Abstract
Nonsteroidal antiinflammatory drugs(NSAIDs) are known as clinically effective agents for treatment of inflammatory diseases. Inhibition of cyclooxygenase has been thought to be a major facet of the pharmacological mechanism of NSAIDs. However, it is difficult to ascribe the antiinflammatory effects of NSAIDs solely to the inhibition of prostaglandin synthesis. Human neutrophil elastase (HNElastase; HNE, EC 3.4.21.37) has been known as a causative factor in inflammatory diseases. To investigate the specific relationship between HNElastase inhibition and specificity of molecular structure of several NSAIDs, HNElastase was purified by Ultrogel AcA54 gel filtration, CM-Sephadex ion exchange, and HPLC (with TSK 250 column) chromatography. HNElastase was inhibited by aspirin and salicylate in a competitive manner and by naproxen, ketoprofen, phenylbutazone, and oxyphenbutazone in a partial competative manner, but not by ibuprofen and tolmetin. HNElastase-phenylbutazone-complex showed strong Raman shifts at 200, 440, 1124, 1194, 1384, 1506, and 1768 cm(-1). The Raman bands 1194, 1384, and 1768 cm(-1) may represent evidences of the conformational change at -N=N-phi radical, pyrazol ring, and -C=O radical of the elastase-drug complex, respectively. Phenylbutazone might be bound to HNElastase by ionic and hydrophobic interaction, and masked the active site. Inhibition of HNElastase could be another mechanism of action of NSAIDs besides cyclooxygenase inhibition in the treatment of inflammatory diseases. Different inhibition characteristics of HNE-lastase by NSAIDs such as aspirin, phenylbutazone-like drugs and ineffective drugs could be important points for drawing the criteria for appropriate drugs in clinical application.
Highlights
We describe the characteristics of HNElastase inhibition by Nonsteroidal antiinflammatory drugs (NSAIDs)
We propose a possible molecular, pharmacological inhibition mechanism of HNElastase by phenylbutazone according to the analysis of Raman spectroscopic data and visualized molecular model of human neutrophil elastase
Three step procedure includes Ultrogel AcA 54 gel filtration, CM-Sephadex ion exchange chromatography, and shallow salt gradient ion exchange chromatography. It is an effective method and the last step is an important step for purification and it is a new method ever reported for purification of HNElastase isozymes
Summary
Human neutrophil elastase (HNElastase, EC 3.4.21.37), is a causative factor in inflammatory diseases such as emphysema and rheumatoid arthritis (Janoff, 1972; Oholson and Olsson, 1977; Starkey, 1980; Fritz et al, 1986; Abramsom & Weissman, 1989), HNElastases are regulated by plasma proteinase inhibitors such as alphalproteinase inhibitor and alpha2-macroglobulin (Janoff, 1972; Oholson and Olsson, 1977; Starkey, 1980; Fritz et al, 1986). Nonsteroidal antiinflammatory drugs (NSAIDs) are known as clinically effective agents for treatment of inflammatory diseases such as rheumatoid arthritis (Lentin et al, 1987; Vane et al, 1987 and 1995; Insel, 1996). In comparison with other methods including IR or NMR, it is reasonable to investigate the mechanism of drug action by conformational change of the elastase-drug complex with Raman spectroscopic method. Since this method has the special advantage with only a small interference from the main biological solvent, water (Keller et al, 1994). We propose a possible molecular, pharmacological inhibition mechanism of HNElastase by phenylbutazone according to the analysis of Raman spectroscopic data and visualized molecular model of human neutrophil elastase
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