FLT3 (IC50 = 0.64 nM); MERTK (IC50 = 1.3 nM)

MRX-2843 treatment induces a dose-dependent suppression of MERTK phosphorylation in the Kasumi-1 cell line. As little as 10 nM shows signs of decreased phosphorylation, and between 100 and 300 nM almost entirely abolishes MERTK activation. In a similar vein, MRX-2843 treatment of Kasumi-1 cells results in downstream signaling pathways crucial for tumor cell survival and proliferation being inhibited. With an IC50 of 143.5±14.1 nM, MRX-2843 treatment causes a decrease in relative cell numbers, suggesting that it significantly inhibits tumor cell survival and/or proliferation. In NOMO-1 cultures treated with 150 nM or 300 nM MRX-2843, respectively, there are 34.1%±5.6% and 67.1%±2.7% apoptotic and dead cells, compared with 6.8%±0.7% in cultures treated with vehicle (P<0.001). When Kasumi-1 cultures are treated with 50 nM and 100 nM MRX-2843, respectively, colony formation is inhibited by 62.3%±6.4% and 84.1%±7.8% (P<0.01). In NOMO-1 cultures, treatment with 100 nM MRX-2843 inhibits colony formation by 54.8%±18.1% (P<0.001). Treatment with MRX-2843 inhibits downstream signaling via STAT5, ERK1/2, and AKT as well as FLT3 phosphorylation in MOLM-14 cells. Treatment with 50 nM MRX-2843 almost completely inhibits FLT3 activation and its signaling pathways, suggesting that it has a slightly stronger cellular potency against FLT3 than MERTK[1].

MRX-2843 is 78% orally bioavailable at a dose of 3 mg/kg with a C_max of 1.3 μM and a t_1/2 of 4.4 hours. Quizartinib and MRX-2843 both improve the median survival in MOLM-14 parental xenografts when compared to mice treated with a vehicle (172.5 days versus 40 days and 121 days versus 36 days, respectively, P<0.001). Although higher doses of MRX-2843 are not evaluated, quizartinib is more effective than MRX-2843 in this model (P<0.005). Quizaratinib increases survival in MOLM-14:D835Y xenografts relative to mice treated with a vehicle, although the difference is not statistically significant (median survival 45 days vs. 36 days, P<0.001). MRX-2843 treatment increases survival in MOLM-14:F691L xenografts by nearly two times in both NSG and NSGS mice (median survival 87 vs. 44.5 days and 87 vs. 48 days, respectively, P<0.005). MRX-2843 treatment is associated with improved survival compared to quizartinib treatment; however, this difference is only statistically significant in NSG mice[1].

MRX-2843 and the control TKI were synthesized as previously described (20). The amount of kinase inhibitor required for 90% inhibition in vivo was estimated using Michaelis-Menton kinetic equations as previously described. Quizartinib (AC220) was obtained as a 1:4 formulation with hydroxybutenyl-β-cyclodextrin (CD). For in vitro studies, stock solutions were prepared in DMSO, and the DMSO vehicle control concentration was equivalent to the highest dose of test agent for the experiment. For in vivo studies, test agents were either dissolved (MRX-2843) or prepared in a homogeneous suspension (quizartinib and CD) in saline[1].

Murine xenograft models.[1]
NOD.Cg-PrkdcscidIl2rgtm1WjlTg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mice and NOD.Cg-PrkdcscidIl2rgtm1Wj1/SzJ (NSG) mice were purchased from The Jackson Laboratory or bred in-house and maintained under sterile conditions. Established leukemia cell lines or mononuclear cells isolated from samples from patients with AML (1 × 106 to 2.5 × 106 per mouse) were suspended in PBS and injected into the tail veins of NSG or NSGS mice to establish xenografts. All mice were 4–6 months of age at the time of injection and were male, with the exception of the NOMO-1, MOLM-14:D835Y, and MOLM-14:F691L NSG xenografts, which were established in female mice. Myeloblasts were detected in peripheral blood (patient-derived xenografts) or bone marrow (MOLM-14 xenografts) samples after staining with a FITC-conjugated anti-human CD45 Ab. Samples were analyzed by flow cytometry using a Gallios flow cytometer and Kaluza software. After engraftment, the mice were weighed and treated once daily with MRX-2843, quizartinib, or vehicle administered by oral gavage in a volume of 10 ml/kg. When mice appeared ill or lost more than 20% of their body weight, they were euthanized.

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Mertk-null (B6;129-Mertktm1Grl/J) mice were backcrossed with C57BL/6J mice for more than 12 generations (Mertk–/–), and Mertk genotype was verified prior to use. WT C57BL/6J and NSG mice were purchased from the Jackson Laboratory and bred in-house. Young mice (aged 2–4 months) were used, given that Mertk–/– mice have the potential to develop autoimmunity after 6 months of age. Arf-null (Arf–/–) BCR-ABL p185+ murine ALL cells expressing GFP were injected via tail vein, and MRX-2843 or vehicle were administered via oral gavage beginning 1 day or 5 days after transplant. Mice with advanced leukemia (>20% weight loss, tachypnea, hind-limb paralysis, minimal activity) were euthanized, and survival was monitored. Alternatively, when mice developed symptoms of leukemia (17–39 days after transplant), they and their cagemates (1 mouse per treatment group) were euthanized, and bone marrow and spleen were harvested for analysis by flow cytometry. Leukemic burden and immune cell infiltration were quantitated on an LSR II flow cytometer and analyzed using FlowJo version X software.[2]

[1]. The MERTK/FLT3 inhibitor MRX-2843 overcomes resistance-conferring FLT3 mutations in acute myeloid leukemia. JCI Insight. 2016 Mar;1(3):e85630.

[2]. MERTK inhibition alters the PD-1 axis and promotes anti-leukemia immunity. JCI Insight . 2018 Nov 2;3(21):e97941.
[3]. MERTK activation drives osimertinib resistance in EGFR-mutant non-small cell lung cancer. J Clin Invest . 2022 Aug 1;132(15):e150517.

Flt3/MerTK Inhibitor MRX-2843 is an orally bioavailable inhibitor of two receptor tyrosine kinases (RTKs), FMS-like tyrosine kinase-3 (Flt3; CD135; fetal liver kinase-2; Flk2) and tyrosine-protein kinase Mer (MerTK; proto-oncogene c-Mer; Mer), with potential antineoplastic activity. Upon administration, MRX-2843 targets and binds to both Flt3 and MerTK. This prevents ligand-dependent phosphorylation and activation of Flt3 and MerTK, which inhibits the activation of their downstream signaling pathways. This induces apoptosis and inhibits proliferation of Flt3- and/or MerTK-overexpressing tumor cells. Flt3 and MerTK, are overexpressed in certain tumor cell types and play key roles in tumor cell proliferation and survival.

C29H40N6O

488.6675

488.326

C, 71.28; H, 8.25; N, 17.20; O, 3.27

1429882-07-4

1429882-07-4;

89495685

Off-white to light yellow solid powder

1.3±0.1 g/cm3

697.3±65.0 °C at 760 mmHg

375.5±34.3 °C

0.0±2.3 mmHg at 25°C

1.696

2.92

2

6

8

36

681

0

OC1CCC(CC1)N1C=C(C2C=CC(=CC=2)CN2CCN(C)CC2)C2=CN=C(N=C12)NCCC1CC1

LBEJYFVJIPQSNX-UHFFFAOYSA-N

InChI=1S/C29H40N6O/c1-33-14-16-34(17-15-33)19-22-4-6-23(7-5-22)27-20-35(24-8-10-25(36)11-9-24)28-26(27)18-31-29(32-28)30-13-12-21-2-3-21/h4-7,18,20-21,24-25,36H,2-3,8-17,19H2,1H3,(H,30,31,32)

4-[2-(2-cyclopropylethylamino)-5-[4-[(4-methylpiperazin-1-yl)methyl]phenyl]pyrrolo[2,3-d]pyrimidin-7-yl]cyclohexan-1-ol

MRX-2843; UNC 2371; UNC2371A; UNC-2371; UNC-2371A; MRX2843; UNC2371; UNC 2371A; UNC-2371A; 2MT30EHI63; UNII-2MT30EHI63; CHEMBL3326007; Cyclohexanol, 4-(2-((2-cyclopropylethyl)amino)-5-(4-((4-methyl-1-piperazinyl)methyl)phenyl)-7H-pyrrolo(2,3-d)pyrimidin-7-yl)-, trans-; trans-4-(2-((2-Cyclopropylethyl)amino)-5-(4-((4-methyl-1-piperazinyl)methyl)phenyl)-7H-pyrrolo(2,3-d)pyrimidin-7-yl)cyclohexanol; MRX 2843

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: 20~98 mg/mL (200.5 mM)
Ethanol: ~25 mg/mL (51.2 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.26 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 2 mg/mL (4.09 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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 3: 2 mg/mL (4.09 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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