Tumors were homogenized in 20mM ammonium acetate, pH 3.5 and extracted in acidified methanol as described above. Samples were vortexed for 10 minutes, centrifuged at 13,000rpm for 10 minutes, and the supernatant was transferred to HPLC vials for analysis. Data are presented as mean ± standard all targets deviation. 2.8. Maximum Tolerated Dose (MTD) Studies Inhibitors,research,lifescience,medical HT-29 cells were subcutaneously injected into the right flank of nude mice at a concentration of 5 million in 0.1mL PBS. When the tumors were approximately 300mm3, mice were given both single and multidose (Q4D × 3, Day 0, 4, 8) intravenous injections

of IT-141 at doses ranging from 10–90mg/kg. Body weight was recorded every other day. The MTD was defined as a dose that caused no greater than a 10% loss in body weight and no treatment-related deaths. 2.9. Antitumor Efficacy Studies HT-29 and HCT-116 colon cancer cells were harvested and resuspended in sterile PBS at Inhibitors,research,lifescience,medical a concentration of 2 million (HT-29) or 4 million (HCT-116)

cells per 0.1mL PBS and injected subcutaneously into the right flank of athymic nude mice. Tumors were allowed to establish logarithmic growth (~7–14 days), and the animals were randomly divided into six to eight mice per group. Drug was Inhibitors,research,lifescience,medical administered by a fast bolus injection of 0.2mL into the mouse tail vein on a schedule of Q4D × 3. Bidimensional tumor measurements were made with calipers once Inhibitors,research,lifescience,medical every other day. Tumor volume was calculated according to the formula: V = (short diameter)2(long diameter)/2. Percent inhibition was calculated using the following formula: 100−Vgroup−Vgroup  0VCtl−VCtl  0×100, (1) where Vgroup is the tumor volume on the final day of the study, Vgroup0 is the tumor volume of the group on day 0, VCtl is the tumor volume of the control group on the final

day of the study, and VCtl0 is the tumor volume of the control group on day 0. Tumor regression was calculated using the following formula:   Vgroup  0−Vgroup100×100, (2) where Vgroup is the tumor volume on the final day of the study and Vgroup0 is the tumor volume of Inhibitors,research,lifescience,medical the group on day 0. Statistical differences in tumor volume between groups were calculated using the Student’s t-test using Microsoft Excel, whereby P < 0.05 was considered statistically significant. Data are presented as mean tumor volume ± standard error. 3. Results ITP-101 is a triblock copolymer consisting of poly(ethylene glycol)-b-poly(aspartic Carfilzomib acid)-b-poly(D-leucine-co-tyrosine). The hydrophobic amino acids provide a core region into which a hydrophobic drug can reside, and the amphiphilic PEG block forms a protective corona around the micelle, giving the delivery system stealth-like properties to avoid protein opsonization and RES uptake (Figure 1). The use of both D and L stereoisomers of amino acids in the leucine/tyrosine core block disrupts the secondary structure of the polypeptide.