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WY-50295 tromethamine: a 5-lipoxygenase inhibitor without activity in human whole blood
Abstract:
The 5-LO inhibitor, WY-50295 tromethamine (T) prevented leukotriene release (LTB4 production) in calcium ionophore stimulated, purified human and rat neutrophils. However, whereas WY-50295T inhibited both in vitro and ex vivo rat whole blood leukocyte LTB4 formation (IC50= 40 microM and oral ED50 of 18 mg/kg, respectively), it did not inhibit LTB4 production in calcium ionophore stimulated human whole blood at concentrations to 200 microM. To reduce binding of WY-50295T to serum albumin, 250 microM of a naphthalene sulfonic acid (> 99.9% binding to albumin primarily at the carboxylic site) and 250 microM sulfanilamide (binding to nonspecific sites) separately or in combination were preincubated in whole blood prior to addition of WY-50295T; however, WY-50295T still did not inhibit 5-LO and free drug blood levels were unchanged. When purified human neutrophils in the presence of fatty acid saturated albumin (fraction V) was employed, the 5-LO inhibitory activity of WY-50295T was prevented. Zileuton (5 microM) inhibited LTB4 production by 99% in the presence of these albumins. Also, rat albumin presented WY-50295T to purified rat neutrophils more effectively than human albumin (i.e. WY-50295T was more active in the presence of rat albumin). These results suggest that the high affinity binding of WY-50295T to human albumin and possibly the reduction of drug uptake (passive diffusion) using purified human vs rat neutrophils may account for the inactivity of WY-50295T in the human whole blood assay.
Insights
The 5-lipoxygenase (5-LO) inhibitor WY-50295 tromethamine effectively reduced leukotriene release in rats but not in humans. High-affinity binding to human albumin likely explains its reduced efficacy in human whole blood assays.
Area of Science:
- Biochemistry
- Pharmacology
- Immunology
Background:
- Leukotriene B4 (LTB4) is a key mediator in inflammatory responses.
- 5-lipoxygenase (5-LO) is a critical enzyme in LTB4 synthesis.
- WY-50295 tromethamine is a novel 5-LO inhibitor.
Purpose of the Study:
- To investigate the efficacy of WY-50295 tromethamine in inhibiting LTB4 production in human and rat neutrophils.
- To elucidate the factors contributing to differential efficacy between species.
Main Methods:
- In vitro and ex vivo assays using purified neutrophils and whole blood from humans and rats.
- Calcium ionophore stimulation to induce leukotriene release.
- Assessment of LTB4 production and 5-LO inhibition.
- Experiments involving albumin binding and drug uptake.
Main Results:
- WY-50295 tromethamine effectively inhibited LTB4 production in rat neutrophils and whole blood.
- It failed to inhibit LTB4 production in human whole blood, even at high concentrations.
- Human serum albumin significantly reduced the inhibitory activity of WY-50295 tromethamine.
- Rat albumin facilitated the presentation of WY-50295 tromethamine to rat neutrophils more effectively than human albumin.
Conclusions:
- High-affinity binding of WY-50295 tromethamine to human albumin is a primary reason for its lack of efficacy in human whole blood.
- Differences in albumin binding and potentially drug uptake contribute to species-specific activity.
- WY-50295 tromethamine shows promise as a 5-LO inhibitor but requires further investigation for human application.