| Size | Price | Stock | Qty |
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| 500mg |
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| Targets |
Receptor-interacting protein 1 (RIP1) kinase. Biochemical apparent inhibition constant (Kiapp) values: human RIP1 (0.00071 μM), cynomolgus monkey RIP1 (0.0013 μM), mouse RIP1 (4.5 μM), rat RIP1 (>10 μM). [3]
Surface plasmon resonance (SPR) determined overall dissociation constant (Kd) of 0.462 nM for the tightly bound complex at 10°C. [3] |
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| ln Vitro |
Inhibition of necroptosis in human colon adenocarcinoma HT29 cells induced by TNF, BV6, and zVAD (TBZ): EC50 = 0.0063 μM, EC90 = 0.020 μM. [3]
Inhibition of necroptosis in mouse L929 cells: IC50 = 0.12 μM, IC90 = 0.74 μM. [3] Inhibition of necroptosis in rat H9c2 cells: IC50 = 0.79 μM, IC90 = 1.2 μM. [3] Inhibition of cell death in primary human colon cells (TBZ-stimulated): IC50 = 0.00072 μM, IC90 = 0.0030 μM. [3] Inhibition of cell death in primary mouse colon cells: IC50 = 0.072 μM, IC90 = 0.64 μM. [3] Inhibition of cell death in primary human kidney epithelial cells: IC50 = 0.0026 μM, IC90 = 0.024 μM. [3] Blocked RIP1 autophosphorylation but did not affect RIP1 protein stability or TNF-stimulated NF-κB and MAPK activation (e.g., IκBα, p38, JNK, ERK phosphorylation) even at concentrations up to 200 μM. [3] In human whole blood ex vivo, inhibited TBZ-stimulated production of CCL3 (MIP-1α), CCL4 (MIP-1β), and IL-1β. IC50/IC90 values: CCL3 (0.0018 ± 0.00034 / 0.0069 ± 0.0027 μM), CCL4 (0.0018 ± 0.00049 / 0.0068 ± 0.0027 μM), IL-1β (0.00086 ± 0.00021 / 0.0046 ± 0.00077 μM). [3] In a Phase 1 healthy volunteer study, ex vivo stimulation of whole blood with TBZ showed GDC-8264 rapidly and completely inhibited CCL4 release. Greater than 90% inhibition of CCL4 was observed at 1.5h post-dose for all single doses (5-225 mg) and was sustained for 14 days with daily dosing (50 and 100 mg). PK-PD modeling estimated an IC50 for CCL4 inhibition of 0.58 ng/mL (~1.8 nM). [2] |
| ln Vivo |
In a TNF plus zVAD (TZ)-driven systemic inflammatory response syndrome (SIRS) mouse model (body temperature as primary endpoint), oral administration of GDC-8264 resulted in dose-dependent inhibition of TZ-induced hypothermia. Total plasma EC20 = 0.43 μM, EC50 = 1.73 μM, EC90 = 15.54 μM. [3]
In a mouse model with intestinal epithelial cell (IEC)-specific NEMO ablation (NEMO IEC-cKO), which causes RIP1 kinase-dependent colitis and ileitis, twice-daily (BID) oral administration of GDC-8264 (1, 5, 15, 50 mg/kg) for 5 days resulted in dose-dependent inhibition of colon and ileum crypt damage and inflammation. The 50 mg/kg BID dose almost completely protected NEMO-deficient intestines. [3] |
| Enzyme Assay |
The biochemical RIP1 assay quantifies RIP1-catalyzed hydrolysis of ATP to ADP using a fluorescence polarization (FP)-based detection method. The reaction mixtures contained HEPES buffer (pH 7.5), MgCl2, dithiothreitol, Brij-35, 50 μM ATP, and 50 nM total RIP1 protein. Inhibitors (GDC-8264) were tested in duplicate at 11 concentrations with a final DMSO concentration of 0.5%. The reaction was initiated by adding RIP1 enzyme to the inhibitor/ATP mixture, incubated at room temperature for 2 hours, then stopped by adding EDTA. An ADP detection mix containing an ADP-specific antibody and a fluorescent ADP tracer was added. After a 1-hour incubation, FP was measured. The ADP concentration was calculated from a standard curve. Fractional activity data were fitted to the tight-binding Morrison equation to calculate the apparent inhibition constant (Kiapp). [3]
Surface plasmon resonance (SPR) chaser assay was performed at 10°C to determine binding kinetics. GDC-8264 binding to RIP1 followed a two-step mechanism: initial formation of a moderate-affinity encounter complex (Kd = 188 nM, kon = 2.46 x 10^5 M^-1 s^-1, koff = 4.87 x 10^-2 s^-1), followed by isomerization to a tightly bound complex (kf = 3.90 x 10^-2 s^-1, kr = 7.93 x 10^-5 s^-1). The overall affinity (Kd) was 0.462 nM, and the dissociation half-life (t1/2) of the tightly bound complex was 4.61 hours. [3] Kinase selectivity was assessed using a binding panel of 468 human kinases (KINOMEscan) at 10 μM GDC-8264. For kinases showing ≥50% inhibition, follow-up Kd determinations were performed. GDC-8264 showed high selectivity, with a Kd of 0.0031 μM for RIP1, while Kd values for other kinases (e.g., Abl1, GRK1, MEK5, NEK3, PI3KCA, PIP5K1C, TBK1) were >10 μM. In a separate 220-member kinase panel (SelectScreen), no kinases were inhibited by ≥50% at 10 μM. [3] |
| Cell Assay |
Cellular necroptosis was assessed using established cell lines (human HT29, mouse L929, rat H9c2) and primary cells. Cells were seeded in 384-well plates and incubated overnight. Necroptosis was induced by adding a mixture of agents: for human and rat cells, TNF (20 ng/mL), the IAP antagonist BV6 (2 μM), and the pan-caspase inhibitor zVAD (20 μM); for mouse cells, TNF (1 ng/mL) and zVAD (20 μM). Cells were immediately treated with a 10-point titration of GDC-8264 or DMSO (0.2% final) using an acoustic dispenser. After 18 (human/mouse) or 24 (rat) hours, CellTiter-Glo reagent was added to quantify ATP levels as a measure of viability. Luminescence was read. The near-total cell death induced by the agents was set to 0% viability, and complete rescue was set to 100% viability. IC50 and IC90 values were calculated by fitting the data to a 4-parameter concentration-response equation. [3]
For Western blot analysis, cells were lysed in a buffer containing Triton X-100, Tris-HCl, NaCl, EDTA, and protease/phosphatase inhibitors. Lysates were centrifuged, and supernatants were analyzed by standard Western blotting using antibodies against RIP1, phospho-RIP1, IκBα, phospho-p38, p38, phospho-JNK, JNK, phospho-ERK, ERK, and actin. [3] In the Phase 1 healthy volunteer study, an ex vivo whole blood stimulation assay was used. Blood samples were collected, and the necroptosis pathway was stimulated with TBZ (TNF-α, BV6, Z-VAD). Levels of CCL4, a downstream biomarker of RIP1 pathway activation, were measured. Percent inhibition of CCL4 was calculated by normalizing to baseline and adjusting to maximum inhibition. [2] |
| Animal Protocol |
For the TZ-induced SIRS model, mice (n=5/group) were orally (PO) administered GDC-8264 (formulated in 10% DMSO/MCT) 15 minutes prior to intravenous (IV) injection of murine TNF (300 μg/kg) and zVAD-FMK (10 mg/kg). Body temperature was measured at 2 and 4 hours post-induction using an infrared ear thermometer. Mice with body temperature below 23.6°C or that were moribund were euthanized. [3]
For the NEMO IEC-cKO colitis/ileitis model, Nemo fl/fl Villin.creERT2 mice were treated with tamoxifen (80 mg/kg, IP) on days 1-3 to induce NEMO deletion. GDC-8264 (formulated in 10% DMSO/MCT) was administered orally (PO), twice daily (BID) at doses of 1, 5, 15, or 50 mg/kg from days 2-6. On day 7, ileum and colon were collected for histology. Tissue sections were scored for crypt damage and inflammation in a blinded fashion. [3] For pharmacokinetic studies in preclinical species, GDC-8264 was formulated as an aqueous suspension in 1% methylcellulose for oral (PO) administration. For intravenous (IV) administration, it was formulated in 10% DMSO, 10% Cremophor EL in saline. [3] A 2-week pilot toxicity study in cynomolgus monkeys (2/sex/group) was conducted. GDC-8264 was administered via oral gavage at doses of 0, 10, 25, and 75 mg/kg once daily for 14 days. Animals were 37-56 months old, with body weights ranging from 3.1-3.8 kg (males) and 2.7-3.7 kg (females). [3] |
| ADME/Pharmacokinetics |
Preclinical PK parameters in C57BL/6 mice (PO 5 mg/kg): Cmax = 61.9 μM, AUC = 242 μM•hr, oral bioavailability (F) = 100%. IV (1 mg/kg): CLp = 2.4 mL/min/kg, Vss = 0.84 L/kg, t1/2 = 12 h. [3]
In Sprague-Dawley rats (PO 5 mg/kg): Cmax = 20.2 μM, AUC = 110 μM•hr, F = 77%. IV (1 mg/kg): CLp = 4.0 mL/min/kg, Vss = 2.59 L/kg, t1/2 = 10 h. [3] In beagle dogs (PO 2 mg/kg): Cmax = 3.36 μM, AUC = 15.6 μM•hr, F = 82%. IV (0.5 mg/kg): CLp = 6.5 mL/min/kg, Vss = 2.31 L/kg, t1/2 = 11 h. [3] In cynomolgus monkeys (PO 2 mg/kg): Cmax = 17.1 μM, AUC = 71.9 μM•hr, F = 82%. IV (0.5 mg/kg): CLp = 1.4 mL/min/kg, Vss = 0.32 L/kg, t1/2 = 5.4 h. [3] In a Phase 1 healthy volunteer study, GDC-8264 showed dose-proportional increases in systemic exposure (5-225 mg). Mean terminal half-life ranged from 10-13 h. After multiple daily doses (50 and 100 mg for 14 days), limited accumulation was observed (accumulation ratio ~1.4). Minimal renal excretion was observed (<0.2% of dose). A high-fat meal had no significant effect on PK parameters. [2] |
| Toxicity/Toxicokinetics |
In vitro safety profiling: GDC-8264 (10 μM) showed no significant binding (>50%) to a panel of 40 non-kinase targets (receptors, ion channels, transporters). [3]
hERG channel inhibition: 2.3% at 1 μM, 3.1% at 10 μM. [3] Human Nav1.5 channel inhibition: 2.8% at 1 μM, 5.3% at 10 μM. [3] Human Cav1.2 channel inhibition: 0.9% at 1 μM, 8.2% at 10 μM. [3] Negative for genotoxicity in the Ames II assay (± rat S9 fraction). [3] Negative for clastogenicity/aneugenicity in the HT-MNT micronucleus assay (± rat S9 fraction). [3] Non-cytotoxic in human hepatocytes (IC50 > 100 μM). [3] Low risk for drug-drug interactions (DDI) – no significant inhibition of CYP3A4, 1A2, 2C19, 2D6, 2D9 (IC50 > 10 μM) and no time-dependent inhibition (IC50 > 10 μM). [3] In a 2-week toxicity study in cynomolgus monkeys, GDC-8264 was well-tolerated at oral doses of 0, 10, 25, and 75 mg/kg/day. No adverse findings were observed in clinical observation, clinical pathology, or anatomic pathology endpoints. [3] In the Phase 1 healthy volunteer study, all adverse events (AEs) in GDC-8264-treated subjects were mild. No deaths or serious AEs occurred. Most frequent AEs in the single-dose stage (5-225 mg) were contact dermatitis (15.8%), headache (15.8%), and nausea (13.2%). In the multiple-dose stage (50 and 100 mg for 14 days), most frequent AEs included abdominal pain, diarrhea, dyspepsia, headache, and dizziness. The incidence of AEs was comparable between GDC-8264 and placebo groups. [2] |
| References | |
| Additional Infomation |
Flizasertib is a small molecule drug. The prefix "-sertib" in its International Nonproprietary Name (INN) indicates that Flizasertib is a serine/threonine kinase inhibitor. Flizasertib is currently being investigated in the clinical trial NCT06602453 (a study evaluating the efficacy and safety of GDC-8264 in the prevention of cardiac surgery-related acute kidney injury (AKI) and major adverse renal events (MAKE)). The monoisotope molecular weight of Flizasertib is 271.11 Da. Flizasertib is an orally potent receptor-interacting serine/threonine protein kinase 1 (RIPK1; receptor-interacting protein 1; RIP1) inhibitor with potential immunomodulatory and anti-inflammatory activities. Oral administration of frizasertib inhibits the RIP1-mediated signaling pathway, thereby protecting intestinal stem cells (ISCs) and preventing RIP1-mediated gastrointestinal (GI) injury in acute graft-versus-host disease (GvHD). RIP1 plays a crucial role in tissue damage and inflammation and cell death caused by pathogen recognition.
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| Molecular Formula |
C15H14FN3O
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|---|---|
| Molecular Weight |
271.289566516876
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| Exact Mass |
271.112
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| Elemental Analysis |
C, 66.41; H, 5.20; F, 7.00; N, 15.49; O, 5.90
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| CAS # |
2268739-68-8
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| PubChem CID |
137408242
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| Appearance |
White to off-white solid at room temperature
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| LogP |
2.5
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
20
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| Complexity |
395
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C(C1CC1)(C1=NN2[C@H](C3=CC=CC=C3)C[C@H](F)C2=N1)=O
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| InChi Key |
LMXPZWQVDDSYHH-RYUDHWBXSA-N
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| InChi Code |
InChI=1S/C15H14FN3O/c16-11-8-12(9-4-2-1-3-5-9)19-15(11)17-14(18-19)13(20)10-6-7-10/h1-5,10-12H,6-8H2/t11-,12-/m0/s1
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| Chemical Name |
cyclopropyl-[(5S,7S)-7-fluoro-5-phenyl-6,7-dihydro-5H-pyrrolo[1,2-b][1,2,4]triazol-2-yl]methanone
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| Synonyms |
Flizasertib; GDC8264; GDC-8264; RG6287; 2268739-68-8;
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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: ~10 mg/mL (36.9 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 | 3.6861 mL | 18.4305 mL | 36.8609 mL | |
| 5 mM | 0.7372 mL | 3.6861 mL | 7.3722 mL | |
| 10 mM | 0.3686 mL | 1.8430 mL | 3.6861 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.
Link: https://clinicaltrials.gov/ct2/show/NCT06602453
Conditions:Acute Kidney InjuryLink: https://clinicaltrials.gov/ct2/show/NCT05673876
Conditions:Acute Graft-versus-host DiseaseProducts are for research use only; We do not sell to patients
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