DHODH/dihydroorotate dehydrogenase

The human DHODH enzyme is selectively and low nanomolarly inhibited by BAY-2402234. It successfully suppresses AML cell line proliferation in vitro in the subnanomolar to low nanomolar range. In cellular mechanistic studies, BAY-2402234 also causes AML cell line differentiation in the subnanomolar to low nanomolar range, indicating the anticipated mode of action [1].

Both patient-derived xenograft (PDX) models of AML and a number of subcutaneous and disseminated AML xenografts showed strong in vivo anti-tumor activity when BAY-2402234 was used as monotherapy. After receiving the new DHODH inhibitor BAY-2402234, tumor and plasma dihydroorotate levels rise, indicating the drug's on-target actions. The increase of differentiated cell surface markers in xenograft and PDX models after inhibitor therapy indicates that BAY-2402234 enhanced AML differentiation in vivo, which is consistent with in vitro results. Furthermore, following a single in vivo dose of BAY-2402234, differentiation-related transcriptome alterations were visible [1].

Biochemical assay:[2]
The DHODH reaction was performed at pH 8.0 at 32°C in buffer containing 50 mM Tris, 0.1% Triton X-100, 150 mM KCl, 2 nM full length human DHODH enzyme, 1 mM DHO, 0.1 mM decylubiquinone, 0.06 mM 2,6-dichlorophenolindophenol and 2% dimethyl sulfoxide. DHODH activity was measured kinetically as a function of decreased 2,6-dichlorophenolindophenol absorbance at 600 nm.

Cell lines studied and their corresponding media conditions:[2]
THP-1, MV4-11 and TF-1 cell lines were acquired from ATCC. MOLM-13, HEL, SKM-1, NOMO-1, UOC-M1 and EOL-1 cell lines were acquired from DSMZ. HL-60 cells were from the NCI-60 panel. All of the in vitro experiments using established human AML cell lines were performed in the following tissue culture media: THP-1, MOLM-13, NOMO-1, MV4-11, HEL, HL-60, SKM-1 and EOL-1 cells were cultured in RPMI containing 10% FBS and 1% penicillin/streptomycin; UOC-M1 cells were cultured in McCoy’s 5a media containing 10% FBS and 1% penicillin/streptomycin; and TF-1 cells were cultured in RPMI containing 10% FBS, 1% penicillin/streptomycin and 2 ng/mL recombinant GM-CSF. Mycoplasma tests are performed regularly.

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In vitro cellular differentiation assays:[2]
MOLM-13, HEL, MV4-11, SKM-1 and THP-1 cells were plated in 96-well plates at 20,000 cells per well in their respective growth media and were treated with BAY 2402234 (concentration range, 10-6 M to 10-10 M) in the presence or absence of 100 μM uridine. After 96 hours of treatment, upregulation of CD11b or CD14 were determined by assessing the binding of an anti-CD11b-APC antibody or an anti-CD14-APC antibody, respectively, using a FACS Canto. TF-1 cells were treated with BAY 2402234 in the presence or absence of 100 μM uridine for 3 or 6 days. Following incubation, cells were stained with anti-CD11b/Mac-1-APC or anti-CD14-FITC antibody and were analyzed on a FACS Calibur cytometer.

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In vitro apoptosis assays:[2]
For the detection of apoptotic cells, the FITC Annexin V/Dead Cell Apoptosis kit was used according to the manufacturer’s instructions. All flow cytometry analysis was performed on a FACS Calibur cytometer and the data were analyzed using FlowJo software V10.4.2.

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In vitro cell cycle assays:[2]
For the assessment of the cell cycle distribution of cells, the PI/RNAse staining kit was used according to the manufacturer’s instructions. All flow cytometry analysis was performed on a FACS Calibur cytometer and the data were analyzed using FlowJo software V10.4.2.

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In vitro proliferation assays:[2]
THP-1, MOLM-13, NOMO-1, MV4-11, HEL, HL-60, UOC-M1, SKM-1 and EOL-1 cells were plated in their respective growth media in 384-well plates at 1,000 cells per well and were treated with BAY 2402234 (concentration range, 10-5 M to 5-12 M) in the presence or absence of 100 µM uridine. After 72 hours, the number of viable cells per well was assessed by Cell Titer-Glo Luminescent Cell Viability Assay. TF-1 cells were plated in 12 well plates at 0.2 x 106 cells/ml. BAY 2402234 was prepared in DMSO at 1,000X concentrations and cells were treated in triplicate at the stated doses in the presence or absence of 100 µM uridine. The number of viable cells per well was assessed every 3 days by counting the cells using the Vi-Cell Cell Viability Analyzer, and cells were re-plated at 0.2 x 106 cells/ml in fresh media plus BAY 2402234.

Tumor cells were injected subcutaneously into the flanks or, for disseminated models, into the tail veins, of 6-12 week old female mice. Xenograft models were performed at Bayer or Charles River (Freiburg, Germany) using SCID CB17 or NOG mice. PDX models AM5512, AM7577 and AM8096 were performed at CrownBioscience Inc., Bejing in NOD/SCID mice and models AML11655 and AML6252 were performed at EPO Berlin-Buch GmbH in NOG mice (Berlin, Germany). All animal studies were conducted in accordance with the animal welfare laws of the respective sites and were approved by local authorities, and all of the PDX studies were accompanied by inform consent from the patients. Treatment was initiated at a predefined tumor size or burden or at a predefined day after cell inoculation, as detailed in the figure legends. Where applicable randomization based on tumor size or burden occurred prior to treatment. Group size was based on experience with the respective model to allow meaningful statistical analysis. Studies were non-blinded. BAY 2402234 was administered orally at indicated doses in PEG400/EtOH 9/1. Subcutaneous tumor volumes were determined by caliper measurement and disseminated tumor burdens were calculated by measurement of human CD45-positive or human HLA-ABC-positive cells in the blood, spleen and bone marrow of the mice and by general health parameters as surrogates for tumor progression[2].

[1]. Andreas Janzer, et al. Abstract DDT02-04: BAY 2402234: A novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of myeloid malignancies. AACR Annual Meeting 2018; April 14-18, 2018; Chicago, IL.
[2]. The novel dihydroorotate dehydrogenase (DHODH) inhibitor BAY 2402234 triggers differentiation and is effective in the treatment of myeloid malignancies. Leukemia . 2019 Oct;33(10):2403-2415.

Orludodstat is an orally available inhibitor of dihydroorotate dehydrogenase (DHODH), with potential antineoplastic activity. Upon administration, orludodstat specifically targets, binds to and prevents the activation of DHODH, thereby preventing the fourth enzymatic step in de novo pyrimidine synthesis. This prevents uridine monophosphate (UMP) formation, DNA synthesis, cell division and cellular proliferation, causes reactive oxygen species (ROS) production, enables differentiation and induces apoptosis in susceptible tumor cells. DHODH, a mitochondrial enzyme, catalyzes the conversion of dihydroorotate (DHO) to orotate in the endogenous synthesis of UMP.

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Acute myeloid leukemia (AML) is a devastating disease, with the majority of patients dying within a year of diagnosis. For patients with relapsed/refractory AML, the prognosis is particularly poor with currently available treatments. Although genetically heterogeneous, AML subtypes share a common differentiation arrest at hematopoietic progenitor stages. Overcoming this differentiation arrest has the potential to improve the long-term survival of patients, as is the case in acute promyelocytic leukemia (APL), which is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. Treatment of APL with all-trans retinoic acid (ATRA) induces terminal differentiation and apoptosis of leukemic promyelocytes, resulting in cure rates of over 80%. Unfortunately, similarly efficacious differentiation therapies have, to date, been lacking outside of APL. Inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway, was recently reported to induce differentiation of diverse AML subtypes. In this report we describe the discovery and characterization of BAY 2402234 - a novel, potent, selective and orally bioavailable DHODH inhibitor that shows monotherapy efficacy and differentiation induction across multiple AML subtypes. Herein, we present the preclinical data that led to initiation of a phase I evaluation of this inhibitor in myeloid malignancies[2].

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Acute myeloid leukemia (AML), the most common acute leukemia in adults, is an aggressive hematologic malignancy resulting in bone marrow failure with a poor outcome; overall survival is approximately 25% at five years. Treatment options, in particular for the elderly population, are limited. Induction chemotherapy of cytarabine and an anthracycline (7+3) remains unchanged standard of care since its introduction in the early 1970s and there is a high medical need for new therapies (Yates et al. Cancer Chemother Rep 1973). DHODH is a key enzyme in the de novo pyrimidine synthesis converting dihydroorotate to orotate. Using a HOXA9 driven phenotypic screen to overcome differentiation arrest in myeloid cells, we have recently identified DHODH as a surprising novel target to overcome differentiation blockade in AML (Sykes et al. Cell 2016). Differentiation therapy already showed its enormous clinical benefit potential in the small subset of patients diagnosed with acute promyelocytic leukemia (APL) following treatment with all-trans retinoic acid with five-year survival exceeding 85% and should be considered the ultimate therapeutic goal for all AML subsets (Lo-Coco et al. NEJM 2013). Here, we disclose for the first time the structure and functional characterization of the novel DHODH inhibitor BAY 2402234. BAY 2402234 is a selective low-nanomolar inhibitor of human DHODH enzymatic activity. In vitro, it potently inhibits proliferation of AML cell lines in the sub-nanomolar to low-nanomolar range. BAY 2402234 induces differentiation of AML cell lines also in a sub-nanomolar to low-nanomolar range, demonstrating the anticipated mode of action in cellular mechanistic assays. In vivo, BAY 2402234 exhibits strong in vivo anti-tumor efficacy in monotherapy in several subcutaneous and disseminated AML xenografts as well as AML patient-derived xenograft (PDX) models. Target engagement of the novel DHODH inhibitor BAY 2402234 can be observed by increase of tumoral and plasma dihydroorotate levels after treatment with the inhibitor. Consistent with the in vitro data BAY 2402234 induces AML differentiation in vivo as detected by upregulation of differentiation cell surface markers in xenograft and PDX models after treatment with the inhibitor. Furthermore, differentiation-associated transcriptomic changes were evident following a single administration of BAY 2402234 in vivo. The start of clinical investigations of BAY 2402234 is planned for early 2018.[1]

C21H18CLF5N4O4

520.8370

520.09

C, 48.43; H, 3.48; Cl, 6.81; F, 18.24; N, 10.76; O, 12.29

2225819-06-5

134470179

White to light yellow dolid powder

4

94.5Ų

FC1=C(NC(C2=CC(F)=C(N3N=C(CO)N(CC)C3=O)C=C2O[C@@H](C)C(F)(F)F)=O)C(Cl)=CC=C1

KNVJMHHAXCPZHF-JTQLQIEISA-N

InChI=1S/C21H18ClF5N4O4/c1-3-30-17(9-32)29-31(20(30)34)15-8-16(35-10(2)21(25,26)27)11(7-14(15)24)19(33)28-18-12(22)5-4-6-13(18)23/h4-8,10,32H,3,9H2,1-2H3,(H,28,33)/t10-/m0/s1

(S)-N-(2-chloro-6-fluorophenyl)-4-(4-ethyl-3-(hydroxymethyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-5-fluoro-2-((1,1,1-trifluoropropan-2-yl)oxy)benzamide

BAY-2402234; BAY 2402234; BAY-2402234; 2225819-06-5; Orludodstat; Orludodstat(inn); (S)-N-(2-Chloro-6-fluorophenyl)-4-(4-ethyl-3-(hydroxymethyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-5-fluoro-2-((1,1,1-trifluoropropan-2-yl)oxy)benzamide; BAY2402234; BAY 2402234; X8GF945GMK; BAY2402234

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 (~240.00 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.99 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 20.8 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: ≥ 2.08 mg/mL (3.99 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.99 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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 (Please use freshly prepared in vivo formulations for optimal results.)