生物活性

PD168393 is a potent, cell-permeable,specific and irreversible inhibitor of EGFR through a specific, covalent modification of the cysteine-733 residuepresent in the catalytic domain of the ATP binding pocket. Does not inhibit any other protein kinases. PD168393 binds to the catalytic domain of the EGFR (EGF receptor tyrosine kinase) with a 1:1 stoichiometry and alkylating Cys773.

Kinaseactivities of PD168393

Receptortyrosine phosphorylation of PD168393[1]

Anti-proliferation of PD168393

体外研究

体内研究

激酶实验

Determination of EGFR tyrosine kinase activity[3]

The epidermal growth factor receptor(EGFR-TK) activity was evaluated in A431 cells treated with epidermal growfactor (EGF, 100 nM) for 60 min in the absence or presence of variousconcentration (0.01-20μM) of PD168393 according to the manufacturer's instructions for theKinase Assay Kit. Tyrosine Kinase Assay Kit is a direct ELISA system for thecolorimetric detection of protein tyrosine phosphotransferase activity using abiotinylated peptide substrate and a monoclonal antiphosphotyrosine- HRPconjugate detection antibody. The results are expressed as concentration ofcompound resulting in 50% inhibition of tyrosine kinase activity (IC50) in A431cell stimulated with EGF.

The EGFR kinase assay was tested as follow.Nunc Maxisorp 96-well plates were incubated overnight at 37°C with 100μl/wellof 0.25 mg/ml poly (L-glutamic acid:Ltyrosine, 4:1) PGT in PBS. Excess PGT wasremoved and the plate was washed three times with wash buffer (Tween 20 (0.1%)in PBS). The kinase reaction was carried out by using 4.5ng/well EGFRaffinity-purified from A431 cells. The compound was added and phosphorylationinitiated by the addition of ATP 50μM. After 8 min at roomtemperature with constant shaking, the reaction was terminated by aspiration ofthe reaction mixture and rinsing the plate four times with wash buffer.Phosphorylated PGT was detected after 2 min incubation with 50μl/wellof HRP-conjugated PY20 anti-phosphotyrosine antibody diluted to 0.2 mg/ml inblocking buffer (3% BSA; 0.05% Tween 20 in PBS). Antibody was removed byaspiration, and the plate washed 4 times with wash buffer. The signals weredeveloped by the addition of 50μl/well of 3,3’,5,5’-tetramethylbenzidine peroxidase substrate andafter blue color development, 50μl of H₂SO₄ (0.09M) was added per well, and plates were read at 450nm using a Bio-Rad ELISAreader.

细胞实验

Cell culture[4]

The DU145 cell line was cultured in DMEMmedium and PC-3 and LNCaP cell lines were cultured in RPMI-1640 medium. MDA PCa2b (an androgen-dependent prostate cancer cell line) was cultured in BRFFHPC1 medium(BRFF Br and TM Media Products). LNCaP and MDA PCa 2b representandrogen-responsive and androgen-dependent cells, respectively. DU145 and PC-3are androgen-independent cells. All media contained 10% FCS, 100 units/mlpenicillin, and 0.1 mg/ml streptomycin.

Combinedtreatment of chemotherapeutic agents and EGFR inhibitors

Cellular chemosensitivity was determined byusing a modified 3-(4, 5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium (MTT)assay in vitro. In brief, cells in 100ml culture medium were seeded into 96-wellmicroplates and incubated at 37^oC for 24 h prior to drug exposure.Cell numbers were titrated to keep control cells growing in the exponentialphase throughout the 72 h incubation period. For the combined treatment, cellswere treated with EGFR inhibitor and chemotherapeutic agent (each in 100 ml ofculture medium) simultaneously and incubated for 72 h. At 72 h, 50 ml of MTT(2mg/ml in RPMI medium) was added to eachwell andincubated for 2.5 h. Blue formazancrystals that formed were pelleted to the bottom of the well by centrifugation,separated from the supernatant, and dissolved in 150ml of dimethylsulfoxymide.The optical density at 492nm was determined by absorbance spectrometry usingamicroplate reader (MRX-2). Three separate experiments with triplicate datawere performed to obtain mean cell viability. Drug concentration that inhibitedcell growth by 50% (IC50) was determined by the dose-effect analysis model andpresented as mean ± standard standarderror of the means (S.E.M.).

动物实验

Animals and surgery[5]

Adult female Sprague-Dawley rats (n = 96;weight 220 to 250 g) were randomly classified into SCI and sham-operated groups.A T10 contusion injury was produced. Briefly, rats were anesthetized byintraperitoneal (ip) injection of ketamine (80mg/kg) and xylazine (10mg/kg).Once rats were confirmed by toe-pinch to be unconscious, a laminectomy wasperformed at thoracic level T10. The spine was immobilized stereotaxically, andweight-drop injury was induced using a standardized instrument releasing aweight (10g, rod diameter of 2mm) from a height of12.5 mm on the exposed duraof the spinal cord, inflicting a moderate contusion injury. Following SCI, ratswere randomly and blindly assigned to either PD168393 or vehicle treatmentgroups. Immediately after injury, an osmotic mini pump placed between theshoulder blades was connected to a 32 gauge catheter and the catheter tip waspositioned subdurally on the dorsal side of the spinal cord over the center of theinjury through a small hole in the dura mater. In the PD168393-treated group,the osmotic pump was filled 2mM PD168393 dissolved in5% DMSO (dissolved in 95%Hank’s balanced salt solution (HBSS)), which was microinfused immediately afterpump placement at a rate of 0.5μl/h for 14 days. Control animals were onlygiven vehicle solution (5% DMSO) lacking PD168393. The overlying back musclesand skin were then sutured. Following surgery, animals were placed andrecovered in a warm environment and were later returned to their originalhousing facility. After 14 days, the pumps were removed and then the wound wasclosed with surgical suture. Sham-operated animals underwent laminectomy alone.Bladders were expressed twice daily until the animals resumed eating anddrinking normally and urinary incontinence disappeared.

LuxolFast Blue staining and cresyl violet eosin staining

Four weeks post-injury, rats weresacrificed, and the tissue was dissected and processed. For myelinquantification, Luxol Fast Blue (LFB) staining was performed on 20μm thickcross-sections (n = 5 in each group). Cryostat sections were processedaccording to the cryo-nerve Fast Blue staining kit protocol.

For quantitative analysis of the totallesion volume in whole spinal cords and neuronal survival following injury inall groups, cresyl violet eosin staining was performed on 20μm thickcross-sections (n = 5 in each group) at the fourth week after SCI.

Serial sections were used for data presentation.Sections were visualized using an Olympus BX-51 light microscope. An unbiasedestimation of the percentage of lesion cavity (or volume of demyelination region)was calculated. The percentage total volume of the injured tissue wascalculated by dividing the total lesion volume by the total spinal cord volume.For rescued motor

neuron counting, sections at 1mm incrementsrostral and caudal to the epicenter were selected and stained with cresyl violetand eosin. Motor neurons located in the ventral horn (VH) with clearly visiblenuclei on both sides of the spinal cord were counted.

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] Fry DW BA, Denny WA, Doherty A, Greis KD, Hicks JL, Hook KE, Keller PR, Leopold WR, Loo JA, McNamara DJ, Nelson JM, Sherwood V, Smaill JB, Trumpp-Kallmeyer S, Dobrusin EM. Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor. Proc Natl Acad Sci U S A. 1998;95(20):12022-12027.
[2] Brahimi F, Rachid Z, Qiu Q, et al. Multiple mechanisms of action of ZR2002 in human breast cancer cells: a novel combi-molecule designed to block signaling mediated by the ERB family of oncogenes and to damage genomic DNA. Int J Cancer. 2004;112(3):484-491.
[3] Tarozzi A, Marchetti C, Nicolini B, et al. Combined inhibition of the EGFR/AKT pathways by a novel conjugate of quinazoline with isothiocyanate. Eur J Med Chem. 2016;117:283-291.
[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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