Development and preclinical efficacy of novel transforming growth factor-ß1 short interfering RNAs for pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a chronic devastating disease of unknown etiology. No therapy is currently available. A growing body of evidence supports the role of transforming growth factor (TGF)-ß1 as the major player in the pathogenesis of the disease. However, attempts to control its expression and to improve the outcome of pulmonary fibrosis have been disappointing. We tested the hypothesis that TGF-ß1 is the dominant factor in the acute and chronic phases of pulmonary fibrosis and developed short interfering (si)RNAs directed toward molecules implicated in the disease. This study developed novel sequences of siRNAs targeting the TGF-ß1 gene and evaluated their therapeutic efficacy in two models of pulmonary fibrosis: a model induced by bleomycin and a novel model of the disease developed spontaneously in mice overexpressing the full length of human TGF-ß1 in the lungs. Intrapulmonary delivery of aerosolized siRNAs of TGF-ß1 with sequences common to humans and rodents significantly inhibited bleomycin-induced pulmonary fibrosis in the acute and chronic phases of the disease and in a dose-dependent manner. Aerosolized human-specific siRNA also efficiently inhibited pulmonary fibrosis, improved lung function, and prolonged survival in human TGF-ß1 transgenic mice. Mice showed no off-target effects after intratracheal administration of siRNA. These results suggest the applicability of these novel siRNAs as tools for treating pulmonary fibrosis in humans.

Enhanced intestinal absorption of drugs by activation of peptide transporter PEPT1 using proton-releasing polymer.

Utilization of carrier-mediated transport systems in the gastrointestinal tract to increase the bioavailability of drugs is of great interest. In the present study, an increased supply of the driving force for peptide transporter PEPT1 by utilizing a proton-releasing polymer, Eudragit L100-55, was employed to increase the intestinal transport activity. Intestinal absorption of zwitterionic cefadroxil and dianionic cefixime was studied in rats by using in situ ileal closed loops and by in vivo oral administration of the drugs concomitantly with Eudragit L100-55. The results showed that Eudragit L100-55 decreased the pH in the intestinal loops, and increased the disappearance of both cefadroxil and cefixime from the loops. In rats, the plasma concentration after oral administration was increased significantly by coadministration of Eudragit L100-55, whereas a proton-nonreleasing analogous polymer, Eudragit RSPO, did not have any effect. Furthermore, the increased absorption of cefixime caused by Eudragit L100-55 was blocked by simultaneous administration of cefadroxil, a PEPT1 substrate/inhibitor, in a concentration-dependent manner. These results demonstrate that improvement of intestinal absorption of peptide-mimetics via a peptide transporter is possible by optimizing the transporter activity through coadministration of a proton-releasing polymer that supplies the driving force for the transporter.

Limited influence of P-glycoprotein on small-intestinal absorption of cilostazol, a high absorptive permeability drug.

Intestinal transport of the type III phosphodiesterase inhibitor cilostazol was characterized to evaluate the influence of secretory transporter. Intestinal absorption of cilostazol measured by the in situ closed loop method, showed regional differences, with high permeability in the upper part of the small intestine. Intestinal secretory transport of cilostazol at the ileum was tended to be decreased by the increase of tested concentration of cilostazol from 10 to 20 microM when evaluated by means of a Ussing-type chamber method with mounted rat intestinal tissues. Transcellular transport of cilostazol in the basolateral-to-apical direction in LLC-GA5-COL150 cells, which overexpress P-glycoprotein, was higher than that in parental LLC-PK1 cells. In addition, cilostazol reduced the basolateral-to-apical transport and increased the accumulation of [(3)H]daunomycin in LLC-GA5-COL150 cells. Accordingly, cilostazol was demonstrated to be transported by P-glycoprotein, while cilostazol is not likely to cause induction of the expression level of P-glycoprotein by the same manner with rifampin. Apical-to-basolateral transport of cilostazol in Caco-2 cells was increased in a low concentration range, followed by a decrease with further increase of the concentration, while the permeability coefficient of cilostazol was above 1 x 10(-6) cm/s at any concentration. Initial uptake of [(14)C]cilostazol by Caco-2 cells was temperature dependent and was reduced in the presence of unlabeled cilostazol, suggesting that apical uptake is also mediated by a transporter(s). In conclusion, intestinal absorption of cilostazol, which has a high absorptive permeability, may not be significantly hampered by efflux transporters, such as P-glycoprotein.

Delivery of peptide drugs to the brain by adenovirus-mediated heterologous expression of human oligopeptide transporter at the blood-brain barrier.

The feasibility of using adenovirus-mediated human oligopeptide transporter (hPEPT1) gene transfer to achieve peptide drug delivery to the brain across the blood-brain barrier was tested by examining the accumulation of model peptides in a rat brain endothelial cell line (RBEC1) and rat brain after transduction with a recombinant adenovirus encoding hPEPT1-enhanced yellow fluorescent protein fusion gene (AdhPEPT1-EYFP). In vitro uptake of [(3)H]GlySar was determined in RBEC1 transduced with AdhPEPT1-EYFP. In vivo, the accumulation of cefadroxil in rat brain was evaluated after transduction of AdhPEPT1-EYFP. At pH 6.0, the uptake of [(3)H]GlySar by RBEC1 transduced with AdhPEPT1-EYFP was increased 4-fold compared with that of nontransduced cells. At pH 7.4, uptake of [(3)H]GlySar in AdhPEPT1-EYFP transduced RBEC1 was 1.5 times higher than that of nontransduced cells. Unlabeled glycylsarcosine (10 mM) reduced the uptake of [(3)H]GlySar to a level comparable with that of nontransduced cells. At 30 min after intravenous administration of cefadroxil to rats transduced with AdhPEPT1-EYFP at 3.2 x 10(9) plaque-forming units/rat by an in situ brain perfusion method, the brain-to-plasma concentration ratio (Kp) of cefadroxil was increased about 2 times compared with that of nontransduced or AdGFP (control vector)-transduced rats, although this was not statistically significant. In contrast, Kp of [(14)C]inulin, a marker for extracellular fluid space, remained unchanged after adenoviral transduction. In conclusion, our results suggest that adenovirus-mediated heterologous expression of hPEPT1 in vivo could be a useful approach to deliver oligopeptides to the brain.

Enhanced delivery of drugs to the liver by adenovirus-mediated heterologous expression of the human oligopeptide transporter PEPT1.

To explore the feasibility of drug delivery to the liver by the use of adenovirus-mediated human oligopeptide transporter (hPEPT1) gene transfer, we examined the accumulation of L-[(3)H]carnosine in the hepatoma cell line (HepG2 and WIFB9) and mouse liver. We constructed a recombinant adenovirus encoding hPEPT1-enhanced yellow fluorescent protein (EYFP) fusion gene (AdhPEPT1-EYFP). In vitro uptake of L-[(3)H]carnosine was determined in HepG2 and WIFB9 cells transduced with AdhPEPT1-EYFP. In vivo, the accumulation of L-[(3)H]carnosine in mouse liver was evaluated after transduction of AdhPEPT1-EYFP. At pH 6.0, the uptake of L-[(3)H]carnosine by HepG2 and WIFB9 cells transduced with AdhPEPT1-EYFP was increased 15- and 2-fold, respectively, compared with the cells without transduction. At pH 7.4, uptake of L-[(3)H]carnosine in AdhPEPT1-EYFP transduced HepG2 cells was 3 times greater than that of nontransduced cells. In the presence of carnosine or glycylsarcosine as an inhibitor at 20 mM, the uptake of L-[(3)H]carnosine was reduced to a level comparable to that of nontransduced cells. At 30 min after intravenous administration of L-[(3)H]carnosine to mice transduced with AdhPEPT1-EYFP at 1 x 10(10) plaque-forming units/mouse, the tissue-to-plasma concentration ratio (K(p)) of L-[(3)H]carnosine in the liver was significantly increased to 7 times that of nontransduced mice. In contrast, the K(p) value of [(14)C]inulin, a marker for extracellular fluid space, remained unchanged after adenoviral transduction suggesting minimal pathological damage of tissues. hPEPT1-EYFP was localized at both the basolateral and apical membranes in HepG2 cells, WIFB9 cells, and mouse liver. In conclusion, our results suggest that delivery of oligopeptide to the liver by adenovirus-mediated heterologous expression of hPEPT1 in vivo is feasible.