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Activated macrophages overexpress a receptor for the vitamin folic acid termed the folate receptor β (FR-β). Because conjugation of folate to low molecular weight drugs, genes, liposomes, nanoparticles, and imaging agents has minor effects on FR binding, the vitamin can be exploited to target both therapeutic and imaging agents to activated macrophages without promoting their uptake by other healthy cells. In this paper, we characterize the binding, internalization, and recycling kinetics of FR-β on activated macrophages in inflamed tissues of rats with adjuvant-induced arthritis. Our results demonstrate that saturation of macrophage FR is achieved at injection doses of ∼150-300 nmol/kg, with more rapidly perfused tissues saturating at lower doses than inflamed appendages. After binding, FR-β internalizes and recycles back to the cell surface every ∼10-20 min, providing empty receptors for additional folate conjugate uptake. Because the half-life of low molecular weight folate conjugates in the vasculature is usually <1 h, these data suggest that targeting of folate conjugates to activated macrophages in vivo can be maximized by frequent dosing at conjugate concentrations that barely saturate FR (∼150 nmol/kg), thereby minimizing nonspecific binding to receptor-negative tissues and maximizing the probability that unoccupied cell surface receptors will be exposed to folate-drug conjugate.

Unlabelled: Folic acid has been frequently exploited to target attached drugs to cells that overexpress a folate receptor (FR). Unfortunately, folic acid and folate-linked drugs bind equally well to both major isoforms of the FR-that is, FR-α, which is primarily expressed on malignant cells, and FR-β, which is upregulated on activated monocytes and macrophages. Because both major isoforms of FR can be expressed simultaneously in the same organism, folic acid cannot enable selective targeting of therapeutic and imaging agents to either tumor masses or sites of inflammation. In an effort to develop a targeting ligand that can selectively deliver attached imaging and therapeutic agents to tumor cells, we constructed a reduced and alkylated form of folic acid, N(5), N(10)-dimethyl tetrahydrofolate (DMTHF) that exhibits selectivity for FR-α., Methods: DMTHF-(99m)Tc was injected into mice bearing FR-α-expressing tumor xenografts and imaged by γ-scintigraphy. The selectivity for FR-α over FR-β in vivo was examined by γ-scintigraphic images of animal models of various inflammatory diseases such as apolipoprotein E-deficient mice with atherosclerosis, DBA/1 LacJ mice with induced arthritis, C57BL/6J mice with muscle injury, and BALB/C mice with both FR-α tumor and ulcerative colitis, by administration of equal doses of DMTHF-(99m)Tc and EC20-(99m)Tc. The uptake of radiochelates in various organs was quantified by biodistribution studies. DMTHF-near-infrared dye conjugate and DMTHF-Oregon green dye conjugates were synthesized and evaluated for FR-α selectivity over FR-β in rat peritoneal macrophages and human peripheral blood monocytes, respectively, by flow cytometry. Fluorescence-guided imaging was also performed using folate and DMTHF dye conjugates., Results: The new targeting ligand was found to bind malignant cells in mice with solid tumor xenografts but not peripheral blood monocytes or inflammatory macrophages in animal models of atherosclerosis, rheumatoid arthritis, muscle injury, or ulcerative colitis. Results from optical and radioimaging studies and biodistribution experiments confirm the differential specificity of this new ligand for malignant masses., Conclusion: The new targeting ligand DMTHF enables selective noninvasive imaging and therapy of tumor tissues in the presence of inflammation.

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.