Ryoko Yumoto

Interaction with P-glycoprotein and transport of erythromycin, midazolam and ketoconazole in Caco-2 cells

The effect of cytochrome P-450 3A (CYP3A) substrates (erythromycin, midazolam) and an inhibitor (ketoconazole) on P-glycoprotein-mediated transport was studied in Caco-2, the human colon adenocarcinoma cell line expressing various functions of differentiated intestinal epithelial cells. The involvement of P-glycoprotein in the transport of these drugs was also examined. The basal-to-apical transport of rhodamine 123, a P-glycoprotein substrate, was inhibited by erythromycin, midazolam and ketoconazole, as well as by P-glycoprotein inhibitors such as verapamil. The apical-to-basal transport of rhodamine 123 was increased by these drugs. The transepithelial transport of erythromycin and midazolam, but not of ketoconazole, was much greater from the basal to apical side than from the apical to basal side. The inhibitory effect of verapamil was observed on the basal to apical transport of erythromycin, but not on midazolam and ketoconazole transport. In conclusion, erythromycin, midazolam and ketoconazole could interact with P-glycoprotein-mediated transport, and P-glycoprotein could be, at least in part, involved in the transport of erythromycin, but not of midazolam and ketoconazole, in the intestinal epithelia.

Expression and function of P-glycoprotein in rats with glycerol-induced acute renal failure

The effect of glycerol-induced acute renal failure on P-glycoprotein expression and function was evaluated in rats. The in vivo function of P-glycoprotein was evaluated by measuring renal secretory and biliary clearance and brain distribution of rhodamine 123 (Rho-123), a P-glycoprotein substrate, under a steady-state plasma concentration. In acute renal failure rats, the P-glycoprotein level increased 2.5-fold in the kidney, but not in the liver and brain. In contrast, P-glycoprotein function in these tissues was suppressed. Interestingly, not only the renal but also the biliary clearance of Rho-123 was correlated with the glomerular filtration rate. In Caco-2 cells, plasma from renal failure rats exhibited a greater inhibitory effect on P-glycoprotein-mediated transport of Rho-123 than did plasma from control rats. In conclusion, P-glycoprotein function was systemically suppressed in acute renal failure, even though the level of P-glycoprotein remained unchanged or rather increased. This may be due to the accumulation of some endogenous P-glycoprotein substrates/modulators in the plasma in disease states.

Effects of endocytosis inhibitors on internalization of human IgG by Caco-2 human intestinal epithelial cells

AIMS The purpose of this study was to characterize the internalization mechanism of human IgG into the epithelial cells of human small intestine, employing human intestinal epithelial cell line Caco-2 as an in vitro model system. MAIN METHODS Real-time PCR analysis and uptake studies of fluorescein isothiocyanate-labeled IgG (FITC-IgG) from human serum were performed using Caco-2 cells. KEY FINDINGS Real-time PCR analysis showed that mRNA level of the neonatal Fc receptor (FcRn) was increased during the differentiation process in Caco-2 cells. The binding of FITC-labeled human IgG to the membrane surface of Caco-2 cells increased with a decrease in pH of incubation buffer. The uptake of FITC-IgG was also stimulated at acidic pH and was time-dependent. The binding and uptake of FITC-IgG at pH 6.0 was partially, but significantly, decreased by human gamma-globulin in a concentration-dependent manner. A mixture of metabolic inhibitors (sodium azide and 2-deoxyglucose) significantly inhibited the uptake, but not the binding, of FITC-IgG. In addition, endosomal acidification inhibitors such as bafilomycin A(1) and chloroquine significantly increased the accumulation of FITC-IgG. Clathrin-dependent endocytosis inhibitors (phenylarsine oxide and chlorpromazine) and caveolin-dependent endocytosis inhibitors (nystatin and indomethacin) did not decrease the uptake of FITC-IgG at pH 6.0. In contrast, macropinocytosis inhibitors such as cytochalasin B and 5-(N-ethyl-N-isopropyl) amiloride significantly decreased the uptake of FITC-IgG at pH 6.0. SIGNIFICANCE The internalization of human IgG in human intestine might be, at least in part, due to FcRn-mediated endocytosis, which could occur by a process other than clathrin- and caveolin-dependent mechanisms.

Interaction of valproic acid and carbapenem antibiotics with multidrug resistance-associated proteins in rat erythrocyte membranes

We recently reported that the decrease in plasma valproic acid (VPA) level by carbapenem antibiotics (CPs) may partly be due to the increased erythrocyte distribution of VPA. In order to clarify the mechanisms underlying altered VPA distribution in erythrocytes, we examined the role of multidrug resistance-associated proteins (Mrps). The uptake of 2,4-dinitrophenyl-S-glutathione (DNP-SG), a substrate of Mrps, by inside-out vesicles (IOVs) prepared from rat erythrocytes was an ATP-dependent, active process. DNP-SG uptake was mediated by high- and low-affinity transport systems, and was inhibited by various Mrp inhibitors such as probenecid and indomethacin. Glutathione stimulated only the high-affinity transport system. VPA inhibited the low-affinity transport of DNP-SG, while panipenem, a CP, inhibited both high- and low-affinity transport. ATP-dependent, Mrp-mediated transport of methotrexate, another Mrp substrate, in IOVs was also observed, and VPA and various CPs inhibited the transport. The uptake of [(3)H]VPA was examined, and found to be ATP-dependent. ATP-dependent uptake of [(3)H]VPA was inhibited by Mrp inhibitors and panipenem, while the inhibition was not observed in the absence of ATP. These results indicate that VPA and CPs interact with Mrp-mediated transport in erythrocyte membranes, and VPA itself is transported by Mrps, which is inhibited by panipenem. Thus, the increased erythrocyte distribution of VPA by CPs observed under in vivo conditions may partly be explained by their interaction with Mrps in erythrocyte membranes.

Biopharmaceutics: Topical Delivery of Keloid Therapeutic Drug, Tranilast, by Combined Use of Oleic Acid and Propylene Glycol as a Penetration Enhancer: Evaluation by Skin Microdialysis in Rats

Topical delivery of tranilast (N‐(3,4‐dimethoxycinnamoyl)anthranic acid), an inhibitor of collagen synthesis and a therapeutic drug for keloid and hypertrophic scar, was examined, in rats, with oleic acid alone or a combination of oleic acid and propylene glycol as penetration enhancer. Evaluation was by measurement of the concentration of tranilast in plasma and in the dialysate from skin microdialysis.

Pharmacokinetic Analysis of the Absorption Enhancing Action of Decanoic Acid and Its Derivatives in Rats

The enhancing action of decanoic acid (C1O) and its derivatives on mucosal absorption of phenolsulfonphthalein (PSP) in the jejunum or colon was analyzed using pharmacokinetics in rats. After administration of a solution containing PSP and an enhancer [C10, 2-hydroxydecanoic acid (2-OHC10), or 3-hydroxydecanoic acid (3-OHC10)] into the jejunal or colonic loop, the amounts of PSP and enhancer remaining in the loop and/or plasma PSP concentration were determined periodically. 2-OHC10 exhibited a greater absorption enhancing potency than C10, while 3-OHC10 was less effective. Disappearance of residual PSP from the loop ceased after complete absorption of the enhancer. The enhancer-induced disappearance rate constant of PSP correlated well with the product of the enhancer disappearance rate and its capacity to sequester calcium ions. In conclusion, the enhancement of PSP mucosal absorption by C10 and its derivatives is consistent with a pharmacokinetic model, assuming that the enhanced membrane permeability of PSP depends on the enhancer disappearance kinetics from the loop and its calcium ion sequestration capacity.

Nanoparticulation of probucol, a poorly water-soluble drug, using a novel wet-milling process to improve in vitro dissolution and in vivo oral absorption

To improve the dissolution and oral absorption properties of probucol, a novel wet-milling process using the ULTRA APEX MILL was investigated. The particle size of bulk probucol powder was 17.1 µm. However, after wet-milling with dispersing agents such as Gelucire 44/14, Gelucire 50/13, vitamin E-TPGS, and Pluronic F-108, the probucol particle sizes decreased to about 77–176 nm. Scanning electron microscopy (SEM) analysis also suggested that the probucol particles were successfully milled into the nanometer range. An in vitro dissolution study showed that the dissolution rates of all nanopowders were several folds higher than those of the corresponding mixed powders. When orally administered to rats, the AUC values of probucol nanopowders treated with Gelucire 44/14 and 50/13, and vitamin E-TPGS were about 3.06–3.54-folds greater than that of the bulk powder. Therefore, through this study, we have developed a new pharmaceutical technique to improve the dissolution rate and oral absorption of probucol using the ULTRA APEX MILL by wet-milling with various dispersing agents.

Biopharmaceutics: Effect of Ointment Bases on Topical and Transdermal Delivery of Salicylic Acid in Rats: Evaluation by Skin Microdialysis

Microdialysis has been used to determine the concentration of salicylic acid in skin tissue and plasma periodically for 4 h to evaluate the effect of ointment bases on topical and transdermal delivery of salicylic acid. The ointment bases examined were solbase (water‐soluble), poloid and white petrolatum (oleaginous), hydrophilic poloid (water in oil (w/o) type emulsion lacking water) and absorptive ointment (w/o‐type emulsion containing water). The ointments (0.1 g) containing 25 μmol salicylic acid were applied for 2 h to the surface of rat skin (1 cm2) with (intact) or without the stratum corneum.

Decanoic acid induced enhancement of rectal absorption of hydrophilic compounds in rats.

The enhancing effect of decanoic acid (CIO) on the rectal absorption of phenolsulfonphtalein (PSP) was analyzed with a pharmacokinetic model. The transfer index of PSP from the rectal lumen to the epithelial layer in the presence of C10 was proportional to the product of the C10 disappearance rate from the rectal lumen and its calcium ion sequestration capacity. The enhancement of rectal PSP absorption by different doses of C10 was also predictable by using a permeability index, Pa, of PSP, which was defined as a proportionality constant relating transfer index and the product value described above. The Pa values of various hydrophilic compounds with different molecular weights were also estimated from their rectal bioavailability in the presence of C10. A linear relationship was observed between Pa values and reciprocal values of the square root of individual molecular weight. These findings suggest that the Pa index is related to the permeant’s diffusion coefficient through paracellular aqueous openings formed by C10.

Mechanism underlying insulin uptake in alveolar epithelial cell line RLE-6TN

For the development of efficient pulmonary delivery systems for protein and peptide drugs, it is important to understand their transport mechanisms in alveolar epithelial cells. In this study, the uptake mechanism for FITC-insulin in cultured alveolar epithelial cell line RLE-6TN was elucidated. FITC-insulin uptake by RLE-6TN cells was time-dependent, temperature-sensitive, and concentration-dependent. The uptake was inhibited by metabolic inhibitors, cytochalasin D, clathrin-mediated endocytosis inhibitors, and dynasore, an inhibitor of dynamin GTPase. On the other hand, no inhibitory effect was observed with caveolae-mediated endocytosis inhibitors and a macropinocytosis inhibitor. Intracellular FITC-insulin was found to be partly transported to the basal side of the epithelial cell monolayers. In addition, colocalization of FITC-insulin and LysoTracker Red was observed on confocal laser scanning microscopy, indicating that FITC-insulin was partly targeted to lysosomes. In accordance with these findings, SDS-PAGE/fluoroimage analysis showed that intact FITC-insulin in the cells was eliminated with time. The possible receptor involved in FITC-insulin uptake by RLE-6TN cells was examined by using siRNA. Transfection of the cells with megalin or insulin receptor siRNA successfully reduced the corresponding mRNA expression. FITC-insulin uptake decreased on the transfection with insulin receptor siRNA, but not that with megalin siRNA. These results suggest that insulin is taken up through endocytosis in RLE-6TN cells, and after the endocytosis, the intracellular insulin is partly degraded in lysosomes and partly transported to the basal side. Insulin receptor, but not megalin, may be involved at least partly in insulin endocytosis in RLE-6TN cells.