The area and number of H1299 and H460 cells grow in a concentration-dependent manner when exposed to salty (0, 3, 10, 30 nM) for 24 hours [2]. Salted ilotasertib (1–1000 nM) exhibits antiproliferative properties [2]. Examine[2]

The area and number of H1299 and H460 cells grow in a concentration-dependent manner when exposed to salty (0, 3, 10, 30 nM) for 24 hours [2]. Salted ilotasertib (1–1000 nM) exhibits antiproliferative properties [2]. Examine[2]

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ABT-348 (Ilorasertib) demonstrates potent antiproliferative activity against a broad panel of cancer cell lines in 3-day or 7-day colony formation assays. IC₅₀ values range from 0.3 nM (MV-4-11 AML cells) to 103 nM (K562 CML cells). It is active against leukemia/lymphoma cell lines including RS4;11 (ALL, IC₅₀ = 3 nM), SEM (ALL, IC₅₀ = 1 nM), and DoHH2 (follicular lymphoma, IC₅₀ = 4 nM).
It inhibits proliferation of solid tumor cell lines such as H1299 (NSCLC, IC₅₀ = 2 nM), H460 (NSCLC, IC₅₀ = 2 nM), MiaPaCa (pancreatic, IC₅₀ = 4 nM), HT1080 (fibrosarcoma, IC₅₀ = 5 nM), and HCT-15 (colorectal with MDR phenotype, IC₅₀ = 6 nM).
It potently inhibits VEGF-stimulated proliferation of HUVECs (IC₅₀ ≤ 0.3 nM) but shows >1000-fold reduced activity against non-proliferating HUVECs (IC₅₀ ~1000 nM), indicating specificity for cycling cells.
In cells, it inhibits autophosphorylation of Aurora A (IC₅₀ = 189 nM in HeLa cells), Aurora B (IC₅₀ = 13 nM in HCT-116 cells), and Aurora C (IC₅₀ = 13 nM in HCT-116 cells).
It induces polyploidy (a hallmark of Aurora B inhibition) in H1299 and H460 NSCLC cell lines in a concentration-dependent manner (EC₅₀ ~5-10 nM).
It inhibits phosphorylation of histone H3 (an Aurora B substrate) in HCT-116 cells with an IC₅₀ of 21 nM.
It shows activity against BCR-ABL expressing cells, including those with the T315I mutation (BaF3-Bcr-Abl T315I IC₅₀ = 260 nM).

In SCID mice bearing MV-4-11 tumors, ilotinib hydrochloride (6.25, 12.5, and 25 mg/kg; interface) exhibited anti-tumor efficacy, with TGIs of 80 at 6.25, 12.5, and 25 mg/kg, correspondingly. With TGIs of 38% and 59%, respectively, iloratib hydrochloride (6.25, 12.5, 25 mg/kg; po) demonstrated tumor anti-tumor efficacy in SCID mice bearing confirmed SKM-1 tumors. 80 percent. Histone H3 phosphorylation in blood-borne tumor cells is inhibited by imolipartib hydrochloride (0, 3.75, 7.5, and 15 mg/kg; ip) after 4–8 hours [2]. In mice, ilotinib hydrochloride (0.2 mg/kg; IV) demonstrates anti-VEGF efficacy [2]. Mice treated with imipratim hydrochloride (20 mg/kg; sidewall once weekly for 3 weeks) demonstrated anticancer action.

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ABT-348 (Ilorasertib) demonstrates antitumor efficacy in multiple human tumor xenograft models.
In an HT1080 fibrosarcoma flank model, dosing at 10 mg/kg intraperitoneally three times a week (MWF) resulted in significant tumor growth inhibition compared to vehicle.
In a MiaPaCa pancreatic carcinoma flank model, a single weekly dose of 20 mg/kg intraperitoneally significantly inhibited tumor growth and caused regression/stabilization of advanced tumors.
In an RS4;11 acute lymphoblastic leukemia (ALL) flank model, a single weekly dose of 20 mg/kg intraperitoneally inhibited growth of established tumors and caused regression of advanced tumors.
In an RS4;11 leukemia engraftment (disseminated) model, intermittent weekly treatment via subcutaneous osmotic minipump (12.5 mg/kg for 24 h) significantly prolonged median survival from 43 days (vehicle) to 72 days.
In a KMS11 multiple myeloma xenograft model, oral administration once weekly (20 mg/kg) showed potent antitumor activity.
It inhibits VEGF-mediated responses in vivo, as shown by potent inhibition of estradiol-induced uterine edema (ED₅₀ ~0.2 mg/kg IV).
Treatment of tumor-bearing mice with ABT-348 induces a dose-proportional elevation in plasma Placental Growth Factor (PLGF) levels, a biomarker of antiangiogenic activity.
In an orthotopic rat glioma model, ABT-348 treatment led to acute and significant reductions in tumor vascular permeability (K^trans) as measured by DCE-MRI, similar to the selective VEGFR/PDGFR inhibitor ABT-869, whereas the Aurora-selective inhibitor AZD1152 had no effect.
It demonstrates target engagement in vivo by inhibiting phosphorylation of histone H3 in circulating leukemia cells (RS4;11 engraftment model) and in solid tumors (HCC827ER model) in a dose- and time-dependent manner, correlating with plasma and tumor drug concentrations.

The potency (IC₅₀ values) of ABT-348 (Ilorasertib) against various kinases was determined using assays with active kinase domains. For tyrosine kinases and Aurora kinases, a homogeneous time-resolved fluorescence (HTRF) assay format was employed. This assay used a biotinylated peptide substrate, 1 mM ATP, a europium cryptate-labeled anti-phosphotyrosine or anti-phospho-peptide antibody, and streptavidin-allophycocyanin. The fluorescence signal was measured to determine inhibition.
For Ser/Thr kinases, assays were performed using 5 μM ATP, [γ-³³P]ATP, and a biotinylated peptide substrate. The incorporation of ³³P into the captured peptide was quantified using a streptavidin-coated flashplate.
The concentration causing 50% inhibition (IC₅₀) and 95% confidence limits were calculated using nonlinear regression analysis of concentration-response data.
A mutant Aurora B (Y156H) kinase was produced by site-directed mutagenesis, expressed, and assayed using the HTRF format described above.

Test[2]
Cell Types: H1299, H460 Cell
Tested Concentrations: 0, 3, 10, 30 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: The degree and number of induced polyploid cells increased in a concentration-dependent manner, with EC50S of 5 and 10 nM respectively. Targets H1299 and H460 cells.

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Cell proliferation assay[2]
Cell Types: MV-4-11, SEM, K562, HCT-15, SW620, H1299, H460 Cell
Tested Concentrations: 1-1000 nM
Incubation Duration:
Experimental Results: Displayed anti-proliferative activity with IC50 of 0.3, 1. 103, 6, 6, 2, and 2 nM for MV-4-11, SEM, K562, HCT-15, SW620, H1299, and H460 cells respectively.

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Cellular antiproliferative activity was assessed using 3-day proliferation assays or 7-10 day colony formation assays.
For the 3-day proliferation assay, carcinoma cells (e.g., 2500 cells/well) or leukemia cells (e.g., 50,000 cells/well) were plated in growth medium overnight. The compound was then added, and cells were incubated for 72 hours. Cell viability/proliferation was measured either by adding a resazurin-based reagent (alamarBlue) followed by fluorescence reading, or by adding a luminescent ATP detection reagent (CellTiter-Glo).
For the colony formation assay, 500 cells/well were seeded into plates. After 24 hours, compounds were added, and cells were cultured for 7 to 10 days. Colonies were then fixed, stained, and counted.
Induction of polyploidy was assessed by cell cycle analysis via flow cytometry. Carcinoma cells (e.g., H1299, H460) were exposed to the compound for 24 hours in complete medium, then stained with a nuclear dye for DNA content analysis.
Phosphorylation of histone H3 (Ser10) was measured in cells. HCT-116 cells were arrested with nocodazole overnight, treated with compound for 1 hour, and lysed. Lysates were analyzed using a specific sandwich ELISA kit for phospho-histone H3 or by Western blot.
For pharmacodynamic studies in circulating tumor cells, blood was collected from mice engrafted with RS4;11 leukemia cells. Cells were lysed, fixed, permeabilized, and stained with fluorescently labeled antibodies against human CD45 and phospho-histone H3 (Ser10). The percentage of CD45-positive cells that were also positive for phospho-histone H3 was determined by flow cytometry.
Receptor phosphorylation (autophosphorylation) assays were performed in various engineered cell lines. Cells were stimulated with appropriate ligands (e.g., VEGF, PDGF) or had constitutive phosphorylation (e.g., FLT-3 in SEM cells). After incubation with the inhibitor, cells were lysed. Phosphorylation levels were quantified using ELISA-based methods on precoded plates or by Western blot analysis with phospho-specific antibodies. Inhibition was calculated by normalizing phospho-signal to total receptor levels.

Animal/Disease Models: Female SCID/beige mouse[2]
Doses: 25 mg/kg
Route of Administration: subcutaneousmini-pump;[2]. 24-hour
Experimental Results: Inhibition of histone H3 phosphorylation, tumor drug concentration is related to 50% inhibition of histone H3 phosphorylation.

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Animal/Disease Models: 22-26 g, female NOD/SCID (severe combined immunodeficient) mouse (multiple myeloma xenograft model (KMS11)) [2]
Doses: 20 mg/kg
Route of Administration: Po; once a week for 3 weeks
Experimental Results: Inhibits tumor growth in mice.

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For most subcutaneous xenograft studies, tumor cells were suspended in PBS, mixed with a basement membrane matrix, and inoculated into the flank of female immunocompromised mice. When mean tumor volume reached approximately 0.4-0.5 cm³, mice were randomized into groups (typically n=10) and treatment began.
ABT-348 was formulated by stepwise addition and mixing of ethanol, a surfactant, polyethylene glycol 400, and an aqueous solution of hydroxypropyl methylcellulose. The dosing volume was 10 ml/kg.
Dosing regimens varied by model: intraperitoneal injection three times per week (Monday, Wednesday, Friday) at 10 mg/kg/day for HT1080 tumors; intraperitoneal injection once weekly at 20 mg/kg for MiaPaCa and RS4;11 (flank) tumors; oral administration once weekly at 20 mg/kg for KMS11 tumors.
Tumor size was measured periodically with calipers, and volume was calculated. The study endpoint was typically when vehicle group tumors reached a predefined size.
For the leukemia engraftment model, female irradiated mice were inoculated intravenously with RS4;11 cells. Treatment began 21 days later. ABT-348 (12.5 or 6.25 mg/kg) or vehicle was administered via a subcutaneously implanted osmotic minipump for 24 hours, once weekly. Survival was monitored daily.
For pharmacokinetic/pharmacodynamic studies in solid tumors, ABT-348 (25 mg/kg) was administered subcutaneously via implanted osmotic minipumps for 24 hours to tumor-bearing mice. Tumor samples were collected at various time points for Western blot analysis of phospho-histone H3 and drug concentration measurement.
For the uterine edema model, mice were pretreated with gonadotropin. They were then randomized, weighed, and given ABT-348 or vehicle intravenously. Thirty minutes later, mice received an intraperitoneal injection of estradiol to induce VEGF-mediated edema. Control animals received sterile water. Mice were euthanized 3 hours after compound administration, and uterine wet weight was measured to calculate percent inhibition of edema.
For DCE-MRI studies in an orthotopic rat glioma model, rats bearing brain tumors were treated with ABT-348 (6.25 mg/kg intraperitoneally twice daily, every 7 days), a selective Aurora inhibitor (AZD1152), or vehicle. MRI was performed at baseline and after treatment cycles to assess changes in the vascular transfer constant (K^trans).
Plasma samples for PLGF measurement or drug concentration were collected at indicated time points from treated mice.

The concentration of ABT-348 (iloracitinib) in plasma was determined by liquid chromatography-mass spectrometry (LC-MS). Proteins in plasma samples were precipitated with acidified methanol, and the supernatant was analyzed. The drug exhibited high protein binding (>99%) in mouse blood, consistent with the efficacy changes observed in cell assays performed in the presence of plasma. In a pharmacodynamic study of a solid tumor (HCC827ER), the tumor drug concentration required to inhibit 50% histone H3 phosphorylation was estimated to be 1–2 μM. In an RS4;11 leukemia transplantation model, the plasma drug concentration required to inhibit 50% histone H3 phosphorylation in circulating tumor cells 4 hours after administration was approximately 3.2 μM.

In vivo tolerability was assessed by monitoring changes in body weight and mortality. In the HT1080 fibrosarcoma model, intraperitoneal injection of 20 mg/kg three times weekly (Monday, Wednesday, and Friday) was associated with increased mortality and was considered intolerable. Intraperitoneal injection of 10 mg/kg at the same regimen resulted in weight loss but did not increase mortality compared to the solvent, and was considered poorly tolerable (weight loss <25%). Conversely, in the MiaPaCa and RS4;11 (lateral ventral) models, once-weekly intraperitoneal injection of 20 mg/kg was effective and did not increase weight loss. In cell culture, ABT-348 showed minimal acute effects (<3 days) on the viability of non-proliferating primary human umbilical vein endothelial cells (HUVECs), indicating a lack of non-specific cytotoxicity to non-proliferating cells.

[1]. Abstract 858: Potent in vivo activity of the aurora kinase inhibitor ABT-348 in human acute myeloid leukemia and myelodysplastic syndrome xenograft models. Cancer Res (2012) 72 (8_Supplement): 858.

[2]. Preclinical characterization of ABT-348, a kinase inhibitor targeting the aurora, vascular endothelial growth factor receptor/platelet-derived growth factor receptor, and Src kinase families. J Pharmacol Exp Ther. 2012 Dec;343(3):617-27.

ABT-348 (Ilorasetine) is a novel ATP-competitive multi-target kinase inhibitor designed to simultaneously target tumor cell proliferation (via Aurora kinases) and tumor microenvironment/angiogenesis (via the VEGFR/PDGFR family). Its unique kinase inhibitory spectrum, combining potent Aurora and VEGFR/PDGFR activity, distinguishes it from more selective Aurora kinase inhibitors and may help overcome the limitations of targeting these pathways separately. It remains active against mutations at the Aurora BY156H catalytic site, potentially reducing the probability of resistance development compared to more selective drugs. It is not a substrate for multidrug resistance protein 1 (MDR1) or breast cancer resistance protein (BCRP) transporters, as evidenced by its sustained activity in cell lines with MDR phenotypes (e.g., HCT-15) and the lack of cross-resistance in relevant models. The biomarker's targeting activities include inhibition of histone H3 phosphorylation (for inhibiting Aurora) and induction of plasma PLGF (for anti-angiogenic activity), both of which are clinically evaluable. Preclinical efficacy and a unique target spectrum provide a theoretical basis for the clinical evaluation of ABT-348 in cancer treatment.

C25H22CLFN6O2S

524.997585773468

524.119

1847485-91-9

Ilorasertib;1227939-82-3

54759636

White to off-white solid powder

5

7

6

36

713

0

Cl.S1C=C(C2C=CC(=CC=2)NC(NC2C=CC=C(C=2)F)=O)C2C(N)=NC=C(C3C=NN(CCO)C=3)C1=2

SUKMSIXFLFTNHO-UHFFFAOYSA-N

InChI=1S/C25H21FN6O2S.ClH/c26-17-2-1-3-19(10-17)31-25(34)30-18-6-4-15(5-7-18)21-14-35-23-20(12-28-24(27)22(21)23)16-11-29-32(13-16)8-9-33;/h1-7,10-14,33H,8-9H2,(H2,27,28)(H2,30,31,34);1H

1-[4-[4-amino-7-[1-(2-hydroxyethyl)pyrazol-4-yl]thieno[3,2-c]pyridin-3-yl]phenyl]-3-(3-fluorophenyl)urea;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~41.67 mg/mL (~79.37 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.96 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 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 (3.96 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 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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 (Please use freshly prepared in vivo formulations for optimal results.)