Thermally Targeted Delivery of a c-Myc Inhibitory Polypeptide Inhibits Tumor Progression and Extends Survival in a Rat Glioma Model

<div><p>Treatment of glioblastoma is complicated by the tumors’ high resistance to chemotherapy, poor penetration of drugs across the blood brain barrier, and damaging effects of chemotherapy and radiation to normal neural tissue. To overcome these limitations, a thermally responsive polypeptide was developed for targeted delivery of therapeutic peptides to brain tumors using focused hyperthermia. The peptide carrier is based on elastin-like polypeptide (ELP), which is a thermally responsive biopolymer that forms aggregates above a characteristic transition temperature. ELP was modified with cell penetrating peptides (CPPs) to enhance delivery to brain tumors and mediate uptake across the tumor cells’ plasma membranes and with a peptide inhibitor of c-Myc (H1). In rats with intracerebral gliomas, brain tumor targeting of ELP following systemic administration was enhanced up to 5-fold by the use of CPPs. When the lead CPP-ELP-fused c-Myc inhibitor was combined with focused hyperthermia of the tumors, an additional 3 fold increase in tumor polypeptide levels was observed, and 80% reduction in tumor volume, delayed onset of tumor-associated neurological deficits, and at least doubled median survival time including complete regression in 80% of animals was achieved. This work demonstrates that a c-Myc inhibitory peptide can be effectively delivered to brain tumors.</p> </div

Inhibition of Glioma Cell Proliferation by CPP-ELP-H1 Polypeptides <i>in vitro</i>.

<p>C6 cells were exposed to various concentrations of Bac-ELP1-H1 (<b>A</b>) or SynB1-ELP1-H1 (<b>B</b>) at 37°C or 42°C for 1 h, and cell number was determined 72 h later. <b>C.</b> C6, D54, or U-87 MG cells were exposed to 40 µM Bac-ELP1-H1 for 1 h at 37°C or 42°C, and cell number was determined 72 h later. Bars, s.e.m. *, Heated and unheated treatments significantly different (p<0.01, Student’s t-test).</p

Heating Intracerebral C6 Tumors with Infrared Light.

<p>Tumor temperature (as monitored by a needle thermocouple in the tumor core) and body temperature was recorded while illuminating the tumor with 950 nm light from an LED light source. Heating was applied for 20 min, followed by a 10 min cooling period, and this protocol was repeated for 4 cycles. Data represent the mean of three rats, bars, s.d.</p

Enhancement of Bac-ELP1-H1 Tumor Uptake by Thermal Targeting.

<p>Following IV administration of Alexa750-labeled Bac-ELP1-H1 or Bac-ELP2-H1 with or without hyperthermia, tumor and organ levels were determined by <i>ex vivo</i> whole organ fluorescence imaging. <b>A.</b> Representative images of brains from each treatment group. <b>B.</b> Quantitation of tumor fluorescence from each group. <b>C.</b> Quantitation of fluorescence from tumor and all major organs. Bars, s.e.m. *, Fluorescence levels are statistically different (p<0.01, one way ANOVA with post hoc Bonferroni, n = 4 rats/group).</p

Tumor Reduction, Delayed Neurological Deficits, and Extended Survival by Thermal Targeting of Bac-ELP1-H1. A.

<p>Representative MRI images of rats from each treatment group. The arrows point to the tumors’ location. ** Animal didn’t survive to the final scan. <b>B.</b> Series of axial sections from a representative animal from saline (top), saline+hyperthermia (middle), and Bac-ELP1-H1+ hyperthermia (bottom) groups. <b>C.</b> Tumor volumes derived from MRI scans in each group. <b>D.</b> Mean Cumulative Neuroligical Score (on a scale of 0–20, 20 being worst deficit) of each treatment group. Bars, s.e. *, Reductions are statistically significant (p<0.01, one way ANOVA with post hoc Bonferroni, n = 6–9 rats/group). <b>E.</b> Kaplan-Meyer survival analysis of rats from each treatment group. *, Survival times are statistically different (p<0.05, log rank test, n = 6–9 rats/group).</p

Biodistribution of CPP-ELPs Following IV Injection.

<p>Organ distribution of rhodamine-labeled ELP1, SynB1-ELP1, or Bac-ELP1 was determined 4 h after IV administration by quantitative fluorescence analysis. Bars, s.e. *, Organ levels are statistically different (p<0.01, one way ANOVA with post hoc Bonferroni, n = 6 rats/group).</p

Plasma Clearance and Tumor Uptake of CPP-ELPs. A.

<p>Plasma levels with time following IV injection of ELP1-Rho, SynB1-ELP1-Rho, or Bac-ELP1-Rho. Data represent the mean ± s.d. of 6 animals per group. <b>B</b>. Representative images of brain sections 4 h after IV injection of rhodamine-labeled polypeptides. <b>C.</b> Tumor levels 4 h after IV administration of rhodamine-labeled ELP or CPP-ELPs. Bars, s.e. *, Tumor levels are significantly enhanced (p<0.01, one way ANOVA with post hoc Bonferroni, n = 6 rats/group). <b>D.</b> Distribution of rhodamine-labeled polypeptides in tumor and normal brain relative to perfused vasculature. Rhodamine fluorescence was used to follow the localization of the polypeptide within the tumor (left panel), and the perfused vasculature was marked by infusion of high molecular weight dextran 1 min prior to euthanasia (middle panel). <b>E.</b> Microscopic images of tumor sections were collected after staining cell nuclei with Hoechst 33342 using a 60× magnification objective.</p