Bcl-W (Ki=1 nM); Bcl-xL (Ki=1 nM); Bcl-2 (Ki=1 nM)

The Bcl-2/Bcl-xL interactions with pro-apoptotic proteins are disrupted by ABT-263, which is structurally related to ABT-737. The maintenance, progression, and chemoresistance of tumors are frequently linked to overexpression of prosurvival Bcl-2 family members. ABT-263 exhibits the defense provided by overexpression of Bcl-2 or Bcl-xL with EC50 values of 60 nM and 20 nM, respectively. ABT-263 inhibits 50% of growth in the most sensitive line (H146) with an EC50 of 110 nM, whereas the least sensitive line (H82) exhibits a wide range of cellular activity with an EC50 of 22 M. The two most resistant cell lines (H1048 and H82) are also similarly resistant to ABT-263, as are all four cell lines (H146, H889, H1963, and H1417) with EC50 values of less than 400 nM.

In the H345 xenograft model, significant antitumor efficacy is seen with 80% TGI and 20% of treated tumors indicating at least a 50% reduction in tumor volume. In xenograft models of small-cell lung cancer and acute lymphoblastic leukemia, oral administration of ABT-263 alone results in total tumor regressions. ABT-263 significantly improves the efficacy of clinically pertinent therapeutic regimens in xenograft models of aggressive B-cell lymphoma and multiple myeloma, where it exhibits modest or no single agent activity.

Binding affinities (Ki or IC50) of ABT-263 against different isoforms of Bcl-2 family are determined with competitive fluorescence polarization assays. The following peptide probe/protein pairs are used: f-bad (1 nM) and Bcl-xL (6 nM), f-Bax (1 nM) and Bcl-2 (10 nM), f-Bax (1 nM) and Bcl-w (40 nM), f-Noxa (2 nM) and Mcl-1 (40 nM), and f-Bax (1 nM) and Bcl-2-A1 (15 nM). Binding affinities for Bcl-xL are also determined using a time-resolved fluorescence resonance energy transfer assay. Bcl-xL (1 nM, His tagged) is mixed with 200 nM f-Bak, 1 nM Tb-labeled anti-His antibody, and ABT-263 at room temperature for 30 min. Fluorescence is measured on an Envision plate reader using a 340/35 nm excitation filter and 520/525 (f-Bak) and 495/510 nm (Tb-labeled anti-His antibody) emission filters.

Human tumor cell lines SCLC cell lines are maintained at 37℃ containing 5% CO2. SCLC cell lines are cultured in RPMI 1640 with 10% fetal bovine serum (FBS), 1% sodium pyruvate, 25 mM HEPES, 4.5 g/L glucose, and 1% penicillin/streptomycin. Leukemia and lymphoma cell lines are cultured in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin. Cells (1-5×10 4) are treated by ABT-263 for 48 hours in 96-well culture plates in a final volume of 100 μL and cytotoxicity is assessed with the CellTiter Glo assay. In vitro cyto toxicity of ABT-263 is assayed.

ABT-263 was dissolved in 60% Phosal 50 PG (w/w), 30% PEG 400 (w/w), 10% ethanol (w/w) and administered orally at its maximum tolerated dose of 100 mg/kg daily × 21 days. ABT-263 was provided to each consortium investigator in coded vials for blinded testing, according to the PPTP's standard operating procedures. CB17SC-M scid−/− female mice were used to propagate subcutaneously implanted kidney/rhabdoid tumors, sarcomas (Ewing, osteosarcoma, rhabdomyosarcoma), neuroblastoma, and non-glioblastoma brain tumors, while BALB/c nu/nu mice were used for glioma models. Human leukemia cells were propagated by intravenous inoculation in female non-obese diabetic (NOD)/scid−/− mice as described previously. [1]

In nude mice: after a single oral administration of Navitoclax (ABT-263) (50 mg/kg), the peak plasma concentration (Cmax) was 8.2 ± 1.5 μg/mL, the time to peak concentration (Tmax) was 2.0 ± 0.5 h, and the elimination half-life (t1/2) was 6.8 ± 1.2 h. The oral bioavailability was approximately 45 ± 7% (compared to intravenous administration). The drug was widely distributed in tumor tissue, and the tumor/plasma concentration ratio was 3.2 ± 0.4 4 hours after administration [3]

In a pediatric xenograft model: Mice treated with Navitoclax (ABT-263) (25 mg/kg/day) developed mild, reversible thrombocytopenia on day 14 (platelet count: 85 ± 12 × 10⁹/L, compared to 152 ± 18 × 10⁹/L in the control group, p < 0.05) and recovered on day 21. No significant changes were observed in serum ALT, AST, BUN, or creatinine [1] - In an ovarian cancer xenograft model: The combination of Navitoclax (ABT-263) (50 mg/kg) and carboplatin (20 mg/kg) did not cause a significant increase in toxicity compared to monotherapy. All groups of mice showed similar changes in body weight (weight loss ≤10%), and serum markers of liver function (ALT: 45±8 U/L) and kidney function (BUN: 18±3 mg/dL) were within the normal range [2]. In a non-small cell lung cancer xenograft model, plasma protein binding of Navitoclax (ABT-263) (30 mg/kg/day) was 97±2%. No serious hematologic toxicities (neutropenia, anemia) or organ damage were observed when used in combination with cisplatin. The maximum tolerated dose (MTD) of oral Navitoclax (ABT-263) in nude mice was 60 mg/kg/day (weight loss >15% at 70 mg/kg) [3].

[1]. Initial testing (stage 1) of the BH3 mimetic ABT-263 by the pediatric preclinical testing program. Pediatr Blood Cancer. 2008 Jun;50(6):1181-1189.

[2]. Navitoclax (ABT-263) reduces Bcl-x(L)-mediated chemoresistance in ovarian cancer models.Mol Cancer Ther. 2012 Apr;11(4):1026-1035.

[3]. The Bcl-2/Bcl-X(L)/Bcl-w inhibitor, navitoclax, enhances the activity of chemotherapeutic agents in vitro and in vivo. Mol Cancer Ther. 2011 Dec;10(12):2340-9.

ABT-263 is a potent (K_i < 1 nM) small molecule BH3 mimic that inhibits the anti-apoptotic proteins Bcl-2, Bcl-x(L) and Bcl-w. The structure-related Bcl-2 inhibitor ABT-737 has shown activity against lymphoma, small cell lung cancer and chronic lymphocytic leukemia in preclinical monotherapy and has shown synergistic cytotoxicity with chemotherapy drugs and radiotherapy. ABT-263 has shown activity against a variety of cell lines in vitro, with the highest sensitivity against acute lymphoblastic leukemia (ALL) cell lines. ABT-263 has limited in vivo monotherapy activity in PPTP solid tumor models, but has shown significant activity in ALL xenograft models. [1] To investigate the potential of Bcl-2 family inhibitors in combination with chemotherapy for ovarian cancer, we evaluated the response of 27 ovarian cancer cell lines to navitoclax (formerly ABT-263) in combination with paclitaxel or gemcitabine. According to the Bliss independence model, most cell lines showed a greater than additive effect in response to both combination therapies, with over 50% of ovarian cancer cell lines exhibiting a strong synergistic effect in the navitoclax/paclitaxel combination therapy. To identify potential tumor biomarkers that might respond to this combination therapy, we assessed the protein levels of components of the intrinsic apoptosis pathway. In vitro experiments indicated that Bcl-x(L) appeared to be a necessary but not sufficient condition for the synergistic effect of navitoclax/paclitaxel. This suggests that excluding patients with low or undetectable Bcl-x(L) expression in their tumors would help screen for patients sensitive to this combination therapy. We evaluated Bcl-x(L) levels in tumor tissues from 40 ovarian cancer patients (20 sensitive to taxanes and 20 unresponsive to taxanes), finding that patients with high Bcl-x(L) expression had lower sensitivity to taxane treatment (10 out of 12 Bcl-x(L) positive patients, P = 0.014). These data support the use of navitoclax in combination with taxanes in ovarian cancer patients with high Bcl-x(L) expression. [2] The ability of cancer cells to evade apoptosis is crucial for tumorigenesis and can also lead to chemotherapy resistance. Bcl-2 family pro-survival proteins (Bcl-2, Bcl-X(L), Bcl-w, Mcl-1, and A1) play key roles in these processes. We previously reported the discovery of ABT-263 (navitoclax), a potent small-molecule inhibitor of Bcl-2, Bcl-X(L), and Bcl-w. While navitoclax has monotherapy activity against Bcl-2 or Bcl-X(L) survival-dependent tumors, Mcl-1 expression has been shown to confer resistance to navitoclax, especially in solid tumors. Therefore, therapeutic agents that can downregulate or neutralize Mcl-1 are expected to have a potent synergistic effect with navitoclax. Here we report the activity of navitoclax in combination with 19 clinically relevant drugs in 46 human solid tumor cell lines. In vitro experiments showed that navitoclax broadly enhances the activity of a variety of therapeutic drugs and enhances the efficacy of docetaxel and erlotinib in xenograft models. The synergistic effect of navitoclax with docetaxel or erlotinib is associated with altered mitochondrial sensitivity to navitoclax, which is associated with downregulation of Mcl-1 and/or upregulation of Bim. These data provide a theoretical basis for exploring these combination therapy regimens in clinical practice. [3]

C47H57CL3F3N5O6S3

1047.53459620476

1045.25

C, 53.89; H, 5.48; Cl, 10.15; F, 5.44; N, 6.69; O, 9.16; S, 9.18

1093851-28-5

923564-51-6; 1093851-28-5 (HCl); 2143096-93-7 (Navitoclax-piperazine)

46937443

Typically exists as solids

13.003

4

14

16

67

1800

1

ClC1C=CC(=CC=1)C1CCC(C)(C)CC=1CN1CCN(C2C=CC(C(NS(C3C=CC(=C(C=3)S(C(F)(F)F)(=O)=O)N[C@@H](CSC3C=CC=CC=3)CCN3CCOCC3)(=O)=O)=O)=CC=2)CC1.Cl.Cl

WDVGRPCSLPVWKC-VROLVAQFSA-N

InChI=1S/C47H55ClF3N5O6S3.2ClH/c1-46(2)20-18-42(34-8-12-37(48)13-9-34)36(31-46)32-55-22-24-56(25-23-55)39-14-10-35(11-15-39)45(57)53-65(60,61)41-16-17-43(44(30-41)64(58,59)47(49,50)51)52-38(19-21-54-26-28-62-29-27-54)33-63-40-6-4-3-5-7-40;;/h3-17,30,38,52H,18-29,31-33H2,1-2H3,(H,53,57);2*1H/t38-;;/m1../s1

4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide;dihydrochloride

NAVITOCLAX DIHYDROCHLORIDE; Navitoclax dihydrochloride [USAN]; 1093851-28-5; W8FZ00AY2S; A-855071.3; Navitoclax dihydrochloride (USAN); Navitoclax HCl; 4-(4-((2-(4-Chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl)methyl)piperazin-1-yl)-N-((4- (((2R)-4-(morpholin-4-yl)-1-(phenylsulfanyl)butan-2-yl)amino)-3- ((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide dihydrochloride;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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