Tumor Targeting with Novel Pyridyl 6-Substituted Pyrrolo[2,3- d]Pyrimidine Antifolates via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of De Novo Purine Nucleotide Biosynthesis.

Tumor-targeted specificities of 6-substituted pyrrolo[2,3- d]pyrimidine analogues of 1, where the phenyl side-chain is replaced by 3',6' (5, 8), 2',5' (6, 9), and 2',6' (7, 10) pyridyls, were analyzed. Proliferation inhibition of isogenic Chinese hamster ovary (CHO) cells expressing folate receptors (FRs) α and ß were in rank order, 6 > 9 > 5 > 7 > 8, with 10 showing no activity, and 6 > 9 > 5 > 8, with 10 and 7 being inactive, respectively. Antiproliferative effects toward FRα- and FRß-expressing cells were reflected in competitive binding with [3H]folic acid. Only compound 6 was active against proton-coupled folate receptor (PCFT)-expressing CHO cells (∼4-fold more potent than 1) and inhibited [3H]methotrexate uptake by PCFT. In KB and IGROV1 tumor cells, 6 showed <1 nM IC50, ∼2-3-fold more potent than 1. Compound 6 inhibited glycinamide ribonucleotide formyltransferase in de novo purine biosynthesis and showed potent in vivo efficacy toward subcutaneous IGROV1 tumor xenografts in SCID mice.

Imaging atherosclerotic plaque inflammation via folate receptor targeting using a novel 18F-folate radiotracer.

Folate receptor ß (FR-ß) is overexpressed on activated, but not resting, macrophages involved in a variety of inflammatory and autoimmune diseases. A pivotal step in atherogenesis is the subendothelial accumulation of macrophages. In nascent lesions, they coordinate the scavenging of lipids and cellular debris to define the likelihood of plaque inflammation and eventually rupture. In this study, we determined the presence of FR-ß-expressing macrophages in atherosclerotic lesions by the use of a fluorine-18-labeled folate-based radiotracer. Human endarterectomized specimens were used to measure gene expression levels of FR-ß and CD68. Increased FR-ß and CD68 levels were found in atherosclerotic plaques compared to normal artery walls by quantitative real-time polymerase chain reaction. Western blotting and immunohistochemistry demonstrated prominent FR-ß protein levels in plaques. FR-ß-positive cells colocalized with activated macrophages (CD68) in plaque tissue. Carotid sections incubated with 3'-aza-2'-[18F]fluorofolic acid displayed increased accumulation in atherosclerotic plaques through in vitro autoradiography. Specific binding of the radiotracer correlated with FR-ß-expressing macrophages. These results demonstrate high FR-ß expression in atherosclerotic lesions of human carotid tissue correlating with CD68-positive macrophages. Areas of high 3'-aza-2'-[18F]fluorofolic acid binding within the lesions represented FR-ß-expressing macrophages. Selectively targeting FR-ß-positive macrophages through folate-based radiopharmaceuticals may be useful for noninvasive imaging of plaque inflammation.

In vivo optical imaging of folate receptor-ß in head and neck squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: Folate receptor (FR) expression, although known to be elevated in many types of cancer and inflammatory cells, has not been well characterized in head and neck squamous cell carcinoma (HNSCC). We hypothesized that tumor infiltrating inflammatory cells expressing FR-ß could allow fluorescent visualization of HNSCC tumors using folate conjugated dyes even when FR expression in cancer cells is low. STUDY DESIGN: Retrospective review of clinical pathologic specimens and in vivo animal study. METHODS: A tissue microarray with tumor and tumor-free tissue from 22 patients with HNSCC was stained with antibodies to FR-α and FR-ß. We characterized FR-ß(+) cells by examining CD45, CD68, CD206, and transforming growth factor (TGF)-ß expression. To investigate fluorescent imaging, mice with orthotopic tumor xenografts were imaged in vivo after intravenous injections of folate conjugated fluorescein isothiocyanate (folate-FITC) and were histologically evaluated ex vivo. RESULTS: All tumor samples demonstrated significant FR-ß staining and negligible FR-α staining. FR-ß(+) cells found in tumors coexpressed CD68 and had increased expression of CD206 and TGF-ß characteristic of tumor-associated macrophages. In the xenograft models, tumors showed strong in vivo fluorescence after folate-FITC injection in contrast to surrounding normal tissues. Histologic examination of the xenograft tissue similarly showed folate-FITC uptake in areas of inflammatory cellular infiltrate. CONCLUSIONS: Although HNSCC tumor cells do not express FR, HNSCC tumors contain a significant population of FR-ß-expressing macrophages. Folate conjugated fluorescent dye is able to specifically target and label tumor xenografts to permit macroscopic fluorescence imaging due to FR-ß expression on the infiltrating inflammatory cells.