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

https://doi.org/10.1016/j.ejpb.2005.02.012Get rights and content

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/N2 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.

Introduction

With the discovery of cytokines and their mechanism of action, there is an increasing interest in their therapeutic use as intestinal mucosal immune modulators. Intestinal mucosal delivery of cytokines would allow a local effect, resulting in fewer side effects than the systemic administration. This encloses the production of an oral formulation of cytokines. Apart from the mere fact that a conventional formulation of recombinant cytokines is extremely expensive, such approaches suffer from many other technical and fundamental obstacles.

Steidler and co-workers [1], [2] described the use of recombinant Lactococcus lactis strains for the delivery of cytokines. As interleukin-10 (IL-10) plays a central role in down-regulating inflammatory cascades, it looked a promising candidate to treat Crohn's disease, a severe, chronic intestinal inflammation [3]. However, IL-10 given through the systemic route was not effective [4]. Local delivery of the cytokine by L. lactis seemed more promising [2]. Murine IL-10 secreting L. lactis (LL-IL-10) were used for the in vivo production and mucosal delivery of this cytokine. Daily intragastric inoculation with these bacteria (LL-IL-10) into mice, in which experimental enterocolitis was installed, could cure or prevent the intestinal inflammation [2].

To evaluate the therapeutic potential of this concept in patients with Crohn's disease, a dry formulation containing the bioengineered human IL-10 (hIL-10) producing L. lactis (Thy12) [5] had to be developed. Since the hIL-10 production is strictly related to the viability of Thy12, a suitable production technique with maintenance of an acceptable level of viability and shelf life had to be selected. The survival of L. lactis has been studied in the human gastrointestinal tract up to the faeces. Klijn et al. [6] showed that the cells recovered in the faeces accounted for approximately 1% of the total number of cells ingested. Moreover, Vesa et al. [7] showed that the survival of L. lactis MG1363 was only 1% at the terminal ileum, demonstrating the detrimental effects of gastric acid and bile salts. This implies the enteric coating of the dosage form.

Research to stabilize biomaterials by formulating them in dry state is a highly active area. The most commonly used method for preparing solid protein pharmaceuticals [8], [9], [10] and dry live (attenuated) bacteria or viruses as vaccines [11], [12] or for food application [13] is freeze-drying. However, there is no universally applicable protocol for the successful freeze-drying of bacteria. Viability loss of lactic acid bacteria, freeze-dried in the same matrix, can change from 1 log for Streptococcus thermophilus to 2 logs for Lactobacillus bulgaricus [14]. This indicated that the survival after freeze-drying is strain dependent [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23] and that for each individual type of bacterium a matrix has to be optimized. Moreover, depending on the cryoprotective agents added, survival after freeze-drying can range from less then 1–67% for S. thermophilus [16]. Besides, most of these studies were conducted mainly in dairy or food industry, they lack the precise data on initial viability, stability and bacterial density. None of them report on freeze drying of bioengineered bacteria and/or maintenance of their properties achieved by bioengineering.

In this study, it was the aim to develop an enteric-coated formulation of freeze-dried Thy12. The first requirement was to develop a freeze-dried powder formulation containing viable and hIL-10 producing Thy12 with an acceptable shelf life. In this study, the optimal growth medium, physiological state and freezing rate have been optimized. Next, the stability of L. lactis was evaluated in order to select acceptable storage conditions. In order to increase the L. lactis concentration in the freeze-dried powder to reach an acceptable dose for oral administration, the culture preparation method was optimized and influence of cell density was evaluated. To increase bacterial viability after freeze-drying and storage, the influence of the incorporated feed components was evaluated. As the second requirement of this study was to protect the bacteria against the detrimental gastric fluid and bile salts, freeze-dried Thy12 was incorporated in enteric-coated capsules. These enteric-coated capsules are especially designed for storage at low temperature and low relative humidity and allowing passage through the stomach with low penetration of gastric fluid [24]. Viability and hIL-10 production in function of storage time and viability after simulated passage through the stomach have been tested.

Section snippets

Strains used in this study

  • L. lactis subsp. cremoris MG1363 [25].

  • Thy 12 (human IL-10 producing L. lactis subsp. cremoris MG1363) [5].

Preparation of the cell suspensions

The non-bioengineered L. lactis subsp. cremoris MG1363 culture was prepared by inoculating a stock suspension, stored at −20 °C in glycerol/GM17 (50/50), 1/1000 in growth medium (further specified). To prevent further activity or growth, the culture was kept on ice until use and in between all manipulations.

To investigate the influence of the freeze-drying matrix, two different growth media

Influence of freeze-drying matrix, physiological state and rate of freezing on viability of L. lactis MG1363 after freeze-drying

L. lactis MG1633 was first freeze-dried in the conventional broth of Lactococus lactis: GM17 (1 freeze-drying run (n=1)). This resulted in an absolute viability of only 9.3±0.8%. Therefore, the bacteria were grown and freeze-dried in an alternative GC-milk matrix. This resulted in a significantly higher viability (60.0±18.0%, n=17). The obtained viability is in accordance with the viability data reported by Carcoba et al. [22] for L. lactis subsp. lactis CECT5130 (44.3%) and Kilara et al. [16]

Conclusion

In this study, a pharmaceutical formulation of biological contained recombinant hIL-10 producing L. lactis was developed. The incorporation of freeze-dried Thy12 in ready-to-use enteric-coated capsules is a promising formulation since after 1 year storage, the enteric properties, an acceptable viability and hIL-10 producing capacity were maintained. This formulation has been used in a preclinical study in which hIL-10 producing L. lactis has been administered to 10 patients suffering from

Acknowledgements

This work was supported by the Research Fund of the Ghent University. The authors gratefully acknowledge Inge Bruggeman for her technical assistance and Dr. Sci. Els Adriaens for her critical review on the statistical analysis of the data.

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    Present address: Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland.

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