Background In a phase I study of angiotensin-(1C7) [Ang-(1C7)], clinical benefit

Background In a phase I study of angiotensin-(1C7) [Ang-(1C7)], clinical benefit was associated with reduction in plasma placental growth factor (PlGF) concentrations. related cancer cell line. Since Ang-(1C7) exerts its anti-angiogenic activity through regulation of different angiogenic hormones depending on cancer type in pre-clinical models, it is possible that clinical changes in plasma anti-angiogenic hormones could vary depending on the type of cancer treated [7,8]. Repression of VEGF has been identified in pre-clinical models, but reduction of plasma VEGF levels was not documented in analysis of the phase I study [9]. If changes in VEGF occurred only in certain cancer types, a significant effect on VEGF could have been underestimated by the analysis. The current study tests whether the type of cancer being treated impacted the likelihood of achieving a biomarker response in the phase I study. Changes in VEGF, PlGF, and basic fibroblast IHG2 growth factor (FGF) were tested for interaction with cancer type. Based on these biomarker results, istudies were performed to confirm activity and evaluate mechanisms of action engaged in a vascular sarcoma cell collection. Methods Study design and dose escalation Individuals with advanced solid tumors refractory to standard therapy were enrolled in the phase I study. The results of this study were previously reported [9]. Patients were ineligible if they were taking angiotensin transforming anzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs). Ang-(1C7) was administered by subcutaneous injection daily for five consecutive days on a 21?day?cycle. Treatment was continued until disease progression or unacceptable toxicity. Planned dose cohorts were: 100 mcg/kg, 200 mcg/kg, 400 mcg/kg, 700 mcg/kg, and 1000 mcg/kg. A standard 3?+?3 dose-escalation strategy was utilized. Maximum tolerated dose was defined as the highest E 2012 dose level at which no more than one of six individuals experienced a dose-limiting toxicity. This study was authorized by the Institutional Review Table of Wake Forest University or college and was E 2012 authorized with the National Tumor Institute PDQ Database and ClinicalTrials.gov while “type”:”clinical-trial”,”attrs”:”text”:”NCT00471562″,”term_id”:”NCT00471562″NCT00471562. Measurement of angiogenic hormone levels Blood samples were drawn at time points immediately prior to treatment as well as 1, 2, 3, 4, and 6?hours after Ang-(1C7) administration. Samples were placed on snow and plasma was extracted within 30?moments of collection. Plasma samples were stored at ?80C prior to performing biomarker analyses. Hemolysis was assessed in all plasma samples and five samples from three individuals were excluded from biomarker modeling due to hemolysis. Aliquots E 2012 of plasma were assayed by a third party merchant (Pierce Biotechnology, Woburn, MA). Searchlight E 2012 ELISA technology was used to prepare standard curves and quantify vascular endothelial growth element (VEGF), placental growth element (PlGF), and fundamental fibroblast growth element (FGF). Samples were blinded prior to shipping. Cell tradition Ang-(1C7) and Ang II peptides were purchased from Bachem (Basel, Switzerland), dissolved in sterile water, and stored at ?20C. EOMA cell lines were purchased from American Type Tradition Collection (Manassas, VA) and E 2012 passaged as recommended. These cells were cultured in Dulbeccos minimal essential media (DMEM) comprising 10% fetal bovine serum (FBS). These cells were cultured inside a humidified incubator at 37C with 5% CO2 and passaged every 3 to 5 5?days. Proliferation assays Cellular proliferation was measured using the CellTiter 96 assay (Promega, Madison, WI). Assays were plated in sextuplicate replicates in 96 well plates at a denseness of 1 1,000 cells per well. Basal absorbance activity was measured immediately prior to treatment. Cells were then treated with Ang-(1C7), Ang II, or untreated like a control. Ang-(1C7) treatments were determined to represent a range of clinically attainable concentrations. Absorbance activity was measured after.