Supplementary MaterialsSupplementary figures mmc1. 90?l of methanol and mixed, and the concentration was measured using HPLC. Stability tests were carried out at room temperature (~?22C) or 4C in triplicates. Vitamin E was added in the formulation to a final concentration of 0.01% or 0.04% (v/v) to evaluate the effect of vitamin E on the chemical stability of DHA-dFdC. To study the effect of temperature on the chemical stability of DHA-dFdC, the DHA-dFdC formulation in crimp-sealed vials under nitrogen was stored RP11-175B12.2 at room temperature, 37C, or 60C, protected from light. Analyses and Sampling were carried out at predetermined time points as described over. The first-order degradation response equation was utilized to calculate the ideals from the response rate continuous (ideals versus 1/to calculate the activation energy (antitumor activity of DHA-dFdC was also examined in feminine C57BL/6 mice (6-8 weeks, Charles River) with SC implanted TC-1 mouse lung tumor cells. Quickly, 5 105 cells in RPMI had been SC injected in the proper flank of feminine C57BL/6 mice. Eight times later, mice had been randomized into 3 organizations (worth of ?.05 (two-tailed) was considered statistically significant. Outcomes Solubility, Partition Coefficient, and Balance of DHA-dFdC Beall et al. reported a strategy to gauge the aqueous solubility of medication substances that are unpredictable in drinking water [46], that was adopted in today’s research. To validate the technique, the aqueous solubility of 4-(worth of DHA-dFdC was discovered to become 2.24??0.25. DHA-dFdC solubilized inside a Tween 80/ethanol/drinking water formulation was discovered isoquercitrin tyrosianse inhibitor to degrade substantially isoquercitrin tyrosianse inhibitor at room temperatures (~?22C) (Shape?1and showed that ARA-dFdC had not been more cytotoxic than dFdC significantly. Uptake of DHA-dFdC by Tumor Cells in Tradition The percent of DHA-dFdC or dFdC that was adopted by Panc-02 cells after 4?hours of incubation is shown in Shape?4. The uptake from the DHA-dFdC was about 10-fold greater than that of dFdC. Open up in another window Shape?4 Uptake of DHA-dFdC by Panc-02 cells in culture. Cells (250,000/well) had been coincubated with DHA-dFdC (10?M in DMSO) or dFdC (10?M in cell tradition moderate) for 4?hours. Data are mean??SD (showed that treatment with DHA-dFdC significantly extended the success from the transgenic mice. To confirm that this Kras Ink4a+/? mice died of pancreatic cancer, mice were euthanized after 10 doses of DHA-dFdC. As shown in Physique?6are body weights of mice that were treated with DHA-dFdC, DHA alone, dFdC alone, or the physical mixture of DHA and dFdC. Open in a separate window Physique?7 Antitumor activity of DHA-dFdC against Panc-1-luc human pancreatic tumors implanted SC in nude male mice. Tumor cells (5 106 isoquercitrin tyrosianse inhibitor cells/mouse) were injected (SC) in the right flank of mice on day 0. On day 5, mice were randomized and treated with DHA-dFdC (50?mg/kg) or molar equivalents of DHA, dFdC, or DHA?+?dFdC. Control mice were injected with an aqueous solution of Tween 80/ethanol in 5% of mannitol as a vehicle control. All treatments were given by IP injection twice a week. (A) Tumor growth curves. (B) Mouse body isoquercitrin tyrosianse inhibitor weights. Data are mean??SD (are the body weights of mice that were treated with DHA-dFdC or dFdC. H&E staining of the tumor tissues revealed that tumors in mice that were left untreated have much larger tumor volume and the tumors are poorly differentiated (Physique?9and and antitumor activity of DHA-dFdC is not limited to pancreatic tumors. Discussion In the present study, we reported the synthesis of DHA-dFdC, an amide, by conjugating DHA and dFdC and presented 1H NMR, MS, and LC/MS data to confirm its structure and purity. DHA-dFdC showed potent and broad-spectrum antitumor activity against all of the NCI-60 human tumor cell lines and several human and mouse pancreatic tumor cell lines (Table?1, Physique?3). Unexpectedly, biodistribution studies revealed that DHA-dFdC had a relatively higher accumulation and slower clearance in mouse pancreas after IV injection when compared with dFdC (Physique?5cytotoxicity of the DHA-dFdC may also be related to the more extensive and/or faster cellular uptake of the DHA-dFdC by tumor cells in culture than the uptake of the dFdC alone (Physique?4) but may.