EFFECT OF 3 LIPOPHILIC METHOTREXATE DERIVATIVES UPON MEDIATOR RELEASE BY LIPOPOLYSACCHARIDE-STIMULATED RAT PERITONEAL-MACROPHAGES

The ability of methotrexate and three lipophilic derivatives (methotrexate gamma-dimyristoylphosphatidylethanolamine (M gamma D), methotrexate-alpha-dimyristoylphosphatidylethanolamine (M alpha D) and methotrexate-alpha-gamma-di-dimyristoylphosphatidylethanolamine (M alpha gamma D) to modulate mediator release by lipopolysaccharide-stimulated rat peritoneal macrophages was investigated. At nontoxic concentrations, approximately 10 nmol/10(5) cells, M alpha D and M gamma D 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 M alpha gamma D 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/10(5) cells had no inhibitory effect upon TNF release (14.7 +/- 0.8%, n = 3). Determination of prostaglandin E(2) (PGE(2)) 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 Mao (3.1 nmol/10(5) cells) and M gamma D (2 5 nmol/10(5) cells) maintained their effects on PGE(2) production with 73 +/- 23 and 71 +/- 2.0% (n = 4) inhibition, respectively. At this lower concentration, however, the diamide M alpha gamma D (3.1 nmol/10(5) cells) was less effective in reducing the amount of PGE(2) released from the macrophages (29 +/- 18%, n=4). Maximal PGE(2) inhibition by each of the conjugates was attained at approximately 5 nmol/10(5) cells. Unconjugated methotrexate (range of 2.5-20 nmol/10(5) cells) did not inhibit the release of PGE(2) from lipopolysaccharide-stimulated macrophages.