Celiac Sprue is a multi-factorial disease characterized by an inflammatory response to ingested wheat
gluten and similar
proteins in rye and barley.
Proline-rich
gluten peptides from wheat, rye, and barley are relatively resistant to gastrointestinal digestion, and therefore persist in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. In this study, we characterize the in vitro
gluten detoxifying properties of a therapeutically promising
prolyl endopeptidase from Myxococcus xanthus (MX PEP), and describe the development of a prototypical enteric-coated
capsule containing a pharmacologically useful dose of this
enzyme. A high-cell density fed-batch fermentation process was developed for overproduction of recombinant MX PEP in E. coli, yielding 0.25-0.4 g/L purified
protein. A simple, scalable purification and lyophilization procedure was established that yields >95% pure, highly active and stable
enzyme as a dry
powder. The dry
powder was blended with
excipients and encapsulated in a hard
gelatin capsule. The resulting
capsule was enteric coated using
Eudragit L30-D55
polymer coat, which provided sufficient resistance to gastric conditions (> 1 h in 0.01 M HCl, pH 2 with
pepsin) and rapid release under duodenal conditions (15-30 min release in pH 6.0 in the presence of
trypsin and
chymotrypsin). In conjunction with pancreatic
enzymes, MX PEP breaks down whole
gluten into a product mixture that is virtually indistinguishable from that generated by the Flavobacterium meningosepticum (FM) PEP as judged by chromatographic assays. Competitive studies involving selected immunogenic
peptides mixed with whole
gluten reveal that both PEPs have a wide range of substrate specificity. Our results support further in vitro and in vivo evaluation of the MX PEP
capsule as an oral therapeutic agent for
Celiac Sprue patients.