Lysosubtilin modification, Fermosorb, designed for polymeric carrier-mediated intestinal delivery of lytic enzymes: pilot-scale preparation and evaluation of this veterinary medicinal product

Abstract

Antimicrobial enzymotherapy/enzymoprophylaxis has potential for use as a measure to overcome problems associated with resistance to commonly applied antibiotics. Lysosubtilin, an authorized veterinary medicinal product, when used per os for the treatment and prophylaxis of intestinal infections in newborn calves, is not always efficient due to partial inactivation of lytic enzymes in the gastric region. In this contribution a simple technology for preparation of pH-dependent reversibly dissociating acid stable enzyme-polymer complex (two-component oral delayed-release lysosubtilin formulation, Fermosorb) designed for intestinal delivery of lytic enzymes is described. The technology is based on immobilization of lytic enzymes, using 1% lysosubtilin solution in 10 mM acetate buffer of pH 5.0, onto commercial highly porous carboxylic cation exchanger Biocarb L (v/w ratio 10:1, process duration 1 h) with after-following procedures of vacuum-filtration, oven-drying and standardization of the enzyme-polymer complex formed. The technology process of pilot-scale Fermosorb fabrication on the whole revealed itself as simply employed and highly repeatable, totalling in the final lytic enzyme activity yield of 40.2% (the average value obtained from the analysis of the 11 batches running) and ∼4000 (3938) kg of Fermosorb (200 batches) produced. The proposed technological approach can be successfully applied for fabrication of other enzyme preparations as well and this was shown in the example of Polyferm, a preparation with both lytic and proteolytic enzyme activities. In vitro evaluation of Fermosorb revealed it was more stable when exposed to the acidic environment as well as in storage when compared with the native lysosubtilin. No negative change in the antimicrobial spectrum of action of Fermosorb versus lysosubtilin, influenced by immobilization of lytic enzymes onto Biocarb L, was observed. Moreover, all six lysosubtilin-resistant microbial strains tested have been found to be Fermosorb-susceptible. In vivo evaluation studies performed on 1200 newborn calves revealed 95.2% therapeutic as well as 95.0% prophylactic efficacy of Fermosorb in respect to colibacillosis versus 74.0 and 80.0% for lysosubtilin, respectively, the differences being statistically significant ( P<0.01). As a consequence of these studies Fermosorb was authorized for use throughout the former Soviet Union. Data collected during postmarketing surveillance of Fermosorb, which was applied for more than 163 000 newborn calves, confirmed high efficacy (92.3 and 95.5% for treatment and prophylaxis, respectively) and safety of this veterinary medicinal product.