Apical Research Articles

Multiple organic cation transporters contribute to the renal transport of sulpiride.

Sulpiride, a selective dopamine D2 receptor blocker, is used widely for the treatment of schizophrenia, depression and gastric/duodenal ulcers. Because the great majority of sulpiride is positively charged at physiological pH7.4, and ~70% of the dose recovered in urine is in the unchanged form after human intravenous administration of sulpiride, it is believed that transporters play an important role in the renal excretion of sulpiride. The aim of the present study was to explore which transporters contribute to the renal disposition of sulpiride. The results demonstrated that sulpiride was a substrate of human carnitine/organic cation transporter 1 (hOCTN1) and 2 (hOCTN2), human organic cation transporter 2 (hOCT2), human multidrug and toxin efflux extrusion protein 1 (hMATE1) and 2-K (hMATE2-K). Sulpiride accumulation from the basolateral (BL) to the apical (AP) side in MDCK-hOCT2/pcDNA3.1 cell monolayers was much greater than that in MDCK-hOCT2/hMATE1 cells, and cimetidine dramatically reduced the intracellular accumulation of sulpiride from BL to AP. In addition, the accumulation of sulpiride in mouse primary renal tubular cells (mPRTCs) was markedly reduced by inhibitors of Oct2 and Octns. The results implied that OCTN1, OCTN2, OCT2, MATE1 and MATE2-K probably contributed to the renal transfer of sulpiride, in which OCT2 mediated the uptake of sulpiride from the bloodstream to the proximal tubular cells, while MATEs contributed to the sulpiride efflux from the proximal tubular cells to the renal lumen, and OCTNs participated in both renal secretion and reabsorption.

The Blood-Brain Barrier Permeability of Six Indole Alkaloids from Uncariae Ramulus Cum Uncis in the MDCK-pHaMDR Cell Monolayer Model.

Uncariae Ramulus Cum Uncis (URCU) is a widely used traditional Chinese medicine, and is reported to have various central nervous system effects. Alkaloids have been demonstrated to be the predominant pharmacological active components of URCU. In order to evaluate the blood-brain barrier (BBB) permeability and transport mechanism of six typical indole alkaloids from URCU, the MDCK-pHaMDR cell monolayer model was used as an in vitro surrogate model for BBB. The samples were analyzed by high-performance liquid chromatography, and the apparent permeability coefficients (Papp) were calculated. Among the six alkaloids, isorhynchophylline (2), isocorynoxeine (4), hirsutine (5) and hirsuteine (6) showed high permeability, with Papp values at 10−5 cm/s level in bidirectional transport. For rhynchophylline (1) and corynoxeine (3), they showed moderate permeability, with Papp values from the apical (AP) side to the basolateral (BL) side at 10−6 cm/s level and efflux ratio (Papp BL→AP/Papp AP→BL) above 2. The time- and concentration-dependency experiments indicated that the main mechanism for 2, 4, 5 and 6 through BBB was passive diffusion. The efflux mechanism involved in the transports of compounds 1 and 3 could be reduced significantly by verapamil, and molecular docking screening also showed that 1 and 3 had strong bindings to P-glycoprotein. This study provides useful information for predicting the BBB permeability for 1–6, as well as better understanding of their central nervous system pharmacological activities.

Bioaccessibility, Intestinal Permeability and Plasma Stability of Isorhamnetin Glycosides from Opuntia ficus-indica (L.).

Isorhamnetin glycosides are representative compounds of Opuntia ficus-indica that possess different biological activities. There is slight information about the changes in bioaccessibility induced by the glycosylation pattern of flavonoids, particularly for isorhamnetin. In this study, the bioaccessibility and permeability of isorhamnetin glycosides extracted from O. ficus-indica were contrasted with an isorhamnetin standard. Also, the plasma stability of these isorhamnetin glycosides after intravenous administration in rats was evaluated. Recoveries of isorhamnetin after oral and gastric digestion were lower than that observed for its glycosides. After intestinal digestion, isorhamnetin glycosides recoveries were reduced to less than 81.0%. The apparent permeability coefficient from apical (AP) to basolateral (BL) direction (Papp(AP-BL)) of isorhamnetin was 2.6 to 4.6-fold higher than those obtained for its glycosides. Isorhamnetin diglycosides showed higher Papp(AP-BL) values than triglycosides. Sugar substituents affected the Papp(AP-BL) of the triglycosides. Isorhamnetin glycosides were better retained in the circulatory system than the aglycone. After intravenous dose of the isorhamnetin standard, the elimination half-life was 0.64 h but increased to 1.08 h when the O. ficus-indica extract was administered. These results suggest that isorhamnetin glycosides naturally found in O. ficus-indica could be a controlled delivery system to maintain a constant plasmatic concentration of this important flavonoid to exert its biological effects in vivo.

Absorption and transport of isoflavonoid compounds from Tongmai formula across human intestinal epithelial (Caco-2) cells in vitro

Tongmai formula (TMF) is a drug combination of three components including Puerariae Lobatae Radix [roots of Pueraria lobata], Salviae Miltiorrhizae Radix (roots of Salvia miltiorrhiza) and Chuanxiong Rhizoma (rhizomes of Ligusticum chuanxiong) in a weight ratio of 1∶1∶1. The absorption and transport of isoflavonoid compounds from Tongmai formula across human intestinal epithelial (Caco-2) cells in vitro were studied in this paper. The assay isoflavonoid compounds include daidzein, formononetin, 5-hydroxylononin, ononin, daidzin, 3'-methoxypuerarin, genistin, puerarin, formononetin-8-C-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, formononetin-7-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, lanceolarin, kakkanin, daidzein-7,4'-di-O-β-D-glucopyranoside, mirificin, 3'-hydroxypuerarin, 3'-methoxydaidzin, formononetin-8-C-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranoside, genistein-8-C-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, genistein-7-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside (ambocin), 3'-hydroxymirificin, 6″-O-β-D-xylosylpuerarin, biochanin A-8-C-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, 3'-methoxydaidzein-7,4'-di-O-β-D-glucopyranoside, daidzein-7-O-β-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside, and daidzein-7-O-α-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside. By using human Caco-2 monolayer as an intestinal epithelial cell model in vitro, the permeability of above-mentioned 25 isoflavonoids in TMF were studied from the apical (AP) side to basolateral (BL) side or from the BL side to AP side. The assay compounds were determined by reversed phased high-performance liquid chromatography (HPLC) coupled with UV detector. Transport parameters and apparent permeability coefficients (Papp) were then calculated and and compared with those of propranolol and atenolol, which are the transcellular transport marker and as a control substance for high and poor permeability, respectively. The Papp values of daidzein and formononetin were (2.55±0.03) ×10⁻⁵,(3.06±0.01) ×10⁻⁵ cm•s⁻¹ from AP side to BL side, respectively, and (2.62±0.00) ×10⁻⁵, (2.65±0.11) ×10⁻⁵ cm•s⁻¹ from BL side to AP side, respectively. Under the condition of this experiment, the Papp value was (2.66±0.32) ×10⁻⁵ cm•s⁻¹ for propranolol and (2.34±0.10) ×10⁻⁷ cm•s⁻¹ for atenolol. The Papp values of daidzein and formononetin were at a same magnitude with those of propranolol. And the Papp values of other 23 isoflavonoid compounds were at a same magnitude with those of atenolol. On the other hand, the rats of Papp AP→BL/Papp BL→AP of daidzein and formononetin on the influx transport were 0.97 and 1.15, respectively. It can be predicted that daidzein and formononetin can be absorbed across intestinal epithelial cells to go to the body circulation by the passive diffusion mechanism and they were assigned to the well-absorbed compounds. Other 23 isoflavonoid compounds were assigned to the poorly absorbed compounds. Because of the rats of Papp AP→BL/Papp BL→AP of 5-hydroxylononin, genistin, lanceolarin, kakkanin, and genistein-7-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside were 0.18, 0.28, 0.45, 0.38, 0.49, they may have been involved in the efflux mechanism in Caco-2 cells monolayer model from the BL side to AP side direction.

P 179 - Antioxidant activity and intestinal absorption of apigenin and its potassium salt derivative in Caco-2 cell monolayers

Apigenin is a common bioactive flavonoid found in a variety of fruits, plants and vegetables. Many pharmacological activities of apigenin have been identified, but its low water solubility limits clinical evidence on its oral bioavailability. This study estimated the antioxidant activity of apigenin and its potassium salt derivative (apigenin-K) and the intestinal permeability of both compounds in Caco-2 cell monolayers. The antioxidant activity was evaluated with different methods: trolox equivalent antioxidant capacity (TEAC) and ferric reducing-antioxidant power (FRAP). Intestinal permeability coefficient was estimated in Caco-2 cell monolayer model with the addition of apigenin or apigenin-K to the apical (AP) or basolateral (BL) site. Apigenin exhibited a higher antioxidant activity than apigenin-K. Intestinal absorption evaluation corroborated the low permeability of both apigenin and apigenin-K. These results indicate that apigenin and apigenin-K may be considered as antioxidant ingredients for oral administration. Their low absorption estimation suggests that encapsulation may be necessary to enhance apigenin bioavailability for oral applications.

Left Ventricular Mechanics Assessed by 2-dimensional Speckle Tracking Echocardiography in Children and Adolescents With Idiopathic Scoliosis.

A retrospective review of data collected prospectively. The study aimed to assess the possible effects of idiopathic scoliosis on left ventricular (LV) myocardial performance in children and adolescent patients. Spine anomaly may impair cardiorespiratory function. It has been confirmed that respiratory function decreased in patients with scoliosis. However, limited study of the effect of scoliosis on heart function has been published, and no assessment of myocardial deformation of these patients has been reported to our knowledge. Forty-one patients with a median age of 16 years and a median Cobb's angle of 75 degrees were studied. LV myocardial deformation was evaluated using 2-dimensional speckle-tracking echocardiography. The results were compared with those of 33 controls. The correlations between Cobb's angle and echocardiographic parameters in patients were also explored. Compared with controls, patients had significantly lower global LV longitudinal systolic strain in all 3 views (4 chamber, 2 chamber, and 3 chamber views), lower global radial and circumferential strains in mitral valve (MV) level and papillary muscle (PM) level; reduced LV longitudinal early diastolic strain rate (SRe) in all 3 longitudinal views and reduced radial SRe in MV level and reduced circumferential SRe in apical (AP) level; decreased LV longitudinal late diastolic strain rate in 4-chamber view and decreased radial ones in all 3 short-axis levels. Among patients, Cobb's angle correlated negatively with LV global radial strain of MV level (r=-0.37, P=0.02), global circumferential strain of MV level (r=-0.35, P=0.03), and global circumferential strain of PM level (r=-0.49, P=0.001), whereas positively with LV longitudinal SRa of 4-chamber view (r=0.46, P=0.003), longitudinal SRa of 2-chamber view (r=0.49, P=0.001), and circumferential SRa of AP level (r=0.35, P=0.02). LV mechanics are impaired in patients with idiopathic scoliosis, which correlate with the severity of scoliosis. Our findings suggest the need and provide a mechanical basis for further studies in these patients.

Transport of a Novel Angiotensin-I-Converting Enzyme Inhibitory Peptide Ala-His-Leu-Leu Across Human Intestinal Epithelial Caco-2 Cells.

The transport behavior and absorption mechanism of Ala-His-Leu-Leu (AHLL) intestinal absorption in Caco-2 cell monolayers were clarified systemically. The safe absorptive concentration of AHLL was 200 μg/mL, which was determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. The permeation of AHLL was concentration dependent in a bidirectional transfer and reached a plateau at 90 min. The efflux ratio was above 0.5, suggesting that AHLL was absorbed by both active transport and passive diffusion. The apparent permeability coefficients (Papp) of AHLL both from the apical (AP) to basolateral (BL) side (PappAB) and from the BL to AP side (PappBA) decreased when the temperature was lowered from 37°C to 4°C.The uptake of AHLL was more at pH 7.4 than at other pHs. Both verapamil and (E)-3-[[[3-[2-(7-chloro-2- quinolinyl) ethenyl] phenyl]-[[(3-dimethyl amino)-3-oxopropyl]thio] methyl] thio]-propanoic acid (MK571) inhibited the absorption of AHLL, indicating that P-glycoprotein and multi-drug resistant proteins (MRPs) were all involved in AHLL secretion, especially multi-drug resistant protein 2 (MRP2). AHLL was transported through both trans- and paracellular pathways across the Caco-2 cell monolayer. This work first elucidates the AHLL absorption mechanism in Caco-2 cells and provides the basis for future studies on the improvement of bioavailability.

Postsystolic Shortening Is Associated with Altered Right Ventricular Function in Children after Tetralogy of Fallot Surgical Repair.

The aim of the study was to determine whether segmental interactions, as expressed by postsystolic shortening (PSS), affects RV mechanics and are connected with impaired systolic and diastolic function in rTOF children. Patients and Methods: 55 rTOF adolescent (study group), and 34 healthy volunteers (control group) were examined using classical Doppler flow (Doppler), Tissue Doppler Imaging (TDI) and Speckle Tracking Echocardiography (STE). PSS was found to occur when time to peak (TTP) was longer than pulmonary valve closure time (PVCT). TTP and strain were derived from RV lateral segments—basal (BL), medial (ML) and apical (AL) in STE. PVCT was measured from the beginning of QRS complex in the ECG to the termination of Doppler flow at the pulmonary valve. TDI was obtained at the lateral tricuspid annulus site and the systolic (S′), early (E′) and late diastolic (A’) peak velocities were measured along with isovolumic contraction (IVCT), and relaxation (IVRT) time. PW was used to measure early tricuspid inflow velocity (E) for calculating the E/E’ ratio. The TDI data in patients with PSS presence (TTP>PVCT) and those in whom it did not occur (TTP≤PVCT) were compared. Results: PSS in BL, ML and AL were observed respectively in: 27(51,9%), 9 (18%), and 8 (16,7%) patients. Mean values of TTP in BL, ML, and AL were respectively: 420.6±55.5ms, 389.8±50.0ms and 366.7±59.0ms. PVCT mean value was 396.6±33.5ms. In the study group, the mean E’ in TTP>PVCT was significantly lower (4.8±1.8 cm/s) compared to mean E’ in TTP≤PVCT (8.4±2.6 cm/s), p<0.01. The average E/E’ was significantly higher in TTP>PCVT than in TTP≤PVCT, respectively 21.6±7.3 vs 12.2±5.1, p<0.05. IVRT was significantly prolonged in TTP>PVCT compared to IVRT in TTP≤PVCT, respectively 95.9±38.7 vs 77.0±35.1, p<0.05. Furthermore, in TTP>PVCT, significantly higher strain in BL (-28.8±8.7%) was observed when compared to that parameter in TTP≤PVCT (-35.3±13.1%), p <0.05. Conclusions: Tissue Doppler Echocardiography and Speckle Tracking Echocardiography are useful techniques for detecting regional systolic and diastolic dysfunction in children after Tetralogy of Fallot surgical repair. Postsystolic shortening in the basal lateral segment is commonly seen in children after the Tetralogy of Fallot surgical repair, and is associated with altered right ventricular systolic and diastolic function.

In Vitro Evaluation of Absorption Characteristics of Peramivir for Oral Delivery.

Peramivir is a novel antiviral agent approved for the treatment of severe influenza. However, the development of oral formulation of peramivir has been severely hurdled by poor bioavailability (human, ≤3%). The present work aims to evaluate oral permeability characteristics of peramivir. In vitro gastrointestinal stability, metabolic stability in human intestinal S9 fraction and Caco-2 permeability were performed. The liquid chromatography with tandem mass spectrometric (LC-MS/MS) was used to quantify peramivir in buffer and biological sample. Using GastroPlus™ software, intestinal effective permeability coefficient (P eff) of peramivir was estimated. Our results indicated that peramivir maintained stability in pH 5.5 and 7.4 buffers, fasted state simulated gastric fluid and fasted state simulated intestinal fluid, and human intestinal S9 fractions. The apparent permeability coefficient (P app) values of peramivir (10μM) were 3.29±0.73×10-7cm/s in a Caco-2 cell model. In vivo intestinal effective permeability coefficient (P eff) was estimated to be 0.06×10-4cm/s. Furthermore, co-incubating with cyclosporine, mitoxantrone, rifampicin, or paroxetine, the apical (AP) to basolateral (BL) flux of peramivir decreased (p<0.05). The efflux and influx of peramivir was not significantly affected with co-incubation with verapamil, MK-571, or diclofenac (p>0.05). These results revealed that carrier-mediated transports, including OATP1B (organic anion transport 1B) and OCT1 (organic cation transport 1), might be involved in the absorption of peramivir. In conclusion, our results provide insight into the poor oral bioavailability of peramivir. Peramivir can be classified as a BCS-III (high solubility/low permeability) and BDDCS-III high solubility/poor metabolism) drug. The oral bioavailability of peramivir primarily depends on its permeability across cell membranes. Both of passive and active transports are involved in the permeability of peramivir.

In Vivo Exposure of Kaempferol Is Driven by Phase II Metabolic Enzymes and Efflux Transporters.

Kaempferol is a well-known flavonoid; however, it lacks extensive pharmacokinetic studies. Phase II metabolic enzymes and efflux transporters play an important role in the disposition of flavonoids. This study aimed to investigate the mechanism by which phase II metabolic enzymes and efflux transporters determine the in vivo exposure of kaempferol. Pharmacokinetic analysis in Sprague-Dawley rats revealed that kaempferol was mostly biotransformed to conjugates, namely, kaempferol-3-glucuronide (K-3-G), kaempferol-7-glucuronide (K-7-G), and kaempferol-7-sulfate, in plasma. K-3-G represented the major metabolite. Compared with that in wild-type mice, pharmacokinetics in knockout FVB mice demonstrated that the absence of multidrug resistance protein 2 (MRP2) and breast cancer resistance protein (BCRP) significantly increased the area under the curve (AUC) of the conjugates. The lack of MRP1 resulted in a much lower AUC of the conjugates. Intestinal perfusion in rats revealed that the glucuronide conjugates were mainly excreted in the small intestine, but 7-sulfate was mainly excreted in the colon. In Caco-2 monolayers, K-7-G efflux toward the apical (AP) side was significantly higher than K-3-G efflux. In contrast, K-3-G efflux toward the basolateral (BL) side was significantly higher than K-7-G efflux. The BL-to-AP efflux was significantly reduced in the presence of the MRP2 inhibitor LTC4. The AP-to-BL efflux was significantly decreased in the presence of the BL-side MRPs inhibitor MK571. TheBCRP inhibitor Ko143 decreased the glucuronide conjugate efflux. Therefore, kaempferol is mainly exposed as K-3-G in vivo, which is driven by phase II metabolic enzymes and efflux transporters (i.e., BCRP and MRPs).

The octanol/water distribution coefficients of ardipusilloside-I and its metabolites, and their permeation characteristics across Caco-2 cell monolayer.

BackgroundArdipusilloside-I (ADS-I) is a triterpenoid saponin extracted from Chinese medicinal herb Ardisiapusill A. DC. Previous studies have demonstrated the potent anti-tumor activities of ADS-I both in vitro and in vivo, and its main metabolites (M1 and M2) from human intestinal bacteria. However, the physicochemical properties and intestinal permeation rate of ADS-I and its metabolites are not understood. In this study, the octanol/water distribution coefficients (logP) of ADS-I and metabolites were investigated using standard shake flask technique, and their permeability properties was investigated across Caco-2 cells monolayer.ResultsThe logP of ADS-I, M1 and M2 was −0.01, 0.95 ± 0.04, 1.57 ± 0.11, respectively. The Papp values of ADS-I, M1 and M2 (in 10 μmol/L) across Caco-2 cell monolayers from the apical (AP) to basolateral (BL) direction were 1.88 ± 0.21 × 10−6 cm·s−1, 4.30 ± 0.43 × 10−6 cm·s−1, 4.74 ± 0.47 × 10−6 cm·s−1, respectively.ConclusionOur data indicated that ADS-I has the poorer intestinal absorption than its metabolites (M1 and M2) in these experimental systems, suggesting that the metabolites of ADS-I may be the predominant products absorbed by the intestine when ADS-I is administered orally.

Four cation-selective transporters contribute to apical uptake and accumulation of metformin in Caco-2 cell monolayers.

Metformin is the frontline therapy for type II diabetes mellitus. The oral bioavailability of metformin is unexpectedly high, between 40 and 60%, given its hydrophilicity and positive charge at all physiologic pH values. Previous studies in Caco-2 cell monolayers, a cellular model of the human intestinal epithelium, showed that during absorptive transport metformin is taken up into the cells via transporters in the apical (AP) membrane; however, predominant transport to the basolateral (BL) side occurs via the paracellular route because intracellular metformin cannot egress across the BL membrane. Furthermore, these studies have suggested that the AP transporters can contribute to intestinal accumulation and absorption of metformin. Transporter-specific inhibitors as well as a novel approach involving a cocktail of transporter inhibitors with overlapping selectivity were used to identify the AP transporters that mediate metformin uptake in Caco-2 cell monolayers; furthermore, the relative contributions of these transporters in metformin AP uptake were also determined. The organic cation transporter 1, plasma membrane monoamine transporter (PMAT), serotonin reuptake transporter, and choline high-affinity transporter contributed to approximately 25%, 20%, 20%, and 15%, respectively, of the AP uptake of metformin. PMAT-knockdown Caco-2 cells were constructed to confirm the contribution of PMAT in metformin AP uptake because a PMAT-selective inhibitor is not available. The identification of four intestinal transporters that contribute to AP uptake and potentially intestinal absorption of metformin is a significant novel finding that can influence our understanding of metformin pharmacology and intestinal drug-drug interactions involving this highly prescribed drug.

Evaluation of physicochemical properties and intestinal permeability of six dietary polyphenols in human intestinal colon adenocarcinoma Caco-2 cells.

Phenolic compounds are common ingredients in many dietary supplements and functional foods. However, data concerning physicochemical properties and permeability of polyphenols on the intestinal epithelial cells are scarce. The aims of this study were to determine the experimental partition coefficient (Log P), and parallel artificial membrane permeability assay (PAMPA), to characterize the bi-directional transport of six phenolic compounds viz. caffeic acid, chrysin, gallic acid, quercetin, resveratrol and rutin in Caco-2 cells. The experimental Log P values of six polyphenols were correlated (R (2) = 0.92) well with the calculated Log P values. The apparent permeability (P app) range of all polyphenols in PAMPA for the apical (AP) to basolateral (BL) was 1.18 ± 0.05 × 10(-6) to 5.90 ± 0.16 × 10(-6) cm/s. The apparent Caco-2 permeability (P app) range for the AP-BL was 0.96 ± 0.03 × 10(-6) to 3.80 ± 0.45 × 10(-6) cm/s. The efflux ratio of P app (BL → AP) to P app (AP → BL) for all phenolics was <2, suggesting greater permeability in the absorptive direction. Six compounds exhibited strong correlations between Log P and PAMPA/Caco-2 cell monolayer permeation data. Dietary six polyphenols were poorly absorbed through PAMPA and Caco-2 cells, and their transepithelial transports were mainly by passive diffusion.

Lack of P-Glycoprotein-Mediated Efflux and the Potential Involvement of an Influx Transport Process Contributing to the Intestinal Uptake of Deltamethrin, cis-Permethrin, and trans-Permethrin

The effectiveness and widespread use of pyrethroid insecticides has lead to concerns regarding their safety. Human ingestion of these potentially neurotoxic compounds is typically through hand-to-mouth contact or consumption of contaminated foods. A substantial proportion of ingested pyrethroids are eliminated in feces, suggesting that absorption is limited, possibly by the action of the efflux transporter P-glycoprotein (P-gp). We utilized caco-2 cells as a model system for intestinal enterocytes and qualitatively and quantitatively assessed the transport of deltamethrin (DLM), cis-permethrin (CPM), and trans-permethrin (TPM). Caco-2 cell uptake of the P-gp substrate R6G was increased by the P-gp inhibitors cyclosporine A (CSA) and ritonavir but not by DLM, CPM, and TPM. Unexpectedly, CSA and ritonavir significantly reduced the uptake of DLM, CPM, and TPM. Permeability coefficients (P app) and directional flux of DLM, CPM, or TPM were greater in the absorptive than the secretory (efflux) direction when measured across caco-2 monolayers grown on Transwell inserts. When CSA was applied to the monolayers' apical (AP) side, the AP to basolateral (BL) P app was significantly reduced, with no change in the BL to AP P app. Kinetic analysis demonstrated saturable transport kinetics for all 3 pyrethroids. These findings indicate that the cellular uptake of DLM, CPM, and TPM is not limited by P-gp efflux but undergo absorptive influx transport as a contributing mechanism for cellular uptake. However, the overall P app values for DLM, CPM, and TPM are consistent with the low permeability/low absorption compound mannitol, suggesting limited gastrointestinal absorption potential.

Kinetics of manganese transport and gene expressions of manganese transport carriers in Caco-2 cell monolayers

Two experiments were conducted to investigate the kinetics of manganese (Mn) transport in Caco-2 cell monolayers and the gene expressions of Mn transport carriers in apical (AP) and basolateral (BL) membranes. In experiment 1, the cells were treated with the medium containing 146 μmol/L of Mn (MnSO4·H2O). Both the uptake and transport of Mn from AP-BL or from BL-AP at different time-points were assessed to determine the optimal time for kinetics of Mn transport. The transport of Mn increased linearly with higher efficiency values in AP-BL than in BL-AP direction, however, the uptake of Mn revealed an asymptotic pattern within 120 min. In experiment 2, the kinetics of Mn transport in AP-BL was determined with media containing Mn concentrations from 0 to 2,500 μmol/L at 40 and 120 min, respectively, and mRNA levels of divalent metal transporter 1 (DMT1) and ferroportin (FPN1) were determined in Caco-2 cells treated with the medium containing 0 or 800 μmol/L of Mn for 120 min. The kinetics of Mn transport showed a carrier-mediated process when Mn concentrations were lower than 1,000 μmol/L and a linear increment when Mn concentrations exceeded 1,000 μmol/L at either 40 or 120 min. Mn treatment decreased (P < 0.01) DMT1 mRNA level and increased (P < 0.01) FPN1 mRNA level. The results from the present study suggested that Mn transport in AP-BL fit both carrier-mediated saturable and non-saturable diffusion processes, and Mn transport carriers DMT1 and FPN1 mediate the apical uptake and basolateral exit of Mn in Caco-2 cells.

Organic Cation Transporter 1 (OCT1/mOct1) Is Localized in the Apical Membrane of Caco-2 Cell Monolayers and Enterocytes

Organic cation transporters (OCTs) are members of the solute carrier 22 family of transporter proteins that are involved in absorption, distribution, and excretion of organic cations. OCT3 is localized in the apical (AP) membrane of enterocytes, but the literature is ambiguous about OCT1 (mOct1) localization, with some evidence suggesting a basolateral (BL) localization in human and mouse enterocytes. This is contrary to our preliminary findings showing AP localization of OCT1 in Caco-2 cell monolayers, an established model of human intestinal epithelium. Therefore, this study aims at determining the localization of OCT1 (mOct1) in Caco-2 cells, and human and mouse enterocytes. Functional studies using OCT1-specific substrate pentamidine showed transporter-mediated AP but not BL uptake in Caco-2 cells and human and mouse intestinal tissues. OCT1 inhibition decreased AP uptake of pentamidine by ∼50% in all three systems with no effect on BL uptake. A short hairpin RNA-mediated OCT1 knockdown in Caco-2 cells decreased AP uptake of pentamidine by ∼50% but did not alter BL uptake. Immunostaining and confocal microscopy in all three systems confirmed AP localization of OCT1 (mOct1). Our studies unequivocally show AP membrane localization of OCT1 (mOct1) in Caco-2 cells and human and mouse intestine. These results are highly significant as they will require reinterpretation of previous drug disposition and drug-drug interaction studies where conclusions were drawn assuming BL localization of OCT1 in enterocytes. Most importantly, these results will require revision of the regulatory guidance for industry in the United States and elsewhere because it has stated that OCT1 is basolaterally localized in enterocytes.

Transport of IRW, an Ovotransferrin-Derived Antihypertensive Peptide, in Human Intestinal Epithelial Caco-2 Cells

IRW is an egg ovotransferrin-derived ACE inhibitory peptide. The purpose of this study was to evaluate the stability and transcellular transport of IRW in Caco-2 cell monolayers. The stability of IRW was monitored on the apical (AP) surface while its transport was studied from AP to basal (BL) and from BL to AP surfaces. The results revealed that IRW is resistant against intestinal peptidase up to 60 min. Transport of IRW was not affected by addition of wortamanin, a transcytosis inhibitor. However, in the presence of cytochalasin D, a gap junction disruptor, transport of IRW was significantly increased, suggesting a possible passive transport from AP to BL surface. A higher transport of IRW from AP to BL surface than that from BL to AP surface suggests a passive-mediated transport. Moreover, in the presence of glycyl-sarcosine, a substrate for peptide transporter PepT 1, transport of IRW was reduced from AP to BL surface. The above observations showed atypical transport of IRW in Caco-2 cell monolayers. Thus, IRW may possibly be absorbed intact into the site of action for controlling hypertension.

Extraction of Antioxidant Components from Bidens pilosa Flowers and Their Uptake by Human Intestinal Caco-2 Cells

Bidens pilosa L. var. radiata (BPR, Asteraceae) is a commonly used folk medicine for treating various disorders such as diabetes, inflammation and hypertension. Recent studies to determine its chemical composition have revealed three di-O-caffeoylquinic acids (DiCQAs) and three polyacetylene glucosides (PGAs) to be among the major bioactive markers. To obtain the major compounds of these two chemical classes, the ethyl acetate fraction (EM) obtained using liquid-liquid partition from the methanol extract resulted in a fraction with the highest total phenolic and total flavonoid contents and antioxidant activities in radical scavenging and ferric reducing power assays. To assess the bioavailability of EM, we examined the in vitro uptake using the Caco-2 human colonic cell line. The apparent permeability coefficient (Papp) for each of the compounds within PGAs measured in both apical (AP) to basolateral (BL) and BL to AP was found to preferentially appear BL to AP direction, indicated that a basolateral to apical efflux system was detected in the study. DiCQAs had a lower efflux ratio than those from PGAs (2.32–3.67 vs. 6.03–78.36). Thus, it strongly implies that most of the DiCQAs are better absorbed than the PGAs.

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Human intestinal Caco-2 cells were differentiated using serum-reduced medium with fetal bovine serum (FBS) added only to the basolateral (BL) medium, and four serum-free media, containing insulin, transferrin, selenium (ITS), or MITO+™ serum extender (ITS plus growth factors), with or without addition of a lipid mixture, respectively. Differentiation was assessed by monitoring monolayer permeability, alkaline phosphatase and sucrase activities, and the transport of digoxin and cephalexin. Notably, the serum-reduced protocol produced results that were comparable to cells differentiated in the control medium and should be recommended as an alternative to the use of 10% FBS in both apical (AP) and BL media. ITS serum-free medium elicited permeability values and cephalexin transport similar to control cells. MITO+™ medium was the most efficient in promoting the two transport activities investigated, and it should be further evaluated with a larger set of substances, although its undisclosed composition represents a limit that may override these advantages.

Study on transport of Yangyin Tongnao granules in main effective fractions

To investigate the absorption characteristics and transportation mechanism Yangyin Tongnao granules in main effective fractions in Caco-2 cell model. The safety concentrations of Yangyin Tongnao granules in main effective fractions in Caco-2 cells. A Caco-2 cell model was established to study the transport situations after the compatibility of Yangyin Tongnao granules in main effective fractions, and the content was determined by high performance liquid chromatography (HPLC). P(app) of puerarin, ligustrazine and astragaloside were less than 1.0 x 10(-6) cm x s(-1), and their P(app) were hard to be close to atenolol. The oral absorption in descending order is shown as the following: puerarin, ligustrazine, astragaloside. After the compatibility between saponins and flavonoids, P(app) of astragaloside was improved obviously, which promoted the transport from apical (AP) to basolateral (BL); the compatibility of puerarin, ligustrazine and astragaloside showed a significant effect in the efflux of astragaloside and no change in the absorption transport of ligustrazine and puerarin at the same time. There is a great difference in bidirectional transport of representative component of each effective fraction, and P(app)(B --> A) was significantly greater than Papp(A --> B), which suggested that the efflux transport from BL side to AP side had an advantage in the three representative components of the three effective fractions in Caco-2 cell monolayer model. Astragaloside, ligustrazine and puerarin may be malabsorptive compounds, and the three compounds may be discharged by the transport protein in small intestine membrane.