Published papers

Cyclosporin A, a cyclic undecapeptide, and FK506 are efficient immunosuppressive agents. They also attract attention as effective P-glycoprotein modulators that inhibit P-glycoprotein from binding to anticancer drugs and overcome multidrug resistance. Cyclosporin A itself interacts with a common binding site of P-glycoprotein to which Vinca alkaloids and verapamil bind. We were interested to determine whether cyclosporin A and FK506 are substrates for P-glycoprotein to transport, and we studied their transcellular transport. In LLC-PK1 cells, derived from porcine kidney proximal tubule and forming a highly polarized epithelium, cyclosporin A was transported in a saturable manner. LLC-GA5-COL300, a transformant cell line derived by transfecting LLC-PK1 with human MDR1 cDNA isolated from normal adrenal gland, expresses P-glycoprotein specifically on the apical surface and shows a typical multidrug-resistant phenotype. LLC-GA5-COL300 cells showed increased transport of cyclosporin A from the basal to the apical side. Kinetic analysis showed that this transport was a typical saturable transport with the calculated apparent Michaelis constant (Kmapp) and the maximum flux (Vmax) as 8.4 μM and 2.4 nmol/mg protein/h, respectively. LLC-GA5-COL300 also snowed increased transport of FK506 from the basal to the apical side. These results indicate that P-glycoprotein transports the immunosuppressive agents cyclosporin A and FK506.

This article represents the first evidence that the renal secretion of the commonly used drug, digoxin, is mediated by P-glycoprotein. In this study, it was demonstrated that digoxin is a substrate of P-glycoprotein, and the mechanism of a clinically important drug interaction, such as digoxin- quinidine, was elucidated. Human P-glycoprotein was expressed on the apical membrane of the porcine kidney epithelial cell line, LLC-PK1 by transfecting with human MDR1 cDNA. The expression and function of P-glycoprotein were confirmed by Southern and Western blotting, RNase protection assay, immunostaining and transporting activity for vinblastine. The transepithelial transport of [3H]digoxin was measured across the cell monolayers grown on microporous polycarbonate membrane filters. The transfectant cells exhibited markedly greater basal-to-apical transport and less apical-to-basal transport than the host cells, and the former was 8-fold greater than the latter. The augmented transepithelial transport resulted from the increased efflux from cells to apical side. This oriented transport was inhibited by the presence of 20 μM vinblastine, quinidine or verapamil. The rate of efflux to the apical side was 2-fold greater than that to the basal side. Quinidine inhibited the efflux to the apical side but did not affect transport into the basal side. These findings demonstrate that digoxin is transported by human P-glycoprotein, which is a previously undiscovered drug transport system in the kidney other than organic cation and anion transport systems, and suggest a molecular mechanism for the renal tubular secretion of digoxin as well as clinically important digoxin-quinidine interaction via P-glycoprotein.

The pharmacokinetics of theophylline in acutely ill patients show wide intraindividual variability associated with the severity of clinical status. To investigate the mechanism of this variability, we studied the effects of leukotriene C4 (LTC4)-induced pathophysiologic changes on the disposition of theophylline. The plasma concentration-time profiles were measured after simultaneous intravenous bolus injection of theophylline and antipyrine in guinea pigs. The animals received 5 μg/kg of LTC4 intravenously 60 min later. The plasma theophylline concentration 30 min after LTC4 treatment was significantly lower (p < 0.05) than that of nontreated control animals, whereas the plasma antipyrine concentration at that time was not affected. In addition, the treated animals showed significantly slower declines in plasma concentrations of both drugs (0.0805 ± 0.0199 and 0.291 ± 0.020 h-1 for theophylline and antipyrine, respectively, mean ± SEM) than did controls (0.197 ± 0.010 and 0.439 ± 0.028 h-1). Leukotriene C4 treatment also induced moderate bronchoconstriction and metabolic acidosis, increased blood hemoglobin concentration and hematocrit, and decreased concentration of serum proteins. In connection with these changes, the plasma unbound fraction of theophylline increased significantly (p < 0.001, 94.4 ± 3.3% in treatment versus 58.2 ± 4.4% in control), but that of antipyrine was unchanged (94.9 ± 3.0% in treatment versus 92.1 ± 0.9% in control). These findings indicated that an increase in the volume of distribution was responsible for the abrupt change in plasma theophylline concentration following LTC4 treatment, and the apparent change in the volume of distribution was estimated as 26.1 ± 5.6%. Results showed that the LTC4 response can influence the disposition kinetics of theophylline and suggested a possible mechanism for the intraindividual variability in acutely ill patients.

The total body and hepatic clearance of two recombinant human tissue-type plasminogen activators (t-PA) produced in mouse C127 cells [t-PA(C127)] and in Chinese hamster ovary cells [t-PA(CHO)] were determined in rats. Recombinant t-PA was administered as a constant iv or intraportal venous infusion for 60 min. The plasma t-PA level over 120 min was measured by an ELISA. Whole-body pharmacokinetics were characterized in terms of the total body clearance (CL(t)), total volume of distribution, and mean residence time in the body. Hepatic disposition was described by the hepatic clearance (CL(hep)) and the mean hepatoportal transit time. Whereas a marked hepatic extraction was observed with both-t-PAs, the estimated CL(hep) accounted for only one-half the CL(t). No elimination took place in the lung, because no difference was observed between iv and intraarterial administration. Extrahepatic elimination, such as that in circulating blood, appeared to play a significant role in the disposition of t-PA in vivo. t-PA(C127) is known to possess the Galα1-3Gal epitope in its complex-type carbohydrate chains, whereas this structure is not involved in t-PA(CHO). In the presence of Bandeiraea simplicifolia lectin I isolectin B4, which is specific to the Galα1-3Gal, the CL(t) of t-PA(C127) was significantly decreased, whereas that of t-PA(CHO) was unchanged. These results imply that the formation of a high molecular weight complex by this carbohydrate epitope effectively reduces the rate of catabolism of t-PA in vivo.

We expressed human MDR1 cDNA isolated from the human adrenal gland in porcine LLC-PK1 cells. A highly polarized epithelium formed by LLC-GA5-COL300 cells that expressed human P-glycoprotein specifically on the apical surface showed a multidrug-resistant phenotype and had 8.3-, 3.4-, and 6.5-fold higher net basal to apical transport of 3H-labeled cortisol, aldosterone, and dexamethasone, respectively, compared with host cells. But progesterone was not transported, although it inhibited azidopine photoaffinity labeling of human P-glycoprotein and increased the sensitivity of multidrug-resistant cells to vinblastine. An excess of progesterone inhibited the transepithelial transport of cortisol by P-glycoprotein. These results suggest that cortisol and aldosterone are physiological substrates for P-glycoprotein in the human adrenal cortex and that substances that efficiently bind to P-glycoprotein are not necessarily transported by P-glycoprotein.

The effect of folinic acid (CF) on the tissue residence and renal excretion of methotrexate (MTX) in the rat model is described in an experiment designed to resemble a clinical regimen of high-dose MTX therapy with CF rescue. Concentrations of MTX in the kidney, liver, and plasma were determined following the sequential killing of the animals up to 24 hr after an iv bolus injection of 100 μmol/kg MTX. The treated group received repeated iv doses of CF (5.86 μmol/kg) for 9.3 hr. The tissue retention property of MTX was characterized by noncompartmental parameters, the mean residence time (MRT), and the single-pass mean residence time (MRTSP) in organs. The values of MRT in the kidney and liver were greater than that in plasma and were shortened by the CF treatment, indicating that CF is effective in reducing the duration of exposure to MTX in those organs. For drugs whose duration of exposure is a key factor in their toxicity, MRTSP was shown to be a useful parameter in toxicokinetic study. The renal clearance of MTX was virtually constant over a wide range of plasma MTX levels (0.1-165 μM) and was inhibited by p-aminohippurate. CF accelerated the renal excretion of MTX, which effect depended on the plasma concentration of MTX. Since reabsorption and filtration were not affected by CF, the increased excretion of MTX was attributed to a stimulation of tubular secretion. These observations show that CF exerts a beneficial effect in accelerating the excretion of MTX residing in the organs, in addition to supplying active folate to aid the resumption of de novo DNA synthesis.

The determining step in transepithelial transport of p-aminohippurate (PAH) in renal tubular secretion has been elucidated in the rat isolated perfused kidney. The method was based upon a multiple indicator dilution experiment and non-compartmental moment analysis. Single-pass dilution curves were obtained from venous and urinary effluents after simultaneous intra-arterial injections of Evans blue with albumin, [3H]inulin and [14C]PAH. Probenecid was used as a transport inhibitor and dissolved in both perfusate and injection solution. The urinary excretion of PAH decreased depending on the probenecid concentration, while that of inulin was not affected. No correlation was observed between the amount of secretion and the mean residence time of secreted PAH in renal epithelial cells [T̄(cell)]. Since T̄(cell) ought to be affected by the rate of secretion from cells to lumen, it was suggested that the secretion rate was independent of the amount secreted. In contrast, the amount of PAH excreted via tubular secretion showed a linear correlation with the volume of distribution in the kidney and the apparent rate constant for tubular uptake of PAH. Since these kinetic parameters reflect the transport from blood into cells across the basolateral membranes of renal epithelial cells, the present results demonstrated that basolateral membrane transport is a determining step in the transcellular transport of PAH and that the major effect of probenecid is the inhibition of transport from blood into cells.

This paper describes a novel method to assess the antiluminal membrane (ALM) and luminal membrane (LM) transport in vivo across renal tubular epithelial cells. The method is based upon a noncompartmental moment analysis of the plasma concentration and urinary excretion rate curves following renal artery injection. Quantitative relationships are represented between the noncompartmental parameters (clearance, volume of distribution, and the mean transit time) and the first-order rate constants associated with transmembrane transport processes. The in vivo transepithelial transport of [14C]p -aminohippurate (PAH) was examined using the rat kidney in the absence or presence of various plasma concentrations of unlabeled PAH, cefazolin, and methotrexate. The tubular secretion intrinsic clearance was reduced with an increase in the plasma concentration of concurrent unlabeled organic onions. The distribution volume of PAH in the kidney decreased in association with a decrease in the amount of PAH secreted, whereas the mean transepithelial (artery-to-lumen) transit time (-Tcell) remained constant. These findings indicate that ALM transport is a capacity-limited process determining the amount of tubular secretion, and that LM transport is linear over the concentration range examined and independent of the amount of secretion. The contribution of ALM and LM transport to transcellular transport was first clarified in vivo. The present method will be useful for analyzing the transmembrane transport processes in vivo for highly diffusible substances in the kidney. © 1991 Plenum Publishing Corporation.

A new method that can assess the kinetics of in vivo transepithelial transport in rat kidney has been established. The method is based upon a multiple-indicator dilution experiment and the moment analysis theory. After simultaneous bolus injections of p-aminohippurate (PAH) and inulin into the right renal artery, blood samples were taken from the carotid artery and urine was separately collected from right and left ureters. The characteristic response for the first passage of drugs through the right kidney was evaluated by taking blood circulation into consideration. To determine the mean artery-tovein transit time and the extraction ratio in the kidney, an intravenous injection was also performed as a reference experiment for deconvolution. The urinary excretion curve corresponding to the first passage was obtained as the difference between both kidneys. The mean artery-tolumen transit time (mean transepithelial transit time, -Tcell)was computed by subtracting the mean urinary transit time of inulin from that of secreted PAH. Sinc transport across the luminal membrane into the lumen from tubular epithelial cells can influence the cellular residence time of drugs, - Tcelland the single-pass mean residence time in epithelial cells (-Tcell.sp)can be thought of describing luminal membrane transport. The value of -Tcell obtained for 0. t mM PAH was 22 sec and it was prolonged to 61 sec in the presence of probenecid, suggesting an inhibitory effect on transport across the luminal membrane. On the other hand, antiluminal membrane transport into cells from blood is characterized by the volume of distribution in the kidney (VdPAH). VdPAH was remarkably decreased by treatment with probenecid, indicating an inhibitory effect on antiluminal membrane transport. The effects of probenecid on both sides of epithelial cell membrane transport were first demonstrated in vivo. The present method is useful for the analysis of in vivo transepithelial transport including antiluminal and luminal membrane transport for drugs excreted via tubular secretion. © 1991 Plenum Publishing Corporation.

The preparation of injectable sustained release microspheres of TRH was investigated. The drug was encapsulated in copoly(dl-lactic/glycolic acid) (PLGA) using an in-water drying method through a w/o/w emulsion. The predominant factors influencing the entrapment ratio of TRH in the microspheres and the release rate of TRH from the microspheres were the loading amount and the dissociation state of the drug. Microspheres of TRH encapsulated as a free base at an adequate concentration provided a small initial burst followed by a zero-order prolonged release regardless of the high water solubility of the drug. The addition of strong acids to the inner aqueous phase interfered with the encapsulation efficacy and caused a large initial burst. The ionic interaction between the basic functional group of TRH and the carboxylic acid end group of PLGA during the preparation is necessary to produce a rigid matrix of the microspheres. This method is applicable to the microencapsulation of other water-soluble basic peptides in PLGA. © 1991.

1991年日本薬剤学会論文賞　受賞論文

A different manner of insufficiency of renal epithelial cell transport between the organic anion and cation, p-aminohippurate and tetraethylammonium, respectively, was observed in the perfused kidney isolated from uranyl nitrate-induced acute renal failure (ARF) rats. The single-pass outflow pattern of the perfused kidney was analyzed by noncompartmental moment analysis. The active tubular secretion was impaired faster than the reduction of glomerular filtration, and the tetraethylammonium secretion decreased at an earlier stage of ARF than p-aminohippurate. The apparent uptake rate constant from blood to cells of p-aminohippurate was reduced with the progress of ARF and associated with the amount of this drug secreted, whereas the uptake rate constant of tetraethylammonium did not change until the late stage of ARF. The mean residence time in renal epithelial cells of tetraethylammonium was prolonged with reduction of the amount to be secreted, while that of p-aminohippurate remained unchanged. Therefore, the uptake of p-aminohippurate across the basolateral membranes decreased gradually, and the transport across the brush border membranes was still unchanged after uranyl nitrate treatment. On the other hand, the secretion of tetraethylammonium from cells to lumen was impaired at first, and then the uptake from blood to cells was impaired. These results suggest that impairment by uranyl nitrate-induced ARF appears at the carrier-mediated transport process of the epithelial cell membranes for both organic anions and cations.

Effects of urine pH on the renal tubular secretion of an organic cation (tetraethylammonium, TEA) and an organic anion (p-aminohippurate, PAH) were investigated using the isolated erythrocyte-perfused rat kidney. The method was based on a multiple indicator dilution experiment and noncompartmental moment analysis. Treatment with sodium bicarbonate and sodium dihydrogen phosphate increased and decreased urine pH, respectively, but affected neither the condition of the perfused kidney nor the renal handling of albumin and inulin. In TEA studies, the increase of urine pH prolonged the mean residence time in renal epithelial cells (T̄(cell)) and reduced the apparent secretion intrinsic clearance, but did not influence the volume of distribution in the kidney (Vd(drug)). The decrease of urine pH did not affect these kinetic parameters. By contrast, PAH secretion was constant against the change of urine pH. Since any change in the basolateral membrane transport is reflected in Vd(drug), the net transport from blood to cells can be regarded as similar under these treatments. On the other hand, the prolonged T̄(cell) of TEA with the increased urine pH suggested a slow transport from cells to lumen across the brush-border membranes. The present results coincide with the hypothelical mechanism that organic cations are secreted via an active transport system, coupled to the countertransport of H+ into cells. In conclusion, the present method is useful to separately evaluate the transmembrane transport across both sides of the renal epithelial cells in a morphologically intact kidney.