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PDF file, 42KB, Pharmacokinetic profiles of plasma and OCI-LY19-Luc tumor tissue in mice treated with a single oral dose of MLN0905.

PDF file, 45KB, MLN0905 yields significant anti-tumor responses in mice bearing subcutaneous OCI LY-19-Luc tumors.

PDF file, 43KB, Evaluating pHisH3 in OCI LY-19-Luc and OCI LY-10 cells following MLN0905 treatment.

Diffuse large B-cell lymphoma (DLBCL) is the most common of the non–Hodgkin lymphomas, accounting for up to 30% of all newly diagnosed lymphoma cases. Current treatment options for this disease are effective, but not always curative; therefore, experimental therapies continue to be investigated. We have discovered an experimental, potent, and selective small-molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL cell lines. In our report, we explored the pharmacokinetic, pharmacodynamic, and antitumor properties of MLN0905 in DLBCL xenograft models grown in mice. These studies indicate that MLN0905 modulates the pharmacodynamic biomarker phosphorylated histone H3 (pHisH3) in tumor tissue. The antitumor activity of MLN0905 was evaluated in three human subcutaneous DLBCL xenograft models, OCI LY-10, OCI LY-19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant antitumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The antitumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI LY-19) model to better mimic human DLBCL disease. In the disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with a standard-of-care agent, rituximab, in the disseminated OCI LY-19 xenograft model. Combining rituximab and MLN0905 provided both a synergistic antitumor effect and a synergistic survival advantage. Our findings indicate that PLK1 inhibition leads to pharmacodynamic pHisH3 modulation and significant antitumor activity in multiple DLBCL models. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anticancer agents in the clinic. Mol Cancer Ther; 11(9); 2045–53. ©2012 AACR.

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PDF file, 44KB, Evaluating cell viability in DLBCL cell lines following MLN0905 treatment.

PDF file, 53KB, MLN0905 yields significant anti-tumor responses in mice bearing subcutaneous OCI LY-10 tumors.

PDF file, 48KB, Plasma and OCI LY-19-Luc tumor pharmacokinetic parameters following a single oral dose of MLN0905 in CB-17 SCID mice.

Purpose: Pediatric glioblastoma multiforme (pGBM) is a highly aggressive tumor in need of novel therapies. Our objective was to demonstrate the therapeutic efficacy of MLN8237 (alisertib), an orally available selective inhibitor of Aurora A kinase (AURKA), and to evaluate which in vitro model system (monolayer or neurosphere) can predict therapeutic efficacy in vivo.Experimental Design: AURKA mRNA expressions were screened with qRT-PCR. In vitro antitumor effects were examined in three matching pairs of monolayer and neurosphere lines established from patient-derived orthotopic xenograft (PDOX) models of the untreated (IC-4687GBM), recurrent (IC-3752GBM), and terminal (IC-R0315GBM) tumors, and in vivo therapeutic efficacy through log rank analysis of survival times in two models (IC-4687GBM and IC-R0315GBM) following MLN8237 treatment (30 mg/kg/day, orally, 12 days). Drug concentrations in vivo and mechanism of action and resistance were also investigated.Results: AURKA mRNA overexpression was detected in 14 pGBM tumors, 10 PDOX models, and 6 cultured pGBM lines as compared with 11 low-grade gliomas and normal brains. MLN8237 penetrated into pGBM xenografts in mouse brains. Significant extension of survival times were achieved in IC-4687GBM of which both neurosphere and monolayer were inhibited in vitro, but not in IC-R0315GBM of which only neurosphere cells responded (similar to IC-3752GBM). Apoptosis-mediated MLN8237 induced cell death, and the presence of AURKA-negative and CD133+ cells appears to have contributed to in vivo therapy resistance.Conclusions: MLN8237 successfully targeted AURKA in a subset of pGBMs. Our data suggest that combination therapy should aim at AURKA-negative and/or CD133+ pGBM cells to prevent tumor recurrence. Clin Cancer Res; 24(9); 2159–70. ©2018 AACR.

Supplementary Figure S1-S3 Figure S1. Anti-proliferative effects of MLN8237 on pGBM cell lines. Paired monolayer (Mono) and neurosphere (NS) of IC-4687GBM, IC-3752GBM and ICR0315GBM were seeded at 2,000 cells/well and exposed to MLN8237 (1 - 4,000 nM) and anti-proliferative effect was assessed by staining with 250 µg/mL of MTT for 4 hrs at day 11. Viable cells were stained with dark colored intracellular crystal. (Bar=100 µm) Figure S2. FCM analysis of cell cycle distribution in vitro and in vivo. A. In vitro analysis was performed in paired monolayer (Mono) and neurosphere (NS) cells of IC-4687GBM, IC-3752GBM and IC-R0315GBM. Both dose-responses (on day 7) (left panel) and time-course change (at 62.5 nM) (right panel) were examined and presented as percentages of G0 /G1 , S and G2 /M phase in alive cells. € P

Purpose: Pediatric glioblastoma multiforme (pGBM) is a highly aggressive tumor in need of novel therapies. Our objective was to demonstrate the therapeutic efficacy of MLN8237 (alisertib), an orally available selective inhibitor of Aurora A kinase (AURKA), and to evaluate which in vitro model system (monolayer or neurosphere) can predict therapeutic efficacy in vivo. Experimental Design: AURKA mRNA expressions were screened with qRT-PCR. In vitro antitumor effects were examined in three matching pairs of monolayer and neurosphere lines established from patient-derived orthotopic xenograft (PDOX) models of the untreated (IC-4687GBM), recurrent (IC-3752GBM), and terminal (IC-R0315GBM) tumors, and in vivo therapeutic efficacy through log rank analysis of survival times in two models (IC-4687GBM and IC-R0315GBM) following MLN8237 treatment (30 mg/kg/day, orally, 12 days). Drug concentrations in vivo and mechanism of action and resistance were also investigated. Results: AURKA mRNA overexpression was detected in 14 pGBM tumors, 10 PDOX models, and 6 cultured pGBM lines as compared with 11 low-grade gliomas and normal brains. MLN8237 penetrated into pGBM xenografts in mouse brains. Significant extension of survival times were achieved in IC-4687GBM of which both neurosphere and monolayer were inhibited in vitro, but not in IC-R0315GBM of which only neurosphere cells responded (similar to IC-3752GBM). Apoptosis-mediated MLN8237 induced cell death, and the presence of AURKA-negative and CD133+ cells appears to have contributed to in vivo therapy resistance. Conclusions: MLN8237 successfully targeted AURKA in a subset of pGBMs. Our data suggest that combination therapy should aim at AURKA-negative and/or CD133+ pGBM cells to prevent tumor recurrence. Clin Cancer Res; 24(9); 2159–70. ©2018 AACR.

RATIONALE Stepwise preparation of calibration standards and quality controls (QCs) is one of the most routine and laborious steps in bioanalysis. An alternative non-contact dispenser using low picoliter digitized dispensing technology is evaluated for its application in non-stepwise preparation of calibration curve and QCs in bioanalysis. METHODS Fluorescein was initially used to assess the accuracy and precision of dispense volumes with fluorescent measurement. Various concentrations of MX-1, an in-house proprietary small molecule compound, in neat solution and in dog plasma were prepared manually with calibrated pipettors and digitally by the digital dispenser. The plasma samples were extracted by protein precipitation. The resultant extracted samples and neat solutions of MX-1 were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using an electrospray ionization (ESI) source in positive ion mode with selected reaction monitoring (SRM) of the mass transitions. RESULTS In the three-day precision and accuracy assessment of dispensing volumes between 13 pL to 411.2 nL, the intra-day precision and accuracy ranged from 1.4% to 10.3% and –12.7% to 12.8%, respectively. The inter-day precision and accuracy ranged from 3.5% to 7.8% and –6.6% to 10.4%, respectively. For real analysis of in vivo study samples, all 49 samples analyzed showed a less than 5% difference between calibrations with digital and manual curve preparations. The resultant pharmacokinetic (PK) parameters were physiologically comparable as well. CONCLUSIONS Using the digitized picoliter dispensing technology, high-speed automated precise and accurate dispense of a wide range of volumes can be achieved and tests for bioanalytical standards and QC preparations passed the stringent criteria set forth for regulated bioanalysis using LC/MS/MS-based technology. The digital dispenser has been found to be a useful tool in drug discovery for automatically preparing standards and QCs in seconds with low consumption of stock solutions and blank matrices. Copyright © 2014 John Wiley & Sons, Ltd.

Stepwise preparation of calibration standards and quality controls (QCs) is one of the most routine and laborious steps in bioanalysis. An alternative non-contact dispenser using low picoliter digitized dispensing technology is evaluated for its application in non-stepwise preparation of calibration curve and QCs in bioanalysis.Fluorescein was initially used to assess the accuracy and precision of dispense volumes with fluorescent measurement. Various concentrations of MX-1, an in-house proprietary small molecule compound, in neat solution and in dog plasma were prepared manually with calibrated pipettors and digitally by the digital dispenser. The plasma samples were extracted by protein precipitation. The resultant extracted samples and neat solutions of MX-1 were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using an electrospray ionization (ESI) source in positive ion mode with selected reaction monitoring (SRM) of the mass transitions.In the three-day precision and accuracy assessment of dispensing volumes between 13 pL to 411.2 nL, the intra-day precision and accuracy ranged from 1.4% to 10.3% and -12.7% to 12.8%, respectively. The inter-day precision and accuracy ranged from 3.5% to 7.8% and -6.6% to 10.4%, respectively. For real analysis of in vivo study samples, all 49 samples analyzed showed a less than 5% difference between calibrations with digital and manual curve preparations. The resultant pharmacokinetic (PK) parameters were physiologically comparable as well.Using the digitized picoliter dispensing technology, high-speed automated precise and accurate dispense of a wide range of volumes can be achieved and tests for bioanalytical standards and QC preparations passed the stringent criteria set forth for regulated bioanalysis using LC/MS/MS-based technology. The digital dispenser has been found to be a useful tool in drug discovery for automatically preparing standards and QCs in seconds with low consumption of stock solutions and blank matrices.

Column-related carryover affects both accuracy and precision in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In this work, a novel straightforward dynamic flush method to reduce the column-related carryover was developed by alternating the column flow direction of liquid chromatographic separation with a Valco switching valve and pristine instrument control software. By alternating the column flow direction, a fresh inlet is always in line to accommodate sample injection and stacking. In addition, the contaminated column inlet from the previous run is switched to the outlet position for a flush with a gradient during the next sample run. In this way, the column-related carryover can be reduced effectively without additional blank runs. It also minimizes the carryover risk between the adjacent unknown samples. The column-related carryover of the tested "sticky" compounds was reduced by 52.3-94.4% compared with the non-dynamic flush method under the same experimental conditions. The performance and reproducibility of high-performance liquid chromatography (HPLC) separation in terms of the retention time shift and peak shape are not compromised under the dynamic flush even after over 300 consecutive injections. The described novel method is simple and easy to implement for compounds with column-related carryover.

Diffuse large B-cell lymphoma (DLBCL) is the most common of the non–Hodgkin lymphomas, accounting for up to 30% of all newly diagnosed lymphoma cases. Current treatment options for this disease are effective, but not always curative; therefore, experimental therapies continue to be investigated. We have discovered an experimental, potent, and selective small-molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL cell lines. In our report, we explored the pharmacokinetic, pharmacodynamic, and antitumor properties of MLN0905 in DLBCL xenograft models grown in mice. These studies indicate that MLN0905 modulates the pharmacodynamic biomarker phosphorylated histone H3 (pHisH3) in tumor tissue. The antitumor activity of MLN0905 was evaluated in three human subcutaneous DLBCL xenograft models, OCI LY-10, OCI LY-19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant antitumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The antitumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI LY-19) model to better mimic human DLBCL disease. In the disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with a standard-of-care agent, rituximab, in the disseminated OCI LY-19 xenograft model. Combining rituximab and MLN0905 provided both a synergistic antitumor effect and a synergistic survival advantage. Our findings indicate that PLK1 inhibition leads to pharmacodynamic pHisH3 modulation and significant antitumor activity in multiple DLBCL models. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anticancer agents in the clinic. Mol Cancer Ther; 11(9); 2045–53. ©2012 AACR.

This article describes the discovery of a series of potent inhibitors of Polo-like kinase 1 (PLK1). Optimization of this benzolactam-derived chemical series produced an orally bioavailable inhibitor of PLK1 (12c, MLN0905). In vivo pharmacokinetic-pharmacodynamic experiments demonstrated prolonged mitotic arrest after oral administration of 12c to tumor bearing nude mice. A subsequent efficacy study in nude mice achieved tumor growth inhibition or regression in a human colon tumor (HT29) xenograft model.

PLK1 is a serine/threonine mitotic kinase that plays a key role in mitotic cell cycle progression, and its over-expression has been linked to poor patient prognosis. We have discovered a potent and selective small molecule inhibitor of PLK1, MLN0905, which reduces cell viability and inhibits cell proliferation in a broad range of human tumor cells. We explored the pharmacokinetic, pharmacodynamic, and anti-tumor properties on MLN0905 in human xenograft models grown in rodents. MLN0905 rapidly and extensively distributed to xenograft tumor with a high tumor-to-plasma exposure ratio. In human xenograft tumor tissue, MLN0905 modulates the pharmacodynamic biomarker phospho-Histone H3 (in a dose dependent fashion), enabling us to track pathway modulation in vivo. MLN0905 demonstrated robust anti-tumor activity (partial and complete responses) in a variety of solid and hematological human xenograft models, including cancers derived from colon (HCT-116, HT29), NSCLC (Calu-6), Ovarian (SKOV3), and Lymphoma (OCI-LY19, OCI-LY10, and the primary lymphoma PHTX-22L). Significant anti-tumor activity was observed when dosing on either a continuous (daily) or intermittent schedule, underscoring the dosing flexibility with this compound. In the SKOV3 and Calu-6 xenograft models, MLN0905 yielded a synergistic anti-tumor response when combined with the standard of care therapy Taxane. This is the first report of a PLK1 inhibitor synergizing with taxane therapy in vivo. In summary, our findings indicate MLN0905 has good drug-like pharmacokinetic properties, modulates the biomarker phospho-Histone H3, and yields significant anti-tumor activity in multiple xenograft models. These preclinical data support further evaluating MLN0905 as a novel anti-cancer agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3573. doi:10.1158/1538-7445.AM2011-3573

Abstract 3922 Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas, accounting for up to 30 percent of newly diagnosed cases. Current treatment options for this disease are effective, but not always curative; therefore experimental therapies are being investigated. Diffuse large B-cell lymphoma cells typically over-express the serine/threonine mitotic kinase PLK1, which plays a key role in mitotic cell cycle progression, and has been linked to poor patient prognosis. We have discovered a potent and selective small molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL lines. We explored the pharmacokinetic, pharmacodynamic, and anti-tumor properties on MLN0905 in mouse models harboring human DLBCL disease. MLN0905 has drug-like pharmacokinetic properties and an acceptable toxicity profile making it a good clinical candidate. In human xenograft tumor tissue, MLN0905 modulates the pharmacodynamic biomarker phospho-Histone H3 (in a dose dependent fashion), enabling us to track pathway modulation in vivo. The anti-tumor activity of MLN0905 was evaluated in three human subcutaneous xenograft models OCI-LY10, OCI-LY19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant anti-tumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The anti-tumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI-LY19) setting to better mimic DLBCL in humans. In this disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with Rituximab in the disseminated OCI-LY19 model. Combining Rituximab and MLN0905 provided both a synergistic anti-tumor effect and a synergistic survival advantage. Our findings indicate for the first time that PLK1 inhibition leads to pharmacodynamic pH3 modulation and significant anti-tumor activity in multiple models of DLBCL. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anti-cancer agents in the clinic. Disclosures: Shi: Millennium: The Takeda Oncology Company: Employment. Lasky:Millennium: The Takeda Oncology Company: Employment. Shinde:Millennium: The Takeda Oncology Company: Employment. Duffey:Millennium: The Takeda Oncology Company: Employment. Stringer:Millennium: The Takeda Oncology Company: Employment. Qian:Millennium: The Takeda Oncology Company: Employment. Liao:Millennium: The Takeda Oncology Company: Employment. Liu:Millennium: The Takeda Oncology Company: Employment. Rao:Millennium: The Takeda Oncology Company: Employment. Vos:Millennium: The Takeda Oncology Company: Employment. D'Amore:Millennium: The Takeda Oncology Company: Employment. Hyer:Millennium: The Takeda Oncology Company: Employment.