Biorelevant refinement of the Caco-2 cell culture model to assess efficacy of paracellular permeability enhancers

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  • Biorelevant Refinement of the CAssess Efficacy of Paracellular P

    , The


    Received 17 January 2007; revised 26 April 2007; accepted 4 June 2007

    Keywords: intestinal absorption; absorption enhancer; Caco-2 cells; paracellulartransport; tight junction; paracellular permeability enhancer; in vivo/in vitro correla-

    example, hydrophilic drugs, containing too many relatively low surface area and restricted passage


    a-cellular permeability enhancer (PPE). In thismethod, cell-to-cell tight junctions are transientlyJournal of Pharmaceutical Sciences, Vol. 97, 19771993 (2008)

    2007 Wiley-Liss, Inc. and the American Pharmacists Associationacross the tight junctions.Among the approaches to improve oral absor

    tion of poorly absorbed compounds is to modify thintestinal barrier by coadministration of a par

    Correspondence to: Dhiren R. Thakker (Telephone: (919)962-0092, Fax: (919) 966-3525;E-mail:

    The oral route is the preferred route of adminis-tering drugs. However, orally administered drugsmay exhibit poor bioavailability because they arenot able to traverse the intestinal epithelium. For

    hydrogen bond donor or acceptor moieties, pre-sence of a charged moiety, or large polar surfacearea, may not partition well into the lipophilic cellmembranes. Therefore, they are relegated to crossthe intestinal epithelium via the aqueous para-cellular pathway, which is inefficient due to thetions (IVIVC); palmitoylcarnitine chloride; hexadecylphosphocholine; sodium caprateABSTRACT: Epithelial cell monolayers are routinely used to evaluate efficacy of para-cellular permeability enhancers (PPEs). The purpose of the present work was toinvestigate how biorelevant refinements to the Caco-2 cell model impact in vitro efficacy(decrease in transepithelial electrical resistance and increase in mannitol permeability)of PPEs. Standard transport buffer was replaced by fasted-state simulated intestinalfluid (FaSSIF) or serum; or stirring was performed to decrease the unstirred water layerthickness. Apical FaSSIF significantly reduced the efficacy of amphiphilic PPEs palmi-toylcarnitine and hexadecylphosphocholine and reduced the amount of these PPEsassociated with cells. In contrast, FaSSIF did not affect efficacy of nonamphiphilicPPEs, ethylenediaminetetraacetic acid or 3-nitrocoumarin. Basolateral serum increasedthe transepithelial flux of PPEs, but did not lessen their potency. Stirring increased theflux of all PPEs, and also enhanced the potency of the amphiphilic PPEs. These resultsshow that inclusion of FaSSIF and agitation in the cellular models significantly alter theefficacy of amphiphilic PPEs but not of hydrophilic or lipophilic PPEs. Future studiesshould be directed at evaluating the ability to these refined in vitro systems to predictin vivo effects of PPEs. 2007 Wiley-Liss, Inc. and the American Pharmacists Association JPharm Sci 97:19771993, 2008ine in Wiley InterScience ( DOI 10.1002/jps.21118Published onlTIMOTHY K. TIPPIN,1,2 DHIREN R. THAKKER1

    1Division of Molecular Pharmaceutics, School of PharmacyChapel Hill, North Carolina

    2Drug Metabolism and Pharmacokinetics Department, MetaGlaxoSmithKline, Research Triangle Park, North Carolina 2JOURNAL Oaco-2 Cell Culture Model toermeability Enhancers

    University of North Carolina at Chapel Hill,

    c Diseases Center of Excellence in Drug Discovery,F PHARMACEUTICAL SCIENCES, VOL. 97, NO. 5, MAY 2008 1977

  • and selectively loosened by coadministration ofa PPE, which opens the paracellular space toallow greater absorption of the poorly absorbedmolecule. This technique has advantages overapproaches that modify the compound, such asmaking a lipophilic prodrug, since it could beapplied generically to diverse hydrophilic com-pounds, including small molecules, as well aslarger peptide-containing molecules. Furthermore,since molecules remain in the paracellular spaces,they would bypass the intracellular metabolizingenzymes and the mucosal membrane efflux trans-porters that can pose a formidable biochemicalbarrier to transcellularly absorbed molecules.1

    The search for a safe and effective PPE has beenunderway for the past 15 years. This search hasbeen greatly aided by the discovery and use ofin vitro models of the intestinal epithelium, suchas Caco-2 cells, which has allowed the rapid

    cant in vivo absorption improvement.6,7 Thereasons for much higher concentrations that areneeded to achieve absorption improvement in vivohave not been thoroughly investigated.

    We hypothesize that the in vitro epithelial cellmodel may be overestimating the potency of theseand other PPEs of the amphiphilic structuralclass, because this system lacks certain in vivocomponents. Indeed, others have shown thatimplementing biorelevant conditions, such asthe use of simulated intestinal fluid or serumcomponents in place of balanced salt solutions,has changed the permeability of certain drugmolecules across cell monolayers.8,9 Therefore, wesought to assess the impact of certain physiologicparameters on the potency of PPEs, particularlyamphiphilic PPEs, by implementing modifiedconditions in the cell-based systems. Threemodifications were examined: standard buffers




    1978 TIPPIN AND THAKKERassessment of structureefficacy relationships formultiple PPE analogs, as well as elucidation ofPPE mechanisms of action. However, the effectiveconcentration of a PPE in cell culture modelscan be much lower compared to that found toincrease absorption of hydrophilic moleculesin vivo in animal models. For example, palmi-toylcarnitine chloride (PCC) was effective in vitroat a concentration of 200mM,2 however, 10- to 100-fold higher concentrations were required toachieve significant in vivo absorption improve-ment.3 Similarly, sodium caprate increased thepermeability of hydrophilic solutes in vitro atconcentrations of 1013 mM,4,5 but required 30- to50-fold higher concentrations to achieve signifi-

    Table 1. Effective Concentration and Putative Mecha

    PPE Effective In Vitro Concentratio

    Amphiphilic PPEsHPC 0.03 mM increased mannitol

    flux 10-foldPCC 0.2 mM increased Lucifer

    Yellow flux 9-fold

    Sodium caprate 10 mM increased mannitolflux 8-fold

    Nonamphiphilic PPEsEDTA 1 mM increased FITC-Dextran

    flux 6-fold

    3-NC 0.03 mM increased mannitolflux 5-foldJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 5, MAY 2008were replaced with simulated intestinal fluid onthe apical side of monolayers or serum on thebasolateral side of monolayers, or monolayerswere agitated at different rates to change theunstirred water layer thickness (Hw). In additionto PCC and sodium caprate, the amphiphilic PPEhexadecylphosphocholine (HPC), as well as twononamphiphilic PPEs, ethylenediaminetetraace-tic acid (EDTA) and 3-nitrocoumarin (3-NC) werealso included in the test set. These five PPEs havewell-established paracellular permeability enhan-cing properties, and represent the diverse chemi-cal space of PPEs studied to date as well as themultiple mechanisms by which PPEs act to opentight junctions (Tab. 1, Fig. 1).

    of Action for Selected PPEs

    Putative Mechanism of Action References

    PLCb inhibition leading toactin disorganization


    Membrane perturbation;no effect on actin organization,Ca2 independent mechanism


    Activation of PLC, increasedintracellular Ca2


    Extracellular chelation ofCa2, leading to disruption ofextracellular E-cadherin


    PLCg inhibition leading tohyperphosphorylation of ZO-2

    10DOI 10.1002/jps



    The synthesis of 3-NC was as describedpreviously.10 D-(1-14C)-mannitol (55 mCi/mmol) waspurchased from American Radiolabeled Chemi-cals, Inc. (St Louis, MO). EDTA was obtainedfrom USB Corp. (Cleveland, OH). Other chemicalsand reagents, including PCC, HPC, sodiumcaprate, phosphatidylcholine, taurocholate, Hanksbalanced salt solution, HEPES buffer, and glucosewere purchased from Sigma Chemical Co. (St

    Figure 1. Structures

    DOI 10.1002/jps JLouis, MO). Cell culture reagents were purchasedfrom Invitrogen (Carlsbad, CA). TranswellsTM

    were purchased from Corning Costar (Cambridge,MA). Fasted-state simulated intestinal fluid(FaSSIF) was prepared as previously reported.11

    The mixture contained 3 mM sodium taurocholateand 0.75 mM phosphatidylcholine in a buffered,isosmotic sodium salt solution (pH 7.1). Malehuman serum was obtained from Bioreclamation,Inc. (Hicksville, NY). The thawed serum wasfiltered through a 0.22 mM filter and adjusted topH 7.1 immediately prior to use.

    of selected PPEs.


  • 1980 TIPPIN AND THAKKEREvaluation of Paracellular PermeabilityEnhancement

    The ability of the selected PPEs to modulateparacellular permeability was measured aftertreatment of cell monolayers on the apical sideof the TranswellTM compartment with increasingconcentrations of PPEs. Two parameters weremeasured: (1) a decrease in TEER, measured atthe end of the treatment period, and (2) anincrease in the amount of the paracellular markercompound, mannitol, appearing in the basolateralside during the PPE treatment period.

    Prior to the addition of PPEs, cell monolayerswere equilibrated for approximately 60 minin transport buffer (Hanks balanced salt solutionsupplemented with 25 mM D-glucose and 25 mMHEPES, pH 7.1) at 378C. At the end of theequilibration period, TEER was measured usingEndohm Voltohmmeter maintained at 378C withslide warmer. This measurement was defined asinitial TEER. Measurements of changes to cellmonolayers due to PPE treatment were initiat-ed upon replacement of the apical transportbuffer with dose solutions (0.4 mL) containing14C-mannitol and the PPE dissolved in transportbuffer, which was modified slightly to improveCell Culture

    The Caco-2 cell line was obtained from AmericanType Culture Collection. Caco-2 cells were grownin 75 cm2 tissue culture flasks in the presence ofcell culture medium (minimum essential mediumwith Earles salts, 10% (v/v) fetal bovine serum,and 1:100 dilutions of 100 stock solutions ofnonessential amino acids and antibioticantimy-cotic agents (penicillin G, streptomycin, ampho-tericin B)). Cells were passaged approximatelyevery 4 days. At approximately 90% confluency,the cells were detached using 0.05% trypsincontaining EDTA. Cells between passage num-bers of 26 and 40 were used for transport studies,except when noted otherwise. Cells were seeded ata density of 120000 cells/cm2 on polycarbonatemembranes of TranswellTM cell culture inserts(pore size 0.4 mm, diameter 1 cm2). Cell culturemedium was replaced every second day. Cellswere considered fully differentiated and ready forPPE experiments after 21 days. TEER values ofmature cell monolayers were between 300 and600 V cm2, as measured using Endohm Voltohm-meter (World Precision Instruments, Sarasota,FL) after correction for filter resistance.JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 5, MAY 2008stability as follows: For amphiphilic compounds,1% FaSSIF (v/v) in transport buffer was used tostabilize dose preparations. Dose solutions ofsodium caprate were prepared in Ca2-freetransport buffer. Dose solutions of 3-NC wereprepared in transport buffer adjusted to pH 5.6.These modifications were also included in controlwells and did not adversely affect the integrity ofthe monolayers. The basolateral compartmentcontained 1.5 mL of prewarmed transport buffer.14C-mannitol that had accumulated in the baso-lateral compartment over the entire treatmentperiod was quantified by mixing 0.5 mL aliquotswith 5 mL of UniverSolTM ES scintillation fluid(ICN, Costa Mesa, CA). Radioactivity in eachsample was measured in a Tri-Carb 2500TRscintillation spectrometer (Perkin-Elmer Lifeand Analytical Sciences, Boston, MA).

    The effect of FaSSIF on the ability of the PPEsto increase paracellular permeability of mannitolor decrease TEER was determined by preparingdosing solutions of each PPE in FaSSIF ratherthan standard transport buffer. Control mono-layers for this condition contained FaSSIF with-out PPE in the apical compartment. Otherprocedures were the same as those describedabove for standard conditions.

    The effect of human serum on the ability ofthe selected PPEs to increase paracellular per-meability of mannitol or decrease TEER wasdetermined by putting 1.5 mL of serum in thebasolateral compartment rather than standardtransport buffer. Control monolayers for thiscondition contained standard transport bufferwithout the PPE in the apical compartment andserum in the basolateral compartment. Dosingsolutions of PPEs as well as other procedures werethe same as those described above for standardconditions.

    The effect of stirring and the associated changesto the unstirred water layer (Hw) on the para-cellular enhancing ability of the selected PPEswere determined in separate experiments in thepresence of standard transport buffer. The Hw wasreduced by agitating monolayers on a rotaryshaker (Lab-line Model 3520 Orbit Shaker) at low(80 rpm), moderate (120 rpm) and high (160 rpm)speeds in the presence of increasing con-centrations of PPEs. The marker compound 3H-testosterone (100 mM) was used to calibrate thechange in Hw at each stirring rate as describedpreviously,12,13 using the equations shown below.In order to simulate the transient exposure to aparticular intestinal segment, the duration of PPEDOI 10.1002/jps

  • plastic support and immersing in 1 mL of ethanol.

    a Sciex API 100 mass spectrometer (Applied

    TEER was measured across control and PPE-treated monolayers at the end of the treatment

    IN VITRO PPE EFFICACY IN BIORELEVANT FLUIDS 1981Biosystems, Foster City, CA) equipped with aTurboionspray interface operated in the positiveionization mode, except for sodium caprate, whichwas detected in negative ionization mode. Nitro-gen was used as both the sheath and drying gas ata pressure of 10 arbitrary units and a flow-rate7 L/min, respectively. The spray voltage was set at5.0 kV and the drying gas temperature was set atThe sample was vortexed and sonicated briefly tolyse cells. Aliquots of the cell extract were furtherdiluted with ethanol and analyzed by LCMS asdescribed below.

    LCMS Analysis of PPEs

    Separation of PPEs from buffer components wasaccomplished with an Agilent high performanceliquid chromatograph (LC, Palo Alto, CA) using aPhenomenex C18 Luna column (2 mm 100 mm,3 mm particle size), and a mobile phase consistingof acetonitrile and 13 mM ammonium formate(pH 3.5) (or ammonium acetate (pH 6.8) for sodi-um caprate analysis) at a flow rate 0.3 mL/min.The percentage of acetonitrile was changed overa 23 min linear gradient so that the PPEswere retained on the LC column for 46 min.This retention allowed the first 3 min of eluateto be diverted to waste. PPEs were detected usingtreatment was reduced to 15 min. Controlmonolayers for this condition contained standardtransport buffer without PPE in the apicalcompartment and were agitated at the samespeeds as PPE-treated monolayers. Dosing solu-tions of PPEs were prepared in the same manneras descri...


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