BRD4 (1/2) (IC50= 77/33 nM)

(+)-JQ-1 represents a strong, highly selective and Kac-competitive inhibitor of the bromodomain BET family. (+)-JQ-1 (100 nM, 48 h) increases squamous development, evidenced by cell spindle formation, flattening, and enhanced keratin expression. (+)-JQ-1 (250 nM) stimulates fast expression of keratin in treated NMC 797 cells compared to (-)-JQ1 (250 nM) and vehicle control, as evaluated by quantitative immunohistochemistry of. (+)-JQ-1 (relative to (-)-JQ1 (250 nM)) causes time-dependent strong (3+) keratin staining in treated NMC 797 cells [1]. Derepression of autophagy genes was seen almost immediately upon addition of (+)-JQ-1 [2]. (+)-JQ-1 is a strong thiophenediazepine inhibitor (Kd=90 nM) of the BET family coactivator protein BRD4, which participates in the development of cancer through the transcriptional regulation of the MYC oncogene. Dose-ranging experiments of (+)-JQ-1 indicated efficient suppression of H4Kac4 binding, with IC50 values of 10 nM for mouse BRDT (1) and 11 nM for human BRDT (1) [3].

Matching mouse cohorts with tumors that had already developed were randomized to receive intraperitoneal injections of either vehicle or (+)-JQ1 (50 mg/kg) every day. FDG-PET imaging was used to assess the mice both four days post-treatment and before to randomization. FDG uptake was shown to be significantly reduced when (+)-JQ1 was administered. Tumor growth was inhibited by JQ1 treatment, as demonstrated by assessments of tumor volume. CD1 mice were used for pharmacokinetic studies of (+)-JQ1 following oral and intravenous dosing. Time profile of the mean plasma concentration of (+)-JQ1 following intravenous injection (5 mg/kg). The half-life (T1/2) of intravenous (+)-JQ1 was approximately one hour, and its pharmacokinetic characteristics demonstrated good drug exposure (AUC=2090 hr*ng/mL). After oral dosage (10 mg/kg), a mean plasma concentration-time profile of (+)-JQ1 was created. Oral (+)-JQ1 pharmacokinetic parameters showed good drug exposure (AUC=2090 hr*ng) /mL), peak plasma concentration (Cmax=1180 ng/mL), and oral bioavailability (F=49%)[1].

Acetyl-Histone Binding Assay. [1]
Assays were performed as described previously51 with minor modifications from the manufacturer’s protocol (PerkinElmer, USA). All reagents were diluted in 50 mM HEPES, 100 mM NaCl, 0.1 % BSA, pH 7.4 supplemented with 0.05 % CHAPS and allowed to equilibrate to room temperature prior to addition to plates. A 24-point 1:2 serial dilution of the ligands was prepared over the range of 150 – 0 μM and 4 μl transferred to low-volume 384-well plates, followed by 4 μl of His-tagged protein (BRD4(1), 250 nM, BRD4(2) and CREBBP, 2000 nM). Plates were sealed and incubated at room temperature for 30 minutes, before the addition of 4 μl of biotinylated peptide at equimolar concentration to the protein [peptide for BRD4(1) & BRD4(2): H4K5acK8acK12acK16ac, HSGRGK( Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH; peptide for CREBBP: H3K36ac, Biotin-KSAPATGGVK(Ac)KPHRYRPGT-OH]. Plates were sealed and incubated for a further 30 minutes, before the addition of 4 μl of streptavidin-coated donor beads (25 μg/ml) and 4 μl nickel chelate acceptor beads (25 μg/ml) under low light conditions. Plates were foil-sealed to protect from light, incubated at room temperature for 60 minutes and read on a PHERAstar FS plate reader using an AlphaScreen 680 excitation/570 emission filter set. IC50 values were calculated in Prism 5 (GraphPad Software, USA) after normalization against corresponding DMSO controls and are given as the final concentration of compound in the 20 μl reaction volume.

Cell Proliferation Assay. [1]
Cells were seeded into white, 384-well microtiter plates (Nunc) at 500 cells per well in a total volume of 50 μl media. The 797, TT and TE10 cells were grown in DMEM containing 1 % penicillin/streptomycin and 10 % FBS. The Per403 cells were grown in DMEM containing 1 % penicillin/streptomycin and 20 % FBS. Patient-derived NMC 11060 cells were grown in RPMI with 10 % FBS and 1% penicillin/streptomycin. Compounds were delivered to microtiter assay plates by robotic pin transfer (PerkinElmer JANUS equipped with a V&P Scientific 100 nl pin tool). Following a 48 h incubation at 37 ºC, cells were lysed and wells were assessed for total ATP content using a commercial proliferation assay. Replicate measurements were analyzed with respect to dose and estimates of IC50 were calculated by logistic regression. [1]

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Cell Growth Assay.[1]
Cells were seeded in 6-well tissue culture dishes at a concentration of 1.5 x 104 cells per well. Cells were grown in 2 ml of either DMEM (797) or RPMI (11060) containing 10 % fetal bovine serum, 1 % penicillin/streptomycin and either 250 nM (+)-JQ1 or the equivalent volume of DMSO (0.025 %). One half of the media in each well was replaced daily. On days 0, 1, 3, 7 and 10, dishes of cells assigned to each time point were trypsinized, mixed in a 1:1 ratio with 0.4 % trypan blue and counted using a Countess automated cell counter.

Xenograft Efficacy Studies.[1]
NMC 797 xenografts were established by injecting NMC 797 cells (107) in 30 % Matrigel (BD Biosciences) into the flank of 6 week-old female NCr nude mice. Twelve days after injection, mice with measureable tumors were divided into cohorts to be treated with JQ1 at 50 mg kg-1 IP or vehicle (5 % DMSO in 5 % dextrose). For FDG-PET studies, mice with established tumors measuring approximately 1 cm in the largest linear dimension underwent baseline CT/PET imaging 1 h after injection of 250 μCi of FDG (Pre-treatment). Mice were then treated with four daily doses of 50 mg kg-1 of racemic JQ1 by intraperitoneal injection. Two hours after the fourth dose of JQ1 or vehicle, mice underwent repeat FDG-PET imaging (Post-treatment). The integrated signal encompassed within the entire tumor volume is expressed as the percent of injected dose per gram (% ID/gm). Tumors were fixed in 10 % buffered formalin for histopathological analysis. For tumor caliper studies, the average size of tumors in the JQ1 treatment group (n = 8) and vehicle group (n = 7) were similar (63.8 ± 17.1 and 73.6 ± 14.4 mm3 respectively) at the start of treatment. Animals were followed daily for clinical symptoms. Tumor measurements were assessed by caliper measurements, and volume calculated using the formula Vol = 0.5 x L x W2. After 2 weeks of treatment, all mice were humanely euthanized, and tumors were fixed in 10 % formalin for histopathological examination. Statistical significance of tumor volumes was calculated by two-sided Students t-test.[1]

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Primary NMC Xenograft Studies.[1]
A primary xenograft model of NMC was established by injecting NCr nude mice with primary cells (107 cells in 100 μl of 30 % Matrigel in 70 % PBS) collected from malignant pleural fluid obtained with IRB approval and informed consent from a patient at the Dana-Farber Cancer Institute and Brigham & Women’s Hospital. As above, four mice with established tumors measuring approximately 1 cm in the largest linear dimension underwent baseline CT/PET imaging 1 h after injection of 250 μCi of FDG (Pre-treatment). Mice were then treated with four daily doses of 50 mg kg-1 of (+)-JQ1 by intraperitoneal injection. Animals were followed daily for clinical symptoms. Two hours after the fourth dose of (+)-JQ1 or vehicle, mice underwent repeat FDG-PET imaging (Post-treatment). The integrated signal encompassed within the entire tumor volume is expressed as the percent of injected dose per gram (%ID/gm). Tumors were fixed in 10 % buffered formalin for histopathological analysis. At the conclusion of the study, all mice were humanely euthanized, and tumors were fixed in 10 % formalin for histopathological examination. Survival (30 days) studies performed with NMC Per403 and 11060 xenografts were initiated as above. For these studies, (+)-JQ1 was administered at a dose of 50 mg kg-1 by daily intraperitoneal injection. The average size of tumors in the (+)-JQ1 treatment group (n = 10) and vehicle group (n = 10) were similar at the start of treatment. Animals were followed daily for clinical symptoms. Tumor measurements were assessed by caliper measurements, and volume calculated using the formula Vol = 0.5xLxW2. Statistical significance of tumor volumes was calculated by two-sided Students t-test. Comparative survival analysis was performed using the Log-rank (Mantel-Cox) Test, and data were presented as a Kaplain-Meier plot annotated with a measure of statistical significance (pvalue). All animal studies were approved by the IACUC of the DFCI.

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Pharmacokinetic Studies in Mice.[1]
Male CD1 mice (24 – 29 gm) were treated with a single dose of (+)-JQ1 at 5 mg kg-1 for intravenous tail vein injection studies and 10 mg kg-1 for oral gavage studies. Approximately 150 μl of blood were taken from animals by retro-orbital puncture under anesthesia with Isoflurane into EDTA tubes at pre-specified time intervals: 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 5, 8 and 24 hours. Three animals were analyzed per time point. Blood samples were put on ice and centrifuged to obtain plasma samples (2000 x.g, 5 min under 4 °C) within 15 minutes post-sampling. Plasma samples were stored at approximately -70 °C until analysis was performed. Mice were provided free access to food and water throughout the study. Compound was formulated for intravenous injection in 10 % DMSO and 10 % HP-β-CD. Pharmcokinetic studies and pharmacologic assay development was performed at ChemPartner (Shanghai, CHINA). Data were analyzed by J.E.B. using Microsoft Excel and GraphPad Prism 5.02.

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In vivo formulations used (reported):

1. Dissolved in 5% dextrose; 50 mg/kg; i.p. injection; Nature. 2010 Dec 23;468(7327):1067-73; 2. Dissolved in 10% DMSO and 90% of a 10% 2-hydroxypropyl-β-cyclodextrin solution; Leukemia. 2017 Oct;31(10):2037-2047.; 3. Dissolved in 1% DMSO+5% Glucose+ddH2O; Cell. 2018 Sep 20;175(1):186-199.e19.; 4. Dissolved in 20% hydroxypropyl-β-cyclodextrin, 5% DMSO, 0.2% Tween-80 in saline; Mol Cancer Ther. 2016 Jun;15(6):1217-26.; 5. Dissolved in 1:1 propylene glycol:water; J Biol Chem. 2016 Nov 4;291(45):23756-23768.; 6. Dissolved in 5% DMSO in 10% 2-hydroxypropyl-β-cyclodextrin solution; Cancer Lett. 2017 Aug 28;402:100-109.

[1]. Selective inhibition of BET bromodomains. Nature. 2010 Dec 23;468(7327):1067-73.

[2]. Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and LysosomalFunction. Mol Cell. 2017 May 18;66(4):517-532.e9.

[3]. Small-molecule inhibition of BRDT for male contraception. Cell. 2012 Aug 17;150(4):673-84.

JQ1 is a member of the class of thienotriazolodiazepines that is the tert-butyl ester of [(6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl]acetic acid. An inhibitor of bromodomain-containing protein 4 that exhibits anti-cancer and cardioprotective properties. It has a role as a bromodomain-containing protein 4 inhibitor, a cardioprotective agent, an antineoplastic agent, an anti-inflammatory agent, an angiogenesis inhibitor, an apoptosis inducer and a ferroptosis inducer. It is a thienotriazolodiazepine, an organochlorine compound, a carboxylic ester and a tert-butyl ester.

C23H25CLN4O2S

456.99

456.138

C, 60.45; H, 5.51; Cl, 7.76; N, 12.26; O, 7.00; S, 7.02

1268524-70-4

(R)-(-)-JQ1 Enantiomer;1268524-71-5;JQ-1 (carboxylic acid);202592-23-2

46907787

White to yellow solid

1.3±0.1 g/cm3

610.4±65.0 °C at 760 mmHg

322.9±34.3 °C

0.0±1.7 mmHg at 25°C

1.657

4.49

0

6

5

31

706

1

ClC1C([H])=C([H])C(=C([H])C=1[H])C1C2C(C([H])([H])[H])=C(C([H])([H])[H])SC=2N2C(C([H])([H])[H])=NN=C2[C@]([H])(C([H])([H])C(=O)OC(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])N=1

DNVXATUJJDPFDM-KRWDZBQOSA-N

InChI=1S/C23H25ClN4O2S/c1-12-13(2)31-22-19(12)20(15-7-9-16(24)10-8-15)25-17(11-18(29)30-23(4,5)6)21-27-26-14(3)28(21)22/h7-10,17H,11H2,1-6H3/t17-/m0/s1

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: ≥ 2.5 mg/mL (5.47 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 (5.47 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 (5.47 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% DMSO+30% PEG 300+5% Tween 80+ddH2O:5mg/mL

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