Saturable Absorptive Transport of the Hydrophilic Organic Cation Ranitidine in Caco-2 Cells: Role of pH-Dependent Organic Cation Uptake System and P-Glycoprotein

Bourdet, David L.; Thakker, Dhiren R.

doi:10.1007/s11095-006-0251-4

Cited by 39 publications

(36 citation statements)

References 38 publications

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“…Cimetidine has been reported to be a substrate for both P-gp and BCRP (Pavek et al, 2005;Taur and Rodriguez-Proteau, 2008), while ranitidine is transported primarily by P-gp (Collett et al, 1999;Bourdet and Thakker, 2006). Our results confirmed that cimetidine was a substrate of both P-gp and BCRP, as the efflux ratio was reduced to unity only in the MDR1/BCRP KO cell line, while ranitidine was identified as a substrate for P-gp alone.…”

Section: Discussionsupporting

confidence: 82%

Zinc Finger Nuclease–Mediated Gene Knockout Results in Loss of Transport Activity for P-Glycoprotein, BCRP, and MRP2 in Caco-2 Cells

Sampson¹,

Brinker²,

Pratt³

et al. 2014

Drug Metab Dispos

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Membrane transporters P-glycoprotein [P-gp; multidrug resistance 1 (MDR1)], multidrug resistance-associated protein (MRP) 2, and breast cancer resistance protein (BCRP) affect drug absorption and disposition and can also mediate drug-drug interactions leading to safety/toxicity concerns in the clinic. Challenges arise with interpreting cell-based transporter assays when substrates or inhibitors affect more than one actively expressed transporter and when endogenous or residual transporter activity remains following overexpression or knockdown of a given transporter. The objective of this study was to selectively knock out three drug efflux transporter genes (MDR1, MRP2, and BCRP), both individually as well as in combination, in a subclone of Caco-2 cells (C2BBe1) using zinc finger nuclease technology. The wild-type parent and knockout cell lines were tested for transporter function in Transwell bidirectional assays using probe substrates at 5 or 10 mM for 2 hours at 37°C. P-gp substrates digoxin and erythromycin, BCRP substrates estrone 3-sulfate and nitrofurantoin, and MRP2 substrate 5-(and-6)-carboxy-29,79-dichlorofluorescein each showed a loss of asymmetric transport in the MDR1, BCRP, and MRP2 knockout cell lines, respectively. Furthermore, transporter interactions were deduced for cimetidine, ranitidine, fexofenadine, and colchicine. Compared with the knockout cell lines, standard transporter inhibitors showed substrate-specific variation in reducing the efflux ratios of the test compounds. These data confirm the generation of a panel of stable Caco-2 cell lines with single or double knockout of human efflux transporter genes and a complete loss of specific transport activity. These cell lines may prove useful in clarifying complex drug-transporter interactions without some of the limitations of current chemical or genetic knockdown approaches.

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Section: Discussionsupporting

confidence: 82%

Zinc Finger Nuclease–Mediated Gene Knockout Results in Loss of Transport Activity for P-Glycoprotein, BCRP, and MRP2 in Caco-2 Cells

Sampson¹,

Brinker²,

Pratt³

et al. 2014

Drug Metab Dispos

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“…To further characterize the transport across apical and basolateral membranes and investigate the possible contribution of carrier-mediated transporters, efflux and trans-stimulation efflux studies were performed (Zhang et al, 1999;Mizuuchi et al, 2000;Bourdet and Thakker, 2006). Figure 6 shows the directional efflux and demonstrates the effect of LY354740 and LY544344, present in the extracellular medium, on the efflux of [ 14 C]LY354740 amount.…”

Section: Varma Et Almentioning

confidence: 99%

Transepithelial Transport of the Group II Metabotropic Glutamate 2/3 Receptor Agonist (1S,2S,5R,6S)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740) and Its Prodrug (1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)propionyl]aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY544344)

Varma¹,

Eriksson²,

Pak³

et al. 2008

Drug Metab Dispos

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ABSTRACT:The limited oral bioavailability of the potent and selective group II metabotropic glutamate (mGlu) 2/3 receptor agonist, (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740), was shown to be improved by its peptidyl prodrug, (1S,2S,5R,6S)-2-[(2S)-(2-amino)propionyl]aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY544344). The purpose of this study was to elucidate the mechanisms of intestinal absorption of LY354740 and its prodrug LY544344. Transepithelial transport and accumulation studies were performed in Caco-2 cell monolayers; the involvement of the peptide transporter 1 (PEPT1) transporter was also examined. In absorptive transport studies, the peptidyl prodrug LY544344 partially hydrolyzed to release LY354740 intracellularly, and both compounds appeared in the basolateral compartment. The absorptive transport rate of LY544344, basolateral appearance rate of LY354740, and their cellular accumulation after incubation with LY544344 were concentrationdependent. PEPT1 inhibition reduced transepithelial transport and cellular accumulation of LY544344 to 22 and 1.1% of control, respectively. LY354740 showed concentration-independent absorptive transport with negligible cellular accumulation. Efflux and transstimulation studies revealed predominantly apical efflux and the existence of specific transporters for LY544344 and intracellularly released LY354740 on the apical and basolateral membranes. LY544344 efflux was also trans-stimulated at the apical side by glycyl-glutamate but not by glycyl-sarcosine. Transport of neither compound was affected by P-glycoprotein-mediated efflux, as shown in transport and uptake inhibition studies in Madin-Darby canine kidney multidrug resistance 1-transfected cell line and inverted membrane vesicles. In conclusion, LY354740 is mainly transported by the paracellular pathway, whereas intestinal absorption of LY544344 is mediated by PEPT1. However, the absorptive transport of LY544344 seems to be modulated by an apical efflux transporter and a rate-limiting transport step across the basolateral membrane.

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“…[8,11,12] To provide insights into the uptake mechanism, the mean fluorescence, and accordingly the uptake/release of pyrene-R, as a function of concentration and time was monitored during incubation with [pyrene-R&1] 6 + and during chase (Figure 4). Figure 4 (top) shows that the uptake of the cage does not increase linearly with incubation time.…”

mentioning

confidence: 99%

Evidence for Drug Release from a Metalla‐Cage Delivery Vector Following Cellular Internalisation

Zava

Mattsson

Therrien

et al. 2010

Chemistry A European J

123

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One of the main challenges in cancer chemotherapy is to develop drugs that selectively target cancer cells in order to reduce general toxicity and consequently side effects. One such targeting method involves using large carrier compounds that release a drug once inside a cancer cell, since large compounds selectively accumulate in cancer tissue due to the "enhanced permeability and retention effect". [1] Recently, we have shown that the encapsulation of a hydrophobic metallo-drug guest in a water-soluble hexacationic arene ruthenium cage delivery vector produces a synergistic effect, due to the modest cytotoxicity of the cage itself, and the cytotoxicity of the encapsulated guest compound. [2] We have now encapsulated in the cavity of [Ru 6 (p-iPrC 6 H 4 Me) 6 A C H T U N G T R E N N U N G (tpt) 2 -A C H T U N G T R E N N U N G (C 6 H 2 O 4 ) 3 ] 6 + (1) (tpt = 2,4,6-tris(pyridin-4-yl)-1,3,5-triazine), an intrinsically fluorescent pyrenyl compound (pyrene-R = 1-(4,6-dichloro-1,3,5-triazin-2-yl)pyrene), thus giving rise to the hexanuclear metalla-prism [pyrene-R&1] 6 + , in which the pyrenyl derivative occupies the cavity of 1. The fluorescence of pyrene-R is quenched inside the metalla-prism cavity. A study of this new system has provided direct evidence that once inside the cell, the hexaruthenium cage releases the fluorescent guest, thus confirming our initial hypothesis that cage 1 can act as a Trojan horse for cancer cells. [2] In addition, experiments into the uptake mechanism indicate that an assisted diffusion pathway is in operation.

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Saturable Absorptive Transport of the Hydrophilic Organic Cation Ranitidine in Caco-2 Cells: Role of pH-Dependent Organic Cation Uptake System and P-Glycoprotein

Cited by 39 publications

References 38 publications

Zinc Finger Nuclease–Mediated Gene Knockout Results in Loss of Transport Activity for P-Glycoprotein, BCRP, and MRP2 in Caco-2 Cells

Zinc Finger Nuclease–Mediated Gene Knockout Results in Loss of Transport Activity for P-Glycoprotein, BCRP, and MRP2 in Caco-2 Cells

Transepithelial Transport of the Group II Metabotropic Glutamate 2/3 Receptor Agonist (1S,2S,5R,6S)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740) and Its Prodrug (1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)propionyl]aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY544344)

Evidence for Drug Release from a Metalla‐Cage Delivery Vector Following Cellular Internalisation

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