Involution of collagen-induced arthritis with an angiogenesis inhibitor, PPI-2458

Abstract

Pannus formation, in both rheumatoid arthritis (RA) and collagen-induced arthritis (CIA), is angiogenesis-dependent. PPI-2458 [(1R)-1-carbamoyl-2-methyl]-carbamic acid-(3R,3S,5S, 6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)oxiranyl]-1-oxaspiro(2*5)oct-6-yl ester], a new fumagillin derivative known to inhibit methionine aminopeptidase 2 (MetAP-2) and endothelial proliferation at the late G(1) phase, was evaluated in CIA rats to study its potential to involute synovitis. Arthritic syngeneic LOU rats received either a vehicle control or various dosages of oral, intravenous, or subcutaneous PPI-2458. Plasma samples were analyzed to determine a pharmacokinetic profile of PPI-2458, and whole blood was evaluated by flow cytometry to assess the effect on lymphocyte subsets. At 15 mg/kg i.v., 30 mg/kg s.c., or 100 mg/kg p.o., there was a significant reduction in clinical severity scores (p < 0.001) and blinded radiographic scores (p < 0.001) compared with vehicle control groups. Structural damage was virtually eliminated with PPI-2458. Continuous inhibition of MetAP-2 was needed to maintain benefits, although pannus involution could be achieved with the inhibitor when escape flares occurred. Pharmacokinetic analysis after a single p.o. dose showed a rapid T(max) value of 15 min followed by biphasic elimination (t(1/2), approximately 20 min and t(1/2), approximately 5 h) and an estimated oral bioavailability of approximately 15%. Flow cytometry revealed a dose-dependent decrease in white blood cells and lymphocytes manifested as decreases in circulating CD3+ T cells and natural killer cells. PPI-2458, however, did not seem to be immunosuppressive, as determined by delayed-type hypersensitivity or IgG antibody assays. These studies indicate that the MetAP-2 inhibitor PPI-2458 can regress established CIA and that angiogenic mechanisms might be important targets in the treatment of other pannus-mediated diseases such as RA.

Fig. 1.

Structure of parent molecule, fumagillin, and modifications of side chain (gray box) for TNP-470 and PPI-2458 (Bernier et al., 2004).

Fig. 2.

In vivo studies. Experimental protocols of PPI-2458, TNP-470, and methotrexate are noted. Rats were immunized with type II collagen on day 0, developed clinical arthritis on day 10, and sacrificed on day 28. The route and frequency are summarized.

Fig. 3.

Pharmacokinetics of PPI-2458. A, AUC, C_max, T_max, half-life, and dose-adjusted bioavailability are shown for intravenous, subcutaneous, and oral routes of administration. B, mean serum levels of PPI-2458 (±S.E.M.) are shown after a single dose of 100 mg/kg p.o. in normal LOU rats.

Fig. 4.

Clinical and radiographic scores (±S.E.M.). The left column (A, C, E, and G) shows longitudinal clinical scores for each dose (on a per kilogram basis) and route of administration evaluated. The right column (B, D, F, and H) depicts mean radiographic scores (±S.E.M.). ^*, statistically different results compared with the respective control group.

Fig. 5.

Clinical arthritis. A, hind limb of a vehicle control rat on day 28 with an arthritis score of 4 (maximum). Erythema and soft tissue swelling is evident at the proximal and distal foot. B, hind limb of a PPI-2458-treated rat on day 28 with an arthritis score of 0.

Fig. 6.

Radiographic damage. Left, a normal limb. Middle, an arthritic control limb on day 28. Periarticular soft tissue swelling (arrows) and severe periosteal reactions/osteolysis/erosions (within the dashed circles) are evident at the tarsus, tarsals, and metatarsophalangeal joints [radiographic score is 3 (maximum)]. Right, a hind limb, day 28 (18 days after arthritis onset on day 10), treated with PPI-2458 (30 mg/kg s.c. q.o.d). No soft tissue swelling or bone destruction is evident, and it seems similar to the normal limb (radiographic score is 0).

Fig. 7.

Joint histology. Left, a normal tarsus joint stained with hematoxylin and eosin (original magnification, 300×). Middle, from a day 28 arthritic control rat. Pannus with numerous blood vessels (circled) are evident along with an erosion of cartilage and subchondral bone (arrows). Right, from a day 28 rat treated with PPI-2458 30 mg/kg s.c. q.o.d. (beginning on day 10). The cartilage and bone seem normal, and no pannus is evident.

Fig. 8.

DTH and antibody. A, DTH responses, on day 28, to native type II collagen in rats administered PPI-2458, TNP-470, or vehicle subcutaneously beginning on day 10. B, antibody levels by ELISA, on day 28, to native type II collagen in rats administered PPI-2458, TNP-470, or vehicle subcutaneously beginning on day 10. Serum was assayed at a 1:2500 dilution and reported as optical density at 490 nm. ^*, statistically different results compared with the vehicle control group.

Fig. 9.

Flow cytometry of day 28 peripheral blood. A, naive rats. B, vehicle control rats. C to E, rats administered the indicated dose of PPI-2458 intravenously beginning on day 10, the onset of clinical arthritis. ^*, statistically different results compared with the vehicle control group.