, alisertib (MLN8237), darussertib (PHA-739358), AT9283, PF-03814735 and AMG 900.
In one embodiment, the alternating electric field is applied at least partially concurrently with the administration of the therapeutic agent.
In one embodiment, the alternating electric field is applied for a duration of at least 72 hours.
In one embodiment, the frequency of the alternating electric field is 180 kHz to 220 kHz, 190 kHz to 210 kHz, 195 kHz to 205 kHz, or about 200 kHz.
In one embodiment, the Aurora kinase inhibitor is administered to the cancer cells at a therapeutically effective concentration, and wherein the alternating electric field has a field strength of at least 1V/cm in at least some of the cancer cells.
In one embodiment, the therapeutically effective concentration of the Aurora kinase inhibitor is reduced by at least 50% relative to a dose of the Aurora kinase inhibitor known to be therapeutically effective in the absence of an alternating electric field.
In one embodiment, the therapeutically effective concentration of the Aurora kinase inhibitor is from about 12.5 nM to about 100 nM.
In one embodiment, the therapeutically effective concentration of the Aurora kinase inhibitor is from 25 nM to 75 nM.
Yet another aspect of the invention relates to a therapeutic agent comprising an Aurora kinase inhibitor for use in the therapeutic treatment of cancer in a subject, wherein the therapeutic agent is administered to a target area of the subject at a therapeutically effective concentration and an alternating electric field having a frequency of 100 kHz to 300 kHz is applied to the target area of the subject.
In one embodiment, the Aurora kinase inhibitor comprises an Aurora a kinase inhibitor.
In one embodiment, the Aurora kinase inhibitor comprises an Aurora B kinase inhibitor.
In one embodiment, the Aurora kinase inhibitor comprises MLN 8237.
In one embodiment, the Aurora kinase inhibitor comprises AZD 1152.
In one embodiment, the cancer comprises a glioblastoma.
In one embodiment, the Aurora kinase inhibitor is selected from AZD1152, alisertib (MLN8237), darussertib (PHA-739358), AT9283, PF-03814735 and AMG 900.
In one embodiment, the alternating electric field is applied at least partially after administration of the therapeutic agent and before the Aurora kinase inhibitor is eliminated or depleted from the body of the subject.
In one embodiment, the alternating electric field is applied for a duration of at least 72 hours.
In one embodiment, the frequency of the alternating electric field is 180 kHz to 220 kHz, 190 kHz to 210 kHz, 195 kHz to 205 kHz, or about 200 kHz.
In one embodiment, the alternating electric field has a field strength of at least 1V/cm in at least some target regions of the subject.
In one embodiment, the therapeutically effective concentration of the Aurora kinase inhibitor is reduced by at least 50% relative to a dose of the Aurora kinase inhibitor known to be therapeutically effective in the absence of an alternating electric field.
In one embodiment, the therapeutically effective concentration of the Aurora kinase inhibitor is from about 12.5 nM to about 100 nM.
In one embodiment, the therapeutically effective concentration of the Aurora kinase inhibitor is from 25 nM to 75 nM.
Brief Description of Drawings
FIGS. 1A-E show TTfields and AZD1152 at various AZD1152 concentrations for U87-MG, U87-MGshp53And U-251 glioma cells.
FIGS. 2A and 2B are U87-MG and U87-MG, respectivelyshp53Exemplary microscopy images of cells showing multinucleated and pycnotic cell formation after treatment with TTFields and AZD 1152.
FIGS. 3A-D show exemplary increases in polyploidy in U87-MG cells after treatment with TTfields at various AZD1152 concentrations.
FIGS. 4A-E show U87-MG after treatment with TTfields at various AZD1152 concentrationsshp53Exemplary increase in polyploidy in cells.
Fig. 5A and 5B show normalized cell counts of U87-MG (fig. 5A) and primary glioblastoma cell line HT12347 (fig. 5B) after treatment with TTFields and AZD1152, alone and in combination, at indicated AZD1152 concentrations and TTField frequencies.
Fig. 6 shows exemplary laser scanning microscopy images of primary glioblastoma cell line HT12347 treated and stained as indicated.
Fig. 7 shows an exemplary light microscopy of primary glioblastoma cell line HT12347 treated as shown, at 2.5X magnification (left) and 10X magnification (right), with bars 200 μ M on the left and bars 100 μ M on the right.
Fig. 8A and 8B show normalized cell counts of U87-MG (fig. 8A) and primary glioblastoma cell line HT12347 (fig. 8B) after treatment with TTFields and MLN8337, alone and in combination.
Fig. 9A is a summary of exemplary PI (propidium iodide) staining results showing DNA content in glioblastoma cell line HT12347 after treatment with controls, TTFields alone, MLN8337 alone and combinations.
FIGS. 9B-9D are cell cycle histograms corresponding to FIG. 9A.
Fig. 10A-10D show exemplary optical microscopy images of primary glioblastoma cells HT12347 after the following treatments: 10A: comparison; 10B: after TTField processing; 10C: after treatment with MLN8237 alone; and 10D: after treatment with TTFields and MLN 8237.
Detailed description of the preferred embodiments
The term "treating" refers to ameliorating, inhibiting, reducing growth, inhibiting metastasis, and prescribing agents to achieve the foregoing behavior. The Aurora B kinase inhibitors described herein may be used in combination with a pharmaceutically acceptable carrier for administrationTo the patient. As used herein, the term "reducing viability" refers to reducing proliferation, inducing apoptosis, or killing cancer cells. As used herein, the term "therapeutically effective concentration" refers to a concentration of one or more drugs that is sufficient to achieve its intended purpose (e.g., the treatment of cancer). See, e.g., Schwartz et al,Phase I study of barasertib (AZD1152), a selective inhibitor of Aurora B kinase, in patients with advanced solid tumorsinvest New drugs, 2013 Apr; (31) (2) 370-80 (maximum tolerated AZD1152 dose of 150 mg administered as a 48 hour continuous infusion and 220 mg administered as two 2 hour infusions (110 mg/day, days 1, 2, 15 and 16)); in Kantariian et al,Phase I study assessing the safety and tolerability of barasertib (AZD1152) with low-dose cytosine arabinoside in elderly patients with AML, Clin Lymphoma Myeloma Leuk. 2013 Oct；13(5):559-67。
introduction to the design reside in
TTField exerts directional forces on polar microtubules and interferes with the assembly of normal mitotic spindles. Such interference with microtubule dynamics results in abnormal spindle formation, and subsequent mitotic arrest or delay. Cells can die at the time of mitotic arrest or progression to cell division. This can lead to the formation of normal or abnormal aneuploid progeny. The formation of tetraploid cells may occur as a result of mitotic withdrawal by slippage, or may occur during inappropriate cell division. Abnormal daughter cells may die at subsequent interphase, may undergo permanent arrest, or may proliferate through additional mitosis in which they will undergo further TTFields attack. See M, GILADI et al, Mitic Spindle navigation by Alternating Electric Fields Leads to Impper Chromosome Segregation and Mitic Catastrophe in Cancer Cells, Scientific Reports, 2015; 5:18046, which is incorporated herein by reference in its entirety.
A promising approach to enhance the efficiency of TTFields is to use drugs that act synergistically with TTFields and prolong the mid-late transition and end-stage. In particular, inhibitors or drugs that interfere with components of the chromosomal passenger complex, particularly those that affect Aurora B kinase, are excellent candidates for use in combination with TTFields.
Aurora B stands for Chromosomal Passenger Protein (CPP). It assembles with the internal centromere proteins (INCENP/INCENP), BIRC 5/survivin and CDCA8/Borealin in a stable complex to construct the Chromosome Passenger Complex (CPC). Aurora B kinase activity is involved in correcting syntelic and merotic microtubule-kinetochore ligation and is therefore an important factor in the biological orientation of sister chromatids towards the opposite spindle pole before the start of the later phase. Aurora B, in cooperation with its CPC partner, protects safely the isolation, is independent of chromosomal integrity of the p53 mutation state, and is therefore important for cell survival. See, R WIEDEMUTH R et al, Janus Face-Like Effects of Aurora B Inhibition, Antitomoral model of Action Versus indication of Aneuploid Progene, Carcinogenesis, 2016; 37(10) 993-1003, which is incorporated herein by reference in its entirety.
The study described herein was run to test the following assumptions: additional inhibition of cytokinesis by chemical inhibition of Aurora kinase may increase the effect of TTFields on tumor cells. More specifically, the study explored the combination of TTFields and the Aurora B kinase inhibitor AZD1152 for the treatment of GBM. In another instance, additional Aurora B kinase inhibitors, including but not limited to dalutasertib (PHA-739358), AT9283, PF-03814735, and AMG 900, may be used before, during, or after treatment with TTfields. Aurora a kinase is associated with centrosome maturation and separation, and affects spindle assembly and stability.See, e.g.Cummings et al,Biphasic activation of Aurora-A kinase during the meiosis I- meiosis II transition in Xenopus oocytes, Mol. Cell. Biol. 23(5): 1703–16。
the effect of combined treatment of TTFields and the Aurora B kinase inhibitor AZD1152 was tested in three different glioma cell lines: U87-MG and U87-MGshp53And U-251. TTfields (1.75V/cm RMS, 200 kHz) were applied for 72 hours using the Inovitro system. AZD1152 was added to the medium at a concentration of up to 100 nmol/L. Determining details at the end of processingCell count, cell cycle and colony formation potential. Microscopic images of cells stained with crystal violet were used to determine the formation of multinucleated cells.
The combined treatment with TTfields and AZD1152 resulted in U251, U87-MG and U87-MG compared to each treatment aloneshp53Significant reduction in the number of cells (2-way ANOVA, p in all three cell lines<0.001). Considering the cytotoxic effect at the end of the treatment and the overall effect of the clonogenic potential, it was confirmed that U87-MG, U87-MG compared to each treatment aloneshp53And a significant reduction in U-251 cells (2-way ANOVA, p in all three cell lines<0.001). U87-MG and U87-MG stained with Crystal Violet after treatmentshp53Microscopy images of cells revealed a high prevalence of multinucleated cells in cells exposed to TTFields and AZD1152 (25 nM) compared to cells treated with AZD1152 (25 nM) alone. Cells treated with TTfields and higher doses of AZD1152 (50-100 nM) demonstrated increased rates of pyknosis.
The results described herein demonstrate that the combination of TTFields and the Aurora B kinase inhibitor AZD1152 can be an effective treatment against glioma cells and that there appears to be a synergy between these two treatment modalities. The results described herein also demonstrate that the combination of TTFields and Aurora a kinase inhibitor MLN8237 may be an effective treatment against glioma cells.
Method
Cell culture and drug
The effect of the combined treatment of TTFields and AZD1152 was tested using the following human glioma cell lines: U87-MG (ATCC), U-251 (ECACC) and U87-MGshp53(supplied by Dr. Achim Time). All cells were grown in humidified incubator supplied with 5% CO 2. Cells were maintained in EMEM supplemented with 10% FBS, 2 mmol/L glutamine, Pen-Strep solution (100 units/ml penicillin and 0.1 mg/ml streptomycin), 1 mmol/L sodium pyruvate and 1% NEAA. Mixing U87-MGshp53Cells were maintained under selection with 400 mg/mL geneticin. AZD1152 was obtained from Sigma of israel.
Cytotoxicity assays and overall Effect
Using the Inovitro seriesTTfields (1.6V/cm RMS, 200 kHz) were applied for 72 hours. At the end of the treatment, the inhibition of tumor cell growth was quantitatively analyzed based on cell count. U87-MG and U87-MGshp53And U-251 was tested for colony formation survival by plating 300 cells/dish in triplicate in 6-well dishes. After 2-3 weeks, cells were stained with crystal violet and the number of clones quantified. The overall effect was calculated by multiplying the percentage of surviving cells at the end of the treatment by the percentage of colonies formed relative to the control.
Flow cytometry
For cell cycle analysis, cells were removed with trypsin immediately after 72 hours of treatment, washed twice with ice-cold PBS with 1% FBS, and fixed with 70% ice-cold ethanol for 30 minutes. After fixation, cells were washed twice with ice-cold PBS with 1% FBS, forming a pellet, and incubated in PBS containing 10 μ g/ml RNase and 7.5 μ g/ml 7-AAD (Sigma-Aldrich) for 30 minutes at 37 ℃. The cell cycle distribution was then quantified using an EC800 flow cytometer (Sony Biotechnology, japan).
Microscopic examination
At the end of the treatment, cells were fixed with 100% methanol, stained with 0.5% crystal violet (Sigma), and imaged under an inverted microscope (Nikon eclipse TS 100).
Statistical analysis
Data are expressed as mean ± SE, and the statistical significance of the differences was assessed using GraphPad Prism 6 Software (GraphPad Software, La Jolla, CA). Differences between groups were compared using 2-way ANOVA and were considered significant at values of 0.05 > p > 0.01, <0.01, and < 0.001.
Results
FIGS. 1A-E depict the effects of TTfields and AZD1152 on glioma cells. U87-MG and U87-MGshp53And U-251 glioma cells were grown at various AZD1152 concentrations and treated with TTfields (200 kHz, 1.6V/cm RMS) for 72 hours. U87-MG demonstrated to be highly sensitive to treatment with AZD1152 at concentrations exceeding 20 nM. As depicted in fig. 1A, the response applied to TTFields alone resulted in an approximately 50% reduction in cell number. At the end of the treatmentThe number of cells was determined and expressed as a percentage of the control. Combined treatment of TTFields and AZD1152 resulted in a significant enhancement of cytotoxic effects (2-way ANOVA, p)<0.001). In addition, as depicted in fig. 1B, the combined treatment of TTFields and AZD1152 resulted in a significant enhancement of the overall effect in U87-MG cells in consideration of both cytotoxic effects and clonogenic responses.
U87-MG compared to the p53 WT counterpart line with an IC-50 of about 50 nMshp53Proved to be less sensitive to treatment with AZD 1152. The response applied to TTfields alone resulted in U87-MGshp53Approximately 40% reduction in cell number was only slightly lower than that observed for its p53 WT counterpart strain (fig. 1C). Combined treatment of TTfields and AZD1152 resulted in U87-MGshp53Cytotoxic effects in cells (2-factor ANOVA, p)<0.001) (fig. 1D) and a significant enhancement of the overall effect (fig. 1D).
It is noteworthy that when AZD1152 is combined with TTFields, the dose of AZD1152 required to obtain the overall effect at a given level can often be reduced by at least 50% relative to the dose of AZD1152 which provides the same overall effect at the given level in the absence of an alternating electric field. For example, the horizontal dashed line in fig. 1D shows that in the absence of TTFields, a 70 nM dose of AZD1152 is required to achieve 38% of the overall effect, whereas when AZD1152 is combined with TTFields, only a 25 nM dose of AZD1152 is required to achieve 38% of the same overall effect.
After treatment with AZD1152, the volume of U-251 cells increased dramatically, which made them impossible to count using flow cytometry. The overall effect on U-251 cells was determined based on a clonogenic assay. The response applied to TTFields alone resulted in an approximately 50% reduction in clonogenic potential of U-251 cells (fig. 1E). Combined treatment of TTFields and AZD1152 resulted in a significant reduction in the number of U-251 colonies (2-way ANOVA, p <0.001) (fig. 1E).
Fig. 2A and 2B depict multinucleated cell formation after combined treatment with TTFields and AZD 1152. FIGS. 2A and 2B are U87-MG and U87-MG, respectivelyshp53Microscopic image of cells showing multinucleated and pycnotic cell formation after combined treatment with TTFields and AZD 1152. At each locationU87-MG and U87-MG grown at AZD1152 concentrationshp53Glioma cells were treated with TTfields (200 kHz, 1.6V/cm RMS) for 72 hours. Cells stained with crystal violet after treatment were imaged under an inverted microscope. The arrow labeled "M" marks the multinucleated cells, while the arrow labeled "P" marks the pycnotic cells. These images revealed a slight increase in the number of U87-MG multinucleated cells after TTFields application (fig. 2A, first row). U87-MG and U87-MG exposed to TTfields and low concentrations of AZD1152 (25 nM) compared to cells treated with AZD1152 alone (25 nM)shp53A high prevalence of multinucleated cells (marked by an arrow labeled "M") was observed in the cells (middle row of fig. 2A, 2B). Cells treated with TTfields and higher doses of AZD1152 (50-100 nM) demonstrated increased rates of solid reduction (marked by the arrow labeled "P") (bottom row of FIGS. 2A, 2B).
FIGS. 3A-D and 4A-E show U87-MG and U87-MG after treatment with TTfields and AZD1152shp53FACS analysis of the DNA content of (1). More specifically, FIGS. 3A-D depict increased polyploidy in U87-MG cells following combined treatment with TTfields (200 kHz, 1.6V/cm RMS) at various AZD1152 concentrations; while FIGS. 4A-E depict U87-MG after combined treatment with TTfields (200 kHz, 1.6V/cm RMS) at various AZD1152 concentrationsshp53Increased polyploidy in cells. Flow cytometry was used to assay for U87-MG and U87-MG based on multinuclear nature observed in microscopy imagesshp53Ploidy analysis of cells revealed an increase in the number of polyploid cells after treatment with TTFields and AZD 1152.
Tables 1 and 2 describe the results for U87-MG and U87-MG, respectivelyshp53Corresponding to the numerical data of figures 3 and 4. The columns in tables 1 and 2 represent DNA copy number (e.g., 2n is the normal chromosome number (23 vs =46), 4n is twice the chromosome number, etc.).
Tables 3 and 4 depict the results for U87-MG and U87-MG, respectively, when exposed to different concentrations of AZD1152, alone and in combination with TTfieldsshp53And the cytotoxicity and overall effect of U-251 cells. This data indicates a synergy between TTFields and AZD1152 because the observed percentage values are lower than the expected percentage values for all non-zero concentrations of AZD 1152.
Alternative methods
In addition to U87-MG cells, primary tumor cell lines were established from surgically obtained glioblastoma tissue. TTfields (1.6V/cm RMS, 200 kHz) were applied for 72 hours using the Inovitro ™ system. AZD1152 was added to the medium at a concentration of up to 100 nmol/L. Cell count, cell cycle and colony formation potential were determined at the end of the treatment.
Primary tumor cell lines were analyzed. In addition, MLN8237, an Aurora A kinase inhibitor, was tested at concentrations up to 50 nmol/l for treatment of U87-MG and primary tumor cell lines to further establish Aurora kinase inhibition as a target for combination therapy with TTfields.
Additional results
The combined treatment of TTFields and AZD1152 resulted in a significant reduction in the number of primary glioblastoma cells compared to each treatment alone (mann whichwas U test, p < 0.001). Fig. 5A and 5B show normalized cell counts of U87-MG (fig. 5A) and primary glioblastoma cell line HT12347 (fig. 5B) after treatment with TTFields and AZD1152, alone and in combination, at indicated AZD1152 concentrations and TTField frequencies. Exemplary boxplots show normalized cell counts after treatment with AZD1152 alone, TTFields alone, and a combination of AZD1152 and TTFields. "x" marks a significant difference where p < 0.001.
Fig. 6 shows exemplary laser scanning microscopy images of primary glioblastoma cell line HT12347 treated and stained as indicated. Confocal laser scanning microscopy of the primary glioblastoma cell line HT18584 showed the following: (1) staining with Hoechst33342 DNA (blue), (2) WGA-Alexa Fluor 647 staining for cell membranes (red), and (3) secondary anti-sheep alpha mouse IgG1 FITC for yH2AX staining. The bars are marked 25 μm. Cells were treated with control, AZD1152 (20 nM), TTFields, or a combination of both. The greatest reduction in cell number was seen with the combined treatment.
Fig. 7 shows an exemplary optical microscopy of primary glioblastoma cell line HT12347 at 2.5X magnification (left) and 10X magnification (right), with bars 200 μ M on the left and bars 100 μ M on the right. Cells were treated with control, AZD1152, TTFields, or a combination of both. The greatest reduction in cell number was seen with the combined treatment.
The combined treatment of MLN8237 and TTFields also resulted in a significant reduction in the cell number of U87-MG as well as primary glioblastoma cell lines compared to each treatment alone (mann wye U-test, p < 0.01). FIGS. 8A-8B show normalized cell counts for U87-MG (FIG. 8A) and primary glioblastoma cell line HT12347 (FIG. 8B) after treatment with TTfields and MLN8237, alone and in combination. Exemplary boxplots show normalized cell counts for U87 cells (fig. 4A) and HT12347 glioblastoma cells (fig. 8B) after treatment with TTFields alone, MLN8237 alone, and a combination of MLN8237 and TTFields. The greatest reduction in the percentage of viable cells relative to the control was shown with the combination of TTFields and MLN 8237.
Fig. 9A-9E show exemplary PI (propidium iodide) staining results showing DNA content in glioblastoma cell line HT12347, with corresponding cell cycle histograms, after treatment with TTFields alone, MLN8337 alone, and combinations. FIG. 9A is a summary, where 2n is light gray, 4n is dark gray, and >4n is black; FIG. 9B is a cell cycle histogram for a control; fig. 9C is a cell cycle histogram for TTField treatment alone; FIG. 9D is a cell cycle histogram for MLN8237 alone; and FIG. 9E is a cell cycle histogram for TTfields in combination with MLN 8237. The combined treatment results in a greater number of multinucleated and polyploid cells.
Fig. 10A-10D show exemplary optical microscopy images of primary glioblastoma cells HT12347 after the following treatments: 10A: comparison; 10B: after TTField processing; 10C: after treatment with MLN8237 alone; 10D: after treatment with TTFields and MLN 8237. The combined treatment showed the lowest number of cells.
The results demonstrate that a combination of TTFields and Aurora kinase inhibitors (e.g., Aurora a kinase inhibitors or Aurora B kinase inhibitors) may be an effective treatment for cancer cells, including glioblastoma cells.
Conclusion
The studies described herein demonstrate that the effect of TTFields on tumor cells can be augmented by chemical inhibition of Aurora kinases (e.g., Aurora a kinase and Aurora B kinase) to otherwise inhibit cytokinesis. More specifically, the study explored the combination of TTFields and Aurora B kinase inhibitor AZD1152 or Aurora a kinase inhibitor MLN8237 for the treatment of GBM. In another instance, additional Aurora kinase inhibitors, including but not limited to dalutasertib (PHA-739358), AT9283, PF-03814735, and AMG 900, may be used before, during, or after treatment with TTfields.
At U87-MG and U87-MGshp53In cells, the combination of TTFields and AZD1125 resulted in a significant reduction in cell number compared to each treatment alone. The overall effect of the combined treatment was significantly higher in all three cell lines tested than each treatment alone. The combination of TTFields and lower doses of AZD1125 resulted in an increase in the number of multinucleated and polyploid cells, similar to the effect observed with higher AZD1152 concentrations.
These results confirm that the viability of cancer cells can be reduced by applying an Aurora B kinase inhibitor to cancer cells and applying an alternating electric field having a frequency of 100 to 300 kHz to the cancer cells. In some cases of the methods described herein, at least a portion of the applying step is performed simultaneously with at least a portion of the applying step.
In the cells tested as described herein, the combination of TTFields and AZD1125 or MLN8237 resulted in a significant reduction in cell number compared to each treatment alone. The overall effect of the combined treatment was significantly higher in all cell lines tested than each treatment alone. The combination of TTFields and MLN8337 resulted in an increase in the number of multinucleated and polyploid cells.
These results confirm that the viability of cancer cells can be reduced by applying an Aurora kinase inhibitor (e.g., Aurora a kinase inhibitor, Aurora B kinase inhibitor) to cancer cells and applying an alternating electric field having a frequency of 100 to 300 kHz to the cancer cells. In some cases of the methods described herein, at least a portion of the applying step is performed simultaneously with at least a portion of the applying step.
It should be noted that although the studies described herein were performed using the frequencies, field strengths, and durations noted above, those parameters may vary. For example, the frequency may be 200 kHz, 180 to 220 kHz, or 100 to 300 kHz; the electric field strength may be 0.5 to 5V/cm, or at least 1V/cm; and the duration may be any time longer than 8 hours.
It should also be noted that although the studies described herein were all performed in vitro, the results of these studies could be extended to an in vivo setting by performing the administration and application to live subjects (e.g., using the optone @system) rather than to cancer cells in vitro.
It should be noted that in an in vitro environment, the first time (t) from the introduction of an Aurora B kinase inhibitor or an Aurora a kinase inhibitor into a container containing cancer cells1) Initially, until the time (t) for the Aurora B kinase inhibitor or Aurora A kinase inhibitor to be removed or exhausted2) Administration of the Aurora B kinase inhibitor or Aurora a kinase inhibitor to cancer cells occurs continuously. As a result, if TTfields are set at t1And t2To cancer cells, the application will be simultaneous with at least a portion of the application. In vivo environmentFrom the first time (t) the Aurora B kinase inhibitor or Aurora A kinase inhibitor circulates in the patient (e.g. after systemic administration thereof), or is introduced in the vicinity of the cancer cells1) Initially, until a time is reached at which the Aurora B kinase inhibitor or Aurora A kinase inhibitor is cleared or depleted from the patient (t)2) Administration of the Aurora B kinase inhibitor or Aurora a kinase inhibitor to cancer cells occurs continuously. As a result, if TTfields are set at t1And t2To cancer cells, the application will be simultaneous with at least a portion of the application.
Although the present invention has been disclosed with reference to certain embodiments, numerous modifications, alterations and changes to the described embodiments are possible without departing from the sphere and scope of the present invention, as defined in the appended claims. Accordingly, it is intended that the invention not be limited to the described embodiments, but that it have the full scope defined by the language of the following claims, and equivalents thereof.
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