Pelabresib (CPI0610)

BRD4-BD1 (IC50=39 nM)

MM cell viability is dose-dependently lowered by CPI-0610 (0-1500 nM; 72 hours; multiple myeloma cell lines and primary MM cells) treatment[2]. G1 cell cycle arrest is caused by CPI-0610 (800 nM; 72 hours; INA6 and MM.1S cells) treatment[2]. After 72 hours, treatment with CPI-0610 (800 nM; INA6 and MM.1S cells) dramatically promotes apoptosis in MM cells[2].

Over the course of the study, CPI-0610 (30–60 mg/kg; oral administration; for 28 days; MV-4-11 mouse xenograft model) treatment significantly suppresses tumor growth (41%, 80%, and 74% tumor growth inhibition, respectively), without causing the animals to lose a significant amount of body weight[1].

Thermal shift assay protocols.
All assays were carried out in 384 well plates. BRD4 BD1 (2 μM) was combined with Sypro Orange (Life Technologies) to a final dye concentration of 5X in 50 mM Tris, 1 mM DTT, pH 8.5. The tube was centrifuged briefly to remove precipitate and the protein:dye solution was then added to a black OptiplateTM plates (Greiner), spun briefly (1 min, 900xg) and then 23 μL transferred to either DMSO controls or fragments plated from 100 mM DMSO stocks at a final compound concentration of 800 μM (0.8% v/v DMSO). Subsequently samples (15 μL) were transferred to LightCycler® 480 plates (Roche Diagnostics), spun (2 min, 900xg) and analyzed on a Roche Lightcycler 480 II using a temperature gradient of 20-85 °C and a scanning rate of 1.2 °C/min. The midpoint of the melting transitions (Tm) were assessed using an application developed in-house measuring the first derivative of the rate of fluorescence change as a function of temperature. Compound induced changes in the melting temperature, Tm, were calculated relative to DMSO controls within the same plate.

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Biochemical assay protocols.
BRD4 protein (either BD1 or BD2) in 5 μL of assay buffer (50 mM HEPES pH 7.5, 1 mM TCEP, 69 uM Brij-35, 150 mM NaCl, and BSA at 0.1 mg/mL) was added to 384 well white Proxiplates (Perkin- Elmer) containing compounds which had previously been dispensed as DMSO stocks for 10 point dose-response titrations (in duplicate). To this was added a biotinylated small molecule ligand to a total volume of 10 μL. After 10 min SurelightTM Streptavidin, and Eu(W1024)-antiHis (both Perkin-Elmer) detection reagents were added as an additional 5 uL aliquot. Final assays were 15 μL total volume in assay buffer with 0.8% (v/v) DMSO, 25 (BD1) or 100 (BD2) nM Surelight Streptavidin (concentration expressed as biotin binding sites), 0.2 nM Eu antibody and either 2.5 nM (BD1) or 10 nM (BD2) with 25 or 100 nM biotin ligand respectively. After a 60 minute room temperature incubation plates were read on the EnVision with appropriate filters employing 20 flashes/well, a 40 μsec delay for data acquisition, and a 16.6 msec delay between flashes. Percent inhibition was calculated relative to positive (no protein) and negative (no inhibitor) controls on a per plate basis. IC50 values were determined by fitting the percent inhibition versus compound concentration. For other non-BET bromodomains assessed by TR-FRET analyses were carried out in a similar manner in the same buffer. Variations with regard to salt, reporter, bioligand,and bromodomain concentrations are found in the table below.

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BET selectivity AlphaLISA assays were conducted in 384 well Proxiplates in assay buffer (40 mM Hepes pH 7.0, 1 mM DTT, 69 uM Brij-35, 40 mM NaCl, and 0.1 mg/mL BSA) with compounds being added as DMSO stocks to a final concentration of 0.8% (v/v) 10 point dose-response titrations. Initial binding reactions were set up with BET protein, a biotinylated JQ1 analog, and compounds in a volume of 9 uL. Samples were incubated for 20 min followed by addition of a 10 uL mixture containing streptavidin donor beads and anti-Flag AlphaLISA acceptor beads (both PerkinElmer) in assay buffer to final concentrations of 10 or 30 nM BET protein, 10 nM biotinylated ligand, and donor and acceptor beads at 15 ug/mL each in assay buffer plus 0.8% DMSO (v/v). Plates were sealed, incubated at room temperature for 90 min, and read on an EnVision 2104 Multilabel Reader equipped with a LANCE Dual Laser 50/200 (PerkinElmer) using AlphaScreen® settings. Percent inhibition was calculated relative to positive and negative controls on a per plate basis. For titration experiments, IC50 values were determined by fitting the percent inhibition versus compound concentration. The conditions for the ATAD2 bromodomain AlphaLISA assay were the same as previously reported.1

Cell Viability Assay [2]
Cell Types: Multiple myeloma (MM) cell lines and primary MM cells
Tested Concentrations: 0 nM, 200 nM, 400 nM, 600 nM, 800 nM, 1000 nM, 1200 nM, or 1500 nM
Incubation Duration: 72 huors
Experimental Results: diminished viability of MM cells in a dose-dependent manner.

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Cell Cycle Analysis[2]
Cell Types: INA6 and MM.1S cells
Tested Concentrations: 800 nM
Incubation Duration: 72 hrs (hours)
Experimental Results: Indeced G1 cell cycle arrest.

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Apoptosis Analysis[2]
Cell Types: INA6 and MM.1S cells
Tested Concentrations: 800 nM
Incubation Duration: 72 hrs (hours)
Experimental Results: MM cells apoptosis was increased after 72 hrs (hours).

Animal/Disease Models: MV-4-11 mouse xenograft model[1]
Doses: 30 mg/kg one time/day, 30 mg/kg twice (two times) daily, or 60 mg/kg one time/day
Route of Administration: Oral administration; for 28 days
Experimental Results: Suppressed of tumor growth, without any significant body weight loss in the animals.

[1]. Albrecht BK, et al. Identification of a Benzoisoxazoloazepine Inhibitor (CPI-0610) of the Bromodomain and Extra-Terminal (BET) Family as a Candidate for Human Clinical Trials. J Med Chem. 2016 Feb 25;59(4):1330-9.
[2]. Siu KT, et al. Preclinical activity of CPI-0610, a novel small-molecule bromodomain and extra-terminal protein inhibitor in the therapy of multiple myeloma. Leukemia. 2017 Aug;31(8):1760-1769.

C20H16CLN3O2

365.81

365.09310

1380087-89-7

White to off-white solid

3

81.5Ų

O=C(N)C[C@@H]1N=C(C2=CC=C(Cl)C=C2)C3=CC=CC=C3C4=C1ON=C4C

GCWIQUVXWZWCLE-INIZCTEOSA-N

InChI=1S/C20H16ClN3O2/c1-11-18-14-4-2-3-5-15(14)19(12-6-8-13(21)9-7-12)23-16(10-17(22)25)20(18)26-24-11/h2-9,16H,10H2,1H3,(H2,22,25)/t16-/m0/s1

2-[(4S)-6-(4-chlorophenyl)-1-methyl-4H-[1,2]oxazolo[5,4-d][2]benzazepin-4-yl]acetamide

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.69 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 (5.69 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 (5.69 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.)