Anti angiogenic therapies using anti VEGF antibodies or inhibitors of VEGF rece

g. 50 mM hydro chloric acid, The low, medium and high con trols were prepared in pooled plasma and in 50 mM HCl. Samples were run in triplicate and recovery was calculated as the percentage of × 100. Stability The stability of CR8 was determined in plasma samples using concentrations of 0. 75, 1. 5, 3. 5 and 7. 0 ug ml, The samples were kept MAPK 癌 at room temperature, 4 C and −20 C and analyzed twice weekly for 2 months. Peak areas of all the samples were compared to peak areas obtained at time zero. Animals All experiments in this study were approved by the Stockholm Southern Ethics Committee for Animal Research and were conducted in accordance with the Animal Protection Law, the Animal Protection Regula tion and the Regulation for the Swedish National Board for Laboratory Animals.<br><br> Female BALB c mice 8 10 weeks old and weighing 18 21 g were obtained from Scanbur, Sollentuna, Sweden. The mice were allowed to acclimatize to their surroundings for one week before starting treatment. The mice were fed pelleted food and water ad libitum. CR8 was dissolved in DMSO: 50 mM HCl, The first group MK-1775 955365-80-7 of mice received i. v. doses of CR8, the second received oral doses of CR8 and three control mice received vehicle alone. Three mice were sacrificed at each time point and blood samples were collected by cardiac puncture into heparinized tubes at 10, 20, 30, 60 min, 2, 4, 6, 8 h post administration. Blood samples were centrifuged immediately after collection at 3000 g for 5 min at 4 C and the plasma was prepared as men tioned previously.<br><br> Supernatants were collected and all samples were frozen buy MS-275 at −20 C directly after preparation until further analysis by HPLC. Other organs including liver, spleen, kidneys, brain, adipose tissue and lungs were removed, washed and snap frozen immediately in liquid nitrogen. Bone marrow was flushed from both fe murs and mononuclear cells were counted and frozen. The samples were stored at −20 C to avoid any meta bolic activity. Tissue sample preparation Samples from different organs were homogenized by probe sonication in sodium perborate for 2 min and vortexed for 1 min. 100 ul of the homogenate was added to 200 ul methanol, vortexed for 1 min and centrifuged at 10000 g for 10 min. 50 ul of the supernatant were injected into the HPLC system. Femurs were removed and cleaned; bone marrow was flushed with 0.<br><br> 3 ml of PBS and single cell suspension was prepared by gentle flushing through needle and syringe. Nucleated cells were counted using Türk solution and bone marrow was stored at −20 C until assay. Quality control samples were run together with the corresponding mouse samples in duplicate. Pharmacokinetics Parameters including distribution volume of the central compartment, elimination rate constant, plasma max imum concentration and micro constants were esti mated.