Disposition Mechanisms of Raloxifene in the Human Intestinal Caco-2 Model

Abstract

The purpose of this study was to determine the mechanisms responsible for transport of raloxifene and its hydrophilic conjugates. Human intestinal Caco-2 cell culture model and Caco-2 cell lysate were used for the studies. The results indicated that absorptive permeability (P AB ) of raloxifene was lower than its secretory permeability (P BA ). As the concentration increased, the efflux ratio (P BA /P AB ) decreased, but P AB increased. P AB was also increased in the presence of verapamil and cyclosporine A, two P-glycoprotein inhibitors. Raloxifene was extensively metabolized into sulfated and glucuronidated conjugates. The extent of metabolism or clearance was decreased as the concentration increased from 3.4 (96%) to 30 (22%) M. Multidrug resistance-related protein inhibitors MK-571 (C 26 H 26 ClN 2 O 3 S 2 ) and leukotriene C 4 significantly decreased (maximal 80%) apical efflux of both conjugates. They also significantly decreased (maximal 85%) basolateral efflux of glucuronides but not sulfates. On the other hand, organic anion transporter (OAT) inhibitor estrone sulfate and estrone glucuronide significantly decreased (maximal 50%) the efflux of sulfate from both sides but had variable effects on glucuronide efflux. Inhibition of conjugate efflux with the OAT inhibitor estrone sulfate was concentration dependent, which resulted in increased transport of intact raloxifene (maximal 90%). This increase in raloxifene transport was also observed in the presence of another OAT inhibitor estrone glucuronide (70%). In conclusion, this is the first report that inhibition of an efflux transporter responsible for the transport of metabolites can result in increase in the transport of the intact compound. It also provides additional explanation why raloxifene has low bioavailability but a long half-life.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). Acc...

Influence of hepatic and intestinal efflux transporters and their genetic variants on the pharmacokinetics and pharmacodynamics of raloxifene in osteoporosis treatment

“…The measured raloxifene Caco2 permeability was higher than reported permeability of estradiol 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 19 and lower than reported permeability of tamoxifen (7.9 ± 1.1 vs 1. (Table 2) showed that raloxifene is actively excreted from the Caco2 cells, while a significant drop of ER to 6.2 observed in the presence of verapamil (a Pgp inhibitor) suggests that raloxifene excretion from Caco2 cells is largely mediated by Pgp, which is in accordance with findings of Jeong et al [12] and Chang et al [11]. On the other hand, no significant changes in efflux Furthermore, M2 and M3 could be MRP2 substrates or inhibitors because of the observed inhibition and activation of MRP2 ATP-ase and significant inhibition of E17βG vesicular uptake.…”

“…Previous studies have 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 shown that raloxifene is probably transported by Pgp [11,12] while the corresponding glucuronides utilize the MRPs and organic anion transporter(s) (OAT) for their transport [12].…”

“…According to the estimations, the fraction of raloxifene absorbed is high, 60 %, but only 2% reaches the systemic circulation in unconjugated form due to the high metabolic turnover during absorption [7]. The rest represents raloxifene conjugated by UDP-glucuronosyltransferases (UGTs) to raloxifene-4'-β-glucuronide (M2), raloxifene-6-β-glucuronide (M1) [7,8] and raloxifene-6,4'-diglucuronide 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 shown that raloxifene is probably transported by Pgp [11,12] while the corresponding glucuronides utilize the MRPs and organic anion transporter(s) (OAT) for their transport [12].…”

Interactions of the human cytosolic sulfotransferases and steroid sulfatase in the metabolism of tibolone and raloxifene

“…As reported previously, SULT1E1 and SULT 2A1 are the major isoforms involved in the sulfation of Tib and its metabolites (10). Raloxifene is sulfated in vitro by at least seven expressed human SULT isoforms including SULTs 1E1 and 2A1 (12,17). SULT1E1 is responsible for the high affinity sulfation of estrogens (14,18,19) while SULT2A1 sulfates hydroxysteroids, bile acids and several therapeutic drugs (10,15,20,21).…”

Breast Cancer Resistance Protein (BCRP) and Sulfotransferases Contribute Significantly to the Disposition of Genistein in Mouse Intestine

“…Sulfation of genistein by S9 fractions was measured using procedures described previously with minor modifications (25). Briefly, S9 fractions (final protein concentration at ∼1 mg/ml) were mixed with genistein in 50 mM potassium phosphate buffer (pH 7.4), and 0.1 mM PAPS solution was added last to the reaction mixture (total volume 200 μl).…”