KLK2 and MYC gene expression is downregulated by ABBV-744 (90 nM; 0~24 h; LNCaP cells) [1]. Senescence is induced by ABBV-744 (90 nM; 0~72 h) on LNCaP cells, which causes cell cycle arrest in the G1 phase [1].

In comparison to ABBV-075 [1], ABBV-744 (4.7 mg/kg; interfacial gavage; 28 days) shown comparable or superior anti-tumor effectiveness and retarded the formation of tumors. /kg; 14) has strong anti-tumor properties. By 20%, ABBV-744 (30 mg/kg) suppresses interference [1].

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In a mouse xenograft model using LNCaP cells, treatment with 4.7 mg kg−1 ABBV-744 (1/16 of the maximum tolerated dose (MTD)) caused a delay in tumour growth that was equivalent to ABBV-075 treatment at the MTD dose of 1 mg kg−1 (Fig. 4a). Comparing efficacious exposure levels of ABBV-744 in LNCaP tumour-bearing mice (4.7 mg kg−1; area under the curve, 1.1 μg h ml−1) and MTD (75 mg kg−1; area under the curve, 13.1 μg h ml−1) demonstrated that ABBV-744 was able to produce significant antitumour activity at 1/12 of the highest tolerable exposure of ABBV-744 (Extended Data Fig. 8a). The activity exhibited by ABBV-744 at 1/16 of the MTD of ABBV-744 was superior to the activities achieved using JQ1 and iBET at their respective MTDs or, in the case of RVX-208, at the highest feasible dose in this model (Extended Data Fig. 8b, c). Similarly, ABBV-744 at 1/16 MTD also displayed equivalent or better antitumour activity compared with ABBV-075 at MTD in the enzalutamide-resistant MDA-PCa-2b xenograft model (Fig. 4b). As a control, lowering the dose of ABBV-075 to 1/2 of the MTD resulted in a significant reduction in antitumour activity to 42% tumour growth inhibition in the LNCaP xenograft model. Even in the xenograft model using OPM2 cells, one of the most sensitive models to DbBi, ABBV-075 at 1/4 of the MTD of ABBV-075 (0.25 mg kg−1) had only marginal antitumour efficacy[1].

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In toxicity studies in rats, ABBV-075 at 3 mg kg−1 (3× the efficacious exposure in the LNCaP mouse xenograft model), caused a 59% reduction in platelets, a decrease in Alcian blue staining of the mucosa and the loss of goblet cells. By contrast, ABBV-744 at 30 mg kg−1 (25× the efficacious exposure) triggered a reduction in platelets of only 20%, and at 60 mg kg−1 (47× the efficacious exposure) did not cause loss of goblet cells or other gross intestinal defects (Fig. 4c and Extended Data Fig. 8a). Similarly, 2.5 mg kg−1 ABBV-075 caused germ cell degeneration in the testes, whereas no microscopic changes in the testes were observed with 25 mg kg−1 ABBV-744. These efficacy and tolerability results collectively suggest that selectively targeting BD2 can induce antitumour activity in some cancer settings while mitigating key tolerability issues of DbBi. These findings support the advancement of ABBV-744 for clinical evaluation (ClinicalTrials.gov identifier NCT03360006) and call for further investigation of BD2-dependent transcription programs to reveal additional therapeutic opportunities.

TR-FRET binding assays. [1]
Alexa647-labeled MS417 was used as the fluorescent probe in assay buffer (20 mM sodium phosphate, pH 6.0, 50 mM NaCl, 1 mM ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-dithiothreitol) containing His-tagged bromodomain, europium-conjugated anti-His antibody and Alexa-647- conjugated probe. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multi-label plate reader.[1]

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SPR binding experiment. [1]
The binding kinetics of compounds were assayed via Surface Plasmon Resonance (SPR) using a Biacore T200 instrument and manufacturer provided software. Brd4 BD1(h)(57-168) and BD2(h)(352-457) coupling solution was prepared by diluting proteins to 50 µg/mL in 10 mM phosphate solution (pH 6.5). Carboxyl groups on the dextran layer of the chip were activated by injecting a 1:1 mixture of 0.4 M N-ethyl-N-(3- dimethylaminopropyl)carbodiimide and 0.1 M N-hydroxysuccinimide for 7 min. The proteins coupling solution was injected over the activated chip surface for 800 sec at 10 l/min to achieve an immobilization level of 3452 and 1640 resonance units (RU) of Brd4 BD1 and BD2 respectively. Remaining free activated carboxyl groups were blocked by injecting a solution 1 M ethanolamine for 7 min. A blank surface was treated similarly but without any protein solution and used as a reference surface during binding assays. Briefly, the running buffer during immobilization was HSB-P+ Buffer (10 mM Hepes, 150 mM NaCl, 0.05% (vol/vol) surfactant P20, pH 7.4) and the coupling procedure was run at a flow rate of 5 l/min. Binding affinity measurements were performed at flow rate of 80 l/min using 10 mM Hepes, 150 mM NaCl, 0.05% (vol/vol) surfactant P20, pH 7.4, containing 15 mM DTT and 1 % DMSO. Compounds were assayed using singlecycle kinetics mode as provided by the Biacore T200 control software and were recorded at a frequency of 10 Hz. The compounds were diluted in the running buffer and injected in a series of increasing concentrations for contact time of 260 sec each and dissociation was monitored for up to 10000 sec. Sensorgrams were processed and analyzed using Biacore T200 evaluation software and the binding curves were fit to determine the equilibrium dissociation constant (Kd).

Western Blot Analysis[1]
Cell Types: LNCaP cells
Tested Concentrations: 90 nM
Incubation Duration: 0~24 hrs (hours)
Experimental Results: Downregulated the expression of KLK2 and MYC genes.

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Cell Cycle Analysis[1]
Cell Types: LNCaP cells
Tested Concentrations: 90 nM
Incubation Duration: 0~72 hrs (hours)
Experimental Results: Induced cell cycle arrest in G1 followed by senescence.

Animal/Disease Models: Mouse
Doses: 4.7 mg/kg (pharmacokinetic/PK/PK analysis) Dosing: po (oral gavage); 28-day
Experimental Results: compared with ABBV-075, caused tumor growth delay and demonstrated the same or better anti- tumor activity.

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Animal/Disease Models: SD (SD (Sprague-Dawley)) rat
Doses: 30 mg/kg (pharmacokinetic/PK/PK analysis) Dosing time: 14 days
Experimental Results: Produced significant anti-tumor activity.

[1]. Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancer. Nature. 2020;578(7794):306-310.

BET Inhibitor ABBV-744 is an orally bioavailable inhibitor of the Bromodomain and Extra-Terminal (BET) family of proteins, with potential antineoplastic activity. Upon oral administration, the BET inhibitor ABBV-744 preferentially binds to the second bromodomain (BD2) of BET proteins, thereby preventing the interaction between the BET proteins and acetylated histones. This disrupts chromatin remodeling and gene expression. Prevention of the expression of certain growth-promoting genes may lead to an inhibition of proliferation in BET-overexpressing tumor cells. BET proteins, comprised of BRD2, BRD3, BRD4 and BRDT, are transcriptional regulators that contain two homologous bromodomains, the BD1 and BD2 domains. They play an important role during development and cellular growth.
See also: Abexinostat Tosylate (annotation moved to).

C28H30FN3O4

491.5539

491.22203

C, 68.42; H, 6.15; F, 3.86; N, 8.55; O, 13.02

2138861-99-9

132010322

White to light yellow solid powder

3.9

3

5

6

36

852

0

FC1C=C(C)C(=C(C)C=1)OC1C=CC(=CC=1C1=CN(C)C(C2=C1C=C(C(NCC)=O)N2)=O)C(C)(C)O

OEDSFMUSNZDJFD-UHFFFAOYSA-N

InChI=1S/C28H30FN3O4/c1-7-30-26(33)22-13-20-21(14-32(6)27(34)24(20)31-22)19-12-17(28(4,5)35)8-9-23(19)36-25-15(2)10-18(29)11-16(25)3/h8-14,31,35H,7H2,1-6H3,(H,30,33)

N-ethyl-4-(2-(4-fluoro-2,6-dimethylphenoxy)-5-(2-hydroxypropan-2-yl)phenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide

ABBV744; ABBV 744; 9MX546E2SF; UNII-9MX546E2SF; ABBV-744.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~203.44 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.09 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 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 (5.09 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 25.0 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.09 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2 mg/mL (4.07 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 2 mg/mL (4.07 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 6: 2.5 mg/mL (5.09 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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