Imaging Sites of Infection Using a 99m Tc-Labeled Folate Conjugate Targeted to Folate Receptor Positive Macrophages Walter A. Henne,* ,†,‡ Ryan Rothenbuhler, † Wilfredo Ayala-Lopez, † Wei Xia, † Bindu Varghese, † and Philip S. Low* ,† † Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States ‡ Division of Science, Governors State University, University Park, Illinois 60484, United States ABSTRACT: EC20, a folate-targeted 99m Tc based radioimaging agent with a high folate receptor (FR) binding affinity, has been used for both the diagnosis and the staging of FR positive malignancies (currently in phase III trials) and also for the localization of inflamed lesions characterized by the accumulation of FR+ macrophages. Because recent evidence has suggested that FR+ macrophages might accumulate at sites of infectious disease, this study evaluated whether EC20 might prove similarly useful for imaging bacterial infection foci. Using gamma scintigraphic imaging, it was demonstrated that EC20 accumulated at sites of Staphylococcus aureus infection with a significant difference (P < 0.0001, n = 12) in enrichment noted between infected and noninfected limbs. Confirmation that the elevated uptake of EC20 in infected limbs was FR-mediated was supported by suppression of EC20 accumulation in the presence of a 200-fold excess of free folic acid (P < 0.0001, n = 12). This study establishes for the first time the use of EC20 to image and localize sites of infectious disease. KEYWORDS: folate, folic acid, EC20, technetium, infection imaging, macrophage, gamma scintigraphy ■ INTRODUCTION The localization of sites of infection remains a significant clinical challenge. Scintigraphic imaging using radiolabeled leukocytes 1 is the current “gold standard” for imaging infection foci; however, preparation of the labeled leukocytes is labor- intensive, may require transport of the patient's blood to distant sites, is time-consuming, and aggravates the risk of infection to both patients and health care workers. 2 Second generation methods for localizing infections have employed radiolabeled murine antibodies (Granuloscint), 3 antibody fragments (Leu- koscan), 4,5 or cytokines (IL-8) 6,7 and have yielded results comparable to those with labeled leukocytes. However, costs and challenges associated with generating protein-based reagents have limited their widespread use. Alternative, nonprotein-based strategies include radiolabeled antimicrobial peptides (e.g., peptide fragments of ubiquicidin) 8,9 and antibiotics (ciprofloxacin) 10,11 among others and are currently in various stages of development. Together, these reports indicate a continuing need for improved methods for localizing and monitoring infectious disease, especially methods that can be easily performed by in primary health care facilities. The folate receptor (FR) and its various isoforms are overexpressed on a variety of cancers (including ovarian, lung, kidney, breast, and endometrial cancers) and on activated macrophages. 12-14 Accordingly, folic acid (MW = 441.4 Da) has been exploited as a targeting ligand to deliver attached imaging and therapeutic cargos to the above pathologic loci. Cargos that have been successfully targeted with folic acid to cancers or inflamed tissues include: (i) chemotherapeutic agents, 15-17 (ii) liposomes with entrapped drugs, 18 (iii) immunotherapeutic agents, 19 and (iv) radio and optical imaging agents. 20-23 Depending on the application, the popularity of folate as a targeting ligand may be due to its ease of conjugation, specificity for pathologic tissues, stability in vitro and in vivo, facile penetration to diseased tissues, rapid internalization by FR-expressing cells, lack of immunogenicity, Received: January 8, 2012 Revised: February 25, 2012 Accepted: March 6, 2012 Published: April 2, 2012 Article pubs.acs.org/molecularpharmaceutics © 2012 American Chemical Society 1435 dx.doi.org/10.1021/mp3000138 | Mol. Pharmaceutics 2012, 9, 1435-1440