John L. Mikitsh

Endothelial targeting of polymeric nanoparticles stably labeled with the PET imaging radioisotope iodine-124

Targeting of therapeutics or imaging agents to the endothelium has the potential to improve specificity and effectiveness of treatment for many diseases. One strategy to achieve this goal is the use of nanoparticles (NPs) targeted to the endothelium by ligands of protein determinants present on this tissue, including cell adhesion molecules, peptidases, and cell receptors. However, detachment of the radiolabel probes from NPs poses a significant problem. In this study, we devised polymeric NPs directly labeled with radioiodine isotopes including the positron emission tomography (PET) isotope (124)I, and characterized their targeting to specific endothelial determinants. This approach provided sizable, targetable probes for specific detection of endothelial surface determinants non-invasively in live animals. Direct conjugation of radiolabel to NPs allowed for stable longitudinal tracking of tissue distribution without label detachment even in an aggressive proteolytic environment. Further, this approach permits tracking of NP pharmacokinetics in real-time and non-invasive imaging of the lung in mice using micro-PET imaging. The use of this strategy will considerably improve investigation of NP interactions with target cells and PET imaging in small animals, which ultimately can aid in the optimization of targeted drug delivery.

Pathways for Small Molecule Delivery to the Central Nervous System Across the Blood-Brain Barrier

The treatment of central nervous system (CNS) disease has long been difficult due to the ineffectiveness of drug delivery across the blood-brain barrier (BBB). This review summarizes important concepts of the BBB in normal versus pathophysiology and how this physical, enzymatic, and efflux barrier provides necessary protection to the CNS during drug delivery, and consequently treatment challenging. Small molecules account for the vast majority of available CNS drugs primarily due to their ability to penetrate the phospholipid membrane of the BBB by passive or carrier-mediated mechanisms. Physiochemical and biological factors relevant for designing small molecules with optimal capabilities for BBB permeability are discussed, as well as the most promising classes of transporters suitable for small-molecule drug delivery. Clinically translatable imaging methodologies for detecting and quantifying drug uptake and targeting in the brain are discussed as a means of further understanding and refining delivery parameters for both drugs and imaging probes in preclinical and clinical domains. This information can be used as a guide to design drugs with preserved drug action and better delivery profiles for improved treatment outcomes over existing therapeutic approaches.

Shape-Controlled Synthesis of Isotopic Yttrium-90-Labeled Rare Earth Fluoride Nanocrystals for Multimodal Imaging

Isotopically labeled nanomaterials have recently attracted much attention in biomedical research, environmental health studies, and clinical medicine because radioactive probes allow the elucidation of in vitro and in vivo cellular transport mechanisms, as well as the unambiguous distribution and localization of nanomaterials in vivo. In addition, nanocrystal-based inorganic materials have a unique capability of customizing size, shape, and composition; with the potential to be designed as multimodal imaging probes. Size and shape of nanocrystals can directly influence interactions with biological systems, hence it is important to develop synthetic methods to design radiolabeled nanocrystals with precise control of size and shape. Here, we report size- and shape-controlled synthesis of rare earth fluoride nanocrystals doped with the β-emitting radioisotope yttrium-90 ((90)Y). Size and shape of nanocrystals are tailored via tight control of reaction parameters and the type of rare earth hosts (e.g., Gd or Y) employed. Radiolabeled nanocrystals are synthesized in high radiochemical yield and purity as well as excellent radiolabel stability in the face of surface modification with different polymeric ligands. We demonstrate the Cerenkov radioluminescence imaging and magnetic resonance imaging capabilities of (90)Y-doped GdF3 nanoplates, which offer unique opportunities as a promising platform for multimodal imaging and targeted therapy.

Development of 124I-Immuno-PET Targeting Tumor Vascular TEM1/Endosialin

Tumor endothelial marker 1 (TEM1/endosialin) is a tumor vascular marker highly overexpressed in multiple human cancers with minimal expression in normal adult tissue. In this study, we report the preparation and evaluation of 124I-MORAb-004, a humanized monoclonal antibody targeting an extracellular epitope of human TEM1 (hTEM1), for its ability to specifically and sensitively detect vascular cells expressing hTEM1 in vivo. Methods: MORAb-004 was directly iodinated with 125I and 124I, and in vitro binding and internalization parameters were characterized. The in vivo behavior of radioiodinated MORAb-004 was characterized in mice bearing subcutaneous ID8 tumors enriched with mouse endothelial cells expressing hTEM1 and by biodistribution and small-animal immuno-PET studies. Results: MORAb-004 was radiolabeled with high efficiency and isolated in high purity. In vitro studies demonstrated specific and sensitive binding of MORAb-004 to MS1 mouse endothelial cells expressing hTEM1, with no binding to control MS1 cells. 125I-MORAb-004 and 124I-MORAb-004 both had an immunoreactivity of approximately 90%. In vivo biodistribution experiments revealed rapid, highly specific and sensitive uptake of MORAb-004 in MS1-TEM1 tumors at 4 h (153.2 ± 22.2 percentage injected dose per gram [%ID/g]), 24 h (127.1 ± 42.9 %ID/g), 48 h (130.3 ± 32.4 %ID/g), 72 h (160.9 ± 32.1 %ID/g), and 6 d (10.7 ± 1.8 %ID/g). Excellent image contrast was observed with 124I-immuno-PET. MORAb-004 uptake was statistically higher in TEM1-positive tumors than in control tumors. Binding specificity was confirmed by blocking studies using excess nonlabeled MORAb-004. Conclusion: In our preclinical model, with hTEM1 exclusively expressed on engineered murine endothelial cells that integrate into the tumor vasculature, 124I-MORAb-004 displays high tumor–to–background tissue contrast for detection of hTEM1 in easily accessible tumor vascular compartments. These studies strongly suggest the clinical utility of 124I-MORAb-004 immuno-PET in assessing TEM1 tumor-status.

Biodistribution and fate of core-labeled 125I polymeric nanocarriers prepared by Flash NanoPrecipitation (FNP)

Non-invasive medical imaging techniques such as positron emission tomography (PET) imaging are powerful platforms to track the fate of radiolabeled materials for diagnostic or drug delivery applications. Polymer-based nanocarriers tagged with non-standard PET radionuclides with relatively long half-lives (e.g. 64Cu: t1/2 = 12.7 h, 76Br: t1/2 = 16.2h, 89Zr: t1/2 = 3.3 d, 124I: t1/2 = 4.2 d) may greatly expand applications of nanomedicines in molecular imaging and therapy. However, radiolabeling strategies that ensure stable in vivo association of the radiolabel with the nanocarrier remain a significant challenge. In this study, we covalently attach radioiodine to the core of pre-fabricated nanocarriers. First, we encapsulated polyvinyl phenol within a poly(ethylene glycol) coating using Flash NanoPrecipitation (FNP) to produce stable 75 nm and 120 nm nanocarriers. Following FNP, we radiolabeled the encapsulated polyvinyl phenol with 125I via electrophilic aromatic substitution in high radiochemical yields (> 90%). Biodistribution studies reveal low radioactivity in the thyroid, indicating minimal leaching of the radiolabel in vivo. Further, PEGylated [125I]PVPh nanocarriers exhibited relatively long circulation half-lives (t1/2 α = 2.9 h, t1/2 β = 34.9 h) and gradual reticuloendothelial clearance, with 31% of injected dose in blood retained at 24 h post-injection.

Abstract B48: Preclinical development of meta-[211At] astatobenzylguanidine ([211At] MABG) targeted radiotherapy for neuroblastoma

Background: Neuroblastoma (NB) is a radiosensitive malignancy accounting for 10% of childhood cancer mortality. NB cells frequently express the norepinephrine transporter (NET) providing a specific mechanism for uptake of NET-ligands. Meta -[ 131 I]iodobenzylguanidine ([ 131 I]MIBG) is a NET-ligand radiotherapeutic that shows single-agent response rates in refractory NB of 40-50%. However, due to the long path lengths of 131 I beta (β)-emission, and low biological effectiveness compared to alpha (α)-emitting radionuclides, [ 131 I]MIBG is generally not curative, perhaps due to non-targeting of isolated circulating tumor cells. Here we report our efforts to optimize NET-targeted radiotherapy by developing relevant preclinical models of refractory NB for α-particle therapeutic [ 211 At] MABG therapy. Methods: We first determined NET (SLC6A2) mRNA and protein expression in 35 human NB cell lines using quantitative RT-PCR and western blotting. We then chose 5 lines with absent to intermediate levels of native NET expression (NB1691, SKNSH, IMR5, NLF and SKNBE2) for dual forced overexpression of human NET and luciferase cDNAs. We used [ 125 I]MIBG for cell-based uptake assays in all isogenic pairs and biodistribution experiments in athymic mice bearing three separate NET-transduced xenografts (N=5 per cell line). These cell lines were also treated with [ 131 I]MIBG and/or external beam radiation (XRT) followed by multi-log cytotoxicity assays. Therapeutic trials of [ 131 I]MIBG (25 mCi/kg) in NB1691 subcutaneous xenograft and metastatic mouse models were also conducted. In parallel, [ 211 At] MABG was synthesized by: (i) cyclotron-production of 211 At via 209 Bi(α,2n) 211 At reaction (ii) distillation of 211 At from the target, and (iii) solid phase no-carrier-added synthesis of [ 211 At] MABG by radioastato-destannylation. [ 211 At] MABG uptake studies were performed in isogenic NB cell lines. Results: Unlike primary human NBs, NET expression was low in the majority of 35 cell-lines studied (median normalized expression value = 0.145; range 0.000-1.005), but all transduced lines showed significant overexpression (0.860-1.107) comparable to human primary tumors. Transduced lines showed 4-10 fold higher uptake of [ 125 I]MIBG than non-transduced isogenic parental cell lines in vitro, and demonstrated significant tumor-specific uptake and retention in vivo with tumor-muscle ratios ranging from 13.80 to 29.48. In vitro cytotoxicity experiments using [ 131 I]MIBG showed NET-expressing cell lines to be more susceptible to treatment compared to non-NET expressing pairs (IC50 of 2.937nCi vs. 15.99 nCi). Treatment of mice bearing NB1691-NET xenografts with [ 131 I]MIBG showed tumor growth delay (p=0.0065), but no significant impact on survival, likely due to de novo radioresistance (1200 cGy of XRT had no impact on NB1691 proliferation; IMR-05 showed 97% decreased cell viability). Lastly, we successfully synthesized [ 211 At] MABG, with radiochemical yields of ∼20% and showed NET specific uptake of [ 211 At] MABG into 1691 NET transfected cells. Conclusions: Development of targeted radiotherapy for neuroblastoma has been limited by the lack of preclinical models and alternative therapeutics. Our development of multiple isogenic pairs with varying NET expression, documentation of de novo radiation sensitivity, and the production of [ 211 At] MABG, will allow for rapid assessment of targeted radiotherapeutic strategies (including combination approaches) to support clinical development of alpha-particle therapeutics in a childhood cancer. Citation Format: V Batra, AM Chacko, M Gagliardi, C Hou, J L. Mikitsh, R H. Freifelder, A Kachur, B C. LeGeyt, A Schmitz, L Toto, G Vaidyanathan, M R. Zalutsky, K K. Matthay, W A. Weiss, W C. Gustafson, D Pryma, J M. Maris. Preclinical development of meta-[211At] astatobenzylguanidine ([211At] MABG) targeted radiotherapy for neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B48.