P-glycoprotein–mediated transport of berberine across Caco-2 cell monolayers

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  • P-GlycoproteinMediated Transport of Berberine acrossCaco-2 Cell Monolayers


    Department of Pharmaceutics, College of Pharmacy, Seoul National University, Seoul 151-742, Korea

    Received 7 March 2002; revised 30 May 2002; accepted 6 July 2002

    ABSTRACT: The objective of this study was to investigate the mechanisms by whichberberine is transported in the secretory and absorptive directions across Caco-2 cellmonolayers. The basolateral-to-apical (B-A) flux was 30-fold greater than the apical-to-basolateral flux and temperature dependent (i.e., drastic decrease at 48C compared with378C). The above results suggest the involvement of a carrier-mediated active transportmechanism for the B-A transport of berberine. However, no significant concentrationdependency for the permeability (Papp) of berberinewas observed forB-A transport over aconcentration range of 5300mM, indicating that theKmvalue of berberine for the carriersystem is greater than 300 mM. Well-documented P-glycoprotein (P-gp) substrates suchas verapamil, daunomycin, and rhodamine123 inhibited the B-A flux of berberine,whereas tetraethylammonium and taurocholate did not, suggesting that P-gp is involv-ed in the transport. For the case of daunomycin, the B-A flux, but not the apical-to-basolateral flux, was significantly increased after pretreatment of the cell monolayerswith berberine. In addition, the uptake of 1 mM daunomycin into Caco-2 cells was de-creased as a result of this pretreatment. These results suggest that the repeatedadministration of berberinemay up-regulate P-gp functions in Caco-2 cells. If this occursin the gastrointestinal epithelial cells, the repeated administration of berberine mayreduce the gastrointestinal absorption of P-gp substrates including chemotherapeuticagents such as daunomycin. 2002 Wiley-Liss, Inc. and the American PharmaceuticalAssociation J Pharm Sci 91:26142621, 2002

    Keywords: berberine; P-gp; Caco-2 cell monolayers; transport


    Berberine (Fig. 1) is an isoquinoline1 that has avaried pharmacology including anti-microbial,2

    anti-motility,3 and anti-secretory activities.46 Ithas been used for more than 2000 years in tradi-tional Eastern medicine as an effective medi-cation for the treatment of gastro-enteritis andsecretory diarrhea.1 It is also effective in theprevention and treatment of diarrhea in animalmodels.79

    It has recently been reported that berberineis readily extruded from microbial cells by the

    multidrug resistance pump of cell membranes,which has a role in protecting the cells from anti-microbials.10 In vitro interactions of berberinewith P-glycoprotein (P-gp) have also been report-ed in hepatoma cells.11 These reports stronglysuggest that berberine is a substrate for P-gp,a 170 kDa membrane glycoprotein encoded bythe multidrug resistance gene (e.g., MDR1 inhumans).

    P-gp confersmultidrug resistance by enhancingthe active efflux of chemotherapeutic agents fromcells,12 and is involved in the transport of a varietyof structurally and pharmacologically unrelatedhydrophobic drugs, including vinca alkaloids,anthracyclines, cyclosporin A, digoxin, and gluco-corticoids.1316 It is expressed in normal tissuessuch as the brush-border membrane of renalproximal tubules, the bile canalicular membrane


    Correspondence to: Chang-Koo Shim (Telephone: 82-2-880-7873; Fax: 82-2-885-8429.; E-mail: shimck@plaza.snu.ac.kr)

    Journal of Pharmaceutical Sciences, Vol. 91, 26142621 (2002) 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association

  • of hepatocytes, intestinal epithelial cells, capillaryendothelial cells of brain and testis, adrenal glandand placental trophoblasts, aswell as inmultidrug-resistant tumor cells.17,18 Thus, it contributes tothe excretion of xenobiotics and endogenous com-pounds into the urine, bile, and intestinal lumen,as well as the formation of biological barriersagainst such compounds in various tissues ororgans including brain, placenta, and tumor cells.For example, intestinal P-gp is thought to limit theabsorption of drugs after oral administration.1922

    If berberine is a substrate of P-gp in the body, itwould be expected to influence the pharmacoki-netics of various substances that are transportedvia the P-gp system. The objective of this study,therefore, was to investigate the mechanisms bywhich berberine is transported in secretory andabsorptive directions across a biological mem-brane. Of particular interest was its possible in-teractionwith the P-gp system on cell membranes.Caco-2 cells were used as a representativemodel of biological membranes, on which P-gp isexpressed.20,23,24



    Berberine chloride (Sigma Chemical Co., St.Louis, MO), [14C]mannitol (50 mCi), [3H]taurocho-late (250 mCi), [3H]daunomycin (250 mCi) (all fromNew England Nuclear, Boston, MA), fetal bovineserum (Hyclone Laboratories, Logan, UT), tryp-sin-EDTA (Gibco Laboratories, Gaithersburg,MD), and Dulbeccos modified Eagles medium,nonessential amino acid solution, penicillin-streptomycin, Hanks balanced salt solution

    (HBSS), HEPES (all from Sigma Chemical Co.)were used as purchased. All other reagents wereof analytical grade.

    Cell Culture

    The human colon adenocarcinoma cell line, Caco-2 (American Type Culture Collection, Rockville,MD), was grown in the form of monolayers inDulbeccos modified Eagles medium, 10% fetalbovine serum, 1% nonessential amino acid solu-tion, 100 U/mL penicillin, and 0.1 mg/mL strepto-mycin at 378C in an atmosphere of 5% CO2 and90% relative humidity. Stock cultures were grownin 75-cm2 tissue culture flasks and were split 1:3at 80 to 90% confluency using 0.02% EDTA and0.05% trypsin. The Caco-2 cells from passagenumbers 46 to 55 were seeded on the permeablepolycarbonate inserts (1-cm2, 0.4-mm pore size;Corning Costar Co., Cambridge, MA) in 12 Trans-well plates (Corning Costar Co.) at a density of2.5 105 to 3.0 105 cells/cm2. The inserts werefed with the incubation medium at 2-day intervalsfor the first week and then at daily intervals,usually for 2 weeks, until they were used for thetransport experiments.

    In experiments to investigate the effect of ber-berine pretreatment on the transport of dauno-mycin, the cell monolayer grown on the insert wasfurther fed with the incubation medium contain-ing berberine (30 mM) for specified time periods(i.e., 1, 2, 3, 5, 7, or 10 days).

    The integrity of the cell monolayers wasevaluated by means of transepithelial electricalresistance (TEER) measurements using anEVOMTM epithelial volt/ohm-meter (World Preci-sion Instruments, Sarasota, FL). When the TEERvalue reached 300700 Ocm2, the cell insertswere used for the transport experiments. Thetransport of [14C]mannitol (5.4 mM)was

  • aspiration.25 In each transport experiment, threeinserts were used. To measure the transport ofberberine from the apical-to-basolateral (A-B)side, 0.5 mL of transport medium (pH 7.4) con-taining 1% (v/v) dimethylsulfoxide and the drugwas added to the apical side, and 1.5 mL of thetransport medium without the drug to the basalside. The inserts were moved to wells containingfresh transport medium at 30-min intervals for2 h. A 300-mL aliquot of the medium, taken at eachtime point, was assayed for berberine by high-performance liquid chromatography (HPLC).

    For measurement of the basolateral-to-apical(B-A) transport of berberine, 1.5 mL of the trans-port medium containing the drug was added to thebasolateral side, and 0.5 mL of the transportmedium without the drug to the apical side. Theinserts were then incubated at 378C, and a 300-mLaliquotwas removed from the apical side at 30-minintervals for 2 h and replaced with 300 mL of thefresh transport medium. A 300-mL aliquot of eachsample was assayed for berberine by HPLC.

    In experiments to investigate the effect of vari-ous compounds on the B-A transport of 20 mM ber-berine, the inhibitors were added to the transportmedium on the basal side of the cell monolayers.

    Berberine levels were determined by the HPLCprocedure described below, whereas [14C]manni-tol, [3H]taurocholate, and [3H]daunomycin weredetermined by liquid scintillation counting using aWallac model 1409 instrument (Wallac, Gaithers-burg, MD).

    HPLC Assay of Berberine

    Themethod of Chen and Chang26 was used for thedetermination of berberine, with minor modifica-tions. To summarize, the HPLC system consistedof a Hitachi L-7110 pump (Hitachi, Japan), aShimadzu SPD-6AV UV-Vis detector (Shimadzu,Japan), a Hitachi L-7200 autosampler (Hitachi),and a Shimadzu C-R6A integrator (Shimadzu).C18 column was used. The mobile phase wasprepared by mixing 60% acetonitrile in a 0.1%phosphoric acid solution and adjusting the pH to6.0 using concentrated ammonia solution. Theeluent resulted in sharp, well-resolved peaks. Thechromatogram was monitored by UV detection ata maximum wavelength of 267 nm. Calibrationcurves for berberine were linear over the con-centration range of 25 1000 ng/mL with acorrelation coefficient of 0.999, but the limit ofdetection which exhibits

  • berberine over the range of 5 300 mM (Fig. 3 forthe B-A transport, data not shown for the A-Btransport), exhibiting no significant concentra-tion dependency in the permeability (Papp) of thetransport in either direction. This indicates thatthe Km of berberine for the responsible carriersystem in Caco-2 cells is >300 mM. Because ofprobable cytotoxicity problems,26,28,29 the trans-port rates for concentrations >300 mM were notpursued. The transport was found to be tempera-ture dependent (i.e., drastic decrease in the flux at48C compared with 378C) for the B-A flux of

    berberine (62.4 1.6 versus 5.7 0.4 pmole/min,meanSD, n 3). Collectively,


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