Bcl-2 (Ki = 0.01 nM); Bcl-xL (Ki = 48 nM); Bcl-W (Ki = 245 nM)

ABT-199 shows less sensitivity to Bcl-xL, Mcl-1 and Bcl-w with Ki of 48 nM, > 444 nM and 245 nM, respectively. ABT-199 exhibits weak activity against FL5.12-Bcl-xL cells with an EC50 of 261 nM, but potently inhibits FL5.12-Bcl-2 cells, RS4;11 cells with EC50s of 4 nM and 8 nM. In RS4;11 cells, ABT-199 causes a rapid apoptosis that is accompanied by the release of cytochrome c, activation of caspase, externalization of phosphatidylserine, and accumulation of sub-G0/G1 DNA. According to quantitative immunoblotting, Bcl-2 expression was strongly correlated with ABT-199 sensitivity in cell lines from NHL, DLBCL, MCL, AML, and ALL. The average EC50 for ABT-199 inducing apoptosis in CLL is 3.0 nM. [1]

ABT-199 (100 mg/kg) causes a maximal tumor growth inhibition of 95% and tumor growth delay of 152% in RS4;11 xenografts. ABT-199 can be used alone or in combination with other drugs, such as SDX-105, to inhibit the growth of xenografts (DoHH2, Granta-519). [1]

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Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compound inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. [2]

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To address antileukemia activities of VEN in individual leukemia samples in a situation more similar to a potential clinical application, we investigated its antileukemia activities in a preclinical phase-II-like trial on different individual, patient-derived xenograft ALL samples in mice (N = 12). Three weeks after transplantation onto recipient mice, ALL-bearing animals were treated with VEN (Venetoclax) for 10 days and times to reoccurrence of full-blown, clinically apparent leukemia after treatment with VEN or vehicle were compared for each leukemia. We observed distinct in vivo antileukemia activities of VEN (Venetoclax) indicated by differences of survival times (‘delta survival’) ranging from minimal effects to prolonged survival without manifestation of ALL for more than 140 days (Fig. ​(Fig.3a).3a). This variation of in vivo responses is similar to the heterogeneity of VEN sensitivities observed ex vivo, and EC50 values analyzed ex vivo showed a moderate association with in vivo survival times.[3]

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Upon leukemia manifestation (presence of 5% human ALL cells in the recipients peripheral blood), mice were treated with either VEN (Venetoclax) or vehicle for 10 days followed by assessment of leukemia-free survival until disease manifestation for each recipient. These results obtained from larger groups of biological replicates precisely reflected the drug responses seen in the preclinical trial and, importantly, clearly corresponded to the degree of BCL-2 dependence assessed by mitochondrial priming: (i) PDX13 showed a minor delay of disease manifestation and low BCL-2 dependence (Fig. ​(Fig.3f,3f, mean survival difference 2.3 days, BAD-HRK priming 18.6%), (ii) in PDX10 we observed a significantly delayed onset of overt leukemia upon VEN therapy in line with clear BCL-2 dependence (Fig. ​(Fig.3g,3g, mean survival difference 43.2 days, BAD-HRK priming 56.8%), and (iii) PDX2 showed a prolonged survival with no leukemia manifestation in the VEN group within the observation period (Fig. ​(Fig.3h,3h, more than 70 days superior survival, BAD-HRK priming 80.3%) corresponding to a strong BCL-2 dependence.[3]

Binding affinities (Ki or IC50) of ABT-199 against different isoforms of Bcl-2 family are determined with competitive fluorescence polarization assays. The peptide probe and protein pairs used are as follows: : 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). A time-resolved fluorescence resonance energy transfer assay is also used to determine the binding affinities for Bcl-xL. At room temperature, for 30 minutes, Bcl-xL (1 nM, His tagged), 200 nM f-Bak, 1 nM Tb-labeled anti-His antibody, and ABT-199 are combined.

RS4;11 cells are treated with ABT-199 (Venetoclax) diluted in half-log steps starting at 1 μM-0.05 nM after being seeded at a density of 5 × 104 per well in 96-well plates. ABT-199 (Venetoclax) is incubated with leukemia and lymphoma cell lines for 48 hours after they have been seeded at 1.5-2 × 104 cells per well in the appropriate medium. Using the Cell TiterGlo reagent, effects on proliferation are assessed. The concentration-response data are analyzed using nonlinear regression to determine the EC50 values.[1]

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Cells from T-ALL cell lines (supplemental Table 1, available on the Blood Web site) were plated at 100 000 cells per well in 96-well plates. The cells were incubated for 48 hours in 100 µL medium with 10% FBS to which 5 µL of the appropriate ABT-199 (Venetoclax) dilution or dimethylsulfoxide (DMSO) was added. [2]

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Cell viability assays were performed upon culturing of cells in RPMI 1640 supplemented with 20% FCS and 1% l-glutamine. Cells were exposed to 11 different concentrations of VEN (Venetoclax) (0.1 nM, 1 nM, 10 nM, 50 nM, 100 nM, 250 nM, 500 nM, 1 µM, 3 µM, 5 µM, and 10 µM) for 72 h (BCP-ALL cell lines) or 24 h (BCP-ALL PDX cells). [3]

Mice: Nonobese diabetic/severe combined immunodeficient γ (NSG) mice are given a 150 µL injection of phosphate-buffered saline containing 5×106 luciferase-labeled LOUCY cells at the age of 6 weeks in the tail vein. The IVIS Lumina II imaging system measures the bioluminescence at regular intervals. After the cells have engrafted and the mice have been randomly split into two groups at 6 weeks (each group contains an equal number of males and females), the treatment is initiated on day 0 of the experiment. Venetoclax (ABT-199) 100 mg/kg body weight or vehicle is administered orally to mice for 4 days in a row. Days 0, 2, and 4 are used to measure the bioluminescene.[1]

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Nonobese diabetic/severe combined immunodeficient γ (NSG) mice were injected at 6 weeks of age in the tail vein with 150 µL phosphate-buffered saline containing 5 × 106 luciferase-labeled LOUCY cells. At regular time points, the bioluminescence was measured using the IVIS Lumina II imaging system. At 6 weeks, the cells were engrafted and the mice were randomly divided into 2 groups (with an equal number of males and females in both groups), and the treatment was started on day 0. Mice were treated with 100 mg ABT-199/kg body weight or with vehicle via oral gavage for 4 consecutive days. Venetoclax (ABT-199) was formulated in 60% phosal 50 propylene glycol, 30% polyethylene glycol 400, and 10% ethanol. At days 0, 2, and 4 the bioluminescene was measured. Before imaging, the mice were injected intraperitoneally with 200 µL of a 15 mg/mL firefly d-luciferin potassium salt solution and anesthetized by inhalation of 5% isoflurane. The mice were imaged 10 minutes after luciferin injection. The total bioluminescence signal in each mouse was calculated via the region of interest tool (total counts) in the Living Image software.[2]

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A xenograft of primary human T-ALL cells from patient 3 was established in NSG mice by retro-orbital injection. Upon establishment of disease, human leukemic cells were isolated from the spleen and retransplanted into secondary recipients. Next, tertiary xenograft injections were performed in a cohort of 10 NSG mice and leukemia engraftment was monitored by human CD45 staining in peripheral blood using FACS analysis with the S3 cell sorter. Upon detection of human CD45+ leukemic blasts in peripheral blood, mice were randomized in 2 groups and treated with vehicle or 100 mg Venetoclax (ABT-199)/kg body weight for 7 consecutive days. After treatment, animals were sacrificed and the percentage human CD45-positive leukemic blasts in bone marrow were determined by FACS as described above.[2]

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Upon transplantation of ALL cells, engraftment of human blasts was monitored in peripheral blood by flow cytometry surface staining for huCD19 and huCD4549,50. Mice were treated with vehicle (60% Phosal 50 PG, 30% polyethylene glycol and 10% ethanol) or VEN (Venetoclax) 100 mg/kg/day orally for 10 days. Treatment was initiated on day 21 post transplantation (Fig. ​(Fig.3a)3a) or upon engraftment of more than 5% blasts in the peripheral blood (Fig. 3f–h). Posttreatment survival times were defined as manifestation of clinically overt leukemia in recipient animals upon initiation of treatment. Manifestation of leukemia was confirmed by flow cytometry staining of bone marrow and spleen cells as described above showing high percentages of human ALL in the respective compartments. For the independent cohort (Fig. ​(Fig.4)4) treatment was carried out as previously described.[3]

Absorption, Distribution and Excretion
Following several oral administrations after a meal, the maximum plasma concentration of venetoclax was reached 5-8 hours after the dose. Venetoclax steady state AUC (area under the curve) increased proportionally over the dose range of 150-800 mg. After a low-fat meal, venetoclax mean (± standard deviation) steady-state Cmax was 2.1 ± 1.1 μg/mL and AUC0-24 was 32.8 ± 16.9 μg•h/mL at the 400 mg once daily dose. When compared with the fasted state, venetoclax exposure increased by 3.4 times when ingested with a low-fat meal and 5.2 times with a high-fat meal. When comparing low versus high fat, the Cmax and AUC were both increased by 50% when ingested with a high-fat meal. The FDA label indicataes that venetoclax should be taken with food,.
After single oral administration of 200 mg radiolabeled [14C]-venetoclax dose to healthy subjects, >99.9% of the dose was found in feces and <0.1% of the dose was excreted in urine within 9 days, suggesting that hepatic elimination is responsible for the clearance of venetoclax from systemic circulation. Unchanged venetoclax accounted for 20.8% of the radioactive dose excreted in feces.
The population estimate for apparent volume of distribution (Vdss/F) of venetoclax ranged from 256-321 L.
Mainly hepatic.

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Metabolism / Metabolites
In vitro studies demonstrated that venetoclax is predominantly metabolized as a substrate of CYP3A4/5,,.

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Biological Half-Life
The half-life of venetoclax is reported to be 19-26 hours, after administration of a single 50-mg dose,.

Hepatotoxicity
In clinical trials in 240 patients with CLL, serum aminotransferase elevations occurred in 20% of subjects treated with venetoclax, but the elevations were generally transient, mild and not associated with jaundice or symptoms. In the preregistration trials, no cases of clinically apparent liver injury attributed to venetoclax were reported and few patients required drug discontinuation for liver test abnormalities. Since approval, venetoclax has had limited clinical use, but has not been implicated in cases of clinically apparent liver injury. Venetoclax decreases total white blood cell counts and can cause lymphopenia in addition to neutropenia. As a consequence, venetoclax may be capable of inducing immune reactions including reactivation of hepatitis B. However, instances of reactivation have not been reported, but neither has detailed information on the effects of venetoclax on hepatitis B virus levels in patients with preexisting hepatitis B or evidence of previous infection.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of venetoclax during breastfeeding. Because venetoclax is more than 99% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is 26 hours and it might accumulate in the infant. Most sources consider breastfeeding to be contraindicated during maternal antineoplastic drug therapy. The manufacturer recommends that breastfeeding be discontinued during vemurafenib therapy and for 1 week after the final dose. Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk. Women who receive chemotherapy during pregnancy are more likely to have difficulty nursing their infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Venetoclax is highly bound to human plasma protein with unbound fraction in plasma <0.01 across a concentration range of 1-30 µM (0.87-26 µg/mL). The mean blood-to-plasma ratio was 0.57.

[1]. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med. 2013 Feb;19(2):202-8.

[2]. ABT-199 mediated inhibition of BCL-2 as a novel therapeutic strategy in T-cell acute lymphoblastic leukemia. Blood. 2014 Dec 11;124(25):3738-47.

[3]. Prediction of venetoclax activity in precursor B-ALL by functional assessment of apoptosis signaling. Cell Death Dis. 2019 Aug; 10(8): 571.

Pharmacodynamics
Venetoclax induces rapid and potent onset apoptosis of CLL cells, powerful enough to act within 24h and to lead to tumor lysis syndrome,,. Selective targeting of BCL2 with venetoclax has demonstrated a manageable safety profile and has been shown to induce significant response in patients with relapsed CLL (chronic lymphocytic leukemia) or SLL (small lymphocytic leukemia), including patients with poor prognostic features. This drug is not expected to have a significant impact on the cardiac QT interval. Venetoclax has demonstrated efficacy in various types of lymphoid malignancies, including relapsed/ refractory CLL harboring deletion 17p, with an overall response rate of approximately 80%.

C45H50CLN7O7S

868.44

867.318

C, 62.24; H, 5.80; Cl, 4.08; N, 11.29; O, 12.90; S, 3.69

1257044-40-8

Venetoclax-d8;1257051-06-1

49846579

Yellow solid powder

1.3±0.1 g/cm3

1.644

10.88

3

11

12

61

1640

0

O=C(NS(=O)(C1=CC=C(NCC2CCOCC2)C([N+]([O-])=O)=C1)=O)C3=CC=C(N4CCN(CC5=C(C6=CC=C(Cl)C=C6)CC(C)(C)CC5)CC4)C=C3OC7=CN=C(NC=C8)C8=C7

LQBVNQSMGBZMKD-UHFFFAOYSA-N

InChI=1S/C45H50ClN7O7S/c1-45(2)15-11-33(39(26-45)31-3-5-34(46)6-4-31)29-51-17-19-52(20-18-51)35-7-9-38(42(24-35)60-36-23-32-12-16-47-43(32)49-28-36)44(54)50-61(57,58)37-8-10-40(41(25-37)53(55)56)48-27-30-13-21-59-22-14-30/h3-10,12,16,23-25,28,30,48H,11,13-15,17-22,26-27,29H2,1-2H3,(H,47,49)(H,50,54)

4-[4-[[2-(4-chlorophenyl)-4,4-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[3-nitro-4-(oxan-4-ylmethylamino)phenyl]sulfonyl-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide

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)

DMSO: ~100 mg/mL (~115.1 mM)
Water: <1 mg/mL(slightly soluble or insoluble)
Ethanol: <1 mg/mL

Solubility in Formulation 1: 5 mg/mL (5.76 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension 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 2: 2.5 mg/mL (2.88 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 3: 2.5 mg/mL (2.88 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
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 4: 5% DMSO+50% PEG 300+5% Tween 80+ddH2O: 5 mg/mL

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Solubility in Formulation 5: 20 mg/mL (23.03 mM) in 60% phosal 50 propylene glycol (PG), 30% polyethylene glycol 400 (PEG400), 10% ethanol (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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