Introduction

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The pH-partition theory states that only the un-ionized form of ionizable drug molecules can diffuse across cell membranes. This assumption is often extended to drug transport across epithelial barriers, such as the intestinal epithelium (Hogben et al., 1959). However, it has become clear from recent studies that the ionized form of drug molecules can also partition into phosphatidylcholine bilayers (Iseki et al., 1992; Ottiger and Wunderli-Allenspach, 1997; Avdeef et al., 1998), although to a much lesser extent than the un-ionized form. In addition, the paracellular pathway through intestinal epithelia has been shown to be more permeable to cationic than to either neutral or anionic drug molecules (e.g., Adson et al., 1994; Karlsson et al., 1994). Therefore, it cannot be ruled out that the intestinal epithelium is also permeable to the ionized form of drug molecules at high degrees of ionization. If this is the case, transport of the ionized form of the drugs should be considered in studies of epithelial permeability to drugs, and the pH-partition theory should be modified accordingly. To enable a rational approach to predictions of the intestinal absorption of proteolytic drugs, information on epithelial permeability to the un-ionized form of the drug, the pK_a of the drug and the effect of molecular charge on drug transport; i.e., the selectivity of the intestinal epithelium for the un-ionized over the ionized form, are needed. Thus, a modification of the pH-partition theory would be particularly important for predictions of intestinal drug absorption from in vitro studies and studies of structure-absorption relationships.

Most experimental investigations on the relationship between pH, drug ionization and intestinal epithelial permeability have been made using animal models. Because of the complexity of these models, it has been difficult to determine the extent of the epithelial selectivity for the un-ionized over the ionized form. Deviations from the pH-partition theory have frequently been observed for both acidic and basic compounds. These deviations have been attributed to the presence of an acidic microclimate close to the intestinal wall and to aqueous boundary effects (Hogben et al., 1959; Högerle and Winne, 1983). Intestinal cell culture models, such as the Caco-2 cell monolayers, offer two major advantages in studies of the relationship between drug ionization and epithelial permeability. First, the absence of villous structures and a mucus layer makes it possible to minimize the influence of an acidic microclimate by using effective stirring and buffers (Shiau et al., 1985). Second, it is possible to determine the cell monolayer permeability unbiased by the aqueous boundary layer (Karlsson and Artursson, 1991).

The aim of this study was to quantify the effect of molecular charge on drug transport across the intestinal epithelium to include this effect in structure-absorption relationships. Therefore, the pH-dependent transport of two model drugs, both with pK_a values in the pH range found in vivo, across Caco-2 cell monolayers was investigated. Alfentanil, which is rapidly transported across Caco-2 cell monolayers, and cimetidine, which is more slowly transported, were selected as model drugs. The cell monolayer permeability coefficients (P_c) of the two basic drugs were determined in the pH range 5.0 to 8.0. For this purpose, a method for determining P_c values under nonsink conditions was developed. The relationship between P_c and the fraction of the drug in un-ionized form (f_u) was established to delineate the P_cs of the un-ionized and ionized forms of the drugs. Finally, the contribution of the transport of the ionized form to the total transport in the studied pH range was investigated.

	Materials and Methods

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Selection of Model Drugs

A comprehensive drug compendium (Craig, 1990) was examined to identify drugs with pK_a values of approximately 6.5. Such drugs were considered as suitable model drugs because they display the largest possible variation in the degree of ionization within the physiological pH-interval of the intestine. In addition, both rapidly and slowly transported drugs were considered because the effect of solute charge on the epithelial permeability to transcellularly and paracellularly transported drugs could be different.

Thirty compounds with pK_a values between 6.2 and 6.8 were identified. These compounds were then evaluated based on levels of toxicity, chemical stability, solubility, active transport mechanisms, intestinal metabolism, and availability. Alfentanil (logarithm of the octanol/water partition coefficient = 2.16), which was confirmed to be stable and sufficiently soluble for the purposes of the study, was selected as an example of a rapidly transported drug. The second model drug, cimetidine (logarithm of the octanol/water partition coefficient = 0.40), has a low transport rate across Caco-2 monolayers (Pade and Stavchansky, 1997) and is believed to be transported across the intestinal epithelium predominantly by a passive diffusion process (Mummaneni and Dressman, 1994). Although affinity for active efflux systems, such as P-glycoprotein and the organic cation exchanger (Miyamoto et al., 1988; Dutt et al., 1992; Pan et al., 1994), has been observed for cimetidine, active efflux of the molecule can be significantly reduced by the addition of verapamil (Pan et al., 1994).

Drug Transport Studies

Drugs and Radiolabeled Markers. Cimetidine and verapamil were purchased from Sigma (St. Louis, MO). Alfentanil was obtained as Rapifen IV solution from Janssen-Cilag through Apoteket AB, Stockholm, Sweden and [¹⁴C]-mannitol (specific radioactivity, 52 mCi/mmol) was purchased from New England Nuclear through Du Pont Scandinavian AB, Norrköping, Sweden.

Cell Culture

Caco-2 cells were obtained from American Tissue Culture Collection (Rockville, MD). The cells were maintained in Dulbecco's modified Eagle's medium, containing 10% heat-inactivated fetal calf serum and 1% nonessential amino acids, in a humidified atmosphere of 90% air and 10% CO₂, as described elsewhere (Artursson, 1990). Cells (5 × 10⁵) (passage number 90-105) were seeded on polycarbonate filter inserts (Transwell Costar, Badhoevedorp, the Netherlands; mean pore size 0.45 µm; diameters 6 and 12 mm) and cultivated in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum, 1% nonessential amino acids, penicillin (100 units/ml), and streptomycin (100 µg/ml). All tissue culture media were obtained from Gibco through Laboratorie Design AB (Lidingö, Sweden). The cells were allowed to grow and differentiate for 22 to 32 days before the monolayers were used in drug transport experiments.

Drug Transport Experiments

The transport experiments were performed in Hanks balanced salt solution (HBSS). The HBSS was buffered with 25 mM acetate (pH 4.0-5.0), 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.0-6.5), or 25 mM HEPES (pH 6.8-8.0), depending on the desired pH on the apical side. These buffers maintain the adjusted pH values during the transport experiments (data not shown; Ellens et al., 1997). The presence of an acidic microclimate at the mucosal surface is dependent on the presence of a mucus layer and is abolished by effective stirring and well-buffered apical solutions (Shiau et al., 1985). Consequently, we assumed that any acidic microclimate produced by the Caco-2 cells would be disrupted under the experimental conditions used in this study. HBSS of pH 7.4 buffered with 25 mM HEPES was always used on the basolateral side of the monolayers.

Transport rates were determined in both the apical-to-basolateral (a-b) and basolateral-to-apical (b-a) directions to detect polarized transport in the absorptive or secretory direction. Drugs were dissolved in the transport buffer to give a final concentration of 0.5 to 10 mM. All solutions were prewarmed to 37°C. The drug solutions were added to the donor side of the monolayers, and buffer without drug was added to the receiver side. The monolayers were incubated in a humidified atmosphere at 37°C. At regular time intervals (4-10 min for alfentanil and 20-30 min for cimetidine), samples were withdrawn from the receiving chamber and frozen (20°C), pending HPLC analysis. All transport experiments were carried out in less than 120 min. The transport of the model compounds was followed at both low (100 rpm) and high (500 rpm) stirring rates using a calibrated plate shaker (IKA-Schüttler MTS4) to obtain P_c values unbiased by the aqueous boundary layer (see below). Monolayer permeability to the paracellular marker [¹⁴C]mannitol was used to investigate the integrity of the monolayers under the experimental conditions.

Analytical Methods

The samples from the drug transport experiments were analyzed using reversed phase HPLC. The system consisted of a Perkin-Elmer Isocratic LC Pump 250, a Perkin-Elmer Advanced LC Sample Processor ISS-200, a Spectra Physics UV100 detector, and the integration software program Chromatography Station for Windows. A Beckman Ultrasphere ODS (250 × 5.6 mm) with a particle size of 5 µm was used as analytical column. Mobile phases consisting of phosphate buffer (pH 3.0; 60 mM KH₂PO₄, 8 mM H₃PO₄), and 20% (cimetidine) or 30% (alfentanil) acetonitrile, resulted in retention times of 6 to 8 min at a flow rate of 1 ml/min. Cimetidine was detected at a wavelength of 225 nm and alfentanil at 232 nm. Samples containing [¹⁴C]mannitol were counted in a liquid scintillation counter (Tricarb 1900 CA; Packard Instruments).

Determination of P_c

At "sink" conditions, the apparent permeability coefficients (P_app, cm/s) were calculated from:

(1)

where k is the transport rate (min¹) defined as the slope obtained by linear regression of the cumulative fraction absorbed as a function of time (min), V_R is the volume in the receiver chamber (ml), and A is the area of the filter (cm²). The fraction absorbed was defined as the ratio between the concentration on the receiver side (C_R) at the end of an interval and the concentration on the donor side (C_D,0) at the beginning of that interval. Alfentanil was transported so rapidly at higher pH values that sink conditions could not be maintained (Fig. 1a). It was therefore necessary to develop a more general method for calculating P_app, one also applicable to nonsink conditions. Taking into account the effect of back flux from the receiver compartment, Fick's law gives:

(2)

where C_D(t) and C_R(t) is the concentration in the donor and receiver compartment, respectively, as a function of time. If the amount of drug in the system (M) is assumed to be constant within each sampling interval, C_D(t) can be expressed as a function of C_R(t). Thus, replacing C_D(t) by this expression in eq. 2 and solving the differential equation for C_R(t) gives:

(3)

where V_D is the volume in the donor compartment, C_R,0 is the drug concentration in the receiver compartment at the beginning of the interval and t is the time from the start of the interval. The sampling procedure necessitates the recalculation of M and C_R,0 for each succeeding interval according to mass balance. P_app values for alfentanil were then determined by nonlinear regression, minimizing the sum of squared residuals ((C_R,i,obs  C_R,i,calc)²), where C_R,i,obs is the observed receiver concentration at the end of interval i and C_R,i,calc is the corresponding concentration calculated according to eq. 3. The importance of the correction for nonsink conditions in calculations of P_app values of alfentanil is shown in Fig. 1a. Sink conditions were maintained in the transport experiments of cimetidine and P_app values of cimetidine calculated according to eqs. 1 and 3 were not different (Fig. 1b).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   a, Amount alfentanil (1.2 mM) transported across Caco-2 cell monolayers at pH 8.0 as a function of time. At a low stirring rate (100 rpm; ), sink conditions (which are maintained when the ratio between receiver and donor concentration, CR/CD, < 0.1) were marginally exceeded in the end of the last time interval (CR/CD = 0.11). There was not a significant difference (p > .05) between Papp of alfentanil at 100 rpm calculated according to eq. 1 (93.5 ± 5.4 · 106 cm/s), which assumes sink conditions, and eq. 3 (105 ± 8 · 106 cm/s), which takes into account the effect of back flux from the receiver compartment. However, at the higher stirring rate (500 rpm; ), sink conditions were exceeded already during the second time interval (CR/CD > 0.12) and the importance of applying a method applicable to nonsink conditions was demonstrated: Calculation of Papp according to eq. 1 (167 ± 6 · 106 cm/s) significantly (p < .01) underestimated the Papp of alfentanil at 500 rpm (197 ± 8 · 106 cm/s) calculated according to eq. 3. b, amount cimetidine (1 mM in the presence of verapamil) transported across Caco-2 cell monolayers at pH 8.0 as a function of time. Sink conditions were maintained both at low (100 rpm; ) and high (500 rpm; ) stirring rates. Thus, Papp values calculated according to eqs. 1 and 3 were not significantly different at 100 rpm (1.68 ± 0.08 · 106 cm/s, according to both equations) or at 500 rpm (1.65 ± 0.17 · 106 cm/s and 1.67 ± 0.17 · 106 cm/s, according to eqs. 1 and 3, respectively). Values are presented as mean ± S.D., n = 3 to 4.

Subsequently, the permeability coefficients of the cell monolayer (P_c,cm/s) for both alfentanil and cimetidine were calculated as described previously (Karlsson and Artursson, 1991):

(4)

where V is the stirring rate, P_f is the calculated P_c of the filter support (Karlsson and Artursson, 1991) and K is a constant.

Theory

Calculation of pK_a Values. Conformational analysis. The conformational preferences of alfentanil and cimetidine were analyzed using molecular mechanics calculations. The MM3 force field, as implemented in the MacroModel program (version 5.5), was used in the analyses of alfentanil. Because parameters for cimetidine were missing in the MM3 force field, the conformational analyses of cimetidine were performed using the Merck Molecular Force Field, also as implemented in MacroModel. Systematic pseudo-Monte Carlo conformational search procedures (BatchMin v 5.5) were used for both compounds in vacuum and simulated water environment.

Calculations of pK_a Using MolSurf. The global minimum conformations found in the conformational analysis of each environment were first geometry optimized using the semiempirical AM1 method as implemented in the Spartan program (version 5.3). Then, a single-point quantum mechanical ab initio calculation using the 3-21G* basis set was performed on the AM1 optimized conformation. The wave function generated by the Spartan program was used by MolSurf to calculate the pK_a values (pK_a,calc) of the compounds (Norinder et al., 1997; Sjöberg, 1997).

Determination of the P_c of Un-ionized and Ionized Drug Forms. In the unmodified pH-partition theory, the following relationship is suggested:

(5)

where f_u is the fraction of the drug in the un-ionized form (f_u = 1 when the drug is completely un-ionized). f_u is calculated from previously reported pK_a values (Table 1):

(6)

and P_c,u is the P_c of the un-ionized species. Thus, according to eq. 5, if the epithelial cell monolayer is impermeable to the ionized form, the ratio P_c/f_u will equal P_c,u and will be independent of pH. However, if we assume that the intestinal epithelium is also permeable to the ionized species, eq. 5 must be expanded to:

(7)

where f_i is the fraction of drug in ionized form and P_c,i the P_c of the ionized species. Because f_i + f_u = 1, eq. 7 can be rewritten as:

(8)

Thus, if an intercept significantly different from zero is obtained when P_c is plotted as a function of f_u, this intercept equals the P_c of the ionized form of the drug and P_c,u can be calculated from the slope of the linear regression line. In this analysis, no assumptions are made with respect to whether transport of the un-ionized or ionized forms occurs via the transcellular or paracellular routes.

Statistics

Values are generally expressed as mean ± one S.D. The differences between mean values were analyzed with Student's t test or one-way ANOVA, followed by Scheffe's F test. When equal variances could not be obtained by logarithmic transformation, the analysis was made using Student's t tests for unequal variances or the Bonferroni correction for multiple comparisons. A level of  = .05 was considered to be statistically significant. In the least-square linear regression analyses on P_c as a function of f_u, a weighting scheme of 1/S.D.² was used.

	Results

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pH-Dependent Integrity of Caco-2 Cell Monolayers. The pH-dependent integrity of the Caco-2 cell monolayers was investigated by studying their permeability to the integrity marker mannitol (Fig. 2). The P_app of mannitol remained constant in the apical pH interval 4.8 to 8.0 (1.51 ± 0.26 · 10⁷ cm/s; n = 32; p > .05). Thus, the integrity of the Caco-2 monolayers was unaffected in this interval, which covers the variability in pH found in the human intestine in vivo (pH 5.5-7.5; Evans et al., 1988; Dressman et al., 1990). However, a significant increase in cell monolayer permeability to mannitol was observed at pH values of 4 (p < .001) and 4.5 (p < .05), indicating an effect on the Caco-2 cell monolayer integrity at pH < 4.8. Based on these results, an apical pH interval of 5.0 to 8.0 was selected for additional studies to include as wide range as possible for f_u of the model drugs (f_u of alfentanil varies from 0.03 to 0.97 and f_u of cimetidine from 0.02 to 0.95 in this pH interval).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Permeability of Caco-2 cell monolayers to the hydrophilic marker mannitol was used to study the effect of pH on the integrity of the monolayers. Papp of mannitol was constant in the pH interval 5.0 to 8.0; i.e., throughout the physiological pH range in the human intestine (pH 5.5-7.5). A significant increase in monolayer permeability was observed at pHs of 4.5 and lower. Values are presented as mean ± S.D., n = 3 to 5. (*p < .05, and ***p < .001)

pH-Dependent Transport of Alfentanil, a Rapidly Transported Drug. To confirm that the transport of alfentanil was predominantly passive, two types of experiments were performed. First, the contribution of polarized transport of alfentanil was investigated by comparing the P_c values of the drug in the a-b (absorption) and b-a (secretion) directions at pH 7.4 in both chambers. A b-a/a-b ratio of 1.0 indicates transport entirely by passive diffusion in Caco-2 monolayers (Karlsson et al., 1993; Burton et al., 1996). The rate of transport of alfentanil (1.2 mM) in the b-a direction was slightly higher than that in the a-b direction (p < .05), giving a b-a/a-b ratio of 1.1, Fig. 3a. However, because this ratio was only marginally higher than 1.0, it was concluded that active secretion is not likely to affect the passive transport of alfentanil in the a-b direction across the Caco-2 monolayers at this pH.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   a, Caco-2 cell monolayer permeability to alfentanil (1.2 mM) at pH 7.4 in the a-b and b-a direction. The b-a/a-b ratio at pH 7.4 was low (1.1) and active secretion (b-a) was not considered to influence the passive transport of the drug in the a-b direction. b, b-a Pc of alfentanil determined at pH 5.0 and 8.0 on the apical side of the monolayers. The b-a permeability was found to be independent of apical pH. Values are presented as mean ± S.D., n = 7 to 12. (comparison between a-b and b-a Pc values: *p < .05)

6

6

(9)

6

6

View larger version (12K): [in this window] [in a new window]   Fig. 4.   pH-dependent transport of the rapidly transported model drug alfentanil across Caco-2 cell monolayers. a, cell monolayer permeability to alfentanil as a function of the fraction of the drug in un-ionized form on the apical side of the monolayers. A linear relationship was obtained (r2 =. 983); the Pc of the ionized form of alfentanil (2.10 ± 0.54 · 106 cm/s; p < .001) was obtained from the intercept. The monolayer permeability to the un-ionized form (323 ± 7 · 106 cm/s) was obtained from the slope of the regression line. b, calculated contribution of un-ionized (dotted line) and ionized forms (dashed line) to the calculated overall permeability of the monolayers to alfentanil (solid line) as a function of pH. (Observed Pc values are represented as .) The contribution of the ionized form to the total transport was small. In the studied pH range, the largest relative contribution of the ionized form to the total transport (17%) was observed at pH 5.0. Experimentally determined values are presented as mean ± S.D., n = 6 to 12.

pH-Dependent Transport of Cimetidine, a Slowly Transported Drug. As with alfentanil, the influence of active secretory mechanisms on the pH-dependent passive transport of cimetidine across the Caco-2 monolayers was studied initially. The Caco-2 monolayers were significantly more permeable to cimetidine (1 mM) in the b-a direction than they were in the a-b direction (p < .001; Fig. 5a). The b-a/a-b ratio was 4.4, indicating that active secretion could have an effect on the a-b transport rate (Karlsson et al., 1993; Burton et al., 1996). When the donor concentration of cimetidine was increased to 10 mM, the b-a/a-b ratio decreased to 2.9. Addition of 0.5 mM verapamil, a compound previously used to inhibit the active efflux of cimetidine (Pan et al., 1994), decreased the b-a/a-b ratio to 1.8. Therefore, subsequent transport experiments were carried out in the presence of verapamil. Interestingly, the decrease in the b-a/a-b ratio at pH 7.4 after addition of verapamil was caused entirely by decreased drug transport in the b-a direction, because there was no effect on a-b transport, Fig. 5a. There were no differences in the rate of b-a transport of cimetidine at apical pHs of 5.0, 7.4, and 8.0 (p > .05; Fig. 5b). These data indicate that the effects of polarized secretion are minimal with this drug, and that reliable estimates of the passive transport of cimetidine in the a-b direction can be obtained. Therefore, cimetidine was chosen as a model drug for studies of the pH-dependent passive transport of slowly transported compounds.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   a, Caco-2 cell monolayer permeability to cimetidine in the a-b and b-a direction at pH 7.4. Pc values obtained at 1 and 10 mM cimetidine and at 1 mM cimetidine in the presence of verapamil are presented. At an apical pH of 7.4, the Pc values determined in the b-a direction was significantly higher than in the a-b direction. The b-a/a-b ratio decreased in the order 1 mM cimetidine > 10 mM cimetidine > 1 mM cimetidine + 0.5 mM verapamil. Therefore, 0.5 mM verapamil was added to limit the influence of active secretion in all subsequent transport experiments. The a-b transport was, however, found to be independent of the b-a/a-b ratio (p > .5). b, cell monolayer permeability to cimetidine in the b-a direction at pH 5.0 and 8.0 on the apical side of the monolayers. The b-a transport was not affected by the pH on the apical side (p > .5). Therefore, the a-b Pc values (obtained in the presence of verapamil) were used as estimates of passive transport in the subsequent analysis of the pH-dependent transport of cimetidine. Values are presented as mean ± S.D., n = 6 to 32. (comparison between a-b and b-a Pc values: **p < .01 and ***p < .001)

6

6

(10)

6

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View larger version (12K): [in this window] [in a new window]   Fig. 6.   pH-dependent transport of the slowly transported model drug cimetidine across Caco-2 cell monolayers. a, cell monolayer permeability to cimetidine as a function of the fraction of the drug in un-ionized form on the apical side of the monolayers. A nearly linear relationship was obtained (r2 =. 946); the Pc of the ionized form of cimetidine (0.045 ± 0.016 × 106 cm/s; p < .001) was obtained from the intercept. The monolayer permeability to the un-ionized form (1.49 ± 0.11 × 106 cm/s) was obtained from the slope of the regression line. b, calculated contribution of un-ionized (dotted line) and ionized forms (dashed line) to the calculated overall permeability of the monolayers to cimetidine (solid line) as a function of pH. (Observed Pc values are represented as .) The contribution of the ionized form to the total transport increased at lower pH values: at pH 5.0, 66% of the total transport was attributable to the ionized form. Experimentally determined values are presented as mean ± S.D., n = 6 to 12.

Calculations of P_c of Un-ionized Form of Drugs. The pH-partition theory suggests that the transport of the ionized form of drugs is negligible. This implies that P_c,u can be calculated from P_c values observed at a single pH value, because the ratio P_c/f_u would be constant and equal P_c,u (eq. 5). The significance of the transport of the ionized form when f_u is small (as it is for the basic drugs in this study at lower pH values) was therefore investigated by plotting P_c/f_u as a function of pH (Fig. 7). At pHs  5.5, the ratio P_c/f_u is significantly higher than P_c,u values calculated according to eq. 8 for both alfentanil (Fig. 7a) and cimetidine (Fig. 7b). Thus, because of the significant contribution of the transport of the ionized form, P_c,u, calculated as P_c/f_u, will be overestimated if the transport experiments are performed when f_u < 0.1. However, the P_c,u of alfentanil calculated as P_c/f_u from P_c values determined at pH 5.0 and 5.5 are only 1.2-fold higher than P_c,u calculated according to eq. 8. For cimetidine, the greater contribution of the ionized form to the total transport results in a 3.1- and 1.4-fold overestimation of P_c,u from P_c/f_u at pH 5.0 and 5.5, respectively. Thus, neglecting the transport of the ionized form in calculations of P_c,u from P_c values obtained at pH values corresponding to f_u < 0.1 leads to overestimation of P_c,u, especially for slowly transported compounds such as cimetidine.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Observed monolayer permeability to: a, alfentanil and b, cimetidine, corrected for the fraction of the drug in un-ionized form, Pc/fu (), as a function of pH. If the epithelium was impermeable to the ionized form, the Pc/fu ratio would equal Pc, u (dotted line) and be independent of pH. However, the simultaneous increase in the contribution of the ionized form of the drug to the total transport and decrease in fu at lower pH values, results in a significant contribution of the ionized form to the Pc/fu ratio (dashed line). Thus, the Pc/fu ratio is increased at lower pH values (<5.5). Therefore, Pc, u is overestimated from Pc values obtained at fu < 0.1 if the transport of ionized drug molecules is not considered. Experimentally determined values are presented as mean ± S.D., n = 6 to 12. (comparison with determined values of Pc, u: *p < .05 and ***p < .001).

Prediction of the P_c of the Un-ionized Form of Drugs from Theoretically Calculated pK_a Values. In the early stages of the drug development process, the absorption of candidate drugs through the intestinal epithelium is determined at single pH values in vitro. Experimental values of pK_a (required to calculate f_u) are not always available at this early stage. We therefore investigated the accuracy of predicting the P_c,u of the model drugs from P_c determined at a single pH value and theoretical estimates of pK_a and f_u. Experimental pK_a values and the pK_a values calculated by the MolSurf program (pK_a,calc) are presented in Table 1. The pK_a values of alfentanil and cimetidine were underestimated by 0.7 to 0.8 U. P_c values determined at pH 6.5 were selected because pH 6.5 is in the middle of the studied pH interval, the acidic microclimate in the small intestine is close to pH 6.5 (Lucas and Blair, 1978), and f_u > 0.1 at this pH. This procedure resulted in a predicted P_c,u for alfentanil of 179 · 10⁶ cm/s, or 1.8-fold lower than the experimentally determined P_c,u. A P_c,u for cimetidine of 0.61 · 10⁶ cm/s was predicted from pK_a,calc. This value is 2.5-fold lower than the experimentally determined P_c,u.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The results of this study support modification of the pH-partition theory to incorporate transport of both un-ionized and ionized forms of drugs across the intestinal epithelium. Transport of the ionized form contributed significantly to the total drug transport at pH values where the f_u of the model drugs was < 0.1. Because many drugs have an f_u < 0.1 in the entire physiological pH range in the intestine, the transport of the ionized forms of drugs should be considered to a greater extent than suggested by the traditional pH-partition theory.

The Caco-2 cell monolayer permeability to the paracellular marker mannitol was unaffected in the apical pH range 5.0 to 8.0. Because the pK_a values of the phosphate groups in the cell membrane phospholipids are approximately 2 (Bangham, 1968), net charge of the cell membrane should be independent of pH in the present study. Only minor deviations from the theoretical relationships were observed in the pH-permeability curves of the model drugs, indicating that pH had no significant effect on the integrity of the cell membrane. Thus, the pH-dependent changes in drug permeation observed in this study were the result of changes in the f_u of the drugs and not of pH-dependent changes in cell membrane properties.

If the cell membrane is assumed to be impermeable to the ionized form, the obtained P_c,i values represent the paracellular transport of the ionized species. P_c,i for alfentanil was considerably higher than that for cimetidine. However, in theory, the larger molecular size of alfentanil (mw = 416) compared with cimetidine (mw = 252), should result in lower paracellular transport of the ionized form of alfentanil (according to a size-restricted diffusion of the paracellular pathway).

One possible explanation for this unexpected result is that the values of pK_a obtained from the literature are not applicable to the experimental conditions used in this study. Because P_c,i is determined by extrapolation of the relationship between f_u and P_c, the method of calculating P_c,i will be sensitive to the values of pK_a used in the analysis, especially for alfentanil. However, the pK_a of alfentanil (6.5) used in the determination of P_c,i is in good agreement with the inflection point of the experimentally obtained pH-permeability curve for alfentanil (6.4; Fig. 4b). Moreover, although the pH-permeability curve for cimetidine (Fig. 6b) indicates a pK_a of 7.1 under these experimental conditions, calculations based on this pK_a value do not result in a markedly different value for P_c,i (0.072 ± 0.029 · 10⁶ cm/s) than that obtained using a pK_a value of 6.8 (P_c,i = 0.045 ± 0.016 · 10⁶ cm/s). Thus, the use of the literature to obtain values of pK_a for use in the calculations does not explain the difference in P_c,i between alfentanil and cimetidine.

Another explanation could be that the ionized form of alfentanil can be transported across the epithelium transcellularly, although at a much slower rate than the un-ionized form. However, the pK_a values of the drug molecules can be different at the amphiphilic membrane interface compared with the aqueous solution pK_a values (Retzinger et al., 1986). Such a shift in pK_a will result in an apparent weaker base at the membrane interface and therefore in an increased transcellular transport. Theoretically, the pK_a-shift is likely to be associated with different conformational preferences in the aqueous solution and at the amphiphilic interface. A less polar environment will promote conformations more prone to form intramolecular hydrogen bonds. Indeed, calculations of pK_a from ionization energies indicated that intramolecular hydrogen bonding significantly influenced the pK_a of the identified low-energy conformations of alfentanil and cimetidine.

The rate of transport of the un-ionized forms of the drugs was 150- and 30-fold higher than the rate of transport of the ionized forms of alfentanil and cimetidine, respectively. In early in vitro studies, the transport of the un-ionized form of weak acids across rat small intestinal tissue was found to be 10,000-fold more rapid than the transport of the ionized form (Tai and Jackson, 1981). The selectivity of the rat gastric mucosa for the un-ionized form of weak bases was, however, found to be considerably smaller, approximately 500-fold (Tai and Jackson, 1982). The lower epithelial selectivity observed for the un-ionized form of bases compared with acids may be the result of the higher paracellular permeability of intestinal epithelia for cationic compared with anionic drug molecules (e.g., Adson et al., 1994; Karlsson et al., 1994).

For the basic model drugs investigated in this study, the intestinal epithelial Caco-2 cell monolayers displayed an even lower selectivity for the un-ionized form than previously observed for rat gastrointestinal epithelia. Because ionized molecules will presumably permeate the epithelium mainly via the paracellular route, the selectivity of the epithelium for the un-ionized form of drugs (P_c,u/P_c,i) is dependent on the paracellular permeability of the epithelium. The paracellular route in Caco-2 monolayers is more permeable than that in gastric mucosa, which might explain the lower selectivity observed in Caco-2 monolayers compared with rat mucosa. However, apart from the level of paracellular permeability, the selectivity of the tissue will also be highly dependent on the level of transcellular permeability to the un-ionized form of drugs (Jackson, 1987). This was also observed in this study, where the Caco-2 monolayers were less selective for the un-ionized form of the hydrophilic drug cimetidine than for the un-ionized form of the more lipophilic drug alfentanil.

An important implication of these results is that a more generally applicable model for predictions of human intestinal permeability to drugs from permeability studies in Caco-2 monolayers or from the molecular structure of the drug must account for the effect of drug ionization. This can be done in at least two ways. One approach is to compensate for the degree of ionization by determination of the P_c,u of the compounds. Because the contribution of the ionized form to the total transport is small, P_c,u can be calculated by correcting the observed P_c for the fraction of the drug in the un-ionized form (P_c/f_u) when f_u > 0.1. However, many drugs are bases with pK_a values > 9 or acids with pK_a values < 4 (Jackson, 1987). For this category of drugs, calculation of P_c,u from P_c determinations at a single pH value poses a problem because f_u of the drugs is < 0.1 throughout the entire pH range of 5.0 to 8.0. For example, in the structurally diversified set of compounds used to investigate the relationship between the fraction absorbed after oral administration to humans and structural descriptors (Palm et al., 1997), 12 of 20 compounds fall into this category. Thus, P_c,u would be difficult to determine for a large number of drugs. Therefore, a relationship between P_c,u and structural descriptors will be difficult to establish for compounds of greater structural diversity.

The second approach to accounting for the degree of ionization is to include a structural descriptor of molecular charge in the structure-permeability models. In the traditional structure-permeability relationship between octanol/water partition coefficients and epithelial permeability to drugs, the charge effect is accounted for by including the apparent distribution coefficients (logD) at the relevant pH. Because organic solvents are more selective for the un-ionized form of the solutes (of the order of 10⁶; see Jackson, 1987) than is the intestinal epithelium (see discussion above), logD values will frequently exaggerate the effect of ionization. The use of an alternative structural descriptor of molecular charge, such as pK_a or f_u, is therefore more attractive. Predictions of intestinal drug absorption from calculated structural descriptors would then depend on computational methods to estimate the pK_a values of the drugs. In this study, the pK_a values calculated from ionization energies gave promising results. However, further validation of this method and other computational methods of calculating pK_a are needed (Leahy et al., 1997).

In summary, the results of this study clearly show that the degree of ionization of the drugs under investigation has a pronounced influence on their permeation through the intestinal epithelium. The implications of this for predictions of the in vivo absorption of proteolytic drugs after oral administration remain to be investigated.