Anne H. Dantzig

Uptake of the cephalosporin, cephalexin, by a dipeptide transport carrier in the human intestinal cell line, Caco-2

The transport of the orally absorbed cephalosporin, cephalexin, was examined in the human epithelial cell line, Caco-2 that possesses intestinal enterocyte-like properties when cultured. In sodium-free buffer, the cells accumulated 1 mM D-[9-14C]cephalexin against a concentration gradient and obtained a distribution ratio of 3.5 within 180 min. Drug uptake was maximal when the extracellular pH was 6.0. Uptake was reduced by metabolic inhibitors and by protonophores indicating that uptake was energy- and proton-dependent. Kinetic analysis of the concentration dependence of the rate of cephalexin uptake showed that a non-saturable component (Kd of 0.18 +/- 0.01 nmol/min per mg protein per mM) and a transport system with a Km of 7.5 +/- 2.8 mM and a Vmax of 6.5 +/- 0.9 nmol/min per mg protein were responsible for drug uptake. Uptake was competitively inhibited by dipeptides. The transport carrier exhibited stereospecificity for the L-isomer of cephalexin. Drug uptake was not affected by the presence of amino acids, organic anions, 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid or 4,4-diisothiocyano-2,2-disulfonic stilbene. Therefore, Caco-2 cells take up cephalexin by a proton-dependent dipeptide transport carrier that closely resembles the transporter present in the intestine. Caco-2 cells represent a cellular model for future studies of the dipeptide transporter.

Intestinal peptide transport systems and oral drug availability

The intestinal peptide transport system has broad substrate specificities. In addition to its physiological function of absorbing di- and tripeptides resulting from the digestion of dietary proteins, this transport system also absorbs some orally administered peptidomimetic drugs, including β-lactam antibiotics, angiotensin converting enzyme inhibitors, renin inhibitors, bestatin, thrombin inhibitors, and thyrotropin-releasing hormone and its analogues. There have been several studies on the mechanism and substrate structure-affinity relationship for this transport system. Rapid progress has been made recently in studies on the molecular basis of the intestinal peptide transport system. A protein apparently involved in peptide transport has been isolated from rabbit small intestines, and genes for human intestinal peptide transporters have been cloned, sequenced and functionally expressed. This review summarizes these studies and addresses the pharmaceutical potential of the intestinal peptide transport system.

The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites

5′-Fluorouracil (5-FU), used in the treatment of colon and breast cancers, is converted intracellularly to 5′-fluoro-2′-deoxyuridine (5-FUdR) by thymidine phosphorylase and is subsequently phosphorylated by thymidine kinase to 5′-fluoro-2′-dUMP (5-FdUMP). This active metabolite, along with the reduced folate cofactor, 5,10-methylenetetrahydrofolate, forms a stable inhibitory complex with thymidylate synthase that blocks cellular growth. The present study shows that the ATP-dependent multidrug resistance protein-5 (MRP5, ABCC5) confers resistance to 5-FU by transporting the monophosphate metabolites. MRP5- and vector-transfected human embryonic kidney (HEK) cells were employed in these studies. In 3-day cytotoxicity assays, MRP5-transfected cells were ∼9-fold resistant to 5-FU and 6-thioguanine. Studies with inside-out membrane vesicles prepared from transfected cells showed that MRP5 mediates ATP-dependent transport of 5 μmol/L [3H]5-FdUMP, [3H]5-FUMP, [3H]dUMP, and not [3H]5-FUdR, or [3H]5-FU. The ATP-dependent transport of 5-FdUMP showed saturation with increasing concentrations and had a Km of 1.1 mmol/L and Vmax of 439 pmol/min/mg protein. Uptake of 250 μmol/L 5-FdUMP was inhibited by dUMP, cyclic nucleotide, cyclic guanosine 3′,5′-monophosphate, amphiphilic anions such as probenecid, MK571, the phosphodiesterase inhibitors, trequinsin, zaprinast, and sildenafil, and by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and glybenclamide. Furthermore, the 5-FU drug sensitivity of HEK-MRP5 cells was partially modulated to that of the HEK-vector by the presence of 40 μmol/L 5-nitro-2-(3-phenylpropylamino)-benzoic acid but not by 2 mmol/L probenecid. Thus, MRP5 transports the monophosphorylated metabolite of this nucleoside and when MRP5 is overexpressed in colorectal and breast tumors, it may contribute to 5-FU drug resistance.

Modulation of P-glycoprotein but not MRP1- or BCRP-mediated drug resistance by LY335979

Our study examines the ability of LY335979 (Zosuquidar trihydrochloride) to modulate 3 distinct ABC transporters that are mechanisms of drug resistance: P‐glycoprotein (Pgp, ABCB1), multidrug resistance associated protein (MRP1, ABCC2) and breast cancer resistance protein (BCRP, ABCG2). Pgp‐mediated resistance can be modulated by coadministration with the highly potent, selective inhibitor, LY335979. Modulation of resistance by mitoxantrone and vinorelbine, 2 drugs used to treat certain solid tumors, was examined in a 3‐day cytotoxicity assay using a panel of HL60 leukemia cell lines or MCF‐7 breast cancer transfectants. LY335979, at 0.5 μM, substantially reversed mitoxantrone resistance and fully reversed vinorelbine resistance of Pgp‐expressing HL60/Vinc cells. However, LY335979 did not modulate drug resistance in the MRP1‐expressing HL60/ADR or drug‐sensitive parental HL60 cells. To ascertain if LY335979 modulates BCRP‐mediated drug resistance, the sensitivity of 26‐fold mitoxantrone resistant, BCRP‐transfected MCF‐7 cells was evaluated. Addition of 5 μM LY335979, a concentration ∼100‐fold higher than the affinity of Pgp, had little to no effect on the BCRP transfectant. [125I]Iodomycin photolabeled Pgp in CEM/VLB100 membranes and was inhibited by 5 μM LY335979 and GF120918. No photolabeling of MRP or BCRP occurred in H69AR or MCF‐7/BCRP membranes, respectively. These results further demonstrate that LY335979 is highly specific for Pgp and does not modulate MRP1‐ or BCRP‐mediated resistance and can be used in combination with mitoxantrone and vinorelbine in tumor cells.

Considerations in the design and development of transport inhibitors as adjuncts to drug therapy.

With the realization of the importance of drug efflux transporters in disease processes and treatment, development of inhibitors to these transporters has been sought for use as adjuncts to therapy. To date, inhibitors that have been best studied are modulators of P-glycoprotein, a transporter important in the removal of anticancer agents from cells and overexpression of this transporter results in multidrug resistance. There is a delicate balance between efficacy and toxicity. This review summarizes key learning points in the development of P-glycoprotein inhibitors. Topics covered include specificity of the inhibitor for the target transporter, effect on metabolism of coadministered drugs, pharmacokinetic interactions, toxicity and the salient features needed for efficacy. These points will have general application to the development of inhibitors of transporters.

Pharmacological characterization of LY335979: A potent cyclopropyldibenzosuberane modulator of P-glycoprotein

The above data indicate that LY335979 displays the following characteristics of an ideal modulator of Pgp-mediated multidrug resistance: high affinity binding to Pgp, high potency for in vitro reversal of drug resistance, high therapeutic index (activity was demonstrated at doses ranging from 1-30 mg/kg) observed in in vivo antitumor efficacy experiments, and a lack of pharmacokinetic interactions that alter the plasma concentration of coadministered oncolytic agents. These desirable features strongly suggest that LY335979 is an exciting new clinical agent to test the hypothesis that inhibition of P-glycoprotein activity will result in reversal of multidrug resistance in human tumors.

Synthesis, Crystallization, and Biological Evaluation of an Orally Active Prodrug of Gemcitabine

The design, synthesis, and biological characterization of an orally active prodrug (3) of gemcitabine are described. Additionally, the identification of a novel co-crystal solid form of the compound is presented. Valproate amide 3 is orally bioavailable and releases gemcitabine into the systemic circulation after passing through the intestinal mucosa. The compound has entered clinical trials and is being evaluated as a potential new anticancer agent.

Mechanism and Kinetics of Transcellular Transport of a New β-Lactam Antibiotic Loracarbef Across an Intestinal Epithelial Membrane Model System (Caco-2)

Various processes involved in the transcellular transport (TT) of loracarbef (LOR) were studied in the Caco-2 cell monolayer, a cell culture model of the small intestinal epithelium. The results provide support for presence of two AP to BL peptide TT pathways in the intestinal epithelial cell monolayer (Caco-2). The H+ gradient-dependent pathway (Km = 0.789 mM, and Jmax = 163 pmol/min per cm2) is relatively “high affinity” and “low capacity” compared to H+ gradient-independent pathway (Km = 8.28 mM, and Jmax = 316 pmol/min per cm2). In addition, TT of LOR in the presence of a H+ gradient was inhibited 77% to 88% (p < 0.05) by 10 mM of cephalexin, enalapril, Gly-Pro and Phe-Pro, while TT of LOR in the absence of a H+ gradient was only inhibited 42% to 48% (p < 0.05) by 10 mM of Gly-Pro and Phe-Pro. Since AP uptake is H+ gradient-dependent and saturable while the BL efflux is mostly nonsaturable and not driven by a H+ gradient, these two transmembrane transport processes must be different, which could be the result of two different peptide carriers. In vivo, these two transport processes must have worked in concert to produce transcellular flux of loracarbef. To explain the differences between kinetic characteristics of AP uptake and TT transport, a cellular pharmacokinetic (PK) model was developed and the results indicate that the PK model appropriately described the kinetics of LOR TT. The use of this PK model may provide an additional advantage to the use of the cell culture model because kinetic parameters at both sides of the intestinal epithelial membrane may be obtained using the same preparation. Taken together, the Caco-2 model system represents an excellent model system for the study of carrier-mediated processes involved in the TT of peptides and peptide-like drugs.

Transport mechanisms responsible for the absorption of loracarbef, cefixime, and cefuroxime axetil into human intestinal Caco-2 cells

Loracarbef, cefixime and cefuroxime axetil are beta-lactam antibiotics that are administered orally. Oral absorption of loracarbef is nearly complete, while that of cefixime and cefuroxime axetil is 30-50%. To investigate this we used the human intestinal cell line Caco-2 that possesses the proton-dependent peptide transporter that takes up cephalexin and cefaclor. Drug uptake was measured at pH 6 by high performance liquid chromatography or with radioactively labelled drug. The initial uptake rate of 1 mM cefixime was lower than that of 1 mM loracarbef. By 2 h both drugs were concentrated intracellularly against a gradient; however, the accumulation of cefixime was only 40% of that of loracarbef. The uptake rate of both drugs was sodium-independent, temperature- and energy-dependent, and was inhibited by dipeptides, cephalexin, cefaclor, but not by amino acids. Kinetic analysis of the concentration-dependence of the uptake rates for loracarbef and cefixime indicated that diffusion and a single transport system were responsible for uptake. The kinetic parameters for loracarbef and cefixime, respectively, were: Km values of 8 and 17 mM and Vmax values of 6.5 and 2 nmol/min per mg protein. Loracarbef and cefixime were competitive inhibitors of each others uptake. By contrast, cefuroxime axetil was taken up and rapidly hydrolyzed to cefuroxime by Caco-2 cells. Cefuroxime axetil uptake was not dependent on energy and was not affected by dipeptides. Thus, cefuroxime axetil apparently enters Caco-2 cells by simple diffusion. By contrast, loracarbef and cefixime share a common transport mechanism, the proton-dependent dipeptide transporter. Cefixime was taken up less well than loracarbef due to a substantial reduction in the turnover rate and decreased affinity of the transporter for cefixime.

Tricyclic isoxazoles are novel inhibitors of the multidrug resistance protein (MRP1)

Tricyclic isoxazoles were identified from a screen as a novel class of selective multidrug resistance protein (MRP1) inhibitors. From a screen lead, SAR efforts resulted in the preparation of LY 402913 (9h), which inhibits MRP1 and reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC(50)=0.90 microM), while showing no inherent cytotoxicity. Additionally, LY 402913 inhibits ATP-dependent, MRP1-mediated LTC(4) uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC(50)=1.8 microM). LY 402913 also shows selectivity ( approximately 22-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, LY 402913 delays the growth of MRP1-overexpressing tumors in vivo.