Intestinal Absorptive Transport of the Hydrophilic Cation Ranitidine: A Kinetic Modeling Approach to Elucidate the Role of Uptake and Efflux Transporters and Paracellular vs. Transcellular Transport in Caco-2 Cells

Purpose The mechanism of intestinal drug transport for hydrophilic cations such as ranitidine is complex, and evidence suggests a role for carrier-mediated apical (AP) uptake and saturable paracellular mechanisms in their overall absorptive transport. The purpose of this study was to develop a model capable of describing the kinetics of cellular accumulation and transport of ranitidine in Caco-2 cells, and to assess the relative contribution of the transcellular and paracellular routes toward overall ranitidine transport. Methods Cellular accumulation and absorptive transport of ranitidine were determined in the absence or presence of uptake and efflux inhibitors and as a function of concentration over 60 min in Caco-2 cells. A three-compartment model was developed, and parameter estimates were utilized to assess the expected relative contribution from transcellular and paracellular transport. Results Under all conditions, ranitidine absorptive transport consisted of significant transcellular and paracellular components. Inhibition of P-glycoprotein decreased the AP efflux rate constant (k₂₁) and increased the relative contribution of the transcellular transport pathway. In the presence of quinidine, both the AP uptake rate constant (k₁₂) and k₂₁ decreased, resulting in a predominantly paracellular contribution to ranitidine transport. Increasing the ranitidine donor concentration decreased k₁₂ and the paracellular rate constant (k₁₃). No significant changes were observed in the relative contribution of the paracellular and transcellular routes as a function of ranitidine concentration. Conclusions These results suggest the importance of uptake and efflux transporters as determinants of the relative contribution of transcellular and paracellular transport for ranitidine, and provide evidence supporting a concentration-dependent paracellular transport mechanism. The modeling approach developed here may also be useful in estimating the relative contribution of paracellular and transcellular transport for a wide array of drugs expected to utilize both pathways.     

Efflux Ratio Cannot Assess P-Glycoprotein-Mediated Attenuation of Absorptive Transport: Asymmetric Effect of P-Glycoprotein on Absorptive and Secretory Transport Across Caco-2 Cell Monolayers

Purpose. The purpose of this work was to determine whether P-glycoprotein (P-gp) modulates absorptive and secretory transport equally across polarized epithelium (i.e., Caco-2 cell monolayers) for structurally diverse P-gp substrates, a requirement for the use of the efflux ratio to quantify P-gp-mediated attenuation of absorption across intestinal epithelium. Methods. Studies were performed in Caco-2 cell monolayers. Apparent permeability (P _app) in absorptive (P _app,AB) and secretory (P _app,BA) directions as well as efflux ratios (P _app,BA / P _app,AB) were determined for substrates as a function of concentration. Transport of these compounds (10 M) was measured under normal conditions and in the presence of the P-gp inhibitor, GW918 (1 M), to dissect the effect of P-gp on absorptive and secretory transport. Apparent biochemical constants of P-gp-mediated efflux activity were calculated for both transport directions. Results. Efflux ratios for rhodamine 123 and digoxin were comparable (approx. 10). However, transport studies in the presence of GW918 revealed that P-gp attenuated absorptive transport of digoxin by approx. 8-fold but had no effect on absorptive transport of rhodamine 123 (presumably because absorptive transport of rhodamine 123 occurs via paracellular route). The apparent K _m for P-gp-mediated efflux of digoxin was >6-fold larger in absorptive vs. secretory direction. For structurally diverse P-gp substrates (acebutolol, colchicine, digoxin, etoposide, methylprednisolone, prednisolone, quinidine, and talinolol) apparent K _m was approximately 3 to 8-fold greater in absorptive vs. secretory transport direction, whereas apparent J _max was somewhat similar in both transport directions. Conclusions. P-gp-mediated efflux activity observed during absorptive and secretory transport was asymmetric for all substrates tested. For substrates that crossed polarized epithelium via transcellular pathway in both directions, this difference appears to be caused by greater apparent K _m of P-gp-mediated efflux activity in absorptive vs. secretory direction. These results clearly suggest that use of efflux ratios could be misleading in predicting the extent to which P-gp attenuates the absorptive transport of substrates.     

Modulation of Organic Cation Transport and Lipid Fluidity by Benzyl Alcohol in Rat Renal Brush-border Membranes

Purpose. Organic cations are actively transported in renal brush-border membranes (BBM) by the H⁺/organic cation antiport system. In the present study, we investigated the relationship between membrane fluidity and organic cation transport in the BBM. Methods. The effects of benzyl alcohol, a membrane fluidizing agent, on the organic cation tetraethylammonium (TEA) uptake were studied using renal BBM vesicles isolated from rat kidney. BBM fluidity was assessed by fluorescence polarization technique. Results. H⁺ gradient-dependent uptake of TEA in BBM vesicles was inhibited by benzyl alcohol in a dose-dependent manner, with an apparent half inhibitory concentration of 18mM. The decrease in fluorescence anisotropy of l,6-diphenyl-l,3,5-hexatriene in BBM, which represents the increase in membrane fluidity, was correlated with the decrease in TEA transport activity. The dissipation rate of H⁺ gradient, a driving force for organic cation transport in BBM, was increased by benzyl alcohol. In addition, H⁺ gradient-independent TEA-TEA exchange was also inhibited by benzyl alcohol. These findings indicate that benzyl alcohol inhibits the uptake of TEA by affecting the intrinsic activity of the organic cation transporter and the H⁺ gradient dissipation rate. Conclusions. The membrane fluidity should be an important determinant for organic cation transport in renal BBM.     

Rhodamine 123 Requires Carrier-Mediated Influx for Its Activity as a P-Glycoprotein Substrate in Caco-2 Cells

Purpose. The purpose of this work was to elucidate transport pathways of the P-glycoprotein (P-gp) substrates rhodamine 123 (R123) and doxorubicin across Caco-2 cells. Methods. Experiments were designed to identify saturable and nonsaturable transport processes and transport barriers for R123 and doxorubicin transport across Caco-2 cells. Confocal laser scanning microscopy (CLSM) imaged R123 transport under normal conditions and in the presence of the P-gp inhibitor, GW918 (used to abolish P-gp-mediated efflux activity). Results. R123 secretory P _app (P _app,BA) showed concentration dependence, whereas R123 absorptive P _app (P _app,AB) did not. Inhibition of P-gp efflux revealed that P-gp-mediated efflux had no effect on R123 or doxorubicin P _app,AB, but enhanced R123 and doxorubicin P _app,BA. In calcium-free medium, R123 P _app,AB increased 15-fold, indicating intercellular junctions are a barrier to R123 absorption. CLSM of R123 fluorescence during absorptive transport under normal conditions and in the presence of GW918 was identical, and was limited to paracellular space, confirming that P-gp is not a barrier to R123 absorption. CLSM revealed that R123 fluorescence during secretory transport under normal conditions and in the presence of GW918 was localized intracellularly and in paracellular space. R123 and doxorubicin uptake across Caco-2 cells basolateral membrane was saturable. Conclusions. R123 absorptive transport occurs primarily by paracellular route, whereas R123 secretory transport involves influx across BL membrane mediated solely by a saturable process followed by apically directed efflux via P-gp. Doxorubicin utilizes similar transport pathways to cross Caco-2 cells.     

Novel Experimental Parameters to Quantify the Modulation of Absorptive and Secretory Transport of Compounds by P-Glycoprotein in Cell Culture Models of Intestinal Epithelium

Purpose. The purpose of this work was to elucidate the asymmetric effect of P-gp on modulation of absorptive and secretory transport of compounds across polarized epithelium, to develop experimental parameters to quantify P-gp-mediated modulation of absorptive and secretory transport, and to elucidate how P-gp-mediated modulation of transport is affected by passive diffusion properties, interaction of the substrate with P-gp, and P-gp expression. Methods. The permeability of a set of P-gp substrates was determined in absorptive and secretory directions in Madine-Darby Canine kidney (MDCK), Caco-2, and MDR-MDCK monolayers. The transport was also determined in the presence of GW918, a non-competitive P-gp inhibitor, to quantify the permeability without the influence of P-gp. From these two experimental permeability values in each direction, two new parameters, absorptive quotient (AQ) and the secretory quotient (SQ), were defined to express the functional activity of P-gp during absorptive and secretory transport, respectively. Western blot analysis was used to quantify P-gp expression in these monolayers and in normal human intestinal. Results. P-gp expression in Caco-2 and MDR-MDCK monolayers was comparable to that in normal intestine, and much less in MDCK cells. For all models, the substrates encompassed a wide range of apparent permeability due to passive diffusion (P _PD). The parameters AQ and SQ, calculated for all compounds, assessed the attenuation in absorptive and enhancement of secretory transport, respectively, normalized to the permeability due to passive diffusion. Analysis of these parameters showed that 1) P-gp affected absorptive and secretory transport differentially and 2) compounds could be stratified into distinct groups with respect to the modulation of their absorptive and secretory transport by P-gp. Compounds could be identified whose absorptive transport was either strongly affected or poorly affected by changes in P-gp expression. For certain compounds, AQ values showed parabolic relationship with respect to passive diffusivity, and for others AQ was unaffected by changes in passive diffusivity. Conclusions. The relationship between attenuation of absorptive transport and enhancement of secretory transport of compounds by P-gp is asymmetric, and different for different sets of compounds. The relationship between attenuation of absorption by P-gp and passive diffusivity of compounds, their interaction potential with P-gp, and levels of P-gp expression is complex; however, compounds can be classified into sets based on these relationships. A classification system that describes the functional activity of P-gp with respect to modulation of absorptive and secretory transport was developed from these results.     

P-Glycoprotein Attenuating Effect of Human Intestinal Fluid

Purpose. To evaluate the effect of human intestinal fluid (HIF) on P-glycoprotein (P-gp)-mediated efflux. Methods. HIF was obtained from eight healthy volunteers by duodenal aspiration. HIF was applied at different concentrations (0-75%) to the apical compartment of the Caco-2 system. Cyclosporin A (CsA) was used as a model compound for P-gp mediated efflux. Results. When the bidirectional transport of CsA across Caco-2 monolayers was assessed, a significant polarity in transport could be observed, the absorptive transport being much lower than the secretory transport. Inclusion of HIF resulted in a moderate increase of the absorptive transport, as well as a significant concentration dependent decrease of the secretory transport, without compromising the integrity of the monolayer. Interestingly, a possible gender difference could be detected as inclusion of HIF obtained from female subjects resulted in a decreased absorptive transport of CsA, whereas inclusion of HIF obtained from male subjects resulted in an increased absorptive transport. The P-gp modulating effect of HIF is not caused by a lack of glucose as an energy source for the efflux mechanism when high concentrations of HIF were present in the buffer used. Conclusions. The results of this study indicate that the contribution of P-gp efflux carriers may be overestimated when using salt buffer solutions as transport media. Additionally, it can be concluded that (presently unidentified) components of HIF may attenuate the P-gp mediated intestinal efflux. The clinical significance of this modulating effect remains to be investigated.     

Effect of ion-pair formation with bile salts on the in vitro cellular transport of berberine

The objective of this study was to examine the effect of ion-pair complexation with endogenous bile salts on the transport of a quarternary ammonium organic cationic (OC) drug, berberine, across the Caco-2 and LLC-PK1 cell monolayers. The basolateral-to-apical (BL-AP) transport of berberine in Caco-2 cells was temperature dependent and 10-fold higher than that of the apical-to-basolateral (AP-BL) transport. Similar results were observed for the transport of berberine across the LLC-PK1 cells. Moreover, the BL-AP transport in the Caco-2 cells was significantly reduced by the cis-presence of P-glycoprotein (P-gp) inhibitors such as cyclosporine A, verapamil, and digoxin. These results suggest that an efflux transporter, probably P-gp, is involved in the Caco-2 cell transport. The K_m and V_max values for the carrier-mediated transport were estimated to be 83.4 mM and 7640 pmole/h/cm², respectively. The apparent partition coefficient (APC) of berberine between n-octanol and a phosphate buffer (pH 7.4) was increased by the presence of an organic anion (OA), taurodeoxycholate (TDC, a bile salt), suggesting the formation of a lipophilic ion-pair complex between an OC (berberine) and an OA (TDC). Despite the ion-pair complexation, however, the BL-AP transport of berberine across the Caco-2 and LLC-PK1 cells was not altered by the cis-presence of bile salts or the rat bile juice. This is consistent with the reportedly unaltered secretory transport of a quarternary ammonium compound, tributylmethylammonium (TBuMA), across the Caco-2 cell monolayers in the cis-presence of bile salts or the rat bile juice, but not with our previous report in which the secretory transport of TBuMA across the LLC-PK1 cell was increased in the cis-presence of TDC. Therefore, the effect of ion-pair formation with the bile components or bile salts on the secretory transport of OCs appears to depend on the molecular properties of OCs (e.g., molecular weight, lipophilicity and affinity to relevant transporters) and the characteristics of cell strains (e.g., expression and contribution of responsible transporters to the transport).     

p-Aminohippurate Transport at the Apical Membrane in the OK Kidney Epithelial Cell Line

Purpose. We investigated the characteristics of transport of an organic anion, p-aminohippurate (PAH), at the apical membrane in a kidney epithelial cell line OK. Methods. Efflux and uptake of [¹⁴C]PAH across the apical membrane were measured using OK cell monolayers grown on microporous membrane filters. Results. PAH efflux to the apical side was greater than that to the basolateral side and significantly inhibited by probenecid. Diethyl pyrocarbonate (DEPC), an inhibitor of potential-sensitive organic anion transport, significantly decreased PAH efflux to the apical side. Moreover, PAH efflux to the apical side was significantly decreased on incubation with high potassium buffer, as compared with control condition. Extracellular pH and Cl^- had no effect on PAH efflux across the apical membrane. PAH uptake from the apical side was inhibited by various organic anions, and the inhibition patterns of PAH uptake from the apical and basolateral sides by various dicarboxylates were similar. Conclusions. These results suggested that PAH efflux to the apical side in OK cells was mediated by a potential-sensitive transport system, but not by an anion exchanger. Moreover, PAH uptake from the apical side was mediated by a specific transport system, which interacts with various organic anions and dicarboxylates.     

Construction of a Functional Transporter Analysis System Using <Emphasis Type="BoldItalic">MDR1</Emphasis> Knockdown Caco-2 Cells

Purpose The efflux transporter, P-glycoprotein (P-gp), located in the brush-border membrane of intestinal absorptive cells, reduces the bioavailability of a wide range of orally administered drugs. Using P-gp inhibitors in transport experiments in Caco-2 cell monolayers is widely accepted as an efficient way to estimate the contribution of P-gp to the intestinal absorption of drugs. However, there still remain some arguments that the inhibitors might affect the function of other proteins. Multidrug resistance 1 gene (MDR1) specifically inhibited Caco-2 cells were constructed, therefore, as a better in vitro evaluation system of intestinal drug absorption.Methods The effective sites of RNAi were selected using siRNA libraries and single siRNAs and MDR1 stable knockdown Caco-2 cells were constructed using a tRNA^val-shRNA expression vector.Results In siRNA stably expressed Caco-2 cells, the expression level of MDR1 was reduced at mRNA and protein levels. Transcellular transport studies using digoxin revealed that the P-gp function was suppressed completely, similar to that in verapamil-treated cells.Conclusions MDR1 stable knockdown Caco-2 cells were successfully constructed by RNAi technology. This will consequently allow the development of a selection system for candidate drugs with improved absorption properties.     

Transepithelial transport of prodrugs of the HIV protease inhibitors saquinavir,indinavir, and nelfinavir across Caco-2 cell monolayers

Purpose. This study is dedicated to the permeation of various amino acid-, D-glucose-, and PEG-conjugates of indinavir, saquinavir, and nelfinavir across monolayers of Caco-2 cells as models of the intestinal barrier. This screening is aimed at detecting the most promising prodrugs for improving the intestinal absorption of these protease inhibitors. Methods. The bidirectional transport of the prodrugs was investigated using P-gp-expressing Caco-2 monolayers grown on membrane inserts using high-performance liquid chromatography for quantitation. Results. The L-valyl, L-leucyl, and L-phenylalanyl ester conjugates led to an enhancement of the absorptive flux of indinavir or saquinavir. These results are likely attributable to an active transport mechanism and/or to a decrease of their efflux by carriers such as P-gp. Connection of tyrosine through its hydroxyl, of D-glucose, or of polyethylene glycol decreased their absorptive and secretory diffusion. Conclusions. Conjugation of the protease inhibitors to amino acids constitutes a most appealing alternative that could improve their intestinal absorption and oral bioavailability. Whether it could improve their delivery into the central nervous system remains to be explored. D-Glucose conjugation will most probably not improve their intestinal absorption or their crossing of the blood-brain barrier. If some pharmacologic benefits are to be expected from PEG-protease inhibitor conjugates, they must then be administered intravenously.     

Stereoselective Interactions of Organic Cations with the Organic Cation Transporter in OK Cells

Recent studies have suggested that certain organic cations, such as pindolol and the diastereomers, quinine and quinidine, may be stereoselectively secreted by the kidney in humans. The goal of this study was to determine if the enantiomers of pindolol, verapamil, and disopyramide and the diastereomers, quinine and quinidine, interact stereoselectively with the organic cation transporter in the brush border membrane of the opossum kidney cell line. All organic cations tested inhibited the uptake of tetraethylammonium (TEA). The IC₅₀ values (mean ± SD) were as follows: quinine (17 ± 2 µM), quinidine (51 ± 13 µM), S-(–)-pindolol (23 ± 4 µM), R-( + )-pindolol (30 ± 4 µM), S-(–)-verapamil (0.4 ± 0.04 µM), R-( + )-verapamil (7 ± 2 µM), R-(–)-disopyramide (27 ± 4 µM), and S-( + )-disopyramide (66 ± 12 µM). Each individual organic cation pair showed significant stereoselective differences in their IC₅₀ values, with quinine, S-(–)-pindolol, S-(–)-verapamil, and R-(–)-disopyramide being the more potent species. Both enantiomers of pindolol, quinine, and quinidine appear to exhibit simple competitive inhibition of TEA uptake based upon a derived slope similar to 1.0, using a sigmoidal inhibition model. The enantiomers of verapamil and disopyramide exhibited a slope of much less than 1.0, suggesting a more complex interaction of these organic cations with the TEA transporter. Our results suggest that organic cations stereoselectively interact with the organic cation transporter in the brush border membrane of OK cells. Stereoselective interactions with the organic cation transporter may be responsible for the stereoselective renal clearance of basic drugs known to occur in humans.     

Carrier-mediated transport in the hepatic distribution and elimination of drugs,with special reference to the category of organic cations

Carrier-mediated transport of drugs occurs in various tissues in the body and may largely affect the rate of distribution and elimination. Saturable translocation mechanisms allowing competitive interactions have been identified in the kidneys (tubular secretion), mucosal cells in the gut (intestinal absorption and secretion), choroid plexus (removal of drug from the cerebrospinal fluid), and liver (hepatobiliary excretion). Drugs with quaternary and tertiary amine groups represent the large category of organic cations that can be transported via such mechanisms. The hepatic and to a lesser extent the intestinal cation carrier systems preferentially recognize relatively large molecular weight amphipathic compounds. In the case of multivalent cationic drugs, efficient transport only occurs if large hydrophobic ring structures provide a sufficient lipophilicity-hydrophilicity balance within the drug molecule. At least two separate carrier systems for hepatic uptake of organic cations have been identified through kinetic and photoaffinity labeling studies. In addition absorptive endocytosis may play a role that along with proton-antiport systems and membrane potential driven transport may lead to intracellular sequestration in lysosomes and mitochondria. Concentration gradients of inorganic ions may represent the driving forces for hepatic uptake and biliary excretion of drugs. Recent studies that aim to the identification of potential membrane carrier proteins indicate multiple carriers for organic anions, cations, and uncharged compounds with molecular weights around 50,000 Da. They may represent a family of closely related proteins exhibiting overlapping substrate specificity or, alternatively, an aspecific transport system that mediates translocation of various forms of drugs coupled with inorganic ions. Consequently, extensive pharmacokinetic interactions can be anticipated at the level of uptake and secretion of drugs regardless of their charge.     

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.     

Evidence for Different ABC-Transporters in Caco-2 Cells Modulating Drug Uptake

Purpose. Secretory systems contribute to drug absorption in the gastrointestinal tract. The purpose of this study was the identification of members of the ATP binding cassette superfamily of secretory transport proteins that may potentially modulate drug absorption in Caco-2 cells, which are an important cellular model predicting enteral absorption of drugs. Methods. Kinetic studies as well as PCR- and Western blot studies with confluent epithelial layers of human Caco-2 cells. Results. The study demonstrates functional expression of multidrug resistance related protein (MRP) and P-glycoprotein (P-gp) in Caco-2 cells: 1) Efflux studies with the MRP specific substrate glutathion-methylfluorescein (GS-MF) showed functional activity of MRP in Caco-2 cells preloaded with the metabolic precursor of GS-MF, chloro-methylfluoresceine-diacetate, CMFDA. Excretion of GS-MF was decreased in presence of the MRP-blocker MK-571.2) Transport experiments with cyclosporin A demonstrated the functional activity of P-gp. Cellular accumulation was increased in presence of the P-gp blocking agent SDZ-PSC 833.3) The expression of the 190 kDa protein MRP and the 170 kDa protein P-gp in Caco-2 cells was shown by Western blot analysis with specific monoclonal antibodies. 4) The expression of MRP-mRNA in Caco-2 cells was detected by RT-PCR and compared with the MRP over-expressing cell line H69AR. MRP primers recognize specifically human MRP1 (GenBank accession number L05628), but not all other published sequences of MRP (MRP2-MRP6). P-gp expression on mRNA-level was also confirmed by RT-PCR. Conclusions. The data demonstrate that besides P-gp, multidrug resistance related protein (MRP) is functionally expressed in Caco-2 cells and contributes to the active excretion of substrates in this cell line.     

Organic Cation Transport in Rabbit Alveolar Epithelial Cell Monolayers

Purpose. To characterize organic cation (OC) transport in primary cultured rabbit alveolar epithelial cell monolayers, using [^l4C]-guanidine as a model substrate. Methods. Type II alveolar epithelial cells from the rabbit lung were isolated by elastase digestion and cultured on permeable filters pre-coated with fibronectin and collagen. Uptake and transport studies of [¹⁴C]-guanidine were conducted in cell monolayers of 5 to 6 days in culture. Results. The cultured alveolar epithelial cell monolayers exhibited the characteristics of a tight barrier. [¹⁴C]-Guanidine uptake was temperature dependent, saturable, and inhibited by OC compounds such as amiloride, cimetidine, clonidine, procainamide, propranolol, tetraethyl-ammonium, and verapamil. Apical guanidine uptake (K_m = 129 ± 41 M, V_max = 718 ± 72 pmol/mg protein/5 min) was kinetically different from basolateral uptake (K_m = 580 ± 125 (M, V_max = 1,600 ± 160 pmol/mg protein/5 min). [¹⁴C]-Guanidine transport across the alveolar epithelial cell monolayer in the apical to basolateral direction revealed a permeability coefficient (P_app) of (7.3 ± 0.4) × 10^-7 cm/sec, about seven times higher than that for the paracellular marker [¹⁴C]-mannitol. Conclusions. Our findings are consistent with the existence of carrier-mediated OC transport in cultured rabbit alveolar epithelial cells.     

Guanidine Transport in a Human Choriocarcinoma Cell Line (JAR)

Purpose. Many endogenous substances and xenobiotics are organic cations. Transplacental transport of organic cations is an important determinant of the delivery of these compounds to the fetus. The aim of this study was to determine the mechanisms of organic cation transport using the human choriocarcinoma cell line (JAR) as a model system with [¹⁴C]guanidine as a ligand. Methods. Uptake studies of [^l4C]guanidine were carried out in JAR cell monolayers on day 2 after plating. Results. [¹⁴C]guanidine uptake was temperature dependent, saturable (K_m = 167 M) and inhibited by many organic cations including amiloride, cimetidine, quinine, quinidine and nicotine. [^l4C]guanidine uptake exhibited a counterflux phenomenon indicative of a carrier-mediated process. The uptake of [¹⁴C]guanidine was sodium and pH-independent and could be driven by an inside-negative membrane potential difference. Conclusions. This is the first demonstration of an electrogenic guanidine transporter in a human cell culture model. This transporter may play a role in the transplacental transport of many clinically used drugs and xenobiotics.     

Influence of Passive Permeability on Apparent P-glycoprotein Kinetics

Purpose. The objectives of this work were to evaluate the importance of moderate passive permeability on apparent P-glycoprotein (P-gp) kinetics, and demonstrate that inspection of basolateral to apical and apical to basolateral (BL-AP/AP-BL) permeability ratios may result in a compound being overlooked as a P-gp substrate and inhibitor of another drug's transport via P-gp inhibition. Methods. The permeability ratios of nicardipine, vinblastine, cimetidine, and ranitidine were determined across Caco-2 monolayers that express P-gp, in the presence and absence of the specific P-gp inhibitor, GF120918. In addition, the permeability ratio of vinblastine was studied after pretreatment of Caco-2 monolayers with nicardipine, ranitidine, or cimetidine. Similar studies were repeated with hMDR1-MDCK monolayers. Results. The permeability ratios for cimetidine and vinblastine were >2. The permeability ratios for nicardipine and ranitidine were close to unity, and were not affected by the addition of GF120918. Based solely on ratios, only compounds with moderate transcellular permeability (vinblastine and cimetidine) would be identified as P-gp substrates. Although the permeability ratios appeared to be unity for nicardipine and ranitidine, both compounds affected the permeability of vinblastine, and were identified as substrates and inhibitors of P-gp. Studies performed in hMDR1-MDCK cells confirmed these experimental results. Data were explained in the context of a kinetic model, where passive permeability and P-gp efflux contribute to overall drug transport. Conclusions. Moderate passive permeability was necessary for P-gp to reduce the AP-BL drug permeability. Inspection of the permeability ratio after directional transport studies did not effectively identify P-gp substrates that affected the P-gp kinetics of vinblastine. Because of the role of passive permeability, drug interaction studies with known P-gp substrates, rather than directional permeability studies, are needed to elucidate a more complete understanding of P-gp kinetics.     

Effects of dietary flavonoids on the transport of cimetidine via P-glycoprotein and cationic transporters in Caco-2 and LLC-PK1 cell models

1.?The hypotheses tested were to study cimetidine as a substrate of P-glycoprotein (P-gp) and organic cation transport systems and the modulatory effects of eight flavonoid aglycones and glycosides on these transport systems using Caco-2 and LLC-PK1 cells.

2.?Transport and uptake experiments of (20 µM) ³H-cimetidine were performed with and without co-exposure to quercetin, quercetrin, rutin, naringenin, naringin, genistein, genistin, and xanthohumol. Co-treatment decreased basolateral to apical (B to A) permeability (P_app) of cimetidine from 2.02 to 1.24 (quercetin), 1.06 (naringenin), 1.24 (genistein), and 0.96 (xanthohumol) × 10^?6 cm s^?1 in Caco-2 cells and from 10.76 to 1.65 (quercetin), 2.05 (naringenin), 2.88 (genistein), and 1.95 (xanthohumol) × 10^?6 cm s^?1 in LLC-PK1 cells. Genistin significantly reduced B to A P_app of cimetidine to 1.24 × 10^?6 cm s^?1 in Caco-2 cells. Basolateral intracellular uptake rate of cimetidine was enhanced 145–295% when co-treated with flavonoids. Co-treatment with P-glycoprotein and organic cation transporter inhibitors, verapamil and phenoxybenzamine, resulted in reduced B to A permeability and slower basolateral intracellular uptake rate of cimetidine. Intracellular uptake rate of ¹⁴C-tetraethylammonium (TEA) was reduced in the presence of quercetin, naringenin and genistein in LLC-PK1 cells.

3.?In conclusion, quercetin, naringenin, genistein, and xanthohumol reduced P-gp-mediated transport and increased the basolateral uptake rate of cimetidine. Quercetin, naringenin, genistein, but not xanthohumol, reduced intracellular uptake rate of TEA in LLC-PK1 cells. These results suggest that flavonoids may have potential to alter the disposition profile of cimetidine and possibly other therapeutics that are mediated by P-gp and/or cation transport systems.     

Substrate Specificity and Mechanism of the Intestinal Clonidine Uptake by Caco-2 Cells

Purpose This study was performed to characterize the substrate specificity and mechanism of the intestinal clonidine transport. Methods Uptake of [³H]clonidine into Caco-2 cells was investigated. Interaction with drugs was studied in competition assays. Results Uptake of [³H]clonidine was linear for up to 2 min, Na⁺-independent, and insensitive to changes in membrane potential, but strongly H⁺-dependent. The uptake rate of clonidine was saturable with kinetic parameters of 0.5 ± 0.1 mM (K_t) and 16.6 ± 1.8 nmol/2 min per mg of protein (V_max) at an outside pH of 7.5. Many drugs such as clonidine, guanabenz, methamphetamine, imipramine, clomipramine, nortriptyline, quinine, xylazine, ephedrine, and diphenhydramine strongly inhibited the [³H]clonidine uptake with K_i values between 0.15 and 1 mM. Conclusions Clonidine is transported by a carrier-mediated process. Substrate specificity and mechanism are very similar to the transport described in blood–brain barrier endothelial cells. The transport characteristics do not correspond to carriers for organic cations of the SLC22 family or the choline transporters CHT1 and CLT1. The system might be identical to the H⁺/tertiary amine antiporter. It interacts with a large number of both hydrophilic and lipophilic cationic drugs, and also, interestingly, with opiates.     

Role of P-Glycoprotein in Restricting Propranolol Transport in Cultured Rabbit Conjunctival Epithelial Cell Layers

Purpose. To determine the role of P-glycoprotein (P-gp) in propranololtransport in cultured rabbit conjunctival epithelial cell layers (RCEC). Methods. The localization of P-gp in the cultured RCEC as well asin the excised conjunctiva was determined by immunofluorescencetechnique. The role of P-gp in transepithelial transport and uptake ofpropranolol in conjunctival epithelial cells cultured on Transwell filterswas evaluated in the presence and absence of P-gp competing substrates, ananti-P-gp monoclonal antibody (4E3 mAb), or a metabolicinhibitor, 2, 4-dinitrophenol (2,4-DNP). Results. Immunofluorescence studies revealed positive staining in theapical membrane of cultured RCEC and in the apical surface of thesuperficial cell layers in the excised conjunctiva, but not the basolateralmembrane of cultured RCEC. Transport of propranolol showed preferencein the basolateral-to-apical direction. The net secretory flux wassaturable with a K_m of 71.5 ± 24.0 nM and a J_max of 1.45 ± 0.17pmol/cm²/hr. Cyclosporin A, progesterone, rhodamine 123, verapamil,4E3 mAb and 2,4-DNP all increased apical 50 nM propranolol uptakeby 43% to 66%. On the other hand, neither -blockers (atenolol,metoprolol, and alprenolol) nor organic cation transporter substrates(tetraethylammonium (TEA) and guanidine), affected apical 50 nMpropranolol uptake. Conclusions. The energy-dependent efflux pump P-gp appears to bepredominantly located on the apical plasma membrane of the conjunctivalepithelium. It may play an important role in restricting the conjunctivalabsorption of some lipophilic drugs.