Development Of Drug Transport Cell Models, Their Application And Preliminary Study On The Associated Mechanism

Despite tremendous innovations in the drug delivery methods in the last few decades, the oral route still remains as the most preferred route of administration for most new chemical entities. With the explosive growth in the field of genomics and combinatorial chemistry coupled with technological innovations in the last few years, synthesizing a large number of potential drug candidates is no longer a bottleneck in the drug discovery process. Varieties of cell monolayers models that mimic in vivo intestinal epithelium in humans have been developed and currently enjoy widespread popularity. Caco-2 cells have been the most extensively characterized and useful cell model in the field of drug permeability and absorption. With respect to passively absorbed drugs, a correlation between Caco-2 cell permeability and human intestinal absorption has been established.In recent years, a number of important transporters have been cloned, and considerable progress has been made in understanding the molecular characteristics of individual transporters. The efflux transporter P-glycoprotein (P-gp) has received the most attention with regard to its role in restricting drug absorption and distribution and as a potential source for variability in drug pharmacokinetics and pharmacodynamics. Consequently, it is important to assess the potential of a compound as a P-gp substrate. The in vitro methods for identifying compounds as substrates of P-gp can be grouped into three general categories of assays: (1) Transport across polarized cell monolayers expressing P-gp on the apical membrane;(2) Drug uptake into cell over expressing P-gp;(3) Direct binding to P-gp using inside-out membrane vesicles or reconstituted P-gp.Many known drug transporters, such as P-glycoprotein, multidrug resistance protein and breast cancer resistance protein et. al, cause multidrug resistance phenomenon by extruding a variety of chemotherapeutic agents from the tumor cells. Proteomics is an increasingly powerful and indispensable technology in molecular cell biology. It can be viewed as a genome-wide assay to differentiate and study cellular states and to determine the molecular mechanisms that control them. A cell model with multidrug resistance phenomenon, which was mainly caused by transporters, was developed. The study of the whole protein expression alterations of tumor cells after becoming multidrug-resistant may provide useful information on drug transport.Propranolol (PPL) was a nonselective p-adrenergic blocking agent and used as racemic mixture clinically. Since S-PPL is about 100 times more potent blocker than R-PPL, significant difference in their absorption and disposition may be important clinically.Now extracts from the leaves of the Ginkgo biloba has become one of the best selling herbal products in the world. But the extract has a low bioavailability, in particular regarding the flavonoidic components. Quercetin, kaempferol, and isorhamnetin were the most important Ginkgo flavonoid constituents. There is limited information available on the role of P-gp in transporting single Ginkgo flavonol. The role of P-gp in affecting cellular level of Ginkgo flavonolbioavailability, however, has not been reported previously.Based on the work as mentioned above, we developed in vitro cell model of theintestinal barrier Caco-2 cell monolayers and a high P-gp expressing cell line Bcap37/MDR 1. An adriamycin-resistant variant of the human leukemia cell line K562 (K562/ADR) was developed in vitro. Two-dimensional gel electrophoresis and mass spectrometry were used to find the proteins which were associated with transport mechanism in K562/ADR cells. Transport studies of propranolol enantiomers were performed in Caco-2 cell monolayers. Accumulated studies of propranolol enantiomers were performed in Caco-2, K562 and K562/ADR cells.Transport studies of Ginkgo flavonols were performed in Caco-2 cell monolayers. Accumulation and efflux studies of Ginkgo flavonols were performed on Bcap37/MDR1 and Bcap37 cells. The interaction of Ginkgo flavonols with P-gp was studied by ATPase assay. The dissertation was developed as following:Part I. Development of in vitro models of drug intestinal absorption -Caco-2 cells and its applicationCaco-2 cells were seeded in Transwell? inserts in 12-well clusters. Cell inserts were used for experiment at 21 days after seeding. Caco-2 monolayer integrity was evaluated by measuring transepithelial electrical resistance (TEER) and Lucifer'>appyellow CH transport. TEER values are more than 500￡2cm2. The Papp of Luciferyellow CH (paracellular transport marker) was 1.65 ± 0.79x10"7cm/s, and the Papp of propranolol (transcellular transport marker) was 1.42 ± 0.18xl0"5cm/s. The degree of morphologic differentiation of Caco-2 cell was observed by transmission electron microscopy. There were microvilli and tight junctions in Caco-2 complete monolayers. Biochemical differentiation and polarity of Caco-2 cells were monitored by measuring the specific activity of alkaline phosphatase, a brush border marker enzyme, at different stages of monolayer maturation. P-gp expression of Caco-2 cells was measured by western blot. The results indicated that Caco-2 cell monolayers represent a valuable transport model system for the small intestinal epithelium.Transport studies of Ginkgo flavonol were performed in Caco-2 cell monolayers. Apparent permeability in absorptive and secretion directions was determined, and quercetin, kaempferol and isorhamnetin displayed polarized transport, with the PaPP.BA being higher than Papp.A-B(p < 0.01 for quercetin, p < 0.001 for kaempferol and isorhamnetin, Student's t-test).A reversed-phase high-performance liquid chromatography assay for S-PPL/R-PPL in transport solution was developed. The mobile phase contained methanol, acetonitrile and pH 4.0 phosphate buffer (10 ! 45 ! 45;v/v/v) at a flow rate of 0.9 ml/min, and the wavelength of UV detector was 220 nm. The calibration curve was linear from 0.2 to 20 |imol/L (r > 0.999) for each propranolol enantiomer. The recovery of the assay was between 98.8-101.8%. The limit of detection was0.005 ^imol/L for S-PPL and R-PPL. The limit of quantitation was 0.2 ^imol/L (RSD < 10%) for each propranolol enantiomer. The intra- and inter-day coefficients of variation were less than 10% (RSD).The transport and accumulation of S-PPL and R-PPL across Caco-2 cell monolayers was determined at several concentrations over the range of 10-500 |imol/L in both AP to BL and BL to AP directions. S-PPL exhibited greater permeability than R-PPL in the AP to BL direction, but in the BL to AP direction amount of S-PPL transported was less than that of R-PPL. Propranolol was stereoselectivity accumulated in Caco-2 cells. The transport and accumulation of S-PPL and R-PPL across Caco-2 cell monolayers was investigated at two pH values (7.4 and 6.5) in AP side. The AP to BL transport and the accumulation of propranolol enantiomer were influences by the pH change. The transport of S-PPL and R-PPL across Caco-2 cell monolayers in the BL to AP direction was not influenced by some inhibitors (verapamil, cyclosporine A, indomethacin, cimetidine, ouabain). The effect of various inhibitors on S-PPL and R-PPL accumulation was investigated. Verapamil increased the S-PPL and R-PPL uptake, but other inhibitors (cyclosporine A, indomethacin, cimetidine, ouabain) had almost no effect.Part II. Establishment of high expressing P-glycoprotein cell line and its applicationThe full-length MDR1 cDNA fragment in plasmid pMDRAl was firstly subcloned into plasmid pET-28a(+), then the MDR1 cDNA was cut from the recombinant plasmid with double-digestion and ligated into the mammalian expression vector pcDNA3.1(+). The recombinant plasmid was confirmed to be inserted in the correct orientation by restrictive enzyme digestion and DNA sequencing. The plasmid pcDNA3.1(+)/MDRl was transfected into breast cancer cell line Bcap37 using the Superfect Transfection Reagent. Several stable transfected clones were obtained after selection with G418. Real-time fluorescent quantitative RT- PCR and Western blot methods were used to detect the expression of P-gp, and the results indicated that P-gp expression in transfected cells was higher than that in control cells. The cellular location of the expressed protein was determined byimmunohistochemical staining, and it showed that the expressed P-gp was localized on cell membrane. The drug sensitivity assay was used to evaluate the biological function of the expressed P-gp. The IC50S for adriamycin and colchicine of the transfected cells were higher than those of the control cells.A reversed-phase high-performance liquid chromatography method was developed to simultaneously determine quercetin, kaempferol and isorhamnetin uptaken by Bcap37 cells. The mobile phase contained phosphate buffer, tetrahydrofuran, methanol and isopropanol (65:15:10:20, v/v/v/v) at a flow rate of 0.5 ml/min. The ultraviolet detector was operated at 380 nm. The calibration curve was linear from 0.1 to 1.0 (xmol/L (r > 0.999) for each flavonol. The mean extraction efficiency was about 70 %. The limit of detection was 0.01 |imol/L for quercetin and kaempferol and 0.05 jimol/L for isorhamnetin. The limit of quantitation was 0.1 |imol/L (RSD < 10%) for each flavonol. The intra- and inter-day coefficients of variation were less than 10% (RSD).Bcap37/MDR1 and control cells were treated with quercetin, kaempferol or isorhamnetin. The concentrations of Ginkgo flavonol in Bcap37/MDR1 cells were lower than those in parent cells (p < 0.05 for quercetin, p < 0.01 for isorhamnetin, Mann Whitney U test). The concentrations of the flavonol in transfected cells increased when incubated with P-gp inhibitor verapamil (p < 0.05 for kaempferol, Mann Whitney U test). A colorometric assay for the ATPase activity was applied to the detection of interaction of flavonol with P-gp. Quercetin and kaempferol inhibited the ATPase activity, and isorhamnetin stimulated the ATPase activity (p < 0.05 for isorhamnetin, Mann Whitney U test).Part III. Development of a K562 multidrug-resistant cell line, its application and study on the proteins with altered expressionAn adriamycin-resistant variant of the human leukemia cell line K562 (K562/ADR) was developed in vitro by continuous exposure to adrimycin. MTT assay was used to determine IC50S of K562/ADR cells to adriamycin (ADR), cisplatin (DDP), 5-fluorouracil (5-FXJ) and vincristin (VCR). The results showed that K562/ADR cells demonstrated cross-resistance to other antineoplastic drugs. TheIC5oS of K562/ADR cells to ADR, DDP, 5-FU, VDR were much higher than those of K562 ip < 0.05 for ADR and p < 0.001 for VCR). Quantitative assessment of P-gp with the P-gp monoclonal antibody by flow cytometry showed that the percentage of positive cells was 0.68% in the K562 cells and 88.79% in the K562/ADR cells.An enantioselective assay for propranolol enantiomers accumulated in K562 cells was developed. The mobile phase contained methanol, acetonitrile and pH 4.0 phosphate buffer (9 I 40 : 51;v/v/v) at a flow rate of 0.9 ml/min, and the wavelength of UV detector was 220 nm. The calibration curve was linear from 0.4 to 2.5 |imol/L (r > 0.995) for each propranolol enantiomer. The recovery of the assay was between 98.5-99.6%. The limit of detection was 0.005 |imol/L for S-PPL and R-PPL. The limit of quantitation was 0.4 fimol/L(RSD < 10%) for each propranolol enantiomer. The intra- and inter-day coefficients of variation were less than 10% (RSD).K562 and K562/ADR cells were incubated at 37 °C with 25 |imol/L or 200 )Limol/L propranolol for 10, 20 and 40min, and cellular accumulation ratio of S-PPL/R-PPL was >1 for K562 and <1 for K562/ADR cells. When the concentration of PPL in the medium was increased from 25 (imol/L to 200 (imol/L, the accumulation ration of S-PPL/R-PPL in K562 cells was >1, and that in K562/ADR cells was <1. K562 and K562/ADR cells were incubated at 4°C (27 °C or 37 °C ) with 25 jimol/L or 200 |J.mol/L propranolol for 30min, and cellular accumulation ratio of S-PPL/R-PPL was >1 for K562 and <1 for K562/ADR cells.The total proteins of K562 and K562/ADR cells were separated by two-dimensional gel electrophoresis and visualized by silver staining. Proteins with significant expression alterations were selected and their peptide mass fingerprints (PMFs) were obtained by matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS). The PMFs were used to search NCBInr database by AutoMS-Fit software. The proteins differentially expressed in the two cell lines were identified as cell cycle-related proteins, zinc finger protein 165, etc. Edman degradation was used to sequence the proteins of spot 2 and spot 6. The amino terminus sequences of proteins were AKEFVLGIDLGTDNS for spot 2 and AVQKPGAKKDFDSSK for spot 6. The sequence of protein was blasted in NCBInrprotein database. The results indicated that spot 2 was HSP70 variant, and spot 6 (it was named p34) was not matched similar protein.Conclusion:1. Caco-2 cell monolayers represent a valuable transport model system for the small intestinal epithelium. Quercetin, kaempferol and isorhamnetin displayed secretory transport in Caco-2 cell monolayers.2. A high P-gp expressing cell line Bcap37/MDR1 was successfully established. Ginkgo flavonols quercetin, kaempferol and isorhamnetin were substrates of P-gp.3. An adriamycin-resistant variant of the human leukemia cell line K562 (K562/ADR) was developed, some proteins were found to be associated with transport mechanism in K562/ADR cells.4. The transport and accumulation of propranolol across Caco-2 cell monolayers were found to be stereoselectivity. Propranolol was stereoselectivity accumulated in K562 and K562/ADR cells.