Aurora A (IC50 = 1.2 nM); Aurora B (IC50 = 396.5 nM)

Alisertib (MLN 8237) induces aberrant mitotic spindles in MM cells, mitotic accumulation, and senescence and death to prevent cell division. Tumor suppressor genes p21 and p27, as well as p53, are upregulated by aleritetib[1]. The enhanced affinity for ATP brought on by cofactor binding to Aurora A may be the cause of Alisertib's (MLN 8237) lower activity for the T217D/W277E Aurora A/TPX2 complex[2]. In various tumor cell lines, aleretitib (MLN 8237) suppresses cell growth with IC50s ranging from 15 to 469 nM[4].

Pharmacodynamic activity of Alisertib/MLN8237 in vivo: increased mitotic index, reduced bipolar mitotic spindles, and increased chromosome alignment abnormalities[4]
Alisertib/MLN8237 dosed orally at 3, 10, and 30 mg/kg in female nude mice bearing HCT-116 colon tumor xenografts resulted in significant bioavailability, as measured by plasma and tumor concentrations (Supplementary Fig. S4). A dose of 30 mg/kg on a once daily schedule was the maximum tolerated dose (MTD).[4]
Analysis of tumor tissue from HCT-116 xenografts treated with increasing doses of alisertib revealed a time-dependent and dose-dependent increase in the mitotic marker pHisH3, suggesting that alisertib inhibited Aurora A (Fig. 2A). The plasma concentration at the time the mitotic marker was declining was approximately 1 to 2 μmol/L, suggesting that this concentration is needed to inhibit AAK in vivo (Supplementary Fig. S4). Moreover, there was no inhibition of pHisH3 at concentrations of approximately 6 μmol/L showing a significant selectivity for Aurora A inhibition over Aurora B in vivo.[4]

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Alisertib/MLN8237 causes tumor growth inhibition in solid tumor xenograft models and regressions in in vivo models of lymphoma[4]
To determine the in vivo antitumor activity of alisertib, mice bearing solid and hematologic human tumor xenografts were administered increasing doses of alisertib. Figure 3A shows average tumor volumes in nude mice bearing subcutaneous HCT-116 tumors after 3 weeks of oral alisertib at 3, 10, or 30 mg/kg once daily. Alisertib treatment resulted in a dose-dependent TGI of 43.3%, 84.2%, and 94.7% for the 3, 10, and 30 mg/kg groups, respectively. The greatest antitumor response in this model was tumor stasis. All doses were well tolerated with the maximum body weight loss of 7.4% in the 30 mg/kg group.[4]

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In the xenograft-murine model of human-MM, alestertib (MLN 8237) (30 mg/kg, po) dramatically lowers tumor burden and improves overall survival[1]. In solid tumor xenograft models, alisertib (3-30 mg/kg; po; once daily for 3 weeks) inhibits the growth of tumors[4].

Protein Kinase Assays and Inhibitors[2]
Alisertib/MLN8237, VX-680, ZM447439 and MLN8054 were used. Chemical structures of these compounds are presented in Figure 1, panel A. To measure Aurora A activity, 25 ng (12.5 nM final concentration, Figure 1, panel B and Supplementary Figure S1) or 250 ng (125 nM final concentration, all other assays) of purified bacterially expressed Aurora A was assayed in the presence of the appropriate inhibitors, using Histone H3 as substrate for 20 min at 30 °C in the presence of 100 μM [γ-32P] ATP. For Aurora A/TPX2 assays, 50 ng of a TPX2 [1−43] peptide, representing a 2-fold molar excess over Aurora A, was included. The Aurora A/TPX2 complex was preformed in kinase reactions prior to subsequent addition of inhibitors and ATP. For Plk4 assays, 250 ng of bacterially expressed, purified His-tagged human catalytic domain (amino acids 1−269) was assayed in the presence of the appropriate inhibitors, using myelin basic protein (MBP) as substrate for 20 min at 30 °C in the presence of 100 μM [γ-32P] ATP. To assess Histone H3 and MBP phosphorylation, radiolabel incorporation was quantified by Cerenkov counting of phosphorylated substrates on p81 phosphocellulose paper, or by phosphorimager after SDS-PAGE. Each experiment was repeated at least three times, with similar results seen on each occasion. To determine the Km [ATP] value for Aurora A and mutants, nonlinear regression analysis was performed on data collated over a range of 1 and 200 μM of [γ-32P] ATP (specific activity 500 cpm pmol−1). Data analysis was performed using Prism software.

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Enzyme and cell-based assays to measure kinase inhibition[4]
Aurora A and Aurora B radioactive Flashplate enzyme assays and cell-based assays were conducted to determine the nature and degree of Alisertib/MLN8237-mediated inhibition in vitro, as described by Manfredi and colleagues. In the cell-based assays, Aurora A activity was determined by measuring autophosphorylation of Aurora A on threonine 288, whereas Aurora B activity was determined by measuring phosphorylation of histone H3 on serine 10 (pHisH3), in both cases, using high content imaging assays and as previously described. The inhibitory activity of 1 μmol/L Alisertib/MLN8237 was also tested against 205 kinases.

Measurement of cell viability and proliferation[1]
MM cell lines, CD138+ tumor cells purified from BM aspirates of patients with MM, and peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were seeded in triplicate 96-well plates in 100 μL complete media at a density of 20 × 104cells/well. MLN8237 was added to each well to give a range of concentrations (0.0001-4μM) in a final volume of 200 μL. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and cell proliferation was measured using 3[H]-thymidine incorporation at 24, 48, and 72 hours of incubation. The absorbance was measured at 570/630 nm by a spectrophotometer.
MM cells were incubated in 96-well plates, alone or in the presence of BM stroma cells, rhIL-6 (10 ng/mL), or rhIGF-1 (25 ng/mL), and then exposed to MLN8237 (0.0001-4μM) for 24, 48, and 72 hours. Cells were pulsed with 3[H]-thymidine (0.5 μCi) for the last 8 hours of incubation, harvested onto glass filters, and counted using LKB Betaplate scintillation counter.
MM cell lines were incubated with DMSO or MLN8237 (0.125-0.5μM) in combination with conventional anti-MM agents melphalan (2.5-5μM), doxorubicin (50-100nM), or dexamethasone (50-100nM); and with novel anti-MM agents bortezomib (2.5-5nM) or lenalidomide (0.5-1μM) for 72 hours. Cell viability was measured by MTT assay. The combination index (CI) was determined by isobologram analysis using CalcuSyn software, Version 2.0 (CI < 1 indicates synergistic effect; CI = 1, additive effect; and CI > 1, no significant combination effect).

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Detection of apoptosis and senescence[1]
Induction of cell death in MM cells triggered by MLN8237 was measured by fluorescein-conjugated annexin V and propidium iodide (PI) costaining. Cells were incubated with 0.5 to 1μM of MLN8237 or DMSO for 24 to 72 hours and stained with fluorescein isothiocyanate-annexin V and PI, according to the manufacturer's protocol. Apoptotic cells were determined by flow cytometric analysis using BDFACS-Canto II and FlowJo Version 7.0 software.
Induction of cell senescence was detected in MM1.S cells and OPM1 cells treated with 0.5μM of MLN8237 for 48 hours using the Senescence β-Galactosidase Staining Kit, according to the manufacturer's protocol. β-Galactosidase positive cells were visualized using a light microscope (original magnification ×20; Leica DMIL) at room temperature.

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Cell-cycle analysis[1]
MM cells were exposed to DMSO or 0.5 to 1μM of MLN8237 for 24 to 72 hours, permeabilized by 70% ethanol at −20°C, and incubated with 50 μg/mL PI and 20 units/mL RNase-A. DNA content was analyzed by flow cytometry using BDFACS-Canto II and FlowJo software.

Animal/Disease Models: Nude mice bearing HCT-116 colon tumor xenograft[4]
Doses: 3, 10, or 30 mg/kg
Route of Administration: Po; one time/day for 3 weeks
Experimental Results: Resulted in a dose-dependent TGI (tumor growth inhibition) of 43.3%, 84.2%, and 94.7% for the 3, 10, and 30 mg/kg groups, respectively.

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In vivo efficacy studies[4]
Nine in vivo tumor models of different histologies grown subcutaneously or disseminated were developed in either nude or severe combined immunodeficient (SCID) mice. The methods for all in vivo studies have been described previously (32), with the exception of the lymphoma tumor models described below. All mice had access to food and water ad libitum and were housed and handled in accordance with the Guide for the Care and Use of Laboratory Animals and Millennium Institutional Animal Care and Use Committee Guidelines. Mice for all models were dosed orally with Alisertib/MLN8237 for approximately 3 weeks and tumor growth inhibition (TGI) was calculated on the last day of treatment. For all studies, Alisertib/MLN8237 was formulated in 10% 2-hydroxypropyl-β-cyclodextrin and 1% sodium bicarbonate and was dosed orally by gavage on a once-daily or twice-daily schedule.
The cell lines OCI-LY7-Luc, OCI-LY19-Luc, and WSU-DLCL2-Luc were used for lymphoma models; tumor cells were inoculated intravenously into 5- to 8-week-old female SCID (nonobese diabetic SCID; Taconic, in study of OCI-LY7-Luc) mice. Mice bearing the disseminated, CD20-positive, non-Hodgkin's lymphoma model OCI-LY19 were treated with vehicle control (10% 2-hydroxypropyl-β-cyclodextrin and 1% sodium bicarbonate was used for all in vivo studies), alisertib at 20 mg/kg twice daily or 30 mg/kg once daily, or the anti-CD20 monoclonal antibody rituximab (Rituxan) at 10 mg/kg once per week. The lymphoma cell lines stably expressed firefly luciferase, and tumor growth over time was measured using whole-body bioluminescent imaging using Xenogen IVIS 200 imaging system. Fifteen minutes before imaging, mice received an intraperitoneal injection of 150 mg/kg of the substrate Luciferin, which when oxidized by luciferase emits light photons. Mice were imaged both dorsally and ventrally, and photon flux values were summed from both views. The antitumor effects of each treatment group were determined by calculating the percent TGI [(Δ control mean tumor photon flux − Δ treated mean tumor photon flux) × 100/Δ control mean tumor photon flux] at the end of treatment.

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Mitotic index, spindle bipolarity, and chromosome alignment assays[4]
Mice bearing HCT-116 xenografts were treated orally with a single dose of 3, 10, and 30 mg/kg Alisertib/MLN8237, and tumor samples were removed at specified time points. Frozen tumor tissue sections were stained for the mitotic marker pHisH3, then visualized using immunofluorescence detection and quantified at the indicated time points. The methods used to stain and quantify pHisH3, which is also an Aurora B substrate, have been described previously.

[1]. A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma Blood June 24, 2010 vol. 115 no. 25 5202-5213.

[2]. Drug-Resistant Aurora A Mutants for Cellular Target Validation of the Small Molecule Kinase Inhibitors MLN8054 and MLN8237 ACS Chem. Biol., 2010, 5 (6), pp 563-576.

[3]. Aurora Kinase Inhibitors: Current Status and Outlook. Front Oncol. 2015 Dec 21;5:278.

[4]. Characterization of Alisertib (MLN8237), an investigational small-molecule inhibitor of aurora A kinase using novel in vivo pharmacodynamic assays.Clin Cancer Res. 2011 Dec 15;17(24):7614-7624.

4-[[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-2-methoxybenzoic acid is a benzazepine.
Alisertib is a novel aurora A kinase inhibitor under investigation for the treatment of various forms of cancer.
Alisertib is a second-generation, orally bioavailable, highly selective small molecule inhibitor of the serine/threonine protein kinase Aurora A kinase with potential antineoplastic activity. Alisertib binds to and inhibits Aurora A kinase, which may result in disruption of the assembly of the mitotic spindle apparatus, disruption of chromosome segregation, and inhibition of cell proliferation. Aurora A kinase localizes to the spindle poles and to spindle microtubules during mitosis, and is thought to regulate spindle assembly. Aberrant expression of Aurora kinases occurs in a wide variety of cancers, including colon and breast cancers.

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Drug Indication
For the treatment of various forms of cancer.

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Aurora-A is a mitotic kinase that regulates mitotic spindle formation and segregation. In multiple myeloma (MM), high Aurora-A gene expression has been correlated with centrosome amplification and proliferation; thus, inhibition of Aurora-A in MM may prove to be therapeutically beneficial. Here we assess the in vitro and in vivo anti-MM activity of MLN8237, a small-molecule Aurora-A kinase inhibitor. Treatment of cultured MM cells with MLN8237 results in mitotic spindle abnormalities, mitotic accumulation, as well as inhibition of cell proliferation through apoptosis and senescence. In addition, MLN8237 up-regulates p53 and tumor suppressor genes p21 and p27. Combining MLN8237 with dexamethasone, doxorubicin, or bortezomib induces synergistic/additive anti-MM activity in vitro. In vivo anti-MM activity of MLN8237 was confirmed using a xenograft-murine model of human-MM. Tumor burden was significantly reduced (P = .007) and overall survival was significantly increased (P < .005) in animals treated with 30 mg/kg MLN8237 for 21 days. Induction of apoptosis and cell death by MLN8237 were confirmed in tumor cells excised from treated animals by TdT-mediated dUTP nick end labeling assay. MLN8237 is currently in phase 1 and phase 2 clinical trials in patients with advanced malignancies, and our preclinical results suggest that MLN8237 may be a promising novel targeted therapy in MM.[1]

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The Aurora kinases regulate multiple aspects of mitotic progression, and their overexpression in diverse tumor types makes them appealing oncology targets. An intensive research effort over the past decade has led to the discovery of chemically distinct families of small molecule Aurora kinase inhibitors, many of which have demonstrated therapeutic potential in model systems. These agents are also important tools to help dissect signaling pathways that are orchestrated by Aurora kinases, and the antiproliferative target of pan-Aurora inhibitors such as VX-680 has been validated using chemical genetic techniques. In many cases the nonspecific nature of Aurora inhibitors toward unrelated kinases is well established, potentially broadening the spectrum of cancers to which these compounds might be applied. However, unambiguously demonstrating the molecular target(s) for clinical kinase inhibitors is an important challenge, one that is absolutely critical for deciphering the molecular basis of compound specificity, resistance, and efficacy. In this paper, we have investigated amino acid requirements for Aurora A sensitivity to the benzazepine-based Aurora inhibitor MLN8054 and the close analogue MLN8237, a second-generation compound that is in phase II clinical trials. A crystallographic analysis facilitated the design and biochemical investigation of a panel of resistant Aurora A mutants, a subset of which were then selected as candidate drug-resistance targets for further evaluation. Using inducible human cell lines, we show that cells expressing near-physiological levels of a functional but partially drug-resistant Aurora A T217D mutant survive in the presence of MLN8054 or MLN8237, authenticating Aurora A as a critical antiproliferative target of these compounds.[2]

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Purpose: Small-molecule inhibitors of Aurora A (AAK) and B (ABK) kinases, which play important roles in mitosis, are currently being pursued in oncology clinical trials. We developed three novel assays to quantitatively measure biomarkers of AAK inhibition in vivo. Here, we describe preclinical characterization of alisertib (MLN8237), a selective AAK inhibitor, incorporating these novel pharmacodynamic assays. Experimental design: We investigated the selectivity of alisertib for AAK and ABK and studied the antitumor and antiproliferative activity of alisertib in vitro and in vivo. Novel assays were used to assess chromosome alignment and mitotic spindle bipolarity in human tumor xenografts using immunofluorescent detection of DNA and alpha-tubulin, respectively. In addition, 18F-3'-fluoro-3'-deoxy-l-thymidine positron emission tomography (FLT-PET) was used to noninvasively measure effects of alisertib on in vivo tumor cell proliferation. Results: Alisertib inhibited AAK over ABK with a selectivity of more than 200-fold in cells and produced a dose-dependent decrease in bipolar and aligned chromosomes in the HCT-116 xenograft model, a phenotype consistent with AAK inhibition. Alisertib inhibited proliferation of human tumor cell lines in vitro and produced tumor growth inhibition in solid tumor xenograft models and regressions in in vivo lymphoma models. In addition, a dose of alisertib that caused tumor stasis, as measured by volume, resulted in a decrease in FLT uptake, suggesting that noninvasive imaging could provide value over traditional measurements of response. Conclusions: Alisertib is a selective and potent inhibitor of AAK. The novel methods of measuring Aurora A pathway inhibition and application of tumor imaging described here may be valuable for clinical evaluation of small-molecule inhibitors.[4]

C27H20CLFN4O4

518.92

518.115

C, 62.49; H, 3.88; Cl, 6.83; F, 3.66; N, 10.80; O, 12.33

1028486-01-2

Alisertib sodium;1028486-06-7; 1208255-63-3 (sodium)

24771867

Light yellow to light pink solid powder

1.4±0.1 g/cm3

729.1±70.0 °C at 760 mmHg

394.8±35.7 °C

0.0±2.5 mmHg at 25°C

1.671

5.56

2

9

6

37

836

0

ZLHFILGSQDJULK-UHFFFAOYSA-N

InChI=1S/C27H20ClFN4O4/c1-36-21-5-3-4-20(29)23(21)25-19-10-15(28)6-8-17(19)24-14(12-30-25)13-31-27(33-24)32-16-7-9-18(26(34)35)22(11-16)37-2/h3-11,13H,12H2,1-2H3,(H,34,35)(H,31,32,33)

4-((9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-benzo[c]pyrimido[4,5-e]azepin-2-yl)amino)-2-methoxybenzoic acid

MLN-8237; alisertib; MLN8237; MLN 8237

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.08 mg/mL (4.01 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.08 mg/mL (4.01 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.01 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 15% Captisol:5mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)