Inflammatory proteases in chronic otitis externa.

OBJECTIVES: Proteases are known to contribute to the pathogenesis of chronic inflammatory skin conditions such as atopic dermatitis and psoriasis. Inhibition of these proteases has shown promise in the treatment of these skin conditions. The purpose of this study was to measure the matrix metalloproteinases (MMP) and human neutrophil elastase (HNE) activities in chronic otitis externa (COE) and to determine whether administration of protease inhibitors recombinant alpha 1-antitrypsin (rAAT) and ilomastat might reduce these protease activities. STUDY DESIGN: Prospective and ex vivo. METHODS: Twenty-five ear canals with COE and 34 with no pathology (i.e., controls) were debrided and filled with saline. After a tragal pump and 1 to 2 minutes, the washes were collected and analyzed for MMP and HNE activities and the inhibitory activity of rAAT and ilomastat on these proteases, respectively. RESULTS: MMP and HNE levels were significantly higher (P = .0057 and .0112) in ears with COE than normal ears. MMP activity greater than 3 mAU/minute was observed in 30% of COE and 0% of controls (P = .0270). HNE activity greater than 3 mAU/minute was found in 77% of COE versus 7% of controls (P < .0001). Ilomastat and rAAT inhibited 60% of MMP and 98% of HNE activity, respectively, in COE ears. CONCLUSIONS: Elevated levels of proteases found in COE, MMP, and HNE may be inhibited with ilomastat and rAAT. The therapeutic potential of these protease inhibitors warrants investigation.

An inhaled matrix metalloprotease inhibitor prevents cigarette smoke-induced emphysema in the mouse.

Inadequately regulated proteolytic activity is responsible for the chronic lung tissue degeneration and irreversible loss of pulmonary function that define emphysema. In this study, we show that an inhaled broad-spectrum matrix metalloprotease inhibitor, ilomastat, can provide protection against the development of emphysema in cigarette smoke-treated mice. Control animals were exposed to daily cigarette smoke for 6 months. As has been reported previously, cigarette smoke was seen to increase significantly the recruitment of macrophages into the lungs of these animals, leading to concomitant alveolar airspace enlargement and emphysema. In animals treated daily with nebulized ilomastat for 6 months, lung macrophage levels were greatly reduced, and neutrophil accumulation was also inhibited. Corresponding reductions in airspace enlargement of up to 96% were observed. These striking observations suggest that delivery of ilomastat directly into the lungs of smoke-treated mice can not only inhibit lung tissue damage mediated by metalloproteases, but may also reduce that component of tissue degeneration mediated by excess neutrophil-derived products. Our data also suggest that the matrix metalloprotease inhibitors may represent a class of drugs that, when delivered by inhalation, could be used practically to treat cigarette smoking-related chronic obstructive pulmonary disease by modifying the course of the disease.

Alpha 1-antitrypsin and ilomastat inhibit inflammatory proteases present in human middle ear effusions.

OBJECTIVES: Proteases of both the serine and metalloproteinase families have been shown to play a role in the pathogenesis of otitis media (OM). Inhibitors of proteases from each of these families have been shown to beneficially impact disease progression in a number of related chronic inflammatory conditions, but their use has not been studied in OM. The purpose of this study was to assess the activity of the protease inhibitors recombinant alpha 1-antitrypsin (rAAT) and ilomastat on inflammatory proteases present in human middle ear effusions (MEEs), with a view to their potential utility in the treatment of OM. STUDY DESIGN: Prospective and ex vivo. METHODS: MEEs were collected from 100 patients presenting for middle ear surgery, most commonly tympanostomy tube placement or treatment of acute posttympanostomy otorrhea (APTO). MEEs were analyzed for the presence of matrix metalloproteinases (MMPs) and human neutrophil elastase (HNE) and the inhibitory activity of rAAT and ilomastat on these proteases, respectively. RESULTS: MMP levels were highest in APTO, and HNE was highest in chronic suppurative OM and APTO. High levels of MMP and HNE (>3 mAU/min) were found in 52% and 37% of MEEs, respectively. Ilomastat and rAAT demonstrated significant inhibition of MMP and HNE activity (>30% reduction), respectively, in 80% and 82% of MEEs with high levels of activity. CONCLUSIONS: Proteases are commonly found in OM. Ilomastat and rAAT are potent inhibitors of proteases in MEEs across a wide range of OM in humans. Investigation into the potential therapeutic benefits of these protease inhibitors is warranted.

Expression of Torpedo californica creatine kinase in Escherichia coli and purification from inclusion bodies.

The pET17 expression vector was used to express creatine kinase from the electric organ of Torpedo californica as inclusion bodies in Escherichia coli BL21(DE3) cells. The insoluble aggregate was dissolved in 8M urea and, following extraction with Triton X-100, the enzyme was refolded by dialysis against Tris buffer (pH 8.0) containing 0.2M NaCl. After two buffer changes, chromatography on Blue Sepharose was used as a final step in the purification procedure. Approximately 54mg active protein was recovered from a 1L culture and the refolded enzyme had a specific activity of 75U/mg. The molecular mass of the purified protein was consistent with that predicted from the amino acid sequence and the CD spectrum of the refolded enzyme was essentially identical to that of creatine kinase from human muscle (HMCK). The K(m) values of ATP and ADP were also similar to those of HMCK, while the K(m) values for both phosphocreatine and creatine were approximately 5-10-fold higher. The purification described here is in marked contrast with earlier attempts at purification of this isozyme where, in a process yielding less than 1mg/L culture, enzyme with a specific activity of ca. 5U/mg was obtained.