Predicting in vivo performance of fenofibrate amorphous solid dispersions using in vitro non-sink dissolution and dissolution permeation setup.

Amorphous solid dispersion (ASD) is emerging as a useful formulation strategy to increase the bioavailability of active pharmaceutical ingredients with poor solubility. In vitro dissolution testing under non-sink conditions has often been used to evaluate the ability of ASDs to generate and maintain supersaturation to predict the in vivo performance. However, such a single compartment dissolution setup can fail to predict the oral bioavailability, due to an interdependence between precipitation and permeation. Hence, the use of two compartment dissolution-permeation setups is emerging. In this study, three ASDs containing fenofibrate as model drug substance were developed using Soluplus®, and Hypromellose Acetate Succinate in two different grades (high and low), respectively. The aim was to compare the use of a small-scale in vitro non-sink dissolution setup and a small-scale in vitro dissolution-permeation setup to predict the in vivo oral exposure of the ASDs in rats. The maximum concentration (Cmax) and area under curve (AUC) obtained in the in vitro studies were used to predict the in vivo rank order of the formulations. The results showed that the two in vitro studies resulted in the same rank order based on both Cmax and AUC. Interestingly, Cmax resulted in a better in vitro/in vivo correlation than the in vitro AUC, and based on the in vitro Cmax, the in vivo rank order was predicted.

Acamprosate Is a Substrate of the Human Organic Anion Transporter (OAT) 1 without OAT3 Inhibitory Properties: Implications for Renal Acamprosate Secretion and Drug-Drug Interactions.

Acamprosate is an anionic drug substance widely used in treating symptoms of alcohol withdrawal. It was recently shown that oral acamprosate absorption is likely due to paracellular transport. In contrast, little is known about the eliminating mechanism clearing acamprosate from the blood in the kidneys, despite the fact that studies have shown renal secretion of acamprosate. The hypothesis of the present study was therefore that renal organic anion transporters (OATs) facilitate the renal excretion of acamprosate in humans. The aim of the present study was to establish and apply OAT1 (gene product of SLC22A6) and OAT3 (gene product of SLC22A8) expressing cell lines to investigate whether acamprosate is a substrate or inhibitor of OAT1 and/or OAT3. The studies were performed in HEK293-Flp-In cells stably transfected with SLC22A6 or SLC22A8. Protein and functional data showed that the established cell lines are useful for studying OAT1- and OAT3-mediated transport in bi-laboratory studies. Acamprosate inhibited OAT1-mediated p-aminohippuric acid (PAH) uptake but did not inhibit substrate uptake via OAT3 expressing cells, neither when applied concomitantly nor after a 3 h preincubation with acamprosate. The uptake of PAH via OAT1 was inhibited in a competitive manner by acamprosate and cellular uptake studies showed that acamprosate is a substrate for OAT1 with a Km-value of approximately 700 µM. Probenecid inhibited OAT1-mediated acamprosate uptake with a Ki-value of approximately 13 µM, which may translate into an estimated clinically significant DDI index. In conclusion, acamprosate was identified as a substrate of OAT1 but not OAT3.

The Permeation of Acamprosate Is Predominantly Caused by Paracellular Diffusion across Caco-2 Cell Monolayers: A Paracellular Modeling Approach.

In drug development, estimating fraction absorbed (Fa) in man for permeability-limited compounds is important but challenging. To model Fa of such compounds from apparent permeabilities (Papp) across filter-grown Caco-2 cell monolayers, it is central to elucidate the intestinal permeation mechanism(s) of the compound. The present study aims to refine a computational permeability model to investigate the relative contribution of paracellular and transcellular routes to the Papp across Caco-2 monolayers of the permeability-limited compound acamprosate having a bioavailability of ∼11%. The Papp values of acamprosate and of several paracellular marker molecules were measured. These Papp values were used to refine system-specific parameters of the Caco-2 monolayers, that is, paracellular pore radius, pore capacity, and potential drop. The refined parameters were subsequently used as an input in modeling the permeability (Pmodeled) of the tested compounds using mathematical models collected from two published permeability models. The experimental data show that acamprosate Papp across Caco-2 monolayers is low and similar in both transport directions. The obtained acamprosate Papp, 1.56 ± 0.28 × 10-7 cm·s-1, is similar to the Papp of molecular markers for paracellular permeability, namely, mannitol (2.72 ± 0.24 × 10-7 cm·s-1), lucifer yellow (1.80 ± 0.35 × 10-7 cm·s-1), and fluorescein (2.10 ± 0.28 × 10-7 cm·s-1), and lower than that of atenolol (7.32 ± 0.60 × 10-7 cm·s-1; mean ± SEM, n = 3-6), while the end-point amount of acamprosate internalized by the cell monolayer, Qmonolayer, was lower than that of mannitol. Acamprosate did not influence the barrier function of the monolayers since it altered neither the Papp of the three paracellular markers nor the transepithelial electrical resistance (TEER) of the cell monolayer. The Pmodeled for all the paracellular markers and acamprosate was dominated by the Ppara component and matched the experimentally obtained Papp. Furthermore, acamprosate did not inhibit the uptake of probe substrates for solute carriers PEPT1, TAUT, PAT1, EAAT1, B0,+AT/rBAT, OATP2B1, and ASBT expressed in Caco-2 cells. Thus, the Pmodeled estimated well Ppara, and the paracellular route appears to be the predominant mechanism for acamprosate Papp across Caco-2 monolayers, while the alternative transcellular routes, mediated by passive diffusion or carriers, are suggested to only play insignificant roles.

Nonionic surfactants modulate the transport activity of ATP-binding cassette (ABC) transporters and solute carriers (SLC): Relevance to oral drug absorption.

Recently, it has become evident that pharmaceutical excipients may interfere with the activity of ATP-binding cassette (ABC) transporters and solute carriers (SLC). The present review aims to provide an overview of surfactants shown to modulate substrate transport via SLCs and ABCs, and to discuss the relevance for oral drug absorption. In vitro, more than hundred surfactants have been suggested to decrease the efflux activity of P-glycoprotein (P-gp, ABCB1), and many of these surfactants also inhibit the breast cancer resistance protein (BCPR, ABCG2), while conflicting results have been reported for multidrug resistance-associated protein 2 (MRP2, ABCC2). In animals, surfactants such as pluronic® P85 and polysorbate 20 have been shown to enhance the oral absorption of P-gp and BCRP substrates. Many surfactants, including cremophor® EL and Solutol® HS 15 inhibiting ABC transporters, were also found to inhibit SLCs in cell cultures. These carriers were SLC16A1, SLC21A3, SLC21A9, SLC15A1-2, and SLC22A1-3. This overlap in specificity of surfactants that inhibit both transporters and carriers might influence the oral absorption of various drug substances, nutrients, and vitamins. Such biopharmaceutical elements may be relevant for future drug formulation design.

Nonionic surfactants increase digoxin absorption in Caco-2 and MDCKII MDR1 cells: Impact on P-glycoprotein inhibition, barrier function, and repeated cellular exposure.

Nonionic surfactants commonly used in pharmaceutical formulations may have P-glycoprotein (P-gp) inhibiting and/or permeation enhancing effects. The present work aims to distinguish these effects and assess the degree of cellular recovery after multiple exposures to nonionic surfactants. The investigated surfactants were polysorbates (PS): PS20, PS40, PS60, PS65, PS80 and PS85; monosaccharide-based: lauroyl methyl glucamide and n-nonyl-ß-D-glucopyranoside; or disaccharide-based: lauryl-ß-D-maltoside and trehalose 6-laurate. Bi-directional permeability studies of digoxin and mannitol, and calcein-AM efflux assay were performed in cell cultures. Cellular recovery was evaluated by continuous measurements of transepithelial electrical resistance (TEER) in Caco-2 cell monolayers. Polysorbates with one fatty acid chain decreased the efflux of digoxin through P-gp inhibition in MDCKII MDR1 cells. Mono- and di-saccharide-based surfactants, in a dose dependent manner, enhanced digoxin absorptive permeability without decreasing the secretory permeability in Caco-2 cells, suggesting that the surfactants had a transcellular permeation enhancing effect. Caco-2 cell monolayers recovered to different degrees of 60-100% of the initial TEER values. Calcein-AM assay was found to be non-predictive to surfactants influence on digoxin permeability across cell monolayers. In conclusion, these results may assist, in a mechanism-based, selection of suitable surfactants for formulating oral dosage forms to enhance the absorption of low bioavailable P-gp substrates.

Polysorbate 20 alters the oral bioavailability of etoposide in wild type and mdr1a deficient Sprague-Dawley rats.

The aim of the present work was to investigate the ability of nonionic surfactants to increase the oral absorption of the P-glycoprotein substrate etoposide in vitro and in vivo. Intestinal absorption was investigated by studying bidirectional permeability of etoposide across filter-grown Caco-2 and MDCKII MDR1 cell monolayers. The oral absorption of etoposide was investigated in wild type (WT) and mdr1a deficient (KO) Sprague-Dawley rats. In cell cultures, polysorbate 20 (PS20) decreased P-glycoprotein mediated efflux of etoposide. When PS20 and etoposide were co-administered to WT rats, the oral absorption of etoposide increased significantly in the presence of 5 and 25% (v/v) PS20. However, in KO rats, the exposure of etoposide after oral co-administration with 5% PS20 was similar to control. Unexpectedly, co-administration of etoposide with 25% PS20 significantly reduced the absorption fraction of etoposide in mdr1a KO rats. In vitro dialysis studies performed on PS20-containing etoposide solutions suggested that the reduced bioavailability may be due to etoposide retention in PS20 micelles and/or through increased viscosity. In conclusion, PS20 increases oral bioavailability of etoposide through inhibition of P-glycoprotein. However, the use of the excipient may be challenged by etoposide retention at higher concentrations.

SGLT1-Mediated Transport in Caco-2 Cells Is Highly Dependent on Cell Bank Origin.

The human colon adenocarcinoma (Caco-2) cell line is a well-established in vitro model for studying transport phenomena for prediction of intestinal nutrient and drug absorption. However, substances depending on transporters such predictions are complicated due to variable transporter expression and limited knowledge about transporter function during multiple cell passaging and cell thawings. In the case of sodium glucose transporter 1 (SGLT1), a key transporter of oral absorption of d-glucose, one reason for compromised prediction could be inadequate expression of SGLT1 in Caco-2 cells and thereby limited sensitivity in the determination of SGLT1-mediated permeability (PSGLT1). Here, the objective is to characterize and compare SGLT1-mediated uptake in Caco-2 cells obtained from different cell banks. SGLT1-mediated uptake of the standard SGLT1 substrate, methyl-α-d-glucopyranoside, in Caco-2 cells was shown to be highly dependent on cell bank origin. The most robust and reliable SGLT1 functionality was identified in Caco-2 cells from Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ), whereas cells from the American Type Culture Collection and European Collection of Authenticated Cell Cultures have lower SGLT1 transport activity. Transepithelial PSGLT1 across Caco-2 cells from DSMZ showed that PSGLT1 likely accounts for approximately 97% of absorptive methyl-α-d-glucopyranoside Papp(a-b). In conclusion, Caco-2 cells from DSMZ provide a robust in vitro model for studying SGLT1-mediated uptake and transport-over multiple cell passages and independent cell stock thawings.

Imidazole-4-acetic acid, a new lead structure for interaction with the taurine transporter in outer blood-retinal barrier cells.

Retinal diseases leading to impaired vision and ultimately blindness are mainly characterized by ischemic and hypoxic stress. Targeting the retinal ρ-containing γ-aminobutyric acid type A receptors (ρ GABAARs) and thereby decreasing the retinal neuronal activity has been proposed as a novel therapeutic approach. The taurine transporter (TAUT) plays a key role in the retinal transport of GABA and has been previously suggested to display a higher functional activity in the retina compared to the brain. TAUT would therefore stand as a suitable target for the selective delivery of ρ GABAAR ligands into the retina. Consequently, an in vitro model of TAUT at the outer blood-retinal barrier (BRB) was developed and characterized using the ARPE-19 cell line. Furthermore, the structural requirements of GABAAR ligands for interacting with TAUT at the BRB were investigated for a series of standard GABAAR ligands by testing their ability to inhibit the TAUT-mediated influx of taurine in ARPE-19 cells. Results showed that taurine influx was seven-fold higher when the ARPE-19 cells were cultured under hyperosmotic conditions and was demonstrated to display saturable kinetics (Km=27.7±2.2µM and Jmax=24.2±0.6pmol/cm2·min). Furthermore, the taurine influx was significantly inhibited in a concentration-dependent manner by GABA and imidazole-4-acetic acid (IAA), which is a naturally occurring metabolite of histamine. These compounds display similar Ki values of 644.2µM and 658.6µM, respectively. Moreover, IAA demonstrated higher inhibitory properties than the other tested GABA analogs: 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), 4,5,6,7-tetrahydropyrazolo[5,4-c]pyridin-3-ol (Aza-THIP), muscimol, and thiomuscimol. These studies demonstrated that IAA interacts with TAUT, which makes IAA a new lead structure in the development of new compounds, which are not only interacting with TAUT but also potent ρ GABAAR ligands.

Interaction between fed gastric media (Ensure Plus®) and different hypromellose based caffeine controlled release tablets: comparison and mechanistic study of caffeine release in fed and fasted media versus water using the USP dissolution apparatus 3.

The aim of the study was to investigate caffeine release in fed and fasted state media from three controlled release matrix tablets containing different HPMC viscosity grades. The biorelevant in vitro dissolution methods utilize the USP 3 dissolution apparatus and biorelevant media to simulate fed and fasted gastro-intestinal dissolution conditions. The effect of tablet reciprocation rate (dip speed) in dissolution media (10 and 15 dips per minute) and media (water, fed and fasted) on caffeine release rate from - and erosion rate of - 100, 4000 and 15,000 mPa s HPMC viscosity tablets was investigated using factorial designed experiments. Furthermore, the mechanism of release in Ensure Plus(®), a nutrition drink similar in composition to the FDA standard meal, was investigated by studying tablet swelling using texture analysis. Altering dip speed has negligible effect on release and erosion rates. Using fasted media instead of water slightly decreases caffeine release from 100 and 4000 mPa s HPMC viscosity tablets as well as erosion rates, while 15,000 mPa s tablets remain unaffected. Fed compared to fasted media decreases caffeine release rate, and the food effect is greater for the 100 mPa s viscosity tablets compared to the 4000 and 15,000 mPa s viscosity tablets. The investigation using texture analysis indicates that Ensure Plus(®) becomes rate-limiting for caffeine release from HPMC tablets by forming a hydrophobic barrier around the tablets. The barrier decreases tablet water permeation, which decreases erosion rate in 100 mPa s viscosity tablets, swelling in 15,000 mPa s viscosity tablets and caffeine release from both tablets. This observed interaction between Ensure Plus(®) and the HPMC tablets may translate into decreased drug release rate in the fed stomach, which may decrease the amount of drug available for absorption in the small intestine and thus reduce systemic drug exposure and maximum plasma concentration.

New insights into the carrier-mediated transport of estrone-3-sulfate in the Caco-2 cell model.

The current studies were undertaken to gain new insights into the interplay and mechanism of membrane transporters involved in the permeability of estrone-3-sulfate (E1S) in Caco-2 cells cultured either on the bottom of multiwell plastic dishes or on filter support. We demonstrate that Caco-2 cells from the "Deutsche sammlung von mikroorganismen und zellkulturen" (DSMZ) exhibit extensive and consistent carrier-mediated uptake of [(3)H]-E1S after a culture period of 11-13 days. The kinetic characterization, the inhibitory profile and the pH dependence for the initial linear uptake permeability (PUP) of [(3)H]-E1S suggest that the organic anion transporting polypeptide (OATP) 2B1 is the main transporter involved in the apical E1S PUP in Caco-2 cells from the DSMZ. Furthermore, our results indicate that the efflux transporter breast cancer resistance protein (BCRP) affects E1S PUP, even when uptake is measured at the initial linear uptake phase. Although almost identical results were obtained for cells cultured on plastic dishes and on filter supports, the OATP2B1 stimulator dexamethasone did not affect the PUP for cells grown on dishes but increased [(3)H]-E1S PUP by more than 2-fold for filter grown cells. The basolateral PUP of [(3)H]-E1S of filter grown cells was inhibited by several inhibitors of the bidirectional transporter organic solute transporter α/ß (OSTα/ß). Efflux studies were performed by loading the cells with either [(3)H]-E1S or [(3)H]-taurocholic acid (TCA) and subsequently measuring the efflux of radio labeled substance in the absence or presence of BCRP or OST α/ß inhibitors. Similar effluxes of [(3)H]-E1S was observed across the apical and basolateral membrane, and the apical efflux was greatly decreased in the presence of the BCRP inhibitor fumitremorgin C. In contrast, efflux of [(3)H]-TCA to the basolateral compartment was clearly larger than to the apical compartment. Trans-stimulation of basolateral [(3)H]-E1S efflux was observed in the presence of taurolithocholic acid (TLC), although none of the applied OSTα/ß inhibitors were able to confirm the existence of carrier-mediated efflux at the basolateral membrane, neither for [(3)H]-E1S nor for [(3)H]-TCA. These results highlight the importance of transporter interplay for E1S and drug compounds in Caco-2 cells and emphasize the importance of identifying the basolateral transporters in these cells.

Wheat germ agglutinin-functionalised crosslinked polyelectrolyte microparticles for local colon delivery of 5-FU: in vitro efficacy and in vivo gastrointestinal distribution.

We have previously reported the development and characterisation of wheat germ agglutinin (WGA)-functionalised chitosan-Ca-alginate (CTS-Ca-ALG) microparticles (MPs) loaded with acid-resistant particles of 5-fluorouracil (5-FU). In the present work, our goal was to evaluate the potential of these carriers for efficient treatment of colon cancer by studying in vitro permeability and cell association of 5-FU and [methyl-³H]thymidine uptake in Caco-2 cells, as well as in vivo gastrointestinal distribution. The amount of 5-FU permeated through Caco-2 cells was 15.1, 7.7 and 6.5% for 5-FU solution, CTS-Ca-ALG MPs and WGA conjugates. The concentration of 5-FU associated with Caco-2 cells was significantly greater when delivered from MPs. By incorporation of 5-FU into MPs and further decoration with WGA, an increased [methyl-³H]thymidine uptake was observed few hours after continuous drug treatment followed by significantly reduced uptake after 6 h. Gastrointestinal distribution was in favour of increased localisation and concentration of the particles in colon region.

Intestinal transporters for endogenic and pharmaceutical organic anions: the challenges of deriving in-vitro kinetic parameters for the prediction of clinically relevant drug-drug interactions.

OBJECTIVES: This review provides an overview of intestinal human transporters for organic anions and stresses the need for standardization of the various in-vitro methods presently employed in drug-drug interaction (DDI) investigations. KEY FINDINGS: Current knowledge on the intestinal expression of the apical sodium-dependent bile acid transporter (ASBT), the breast cancer resistance protein (BCRP), the monocarboxylate transporters (MCT) 1, MCT3-5, the multidrug resistance associated proteins (MRP) 1-6, the organic anion transporting polypetides (OATP) 2B1, 1A2, 3A1 and 4A1, and the organic solute transporter α/ß (OSTα/ß) has been covered along with an overview of their substrates and inhibitors. Furthermore, the many challenges in predicting clinically relevant DDIs from in-vitro studies have been discussed with focus on intestinal transporters and the various methods for deducting in-vitro parameters for transporters (K(m) /K(i) /IC50, efflux ratio). The applicability of using a cut-off value (estimated based on the intestinal drug concentration divided by the K(i) or IC50) has also been considered. SUMMARY: A re-evaluation of the current approaches for the prediction of DDIs is necessary when considering the involvement of other transporters than P-glycoprotein. Moreover, the interplay between various processes that a drug is subject to in-vivo such as translocation by several transporters and dissolution should be considered.

Estimating intestinal absorption of inorganic and organic selenium compounds by in vitro flux and biotransformation studies in Caco-2 cells and ICP-MS detection.

The aim of the present work was to compare and estimate absorption and biotransformation of selected selenium compounds by studying their fluxes across Caco-2 cells. Five different selenium compounds, selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), selenate, selenite, and methylseleninic acid (MeSeA), were applied to Caco-2 cells in a concentration of 10 µM, and fluxes in both directions were studied for 2 h. Fluxes of selenite and MeSeA in the presence of excess reduced glutathione (selenite + GSH and MeSeA + GSH) and flux of MeSeA in the presence of excess cysteine (MeSeA + Cys) were also studied. Selenium absorptive and exsorptive fluxes and accumulation in cell cytosol were analyzed by means of flow injection inductively coupled plasma mass spectrometry (ICP-MS). Absorptive flux of SeMet, MeSeCys, and selenate showed values correlating to complete in vivo absorption, while selenite and MeSeA fluxes correlated to poor in vivo absorption. Speciation analysis of cell lysate and donor and receptor solutions by LC-ICP-MS showed limited transformation of all selenium compounds. Extensive transformation as well as significantly increased absorptive flux was observed when co-administering selenite with glutathione compared to administering selenite alone. These observations are possibly due to formation of selenodiglutathione (GS-Se-SG) which may be absorbed differently than selenite. Concomitant application of GSH or cysteine with MeSeA resulted in extensive transformation of MeSeA, including volatile species, whereas no significant increases in fluxes were observed. In summary, the absorption of selenite selenate and the selenoamino acids is considered complete under physiological conditions, but the absorption mechanisms and metabolism of the compounds are different.

Simulating kinetic parameters in transporter mediated permeability across Caco-2 cells. A case study of estrone-3-sulfate.

Substances that compete for the same saturable intestinal transporters may when dosed together lead to altered permeability and hence influence bioavailability. The aim was to simulate kinetic parameters, i.e. K(m) and J(max), for transporter mediated E(1)S permeability across Caco-2 cells by a combined experimental modeling approach. 4 classes of transporters were suggested to be involved in the permeability of E(1)S, i.e. apical influx (T(I)) and efflux (T(III)) as well as basolateral efflux (T(II)) and influx (T(IV)). Efflux ratio of E(1)S was determined to 6.8. E(1)S is suggested to have highest affinity to T(III). T(IV) is however suggested to be rate limiting in exsorptive P(APP) due to lower J(max) of T(IV), compared to T(III). Possible interactions between E(1)S and the excipients erythrosine and Brij35 on these 4 classes of transporters were also studied. From these studies it is suggested that erythrosine does interact with E(1)S on apical efflux transporter T(III) by competitive inhibition. Furthermore interaction between erythrosine and E(1)S is suggested on apical influx transporter (T(I)). Brij35 does not seem to interact with E(1)S on apical transporters. The present model seem to be a valuable tool to simulate kinetic parameters for compounds being substrates to multiple transporters as well as to estimate kinetic parameters for compounds interacting on the same transporters.

The tandem chain extension aldol reaction used for synthesis of ketomethylene tripeptidomimetics targeting hPEPT1.

The rationale for targeting the human di-/tripeptide transporter hPEPT1 for oral drug delivery has been well established by several drug and prodrug cases. The aim of this study was to synthesize novel ketomethylene modified tripeptidomimetics and to investigate their binding affinity for hPEPT1. Three related tripeptidomimetics of the structure H-Phe-ψ[COCH(2)]-Ser(Bz)-X(aa)-OH were synthesized applying the tandem chain extension aldol reaction, where amino acid derived ß-keto imides were stereoselectively converted to α-substituted γ-keto imides. In addition, three corresponding tripeptides, composed of amide bonds, were synthesized for comparison of binding affinities. The six investigated compounds were all defined as high affinity ligands (K(i)-values <0.5 mM) for hPEPT1 by measuring the concentration dependent inhibition of apical [(14)C]Gly-Sar uptake in Caco-2 cells. Consequently, the ketomethylene replacement for the natural amide bond and α-side chain modifications appears to offer a promising strategy to modify tripeptidic structures while maintaining a high affinity for hPEPT1.

Interactions between organic anions on multiple transporters in Caco-2 cells.

In drug development, Caco-2 cells are often employed to study the influence of membrane transporters on drug permeability. The aim of the current study was to characterize permeability and kinetic parameters of selected organic anionic compounds in Caco-2 cells, and to investigate whether the Caco-2 cell line may be used as an overall model to predict interactions on multiple membrane transporters in the intestine. Taurocholic acid (TCA) and estrone-3-sulfate (E(1) S) were used as model substrates. Possible inhibitors studied were TCA, E(1) S, taurolithocholic acid, fluvastatin, and glipizide. The effects of these compounds on initial uptake, apparent permeability, and intracellular end-point accumulations of the probe substrates were studied. Both interactions on apical and basolateral influx transporters were observed. These interactions were proposed to be mediated mainly by the apical sodium-dependent bile acid transporter and the organic solute transporter α/ß, and to less extent by the organic anion transporting polypeptide 2B1. However, interactions on efflux transporters were not detected, although they were expected from the literature on the investigated compounds. Biosimulation methods may be the key to further distinguish between interactions on multiple transporters, including the involvement of efflux transporters, by taking into account the changes in dynamics that drug interactions may cause.

A quantitative structure-activity relationship for translocation of tripeptides via the human proton-coupled peptide transporter, hPEPT1 (SLC15A1).

The human intestinal proton-coupled peptide transporter, hPEPT1 (SLC15A1), has been identified as an absorptive transporter for both drug substances and prodrugs. An understanding of the prerequisites for transport has so far been obtained from models based on competition experiments. These models have limited value for predicting substrate translocation via hPEPT1. The aim of the present study was to investigate the requirements for translocation via hPEPT1. A set of 55 tripeptides was selected from a principal component analysis based on VolSurf descriptors using a statistical design. The majority of theses tripeptides have not previously been investigated. Translocation of the tripeptides via hPEPT1 was determined in a MDCK/hPEPT1 cell-based translocation assay measuring substrate-induced changes in fluorescence of a membrane potential-sensitive probe. Affinities for hPEPT1 of relevant tripeptides were determined by competition studies with [14C]Gly-Sar in MDCK/hPEPT1 cells. Forty tripeptides were found to be substrates for hPEPT1, having K(m)(app) values in the range 0.4-28 mM. Eight tripeptides were not able to cause a substrate-induced change in fluorescence in the translocation assay and seven tripeptides interacted with the probe itself. The conformationally restricted tripeptide Met-Pro-Pro was identified as a novel high-affinity inhibitor of hPEPT1. We also discovered the first tripeptide (Asp-Ile-Arg) that was neither a substrate nor an inhibitor of hPEPT1. To rationalise the requirements for transport, a quantitative structure-activity relationship model correlating K(m)(app) values with VolSurf descriptors was constructed. This is, to our knowledge, the first predictive model for the translocation of tripeptides via hPEPT1.

Impact of carriers in oral absorption: Permeation across Caco-2 cells for the organic anions estrone-3-sulfate and glipizide.

Carriers may mediate the permeation across enterocytes for drug substances being organic anions. Carrier mediated permeation for the organic anions estrone-3-sulfate (ES) and glipizide across Caco-2 cells were investigated kinetically, and interactions on involved carriers evaluated. Initial uptakes (P(UP)) at apical and basolateral membranes, apparent permeabilities (P(APP)) and corresponding intracellular end-point accumulations (P(EPA)) of radioactive labeled compounds were studied. Possible effects of other anionic compounds were investigated. Apical P(UP) and absorptive P(APP) for ES were inhibited and its absorptive P(EPA) prevented in presence of the investigated organic anions and apical P(UP) was saturable with K(m) 23microM. Basolateral P(UP) and exsorptive P(APP) were inhibited, its exsorptive P(EPA) was prevented, and basolateral P(UP) and exsorptive P(APP) were saturable with K(m) 44microM and 38microM, respectively. BCRP inhibition affected both absorptive an exsorptive P(EPA) and P(APP) for ES. Glipizide apical P(UP) and absorptive P(APP) were not inhibitable. Basolateral P(UP) for glipizide was inhibitable, its P(EPA) prevented, and P(UP) was saturable with K(m) 56microM, but exsorptive P(APP) was not affected. Carrier mediated exsorption kinetics for ES are seen at both apical and basolateral membranes, resulting in predominant exsorption despite presence of absorptive carrier(s). Carrier mediated basolateral P(UP) for glipizide was observed, but glipizide P(APP) was not described by carrier kinetics. However, glipizide is affecting exsorption for ES, due to interactions on basolateral carrier. The study confirms that estrone-3-sulfate can be used to characterize anionic carrier kinetics. Furthermore it is suggested that estrone-3-sulfate may be used to identify compounds which may interact on anionic carriers.

Impact of transporters in oral absorption: a case study of in vitro and in vivo organic anion absorption.

A key determinant for oral bioavailability of a drug candidate is the intestinal epithelial permeation of the drug candidate. This intestinal permeation may be affected by interactions on membrane transporters expressed in the intestinal epithelial cells. The purpose of the present study was to investigate whether transporters were involved in the intestinal absorption of an organic anion A275 and to compare the impact of interactions related to transporters in the Caco-2 cell model versus the in vivo rat model of intestinal absorption. In both models, it was investigated whether intestinal permeation of A275 was concentration dependent and affected by inhibitors or competitive organic anions. Interactions related to transporters in intestinal permeation was clearly demonstrated in the Caco-2 cell model but was not directly evident for in vivo rat absorption. However, an observed biphasic in vivo absorption and a large intervariability between rats might mask a dose-dependent absorption of A275. To avoid these suggested interactions, a dose of at least 10 mg/kg, which saturates the intestinal transporters involved in A275 absorption, should be administered, but at doses below that the risk of such drug interactions should be taken into account.