| 1. |
- Antonescu, Irina E., et al.
(author)
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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
- 2020
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In: Pharmaceutics. - : MDPI. - 1999-4923. ; 12:4
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Journal article (peer-reviewed)abstract
- 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 K-m-value of approximately 700 mu M. Probenecid inhibited OAT1-mediated acamprosate uptake with a K-i-value of approximately 13 mu M, which may translate into an estimated clinically significant DDI index. In conclusion, acamprosate was identified as a substrate of OAT1 but not OAT3.
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| 2. |
- Antonescu, Irina E, et al.
(author)
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The Permeation of Acamprosate Is Predominantly Caused by Paracellular Diffusion across Caco-2 Cell Monolayers : A Paracellular Modeling Approach
- 2019
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In: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 16:11, s. 4636-4650
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Journal article (peer-reviewed)abstract
- 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.
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| 3. |
- Dahlgren, David
(author)
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Biopharmaceutical aspects of intestinal drug absorption : Regional permeability and absorption-modifying excipients
- 2018
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Doctoral thesis (other academic/artistic)abstract
- Before an orally administered drug reaches the systemic circulation, it has to dissolve in the intestinal fluids, permeate across the intestinal epithelial cell barrier, and pass through the liver. The permeation rate of drug compounds can be low and show regional differences.The thesis had two general aims. The first of these was, to determine and compare regional intestinal permeability values of model compounds in human and dog. The second was to understand the possible effects of absorption-modifying pharmaceutical excipients (AMEs) on the intestinal permeability of the model compounds. The usefulness of several preclinical animal models for predicting the impact of regional intestinal permeability and AMEs in human was also investigated.There was a good correlation between human and dog permeability values in the small intestines for the model compounds. The colon in dog was substantially more permeable than the human colon to the low permeability drug, atenolol. This difference in colonic permeability may have implications for the use of dog as a model species for prediction of human intestinal drug absorption.There were no effects of AMEs on the intestinal permeability of any of the high permeability compounds, in any animal model. In the rat single-pass intestinal perfusion model, there was a substantial increase in permeability of all low permeability drugs, induced by two AMEs, chitosan and SDS. This AME-induced increase was substantially lower in the more in vivo relevant rat and dog intraintestinal bolus models. A shorter AME exposure-time in the rat single-pass intestinal perfusion model (15 vs. 75 min) could, however, predict the result from the bolus studies in rat and dog. This illustrates the impact of intestinal transit and mucosal exposure time on AME effects in vivo. The intestinal luminal conditions and enteric neural activity also had an impact on determinations of drug permeability in the rat single-pass intestinal perfusion model, which can have implications for its in vivo relevance.In summary, this thesis used multiple in vivo models to evaluate the impact of several biopharmaceutical processes on intestinal drug absorption. This has led to an increased understanding of these absorption mechanisms.
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| 4. |
- Gram, Luise K, et al.
(author)
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Impact of carriers in oral absorption : Permeation across Caco-2 cells for the organic anions estrone-3-sulfate and glipizide
- 2009
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In: European Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0928-0987 .- 1879-0720. ; 37:3-4, s. 378-386
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Journal article (peer-reviewed)abstract
- 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.
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| 5. |
- Grandvuinet, Anne Sophie, et al.
(author)
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New Insights into the Carrier-Mediated Transport of Estrone-3-sulfate in the Caco-2 Cell Model
- 2013
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In: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8392 .- 1543-8384. ; 10:9, s. 3285-3295
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Journal article (peer-reviewed)abstract
- 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 [H-3]-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 (P-UP) of [H-3]-E1S suggest that the organic anion transporting. polypeptide (OATP) 2B1 is the Main transporter involved in the apical E1S P-UP 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 [H-3]-E1S P-UP by more than 2-fold for filter grown cells. The basolateral P-UP of [H-3]-E1S of filter grown cells was inhibited by several inhibitors of the bidirectional, transporter organic solute transporter alpha/beta (OST alpha/beta). Efflux studies were performed by loading the cells with either [H-3]-E1S or [H-3]-taurocholic acid (TCA) and subsequently measuring the efflux of radio labeled' substance in the absence or presence of BCRP or OST alpha/beta inhibitors. Similar effluxes of [H-3]-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 [H-3]-TCA to the basolateral compartment was clearly larger than to the apical compartment. Trans-stimulation of basolateral [H-3]-E1S efflux was observed in the presence of taurolithocholic acid (TLC), although none of the applied OST alpha/beta inhibitors were able to confirm the existence of carrier mediated efflux at the basolateral membrane, neither for [H-3]-E1S nor for [H-3]-TCA. These results highlight the importance of transporter interplay for EIS and drug compounds in Caco-2 cells and emphasize the importance of identifying the basolateral transporters in these cells.
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| 6. |
- Våbenø, Jon, et al.
(author)
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Conformational restrictions in ligand binding to the human intestinal di-/tripeptide transporter: implications for design of hPEPT1 targeted prodrugs.
- 2005
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In: Bioorganic & medicinal chemistry. - : Elsevier BV. - 0968-0896. ; 13:6, s. 1977-88
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Journal article (peer-reviewed)abstract
- The aim of the present study was to develop a computational method aiding the design of dipeptidomimetic pro-moieties targeting the human intestinal di-/tripeptide transporter hPEPT1. First, the conformation in which substrates bind to hPEPT1 (the bioactive conformation) was identified by conformational analysis and 2D dihedral driving analysis of 15 hPEPT1 substrates, which suggested that psi(1) approximately 165 degrees , omega(1) approximately 180 degrees , and phi(2) approximately 280 degrees were descriptive of the bioactive conformation. Subsequently, the conformational energy required to change the peptide backbone conformation (DeltaE(bbone)) from the global energy minimum conformation to the identified bioactive conformation was calculated for 20 hPEPT1 targeted model prodrugs with known K(i) values. Quantitatively, an inverse linear relationship (r(2)=0.81, q(2)=0.80) was obtained between DeltaE(bbone) and log1/K(i), showing that DeltaE(bbone) contributes significantly to the experimentally observed affinity for hPEPT1 ligands. Qualitatively, the results revealed that compounds classified as high affinity ligands (K(i)<0.5 mM) all have a calculated DeltaE(bbone)<1 kcal/mol, whereas medium and low-affinity compounds (0.5 mM
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| 7. |
- Våbenø, Jon, et al.
(author)
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Dipeptidomimetic ketomethylene isosteres as pro-moieties for drug transport via the human intestinal di-/tripeptide transporter hPEPT1: design, synthesis, stability, and biological investigations.
- 2004
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In: Journal of medicinal chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 47:19, s. 4755-65
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Journal article (peer-reviewed)abstract
- Five dipeptidomimetic-based model prodrugs containing ketomethylene amide bond replacements were synthesized from readily available alpha,beta-unsaturated gamma-ketoesters. The model drug (BnOH) was attached to the C-terminus or to one of the side chain positions of the dipeptidomimetic. The stability, the affinity for the di-/tripeptide transporter hPEPT1, and the transepithelial transport properties of the model prodrugs were investigated. ValPsi[COCH(2)]Asp(OBn) was the compound with highest chemical stability in buffers at pH 6.0 and 7.4, with half-lives of 190 and 43 h, respectively. All five compounds showed high affinity for hPEPT1 (K(i) values < 1 mM), and PhePsi[COCH(2)]Asp(OBn) and ValPsi[COCH(2)]Asp(OBn) had the highest affinities with K(i) values of 68 and 19 microM, respectively. An hPEPT1-mediated transport component was demonstrated for the transepithelial transport of three compounds, a finding that was corroborated by hPEPT1-mediated intracellular uptake. The results indicate that the stabilized Phe-Asp and Val-Asp derivatives are promising pro-moieties in a prodrug approach targeting hPEPT1.
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| 8. |
- Våbenø, Jon, et al.
(author)
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Phe-Gly dipeptidomimetics designed for the di-/tripeptide transporters PEPT1 and PEPT2: synthesis and biological investigations.
- 2004
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In: Journal of medicinal chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 47:4, s. 1060-9
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Journal article (peer-reviewed)abstract
- A series of five Phe-Gly dipeptidomimetics containing different amide bond replacements have been synthesized in a facile way from the readily available unsaturated ketoester 1, and their affinities for the di-/tripeptide transporters hPEPT1 (Caco-2 cells) and rPEPT2 (SKPT cells) were tested. The compounds contained the amide bond isosteres ketomethylene (2a), (R)- and (S)-hydroxyethylidene (3a and 4a), and (R)- and (S)-hydroxyethylene (5a and 6a) to provide information on the conformational and stereochemical requirements for hPEPT1 and rPEPT2 affinity. The affinity studies showed that for rPEPT2 there is no significant difference in affinity between the ketomethylene isostere 2a and the natural substrate Phe-Gly (K(i) values of 18.8 and 14.6 microM, respectively). Also the affinities for hPEPT1 are in the same range (K(i) values of 0.40 and 0.20 mM, respectively). This corroborates earlier findings that the amide bond as such is not essential for binding to PEPTX, but the results also reveal possible differences in the binding of ketomethylene isosteres to hPEPT1 and rPEPT2. The trans-hydroxyethylidene and hydroxyethylene isosteres proved to be poor substrates for PEPTX. These results provide new information about the importance of flexibility and of the stereochemistry at the C(4)-position for this class of compounds. Furthermore, the intracellular uptake of 2a-4a in Caco-2 cells was investigated, showing a 3-fold reduction of the uptake of 2a in the presence of the competetive inhibitor Gly-Pro, indicating contribution from an active transport component. No active uptake of 3a and 4a was observed. Transepithelial transport studies also indicated active transport of 2a across Caco-2 monolayers.
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