AKN-028 is a novel tyrosine kinase inhibitor. Its primary identified target is FMS-like receptor tyrosine kinase 3 (FLT3), with an IC50 of 6 nM for FLT3 enzyme inhibition. [1]
In a panel screen of 320 kinases, at a concentration of 1 µM, AKN-028 showed less than 20% remaining enzyme activity for FLT3, CDC-like kinase 1 (CLK1), and Ribosomal Protein S6 Kinase (RPS6K). [1]
Further characterization yielded IC50 values: CLK1 = 140 nM, RPS6K = 220 nM, Fibroblast growth factor receptor 2 (FGFR2) = 1200 nM, and Vascular endothelial growth factor receptor 2 (VEGFR2) = 520 nM. [1]
In cellular assays, AKN-028 was shown to inhibit autophosphorylation of FLT3 (wild-type, ITD, and TKD mutants) and KIT. [1]

In a dose-dependent manner, AKN-028 (0.1 nM-100 μM; 15 hours) suppresses FLT3 and KIT autophosphorylation in mouse embryonic fibroblasts and human acute megakaryocytic leukemia M07 cells [1]. AML cell lines are cytotoxic to AKN-028 (10 μM; 72 hours; malignant cell lines) and AKN-028 causes apoptosis in the AML cell line MV4-11 [1].

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AKN-028 inhibited the FLT3 enzyme in a dose-dependent manner with an IC50 of 6 nM. [1]
In a panel of 17 cell lines, AKN-028 showed cytotoxic activity against all five AML cell lines tested. The highest sensitivity was observed in FLT3-ITD-mutated cell lines MV4-11 and MOLM-13, with IC50 values < 50 nM. Other AML cell lines (Kasumi-1, HL-60, KG1a) showed IC50 values ranging from 0.5 to 6 µM. Almost no cytotoxic effect was observed in the solid tumor cell lines HeLa, ACHN, and NCI-H69. [1]
In the MV4-11 AML cell line, AKN-028 at 10 µM induced apoptosis, evidenced by the activation of caspase 3. [1]
In primary AML patient samples (n=15), AKN-028 induced a clear dose-dependent cytotoxic response with a mean IC50 of 1 µM. No significant correlation was observed between the cytotoxic response and FLT3 mutation status, allelic burden, or quantitative FLT3 expression levels. [1]
In an additional 11 primary AML samples, AKN-028 demonstrated a consistent dose-dependent response, reaching IC50 in all samples tested. In contrast, the specific FLT3 inhibitor AC220 showed variable responses, often with flat dose-response curves that plateaued. [1]
In combination studies with cytarabine or daunorubicin in MV4-11 cells, median-effect analysis revealed a sequence-dependent synergy. Synergy (mean CI < 0.7) was observed when cells were treated with chemotherapy simultaneously or 24 hours prior to AKN-028. Antagonism was observed when cells were pretreated with AKN-028 for 24 hours before adding chemotherapy. [1]
Using a radiometric protein kinase assay, AKN-028 did not directly inhibit the downstream targets AKT 1, 2, 3 or ERK 1, 2 at a concentration of 1 µM. [1]

Primary AML and MV4-11 cell proliferation in mice is inhibited by AKN-028 (15 mg/kg; IV; twice daily for 6 days; male C57 black mice with MV4-11 xenografts) [1].

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In the mouse hollow-fiber model, NMRI mice were administered AKN-028 subcutaneously twice daily at a dose of 15 mg/kg for 6 days. This treatment significantly inhibited net growth of the MV4-11 cell line in vivo. Of the two primary AML samples tested, a significant inhibition of net growth was observed in one sample (UPN26), while the other (UPN25) showed very modest growth with no significant inhibition. [1]
No major toxicity was observed in the animals during the hollow-fiber experiment. Body weight and hematological profiles (white blood cell count, red blood cell count, hemoglobin, hematocrit, and platelet count) were not significantly affected. [1]

FLT3 Enzyme Inhibition Assay: The inhibitory effect of AKN-028 on the FLT3 enzyme was evaluated using an immobilized metal ion affinity-based fluorescence polarization (IMAP) technique. The assay was performed on the tyrosine kinase domain of FLT3. [1]
Kinase Panel Screen: The kinase inhibitory profile of AKN-028 at a concentration of 1 µM was evaluated over a panel of 320 kinases. This profiling was performed using a radiometric protein kinase assay. The assay was carried out in a 50 µL reaction volume using FlashPlates. The percentage of remaining kinase activity was determined after a 60-minute incubation with the test compound. Reference compounds AC220 and staurosporine were used for comparison. [1]
Radiometric Protein Kinase Assay for Downstream Targets: The inhibition of AKT 1, 2, 3 and ERK 1, 2 was measured by a radiometric protein kinase assay. The percentage of kinase activity inhibition was determined after a 60-minute incubation with 1 µM of AKN-028 or the reference compounds. [1]

Western Blot Analysis[1]
Cell Types: mouse embryonic fibroblasts overexpressing FLT-wt, FLT3-TKD or FLT3-ITD and human acute megakaryocytic leukemia M07 cells overexpressing KIT
Tested Concentrations: 0.1 nM-100 μM
Incubation Duration:15 hrs (hours)
Experimental Results: FLT3 is inhibited and KIT autophosphorylated.

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Cytotoxicity assay[1]
Cell Types: Tumor cell line
Tested Concentrations: 10 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: MV4-11 and MOLM-13 had the highest cytotoxic activity (IC50<50 nM), followed by the other three AML cell lines (IC50=0.5-6 μM).

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Cytotoxicity Assay (FMCA): The cytotoxic activity of AKN-028 was evaluated using the fluorometric microculture cytotoxicity assay (FMCA). Cells were seeded into drug-prepared microplates at varying densities. After a 72-hour incubation period, the density of living cells was assessed. Results were presented as a survival index (%), defined as the fluorescence in test wells as a percentage of control cultures. IC50 values were determined from log concentration-effect curves using nonlinear regression analysis. This assay was used for cell line panels and primary patient samples. For the MOLM-13 cell line, cytotoxic activity was analyzed by Alamar Blue assay. [1]
Apoptosis Assay: The cell-death characteristics of AKN-028 were studied by measuring caspase-3 activation. MV4-11 cells were seeded into 96-well optic plates and treated with 10 µM of the compound. A probe that stains cells with active caspase 3 was added to each well. A caspase-3 inhibitor was used as a control. Cells were analyzed over 48 hours using a live-cell imaging instrument. Etoposide was used as a positive control. [1]
Phospho-ELISA for Autophosphorylation: The inhibition of FLT3 (wild-type, ITD, TKD) and KIT autophosphorylation by AKN-028 was assessed via phospho-ELISA. Cells overexpressing these targets were incubated for 5 minutes with eight concentrations of the compound. Following cell lysis, phosphorylation levels were determined using a sandwich ELISA. Sunitinib and AC220 were used for comparison. [1]
Western Blot Analysis: Inhibition of FLT3 autophosphorylation in MV4-11 cells after exposure to AKN-028 for 15 hours was evaluated by western blot analysis. [1]
Combination Study: The combination of AKN-028 with cytarabine or daunorubicin was studied in MV4-11 cells using a fixed molar ratio (cytarabine:AKN-028 = 2.5:1; daunorubicin:AKN-028 = 1:20). Drugs were added in three different sequences: (I) pre-treatment with chemotherapy for 24 hours, followed by AKN-028; (II) simultaneous treatment; (III) pre-treatment with AKN-028 for 24 hours, followed by chemotherapy. Cytotoxic activity was assessed by the FMCA after a total incubation time of 72 hours. Drug interactions were analyzed using median-effect analysis with CalcuSyn software, and the combination index (CI) was calculated. A CI < 0.7 indicated synergy, CI ≈ 1 indicated additivity, and CI > 1.45 indicated antagonism. [1]
Quantitative FLT3 Expression Analysis: Total RNA was isolated from primary AML cells and reverse-transcribed to cDNA. FLT3 mRNA transcripts were quantified by quantitative real-time reverse transcriptase-PCR (qRT-PCR). A standard curve for FLT3 was created using a plasmid containing wild-type FLT3. GUSB was used as a reference gene. The amount of FLT3 transcripts was expressed as a ratio of FLT3 copy number relative to 100 GUS copies. [1]

Animal/Disease Models: Male C57 black mice with MV4-11 xenografts [1]
Doses: 15 mg/kg
Route of Administration: subcutaneous injection; twice (two times) daily for 6 days
Experimental Results: Inhibited tumor growth without affecting body weight .

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Hollow-Fiber Mouse Model for In Vivo Efficacy:** The in vivo activity of AKN-028 was assessed in the hollow-fiber mouse model. NMRI male mice were implanted subcutaneously with hollow fibers containing either MV4-11 cells or primary AML cells (from patients UPN25 and UPN26). AKN-028 was administered subcutaneously twice daily (2×) at a dose of 15 mg/kg. A vehicle-only group served as control. The treatment was given for 6 days, with eight animals per group and three fibers per animal. After 6 days, the fibers were extracted and the living cell mass was assessed using the MTT assay. [1]
**Pharmacokinetic Study:** The pharmacokinetic properties of AKN-028 were evaluated in male C57 black mice. The details of this study were provided in a supplementary section. The predicted plasma profile in the hollow-fiber experiment was based on these pharmacokinetic data, estimating a target exposure of 1-3 µM. [1]

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Hollow-Fiber Mouse Model for In Vivo Efficacy: The in vivo activity of AKN-028 was assessed in the hollow-fiber mouse model. NMRI male mice were implanted subcutaneously with hollow fibers containing either MV4-11 cells or primary AML cells (from patients UPN25 and UPN26). AKN-028 was administered subcutaneously twice daily (2×) at a dose of 15 mg/kg. A vehicle-only group served as control. The treatment was given for 6 days, with eight animals per group and three fibers per animal. After 6 days, the fibers were extracted and the living cell mass was assessed using the MTT assay. [1]
Pharmacokinetic Study: The pharmacokinetic properties of AKN-028 were evaluated in male C57 black mice. The details of this study were provided in a supplementary section. The predicted plasma profile in the hollow-fiber experiment was based on these pharmacokinetic data, estimating a target exposure of 1-3 µM. [1]

AKN-028 demonstrated high oral bioavailability in mice. [1]
Plasma protein binding of AKN-028 was determined to be 96.5%. [1]
Based on pharmacokinetic studies in C57 black mice, the predicted area under the curve (AUC) for the hollow-fiber experiment was 6 µM·h, assuming linear pharmacokinetics and no strain difference. The predicted plasma profile indicated approximately 1 hour of exposure above the plasma target concentration (1-3 µM) twice daily. [1]

In the hollow-fiber mouse model, following twice-daily subcutaneous administration of 15 mg/kg AKN-028 for 6 days, no major toxicity was observed. Body weight development of the animals was monitored over time and was not adversely affected. A hematological profile analyzed on day 6 (including white blood cell count, red blood cell count, hemoglobin, hematocrit, and platelet count) showed no significant toxicity compared to the vehicle control group. [1]

[1]. The Novel Tyrosine Kinase Inhibitor AKN-028 Has Significant Antileukemic Activity in Cell Lines and Primary Cultures of Acute Myeloid Leukemia. Blood Cancer J. 2012 Aug 3;2(8):e81.

AKN-028 has been used in clinical trials for the treatment of acute myeloid leukemia. The FLT3/KIT kinase inhibitor AKN-028 is an orally bioavailable protein tyrosine kinase inhibitor that inhibits FMS-associated tyrosine kinase 3 (FLT3; STK1) and stem cell factor receptor (SCFR; KIT), exhibiting potential antitumor activity. The FLT3/KIT kinase inhibitor AKN-028 binds to and inhibits both wild-type and mutant FLT3 and SCFR. This may lead to suppression of tumor cell proliferation in cancer cell types that overexpress these receptor tyrosine kinases.

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AKN-028 (N-3-(1H-indol-5-yl)-5-pyridin-4-yl-pyrazine-2,3-diamine) is a novel small-molecule tyrosine kinase inhibitor identified from a 2-aminopyrazine series. It was found to have potent antileukemic activity in acute myeloid leukemia (AML) cell lines and primary patient cultures, irrespective of FLT3 mutation status. The compound was selected for further development based on its kinase inhibition profile, pharmacokinetic properties, and in vitro activity. The study suggests that its mechanism of action may involve targeting multiple pathways beyond FLT3, including the inhibition of KIT, CLK1, and RPS6K, which could contribute to its efficacy in FLT3 wild-type AML. An international two-part multicenter phase-I study of AKN-028 in patients with AML began in January 2012 (ClinicalTrials.gov NCT01573247). [1] In the hollow-fiber mouse model, following twice-daily subcutaneous administration of 15 mg/kg AKN-028 for 6 days, no major toxicity was observed. Body weight development of the animals was monitored over time and was not adversely affected. A hematological profile analyzed on day 6 (including white blood cell count, red blood cell count, hemoglobin, hematocrit, and platelet count) showed no significant toxicity compared to the vehicle control group. [1]

C17H14N6

302.33

302.128

1175017-90-9

AKN-028 acetate

44177328

Typically exists as solid at room temperature

3.348

3

5

3

23

385

0

C1(N)=NC=C(C2C=CN=CC=2)N=C1NC1C=CC2=C(C=1)C=CN2

JLRIJKVMMZEKDF-UHFFFAOYSA-N

InChI=1S/C17H14N6/c18-16-17(22-13-1-2-14-12(9-13)5-8-20-14)23-15(10-21-16)11-3-6-19-7-4-11/h1-10,20H,(H2,18,21)(H,22,23)

3-N-(1H-indol-5-yl)-5-pyridin-4-ylpyrazine-2,3-diamine

AKN 028 AKN028AKN-028

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)

DMSO : ~125 mg/mL (~413.46 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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