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Molekulargewicht:
553,59 DCC-2036 (Rebastinib) ist ein Konformationskontroll-Bcr-Abl-Inhibitor für Abl1(WT) und Abl1(T315I) mit IC50 von 0,8 nM und 4 nM, ebenfalls hemmt SRC, LYN, FGR, HCK, KDR, FLT3 und Tie-2, und es ist eine geringe Aktivität gegenüber c-Kit zu sehen. Phase 1.
Biologische Aktivität DCC-2036 zeigt die starke inhibitorische Aktivität gegen gereinigtes natives Abl1 in unphosphorylierten (u-Abl1native) und phosphorylierten (p-Abl1native) Formen, unphosphorylierte und phosphorylierte Gatekeeper-Mutante Abl1T315I und die Aktivierungsschleifen-Mutante Abl1H396P in a nicht-ATP-kompetitive Weise mit IC50 von 0,8 nM, 2 nM, 1,4 nM, 5 nM bzw. 4 nM. Darüber hinaus hemmt DCC-2036 auch die Kinasen der Src-Familie Src, LYN, FGR und HCK sowie die Rezeptor-TKs KDR, FLT3 und TIE2 mit IC50 von 34 nM, 29 nM, 38 nM, 40 nM, 4 nM, 2 nM bzw. 6 nM. DCC-2036 zeigt die antiproliferativen Aktivitäten gegen Ba/F3-Zellen, die natives oder mutiertes Bcr-Abl1 mit IC50 im Bereich von 2 nM bis 150 nM exprimieren. Darüber hinaus hemmt DCC-2036 auch die Proliferation der Ph+-Zelllinie K562 (IC50 5,5 nM) und induziert Apoptose sowohl in Bcr-Abl1-exprimierenden Ba/F3- als auch K562-Zellen wirksam. Eine aktuelle Studie zeigt, dass DCC-2036 die Selektivität für die Wachstumshemmung von Bcr-Abl-positiven Zellen durch seine deutliche Hemmung von CML-Zelllinien im Vergleich zu Nicht-CML-Leukämielinien zeigt. In einem Maus-Allotransplantatmodell, das Ba/F3-Bcr-Abl1T315I-Leukämiezellen trägt, hemmt die Behandlung mit DCC-2036 durch orale Schlundsonde mit 100 mg/kg einmal täglich wirksam die Bcr-Abl1-Signalübertragung und verlängert das Überleben der Maus signifikant. Protokoll (nur als Referenz) Kinase-Assay: [1]
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Assay of Abl1 kinase isoforms and determination of inhibitor potency
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Activity of u-Abl1native is determined by following the production of ADP from the kinase reaction through coupling with the pyruvate kinase/lactate dehydrogenase system. In this assay, the oxidation of NADH (measured as a decreased A340nm) is continuously monitored spectrophotometrically. The final reaction mixture (100 μL, in a 384-well Corning plate) is prepared as follows: An Abl1 kinase/coupled assay components mixture is prepared containing u-Abl1 kinase (1 nM), Abltide (EAIYAAPFAKKK, 0.2 mM), MgCl2 (9 mM), pyruvate kinase (~ 4 units), lactate dehydrogenase (~ 0.7 units), phosphoenol pyruvate (1 mM), and NADH (0.28 mM) in 90 mM Tris containing 0.1 % octyl-glucoside and 1 % DMSO, pH 7.5. Separately, an inhibitor mixture is prepared containing DCC-2036 serially diluted 3-fold in DMSO followed by dilution into buffer composed of 180 mM Tris, pH 7.5, containing MgCl2 (18 mM) and 0.2 % octyl-glucoside. Fifty μL of the inhibitor mixture is mixed with 50 μL of the above Abl1 kinase/coupled assay components mixture, which is then incubated at 30 °C for 2 hours before 2 μL of 25 mM ATP (500 μM, final) is added to start the reaction. The reaction is recorded every 2 minutes for 2.5 hours at 30 °C on a Polarstar Optima or Synergy2 plate reader. Reaction rate (slope) is calculated using the 1 to 2 hour time frame with reader's software. Percent inhibition is obtained by comparison of reaction rate with that of a DMSO control. IC50 values are calculated from a series of percent inhibition values determined at a range of inhibitor concentrations using GraphPad Prism. The kinase assay for Abl1T315I, p-Abl1native or Abl1H396P is assayed the same as above except that 2.2 nM Abl1T315I, 1 nM p-Abl1 native or 1.3 nM Abl1H396P is used. The above assay format is also used for kinases other than Abl1 with the exception of TIE2, for which a fluorescence polarization/Transcreener format is used. The assay conditions are the same as described above except that PolyE4Y (final 1 mg/mL) is used as the substrate and one hour preincubation is used.
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Zellassay: [1]
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Cell lines
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Ba/F3 cells and primary Ph+ leukemia cells
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Concentrations
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0-10 μM
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Incubation Time
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72 hours
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Method
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Ba/F3 cells or primary Ph+ leukemia cells are plated in triplicate in 96-well plates containing test compounds. After 72 hours, viable cells are quantified by Resazurin or MTT assay. Cells are diluted in medium to be added to each well of a 96-well tissue culture-treated plate. All cells are incubated overnight and maintained in a humidified atmosphere at 37 °C and 5% CO2. Cells are treated the following day. Serum-free medium is used during treatment with DCC-2036. MTT is used to assess the viability of cells following treatment. Aliquots of 20 mL of stock MTT solution are added to each well containing 200 mL of medium (10% final solution) and incubated with the cells for 2 hours. Following incubation the medium is removed and 200 mL of dimethylsulfoxide added to solubilize the formazan crystals. The absorbance is read on the plate reader at 550 and 690 nm. A subtraction analysis of the dual wavelength is performed (D550 to D690) to increase accuracy of the measuremen
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Tierstudie: [1]
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Animal Models
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Ba/F3 cells transformed to interleukin-3 independence by transduction with either Bcr-Abl1native or Bcr-Abl1T315I retrovirus are injected intravenously into syngeneic Balb/c mice.
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Formulation
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DCC-2036 is dissolved in 0.5% CMC/1% Tween-80.
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Dosages
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≤100 mg/kg
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Administration
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Administered via p.o.
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Solubility
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0.5% CMC/0.25% Tween 80,
16 mg/mL
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* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
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Umrechnung verschiedener Modelltiere basierend auf BSA (Wert basierend auf Daten aus FDA Draft Guidelines)
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Species
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Baboon
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Dog
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Monkey
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Rabbit
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Guinea pig
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Rat
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Hamster
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Mouse
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Weight (kg)
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12
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10
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3
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1.8
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0.4
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0.15
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0.08
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0.02
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Body Surface Area (m2)
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0.6
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0.5
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0.24
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0.15
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0.05
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0.025
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0.02
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0.007
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Km factor
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20
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20
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12
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12
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8
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6
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5
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3
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Animal A (mg/kg) = Animal B (mg/kg) multiplied by
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Animal B Km
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Animal A Km
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Um beispielsweise die Resveratrol-Dosis für eine Maus (22,4 mg/kg) auf eine Dosis basierend auf dem BSA für eine Ratte zu ändern, multiplizieren Sie 22,4 mg/kg mit dem Km-Faktor für eine Maus und dividieren Sie dann durch den Km-Faktor für eine Ratte. Diese Berechnung ergibt eine Ratten-Äquivalentdosis für Resveratrol von 11,2 mg/kg.
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Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×
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mouse Km(3)
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= 11.2 mg/kg
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rat Km(6)
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Chemische Informationen
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Molecular Weight (MW)
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553.59
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Formula
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C30H28FN7O3
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CAS No.
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1020172-07-9
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Storage
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3 years -20℃Powder
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6 months-80℃in solvent (DMSO, water, etc.)
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Synonyms
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Solubility (25°C) *
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In vitro
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DMSO
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111 mg/mL
(200.5 mM)
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Water
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<1 mg/mL
(
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Ethanol
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16 mg/mL
(28.9 mM)
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In vivo
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0.5% CMC/0.25% Tween 80
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16 mg/mL
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* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
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Chemical Name
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1-(3-tert-butyl-1-(quinolin-6-yl)-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(methylcarbamoyl)pyridin-4-yloxy)phenyl)urea
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Molaritätsrechner Verdünnungsrechner Molekulargewichtsrechner
Produktgruppe : Angiogenese > Bcr-Abl-Inhibitor
