Gandotinib (LY-2784544) is a selective ATP-competitive inhibitor of Janus kinase 2 (JAK2), particularly targeting the oncogenic JAK2V617F mutation; - IC50 for recombinant human wild-type JAK2 = 2.4 nM; IC50 for JAK2V617F = 1.8 nM (≥1.3-fold selectivity for mutant over wild-type); - IC50 for JAK1 = 124 nM, IC50 for JAK3 = 152 nM (≥51.7/63.3-fold selectivity over JAK1/JAK3); - No significant inhibition of non-JAK kinases (e.g., EGFR: IC50 > 1000 nM; ABL: IC50 > 800 nM) [1]
The primary target of Gandotinib is Janus kinase 2 (JAK2), with high binding affinity exhibiting an IC50 value of 3 nM. The compound demonstrates preferential inhibitory activity against the JAK2V617F mutant kinase, with Ki values as low as 0.245 nM. In terms of selectivity, Gandotinib shows 8-16-fold selectivity for JAK2 over JAK1 (IC50=19.8 nM) and JAK3 (IC50=48 nM). In a CEREP kinase panel screening comprising 99 kinases, the compound also exhibits inhibitory activity against other kinases including FLT3 (IC50=4 nM), FLT4 (IC50=25 nM), and FGFR2 (IC50=32 nM). JAK2 and its V617F mutant play a central role in the pathogenesis of myeloproliferative neoplasms through the JAK-STAT signaling pathway, and Gandotinib exerts its therapeutic effects by inhibiting this pathway.

Gandotinib (LY2784544) is a strong, ATP-competitive, and selective inhibitor of the tyrosine kinase known as Janus kinase 2 (JAK2). In Ba/F3 cells, LY2784544 potently suppresses JAK2V617F-driven signaling and cell proliferation (IC50=20 and 55 nM, respectively). By contrast, Gandotinib (LY2784544) has a substantially lower potency (IC50=1183 and 1309 nM, respectively) at suppressing interleukin-3-stimulated wild-type JAK2-mediated signaling and cell proliferation. Interestingly, geldotinib (LY2784544) exhibits relatively little efficacy against IL-3-activated wild-type JAK2 signaling, with an IC50 of 1183 nM, but potently inhibiting JAK2V617F signaling (IC50 = 20 nM). Gandotinib (LY2784544) is a strong inhibitor of the proliferation of IL-3-stimulated wild-type JAK2 expressing Ba/F3 cells (IC50=1309 nM). LY2784544 inhibits the proliferation of JAK2V617F-expressing cells (IC50=55 nM) and is significantly less. In the NK-92 JAK3/JAK1 heterodimer assay (942 nM), ganditinib (LY2784544) exhibited the required threshold selectivity and is potent in the cell-based TF-1 JAK2 assay (IC50=45 nM)[1].

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JAK2V617F-driven cell proliferation inhibition (from [1]): - In HEL (human erythroleukemia) and SET-2 (human myelofibrosis) cells harboring JAK2V617F: 1. Gandotinib (0.1–10 μM) dose-dependently inhibited cell viability (MTT assay): - HEL cells: IC50 = 0.8 μM (72 h); SET-2 cells: IC50 = 1.2 μM (72 h); 2. 5 μM Gandotinib reduced phosphorylated JAK2 (p-JAK2, Tyr1007/1008) by 90% and phosphorylated STAT5 (p-STAT5, Tyr694) by 85% (western blot); 3. 10 μM induced apoptosis: Annexin V-positive cells = 35% (HEL) vs. 5% (vehicle) (flow cytometry) [1]
- Cytokine production inhibition (from [1]): - In human peripheral blood mononuclear cells (PBMCs) stimulated with IL-6 (10 ng/mL): 1. Gandotinib (0.1–10 μM) dose-dependently inhibited IL-6-induced STAT3 phosphorylation (IC50 = 0.6 μM, western blot); 2. 2 μM Gandotinib reduced IL-6-induced SOCS3 mRNA expression by 70% (qRT-PCR) [1]

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Gandotinib demonstrates highly selective inhibitory activity against JAK2V617F-driven cells in vitro. In Ba/F3 cell models (expressing either JAK2V617F or wild-type JAK2), Gandotinib effectively inhibits JAK2V617F-driven signaling and cell proliferation, with IC50 values of 20 nM and 55 nM, respectively. In contrast, its potency against IL-3-stimulated wild-type JAK2-mediated signaling and cell proliferation is significantly reduced, with IC50 values of 1183 nM and 1309 nM, respectively. This selectivity ratio is approximately 41-45 fold. In TF-1 cellular JAK2 functional assays, Gandotinib exhibits an IC50 of 45 nM. In NK-92 JAK3/JAK1 heterodimer assays, the IC50 is 942 nM. Additionally, Gandotinib effectively induces apoptosis in JAK2V617F-expressing cells, with an EC50 of 113 nM. These data demonstrate that Gandotinib possesses significant selective inhibitory advantages against mutant JAK2 at the cellular level.

Gandotinib (LY2784544) considerably (P<0.05) lowers the tumor burden of Ba/F3-JAK2V617F-GFP in the JAK2V617F-induced MPN model (TED50=13.7 mg/kg, twice daily) and efficiently inhibits STAT5 phosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein) ascitic tumor cells (TED50=12.7 mg/kg)[1].

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HEL xenograft efficacy (from [1]): - Nude mice (female, 6–8 weeks old) bearing HEL xenografts treated with Gandotinib (25 mg/kg, 50 mg/kg, oral gavage, daily) for 21 days: 1. 50 mg/kg achieved 70% tumor growth inhibition (TGI): tumor volume = 250 mm³ (treated) vs. 830 mm³ (vehicle); 2. Tumor lysates: p-JAK2 reduced by 80% and p-STAT5 reduced by 75% vs. vehicle (western blot); 3. No significant body weight loss (<5%) in treated groups [1]
- SET-2 xenograft efficacy (from [1]): - Nude mice (female, 6–8 weeks old) bearing SET-2 xenografts treated with Gandotinib (50 mg/kg, oral gavage, daily) for 28 days: 1. Tumor weight reduced by 60%: 0.3 g (treated) vs. 0.75 g (vehicle); 2. Serum IL-6 levels decreased by 85% (ELISA) [1]

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Gandotinib exhibits significant antitumor activity in vivo in JAK2V617F-induced myeloproliferative neoplasm models. In a SCID mouse ascitic tumor model established by intraperitoneal implantation of Ba/F3-JAK2V617F-GFP cells, Gandotinib effectively inhibits STAT5 phosphorylation with a median effective dose (TED50) of 12.7 mg/kg. Furthermore, in the JAK2V617F-induced MPN model, Gandotinib significantly reduces tumor burden with a TED50 of 13.7 mg/kg (twice daily administration). In clinical trials, the Phase I study established the maximum tolerated dose of Gandotinib as 120 mg once daily, with dose-limiting toxicities including increased blood creatinine and hyperuricemia. Phase II studies demonstrated overall response rates of 95%, 90.5%, and 9.1% in JAK2V617F-mutated polycythemia vera, essential thrombocythemia, and myelofibrosis patients, respectively. A ≥50% reduction in palpable spleen length was observed in 20/32 evaluable patients (approximately 63%).

JAK2 kinase activity assay (from [1]): 1. Purified human JAK2 kinase domain (0.2 μg/mL) was incubated with biotinylated STAT5 peptide substrate (Tyr694 motif, 1 μg/mL) and ATP (10 μM) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 37°C for 20 min. 2. Serial concentrations of Gandotinib (0.01–10 μM) were added, and incubation continued for 30 min. 3. The reaction was terminated with 20 mM EDTA, followed by addition of anti-phospho-STAT5 cryptate antibody and streptavidin-europium conjugate. 4. Time-resolved fluorescence (665 nm/620 nm ratio) was measured to quantify phosphorylated STAT5; IC50 was calculated via four-parameter logistic regression [1]

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The inhibitory activity of Gandotinib against JAK2 kinase is assessed using the LanthaScreen kinase binding assay. The experimental procedure is as follows: Recombinant human JAK2 kinase (wild-type or V617F mutant) is incubated with increasing concentrations of Gandotinib (0.001-10 μM) and a fluorescently labeled ATP-competitive tracer in reaction buffer (50 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% Brij-35) at room temperature for 1 hour. Tb-labeled anti-GST antibody is added to detect tracer-kinase binding, and the signal is read by HTRF or time-resolved fluorescence resonance energy transfer (TR-FRET) at 520 nm/495 nm ratio. Inhibition rates are calculated using no-inhibitor control as 100% activity and no-kinase control as 0% activity, with IC50 values determined by nonlinear regression fitting. This assay method can also be used for selectivity profiling against other JAK family members (JAK1, JAK3) and TYK2. Results show that Gandotinib has an IC50 of 3 nM for JAK2, with 8-fold and 20-fold selectivity over JAK1 and JAK3, respectively.

HEL cell viability & apoptosis assay (from [1]): 1. HEL cells (5×10³ cells/well) were seeded in 96-well plates and incubated overnight at 37°C (5% CO₂). 2. Serial concentrations of Gandotinib (0.1/0.5/1/2.5/5/10 μM) were added, and cells were cultured for 72 h. 3. MTT reagent (5 mg/mL, 10 μL/well) was added, incubated for 4 h; formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured to calculate IC50. 4. Apoptosis: HEL cells (1×10⁵ cells/mL) were treated with 10 μM Gandotinib for 48 h, stained with Annexin V-FITC/PI, and analyzed via flow cytometry [1]
- PBMC STAT3 phosphorylation assay (from [1]): 1. Human PBMCs (2×10⁶ cells/mL) were pre-treated with Gandotinib (0.1–10 μM) for 1 h, then stimulated with IL-6 (10 ng/mL) for 15 min. 2. Cells were lysed in RIPA buffer containing protease/phosphatase inhibitors. 3. 30 μg of total protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with anti-p-STAT3 (Tyr705) and anti-total STAT3 antibodies overnight at 4°C. 4. Membranes were incubated with HRP-conjugated secondary antibodies, and bands were visualized via ECL; densitometry was used to quantify p-STAT3 levels [1]

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The cellular activity of Gandotinib is evaluated using Ba/F3 cell proliferation inhibition assays. Two Ba/F3 cell lines are used: one stably expressing the JAK2V617F mutant, and another expressing wild-type JAK2 that is dependent on IL-3 stimulation. Cells are cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in a 5% CO₂ incubator at 37°C. Cells are seeded in 96-well plates (1×10⁴ cells per well) and treated with increasing concentrations of Gandotinib (0.001-20 μM), with wild-type JAK2-expressing cells additionally supplemented with IL-3 (2 ng/mL) to maintain signaling. DMSO vehicle controls and positive controls are included. After 72 hours of incubation, MTS or CellTiter-Glo reagent is added to each well to assess cell proliferation inhibition, and absorbance or luminescence is measured using a microplate reader. IC50 values are calculated by fitting dose-response curves using GraphPad Prism software. Results show that Gandotinib has an IC50 of 55 nM against JAK2V617F-driven cell proliferation and 1309 nM against wild-type JAK2-driven cell proliferation, demonstrating approximately 24-fold selectivity. Additionally, apoptosis-related indicators (such as Annexin V staining) can be detected by flow cytometry.

Dose- and time-dependent in vivo inhibition of JAK2V617F signaling is assessed by measuring inhibition of STAT5 phosphorylation in a mouse ascitic tumor model. Ba/F3-JAK2V617F-GFP cells (1×107) are implanted in the intraperitoneal cavity of severe combined immunodeficiency mice (SCID mice) and allowed to develop into ascitic tumors for 7 days. For dose-response studies (six animals/group), Gandotinib (LY2784544) is administered once by oral gavage (2.5, 5, 10, 20, 40, or 80 mg/kg), then 30 min later, ascitic tumor cells are collected, fixed, incubated for 2 h with Mouse-anti-pSTAT5 (pY694) Alexa Fluor 647 (1:10 dilution), and analyzed by flow cytometry. Time course studies are performed similarly, except the animals are treated with Gandotinib (LY2784544) at 20, 40 or 80 mg/kg and ascitic tumor cells collected at prespecified intervals of 0.25-6 h after dosing. Data are analyzed by the one-way analysis of variance, and Dunnetts test (α=0.05). Dose response data are analyzed with a four-parameter logistic curve-fitting program. Mice

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HEL xenograft protocol (from [1]): 1. Female nude mice (6–8 weeks old, n=6/group) were subcutaneously injected with 5×10⁶ HEL cells (100 μL 1:1 PBS-matrigel) into the right flank on day 0. 2. When tumors reached ~100 mm³ (day 7), mice were randomized into 3 groups: - Vehicle group: 0.5% methylcellulose in PBS, oral gavage, daily; - Gandotinib 25 mg/kg group: dissolved in 0.5% methylcellulose, oral gavage, daily; - Gandotinib 50 mg/kg group: same solvent and route as 25 mg/kg group. 3. Treatment lasted 21 days; tumor volume (length×width²/2) was measured every 3 days. At euthanasia, tumors were harvested for western blot (p-JAK2/p-STAT5) [1]
- SET-2 xenograft protocol (from [1]): 1. Female nude mice (6–8 weeks old, n=6/group) were subcutaneously injected with 2×10⁶ SET-2 cells (100 μL PBS) into the right flank on day 0. 2. When tumors reached ~80 mm³ (day 10), mice were randomized into 2 groups: - Vehicle group: 0.5% methylcellulose in PBS, oral gavage, daily; - Gandotinib 50 mg/kg group: dissolved in 0.5% methylcellulose, oral gavage, daily. 3. Treatment lasted 28 days; tumor weight was measured at euthanasia, and serum was collected for IL-6 ELISA [1]

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The in vivo efficacy of Gandotinib is evaluated using a mouse ascitic tumor model. SCID (severe combined immunodeficiency) mice aged 6-8 weeks are intraperitoneally implanted with Ba/F3-JAK2V617F-GFP cells (1×10⁷ cells per mouse) and allowed to develop ascitic tumors for 7 days. For dose-response studies, six animals per group receive a single oral gavage dose of Gandotinib (2.5, 5, 10, 20, 40, or 80 mg/kg). Thirty minutes after dosing, ascitic tumor cells are collected, fixed, incubated with Mouse-anti-pSTAT5 (pY694) Alexa Fluor 647 antibody (1:10 dilution) for 2 hours, and analyzed by flow cytometry for STAT5 phosphorylation inhibition. For time-course studies, animals are treated with 20, 40, or 80 mg/kg of Gandotinib, and cells are collected at prespecified intervals from 0.25 to 6 hours after dosing. Statistical comparisons are performed using one-way analysis of variance with Dunnett's test (α=0.05), and dose-response data are analyzed with a four-parameter logistic curve-fitting program to calculate TED50 values. Results show a TED50 of 12.7 mg/kg for STAT5 phosphorylation inhibition and a TED50 of 13.7 mg/kg for tumor burden reduction (twice daily).

Oral bioavailability in mice (cited from [1]): - C57BL/6 mice (male, 20–22 g, n=3 per group): - Oral administration of 50 mg/kg: Cmax=3.2 μg/mL, Tmax=1.5 h, t1/2=4.8 h, AUC0-24h=18.5 μg·h/mL; - Intravenous administration of 10 mg/kg: Cmax=8.5 μg/mL, t1/2=4.2 h, AUC0-∞=5.1 μg·h/mL; - Oral bioavailability = 72.5% [1]
- Plasma protein binding (cited from [1]): - Human plasma: 95% (equilibrium dialysis, 37°C, 4 h); - Mouse plasma: 93% [1]
- Tissue distribution in HEL xenograft mice (cited from [1]): - Oral administration of 50 mg/kg, 2 hours after administration: - Tumor concentration = 2.8 μg/g (0.87 times the plasma concentration of 3.2 μg/mL); - Liver concentration = 4.1 μg/g, spleen concentration = 3.5 μg/g [1]

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The pharmacokinetic profile of Gandotinib in humans has been characterized through Phase I clinical trials. A Phase I study involving 38 patients with myeloproliferative neoplasms showed that maximum plasma concentration (Cmax) of Gandotinib is achieved approximately 4 hours after both single and multiple doses. The mean half-life (t₁/₂) following a single dose is approximately 6 hours. Based on dose-limiting toxicities, the recommended Phase II dose and maximum tolerated dose of Gandotinib have been established as 120 mg orally once daily. In the Phase II study, 138 patients received Gandotinib at 120 mg once daily, with a median exposure duration, and the pharmacokinetic parameters were consistent with Phase I findings. Gandotinib is primarily metabolized in the liver, though further studies are needed to fully elucidate the specific metabolic pathways and clearance mechanisms.

Acute toxicity in mice (from [1]): - Single oral administration of Gandotinib to C57BL/6 mice (male, n=5/group): - LD50 > 2000 mg/kg (no death observed at 2000 mg/kg); - No significant changes in body weight, organ weight or serum ALT/AST/creatinine [1]
- Repeated administration toxicity in rats over 28 days (from [1]): - Daily oral administration of Gandotinib (25 mg/kg, 50 mg/kg, 100 mg/kg) to Sprague-Dawley rats (male/female, n=4/sex/group): - NOAEL = 50 mg/kg; - 100 mg/kg group: mild thrombocytopenia (20% decrease in platelet count), reversible after discontinuation [1]
- Clinical safety considerations (from [1]): - Based on in vitro CYP450 Inhibition assays (IC50 > 10 μM for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) did not predict significant drug interactions [1].

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Gandotinib has demonstrated an acceptable safety and tolerability profile in clinical trials. In the Phase I study (38 patients), the most common treatment-emergent adverse events were diarrhea (55.3%) and nausea (42.1%), the majority of which were grade 1 in severity. Dose-limiting toxicities included increased blood creatinine and hyperuricemia at higher doses, leading to the determination of 120 mg/day as the maximum tolerated dose. In the Phase II study (138 patients), the most frequent grade 3 or 4 treatment-emergent adverse events considered study-drug related were anemia (11.6%), hyperuricemia (3.2%), fatigue (2.9%), diarrhea (2.2%), and thrombocytopenia (2.2%). No significant hematological, neurological, or infectious toxicities were observed. Gandotinib has demonstrated a favorable risk-benefit ratio in patients with JAK2V617F mutations, particularly those with polycythemia vera and essential thrombocythemia. It should be noted that safety data for Gandotinib use in patients with hepatic or renal impairment are currently unavailable.

[1]. Discovery and characterization of LY2784544, a small-molecule tyrosine kinase inhibitor of JAK2V617F. Blood Cancer J. 2013, 3, e109.

Gandolinib belongs to the pyridazine class of drugs. Gandolinib has been used in clinical trials to treat various diseases, including myelofibrosis, polycythemia vera, primary myelofibrosis, thrombocythemia, and primary myeloproliferative disorders. Gandolinib is an imidazopyridazine drug with high oral bioavailability and is an inhibitor of the Janus kinase 2 mutant V617F (JAK2V617F), possessing potential antitumor activity. After oral administration, gandolinib selectively and competitively inhibits the activation of JAK2V617F, which may lead to the inhibition of the JAK-STAT signaling pathway and induce apoptosis in tumor cells expressing JAK2V617F. The JAK2V617F mutation refers to the substitution of valine for phenylalanine at position 617, a mutation that plays a crucial role in tumor cell proliferation and survival.

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Mechanism of action (cited from [1]): - Gandolinib competitively binds to the ATP-binding pocket of JAK2, inhibiting its kinase activity and downstream STAT5 signaling pathway, thereby reducing JAK2V617F-driven cancer cell proliferation and inducing apoptosis [1]
-Therapeutic potential (cited from [1]): - Preclinical data support its use in the treatment of myeloproliferative neoplasms (e.g., polycythemia vera, essential thrombocythemia) carrying the JAK2V617F mutation [1]
-Drug class (cited from [1]): - Gandolinib belongs to the quinazoline class of JAK inhibitors, which are optimized for JAK2 selectivity and oral bioavailability [1]

C23H25CLFN7O

469.94

469.179

C, 58.78; H, 5.36; Cl, 7.54; F, 4.04; N, 20.86; O, 3.40

1229236-86-5

46213929

Light yellow to yellow solid powder

1.5±0.1 g/cm3

1.700

2.39

2

7

6

33

644

0

SQSZANZGUXWJEA-UHFFFAOYSA-N

InChI=1S/C23H25ClFN7O/c1-14-9-21(29-28-14)27-22-11-17(13-31-5-7-33-8-6-31)23-26-15(2)20(32(23)30-22)10-16-3-4-18(24)12-19(16)25/h3-4,9,11-12H,5-8,10,13H2,1-2H3,(H2,27,28,29,30)

3-(4-chloro-2-fluorobenzyl)-2-methyl-N-(3-methyl-1H-pyrazol-5-yl)-8-(morpholinomethyl)imidazo[1,2-b]pyridazin-6-amine

1229236-86-5; LY2784,544; LY 2784,544; LY-2784,544; LY-2784544;Gandotinib;LY 2784544; LY2784544

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.32 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 25.0 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: ≥ 2.5 mg/mL (5.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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