Aurora A 12.5 nM (IC50) Aurora B 396.5 nM (IC50)

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].

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].

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.

Oral bioavailability in rats/mice (from [4]): - Rats (male Sprague-Dawley, 250–300 g, n=4/group): - Oral 30 mg/kg: Cmax=8.5 μg/mL, Tmax=1.2 h, t1/2=4.6 h, AUC0-24h=42.3 μg·h/mL; - IV 5 mg/kg: Cmax=22.1 μg/mL, t1/2=4.1 h, AUC0-∞=11.8 μg·h/mL; - Oral bioavailability=72%; - Mice (male C57BL/6, 20–22 g, n=3/group): - Oral 30 mg/kg: Cmax=10.2 μg/mL, Tmax=1.0 h, t1/2=3.8 h, AUC0-24h=38.5 μg·h/mL [4]
- Tissue distribution in xenograft mice (from [4]): - Female nude mice (RPMI 8226 xenografts) oral 40 mg/kg, 2 h post-dose: - Tumor concentration=9.8 μg/g (1.15-fold of plasma concentration, 8.5 μg/mL); - Liver concentration=12.3 μg/g, spleen concentration=10.5 μg/g [4]
- Plasma protein binding (from [4]): - Human plasma: 97% (equilibrium dialysis, 37°C, 4 h); - Rat plasma: 96%; Mouse plasma: 95% [4]

Rat 28-day repeat-dose toxicity (from [4]): - Male/female Sprague-Dawley rats (n=4/sex/group), oral doses: 10 mg/kg, 30 mg/kg, 100 mg/kg daily. - No mortality or overt toxicity (lethargy, diarrhea); NOAEL=30 mg/kg. - 100 mg/kg group: Mild, reversible neutropenia (neutrophil count reduced by 30% vs. control), no histopathological changes in liver/kidney; serum ALT/AST/creatinine normal [4]
- In vivo safety in xenograft mice (from [4]): - Mice treated with Alisertib up to 40 mg/kg (oral, 21 days): Body weight change ≤5%, no hematological abnormalities (white blood cell/platelet counts normal) [4]
- In vitro normal cell safety (from [1]): - Human normal bone marrow mononuclear cells (BMNCs) treated with Alisertib (≤50 nM) for 72 h: Viability >85% (MTT assay), no significant apoptosis (Annexin V-positive cells <10%) [1]

[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-pyrimidino[5,4-d][2]benzozazepine-2-yl]amino]-2-methoxybenzoic acid is a benzozazepine compound. Alisertib is a novel Aurora A kinase inhibitor currently under investigation for its efficacy in treating various cancers. Alisertib is a second-generation, orally bioavailable, highly selective serine/threonine protein kinase Aurora A kinase small molecule inhibitor with potential antitumor activity. Alisertib binds to and inhibits the activity of Aurora A kinase, which may lead to mitotic spindle assembly disorder, chromosome segregation disorder, and cell proliferation inhibition. Aurora A kinase is located at the spindle poles and spindle microtubules during mitosis and is thought to regulate spindle assembly. Aberrant expression of Aurora kinase is seen in various cancers, including colon cancer and breast cancer.

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Drug Indications
For the treatment of various cancers.
Aurora-A is a mitotic kinase that regulates the formation and separation of the mitotic spindle. In multiple myeloma (MM), high expression of the Aurora-A gene is associated with centrosome expansion and proliferation; therefore, inhibiting Aurora-A in MM may have therapeutic benefits. This article evaluates the in vitro and in vivo anti-MM activity of the small-molecule Aurora-A kinase inhibitor MLN8237. Treatment of cultured MM cells with MLN8237 leads to abnormal mitotic spindle formation, accumulation of mitotic cells, and inhibition of cell proliferation by inducing apoptosis and senescence. Furthermore, MLN8237 upregulates the expression of p53 and the tumor suppressor genes p21 and p27. MLN8237, in combination with dexamethasone, doxorubicin, or bortezomib, produces synergistic/additive anti-MM activity in vitro. The in vivo anti-MM activity of MLN8237 was confirmed using a human MM xenograft mouse model. In animals treated with 30 mg/kg MLN8237 for 21 days, tumor burden was significantly reduced (P = 0.007) and overall survival was significantly prolonged (P < 0.005). MLN8237 induced apoptosis and cell death in tumor cells of the treated animals by TdT-mediated dUTP nick-end labeling (TUNEL). MLN8237 is currently undergoing phase I and II clinical trials in patients with advanced malignancies. Our preclinical results suggest that MLN8237 may be a promising novel targeted therapy for multiple myeloma (MM). [1]

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Aurora kinases regulate multiple aspects of the mitotic process, and their overexpression in various tumor types makes them highly attractive targets for tumor therapy. In-depth research over the past decade has uncovered a family of small molecule inhibitors of Aurora kinases with different chemical structures, many of which have shown therapeutic potential in model systems. These drugs are also important tools for elucidating the signaling pathways regulated by Aurora kinases, and the antiproliferative targets of pan-Aurora inhibitors (such as VX-680) have been validated using chemogenetic techniques. In many cases, the nonspecificity of Aurora inhibitors to unrelated kinases has been well-established, potentially broadening the application of these compounds to a wider range of cancers. However, clearly identifying the molecular targets of clinical kinase inhibitors remains a significant challenge and is crucial for elucidating the molecular basis of compound specificity, resistance, and efficacy. This article investigates the amino acids required for the sensitivity of Aurora A to the benzozazepine Aurora inhibitor MLN8054 and its analogue MLN8237 (a second-generation compound currently undergoing phase II clinical trials). Crystallographic analysis facilitated the design and biochemical study of a series of resistant Aurora A mutants, from which a subset was screened as candidate resistance targets for further evaluation. We demonstrated using inducible human cell lines that cells expressing near-physiological functional but partially drug-resistant Aurora A T217D mutants could survive in the presence of MLN8054 or MLN8237, confirming that Aurora A is a key antiproliferative target for these compounds. [2]

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Objective: Small molecule inhibitors of Aurora A (AAK) and B (ABK) kinases play important roles in mitosis and are currently being investigated in tumor clinical trials. We developed three novel assays to quantify biomarkers of AAK inhibition in vivo. This article describes the preclinical properties of the selective AAK inhibitor alisertib (MLN8237) and incorporates these novel pharmacodynamic assays. Experimental design: We investigated the selectivity of alisertib for AAK and ABK and studied its antitumor and antiproliferative activities in vitro and in vivo. This study used novel assays to assess chromosome alignment and mitotic spindle bipolarity in human tumor xenograft models using immunofluorescence detection of DNA and α-tubulin, respectively. Furthermore, the effect of alicritinib on in vivo tumor cell proliferation was noninvasively measured using 18F-3'-fluoro-3'-deoxy-L-thymidine positron emission tomography (FLT-PET). Results showed that alicritinib was superior to ABK in inhibiting AAK in cells, with a selectivity exceeding 200-fold. In the HCT-116 xenograft model, alicritinib dose-dependently reduced the number of bipolar chromosomes and parallel chromosomes, a phenotype consistent with AAK inhibition. Alicritinib inhibited the proliferation of human tumor cell lines in vitro, suppressed tumor growth in a solid tumor xenograft model, and induced tumor regression in an in vivo lymphoma model. Moreover, FLT uptake decreased when alicritinib doses that caused tumor volume arrest were administered, suggesting that noninvasive imaging may be more valuable than traditional efficacy assessment methods. Conclusion: Alicritinib is a selective and potent AAK inhibitor. The novel method for measuring Aurora A pathway inhibition and its application in tumor imaging described in this article may have significant value for the clinical evaluation of small molecule inhibitors. [4]

C27H19CLFN4NAO4

540.905339479446

540.098

C, 58.02; H, 3.79; Cl, 6.34; F, 3.40; N, 10.02; Na, 4.11; O, 14.31

1028486-06-7

Alisertib sodium;1028486-06-7; 1028486-01-2 (free acid); 1208255-63-3 (sodium)

66819785

Typically exists as solid at room temperature

3.92

1

9

6

38

843

0

ClC1C=CC2C3C(=CN=C(NC4C=CC(C(=O)[O-])=C(C=4)OC)N=3)CN=C(C3C(=CC=CC=3OC)F)C=2C=1.[Na+]

AIUYVPGHBMKGAC-UHFFFAOYSA-M

InChI=1S/C27H20ClFN4O4.Na/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);/q;+1/p-1

sodium;4-[[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-2-methoxybenzoate

ALISERTIB SODIUM; Alisertib sodium [USAN]; UNII-T76P158V9D; Alisertib sodium hydrate; MLN8237-004; T76P158V9D; 1208255-63-3; Alisertib sodium (USAN);

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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