生物活性

Flavopiridol is a synthetic flavonoid based on an extract from Dysoxylum binectariferum which has shown potential for treatment of cancer in vitro. Flavopiridol hydrochloride is a potent inhibitor of cyclin-dependent kinases (primarily Cdk1, Cdk2, and Cdk4) with an IC50 of 100 nM. The (-)-cis form induces apoptosis in particular tumor cells. At nanomolar concentrations, flavoporidol hydrochloride has been shown to reduce rhabdoid cell growth with an IC50 ranging from 50 to 200 nM. Treatment of flavopiridol decreased the activities of Cdk2 and potently prevented cell proliferation with an IC50 ranging from 43 to 83 nM in HNSCC cells.

The flavopiridol inhibition of macromolecular synthesis in breast carcinoma cells[1]

Kinases activity offlavopiridol

The IC50 value for flavopiridol was calculated to be 1μM for muscle GP-b in the presence of 0.8mM AMP and 2.5μM for muscle-GP-a in the absence of AMP.[5]

Effect of flavopiridol on breast and lung carcinoma cell line growth[1]

Drug resistance profile of flavopiridol aganist acute lymphoblastic leukaemia (ALL) cell lines (IC50 in ng/ml)[6]

Cell viability for tumour cell lines of flavopiridol

体外研究

体内研究

30% propylene glycol, 5% Tween 80, 65% D5W(5%葡萄糖水溶液)

激酶实验

Recombinant CDK4 Rimisi- Reactions[12]

CDK4-cyclin D1 kinase activity was determined in microtiter plates as follows. 40μg Gst-Rb were mixed with different amounts of Flavopiridol and unlabeled ATP. Reactions were then started by the addition of an ammonium sulfate cut of the S100 fraction obtained from insect cells expressing recombinant human CDK4 and cyclin D1. The final reaction conditions were 10mM MgCl2, 50 Mm Tris-HCl (pH 7.5), and 1 mM DTT. The final concentration of ATP was adjusted accordingly. Radiolabeled ATP was used as a phosphoryl donor. The reaction was carried out for 2.5 min at 30°C after addition of enzyme and then terminated with the addition of EDTA. The Gst-Rb was then captured with glutathione-Sepharose, and the incorporated radioactivity was determined by liquid scintillation counting.

GP enzyme assay with purified muscle GP[5]

For GP enzyme assays purified rabbit muscle-GP-a and muscle GP-b were dissolved in 40 mMβ-glycerophosphate (pH 6.8), 80 mM cysteine, and stored at 15 °C. GP enzyme activity was determined using a modified protocol. This method is based on the breakdown of glycogen in the presence of orthophosphate to glucose-1-phosphate, which is then converted to glucose-6-phosphate. Glucose-6-phosphate is dehydrogenated to 6-phosphogluconate thereby reducing NADP to NADPH. GP-activity was measured over a 9-min period spectrophotometrically at 340 nm. GP enzyme activity was calculated as mmol NADP reduced/min/mg GP.

For GP enzyme assays 1.5 mg purified muscle-GP-a or muscle-GP-b were diluted in 900 ml assay medium containing 20 mM sodium phosphate (pH 7.2), 2 mM MgSO4, 1 mM NADP, 0.8 mM AMP (only added when indicated), 1.4 units/ml glucose-6-phosphate-dehydrogenase, and 3 units/ml phosphoglucomutase. The enzyme reaction was started by adding 100 μl glycogen (10 mg/ml) yielding a final glycogen concentration of 0.1%. All enzyme assays were performed at 25 °C.

细胞实验

Drugs[13]

Murine 2.5S NGF (mNGF) purified from male mouse submaxillary glands. Flavopiridol hydrochloride (Flav) and sodium selenite (Na2SeO3; Sel) were prepared. Stock solutions of Flav (1 mM) and Sel (100 mM) were prepared in sterile distilled water and stored at –20oC.

Cell Cultures and Treatments

PC12 cells (clone 615) overexpressing the TrkA NGF receptor were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5% heat-inactivated horse serum (HS), 2 mM L-glutamine, 100μg/ml streptomycin, 100 U/ml penicillin, and 200μg/ml G418, in a humidified atmosphere of 95% air 5% CO2at 37OC.

All experiments were performed on PC12 cells differentiated with 2.5S mNGF (10 ng/ml) for 6 days in low-serum medium (DMEM supplemented with 1% FBS and0.5% heat-inactivated HS). The medium was changed every other day and immediately before treatments, which were performed in low-serum medium.

Cell Viability

Cell survival was analyzed by MTT assay. Reduction of the yellow tetrazolium salts (MTT) to the purple formazan is dependent on the activity of mitochondrial dehydrogenases by intact mitochondria and can also be taken as an index of metabolic mitochondrial activity. The assay was performed according to the specifications of the manufacturer. Briefly, PC12 cells (5,000 cells/well) were differentiated in 96-well plates precoated with poly-Llysine (1 mg/ml). After treatments, tetrazolium salts (0.5 mg/ ml) were added directly to the culture medium for 4 hr at37OC in a humidified atmosphere. After incubation, 100μl of the MTT solubilization solution was added to each well for18 hr. The absorbance of samples was measured at wavelength570 nm (700 nm reference wavelength) with a Microplate Reader. MTT conversion levels were expressed as a percentage of control.

动物实验

Animal model[10]

Forty-six female Fischer 344 rats (aged 6-7 weeks and weighing 120-150 g) were purchased from Charles River and housed 5 rats per cage at a temperature of 23oC in a controlled dark-light-rhythm of 12 h under pathogen-low conditions. All rats used in the experiments were acclimatized for 2 weeks under routine laboratory conditions before starting any experiments.

Treatment schedules

The animals were anesthetized with ether inhalation narcosis prior to intraperitoneal (i.p.) injection of flavopiridol. A dose of 0.1 mg/kg flavopiridol dissolved in normal saline was administered, beginning on the 16th week after tumor initiation, daily for 5 days (group A), or 0.1 mg/kg, 3 times weekly over a total of 3 weeks (group B). As control, rats with tumor were treated by intravesical instillation of normal saline in the same schedules. The animals were sacrificed 2 days after the end of therapy by carbon dioxide narcosis. The urinary bladder was excised completely. All organs were fixed in isopentane and then frozen in liquid nitrogen. Sections were cut transversely through the midportion of the bladder, and 4-μm sections were taken to adequately sample the entire bladder. Comparison of the treatment results was done by evaluating proliferation and apoptosis by MIB-1 (KI-67 antigen) immunostaining and TUNEL assay, respectively.

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] Kaur G S-SM, Sebers S, Worland P, Sedlacek H, Myers C, Czech J, Naik R, Sausville E. Growth inhibition with reversible cell cycle arrest of carcinoma cells by flavone L86-8275. J Natl Cancer Inst. 1992;84(22):1736-1740.
[2] AM S. The cell cycle as a target for cancer therapy: basic and clinical findings with the small molecule inhibitors flavopiridol and UCN-01. Oncologist. 2002;7 (Suppl 3):12-19.
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体内溶解度
约26 mg/mL

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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