Adenosine 5'-triphosphate (ATP) supplements are not orally bioavailable: a randomized, placebo-controlled cross-over trial in healthy humans.

BACKGROUND: Nutritional supplements designed to increase adenosine 5'-triphosphate (ATP) concentrations are commonly used by athletes as ergogenic aids. ATP is the primary source of energy for the cells, and supplementation may enhance the ability to maintain high ATP turnover during high-intensity exercise. Oral ATP supplements have beneficial effects in some but not all studies examining physical performance. One of the remaining questions is whether orally administered ATP is bioavailable. We investigated whether acute supplementation with oral ATP administered as enteric-coated pellets led to increased concentrations of ATP or its metabolites in the circulation. METHODS: Eight healthy volunteers participated in a cross-over study. Participants were given in random order single doses of 5000 mg ATP or placebo. To prevent degradation of ATP in the acidic environment of the stomach, the supplement was administered via two types of pH-sensitive, enteric-coated pellets (targeted at release in the proximal or distal small intestine), or via a naso-duodenal tube. Blood ATP and metabolite concentrations were monitored by HPLC for 4.5 h (naso-duodenal tube) or 7 h (pellets) post-administration. Areas under the concentration vs. time curve were calculated and compared by paired-samples t-tests. RESULTS: ATP concentrations in blood did not increase after ATP supplementation via enteric-coated pellets or naso-duodenal tube. In contrast, concentrations of the final catabolic product of ATP, uric acid, were significantly increased compared to placebo by ~50% after administration via proximal-release pellets (P = 0.003) and naso-duodenal tube (P = 0.001), but not after administration via distal-release pellets. CONCLUSIONS: A single dose of orally administered ATP is not bioavailable, and this may explain why several studies did not find ergogenic effects of oral ATP supplementation. On the other hand, increases in uric acid after release of ATP in the proximal part of the small intestine suggest that ATP or one of its metabolites is absorbed and metabolized. Uric acid itself may have ergogenic effects, but this needs further study. Also, more studies are needed to determine whether chronic administration of ATP will enhance its oral bioavailability.

Optimisation of an enteric coated, layered multi-particulate formulation for ileal delivery of viable recombinant Lactococcus lactis.

Layering of pellets with recombinant Lactococcus lactis Thy 12 was optimised for the production of a dosage form with a high load of viable recombinant L. lactis. Shear stress induced during the atomisation and the type of carrier used for the layering process did not influence the viability. A 5% lactose matrix resulted in the highest viability of L. lactis (8.9+/-1.7%) which could be maintained for at least 12 months at -20 degrees C. A higher bacterial cell load on the pellets was obtained using a longer process time, but the addition of 10% skim milk was essential to maintain the stabilising capacity of the matrix. Increasing the load of viable L. lactis was also possible using a higher bacterial cell concentration of the layering suspension and increasing the amount of stabilising matrix to 10% lactose/20% skim milk, yielding a formulation with 1.7 x 10(9)cfu/100 mg pellets. To protect the bacteria during gastric passage and to obtain ileum targeting, the formulation was enteric coated with 5% Eudragit FS30D, but after coating and gastric residence for 2 h HCl about 1% of the bacteria remained viable. Application of a subcoating, previous to enteric coating, did not result in a higher viability.

A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease.

BACKGROUND & AIMS: The use of living, genetically modified bacteria is an effective approach for topical delivery of immunomodulatory proteins. This strategy circumvents systemic side effects and allows long-term treatment of chronic diseases. However, treatment of patients with a living, genetically modified bacterium raises questions about the safety for human subjects per se and the biologic containment of the transgene. METHODS: We treated Crohn's disease patients with genetically modified Lactococcus lactis (LL-Thy12) in which the thymidylate synthase gene was replaced with a synthetic sequence encoding mature human interleukin-10. Ten patients were included in a placebo-uncontrolled trial. Patients were assessed daily for the presence of potential adverse effects by direct questioning and assessment of disease activity. We evaluated the presence and kinetics of LL-Thy12 release in the stool of patients by conventional culturing and quantitative polymerase chain reaction of LL-Thy12 gene sequences. RESULTS: Treatment with LL-Thy12 was safe because only minor adverse events were present, and a decrease in disease activity was observed. Moreover, fecally recovered LL-Thy12 bacteria were dependent on thymidine for growth and interleukin-10 production, indicating that the containment strategy was effective. CONCLUSIONS: Here we show that the use of genetically modified bacteria for mucosal delivery of proteins is a feasible strategy in human beings. This novel strategy avoids systemic side effects and is biologically contained; therefore it is suitable as maintenance treatment for chronic intestinal disease.

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

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 degrees 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 degrees C, respectively, as well as its hIL-10 producing capacity.

Development of an enteric-coated, layered multi-particulate formulation for ileal delivery of viable recombinant Lactococcus lactis.

Layering of recombinant hIL-10 producing Lactococcus lactis (L. lactis Thy12) on inert carriers is a promising technique for the preparation of a multi-particulate formulation of viable, hIL-10 producing L. lactis. To improve viability after layering and storage, L. lactis Thy12 was layered in different matrices (10% skim milk and/or 2.5, 5, 10% inulin). After layering, the highest viability was obtained in the 10% skim milk supplemented with 5% inulin matrix (8.7%). However, upon storage, 10% skim milk alone yielded the highest viability. Thereby, layered L. lactis Thy12 showed superior long term stability in comparison with freeze-dried L. lactis Thy12. The layering process was performed during 3h without encountering technical problems, with good layer consistence and constant viability. Enteric properties were obtained with a 30% Eudragit L30D-55 or 15% Eudragit FS30D coating and maintained during an initial six months storage period (-20 degrees C/20% RH). After in vitro simulation of the gastric stage, only 5% of the bacteria remained viable in Eudragit L30D-55 coated pellets, contrary to 85% in Eudragit FS30D coated pellets, indicating its superior protective capacity against gastric fluid. After eight months storage (-20 degrees C), 80% of the initial L. lactis Thy12 remained viable in the Eudragit FS30D coated pellets.

In vitro evaluation of coating polymers for enteric coating and human ileal targeting.

Recombinant interleukin-10 producing Lactococcus lactis is an alternative therapy for Crohn's disease. For in vivo interleukin-10 production, thymidine, the essential feed component of these recombinant bacteria should be coadministered. Different coating polymers were evaluated in vitro for enteric properties and targeting suitability to the ileum, the major site of inflammation in Crohn's disease. To guarantee ileal delivery, the polymer must dissolve from pH 6.8 and allow complete release within 40 min. Aqoat AS-HF coated pellets (15%) showed poor enteric properties and thymidine was released below pH 6.8. Eudragit FS30D coated pellets (15%) showed good enteric properties, but no thymidine was released within 40 min at pH 6.8. Eudragit S coated pellets (15%) showed good enteric properties after curing at elevated temperature while no thymidine was released within 40 min at pH 6.8. In another approach to pass the proximal small intestine intact, pellets were coated with 30% Eudragit L30D-55. At pH 6.0, they showed a lag-phase of 20 min. No influence of layer thickness was seen above pH 6.5. Alternatively, pellets were coated with a mixture of Eudragit FS30D/L30D-55 but they showed poor enteric properties and thymidine was released below pH 6.8. In conclusion, none of the tested polymers/mixtures ensured enteric properties and ileal targeting.

Evaluation of extrusion/spheronisation, layering and compaction for the preparation of an oral, multi-particulate formulation of viable, hIL-10 producing Lactococcus lactis.

Three formulation techniques were compared in order to develop a multi-particulate formulation of viable, interleukin-10 producing Lactococcus lactis Thy12. First, freeze-dried L. lactis was compacted into mini-tablets. Next, liquid L. lactis culture was used as the granulation fluid for the production of pellets by extrusion/spheronisation. Finally, liquid L. lactis culture was layered on inert pellets as an alternative technique for the production of pellets. L. lactis viability and interleukin-10 production was evaluated. Viability dropped to 15.7% after compaction of freeze-dried L. lactis and to 1.0% after pelletisation of liquid L. lactis by extrusion/spheronisation. The viability in the mini-tablets and pellets, stored for 1 week at RT and 10% RH was reduced to 23 and 0.5% of initial viability, respectively. Storage for 1 week at RT and 60% RH resulted in complete loss of viability. Layering of L. lactis on inert pellets resulted in low viability (4.86%), but 1 week after storage at RT and 10% RH, 68% of initial viability was maintained. Increasing product temperature and cell density of L. lactis in the layering suspension did not significantly change viability after layering and storage. Interleukin-10 production capacity of L. lactis Thy12 was maintained after layering.

Alternative method for enteric coating of HPMC capsules resulting in ready-to-use enteric-coated capsules.

The aim of this study was to develop an alternative method for enteric coating of HPMC capsules that avoids the sealing step before coating, resulting in ready-to-use enteric-coated capsules for the use in retail or hospital pharmacy or R&D sections of pharmaceutical industry and for the production of enteric-coated heat and moisture sensitive biomaterials. HPMC caps and bodies 00 (Vcaps, Capsugel) were coated separately in a fluid bed apparatus prior to filling (GPCG-1, Glatt) with Eudragit L30D-55 or Eudragit FS 30 D (Röhm), Aqoat AS-HF (Shin-Etsu) and Sureteric (Colorcon), using an optimised coating process. The coated bodies were filled and closed with the coated caps without encountering problems of coating damage. The release in 0.1N HCl after 2h from capsules coated with Eudragit L30D-55, Eudragit FS 30 D, Aqoat AS-HF and Sureteric was 0.6+/-.03, 0.6+/-0.3, 1.2+/-0.2 and 7.3+/-1.9%, respectively. The alternative method was reproducible and offered a way to overcome the time-consuming and expensive sealing step required using the conventional coating procedure. The obtained enteric-coated HPMC capsules can be stored (un)-filled for at least 6 months without loosing enteric properties.

Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10.

Genetically modified Lactococcus lactis secreting interleukin 10 provides a therapeutic approach for inflammatory bowel disease. However, the release of such genetically modified organisms through clinical use raises safety concerns. In an effort to address this problem, we replaced the thymidylate synthase gene thyA of L. lactis with a synthetic human IL10 gene. This thyA- hIL10+ L. lactis strain produced human IL-10 (hIL-10), and when deprived of thymidine or thymine, its viability dropped by several orders of magnitude, essentially preventing its accumulation in the environment. The biological containment system and the bacterium's capacity to secrete hIL-10 were validated in vivo in pigs. Our approach is a promising one for transgene containment because, in the unlikely event that the engineered L. lactis strain acquired an intact thyA gene from a donor such as L. lactis subsp. cremoris, the transgene would be eliminated from the genome.