l-660-711 and verlukast

Cornea is considered as a major barrier for ocular drug delivery. Low ocular bioavailability of drugs has been attributed primarily to low permeability across corneal epithelium, thus leading to sub-therapeutic concentrations of drug in the eye and treatment failure. The role of drug efflux proteins, particularly the P-glycoprotein (P-gp) in ocular drug bioavailability has been reported. The objective of this research was to determine whether human corneal epithelium expresses multidrug resistance associated proteins (MRPs) contributing to drug efflux by employing both cultured corneal cells and freshly excised rabbit cornea. SV40-HCEC and rPCEC were selected for in vitro testing. SV40-HCEC and freshly excised rabbit corneas were utilized for transport studies. [(3)H]-cyclosporine-A and [(14)C]-erythromycin, which are known substrates for ABCC2 and MK-571, a specific inhibitor for MRP were applied in this study. RT-PCR indicated a unique and distinct band at approximately 272 bp corresponding to ABCC2 in HCEC, SV40-HCEC, rabbit cornea, rPCEC, and MDCKII-MRP2 cells. Also RT-PCR indicated a unique band approximately 181 bp for HCEC and SV40-HCEC. Immunoprecipitation followed by Western Blot analysis revealed a specific band at approximately 190 kDa in membrane fraction of SV40-HCEC, MDCKII-MRP2 and no band with isotype control. Uptake of [(3)H]-cyclosporine-A and [(14)C]-erythromycin in the presence of MK-571 was significantly enhanced than control in both SV40-HCEC and rPCEC. Similarly a significant elevation in (A-->B) permeability of [(3)H]-cyclosporine-A and [(14)C]-erythromycin was observed in the presence of MK-571 in SV40-HCEC. A-->B transport of [(3)H]-cyclosporine-A was elevated in the presence of MK-571 in freshly excised rabbit cornea indicating potential role of this efflux transporter and high clinical significance of this finding.

Topics: Animals; Biological Transport; Cell Line; Cell Membrane; Cells, Cultured; Cornea; Cyclosporine; Dogs; Epithelial Cells; Epithelium, Corneal; Erythromycin; Gene Expression; Humans; Male; Membrane Transport Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Pharmaceutical Preparations; Propionates; Quinolines; Rabbits

Based upon several previous reports, no consistent relationship between multidrug resistance protein 1 (MRP1, ABCC1) expression and cellular sensitivity to mitoxantrone (MX) toxicity can be ascertained; thus, the role of MRP1 in MX resistance remains controversial. The present study, using paired parental, MRP1-poor, and transduced MRP1-overexpressing MCF7 cells, unequivocally demonstrates that MRP1 confers resistance to MX cytotoxicity and that resistance is associated with reduced cellular accumulation of MX. This MRP1-associated reduced accumulation of MX was partially reversed by treatment of cells with 50 microM MK571 [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid]-an MRP inhibitor that increased MX accumulation in MRP1-expressing MCF7 cells but had no effect on MRP-poor MCF7 cells. Moreover, in vitro experiments using inside-out membrane vesicles show that MRP1 supports ATP-dependent, osmotically sensitive uptake of MX. Unlike ABCG2 (breast cancer resistance protein, mitoxantrone-resistant protein), MRP1-mediated MX transport is dependent upon the presence of glutathione or its S-methyl analog. In addition, MX stimulates transport of [3H]glutathione. Together, these data are consistent with the interpretation that MX efflux by MRP1 involves cotransport of MX and glutathione. The results suggest that MRP1-like the alternative MX transporters ABCG2 and ABCB1 (MDR1, P-glycoprotein)-can significantly influence tumor cell sensitivity to and pharmacological disposition of MX.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Cell Line, Tumor; Drug Resistance, Neoplasm; Glutathione; Humans; Mitoxantrone; Multidrug Resistance-Associated Proteins; Propionates; Quinolines

We investigated the effects of grapefruit juice (GFJ) and orange juice (OJ) on drug transport by MDR1 P-glycoprotein (P-gp) and multidrug resistance protein 2 (MRP2), which are efflux transporters expressed in human small intestine. We examined the transcellular transport and uptake of [(3)H]vinblastine (VBL) and [(14)C]saquinavir in a human colon carcinoma cell line (Caco-2) and in porcine kidney epithelial cell lines transfected with human MDR1 cDNA and human MRP2 cDNA, LLC-GA5-COL150, and LLC-MRP2, respectively. In Caco-2 cells, the basal-to-apical transports of [(3)H]VBL and [(14)C]saquinavir were greater than those in the opposite direction. The ratio of basal-to-apical transport to apical-to-basal transport of [(3)H]VBL and [(14)C]saquinavir by Caco-2 cells was reduced in the presence of MK571 (MRPs inhibitor), verapamil (P-gp inhibitor), cyclosporin A (inhibitor of both), 50% ethyl acetate extracts of GFJ and OJ, or their components (6',7'-dihydroxybergamottin, bergamottin, tangeretin, hepatomethoxyflavone, and nobiletin). Studies of transport and uptake of [(3)H]VBL and [(14)C]saquinavir with MDR1 and MRP2 transfectants showed that VBL and saquinavir are transported by both P-gp and MRP2. GFJ and OJ components inhibited the transport by MRP2 as well as P-gp. However, their inhibitory potencies for P-gp or MRP2 were substrate-dependent. The present study has revealed that GFJ and OJ interact with not only P-gp but also MRP2, both of which are expressed at apical membranes and limit the apical-to-basal transport of VBL and saquinavir in Caco-2 cells.

Topics: Algorithms; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Beverages; Biological Transport, Active; Caco-2 Cells; Calcium Channel Blockers; Citrus; Cyclosporine; Food-Drug Interactions; HIV Protease Inhibitors; Humans; Immunosuppressive Agents; Membrane Transport Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Plant Extracts; Propionates; Quinolines; Saquinavir; Transfection; Verapamil; Vinblastine

The canalicular multispecific organic anion transporter (cMOAT), also termed MRP2, is a recently identified ATP-binding cassette transporter. We previously established stable human cMOAT cDNA-transfected cells, LLC/cMOAT-1 from LLC-PK1 cells, and LLC/CMV cells that were transfected with an empty vector. We found that LLC/cMOAT-1 cells have increased resistance to vincristine (VCR), 7-ethyl-10-hydroxy-camptothecin, and cisplatin but not to etoposide. The multidrug resistance-reversing agents cyclosporin A (CsA) and 2-[4-(diphenylmethyl)-1-piperazinyl]-5-(trans-4,6-dimethyl-1,3, 2-dioxaphosphorinan-2-yl)-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P) almost completely reversed the resistance to VCR, 7-ethyl-10-hydroxy-camptothecin, and cisplatin of LLC/cMOAT-1 cells; and DL-buthionine-(S,R)-sulfoximine, (3'-oxo-4-butenyl-4-methyl-threonine(1), (valine(2)) cyclosporin (PSC833), and 3-([(3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl)-((3-dimethylamino-3- oxopropyl)-thio)-methyl]thio)propanoic acid (MK571) partially reversed the resistance to these drugs. CsA and PAK-104P at 10 microM enhanced the accumulation of VCR in LLC/cMOAT-1 cells almost to the level in LLC/CMV cells without the agents. The efflux of VCR from LLC/cMOAT-1 cells was enhanced compared with LLC/CMV cells and inhibited by CsA and PAK-104P. Transport of leukotriene C(4) (LTC(4)) and S-(2, 4-dinitrophenyl)glutathione also was studied with membrane vesicles prepared from these cells. LTC(4) and S-(2, 4-dinitrophenyl)glutathione were actively transported into membrane vesicles prepared from LLC/cMOAT-1 cells. The K(m) and V(max) values for the uptake of LTC(4) by the LLC/cMOAT-1 membrane vesicles were 0. 26 +/- 0.05 microM and 7.48 +/- 0.67 pmol/min/mg protein, respectively. LTC(4) transport was competitively inhibited by PAK-104P, CsA, MK571, and PSC833, with K(i) values of 3.7, 4.7, 13.1, and 28.9 microM, respectively. These findings demonstrate that cMOAT confers a novel drug-resistance phenotype. CsA and PAK-104P may be useful for reversing cMOAT-mediated drug resistance in tumors.

Topics: Adenosine Triphosphate; Animals; Anion Transport Proteins; Antineoplastic Agents; Biological Transport; Carrier Proteins; Cells, Cultured; Cyclic P-Oxides; Cyclosporine; Drug Interactions; Drug Resistance, Multiple; Glutathione; Humans; Kinetics; Leukotriene C4; Nicotinic Acids; Osmolar Concentration; Propionates; Quinolines; Swine; Transfection; Tritium; Vincristine

Based on LTD4 receptor antagonist activity of 3-(2-quinolinyl-(E)-ethenyl)pyridine (2) found in broad screening, structure-activity studies were carried out which led to the identification of 3-[[[3-[2-(7-chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3- (dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid (1, MK-571) as a potent and orally active LTD4 receptor antagonist. These studies demonstrated that a phenyl ring could replace the pyridine in 2 without loss of activity, that 7-halogen substitution in the quinoline group was optimal for binding, that the (E)-ethenyl linkage was optimal, that binding was enhanced by incorporation of a polar acidic group or groups in the 3-position of the aryl ring, and that two acidic groups could be incorporated via a dithioacetal formed from thiopropionic acid and the corresponding styrylquinoline 3-aldehyde to yield compounds such as 20 (IC50 = 3 nM vs [3H]LTD4 binding to the guinea pig lung membrane). It was found that one of the acidic groups could be transformed into a variety of the amides without loss of potency and that the dimethylamide 1 embodied the optimal properties of intrinsic potency (IC50 = 0.8 nM on guinea pig lung LTD4 receptor) and oral in vivo potency in the guinea pig, hyperreactive rat, and squirrel monkey. The evolution of 2 to 1 involves the increase of > 6000-fold in competition for [3H]LTD4 binding to guinea pig lung membrane and a > 40-fold increase in oral activity as measured by inhibition of antigen-induced dyspnea in hyperreactive rats.

Topics: Animals; Bronchial Hyperreactivity; Guinea Pigs; Magnetic Resonance Spectroscopy; Propionates; Quinolines; Rats; Receptors, Immunologic; Receptors, Leukotriene; Structure-Activity Relationship