ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1992, p. 1374-1381 Vol. 36, No. 70066-4804/92/071374-08$02.00/0Copyright © 1992, American Society for Microbiology

Epithelial Cells in Culture as a Model for the IntestinalTransport of Antimicrobial Agents

GIULIA RANALDI,' KHALID ISLAM,2 AND YULA SAMBUYl*Istituto Nazionale della Nutrizione, Rome, 1 and Lepetit Research Center, Marion Merrell-Dow

Research Institute, Gerenzano, Varese, 2 Italy

Received 26 November 1991/Accepted 21 April 1992

The bioavailabilities of orally administered drugs depend to a great extent on their capability of beingtransported across the intestinal mucosa. In an attempt to develop an in vitro model for studying the intestinaltransport of drugs, we used an intestinal epithelial cell line (Caco 2) derived from a human colonadenocarcinoma. A renal epithelial cell line (MDCK) was also used to determine the tissue specificity of drugtransport. These cell lines, which were grown on filters, form a monolayer of well-polarized cells coupled bytight junctions and can be used for transcellular transport experiments. We studied the transport of nineantimicrobial agents with different physicochemical and pharmacokinetic characteristics using these epithelialcell monolayers to determine whether this model could be predictive of oral bioavailability. The transepithelialpassage was assayed from the apical (AP) to the basolateral (BL) side and in the opposite direction (BL to AP)in both cell lines. Radioactively labeled mannitol was used to monitor the intactness of the cell monolayerduring drug passage. The results indicated that all antimicrobial agents tested tended to behave in vitrogenerally according to their known in vivo absorptive characteristics. In addition, the use of epithelia fromdifferent tissues enabled us to divide the drugs into four groups according to their behaviors and suggested theexistence of different transport mechanisms. In particular, two antibiotics, gentamicin and teicoplanin, showedno passage in either direction or cell line, in accordance with their very poor in vivo absorbances after oraladministration. In contrast, rifapentine, rifampin, and nalidixic acid passed very efficiently at similar rates inboth directions and cell lines in a concentration-dependent, nonsaturable manner, which is suggestive of passivediffusion down a concentration gradient. Of the remaining drugs, isoniazid and novobiocin sodium showedsome differences in passage between the two cell lines and, given their ionized state at the pH that was used,may use the paracellular route. Finally, trimethoprim and D-cycloserine exhibited differences in passage bothwith respect to polarity and cell line; in particular, trimethoprim had a faster rate of passage only in Caco 2cells and in the BL to AP direction, while D-cycloserine was exclusively transported by Caco 2 cells in the APto BL direction. In both cases it is possible that active transport mechanisms are involved.

The enteral (oral) route of drug administration is the mostconvenient and economical (15), especially when one con-siders over-the-counter drugs prescribed by general practi-tioners. For orally administered drugs, the small intestinerepresents the major site of absorption because of its exten-sion, the functional specialization of the cells lining themucosa, and the long transit time (23). Knowledge of theabsorptive functions of this intestinal tract comes from invivo studies or in vitro investigations which used evertedintestinal sacs, mucosal tracts mounted in Ussing chambers,brush border membrane vesicles, and luminally or vascu-larly perfused intestine (18, 24, 36, 37). Because in vivostudies are restricted to animals, because they are expen-sive, and because it is difficult to control the experimentalconditions and to dissect the various events that contributeto net absorption along the entire gastrointestinal tract,several workers have attempted to develop in vitro systemsthat are easier to use. Furthermore, it is not feasible toscreen large numbers of new antimicrobial agents in vivo orto test synthetic derivatives to select for orally bioavailablesubstances. In vitro studies would be advantageous for thepurpose of screening new compounds for their absorptiveproperties, but the systems that have traditionally been usedby gastrointestinal physiologists have many disadvantages:

* Corresponding author.

they are laborious, difficult to reproduce, and often short-lived.

Epithelial cells from different organs can be grown inculture and maintain polarized morphological and functionalcharacteristics that are typical of their role in vivo, wherethey form a barrier between the external and the internalenvironment (34). The MDCK cell line is frequently used tostudy the development and maintenance of epithelial polar-ized morphology and functions (26, 30). In addition, therecent observation that intestinal cell lines derived fromcolon carcinomas can be induced, under controlled cultureconditions, to differentiate into mature absorptive entero-cytes has opened the possibility of developing new in vitromodels for intestinal absorption and metabolism (21, 31).One of these cell lines, Caco 2, which was obtained from ahuman colon adenocarcinoma (14), grows as a monolayer ofcells that, at confluency, initiates a process of differentiation.This leads to the formation of a brush border with well-developed microvilli, tight apical junctions, and a polarizeddistribution of membrane components, including enzymes,receptors, transport systems, ion channels, and lipid mole-cules, in the opposite membrane domains similar to thosefound in absorptive small intestinal epithelial cells in vivo(16, 21, 31, 32, 46). The transport and metabolism of severalsubstances, both components of the diet and xenobioticcompounds, including amino acids, bile acids, vitamins, andother compounds, have been studied in Caco 2 cells (11, 19,20, 40, 43). More recently Caco 2 cells have been proposed

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IN VITRO MODEL FOR INTESTINAL DRUG TRANSPORT

as a model for the transport of drugs belonging to differentclasses (2-5, 10, 22, 44).Few studies have attempted to determine the transport of

antimicrobial agents in these epithelial cell systems (10). Wetherefore undertook a study of the transport of severalantimicrobial agents with very different molecular character-istics whose in vivo absorption properties have been de-scribed previously in order to determine whether these cellmodels can be used to evaluate the oral bioavailability ofnew antimicrobial agents.

MATERIALS AND METHODS

Cell culture. The Caco 2 cell line was donated by A.Zweibaum (Institut National de la Sante et de la RechercheMedicale, Villejuif, France) and was used between passages80 and 100. The cells were routinely grown in plastic tissueculture flasks (75-cm2 growth area; Falcon; Becton Dickin-son Italia, Milan, Italy) in Dulbecco modified minimumessential medium (DMEM) containing 25 mM glucose and3.7 g of NaHCO3 liter-' and supplemented with 4 mML-glutamine, 10% fetal calf serum, 1% nonessential aminoacids, 100 U of penicillin ml-', and 100 ,ug of streptomycinml-1. The MDCK type II cell line was donated by E.Rodriguez-Boulan (Cornell University Medical College,New York, N.Y.) and was grown in DMEM-25 mM glu-cose-3.7 g of NaHCO3 liter-' supplemented with 4 mML-glutamine, 10% donor horse serum, 100 U of penicillinml'-, and 100 ,ug of streptomycin ml-'. Both cell lines weremaintained at 37°C in an atmosphere of 5% C02-95% air at90% relative humidity. The medium was changed three timesa week, and at confluency the cells were passaged bydetaching them with 0.25% trypsin (1:250) and 10mM EDTAin calcium-free and magnesium-free phosphate-buffered sa-line (PBS-). All cell culture reagents were from FlowLaboratories International (Opera, Milan, Italy). The fluo-rescent dye bisbenzimide (H 33258; Boehringer Mannheim,Milan, Italy) was routinely used to screen cells for myco-plasma contamination (9).For drug transport experiments, the cells were seeded

onto polycarbonate filter cell culture chamber inserts (diam-eter, 24 mm; area, 4.7 cm2; pore diameter, 0.4 ,um; Tran-swell; Costar Europe, Badhoevedorp, The Netherlands) at adensity of 2 x 10 cells per filter; the high seeding densityallowed confluency to be reached within 48 h. Caco 2 cellswere allowed to differentiate at confluency for 14 to 18 days,while MDCK cells were used 5 to 10 days after seeding; themedium was regularly changed three times a week. Figure 1shows a diagram of the system that was used to growepithelial cells, with the apical (AP) compartment separated

APICALCOMPARTMENT

MONOLAYER COMPARTMENTFIG. 1. Schematic representation of the filter system used for

transport experiments with cell monolayers. The filter divides theAP from the BL compartments.

from the basolateral (BL) compartment by the cell mono-layer and the filter.Drug transport experiments. The intactness of filter-grown

cell monolayers was monitored by determining the transepi-thelial passage of the radiolabeled markers [3H(G)]inulin(specific activity, 12.4 GBq/mmol) or D-1[3H(N)]mannitol(specific activity, 706.7 GBq/mmol) (NEN Research Prod-ucts, Florence, Italy). Briefly, the radioactive compounds incomplete growth medium were added to the AP compart-ment, and after 2 h of incubation at 37°C, the radioactivity inthe BL compartment was measured in a liquid scintillationcounter (LS 1801; Beckman Instruments, Inc., Irvine, Calif.).The antimicrobial agents used for the experiments belong

to different classes, including aminoglycosides (gentamicin),glycopeptides (teicoplanin), rifamycins (rifapentine and ri-fampin), antibiotics from Streptomyces spp. (D-cycloserineand novobiocin sodium), a synthetic pyrimidine analog (tri-methoprim), and two synthetic antibacterial agents (iso-niazid and nalidixic acid). The molecular weights, chemicalformulas, and absorbance characteristics of these sub-stances are listed in Table 1, and the structures are shown inFig. 2. Teicoplanin, rifapentine, and rifampin were obtainedfrom Marion Merrell-Dow (Gerenzano, Varese, Italy); gen-tamicin was from Schering Plough (Comazzo, Milan, Italy),isoniazid was from Fluka Chemie AG (Buchs, Switzerland),and the remaining drugs were from Sigma Chemical Com-pany (St. Louis, Mo.).The plates containing the filter inserts were transferred to

a water bath that was kept at 37°C, and the cell monolayerwas carefully washed with PBS containing 1 mM MgCl2 and

TABLE 1. Antimicrobial agents tested in this study

Drug Mol wt Chemical Absorption maximum e (dm3 mol-I cm-')Drug ~~~~~~~~~~formula(nmM)a (dnmo1c )

Gentamicin complex Cl-C2-Cla 449478 C19_2OH39-43N5O7 NATeicoplanin complex TA2-1,5 1,562-1,891 C88-89H95-99Cl2N9O33 NARifampin 823 C43H58N4012 332 18,517Rifapentine 877 C47H64N4012 332 17,540Trimethoprim 290 C14H18N403 278 5,568D-Cycloserine 102 C3H6N202 230 3,570Novobiocin sodium 612 C3jH35N2NaOjj 300 15,912Isoniazid 137 C6H7N30 266 4,315Nalidixic acid 232 C12H12N203 332 11,136

a NA, does not absorb.

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ANTIMICROB. AGENTS CHEMOTHER.

GENTAMICIN

NH2

N H2

CH2OH

HO 0

00

HO-RNHCHR

OH

H3C HH HO

OH

RIFAMPIN

,O CH3 CH3 CH3H3C-CO O

Hp CH NCH3

TEICOPLANIN A2

RIFAPENTINE

ISONIAZID

N

CONHNH2

NAUDIXIC ACID0

COOH

H3C NN

CH2CH3

Na-NOVOBIOCIN

CH3CH3 0

11NHCOJ1 C)C(CH3)2

IO OHNHCO

H2NCOO OH

TRIMETHOPRIM D-CYCLOSERINE

H3CO

H3CO e CH2 < NH2 NH

H3CO NH2

FIG. 2. Structures of the antimicrobial agents used in this study.

1376 RANALDI ET AL.

0 CH3 CH3 CH3aH3C- CO

OH OH

CH3OH OH H

H3C CH3 N CH3

0

/-\0 Hm N-N N

j OH

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IN VITRO MODEL FOR INTESTINAL DRUG TRANSPORT 1377

1 mM CaCl2 (PBS+) or with DMEM and 25 mM HEPES(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid)with-out NaHCO3, fetal calf serum, or antibiotics. The antibioticswere directly dissolved in PBS+ or, when required, indimethyl sulfoxide or ethanol at 10 mg ml-1 and were thendiluted to the working concentration with PBS+ or DMEMbefore addition to the donor compartment; the final dimethylsulfoxide or ethanol concentrations given to the cells neverexceeded 1% and did not affect the intactness of the mono-layer as monitored by morphology and [3H]mannitol passage(data not shown). Fresh PBS+ or DMEM was added to theacceptor compartment at the start of the transport experi-ment. All assays with spectrophotometric determinationswere performed in PBS+; gentamicin, rifampin, rifapentineand teicoplanin, when assessed microbiologically, weretested in DMEM. Since we were especially interested inreproducing the conditions for transmucosal drug transportin the luminal to serosal direction, we adjusted the donorcompartment to pH 6.0 and the acceptor compartment to pH7.5, in analogy with the pH conditions of the small intestinallumen and the submucosal compartment (45). The pHs of thedonor and acceptor compartment were also determined atthe end of the incubation period, and no pH alterations wereobserved. In order to minimize the well-known effects of pHon the transport of drugs which are weak acids or bases (23),we maintained the donor compartment at pH 6.0 and theacceptor compartment at pH 7.5, irrespective of the direc-tion of transport across the cell monolayer (AP to BL or BLto AP), as a way of controlling the specificity and polarity oftransport.

Prior to the drug transport experiments, the cells werepreequilibrated for 10 min in the presence of the drug, afterwhich the donor and acceptor solutions were replaced. Inorder to avoid drug back flow, the acceptor medium wasreplaced with fresh prewarmed medium after each timepoint. The initial rates were calculated under "sink" condi-tions (2) from the linear portion of the drug appearance curve(i.e., before >10% of the drug had been transported).However, when long time periods were used and when morethan 10% of the drug in the donor compartment was ex-pected to be transported, both the AP and BL media werereplaced after each time point.

In all experiments, antibiotic recoveries from the donorand acceptor compartments were determined to ascertainany loss due to adsorption to the cell surface or to the filter,intracellular accumulation, and/or degradation; generally,after the preequilibration period, full recovery of the drugswas obtained in the course of the experiment.

Determination of drug concentrations. The drug concentra-tions were determined by microbiological and spectrophoto-metric methods. An agar plate diffusion assay was used forboth rifapentine and rifampin by using Sarcina lutea ATCC9341 grown overnight at 30°C in antibiotic medium 1 (DifcoLaboratories, Detroit, Mich.) at a density of 3 x 105 cells perml. Gentamicin and teicoplanin were similarly tested byusing Bacillus subtilis ATCC 6633 grown overnight at 37°C atthe same density in antibiotic medium 2 (Difco). Rifampin,rifapentine, gentamicin, and teicoplanin could be detected at0.15, 0.125, 5.0, and 1.25 ,ug/ml, respectively, in this assay.Spectrophotometric assays for all drugs used the peakabsorbance of the compound in PBS+ (Table 1), and theconcentrations were determined by using the extinctioncoefficients (£) given in Table 1. Gentamicin and teicoplaninwere only assayed microbiologically.

V'

0.5.

4cI

-J

In

4

0.4 -

0.3 -

0.2-

0.1

0

30-tu

IJm

44 20'-a.co

4c

50

--w-- MANNITOL

0 20 40 60min

100

80180

150

minFIG. 3. Time course of passage of [3H]mannitol and [3H]inulin

across monolayers of Caco 2 cells. (Inset) Passage of [3H]mannitolacross cell-free Transwell filter inserts. Each point represents themean of cumulative transport across three filters and is expressed asa percentage of the total radioactivity applied to the donor compart-ment.

RESULTS

The Caco 2 and the MDCK cell lines grown on polycar-bonate filters form a monolayer of cells coupled by tightjunctions; this process is critical to the use of these cellmodels to study the transepithelial transport of molecules.The efficiency of junctional assembly was assessed by mea-suring the passage of radioactively labeled molecules whichare not absorbed or metabolized by cells, namely, inulin andmannitol. The time course of [3H]mannitol and [3H]inulinpassage across monolayers of Caco 2 cells maintained atconfluency for 14 days is shown in Fig. 3. There was littlepassage of mannitol and no passage of inulin under theseconditions (the [3H]inulin used in these experiments wasfractionated on a Sephadex G25 column to eliminate anylow-molecular-weight degradation products that could crossthe cell monolayer). By contrast, [3H]mannitol passed rap-idly across the filter in the absence of cells (Fig. 3, inset).Similar curves were obtained with the MDCK cell monolay-ers (data not shown). Consequently, for drug transportexperiments, cell monolayers were considered to be "tight"or intact when less than 0.5% mannitol passed from the APto the BL compartment in 2 h.The transepithelial passage of three antibiotics, namely,

rifampin, rifapentine, and gentamicin, across MDCK andCaco 2 cells was investigated. These antibiotics have differ-ent molecular weights, physicochemical characteristics, andpharmacokinetics (25, 35, 40). The time course of AP to BLpassage of these antibiotics at an initial concentration of 100,ug ml-' is shown in Fig. 4. Both of the rifamycins, rifampinand rifapentine, crossed the cell monolayer, and the rate ofpassage appeared to be relatively linear for up to 30 to 60min. Gentamicin failed to cross the cell monolayer under thesame conditions. The same time course of passage of theantibiotics was also observed over the concentration rangeof 25 to 150 p,g ml-l; in all cases the rates of appearance

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ANTIMICROB. AGENTS CHEMOTHER.

40

30

20

0c

z4

4c

a.

43

C,

a

10

300

Ia

w

0z

w

.4

0 O , _ I_.

0 50 100 150

TIME (min)

FIG. 4. Time course of AP to BL passage of rifampin (-),rifapentine (Li), and gentamicin (A) across monolayers of Caco 2cells. The antibiotics were applied to the donor compartment inPBS+ (for the spectrophotometric assay) or DMEM (for the micro--biological assay) at an initial concentration of 100 p,g ml-'. Eachpoint represents the mean + standard deviation of three to fourfilters.

were linear from 30 to 60 min (data not shown). However, athigher concentrations both of the rifamycins disrupted theintactness of the monolayer, as monitored by [3H]mannitolpassage. By contrast, little or no [3H]mannitol passage wasobserved with gentamicin concentrations of up to 400 ,ugml-' and 2 h of incubation. The transport of the rifamycinswas also measured from the BL side to the AP side, and nomajor differences were detected in the kinetics of passage ineither direction (Table 2).The initial rates of passage of the antibiotics were deter-

mined by using the linear portion of the curve at severaldifferent concentrations (25 to 150 ,ug ml-'). Figure 5 showsa plot of the initial rates of appearance in the acceptorcompartment as a function of the initial antibiotic concen-tration. The rifamycins rifapentine and rifampin passed veryefficiently, showing linear first-order kinetics at all concen-trations tested. Generally, rifapentine appeared to crossmuch more efficiently compared with rifampin (18 versus 5%

200

100

R 0.99

R a 0.93

200

ANTIBIOTIC (gI/ml)FIG. 5. AP to BL passage rates of rifampin (-), rifapentine (L),

and gentamicin (A) across Caco 2 cell monolayers versus initialantibiotic concentrations. Rifampin and rifapentine were assayedboth spectrophotometrically in PBS+ and microbiologically inDMEM, while gentamicin was assayed microbiologically only.Linear regression analysis of initial linear velocities calculated atevery concentration from kinetic experiments (see Fig. 4) wasperformed; each point represents a single filter experiment per-formed in duplicate.

of the initial concentration in 60 min for rifapentine andrifampicin, respectively; Fig. 4 and Table 2). Indeed, theinitial rate of passage of rifapentine exceeded that of ri-fampin by about threefold (Fig. 5). In contrast, gentamicindid not exhibit any significant passage at any of the concen-trations tested (Fig. 4 and 5) (for concentrations up to 400 p,gml-1, data not shown). To determine whether the antibioticsthat were transported across, the cell monolayer maintainedtheir biological activity, the drug concentrations, both in theAP and BL media, were also assayed microbiologically.Spectrophotometric and microbiological assays gave identi-cal results. The rates of passage of these antibiotics werealso determined in MDCK cells, and no major differences inbehavior were observed between the two cell lines (Table 2).The transport of the orally administered antituberculosis

agent isoniazid was also investigated. Isoniazid was found tobe efficiently transported across Caco 2 cell monolayers.Linear kinetics were observed at initial drug concentrations

TABLE 2. Drug transport across epithelial cell monolayersa

% Passage/h (mean ± SD)Concn In vivoDrug range Caco 2 cells MDCKcells bioavailability Reference

(pg/ml) AP to BL BL to AP AP to BL BL to AP

Gentamicin 100-400 0 0 0 0 No 35Teicoplanin 100-200 0 0 0 0 No 12Rifampin 5-200 5.2 ± 0.2 6.7 ± 0.2 4.0 ± 0.3 5.6 ± 0.2 Yes 25Rifapentine 5-150 18.1 ± 1.8 19.2 + 1.3 20.3 ± 1.7 20.2 ± 0.8 Yes 41Trimethoprim 100 3.3 ± 0.4 8.4 ± 0.8 3.9 ± 0.2 5.1 ± 0.6 Yes 28, 33D-Cycloserine 25-100 14.3 ± 1.8 1.5 ± 0.1 0.7 ± 0.2 1.1 ± 0.2 Yes 13Novobiocin sodium 100 19.4 ± 2.4 19.8 ± 3.3 9.3 ± 0.7 9.3 ± 0.7 Yes 33Isoniazid 70-7,000 35.6 ± 3.5 36.2 ± 3.1 21.7 ± 3.5 17.1 ± 2.2 Yes 7Nalidixic acid 100 132.7 ± 21.1 131.0 ± 10.8 143.2 ± 16.8 129.0 ± 17.8 Yes 17, 33

a The drugs were applied to the donor compartment within the indicated concentration ranges, and the passage is expressed as a percentage of the initialconcentration from the linear portions of transport curves (see text). The data represent the means of ± standard deviations 8 to 12 determinations. Informationon bioavailability in vivo in humans is given in the indicated references.

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IN VITRO MODEL FOR INTESTINAL DRUG TRANSPORT 1379

R - 1.00 DISCUSSION

c~~~~~~~~~E 40-

E

ul 20-z

0. 10-

0

0 2 4 6 8

ISONIAZID mg/ml

FIG. 6. Linear regression analysis of AP to BL isoniazid passagerates across monolayers of Caco 2 (A) and MDCK (C1) cells atdifferent initial drug concentrations. Isoniazid was assayed spectro-photometrically in PBS+; each point represents the average initialvelocity of duplicate filters.

of between 0.07 and 7 mg ml-', suggesting that transportoccurred by passive diffusion (Fig. 6). In order to determinethe cell specificity of isoniazid transport, we determined thepassage of the drug across monolayers of MDCK cells. Incontrast to the transport of the antibiotics rifampin andrifapentine in the two cell lines, the rate of passage ofisoniazid across Caco 2 monolayers was approximatelytwice that observed in MDCK cells at all initial concentra-tions tested (Fig. 6).These data suggested that the intestinal cell line Caco 2

may exhibit different behavioral properties compared withthe control (MDCK) cell line. We therefore investigated thetransepithelial passage of other antimicrobial agents withdifferent characteristics in both cell lines. The drugs hadvarious molecular weights and physicochemical characteris-tics (Tables 1 and 2; Fig. 2). The rates of passage of thesesubstances in the AP to BL and the BL to AP directions inboth cell lines, expressed as a percentage of the initialconcentration, are given in Table 2. Although the rates ofpassage of the different antimicrobial agents across the cellmonolayers differed widely, only two, namely, gentamicinand teicoplanin, failed to cross the cell monolayer, evenwhen they were used at high initial concentrations (200 to400 ug/ml) in both cell lines. Similar appearance rates wereobserved for rifapentine, rifampin, and nalidixic acid in bothdirections and in both cell lines. By contrast, novobiocinsodium and isoniazid exhibited differences in passage be-tween the two cell lines, with a higher rate of passage inCaco 2 cells compared with that in MDCK cells. Trimetho-prim showed some differences in the passage in the twodirections, but only in the Caco 2 cells (higher rate fromthe BL side to the AP side). Finally, D-cycloserine wasonly transported in the AP to BL direction by Caco 2 cellsand showed hardly any passage at all in the oppositedirection or in MDCK cells. The passage of D-cycloserinefrom the AP side to the BL side was similar for all concen-trations from 25 to 100 ,ug/ml (14.3% + 1.8%/h), but it tendedto decrease at higher concentrations (400 p,g/ml; 8.9% +1.5%/h).

We studied the passage of different antimicrobial agentswith known physicochemical and pharmacokinetic charac-teristics across epithelial cells in culture. Several of these arevery well absorbed in vivo after oral administration, andothers are not absorbed at the level of the gastrointestinaltract (see the references listed in Table 2).

Rifampin and rifapentine belong to the rifamycin class ofsemisynthetic antibiotics which show very good activityagainst Mycobactenium tuberculosis and many other bacte-ria by inhibiting the DNA-dependent RNA polymerase ofbacterial origin (25, 41). Isoniazid is an antimicrobial agentused, often in synergy with rifampin, for the treatment oftuberculosis. Isoniazid is preferentially administered orally,and it is rapidly absorbed along the gastrointestinal tract (7).Gentamicin was selected as a control drug in our studybecause it is not bioavailable after oral administration.Gentamicin is a complex of three basic and water-solubleaminoglycosides that are effective against a wide variety ofgram-negative and gram-positive bacteria (35).The passage of these drugs across Caco 2 and MDCK cells

measured in vitro reflects their in vivo absorptive character-istics. Rifapentine, rifampin, and isoniazid diffused throughthe cells and were found in the BL compartment, whilegentamicin failed to cross the monolayer and was recoveredcompletely from the AP compartment (Fig. 4 and 5). Inaddition, we observed a faster passage rate for rifapentinecompared with that of rifampin (Fig. 4 and 5). Although invivo pharmacokinetic data show similar peak levels in serumafter oral administration under fasting conditions, the bio-availability of rifapentine is increased two to three timesafter a meal (8). Since the drug is not readily water solubleover a wide pH range, increased solubilization because ofthe direct or indirect effects of food (i.e., increased bile flow)may actually explain its enhanced absorption when it isadministered after a meal. In contrast, rifampin bioavailabil-ity is adversely affected by food (25). In fact, what ismeasured in vitro is the capability of the substance, once itis present in its soluble form, of crossing the epithelialbarrier, which is considered the rate-limiting step in intesti-nal drug transport (23).

In the case of isoniazid, its rapid passage across theintestinal cell monolayer reflects its fast in vivo absorptionafter oral administration. However, the physicochemicalcharacteristics of the molecule are such that at intestinal pHit is present mostly in the ionized form (pK., 10.8), and thus,it is not permeable across the lipid bilayer. An in vitro studyusing perfused rat intestine showed that isoniazid was trans-ported across the intestinal mucosa with complex kineticssuggestive of passive and capacity-limited transport compo-nents (39); previously, other investigators had proposed anexclusively passive transport mechanism for isoniazid (6).Under the conditions used in this study (0.07 to 7 mg/ml),isoniazid crossed the epithelial barrier by a passive, nonsat-urable mechanism, and no capacity-limited transport com-ponent was observed.

It therefore appears that, in general, the antimicrobialagents examined in this study behaved in epithelial cellmonolayers in a fashion similar to the in vivo situation,suggesting that these cell lines may prove to be a usefulmodel for studying drug transport. Such systems would beextremely useful for screening semisynthetic or syntheticderivatives and as a rapid means of selecting orally bioavail-able drugs.To determine the extent to which these cell models could

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ANTIMICROB. AGENTS CHEMOTHER.

be used as a more general system to screen drugs belongingto different classes, we also assessed the behaviors ofantimicrobial agents which are totally unrelated to thosedescribed above. The different classes of antimicrobialagents also showed properties in these cell models similar tothose observed in vivo. Significant and interesting differ-ences in passage were observed with the different classes ofantimicrobial agents. We can generally classify the ninedifferent antimicrobial agents into four distinct groups on thebasis of their transport characteristics in the two cell lines.The first group consists of antibiotics, namely, gentamicin

and teicoplanin, which did not pass in either direction or cellline. Both are large hydrophilic molecules which are unlikelyto diffuse across membranes or to permeate the paracellularpathway via the tight junctions.The second group of drugs includes rifapentine, rifampin,

and nalidixic acid, which all efficiently passed at similar ratesin both directions and cell lines. This behavior reflects thehydrophobicity of the molecules, which allows passive dif-fusion down a concentration gradient across the monolayer.

Interesting differences in the passage rates in the MDCKand Caco 2 cell lines were observed for a third group of drugsthat included isoniazid and novobiocin sodium. The lipidcomposition of the AP and BL plasma membranes has beenshown to be very similar in MDCK and Caco 2 cells (38, 42)and generally to correspond to the membrane composition ofthe cells in their tissues of origin. By contrast, tight junc-tional permeability assessed by transepithelial electrical re-sistance and morphological criteria is different in the two celllines (16, 29). Isoniazid and novobiocin sodium differ in theirmolecular weights (Table 1), but at the pH of the donorcompartment, both antibiotics are fully ionized and carry apositive charge. It has recently been shown that tight junc-tions can regulate paracellular transport of ions and nutrients(27) and that they exhibit cation selectivity (23), although thecharge interactions with fixed sites within the junctionalchannel may be attenuated by the aqueous environment.Furthermore, in Caco 2 cells it has been shown that, forhydrophobic drugs, the contribution of the paracellular routeto net passage is very small, irrespective of the permeabilityof the tight junctions (4). It is therefore more likely that thedifferent behaviors in the transport of isoniazid and novobi-ocin sodium in the two cell lines may be related to differ-ences in tight junctional permeability rather than to differ-ences in membrane composition. Indeed, the observationthat hydrophobic molecules pass at similar rates (see above)in both the cell lines would be consistent with such aninterpretation.

Finally, in a last group of antibiotics, D-cycloserine andtrimethoprim, differences both in cell lines and in the polar-ity of the passage were observed. Trimethoprim showedsimilar passage in both directions in MDCK cells, but itexhibited a faster rate in the BL to AP direction only in Caco2 cells. This may reflect an additional transport componentwhich is not consistent with diffusion through either themembrane or the paracellular pathway. More studies arerequired to further characterize this transport component. Incontrast, D-cycloserine was transported from the AP side tothe BL side only in Caco 2 cells, but it did not pass to asignificant extent in MDCK cells, nor did it pass in the BL toAP direction in Caco 2 cells. This behavior is suggestive ofsome transport mechanism which is present only in theapical membranes of Caco 2 cells. We are investigating thekinetics of this transport and the possible mechanisms thatare involved.Our studies and the classification of the drugs in groups

highlight the importance of using more than one cell line ofdifferent tissue origin for transport studies. For drugs thatare mainly transported through the membrane by passivediffusion, a single cell line may be sufficient, but it is crucialthat more than one cell line be used to differentiate betweenthose drugs that are not orally absorbed and those whichmay use some specific transport component that may betissue or cell specific. Since other intestinal cell lines whichcan form tight monolayers in culture are available (e.g.,HT29 and T84 [31]), it is important to undertake similarstudies with these cell lines. Those studies should be aimedat determining which active transport mechanisms are ex-pressed in the in vitro situation and how these transportmechanisms resemble those present in the mucosa of thesmall intestine.

ACKNOWLEDGMENTS

We thank M. Denaro and E. Robotti (Lepetit Research Center)and S. Gaetani (Istituto Nazionale della Nutrizione) for criticalcomments on the manuscript. We also thank M. Berti and B.Goldstein (Lepetit Research Center) for helpful suggestions formicrobiological assays. We also acknowledge the kind technicalassistance of G. Crocchioni (Istituto Nazionale della Nutrizione).This work was supported by CNR Target Project "Biotechnology

and Bioinstrumentation" and by Special Project RAISA, subproject4, paper no. 323.

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