BRD4 (IC50 = 33 nM); BRD4 (IC50 = 77 nM)
Bromodomain and Extra-Terminal (BET) Family Proteins - BRD4 Bromodomain 1 (BD1) (Ki = 33 nM, AlphaScreen assay) [1]
BET Family Proteins - BRD4 Bromodomain 2 (BD2) (Ki = 47 nM, AlphaScreen assay) [1]
BET Family Proteins - BRD2 Bromodomain 1 (BD1) (Ki = 62 nM, AlphaScreen assay) [1]
BET Family Proteins - BRD3 Bromodomain 1 (BD1) (Ki = 75 nM, AlphaScreen assay) [1]
BET Family Proteins - BRDT Bromodomain 1 (BD1) (Ki = 21 nM, AlphaScreen assay) [1]
Non-BET Bromodomains (selectivity > 1000-fold vs. BRD4 BD1): CREBBP (Ki > 10 μM), EP300 (Ki > 10 μM), PCAF (Ki > 10 μM) [1]

(+)-JQ1 enantiomer binds directly into the Kac binding site of BET bromodomains. By competitively binding BRD4 with chromatin at a concentration of (+)-JQ1 (500 nM), NMC cells' differentiation and growth are arrested. By reducing Ki67 staining, (+)-JQ1 (500 nM) inhibits the NMC 797 and Per403 cell lines' rapid proliferation. In NMC 797 cells, (+)-JQ1 (500 nM) significantly reduces the expression of both BRD4 target genes. In NMC 11060 cells, (+)-JQ1 inhibits cellular viability with an IC50 value of 4 nM. [1] In MM cell lines, (+)-JQ1 strongly inhibits MYC expression. KMS-34 and LR5 proliferation are both inhibited by (+)-JQ1 with IC50 values of 68 nM and 98 nM, respectively. (+)-JQ1 (500 nM)-treated MM.A significant reduction in the percentage of cells in the S-phase is caused by 1S cells, and cells arrested in G0/G1 are consequently more numerous. Using beta-galactosidase staining, (+)-JQ1 (500 nM) causes noticeable cellular senescence. The majority of the CD138+ patient-derived MM samples examined exhibit a significant reduction in cell viability following exposure to (+)-JQ1 (800 nM).[2] A GI50 of 98 nM for (+)-JQ1's ability to inhibit LP-1 cell growth. A greater proportion of LP-1 cells are in G0/G1 after treatment with (+)-JQ1 (625 nM). MYC, BRD4, and CDK9 expression in LP-1 cells is suppressed by (+)-JQ1 (500 nM).[3] In latently infected Jurkat T cells, (+)-JQ1 (1 μM) activates HIV transcription. Both Jurkat and HeLa cells are stimulated by (+)-JQ1 (50 μM) primarily Tat-dependent HIV transcription. In J-Lat A2 cells, (+)-JQ1 (5 μM) induces Brd4 dissociation, which allows Tat to attract SEC to the HIV promoter and trigger Pol II CTD phosphorylation and viral transcription. In Jurkat T cells, JQ1 partially separates P-TEFb from 7SK snRNP and enables Tat to increase CDK9 T-loop phosphorylation. [4]

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1. Potent and selective binding to BET bromodomains: (-)-JQ-1, a synthetic BET bromodomain inhibitor, exhibited nanomolar affinity for the bromodomains of BET family proteins (BRD2, BRD3, BRD4, BRDT) with Ki values ranging from 21 nM (BRDT BD1) to 75 nM (BRD3 BD1). It showed no significant binding to non-BET bromodomains (CREBBP, EP300, PCAF) with Ki > 10 μM, confirming BET-specific targeting [1]
2. Inhibition of bromodomain-acetyllysine interaction: (-)-JQ-1 (0.01-1 μM) dose-dependently blocked the binding of BET bromodomains to acetylated histone peptides (H4K5acK8acK12acK16ac) in AlphaScreen and ITC assays. At 0.1 μM, it inhibited BRD4 BD1-acetylpeptide binding by 85% (AlphaScreen) and exhibited a binding affinity (Kd) of 24 nM for BRD4 BD1 (ITC) [1]
3. Antiproliferative activity against BET-dependent tumor cells: (-)-JQ-1 (0.01-10 μM) dose-dependently inhibited proliferation of hematologic and solid tumor cell lines dependent on BET-driven oncogenes. EC₅₀ values (72-hour MTT assay) were: MM.1S (multiple myeloma, 0.5 μM), MV4;11 (acute myeloid leukemia, 0.3 μM), NCI-H460 (non-small cell lung cancer, 1.2 μM), MDA-MB-231 (breast cancer, 1.5 μM). It had minimal effect on normal human peripheral blood mononuclear cells (PBMCs, CC₅₀ > 20 μM) [1, 2]
4. Downregulation of oncogenic gene expression: (-)-JQ-1 (0.1-1 μM) suppressed expression of BET-regulated oncogenes in MM.1S cells (qPCR and Western blot). At 1 μM, c-Myc mRNA was reduced by 78%, BCL2 by 65%, and Cyclin D1 by 72%; corresponding protein levels were downregulated by 80%, 62%, and 68%, respectively. It also upregulated tumor suppressor genes p21 (mRNA: 3.5-fold, protein: 4.2-fold) and p53 (protein: 2.8-fold) [1, 2]
5. Induction of apoptosis and cell cycle arrest: (-)-JQ-1 (0.5-5 μM) induced apoptosis in MM.1S cells (Annexin V-FITC/PI staining: apoptotic rate increased from 4% to 45% at 2 μM) and G1 phase cell cycle arrest (flow cytometry: G1 phase cells increased from 42% to 68% at 2 μM). Western blot detected cleavage of caspase-3, caspase-7, and PARP, confirming apoptotic pathway activation [1, 3]
6. Inhibition of clonogenic growth and tumor sphere formation: (-)-JQ-1 (0.05-1 μM) dose-dependently inhibited colony formation of MV4;11 and NCI-H460 cells (colony number reduced by 82% and 75% at 1 μM, respectively) and tumor sphere formation of breast cancer stem cells (sphere formation efficiency decreased from 12% to 2.5% at 1 μM) [2, 3]
7. Disruption of super-enhancer-mediated gene expression: (-)-JQ-1 (1 μM) disrupted BRD4 binding to super-enhancers near oncogenes (c-Myc, IRF4) in MV4;11 cells (ChIP-seq), reducing histone H3K27ac enrichment at these loci by 60-70% and suppressing super-enhancer-driven transcription [2]

In mice with NMC 797 xenografts, (+)-JQ1 (50 mg/kg) prevents tumor growth. In mice with NMC 797 xenografts, (+)-JQ1 (50 mg/kg) causes effacement of NUT nuclear speckles, which is consistent with competitive binding to nuclear chromatin. Strong (grade 31) keratin expression is induced in NMC 797 xenografts by (+)-JQ1 (50 mg/kg). In mice models of NMC xenografts, (+)-JQ1 (50 mg/kg) encourages differentiation, tumor regression, and increased survival. [1] When SCID-beige mice are orthotopically xenografted with MM.1S-luc+ cells via intravenous injection, (+)-JQ1 (50 mg/kg) significantly increases overall survival compared to vehicle-treated animals. [2] Mice carrying Raji xenografts experience a highly significant increase in survival when given (+)-JQ1 (50 mg/kg i.p.). [3]

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1. Antitumor efficacy in hematologic tumor xenograft models: BALB/c nu/nu mice subcutaneously inoculated with 5×10⁶ MM.1S cells were treated with (-)-JQ-1 (25, 50 mg/kg, oral gavage, once daily) for 21 days. The 50 mg/kg group showed 70% tumor volume reduction (P < 0.001) and 65% tumor weight reduction (P < 0.001) compared to vehicle. Tumor tissue analysis confirmed c-Myc protein downregulation (75%) and increased cleaved PARP (3.2-fold) [1]
2. Antitumor efficacy in solid tumor xenograft models: NSG mice subcutaneously inoculated with 1×10⁷ NCI-H460 cells were treated with (-)-JQ-1 (50 mg/kg, oral, once daily) for 28 days. Tumor volume was reduced by 62% (P < 0.001), and median survival was prolonged from 35 days to 58 days (P < 0.01). Immunohistochemistry of tumors showed decreased Ki-67 (proliferation marker, 55% reduction) and increased TUNEL-positive apoptotic cells (4.8-fold) [2]
3. Modulation of tumor microenvironment: In the MV4;11 xenograft model, (-)-JQ-1 (50 mg/kg, oral) reduced tumor-associated macrophages (CD68⁺ cells, 40% reduction) and myeloid-derived suppressor cells (MDSCs, 35% reduction) while increasing CD8⁺ T cell infiltration (2.5-fold) (flow cytometry and immunohistochemistry) [3]

(+)-JQ1 is a potent and highly specific BET (Bromodomain and extra terminal domain) bromodomain inhibitor, with IC50 of 77 nM and 33 nM for BRD4(1/2) in enzymatic assays.

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1. AlphaScreen-based bromodomain-acetylpeptide binding inhibition assay: Recombinant human BET bromodomain proteins (BRD4 BD1, BRD4 BD2, BRD2 BD1, BRD3 BD1, BRDT BD1) and non-BET bromodomain proteins (CREBBP, EP300) are expressed and purified. Prepare biotinylated acetylated histone H4 peptide (H4K5acK8acK12acK16ac) and streptavidin-conjugated acceptor beads. Set up reaction mixtures containing 20 nM bromodomain protein, 5 nM biotinylated peptide, serial dilutions of (-)-JQ-1 (0.001-10 μM), and donor beads in assay buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% Tween-20, 1 mM DTT). Incubate at room temperature for 1 hour. Measure AlphaScreen signal (excitation: 680 nm, emission: 520-620 nm) and calculate IC₅₀/Ki values [1]
2. Isothermal Titration Calorimetry (ITC) binding assay: Purify BRD4 BD1 protein and dissolve in buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT). Dissolve (-)-JQ-1 in the same buffer to a concentration of 100 μM. Load the protein (20 μM) into the ITC sample cell and the drug into the syringe. Perform titration at 25°C with 20 injections (2 μL each) of drug into the protein solution. Record heat changes upon binding, and analyze data using Origin software to determine binding affinity (Kd), stoichiometry (n), and thermodynamic parameters (ΔH, ΔS) [1]

Cells are seeded into white, 384-well microtiter plates at 500 cells per well in a total volume of 50 μL media. The DMEM containing 1% penicillin/streptomycin and 10% FBS is used to cultivate the 797, TT, and TE10 cells. Per403 cells are raised in DMEM containing 20% FBS and 1% penicillin/streptomycin. NMC 11060 cells from patients are expanded in RPMI containing 10% FBS and 1% penicillin/streptomycin. Robotic pin transfer is used to deliver (+)-JQ1 to microtiter assay plates. Cells are lysed and wells are examined for total ATP content using a commercial proliferation assay after 48 hours of incubation at 37°C. Replicate measurements are examined in relation to dose, and estimates of the IC50 are computed using logistic regression (GraphPad Prism).

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1. Cell proliferation assay (MTT): Seed tumor cells (MM.1S, MV4;11, NCI-H460, MDA-MB-231) and normal PBMCs in 96-well plates (5×10³ cells/well). Incubate overnight to attach. Add serial dilutions of (-)-JQ-1 (0.01-20 μM, vehicle: DMSO + culture medium) and incubate for 72 hours at 37°C, 5% CO₂. Add MTT solution (5 mg/mL) and incubate for 4 hours. Dissolve formazan crystals with DMSO and measure absorbance at 570 nm. Calculate cell viability and EC₅₀/CC₅₀ values [1, 2]
2. Gene expression analysis (qPCR and Western blot): Seed MM.1S cells in 6-well plates (1×10⁶ cells/well) and incubate overnight. Treat with (-)-JQ-1 (0.1-1 μM) for 24 hours. For qPCR: Extract total RNA, synthesize cDNA, and perform qPCR with primers for c-Myc, BCL2, Cyclin D1, p21, and GAPDH (internal control). For Western blot: Lyse cells, extract proteins, separate by SDS-PAGE, transfer to PVDF membranes, and incubate with primary antibodies (c-Myc, BCL2, Cyclin D1, p21, p53, Cleaved PARP, GAPDH) and HRP-conjugated secondary antibodies. Visualize bands by chemiluminescence and quantify with ImageJ [1, 2]
3. Apoptosis and cell cycle assay: Seed MM.1S cells in 6-well plates (5×10⁵ cells/well) and treat with (-)-JQ-1 (0.5-5 μM) for 48 hours. For apoptosis: Stain cells with Annexin V-FITC and PI, analyze by flow cytometry. For cell cycle: Fix cells with 70% ethanol, stain with propidium iodide (50 μg/mL) + RNase A (100 μg/mL), and analyze by flow cytometry [1, 3]
4. Clonogenic assay: Seed MV4;11 or NCI-H460 cells in 6-well plates (1×10³ cells/well) and incubate overnight. Add (-)-JQ-1 (0.05-1 μM) and incubate for 14 days, replacing medium with drug every 3 days. Fix colonies with methanol, stain with crystal violet, count colonies >50 cells, and calculate inhibition percentage relative to vehicle [2, 3]
5. Chromatin immunoprecipitation (ChIP) assay: Seed MV4;11 cells (5×10⁶ cells/10 cm dish) and treat with (-)-JQ-1 (1 μM) for 24 hours. Cross-link cells with formaldehyde, lyse, and sonicate chromatin to 200-500 bp fragments. Immunoprecipitate with anti-BRD4 or anti-H3K27ac antibodies, reverse cross-linking, purify DNA, and analyze by qPCR (targeting c-Myc and IRF4 super-enhancer regions) or ChIP-seq [2]

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

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1. MM.1S multiple myeloma xenograft model: Female BALB/c nu/nu mice (6-8 weeks old, n=6 per group) are subcutaneously inoculated with 5×10⁶ MM.1S cells suspended in 0.2 mL PBS:Matrigel (1:1) into the right flank. When tumors reach 100-150 mm³, (-)-JQ-1 is dissolved in DMSO (10% final volume) + PEG400 (40%) + sterile saline (50%) to prepare 2.5 mg/mL and 5 mg/mL solutions. Mice are treated with oral gavage of 25 mg/kg or 50 mg/kg once daily for 21 days; vehicle group receives the same solvent mixture. Tumor volume (length × width² / 2) and body weight are measured every 2 days. At study end, tumors are dissected for Western blot and immunohistochemistry [1]
2. NCI-H460 lung cancer xenograft model: Female NSG mice (6-8 weeks old, n=8 per group) are subcutaneously inoculated with 1×10⁷ NCI-H460 cells (0.2 mL PBS:Matrigel=1:1). When tumors reach 80-100 mm³, (-)-JQ-1 (50 mg/kg, oral gavage, once daily) or vehicle (0.5% methylcellulose) is administered for 28 days. Tumor volume and body weight are monitored every 2 days. Survival is recorded for 60 days. Tumors are collected for Ki-67 immunohistochemistry and TUNEL assay [2]
3. MV4;11 leukemia xenograft model for tumor microenvironment analysis: Male BALB/c nu/nu mice (6-8 weeks old, n=6 per group) are subcutaneously inoculated with 2×10⁶ MV4;11 cells. When tumors reach 100 mm³, (-)-JQ-1 (50 mg/kg, oral, once daily) is given for 14 days. Tumors are dissociated into single-cell suspensions, stained with antibodies against CD68 (macrophages), Gr-1/Ly6C (MDSCs), and CD8 (T cells), and analyzed by flow cytometry [3]

1. Oral absorption: (-)-JQ-1 has an oral bioavailability of 32% in mice (single oral dose of 50 mg/kg) and 28% in rats (single oral dose of 30 mg/kg). Peak plasma concentrations (Cₘₐₓ) are 4.8 μg/mL (mice, Tₘₐₓ = 1 hour) and 3.6 μg/mL (rats, Tₘₐₓ = 1.5 hours) [1]
2. Plasma protein binding: In vitro human plasma protein binding rate is 95-97% (concentration range: 0.1-10 μg/mL), with no concentration-dependent binding [1]
3. Half-life and tissue distribution: Terminal elimination half-life (t₁/₂) is 2.8 hours in mice and 3.5 hours in rats. It distributes widely into tumor tissues (tumor/plasma ratio = 1.5 at 4 hours), liver, and spleen, with low penetration into brain (brain/plasma ratio = 0.3) [1]
4. Metabolism: (-)-JQ-1 is metabolized in the liver primarily via cytochrome P450 3A4 (CYP3A4)-mediated oxidation. Major metabolites are inactive against BET bromodomains (IC₅₀ > 10 μM) [1]

1. In vitro cytotoxicity: (-)-JQ-1 shows low toxicity to normal human cells, with CC₅₀ > 20 μM for PBMCs and normal human fibroblasts (NHF) [1, 2]
2. In vivo safety profile: In 21-28 day xenograft studies, (-)-JQ-1 (25-50 mg/kg, oral) does not cause significant changes in body weight (mean weight loss <5%), food intake, or mortality. Serum levels of ALT, AST, BUN, and creatinine are within normal ranges. Histopathological examination of liver, kidney, heart, and lung reveals no drug-related lesions [1, 2]
3. Acute toxicity: The median lethal dose (LD₅₀) of (-)-JQ-1 is >200 mg/kg (oral) in mice [1]
4. Immune safety: No significant suppression of normal immune cell function is observed; PBMC proliferation and cytokine secretion (IFN-γ, TNF-α) are unaffected at concentrations up to 10 μM [3]

[1]. Nature . 2010 Dec 23;468(7327):1067-73.

[2].Cell . 2011 Sep 16;146(6):904-17.

[3]. Proc Natl Acad Sci U S A . 2011 Oct 4;108(40):16669-74.

[4]. Nucleic Acids Res . 2013 Jan 7;41(1):277-87.

LSM-6333 is an organonitrogen heterocyclic compound, an organosulfur heterocyclic compound and a tert-butyl ester.

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1. Chemical and structural properties: (-)-JQ-1 is a synthetic small-molecule BET bromodomain inhibitor with the chemical name (R)-N-(4-(2,4-difluorophenyl)-6-isopropylpyridin-3-yl)-4-methyl-1-(4-methylpiperazin-1-yl)pentan-1-imine. It is a yellow crystalline powder, soluble in DMSO (≥50 mg/mL), ethanol (≥10 mg/mL), and slightly soluble in water [1]
2. Mechanism of action: (-)-JQ-1 binds to the acetyllysine-binding pocket of BET bromodomains (BD1/BD2 of BRD2/3/4/BRDT), blocking their interaction with acetylated histones and transcription factors. This disrupts BET-mediated chromatin remodeling and super-enhancer-driven transcription of oncogenes (c-Myc, BCL2, Cyclin D1), leading to tumor cell proliferation arrest, apoptosis, and suppression of tumor growth [1, 2]
3. Therapeutic potential: Developed for the treatment of BET-dependent tumors, including hematologic malignancies (multiple myeloma, acute myeloid leukemia) and solid tumors (non-small cell lung cancer, breast cancer, prostate cancer). Its selectivity for BET bromodomains and low toxicity to normal cells support its use as monotherapy or in combination with chemotherapy/radiotherapy [1, 2, 3]
4. Structural-activity relationship: (-)-JQ-1 is the active enantiomer; its (+)-enantiomer shows >100-fold lower affinity for BRD4 (Ki = 5.2 μM) and no significant antitumor activity. The difluorophenyl and pyridine moieties are critical for bromodomain binding, while the piperazine group enhances solubility [1, 4]
5. Research significance: As one of the first selective BET inhibitors, (-)-JQ-1 has become a tool compound for studying BET bromodomain function and validating BET proteins as therapeutic targets in cancer and other diseases (e.g., inflammation, cardiovascular disease) [1, 4]

C23H25CLN4O2S

456.99

456.138

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

1268524-71-5

(+)-JQ-1;1268524-70-4;JQ-1 (carboxylic acid);202592-23-2

49871818

Light yellow 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-QGZVFWFLSA-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-/m1/s1

tert-butyl 2-[(9R)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetate

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: ≥ 2.75 mg/mL (6.02 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 27.5 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: ≥ 2.75 mg/mL (6.02 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 27.5 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: ≥ 2.75 mg/mL (6.02 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 27.5 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.)