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1.
Oral Oncol ; 46(7): 564-70, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20542722

RESUMO

Non-clinical studies, focusing on the pharmacodynamics (PD), pharmacokinetics (PK) and safety pharmacology of genetically modified Lactococcus lactis (L. lactis) bacteria, engineered to secrete human Trefoil Factor 1 (hTFF1), were performed to provide proof-of-concept for the treatment of oral mucositis (OM) patients. L. lactis strain sAGX0085 was constructed by stably inserting an htff1 expression cassette into the bacterial genome, and clinically formulated as a mouth rinse (coded AG013). PD studies, using different oral dosing regimens, were performed in a clinically relevant hamster model for radiation-induced OM. The PK profile was assessed in healthy hamsters and in hamsters with radiation-induced OM. In addition, in vitro and in vivo safety pharmacology studies were conducted, in pooled, complement-preserved human serum, and in neutropenic hamsters and rats respectively. Topical administration of L. lactis sAGX0085/AG013 to the oral mucosa significantly reduced the severity and course of radiation-induced OM. PK studies demonstrated that both living L. lactis bacteria, as well as the hTFF1 secreted, could be recovered from the administration site for maximum 24h post-dosing, without systemic exposure. The in vitro and in vivo safety pharmacology studies confirmed that L. lactis sAGX0085 could not survive in systemic circulation, not even under neutropenic conditions. The results from the PD, PK and safety pharmacology studies reported here indicate that in situ secretion of hTFF1 by topically administered L. lactis bacteria provides a safe and efficacious therapeutic tool for the prevention and treatment of OM.


Assuntos
Lactococcus lactis/metabolismo , Antissépticos Bucais/metabolismo , Peptídeos/metabolismo , Estomatite/tratamento farmacológico , Animais , Cricetinae , Humanos , Antissépticos Bucais/farmacocinética , Peptídeos/farmacocinética , Ratos , Resultado do Tratamento , Fator Trefoil-2
2.
Appl Environ Microbiol ; 74(20): 6271-9, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18689518

RESUMO

The apuB gene of Bifidobacterium breve UCC2003 was shown to encode an extracellular amylopullulanase. ApuB is composed of a distinct N-terminally located alpha-amylase-containing domain which hydrolyzes alpha-1,4-glucosidic linkages in starch and related polysaccharides and a C-terminally located pullulanase-containing domain which hydrolyzes alpha-1,6 linkages in pullulan, allowing the classification of this enzyme as a bifunctional class II pullulanase. A knockout mutation of the apuB gene in B. breve UCC2003 rendered the resulting mutant incapable of growth in medium containing starch, amylopectin, glycogen, or pullulan as the sole carbon and energy source, confirming the crucial physiological role of this gene in starch metabolism.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bifidobacterium/enzimologia , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Amilopectina/metabolismo , Bifidobacterium/genética , Bifidobacterium/crescimento & desenvolvimento , Bifidobacterium/metabolismo , Deleção de Genes , Glucanos/metabolismo , Glicogênio/metabolismo , Estrutura Terciária de Proteína , Amido/metabolismo
3.
Inflamm Bowel Dis ; 14(4): 471-9, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18092343

RESUMO

BACKGROUND: Genetically modified Lactococcus lactis secreting interleukin-10 (IL-10) has been demonstrated to provide localized delivery of a therapeutic agent through active in situ synthesis in murine colitis. At present, many aspects of the exact mechanism by which the beneficial effect of the IL-10-producing L. lactis on the mucosa is mediated remain to be clarified. METHODS: Our aim was to determine the interaction of L. lactis with the intestinal mucosa. Therefore, we administered IL-10-producing L. lactis to healthy mice and in 2 mouse models of chronic colitis. Paraffin sections of ileum and colon samples were examined with confocal and transmission electron microscopy. Ileum and colon homogenates were prepared after flushing and after removal of mucus layer and epithelium. These homogenates and homogenates of mesenteric lymph nodes and spleen were plated on agar and immunoblotting for L. lactis and IL-10 was performed. RESULTS: Both confocal and electron microscopy showed the presence of lactococci in inflamed intestinal mucosa of mice with colitis. We recovered viable bacteria that could still produce IL-10 from homogenates of inflamed ileum and colon of which mucous and epithelial layers were removed. We did not find lactococci in mesenteric lymph nodes or in the spleen of mice with colitis. CONCLUSIONS: This study demonstrates uptake of IL-10-secreting L. lactis by the paracellular route in inflamed mucosal tissue. We suggest that IL-10 production by L. lactis residing inside the mucosa in the vicinity of responsive cells can improve the local action of interleukin-10 in inflamed tissue and the efficiency of the treatment.


Assuntos
Colite/patologia , Interleucina-10/biossíntese , Lactococcus lactis/metabolismo , Animais , Colite/microbiologia , Colite/terapia , Colo/microbiologia , Colo/patologia , Engenharia Genética , Íleo/microbiologia , Íleo/patologia , Interleucina-10/uso terapêutico , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Lactococcus lactis/genética , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Microscopia Eletrônica de Transmissão
4.
Appl Environ Microbiol ; 72(12): 7694-700, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17028239

RESUMO

Interleukin-10 (IL-10) is a promising candidate for the treatment of inflammatory bowel disease. Intragastric administration of Lactococcus lactis genetically modified to secrete IL-10 in situ in the intestine was shown to be effective in healing and preventing chronic colitis in mice. However, its use in humans is hindered by the sensitivity of L. lactis to freeze-drying and its poor survival in the gastrointestinal tract. We expressed the trehalose synthesizing genes from Escherichia coli under control of the nisin-inducible promoter in L. lactis. Induced cells accumulated intracellular trehalose and retained nearly 100% viability after freeze-drying, together with a markedly prolonged shelf life. Remarkably, cells producing trehalose were resistant to bile, and their viability in human gastric juice was enhanced. None of these effects were seen with exogenously added trehalose. Trehalose accumulation did not interfere with IL-10 secretion or with therapeutic efficacy in murine colitis. The newly acquired properties should enable a larger proportion of the administered bacteria to reach the gastrointestinal tract in a bioactive form, providing a means for more effective mucosal delivery of therapeutics.


Assuntos
Colite/terapia , Trato Gastrointestinal/microbiologia , Lactococcus lactis/crescimento & desenvolvimento , Trealose/metabolismo , Animais , Bile/química , Doença Crônica , Colite/microbiologia , Crioprotetores/metabolismo , Crioprotetores/farmacologia , Sistemas de Liberação de Medicamentos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Feminino , Liofilização , Suco Gástrico/química , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trealose/farmacologia
5.
Clin Gastroenterol Hepatol ; 4(6): 754-9, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16716759

RESUMO

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.


Assuntos
Doença de Crohn/terapia , Terapia Genética , Interleucina-10/administração & dosagem , Lactococcus lactis , Proteína C-Reativa/análise , Doença de Crohn/patologia , Humanos , Interleucina-10/genética , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Organismos Geneticamente Modificados , Comprimidos com Revestimento Entérico , Timidilato Sintase/genética , Transgenes
7.
Eur J Pharm Biopharm ; 60(3): 349-59, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15927456

RESUMO

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.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Íleo/metabolismo , Interleucina-10/administração & dosagem , Mucosa Intestinal/metabolismo , Lactococcus lactis/crescimento & desenvolvimento , Proteínas Recombinantes/administração & dosagem , Composição de Medicamentos , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Liofilização , Humanos , Íleo/microbiologia , Interleucina-10/genética , Mucosa Intestinal/microbiologia , Lactococcus lactis/genética , Proteínas Recombinantes/genética , Comprimidos com Revestimento Entérico
8.
Eur J Pharm Biopharm ; 59(1): 9-15, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15567296

RESUMO

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.


Assuntos
Interleucina-10/síntese química , Lactococcus lactis/química , Tecnologia Farmacêutica/métodos , Administração Oral , Química Farmacêutica , Humanos , Interleucina-10/administração & dosagem , Interleucina-10/biossíntese , Lactococcus lactis/metabolismo
9.
Gastroenterology ; 127(2): 502-13, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15300583

RESUMO

BACKGROUND & AIMS: Effective therapeutics for treating acute colitis, caused by disruption of the intestinal epithelial barrier, are scarce. Trefoil factors (TFF) are cytoprotective and promote epithelial wound healing and reconstitution of the gastrointestinal tract, which makes them good candidate therapeutics for acute colitis. However, orally administered TFF stick to the mucus of the small intestine and are absorbed at the cecum. METHODS: We have engineered the food-grade bacterium Lactococcus lactis to secrete bioactive murine TFF. The protective and therapeutic potentials of these TFF-secreting L. lactis were evaluated in parallel with purified TFF in the dextran sodium sulfate (DSS)-induced murine model for acute colitis and in established chronic colitis in interleukin (IL)-10(-/-) mice. Disease was evaluated by blinded macroscopic and microscopic inflammatory scores and by myeloperoxidase activity. RESULTS: Intragastric administration of TFF-secreting L. lactis led to active delivery of TFF at the mucosa of the colon and, in contrast to administration of purified TFF, proved to be very effective in prevention and healing of acute DSS-induced colitis. The in situ secreted murine TFF significantly decreased morbidity and mortality and stimulated prostaglandin-endoperoxide synthase 2 expression, which represents a major therapeutic pathway. In addition, this approach was successful in improving established chronic colitis in IL-10(-/-) mice. CONCLUSIONS: We have positively evaluated a new therapeutic approach for acute and chronic colitis that involves in situ secretion of murine TFF by orally administered L. lactis. This novel approach may lead to effective management of acute and chronic colitis and epithelial damage in humans.


Assuntos
Colite/terapia , Terapia Genética/métodos , Lactococcus lactis/genética , Mucinas , Proteínas Musculares , Neuropeptídeos , Peptídeos/genética , Doença Aguda , Administração Oral , Animais , Doença Crônica , Colite/prevenção & controle , Ciclo-Oxigenase 2 , Feminino , Regulação Enzimológica da Expressão Gênica , Técnicas In Vitro , Isoenzimas/genética , Lactococcus lactis/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/metabolismo , Peptídeos/farmacologia , Prostaglandina-Endoperóxido Sintases/genética , Proteínas Recombinantes/genética , Fator Trefoil-2 , Fator Trefoil-3 , Cicatrização
10.
Nat Biotechnol ; 21(7): 785-9, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12808464

RESUMO

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.


Assuntos
Regulação Bacteriana da Expressão Gênica/fisiologia , Íleo/microbiologia , Interleucina-10/genética , Interleucina-10/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Engenharia de Proteínas/métodos , Timidilato Sintase/deficiência , Sequência de Aminoácidos , Animais , Divisão Celular , Sobrevivência Celular , Colite/microbiologia , Colite/terapia , Sistemas de Liberação de Medicamentos/métodos , Engenharia Genética/métodos , Humanos , Interleucina-10/uso terapêutico , Lactococcus lactis/citologia , Lactococcus lactis/crescimento & desenvolvimento , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Probióticos/uso terapêutico , Suínos , Timidilato Sintase/genética , Timidilato Sintase/metabolismo
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