Characterization of Drug Transport Through Tight-Junctional Pathway in Caco-2 Monolayer: Comparison with Isolated Rat Jejunum and Colon

Drug transport through the tight-junctional pathway in Caco-2 monolayer was studied by examining the relationship between its permeability to hydrophilic drugs and membrane conductance. Compared with the rat isolated jejunum or colon, Caco-2 monolayer displayed high electrical resistance and low conductance, as well as low permeability to sulfanilic acid and FITC-dextran (M.W. 4000). However, there was a linear relationship between the drug permeability and partial Cl^– ion conductance for Caco-2 monolayer, rat jejunum and colon. Hence, the permeability to those drugs per unit of Cl^– conductance is similar in the three membranes, suggesting that the size (radius) of the tight-junctional pathway in the three membranes is similar. In addition, when the electrical resistance of Caco-2 monolayer was reduced to the same level as that of the jejunum or colon by pretreatment with disodium ethylenediamine-tetraacetate, its permeability to FITC-dextran became significantly higher than that of other membranes. Accordingly, the high resistance and the low permeability of Caco-2 monolayer compared with rat intestinal membrane may be due to structural differences between the membranes, rather than a difference in the tightness of the junction.     

Transport of Pregabalin in Rat Intestine and Caco-2 Monolayers

Purpose. The purpose of this study was to determine if the intestinal transport of pregabalin (isobutyl --aminobutyric acid, isobutyl GAB A), a new anticonvulsant drug, was mediated by amino acid carriers with affinity for large neutral amino acids (LNAA). Methods. Pregabalin transport was studied in rat intestine and Caco-2 monolayers. An in vitro Ussing/diffusion chamber model and an in situ single-pass perfusion model were used to study rat intestinal transport. An in vitro diffusion chamber model was used to evaluate Caco-2 transport. Results. In rat ileum, pregabalin transport was saturable and inhibited by substrates of intestinal LNAA carriers including neurontin (gabapentin), phenylalanine, and proline. Weak substrates of intestinal LNAA carriers (-alanine, --aminobutyric acid, and methyl aminoisobutyric acid) did not significantly change pregabalin transport. In Caco-2 mono-layers that showed a high capacity for phenylalanine transport, pregabalin transport was concentration- and direction-independent and equivalent in magnitude to the paracellular marker, mannitol. The in vitro and in situ rat ileal permeabilities of the LNAA carrier-mediated compounds neurontin, pregabalin, and phenylalanine correlated well with the corresponding in vivo human oral absorption. Conclusions. The transport of pregabalin was mediated by LNAA carriers in rat ileum but not in Caco-2 monolayers. Caco-2 was not an appropriate model for evaluating the in vivo human oral absorption of pregabalin and neurontin.     

Transepithelial Transport of Diphenhydramine Across Monolayers of the Human Intestinal Epithelial Cell Line Caco-2

Purpose. The transepithelial transport characteristics of theantihistamine, diphenhydramine, were studied in human intestinal Caco-2 cellmonolayers to elucidate the mechanisms of its intestinal absorption. Methods. The transepithelial transport and the cellular accumulationof diphenhydramine were measured using Caco-2 cell monolayersgrown in Transwell chambers. Results. The transepithelial transport of diphenhydramine from theapical to basolateral side was saturable, and the flux and cellularaccumulation of diphenhydramine were dependent on the apicalextracellular pH (pH 7.4 > 6.5 > 5.5). Transport and accumulation ofdiphenhydramine from the apical side were inhibited by anotherantihistamine, chlorpheniramine, while typical substrates for the renal organiccation transport system such as tetraethylammonium, cimetidine andguanidine had no effect. The transepithelial transport and cellularaccumulation of diphenhydramine from the basolateral side were alsopH-dependent and inhibited by chlorpheniramine. In addition, intracellulardiphenhydramine preloaded was preferentially effluxed to the apicalside, suggesting the involvement of the secretory pathway indiphenhydramine transport. Furthermore, diphenhydramine uptake from boththe apical and basolateral sides was stimulated by preloadingmonolayers with chlorpheniramine (trans-stimulation effect). Conclusions. Transepithelial transport of diphenhydramine acrossCaco-2 cells is mediated by pH-dependent, specific transport systemsthat exist in both the apical and basolateral membranes.     

Characterization of the Regional Intestinal Kinetics of Drug Efflux in Rat and Human Intestine and in Caco-2 Cells

Purpose. The aim of the present study was to investigate the transport kinetics of intestinal secretory processes in the jejunum, ileum and colon of rats and humans and in Caco-2 cells, in vitro. Methods. Etoposide, vinblastine sulphate and verapamil hydrochloride were chosen as model substrates since they have been reported to undergo efflux in various other tissues. The concentration dependence, inhibition, directionality, temperature dependence, proton/sodium dependence, and ATP dependence of efflux were studied using side-by-side diffusion chambers and brush border membrane vesicles (BBMVs). Intestinal tissue from rats and humans and Caco-2 cells (passage no. 26) were used. Directional steady state effective permeabilities were calculated from drug appearance in the apical (AP) or basolateral (BL) chambers. Kinetic studies were carried out by investigating substrate efflux at concentrations ranging from 0.2 M to 1000 M. Since substrate efflux may be a result of more than one transporter, the hybrid efflux Km (Michaelis-constant), Pc (carrier-mediated permeability), and Pm (passive permeability) were determined as a function of intestinal region. Inhibitor studies were performed using quinidine (0.2 mM), a mixed inhibitor of P-glycoprotein (Pgp) and Multidrug Resistance-Associated Protein (MRP), and Leukotriene C₄(100 nM), an inhibitor of MRP and the canalicular multispecific organic anion transporter (cMOAT). Temperature dependent efflux was determined by investigating the BL to AP transport at temperatures ranging from 3°C to 37°C. Energies of activation (Ea) were determined from an Arrhenius analysis. Sodium, proton, and ATP dependence were determined using BBMVs. Immunoquantitation of Pgp, MRP and Lung Resistance Protein (LRP) in Caco-2 cells were carried out using Western blot analysis. Results. Active efflux of all substrates was observed in all regions of rat and human intestine and in Caco-2 cells. Directionality was observed with BL to AP transport exceeding AP to BL transport. The BL to AP/AP to BL permeability ratio, the efflux ratio, ranged from 1.4 to 19.8. Heal efflux was significantly higher (p < 0.001) than in other regions. Kinetic studies revealed that hybrid efflux Km values ranged from 4 to 350 M. In some cases, efflux was not saturable due to the solubility limits of the compounds utilized in this study. In presence of inhibitors, efflux ratios approached 1. BL to AP transport was temperature dependent in rat ileum for all substrates. Ea of intestinal efflux was found to be 11.6, 8.3, and 15.8 kcal/mole for etoposide, vinblastine and verapamil, respectively, suggesting an active, energy-dependent efflux mechanism. Substrate efflux was not sodium or proton dependent but was dependent on ATP. Using Western blot analysis the presence of Pgp, MRP, and LRP was demonstrated in Caco-2 cells and the amount of each transport protein varied as a function of passage number. Conclusions. Using multiple putative efflux substrates, the current results demonstrate that intestinal efflux was regionally dependent, mediated by multiple efflux transporters, the Kms were in the micro-molar range, and involved an energy dependent mechanism(s).     

Increased Transport of Resveratrol Across Monolayers of the Human Intestinal Caco-2 Cells is Mediated by Inhibition and Saturation of Metabolites

Purpose The study's aim was to investigate the dose-dependent effect of sulfation and glucuronidation on intestinal absorption of resveratrol, a dietary constituent found in grapes and various medical plants.Materials and Methods The intestinal epithelial membrane transport kinetics and metabolism of resveratrol (10–200 μM) was studied using Caco-2 monolayers cultured in Transwells.Results Along with resveratrol it was possible to identify three metabolites, namely, resveratrol-4′-O-glucuronide (M1), resveratrol 3-O-gucuronide (M2), and resveratrol-3-O-sulfate (M3) by LC/MS and NMR. Efflux of the glucuronides M1 and M2 followed Michaelis–Menten kinetics significantly favouring basolateral efflux. The predominant metabolite was the monosulfate M3, however, its formation was strongly inhibited at higher resveratrol concentrations. As biotransformation was either inhibited or saturated, total amount of resveratrol transported across the Caco-2 monolayers increased as much as 3.5-fold at 200 μM resveratrol. This value might be even higher when taking into account the high intracellular concentration of resveratrol, which accounted for up to 61% of the applied dose.Conclusions Our data demonstrate a concentration-dependent biotransformation of resveratrol in Caco-2 cells, which may also apply to human enterocytes affecting oral bioavailability.     

Heparin Absorption Across the Intestine: Effects of Sodium N-[8-(2-Hydroxybenzoyl)Amino]Caprylate in Rat In Situ Intestinal Instillations and in Caco-2 Monolayers

Purpose. The effects of sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) on heparin intestinal absorption were studied using rat in situ ileal and colonic instillations and Caco-2 monolayers. Methods. The flux of heparin was tested in the following groups: i) heparin alone, ii) heparin in the presence of SNAC, iii) heparin in the presence of propylene glycol (PG), and iv) heparin in the presence of SNAC and PG. Heparin absorption was measured by the APTT assay in the in situ models and by the anti-Factor Xa assay in Caco-2. SNAC and [³H]-SNAC fluxes were assessed by HPLC and by scintillation counting respectively. Results. In the rat ileal and colonic in situ instillations SNAC (17–35mg) promoted heparin absorption in the presence and absence of PG without damaging the tissue. PG alone did not alter heparin absorption in situ, but it amplified the effect of SNAC. In Caco-2, enhanced heparin fluxes were variable in the presence of non-cytotoxic concentrations of SNAC (< l0mg/ml) and these effects could not be discriminated from those of PG. Papp values for SNAC alone were 2.2 × 10^–5 cm/s and 2.0 × 10^–5 cm/s in the mucosal-to-serosal and serosal-to-mucosal directions respectively, suggesting a substantial passive transcellular flux. Transport of SNAC was significantly reduced in the presence of heparin and/or PG, perhaps indicating physical association between the agents. Conclusions. SNAC augmented heparin absorption alone and in combination with PG in the rat in situ models without causing toxicity. Caco-2 had limitations for testing increased heparin absorption due to cytotoxic effects of high concentrations of SNAC and PG. However, SNAC itself was well absorbed across Caco-2 and its mechanism of permeation was determined.     

Quantification and Imaging of Mannitol Transport Through Caco-2 Cell Monolayers Using a Positron-Emitting Tracer

Pharmaceutical Research -     

Comparison of the Disposition of Ester Prodrugs of the Antiviral Agent 9-(2-phosphonylmethoxyethyl)adenine [PMEA] in Caco-2 Monolayers

Purpose. To evaluate the potential of several bis-ester prodrugs of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine (PMEA, adefovir) to enhance the oral absorption of PMEA. Methods. Caco-2 monolayers were used to estimate intestinal transport and metabolism of the bis(pivaloyloxymethyl)-ester [bis(POM)-] and a series of bis(S-acyl-2-thioethyl)-esters [bis(SATE)-] of PMEA. An LC-MS method was used for the identification of unknown metabolites which were formed from the SATE-esters. Results. During transport across Caco-2 monolayers, all esters were extensively degraded as could be concluded from the appearance of the mono-ester and free PMEA in apical as well as basolateral compartments. Incubation of SATE-esters with the monolayers resulted in the formation of two additional metabolites, which were identified as 2-thioethyl-PMEA and its dimerisation product. All ester prodrugs resulted in enhanced transepithelial transport of total PMEA (i.e. the bis-esters and their corresponding metabolites, including PMEA), but significant differences could be observed between the various esters. Transport of total PMEA ranged from 0.4 ± 0.1 % for the bis[S(methyl) ATE]-ester to 15.3 ± 0.9% for the more lipophilic bis[S(phenyl)ATE]-PMEA. A relationship between total transport of the esters and their lipophilicity (as estimated by their octanol/water partition coefficient) was established (r² = 0.87). Incubation of prodrug esters with homogenates from Caco-2 cells showed large differences in susceptibility of the compounds to esterases, the half-lives of the bis-esters varying from 4.3 ± 0.3 min for the bis[S(phenyl)ATE]-PMEA to 41.5 ± 0.8 min for its methyl analogue. In addition, intracellularly formed PMEA was observed to be further converted by the cells to the diphosphorylated PMEA (PMEApp). Conclusions. Several SATE-esters of PMEA can be considered as potential alternatives to bis(POM)-PMEA, due to enhanced epithelial transport, sufficient chemical and enzymatic stability and adequate release of PMEA. Toxicological studies as well as in vivo experiments are required in order to further explore the potential of those SATE-esters as prodrugs for oral delivery of PMEA.     

Messenger RNA Expression of Transporter and Ion Channel Genes in Undifferentiated and Differentiated Caco-2 Cells Compared to Human Intestines

Purpose. The purpose of this work was to study the influence of cell differentiation on the mRNA expression of transporters and channels in Caco-2 cells and to assess Caco-2 cells as a model for carrier-mediated drug transport in the intestines. Method. Gene mRNA expression was measured using a custom-designed microarray chip with 750 deoxyoligonucleotide probes (70mers). Each oligomer was printed four times on poly-lysine-coated glass slides. Expression profiles were expressed as ratio values between fluorescence intensities of Cy3 and Cy5 dye-labeled cDNA derived from poly(A) + RNA samples of Caco-2 cells and total RNA of human intestines. Results. Significant differences in the mRNA expression profile of transporters and channels were observed upon differentiation of Caco-2 cells from 5 days to 2 weeks in culture, including changes for MAT8, S-protein, and Nramp2. Comparing Caco-2 cells of different passage number revealed few changes in mRNAs except for GLUT3, which was down-regulated 2.4-fold within 13 passage numbers. Caco-2 cells had a similar expression profile when either cultured in flasks or on filters but differed more strongly from human small and large intestine, regardless of the differentiation state of Caco-2 cells. Expression of several genes highly transcribed in small or large intestines differed fourfold or more in Caco-2 cells. Conclusions. Although Caco-2 cells have proven a suitable model for studying carrier-mediated transport in human intestines, the expression of specific transporter and ion channel genes may differ substantially.     

Profound Effect of Plasma Protein Binding on the Polarized Transport of Furosemide and Verapamil in the Caco-2 Model

Pharmaceutical Research -     

Selective Paracellular Permeability in Two Models of Intestinal Absorption: Cultured Monolayers of Human Intestinal Epithelial Cells and Rat Intestinal Segments

New data on the permeabilities of hydrophilic markers in two commonly used in vitro models, i.e., excised intestinal segments from the rat and monolayers of Caco-2 cells, are presented. The results are compared to human in vivo data. Two groups of hydrophilic marker molecules were tested: (1) monodisperse polyethylene glycols of molecular weights ranging from 194 to 502 g/mol and (2) a heterogeneous group of molecules consisting of urea, creatinine, erythritol, and mannitol (60–182 g/mol). The permeabilities of the marker molecules showed a nonlinear dependence on the molecular weight and decreased in the order rat ileum > rat colon > Caco-2 cells. Surprisingly, the polyethylene glycols permeated more easily than the other marker molecules, indicating that characteristics other than molecular weight, e.g., the flexibility of the structure, may also be important for permeation through the membrane. Comparisons with the published permeability profiles of polyethylene glycols in human intestinal segments in vivo (i.e., calculated permeability coefficients as a function of molecular weight) indicate that the human intestine is more permeable than the in vitro models. However, the permeability profiles of the corresponding segments in the human intestine and the in vitro models were comparable. Thus, good correlations were established between permeabilities of the human ileum and rat ileum and between those of human colon, rat colon, and the Caco-2 cells. We conclude that the paracellular absorption in humans can be studied mechanistically in these in vitro models.     

大鼠小肠原位灌流与Caco-2细胞法研究柠檬苦素吸收机制

柠檬苦素广泛存在于柑橘类水果中, 具有抗菌、抗病毒、镇痛、抗炎和抗癌等活性, 但其口服生物利用度较低。本文意在研究柠檬苦素在肠道内的吸收机制, 为其今后的研究奠定基础。实验通过大鼠原位肠灌流和体外Caco-2细胞法进行。大鼠原位肠灌流结果显示, 柠檬苦素可能通过肠道促进扩散机制吸收, 吸收差且在全肠段都有吸收, 没有部位选择性。Caco-2细胞实验结果显示, 维拉帕米和酮康唑能显著提高柠檬苦素的吸收, 而丙磺舒的影响不明显。柠檬苦素吸收较低和生物利用度较差, 可能是P-gp外排以及CYP3A4代谢共同参与的结果。柠檬苦素的肠吸收机制研究将为其剂型设计和临床应用提供重要的参考。     

Binding and Transport of Some Bioadhesive Plant Lectins Across Caco-2 Cell Monolayers

Pharmaceutical Research -     

白花前胡丁素在人结肠腺癌细胞系细胞模型上的吸收转运特征研究

目的研究白花前胡丁素在人结肠腺癌细胞系(Caco-2)细胞模型上的转运特征。方法利用Caco-2单层细胞模型研究白花前胡丁素的双向转运,采用高效液相色谱(HPLC)法测定药物的转运量,计算其表观渗透系数(Papp),并考察时间和药物质量浓度对其转运的影响。结果白花前胡丁素在Caco-2细胞模型上的双向转运量在120min内均随时间与浓度的增加而增大,其从肠腔侧(AP)向基底侧(BL)和从BL向AP的Papp介于2.0×10-6cm/s～5.0×10-6cm/s,Papp(BL-AP)/Papp(AP-BL)<1.5。结论白花前胡丁素为吸收较差的药物,主要以被动扩散方式经肠道吸收。     

克拉霉素对Caco-2细胞转运他克莫司的影响及机制研究

目的 探讨克拉霉素对Caco-2细胞转运他克莫司的影响及机制。方法 用化学发光免疫分析法测定他克莫司药物浓度；建立Caco-2细胞单层模型，计算表观渗透系数，比较不同浓度他克莫司在Caco-2细胞单层中转运及不同浓度克拉霉素抑制他克莫司的转运。结果 他克莫司浓度为20，40和80 μg·mL^-1时吸收渗透系数P_appAP-BL分别为（2.47±0.09）×10^-6，（3.91±0.17）×10^-6和（4.49±0.16）×10^-6 cm·s^-1；分泌渗透系数P_appBL-AP分别为（6.05±0.21）×10^-6，（9.86±0.70）×10^-6和（11.75±0.28）×10^-6 cm·s^-1；外流比（apparent permeability ratio，PDR）分别为2.45±0.03，2.52±0.12和2.62±0.11；加入不同浓度的克拉霉素（15，30，60 μg·mL^-1）后，分泌渗透系数显著降低，而吸收系数影响不大，PDR随着克拉霉素浓度增加显著降低。结论 Caco-2细胞外排转运体可能参与了他克莫司的转运，克拉霉素合用他克莫司可显著影响他克莫司的吸收。     

Transport of Poly(Amidoamine) Dendrimers across Caco-2 Cell Monolayers: Influence of Size, Charge and Fluorescent Labeling

Purpose To investigate the transport of poly(amidoamine) (PAMAM) dendrimers with positive, neutral and negatively charged surface groups across Caco-2 cell monolayers. Methods Cationic PAMAM-NH₂ (G2 and G4), neutral PAMAM-OH (G2), and anionic PAMAM-COOH (G1.5–G3.5) dendrimers were conjugated to fluorescein isothiocyanate (FITC). The permeability of fluorescently labeled PAMAM dendrimers was measured in the apical-to-basolateral direction. ¹⁴C-Mannitol permeability was measured in the presence of unlabeled and FITC labeled PAMAM dendrimers. Caco-2 cells were incubated with the dendrimers followed by mouse anti-occludin or rhodamine phalloidin, and visualized using confocal laser scanning microscopy to examine tight junction integrity. Results The overall rank order of PAMAM permeability was G3.5COOH > G2NH₂ > G2.5COOH > G1.5COOH > G2OH. ¹⁴C-Mannitol permeability significantly increased in the presence of cationic and anionic PAMAM dendrimers with significantly greater permeability in the presence of labeled dendrimers compared to unlabeled. PAMAM dendrimers had a significant influence on tight junction proteins occludin and actin, which was microscopically evidenced by disruption in the occludin and rhodamine phalloidin staining patterns. Conclusions These studies demonstrate that enhanced PAMAM permeability is in part due to opening of tight junctions, and that by appropriate engineering of PAMAM surface chemistry it is possible to increase polymer transepithelial transport for oral drug delivery applications.     

Characterization of the Unstirred Water Layer in Caco-2 Cell Monolayers Using a Novel Diffusion Apparatus

Caco-2 monolayers grown on Transwell polycarbonate membranes have been characterized as a valuable tool in drug transport studies. Despite the clear advantages of this system, the lack of stirring may create an unstirred water layer (UWL) whose resistance may limit the transcellular transport of lipophilic molecules. The objective of this study was to evaluate a novel diffusion cell where the transport buffer is mixed by gas lift and to determine the mixing flow rate needed to reduce the thickness (h) of the UWL adjacent to cell monolayers. The transport of the leakage marker, mannitol, remained at least 15-fold lower than the flux of testosterone, indicating that the stirring flow rates used did not affect the integrity of the monolayers. The permeability (P) of testosterone (log PC 3.13) across monolayers mounted on this diffusion cell was 4.07, 10.90, and 14.18 × 10^–5 cm/sec at flow rates of 0, 15, and 40 ml/min, respectively, and the apparent UWLs were calculated to be 1966, 733, and 564µm. P and h in the stagnant Transwell were 3.08 × 10^–5 cm/sec and 2597 µm, respectively. On the other hand, h was significantly smaller in the unstirred, cell-free membranes than in their cell-containing counterparts. P was correlated with lipophilicity and, in the case of the more lipophilic compounds, with the mixing flow rate.     

肠道转运Caco-2细胞单层模型的建立及验证评价

目的建立Caco-2细胞单层模型用于药物转运研究。方法按照常规的细胞培养方法,将Caco-2细胞接种到Millicell小室内(接种密度1×10⁶个·mL^-1),培养21 d。定期用细胞电位仪监测跨上皮细胞电阻（TEER）,评价细胞单层的紧密性与完整性;通过荧光黄转运实验检查Caco-2细胞单层模型细胞旁路转运通透性;通过普萘洛尔转运实验验证Caco-2细胞单层模型跨细胞被动转运通透性。结果培养21 d后,TEER值达到（981±123）Ω·cm²,荧光黄和普萘洛尔的表观通透系数分别为0.33×10及16.7×10^-6cm·s^-1。结论本研究建立的Caco-2细胞单层模型紧密、完整,具有良好通透性,可用于药物转运研究。     

Mechanisms of Transport and Structure-Permeability Relationship of Sulfasalazine and Its Analogs in Caco-2 Cell Monolayers

Purpose. To investigate the mechanisms involved in transport of sulfasalazine in Caco-2 cells. Methods. Permeability coefficients of sulfasalazine and its analogs across Caco-2 cell monolayers were measured as a function of direction of transport, energy and concentration dependence, and in the presence of inhibitors of various cellular efflux pumps and transporters. Results. Permeability coefficients of sulfasalazine across Caco-2 cell monolayers were approximately 342-, 261-, and 176-fold higher from basolateral to apical direction (BLAP) than from apical to basolateral direction (APBL) at 100, 200, and 500 M, respectively. Carrier permeability coefficient, non-saturable membrane permeability coefficient, and Michaelis constant were estimated to be 1.4×10^–5 cm/s, 1.9×10^–8 cm/s, and 369 M, respectively. The efflux of sulfasalazine was completely blocked at 4°C and in the presence of an uncoupler of oxidative phosphorylation. Using cellular efflux inhibitors, the permeability of sulfasalazine was shown to depend on multidrug resistance-associated protein and anion sensitive transport mechanisms. Structure-permeability studies showed that the affinity of sulfasalazine for the cellular efflux pumps and transporters in Caco-2 cells depended strongly on the carboxylic acid functional group. Conclusions. The permeability of sulfasalazine across Caco-2 cell monolayer is very low due to its strong interaction with multiple cellular efflux pumps and transporters. This may partially explain its low absorption in vivo.     

Comparison of ceftibuten transport across Caco-2 cells and rat jejunum mounted on modified Ussing chambers

Ceftibuten uptake into Caco-2 cells and intestinal brush border membrane vesicles is mediated by the dipeptide transport system (PEPT1). The apical to basolateral transport characteristics of ceftibuten across Caco-2 cells and rat jejunum mounted on a modified Ussing chamber was examined. Mannitol was used as a paracellular marker along with trans-epithelial electrical resistance (TEER) for monitoring tight junction permeability. Transport across Caco-2 cells and rat jejunum mounted on a modified Ussing chamber was linear across the concentration range 0.25-10 mM. The net flux of mannitol and ceftibuten was higher across rat jejunum compared with Caco-2 cells. At a donor concentration of 0.25 mM, ceftibuten transport across Caco-2 cells was found to be pH dependent. Glycyl proline, a dipeptide, and 2,4- dinitrophenol, an energy poison, caused a reduction in the permeability of 0.25 mM ceftibuten across Caco-2 cells. Benzoic acid and adipic acid also inhibited transcellular transport of ceftibuten. At a donor concentration of 0.25 mM, passive paracellular transport accounts for about 60% and the active carrier mediated mechanism accounts for about 40% of ceftibuten transport across Caco-2 cells. None of the inhibitors however, had a significant effect on ceftibuten transport across rat jejunum mounted on a modified Ussing chamber at a donor concentration of 0.25 mM. In the concentration range 0.25-10 mM, ceftibuten is predominantly transported by paracellular mechanisms across rat jejunum and a mixture of active and passive transport across Caco-2 cells.