Treatment of experimental adjuvant arthritis with a novel folate receptor-targeted folic acid-aminopterin conjugate

Abstract

IntroductionFolate receptor (FR)-expressing macrophages have been shown to accumulate at sites of inflammation, where they promote development of inflammatory symptoms. To target such a macrophage population, we designed and evaluated the biologic activity of EC0746, a novel folic acid conjugate of the highly potent antifolate, aminopterin.MethodsUsing a FR-positive subclone of murine macrophage-derived RAW264.7 cells and rat thioglycollate-elicited macrophages, we studied the effect of EC0746 on dihydrofolate reductase activity, cell proliferation, and cellular response towards bacterial lipopolysaccharide as well as IFNγ activation. The EC0746 anti-inflammatory activity, pharmacokinetics, and toxicity were also evaluated in normal rats or in rats with adjuvant-induced arthritis; that is, a FR-positive macrophage model that closely resembles rheumatoid arthritis in humans.ResultsEC0746 suppresses the proliferation of RAW264.7 cells and prevents the ability of nonproliferating rat macrophages to respond to inflammatory stimuli. In the macrophage-rich rat arthritis model, brief treatment with subcutaneously administered EC0746 is shown to mediate an FR-specific anti-inflammatory response that is more potent than either orally administered methotrexate or subcutaneously delivered etanercept. More importantly, EC0746 therapy is also shown to be ~40-fold less toxic than unmodified aminopterin, with fewer bone marrow and gastrointestinal problems.ConclusionsEC0746 is the first high FR-binding dihydrofolate reductase inhibitor that demonstrates FR-specific anti-inflammatory activities both in vitro and in vivo. Our data reveal that a relatively toxic anti-inflammatory drug, such as aminopterin, can be targeted with folic acid to inflammatory macrophages and thereby relieve inflammatory symptoms with greatly reduced toxicity.

Folate-Conjugated Rapamycin Slows Progression of Polycystic Kidney Disease

“…Of note, many previous studies with other folate-conjugated compounds have shown that their effects are rather specific to FR-expressing diseased tissues without exerting appreciable off-target effects. 22,40 Our findings suggest that, in addition to ADPKD, the targeting of folate-conjugated compounds to kidneys may be a useful approach for numerous other renal epithelial disorders. As long as the high expression level of the FR is maintained in the diseased target cells, it may be possible to achieve specific drug delivery.…”

Antiinflammatory Activity of a Novel Folic Acid Targeted Conjugate of the mTOR Inhibitor Everolimus

“…The thioglycollate (TG)-elicited macrophages (TG-mac) were harvested from rats intraperitoneally dosed with an aged TG medium. After a 2-h plastic adherence, the respective macrophage populations were ~90% pure based on CD11b/c expression, and ~70% of the CD11b/c cells expressed a functional FR (17).…”

“…Rat adjuvant arthritis (AIA) was induced by intradermal inoculation (at the base of the tail) of 0.5 mg heat-killed Mycobacteria butyricum (BD Diagnostic Systems) in 100 μL light mineral oil (Sigma). During the course of disease development, rats were scored three times a week according to the previously established criteria (17). Each paw was graded with a score of 0-4, with a maximum possible score of 16 for each animal.…”

“…RAW264.7 cells is a murine macrophage-derived tumor cell line grown in folate-deficient medium (17). The arthritic macrophages (AIAmac) were obtained from peritoneal lavage of arthritic rats at the peak of the disease (~18-22 d after induction).…”

“…Thus, FR-β-expressing macrophages have emerged as a promising target for antibody-or ligand-mediated drug delivery to control local and systemic inflammation (11). In animal models, anti-FR-β immunotoxins (12)(13)(14), folate-hapten immunotherapy (15,16) and folate-targeted aminopterin therapy (17,18) were found effective in treating models of arthritis (15)(16)(17), autoimmune uveitis (18), autoimmune encephalomyelitis (18), experimental fibrosis (13) and atherosclerosis (14). In early clinical trials, Tc-EC20 (etarfolatide), a FR-specific radioimaging agent, was shown to preferentially accumulate in inflamed arthritic joints in patients with RA (19) and osteoarthritis (20).…”