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A cotton-bound serine protease inhibitor of elastase (fiber-inhibitor) has been formulated for in vitro evaluation in chronic wound fluid. As a model to understand the properties of the inhibitor in wound dressings, the kinetic profile and in vitro release of the fiber-inhibitor formulation have been examined. The elastase inhibitor N-Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone was modified onto cotton cellulose fibers and assayed as a colloidal system. Amino acid analysis and reversed phase high performance liquid chromatography were compared as semiquantitative methods to assess elastase inhibitor release from the cotton fibers. The kinetics of inhibition was assessed on treated fibers of synthetic dressings such that a colloidal suspension of the fiber-inhibitor and elastase was employed as an assay. A dose–response relationship was observed in the kinetics of substrate hydrolysis catalyzed by three elastases: porcine pancreatic elastase, which was employed to model this approach; human leukocyte elastase; and elastase in human chronic wound fluid. Both freely dissolved and fiber-bound inhibitors were studied. The initial rates of substrate hydrolysis were inversely linear with freely dissolved inhibitor dose. The apparent first order rate constants, k[sub obs], for the elastase-inhibitor complex were calculated from the kinetic profiles. The k[sub obs] for inhibitor bound enzyme varied as a function of inhibitor vs. enzyme concentration and based on the order of mixing of substrate, inhibitor and enzyme in the assay. Enzyme inhibition by the fiber-inhibitor was measured as inhibitor concentration at 50% inhibition (I[sub 50]). I[sub 50] values measured from the colloidal assay with fiber-released inhibitor were within the same range to those for freely dissolved inhibitor. Inhibition of elastase activity in chronic wound fluid was observed with 1–5 mg of fiber-inhibitor formulation. This approach constitutes an in vitro assessment ... [ABSTRACT FROM AUTHOR]

Elastase-sequestering wound dressings were prepared as the fructose and glucose citrate–cellulose conjugates on the cotton fibers of gauze dressings. The modified dressings were designed for chronic wounds, which contain destructively high levels of neutrophil elastase. The design of the modified gauzes was based on anionic citrate-esterified cellulose and the open chain isomers of the hemiketal and hemiacetal of fructose and glucose, which possess partial ketone, and aldehyde functionality as is found in synthetic elastase inhibitors. The cellulose–citrate conjugates of glucose and fructose were prepared on cotton gauze by rapid acid-catalyzed esterification of fibrous cellulose with citric acid as an ester cross-linking agent. The glucose and fructose citrate ester conjugates of cellulose were characterized as both intermolecular ester bonds of citrate-cross-linked cellulose and as citrate esters of glucose and fructose. Glucose and fructose were released from the fabric through hydrolysis of the citrate ester of the monosaccharide and were characterized using high performance anion exchange chromatography with pulsed amperometric detection and a modified Fehling''s test. Chromatographic analysis of the hydrolyzed glucose and fructose ester products revealed the cellulose conjugates to be formed at levels of 4 mg of glucose per gram of cotton and 0.9 mg of fructose per gram of cotton. Fourier transform infrared spectroscopy was employed to detect the citrate-esterified conjugates on the cotton fiber. The modified cotton gauzes containing the monosaccharide ester conjugates and citrate-cross-linked cellulose were compared with untreated gauze for reduction of elastase activity in buffered saline, and in solutions of chronic wound fluid. The monosaccharide and citric acid conjugates of cellulose demonstrate a sequestration effect by extracting elastase from solution. The order of elastase-lowering potency of the monosaccharide–citrate cellulose conjugates in wound fluid was judged to be fructose–citrate>glucose–citrate=citrate. Elastase-lowering activity displayed a linear response over a range of 10–75 mg of gauze samples with the fructose–citrate cellulose conjugate (11.5–0.5 μmol of sugar/unit of elastase activity). [Copyright &y& Elsevier]