In vitro study on the transport of zinc across intestinal epithelial cells using Caco-2 monolayers and isolated rat intestinal membranes

The variety of physiologic and biologic functions of zinc is fascinating and could be applicable to medicine. Our previous studies demonstrated that the absorption of zinc after oral administration to rats is dose-dependent. In order to clarify the detailed mechanism of the dose-dependent in vivo absorption, the transport of zinc across intestinal epithelial cells was investigated using Caco-2 monolayers and isolated rat intestinal membranes. The permeation of zinc across Caco-2 monolayers is concentration-dependent, and both saturable and nonsaturable components are involved. The Michaelis constant and maximum transport rate for saturable transport are 11.7 μM and 31.8 pmol min(-1) cm(-2), respectively; the permeability coefficient for nonsaturable trasnport is 2.37×10(-6) cm s(-1). These parameters for permeation across membranes isolated from duodenum, ileum, and jejunum of rats are similar with those of Caco-2 cells. The comparison of the parameters for permeation across isolated intestinal membrane suggests that the major site of intestinal zinc absorption in rats is the duodenum. The maximum rate of zinc transport across the isolated intestinal membrane (V(max)) shows no correlation with mRNA expression of ZIP4, ZIP5 or ZnT1 in rats, but shows an inverse correlation with that of metallothioneins (MTs). This finding may be partly explained by the buffering role of metallothionein in intestinal absorption. The saturable transport of zinc is not simply determined only by the influx transporter, ZIP4, since three influx and efflux transporters are involved in the transport of zinc.     

Transcellular transport of aconitine across human intestinal Caco-2 cells

Aconitine (AC) is a highly toxic compound present in plants of the genus Aconitum. The transcellular transport mechanism of AC was investigated using Caco-2 cells. The flux of AC was time- and concentration-dependent in both apical-to-basolateral and the reverse direction. The efflux of AC was more than two-fold that in the opposite direction. The influx of AC was temperature-, pH- and Na⁺-dependent. Glucose markedly decreased the absorption of AC. However, the efflux of AC was temperature- and pH-dependent, but Na⁺-independent. Cyclosporin A and verapamil, both inhibitors of P-glycoprotein (P-gp), significantly decreased the efflux of AC. In addition, MK-571, an inhibitor of multidrug resistance-associated protein 2 (MRP2), exhibited the same trend but to a lesser extent. These results indicate that both the influx and efflux of AC across Caco-2 monolayers were through an active process. A pH-dependent carrier-mediated transport system was the major absorption mechanism and a sodium-dependent glucose transporter may be involved. The active efflux of AC across Caco-2 cells was mediated mainly by ABC-transporter P-gp. It is involved in reducing the toxicity of AC to organisms and is the major reasons for the poor absorption of AC in vivo.     

Transcellular transport of domoic acid across intestinal Caco-2 cell monolayers

The intestinal absorption mechanism of domoic acid (DA) was investigated using Caco-2 cells. DA is a tricarboxylic amino acid that contains a glutamic acid moiety, and causes deficits in short-term memory by binding to glutamate receptors as an agonist of glutamic acid. Caco-2 cell monolayers cultured on permeable membranes were incubated with 100 μM DA on either the apical or basolateral side, and the transcellular transport of DA was measured. The transcellular transport of DA from the apical to basolateral side was about twofold that in the opposite direction. The transcellular transport of DA from the apical side was optimal at a neutral pH, and was temperature- and Cl⁻-dependent, but was Na⁺-independent. Coincubation of DA with 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), an anion exchange inhibitor, significantly decreased the apical-to-basolateral transport of DA by 48%, and coincubation with probenecid (a non-specific anion transport inhibitor) significantly decreased the transport of DA by 31%. In contrast, coincubation with glutamic acid, succinic acid (a dicarboxylic acid), or citric acid (a tricarboxylic acid) did not decrease the transport of DA. These results suggest that the apical-to-basolateral transport of DA across the Caco-2 cell monolayers is mediated by DIDS-sensitive anion transporters.     

Pharmacokinetic analysis of transcellular transport of quinidine across monolayers of human intestinal epithelial Caco-2 cells

To investigate the mechanism responsible for the intestinal absorption of a lipophilic organic cation, quinidine, we performed a pharmacokinetic analysis of transcellular transport across Caco-2 cell monolayers grown on a porous membrane. Basolateral-to-apical transport of the drug was almost constant in the concentration range of 100 nM-100 microM. Transcellular transport was greater in the apical-to-basolateral direction than in the opposite direction. Apical-to-basolateral transport was greater at a concentration of 100 microM than 100 nM. The calculated influx clearance value of the apical membrane was much greater than the other influx/efflux clearance values of cell membranes, and was 5.6-fold the influx clearance value of the basolateral membrane at the drug concentration of 100 microM. We also investigated the uptake of quinidine at the apical membrane of Caco-2 cells grown on plastic dishes. The uptake was markedly increased by alkalization of the apical medium at 37 degrees C, and was decreased at low temperature (4 degrees C). In addition, it was inhibited by diphenhydramine and levofloxacin, but not by carvedilol, rifamycin SV, or L-carnitine. These findings indicated that the influx at the apical membrane was the direction-determining step in the transcellular transport of quinidine across Caco-2 cell monolayers, and that some specific transport system was involved in this influx.     

Gamma-Aminobutyric acid (GABA) transport across human intestinal epithelial (Caco-2) cell monolayers

1. Transintestinal absorption of gamma-aminobutyric acid (GABA) via a pH-dependent mechanism is demonstrated in the model human intestinal epithelial cell line Caco-2. 2. Experiments with BCECF [2',7',-bis(2-carboxyethyl)-5(6)- carboxyfluorescein]-loaded Caco-2 cells demonstrate that GABA transport across the apical membrane is coupled to proton flow into the cell. 3. Short-circuit current (ISC) measurements using Caco-2 cell monolayers under voltage-clamped conditions demonstrate that pH-dependent GABA transport is a rheogenic process even in the absence of extracellular Na+, consistent with H+/GABA symport. 4. A range of GABA analogues were tested for their abilities to: (a) inhibit pH-dependent [3H]GABA uptake across the apical membrane; (b) stimulate H+ flow across the apical surface of BCECF-loaded Caco-2 cell monolayers; (c) increase inward ISC across voltage-clamped Caco-2 cell monolayers. 5. Nipecotic acid, isonipecotic acid, D,L-beta-aminobutyric acid, and 3-amino-1-propanesulphonic acid each caused a marked acidification of intracellular pH and an increase in ISC when superfused at the apical surface of Caco-2 cell monolayers. In contrast L-alpha-amino-n-butyric acid failed to induce proton flow or ISC. The ability of these compounds to induce proton or current flow across the apical surface of this intestinal epithelium was closely related to the relative inhibitory effects on [3H]GABA uptake. 6. These observations demonstrate H+/GABA symport and suggest that this transport mechanism may be accessible as a route for oral absorption of therapeutically-useful GABA analogues.     

Mechanisms for membrane transport of metformin in human intestinal epithelial Caco-2 cells

The aim of the present study was to investigate the mechanisms for membrane transport of metformin in human intestinal epithelial Caco-2 cells. The mRNA of not only organic cation transporter (OCT) 3, but also OCT1 and OCT2, was expressed in Caco-2 cells. The uptake of 100 μm metformin at the apical membrane of Caco-2 cells grown on porous filter membrane was significantly greater than that at the basolateral membrane. The apical uptake of 100 μm metformin in Caco-2 cells grown on plastic dishes was inhibited significantly by 1 mm unlabeled metformin, quinidine and pyrilamine, indicating that a specific transport system is involved in the apical uptake of metformin in Caco-2 cells. The apical uptake of 100 μm metformin in Caco-2 cells was decreased by acidification of the medium, but not increased by alkalization. In addition, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (a protonophore) had no effect on the apical uptake of metformin in Caco-2 cells at apical medium pH 8.4. These findings suggested that the apical uptake of metformin in Caco-2 cells is mediated at least partly by OCTs, but that the postulated H(+) /tertiary amine antiport system is not responsible for the apical uptake of metformin.     

Secretory transport of methylprednisolone possibly mediated by P-glycoprotein in Caco-2 cells

We recently reported that P-glycoprotein (MDR1) is capable of interfering with the absorption of methylprednisolone in the rat small intestine. This study was undertaken to examine the interaction between methylprednisolone and MDR1 using Caco-2 cells. The permeation of various steroid hormones (hydrocortisone, prednisolone, progesterone, beta-estradiol, and testosterone) was compared. The basolateral-to-apical (secretory) permeation of methylprednisolone was more than 3-fold greater than the apical-to-basolateral (absorptive) permeation. When verapamil (0.1 mm), a potent modulator of MDR1, was added to both apical and basolateral sides of Caco-2 cells, the absorptive permeation of methylprednisolone was increased and its secretory permeation was decreased. As a result, the secretory-oriented manner of methylprednisolone permeation almost completely disappeared. Prednisolone and hydrocortisone exhibited weaker secretory-oriented movement than did methylprednisolone. The secretory-oriented permeation of prednisolone and hydrocortisone was also diminished by the addition of verapamil. There was no significant directionality in progesterone permeation and the permeation of beta-estradiol and testosterone tended to be absorptive. These results appear to suggest that methylprednisolone, prednisolone, and hydrocortisone interact with MDR1 as the substrates. In contrast, there was no evidence that MDR1 was capable of potently interfering with the absorption of the sex hormones tested in this study, supporting our previous findings in the rat. It was further found that apically-added verapamil demonstrated a modulating effect on MDR1 function even at 5 microM.     

Lectin-mediated transport of nanoparticles across Caco-2 and OK cells

Recent experiments by a number of workers have suggested that it may be possible to use various targeting molecules, which bind to the intestinal epithelium, to promote the uptake and transport of nanoparticles from the intestine to the circulation. We have used commercial nanoparticles to examine the effect of size, density and inhibitors on uptake of lectin-coated nanoparticles by epithelial cells. The degree of uptake was most influenced by the density of lectin on the particle, with size and type of lectin being less important. Uptake could be inhibited by the presence of specific sugars or free lectin. These studies should provide a good basis for the design of targetable biodegradable drug-loadable particles suitable for oral delivery.     

Deoxynivalenol transport across human intestinal Caco-2 cells and its effects on cellular metabolism at realistic intestinal concentrations

Deoxynivalenol (DON) is a mycotoxin of the trichothecenes family to which human exposure levels can be high. Epidemiological studies suggest a link between DON and gastrointestinal illness. We investigated the interaction of DON with Caco-2 cells, a widely used in vitro model of the human intestinal barrier. The apical to basolateral (absorption) and basolateral to apical (excretion) transports of DON were found strictly proportional to both the initial concentration and the duration of the incubation. The absorption and excretion mean rates were similar to those of mannitol and were increased in the presence of EGTA, a calcium chelator. These data suggest that DON crosses the intestinal mucosa by a paracellular pathway through the tight junctions although some passive transcellular diffusion may not be ruled out. The DON transport was not affected by P-glycoprotein (PgP) or multidrug resistance-associated proteins (MRPs) inhibitors. A prolonged exposure to DON provokes the phosphorylation of the mitogen-activated protein kinases (MAPKs) Erk1/2, p38 and SAPK/JNK, as well as a decrease of the transepithelial resistance, suggesting that DON could trigger intestinal inflammation. These data imply that a chronic exposure to DON contaminated foods may negatively affect human health by altering the intestinal mucosa integrity and by inducing the MAPKs implicated in inflammation.     

Zolmitriptan uptake by human intestinal epithelial Caco-2 cells

The oral uptake of zolmitriptan, a novel and highly selectively 5-HT 1B/1D receptor agonist, was evaluated in the human epithelial cell line caco-2 that possesses intestinal enterocyte-like properties when cultured in vitro. The study demonstrated that zolmitriptan uptake significantly depended upon the extracelluar temperature and pH in the Caco-2 cell. The zolmitriptan uptake at 39 degrees C was 2.1 fold as that at 23 degrees C and the zolmitriptan uptake at pH 8.0 was 2.7 fold as that at pH 6.0. The uptake rates of zolmitriptan on both sides increased with increasing zolmitriptan concentration from 0.1 to 10 mmol x L(-1), and it shows concave concentration-dependency at high concentration. The uptake rates of zolmitriptan on the basolateral side (BL) were 3-7 times higher than that on the apical side (AP). Verapamil, nimodipine, nifedipine, flunarizine, amiloride and sumatriptan significantly increased the uptake rates of zolmitriptan on the apical sides. Propafenone significantly inhibited the uptake rate of zolmitriptan on both sides. Propranolol and aspirin have no significant effect. The results indicated that the zolmitriptan uptake in Caco-2 cells was temperature, pH and concentration dependent, and was partially counteracted by the action of an outwardly directed efflux pump, presumably p-glycoprotein. Absorption interactions should be considered when P-gp substrates or inhibitors, Na+ -H+ exchange inhibitors, P-gp ATPase agonists or inhibitors are co-administered with zomitriptan in clinical practice.     

Resveratrol transport and metabolism by human intestinal Caco-2 cells

Resveratrol is a dietary constituent suggested to have protective effects against cancer as well as cardiovascular disease. The purpose of the study was to learn whether this agent could be absorbed in man and enter the systemic circulation. This was examined by measuring transport and metabolism of resveratrol (5-40 microM) by the human intestinal epithelial cell line Caco-2 cultured in Transwells. Transport across the Caco-2 monolayer occurred in a direction-independent manner with P(app) values of approximately 7 x 10(-6) cm s(-1), much higher than for the paracellular transport marker mannitol (approximately 0.4 x 10(-6) cm s(-1)), suggesting efficient absorption in-vivo. At the highest resveratrol concentration, the absorption increased, possibly due to saturation of metabolism. In sharp contrast to previous findings in the rat, the metabolism of resveratrol in Caco-2 cells involved mainly sulfation and, to a minor extent, glucuronidation. At low resveratrol concentrations, most of the sulfate conjugate was exported to the apical side, presumably by MRP2, which is well expressed in these cells. At high concentrations, there was a shift towards the basolateral side, possibly involving MRP3, which was recently shown also to be expressed in Caco-2 cells. These results indicate that absorption of resveratrol in-vivo may be high but with limited bioavailability due to efficient sulfate conjugation. Extensive accumulation of resveratrol in the Caco-2 cells, demonstrated in additional experiments, suggests enterocytes as a major target site for this cancer preventive agent.     

Transepithelial transport of 4-chloro-2-methylphenoxyacetic acid (MCPA) across human intestinal Caco-2 cell monolayers

Mechanisms of transcellular transport of 4-chloro-2-methylphenoxyacetic acid (MCPA) across the small intestine were investigated using Caco-2 cells cultured on permeable membranes. The cell monolayers were incubated with MCPA, either from apical side at pH 6.0 or 7.4, or basolateral side at pH 7.4. The accumulation and apical-to-basolateral transport of MCPA were markedly stimulated by the acidic pH on the apical side (inwardly directed H(+) gradient), dependent on metabolic energy and inhibited by co-incubation with acetic acid or benzoic acid. Without the H(+) gradient, on the other hand, the basolateral-to-apical transport of MCPA (secretory transport) was higher than the apical-to-basolateral transport (absorptive transport), although the secretory transport of MCPA was markedly lower than the absorptive transport under the H(+) gradient. Co-incubation of MCPA with probenecid from the basolateral side significantly inhibited the accumulation and transport of MCPA, whereas co-incubation with p-aminohippuric acid did not. These results suggest that the absorptive transport of MCPA is mediated by H(+)-linked monocarboxylic acid transporters expressed on the apical membranes, while secretory transport is mediated by a probenecid-sensitive transporter expressed on the basolateral membranes of Caco-2 cell monolayers.     

8-异丙胺亚甲基橙皮素(IPHP)在Caco-2细胞模型上跨膜转运的研究

目的利用Caco-2细胞模型研究8-异丙胺亚甲基橙皮素(IPHP)在小肠吸收转运的机制。方法在Caco-2细胞模型上进行IPHP的跨膜转运实验,探讨药物浓度、p H、温度、P-gp抑制剂维拉帕米、MRP2抑制剂MK-571和丙磺舒对IPHP在体外细胞模型上跨膜转运的影响。结果 IPHP在Caco-2细胞模型上的转运具有一定的浓度依赖性,IPHP不同浓度从A侧到B侧的渗透系数Papp(AP-BL)(×10-5)分别为:(2.21±0.200)、(3.56±0.306)、(3.81±0.179)、(4.23±0.229)、(4.17±0.262)cm·s-1,B侧到A侧的渗透系数Papp(BL-AP)(×10-5)分别为:(3.57±0.209)、(4.51±0.113)、(4.97±0.229)、(5.24±0.550)、(5.07±0.557)cm·s-1,外排率分别为:1.61、1.26、1.3、1.23、1.21。温度和p H对其转运均有影响,而P-gp抑制剂对于IPHP的转运没有明显的影响,MRP2抑制剂在一定程度上增加了IPHP的转运量(P<0.05)。结论 IPHP在Caco-2细胞模型上的转运方式主要是被动扩散,且其转运不受P-gp外排蛋白影响,而外排蛋白MRP2可能参与了IPHP的外排转运。     

Transport of parthenolide across human intestinal cells (Caco-2)

This study examined the intestinal epithelial membrane transport of the sesquiterpene lactone parthenolide, a bioactive compound present in the migraine prophylactic herb feverfew. The Caco-2 human colonic cell line was used as an in vitro model of the human intestinal mucosal barrier. The bidirectional transport (apical to basolateral and basolateral to apical) of parthenolide was investigated using Caco-2 monolayers grown on Transwell inserts. Quantitation of parthenolide was performed using high performance liquid chromatography (HPLC). Apical to basolateral and basolateral to apical permeability coefficients and percent transport were calculated and a potential bioavailability of parthenolide was determined. Sodium fluorescein was used as a marker for paracellular leakage. Parthenolide, at a concentration of 250 microM, demonstrated substantial linear transport across the monolayer. The transport parameters were not affected by the presence of MK-571, an inhibitor of multidrug resistance transporter P-glycoprotein (MRP). Upon comparison of the transport parameters of parthenolide with atenolol under identical conditions and the reported values for model compounds like mannitol and propranolol, it is concluded that parthenolide is effectively absorbed through the intestinal mucosa via a passive diffusion mechanism.     

Enhanced transport of a novel anti-HIV agent--cosalane and its congeners across human intestinal epithelial (Caco-2) cell monolayers

PURPOSE: Cosalane is a potent inhibitor of HIV replication with activity against a broad range of viral targets. However, oral bioavailability of this highly lipophilic compound is extremely poor (<1%). The purpose of this study is to screen a variety of permeation enhancers (cyclodextrin derivatives, cremophor EL, bile salts and mixed micelles) for their ability to enhance the transport of cosalane and its analogs/prodrugs across Caco-2 cell monolayers. METHODS: Cosalane and its different analogs/prodrugs were synthesized and their physicochemical properties were determined. Caco-2 cells were cultured at a density of 66,000 cells/cm(2) either on collagen coated clear polyester membranes or Transwell inserts. Side-bi-side diffusion cells and Transwell inserts were employed to study for the transport of cosalane and its analogs/prodrugs with various permeation enhancers across Caco-2 cell monolayers. RESULTS: Permeabilities of EH-3-39, EH-3-55 and EH-3-57 significantly improved compared to that of cosalane in the presence of bile salt, sodium desoxycholate. Among the various cyclodextrins studied, hydroxypropyl beta cyclodextrin (HP-beta-CD) and dimethyl beta cyclodextrin (DM-beta-CD) exhibited 22.3-fold and 19-fold permeability enhancement of cosalane respectively across Caco-2 cell monolayers. Sodium desoxycholate (10 mM) also showed a remarkable (105-fold) enhancement on the permeability of cosalane (P(app) 11.72+/-3.31 x 10(-6) cm/s) without causing any measurable cellular damage. Cremophor EL resulted in higher transport of 14C mannitol. The mechanism of enhancement effect can be mainly attributed to the alteration of membrane fluidity by cyclodextrin and opening of tight junctions by cremophor EL. CONCLUSIONS: Among the enhancers tested, 10 mM sodium desoxycholate and HP-beta-CD appear to be viable candidates for further development of an oral formulation of cosalane and its congeners.     

Angiotensin-converting enzyme (ACE) inhibitor transport in human intestinal epithelial (Caco-2) cells.

1. The role of proton-linked solute transport in the absorption of the angiotensin-converting enzyme (ACE) inhibitors captopril, enalapril maleate and lisinopril has been investigated in human intestinal epithelial (Caco-2) cell monolayers. 2. In Caco-2 cell monolayers the transepithelial apical-to-basal transport and intracellular accumulation (across the apical membrane) of the hydrolysis-resistant dipeptide, glycylsarcosine (Gly-Sar), were stimulated by acidification (pH 6.0) of the apical environment. In contrast, transport and intracellular accumulation of the angiotensin-converting enzyme (ACE) inhibitor, lisinopril, were low (lower than the paracellular marker mannitol) and were not stimulated by apical acidification. Furthermore, [14C]-lisinopril transport showed little reduction when excess unlabelled lisinopril (20 mM) was added. 3. pH-dependent [14C]-Gly-Sar transport was inhibited by the orally-active ACE inhibitors, enalapril maleate and captopril (both at 20 mM). Lisinopril (20 mM) had a relatively small inhibitory effect on [14C]-Gly-Sar transport. pH-dependent [3H]-proline transport was not inhibited by captopril, enalapril maleate or lisinopril. 4. Experiments with BCECF[2',7',-bis(2-carboxyethyl)-5(6)-carboxyfluorescein]-loaded Caco-2 cells demonstrate that dipeptide transport across the apical membrane is associated with proton flow into the cell. The dipeptide, carnosine (beta-alanyl-L-histidine) and the ACE inhibitors enalapril maleate and captopril, all lowered intracellular pH when perfused at the apical surface of Caco-2 cell monolayers. However, lisinopril was without effect. 5. The effects of enalapril maleate and captopril on [14C]-Gly-Sar transport and pHi suggest that these two ACE inhibitors share the H(+)-coupled mechanism involved in dipeptide transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

D-cycloserine transport in human intestinal epithelial (Caco-2) cells: mediation by a H(+)-coupled amino acid transporter.

1. The ability of D-cycloserine to act as a substrate for H+/amino acid symport has been tested in epithelial layers of Caco-2 human intestinal cells. 2. In Na(+)-free media with the apical bathing media held at pH 6.0, D-cycloserine (20 mM) is an effective inhibitor of net transepithelial transport (Jnet) of L-alanine (100 microM) and its accumulation (across the apical membrane) in a similar manner to amino acid substrates (L-alanine, beta-alanine, L-proline and glycine). In contrast L-valine was ineffective as an inhibitor for H+/amino acid symport. Both inhibition of L-alanine Jnet and its accumulation by D-cycloserine were dose-dependent, maximal inhibition being achieved by 5-10 mM. 3. Both D-cycloserine and known substrates for H+/amino acid symport stimulated an inward short circuit current (Isc) when voltage-clamped monolayers of Caco-2 epithelia, mounted in Ussing chambers, were exposed to apical substrate in Na(+)-free media, with apical pH held at 6.0. The D-cycloserine dependent increase in Isc was dose-dependent with an apparent Km = 15.8 +/- 2.0 (mean +/- s.e. mean) mM, and Vmax = 373 +/- 21 nmol cm-2h-1. 4. D-Cycloserine (20 mM) induced a prompt acidification of Caco-2 cell cytosol when superfused at the apical surface in both Na+ and Na(+)-free conditions. Cytosolic acidification in response to D-cycloserine was dependent upon superfusate pH, being attenuated at pH 8 and enhanced in acidic media. 5. The increment in Isc with 20 mM D-cycloserine was non-additive with other amino acid substrates for H+/amino acid symport.     

Vitamin B12-mediated transport of nanoparticles across Caco-2 cells

Several studies have shown that Caco-2 cells have the capability to transport peptides and proteins from their apical to basal surfaces when these molecules are linked to vitamin B12 (VB12). In this study we have extended these studies and have shown that Caco-2 cells are also able to internalize and transport VB12-modifed nanoparticles from their apical to basal surfaces. Uptake and transport of nanoparticles was found to occur in both a VB12-dependent intrinsic factor (IF)-independent manner as well as in a VB12-dependent IF-dependent manner. Both IF-independent and IF-dependent VB12-mediated uptake and transport were dependent upon the surface density of VB12 as a reduction in surface modification of the nanoparticles with VB12 resulted in a reduced level of both VB12-mediated and IF-mediated uptake. At lower levels of VB12 modification there was no apparent non-IF-mediated uptake; however, VB12-IF-mediated uptake was still measurable. These studies show that Caco-2 cell cultures are a suitable model for the study of VB12-mediated uptake and transport of nanoparticles, and suggest that for effective oral uptake of VB12-coated nanoparticles high surface densities of VB12 are required.Copyright     

Transport characteristics of ebastine and its metabolites across human intestinal epithelial Caco-2 cell monolayers

The transport characteristics of a selective peripheral H1 receptor antagonist, ebastine, a substrate for cytochrome P450 3A4, and its three major metabolites, i.e., the hydroxy metabolite of ebastine (M-OH), the pharmacologically active metabolite carebastine (Car), and the desbutyrophenone metabolite (des-BP), were studied in cultured human intestinal Caco-2 cells expressing a drug efflux pump, P-glycoprotein (P-gp), on the apical membrane. The polarized transport of [3H]cyclosporin A (CyA), mediated by P-gp in the basolateral to apical direction across the Caco-2 cell monolayers, was affected by the presence of ebastine in a concentration-dependent manner and significant inhibition was observed at high concentrations (>50 microM). M-OH (300 microM) also significantly inhibited whereas Car and des-BP did not. Although no marked polarized transport of [14C]ebastine in a secretory direction was observed in the Caco-2 systems, the flux in the basolateral to apical direction was slightly higher than that in the opposite direction at concentrations less than 30 microm. [14C]Ebastine (2 microM) uptake from the apical side was significantly increased in the presence of an excess of cold CyA, suggesting that the efflux process mediated by P-gp may be involved in the ebastine uptake by Caco-2 cells. Collectively, these results indicate that ebastine (and presumably M-OH) is transported via P-gp in Caco-2 cells, however, the affinity for P-gp is very low. It is unlikely that the secretory transport of ebastine mediated by P-gp will dramatically affect overall intestinal absorption in vivo because efficient passive diffusion of this drug should occur due to its high lipophilicity. However, it may be advantageous for its efficient first-pass metabolism.