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Effect of three lipophilic methotrexate derivatives upon mediator release by lipopolysaccharide-stimulated rat
A S Williams1, N Topley, N Amos
1Rheumatology Research Laboratory, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Abstract:
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 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 M alpha (3.1 nmol/10(5) cells) and M gamma D (2.5 nmol/10(5) 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 M alpha gamma D (3.1 nmol/10(5) 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/10(5) cells. Unconjugated methotrexate (range of 2.5-20 nmol/10(5) cells) did not inhibit the release of PGE2 from lipopolysaccharide-stimulated macrophages.
Insights
Lipophilic methotrexate derivatives effectively inhibited tumor necrosis factor (TNF) and prostaglandin E2 (PGE2) release from stimulated macrophages. Methotrexate-gamma-dimyristoylphosphatidylethanolamine (M gamma D) showed potent inhibition, while M alpha D and M alpha gamma D also demonstrated significant effects.
Area of Science:
- Immunology
- Pharmacology
- Cell Biology
Background:
- Macrophages play a crucial role in inflammatory responses.
- Lipopolysaccharide (LPS) is a potent stimulator of macrophage activation.
- Tumor necrosis factor (TNF) and prostaglandin E2 (PGE2) are key inflammatory mediators released by macrophages.
Purpose of the Study:
- To investigate the immunomodulatory effects of methotrexate and its lipophilic derivatives on mediator release from LPS-stimulated rat peritoneal macrophages.
- To compare the inhibitory potential of different methotrexate conjugates on TNF and PGE2 production.
Main Methods:
- Rat peritoneal macrophages were stimulated with lipopolysaccharide (LPS).
- The release of TNF and PGE2 was measured in the presence of varying concentrations of methotrexate and its lipophilic derivatives: M gamma D, M alpha D, and M alpha gamma D.
- Cellular ATP levels were assessed to evaluate cytotoxicity.
Main Results:
- Lipophilic methotrexate derivatives, particularly M gamma D and M alpha D, significantly inhibited TNF release at nontoxic concentrations.
- All three methotrexate conjugates potently inhibited PGE2 production in a dose-dependent manner.
- M alpha gamma D showed reduced efficacy in inhibiting PGE2 at lower concentrations and significantly impacted cellular ATP levels.
- Unconjugated methotrexate did not inhibit TNF or PGE2 release.
Conclusions:
- Lipophilic modification of methotrexate enhances its ability to inhibit inflammatory mediator release from macrophages.
- Methotrexate derivatives, especially M gamma D, show promise as modulators of macrophage-mediated inflammation.
- The efficacy and potential toxicity of methotrexate derivatives are dependent on their lipophilic structure and concentration.