FLT3 (IC50 = 0.29 nM); LTK (IC50 = 0.35 nM); AXL (IC50 = 0.73 nM); EML4-ALK (IC50 = 1.2 nM); c-KIT (IC50 = 230 nM)
The targets of Gilteritinib hemifumarate (active ingredient: Gilteritinib/ASP2215) are Fms-like tyrosine kinase 3 (FLT3) and AXL receptor tyrosine kinase; it is a type I tyrosine kinase inhibitor with high selectivity for FLT3 and AXL, and weak activity against c-KIT. [2]
Gilteritinib hemifumarate inhibits mutated forms of FLT3 including internal tandem duplication (FLT3-ITD), FLT3-D835Y point mutation, and FLT3-F691 mutation (weaker inhibitory activity against FLT3-F691); [2]

After administering oral Gilteritinib at a dose of 10 mg/kg for four days, the concentration of the drug in tumors in MV4-11 xenografted mice was found to be over 20 times higher than that in plasma. MV4-11 tumor growth is dose-dependently inhibited during a 28-day treatment with gelderitinib, and at doses greater than 6 mg/kg, total tumor regression is induced. Furthermore, gerteritinib prolongs the survival of mice receiving intravenous MV4-11 cell transplants and reduces the tumor burden in the bone marrow[1].

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1. Plasma and tumor distribution: Nude mice subcutaneously xenografted with MV4-11 cells were administered a single oral dose of Gilteritinib (from Gilteritinib hemifumarate) at 1 mg/kg, 6 mg/kg, or 10 mg/kg. Plasma and tumor concentrations of gilteritinib were determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), with high intratumor distribution observed (mean ± SD: 2-3 animals for plasma, 3 animals for tumor) [2]
2. Inhibition of FLT3/STAT5 phosphorylation in xenograft tumors: Male mice xenografted with MV4-11 cells were orally treated with vehicle control or increasing concentrations of Gilteritinib (from Gilteritinib hemifumarate). Over a 24-hour time course, tumor protein lysates were collected to measure: (1) the ratio of phospho-FLT3 to total FLT3 by ELISA (normalized to vehicle control); (2) the ratio of phospho-STAT5 to total STAT5 by immunoblot (normalized to vehicle control) [2]
3. Antitumor activity in MV4-11 xenograft model: Male mice xenografted with MV4-11 cells were orally treated with vehicle control or Gilteritinib (from Gilteritinib hemifumarate) at 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg once daily for 28 days. Gilteritinib dose-dependently reduced tumor volume (mean ± SEM, n=6 mice/group; P < 0.05, P < 0.01, P < 0.001 vs control on Day 28, Dunnett’s test), with no significant changes in body weight [2]
4. Tumor regression in FLT3 mutant xenograft models: Male nude mice xenografted with Ba/F3 cells expressing FLT3-ITD, FLT3-D835Y, or FLT3-ITD-D835Y were treated with 10 mg/kg or 30 mg/kg of oral Gilteritinib (from Gilteritinib hemifumarate) once daily for up to 7 days after confirmed tumor growth. Gilteritinib induced significant tumor regression in all three models (mean ± SEM, n=5 mice/group; P < 0.01, P < 0.001 vs control on Day 7, Dunnett’s test) [2]
5. Reduction of leukemic burden and improved survival: Female NOD-SCID mice engrafted with MV4-11-luc cells were treated with 30 mg/kg of oral Gilteritinib (from Gilteritinib hemifumarate) once daily for 56 days starting on Day 15. Whole-body imaging showed significant reduction in bone marrow infiltration of MV4-11-luc cells (mean ± SEM, n=10 mice/group; P < 0.001 vs control on Day 42, Student’s t-test). Kaplan–Meier analysis confirmed improved survival in gilteritinib-treated mice (P < 0.001 vs control, Log-rank test) [2]

TK-ELISA or off-chip mobility shift assays are used to test the kinase inhibitory activity of Gilteritinib against a panel of 78 tested kinases, with ATP concentrations roughly equivalent to each kinase's Km value. Initially, the inhibitory effect of each compound on TK activity is evaluated at two concentrations of Gilteritinib (1 nM and 5 nM). Next, additional research employing a range of Gilteritinib doses is carried out to ascertain IC50 values for c-KIT and kinases whose activity is inhibited by more than half at 1 nM Gilteritinib. FLT3, LTK, AXL, and c-KIT IC50 studies are carried out using TK-ELISA and MSA assays; the HTRF KinEASE-TK assay is used to determine the IC50 value of echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK)[2].

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Kinase selectivity assays were performed to evaluate Gilteritinib (active component of Gilteritinib hemifumarate). Purified FLT3, AXL, c-KIT, and other tyrosine kinases were incubated with gilteritinib in the presence of ATP and specific substrates (substrate details not provided) under optimized reaction conditions (temperature and incubation time not specified). Kinase activity was measured using an unspecified detection method to assess the inhibitory effect of gilteritinib on each kinase. The results confirmed high selectivity for FLT3 and AXL, with weak activity against c-KIT; [2]

The CellTiter-Glo Luminescent Cell Viability Assay is used to evaluate the impact of gelderitinib on MV4-11 and MOLM-13 cells. Further research is done to investigate the impact of quizartinib and gilteritinib on Ba/F3 cells that express FLT3-ITD, FLT3-D835Y, FLT3-ITD-D835Y, FLT3-ITD-F691 L, or FLT3-ITD-F691I. For five days, DMSO or increasing concentrations of Gilteritinib (0.01, 0.1, 1, 10, and 100 nM) are applied to MV4-11 and MOLM-13 cells. CellTiter-Glo is used to measure the viability of the cells[2].

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1. AML cell viability assay:
- Step 1: MV4-11 and MOLM-13 AML cells were seeded in appropriate culture plates at an unspecified density and cultured under standard conditions to ensure logarithmic growth.
- Step 2: Cells were treated with DMSO (control) or serial concentrations of Gilteritinib (from Gilteritinib hemifumarate) and incubated for 5 days.
- Step 3: Cell viability was quantified using the CellTiter-Glo assay (detection principle not specified), with data collected in quadruplicate and presented as mean ± SEM. The experiment was repeated three times, with a representative result reported [2]
2. Immunoprecipitation and immunoblot for FLT3 phosphorylation:
- Step 1: MV4-11 cells were cultured to the desired confluency and treated with DMSO or increasing concentrations of Gilteritinib (from Gilteritinib hemifumarate) for 2 hours.
- Step 2: Cells were harvested and lysed with an unspecified lysis buffer; protein concentration was determined by an unspecified method.
- Step 3: FLT3 protein was immunoprecipitated using a FLT3-specific antibody (details not provided), and immunoprecipitates were separated by SDS-PAGE (gel concentration not specified).
- Step 4: Separated proteins were transferred to an unspecified membrane, probed with primary antibodies against phosphorylated and total FLT3, followed by secondary antibody incubation.
- Step 5: Protein bands were detected by an unspecified method, and densitometry analysis was performed to quantify band intensities (values listed below blots). The experiment was conducted in triplicate [2]
3. Immunoblot for FLT3 downstream signaling proteins:
- Step 1: MV4-11 cells were treated with DMSO or increasing concentrations of Gilteritinib (from Gilteritinib hemifumarate) for 2 hours as described above.
- Step 2: Cell lysates were prepared, and equal amounts of protein were separated by SDS-PAGE, transferred to a membrane, and probed with primary antibodies against phosphorylated AKT, ERK, STAT5, and their total forms (loading control not specified).
- Step 3: Protein bands were detected and analyzed (triplicate experiments) [2]

Mice: In nude mice transplanted with MV4-11 AML cells, antitumor activity is assessed. Additionally, the pharmacokinetics in xenografted mice are examined. Gilteritinib (10 mg/kg) is given orally to MV4-11 xenografted mice for a period of four days as part of their treatment. Gilteritinib treatment for 28 days causes total tumor regression at doses greater than 6 mg/kg and dose-dependent inhibition of MV4-11 tumor growth[1].

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1. MV4-11 xenograft model (pharmacokinetic analysis):
- Step 1: MV4-11 AML cells were subcutaneously injected into nude mice (gender/age not specified) at an unspecified cell number to establish xenograft models.
- Step 2: When tumors reached an unspecified volume, mice were given a single oral dose of Gilteritinib (from Gilteritinib hemifumarate) at 1 mg/kg, 6 mg/kg, or 10 mg/kg (drug formulation/vehicle not specified).
- Step 3: Blood and tumor samples were collected at unspecified time points after administration.
- Step 4: Plasma and tumor concentrations of gilteritinib were measured by HPLC-MS/MS, with data presented as mean ± SD (2-3 animals for plasma, 3 animals for tumor) [2]
2. MV4-11 xenograft model (pharmacodynamic/efficacy analysis):
- Step 1: MV4-11 cells were subcutaneously implanted into male nude mice (n=6 per group) to establish xenograft models.
- Step 2: After tumor establishment (unspecified volume), mice were orally administered vehicle control or Gilteritinib (from Gilteritinib hemifumarate) at 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg once daily for 28 days (drug formulation not specified).
- Step 3: For pharmacodynamic analysis: Tumor tissues were collected over a 24-hour time course, protein lysates were prepared, and the ratios of phospho-FLT3/total FLT3 (ELISA) and phospho-STAT5/total STAT5 (immunoblot) were determined.
- Step 4: For efficacy analysis: Tumor volume and body weight were measured at unspecified intervals, with statistical analysis (Dunnett’s test) performed on Day 28 [2]
3. Ba/F3-FLT3 mutant xenograft model:
- Step 1: Ba/F3 cells expressing FLT3-ITD, FLT3-D835Y, or FLT3-ITD-D835Y were subcutaneously injected into male nude mice (n=5 per group) to establish xenograft models.
- Step 2: After confirmed tumor growth (unspecified volume), mice were orally administered vehicle control, 10 mg/kg, or 30 mg/kg of Gilteritinib (from Gilteritinib hemifumarate) once daily for up to 7 days (drug formulation not specified).
- Step 3: Tumor volume was measured at unspecified time points, with statistical analysis (Dunnett’s test) performed on Day 7 [2]
4. Intra-bone marrow transplantation model:
- Step 1: MV4-11-luc cells (luciferase-expressing MV4-11 cells) were engrafted into female NOD-SCID mice via intra-bone marrow injection (injection site/cell number not specified).
- Step 2: Starting on Day 15 after engraftment, mice (n=10 per group) were orally administered vehicle control or 30 mg/kg of Gilteritinib (from Gilteritinib hemifumarate) once daily for 56 days (drug formulation not specified).
- Step 3: Whole-body bioluminescence imaging was performed at unspecified time points (e.g., Day 21, Day 42) to monitor bone marrow infiltration of MV4-11-luc cells (imaging parameters not specified).
- Step 4: Mouse survival was recorded, and Kaplan–Meier analysis (Log-rank test) was conducted [2]

1. Absorption and Distribution: Following oral administration, gipritinib (the active ingredient of gipritinib hemifumarate) was rapidly absorbed in nude mice carrying MV4-11 xenografts and widely distributed in tumor tissues. Plasma and tumor tissue concentrations were determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) after a single oral administration (1 mg/kg, 6 mg/kg, 10 mg/kg) [2]. 2. Metabolism, Excretion, Half-life, and Bioavailability: Information on the metabolism, excretion, half-life, or oral bioavailability of gipritinib hemifumarate was not provided [2].

1. In vivo toxicity: No significant toxicity was observed in mouse models treated with gipritinib (derived from gipritinib hemifumarate). In the MV4-11 xenograft model, mice were well-tolerated after 28 days of treatment with gipritinib (1-10 mg/kg once daily) without significant changes in body weight [2].

[1]. ASP2215, a novel FLT3/AXL inhibitor: Preclinical evaluation in acute myeloid leukemia (AML). 2014 ASCO Annual Meeting.

[2]. Gilteritinib, a FLT3/AXL inhibitor, shows antileukemic activity in mouse models of FLT3 mutated acute myeloid leukemia. Invest New Drugs. 2017 Oct;35(5):556-565.

Gilteritinibib fumarate, the fumarate form of Gilteritinibib, is an orally bioavailable receptor tyrosine kinase (RTK) inhibitor, including FMS-like tyrosine kinase 3 (FLT3; STK1; FLK2), AXL (UFO; JTK11), anaplastic lymphoma kinase (ALK; CD246), and leukocyte receptor tyrosine kinase (LTK), possessing potential antitumor activity. After administration, Gilteritinibib binds to and inhibits the activity of wild-type and mutant FLT3, AXL, ALK, and LTK. This may lead to inhibition of FLT3, AXL, ALK, and LTK-mediated signal transduction pathways and reduce the proliferation of cancer cells overexpressing these RTKs. FLT3, AXL, ALK, and LTK are overexpressed or mutated in various cancer cell types and play a crucial role in tumor cell growth and survival.
See also: Gilteritinibib (with active fraction).

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Drug Indications
Xospata is indicated for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with FLT3 mutations, as a monotherapy. 1. Background and Composition: Gifitinib hemifumarate is the salt form of gifitinib (ASP2215), a novel type I tyrosine kinase inhibitor with high selectivity for FLT3 and AXL. FLT3 is one of the most common mutated genes in acute myeloid leukemia (AML), and FLT3 mutation is associated with poor overall survival; AXL is involved in FLT3 activation and AML pathogenesis[2]
2. Mechanism of action: The active ingredient giretinib hemifumarate exerts anti-leukemic activity by inhibiting the phosphorylation of mutated FLT3 and its downstream signaling pathways (AKT, ERK, STAT5), thereby inhibiting AML cell proliferation and inducing tumor regression in vivo[2]
3. Clinical potential: Preclinical studies have shown that giretinib hemifumarate (through its active ingredient giretinib) has strong anti-leukemic activity in FLT3-mutated AML cell lines and mouse models (xenograft and intramedullary transplantation models) without significant toxicity, suggesting that it may be a promising next-generation FLT3 inhibitor for the treatment of FLT3-mutant positive AML[2]
4. Computational model: The binding of giretinib (the active ingredient of giretinib hemifumarate) to wild-type FLT3 was computationally modeled. Gilteritinibib was visualized using a ball-and-stick model (atoms were colored by element type), and the protein surface was colored by electrostatic potential; the gated residue F691 was highlighted [2].

C29H44N8O3₀.₅C₄H₄O₄

610.75

1220.718

C, 60.96; H, 7.59; N, 18.35; O, 13.10

1254053-84-3

Gilteritinib;1254053-43-4

76970819

White to yellow solid powder

8

24

20

88

904

0

CCC1=C(N=C(C(=N1)C(=O)N)NC2=CC(=C(C=C2)N3CCC(CC3)N4CCN(CC4)C)OC)NC5CCOCC5.CCC1=C(N=C(C(=N1)C(=O)N)NC2=CC(=C(C=C2)N3CCC(CC3)N4CCN(CC4)C)OC)NC5CCOCC5.C(=C/C(=O)O)\C(=O)O

UJOUWHLYTQFUCU-WXXKFALUSA-N

InChI=1S/2C29H44N8O3.C4H4O4/c2*1-4-23-28(31-20-9-17-40-18-10-20)34-29(26(33-23)27(30)38)32-21-5-6-24(25(19-21)39-3)37-11-7-22(8-12-37)36-15-13-35(2)14-16-36;5-3(6)1-2-4(7)8/h2*5-6,19-20,22H,4,7-18H2,1-3H3,(H2,30,38)(H2,31,32,34);1-2H,(H,5,6)(H,7,8)/b;;2-1+

(E)-but-2-enedioic acid;6-ethyl-3-[3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]anilino]-5-(oxan-4-ylamino)pyrazine-2-carboxamide

ASP2215 hemifumarate; ASP-2215 hemifumarate; ASP 2215 hemifumarate; Gilteritinib hemifumarate; Trade name: Xospata

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 (e.g. under nitrogen), 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)

Solubility in Formulation 1: 10 mg/mL (16.37 mM) in 50% PEG300 +50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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