生物活性

Crenolanib (CP-868596) is an orallybioavailable specific tyrosine kinase inhibitor that targets and inhibits thekinase activity of PDGFR and the FMS-related tyrosine kinase 3 (FLT3). Crenolanib is significantly more potent than imatinib in inhibiting the kinase activity of imatinib-resistant PDGFRα kinases (D842I, D842V, D842Y, D1842-843IM, and deletion I843). Crenolanib is 135-fold more otent than imatinib against D842V in the isogenic model system, with an IC50 of approximately 10 nM. Crenolanib inhibits the kinase activity of the fusion oncogene in EOL-1 cell line, which is derived from a patient with chronic eosinophilic leukemia and expresses the constitutively activated FIP1L1- PDGFRα fusion kinase, with IC50 = 21 nM. Crenolanib also inhibits the proliferation of EOL-1 cells with IC50 = 0.2 pM. Crenolanib inhibits the activation of V561D or D842V-mutant kinases expressed in BaF3 cells with IC50 with 85 nM or 272 nM, respectively. Crenolanib is a potent and selective inhibitor of PDGFRα/β with Kd of 2.1 nM/3.2 nM, also potently inhibits FLT3, sensitive to D842V mutation not V561D mutation, >100-fold more selective for PDGFR than c-Kit, VEGFR-2, TIE-2, FGFR-2, EGFR, erbB2, and Src. IC50 value: 2.1 nM/3.2 nM(PDGFRα/β).

Kinaseactivity[1]

The inhibitor binding constant [Kd (nM)] ofcrenolanib

Inhibitoryactivity of crenolanib[4]

Cellviability in vitro of crenolanib

体外研究

体内研究

激酶实验

Biochemical assessment of PDGFRα/KIT kinase activity[2]

Chinese hamster ovary (CHO) cells weretransiently transfected with mutated KIT or PDGFRα cDNAconstructs and treated with various concentrations of crenolanib. Experimentsinvolving recombinant DNA were carried out using biosafety level 2 conditionsin accordance with published guidelines. Protein lysates from cell lines were preparedand subjected to immunoprecipitation using anti-KIT or anti-PDGFRα antibodies followed by sequential immunoblotting for phospho-KIT and total KIT,or phosphotyrosine or total PDGFRα. Densitometry was carried outto quantify drug effect using Photoshop 5.1 software, with the level ofphospho-KIT or phospho-PDGFRα normalized to total protein. Densitometry and proliferation experimentalresults were analyzed using Calcusyn 2.1 software to mathematically determinethe IC50 values. 

细胞实验

Celllines[5]

Vincristine-selected HL60/VCR cells,overexpressing ABCB1, were obtained from doxorubicin-selected HL60/ADR cells,overexpressing ABCC1 and mitoxantrone-selected 8226/MR20 myeloma cells,overexpressing wild-type ABCG2 HL60/VCR cells were maintained in drug-free RPMI1640 medium with 10 % fetal bovine serum (FBS) and 8226/MR20 in RPMI 1640medium with 10 % FBS and 20 nM mitoxantrone. Transfected K562 cells stablyoverexpressing ABCB1 or wild-type ABCG2 were cultured in RPMI 1640, pH 7.4,supplemented with 10 % FBS at 37 °C in a humidified atmosphere containing 5 %CO₂. Parental HL60 and K562 cells and MV4-11 and MOLM-14 cells, withFLT3-ITD, were obtained.

Materials

Crenolanib was stored at −80 °C as a 100mM stock solution in dimethyl sulfoxide.

Cellviability assay

Viable cell numbers following drugtreatment were measured using the WST-1 assay. Briefly, 1×10³ cellswere seeded in 100μL complete medium per well in 96-well tissue culture plates andincubated with crenolanib (0-10μM) at 37°Cin5% CO₂ for 96 h. 10μLWST-1reagent was then added to each well, incubation was continued for twoadditional hours and the color developedwas quantified according to themanufacturer’s instructions. Each experiment wasperformed in triplicate. IC50 concentrations were calculated by the leastsquare fit of dose-response inhibition in a non-linear regression model using GraphPadPrism V software.

动物实验

Mice[1]

B6. 129S4-Pdgfra^{tm11 (EGFP)Sor}/J (PDGFRα-GFP) andWild-type C57BL/6J mice were used during the experiment.

Ang II-induced fibrosis mice model

Alzet osmotic miniature pumps deliveringAng at a rate of 1000ng/kg/min (pressor dose) or PBS, were implantedsubcutaneously on the back of 8-week old mice. Crenolanib was dissolved in 5%glycerol formal at the concentration of 1 mg/ml. Crenolanib 15mg/kg viaintraperitoneal injection was carried out daily for 1 or 2 weeks. At the end ofexperiment mice were euthanized and the heart and skin surrounding the pumpoutlet were collected.

Histologic assessment

5 mm thick skin samples were stained withhaematoxylin and eosin (HE). Dermal thickness was evaluated by measuring thedistance between the epidermal-dermal junction and the dermal-fat junction inHE sections. Heart samples were stained with picrosirius red to detect collagendeposition. Collagen content was determined by picrosirius red-positive areaper total area. Densitometric quantification was done using Image J.

Hydroxyproline assay

Collagen deposition was determined bymeasuring total hydroxyproline content in 8mm skin punch biopsies obtained fromPBS and Ang II infusion sites.

Efficacy studies in a mouse xenograft model invivo[6]

A549 cells were injected into the axillaryregions of the athymic nude mice (2×10⁶cells/mouse). When the tumorvolumes reached 70mm³, the mice were randomly allocated to thecontrol group, low-dose crenolanib group (10mg/kg), or high-dose crenolanibgroup (20mg/kg) (n=6 per group). The vehicle for crenolanib treatment consistsof 10% 1-methyl-2-pyrrolidinone and 90% polyethylene glycol 300. The tumor sizeand mouse body weight were measured every other day for about 2 weeks. Thetumor volume was calculated as follows: (mm³) = (width × width ×length)/2. After treatment, the mice were euthanized using carbon dioxide, andthe tumors were harvested and analyzed.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Makino K, Makino T, Stawski L, et al. Blockade of PDGF receptors by crenolanib has therapeutic effect in patient fibroblasts and in preclinical models of systemic sclerosis. J Invest Dermatol. 2017.
[2] Heinrich MC, Griffith D, McKinley A, et al. Crenolanib inhibits the drug-resistant PDGFRA D842V mutation associated with imatinib-resistant gastrointestinal stromal tumors. Clin Cancer Res. 2012;18(16):4375-4384.
[3] Zimmerman EI, Turner DC, Buaboonnam J, et al. Crenolanib is active against models of drug-resistant FLT3-ITD-positive acute myeloid leukemia. Blood. 2013;122(22):3607-3615.
[more]

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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