Study design and dose escalation
Patients with advanced solid tumors refractory to standard therapy were enrolled in the phase I study. The results of this study were previously reported
. Patients were ineligible if they were taking angiotensin converting anzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs).
Ang-(1–7) 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 dose level at which no more than one of six patients experienced a dose-limiting toxicity. This study was approved by the Institutional Review Board of Wake Forest University and was registered with the National Cancer Institute PDQ Database and ClinicalTrials.gov as 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-(1–7) administration. Samples were placed on ice and plasma was extracted within 30 minutes of collection. Plasma samples were stored at −80°C prior to performing biomarker analyses.
Hemolysis was assessed in all plasma samples and five samples from three patients were excluded from biomarker modeling due to hemolysis. Aliquots of plasma were assayed by a third party vendor (Pierce Biotechnology, Woburn, MA). Searchlight ELISA technology was used to prepare standard curves and quantify vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and basic fibroblast growth factor (βFGF). Samples were blinded prior to shipping.
Ang-(1–7) and Ang II peptides were purchased from Bachem (Basel, Switzerland), dissolved in sterile water, and stored at −20°C. EOMA cell lines were purchased from American Type Culture Collection (Manassas, VA) and passaged as recommended. These cells were cultured in Dulbecco’s minimal essential media (DMEM) containing 10% fetal bovine serum (FBS). These cells were cultured in a humidified incubator at 37°C with 5% CO2 and passaged every 3 to 5 days.
Cellular proliferation was measured using the CellTiter 96 assay (Promega, Madison, WI). Assays were plated in sextuplicate replicates in 96 well plates at a density of 1,000 cells per well. Basal absorbance activity was measured immediately prior to treatment. Cells were then treated with Ang-(1–7), Ang II, or untreated as a control. Ang-(1–7) treatments were selected to represent a range of clinically achievable concentrations. Absorbance activity was measured after 72 hours in Ang-(1–7) treated, Ang II treated, and control cells. Proliferation was calculated by subtracting the baseline absorbance from absorbance measured following angiotensin or control treatments. Proliferation rates in treated cells were normalized to untreated control cells.
EOMA cells were independently treated with Ang-(1–7) at a concentration of 500 nM or untreated as a control. Cells were harvested after 24 hours and RNA was extracted using TriReagent (Invitrogen, Carlsbad, CA). RNA concentrations were measured, and RNA integrity was inspected by assessing 18S/28S ratios. Murine angiogenesis and murine endothelial cell biology PCR Arrays were purchased from SABiosciences (Frederick, MD) and real-time RT-PCR reactions were performed per the manufacturer’s protocol. A four-fold change in gene expression was used to identify genes that were regulated by Ang-(1–7) treatment.
Custom PCR Arrays (SABiosciences) were then designed to measure genes identified by these initial PCR Arrays as well as the VEGF and PlGF angiogenic hormones in quadruplicate replicate. Real-time PCR reactions were performed according to the manufacturer’s protocol. Outlying values defined as those greater than 2 standard deviations from the mean were excluded from statistical analyses. In cases where outlying values were observed, PCR Array experiments were repeated to confirm the results.
Biomarker levels over time were modeled after log-transformation, consideration of quadratic effects of time (after centering), and adjustment for plasma drug levels. A mixed effects model was used to tests for interaction of cancer type and biomarker effect. This potential interaction was controlled for presence or absence of clinical benefit and plasma drug levels. In cases where a significant interaction between biomarker and cancer type was observed, univariate regression analyses were performed separately for each cancer type. These regression analyses examined linear biomarker changes over time following drug administration. Two sample t-tests were performed to compare in vitro proliferation rates at each dose level of Ang-(1–7) and Ang II to untreated controls. Two sample t-tests were also performed to compare PCR Array measurements from Ang-(1–7) treated and untreated cells. All analyses were two-sided, and a P-value < 0.05 was considered statistically significant. P values were not corrected for multiple comparisons. Clinical biomarker analyses were performed using SAS v9.1.3 (SAS Institute, Cary, NC) and Stata v10.1 (StataCorp, College Station, TX).