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Purity: = 98.67%
H3B-8800 is a novel, potent and orally bioavailable modulator of the SF3b complex. H3B-8800 potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers. Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor-encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function. Cancer cells bearing these mutations are preferentially dependent on wild-type (WT) spliceosome function, but clinically relevant means to therapeutically target the spliceosome do not currently exist.
Targets |
SF3B splicing
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ln Vitro |
H3B-8800 potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components.
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ln Vivo |
Oral administration of 2 or 4 mg H3B-8800 per kg body weight (mg/kg) daily slowed the growth of xenografts with SF3B1K700E (P < 0.003, two-way ANOVA followed by Dunnett’s multiple comparison test) but had no effect on SF3B1WT xenografts. Although a dose of 8 mg/kg did slow the growth of SF3B1WT tumors, it completely abrogated growth of SF3B1K700E tumors (P < 0.003).b). H3B-8800 similarly demonstrated significant antitumor activity against xenografts of human HNT-34 acute myeloid leukemia (AML) cells bearing the endogenous mutation encoding SF3B1-K700E. H3B-8800 reached similar concentrations in plasma as well as in tumors of mice harboring SF3B1WT or SF3B1K700E xenografts, and H3B-8800 modulated both canonical and aberrant splicing in a dose-dependent manner. Splicing modulation was observed as early as 1 h following treatment, and splicing returned to pretreatment levels within 24 h following treatment, in accordance with the H3B-8800’s pharmacokinetic profile.
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Enzyme Assay |
SF3B complexes were immunoprecipitated from nuclear extracts prepared from 293F cells overexpressing Flag-tagged SF3B1. First, batch immobilization of antibody to beads was performed through incubating 80 μg of anti-SF3B1 antibody (MBL International D221–3, Anti-Sap155 monoclonal antibody) and 24 mg anti-mouse PVT scintillation proximity assay (SPA) beads (PerkinElmer) for 30 min. After centrifugation (14,000 r.p.m. for 5 min at 4 °C), the antibody–bead mixture was resuspended in PBS supplemented with PhosSTOP phosphatase inhibitor cocktail (Roche) and cOmplete ULTRA protease inhibitor cocktail (Roche). Nuclear extracts were prepared through diluting 40 mg into a total volume of 16 ml PBS with phosphatase and protease inhibitors, and the mixture was centrifuged (14,000 r.p.m. for 10 min at 4 °C). The supernatant was transferred into a clean tube, and the antibody–bead mixture was added and incubated for 2 h. The beads were collected via centrifuging, washed twice with PBS + 0.1% Triton X-100, and resuspended with 4.8 ml of PBS. 100 μl binding reactions were prepared using slurry and varying concentrations of H3B-8800. After 15 min preincubation at room temperature, 1 nM 3H-probe used in Kotake et al.20 was added. The mixture was incubated at room temperature for 15 min, and luminescence signals were read using a MicroBeta2 Plate Counter (PerkinElmer).
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Cell Assay |
Pancreatic cancer cells were seeded at 750 cells per well in a 384 well plate and treated with compound on day 2. K562 isogenic cells (K562-SF3B1K700E and K562-SF3B1K700K) were seeded at 10,000 cells per well in 96-well plates and treated with compound 4 h later. The relative numbers of viable and apoptotic cells were measured via luminescence using CellTiter-Glo or Caspase-Glo 3/7 (Promega) at the indicated time points as instructed.
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Animal Protocol |
K562 isogenic lines, HNT-34 and K052 xenograft models efficacy and pharmacokinetic/pharmacodynamic modeling.
1 × 107 K562 isogenic, HNT-34, or K052 cells were subcutaneously implanted into the flank of female NSG or CB17-SCID mice of 6–8 weeks of age. Mice were treated with H3B-8800 (10% ethanol, 5% Tween-80, 85% saline) or vehicle control. For the efficacy studies, the mice were orally dosed daily, and the mice were monitored until they reached either of the following endpoints: (i) excessive tumor volume (≥20 mm in its longest diameter), which was measured three times a week (tumor volume calculated by using the ellipsoid formula: (length × width2) / 2), or (ii) development of any health problem, such as paralysis or excessive body weight loss. The differences in tumor volume during the study period between the vehicle-treated and H3B-8800-treated groups were analyzed by two-way analysis of variance (ANOVA) followed by the Dunnett’s multiple comparison test. For the pharmacokinetic/pharmacodynamic modeling (PK/PD) studies, the mice were treated with one dose, and the tumors were collected at the indicated times (in Figs. 2 and and3)3) after treatment for further analysis. RNA was isolated using RiboPure RNA purification kit (Ambion) and used for TLDA. All mouse studies were carried out under protocols approved by the Institutional Animal Care and Use Committee at H3 Biomedicine.
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References | |
Additional Infomation |
H3B-8800 is a novel spliceosome inhibitor developed by H3 Biomedicine. It offers the benefit of preferentially killing spliceosome-mutant cancer cells whereas other splicesome inhibitors, such as the pladienolide analogue E7107, show no such preferential targeting. H3B-8800 was granted orphan drug status by the FDA in August 2017 and is in clinical trials for the treatment of acute myelogenous leukemia and chronic myelomonocytic leukemia.
Splicing Inhibitor H3B-8800 is an orally bioavailable inhibitor of the splicing factor 3B subunit 1 (SF3B1), with potential antineoplastic activity. Upon administration, H3B-8800 binds to and blocks the activity of SF3B1, a core spliceosome protein that is mutated in various cancer cells. This modulates RNA splicing by preventing aberrant mRNA splicing by the spliceosome, blocks RNA mis-splicing, enhances proper RNA splicing and prevents the expression of certain tumor-associated genes. This leads to an induction of apoptosis and prevents tumor cell proliferation. In many cancer cells, core spliceosome proteins, including SF3B1, U2 small nuclear ribonucleoprotein auxiliary factor 1 (U2AF1), serine/arginine-rich splicing factor 2 (SRSF2) and U2 small nuclear ribonucleoprotein auxiliary factor subunit-related protein 2 (ZRSR2), are mutated and aberrantly activated leading to a dysregulation of mRNA splicing. Mechanism of Action H3B-8800 is thought to bind to a site similar to pladienolides on the SF3B complex within the spliceosome. Once bound it induces increased retention of short (<300 nucleotide) GC-rich introns through modulation of pre-mRNA processing. These intron-retained mRNA sequences are then thought to be destroyed through the nonsense-mediated decay pathway. It has been suggested that modulation by H3B-8800 is mediated by disruption of branchpoint sequence recognition by the SF3B complex as there is overall less preference for adenosine as the branchpoint nucleotide and a greater amount of sequences with weaker association to the SFB3 in introns retained with H3B-8800. It was found that 41 of 404 genes encoding spliceosome proteins contained GC-rich sequences whose retention was induced by H3B-8800. It is suggested that this is key to the specificity of H3B-8800's lethality as cells with spliceosome-mutant cells are dependent on the expression of wild-type spliceosome components for survival. Since cancer cells, as in myelodysplasia, experience SF3B1 mutations much more frequently than host cells, this allows H3B-8800 to be used to preferentially target these cells by inducing intron-retention in critical spliceosome component pre-mRNA leading to destruction of the now nonsense mature RNA ultimately cell-death due to the lack of these critical proteins. Pharmacodynamics H3B-8800 preferentially targets cells with spliceosome complexes containing mutant splicing factor 3B1 (SF3B1) protein, modulating intron splicing leading to increased death in cancer cells while having little effect on the viability cells with wild-type SF3B1. Both normal and aberrant mature mRNA are suppressed in mutant and wild-type cells, the selectivity of the lethal effect is thought to be due to the presence of mutant SF3B1 and its implications rather than a change in mechanism or potency of effect on the mutant protein over the wild-type [A32749 . Since SF3B1 is frequently mutated in cancer, this allows preferential targeting of cancer cells over host cells. |
Molecular Formula |
C31H45N3O6
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Molecular Weight |
555.705508947372
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Exact Mass |
555.33
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Elemental Analysis |
C, 67.00; H, 8.16; N, 7.56; O, 17.27
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CAS # |
1825302-42-8
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Related CAS # |
1825302-42-8;
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PubChem CID |
92135969
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Appearance |
Yellow to orange solid
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Density |
1.2±0.1 g/cm3
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Boiling Point |
710.6±60.0 °C at 760 mmHg
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Flash Point |
383.6±32.9 °C
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Vapour Pressure |
0.0±2.4 mmHg at 25°C
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Index of Refraction |
1.581
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LogP |
2.22
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Hydrogen Bond Donor Count |
2
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Hydrogen Bond Acceptor Count |
8
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Rotatable Bond Count |
6
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Heavy Atom Count |
40
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Complexity |
928
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Defined Atom Stereocenter Count |
6
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SMILES |
O(C(N1CCN(C)CC1)=O)[C@H]1C=C[C@H](C)[C@@H](/C(=C/C=C/[C@@H](C)C2C=CC=CN=2)/C)OC(C[C@@H](CC[C@@]1(C)O)O)=O |t:12|
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InChi Key |
YOIQWBAHJZGRFW-WVRLKXNASA-N
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InChi Code |
InChI=1S/C31H45N3O6/c1-22(26-11-6-7-16-32-26)9-8-10-23(2)29-24(3)12-13-27(39-30(37)34-19-17-33(5)18-20-34)31(4,38)15-14-25(35)21-28(36)40-29/h6-13,16,22,24-25,27,29,35,38H,14-15,17-21H2,1-5H3/b9-8+,13-12+,23-10+/t22-,24+,25-,27+,29-,31-/m1/s1
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Chemical Name |
(2S,3S,6S,7R,10R,E)-7,10-Dihydroxy-3,7-dimethyl-12-oxo-2-((R,2E,4E)-6-(pyridin-2-yl)hepta-2,4-dien-2-yl)oxacyclododec-4-en-6-yl 4-methylpiperazine-1-carboxylate
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Synonyms |
H3B-8800; H3B8800; RVT-2001; UNII-90YLS47BRX; RVT2001; 1825302-42-8; 90YLS47BRX; 1-Piperazinecarboxylic acid, 4-methyl-, (2S,3S,4E,6S,7R,10R)-7,10-dihydroxy-3,7-dimethyl-2-((1E,3E,5R)-1-methyl-5-(2-pyridinyl)-1,3-hexadien-1-yl)-12-oxooxacyclododec-4-en-6-yl ester; SCHEMBL17255784; EX-A8015; H3B 8800 [WHO-DD]; H3B 8800
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HS Tariff Code |
2934.99.9001
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Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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Solubility (In Vitro) |
DMSO: ≥ 100 mg/mL (~180 mM)
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Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80: Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400  (Please use freshly prepared in vivo formulations for optimal results.) |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 1.7995 mL | 8.9975 mL | 17.9950 mL | |
5 mM | 0.3599 mL | 1.7995 mL | 3.5990 mL | |
10 mM | 0.1800 mL | 0.8997 mL | 1.7995 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
![]() H3B-8800 modulates splicing of WT and mutant SF3B1 spliceosomesin vitroand preferentially kills SF3B1-mutant cells.Nat Med.2018May;24(4):497-504. th> |
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![]() H3B-8800 modulates splicing and selectively kills SF3B1-mutant leukemia cellsin vivo.Nat Med.2018May;24(4):497-504. td> |
![]() H3B-8800 demonstrates preferential activity on SRSF2-mutant leukemia in PDX mice.Nat Med.2018May;24(4):497-504. td> |