Interaction of hydrophobic components in female urine before and after childbirth with P-glycoprotein in vitro.

The first urine in the morning (total 15 samples) and whole day urine (total 4 days, 17 samples) were collected from a young healthy woman during the pregnancy and lactation period, to examine the possible interactions of urine components (methanol extracts) with P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs). The interaction was evaluated by measuring the intracellular accumulation of rhodamine123, a P-gp substrate, in LLC-GA5-COL150 cells, or calcein, an MRP substrate, in Caco-2 cells in the absence and presence of urine components. Four first urine samples out of 12 collected before childbirth and one sample out of three collected after childbirth suppressed P-gp function significantly. The effect of pregnancy and lactation on P-gp inhibitory potencies of urine components was not observed. The whole day urine samples showed a clear circadian rhythm, in which three first urine samples in the morning out of four showed greater P-gp inhibitory potencies than other daytime samples. Interaction of urine components with MRPs was not detected. In conclusion, the concentration of endogenous P-gp inhibitor(s) was higher in the first urine in the morning, showing a clear circadian rhythm. Normal pregnancy and lactation appeared not to significantly affect the P-gp inhibitory potencies of urine components.

Characterization of intestinal absorption of quinidine, a P-glycoprotein substrate, given as a powder in rats.

The characteristics of intestinal absorption of quinidine, a P-glycoprotein (P-gp) substrate in biopharmaceutics classification system (BCS) Class I, after oral administration as a powder in No. 9 HPMC capsule (diameter 2.6 mm; length 8.4 mm, volume 25 microl) was examined in rats from the following viewpoints: (i) main absorption site of quinidine, (ii) effect of dosage amounts (or luminal concentrations) of quinidine (10 mg vs 0.1 mg/kg), (iii) contribution of P-gp in quinidine absorption (0.1 mg/kg), and (iv) effect of gastric pH on quinidine absorption. Quinidine administered orally at a dose of 10 mg/kg was discharged from the stomach steadily with time and disappeared rapidly from the proximal intestine, where P-gp expression was low. In contrast, quinidine administered at a dose of 0.1 mg/kg remained longer in the gastrointestinal lumen than that administered at a dose of 10 mg/kg. The pretreatment with cyclosporine A, a P-gp inhibitor, greatly increased the intestinal absorption of quinidine given at a dose of 0.1 mg/kg. The gastric pH in untreated control rats was pH 3.6, and the treatment with ranitidine (10mg/kg, ip), a H2 blocker, increased to pH 6.4. The recovered amounts of quinidine 30 min after administration were 21.1% of dose in control rats and 94.7% in ranitidine-treated rats. The value of plasma AUC(0-6h) of quinidine in ranitidine-treated rats was about 40% that in untreated control rats. In conclusion, quinidine was rapidly and efficiently absorbed at the proximal intestine under ordinary circumstances. However, the oral bioavailability was modulated by various factors including the dose (or luminal concentration at the absorption site), presence of P-gp inhibitors, and gastrointestinal pH.

Modulation in concentrative nucleoside transporters-mediated intestinal absorption of mizoribine, an immunosuppressive agent, in lipopolysaccharide-treated rats.

The characteristics of intestinal absorption of mizoribine and cephalexin, that are mediated by concentrative nucleoside transporters (CNTs) and PEPT1, respectively, was examined in lipopolysaccharide (LPS)-treated rats. LPS treatment is known to modify the expression of some transporters and induce cholestasis. At 24 h after the LPS treatment, averaged concentrations of IL-6 and total bile acids in plasma were 15-fold and 2-fold that in untreated control rats, respectively, and bile flow rate decreased by 40% of control, indicating the induction of inflammatory and cholestatic states. The oral bioavailability, estimated by urinary excretion percentage of unchanged form, of mizoribine in LPS-treated rats was 1.5-fold higher than that in control rats, whereas the bioavailability of cephalexin remained unchanged. When mizoribine and cephalexin were administered into in-situ jejunum loops, there were no differences in the absorption rates between control and LPS-treated rats. These results indicated that the functional expression of CNT1, CNT2, and PEPT1 were not modulated by LPS treatment. When mizoribine (a CNT1/CNT2 substrate) and gemcitabin (a CNT1 substrate) were administered as a solution dissolved in bile into the intestinal loop, their absorption rates decreased significantly. In contrast, the absorption rate of ribavirin (a CNT2 substrate) remained unchanged. In conclusion, LPS treatment exerted no significant effect on the expression of CNT1 and CNT2 in the intestine. Bile was found to suppress the CNT1-mediated intestinal absorption of mizoribine and gemcitabin. The increased oral bioavailability of mizoribine in LPS-treated rats could be ascribed to the less amount of bile or bile acids in the intestine under cholestatic state of rats.

Study on absorption sites of quinidine and methotrexate in rat intestine.

Influx or efflux transporter(s), or both, are frequently involved in the intestinal absorption of various therapeutic drugs. In the present study, the effects of altered gastric emptying rates (GER) on intestinal absorption of quinidine (a substrate for P-glycoprotein, P-gp) and methotrexate (a substrate for multiple-transporters including proton-coupled folate transporter, PCFT) were examined to find their main absorption sites along the intestine employing rats. In untreated control rats, quinidine administered orally was rapidly absorbed from the proximal intestine, where P-gp is less expressed. Increased GER, which transferred an unabsorbable model compound to the middle intestine within 15 min after oral administration, exerted no significant effects on the extent of oral bioavailability of quinidine, whereas it increased the initial absorption rate greatly. Decreased GER, in which more than 50% of a model compound administered was retained in the stomach even 1 h after administration, decreased the onset time of intestinal absorption, but not the extent of oral bioavailability of quinidine. In untreated control rats, methotrexate was absorbed efficiently from the proximal intestine under acidic conditions, where PCFT is abundantly expressed. Increased GER significantly decreased, and decreased GER slightly increased the oral bioavailability of methotrexate. In conclusion, altered GER was found to affect the transporter-mediated intestinal absorption of drugs in different manners, depending on the solubility, membrane permeability, luminal concentration of the drug, luminal pH, substrate specificity, and the expression sites of transporter.

Increased intestinal absorption of mizoribine, an immunosuppressive agent, in cholestatic rats.

The intestinal absorption of mizoribine, an imidazole nucleoside, is mediated by concentrative nucleoside transporter (CNT)1 and CNT2 in rat. Previously, bile and bile salts such as sodium glycocholate were found to suppress the intestinal absorption of mizoribine. In the present study, the contribution of bile on the intestinal absorption of mizoribine was further evaluated in rats. Cholestatic states were induced by an intraperitoneal injection (2 ml/kg) of 50% carbon tetrachloride (CCl4) dissolved in olive oil, or oral administration (100 mg/2 ml/kg) of alpha-naphthylisothiocyanate (ANIT) dissolved in olive oil. The animals were subjected to absorption studies 24 h after treatment. Cholestatic states were confirmed by measuring plasma concentrations of bile acids and bile flow rates. When oral bioavailability of mizoribine was estimated by the recovery amount in the urine, rats under cholestatic states exhibited significantly higher oral bioavailabilities than untreated control rats. In contrast, the intestinal absorption percentages of mizoribine from in-situ lavaged intestinal loops were the same magnitudes among untreated control, CCl4- and ANIT-treated rats. These results indicated that the increased oral bioavailability of mizoribine in cholestatic rats was not ascribed to the modulation of nucleoside transporter's expression. In conclusion, various diseased states accompanied with cholestasis may increase the oral bioavailability of mizoribine, possibly due to its less amounts of bile in the intestinal lumen.

Interaction of Rhei Rhizoma extract with cytochrome P450 3A and efflux transporters in rats.

Traditional Chinese herbal medicines are frequently prescribed in pharmacotherapy in Japan. In the present study, we evaluated the possible interaction of several herbal extracts including Rhei Rhizoma extract with cytochrome P450 (CYP) 3A and efflux transporters such as P-glycoprotein and multidrug resistance-associated protein (MRP) 2. Rhei Rhizoma extract (100 microg/ml) significantly suppressed the CYP3A-mediated 6beta-hydroxylation of testosterone in hepatic microsomes, and increased the extent of bioavailability of midazolam, a typical CYP3A substrate, in rats. Also, Rhei Rhizoma extract (300 microg/ml) significantly suppressed P-glycoprotein-mediated efflux transport of rhodamine 123 (Rho123) in rat everted intestine. In an in-vivo study, Rhei Rhizoma extract added to intestinal perfusate at a concentration of 300 microg/ml significantly suppressed the intestinal exsorption of Rho123, though it exerted no effect on the biliary excretion of Rho123. Furthermore, the in-vitro and in-vivo MRP2-mediated intestinal efflux of 2,4-dinitrophenyl-S-glutathione was significantly suppressed by Rhei Rhizoma extract (1000 microg/ml). In conclusion, Rhei Rhizoma extract, which is taken orally at doses of 0.5-1 g each or 1-3 g daily in clinical practice, may cause pharmacokinetic herb-drug interactions in the process of the intestinal and/or hepatic CYP3A-mediated drug metabolism and P-glycoprotein- and/or MRP2-mediated efflux transport in the intestine.

Fate of quinidine, a P-glycoprotein substrate, in the gastrointestinal tract after oral administration in rats.

P-Glycoprotein (P-gp), an ATP-dependent efflux transporter, is expressed in brush-border membranes in the intestines of humans and rodents. In this study, the fate of quinidine, a P-gp substrate, in the gastrointestinal tract after oral administration was examined in conscious rats. The animals received quinidine (3 mg/ml/kg) or FITC-dextran of molecular weight of 10,000 (FD-10S, 5 mg/ml/kg, a poorly absorbable compound) orally, and the remaining amount of the compound in the upper gastrointestinal tract was measured at designated time intervals. As a control, FD-10S was distributed almost evenly throughout the gastrointestinal tract at 30 min, and most of FD-10S was accumulated in the distal small intestine at 60 min after administration. In contrast, most of the orally administered quinidine disappeared at the proximal intestine, and only small amounts reached the distal region. Also, the gastrointestinal transit of FD-10S was markedly slowed by stopping or inhibiting the bile flow, indicating that bile flow significantly affects the transit of drugs in the gastrointestinal tract. In conclusion, this study has demonstrated that compounds with high solubility and high permeability, such as quinidine, can be absorbed rapidly at the proximal intestine,escaping the barrier function of P-gp, because P-gp is mostly expressed in the distal intestine.