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

Abstract

Introduction: Folate 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.

Methods: Using 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.

Results: EC0746 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.

Conclusions: EC0746 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.

Figure 1

EC0746 folate receptor binding affinities. (a) Chemical structure of EC0746. There are four separate functional components to this novel construct: the folate receptor (FR)-targeting moiety folic acid (FA; black), the drug moiety aminopterin (AMT; red), a saccharo-amino acid peptide-based spacer of ((saccharo-γGlu)-γGlu)₂-γCys (blue), and a hydrazide/disulfide-containing linker (green). (b) Relative binding affinities of EC0746 in comparison with AMT and methotrexate (MTX) using FRα-expressing KB cells and FRβ-expressing CHO-FRβ cells. The assays were performed in triplicate at 37°C using each compound as a competitor to displace [³H]FA from binding to FR-expressing cells. Numbers shown next to each test article are relative affinity values with FA itself set at 1.

Figure 2

EC0746 is a folate-receptor-specific dihydrofolate reductase inhibitor with potent cytostatic effect on RAW264.7 macrophages. (a) RAW264.7 cells were given a 2-hour pulse of 100 nM EC0746 ± 10 μM folic acid (FA) followed by a 22-hour chase. Aminopterin (AMT) and methotrexate (MTX) were allowed to incubate for 24 hours. The dihydrofolate reductase activities in whole cell lysates (in duplicate) were normalized to untreated control cells (mean ± standard error of the mean). *P < 0.05. (b), (c) RAW264.7 cells were subjected to a 2-hour pulse followed by a 70-hour chase of a 10-fold serial dilution of EC0746 ± 100-fold molar excess of FA. Free AMT was allowed to incubate for 72 hours continuously. Four hours prior to the end of incubation, lipopolysaccharide (100 ng/ml) was added to stimulate TNFα production. The (b) cell viability and (c) TNFα in culture media were determined by XTT and ELISA assays, respectively. Results expressed as the percentage of control in absorbance (mean ± standard error of the mean in triplicates). (d) RAW264.7 cells were treated with indicated concentrations of EC0746 ± excess FA (2-hour pulse plus a 70-hour chase). The surviving cells were redistributed in equal numbers in fresh medium and allowed to incubate further for 72 hours. The cell proliferation was again determined by the XTT assay.

Figure 3

EC0746 has an immunomodulatory effect on folate-receptor-expressing rat TG-macs. Rat TG-macs were treated with media only, 100 nM EC0746 ± 10 μM folic acid (FA), or FA alone (10 μM) for 2 hours followed by a 70-hour chase. In comparison, the cells were also treated with 100 nM free aminopterin (AMT) and methotrexate (MTX) for 72 hours. At 24 hours before the end of incubation, all cells were stimulated with lipopolysaccharide (LPS) (5 μg/ml) plus IFNγ (100 ng/ml). The cytokines/chemokines produced in culture supernatants were detected using a rat cytokine array. (a) Cytokine release profiles of rat TG-macs stimulated with LPS and IFNγ with or without drug treatment. (b) Cytokine array map. (c) Mean pixel intensity (y axis) determined for each array position and plotted for the 11 products, which were detected at three times above background levels and in at least three independent experiments. Data shown are mean ± standard error of the mean. *P < 0.05 when compared with its corresponding cytokine level in the media only sample. CINC, cytokine-induced neutrophil chemoattractant; LIX, LPS-induced CXC chemokine; MIG, monokine induced by IFNγ; MIP-1α, macrophage inflammatory protein-1α; RANTES, regulated upon activation, normal T-cell expressed and secreted; sICAM, soluble intracellular adhesion molecule; TIMP-1, tissue inhibitor of metalloproteinase 1; VEGF, vascular endothelial growth factor.

Figure 4

EC0746 demonstrates folate-receptor-specific anti-inflammatory activities in vivo. Starting on day 10 after arthritis induction, rats with developing adjuvant-induced arthritis (n = 5) were given a biweekly subcutaneous dosing regimen of EC0746 (250 nmol/kg) without or with a 500-fold molar excess of EC0923 as the folate competitor. For comparison purposes, methotrexate (MTX) (250 nmol/kg) was dosed orally following the same schedule as EC0746, and etanercept (10 mg/kg) was given subcutaneously on days 10, 13, 16, 19, and 22. Multiple endpoints are shown: (a) arthritis score; (b) change in body weight; (c) percentage increase in paw weight; (d) percentage increase in spleen weight; (e) representative X-ray images of arthritic hind paws taken using a Kodak Imaging Station; and (f) radiographic score of arthritic hind paws. *P < 0.05 when compared with the arthritic control group.

Figure 5

Histopathological assessment. The histopathological analysis was performed on formalin-fixed arthritic hind paws by Bolder BioPATH Inc. (Boulder, CO, USA). (a) Individual histological scores of ankle joints on a scale of 0 to 5 for inflammation, bone resorption, pannus formation, and cartilage damage with a maximal histology score of 20 per foot. (b) Representative photomicrographs (16x) of the ankle closest to the (c) mean summed histological score. (d) Dorsal to ventral paw thickness for each group. Notably, the arthritic control animal showed very severe inflammation (S), bone resorption (arrowhead) with mild pannus (small arrow), and cartilage damage (large arrow). *P < 0.05 when compared with the arthritic control group. MTX, methotrexate.

Figure 6

Methotrexate displays a lack of competition for folate-receptor-binding sites and a nonfolate-receptor-targeted anti-arthritic activity. In an identical dosing fashion as was carried out for EC0746 in Figure 4, adjuvant-induced arthritis rats (n = 5) were treated subcutaneously with methotrexate (MTX) (250 nmol/kg biweekly) for 2 weeks without or with a 500-fold molar excess of EC0923 as the folate competitor. Anti-arthritic activities are shown: (a) arthritis score; (b) percentage increases in paw and spleen weights; and (c) percentage change in body weight. ns, not significant.