Apical-to-basolateral Research Articles

Short peptide sequence enhances epithelial permeability through interaction with protein kinase C

We have identified a short peptide sequence (L-R5) acting as partial inhibitor of intracellular protein kinase C, capable of tight junction modulation in terms of reversible and non-toxic drug permeation enhancement. L-R5 is a pentapeptide with a cell-penetrating group at the N-terminus and of the sequence myristoyl-ARRWR. Apically applied in vitro, L-R5 transiently increased epithelial permeability within minutes, enhancing apical-to-basolateral (AB) transport of 4-kDa dextran and BCS class III drug naloxone. L-R5 was shown to be stable and effective at 37°C over a period of 24 hours. L-R5 was shown to be non-cytotoxic in consecutive exposure studies on primary human nasal epithelial cells by LDH release assay and ciliary beating frequency test. Finally, L-R5 by itself showed very low diffusion across epithelial monolayers, which is of advantage with regard to its expected negligible systemic bioavailability and side effects. Taken together, these data demonstrate the potential of short peptide partial inhibitor L-R5 to enhance the epithelial paracellular permeability via a reversible mechanism, and in a non-toxic manner.

Improving the bioaccessibility and in vitro absorption of 5-demethylnobiletin from chenpi by se-enriched peanut protein nanoparticles-stabilized pickering emulsion

Abstract 5-demethylnobiletin (5DN) in aged citrus peel (chenpi) is a unique polymethoxyflavone that has been shown to exhibit superior anti-cancer effects, but its low water-solubility and poor oral bioavailability may limit its application as a potent nutraceutical. In this study, Se-enriched peanut protein nanoparticles-stabilized Pickering emulsions (PPEs) were used to encapsulate 5DN to enhance its bioaccessibility, cellular uptake and transport rate. The preparation of PPEs, and their digestion profiles in gastrointestinal tract, cell uptake and transport were investigated. Results showed that the bioaccessibility of 5DN was significantly higher within PPEs (18.3%) than in bulk oil (9.2%). Besides, PPEs-encapsulated 5DN has a greater apical-to-basolateral (AP-BL) transport rate (26.9 × 10−6 cm s−1) than 5DN (21.7 × 10−6 cm s−1). It was proved PPEs may promote the bioavailability of 5DN, and this type of delivery system may be useful for the application of 5DN and other crystalline nutraceuticals in functional foods and beverages.

An intestinal paracellular pathway biased toward positively-charged macromolecules

Lacking an effective mechanism to safely and consistently enhance macromolecular uptake across the intestinal epithelium, prospects for successful development of oral therapeutic peptide drugs remain unlikely. We previously addressed this challenge by identifying an endogenous mechanism that controls intestinal paracellular permeability that can be activated by a peptide, termed PIP 640, which can increase cellular levels of phosphorylated myosin light chain at position S19 (MLC-pS19). Apical application in vitro or luminal application in vivo was shown to increase macromolecular solute transport within minutes that recovered completely within a few hours after removal. We now examine the nature of PIP 640-mediated permeability changes. Confluent Caco-2 cell monolayers treated with PIP 640 enhanced apical-to-basolateral (AB) transport of 4-kDa, but not 10-kDa, dextran. Expression and/or cellular distribution changes of tight junction (TJ) proteins were restricted to increased claudin-2 over a time course that correlated with an apparent shift in its distribution from the nucleus to the membrane fraction of the cell. PIP 640-mediated epithelial changes were distinct from the combined actions of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). While TNF-α/IFN-γ treatment also increased MLC-pS19 levels, these cytokines enhanced AB transport for 70-kDa dextran and decreased occludin expression at TJs. Claudin-2-dependent changes induced by PIP 640 resulted in an AB transport bias for positively-charged macromolecules demonstrated in vitro using charge variants of 4-kDa dextrans and by comparing transport of salmon calcitonin to exenatide. Comparable outcomes of increased TJ localization of claudin-2 and enhanced transport of these therapeutic peptides that biased toward cationic characteristics was demonstrated in vivo following after intra-luminal injection into rat jejunum. Together, these data have shown a potential mechanism for PIP 640 to enhance paracellular permeability of solutes in the size range of small therapeutic peptides that is biased toward positively-charged solutes.

Enhancing Oral Bioavailability of Cyclic RGD Hexa-peptides by the Lipophilic Prodrug Charge Masking Approach: Redirection of Peptide Intestinal Permeability from a Paracellular to Transcellular Pathway.

Hydrophilic peptides constitute most of the active peptides. They mostly permeate via tight junctions (paracellular pathway) in the intestine. This permeability mechanism restricts the magnitude of their oral absorption and bioavailability. We hypothesized that concealing the hydrophilic residues of the peptide using the lipophilic prodrug charge masking approach (LPCM) can improve the bioavailability of hydrophilic peptides. To test this hypothesis, a cyclic N-methylated hexapeptide containing Arg-Gly-Asp (RGD) and its prodrug derivatives, masking the Arg and Asp charged side chains, were synthesized. The library was evaluated for intestinal permeability in vitro using the Caco-2 model. Further investigation of metabolic stability ex vivo models in rat plasma, brush border membrane vesicles (BBMVs), and isolated CYP3A4 microsomes and pharmacokinetic studies was performed on a selected peptide and its prodrug (peptide 12). The parent drug analogues were found to have a low permeability rate in vitro, corresponding to atenolol, a marker for paracellular permeability. Moreover, palmitoyl carnitine increased the Papp of peptide 12 by 4-fold, indicating paracellular permeability. The Papp of the prodrug derivatives was much higher than that of their parent peptides. For instance, the Papp of the prodrug 12P was 20-fold higher than the Papp of peptide 12 in the apical to basolateral (AB) direction. Whereas the permeability in the opposite direction (BA of the Caco-2 model) was significantly faster than the Papp AB, indicating the involvement of an efflux system. These results were corroborated when verapamil, a P-gp inhibitor, was added to the Caco-2 model and increased the Papp AB of prodrug 12P by 3-fold. The prodrug 12P was stable in the BBMVs environment, yet degraded quickly (less than 5 min) in the plasma into the parent peptide 12. Pharmacokinetic studies in rats showed an increase in the bioavailability of peptide 12 > 70-fold (from 0.58 ± 0.11% to 43.8 ± 14.9%) after applying the LPCM method to peptide 12 and converting it to the prodrug 12P. To conclude, the LPCM approach converted the absorption mechanism of the polar peptides from a paracellular to transcellular pathway that tremendously affects their oral bioavailability. The LPCM method provides a solution for the poor bioavailability of RGD cyclohexapeptides and paves the way for other active hydrophilic and charged peptides with poor oral bioavailability.

Elucidation of the Intestinal Absorption Mechanism of Loganin in the Human Intestinal Caco-2 Cell Model.

Loganin, iridoid glycosides, is the main bioactive ingredients in the plant Strychnos nux-vomica L. and demonstrates various pharmacological effects, though poor oral bioavailability in rats. In this study, the intestinal absorption mechanism of loganin was investigated using the human intestinal Caco-2 cell monolayer model in both the apical-to-basolateral (A-B) and the basolateral-to-apical (B-A) direction; additionally, transport characteristics were systematically investigated at different concentrations, pHs, temperatures, and potential transporters. The absorption permeability (PappAB) of loganin, which ranged from 12.17 to 14.78 × 10−6cm/s, was high at four tested concentrations (5, 20, 40, and 80μM), while the major permeation mechanism of loganin was found to be passive diffusion with active efflux mediated by multidrug resistance-associated protein (MRP) and breast cancer resistance protein (BCRP). In addition, it was found that loganin was not the substrate of efflux transporter P-glycoprotein (P-gp) since the selective inhibitor (verapamil) of the efflux transporter exhibited little effects on the transport of loganin in the human intestinal Caco-2 cells. Meanwhile, transport from the apical to the basolateral side increased 2.09-fold after addition of a MRP inhibitor and 2.32-fold after addition of a BCRP inhibitor. In summary, our results clearly demonstrate, for the first time, a good permeability of loganin in the human intestinal Caco-2 cell model and elucidate, in detail, the intestinal absorption mechanism and the effects of transporters on iridoid glycosides compounds.

Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates

Recently, we transfected the porcine intestinal cell line IPEC-J2, with human P-glycoprotein (P-gp, ABCB1). The resulting cell line, iP-gp, has a high expression of functional human P-gp in the apical membrane, and a low expression of nonhuman ATP-binding cassette (ABC) transporters. The aim of the present work was to investigate the usability of iP-gp cell line for determining transepithelial transport kinetics of the prototypical P-gp substrates digoxin and rhodamine 123.The cell line generated tight monolayers after 16days of culture, reflected by high transepithelial electrical resistance values (TEER>15,000Ω·cm2), immunocytochemistry and low fluxes of the paracellular flux marker [14C]-mannitol. Monolayer integrity was not affected the common solvents dimethyl sulfoxide (DMSO), methanol and ethanol in concentrations up to 2% (v/v).Transepithelial fluxes of [3H]-labeled digoxin and rhodamine 123 were measured at varying donor concentrations, and kinetic parameters were estimated. Km and Vmax of P-gp mediated basolateral-to-apical (B-A) flux of rhodamine 123 were estimated to 332±124μM and 111±16pmol·cm−2·min−1 (n=3, total N=6), respectively. Vmax and Km of digoxin B-A flux could not be estimated due to the low aqueous solubility of digoxin. The half maximal inhibitory concentrations (IC50) of the selective P-gp inhibitor, zosuquidar (LY-335979), were estimated to 0.05±0.01μM (n=3, total N=6) and 0.04±0.01μM (n=3, total N=6) in transport experiments with digoxin and rhodamine 123 as substrates, respectively. Bidirectional fluxes of digoxin and rhodamine 123 were measured in transfected Madin Darby canine kidney cells (MDCK II MDR1) and compared with the fluxes obtained with the iP-gp cell monolayers. Efflux ratios were highest in the iP-gp cells, due to a tighter paracellular pathway. In conclusion, both digoxin and rhodamine 123 could be used to obtain IC50 values of inhibition, Ki values were only possible to obtain using rhodamine 123. The observed tightness, robustness towards solvents and the high efflux ratios confirmed that the iP-gp cell line may serve as a useful screening tool for investigations of substrate-P-gp interactions and modulation of P-gp function.

Esterification of Quercetin Increases Its Transport Across Human Caco-2 Cells.

Plant polyphenols showed useful biochemical characteristics in vitro; however, the assessments of their clinical applications in vivo are restricted by their limited bioavailability due to their strong resistance to 1st-pass effectsduring absorption. In order to improve the bioavailability of quercetin (QU), the ester derivative of QU (3,3',4',5,7-pentahydroxy flavones, TAQU) was synthesized, followed by examining the oil-water partition coefficient as well as the transport mechanisms of QU and its ester derivative (TAQU) using human Caco-2 cells. The transport characteristics of QU and TAQU transport under different conditions (different concentrations, time, pH, temperature, tight junctions, and potential transporters) were systematically investigated. Results showed that QU had a lower permeability coefficient (2.82 × 10(-6) cm/s) for apical-to-basolateral (AP-BL) transport over 5 to 50 μM, whereas the transport rate for AP to BL flux of TAQU (5.23 × 10(-6) cm/s) was significantly greater than that of QU. Paracellular pathways were not involved during the transport of both QU and TAQU. QU was poorly absorbed by active transport, whereas TAQU was mostly absorbed by passive diffusion. Efflux transporters, P-glycoproteins, multidrug resistance proteins were proven to participate in the transport process of QU, but not in that of TAQU. These results suggested that improving the lipophicity of QU by esterification could increase the transport of QU across Caco-2 cells.

Apomorphine and its esters: Differences in Caco-2 cell permeability and chylomicron affinity

Oral delivery of apomorphine via prodrug principle may be a potential treatment for Parkinson’s disease. The purpose of this study was to investigate the transport and stability of apomorphine and its esters across Caco-2 cell monolayer and their affinity towards chylomicrons. Apomorphine, monolauroyl apomorphine (MLA) and dilauroyl apomorphine (DLA) were subjected to apical to basolateral (A-B) and basolateral to apical (B-A) transport across Caco-2 cell monolayer. The stability of these compounds was also assessed by incubation at intestinal pH and physiological pH with and without Caco-2 cells. Molecular dynamics (MD) simulations were performed to understand the stability of the esters on a molecular level. The affinity of the compounds towards plasma derived chylomicrons was assessed. The A-B transport of intact DLA was about 150 times lower than the transport of apomorphine. In contrast, MLA was highly unstable in the aqueous media leading to apomorphine appearance basolaterally. MD simulations possibly explained the differences in hydrolysis susceptibilities of DLA and MLA. The affinity of apomorphine diesters towards plasma derived chylomicrons provided an understanding of their potential lymphatic transport. The intact DLA transport is not favorable; therefore, the conversion of DLA to MLA is an important step for intestinal apomorphine absorption.

Poor permeability and absorption affect the activity of four alkaloids from Coptis.

Coptidis rhizoma (Coptis) and its alkaloids exert various pharmacological functions in cells and tissues; however, the oral absorption of these alkaloids requires further elucidation. The present study aimed to examine the mechanism underlying the poor absorption of alkaloids, including berberine (BER), coptisine (COP), palmatine (PAL) and jatrorrhizine (JAT). An ultra‑performance liquid chromatography (UPLC) method was validated for the determination of BER, COP, PAL and JAT in the above experimental medium. In addition, the apparent oil‑water partition coefficient (Po/w); apparent permeability coefficient (Papp), determined using a parallel artificial membrane permeability assay (PAMPA) plate; membrane retention coefficient (R %); and effect of P‑glycoprotein (P‑gp) inhibitor on the Papp of the four alkaloids were investigated. The intestinal absorption rate constant (Ka) and absorption percentage (A %) of the four alkaloids were also determined. The results of the present study demonstrated that the Po/w of the four alkaloids in 0.1 mol·l‑1 HCl medium was significantly higher (P<0.01), compared with those of the alkaloids in phosphate buffer (pH 7.4). The Papp of BER was 1.0‑1.2x10‑6 cm·s‑1, determined using a PAMPA plate, and the Papp of BER, COP, PAL and JAT decreased sequentially. The concentrations of the four alkaloids on the apical‑to‑basolateral (AP‑BL) surface and the basolateral‑to‑apical (BL‑AP) surface increased in a linear manner, with increasing concentrations between 10 and 100 µmol. In addition, the transportation of BER on the BL‑AP surface was significantly faster (P<0.01), compared with that on the AP‑BL surface and, following the addition of verpamil (a P‑gp inhibitor), the Papp (AP‑BL) of the four alkaloids increased, whereas the Papp (BL‑AP) was significantly decreased (P<0.01). The rat intestinal perfusion experiment demonstrated that the four alkaloids were poorly absorbed; however, the Ka of BER was significantly higher, compared with the three other alkaloids. Furthermore, the A % and Ka provided evidence that the absorption of BER was increased in the jejunum, compared with in the ileum. In conclusion, the four alkaloids from Coptis appeared to be poorly absorbed, determined using a shake flask, pre‑coated PAMPA plates, a Caco‑2 cell monolayer model and intestinal perfusion; however, absorption was higher in the jejunum than in the ileum. Among the four alkaloids, the permeability of BER was markedly higher than the others, and P‑gp efflux had a significant effect on the absorption of those alkaloids.

Biopharmaceutic Risk Assessment of Brand and Generic Lamotrigine Tablets.

The therapeutic equivalence of generic and brand name antiepileptic drugs has been questioned by neurologists and the epilepsy community. A potential contributor to such concerns is pharmaceutical quality. The objective was to assess the biopharmaceutic risk of brand name Lamictal 100 mg tablets and generic lamotrigine 100 mg tablets from several manufacturers. Lamotrigine was characterized in terms of the Biopharmaceutics Classification System (BCS), including aqueous solubility and Caco-2 permeability. A panel of pharmaceutical quality tests was also performed on three batches of Lamictal, three batches of Teva generic, and one batch of each of four other generics: appearance, identity, assay, impurity, uniformity of dosage units, disintegration, dissolution, friability, and loss on drying. These market surveillance results indicate that all brand name and generic lamotrigine 100 mg tablets passed all tests and showed acceptable pharmaceutical quality and low biopharmaceutic risk. Lamotrigine was classified as a BCS class IIb drug, exhibiting pH-dependent aqueous solubility and dissolution. At pH 1.2 and 4.5, lamotrigine exhibited high solubility, whereas lamotrigine exhibited low solubility at pH 6.8, including non-sink dissolution. Lamotrigine showed high Caco-2 permeability. The apparent permeability (Papp) of lamotrigine was (73.7 ± 8.7) × 10(-6) cm/s in the apical-to-basolateral (AP-BL) direction and (41.4 ± 1.6) × 10(-6) cm/s in the BL-AP direction, which were higher than metoprolol's AP-BL Papp of (21.2 ± 0.9) × 10(-6) cm/s and BL-AP Papp of (34.6 ± 4.6) × 10(-6) cm/s. Overall, lamotrigine's favorable biopharmaceutics from a drug substance perspective and favorable quality characteristics from a tablet formulation perspective suggest that multisource lamotrigine tablets exhibit a low biopharmaceutic risk.

Esterification Enhanced Intestinal Absorption of Ginsenoside Rh2 in Caco-2 Cells without Impacts on Its Protective Effects against H2O2-Induced Cell Injury in Human Umbilical Vein Endothelial Cells (HUVECs)

Ginsenoside Rh2 and its octyl ester derivative (Rh2-O) were investigated for their transcellular transport in the Caco-2 cell system and their protective effect against oxidative stress in human umbilical vein endothelial cells (HUVECs). Results showed that the transport rates for apical-to-basolateral (AP-BL) flux of Rh2 (0.21 × 10⁻⁶ cm/s) was enhanced by the synthesis of its esterified derivative Rh2-O (1.93 × 10⁻⁶ cm/s) over the concentrations of 10-50 μM. In addition, both Rh2 and its esterified derivative Rh2-O exhibited similar protective effects against oxidative damage induced by H₂O₂. Pretreatment of Rh2 and Rh2-O significantly decreased the activation of caspase-3 known to play a key role in H₂O₂-induced cell apoptosis. These results were consistent with that of a flow cytometry assay analyzing HUVECs apoptosis. The present study demonstrated that the absorption of ginsenoside Rh2 in vitro can be significantly enhanced by synthesis of its ester derivative. Meanwhile, no significant discrepancy between Rh2 and Rh2-O on their bioactivities against the oxidative damage induced by H₂O₂ was observed, which means that esterification of Rh2 might have a higher bioavailability than Rh2 in vitro without impacts on pharmaceutical actions.

Study on the Mechanism of Intestinal Absorption of Epimedins A, B and C in the Caco-2 Cell Model

Epimedium spp. is commonly used in Traditional Chinese Medicine. Epimedins A, B, and C are three major bioactive flavonoids found in Epimedium spp. that share similar chemical structures. In this study, the intestinal absorption mechanism of these three compounds was investigated using the Caco-2 cell monolayer model in both the apical-to-basolateral (A-B) and the basolateral-to-apical (B-A) direction. The absorption permeability (PAB) of epimedins A, B, and C were extremely low and increased as the concentration of the epimedins increased from 5 to 20 μM, but, at 40 μM, the PAB values were reduced. Meanwhile, the amount of transported compounds increased in a time-dependent manner. The PAB of epimedins A and C were significantly increased and efflux ratios decreased in the presence of verapamil (an inhibitor of P-glycoprotein) and dipyridamole (an inhibitor of breast cancer resistance protein) while, in the presence of MK571 (an inhibitor of multidrug resistance proteins), the absorption of epimedins A and C did not change significantly, indicating that P-gp and BCRP might be involved in the transport of epimedins A and C. The PAB of epimedin B significantly increased while its secretory permeability (PBA) significantly decreased in the presence of dipyridamole, indicating that BCRP might be involved in the transport of epimedin B. No obvious changes in the transport of epimedin B were observed in the presence of verapamil and MK571. In summary, our results clearly demonstrate, for the first time, that poor bioavailability of these three prenylated flavonoids is the result of poor intrinsic permeability and efflux by apical efflux transporters.

The absorptive flux of the anti-epileptic drug substance vigabatrin is carrier-mediated across Caco-2 cell monolayers

Vigabatrin is an anti-epileptic drug substance. The oral bioavailability of vigabatrin is high (60–70%), however, little is known about the mechanism(s) mediating the intestinal absorption. The aim of the present study was to identify which solute carrier(s) are involved in the absorption of vigabatrin in Caco-2 cells, a cell culture model of the small intestinal epithelium. The uptake and transepithelial flux of vigabatrin was measured using an LC-MS method for quantification. Transepithelial transport of vigabatrin was shown to be proton-dependent and polarized in the apical-to-basolateral (A-B) direction. The A-B flux of vigabatrin had a saturable component and a passive component, indicating the presence of a carrier system in parallel with a passive permeability. The Michaelis constant, Km, of the transepithelial A-B flux of vigabatrin was estimated to be 32.8±7.4mM (n=3–5), whereas the Km of the apical uptake was found to be 12.7±3.7mM (n=3). The carrier-mediated transepithelial A-B flux of vigabatrin accounted for 80–95% (50.0–1.0mM) of the total A-B flux. The transepithelial A-B flux (as well as apical uptake) of vigabatrin was significantly decreased upon addition of substrates or inhibitors of the human proton-coupled amino acid transporter (hPAT1) to the apical solution. The present study indicates that the transepithelial A-B flux of vigabatrin is mainly mediated by hPAT1 in Caco-2 cells at dose-relevant concentrations.

Absorption &amp; Transport of the Pyrethroid Insecticide Deltamethrin (DLM) by Caco‐2 Cells

Our objective was to determine the transport and absorption of the pyrethroid insecticide, DLM, using Caco‐2 cells as a model for human enterocytes. Flux of 14C‐DLM (3μM) in the apical to basolateral (AP‐BL) and BL‐AP directions was monitored for 120 min at 37°C in Caco‐2 monolayers grown on Transwell® membranes. DLM was quantified in receiver chambers by GC‐MS. Directional flux of DLM in the AP to BL (absorptive) direction (Papp = 3.4 × 10−6 cm/sec) was greater than in the BL to AP (secretory) (7.3 × 10−7 cm/sec) direction, with a resulting transport ratio (BL to AP/AP to BL) of 0.22. Flux studies with the carboxylesterase inhibitor bis‐p‐nitrophenol (BNPP) revealed no change in Papp nor in metabolites. The presence of cyclosporine (CSA), a P‐gp inhibitor, reduced cellular DLM uptake ~3‐fold, but the MRP inhibitor MK571 had no effect. The influx of DLM is suggestive of an absorptive transport mechanism. DLM may be a substrate for an AP SLC transporter or a BL ABC transporter. CSA is a well‐established P‐gp inhibitor, but there is evidence it can also inhibit SLC transporters. The decrease in DLM uptake with CSA may be indicative of action of an AP influx transporter in GI absorption of DLM. (Supported by the Council for the Advancement of Pyrethroid Human Risk Assessment)

Comparison of the Permeability of Metoprolol and Labetalol in Rat, Mouse, and Caco-2 Cells: Use as a Reference Standard for BCS Classification

The purpose of this study was to investigate labetalol as a potential high permeability reference standard for the application of Biopharmaceutics Classification Systems (BCS). Permeabilities of labetalol and metoprolol were investigated in animal intestinal perfusion models and Caco-2 cell monolayers. After isolating specific intestinal segments, in situ single-pass intestinal perfusions (SPIP) were performed in rats and mice. The effective permeabilities (Peff) of labetalol and metoprolol, an FDA standard for the low/high Peff class boundary, were investigated in two different segments of rat intestine (proximal jejunum and distal ileum) and in the proximal jejunum of mouse. No significant difference was found between Peff of metoprolol and labetalol in the jejunum and ileum of rat (0.33 ± 0.11 × 10(-4) vs 0.38 ± 0.06 × 10(-4) and 0.57 ± 0.17 × 10(-4) vs 0.64 ± 0.30 × 10(-4) cm/s, respectively) and in the jejunum of mouse (0.55 ± 0.05 × 10(-4) vs 0.59 ± 0.13 × 10(-4) cm/s). However, Peff of metoprolol and labetalol were 1.7 and 1.6 times higher in the jejunum of mouse, compared to the jejunum of rat, respectively. Metoprolol and labetalol showed segmental-dependent permeability through the rat intestine, with increased Peff in the distal ileum in comparison to the proximal jejunum. Most significantly, Peff of labetalol was found to be concentration-dependent. Decreasing concentrations of labetalol in the perfusate resulted in decreased Peff compared to Peff of metoprolol. The intestinal epithelial permeability of labetalol was lower than that of metoprolol in Caco-2 cells at both apical pH 6.5 and 7.5 (5.96 ± 1.96 × 10(-6) vs 9.44 ± 3.44 × 10(-6) and 15.9 ± 2.2 × 10(-6) vs 23.2 ± 7.1 × 10(-6) cm/s, respectively). Labetalol exhibited higher permeability in basolateral to apical (BL-AP) compared to AP-BL direction in Caco-2 cells at 0.1 times the highest dose strength (HDS) (46.7 ± 6.5 × 10(-6) vs 14.2 ± 1.5 × 10(-6) cm/s). The P-gp inhibitor, verapamil, significantly increased AP-BL and decreased BL-AP direction transport of labetalol. Overall, labetalol showed high Peff in rat and mouse intestinal perfusion models similar to metoprolol at a concentration based on HDS. However, the concentration-dependent permeability of labetalol in mice due to P-gp and the inhibition study with verapamil in Caco-2 cells indicated that labetalol is not an ideal reference standard for BCS classification.

Absorption and Interaction of the Main Constituents from the Traditional Chinese Drug Pair Shaoyao-Gancao via a Caco-2 Cell Monolayer Model

Shaoyao-Gancao (Paeoniae Radix Alba and Glycyrrhizae Radix et Rhizoma) is a traditional Chinese drug pair widely used in decoctions for relieving pains, especially abdominal pain. We aimed to determine the intestinal absorption and interaction of three active compounds (glycyrrhizic acid, liquiritin, and paeoniflorin) in this drug pair. We investigated the transport of these compounds across intestinal epithelial cells by using the Caco-2 cell monolayer in both the apical-to-basolateral (A-B) and B-A directions. All compounds could only travel through the Caco-2 cell monolayer at a low level when the cells were treated with single component solutions. In the presence of verapamil, an inhibitor of P-glycoprotein (P-gp), the absorptive permeability (PAB) of paeoniflorin and liquiritin increased significantly (p < 0.05) and efflux ratios decreased, while the absorption of glycyrrhizic acid did not change significantly, which indicated that paeoniflorin and liquiritin might be P-gp substrates. In addition, when liquiritin and glycyrrhizic acid in Gancao extract and paeoniflorin in Shaoyao extract were examined, PAB of paeoniflorin and liquiritin were significantly higher, while glycyrrhizic acid retained the same absorption level compared to the corresponding single component solutions, which suggested that some certain ingredients in the extracts can promote the absorption of paeoniflorin and liquiritin, but not that of glycyrrhizic acid. Furthermore, compared to the results of treatment with individual extracts, treatment of cells with a mixture of the two extracts considerably increased (p < 0.05) the absorption of glycyrrhizic acid and paeoniflorin and showed no change in the absorption of liquiritin, which implied that the transport of glycyrrhizic acid and paeoniflorin is increased by some ingredients from the complementary drug in the drug pair, while that of liquiritin remains unaffected.

The absorption and transport of magnolol in Caco-2 cell model

To investigate the absorption and transport mechanism of magnolol in Caco-2 cell model. A human intestinal epithelial cell model Caco-2 cell in vitro cultured was applied to study the absorption and transport of magnolol, the effects of time, donor concentration, P-gp inhibitor verapamil, pH and temperature on the absorption and transport of magnolol were investigated. The determination of magnolol was performed by high performance liquid chromatography, then the values of apparent permeability coefficient (P app ) and P ratio Basolateral-to-Apical (BL-to-AP)/Apical-to-Basolateral (AP-to-BL) were calculated. In Caco-2 cell model, comparing the amounts of transport of AP-to-BL and BL-to-AP, the latter was larger. At the same donor concentration, either the amounts of transport of AP-to-BL or BL-to-AP increased with increase in donor concentration and incubation time. Verapamil could significantly improve the amounts of transport of AP-to-BL. The transport of AP-to-BL and BL-to-AP depended on temperature, and there was no significant effect of pH on the transport of AP-to-BL. Magnolol could be transported through the intestinal mucosa via a passive diffusion mechanism primarily, coexisting with a carrier-mediated transport, at the same time, the efflux mechanism could be involved.

Trans-epithelial transport of the betalain pigments indicaxanthin and betanin across Caco-2 cell monolayers and influence of food matrix

This study investigated the absorption mechanism of the phytochemicals indicaxanthin and betanin and the influence of their food matrix (cactus pear and red beet) on the intestinal transport. Trans-epithelial transport of dietary-consistent amounts of indicaxanthin and betanin in Caco-2 cell monolayers seeded on Transwell(R) inserts was measured in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under an inwardly directed pH gradient (pH 6.0/7.4, AP/BL) mimicking luminal and serosal sides of human intestinal epithelium. The effect of inhibitors of membrane transporters on the absorption was also evaluated. Contribution of the paracellular route was investigated after EDTA treatment of the cell monolayer. In vitro digestion of betalainic food was performed to provide a post-intestinal fraction containing bioaccessible pigments. Apparent permeability coefficients (P(app)) in the absorptive direction were (4.4 ± 0.4) × 10⁻⁶ and (3.2 ± 0.3) × 10⁻⁶ cm s⁻¹ for indicaxanthin and betanin, respectively. Transport of indicaxanthin was non-polarized, linear as a function of time and concentration, and unaffected by inhibitors of membrane transporters. Betanin exhibited significantly different bidirectional P(app) values and non-linear efflux kinetics. The concentration-dependent betanin efflux was described by a kinetic model including one non-saturable (K(d) = 0.042 μL cm⁻² min⁻¹) and one saturable component identified as the apical multidrug resistance-associated protein 2 (MRP2; K(m) = 275 μM; J(max) = 42 pmol min⁻¹ cm⁻²). Permeation of both betalains increased remarkably after EDTA treatment of the cell monolayer. Neither indicaxanthin nor betanin underwent metabolic transformation. Food matrix did not affect trans-epithelial transfer of indicaxanthin, but reduced the absorption rate of betanin, red beet more than cactus pear. Dietary indicaxanthin and betanin can substantially be absorbed through paracellular junctions of intestinal epithelial cells. Additional trans-membrane permeation can be considered for betanin, whose absorption is limited by a MRP2-mediated efflux and negatively affected by its food matrix. Present findings are consistent with the quite higher bioavailability of indicaxanthin over betanin established in humans.

Intestinal absorption of forsythoside A in in situ single-pass intestinal perfusion and in vitro Caco-2 cell models

To investigate the mechanisms underlying the intestinal absorption of the major bioactive component forsythoside A (FTA) extracted from Forsythiae fructus. An in vitro Caco-2 cell model and a single-pass intestinal perfusion in situ model in SD rats were used. In the in vitro Caco-2 cell model, the mean apparent permeability value (P(app)-value) was 4.15×10(-7) cm/s in the apical-to-basolateral (AP-BL) direction. At the concentrations of 2.6-10.4 μg/mL, the efflux ratio of FTA in the bi-directional transport experiments was approximately 1.00. After the transport, >96% of the apically loaded FTA was retained on the apical side, while >97% of the basolaterally loaded FTA was retained on the basolateral side. The P(app)-values of FTA were inversely correlated with the transepithelial electrical resistance. The paracellular permeability enhancers sodium caprate and EDTA, the P-gp inhibitor verapamil and the multidrug resistance related protein (MRP) inhibitors cyclosporine and MK571 could concentration-dependently increase the Papp-values, while the uptake (OATP) transporter inhibitors diclofenac sodium and indomethacin could concentration-dependently decrease the P(app)-values. The intake transporter SGLT1 inhibitor mannitol did not cause significant change in the P(app)-values. In the in situ intestinal perfusion model, both the absorption rate constant (K(a)) and the effective permeability (P(eff)-values) following perfusion of FTA 2.6, 5.2 and 10.4 μg/mL via the duodenum, jejunum and ileum had no significant difference, although the values were slightly higher for the duodenum as compared to those in the jejunum and ileum. The low, medium and high concentrations of verapamil caused the largest increase in the P(eff)-values for duodenum, jejunum and ileum, respectively. Sodium caprate, EDTA and cyclosporine resulted in concentration-dependent increase in the P(eff)-values. Diclofenac sodium and indomethacin caused concentration-dependent decrease in the Peff-values. Mannitol did not cause significant change in the P(app)-values for the duodenum, jejunum or ileum. The results suggest that the intestinal absorption of FTA may occur through passive diffusion, and the predominant absorption site may be in the upper part of small intestine. Paracellular transport route is also involved. P-gp, MRPs and OATP may participate in the absorption of FTA in the intestine. The low permeability of FTA contributes to its low oral bioavailability.

Abstract 5473: The contribution of P-glycoprotein to clinical pharmacokinetic interactions between lenalidomide and temsirolimus

Abstract Purpose: To evaluate the source of an apparent clinical pharmacokinetic interaction between lenalidomide (an Immunomodulatory Drug, IMiD) and temsirolimus (mTOR inhibitor). Background: The combination of lenalidomide with temsirolimus demonstrated a synergistic effect in in vitro models of Multiple Myeloma (MM), thus providing rationale for this combination in patients with relapsed MM. However pharmacokinetic interactions between these two agents have not been reported. In the current study, clinical pharmacokinetic interactions of lenalidomide with temsirolimus and the underlying mechanisms were investigated. Methods: A phase I clinical trial was completed for patients with relapsed multiple myeloma with oral lenalidomide (15-25 mg) days 1-21 of a 28 day cycle and weekly × 4 intravenous temsirolimus (15-20 mg). Pharmacokinetic data for both agents were obtained and potential drug-drug interactions were further evaluated using in vitro transport and uptake studies. An apparent clinical interaction was observed between lenalidomide and temsirolimus as demonstrated by statistically significant changes in clearance, maximum concentrations (Cmax), and area under the concentration-time curves (AUCs) for constant doses of lenalidomide with increased doses of temsirolimus. Similar and significant changes for temsirolimus pharmacokinetics at a fixed dose were also observed when lenalidomide doses were increased. In vitro studies indicated a higher basolateral-to-apical (BL-to-AP) flux compared to AP-to-BL flux for lenalidomide in MDCKII monolayers, suggesting an apically directed active transport of lenalidomide. Further evaluation of intracellular lenalidomide uptake showed an approximately 2-fold higher accumulation of lenalidomide in HL-60 than in P-glycoprotein (P-gp, ABCB1, MDR-1)-overexpressing HL-60/VCR cells (38.4 ± 13.2 vs. 19.2 ± 9.6 pmol/mg protein, respectively, P=0.005). Co-incubation with temsirolimus, a known P-gp substrate and inhibitor, resulted in increased lenalidomide accumulation (37.2 ± 6.0 vs. 19.2 ± 9.6 pmol/mg protein, respectively, P=0.002) in HL-60/VCR cells, which was equivalent to that in HL-60 cells. Clinical significance of this pharmacokinetic interaction in terms of either toxicity or response has also been evaluated. Conclusions: These results suggest that lenalidomide is a P-gp substrate, explaining the pharmacokinetic interaction between lenalidomide and temsirolimus. Quantifying the interaction of lenalidomide with other P-gp substrates, e.g. dexamethasone, is warranted to optimize dose administration of IMiD-based therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5473. doi:10.1158/1538-7445.AM2011-5473