| 包装 | 价格(元) |
| 10mM (in 1mL DMSO) | 电议 |
| 2mg | 电议 |
| 5mg | 电议 |
| 10mg | 电议 |
| 50mg | 电议 |
| 100mg | 电议 |
Kinase experiment: | Cells are plated overnight and treated with 100 nM AZD3965 or vehicle for 24 hours. The cells are then washed, once in PBS and twice with lysis buffer (50 mM Mops, 100 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.1 mM DTT, 1 mM PMSF supplemented with 1× mini complete protease inhibitor cocktail tablets. The cells are harvested by scraping and centrifugation, and the pellet snap frozen without buffer in liquid nitrogen. Frozen aliquots of cells are thawed on ice and re-suspended in lysis buffer. Cells are lysed by 3 rounds of freezing in liquid nitrogen and thawing at 37℃ for 2 minutes each. Lysates are subsequently centrifuged (13000 g, 10min, 4℃) until clear and then stored on ice. Enzyme activity in the cell lysates is determined using a micro-plate reader to measure either production or depletion of NADH/NADPH, through its absorbance at 340/10 nm, occurring as a result of direct or coupled enzyme reactions. The 96 well plates used for the assays are pre-heated to 37℃ for 10 minutes prior to starting reactions, initiated by the addition of 5 μL cell lysate to 95 μL of reaction buffer (50 mM Mops pH 7.4, 100 mM KCl, 5 mM free magnesium). The standard reaction buffer is supplemented to assay the kinetics of the different enzymes. Absorbance values for controls are subtracted from absorbance of corresponding reactions. Graphpad prism 6 is used to plot V0 values against substrate concentration and determine Vmax and Km values. The Vmax is then normalised to the protein concentration in the cell lysate[1]. |
Animal experiment: | Mice[2] COR-L103 xenografts are grown by subcutaneous injection of 5×106 cells in 0.2 mL of 1:1 serum-free RPMI:Matrigel into the mid-dorsal flank of 8 to 14-week-old male NOD scid gamma mice. Mice are housed in individually vented caging systems in a 12-hour light/12-hour dark environment and maintained at uniform temperature and humidity. Tumor size is measured twice a week using calipers and the volume calculated as tumor length×tumor width2/2. 30 days after implantation, mice bearing tumors between 150 and 250mm3 are randomized into two groups of six and treated with 100 mg/kg BID AZD3965 in 0.5% hydroxypropyl methyl cellulose, 0.1% tween 80 or vehicle only by oral gavage for 21 days. Measurements are continued 3 times a week for the duration of drug treatment to assess tumor growth kinetics. At sacrifice tumors are collected to determine intra-tumor lactate concentration. |
| 产品描述 | Ki = 1.6 nM for MCT1 AZD 3965 is a potent inhibitor of monocarboxylate transporter 1 (MCT1). Inhibition of monocarboxylate transporter 1 (MCT1) is currently considered as a promosing therapeutic way to block lactate shuttling in tumor cells lacking monocarboxylate transporter 4. In vitro: Previous study found that the in-vitro AZD3965 sensitivity varied and was highest in hypoxia. To further support that AZD3965 targeted MCT1, NCIH1048 cells were engineered to inducibly overexpress MCT1. It was found that when MCT1 was overexpressed, the EC50 of AZD 3965 against NCI-H1048 was increased from 0.14 to 10.5 nM, which was consistent with AZD3965 acting through MCT1 inhibition [1]. In vivo: COR-L103 xenograft studies were conducted to test whether the in-vitro effect of AZD3965 could be recapitulated in vivo. COR-L103 tumor-bearing mice were treated with AZD3965 at 100 mg/kg BID for 21 days. The pharmacokinetic analyses showed that AZD3965 at 100 mg/kg BID led to free concentrations of AZD3965 predicted to inhibit lactate transport. Moreover, AZD3965 treatment was able to reduce the growth of CORL103 tumors significantly, though tumor regression was not observed, which was consistent with AZD3965 only targeting the hypoxic fraction of the tumor [1]. Clinical trial: A phase I trial of AZD3965 in patients with advanced cancer is currently recruiting participants [2]. References: |
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