Development of an enteric-coated formulation containing freeze-dried, viable recombinant Lactococcus lactis for the ileal mucosal delivery of human interleukin-10

Abstract Recombinant hIL-10 producing Lactococcus lactis (Thy12) looks a promising intestinal mucosal delivery system for treatment of Crohn's disease [L. Steidler, W. Hans, L. Schotte, S. Neirynck, F. Obermeirer, W. Falk, W. Fiers, E. Remaut, Treatment of murine colitis by L. lactis secreting interleukin-10, Science 289 (2000) 1352–1355. L. Steidler, S. Neirynck, N. Huyghebaert, V. Snoeck, A. Vermeire, B.M. Goddeeris, E. Cox, J.P. Remon, and E. Remaut, Biological containment of genetically modified L. lactis for intestinal delivery of human interleukin-10, Nat. Biotechnol. 21 (7) (2003) 785–789]. As the hIL-10 production is strictly related to Thy12's viability and gastric fluid negatively influences this viability, an enteric-coated formulation had to be developed with maintenance of its viability after production and storage. L. lactis MG1363, used for optimization, was grown until stationary phase in milk (glucose/casiton supplemented) and freeze-dried. This resulted in a viability of about 60%. Storage at different conditions showed that viability remained highest at 8 °C/N 2 atmosphere (32.5% of initial remained viable after 6 months). To increase the concentration of bacteria in the freeze-dried powder, they were concentrated by centrifugation. L. lactis tolerated this procedure. However, the concentration factor was limited to 10. Freeze-dried Thy12 was filled in ready-to-use enteric-coated capsules. Despite the good enteric properties of the capsules, viability of Thy12 dropped to about 43 and 28% after gastric fluid stage, depending on the enteric polymer used. Freeze-dried Thy12 filled in ready-to-use enteric-coated capsules, packed in Alu sachets (sealed at 20% RH) maintained 6.1 and 44.3% of initial viability after storage for 1 year at 8 and −20 °C, respectively, as well as its hIL-10 producing capacity.