Raloxifene, a selective estrogen receptor modulator (Colacurci et al., 2003; Duschek et al., 2003), is used for the treatment of osteoporosis. Raloxifene blocks the adverse effects of estrogen in breast tissues and uterine endometrium while mimicking the beneficial estrogen effects on bone and lipid metabolism (MacGregor and Jordan, 1998; Scott et al., 1999). Data from both animal and human studies demonstrated that raloxifene has minimal effects on the uterus and caused no significant changes in the histological appearance of the endometrium (Scott et al., 1999 and references therein). Raloxifene is being considered as a chemopreventive agent for breast cancer (Delmas et al., 1997). Short-term clinical trials showed that raloxifene did not increase the risk of breast cancer and long-term multicenter trials are currently ongoing (Jordan and Morrow, 1999; Scott et al., 1999).

Raloxifene is reported by its manufacturer to be rapidly absorbed after oral administration, but its absolute bioavailability is only 2%. This poor bioavailability is thought to be the result of extensive phase II metabolism, since it is absorbed rapidly and approximately 60% of dose was absorbed after oral dosing (Hochner-Celnikier, 1999). According to its manufacturer, raloxifene exhibits linear pharmacokinetics with high within-subject variability (approximately 30% CV) (Hochner-Celnikier, 1999). It undergoes extensive phase II biotransformation, primarily glucuronidation, but was not metabolized by the cytochrome P450s (Eli Lilly, 1998). Raloxifene has a plasma elimination half-life of approximately 27 h, which is attributed to its reversible systemic metabolism and significant enterohepatic cycling (Eli Lilly, 1998). The major human intestinal enzymes responsible for its phase II conjugation have been shown to be UGT1A8 and UGT1A10 (Kemp et al., 2002).

Although a variety of pharmacokinetic information has been provided by its manufacturer (as summarized previously), mechanisms responsible for absorption of the raloxifene and excretion of raloxifene conjugates have not been reported. Studies have not established the dynamic relationship among absorption, metabolism, and excretion of raloxifene in the intestine, which deserves more attention since it is likely to be the main site of presystemic clearance (Kemp et al., 2002). In the present research, we focused on the study of absorption/excretion pathways responsible for intestinal disposition of raloxifene using the human intestinal Caco-2 cell model employing the cloned TC7 variant.

We chose Caco-2 TC7 cells, one of the cloned Caco-2 variants, for increased homogeneity and stability of cell population (Caro et al., 1995; Ranaldi et al., 2003). TC variant exhibits morphological characteristics similar to those of the parental Caco-2 cells (Gres et al., 1998) and shows similar expression of MDR1 and MRP1–5 as human jejunal biopsies (Pfrunder et al., 2003). It has been used by many investigators to study human intestinal disposition (Hu et al., 1999, 2000, 2003; Sabolovic et al., 2000; Pontier et al., 2001; Bohets et al., 2001).

Therefore, the objective of the present study was to determine whether enteric recycling and other intestinal disposition events could help explain why raloxifene has a low bioavailability but a long half-life. The process of enteric recycling, which was first proposed to explain the disposition of flavonoids (Liu and Hu, 2002), is analogous to enterohepatic recycling except the phase II conjugates are produced and excreted by the enterocytes instead of hepatocytes.

Materials and Methods

Materials. Cloned Caco-2 cells (TC7) were a kind gift from Dr. Moniqué Rousset (Institute National de la Santé et de la Recherche zU178, Villejuit, France). Raloxifene was extracted from Evista tablet (Eli Lilly & Co., Indianapolis, IN) using 100% ethanol, and concentration was then verified by using raloxifene hydrochloride purchased from National Cancer Institute Chemical Standard Repository managed by Midwest Research Institute (Kansas City, MO). β-Glucuronidase, sulfatase, Hank's balanced salt solution (HBSS; powder form), cyclosporin A, verapamil, estrone glucuronide, and estrone sulfate were purchased from Sigma-Aldrich (St. Louis, MO). Leukotriene C₄ and MK-571 were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). All other materials were analytical grade or better and used as received.

Cell Culture. Cell culture conditions for growing Caco-2 cells have been described previously (Hu et al., 1994a, 1994b; Liu and Hu, 2002; Chen et al., 2003). The seeding density was 100,000 cells/cm² (4.2 cm²/monolayer), and Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum was used as growth media. Quality control criteria were the same as described previously (Hu et al., 1994a, 1994b). Cell monolayers from 19 to 22 days past seeding were used for the experiments.

Transport Experiments in the Caco-2 Cell Model. The protocols for performing transport experiments were similar to those described previously (Liu and Hu, 2002). In brief, the cell monolayers were washed three times with 37°C HBSS (pH 7.4). The transepithelial electrical resistance values were measured, and those with the values less than 420 Ω/cm² were discarded. Various concentrations (ranging from 1.5–30 μM) of raloxifene were loaded at the apical or basolateral side of the Caco-2 cell monolayer, and the concentration of raloxifene and its metabolites at both sides were measured by HPLC. Inhibitors, when used, were loaded at the apical, basolateral, or both sides of the monolayer. Six samples (650 μl each) were taken at different times (0, 1, 2, 3, 4, and 7 or 24 h after incubation) from both donor and receiver side (total volume of each chamber was 2.5 ml), and the same volume of donor solution (containing raloxifene) or transport medium was replaced after each sampling. Forty-five microliters of internal standard solution (100 μM testosterone in methanol) was immediately added to 200 μl of samples to stabilize them until analysis.

Extraction of Raloxifene from Caco-2 Cell Monolayers. After the transport experiments, cells were washed three times with ice-cold saline solution, and the polycarbonate membranes containing cells were removed from the insert (4.2 cm²; Nalge Nunc International, Naperville, IL). One milliliter of HBSS (pH 7.4) was added to each membrane, and the solution was frozen (in liquid nitrogen) and thawed (in 37°C water bath) three times to disrupt cell membranes. After centrifugation at 13,000 rpm for 8 min, 0.5 ml of supernatant was collected. Methanol (0.5 ml) was added to the remaining suspension, and the tubes were centrifuged again to extract the remaining raloxifene.

Preparation of Caco-2 Cell Lysate. After six mature (19–22 days post-seeding) Caco-2 cell monolayers were washed twice with 3 ml of 37°C HBSS (pH 7.4), they were cut out together with the porous polycarbonate membranes, immersed into 6 ml of 50 mM potassium phosphate buffer (pH 7.4), and sonicated in an ice bath (4°C) for 30 min as described previously (Hu et al., 2003). Afterward, the cell lysate was centrifuged at 1000 rpm for 5 min to remove the polycarbonate membrane. The protein concentration of the cell lysate was then determined using a commercial protein assay kit (Bio-Rad, Hercules, CA).

Glucuronidation of Raloxifene in Caco-2 Cell Lysate. Glucuronidation of raloxifene by Caco-2 cell lysate was measured using procedures described previously (Hu et al., 2003). The cell lysate (final concentration ≈ 0.65 mg/ml) was mixed with magnesium chloride (0.88 mM), saccharolactone (4.4 mM), and alamethicin (0.022 mg/ml). Raloxifene or raloxifene plus estrone sulfate in 50 mM potassium phosphate buffer (pH 7.4) were then added. Uridine diphosphoglucuronic acid (3.5 mM) was added last to the reaction mixture (total volume 200 μl), and the mixture was incubated in a 37°C shaking (200 rpm) water bath for 4 h. The reaction was stopped by the addition of 50 μl solution of 94% acetonitrile/6% glacial acetic acid containing 100 μM testosterone as the internal standard.

Sulfation of Raloxifene in Caco-2 Cell Lysate. The cell lysate (final concentration of about 0.91 mg/ml) was mixed with raloxifene or raloxifene plus estrone sulfate in 50 mM potassium phosphate buffer (pH 7.4). The cofactor 3′-phosphoadenosine 5′-phosphosulfate (0.1 mM) was added last to the reaction mixture (total volume 200 μl), and the mixture was incubated in a 37°C shaking (200 rpm) water bath for 4 h. The reaction was stopped by the addition of 50 μl solution of 94% acetonitrile/6% glacial acetic acid containing 100 μM testosterone as the internal standard.

Hydrolysis of Raloxifene Metabolites by Hydrolases. A portion of 24-h samples of raloxifene (7 μM) transport and metabolism experiments was extracted with methylene chloride to remove intact raloxifene. The remaining aqueous phase was incubated with glucuronidase (20 units per reaction) or sulfatase (0.5 unit per reaction) at 37°C for 4 h to reconvert conjugated raloxifene to raloxifene, which is used to identify the raloxifene metabolite peaks and amounts of metabolites (via reconversion) in HPLC chromatograms (Fig. 1).

HPLC Analysis of Raloxifene and Its Conjugates. The HPLC conditions were as follows: system, Agilent 1090 controlled by Chemstation with a dioarray detector and an autosampler (Agilent Technologies, Palo Alto, CA); column, Aqua 5 μm, 150 × 4.60 mm (Phenomenex, Torrance, CA); mobile phase A, 0.04% (v/v) phosphoric acid plus 0.06% (v/v) triethylamine in water (pH 2.8); mobile phase B, 100% acetonitrile; gradient, 0 to 3 min, 80% A, 3 to 22 min, 80% to 51.5% A, 22 to 25 min, 51.5% A, 25 to 26 min, 51.5% to 80% A, 26 to 28 min 80% A; wavelength, 288 nm for raloxifene and 254 nm for the internal standard; and injection volume, 200 μl. The retention times for raloxifene, glucuronide, sulfate, and internal standard were 14.4, 9.7, 12.1, and 21.5 min, respectively (Fig. 1).

Data Analysis. Rates of transport (B_t) were obtained using rate of change in concentration of transported raloxifene or its metabolites as a function of time and volume of the sampling chamber (V) (eq. 1). Permeability (P) across a cellular membrane was calculated using the rate of transport divided by the surface area (A) of the monolayer and the initial concentration of raloxifene at the loading side (C_i) (eq. 2). Apparent metabolic clearance (CL) of raloxifene was calculated using rate of excretion (B_ex) divided by the initial concentration of raloxifene at the loading side (C_i) (eq. 3). This is based on the assumption that the rate of excretion is related to initial concentration of raloxifene. It is also based on practical consideration in that intracellular concentrations of metabolites (e.g., glucuronides) are sometimes too low to measure (see Results). Therefore, if the rate of excretion is always proportional to the initial concentration, the clearance will be a constant. Otherwise, the clearance will change as concentration changes.

Statistical Analysis. One-way ANOVA or Student's t test (Microsoft Excel) was used to analyze the data. The prior level of significance was set at 5%, or P < 0.05.

Results

Time Course of Transport and Metabolism of Raloxifene. We monitored amounts of raloxifene and its metabolites in the apical and basolateral media as a function of time for 4 h after apical or basolateral loading. The results indicated that amounts of raloxifene and its metabolites appeared in the receiver side increased linearly with time (Fig. 2, A and C). The amounts of metabolites in the donor side also increased linearly with time (Fig. 2, B and D), but the amount of raloxifene in the donor side did not change significantly (Fig. 2, B and D) since we replenished it after each sampling.

The rates of raloxifene transport were determined with respect to the direction of transport (Table 1). Secretory (basolateral to apical) rates of transport (B_BA) were always higher than absorptive (apical to basolateral) rates of transport (B_AB)(P < 0.05) (Table 1). As concentration of raloxifene increased, the ratio of B_BA to B_AB decreased from 3.5 to 1.5. Pretreatment of Caco-2 cells with 17 μM raloxifene to saturate the conjugating enzymes increased (P < 0.05) the secretory rate of transport, but the extent of increase was small (Table 1).

Effect of Raloxifene Concentration on the Transport of Raloxifene. Raloxifene has low absorptive permeability (P_AB = 0.39 × 10^-6∼4.1 × 10^-6, 1.5∼30 μM) when compared with genistein (P_AB = 31 × 10^-6, 35 μM) and apigenin (P_AB = 17 × 10^-6, 35 μM) (Table 2), two compounds with extensive metabolism in the intestine (Chen et al., 2003; Hu et al., 2003). Permeability of raloxifene increased linearly with rising raloxifene concentration (Fig. 3A). On the other hand, the rate of transport increased with the square of concentration (Fig. 3B).

Effect of Raloxifene Concentration on the Excretion of Its Metabolites. Significant amounts of raloxifene were metabolized into glucuronidated and sulfated metabolites during transport across the Caco-2 cell monolayers, with the sulfates being the major metabolites (Fig. 2; Table 2). The extent of metabolism decreased as the concentration of raloxifene increased (Table 2).

The extensive metabolism of raloxifene at lower concentration can be demonstrated by its high clearance value. The clearance values for apical efflux of glucuronide and sulfate (at a raloxifene concentration of 7 μM) were 10.04 ± 1.37 and 26.57 ± 1.12 μl/h, respectively. For basolateral efflux, they were roughly equal, or about 11 μl/h. Taken together, the total clearance values were 37.57 μl/h for sulfates and 21 μl/h for glucuronides, respectively. With a cellular water volume of about 3.66 μl/mg protein, the total monolayer cellular water volume is about 3.66 μl (Hu et al., 1994b). Therefore, the whole “cellular sulfate volume” is replaced about 10 times/h or once every 6 min, whereas the whole “cellular glucuronide volume” is replaced about 5 times/h or once every 12 min.

Clearance of raloxifene glucuronide into both apical and basolateral sides were similar and both decreased significantly (P < 0.05 according to one-way ANOVA) with an increase in raloxifene concentration (Fig. 4, A and B).

Clearance of raloxifene sulfate, on the other hand, was polarized and mainly (up to 3.5 times more) to the apical side (P < 0.05). Similar to clearance of glucuronides, both apical and basolateral clearance also decreased significantly (P < 0.05 according to one-way ANOVA) with an increase in raloxifene concentration (Fig. 4, A and B).

When comparing the clearance of these two conjugates from the same side of the cell monolayers, apical clearance of raloxifene sulfate was higher (2.7∼23 times) than that of glucuronide at raloxifene concentration between 1.5 and 17 μM(P < 0.05) (Fig. 4A). However, this difference in clearance decreased as raloxifene concentration increased and eventually became minimal at 30 μM. On the other hand, basolateral clearance of the two metabolites was similar at all the concentrations measured (Fig. 4B).

Effect of Multidrug Resistance-Related Protein (MRP) and P-Glycoprotein Inhibitors on the Transport of Raloxifene and Excretion of Its Metabolites. MRP inhibitor MK-571 (50 μM) and leukotriene C₄ (0.1 μM) (Rappa et al., 1999; Wheeler et al., 2000; Hu et al., 2003) or P-glycoprotein inhibitors cyclosporin A or verapamil were added to both sides of Caco-2 cell monolayer to determine whether MRP or P-glycoprotein is involved in the transport of raloxifene and the excretion of its metabolites. Previously, it was shown that leukotriene C₄ was effective in decreasing the efflux of apigenin sulfate and glucuronide (Hu et al., 2003), and MK 571 was effective in decreasing the efflux of both apigenin and chrysin conjugates from the Caco-2 cells (Walle et al., 1999; Hu et al., 2003). MRP inhibitors significantly decreased P_BA of raloxifene (7 μM) (P < 0.05) (Fig. 5A) but had no effect on P_AB of raloxifene (Fig. 5A). In contrast, P-glycoprotein inhibitors significantly increased P_AB of raloxifene (7 μM) (P < 0.05) (Fig. 5A) but had no effect on P_BA of raloxifene (Fig. 5A).

In addition, MRP inhibitors also significantly decreased the clearance of glucuronide from both basolateral and apical sides (P < 0.05) (Fig. 5B) and the clearance of sulfate from the apical side (Fig. 5C). However, they did not change the basolateral clearance of sulfate (Fig. 5C). P-glycoprotein inhibitors had no significant effect on the clearance of glucuronides and sulfates from both sides of Caco-2 cells (Fig. 5, B and C) except for cyclosporin A, which increased the basolateral clearance of sulfate when raloxifene was added to the apical side (Fig. 5C).

Effect of Organic Anion Transporter (OAT) Inhibitors on the Transport of Raloxifene and Excretion of Its Metabolites. Estrone sulfate (an organic anionic transporter or OAT substrate and inhibitor) (Sekine et al., 2000) was shown to be effective in decreasing the efflux of apigenin sulfate from the Caco-2 cells (Hu et al., 2003). Therefore, OAT inhibitors estrone glucuronide (50 μM) or estrone sulfate (50 μM) were used to study the possible involvement of OAT in the transport of raloxifene (7 μM) and in the excretion of raloxifene metabolites. Both of them increased the rate of transport for raloxifene (70% and 90% for estrone glucuronide and estrone sulfate, respectively, P < 0.05) when they were added to both the apical and the basolateral media. There was no significant difference between the effects of these two OAT inhibitors (Fig. 6A).

For raloxifene glucuronide, estrone sulfate significantly decreased (40%) the basolateral clearance without affecting its apical clearance, whereas estrone glucuronide significantly increased (75%) the apical clearance without affecting its basolateral clearance (Fig. 6B). For raloxifene sulfate, these two inhibitors significantly decreased its clearance from both sides of the Caco-2 cell monolayer (25%∼50%) (Fig. 6C).

Effect of Estrone Sulfate on the Transport and Metabolism of Raloxifene as a Function of Concentration. Increasing concentration of estrone sulfate (1∼50 μM) significantly increased (135%∼190%) the rate of transport of raloxifene (7 μM) through Caco-2 cells (P < 0.05) (Fig. 7A) with an apparent EC₅₀ value of 16.4 μM. On the other hand, increasing concentration of estrone sulfate significantly decreased the basolateral clearance of raloxifene glucuronide with a maximum decrease of ≈40% but did not significantly affect the apical clearance of glucuronide (Fig. 7B). Estrone sulfate inhibited both apical and basolateral clearance of raloxifene sulfate with a maximum decrease of ≈50% (Fig. 7C).

Effect of Estrone Sulfate on the Metabolism of Raloxifene by Caco-2 Cell Lysate. Twenty micromolar estrone sulfate decreased in vitro formation of raloxifene glucuronide and sulfate (using Caco-2 cell lysate) by about 30% (P < 0.05) (Fig. 8). Excretion rates (B_ex) of both metabolites from the Caco-2 cells monolayer were higher (3.4 and 1.3 times for glucuronide and sulfate, respectively) than the rates of metabolism in the cell lysate (B_met) (Fig. 8). Estrone sulfate decreased the ratio of excretion rate over metabolism rate (B_ex/B_met) for raloxifene sulfate by 40% (P < 0.05) (Fig. 8B) but did not affect this ratio for raloxifene glucuronide (Fig. 8A).

Cellular Accumulation of Raloxifene and Its Metabolites in Caco-2 Cells. Raloxifene was extensively bound to Caco-2 cells (up to 73% of dose after 24 h of incubation with 6 μM raloxifene). Among the conjugated metabolites, only sulfate was detected in the cell, whereas glucuronide was not detected. The amount of raloxifene taken up by or bound to cells was significantly decreased after 24 h of incubation compared with those after 4 h of incubation, whereas the amount of sulfate found inside the cells was significantly increased after 24 h of incubation (data not shown). The amount of raloxifene taken up by the Caco-2 cell increased significantly as concentrations increased (up to 7 μM) but did not increase further after that concentration (Fig. 9A). The uptake trend of raloxifene sulfate was similar to raloxifene, but the extent of uptake was 3.5 times smaller (Fig. 9B).

Discussion

Raloxifene has the potential to become the drug for breast cancer prevention. However, raloxifene has poor bioavailability (Lindstrom et al., 1984). We hypothesized that extensive intestinal metabolism and enteric and enterohepatic recycling of raloxifene conjugates could explain why this drug has poor oral bioavailability but a long half-life (Fig. 10). We further hypothesized that bioavailability of raloxifene could be enhanced via inhibition of phase II metabolism and/or inhibition of efflux transporters responsible for pumping out the intracellular phase II metabolites. In this study, we investigated the mechanisms responsible for the absorption and excretion of raloxifene and its metabolites in the Caco-2 cell culture model.

Our results suggest that raloxifene is subjected to efflux because: 1) as concentration increased, the permeability of raloxifene across the Caco-2 cell monolayer increased and the rate of transport increased parabolically (Fig. 3), consistent with saturation of efflux transporters; 2) secretory transport of raloxifene was faster than absorptive transport (Table 1); and 3) the ratio of secretory to absorptive transport rate (B_BA/B_AB) was decreased as the concentration of raloxifene increased (Table 1). The efflux appeared to be mediated by MRP and P-glycoprotein since secretory transport was diminished when MRP inhibitors were added to the media and absorptive transport was augmented when P-glycoprotein inhibitors were added to the media (Fig. 5). The decrease by MRP inhibitors was achieved even though blockage of conjugate efflux has the tendency to increase the amounts of intact compound transported. Therefore, our data showed for the first time that one of the reasons why intestinal absorption of raloxifene is incomplete was due to its efflux by MRP and P-glycoprotein. This represents an important discovery about the transport of raloxifene in the enterocytes. Previously, absorption mechanisms of raloxifene were unknown. Usually, the passive diffusion process was implied as the mechanism of absorption since there is no demonstrated nonlinear relationship between dose and absorption.

Our results showed that raloxifene undergoes extensive intestinal metabolism during transport across the Caco-2 cells (Fig. 1; Table 2). At low concentration of 7 μM raloxifene, the total clearance value for both conjugates added together was about 59 μl/h, which is enough to clear the cellular metabolite volume 16 times/h. The extensive metabolism is consistent with reported extensive first pass metabolism of raloxifene following oral administration (Hochner-Celnikier, 1999). The major metabolites in the Caco-2 cells are mainly sulfates followed by glucuronides. In addition, sulfates are rapidly and preferentially excreted into the apical side, which diminishes their transport into the systemic circulation. Furthermore, percentages of glucuronides increased as the concentration of raloxifene increased. Therefore, our results are consistent with the earlier observation that the main metabolite in human plasma is the glucuronides including the primary metabolite raloxifene-4′-glucuronide (Hochner-Celnikier, 1999). They are also consistent with a recent discovery that intestinal glucuronidation is the most important contributor to the presystemic clearance of raloxifene (Kemp et al., 2002).

We then proceeded to determine the mechanisms responsible for the efflux of raloxifene conjugates since metabolic clearance decreased as concentration increased (Fig. 4), suggesting the involvement of a saturable process. The results indicated that excretion of raloxifene glucuronide and sulfate was mediated by efflux transporters such as MRP and/or OAT because: 1) clearance of metabolites was polarized and saturable (Fig. 4), and 2) clearances into apical and basolateral media were changed in the presence of MK-571 and leukotriene C₄, estrone glucuronide, or estrone sulfate (Figs. 5 and 6). Taken together, these results have elucidated for the first time the mechanisms responsible for efflux of a glucuronide and a sulfate metabolite of a drug from the intestinal cells. Previously, we have reported the efflux mechanism responsible for the efflux of apigenin sulfate and apigenin glucuronide (Hu et al., 2003), whereas Walle and his coworker reported efflux of chrysin conjugates were inhibited by MK-571, an inhibitor of both glucuronidation and MRP (Walle et al., 1999; Hu et al., 2003).

We further analyzed the results of various chemical inhibitors on the excretion to determine which transporter(s) may be mainly responsible for the excretion of raloxifene sulfate and glucuronide. The main pathway for glucuronide (Fig. 10) appears to be MRP since use of two MRP inhibitors inhibited the excretion by more than 80% (Fig. 5), with minor contribution from basolateral OAT (Fig. 6). The main pathway for sulfate is less well defined since its excretion was never inhibited by more than 55%. Contributions from both apical MRP and OAT and basolateral OAT are likely, and additional contributions from other transporter(s) are possible (Fig. 10). In comparison with the efflux of apigenin conjugates (Hu et al., 2003), the efflux of raloxifene glucuronide is much better defined than apigenin conjugate (about 60% attributable to MRP and OAT), but the efflux of sulfate is less well defined than apigenin sulfate (>80% attributable to MRP and OAT).

Further analysis of the inhibitor results suggests that MDR1 or P-glycoprotein is not involved in limiting the efflux of conjugates since neither cyclosporine A nor verapamil decreased the efflux of any conjugates. However, it is not presently clear why cyclosporine A increased the basolateral efflux of sulfate. To probe the possible mechanisms, we determined if cyclosporine A would increase sulfate formation in Caco-2 cell lysate. The result indicated a slight decrease in the formation of raloxifene sulfate in the presence of 20 μM cyclosporine A using Caco-2 cell lysate (27% decrease), which suggests that cyclosporine A did not enhance the metabolite formation. The actual mechanism for sulfate efflux enhancement remains to be determined.

Our discovery that raloxifene and its metabolites are excreted by the enterocyte efflux transporters is novel. The excretion of raloxifene metabolites is the result of direct coupling of efflux transporter and phase II metabolic enzymes, which has not been demonstrated for the disposition of a drug in the intestine previously. The direct coupling is demonstrated by the fact that inhibition of conjugate efflux could result in increase in raloxifene transport (Figs. 6 and 7). The direct coupling is clearly the enabling process for enteric recycling and may also be used to explain why raloxifene has a low oral bioavailability but a long half-life.

The coupling of conjugating enzymes that produce hydrophilic metabolites and efflux transporters is more complex than the coupling of CYP3A4 and P-glycoprotein (Cummins et al., 2002) because these conjugates cannot exit cells via passive diffusion. Hence, the action of the efflux transporters can change cellular equilibrium between parent compound and its conjugates. This change in equilibrium has at least two possible consequences, both of which could lead to a higher than expected excretion of raloxifene conjugates (Fig. 8). The first consequence is less accumulation of conjugates in the cells because of rapid excretion of conjugates that can inhibit phase II conjugation via product inhibition. In other words, when the efflux dominates the cellular excretion, very little conjugate is accumulated by the cells. In the present study, the excretion rate of raloxifene glucuronide was much faster (3.4 times) than its formation rate, resulting in no measurable cellular accumulation of this metabolite even after 24 h. On the other hand, sulfate excretion rate was comparable with sulfate formation rates, resulting in significant accumulation of sulfate in cells after 24 h (Fig. 9). The second consequence is less hydrolysis of the conjugates to parent drug via the action of glucuronidases and sulfatases as proposed (Hochner-Celnikier, 1999), resulting in higher than expected excretion of conjugates (Fig. 8). Taken together, these two consequences will result in higher apparent excretion rate than the formation rate when efflux inhibitor was absent (Fig. 8). When the efflux transporter function was inhibited by estrone sulfate, the excretion of sulfates became slower than metabolism of sulfate as expected. Taken together, these results support the coupling hypothesis that predicts the modulation of cellular excretion of hydrophilic conjugates by the efflux transporters.

The coupling of efflux transporters and conjugating enzymes also support our hypothesis that enteric recycling (Liu and Hu, 2002) is important for the in vivo metabolism of raloxifene. This coupling can explain why substantial amounts of raloxifene conjugates are produced and excreted by the enterocytes. Because this coupling appears to function better at the apical side, at least in the case of raloxifene, it increases the importance of enteric recycling in the disposition of raloxifene. Because the apical side was the preferred side of excretion for the raloxifene sulfate, it may be used to explain why smaller amounts of sulfated metabolites are recovered in plasma since excreted metabolites are expected to be eliminated in feces or hydrolyzed by the microflora.

Lastly, we also hypothesized that we could increase the bioavailability of raloxifene by inhibiting the efflux transporters responsible for the excretion of raloxifene conjugates. OAT inhibitors we used are not known to interact with MRP, even though they could slightly decrease the formation rates of conjugates. Therefore, increase in the intestinal transport of intact raloxifene (Fig. 6) can be viewed mainly as the result of inhibiting the efflux of conjugated metabolites, which in turn decreased the amounts of metabolites formed and allowed more intact raloxifene to permeate the Caco-2 cell monolayers intact. This is an important discovery since we have shown that inhibition of an efflux transporter(s) that are tightly coupled to conjugating enzymes can decrease the extent of metabolism and improve the transport of the intact compound. If such an effect can be shown in vivo, it would provide a new mechanism to improve the oral bioavailability of drugs and to explain drug interactions in the clinic.

In conclusion, raloxifene and its phase II conjugates are excreted by the intestinal cells via the actions of P-glycoprotein, MRP, and/or OAT. Coupling of efflux transporters and conjugating enzymes enhances intestinal excretion and renders the enteric recycling. On the other hand, the inhibition of OAT responsible for conjugate efflux can increase transport of intact raloxifene and has the potential to increase its bioavailability pending the actual demonstration in vivo. Taken together with enterohepatic recycling, intestinal disposition may be used to explain why this drug has low bioavailability but a long half-life.