Risperidone and paliperidone inhibit p-glycoprotein activity in vitro.

Risperidone (RSP) and its major active metabolite, 9-hydroxy-risperidone (paliperidone, PALI), are substrates of the drug transporter P-glycoprotein (P-gp). The goal of this study was to examine the in vitro effects of RSP and PALI on P-gp-mediated transport. The intracellular accumulation of rhodamine123 (Rh123) and doxorubicin (DOX) were examined in LLC-PK1/MDR1 cells to evaluate P-gp inhibition by RSP and PALI. Both compounds significantly increased the intracellular accumulation of Rh123 and DOX in a concentration-dependent manner. The IC(50) values of RSP for inhibiting P-gp-mediated transport of Rh123 and DOX were 63.26 and 15.78 microM, respectively, whereas the IC(50) values of PALI were >100 microM, indicating that PALI is a less potent P-gp inhibitor. Caco-2 and primary cultured rat brain microvessel endothelial cells (RBMECs) were utilized to investigate the possible influence of RSP on intestinal absorption and blood-brain barrier (BBB) transport of coadministered drugs that are P-gp substrates. RSP, 1-50 microM, significantly enhanced the intracellular accumulation of Rh123 in Caco-2 cells by inhibiting P-gp activity with an IC(50) value of 5.87 microM. Following exposure to 10 microM RSP, the apparent permeability coefficient of Rh123 across Caco-2 and RBMECs monolayers was increased to 2.02 and 2.63-fold in the apical to basolateral direction, but decreased to 0.37 and 0.21-fold in the basolateral to apical direction, respectively. These data suggest that RSP and PALI, to a lesser extent, have a potential to influence the pharmacokinetics and hence the pharmacodynamics of coadministered drugs via inhibition of P-gp-mediated transport. However, no human data exist that address this issue. In particular, RSP may interact with its own active metabolite PALI by promoting its brain concentration through inhibiting P-gp-mediated efflux of PALI across endothelial cells of the BBB.     

Secretory transport of p-aminohippuric acid across intestinal epithelial cells in Caco-2 cells and isolated intestinal tissue

The intestinal transport of an organic anion, p-aminohippuric acid (PAH), was studied in Caco-2 cell monolayers and rat intestinal tissue mounted in Ussing chambers. In both experimental methods, PAH exhibited vectorial transport with significantly greater permeability in the secretory direction than the absorptive direction, indicating net secretion. This secretory transport required metabolic energy, but protons or hydroxyl ions were not involved as the driving force. In Caco-2 monolayers, secretory transport of [3H]PAH was decreased, and the intracellular accumulation of PAH was increased with increasing concentration of unlabelled PAH at the basolateral side. Addition of probenecid and genistein at the basolateral side decreased the secretory transport of [3H]PAH; the accumulation was not changed by probenecid, but was increased by genistein. In addition, the initial uptake rate of [3H]PAH from the basolateral side was decreased by both PAH and probenecid, but not by genistein. Therefore, it is suggested that the transport of PAH in Caco-2 cells is regulated by several transporters: a genistein-sensitive transporter on the apical membrane and probenecid-sensitive transporters on both the basolateral and apical membranes. In rat intestinal tissues, the transport rate of PAH showed regional variation (ileum > jejunum > duodenum), suggesting that secretory transporters with high activity exist predominantly in the lower region of the small intestine. The results suggest that PAH transport in both Caco-2 cells and rat intestinal tissues is regulated by multiple transporters on the apical and basolateral membranes, and these transporters have different characteristics.     

P-glycoprotein interactions of nefazodone and trazodone in cell culture.

This study investigated the effects of nefazodone (NFZ) and trazodone (TZD) on P-glycoprotein (P-gp) activity and expression in cell culture. NFZ and TZD showed no differential transport between the basolateral to apical and apical to basolateral direction across Caco-2 cell monolayers. Transport in either direction was not affected by verapamil. NFZ was a potent inhibitor (IC50 = 4.7 microM) of rhodamine123 (Rh123) B to A transport across Caco-2 cell monolayers, while TZD had minimal effect. Following 72-hour exposure of LS180V cells to NFZ and TZD (10 microM), a twofold increase in immunoreactive P-gp was observed. Rh123 accumulation into these cells was reduced to 65% and 74% of control by NFZ and TZD (10 microM), respectively. It was concluded that differential rates of transport of NFZ and TZD in Caco-2 cells were not evident. However, NFZ is an inhibitor of P-gp activity at clinically relevant in vivo concentrations and may have the potential to increase bioavailability of coadministered compounds that are substrates for transport. Concentrations of NFZ and TZD achieved in the intestine after chronic oral dosing may induce P-gp expression and reduce absorption of coadministered drugs.     

Transport characteristics of rutin deca (H-) sulfonate sodium across Caco-2 cell monolayers

The aim of this study was to explore potential transport mechanisms of rutin deca (H-) sulfonate sodium (RDS) across Caco-2 cell monolayers. As an in-vitro model of human intestinal epithelial membrane, Caco-2 cells were utilized to evaluate the transepithelial transport characteristics of this hydrophilic macromolecular compound. Bi-directional transport study of RDS demonstrated that the apparent permeability (P(app)) in the secretory direction was 1.4 approximately 4.5-fold greater than the corresponding absorptive P(app) at concentrations in the range 50.0 approximately 2,000 microM. The transport of RDS was shown to be concentration, temperature and pH dependent. In the presence of ciclosporin and verapamil, potent inhibitors of P-glycoprotein (P-gp)/MRP2, the absorptive transport was enhanced and secretory efflux was diminished. RDS significantly reduced the efflux ratio of the P-gp substrate rhodamine-123 in a fashion indicative of P-gp activity suppression, while rhodamine-123 competitively inhibited the polarized transport of the compound. In conclusion, the results indicated that RDS was likely a substrate of P-gp. Several efflux transporters, including P-gp, participated in the absorption and efflux of RDS and they might play significant roles in limiting the oral absorption of the compound. These observations offered important information for the pharmacokinetics of RDS.     

Methadone inhibits rhodamine123 transport in Caco-2 cells.

This study investigated the effects of racemic methadone (MET) on P-glycoprotein (P-gp) activity in cell culture. MET showed no differential rates of passage between the basolateral to apical (B to A) and apical to basolateral (A to B) direction across Caco-2 cell monolayers in a transwell system. MET transport in either direction was not importantly influenced by the P-gp inhibitor verapamil. However, MET was a potent inhibitor (IC(50) = 7.5 microM) of rhodamine123 B to A transport across Caco-2 cell monolayers, causing a reduction to 25% of control at 100 microM MET. In this model of Caco-2 monolayers, rates of MET passage between B to A and A to B directions could not be distinguished. However, MET can inhibit P-gp activity at intraluminal concentrations that might be achieved clinically. This may lead to increased bioavailability of coadministered compounds.     

Functional assessment of multiple P-glycoprotein (P-gp) probe substrates: influence of cell line and modulator concentration on P-gp activity.

Compounds known to modulate P-glycoprotein (P-gp) activity were evaluated in cell monolayers expressing P-gp for their effects on the secretory transport of P-gp substrates paclitaxel, vinblastine, and digoxin. Paclitaxel has been proposed to selectively interact with a binding site on P-gp that is distinct from the vinblastine and digoxin-binding site. Using Madin-Darby canine kidney (MDCK)-multidrug resistance-1 (MDR1), MDCK-wild-type (WT), and Caco-2 cell monolayers, the basal-to-apical (BL-AP) apparent permeability (Papp) of [3H]paclitaxel, [3H]vinblastine, and [3H]digoxin in the presence of various concentrations of a series of structurally diverse P-gp substrates and modulators of P-gp function were determined. MDCK-WT cell monolayers demonstrated active secretory transport of all P-gp substrate probes, although the sensitivity to inhibition by verapamil was lower than that demonstrated in MDCK-MDR1 cell monolayers. When evaluated as competitive inhibitors, several known P-gp substrates had no effect or only a slight modulatory effect on the BL-AP Papp of all probe substrates in MDCK-MDR1 cells. The secretory transport of P-gp substrates in MDCK-WT cells was more sensitive to inhibition by known P-gp modulators compared with MDCK-MDR1 cells. Low concentrations of ketoconazole (1-3 microM) activated the BL-AP Papp of [3H]vinblastine and [3H]digoxin in MDCK-MDR1 cells but not in MDCK-WT or Caco-2 cells. Determination of secretory transport in P-gp expressing cell monolayers, such as MDCK-MDR1 and Caco-2, may be complicated by substrate cooperativity and allosteric binding, which may result in the activation of P-gp. In addition, expression of other efflux transporters in these cell lines introduces additional complexity in distinguishing which transporter is responsible for substrate recognition and transport.     

Circumventing P-glycoprotein-mediated cellular efflux of quinidine by prodrug derivatization

The objective of this study is to investigate whether transporter-targeted prodrug derivatization of quinidine, a model P-glycoprotein (P-gp) substrate, can circumvent P-gp-mediated efflux. The L-valine ester of quinidine (val-quinidine) was synthesized in our laboratory. Uptake and transport studies were carried out using the MDCKII-MDRI cell line at 37 degrees C for 10 min and 3 h, respectively. [3H]Ritonavir and cyclosporine were also used as model P-gp substrates to delineate the kinetics of translocation of val-quinidine across the MDCKII-MDRI cell monolayer. The rate of uptake of [3H]ritonavir by MDCKII-MDRI cells exhibited a 2-fold increase in the presence of 75 microM quinidine, but 75 microM val-quinidine did not demonstrate any effect on [3H]ritonavir uptake. The rate of transport of quinidine from the basolateral to the apical membrane [(18.3 +/- 1.25) x 10(-6) cm s(-1)] and from the apical to the basolateral membrane [(6.5 +/- 0.66) x 10(-6) cm s(-1)] exhibited a 3-fold difference. However, transport of val-quinidine from the apical to the basolateral membrane [(5.13 +/- 0.49) x 10(-6) cm s(-1)] and from the basolateral to the apical membrane [(6.17 +/- 1.28) x 10(-6) cm s(-1)] did not demonstrate any statistically significant difference. Moreover, cyclosporine, a potent P-gp substrate and/or inhibitor, did not alter the transport kinetics of val-quinidine. The rates of uptake of [3H]Gly-Sar and various amino acid model substrates were reduced in the presence of 200 microM val-quinidine. Results from this study clearly indicate that prodrug derivatization of quinidine into val-quinidine can overcome P-gp-mediated efflux. Val-quinidine once bound to a peptide or amino acid transporter is probably not recognized and cannot be accessed by the P-gp efflux pump. Transporter-targeted prodrug derivatization seems to be a viable strategy for overcoming P-gp-mediated efflux.     

Relevance of p-glycoprotein for the enteral absorption of cyclosporin A: in vitro-in vivo correlation.

1. The interaction of cyclosporin A (CyA) with p-glycoprotein during intestinal uptake was investigated by a combination of in vitro experiments with human Caco-2 cells and an intubation study in healthy volunteers. 2. CyA uptake into the cells was not saturable and exhibited only a low temperature sensitivity, suggesting passive diffusion. When the permeation of CyA across Caco-2 monolayers from the apical to the basolateral side was determined, overall transport had an apparently saturable component up to a concentration of 1 microM. At higher concentrations permeation increased over-proportionally. Calculation of the kinetic parameters of apical to basolateral permeation suggested a diffusional process with a KD of 0.5 microliter min-1 per filter, which was overlayed by an active system in basolateral to apical direction with a KM of 3.8 microM and a Jmax of 6.5 picomol min-1 per filter. 3. CyA permeation was significantly higher when the drug was given from the basolateral side as compared to the permeation from the apical side. Apical to basolateral transport of CyA was increased in the presence of vinblastine, daunomycin and a non-immunosuppressive CyA-derivative. All compounds inhibit p-glycoprotein-mediated transport processes. Basolateral to apical permeation of CyA showed a dose-dependent decrease in the presence of vinblastine. Permeation of daunomycin across Caco-2 cell monolayers was also higher from the basolateral to the apical side than vice versa. Basolateral to apical permeation was decreased in the presence of SDZ PSC 833 and cyclosporin A. 4. Western blot analysis of Caco-2 cells with the monoclonal antibody C219 confirmed the presence of p-glycoprotein in the used cell system. 5. When the absorption of CyA in the gastrointestinal (GI)-tract of healthy volunteers was determined, a remarkable decrease of the plasma AUC could be observed dependent on the location of absorption in the rank order stomach > jejunum/ileum > colon. The decrease in absorption exhibited a marked correlation (r = 0.994) to the expression of mRNA for p-glycoprotein over the GI-tract (stomach < jejunum < colon). 6. All data provide evidence that CyA is a substrate of p-glycoprotein in the GI-tract, which might explain the local differences and the high variability in cyclosporin absorption found in vivo.     

Transport characteristics of 9-nitrocamptothecin in the human intestinal cell line Caco-2 and everted gut sacs

The intestinal absorptive characteristics and the efflux mechanisms of 9-nitrocamptothecin (9-NC), a novel water-insoluble camptothecin (CPT) derivative, were investigated. The Caco-2 cells and the everted gut sacs were used as models of the intestinal mucosa to assess transepithelial transport of 9-NC. The determination of 9-NC was performed by HPLC. In the Caco-2 cells, the absorptive transport of 9-NC was pH dependent and the transport was enhanced at weakly acidic pH on the apical side. No concentration dependence and saturation were observed for the absorptive transport of 9-NC at concentrations up to 250 microM, while secretory transport were concentration dependent and saturable process (K(m) was 49.8 +/- 1.2 microM, V(max) was 38.28 +/- 0.8 ng/cm(2)/min). In the presence of verapamil (100 microM) and CsA (10 microM), potent inhibitors of P-glyprotein (P-gp)/MRP2 (cMOAT), the P(appBL-AP)/P(appAP-BL) ratio was decreased from 3.4 to 1.4 and 1.3, respectively, and permeation of apical to basolateral was enhanced approximately two-fold. In the everted gut sacs, the absorption of 9-NC was passive diffusion and had no significant difference in different gut regions. Adding verapamil in the everted gut sacs over a concentration ranging from 10 to 100 microM, the absorption of 9-NC was significantly enhanced, especially more markedly in lower small intestine (P < 0.05). Overall, the current study suggests that pH and efflux transporters are capable of mediating the absorption and efflux of 9-NC, and they may play significant roles in limiting the oral absorption of 9-NC.     

The apical uptake transporter of levofloxacin is distinct from the peptide transporter in human intestinal epithelial Caco-2 cells

The aim of this study was to investigate the involvement of the peptide transporter for absorption of levofloxacin in Caco-2 cells. To evaluate the activity of apical and basolateral peptide transport, we first performed pharmacokinetic analysis of transcellular transport of glycylsarcosine (Gly-Sar) in cell monolayers grown on porous membrane filters. Transcellular transport of Gly-Sar at the medium pH 6 was greater in the apical-to-basolateral direction than in the opposite direction. Influx clearance of Gly-Sar at the apical membrane was much greater than basolateral influx and efflux clearance, indicating that the apical peptide transporter plays an important role in directional transcellular transport of the dipeptide across Caco-2 cell monolayers. We then evaluated the effect of various compounds on the uptake of Gly-Sar and levofloxacin at the apical membrane of Caco-2 cells. The apical uptake of [3H]Gly-Sar was significantly inhibited by Ala-Ala, Gly-Sar, and also levofloxacin, whereas that of [14C]levofloxacin was not inhibited by Ala-Ala and Gly-Sar. On the other hand, the apical uptake of [14C]levofloxacin was inhibited by nicotine, enalapril, fexofenadine, and L-carnitine. These findings indicated that the apical uptake transporter of levofloxacin is distinct from the peptide transporter in Caco-2 cells.     

Vectorial transport of fexofenadine across Caco-2 cells: involvement of apical uptake and basolateral efflux transporters

Fexofenadine is a nonsedative antihistamine that exhibits good oral bioavailability despite its zwitterionic chemical structure and efflux by P-gp. Evidence exists that multiple uptake and efflux transporters play a role in hepatic disposition of fexofenadine. However, the roles of specific transporters and their interrelationship in intestinal absorption of this drug are unclear. This study was designed to elucidate vectorial absorptive transport of fexofenadine across Caco-2 cells involving specific apical uptake and efflux transporters as well as basolateral efflux transporters. Studies with cellular models expressing single transporters showed that OATP2B1 expression stimulated uptake of fexofenadine at pH 6.0. Apical uptake of fexofenadine into Caco-2 cells was decreased by 45% by pretreatment with estrone 3-sulfate, an OATP inhibitor, at pH 6.0 but not at pH 7.4, indicating that OATP2B1 mediates apical uptake of fexofenadine into these cells. Examination of fexofenadine efflux from preloaded Caco-2 cells in the presence or absence of (i) the MRP inhibitor MK-571 and (ii) the P-gp inhibitor GW918 showed that apical efflux is predominantly mediated by P-gp, with a small contribution by MRP2, whereas basolateral efflux is predominantly mediated by MRP3. These results also showed that while OSTαβ is functionally active in the basolateral membrane of Caco-2 cells, it does not play a role in the export of fexofenadine. MK-571 decreased the absorptive transport of fexofenadine by 17%. However, the decrease in absorptive transport by MK-571 was 42% when P-gp was inhibited by GW918. The results provide a novel insight into a vectorial transport system mainly consisting of apical OATP2B1 and basolateral MRP3 that may play an important role in delivering hydrophilic anionic and zwitterionic drugs such as pravastatin and fexofenadine into systemic circulation upon oral administration.     

P-glycoprotein-mediated transport of morphine in brain capillary endothelial cells.

Cell accumulation, transendothelial permeability, and efflux studies were conducted in bovine brain capillary endothelial cells (BBCECs) to assess the role of P-glycoprotein (P-gp) in the blood-brain barrier (BBB) transport of morphine in the presence and absence of P-gp inhibitors. Cellular accumulation of morphine and rhodamine 123 was enhanced by the addition of the P-gp inhibitors N-{4-[2-(1,2,3,4-tetrahydro-6,7dimethoxy-2-isoquinolinyl)-ethyl]-phenyl}-9,10-dihydro-5-methoxy-9- carboxamide (GF120918), verapamil, and cyclosporin A. Positive (rhodamine 123) and negative (sucrose and propranolol) controls for P-gp transport also were assessed. Morphine glucuronidation was not detected, and no alterations in the accumulation of propranolol or sucrose were observed. Transendothelial permeability studies of morphine and rhodamine 123 demonstrated vectorial transport. The basolateral to apical (B:A) fluxes of morphine (50 microM) and rhodamine (1 microM) were approximately 50 and 100% higher than the fluxes from the apical to the basolateral direction (A:B), respectively. Decreasing the extracellular concentration of morphine to 0.1 microM resulted in a 120% difference between the B:A and A:B permeabilities. The addition of GF120918 abolished any significant directionality in transport rates across the endothelial cells. Efflux studies showed that the loss of morphine from BBCECs was temperature- and energy-dependent and was reduced in the presence of P-gp inhibitors. These observations indicate that morphine is transported by P-gp out of the brain capillary endothelium and that the BBB permeability of morphine may be altered in the presence of P-gp inhibitors.     

Transport Characteristics of Fexofenadine in the Caco-2 Cell Model

PURPOSE: To investigate the membrane transport mechanisms of fexofenadine in the Caco-2 model. METHODS: Transport studies were performed in Caco-2 cell monolayers 21-25 days after seeding. The apparent permeability (Papp) of fexofenadine was determined in the concentration range 10-1000 microM in the basolateral-to-apical (b-a) and 50-1000 microM in the apical-to-basolateral (a-b) direction. The concentration-dependent effects of various inhibitors of P-glycoprotein (P-gp) (GF120918, ketoconazole, verapamil, erythromycin), multidrug resistant associated protein (MRP) (indomethacin, probenecid), and organic anion transporting polypeptide (OATP) (rifamycin SV) on the bidirectional transport of 150 microM fexofenadine were also examined. RESULTS: Fexofenadine displayed polarized transport, with the Pappb-a being 28- to 85-fold higher than the Papp(a-b). The Papp(a-b) was independent of the concentration applied, whereas Pappb-a decreased with increasing concentration (Vmax = 5.21 nmol cm(-2)s(-1) and K(M) = 150 microM), suggesting saturation of an apical efflux transporter. All four P-gp inhibitors had a strong, concentration-dependent effect on the Papp of fexofenadine in both directions, with GF 120918 being the most specific among them. The IC50 of verapamil was 8.44 microM on the P-gp-mediated secretion of fexofenadine. The inhibitors of OATP or MRP appeared not to affect the Papp(a-b) of fexofenadine in the Caco-2 model. CONCLUSIONS: This study clearly indicates that P-gp was the main transport protein of fexofenadine in the Caco-2 model. Even though P-gp was completely inhibited, fexofenadine was predicted to have a low fraction dose absorbed in humans due to poor intestinal permeability, and low passive diffusion seems to be the major absorption mechanism.     

Absorption of fumonisin B1 and aminopentol on an in vitro model of intestinal epithelium; the role of P-glycoprotein.

The aim of the present paper is to evaluate the absorption of fumonisin B1 and its principal metabolite, aminopentol on a human intestinal model, Caco-2 cells, cultured on semi-permeable inserts, that reproduces the two different intestinal compartments: luminal (apical) and serosal (basolateral) side. Following separate exposure in apical and in basolateral compartments, aminopentol passage through the cell layer (in particular from basolateral to apical direction) was shown, while it was not observed for the parent compound. The different aminopentol distribution between the two compartments of the culture system, and its variation in presence of verapamil or probenecid (P-gp and MRP inhibitors respectively), strongly suggests the involvement of P-glycoprotein in the influx/efflux mechanisms of aminopentol in the intestinal cells, reducing its oral bioavailability.     

Phorbol 12-myristate 13-acetate inhibits P-glycoprotein-mediated efflux of digoxin in MDCKII- MDR1 and Caco.2 cell monolayer models

Aim： To investigate the effects of phorbol 12-myristate 13-acetate （PMA）, a PKC activator, on P-glycoprotein-mediated efflux of digoxin in two cell transport models. Methods： Caco-2 cells, wild MDCKII cells （MDCKII-WT） and MDCKII cells transfected stably with human MDRl-gene encoding P-gp （MDCKII-MDR1） were examined. Cell viability was evaluated with MTI- assay. Bidirectional transport of digoxin was evaluated in these cells. Intracellular ATP level was measured using ATP assay. P-gp ATPase activity was analyzed using a Pgp-GIoTM assay. Results： PMA （10 pmol/L） did not reduce the viability of the 3 types of cells. In Caco-2 and MDCKII-MDR1 cell monolayers, PMA （1, 10 and 100 nmol/L） dose-dependently inhibited the basolateral to apical transport of digoxin, but did not change the apical to basolatera transport. In addition, PMA did not affect both the basolateral to apical and apical to basolateral transport of digoxin in MDCKII-WT ce monolayer. In agreement with the above results, PMA dose-dependently reduced intracellular ATP level and stimulated P-gp ATPase activity in both Caco-2 and MDCKII-MDR1 cells. Verapamil （a positive control, 100 pmol/L） caused similar inhibition on digoxin efflux as PMA did, whereas 4c（-PMA （a negative control, 100 nmol/L） had no effect. Conclusion： PMA significantly inhibited P-gp-mediated efflux of digoxin in both Caco-2 and MDCKII-MDR1 cell monolayers via PKC activation.     

Intestinal epithelial cell accumulation of the cancer preventive polyphenol ellagic acid--extensive binding to protein and DNA

Ellagic acid (EA), a polyphenol present in many berries, has been demonstrated to be preventive of esophageal cancer in animals both at the initiation and promotion stages. To be able to extrapolate these findings to humans we have studied the transcellular absorption and epithelial cell accumulation of [14C]EA in the human intestinal Caco-2 cells. The apical (mucosal) to basolateral (serosal) transcellular transport of 10 microM [14C]EA was minimal with a P(app) of only 0.13 x 10(-6)cm/s, which is less than for the paracellular transport marker mannitol. In spite of observations of basolateral to apical efflux, Caco-2 cell uptake studies showed high accumulation of EA in the cells (1054+/-136 pmol/mg protein), indicating facile absorptive transport across the apical membrane. Surprisingly, as much as 93% of the cellular EA was irreversibly bound to macromolecules (982+/-151 pmol/mg protein). To confirm the irreversible nature of the binding to protein, Caco-2 cells treated with 10 microM [14C]EA were subjected to SDS-PAGE analysis. This resulted in radiolabeled protein bands trapped in the stacking gel, consistent with [14C]EA-crosslinked proteins. Treatment of Caco-2 cells with 10 microM [14C]EA also revealed irreversible binding of EA to cellular DNA as much as five times higher than for protein (5020+/-773 pmol/mg DNA). Whereas the irreversible binding to protein required oxidation of EA by reactive oxygen species, this did not seem to be the case with the DNA binding. The avid irreversible binding to cellular DNA and protein may be the reason for its highly limited transcellular absorption. Thus, EA appears to accumulate selectively in the epithelial cells of the aerodigestive tract, where its cancer preventive actions may be displayed.     

Pharmacokinetic analysis of transcellular transport of levofloxacin across LLC-PK1 and Caco-2 cell monolayers

To characterize the membrane transport responsible for the renal excretion and intestinal absorption of levofloxacin, we performed pharmacokinetic analysis of transcellular transport across LLC-PK(1) and Caco-2 cell monolayers. Transcellular transport of levofloxacin in LLC-PK(1) cells was greater in the basolateral-to-apical direction than in the opposite direction. Pharmacokinetic analysis indicated that basolateral uptake was the direction-determining step for the transcellular transport of levofloxacin in LLC-PK(1) cells. The apical efflux clearance of levofloxacin in LLC-PK(1) cells was increased at the medium pH 6 as compared with at pH 8, suggesting that membrane transport characteristics of levofloxacin are apparently similar to those of a prototypical organic cation, tetraethylammonium. On the other hand, transcellular transport of levofloxacin in Caco-2 cells was only slightly greater in the basolateral-to-apical direction than in the opposite direction. The apical efflux clearance of levofloxacin in Caco-2 cells was greater than basolateral efflux clearance, and apical influx clearance was greater than any other membrane transport clearance. In addition, the apical uptake of levofloxacin as well as quinidine in Caco-2 cells was inhibited significantly by nicotine and imipramine. The findings indicated that some transporters are responsible not only for the efflux but also for the influx of levofloxacin at the apical membrane of Caco-2 cells.     

Intestinal absorption of Stemona alkaloids in a Caco-2 cell model

The intestinal absorption of neotuberostemonine and neostenine, two major bioactive alkaloids of the commonly used antitussive traditional Chinese medicine Stemona tuberosa Lour, was investigated using a Caco-2 monolayer model. Both alkaloids exhibited a high absorptive permeability which was higher for neostenine [P(app(AB)) = 12.03 +/- 1.14 x 10 (-6) cm/s] than for neotuberostemonine [P(app(AB)) = 9.27 +/- 0.79 x 10 (-6) cm/s], indicating that they are likely to be well absorbed and orally active. Furthermore, both alkaloids were identified to be the substrates of P-glycoprotein and have a transport preference from the basolateral to apical direction with efflux ratios between 2 and 3. Cyclosporin A dose-dependently inhibited the secretory permeability of these alkaloids and abolished their active efflux transport.     

Effect of organophosphate pesticide diazinon on expression and activity of intestinal P-glycoprotein

Organophosphate insecticide diazinon is widely used in agricultural practices, submitting farmers to repeated exposure. Because efflux pumps, as P-glycoprotein (P-gp), serve both as natural defense mechanisms and influence the bioavailability and disposition of drugs, we analyzed the ability of diazinon to act as efflux modulator. Oral administration of diazinon (2-20 mg/kg, 5 days, or 10 mg/kg, 2-12 days) increased intestinal mdr1a mRNA of rats, in both dose- and time-dependent manner, and increased the expression of intestinal P-gp. Using the intestinal cell-line Caco-2, we found that 100 microM diazinon significantly inhibited digoxin and vinblastine secretive flux through the cell monolayers, whereas digoxin and vinblastine absorptive flux increased. The 25 microM diazinon was transported preferentially in basolateral (BL) to apical (AP) direction, suggesting a net secretion. The efflux rate significantly decreased in the presence of metabolic inhibitors sodium azide and 2-deoxy-d-glucose, P-gp inhibitors cyclosporin A and valspodar, but not in the presence of MRPs inhibitor MK571. Repeated exposure of Caco-2 cells to diazinon increased P-glycoprotein expression and activity. These results suggested the involvement of P-gp in the transfer of diazinon, leading to potential consequences for xenobiotic interactions, and showed that repeated exposure to low doses of pesticide may lead to up-regulated P-gp functions in the intestine of mammals.