Abstract

The relative anti-inflammatory activities of the immunomodulators HR325 and leflunomide, or its active metabolite A77 1726, were examined by determining potencies in vitro on prostaglandin endoperoxide H synthase (PGHS) and in vivo in rat air pouch inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs) were used as comparators. HR325 was more potent than A77 1726 as an inhibitor of PGHS in guinea pig polymorphonuclear leukocytes (IC₅₀ = 415 and 4400 nM, respectively) and on isolated ovine PGHS-1 (IC₅₀ = 64 and 742 μM) and PGHS-2 (IC₅₀ = 100 and 2766 μM). In vivo, in rat carrageenan air pouch inflammation, HR325 but not leflunomide at 25 mg/kg inhibited accumulation of leukocytes (48%) and PGE₂ (61%). HR325 was also more potent than A77 1726 against human peripheral blood mononuclear cell PGHS-1 [IC₅₀ = 1.6 and 25.6 μM (thromboxane B₂production) or 1.1 and 8 μM (PGE₂ production)] and lipopolysaccharide-induced PGHS-2 in human adherent peripheral blood mononuclear cells (IC₅₀ = 435 nM and 9.5 μM) and peripheral blood polymorphonuclear leukocytes (IC₅₀ = 91 nM and 3.2 μM). HR325 had low PGHS-2 selectivity in the human (2.5–12-fold) and was a more potent PGHS-2 inhibitor than naproxen, ibuprofen and piroxicam (2–8-fold). Assays using endogenous arachidonic acid as substrate yielded IC₅₀ values for NSAIDs that were in general markedly lower than those published for assays using 10 μM substrate. With this approach, piroxicam had reasonable activity on human PGHS-2 (IC₅₀ = 260–290 nM). Only NS398 and flufenamic acid were PGHS-2 selective in the human (90–330-fold and 37–60-fold, respectively); the other NSAIDs were either PGHS-1-selective (naproxen, ibuprofen, flurbiprofen and indomethacin) or nonselective (piroxicam and diclofenac). Inclusion of 10% human plasma reduced HR325 potency against PGHS-1 in human peripheral blood mononuclear cells approximately 32-fold (IC₅₀ = 36 μM). Plasma protein binding further reduced HR325 potency (IC₅₀ = 164 μM) and minimized the difference between HR325 and A77 1726 (IC₅₀ = 292 μM) in a whole blood PGHS assay. Whether the greater activity against human PGHS-2 would allow HR325 to exhibit NSAID-like therapeutic effects in humans remains unclear.