ASLAN003 targets human dihydroorotate dehydrogenase (DHODH) with a half maximal inhibitory concentration (IC50) of 35 nM. [1]

For 48 hours, farudodstat (0.01-100 μM) suppresses the growth of leukemia cells. 50% or more of the cell viability is maintained at concentrations of Farudodstat [1]. Cleaved caspase 8 is considerably increased by farudodstat (0.5, 1 μM) for 48 hours [1]. Farudodstat (2, 4 μM; for 96 hours) decreases viability and causes differentiation in myelodysplastic syndrome samples and primary acute myeloid leukemia blasts [1]. It was demonstrated that in MOLM-14 and KG-1 cells, fardodstat (1, 2 μM; 1 hour pretreatment before OPP, 1 hour on) slows protein synthesis and decreases incorporation of O-propargylpuromycin (OPP) into protein translation sites. RPL6 and EIF4B protein downregulation is brought on by farudodstat [1].

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ASLAN003 inhibits proliferation of THP-1, MOLM-14 and KG-1 cells with IC50 values of 152 nM, 582 nM, and 382 nM, respectively. Cell viability was maintained at ~50% at 1 µM and higher, indicating a mode of action distinct from cytotoxic drugs. [1]
ASLAN003 induces differentiation: treatment increased CD11b+ cells by 86.7% (THP-1), 63.9% (MOLM-14) and 86.5% (KG-1) at 100 nM after normalization to DMSO (P<0.001); CD14+ cells were also significantly increased (P<0.01). Morphological changes (lower nucleocytoplasmic ratio, condensed chromatin, increased nuclear lobulation) characteristic of myeloid maturation were observed. NBT reduction assay showed 95.2% (THP-1), 62.4% (MOLM-14) and 93.6% (KG-1) positive cells after 96h treatment with 100 nM ASLAN003 (P<0.001); at 50 nM, >50% of THP-1 cells showed increased NBT reduction (P<0.001). Time-dependent experiment: after 24 or 48h exposure to 100 nM ASLAN003, almost 100% of THP-1 cells became CD11b+. [1]
Comparison with brequinar: at 100 nM, MOLM-14 cells treated with brequinar had 33.1% CD11b+ cells, while ASLAN003 gave 63.9% CD11b+ cells (P<0.05), showing nearly two-fold higher potency of ASLAN003. [1]
Uridine rescue: Addition of 50 µM uridine completely rescued ASLAN003-mediated differentiation and also rescued cell viability (P<0.05) in MOLM-14 and THP-1 cells, demonstrating on-target specificity. [1]
Primary AML and MDS samples: ASLAN003 induced differentiation and cell death in primary AML blasts (e.g., UPN1: 22% and 30% increase in CD11b+ at 2 µM and 4 µM, respectively; UPN6: 18% and 23% increase in CD13/CD33 double positive cells at 2 µM and 4 µM; UPN5 (del7): 62% increase in CD11b+ at 1 µM). Among AML samples, 43% sensitive, 43% moderately sensitive, 14% resistant; among MDS samples, 50% sensitive, 50% moderately sensitive. ASLAN003 had negligible impact on mononuclear cells from a healthy donor. [1]
Transcriptome analysis (RNA-seq) of KG-1 and MOLM-14 cells treated with ASLAN003 revealed 320 upregulated and 225 downregulated genes. Upregulated genes related to myeloid differentiation (8.4%), cell surface antigens (4.7%), apoptosis (2.5%). Downregulated genes enriched for ribosome family (19.6%) and metabolism-related (8.4%). Gene set enrichment analysis showed enrichment of “myeloid differentiation_up”, “hematopoietic stem cell_down”, “targets of HoxA9 and Meis1_down” signatures, and suppression of “pyrimidine ribonucleoside triphosphate metabolic process”. [1]
Apoptosis: ASLAN003 triggered extrinsic pathway (increased cleaved caspase 8) and intrinsic pathway (loss of mitochondrial membrane potential in a dose-dependent manner in THP-1, MOLM-14 and KG-1 cells; increased cytochrome c leakage; cleaved caspase-3 and -7). [1]
Protein synthesis inhibition: ASLAN003 inhibited protein synthesis as shown by reduced O-propargyl-puromycin (OPP) incorporation in MOLM-14 and KG-1 cells. Downregulation of EIF4B and RPL6 proteins was confirmed by western blot. [1]
AP-1 activation: ASLAN003 increased c-FOS protein levels. The AP-1 inhibitor T-5224 (20 µM) completely abolished ASLAN003-mediated differentiation in KG-1 cells and dampened it by half in MOLM-14 cells (P<0.001), indicating that AP-1 activation partially facilitates differentiation. [1]

The number of disseminated tumors is significantly decreased and survival is prolonged when farudstat (50 mg/kg; oral gavage; once daily; days 3 to 30) is administered [1].

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In MOLM-14 and THP-1 xenograft models (NGS mice, female, 4-6 weeks old), ASLAN003 (50 mg/kg, once daily oral gavage) significantly prolonged survival compared to vehicle control (P=0.03 for MOLM-14, P<0.001 for THP-1). ASLAN003 substantially reduced the number and size of disseminated tumors in the MOLM-14 model, and prevented leukemic infiltration into livers in the THP-1 model. No significant differences in body weight, hemoglobin concentration or platelet counts were observed between treated and control groups. [1]
In the same models, ASLAN003 significantly reduced human CD45+ cells in bone marrow, peripheral blood, spleen and liver, and significantly increased CD11b+ and CD14+ cells in bone marrow of treated mice (both models). [1]
In AML-14 patient-derived xenograft (indolent line), ASLAN003 (50 mg/kg, once daily oral gavage) significantly reduced leukemic burden (human CD45+ cells) compared to vehicle (P=0.04). All mice remained alive with no obvious signs of disease; body weight increased similarly in both groups. [1]
In AML-23 patient-derived xenograft (aggressive line), ASLAN003 (50 mg/kg, once daily oral gavage) extended median survival (50% of treated mice alive at day 37 vs. all vehicle-treated mice succumbed within a month, median survival 20 days; P=0.0002). Percentages of human CD45+ leukemia cells were significantly reduced in bone marrow, peripheral blood, spleen, and liver, and increased human CD11b+ and CD14+ cells were observed in bone marrow. No statistical difference in body weight between groups (P=0.73). [1]

Cell Proliferation Assay[1]
Cell Types: THP-1, MOLM-14 and KG-1 Cell
Tested Concentrations: 0.01, 0.1, 1, 10, 100 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: THP-1, MOLM-14 and KG- 1 Cell Inhibition of Leukemia Cell Proliferation The IC50 values of MOLM-14 and KG-1 are 152 nM, 582 nM and 382 nM respectively.

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Western Blot Analysis[1]
Cell Types: KG-1 and MOLM-14 Cell
Tested Concentrations: 0.5, 1 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Significant increase in cleaved caspase 8 and leakage of cytochrome c from mitochondria into the cytosol and induces cleavage of caspase-3 and -7.

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Cell viability assays: Cells were exposed to ASLAN003 at various concentrations for 96 h, and viability was assessed using standard colorimetric or luminescent methods (details in Online Supplementary Methods). [1]
Western blot analysis: Whole-cell lysates or cytosolic fractions from KG-1 and MOLM-14 cells treated with DMSO, 0.5 µM or 1 µM ASLAN003 for 48 h were used to measure apoptosis-related proteins (cleaved caspase 8, caspase-3, caspase-7, cytochrome c) and protein synthesis markers (EIF4B, RPL6, c-FOS) via standard immunoblotting with GAPDH as loading control. [1]
FACS analysis of myeloid differentiation: Cells were treated with ASLAN003 or DMSO for 96 h (or time-course up to 48 h), then stained with fluorescently labeled antibodies against human CD11b, CD14, CD13, CD33 and analyzed on a flow cytometer. For primary AML/MDS samples, response was classified based on percentage increase of these markers. [1]
Wright-Giemsa staining and nitro blue tetrazolium (NBT) reduction assay: After 96 h exposure to ASLAN003 or DMSO, 1×10^6 AML cells were harvested and equally divided for cytospin preparation followed by Wright-Giemsa staining to assess morphological changes, and for NBT reduction assay to evaluate functional myeloid maturation (cells reducing NBT to formazan were counted). [1]
Mitochondrial membrane potential (MMP) assessment: THP-1, MOLM-14 and KG-1 cells were exposed to 1 µM or 2 µM ASLAN003 or DMSO for 48 h, then stained with JC-10 dye and analyzed by flow cytometry to quantify depolarization of MMP. [1]
RNA sequencing and analysis: KG-1 and MOLM-14 cells treated with ASLAN003 or DMSO were subjected to RNA extraction, library preparation, and sequencing. Differential expression analysis was performed (posterior probability of differential expression 0.95-1, fold-change cutoff 1.5). Gene ontology and single-sample gene set enrichment analysis were conducted. [1]
Protein synthesis assay (Click-iT): MOLM-14 and KG-1 cells were exposed to 1 µM or 2 µM ASLAN003 for 1 h, then O-propargyl-puromycin (OPP, 20 mM) was added for 1 h. Cells were washed, fixed, permeabilized, and analyzed by flow cytometry for OPP incorporation (Alexa Fluor 488). [1]
qRT-PCR: Total RNA was extracted from ASLAN003-treated cells, reverse transcribed, and quantitative PCR performed using specific primer sequences (Online Supplementary Table S1) to confirm expression changes of selected genes. [1]

Animal/Disease Models: Female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, NGS mice (4-6 weeks old) with MOLM-14 cells [1]
Doses: 50 mg/kg
Route of Administration: po (oral gavage); one time/day; Results from day 3 to day 30: The number of disseminated tumors and tumor size were Dramatically diminished. Survival period was Dramatically prolonged.

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Human AML cell line xenograft model: Female NOD.Cg-Prkdc-scid Il2rg-tm1Wjl/SzJ (NSG) mice (4-6 weeks old) were injected intravenously (tail vein) with 3×10^6 exponentially growing THP-1 or MOLM-14 cells. From the second day after inoculation, mice were administered either vehicle control or ASLAN003 at 50 mg/kg by oral gavage once daily in a 200 µL volume. Treatment continued for 2 to 11 weeks depending on the model. Body weight, hemoglobin concentration, and platelet counts were monitored. Survival was analyzed by Kaplan-Meier and log-rank test. At endpoint, bone marrow, peripheral blood, spleen, and liver were harvested for FACS analysis of human CD45+, CD11b+, and CD14+ cells. [1]
Human AML patient-derived xenograft (PDX) models: AML-14 PDX line (from AML-M4, normal karyotype) and AML-23 PDX line (from chronic myeloid leukemia in accelerated phase) were established. NSG mice were engrafted with patient-derived AML cells and then treated with vehicle or ASLAN003 at 50 mg/kg once daily oral gavage. Treatment schedules and endpoints were similar to the cell line xenograft models. Leukemic burden (human CD45+ cells) and differentiation markers (CD11b+, CD14+) were assessed in bone marrow, peripheral blood, spleen, and liver by FACS. Body weight was monitored throughout. [1]

ASLAN003 exhibits high plasma protein binding (>99%). In phase I single and multiple ascending dose clinical trials, it has been shown to be tolerated by healthy volunteers. [1]

ASLAN003 showed no evident effect on normal hematopoietic cells; it had negligible impact on cell viability and differentiation status of mononuclear cells from a healthy donor at concentrations of 2 µM and 4 µM. [1]
In CD34+CD38+ normal bone marrow myeloid progenitor cells (dividing cells), the IC50 of ASLAN003 was 5.22 µM, and on average ASLAN003 was 11-fold more active in AML cells than in these normal cells, suggesting a favorable therapeutic index. [1]
In mouse xenograft models (MOLM-14 and THP-1), treatment with ASLAN003 (50 mg/kg, once daily oral gavage) was well tolerated: there were no significant differences in body weight, hemoglobin concentration or platelet counts between vehicle control and treated groups. In PDX models (AML-14 and AML-23), body weight of treated mice did not differ statistically from controls (P=0.42 for AML-14, P=0.73 for AML-23). The drug exhibited excellent safety profiles in mice even after prolonged administration. [1]

[1]. ASLAN003, a potent dihydroorotate dehydrogenase inhibitor for differentiation of acute myeloid leukemia. Haematologica. 2019 Nov7;haematol.2019.230482.

[2]. Human Dihydroorotate Dehydrogenase (hDHODH) as a new target on Acute Myelogenous Leukemia (AML): Targeting Myeloid Differentiation using Potent and Innovative hDHODH Inhibitors. 23rd Swedish Conference on Macromolecular Structure an.

Farudodstat (ASLAN003) is currently undergoing clinical trial NCT03451084 (ASLAN003 Dosage Optimization Study for Acute Myeloid Leukemia). Farudodstat is an oral dihydroorotate dehydrogenase (DHODH) inhibitor with potential antitumor activity. After administration, Farudodstat specifically targets and binds to DHODH, inhibiting its activation and blocking the fourth enzymatic step of de novo pyrimidine synthesis. This inhibits uridine monophosphate (UMP) production, DNA synthesis, cell division and proliferation, leading to reactive oxygen species (ROS) production, promoting differentiation of susceptible tumor cells, and inducing apoptosis. DHODH is a mitochondrial enzyme that catalyzes the conversion of dihydroorotate (DHO) to orotate, participating in the synthesis of endogenous UMPs.

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ASLAN003 is a potent DHODH inhibitor that induces differentiation of AML cells. It triggers apoptotic pathways, inhibits protein synthesis, and activates AP-1 transcription factors, contributing to its differentiation capacity. The drug substantially reduces leukemic burden and prolongs survival in AML xenograft and PDX models. [1]
ASLAN003 has been granted orphan drug designation for the treatment of AML by the US Food and Drug Administration and is currently being evaluated in a Phase IIa clinical trial in patients with AML (ClinicalTrials.gov: NCT03451084). [1]
In a separate report, ASLAN003 is mentioned as one of three DHODH inhibitors in AML-related clinical trials (Phase II). [2]

C19H14F2N2O3

356.322871685028

356.097

1035688-66-4

24986824

White to off-white solid powder

4.6

2

7

5

26

470

0

FC1C=C(C=C(C=1NC1C(C(=O)O)=CC=CN=1)F)C1C=CC=C(C=1)OC

OMPATGZMNFWVOH-UHFFFAOYSA-N

InChI=1S/C19H14F2N2O3/c1-26-13-5-2-4-11(8-13)12-9-15(20)17(16(21)10-12)23-18-14(19(24)25)6-3-7-22-18/h2-10H,1H3,(H,22,23)(H,24,25)

2-((3,5-difluoro-3'-methoxy-[1,1'-biphenyl]-4-yl)amino)nicotinic acid

ASLAN003 ASLAN 003 ASLAN-003 Aslan-003.

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 (~350.81 mM)

Solubility in Formulation 1: ≥ 2.17 mg/mL (6.09 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 21.7 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: ≥ 2.17 mg/mL (6.09 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 21.7 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.)