RESUMO
Intestinal transporter proteins and metabolizing enzymes play a crucial role in the oral absorption of a wide variety of drugs. The aim of the current study was to characterize better available intestinal in vitro models by comparing expression levels of these proteins and enzymes between porcine intestine, human intestine, and Caco-2 cells. We therefore determined the absolute protein expression of 19 drug transporters and the mRNA expression of 12 metabolic enzymes along the pig intestinal tract (duodenum, jejunum, ileum; N = 4), in human intestine (jejunum; N = 9), and Caco-2 cells. Expression of the included transporters and enzymes was in general well comparable between porcine and human intestinal tissue, although breast cancer resistance protein, monocarboxylate transporter 5, multidrug resistance protein (MRP) 1, MRP1, MRP3 (â¼2-fold), and organic anion-transporting polypeptide (OATP) 4A1 (â¼6-fold) was higher expressed in pig compared with human jejunum. Alternatively, expression level of relevant transporter proteins (glucose transporter 1, OATP4A1, MRP2, MRP1, and OATP2B1) was significantly higher (3- to 130-fold) in Caco-2 cells compared with human jejunum. Moreover, all examined CYPs showed at least a fivefold lower gene expression in Caco-2 cells compared with human jejunum, with the smallest differences for CYP1A1 and CYP3A5 and the largest difference for CYP3A4 (871-fold higher expression in human jejunum compared with Caco-2 cells). In conclusion, a comprehensive overview is provided of the expression levels of clinically relevant transporter proteins and metabolic enzymes in porcine and human intestinal tissue and Caco-2 cells, which may assist in deciding upon the most suitable model to further improve our understanding of processes that determine intestinal absorption of compounds.
Assuntos
Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Absorção Intestinal/fisiologia , Mucosa Intestinal/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Células CACO-2 , Membrana Celular/metabolismo , Feminino , Glucuronosiltransferase/metabolismo , Humanos , Masculino , RNA Mensageiro/metabolismo , Sus scrofa/metabolismo , SuínosRESUMO
Inflammation is an important therapeutic target. Due to their potency, steroidal drugs dominate the current treatment of inflammatory disorders. However, steroidal drugs can also exert a broad range of side effects and appear not always effective. This calls for the development of alternative drugs with a different mechanism of action, which are likely to be found in the field of natural products (NPs). For many NPs strong anti-inflammatory effects have been described, but usually investigating a single compound in a single assay. In this study, eight promising NPs were selected and tested against the strong anti-inflammatory drug prednisolone. For this head-to-head comparison, in vitro assays were used which represent different pathways of the inflammatory response: TNF-α and IL-6 expression by macrophages, IL-8 expression by colon epithelial cells, ROS production in polymorphonuclear leukocytes and platelet activation in whole blood. Performance profiles were established which allowed us to identify curcumin, berberine chloride and epigallocatechin gallate as potential alternatives for prednisolone or other glucocorticoids in inflammation.
Assuntos
Anti-Inflamatórios/farmacologia , Berberina/farmacologia , Produtos Biológicos/farmacologia , Catequina/análogos & derivados , Curcumina/farmacologia , Neutrófilos/efeitos dos fármacos , Acetofenonas/farmacologia , Animais , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Células CACO-2 , Catequina/farmacologia , Linhagem Celular , Humanos , Interleucina-6/antagonistas & inibidores , Interleucina-6/biossíntese , Interleucina-6/imunologia , Interleucina-8/antagonistas & inibidores , Interleucina-8/biossíntese , Interleucina-8/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Neutrófilos/citologia , Neutrófilos/imunologia , Ativação Plaquetária/efeitos dos fármacos , Pravastatina/farmacologia , Prednisolona/farmacologia , Cultura Primária de Células , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Estilbenos/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: Inherited apolipoprotein (Apo) A-I deficiency is an orphan disorder characterized by high-density lipoprotein (HDL)-cholesterol deficiency and premature atherosclerosis. Constitutive over-expression of ApoA-I might provide a means to treat this disease. The present study provides a comprehensive evaluation of adeno-associated virus (AAV)-mediated ApoA-I gene delivery to express human (h)ApoA-I and correct the low HDL-cholesterol phenotype associated with ApoA-I deficiency. METHODS: In an effort to maximize AAV-mediated gene expression, we performed head-to-head comparisons of recombinant AAVs with pseudotype capsids 1, 2, 6 and 8 administered by different routes with the use of five different liver-specific promoters in addition to cytomegalovirus as single-stranded or as self-complementary (sc) AAV vectors. RESULTS: Intravenous administration of 1 x 10(13) gc/kg scAAV8, in combination with the liver-specific promoter LP1, in female ApoA-I(-/-) mice resulted in hApoA-I expression levels of 634 +/- 69 mg/l, which persisted for the duration of the study (15 weeks). This treatment resulted in full recovery of HDL-cholesterol levels with correction of HDL particle size and apolipoprotein composition. In addition, we observed increased adrenal cholesterol content and a significant increase in bodyweight in treated mice. CONCLUSIONS: The present study demonstrates that systemic delivery of a scAAV8 vector provides a means for efficient liver expression of hApoA-I, thereby correcting the lipid abnormalities associated with murine ApoA-I deficiency. Importantly, the study demonstrates that AAV-based gene therapy can be used to express therapeutic proteins at a high level for a prolonged period of time and, as such, provides a basis for further development of this strategy to treat hApoA-I deficiency.
Assuntos
Apolipoproteína A-I/sangue , Apolipoproteína A-I/deficiência , HDL-Colesterol/sangue , Dependovirus/genética , Terapia Genética , Animais , Apolipoproteína A-I/genética , Peso Corporal , Citomegalovirus/genética , Dependovirus/classificação , Elementos Facilitadores Genéticos/genética , Vetores Genéticos/genética , Humanos , Injeções Intravenosas , Fígado/metabolismo , Camundongos , Mutagênese Insercional , Especificidade de Órgãos , Fenótipo , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Sorotipagem , Aumento de PesoRESUMO
Variation in the apolipoprotein A5 (APOA5) gene has consistently been associated with increased plasma triglyceride (TG) levels in epidemiological studies. In vivo functionality of these variations, however, has thus far not been tested. Using adenoviral over-expression, we evaluated plasma expression levels and TG-lowering efficacies of wild-type human apoAV, two human apoAV variants associated with increased TG (S19W, G185C) and one variant (Q341H) that is predicted to have altered protein function. Injection of mice with adenovirus encoding wild-type or mutant apoAV resulted in an identical dose-dependent elevation of human apoAV levels in plasma. The increase in apoAV levels resulted in pronounced lowering of plasma TG levels at two viral dosages. Unexpectedly, the TG-lowering efficacy of all three apoAV variants was similar to wild-type apoAV. In addition, no effect on TG-hydrolysis-related plasma parameters (free fatty acids, glycerol and post-heparin lipoprotein lipase activity) was apparent upon expression of all apoAV variants. In conclusion, our data indicate that despite their association with hypertriglyceridemia and/or predicted protein dysfunction, the 19W, 185C and 341H apoAV variants are equally effective in reducing plasma TG levels in mice.
Assuntos
Apolipoproteínas A/metabolismo , Hipertrigliceridemia/metabolismo , Triglicerídeos/sangue , Animais , Apolipoproteína A-V , Apolipoproteínas A/sangue , Apolipoproteínas A/genética , Humanos , Hipertrigliceridemia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Overexpression of lipoprotein lipase (LPL) protects against atherosclerosis in genetically engineered mice. We tested whether a gene therapy vector that delivers human (h) LPL(S447X) cDNA to skeletal muscle could induce similar effects. METHODS: LDL receptor knockout (LDLr-/-) mice were injected intramuscular (i.m.) with adeno-associated virus serotype 1 (AAV1) LPL(S447X) or PBS. Four weeks later they were started on an atherogenic diet for 12 weeks. After termination, atherosclerosis was assessed and homogenates of muscle and liver tissue were analyzed. RESULTS: AAV1-treated mice showed hLPL concentrations of 768+/-293 ng/mL in post-heparin plasma associated with 48% reductions of fasting triglycerides (TG) levels (p<0.0001). In the absence of an effect on total cholesterol (TC) levels, no effects on atherosclerosis were found. An increase in lipid content of injected muscles was accompanied by a significant decrease of TG (-20%, p<0.0001) and free cholesterol (FC) content (-24%, p<0.0001) in liver homogenates. CONCLUSIONS: The data show that transgenic hLPL(S447X) on top of endogenous murine LPL reduces fasting TG levels in plasma but has no effect on atherosclerosis in LDLr-/- mice. While lipid accumulation in the injected muscle was anticipated, this coincided with an interesting decrease of both TG and FC in liver homogenates.
Assuntos
Aterosclerose/terapia , Dependovirus/genética , Terapia Genética/métodos , Lipase Lipoproteica/genética , Receptores de LDL/genética , Animais , Aterosclerose/genética , Colesterol/sangue , Gorduras na Dieta/farmacologia , Emulsões Gordurosas Intravenosas/farmacologia , Feminino , Vetores Genéticos/genética , Humanos , Injeções Intramusculares , Lipase Lipoproteica/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Knockout , Músculo Esquelético/fisiologia , Triglicerídeos/sangueRESUMO
CD2F1 mice were inoculated with C26 adenocarcinoma cells, followed by assessment of ex vivo muscular function. Muscles from tumor-bearing mice had a significantly lower force output during a single maximal contraction and during repeated contractions than control muscles. The relative force output, however, did not differ when corrected for muscle mass. Thus, cachexia significantly reduces absolute skeletal muscle function, but muscle "quality" appears unaltered.