Epithelial Transport of Drugs in Cell Culture. VII: Effects of Pharmaceutical Surfactant Excipients and Bile Acids on Transepithelial Permeability in Monolayers of Human Intestinal Epithelial (Caco‐2) Cells

Epithelial Transport of Drugs in Cell Culture. VII: Effects of Pharmaceutical Surfactant Excipients and Bile Acids on Transepithelial Permeability in Monolayers of Human Intestinal Epithelial (Caco-2) Cells EVAKARIN ANDERBERG,CHRISTER NYSTR~M, AND

PER

ARTURSSON~

Received July 8, 1991, from the Department of Pharmaceutics, Biomedical Centre, Uppsala University, Box 580, Accepted for publication December 5, 1991. 751 23 Uppala, Sweden. Abstract 0 The effects of anionic (sodium dodecyl sulfate and sodium dioctyl sulfosuccinate) and nonionic (polysorbate 80 and polyoxyl 40 hydrogenated castor oil) synthetic surfactants and bile acids (sodium

taurocholate, sodium taurodeoxycholate, and sodium taurodihydrofusidate) on epithelial integrity were studied in monolayers of human intestinal epithelial (Cam-2)cells grown on microporous polycarbonate filters. The effects of the surfactants on intracellular enzyme activity, cell monolayer permeability, and morphology were studied. The effects on permeability were studied by two methods: measurements of transport of marker molecules (mannitol and polyethylene glycol) and measurements of transepithelial electrical resistance. All surfactants demonstrated concentration-dependenteffects on intracellular enzyme activities, permeability,and morphology.The effects of the anionic surfactants were more pronounced than those of the nonionic surfactants. The effects on transepithelial electrical resistance correlated with intracellular dehydrogenase activity. Fluxes of marker molecules were the most sensitive measure of epithelial integrity. The results indicate that the hydrophilic marker molecules permeate the epithelial monolayers through different pathways at different concentrationsof the surfactants. The effects of the surfactants were reversible at intermediate concentrations, even though the morphology of the monolayers had changed. The results agree with published data obtained with experimental animals and indicate that Caco-2 cells can be used to study the concentration-dependenteffects of surfactants and other pharmaceutlcal additives on Intestinal epithelial permeability.

Structurally different surfactants have been used to enhance drug absorption across various epithelia.l Among these are surfactants routinely used in solid oral dosage forms, including anionic surfactants such as sodium dodecyl sulphate and sodium dioctyl sulfosuccinate, as well as nonionic surfactants such as polysorbates and hydrogenated castor oils. Sodium dodecyl sulfate and dioctyl sodium sulfosuccinate increase the permeability of the intestinal epithelium to various drugs, inhibit transport of nutrients and water, and alter the activity of membrane-bound enzymes.2-6The effects of the anionic surfactants are similar to those of the extensively studied, naturally occurring anionic surfactants (i.e., the bile salts-). Nonionic surfactants also change the permeability of the intestinal epithe1ium.QThe mechanisms of action of the surfactants on the intestinal epithelium a t the cellular level are not yet fully elucidated. The effects of surfactants on intestinal permeability are usually studied in situ or in excised intestinal segments.1Jj These models give important information on the interaction between surfactants and the intestinal epithelial barrier. However, in most studies, relatively high surfactant concentrations (i.e., concentrations a t which the surfactants solubilize the cell membranes and cause cell death, cell extrusion, and increased permeability) have been used.2 Studies over wider concentration ranges are not usually performed. In Oo22-3~9/92/0900-0879$02. MI0 8 1992, American Pharmaceutical Association

addition, the results of experiments carried out in heterogeneous, whole-tissue models have often been difficult to interpret.@Therefore, simpler models based on preparations of brush border membrane vesicles have been developed.10 Although these models have a larger capacity, they can be used to study only the effects on the cell membranes. No information about possible effects on the permeability across the tight junctions is obtained from such models. In this study, we introduce a new model for studying changes in epithelial permeability based on a human intestinal epithelial cell line, Caco-2. The Caco-2 cell line, which is derived from a human colorectal carcinoma, is the only human intestinal epithelial cell line that differentiates spontaneously to enterocytelike cells under conventional cell culture conditions.11-13 The enterocytelike properties of this cell line render it suitable for studies of drug and peptide absorption.14-19 After a cultivation period of -2 weeks on permeable polycarbonate filters, polarized monolayers with apical brush border and well-developed tight junctions are obtained.14 Thus, with Caco-2 monolayers, the effects of surfactants on the cell membranes and also on the tight junctions may be studied. Caco-2 cells and other epithelial cell lines have been used recently to study the effects of various hormones and ionophores on epithelial integrity.2w23 Another recent study24 suggests that this model is suitable for studies of absorption enhancers as well. Monolayers of Caco-2 cells grown on permeable polycarbonate filters were used to study the dose-dependenteffects on the intestinal epithelium of synthetic surfactants (sodium dodecyl sulfate, sodium dioctyl sulfosuccinate, polysorbate 80, and polyoxyl 40 hydrogenated castor oil), two bile acids (sodium taurocholate and sodium taurodeoxycholate), and a bile acid derivative commonly used as an absorption enhancer (sodium taurodihydrofusidate26). The permeabilities of the cell monolayer were studied by two different approaches: measurements of transport of marker molecules and measurements of transepithelial electrical resistance. Two hydrophilic markers of different molecular weight were used: 182)and polyethylene glycol (M,., 4000). These mannitol (M,., very hydrophilic markers do not partition into cell membranes and have therefore been used extensively to study paracellular permeability in intact and diseased epithelia (for a review, see ref 26). The effects of the surfactants on intracellular enzyme activities were also studied. In addition, scanning electron microscopy was used to examine the surface morphology of the surfactant-treated cells.

Experimental Section M ate r ial~3H]M annitl(Mr, 182; specific radioactivity, 105 CUg) and [“CJpolyethylene glycol ([14ClPEG;M,,4000; specific radioactivity, 11.0mCi/g) were obtained from New England Nuclear, Boston, Journal of Pharmaceutical Sciences I 879 Vol. 81, No. 9, September 1992

MA, through Du Pont Scandinavia A.B., Kista, Sweden. Polysorbate 80 (Tween 80; P80, ICI Specialty Chemicals, pharmacopoeia1 grade) was a gift from Kemi-Intressen A.B., Stockholm, Sweden, and polyoxyl40 hydrogenated castor oil (Cremophor RH40; HCO, pharmacopoeial grade; BASF, Ludwigshafen, Germany) was a gift from BASF Svenska AB, Gtiteborg, Sweden. Sodium dodecyl sulfate (SDS; 99% purity), sodium dioctyl sulfosuccinate (DS; 99% purity), sodium taurocholate (STC; 98% purity), sodium hurodeoxycholate (STDC; 98% purity), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; 98%purity), and mannitol were obtained from Sigma Chemical Company, St. Louis, MO. Polyethylene glycol (PEG; M, 4000) was obtained from Kebo Lab A.B., Splnga, Sweden. Sodium taurodihydrofusidate (STDHF; 98.8% purity) was a gift from Dr. Kurt Larsen, Leo Pharmaceutical Products, Ballerup, Denmark. Cells-Caco-2 cells originating from a human colorectal carcinoma12 were obtained from American Tissue Culture Collection, Rockville, MD. The cells were cultivated on polycarbonate filters (Costar Transwell cell culture inserts; mean pore diameter, 0.45 prn) as described elsewhere.14 Cells of passage number 85-100 were used throughout. Determination of Critical Micelle Concentrations-A DuNouy interfacial tensiometer (A. Kruss Optish-Mechanishe Werkstlitten, Hamburg, Germany) was used to determine the critical micelle concentration (CMC) of the surfactants in Dulbecco's modified Eagle medium (DMEM;pH 7.3) containing 1% nonessential amino acids, 10 mM N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES) buffer, and 0.1% human serum albumin (i.e., the same medium as that used in the absorption experiments). The CMC values were obtained from plots of surface tension versus the logarithm of surfactant concentration. The CMC determinations were performed at 22 f 1"C. Osrnolality-The osmolalities of surfactant solutions were measured in a 5500 vapor pressure osmometer (Wescor, Inc., Logan, UT). All studies were performed with isoosmotic solutions. Intracellular Enzyme Activities-The effect of the surfactants on the intracellular dehydrogenase activity was determined by the M" method.27.m MTT is a tetrazolium salt that is cleaved by mitochondrial dehydrogenases in living, but not dead, cells to give a dark-blue product. Cells were seeded in 96-well tissue culture plates (Flow Laboratories, Irvine, U.K.) and were incubated for 24 h. Calibration experiments showed that the dehydrogenase activity was linearly correlated with the cell number in the range 8000-62 500 celldwell. Therefore, a cell concentration of 50 000 celldwell was used in all experiments. The cells were incubated for 10 min or 24 h with different concentrations of surfactants, MTT solution (5mg/mL in cell culture medium) was added, and the plates were incubated for another 90 min. The color was measured in a multiwell scanning spectrophotometer (Multiscan MCC1340, Labsystems Oy, Helsinki, Finland). The surfactant concentration that produced a 50% inhibition of the dehydrogenase enzyme activity (IC,) was obtained from the concentration-absorbance curves. Measurements of Transepithelial Electrical R e s i s t a n c e A decrease in transepithelial resistance indicates that the passive permeability to ions (mainly sodium and chloride ionem) has increased. The transepithelial electrical resistance (expressed as ohms x cm2) was measured as previously described." The monolayers were exposed to increasing concentrations of the surfactants in the same medium as that used in the absorption experiments for 5 min at 37 "C before each measurement. The concentration of surfactant that decreased the transepithelial electrical resistance of the cell monolayers by 50% (RC6,J was obtained from the concentration-resistance curves. The transepithelial electrical resistance was also measured after each drug transport experiment. The transepithelial electrical resistance of control monolayers was 480 2 81 [mean 2 standard deviation (SD); n = 301. Absorption Studies-The absorptions of ['Hlmannitol (1 x lo-' M) and ["CIPEG (1 x lo-' M) were measured simultaneously. In general, the absorption studies were performed in air at 95% relative humidity and at 37 "C in DMEM (pH 7.3) containing 1% nonessential amino acids, 10 mM HEPES buffer, and 0.1% human serum albumin. Under these conditions, the integrity of the monolayers was intact for at least 6 h.14 Studies of the recovery of the cell monolayers after exposure to surfactants were performed in DMEM containing 10% fetal calf serum and 1% nonessential amino acids. The monolayers were agitated on a microscope slide mixer (Relax 3, Kebo Lab, Sweden) a t 10 rpm and at a n angle of 2.5".The aqueous boundary 880 I Journal of Pharmaceutical Sciences Vol. 81, No. 9, September 1992

layer did not influence the permeability coefficient for mannitol and PEG under these conditions.31 The two hydrophilic markers were studied at three concentrations for each surfactant 0.060,0.58, and 2.0 mM SDS 0.15,0.48, and 1.6

mMDS;0.0053,0.047,and3.8mMP80;0.010,0.27,and7.1mMHCO; 9.4, 21, and 50 mM STC;1.2, 3.3, and 5.0 mM STDC; and 0.94, 2.7, and 5.0 mM STDHF. ['HIMannitol, ["CIPEG, and surfactant were added to the apical chamber, and the samples were agitated for 30 s. At this time, the initial concentrations in the donor chamber and the receiving chamber were determined from a lo@& sample. The absorption was followed for up to 240 min, and a 100-p.L sample was taken from the receiving chamber every 60 min. The integrity of the monolayers was checked at the end of each experiment by measuring the transepithelial electrical resistance. The results were corrected for dilution and expressed as a fraction of initial dose transported at time t. The amount of absorbed marker versus t showed a linear relationship. The apparent permeability coefficient PaPJ was determined according to the following equation:

where dQldt is the permeability rate (steady-state flux;moYs), C, is the initial concentration in the donor chamber (mollmL), and A is the surface area of the membrane (cm2).Pappvalues were calculated for time intervals during which
Results Surfactants in Oral Drug Products-The compositions of registered solid oral drug products were obtained from the Medical Produds Agency, Uppsala, Sweden (Table U.36 In general, relatively small amounts of the surfactants have been added to the solid oral dosage forms, presumably as wetting agents. In one product that contains a large amount of DS, the surfactant acts as a laxative. Approximate mean concentrations of the surfactants after dissolusion of the dosage forms were calculated by assuming a luminal volume of 260 m L . 3 6 The calculated luminal concentrations of the surfactants were generally lower than their CMC values (Table 11). Effect of Surfactants on Intracellular Enzyme Activities and Transepithelial Electrical Resistance-A dosedependent decrease in dehydrogenase activity was observed for all surfactants (Figure 1 and Table 11). The effects of the anionic surfactants were more pronounced than those of the nonionic surfactants. The ICso values for the anionic surfactants were generally similar to the measured CMC values. However, the CMC for SDS was -10 times higher than the ICs0 value for SDS. The reason for this discrepancy is not known but may be related to interactions between SDS and some component in the cell culture medium.9The CMC values of SDS and other surfactants vary with buffer composition.37 Indeed, the measured CMC values for SDS, as well as those

Table CSurfactanta In Regldered Solld Oral Drug Productr* Surfactant Dosage per Unit Surfactant

SDS DS P80 HCO

Number of Drug Products

Median

92 27 61 4

Quartile Deviation

Maximum

mg

mMb

mg

mMb

0.98 0.85 0.10 0.21

1.4 X 7.6 x 10-3 3.1 x 10-4 4.0 x 10-4

1.1 0.25 0.14

1.5 X 2.2 x 10-3 4.3 x 1 0 - 4

-

mg

20 121 35

-

C

mMb

2.8 X lo-' 1.1 1.1 x 10-1

04

7 6 x 10-7

The number of solid oral drug products for human use registered in Sweden In 1990 containingsurfactants (SDS, DS, P80, or HCO) and the amount used in each product.= bThe concentrations calculated from the solid oral dosage forms dissolved in a luminal volume of 250 mL.= =-, Not determined. Table ICCMC, lCm and RC- Valuer for Surfactant8 Surfactant Synthetic anionic SDS DS Synthetic nonionic P80 HCO Bile acids STC STDC STDHF

CMC, mMa

4.1 0.32 0.24 1.2

5.6 1.9 2Sd

C ,I,

mMb

10min

24h

0.36 0.36

0.32

>34 10 10 1.3 1.4

0.30

0.69 6.2 9.7 1.3 1.4

RC ,,

mMc

0.58 0.48 >34 >21 21 2.3 2.7

-4

0

-2

2

6

4

log concentration SDS, mM

Measurements made in serum-free medium (DMEM; pH 7.3) containing 1 % nonessential amino acids, 10 mM HEPES buffer, and 0.1% human serum albumin with a DuNouy tensiometer. Concentration of surfactant that decreased the absorbance value by 50% in M l l tests; cells incubated for 10 min or 24 h; mean values of 6 4 determinations; SD for repeated determinations of ICw generally
for the other surfactants, are within the range of their CMC values in different b d e r systems.37~38The exposure time (10 min or 24 h) had no effect on the IC,, values of the anionic surfactants (Table 11). No correlation was found between the IC,, values of the nonionic surfactanta and CMC. Inhibition of dehydrogenase activity was obtained at concentrations above the CMC. The IC, values of nonionic surfactants (unlike those of anionic surfactants) depended on the exposure time. Exposure to P80 for 10 min did not reduce the dehydrogenase activity significantly. However, after 24 h of exposure, a relatively low IC, value of 0.69 mM was obtained. The surfactants showed a dose-dependent effect on electrical resistance (Figure 2). Measurements of transepithelial electrical resistance were not as precise as the MTI' method. The lower precision was caused by a relatively large variation in the electrical resistance of individual monolayers.14~39 The results of the resistance measurements agreed with those obtained by the M" method (Table II). A good correlation (correlation coefficient, 0.992) between the RC,, and the IC,, (10-min incubation) values was obtained for the anionic surfactants (Figure 3). Effects of Surfactants on Absorption Rate-The effects on the absorption rates of PEG and mannitol were studied at three different concentrations of each surfactant. The concentrations of the anionic surfactants were chosen from the curves of the transepithelial electrical resistance versus surfactant concentration. The lowest concentration was equal to the highest concentration that did not significantly affect the resistance multiplied with a factor of 0.76. The factor 0.76 was introduced to ensure that the lowest surfactant concen-

-

4 2

-

2

0

2

4

6

8

log concentration STDHF, m M

-6

-4 3

-2

0

2

4

log concentration P80, mM

Figure 1-Effect of surfactants on intracellulardehydrogenase activities: (Top) SDS; (Mlddk) STDHF; (Bottom) P80. The IC- values are indicated with arrows (mean f SD; n = 8).

tration did not affect the resistance. This concentration was lower than or similar to the CMC of the surfactant. The intermediate concentration was equal to the RC60 values in Table 11, and the highest concentration was equal to a Journal of Pharmaceutical Sciences I 881 Vol. 81, No. 9, September 1992

30

1

f a9

-ec'

I

20-

I I I l I

.-0

0

9 O ! -8

I

I

-6

-4

I

I

1

-2

0

2

0

0

log concentration SDS, mM

150

Ei

10-

//

100

200

300

200

300

Tlme, mln

1

I

I

0

I

-6

I

I

I

1

-4 -2 0 2 4 log cocentration STDHF, mM I

0

100 lime,

min Flgure &Effect of STDHF on the absorption of (Top) mannitol and (Bottom) PEG. Key: (0)control; (m) 0.94 mM STDHF; (A) 2.7 mM STDHF; (A)5.0 mM STDHF (the symbols 0 and W are superimposed). Values are expressed as mean 2 SD (n = 3).

O ! -go

i

I

-4 -2 0 2 log concentration P80, mM

4

Flgure 2-Effect of surfactants on transepithelial electrical resistance: (Top) SDS; (Mlddle) STDHF; (Bottom) P80. The RCK, values are indicated with arrows (mean t SD; n = 2-3).

zE-

m

I*: lo 0

0"

90

11

b o

882 t Journal of Pharmaceutical Sciences Val. 81, No. 9, September 1992

concentration that decreased the resistance by >50%. For the nonionic surfactants, for which no RC,, values were obtained, concentrations below, equal to, and greatly exceeding CMC were chosen. Results of typical absorption experiments are shown in Figure 4. A clear concentration-dependent effect of STDHF on absorption rates was observed (p = 0.0001, ANOVA). The lowest concentration of STDHF (0.94 mM) had no effect on the absorption of the markers or the resistance. At the intermediate concentration of STDHF (2.7 mM), the resistance decreased significantly (p < 0.01, ANOVA), but the absorption rates of mannitol and PEG were not significantly different from the control values. However, when the absorption rate was compared with that of control monolayers, a significant increase was obtained (p < 0.01, t test). The highest concentration of STDHF (5.0 mM) significantly affected both the resistance and the absorption of mannitol and PEG. The effects of three concentrations of the surfactants on the permeability of the cell monolayers are summarized in Tables 111-V. No increase in mannitol and PEG permeability was observed at the lowest concentrations of SDS and DS (Table III). At concentrations equal to the RC,, values, a clear decrease in transepithelial electrical resistance and an increase in permeability to mannitol and PEG were observed. However, the decrease in transepithelial electrical resistance was larger than the expected 50%. Thus, 0.58 mM SDS decreased the resistance by -80%, and 0.48 mM DS decreased the resistance by -95%. The differences between the resis-

Tabk IlcEffeCts of Synthetic Anionic Surfactantson PTranaeplthdlalE M c a l Realstanct,

surfadant

mh- Resistance,

tion,mM

fm x 106,

and *

s-'

Mannitol

PEG

0.110 f 0.001 0.118 f 0.002 3.02 f 0.18' >15.3 f 0.5' 0.117 f 0.011 0.299f 0.025 15.6 f 3.5' >20.3 f 0.P

0.079 f 0.008 0.072 f 0.003 0.681 f 0.023 11.2 f 0.7' 0.064f 0.007 0.107 f 0.007 5.20 f 0.28' 5.65 f 0.38'

Tebk V-Effects of SyntheUc Nonbnk Surfactants on Pw and Transeplthellal Ekctrlcal R e a m

Surfactant

Concentra- Resistance, tion, mM

%ab

f-

x 108,cm.s-lb

Mannitol

PEG ~______

SDS

DS

0 0.060 0.58 2.0 0 0.15 0.48 1.6

100 f 4 101 f 6 21 f 4' 10 f 3' 100 f 12 119 f 5 6 f0 ' 3 f 3'

Resistance measured after the absorption experiments and expressed as a percentage of the resistance of control monolayers. Values expressed as mean f SD. Statistically significantly different from control (p < 0,001). tance values in Tables 11and 111are presumably due to a sharp decrease in the transepithelial electrical resistance over a narrow concentration interval; the reproducibility in this part of the concentration-resistance curve is reduced, as supported by the finding that the effects of 0.58mM SDS were reversible in some experiments, while in others, the monolayers could not be recovered (data not shown). The effects of the bile acids on the epithelial permeability were similar to those of the synthetic anionic surfactants (Table IV).However, for the resistance values, the reproducibility in the determinations ofPappvalues was limited a t the intermediate concentrations. Exposure to intermediate concentrations (RC,,) of STC, STDHF, and STDC resulted in transepithelial electrical resistances decreasing to 71,76, and 8%, respectively. These results agree with those from the absorption experiments. Exposure to STC and STDHF resulted in 3-20-fold increases in permeability to the markers, whereas exposure to STDC resulted in a >100-fold increase in permeability to the markers (Table IV). These results indicate that marker permeability and transepithelial electrical resistance are good indicators of larger changes in epithelial permeability. A significant effect of P80 on the epithelial permeability was obtained only at the highest concentration of the surfactant (Table V). Thus, although 3.8mM of P80 did not reduce the transepithelial electrical resistance, it increased permeabilities to mannitol and PEG by approximately four- and Table IV-€ffect8

of Bile Acklr on PW and TransepMte~llalElectrkal

lbabmwe Resistance, YO*b

STC

STDC

STDHF

0 9.4 21 50 0 1.2 2.3 5.0 0

0.94 2.7 5.0

10057 113 f 2 71 f 18 f 6' 100f30 98520 8 f 6' 3f1' 10057 101 2 6 76 f 3' 11 f 6 '

f-x

106,cm.s-lb

Mannitol

PEG

0.103 f 0.004 0.118 f 0.009 0.489f 0.015 >41.6 f 2.6' 0.156 f 0.027 0.391 f 0.212 27.2 f 8.9' >32.5 f 2.7' 0.079 f 0.003 0.103 f 0.007 1.70 f 0.15 >28.9 f 2.1

0.046f 0.007 0.074 f 0.007 0.126 f 0.023 >30.5 f 0.6' 0.068f 0.005 0.123 f 0.061 >9.39 f 2.58" B22.6 f 2 . p 0.058 0.004 0.0822 0.007 0.268f 0.027 10.9 f 0.6'

'

*

a Resistance measured after the absorption experiments and expressed as a percentage of the resistance of control monolayers. Values expressed as mean f SD. 'Statistically significantiy different from control (p < 0.01). Statistically significantiy different from control (p < 0.001).

P80

0

0.0053 HCO

0.047 3.8 0 0.010 0.27 7.1

100214 97 f 8

90f5 8856 10026 9424 101 2 2 98211

0.176 2 0.014 0.041 f 0.005 0.179 f 0.002 0.035f 0.001 0.182 f 0.018 0.040f 0.004 0.630 f 0.089' 0.131 f 0.013' 0.233f 0.018 0.061 f 0.006 0.257 f 0.032 0.074 f 0.005 0.189 f 0.015 0.052 f 0.005 0.555 f 0.372 0.422 f 0.371

a Resistance measured after the absorption experiments and expressed as a percentage of the resistance of control monolayers. Values expressed as mean f SD. 'Statistically significantly different from control (p < 0.001).

threefold, respectively. HCO had no effect on transepithelial electrical resistance or permeability of the markers. Results of initial studies of the recovery of the cell monolayers after exposure to the surfactants are presented in Figure 5. Concentrations of surfactants giving a marked decrease in resistance and a corresponding increase in marker permeability were chosen. Thus, intermediate (RC,,) concentrations of SDS and STDHF and a high concentration of P80 were used. Monolayers exposed to STDHF and P80 for 2 h recovered within 6 h (p < 0.001 and p < 0.05, respectively; Figure 5).Monolayers exposed to 0.58mM SDS did not always recover (data not shown). However, when the SDS concentration was reduced to 0.40 mM and the incubation time was reduced to 20 min, a rapid and reproducible recovery was obtained (p < 0.001; Figure 5, Right). Effects on Monolayer Morphology-The morphologies of surfactant-treated and untreated monolayers were compared by scanning electron microscopy. Monolayera exposed to RC,, concentrations of the anionic surfactants were investigated. Because no RCso value was obtained for P80,the concentration of this surfactant was chosen from the right portion of the plateau in Figure 2, Bottom. The concentration of HCO was chosen from a similar curve (not shown). The morphologies of surfactant-exposed Caco-2 monolayers are summarized in Table VI. The apical side of untreated Caco-2 monolayers was mainly covered by relatively dense and uniform microvilli. However, small areas with a low density of microvilli were also found; such areas reflect the polyclonal properties of the cell line (Figure 61.40 In some areas, microvilli were sparse and the cell membrane was exposed. Occasionally, traces of extracellular deposits were seen. f i r exposure to 0.58 mM SDS for 4 h, the morphology of the monolayers changed (Figure 7). Microvilli were absent from large areas of the monolayer surface. In some areas, the microvilli were aggregated. Further, membrane wounds of different sizes were found. More extracellular deposits were found in treated monolayers than in control monolayers. No signs of epithelial cell detachment were observed. The morphological changes induced by DS were similar to, but not as pronounced as,those obtained with SDS (Table VI). Monolayers exposed to 2.7 mM STDHF for 4 h were also affected, but the morphological changes were not as marked as those for SDS and DS (Figure 8). In some areas, the monolayers looked normal, whereas in others, a decrease in the density of microvilli, sometimes in combination with small membrane wounds, was observed. The morphology of monolayers exposed to STC was similar to that of monolayers exposed to STDHF. Exposure to STDC resulted in large areas with complete loss of microvilli and extensive extracellular Journal of fhannaceutical Sciences I 883 Vol. 81, No. 9, September 1992

mfim

20

T

T

T

\ \ \

,,, ,,, 1

3

2

1

2

I

,,, 3

I

2

3

Flgure 5-Reversibility of effect of surfactants on mannitol absorption: (Left) 3.8 mM P80; (Mlddle) 2.7 mM STDHF; (Right) 0.40 mM SDS. The P values were measured (1) before and (2) during exposure to the surfactants and (3) after recovery of the monolayers. The exposure time was for P80 and STDHF and 20 min for SDS. The recovery period was 6 h for P80 and STDHF and 90 min for SDS (mean k SD; n = 3).

8

Table VCMorphoiogy of Monolayers after 4 h of Exposure to Surtactants Morphologic Score' incubation Treatment No treatment (control) SDS (0.58 mM) DS (0.48 mM) P80 (3.8 mM) HCO (7.1 mM) STC (21 mM) STDC (2.3 mM) STDHF (2.7 mM)

*Wegated and Shortened Microvilli

Areas with Decreased Microvilli Density

Membrane Woundsb

0

1

0

3

3 2 2

3

1 2 3 2

1 1 1 1

2 2 2 1

2 1

1 1

a Scoring from 0 to 3, where 0 indicates total absence of the specified morphological parameter. Score of 1, 2, or 3 indicating membrane wound with a diameter of <0.5, 0.5-1, or >1 pm, respectively.

Figure &Scanning electron micrographs of control monolayers. The bars indicate (Left) 5 pm and (Rlght) 1 pm.

deposits. All monolayers exposed to the bile acids were continuous, and no signs of cell losses were observed. Exposure to 3.8 mM P80 resulted in a slightly different monolayer morphology (Figure 9). Areas with sparse microvilli were relatively common, but the remaining microvilli were more aggregated, and large amounts of extracellular deposits were observed. Small wounds were found in the cell membranes. Monolayers incubated with 7.1 mM HCO were generally less affected than monolayers incubated with P80, but the morphological changes were similar. No signs of 004 I Journal of Pharmaceutical Sciences Vol. 81, No. 9, September 1992

Figure 7-Scanning electron micrographs of cells exposed to 0.58mM h. The bars indicate (Lett) pm and (RlgM) prn.

sDs for

Figure &Scanning electron micrographs of cells exposed to 2.7 mM STDHF for 4 h. The bars indicate (Left) 1 prn and (Right) 5 prn.

epithelial cell detachment were found with the nonionic surfactants.

Discussion Monolayers of intestinal epithelial cells were used to study the effects of luminal exposure to seven pharmaceutically relevant surfactants on epithelial permeability.l6,21~23.24.41,42 Initially, dose-response relationships were established by two methods: M I T assay and measurements of transepithelial electrical resistance. These methods measure different responses and are performed with Caco-2 cells of different ages. Thus, in the M I T method, 1-day-old cells grown in

flgure &Scanning electron micrographs of cells exposed to 3.8 rnM P80 for 4 h. The bars indicate (Left) 1 pm and (Rlght) 2.5 pm.

conventional microtiter plates as subconfluent monolayers are used,28.43 whereas in the resistance assay, confluent monolayers cultivated on filters for at least 2 weeks are used.14 Despite these differences, comparable dose-response curves were obtained for the two methods, and a particularly good correlation was established between the IC,, and RCso values of the anionic surfactants. This agreement indicates that the simple M2T method can be used as an initial screening method to characterize the effects of surfactants on epithelial cell monolayers. However, when more subtle changes in permeability were studied, both assays were insensitive. Measurements of permeabilities of radiolabeled mannitol and PEG were generally more sensitive. Because these marker molecules permeate intact Caco-2 monolayers through the intercellular spaces, their Pappvalues are very low. Small changes in permeability can therefore be readily detected. These results agree with those of a recent study by Milton and Knutson,44 who found that permeability coefficients of radiolabeled marker molecules were more sensitive indicators of the integrity of cell monolayers compared with transepithelial electrical resistance. The permeabilities to both mannitol and PEG were studied to investigate whether permeability depended on molecular weight. The ratio of the aqueous diffusion coefficients of mannitol and PEG is -2.8. Thus, if the two markers permeate the epithelium through the same aqueous environment, the Pa of mannitol would be 2.8 times higher than that of PEG. R e ratios of the permeabilities of mannitol and PEG varied by a factor of three in different control experiments (Tables 111-V). Similar variations were observed in monolayers treated with surfactants. The relatively large variations were only partly dependent on differences between individual monolayers. The radiolabeled probes were more significant variables. Thus, a ratio of c2.8, observed in some experiments, indicates that PEG was polydisperse and probably contained lowmolecular-weight species that could permeate the epithelium more rapidly than expected from the mean molecular weight. Further, different batches of radiolabeled mannitol gave slightly different P , values. The interaction of surfactants with the intestinal epithelium has previously been studied in whole-tissue models. Comparisons between such studies and the results presented here are complicated by the fact that, in the earlier studies, relatively high concentrations of the surfactants have generally been used. SDS and DS have usually been studied at concentrations of >2 mM (i.e., at concentrations leading to partial denudation of the epithelium both in situ and in the Caco-2 mode1)3.46?46,suggesting that the enhanced permeabil-

ity obtained in the earlier studies is associated with severe epithelial damage. However, in some studies, lower concentrations were used. Thus, 1 mM SDS increased the permeability twofold in rat small intestinal mucosa,47 and Sund48 reported that SDS and DS at concentrations of <1.7 mM gave subtle effects on water and sodium secretion in jejunal loops of rats. In this study, the Caco-2 monolayers were slightly more sensitive to the surfactants compared with the wholetissue models. Thus, a distinct effect on the intestinal permeability was obtained with 0.5 mM SDS and DS. Many factors may contribute to this difference. For instance, no extracellular debris can bind and neutralize the surfactants in the Caco-2 model. Further, Caco-2 monolayers do not produce a protective mucus coat. Thus, in the Caco-2 model, the surfactants have free access to the apical cell membranes. As with SDS and DS, the nonionic surfactants studied in other models were used in concentrations usually in excess of those used in this study. When methotrexate and either 15 or 45 mM P80 were given orally to mice, the absorption of the drug increased 1.6-3 times.9 A similar increase in the rectal permeability of sulfanilic acid in rats was obtained with 38 mM P80.a These two studies clearly indicate that large doses of P80 are needed to produce detectable changes in permeability. This finding agrees with those from the Caco-2 model, in which relatively high concentrations of P80 were needed to obtain modest increases in permeability. The effects of natural bile acids on the intestinal mucosa have been extensively studied. In general, an increase in epithelial permeability is observed at concentrations similar to the CMC ~alues.7.8.49~50 Comparable results were obtained in this study. Thus, the dost+response curves for STC and STDC in the Caco-2 model were similar to those obtained in whole-tissue models, in contrast to the findings with SDS and DS. The difference may be due to the fact that SDS and DS are effective a t lower concentrations compared with the bile acids. Thus, nonspecific interactions with luminal contents and mucus in the whole-tissue models may neutralize a relatively large fraction of the amounts of SDS and DS added; such neutralization could compromise the effect of these surfactants a t the epithelial interface. STDHF is a synthetic bile acid derivative claimed to have a milder effect on epithelia compared with natural bile acids. Thus, red blood cells are relatively insensitive to lysis when exposed to STDHF.25 The results obtained in the MTT and the resistance assays did not reveal any differences between STDHF and the other bile acids. All three compounds had IC,, and RC,, values in the region of their CMC values. Likewise, the morphological appearance and the mannitol and PEG permeabilities were comparable. These results suggest that the effects of STDHF, STC, and STDC on the Caco-2 monolayers are comparable. The effects of STDHF on the intestinal mucosa have only been studied at concentrations far above CMC (i.e., at concentrations that produced cell denudation in the Caco-2 mode161.62). Therefore, a direct comparison with the results of this study is not possible. Hydrophilic marker molecules, such as mannitol and PEG, permeate the normal epithelial cell monolayer through the intercellular spaces.26.53 After exposure to surfactants, the permeation may occur a t three different sites: (1)through the intercellular spaces, (2) across unusually leaky cell membranes, or (3) a t areas of cell denudation. The literature and the results of this study indicate that the relative contributions of these pathways vary with surfactant concentration. At the lowest concentrations, the surfactant-to-lipid ratios were low, and therefore, the surfactants are expected to bind to the cell membranes and stabilize, rather than increase, their permeability.2.7 Indeed, for some surfactants, a small increase in transepithelial resistance was observed immediately after exposure to a low surfactant concentration (data Journal of Pharmaceutical Sciences I 885 Vol. 81, No. 9, September 1992

not shown). However, small increases in the marker permeabilities were sometimes observed after longer exposures. The marker molecules permeated the treated and untreated epithelia by the same route (i.e., through the intercellular spaces). At the intermediate concentrations of the anionic surfactant and a t the highest concentration of P80, the monolayers remained intact (no cell losses), and the changes in permeabilities were reversible. Therefore, the possibility that the increased permeation occurred at sites of cell denudation is excluded. The morphological data, together with the effects on the intracellular enzyme activities, indicate that the surfactants rendered the cell membranes more leaky and facilitated the permeation of the markers through the apical cell membrane. However, a more leaky apical cell membrane does not necessarily indicate that the markers permeated the epithelial cells by the transcellular route, because the basal cell membrane may have remained intact. McNeil and It064 recently showed that normal intestinal epithelial cells continuously develop apical membrane wounds in situ and that the cells reseal the wounds and survive. When fluoresceinlabeled macromolecules are incubated with wounded cells, the marker is trapped inside the cells after the wounds are resealed. Thus, fluorescein-labeleddextran with a molecular weight of 5000 (similar to that of the PEG used in this study) is retained inside the epithelial cells for several hours.64 Although the possibility that the marker molecules passed the Caco-2 cell monolayers through unusually leaky cell membranes cannot be excluded, the results reported by McNeil and It054 suggest that both apical and basal cell membranes must be damaged before transcellular passage of PEG can occur. Whether epithelial cells can survive such extensive perforation is not known. A more obvious target for the surfactants is the tight junctions,65 as evidenced by transmission electron microscopic studies, which showed an increased paracellular permeability to the electron-dense marker lanthanum after exposure to bile acids.7 Further, studies by Free1 et al.49 showed that the ratio of the permeabilities of sodium and mannitol in the rabbit colon after exposure to bile acid concentrations that decreased the transepithelial electrical resistance and increased the flux of the two markers was the same as in control tissues. Thus the markers presumably permeated the control and treated epithelia by similar pathways (i.e., a paracellular pathway). Results of a preliminary study56 on the effects of another surfactant, palmitoyl carnitine, on Caco-2 monolayers suggest that this agent expanded the paracellular pathway. The same study56 shows that microscopic techniques more refined than those used in the present study are required to investigate the relative importance of paracellular and transcellular permeation. At higher surfactant concentrations, membrane solubilization followed by cell lysis and extrusion of the cells from the villus tips has been 0bserved.6.7~67"g Denudation of the epithelial cells a t the villus tips results in a decrease in electrical resistance as well as an increase in marker permeability. The very large increases in permeability observed for the anionic surfactants at the highest concentrations, together with a profound decrease in resistance, indicated cell detachment, which was verified by light microscopy. The results clearly indicate that commonly used wetting agents induce concentration-dependent changes in intestinal permeability. Thus, the surfactants used in oral dosage forms may be associated with mucosal damage, although such damage may not necessarily exclude clinical applications. Several suppository bases that are commonly used in humans have been found to have profound effects on the rectal mucosa.60361 Examination of the contents of surfactants that are nor886 I Journal of Pharmaceutical Sciences Vol. 87, No. 9, September 7992

mally incorporated into solid oral dosage forms indicates that the amounts are generally too small to affect the intestinal mucosa. Concentrations many times higher than those obtained after dissolution of the solid oral dosage forms were normally required to impair epithelial integrity. However, relatively large amounts of the anionic surfactants are used in a few dosage forms. In these cases, estimates of the concentrations obtained after dissolution of the dosage form approached (SDS) or were even higher (DS) than the concentrations that gave distinct effects in the Caco-2 model. The results from the reversibility experiments indicate that Caco-2 monolayers recover from the treatment with the surfactants. Relatively long recovery periods were used, and therefore, no detailed information about the rate of recovery was obtained. Normal intestinal epithelium recovers from surfactant treatment within minutes to a few hours.3.57 SDS-treated monolayers recovered within 90 min; thus, the kinetics of the recovery of the Caco-2 monolayers in vitro are comparable with those in vivo. In summary, the results obtained with the Caco-2 monolayers agree with the results obtained in experimental animals. Further, the results indicate that the hydrophilic marker molecules permeate the epithelial monolayers by different pathways at different concentrations of the surfactants. At low concentrations, the surfactants have only a slight effect on the epithelial permeability of the marker molecules. At concentrations that induce a reversible increase in marker permeability, the markers permeate via expanded paracellular spaces. However, permeation through unusually leaky apical and basal cell membranes cannot be ruled out. At high concentrations, the surfactants cause cell death with enhanced permeation at sites of cell denudation. The results indicate that Caco-2 monolayers can be used to study the effects of surfactants, as well as other pharmaceutical additives, on epithelial integrity and drug absorption.

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b.

Acknowledgments We are ateful to Tapio Nikkilii, Swedish Veterin Institute, Uppsala, & d e n , for re arin the electron micmgraTs. We also thank LGvens Kemisfe gabrif, Denmark, for supplyin sodium taurodihydrofusidate. This work was supported by grants from The Swedish Medical Research Council (094781, The Swedish Fund for Scientific Research without Animal Experiments, and Centrala Forsiiks4jursniimnden (FN L-90-04).

Journal of Pharmaceutical Sciences I 887 Vol. 81, No. 9, September 1992