Masako Oda

Secretory Transport of Methylprednisolone Possibly Mediated by P-Glycoprotein in Caco-2 Cells

We recently reported that P-glycoprotein (MDR1) is capable of interfering with the absorption of methylprednisolone in the rat small intestine. This study was undertaken to examine the interaction between methylprednisolone and MDR1 using Caco-2 cells. The permeation of various steroid hormones (hydrocortisone, prednisolone, progesterone, beta-estradiol, and testosterone) was compared. The basolateral-to-apical (secretory) permeation of methylprednisolone was more than 3-fold greater than the apical-to-basolateral (absorptive) permeation. When verapamil (0.1 mm), a potent modulator of MDR1, was added to both apical and basolateral sides of Caco-2 cells, the absorptive permeation of methylprednisolone was increased and its secretory permeation was decreased. As a result, the secretory-oriented manner of methylprednisolone permeation almost completely disappeared. Prednisolone and hydrocortisone exhibited weaker secretory-oriented movement than did methylprednisolone. The secretory-oriented permeation of prednisolone and hydrocortisone was also diminished by the addition of verapamil. There was no significant directionality in progesterone permeation and the permeation of beta-estradiol and testosterone tended to be absorptive. These results appear to suggest that methylprednisolone, prednisolone, and hydrocortisone interact with MDR1 as the substrates. In contrast, there was no evidence that MDR1 was capable of potently interfering with the absorption of the sex hormones tested in this study, supporting our previous findings in the rat. It was further found that apically-added verapamil demonstrated a modulating effect on MDR1 function even at 5 microM.

Region-dependent disappearance of vinblastine in rat small intestine and characterization of its P-glycoprotein-mediated efflux system

This study was aimed to characterize the absorption behavior of vinblastine (VLB), a well-known substrate of P-glycoprotein (P-gp), from rat small intestine, especially focusing on the regional-dependence of its efflux mediated by P-gp. VLB disappeared from duodenal and ileal loops of male Wistar rats fairly rapidly (30-60% in 30 min). In contrast, its disappearance from the jejunal loop was almost negligible and in some rats >100% of the jejunal dose was recovered. The radioactivity derived from [3H]VLB, which was absorbed from duodenum and ileum, was detected in the jejunal region. The jejunal appearance of radioactivity was increased when unlabeled VLB was present in the region in advance. The basolateral-to-apical transport of [3H]VLB across Caco-2 cell monolayers was greater when unlabeled VLB was added to the apical medium than when VLB-free buffer was applied to the apical side. When verapamil or cyclosporin A, potent modulators of P-gp, was added to the apical medium together with unlabeled VLB, enhanced basolateral-to-apical transport of [3H]VLB was disappeared. It is suggested that VLB absorption is strongly restricted by P-gp, especially in the jejunal region of the rat small intestine, and that the secretory transport via intestinal P-gp may be subject to trans-stimulation. Moreover, intravenously administered methylprednisolone and intramuscularly administered progesterone significantly enhanced the absorption of VLB, suggesting that parenterally administered P-gp modulators could influence the intestinal absorption of P-gp substrates.

External control of drug release and penetration: enhancement of the transdermal absorption of indomethacin by ultrasound irradiation

Abstract— The effect of ultrasound (1 MHz) on transdermal absorption of indomethacin from an ointment has been studied in rats. Ultrasound energy was supplied for 10 min at a range of intensities (0.25, 0.5 and 0.75 W cm−2), energy levels commonly used for therapeutic purposes. The pronounced effect of ultrasound on transdermal absorption of indomethacin was observed for all three intensities studied. The mean AUC value (33.22 μg h mL−1) after irradiation at 0.75 W cm−2 was 3.4 times the control value (9.70 μg h mL−1).

Relative contribution of absorptive and secretory transporters to the intestinal absorption of fexofenadine in rats

It has been shown that fexofenadine, a selective non-sedating histamine H(1)-receptor antagonist, is a substrate for P-glycoprotein (P-gp) and an organic anion transporting peptide (OATP). This study was undertaken to investigate the relative contribution of these absorptive and secretory transporters to the intestinal absorption of fexofenadine in rats. When 0.1mM fexofenadine was introduced into duodenal, jejunal, and ileal loops, its disappearance was around 10% over 30 min. Cyclosporine A, but not ketoconazole, probenecid or mitoxantron, significantly increased fexofenadine disappearance in the ileal loops. The serosal-to-mucosal permeation of fexofenadine across the rat ileal segments was approximately 18-fold greater than its mucosal-to-serosal permeation. The secretory orientation of the ileal permeation of fexofenadine was weakened significantly in the presence of cyclosporine A, moderately in the presence of ketoconazole, but was unchanged in the presence of probenecid. When fexofenadine (0.1 or 0.5mM) was administered to rats intraluminally, plasma concentrations increased linearly up to 120 min. The magnitude of the increase in plasma fexofenadine concentrations in the presence of cyclosporine A was more remarkable at 0.5mM than at 0.1mM. The results obtained in this study suggest that the intestinal absorption of fexofenadine is relatively small in rats even if OATP functions as an absorptive transporter for fexofenadine. Low absorption of fexofenadine in rats is attributed to potent secretory transport mediated by P-gp.

Plasma concentrations of anionic uremic toxins in hemodialysis patients and their effects on protein binding of pravastatin

Uremic toxins (UTs) accumulate in the body of hemodialysis patients. UTs often exert unfavorable effects on patients and cause significant interactions with clinically relevant drugs. In this study, we assayed plasma concentrations of three typical anionic UTs, indoxyl sulfate (IS), 3-indoleacetic acid (IA), and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF), in 20 hemodialysis patients and 5 healthy volunteers. Moreover, the effects of these anionic UTs on the binding of pravastatin to human serum albumin (HSA) were also evaluated. CMPF concentrations in the plasma of patients were unchanged before and after dialysis (63.0 ± 6.3 μM and 65.1 ± 6.7 μM, respectively), and these values were about 5-fold greater compared with those in healthy volunteers. Although dialysis decreased the plasma IS concentration from 157.9 ± 19.9 μM to 103.8 ± 13.3 μM, the value after hemodialysis was still ca. 27-fold greater than that in healthy volunteers. IA concentrations before and after hemodialysis were almost identical to those in healthy volunteers. There were no significant differences in the plasma concentrations of the three anionic UTs between male and female patients. The magnitude of protein binding was in the order CMPF>IS>IA, indicating that hemodialysis clearance of these anionic UTs was dependent on their protein binding capacities. The ability of IS to reduce pravastatin binding to HSA was much greater than that of CMPF. The present results suggest that statins bind to site II on HSA, and that their binding is modulated by IS when elevated in hemodialysis patients.

Arbekacin is actively secreted in the rat intestine via a different efflux system from P-glycoprotein.

This study was undertaken to examine the secretory transport of arbekacin, an aminoglycoside antibiotic, in the rat small intestine and to compare it with those in Caco-2 and LLC-PK1 cells. In vitro permeation of arbekacin was examined using an Ussing chamber technique. Serosal-to-mucosal (secretory)/mucosal-to-serosal (absorptive) permeation ratios of 0.5 mM arbekacin were 2.8 in the jejunum and 7.0 in the ileum, respectively, indicating that arbekacin permeation was highly secretory-oriented. In the ileum, the ratios became smaller with increase in arbekacin concentration applied. When D-glucose was replaced with 3-o-methyl-D-glucose in the experimental medium, the directionality of the arbekacin permeation disappeared almost completely. Absorptive permeation of arbekacin was not significantly influenced by verapamil, cyclosporin A, or probenecid. On the other hand, when gentamicin sulfate was added to the serosal medium, secretory transport of arbekacin was significantly inhibited. The results of this study strongly suggest that a specialized efflux system other than P-glycoprotein and multidrug resistance proteins was involved in the secretory transport of arbekacin in the rat intestine. There was no directionality in arbekacin permeation across Caco-2 cell monolayers, suggesting the absence or very slight expression of the secretory system for arbekacin in this cell line.

Limited Effect of Intravenously Administered Indoxyl Sulfate, a Uremic Toxin, on the Hepatic Transport of Pravastatin in Normal Rats

Indoxyl sulfate (IS) is a typical uremic toxin that extensively accumulates in the plasma of patients with seriously impaired renal function. This study seeks to clarify whether IS exerts a potent modulating effect on the hepatic transport of pravastatin, which is a substrate of both organic anion transporting peptides (OATPs) and multidrug resistance-associated protein (Mrp) 2 in rats. When IS is administered intravenously to the normal rats at a dose of 120 μmol/kg; plasma IS levels are approximately 600 μM after 2 min and 100 μM after 120 min. In rats with acute renal failure (ARF) induced by cisplatin, the area under the curve (AUC) was more than 2.5-fold greater compared with that in the normal rats, indicating that IS accumulates in ARF rats. Intravenously administered pravastatin almost disappeared from the plasma by 60 min post-administration and approximately 55% of dose was excreted in the bile within 60 min. This result suggested that pravastatin was efficiently taken up from the sinusoid into hepatocytes via rat OATPs on the sinusoidal membrane and preferentially transported in the bile mediated by Mrp2 on the canalicular membrane. IS administered intravenously at a dose of 120 μmol/kg caused neither an increase in plasma pravastatin levels nor a decrease in its biliary excretion. In conclusion, the present results demonstrate that single intravenous administration of IS does not interfere with the hepatic transport of pravastatin directly in vivo, which is at variance with the results of previous in vitro studies.

Investigation on the Interactions between Various Drugs and Aojiru (Green Juice) Using a Simple Centrifugation Method

The objective of this study was to evaluate the interactions between various drugs and aojiru (green juice), a popular health food in Japan, using a simple centrifugation method. The mixture of drug solution and aojiru suspension was gently shaken and centrifuged. The drug concentration in the supernatant fluid was then determined by HPLC. The concentration of rhodamine 123 (Rho-123), a model compound, in the supernatant fluid significantly decreased after mixing with aojiru, indicating extensive binding of Rho-123 to the insoluble components of aojiru. When administered into rat small intestinal loops together with aojiru, the plasma Rho-123 concentrations became much smaller than those when administered alone. This result strongly suggested that a strong interaction observed in vitro was well reflected in modulated absorption. Among seven drugs tested, chlorpromazine and imipramine exerted binding properties to aojiru similar to or greater than Rho-123. As a small part of both Rho-123 and imipramine was released when the aojiru precipitate was resuspended, their binding to aojiru was considered to be tight. The binding of diltiazem, fexofenadine, glibenclamide, metformin, and norfloxacin to aojiru was much weaker or almost negligible compared with that of chlorpromazine and imipramine. The present results suggest that aojiru can decrease the intestinal absorption of some clinically relevant drugs through tight binding in the small intestine and that the present centrifugation method is useful for predicting in vivo interactions between drugs and aojiru.

Correlations between Plasma Levels of Anionic Uremic Toxins and Clinical Parameters in Hemodialysis Patients

When the kidney is seriously impaired, various uremic toxins (UTs) accumulate in the body, often exerting unfavorable effects on physiological functions and drug pharmacokinetics. To prevent this, it is important to determine plasma UT levels accurately in chronic kidney disease patients. Although attempts to predict plasma UT levels using biomarkers have been made, the correlation between UT levels and the markers is not yet fully understood. In this study, we assessed the correlations among plasma levels of indoxyl sulfate (IS), indoleacetic acid (IA), and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) in 20 hemodialysis patients and evaluated the relationship between the plasma levels of UTs and clinical parameters, such as serum creatinine (Scr), blood urea nitrogen, and estimated glomerular filtration rate (eGFR), with special focus on IS. There were no correlations among the plasma levels of the three UTs before and immediately after hemodialysis. However, a significant correlation was observed between plasma IS levels and Scr before hemodialysis (r=0.643, p=0.002), with the correlation becoming much stronger when using the data obtained immediately after hemodialysis (r=0.744, p<0.001). Further, plasma IS levels showed a significant negative correlation with eGFR (r=-0.558, p=0.011). However, no correlations were observed for IA or CMPF. The results obtained from this study suggest that plasma IS levels can be predicted from Scr values, although the precise mechanism behind the correlation remains to be clarified.

Region-dependent absorption of faropenem shared with foscarnet, a phosphate transporter substrate, in the rat small intestine

Faropenem, a penem antibiotic, is orally active despite its hydrophilic nature. However, its intestinal absorption has not yet been characterised in detail. This study was undertaken to determine the factors regulating faropenem absorption using intestinal loops prepared in the rat duodenum, jejunum and terminal ileum. Faropenem disappearance was much greater than that of cefotaxime and meropenem, and faropenem disappeared more extensively from the terminal ileum than from the jejunum or duodenum. In contrast to faropenem, the disappearance of ceftibuten was much greater from the duodenum and jejunum than from the terminal ileum. As the accumulation and enzymatic degradation of faropenem was minimal in the intestinal mucosa, faropenem was considered to enter the portal vein smoothly after its disappearance from the intestinal loops. Faropenem disappearance was not significantly influenced by the presence of monocarboxylic acids, amino acids or bile acid. Dipeptides such as L-carnosine and glycylglycine slightly but significantly lowered faropenem disappearance from the terminal ileum. On the other hand, foscarnet exerted a marked inhibitory effect on faropenem disappearance, but the antiviral agent did not modulate ceftibuten absorption. The present results suggest that faropenem is in part absorbed via a phosphate transporter present in the rat small intestine.