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)

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].

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].

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].

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].

Absorption, Distribution and Excretion
In preclinical trials, the maximal plasma concentration of gilteritinib was observed 2 hours after oral administration and followed by a maximal intratumor concentration after 4-8 hours. The maximum concentration, as well as the AUC, were modified correspondingly with the dose and were reported to be 374 ng/ml and 6943 ng.h/ml, respectively. The steady-state plasma level is reached within 15 days of dosing with an approximate 10-fold bioaccumulation. In a fasted state in humans, the tmax is reported to be of 4-6 hours. The Cmax and AUC were decreased by 26% and 10% respectively by the co-ingestion of a high-fat meal with a tmax delay of 2 hours.
From the administered dose, gilteritinib is mainly excreted in feces which represents 64.5% of the administered dose while 16.4% is recovered in urine either as the unchanged drug or as its metabolites.
The estimated apparent central and peripheral volume of distribution is 1092 L and 1100 L respectively. This value indicated an extensive tissue distribution.
The estimated clearance of gilteritinib is 14.85 L/h.

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Metabolism / Metabolites
Gilteritinib is primarily metabolized in the liver by the activity of CYP3A4. Its metabolism is driven by reactions of N-dealkylation and oxidation which forms the metabolite M17, M16 and M10. From the plasma concentration, the major form is the unchanged drug.

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Biological Half-Life
The reported median half-life of gilteritinib was of approximate 45-159 hours.

Hepatotoxicity
Elevations in serum aminotransferase levels are common during gilteritinib therapy occurring in 78% of patients and rising above 5 times the upper limit of the normal range in 12%. Gilteritinib has had limited clinical use but has not been linked to instances of acute liver injury with symptoms or jaundice. Because of the limited clinical experience with the use of FLT3 inhibitors, their potential for causing liver injury is not well defined.
Likelihood score: E* (unproved but suspected cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on gilteritinib during breastfeeding. It is 94% bound to plasma proteins, so amounts in milk are likely to be low; however, it has a long half-life of about 113 hours. The manufacturer recommends that breastfeeding be discontinued during gilteritinib therapy and for 2 months after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Gilteritinib is reported to be highly bound to plasma proteins, representing 94% of the dose. From this ratio, the main protein-bound is serum albumin.

[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.

Gilteritinib is a member of the class of pyrazines that is pyrazine-2-carboxamide which is substituted by {3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}nitrilo, (oxan-4-yl)nitrilo and ethyl groups at positions 3,5 and 6, respectively. It is a potent inhibitor of FLT3 and AXL tyrosine kinase receptors (IC50 = 0.29 nM and 0.73 nM, respectively). Approved by the FDA for the treatment of acute myeloid leukemia in patients who have a FLT3 gene mutation. It has a role as an apoptosis inducer, an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor and an antineoplastic agent. It is a N-methylpiperazine, a member of piperidines, a secondary amino compound, a monomethoxybenzene, a member of pyrazines, a primary carboxamide, an aromatic amine and a member of oxanes.
Gilteritinib, also known as ASP2215, is a small molecule part of the FLT3 tyrosine kinase inhibitors that presented a greater selectivity and potency when compared with other agents from this group. It is a pyrazinecarboxamide derivative that showed high selectivity to FLT3 preventing the c-Kit -driven myelosuppression observed in other therapies. Gilteritinib was developed by Astellas Pharma and FDA approved on November 28, 2018. This drug was approved after being designed as an orphan drug with a fast track and priority review status.
Gilteritinib is an orally available small molecule inhibitor of FMS-like tyrosine kinase 3 (FLT3) which is used as an antineoplastic agent in the treatment of acute myeloid leukemia with FLT3 mutations. Gilteritinib is associated with a moderate rate of serum aminotransferase elevations during therapy and is suspected to cause rare instances of clinically apparent acute liver injury.
Gilteritinib is an orally bioavailable inhibitor of the receptor tyrosine kinases (RTKs) FMS-like tyrosine kinase 3 (FLT3; STK1; FLK2), AXL (UFO; JTK11), anaplastic lymphoma kinase (ALK; CD246), and leukocyte receptor tyrosine kinase (LTK), with potential antineoplastic activity. Upon administration, gilteritinib binds to and inhibits both the wild-type and mutated forms of FLT3, AXL, ALK and LTK. This may result in an inhibition of FLT3-, AXL-, ALK-, and LTK-mediated signal transduction pathways and reduced proliferation in cancer cells that overexpress these RTKs. FLT3, AXL, ALK, and LTK, which are overexpressed or mutated in a variety of cancer cell types, play key roles in tumor cell growth and survival.
See also: Gilteritinib Fumarate (has salt form).

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Drug Indication
Gilteritinib is indicated for the treatment of adult patients who have relapsed or refractory acute myeloid leukemia with an FLT3 mutation detected by an FDA-approved test. This indication was expanded for a companion diagnostic to include use with gilteritinib such as the LeukoStrat CDx FLT3 Mutation Assay. Acute myeloid leukemia is cancer that impacts the blood and bone marrow with a rapid progression. This condition produces low numbers of normal blood cells and the requirement of continuous need for transfusions.
Xospata is indicated as monotherapy for the treatment of adult patients who have relapsed or refractory acute myeloid leukaemia (AML) with a FLT3 mutation. ,

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Mechanism of Action
Gilteritinib is a potent selective inhibitor of both of the mutations, internal tandem duplication (ITD) and tyrosine kinase domain (TKD), of the FLT3 receptor. In the same note, gilteritinib also inhibits AXL and ALK tyrosine kinases. FLT3 and AXL are molecules involved in the growth of cancer cells. The activity of gilteritinib permits an inhibition of the phosphorylation of FLT3 and its downstream targets such as STAT5, ERK and AKT. The interest in FLT3 transmembrane tyrosine kinases was raised when studies reported that approximately 30% of the patients with acute myeloid leukemia presented a mutationally activated isoform. As well, the mutation ITD is associated with poor patient outcomes while the mutation TKD produces a resistance mechanism to FLT3 tyrosine kinase inhibitors and the AXL tyrosine kinase tends to produce a resistance mechanism to chemotherapies.

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.

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

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