Effect of Three Lipophilic Methotrexate Derivatives Upon Mediator Release by Lipopolysaccharide‐stimulated Rat Peritoneal Macrophages

— The ability of methotrexate and three lipophilic derivatives (methotrexate-γ-dimyristoylphos-phatidylethanolamine (MγD), methotrexate-α-dimyristoylphosphatidylethanolamine (MαD) and metho-trexate-α-γ-di-dimyristoylphosphatidylethanolamine (MαγD) to modulate mediator release by lipopolysaccharide-stimulated rat peritoneal macrophages was investigated. At nontoxic concentrations, approximately 10 nmol/105 cells, MαD and MγD produced 11·06±1·0 and 75·6 ± 5·2%, respectively, inhibition of tumour necrosis factor (TNF) release (mean ± s.e.m., n = 4). At this same dose MαγD resulted in 68·8 ± 2·1 % inhibition of TNF but cellular ATP levels were reduced by 80%. The inhibitory activity of all three derivatives was dose-dependent. Non-derivatized methotrexate at a concentration of 25 nmol/105 cells had no inhibitory effect upon TNF release (14·7 ±0·8%, n = 3). Determination of prostaglandin E2 (PGE2) levels in the same samples demonstrated that all three conjugates were powerful inhibitors of prostaglandin release. At a quarter of the conjugate concentrations described above the monoamides MαD (31 nmol/105 cells) and MγD (2·5 nmol/105 cells) maintained their effects on PGE2 production with 73±2·3 and 71±2·0% (n=4) inhibition, respectively. At this lower concentration, however, the diamide MαγD (3·1 nmol/105 cells) was less effective in reducing the amount of PGE2 released from the macrophages (29 ± 18%, n = 4). Maximal PGE2 inhibition by each of the conjugates was attained at approximately 5 nmol/105 cells. Unconjugated methotrexate (range of 2·5–20 nmol/105 cells) did not inhibit the release of PGE2 from lipopolysaccharide-stimulated macrophages.