Erik C. Wiener

Targeting dendrimer-chelates to tumors and tumor cells expressing the high-affinity folate receptor.

RATIONALE AND OBJECTIVES The authors developed a new method for delivering contrast agents to tumors and tumor cells. Gadolinium complexes of folate-conjugated dendrimer-chelates increased the longitudinal relaxation rate of tumor cells expressing the high-affinity folate receptor, hFR. The coupling of folate to polymeric chelates, composed of a dendrimer backbone, targets these chelates to endogenous folate binding proteins. These proteins exist in both the serum of patients with cancer and on the cell surface of many human cancers of epithelial origin. METHODS The authors attached folic acid to a generation four ammonia core polyamidoamine dendrimer. The folate-dendrimer was reacted with 2-(4-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid to form the polymeric chelate f-PAMAM-TU-DTPA. For fluorescent studies, the generation four dendrimer was reacted with fluorescein-5-isothiocyanate and carboxytetramethylrhodamine succinimidyl ester, followed by capping the remaining amines with succinic anhydride. RESULTS The study results show that cells accumulate the folate-conjugated dendrimer in a receptor specific manner. Tumor cells expressing the high-affinity folate receptor showed a 650% increase in the mean fluorescence. This increase occurred with a rapid rise to 325%, followed by a slow increase to 650%. It required both the expression of the hFR and the coupling of folic acid to the dendrimer. Excess free folic acid inhibited the binding of the folate conjugated polymer. Fluorescent microscopic study showed that the folate-conjugated dendrimer binds to the cell surface and is accumulated within the cells. Treatment of tumor cells that express the hFR with gadolinium complexes of the folate-conjugated polymeric chelate increases the longitudinal relaxation rate by 110%. This increase was inhibited by an excess of free folic acid. CONCLUSIONS These data demonstrate that folate-conjugated magnetic resonance imaging contrast agents represent a promising new approach to tumor targeting.

Specific targeting of folate-dendrimer MRI contrast agents to the high affinity folate receptor expressed in ovarian tumor xenografts.

The need to develop target-specific MRI contrast agents to aid in disease characterization remains highly essential. In this study, we present a generation four polyamidoamine (PAMAM) folate-dendrimer that specifically targets the high affinity folate receptor (hFR) overexpressed on more than 80% of ovarian tumors. In vitro, mouse erythroleukemia cells expressing the hFR bind the radiolabeled folate-dendrimer chelate resulting in over 2700% increase in binding compared with untreated cells. The binding was inhibited by free folic acid to levels observed on folate-receptor-negative cells. In vivo, ovarian tumor xenografts resulted in a 33% contrast enhancement, following the folate-dendrimer chelate administration, that was significantly different compared with results obtained with a non-specific, extracellular fluid space agent, Gd-HP-DO3A. In addition, this contrast enhancement was absent in saline-treated animals, folate-receptor-negative tumors, and was inhibited by free folic acid. Results suggest that a macromolecular, dendrimeric MRI agent with high molecular relaxivities (1646 mM−1 s−1) can be used in specifically targeting the hFR on tumor cells and ovarian tumors.

Development of a tumor-targeting MR contrast agent using the high- affinity folate receptor: Work in progress

RATIONALE AND OBJECTIVES Macromolecular contrast agents enhance tumors by means of active or passive targeting. Active targeting requires surface receptors. Many tumors of epithelial origin express the high-affinity folate receptor (hFR), including ovarian tumors. The objective of this research was to enhance tumors that express hFR using macromolecular contrast agents conjugated to folic acid. METHODS The authors prepared a folate-conjugated dendrimer polychelate by attaching folic acid to a fourth-generation ammonia-core polyamidoamine dendrimer. The remaining amines were reacted with 2-(4-isothiocyanatobenzyl)-6-methyldiethylenetriamine pentaacetic acid. Relaxivity measurements (r1 and r2) and MRI were conducted at 4.7 T. RESULTS The dendrimer r2 exceeded that of Gd-HP-DO3A by 8.2 times at 4.7 T. It increased the tumor percentage contrast enhancement, 24 hours after injection, of T2-weighted images by 33%. CONCLUSIONS This new agent accumulates in tumors expressing hFR. These results do not differentiate between active and passive targeting mechanisms. Receptor-negative tumors suggest a mechanism other than a nonspecific blood pool effect.

Biodistribution of a 153Gd-Folate Dendrimer, Generation = 4, in Mice With Folate-Receptor Positive and Negative Ovarian Tumor Xenografts

Konda SD, Wang S, Brechbiel M, et al. Biodistribution of a 153Gd-folate dendrimer, generation = 4, in mice with folate-receptor positive and negative ovarian tumor xenografts. Invest Radiol 2002;37:199–204. rationale and objectives. An important characteristic of targeted contrast agents is how they are tolerated in a biologic environment and their localization in the surrounding tissues in addition to target tissue. We evaluate the biodistribution of a gadolinium Gd 153-folate-dendrimer in high affinity folate-receptor (hFR) positive and negative ovarian tumor xenografts. methods. The 153Gd-folate-dendrimer chelate was prepared by exchanging 153Gd with nonradioactive gadolinium for 1 week, followed by extensive filtration. Athymic mice with hFR-positive (n = 3) and negative tumors (n = 3) were injected intravenously and counted using a whole-body counting system with a 80 to 150 keV counting window. results. The hFR-positive tumors accumulate 3.6% ± 2.8% injected dose/g, whereas only background counts were found in hFR-negative tumors. The folate-dendrimer’s tumor-to-blood ratio of 12.6, in hFR-positive tumors, was ∼5.7 to 17.0 fold better than those obtained with monoclonal antibodies targeted to the folate receptor. conclusions. Biodistribution studies confirm previous MRI findings and show that the accumulation of the folate-dendrimer requires the expression of the hFR.

Characteristics of a new MRI contrast agent prepared from polypropyleneimine dendrimers, generation 2.

Rationale and objectives:Dendrimer-based magnetic resonance imaging (MRI) contrast agents offer many advantages including high levels of amplification. The objective of this research was to test the adequacy and viability of a new family of dendrimers for use as MRI contrast agents in vitro and in vivo. Methods:Dendrimers based on 1,4-diaminobutane core polypropyleneimine (PPI) generation 2 and ammonia core polyamidoamine dendrimers had the free surface amines conjugated to a diethylenetriaminepentaacetic acid derivative followed by complex formation with gadolinium. Relaxivity measurements were made on an IBM Field Cycling Relaxometer. Biodistribution and pharmacokinetic studies were examined with the radiotracer 153Gd in rats and a counting window of 95 to 105 keV. MRI images were conducted at 4.7 T. Results:The relaxivity of the PPI agent exceeded that of the corresponding generation polyamidoamine (PAMAM) agent. Uptake occurred in the liver, spleen, and kidney. Pharmacokinetic studies showed a biexponential decay with excretion half-lives of 3 hours and 33.6 days respectively. The agent increased the contrast enhancement, 1 hour after injection, of T1-weighted images by 52%. Conclusions:This PPI agent resulted in significant contrast signal enhancement. This family of agent may also provide a valuable contrast agent backbone.

ZD6474, a Multitargeted Inhibitor for Receptor Tyrosine Kinases, Suppresses Growth of Gliomas Expressing an Epidermal Growth Factor Receptor Mutant, EGFRvIII, in the Brain

Epidermal growth factor receptor (EGFR) vIII is a mutated EGFR that is frequently overexpressed in glioblastomas and implicated in response to receptor tyrosine kinase inhibitors. In this study, we investigate the effect of ZD6474 (ZACTIMA, vandetanib), a dual inhibitor for vascular endothelial growth factor receptor 2 and EGFR on growth and angiogenesis of gliomas expressing EGFRvIII. We used two glioma xenograft models, U87MG cells overexpressing EGFRvIII and short-term cultured primary glioma GBM8 cells with EGFRvIII. ZD6474 inhibited tumor growth and angiogenesis and induced cell apoptosis in various brain gliomas. Moreover, significant inhibition of EGFRvIII-expressing U87MG and GBM8 gliomas was observed compared with their controls. Magnetic resonance imaging analysis using the apparent diffusion coefficient and three-dimensional T2*weighed measurements validated ZD6474 inhibition on tumor growth and angiogenesis in EGFRvIII-expressing GBM8 gliomas. Mechanistically, ZD6474 shows better inhibition of cell growth and survival of U87MG/EGFRvIII, GBM6, and GBM8 cells that express EGFRvIII than U87MG or GBM14 cells that have nondetectable EGFRvIII through attenuation of activated phosphorylation of signal transducer and activator of transcription 3, Akt, and Bcl-XL expression. Albeit in lesser extent, ZD6474 also displays suppressions of U87MG/EGFR and GBM12 cells that overexpress wild-type EGFR. Additionally, ZD6474 inhibits activation of extracellular signal-regulated kinase 1/2 in both types of cells, and expression of a constitutively active phosphoinositide 3-kinases partially rescued ZD6474 inhibition in U87MG/EGFRvIII cells. Taken together, these data show that ZD6474 significantly inhibited growth and angiogenesis of gliomas expressing EGFRvIII by specifically blocking EGFRvIII-activated signaling mediators, suggesting a potential application of ZD6474 in treatments for glioblastomas that overexpress EGFRvIII. Mol Cancer Ther; 9(4); 929–41. ©2010 AACR.

Comparison of tumor histology to dynamic contrast enhanced magnetic resonance imaging-based physiological estimates

PURPOSE The purpose of this study was to compare histologically determined cellularity and extracellular space to dynamic contrast-enhanced magnetic resonance imaging (DCE MRI)-based maps of a two-compartment models parameters describing tumor contrast agent extravasation, specifically tumor extravascular extracellular space (EES) volume fraction (ve), tumor plasma volume fraction (vp) and volume-normalized contrast agent transfer rate between tumor plasma and interstitium (KTRANS/VT). MATERIALS AND METHODS Obtained ve, vp and KTRANS/VT maps were estimated from gadolinium diethylenetriamine penta-acetic acid DCE T1-weighted gradient-echo images at resolutions of 469, 938 and 2500 microm. These parameter maps were compared at each resolution to histologically determined tumor type, and the high-resolution 469-microm maps were compared with automated cell counting using Otsus method and a color-thresholding method for estimated intracellular (Vintracellular) and extracellular (Vextracellular) space fractions. RESULTS The top five KTRANS/VT values obtained from each tumor at 469 and 938 microm resolutions are significantly different from those obtained at 2500 microm (P<.0001) and from one another (P=.0014). Using these top five KTRANS/VT values and the corresponding tumor EES volume fractions ve, we can statistically differentiate invasive ductal carcinomas from noninvasive papillary carcinomas for the 469- and 938-microm resolutions (P=.0017 and P=.0047, respectively), but not for the 2500-microm resolution (P=.9008). The color-thresholding method demonstrated that ve measured by DCE MRI is statistically similar to histologically determined EES. The Vextracellular obtained from the color-thresholding method was statistically similar to the ve measured with DCE MRI for the top 10 KTRANS/VT values (P>.05). DCE MRI-based KTRANS/VT estimates are not statistically correlated with histologically determined cellularity. CONCLUSION DCE MRI estimates of tumor physiology are a limited representation of tumor histological features. Extracellular spaces measured by both DCE MRI and microscopic analysis are statistically similar. Tumor typing by DCE MRI is spatial resolution dependent, as lower resolutions average out contributions to voxel-based estimates of KTRANS/VT. Thus, an appropriate resolution window is essential for DCE MRI tumor diagnosis. Within this resolution window, the top KTRANS/VT values with corresponding ve are diagnostic for the tumor types analyzed in this study.

Identifying tumor vascular permeability heterogeneity with magnetic resonance imaging contrast agents

Aref M, Brechbiel M, Wiener E. Identifying tumor vascular permeability heterogeneity with magnetic resonance imaging contrast agents. Invest Radiol 2002;37:178–192. rationale and objectives. Dynamic contrast enhanced (DCE) MR mammography (MRM) uses tumor capillary density differences for prognosis. The heterogeneous response of permeability-surface area products (PS = Kp↔t) was examined in mammary tumors, as a function of contrast agent size, to determine what effect ROI size might have on PS and prognosis. methods. DCE FLASH signal intensities were converted to gadolinium concentrations by a standard curve, which was fitted by a two-compartment model for the tumor’s extravascular extracellular space (EES) volume fraction (ve), and the tumor volume normalized transfer rate between plasma and EES (Kp↔t/VT). results. For Gd-DTPA ve = 9% to 13% Kp↔t/VT = 0.01 to 0.06 minutes−1, and the macromolecular agent, PAMAM-TU-DTPA G = 4 ve = 0.8% to 1% Kp↔t/VT = 0.008 to 0.04 minutes−1. Significant differences in Kp↔t/VT for local regions were found for both agents relative to the whole tumor and the macromolecular agent had greater dynamic range. conclusions. Smaller ROI values or pixels should yield more accurate assessment of neovascularization.

Combined imaging biomarkers for therapy evaluation in glioblastoma multiforme: correlating sodium MRI and F-18 FLT PET on a voxel-wise basis

We evaluate novel magnetic resonance imaging (MRI) and positron emission tomography (PET) quantitative imaging biomarkers and associated multimodality, serial-time-point analysis methodologies, with the ultimate aim of providing clinically feasible, predictive measures for early assessment of response to cancer therapy. A focus of this work is method development and an investigation of the relationship between the information content of the two modalities. Imaging studies were conducted on subjects who were enrolled in glioblastoma multiforme (GBM) therapeutic clinical trials. Data were acquired, analyzed and displayed using methods that could be adapted for clinical use. Subjects underwent dynamic [(18)F]fluorothymidine (F-18 FLT) PET, sodium ((23)Na) MRI and 3-T structural MRI scans at baseline (before initiation of therapy), at an early time point after beginning therapy and at a late follow-up time point after therapy. Sodium MRI and F-18 FLT PET images were registered to the structural MRI. F-18 FLT PET tracer distribution volumes and sodium MRI concentrations were calculated on a voxel-wise basis to address the heterogeneity of tumor physiology. Changes in, and differences between, these quantities as a function of scan timing were tracked. While both modalities independently show a change in tissue status as a function of scan time point, results illustrate that the two modalities may provide complementary information regarding tumor progression and response. Additionally, tumor status changes were found to vary in different regions of tumor. The degree to which these methods are useful for GBM therapy response assessment and particularly for differentiating true progression from pseudoprogression requires additional patient data and correlation of these imaging biomarker changes with clinical outcome.

A Fluorinated Dendrimer-Based Nanotechnology Platform: New Contrast Agents for High Field Imaging

Objectives:We aimed to develop a directly detected magnetic resonance imaging (MRI) contrast agent for use with high fields based on a nanoscale fluorinated dendrimer-based platform for 19F MRI and overcome some of the problems with 19F MRI. Materials and Methods:The dendrimers were prepared in a convergent manner by making the appropriate dendron, followed by coupling to a central core. The dendrons were prepared by attaching 3 equivalents of the fluorinated amino acid to the 3 carboxylic acids of the repeat branch unit followed by deprotection of the amine branch point, and either coupling to another repeat branch unit (increasing the generation G) or used directly allowing the precise growth of the dendrimer. The size of the dendrimers was determined by diffusion nuclear magnetic resonance (NMR) spectroscopy. The toxicity of the dendrimers was measured using the MTT assay. Fluorine longitudinal relaxation time measurements were performed on a Bruker ACP-500 NMR using a saturation recovery experiment at 470.59 MHz frequency. Healthy 150 g Sprague-Dawley female rats were imaged using a dendrimer solution. Results:The size of the dendrimers is generally less than 3 nm, 2 orders of magnitude smaller than the size of the perfluorocarbon nanoparticles (about 200 nm). The longitudinal relaxation time, T1, decreases with increasing dendrimer generation. A significant improvement in relaxation rate and signal-to-noise ratio can be achieved by either the chemical modification of the dendrimer with a gadolinium-chelate or by the physical addition of exogenous contrast agent. Although the dendrimers with fluorine in the surface layer are toxic, this toxicity is easily reduced by burying the fluorine further into the dendrimer interior. 19F MR images of the rat using the dendrimer solution were rapidly obtained at 7 Tesla, the strong contrast in the heart generated by the dendrimer can be seen. Conclusions:A novel fluorinated dendrimer-based nanotechnology platform in 19F MRI and a new bifunctional DOTA chelate were prepared and characterized. We introduce 2 methods for reducing the 19F longitudinal relaxation time: (a) Increasing the generation; (b) covalent and noncovalent introduction of Gd(III)-chelates. A new bifunctional Gd(III)-chelate is presented. The investigations of imaging on rats suggest potential importance of the dendrimers in 19F MRI application.