WO2022270621A1 - 骨肉腫及び神経膠腫の転移と増殖を抑制する新規治療薬 - Google Patents

骨肉腫及び神経膠腫の転移と増殖を抑制する新規治療薬 Download PDF

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WO2022270621A1
WO2022270621A1 PCT/JP2022/025309 JP2022025309W WO2022270621A1 WO 2022270621 A1 WO2022270621 A1 WO 2022270621A1 JP 2022025309 W JP2022025309 W JP 2022025309W WO 2022270621 A1 WO2022270621 A1 WO 2022270621A1
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lpar1
osteosarcoma
cells
expression
glioma
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聡 高木
量平 片山
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Japanese Foundation for Cancer Research
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Definitions

  • the present invention relates to a novel therapeutic drug that suppresses the metastasis and growth of osteosarcoma and glioma.
  • it relates to therapeutic agents for osteosarcoma and glioma that target LPAR1.
  • Osteosarcoma is a malignant tumor that forms in the bone, and is a histologically and genetically heterogeneous tumor. Osteosarcoma is the most frequently occurring malignant tumor that directly develops in the bone, but the frequency of occurrence is as low as 1 to 3 per 1,000,000 people, and it is said to be a rare cancer. However, since the most common age of onset is children and the AYA generation, and large amounts of chemotherapeutic agents are administered before and after surgery, the effects on growth and fertility, which are problems specific to the AYA generation, and new problems after adulthood. There is concern about the onset of cancer.
  • Osteosarcoma is said to first cause chromosomal instability due to the occurrence of TP53 or RB1 gene mutations, and then to develop polyclonal tumors with metastasis due to the occurrence of other oncogenic mutations (non Patent Documents 1 and 2). Therefore, it is a highly heterogeneous tumor that makes it difficult to treat.
  • glioma is a malignant brain tumor that develops from glial cells, and the most malignant glioma is called glioblastoma.
  • various symptoms such as paralysis of limbs, visual field and visual impairment occur depending on the site where the tumor occurs and grows.
  • Glioma is considered to be one of the tumors with poor prognosis because it is difficult to completely remove it by surgery when tumor cells invade normal brain tissue.
  • glioblastoma which is the most malignant type of glioma, causative gene mutations such as IDH and p53 gene mutations have been reported, but no molecular targets linked to treatment have been reported.
  • the treatment for glioma is surgical treatment to remove as much of the tumor as possible while preserving motor and language functions.
  • radiotherapy and chemotherapy are performed to prevent recurrence.
  • Treatment with temozolomide or bevacizumab administration is performed in combination with radiotherapy, but there are still many cases of recurrence. Therefore, the development of more effective therapeutic agents, particularly molecular-targeted agents, is desired.
  • An object of the present invention is to provide effective therapeutic agents and therapeutic methods for osteosarcoma and glioma.
  • it is an object to provide a therapeutic drug that suppresses metastasis and tumor growth.
  • Osteosarcoma metastasizes to the lung at a relatively high rate, and there is no effective treatment for patients with lung metastasis. If metastasis can be suppressed, the prognosis can be improved.
  • development of a therapeutic drug that suppresses tumor growth is expected. Since no effective molecular-targeted drugs have been developed for osteosarcoma and glioma, discovering molecules that are specifically expressed in these tumors and suppressing their function could lead to the development of new therapeutic drugs. It becomes possible.
  • LPAR1 expression is high in osteosarcoma. Furthermore, since LPAR1 expression is also high in glioma, the effects of LPAR1 antagonists were analyzed. It becomes possible to provide an effective therapeutic agent for osteosarcoma and glioma, for which there has been no effective therapeutic agent until now.
  • the present invention relates to the following pharmaceutical composition, method for assisting prognosis diagnosis, and method for selecting a drug.
  • the LPAR1 antagonist is ONO-7300243, BMS-986020, Ki16425, ONO-3080573, ONO-9780307, ONO-9910539, Ki16198, AM095, AM966, SAR100842, BMS-986278 and analogs thereof;
  • a method for assisting prognosis in patients with osteosarcoma comprising analyzing LPAR1 expression in diseased tissue obtained from the patient, and determining that high LPAR1 expression indicates a high risk of metastasis.
  • a method of selecting a drug comprising analyzing LPAR1 expression in diseased tissue, and determining that an LPAR1 antagonist is to be administered when LPAR1 expression is detected.
  • a screening method for a pharmaceutical composition for the treatment of osteosarcoma or glioma comprising the steps of contacting a candidate substance with cells in which LPAR1 expression is observed, and measuring changes in LPAR1 expression. screening method.
  • A shows the platelet aggregation ability of various osteosarcoma cells.
  • B) and C show that platelet releasates from platelets enhance the infiltration ability of osteosarcoma cells.
  • B) is a micrograph showing invaded cells, and
  • C) is a diagram showing the number of invaded cells.
  • A) shows an analysis of LPAR1 mRNA expression levels using RNA sequence data from The Cancer Genome Atlas (TCGA) and TARGET.
  • FIG. 1 shows the results of analysis of LPAR1 mRNA expression in osteosarcoma cell lines by qPCR, and (C) analysis of protein expression by Western blotting.
  • D shows protein expression of LPAR1 in osteosarcoma patient-derived xenografts.
  • A shows an ELISA analysis of the release of LPA from platelets by adding MG-63 osteosarcoma cells to a platelet suspension.
  • (B) is a fluorescence micrograph showing the analysis of the localization of phosphorylated AKT and F-actin in MG-63 osteosarcoma cells stimulated with LPA.
  • (C) shows the effect of LPA on cell migration ability in various osteosarcoma cells and that the effect of LPA is canceled by the LPAR antagonist Ki16425; Cancels the effect of platelet releasate.
  • (E) shows an analysis of the effect of Ki16425 on the growth of osteosarcoma cells. A diagram showing that the LPA-LPAR1 interaction is important for osteosarcoma cell invasion.
  • (A) shows the establishment of LPAR1 knockout cells using MG-63 osteosarcoma cells.
  • (B) and (C) show the effect of LPA on the invasion ability of LPAR1 knockout cells.
  • (D) Effect of platelet releasate on invasion ability in LPAR1 knockout cells.
  • a diagram showing that LPAR1 has an important role in lung metastasis of osteosarcoma is the luminescence of MG-63 osteosarcoma cells introduced with Akaluc luciferase (MG-63/Akaluc/sgCTRL) and LPAR1 knockout MG-63 osteosarcoma cells (MG-63/Akaluc/sgLPAR#1); B) shows the cell proliferation ability.
  • MG-63/Akaluc/sgCTRL and MG-63/Akaluc/sgLPAR#1 cells were injected intravenously, and AkaLamine was administered 0 days later (on the day of injection) and 7 days later.
  • a diagram showing that LPAR1 antagonists suppress osteosarcoma lung metastasis (A) is a diagram showing the procedure of the experiment. (B) and (C) show the results of in vivo imaging analysis showing that administration of the LPAR1 antagonist ONO-7300243 and injection of HuO9/Akaluc osteosarcoma cells suppress lung metastasis. A diagram showing that LPAR1 is involved in the proliferation of osteosarcoma cells. (A) is a diagram showing that cell proliferation is suppressed in clone lines obtained by knocking out LPAR1 of osteosarcoma cells MG-63 and G-292 clone A141B1 (G-292) cells.
  • FIG. 4 shows that administration of an LPAR1 antagonist exerts an antitumor effect in an osteosarcoma xenograft model.
  • A shows the LPAR1 administration schedule, and (B) shows changes in tumor volume over time.
  • FIG. 1 shows that the motility of glioblastoma cells is updated in the presence of LPA.
  • A LPAR1 antagonists reduce glioblastoma cell viability.
  • B LPAR1 knockdown reduces glioblastoma cell viability.
  • FIG. 1 shows that administration of an LPAR1 antagonist exerts an antitumor effect in a glioblastoma xenograft model.
  • A) shows the LPAR1 antagonist administration schedule, and (B) shows the time course of relative tumor volume.
  • the present inventors found that osteosarcoma cells have a high ability to aggregate platelets, and found that platelet releasates released from activated platelets enhance the invasive ability of osteosarcoma cells. Further analysis revealed that lysophosphatidic acid (LPA) released from activated platelets functions as a mediator that enhances the invasive ability of osteosarcoma. In addition, the LPA receptor LPAR1 was found to be significantly expressed in osteosarcoma cells and patient-derived xenografts, suggesting that the LPA-LPAR1 interaction is involved in distant metastasis of osteosarcoma. concluded.
  • LPA lysophosphatidic acid
  • LPAR1 antagonists specifically ONO-7300243, inhibited lung metastasis, and BMS986020 inhibited tumor growth.
  • ONO-7300243 the growth of cells in which LPAR1 expression is knocked out or knocked down is suppressed and apoptosis is induced.
  • osteosarcoma the growth of osteosarcoma can be suppressed by suppressing the expression of LPAR1.
  • LPAR1 is also highly expressed in glioma, they analyzed the effects of LPAR1 antagonists and found that they exhibit antitumor effects.
  • any LPAR1 antagonist that suppresses metastasis and proliferation may be used.
  • low-molecular-weight compounds include ONO-3080573, ONO-9780307, ONO-9910539, ONO-9910539, Ki16198, AM095, AM966, SAR100842, BMS-986278 and analogues thereof.
  • Antibodies and polypeptides that bind to LPAR1 and inhibit its function can also be used.
  • LPAR1 expression itself may be suppressed by nucleic acids such as siRNA, antisense RNA, shRNA and miRNA.
  • LPAR1 antagonists not only existing LPAR1 antagonists, but also compounds that suppress LPAR1 expression may be screened and used.
  • a compound that suppresses LPAR1 expression can be screened by adding a candidate substance to the culture medium of LPAR1-expressing cells, specifically osteosarcoma cells and glioma cells, and using a decrease in LPAR1 expression as an index. .
  • LPAR1 expression is deeply involved in bone metastasis, it is possible to determine the risk of distant metastasis, that is, prognosis, by measuring LPAR1 expression in tumor tissue. . Specifically, LPAR1 expression in osteosarcoma tissue obtained by surgery or biopsy can be detected, and when LPAR1 expression is high, it can be determined that the possibility of distant metastasis is high. When the possibility of distant metastasis is high, preventive measures such as administering an LPAR1 antagonist can prevent distant metastasis. In addition, if LPAR1 expression is high not only in osteosarcoma and glioma, but also in other cancer types, LPAR1 inhibitors are considered to be effective. , to determine which patients should be treated with LPAR1 inhibitors.
  • Platelets were obtained from healthy subjects who had not taken antiplatelet drugs for at least 10 days before blood collection, and were isolated by a conventional method. Isolated platelets were washed at 2 ⁇ 10 8 /mL in modified Tyrode's buffer (137 mM NaCl, 11.9 mM NaHCO 3 , 0.4 mM Na 2 HPO 4 , 2.7 mM KCl, 1.1 mM MgCl 2 , 5.6 mM glucose), 1.2 mM CaCl 2 was added and used for platelet aggregation assay. Cell suspension (10 ⁇ L of 5 ⁇ 10 6 cells/mL cell suspension) or PBS was added to 200 ⁇ L of platelet suspension, and analysis was performed at 37° C. for 30-60 minutes. Platelet aggregation was measured using a platelet aggregometer (MCM HEMA Tracer 313M, SSR Engineering) (Fig. 1(A)).
  • MCM HEMA Tracer 313M, SSR Engineering Fig. 1(A)
  • All osteosarcoma cells had a higher ability to activate platelets than the lung adenocarcinoma cell A549 used as a negative control.
  • Collagen (10 ⁇ g/mL, 10 ⁇ L added) is a positive control.
  • the reaction solution subjected to the platelet aggregation assay was collected, 0.5 ⁇ M prostaglandin I2 was added and centrifuged, and the centrifugal supernatant was collected. Centrifuge supernatant was used as platelet releasate containing bioactive molecules released from activated platelets.
  • Osteosarcoma cells MG-63, HuO9, and G-292 were each seeded at 1.5 ⁇ 10 5 cells/0.5 mL in the insert (upper chamber) of Matrigel invasion chamber (Corning), and in the lower chamber. placed the platelet releasate and allowed to stand at 37° C. for 22-24 hours, then completely wiped the cells on the top surface of the insert, fixed with 4% paraformaldehyde, and removed the cells on the bottom surface of the membrane of the insert, that is, infiltrated. Cells containing the cells were stained with 1% crystal violet (Fig. 1(B)). The number of invading cells was counted and the relative cell ratio is shown in FIG. 1(C). In both osteosarcoma cells, the number of invading cells was significantly increased when platelet releasate was added.
  • Non-Patent Documents 9 and 10 It has been reported from mass spectrometric analysis that lipid mediators such as TxA2, S1P, and LPA are released from activated platelets (Non-Patent Documents 9 and 10). Although data are not shown here, the expression of these lipid mediator receptors (TBA2R, S1PR1-5, LPAR1-6) in various tumors was examined using RNA sequence data from the TCGA and TARGET databases. LPAR1, LPAR6, S1PR1 and S1PR3 were found to be highly expressed in osteosarcoma. Furthermore, when various cancer types were compared, it was confirmed that LPAR1 was most highly expressed in osteosarcoma (OS) and sarcoma (SARC) (Fig. 2(A)).
  • OS osteosarcoma
  • SARC sarcoma
  • TCGA and TARGET are data of tumor tissue, they also include data of stromal cells, epithelial cells, immune cells, etc., in addition to tumor cells. Therefore, although data are not shown here, examination was performed using the Cancer Cell Line Encyclopedia (CCLE) database, which reflects the gene expression of cancer cell lines. LPAR1 expression in several osteosarcoma cell lines showed significantly higher expression compared to the Ewing's sarcoma family tumor, chondrosarcoma.
  • CCLE Cancer Cell Line Encyclopedia
  • ELISA human lysophosphatidic acid ELISA kit, Cusabio
  • ELISA human lysophosphatidic acid ELISA kit, Cusabio
  • 200 ⁇ L of platelet suspension and 5 ⁇ 10 4 MG-63 cells, or MG-63 cells alone were allowed to stand at 37° C. for 30 minutes.
  • Prostaglandin I2 was added to 0.5 ⁇ M, and after centrifugation, LPA was detected by ELISA.
  • FIG. 3(A) LPA was not detected in the supernatant of MG-63 cells alone, whereas the mixture of MG-63 cells and platelets contained a large amount of LPA. It was shown that It was also shown that LPA was significantly released compared to the centrifugal supernatant of non-activated platelets (control).
  • LPA receptors are G protein-coupled receptors, and LPAR1 activates three G proteins, G ⁇ i /0 , G ⁇ q /11 , and G ⁇ 12/13, and activates signal transduction systems including the PI3K/AKT pathway. known to do.
  • MG-63 cells were cultured overnight in serum-free MEM medium, treated with 100 nM LPA for 4 hours, and treated with anti-phosphorylated AKT antibody (an antibody that detects phosphorylation of S473), rhodamine-labeled phalloidin (anti-F actin binding), immunostaining was performed using Hoechst 33342 (staining the nucleus), and observed under a microscope (Fig. 3(B)).
  • Ki16425 has an inhibitory effect on all of LPAR1, LPAR2, and LPAR3, although the degree of inhibition differs.
  • LPAR1 knockout cells were established from MG-63 cells using the CRISPR/CAS9 system. The established cells are called sgLPAR1#1-3. Loss of LPAR1 expression was confirmed by Western blotting for all sgLPAR1#1 to 3 (FIG. 4(A)).
  • knockout cells were used to analyze invasion ability. 1 ⁇ 10 5 of each cell was seeded in the insert, 10 nM LPA was added to the bottom layer of the Matrigel invasion chamber, fixed after 22-24 hours, stained with crystal violet, and the number of cells was counted ( 4(B) and (C)). All knockout cells had significantly reduced invasive ability compared to the control.
  • mice Female SCID-beige mice (CB-lgh-1b/GbmsTac-Prkdc scid -Lyst bg N7, Charles River Laboratories Japan, Inc.) were injected with 1 ⁇ 10 6 MG-63/Akaluc/sgCTRL or MG- 63/Akaluc/sgLPAR#1 was intravenously injected, and 3 hours and 7 days later, 100 ⁇ L of 5 mM AkaLumine-HCL was intraperitoneally injected, and bioluminescence imaging (BLI) was performed in vivo. BLI was performed using an IVIS imaging system (PerkinElmer). FIG. 5(C) shows the image, and (D) shows the total flux measured by IVIS.
  • IVIS imaging system PerkinElmer
  • ONO-7300243 (Cayman Chemical), which is one of the LPAR1 inhibitors.
  • ONO-7300243 was tested at different doses of 10 mg/kg and 30 mg/kg.
  • Total flux measured by IVIS showed no significant difference in any group on the day of cell injection, but a significant difference was observed between the solvent-administered group and the 30 mg/kg-administered group one day after the injection. A significant difference was also observed between the 10 mg/kg administration group and the 30 mg/kg administration group after 30 days, and a dose-dependent decrease in osteosarcoma cells trapped in the lung was observed.
  • LPAR1 was knocked down in MG-63 and HuO9 cells using LPAR-targeted siRNA (Dharmacon). Since the knockdown of LPAR1 was confirmed to increase the apoptosis marker truncated PARP (Cl-PARP), it is considered that the knockdown of LPAR1 induces apoptosis. Therefore, not only the LPAR1 antagonist but also the suppression of LPAR1 expression by siRNA induces apoptosis of osteosarcoma cells and is recognized to exert an antitumor effect.
  • mice Female SCID-beige mice were subcutaneously transplanted with 8.2 ⁇ 10 5 osteosarcoma cells G-292 (Day 0), and the day after transplantation (Day 1), the LPAR1 antagonist BMS-986020 was administered at 30 mg/kg for 5 consecutive days. , was orally administered on a dosing schedule of 2 days off (Fig. 8(A)). Tumor growth was analyzed by measuring the major axis and minor axis of the tumor and calculating the tumor volume as major axis (mm) ⁇ [minor axis (mm)] 2 ⁇ 1/2.
  • the tumor volume was measured from day 14 after tumor transplantation, and a significant tumor growth inhibitory effect was observed from day 14 in the LPAR1 antagonist administration group (Fig. 8 (B)). Since the LPAR1 antagonist was found to have an anti-tumor effect, it can be expected not only to suppress lung metastasis of osteosarcoma but also to lead to remission.
  • LPAR1 is highly expressed in glioblastoma (GBM) and glioma (LGG) following osteosarcoma and sarcoma. Therefore, the effect of suppressing metastasis and cell proliferation by LPAR1 antagonists is also expected for glioma.
  • glioblastoma is the most malignant tumor among gliomas, so glioblastoma cell lines were used to analyze migration ability by adding LPA. It was analyzed whether the motility of glioblastoma cells was enhanced in the presence of LPA.
  • LPAR1 antagonists have an effect on the survival rate of glioblastoma cells. 1,500 to 3,000 human glioblastoma cells Onda7, YKG-1, 42-MG-BA were seeded in a 96-well plate, treated with varying concentrations of LPAR1 antagonists Ki16425, BMS-986020, and ONO-7300243 for 72 hours. Subsequent cell viability was measured by CellTiter-Groreagent (Fig. 10(A)). A decrease in cell viability was observed with any cell and any LPAR1 antagonist.
  • LPAR1 antagonists can be used for therapy as molecular targeted drugs.
  • LPAR1 antagonist has an antitumor effect.
  • 2 ⁇ 10 5 Onda7 cells transfected with Akaluc luciferase were intracranially implanted into female SCID-beige mice.
  • the LPAR1 antagonist BMS-986020 was orally administered at 50 mg/kg or 100 mg/kg according to a dosing schedule of continuous administration for 5 days followed by rest for 2 days (FIG. 11(A)).
  • the tumor volume was determined by quantifying the enzymatic activity of AkaLuc luciferase stably expressed in Onda7 cells using an IVIS imaging system (Fig. 11(B)).
  • LPAR1 is greatly involved in the metastasis mechanism of osteosarcoma. Furthermore, based on the elucidation of the mechanism of metastasis, a mouse model was used to demonstrate that administration of an LPAR1 antagonist can suppress metastasis. As described above, osteosarcoma metastasizes to the lungs at a high rate, there is no effective treatment for patients with osteosarcoma that has metastasized to the lungs, and respiratory failure due to lung metastasis is the cause of most patient deaths. Taking this into consideration, the ability to suppress lung metastasis of osteosarcoma by an LPAR1 antagonist is extremely useful for providing a new therapeutic method for osteosarcoma and improving prognosis.
  • LPAR1 inhibition inhibits the proliferation of osteosarcoma cells and induces apoptosis. That is, administration of an LPAR1 antagonist suppresses not only metastasis but also growth of osteosarcoma. Treatment of osteosarcoma by inhibiting the LPA-LPAR1 interaction is considered to be a new therapeutic method capable of inhibiting metastasis and growth of osteosarcoma.
  • LPAR1 antagonists are effective not only in osteosarcoma, but also in glioma, in which LPAR1 is highly expressed.
  • Glioma is a refractory tumor that is difficult to treat surgically depending on the site where it occurs, and it is difficult to completely remove tumor cells.
  • LPAR1 antagonists are also considered to be a new therapeutic option for glioma.

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