Regeneration and maintenance of bile canalicular networks in collagen-sandwiched hepatocytes.

The morphological and cytoskeletal reorganization of collagen-sandwiched rat hepatocytes during the de novo formation of complete canalicular networks was examined by phase, fluorescence and electron microscopy. During the initial stages of membrane repolarization, there was a marked accumulation of both microfilaments and microtubules at the sites of canalicular generation. Microtubule-disrupting agents (colchicine, nocodazole) inhibited the localization of actin filaments at cell margins and the initiation and branching of canalicular networks. After removal of microtubule-disrupting agents, microfilaments relocalized to the canalicular borders and microtubules nucleated along the margins of the bile canaliculi at sites distinct from the peri-canalicular actin networks. Microfilament-perturbing agents (cytochalasin D, phalloidin) did not affect the de novo initiation of bile canaliculi and only slightly impaired the development of canalicular lumina into networks. In established cultures with complete canalicular networks, subsequent treatment with microtubule-disrupting agents did not acutely affect the integrity of preformed canalicular networks. In contrast, treatment with microfilament-perturbing agents caused a marked dilation of most canaliculi. These results illustrate the differential role of the cytoskeleton in the regeneration and maintenance of bile canalicular networks by collagen-sandwiched hepatocytes. Moreover, this study shows the utility of this system as an in vitro model for examining the regulation of cell and membrane polarity.

In vitro and in vivo evaluation of effects of sodium caprate on enteral peptide absorption and on mucosal morphology.

Sodium salts of medium-chain fatty acids, sodium caprate (C10) in particular, have been used as absorption-enhancing agents to promote transmucosal drug absorption. In this study, we conducted both in vitro and in vivo experiments to investigate the effects of C10 on intestinal permeabilities and mucosal morphology. Mucosal addition of C10 (13-25 mM) reduced the transepithelial electric resistance (TEER) of cultured monolayers of the human intestinal cell line Caco-2 by 40-65% and, upon removal of C10, a marked tendency of TEER recovery was recorded. C10 added mucosally at 13-50 mM increased the transports of mannitol and polyethylene glycol (PEG) 900 across Caco-2 in a dose-dependent manner. In contrast, the transport of a model D-decapeptide was maximally enhanced with 20-25 mM C10. No noticeable morphological alteration of the Caco-2 monolayers was observed after a 1-h mucosal pretreatment with C10. Co-delivery with C10 (0.05-0.5 mmol/kg) into the rat terminal ileum increased the D-decapeptide bioavailability (BA) dose-dependently. With 0.5 mmol/kg C10 co-administered, D-decapeptide percent BA was elevated from 2 to 11%. Following a 1-h incubation with 0.5 mmol/kg C10 (in liquid or powder form) non-invasively delivered into the rectal lumen, no signs of histological change in the rectal mucosa were detected. These results demonstrate that C10 can promote intestinal absorption of a small peptide without causing detrimental alterations of the intestinal mucosa. C10 thus seems to be a good candidate as an enhancing agent for improving the oral BA of small therapeutic peptides.

Enhancement of intestinal model compound transport by DS-1, a modified Quillaja saponin.

DS-1, a modified Quillaja saponin, has recently been shown to promote the absorption of insulin and aminoglycoside antibiotics via the ocular and nasal route. The purpose of this study is to investigate the effect of DS-1 on intestinal permeability, the mechanism of its action, and reversibility of the effect. The permeation-enhancing activity of DS-1 was evaluated in cultured monolayers of the Caco-2 intestinal epithelial cells by examining its effect on the transepithelial electric resistance (TEER) and on transport of mannitol and a model D-decapeptide. Mucosal addition of DS-1 promptly reduced the TEER of the Caco-2 monolayers, and a propensity of recovery of the TEER was observed upon its removal. DS-1 added at 0.01-0.1% (w/v) increased the transports of both mannitol and D-decapeptide in a dose-dependent manner; a relatively "flat" concentration-dependence was seen at 0.1-0.2%. Visualization studies conducted by confocal laser scanning microscopy (CLSM) seem to suggest that DS-1 enhances the Caco-2 permeability mainly via a transcellular route. Histological examination failed to reveal noticeable morphological alterations in the cell monolayers pretreated with DS-1. The integrity of the Caco-2 monolayers, as assessed by their permeability to mannitol, was found to be recoverable following the mucosal pretreatment of DS-1. These results suggest that DS-1 is an efficacious intestinal permeation-enhancing agent with low adverse effect on the epithelial viability and barrier function.

Molecular weight-dependent paracellular transport of fluorescent model compounds induced by palmitoylcarnitine chloride across the human intestinal epithelial cell line Caco-2.

Long-chain acylcarnitines, such as palmitoylcarnitine chloride (PCC), are endogenous compounds which have been shown to increase intestinal transport of small hydrophilic compounds (including some pharmaceutical agents) through the paracellular pathway. However, the size range of the compounds whose absorption can be improved by PCC has not been fully investigated. In the present study, we systematically examined the effect of PCC on the transport rate of a series of hydrophilic fluorescent model compounds of varying molecular weights (0.3-71.2 kD) across cultured monolayers of the human intestinal epithelial cells Caco-2. Mucosal addition of 100 or 200 microM PCC resulted in comparable time-dependent decreases in the transepithelial electric resistance (T1/2, approximately 15 min). PCC addition induced a striking increase in the transport of sodium fluorescein (Flu-Na; 0.3 kD) and a slight or moderate increase in transports of fluorescent compounds of 0.6-11 kD. The effect of PCC on transport of compounds with molecular weights of > or = 17 kD appeared to be negligible. Examination by confocal laser scanning microscopy clearly revealed dilated paracellular spaces in Caco-2 monolayers which had been mucosally pretreated with PCC, confirming that PCC increases intestinal permeability by opening a paracellular transport pathway. Our results suggest that PCC is particularly effective in enhancing intestinal absorption of small hydrophilic compound like Flu-Na and may also have limited use in promoting the transport of compounds of < or = 10 kD.

Adenovirus-mediated transduction of intestinal cells in vivo.

The intestinal tract has many features that make it an attractive target for therapeutic gene transfer. In this study, replication-defective adenoviral vectors were used to explore parameters that may be important in administering gene therapy vectors to the intestine. After surgically accessing the intestine, an E1-, E3-deleted adenoviral vector encoding beta-galactosidase (beta-Gal) was directly injected into various regions of the small and large intestine of rats and rabbits. Significant transduction of the tissue was observed and histochemical staining was used to identify enterocytes as the primary targets of gene transfer. Expression of beta-Gal did not differ substantially when the virus was administered to the duodenum, ileum, or colon. When the vector was directly administered to segments of the distal ileum containing a Peyer's patch, transgene expression was approximately 10-fold higher than in segments lacking a Peyer's patch. In the Peyer's patches, a high level of expression was localized to epithelial cells, potentially M cells, overlying the lymphoid follicle domes. Transduction of these cells could have application in DNA-mediated oral vaccination. Administration of an adenoviral vector encoding a secreted alkaline phosphatase to the lumen resulted in expression and secretion of this gene product into the circulation. This finding demonstrates the potential of enterocytes to serve as heterotopic sites for the synthesis of heterologous gene products that would be secreted into the lumen of the intestinal tract or into the bloodstream.

Strategies for delivery of peptides utilizing absorption-enhancing agents.

The development of oral formulations for the effective delivery of peptides and proteins has been an elusive target. Although some success has been achieved (e.g., with cyclosporine), progress has been slow compared with what has been achieved with more traditional, organic drug molecules. Poor membrane permeability, enzymatic instability, and large molecular size are three factors that have remained major hurdles for peptide formulators. Absorption-enhancing agents that have been effective, at least in research environments, with smaller drug candidates, have also shown some limited efficacy in small animal models with certain peptides. In most cases, however, effective formulations have only achieved fairly low peptide absorption (< 10%) and have also resulted in significant alterations in the normal cellular morphology of the gastrointestinal tract, at least on a transient basis. Both literature and current data are reviewed in this report. Taken as a whole, the data suggest that the successful development of oral peptide formulations remains a significant challenge. Where successes are achieved, they will most likely be on a case-by-case basis and will reflect a balance between absorption-promoting efficacy of the formulation and the extent to which transient alteration of cell or tissue morphology occurs.

Oral controlled release technology for peptides: status and future prospects.

In spite of significant efforts in academic and commercial laboratories, major breakthroughs in oral peptide and protein formulation have not been achieved. The major barriers to developing oral formulations for peptides and proteins include poor intrinsic permeability, lumenal and cellular enzymatic degradation, rapid clearance, and chemical and conformational stability. Pharmaceutical approaches to address these barriers, which have been successful with traditional, small, organic drug molecules, have not readily translated into effective peptide and protein formulations. The success achieved by Sandoz with cyclosporin formulations remains one clear example of what can be achieved, although it is likely that effective oral formulations for peptides and proteins will remain highly compound specific. Although the challenges are significant, the potential therapeutic benefit remains high, particularly with the increasing identification of potential peptide and protein drug candidates emerging from the biotechnology arena. Successful formulations will most likely require a systematic and careful merger of formulation and design delivery systems which maximize the potential for absorption across the epithelial cell layer.

Assessment of a model for measuring drug diffusion through implant-generated fibrous capsule membranes.

Fibrous tissue, which encapsulates subcutaneously implanted silastic, vinyl, polyurethane and Teflon discs in rats, has been isolated, characterized and tested for drug permeability in order to develop an in vitro model for determining the effect of this tissue on drug disposition from implant sites. With all materials, capsule tissue thickness and collagen content (approximately 59%) was consistent from 2 to 4 months after implantation. Silastic implants afforded the most consistent and usable tissue in terms of thickness and lack of vascularity, and these capsule membranes were used for determining the transport of three model compounds in an in vitro diffusion cell model. The rank ordering of permeability through these membranes was estrone (60.2 x 10(-6) cm s-1) > 3-O-methylglucose (18.7 x 10(-6) cm s-1) > dextran of molecular weight 70 000 (5.6 x 10(-6) cm s-1), which is consistent with expectations based on the molecular weights and partitioning behaviour of the model compounds. The results of these studies indicate that implant-generated encapsulating membranes can be successfully isolated and employed to study drug diffusion in an in vitro model, providing a direct assessment of the barrier properties of encapsulating membranes.

Use of everted intestinal rings for in vitro examination of oral absorption potential.

The ability to predict in vivo oral absorption potential based on ex vivo screening in an everted intestinal ring model was examined. In vitro drug accumulation in cross sectional rings of everted rat jejunum was determined with 12 compounds whose in vivo absorptions (as distinct from bioavailabilities) are well characterized. The compounds examined ranged from well- to poorly-absorbed and included compounds absorbed by active and passive mechanisms. The effects of drug concentration, pH, cosolvents, and tissue origin site on drug accumulation were determined. Light microscopic observation indicated that the mucosal tissue remained intact up to 3 h after the intestine was excised. Accumulations of two nonabsorbable markers were also determined as measures of tissue integrity. A strong correlation (slope = 23 pmol/mg of tissue weight per percent oral absorption, r2 = 0.9430 by linear regression analysis) of in vitro uptake into everted rings from a 10 mM drug solution versus the known in vivo bioavailability for each compound was observed. These results indicated that under appropriate conditions, in vitro uptake of drug by the everted intestinal ring model closely paralleled known in vivo bioavailability and was relatively independent of pH, cosolvent, and tissue origin.

Interspecies differences in systemic drug availability following subcutaneous pulsatile administration in cattle, sheep, dogs, and rats.

Rats, dogs, sheep, and cattle were implanted subcutaneously with stainless-steel tissue cages. Bolus injections of cefoxitin and ivermectin were administered to the interiors of the tissue cages 11, 32, and 60 days after implantation to simulate pulsatile drug release from an implanted device. Plasma drug levels were determined for 6 h for cefoxitin and up to 8 days for ivermectin. Tissue cages were retrieved 3 and 6 months after implantation for macroscopic and microscopic examination. In dogs and rats, plasma levels of both drugs following administrations to the tissue cages were significantly lower than those following subcutaneous injection, suggesting that the tissue growth around and in the cages posed a barrier to systemic drug availability in those species. In cattle and sheep, the tissue cages and associated tissue did not inhibit systemic availability of either drug as compared with routine subcutaneous administration.

In vitro and in vivo analysis of the mechanism of absorption enhancement by palmitoylcarnitine.

Long-chain acylcarnitines (12-18 carbon fatty acid esters) dramatically enhance the absorption of hydrophilic drugs across intestinal mucosa without altering the morphology of the epithelium. The mechanism underlying these effects was studied using the colon carcinoma cell line Caco-2. Caco-2 monolayers treated with 0.2 mM palmitoylcarnitine (PCC) show dramatic increases in the transport of hydrophilic markers. This enhanced transport coincides with a rapid drop in transepithelial electrical resistance (TER). The drop in TER is initiated within the first minute after PCC addition and continues for approximately 20 min to a 70 to 85% drop of the initial TER values. This effect is reversible after removing the PCC and does not appear to involve lysis of the apical membrane. Instead PCC's effect appears to be due to loosening of tight junctions as indicated by the accumulation of fluorescent dextrans and the electron dense marker lanthanum nitrate in paracellular spaces. Moreover transmission electron microscopy and freeze fracture electron microscopy indicate that PCC produces significant structural alterations to tight junctions. In contrast to many other tight junction disrupting agents, PCC effects appear to be Ca(++)-independent and PCC does not induce significant disruption of actin filament distribution in Caco-2 cells.

Enhanced bioavailability of cefoxitin using palmitoylcarnitine. II. Use of directly compressed tablet formulations in the rat and dog.

The performance of tablets containing the absorption enhancer palmitoylcarnitine chloride (PCC) and the antibiotic cefoxitin (CEF) was determined by direct placement of tablets in the rat stomach, small intestine, and colon. While the bioavailability (F) of tablets containing 12 mg CEF without PCC ranged from 0.6 to 3.9%, the addition of 24 mg PCC resulted in an enhanced CEF bioavailability in the rat colon (mean +/- SD: F = 57 +/- 19%) and rat jejunum (F = 71 +/- 16%) but not in the rat stomach. Following oral administration to dogs, tablets of 200 mg CEF without or with 600 mg PCC resulted in the same low bioavailabilities (7.0 +/- 10.3 and 7.0 +/- 3.6%, respectively). However, when these tablets were enteric coated, PCC improved CEF bioavailability from 2.44 +/- 1.84 to 29.0 +/- 13.4%. Therefore, the use of enteric-coated direct compressed tablets containing PCC and direct compression excipients improved the peroral bioavailability of a poorly absorbed compound.

In vitro effects of long-chain acylcarnitines on the permeability, transepithelial electrical resistance and morphology of rat colonic mucosa.

Absorption-enhancing properties of the long-chain acylcarnitines (C12-C18) were examined in vitro utilizing a modified Sweetana-Grass diffusion cell system. Transepithelial electrical resistance (TEER) and drug permeability (P) of rat colonic mucosa were used as parameters to determine the potency, selectivity and reversibility of acylcarnitine-enhancing effects. All long-chain acylcarnitines induced a rapid, concentration-dependent decrease in mucosal TEER. The minimum or threshold concentration (Ct) which produced a rapid decrease in mucosal TEER was determined for each acylcarnitine. Tissues treated with either palmitoyl or lauroyl carnitine at their Ct showed significantly better recovery of TEER after removal of the enhancers from the diffusion cells as compared to the other long-chain acylcarnitines. In addition, palmitoyl and lauroyl carnitine treatment significantly enhanced the mucosal permeability to small, hydrophilic markers without adversely altering tissue morphology. Following enhancer replacement with buffer alone and the subsequent recovery of TEER, mucosal permeability was observed to return to near control levels. At higher concentrations of palmitoyl and lauroyl (5 x Ct), P increased nearly 2-fold over that observed at the Ct; however, epithelial barrier morphology was compromised. This study shows that colonic mucosal tissue mounted in modified Sweetana-Grass diffusion cells may be a useful model for examining the mechanism(s) of absorption enhancer activity. Moreover, the results present evidence that the long-chain acylcarnitines may enhance drug absorption via two different concentration-dependent pathways.

Enhanced bioavailability of cefoxitin using palmitoyl L-carnitine. I. Enhancer activity in different intestinal regions.

The conditions under which the absorption enhancer palmitoyl L-carnitine chloride (PCC) improved the bioavailability of the poorly absorbed antibiotic cefoxitin throughout the rat intestine has been studied. Cefoxitin alone was appreciably absorbed only in the duodenum (31% vs less than 7% elsewhere). PCC solutions (3 mg/rat, pH 4.0) enhanced cefoxitin bioavailability (F) by 0-, 22-, 16-, and greater than 32-fold in the duodenum, jejunum, ileum, and colon regions, respectively. The inability of PCC to improve F in the duodenum could not likely be attributed to enzymatic degradation of the enhancer, since coadministration with protease and esterase inhibitors produced similar results (F = 30%). Coadministration of PCC solution with cefoxitin in the unligated or ligated colon, increased F to 33 and 76%, respectively. Qualitatively similar results were seen with PCC suspensions (3 mg/rat, pH 6.0). Maintaining a high concentration of cefoxitin and PCC in a restricted region (i.e., by ligating a 2- to 3-cm section of the colon) afforded a two- to threefold advantage over an unligated colon section. The difference in cefoxitin bioavailability between ligated and unligated colon was probably due to sample spreading and subsequent/simultaneous dilution.

Enhancement of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor efficacy through administration of a controlled-porosity osmotic pump dosage form.

An extended-release osmotic dosage form was designed for gastrointestinal delivery of the water-soluble tromethamine salt of the beta-hydroxyacid form of simvastatin, a potent HMG-CoA reductase inhibitor and cholesterol lowering agent. The cholesterol lowering efficacy and systemic plasma drug levels resulting from peroral administration of this dosage form, relative to a powder-filled capsule oral bolus, were evaluated in dogs. A twofold improvement in cholesterol lowering efficacy was realized with the controlled-release dosage form that was accompanied by a drug AUC and Cmax that were 67 and 16%, respectively, of those achieved with the bolus dosage form. These results suggest that extended-release dosage forms have the potential for a dose-sparing advantage in the administration of HMG-CoA reductase inhibitors for the treatment of hypercholesterolemia.

A comparison of oral and rectal absorption of L-dopa esters in rats and mice.

Short-chain alkyl esters of L-dopa were administered to rats and mice via oral and rectal routes. Plasma L-dopa esters and L-dopa were determined in the systemic and portal circulation by HPLC. A comparison of isopropyl, butyl, and 4-hydroxybutyl esters of L-dopa demonstrated significantly higher levels of the esters in both systemic and portal blood samples following rectal administration than following oral administration. In most cases, oral administration resulted in nondetectable (less than 0.01 micrograms/ml) levels of the esters in plasma. Correspondingly, the plasma levels of L-dopa itself were consistently higher following rectal administration. At very high oral doses (500 mg L-dopa equivalents/kg body weight), systemic plasma levels of the butyl ester could be detected (1.25 micrograms/ml at 10 min), which might indicate saturation of the esterase activity of the small intestine. These studies indicate that the systemic availability of L-dopa from short-chain alkyl esters of L-dopa may be best optimized by rectal administration, which avoids the relatively high esterase activity characteristic of the small intestine.