Mikio Tomita

Enhancement of Colonic Drug Absorption by the Transcellular Permeation Route

The effects of sodium caprate and sodium caprylate on transcellular permeation routes were examined in rats. The release of membrane phospholipids was significantly increased only by caprate, while protein release did not change from the control in the presence of caprate or caprylate, indicating that the extent of membrane disruption was insufficient to account for enhanced permeation. Using brush border membrane (BBM) vesicles prepared from colon, with their protein and lipid component labeled by fluorescent probes, the perturbing actions of caprate and caprylate toward the membrane were examined by fluorescence polarization. Caprate interacted with membrane protein and lipids, and caprylate mainly with protein, causing perturbation to the membrane. The release of 5(6)-carboxyfluorescein previously included in BBM vesicles was increased by caprate but not by caprylate. These results suggest that caprate enhances permeability via the transcellular route through membrane perturbation.

Physiological mechanism for enhancement of paracellular drug transport.

We examined the action mechanisms of enhancers that improve paracellular drug transport. For sodium caprate (C10), the increase in the intracellular calcium level was considered to induce the contraction of calmodulin-dependent actin filaments, followed by dilation of the paracellular pathway. Although decanoylcarnitine (DC) also increased the intracellular calcium level, the action was independent of calmodulin and thus, the action mechanism of acylcarnitines was considered to differ from that of C10. Other acylcarnitines, lauroylcarnitine (LC) and palmitoylcarnitine (PC) and organic acids, tartaric acid (TA) and citric acid (CA) decreased the intracellular ATP level and the intracellular pH. From these results, it was considered that one of the action mechanism of acylcarnitines and organic acids is that the intracellular acidosis increases the calcium level through the decrease in ATP levels, followed by opening the tight junction. Membrane dysfunction which was expected from the above mechanism was assessed by the transport function of electrolytes. Membrane conductance, which was increased by C10, LC and PC, returned to the control value during a 3- to 6-h recovery period. On the other hand, Cl(-) ion secretion, which was obtained from short-circuit current (I(sc)), was decreased by these enhancers, but was normalized by C10 but not by LC and PC. Accordingly, C10 can be considered a safer enhancer than acylcarnitines.

Role of Paracellular Pathway in Nonelectrolyte Permeation Across Rat Colon Epithelium Enhanced by Sodium Caprate and Sodium Caprylate

The enhancing effects of 0.25% sodium caprate (C10) and sodium caprylate (C8) on the paracellular permeation of seven water-soluble nonelectrolytes (inulin, polyethylene glycol 900, mannitol, erythritol, glycerol, thiourea, and urea) across the isolated rat colonic epithelium were examined using the Ussing-type chamber technique. The paracellular changes were also measured by impedance analysis. In both the presence and the absence of enhancers, the permeation clearances (Pm) for inulin (12–15 Å in molecular radius) to erythritol (3.2 Å) increased linearly with the increase in their free diffusion coefficients (Dfr), showing the existence of a paracellular shunt pathway unrestricted to any molecular size. Glycerol (2.9 Å), thiourea (2.6 Å), and urea (2.3 Å) had higher clearances than the expected linear values, showing the existence of a restricted paracellular or transcellular pathway. Both C10 and C8 increased the permeabilities in the two pathways, but C10 was more effective than C8. The increase in the permeabilities via the shunt pathway caused by the enhancers was greater than that via the restricted pathway, and thus, the two-phase pattern in the relationship of Pm and Dfr was similar to that in the absence of enhancers. The transcellular permeabilities for urea and thiourea, which were obtained from the efflux experiments, were increased by the enhancers. However, the relative increase caused by C10 was smaller than that of the paracellular-restricted permeabilities. The paracellular changes probably were due to the increase in pore area per unit diffusive path length. A decrease in the resistance of the intercellular junctions involving a simultaneous increase of membrane capacitance was observed in the presence of C10, corresponding to an increase of pore area per unit path length. The effect of C10 on the paracellular permeability was reversible, and the junctional resistance, membrane capacitance, and Pm of mannitol returned to the control level following the removal of C10.

Absorption-Enhancing Effects of Sodium Caprate and Palmitoyl Carnitine in Rat and Human Colons

We examined the enhancing action of sodiumcaprate and palmitoylcarnitine on the permeability offluorescein isothiocyanate dextran 4000 as aparacellular permeant compound in isolated rat and humancolon samples using the Ussing-type chamber method.In the absence of an enhancer, the permeation clearanceof fluorescein isothiocyanate dextran 4000 was notsignificantly different in the rat and human colons, but the electric membrane resistance wassmaller in the rat colon than in the human colon. Sodiumcaprate and palmitoylcarnitine increased permeationclearance and decreased electric membrane resistance in both types of colonic membrane, showing thatthe rat colon can be used as a model of the human colonfor studies of enhancer effects. A calmodulin antagonistsignificantly inhibited the action of sodium caprate in both colonic membranes. However, ittended to promote the effects of palmitoylcarnitine onpermeation clearance and electric membrane resistance.These results suggest that sodium caprate induces the contraction of the perijunctionalactomyosin ring to widen the tight junction and that themechanism of palmitoylcarnitine is different from thatof sodium caprate in the human colon, as reportedpreviously for Caco-2 cell monolayers.

Differences in the enhancing effects of sodium caprate on colonic and jejunal drug absorption.

We examined the enhancing effect of sodium caprate (C10) on the jejunal absorption of a poorly absorbed drug, cefmetazole, in rats, in comparison with its colonic absorption (Pharm. Res. 5, 341–346, 1988). Jejunal absorption was significantly enhanced by C10, but to a smaller extent than colonic absorption. Membrane perturbation, caused by the interaction between C10 and membrane proteins or lipids, was shown to increase transcellular drug permeability, as reported in the colon. Paracellular permeabilities, obtained from the permeabilities of water-soluble nonelectrolytes of various molecular weights, showed a two-phase pattern against their free diffusion coefficients, suggesting the existence of at least two pore routes similar to those in the colon. C10 increased paracellular permeability in the colon but not in the jejunum. Impedance analysis and voltage clamp technique in the jejunum showed no significant effect of C10 on paracellular permeability, such as found in the colon. Accordingly, the difference in the effects of C10 on the jejunal and colonic absorption of cefmetazole was due mainly to the difference in its effects on the paracellular pathway.

Transcellular and paracellular contribution to transport processes in the colorectal route

Abstract The two permeation pathways for colorectal drug absorption, the transcellular and paracellular pathways, were examined. The transcellular pathway is generally a principal route for drugs with some degree of lipophilicity. The contribution of the paracellular pathway to drug absorption is significant when absorption enhancers such as capric acid (C10) and decanoylcarnitine (DC) are used. The action mechanism of C10 on the transcellular pathway was examined by membrane perturbation. The following in vitro effects of C10 on the paracellular pathway were observed: (i) an increase in the equivalent pore radius; (ii) an increase in the permeabilities of water-soluble non-electrolytes and ionic drugs; (iii) a decrease in the junctional resistance and an increase in the membrane capacitance. The action mechanism of C10 in the paracellular pathway was especially elucidated by stimulation to the contraction of the perijunctional actomyosin ring. Finally, an in vivo C10 effect was also observed, as an enhancement of rectal drug absorption from suppositories in rats.

Effects of nitric oxide on mucosal barrier dysfunction during early phase of intestinal ischemia/reperfusion

Ischemia/reperfusion (I/R) injury must be overcome for successful small intestinal transplantation. During intestinal I/R, the expression level of nitric oxide (NO) is increased, and vermiculation of the mucosal tract is induced by NO. Although NO has many beneficial effects on intestinal I/R injury, its role in intestinal I/R injury is controversial. Therefore, in the present study, we examined changes in the tight junctions (TJ) and P-glycoprotein (P-gp) by aminoguanidine (AG), which can be considered a selective inducible NO synthase inhibitor during intestinal I/R, to clarify the effect of NO on mucosal barrier dysfunction during intestinal I/R. A mucosal lesion was induced by intestinal I/R. The protein expression levels of the claudin family organizing TJ and P-gp, were decreased, and their functions were also decreased. Through the inhibition of NO generation by AG in the above mucosal lesion, TJ and P-gp dysfunction was significantly inhibited. NO participated in opening TJ and decreasing P-gp function and expression induced during intestinal I/R. Therefore, it is important to consider the level of NO generation in the ileal mucosa in drug therapy for intestinal I/R injury.

Effect of intestinal ischaemia/reperfusion on P-glycoprotein-mediated ileal excretion of rhodamine 123 in the rat

Objectives We have shown that ischaemia/reperfusion in the small intestine at an early phase, such as 1 h after reperfusion, induced not only functional changes in the membrane, such as P‐glycoprotein (P‐gp) dysfunction, but also decreased expression of P‐gp protein and mdr1a mRNA. In the present study we examined whether intestinal ischaemia/reperfusion modifies the P‐gp‐mediated ileal excretion transport system in rats beyond 1 h after reperfusion.

Nonlinear absorption of methylprednisolone by absorptive and secretory transporters.

The intestinal absorption rate constant of methylprednisolone (MP) evaluated by the loop method increased significantly with increasingly higher concentrations of the drug up to 500 microM in a nonlinear fashion but did not increase further at higher concentrations. Mucosal-to-serosal directed permeation of MP across rat jejunal sheets also increased in a nonlinear fashion in a low concentration range (100-150 microM), followed by a decrease as the concentration increased further, whereas serosal-to-mucosal directed permeation decreased in a concentration-dependent manner. Vectorial transport of MP across Caco-2 cell monolayers was observed, with greater transport in the basolateral-to-apical direction at 37 degrees C. These observations suggest that MP is taken up in the intestinal epithelial cells by a carrier-mediated transport mechanism. The absorptive and secretory clearance of MP increased and decreased with P-glycoprotein (P-gp) inhibitors, respectively. These results strongly suggest that MP is secreted into the intestinal lumen predominantly by P-gp. We conclude that intestinal transport of MP involves P-gp or some other transporters in both the absorptive and secretory directions, and complex nonlinear intestinal absorption characteristics can be ascribed to the existence of multiple transport mechanisms.

RETRACTED: Suppression of efflux transporters in the intestines of endotoxin-treated rats

Infection and inflammation suppress the expression and activity of several drug transporters in the liver. In the intestine, P-glycoprotein (PGP/mdr1) and the multidrug resistance-associated protein 2 (MRP2) are important barriers to the absorption of many clinically important drugs. The protein expression and activity of these transporters were examined during inflammation induced by lipopolysaccharide (LPS). The transport of rhodamine123 (Rho123) and 5-carboxyfluorescein (5-CF) was determined in isolated ileal segments from endotoxin-treated or control rats in the presence or absence of inhibitors. The reverse transcription-polymerase chain reaction was used to measure mRNA levels. Compared with the controls, the mRNA levels of mdr1a and mrp2 were significantly decreased by approximately 50% in the ilea of the LPS-treated rats. Corresponding reductions in the basolateral-apical efflux of Rho123 and 5-CF were observed, resulting in significant increases in the apical-basolateral absorption of these compounds. Neither the permeability of fluorescein isothiocyanate labeled dextran 4000 (FD-4), a paracellular marker, nor membrane resistance was altered. These results indicate that endotoxin-induced inflammation reduces the intestinal expression and activity of PGP and MRP2 in rats, which eliciting corresponding changes in the intestinal transport of their substrates. Hence, infection and inflammatory diseases may induce variability in drug bioavailability through alterations in the intestinal expression and activity of drug transporters.