Folate receptor-targeted imaging agents for cancer and inflammatory diseases

Folate receptor-targeted imaging agents have previously been utilized to image and diagnose both cancer and inflammatory diseases in animals and humans. These imaging agents are now being further developed for novel applications. Fluorescence guided surgery is one such application that allows for the real time removal of FR+ tumors by surgeons. In Chapter 5, we explore the use of tumor-specific targeting ligands to deliver near-infrared (NIR) fluorescent dyes specifically to FR expressing cancers, thereby rendering only the malignant cells highly fluorescent. We report here upon intravenous injection into tumor-bearing mice with metastatic disease, these ligand−NIR dye conjugates render receptor-expressing tumor tissues fluorescent, enabling their facile resection with minimal contamination from healthy tissues. A major objective of personalized medicine for inflammatory and autoimmune diseases lies in developing methods for identifying patients who will eventually benefit from a therapy. The ability to select patients who will respond to therapies for these diseases is especially acute, because of the cost and damage associated with failed therapies. We describe in Chapters 2 and 3 a clinical test that will rapidly predict the response of a patient with an autoimmune/inflammatory disease to any of the most commonly employed therapies. This test involves quantitative assessment of uptake of folate receptor-targeted imaging agents by FR+ macrophages that accumulate at sites of inflammation. Murine models of a variety of inflammatory diseases show markedly decreased uptake of folate receptor-targeted radioimaging agents in inflamed lesions upon initiation of a successful therapy, but no decrease in uptake upon administration of an ineffective therapy, both long before any change in clinical symptoms can be detected. Once FR-targeted imaging agents have been employed to identify patients with inflammatory diseases, the next logical step is to treat those patients with FR-targeted therapies. In Chapter 4 we use a folate-targeted imaging agent to assess disease in rats induced to develop osteoarthritis. These rats were also treated with a FR-targeted anti-inflammatory drug. Treated rats were found to have significant attenuation of joint damage in the knees of MIA rats when compared to saline treated controls. With the rapid emergence of targeted nanoparticples in recent years, we found it important to study the effect of size on nanoparticle accumulation in solid tumors. As discussed in Chapter 6, we demonstrate that the low molecular weight folate-fluorescein conjugate accumulated in the tumor mass by the two hour time point ∼40-fold more readily on a molar basis than the high molecular weight folate-BSA-fluorescein conjugate. Because the effects of nanocarrier size, shape, chemistry, and targeting ligand are interconnected and complex, we suggest that these parameters must be carefully optimized for each nanocarrier to ensure optimal drug delivery in vivo

Effect of Folate-Targeted Nanoparticle Size on Their Rates of Penetration into Solid Tumors

Targeted therapies are emerging as a preferred strategy for the treatment of cancer and other diseases. To evaluate the impact of a high affinity targeting ligand on the rate and extent of tumor penetration of different sized nanomedicines, we have used intravital multiphoton microscopy to quantitate the kinetics of tumor accumulation of a homologous series of folate-PEG-rhodamine conjugates prepared with polyethylene glycols (PEG) of different molecular weights. We demonstrate that increasing the size of the folate-PEG-rhodamine conjugates results in both longer circulation times and slower tumor penetration rates. Although a “binding site barrier” is observed with the folate-linked polymers in folate receptor expressing tumors, ligand targeting eventually leads to increased tumor accumulation, with endocytosis of the targeted nanocarriers contributing to their enhanced tumor retention. Because the effects of nanocarrier size, shape, chemistry, and targeting ligand are interconnected and complex, we suggest that these parameters must be carefully optimized for each nanocarrier to ensure optimal drug delivery <i>in vivo</i>

Development of Tumor-Targeted Near Infrared Probes for Fluorescence Guided Surgery

Complete surgical resection of malignant disease is the only reliable method to cure cancer. Unfortunately, quantitative tumor resection is often limited by a surgeon’s ability to locate all malignant disease and distinguish it from healthy tissue. Fluorescence-guided surgery has emerged as a tool to aid surgeons in the identification and removal of malignant lesions. While nontargeted fluorescent dyes have been shown to passively accumulate in some tumors, the resulting tumor-to-background ratios are often poor, and the boundaries between malignant and healthy tissues can be difficult to define. To circumvent these problems, our laboratory has developed high affinity tumor targeting ligands that bind to receptors that are overexpressed on cancer cells and deliver attached molecules selectively into these cells. In this study, we explore the use of two tumor-specific targeting ligands (i.e., folic acid that targets the folate receptor (FR) and DUPA that targets prostate specific membrane antigen (PSMA)) to deliver near-infrared (NIR) fluorescent dyes specifically to FR and PSMA expressing cancers, thereby rendering only the malignant cells highly fluorescent. We report here that all FR- and PSMA-targeted NIR probes examined bind cultured cancer cells in the low nanomolar range. Moreover, upon intravenous injection into tumor-bearing mice with metastatic disease, these same ligand–NIR dye conjugates render receptor-expressing tumor tissues fluorescent, enabling their facile resection with minimal contamination from healthy tissues

Automated, Resin-Based Method to Enhance the Specific Activity of Fluorine-18 Clicked PET Radiotracers

Radiolabeling of substrates with 2-[¹⁸F]­fluoroethylazide exploits the rapid kinetics, chemical selectivity, and mild conditions of the copper-catalyzed azide–alkyne cycloaddition reaction. While this methodology has proven to result in near-quantitative labeling of alkyne-tagged precursors, the relatively small size of the fluoroethylazide group makes separation of the ¹⁸F-labeled radiotracer and the unreacted precursor challenging, particularly with precursors >500 Da (e.g., peptides). We have developed an inexpensive azide-functionalized resin to rapidly remove unreacted alkyne precursor following the fluoroethylazide labeling reaction and integrated it into a fully automated radiosynthesis platform. We have carried out 2-[¹⁸F]­fluoroethylazide labeling of four different alkynes ranging from <300 Da to >1700 Da and found that >98% of the unreacted alkyne was removed in less than 20 min at room temperature to afford the final radiotracers at >99% radiochemical purity with specific activities up to >200 GBq/μmol. We have applied this technique to label a novel cyclic peptide previously evolved to bind the Her2 receptor with high affinity, and demonstrated tumor-specific uptake and low nonspecific background by PET/CT. This resin-based methodology is automated, rapid, mild, and general allowing peptide-based fluorine-18 radiotracers to be obtained with clinically relevant specific activities without chromatographic separation and with only a minimal increase in total synthesis time