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 (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 (ASP2215) inhibits mutated forms of FLT3 including internal tandem duplication (FLT3-ITD), FLT3-D835Y point mutation, and FLT3-F691 mutation (inhibitory activity against FLT3-F691 is weaker); [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].

---

1. Plasma and tumor distribution in MV4-11 xenograft model: Nude mice subcutaneously xenografted with MV4-11 cells were treated with a single oral dose of Gilteritinib (ASP2215) (1 mg/kg, 6 mg/kg, 10 mg/kg). Plasma and tumor concentrations of gilteritinib were determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The results showed high intratumor distribution of gilteritinib, with data presented as mean ± SD (2 or 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 (ASP2215). Protein lysates were collected over a 24-hour time course: (1) The ratio of phospho-FLT3 to total FLT3 was determined by ELISA, with results presented as the ratio of phosphorylated protein levels normalized to total protein levels relative to vehicle control; (2) The ratio of phospho-STAT5 to total STAT5 was measured by immunoblot, with the same normalization method applied [2]
3. Antitumor activity in MV4-11 xenograft model: Male mice xenografted with MV4-11 cells were orally treated with vehicle control or increasing concentrations of Gilteritinib (ASP2215) (1 mg/kg, 3 mg/kg, 6 mg/kg, 10 mg/kg) 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), while no significant changes in body weight were observed, indicating no overt toxicity [2]
4. Tumor regression in FLT3 mutant-expressing 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 once-daily oral Gilteritinib (ASP2215) 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 in intra-bone marrow transplantation model: Female NOD-SCID mice engrafted with MV4-11-luc cells were treated once daily with vehicle or 30 mg/kg Gilteritinib (ASP2215) for 56 days starting on day 15. Whole-body imaging showed that gilteritinib significantly decreased MV4-11-luc cell bone marrow infiltration (mean ± SEM, n=10 mice/group; P < 0.001 vs control on day 42, Student’s t-test). Kaplan–Meier analysis demonstrated that gilteritinib significantly improved mouse survival (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].

---

Initial kinase activity assays were conducted to evaluate the selectivity of Gilteritinib (ASP2215). The assay system included purified FLT3, AXL, c-KIT, and other tyrosine kinases (specific kinases not specified). Gilteritinib was incubated with the kinases in the presence of ATP and specific substrates (substrate details not provided) under optimized reaction conditions (temperature, incubation time not specified). The kinase activity was measured using a detection method (not specified) to determine the inhibitory effect of gilteritinib on each kinase. The results confirmed high selectivity of gilteritinib 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].

---

1. AML cell viability assay:
- Step 1: MV4-11 and MOLM-13 AML cells were seeded in suitable culture plates at a specific density (not specified) and cultured under standard conditions (temperature, CO2 concentration not mentioned) to ensure cell adherence and logarithmic growth.
- Step 2: The cells were treated with DMSO (control) or serial concentrations of Gilteritinib (ASP2215) 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, and a representative result was reported [2]
2. Immunoprecipitation and immunoblot for FLT3 phosphorylation:
- Step 1: MV4-11 cells were cultured in complete medium until reaching the desired confluency (not specified), then treated with DMSO or increasing concentrations of Gilteritinib (ASP2215) for 2 hours.
- Step 2: Cells were harvested and lysed with a lysis buffer (components not specified), and protein concentration was determined (method not specified).
- Step 3: FLT3 protein was immunoprecipitated from cell lysates using a specific antibody against FLT3 (antibody details not provided), and the immunoprecipitates were separated by SDS-PAGE (gel concentration not specified).
- Step 4: The separated proteins were transferred to a membrane (type not specified), probed with primary antibodies against phosphorylated FLT3 and total FLT3, followed by secondary antibody incubation.
- Step 5: Protein bands were detected (detection method not specified), 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 (ASP2215) 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 total forms of these proteins (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].

---

1. MV4-11 subcutaneous xenograft model (pharmacokinetic analysis):
- Step 1: MV4-11 AML cells were subcutaneously injected into nude mice (gender/age not specified) at a specific cell number (not specified) to establish xenograft models.
- Step 2: When tumors reached a certain volume (not specified), mice were administered a single oral dose of Gilteritinib (ASP2215) at 1 mg/kg, 6 mg/kg, or 10 mg/kg (drug dissolution formula/vehicle not specified).
- Step 3: Blood and tumor samples were collected at predetermined time points (not specified) after administration.
- Step 4: Plasma and tumor concentrations of gilteritinib were measured by HPLC-MS/MS, with data presented as mean ± SD (2 or 3 animals for plasma, 3 animals for tumor) [2]
2. MV4-11 subcutaneous xenograft model (pharmacodynamic and 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 (specific volume not specified), mice were orally administered vehicle control or Gilteritinib (ASP2215) 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 ratio of phospho-FLT3/total FLT3 (ELISA) and phospho-STAT5/total STAT5 (immunoblot) was determined.
- Step 4: For efficacy analysis: Tumor volume and body weight were measured regularly (interval not specified) over the 28-day treatment period, with statistical analysis (Dunnett’s test) performed on Day 28 [2]
3. Ba/F3-FLT3 mutant subcutaneous 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 (specific volume not specified), mice were orally administered vehicle control, 10 mg/kg, or 30 mg/kg Gilteritinib (ASP2215) once daily for up to 7 days (drug formulation not specified).
- Step 3: Tumor volume was measured at specified time points (not specified), and statistical analysis (Dunnett’s test) was performed on Day 7 [2]
4. Intra-bone marrow transplantation model of AML:
- 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 Gilteritinib (ASP2215) once daily for 56 days (drug formulation not specified).
- Step 3: Whole-body bioluminescence imaging was performed at specified time points (e.g., day 21, day 42) to monitor MV4-11-luc cell bone marrow infiltration (imaging parameters not specified).
- Step 4: Mouse survival was recorded, and Kaplan–Meier analysis (Log-rank test) was conducted to evaluate the effect of gilteritinib on survival [2]

Absorption, Distribution and Excretion
In preclinical trials, peak plasma concentrations of gipritinib were reached 2 hours after oral administration, followed by peak intratumoral concentrations 4–8 hours later. The maximum concentration and AUC varied with dose, reaching 374 ng/ml and 6943 ng·h/ml, respectively. Steady-state plasma concentrations were reached within 15 days after administration, with a bioaccumulation of approximately 10-fold. The time to peak concentration (tmax) in humans under fasting conditions was reported to be 4–6 hours. Concomitant intake with a high-fat meal reduced Cmax and AUC by 26% and 10%, respectively, and delayed tmax by 2 hours. Gipritinib is primarily excreted in feces, accounting for 64.5% of the administered dose, with the remaining 16.4% excreted unchanged or as metabolites in urine. The estimated apparent central and peripheral volumes of distribution were 1092 L and 1100 L, respectively. These values indicate that gipritinib has broad tissue distribution.
The estimated clearance of gipritinib is 14.85 L/h.

---

Metabolism/Metabolites
Gipritinib is primarily metabolized in the liver via CYP3A4. Its metabolism is mainly driven by N-dealkylation and oxidation reactions, producing metabolites M17, M16, and M10. Depending on plasma concentration, the main form is the parent drug.

---

Biological Half-Life
The median half-life of gipritinib has been reported to be approximately 45–159 hours.

---

1. Absorption and Distribution: Gipritinib (ASP2215) was orally administered to nude mice carrying MV4-11 xenografts, and the results showed rapid absorption (Tmax data not provided) and extensive distribution within the xenografts. Following a single oral administration (1 mg/kg, 6 mg/kg, 10 mg/kg), plasma and tumor concentrations were determined by HPLC-MS/MS, and higher drug concentrations were observed within the tumor; [2]

Hepatotoxicity
Elevated serum transaminase levels are common during gipritinib treatment, occurring in 78% of patients, with 12% of these patients having transaminase levels exceeding 5 times the upper limit of normal. Clinical use of gipritinib is limited, and no cases of acute liver injury with symptomatic or jaundice have been identified. Due to limited clinical experience with FLT3 inhibitors, the likelihood of liver injury is unclear. Probability score: E (Unproven, but suspected cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the use of gipritinib during lactation. Gipritinib binds to plasma proteins at a rate of 94%, therefore its concentration in breast milk may be low; however, its half-life is relatively long, approximately 113 hours. The manufacturer recommends discontinuing breastfeeding during gipritinib treatment and for 2 months after the last dose.
◉ Effects on breastfed infants
No relevant published information was found as of the revision date.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

---

Protein binding
It has been reported that giretinib is highly bound to plasma proteins, accounting for 94% of the dose. Based on this proportion, the main protein bound is serum albumin.

---

1. In vivo toxicity in mouse models: No significant toxicity was observed in mouse models treated with giretinib (ASP2215). In the MV4-11 xenograft model, after 28 days of treatment with giretinib (1-10 mg/kg, once daily), there was no significant change in mouse body weight, indicating that it was well tolerated [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 belongs to the pyrazine class of compounds. Its structure is pyrazine-2-carboxamide, with substituted {3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}nitroso, (oxecyclohexane-4-yl)nitroso, and ethyl groups at positions 3, 5, and 6, respectively. It is a potent inhibitor of the FLT3 and AXL tyrosine kinase receptors (IC50 values of 0.29 nM and 0.73 nM, respectively). Gilteritinibib has been approved by the FDA for the treatment of patients with acute myeloid leukemia harboring FLT3 gene mutations. It exhibits dual effects, including inducing apoptosis, inhibiting EC 2.7.10.1 (receptor protein tyrosine kinase), and antitumor activity. It is an N-methylpiperazine compound, belonging to the piperidine, secondary amino, monomethoxybenzene, pyrazine, primary carboxamide, aromatic amine, and oxecyclohexane classes. Gilteritinib, also known as ASP2215, is a small molecule FLT3 tyrosine kinase inhibitor with higher selectivity and potency compared to other drugs in its class. It is a pyrazine carboxamide derivative with high selectivity for FLT3, avoiding c-Kit-mediated myelosuppression observed in other therapies. Developed by Astellas Pharma, gilteritinib was approved by the U.S. Food and Drug Administration (FDA) on November 28, 2018. The drug was initially designed as an orphan drug and received Fast Track and Priority Review designations. Gilteritinib is an oral small molecule FMS-like tyrosine kinase 3 (FLT3) inhibitor used to treat acute myeloid leukemia with FLT3 mutations; it is an anti-tumor drug. Elevated serum transaminases occur moderately during gilteritinib treatment and may cause rare, clinically significant acute liver injury.
Gidateinib 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), with potential antitumor activity. After administration, gidateinib 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 that overexpress 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: Gidateinib fumarate (salt form).

---

Drug Indications
Gipretinib is indicated for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) who have an FLT3 mutation detected by an FDA-approved assay. This indication has been expanded to include companion diagnostics, such as the LeukoStrat CDx FLT3 mutation assay, for use in combination with gipretinib. Acute myeloid leukemia is a rapidly progressing cancer that affects the blood and bone marrow. This disease leads to a reduction in the number of normal blood cells, requiring continuous blood transfusions.
Xospata is indicated for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with an FLT3 mutation, as a monotherapy.

---

Mechanism of Action
Gipretinib is a potent and selective inhibitor that inhibits two mutations in the FLT3 receptor: internal tandem repeat (ITD) and tyrosine kinase domain (TKD). Gipretinib also inhibits AXL and ALK tyrosine kinases. FLT3 and AXL are molecules involved in cancer cell growth. The mechanism of action of gipretinib is to inhibit the phosphorylation of FLT3 and its downstream targets (such as STAT5, ERK, and AKT). Interest in FLT3 transmembrane tyrosine kinases has grown significantly since studies reported that approximately 30% of acute myeloid leukemia patients have mutant-activated FLT3 subtypes. Furthermore, ITD mutations are associated with poor patient prognosis, while TKD mutations lead to resistance mechanisms against FLT3 tyrosine kinase inhibitors, and AXL tyrosine kinases tend to lead to resistance mechanisms against chemotherapy.

---

1. Background and Classification: Gipretinib (ASP2215) is a novel type I tyrosine kinase inhibitor with high selectivity for both 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 plays a role in FLT3 activation and AML pathogenesis [2]. 2. Mechanism of action: Giratinib (ASP2215) exerts anti-leukemic activity by inhibiting phosphorylation of mutant FLT3 and its downstream signaling pathways (AKT, ERK, STAT5), thereby inhibiting AML cell proliferation and inducing tumor regression in vivo [2]. 3. Clinical application potential: Preclinical studies have shown that giratinib (ASP2215) has significant anti-leukemic activity in FLT3 mutant AML cell lines and mouse models (xenograft and intramedullary transplantation models) without significant toxicity. These results suggest that giratinib may be an important next-generation FLT3 inhibitor for the treatment of FLT3 mutation-positive AML [2]. 4. Computer model: A computer model of the binding of giratinib (ASP2215) to wild-type FLT3 was constructed. Gilteritinibib was modeled as a ball-and-stick model, with atoms colored by element type and the protein surface colored by electrostatic potential. Gated residue F691 was highlighted, and the binding mode of Gilteritinibib to FLT3 was visualized [2].

C29H44N8O3

552.71

552.353

C, 63.02; H, 8.02; N, 20.27; O, 8.68

1254053-43-4

Gilteritinib hemifumarate;1254053-84-3;Gilteritinib-d8;2377109-74-3

49803313

Yellow solid powder

1.2±0.1 g/cm3

696.9±55.0 °C at 760 mmHg

375.3±31.5 °C

0.0±2.2 mmHg at 25°C

1.627

4.35

3

10

9

40

785

0

O(C([H])([H])[H])C1C([H])=C(C([H])=C([H])C=1N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H])N([H])C1C(C(N([H])[H])=O)=NC(C([H])([H])C([H])([H])[H])=C(N=1)N([H])C1([H])C([H])([H])C([H])([H])OC([H])([H])C1([H])[H]

GYQYAJJFPNQOOW-UHFFFAOYSA-N

InChI=1S/C29H44N8O3/c1-4-23-28(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/h5-6,19-20,22H,4,7-18H2,1-3H3,(H2,30,38)(H2,31,32,34)

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

ASP-2215; ASP2215; ASP 2215; Gilteritinib; 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 (18.09 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 0.2mg/ml

---

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