The aim of the present study was to examine conditions for preserving isolated human colon tissues when assessing colonic drug absorption. Isolated human colon tissues were kept in various conditions. And electrophysiological analysis at 37°C was then carried out to assess the integrity of the paracellular and transcellular route using Ussing-type chambers. The membrane resistance (Rm) of isolated human colon preserved at 4°C for 3, 6 or 9 hours increased up to 90 min and was thereafter constant for at least 40 min. The permeability of FD-4, a paracellular permeable compound, was restricted at constant Rm. On the other hand, the value of potential difference and of short circuit current as well as that of Rm was constant for at least 40 min. Neither preservation for 12 hours at 4°C nor that for 3 hours at room temperature was suitable when evaluated in terms of the recovery of Rm. These results indicate that cell viability was guaranteed for 9 hours provided that isolated human colon tissues were preserved at 4°C. Furthermore, it is necessary to preincubate for at least 90 min to obtain a structural and functional recovery of the paracellular and transcellular route.

View full abstract

Itraconazole is known to interact with digoxin, a substrate of P-glycoprotein, in clinical situations. In this study, the interactions of the azole antifungal agents, miconazole, fluconazole, ketoconazole as well as itraconazole, with P-glycoprotein were examined using a transfected kidney epithelial cell line, LLC-GA5-COL150, which expresses human P-glycoprotein on the apical membrane.
Itraconazole decreased the transcellular transport of digoxin from the basolateral to apical side in LLC-GA5-COL150 cell monolayers, that was accompanied by increased cellular accumulation. The basolateralto-apical transcellular transport of digoxin in the host LLC-PK₁ cell monolayers was not affected by itraconazole. Ketoconazole and fluconazole also significantly inhibited P-glycoprotein-mediated transport of digoxin, although miconazole was not effective. The inhibitory effects of itraconazole and ketoconazole on digoxin transport in LLC-GA5-COL150 monolayers were as strong as that of cyclosporin A, a typical P-glycoprotein modulator, and neither quinidine nor cimetidine inhibited digoxin transport at the same concentration (10 μM). In addition, the viability of LLC-GA5-COL150 cells against itraconazole was higher than that of LLC-PK₁ cells, but viability against the other three azoles was similar to that of LLC-PK₁ cells.
In conclusion, ketoconazole and fluconazole as well as itraconazole inhibited the transport of digoxin via human P-glycoprotein, and itraconazole could be a substrate of P-glycoprotein.

View full abstract

Research history and recent progress on the mechanism by which xenobiotics induce cytochromes P450 belonging to the CYP2B subfamily are briefly reviewed. One of the major interests focused here is that what is the initial target site for exogenous inducers? In this topics, we discussed the validity of the concept for the receptor-mediated induction, and for a mechanism in which xenobiotic inducers cancel the effect with endogenous suppressors. Recent studies have revealed a crucial role of nuclear receptors, CAR (Constitutive Androstane Receptor) and RXR (Retinoid X-Receptor), in trans-activating a specific region located 5'-Upstream of the CYP2B genes. We also reviewed the role of DNA elements participating in transactivation, and those of the trans-factors involving not only CAR/RXR but also other factors such as Barbie box and RBP-Jκ.

View full abstract

The purpose of this study was to clarify the mechanism (s) responsible for the growth hormone (GH)-induced expression of the CYP2C12 gene. To identify a functional GH-responsive element (GHRE) in vivo, we performed the direct injection of promoter-luciferase chimeric genes into female rat livers. The results showed that the luciferase activity was decreased to approximately 20% by the deletion of the sequence between nucleotides -4213 and -4161. Within this region, two copies of a possible GHRE were present. The sequence of the GHRE was overlapped with that of an interferon-y-activated sequence (GAS), known to be recognized by the signal transducer and activator of transcription (STAT) proteins. In fact, a super shift assay showed that STAT5 was capable of binding to the core sequence of the GHRE. Furthermore, a luciferase assay with reporter plasmids, which lacks HNF4 or HNF6-binding sites, revealed that the GH-stimulated expression of the CYP2C12 gene was regulated cooperatively by STAT5, HNF-4, HNF-6 and the factor(s) which binds to the elements, 2C12-I (-4095 to -4074) and 2C12-II (-4072 to -4045). The cooperative regulation by STAT5 and the liver-enriched transcription factors account for the GH-dependent and the liver-specific expression of the CYP2C12 gene in female rats.

View full abstract

Role of denitration in the pharmacokinetics (PK) and pharmacodynamics (PD) of organic nitrates was reviewed. The PK/PD relationship following i.v. bolus injection of stereoisomeric organic nitrates can be described using simple compartment and sigmoidal Emax models. Because the nitrates are prodrugs of nitric oxide and eliminate mainly as denitrated metabolites, both potency and duration of vasodilation depend on the extent of denitration. At present, the mechanism of nitrate tolerance remains poorly understood. However, the most of the important hypotheses are counter-regulative, so that the denitration may play an important role in the development of tolerance. It is desired to establish the PK/PD relationship of organic nitrates involving tolerance period and improve nitrate-therapy based on the relationship.

View full abstract

Drug-induced parkinsonism is generally known as a side effect of antipsychotic drugs. Recently, the induction of parkinsonism has been reported with the use of various peripherally acting drugs such as calcium channel blockers, antiarrhythmic agents and so on. Therefore, it is eagerly required to quantitatively assess the extent of drug-induced parkinsonism prior to the dispensing of drugs. We have developed a strategy to quantitatively predict the intensity of drug-induced parkinsonism based on the receptor-occupancies of the dopamine D₁, D₂ and muscarinic acetylcholine (mACh) receptors in vivo and in vitro. First, we assessed the in vivo dopamine D₁, D₂ and mACh receptor occupancies in the striatum in mice after drug administration for twenty five drugs. Simultaneously, the intensity of catalepsy induced by these drugs were assessed in the same animals. The intensity of catalepsy can be predicted by in vivo occupancies of the dopamine D₁, D₂ and mACh receptors using a pharmacodynamic model. Second, we applied this pharmacodynamic model to predict quantitatively the risk of drug-induced parkinsonism in humans. The in vivo dopamine D₁, D₂ and mACh receptor occupancies of each drug in humans were estimated from the pharmacokinetic data in humans and the in vitro affinities for the receptors in mice. The estimated risk of each drug to induce parkinsonism well coincided with the incidence of parkinsonism reported during the clinical trial period. The pharmacodynamic model was applicable not only to an individual drug but also to a prescription. The clinical risk of every prescription to induce parkinsonism can be quantitatively predicted by this strategy from the set of pharmacokinetic data in humans and the affinities for the receptors of each drug in the prescription. Finally, we incorporated this model into a prescription-ordering system to develop a computer-aided drug information system for rational prescription. Developed system can display the risk of parkinsonism for an individual prescription and provide essential drug informations to the physician to prescribe. The developed system may be also applicable to quantitatively predict other receptor-mediated adverse reactions of drugs.

View full abstract

In the kidney, various compounds in the plasma are filtrated through glomeruli. Though the glomerular filtration of many plasma proteins is restricted because of their large molecular weights, still significant amount of proteins is filtered. The proteins filtered are reabsorbed in the proximal tubular cells almost completely. The mechanism involved in protein reabsorption is receptor-mediated endocytosis. Not only proteins but also drugs such as aminoglycoside antibiotics are taken up by receptor-mediated endocytosis in renal proximal tubules. Megalin is a large (ca. 600 kDa) endocytic receptor, which is abundantly expressed in renal proximal tubules, and recognizes various compounds such as lipoprotein lipase, aprotinin, transcobalamin-vitamin B₁₂, apolipoprotein J/clusterin, RAP (receptor-associated protein), 25-(OH) vitamin D₃-vitamin D binding protein complex as substrates. Albumin, a major plasma protein, was initially reported as a substrate for megalin, but recently the involvement of another protein, cubilin, in albumin endocytosis was indicated. Namely, albumin binds to cubilin, and albumin-cubilin complex binds to megalin for endocytosis; the hypothesis which should be tested further. Megalin is also suggested to be the receptor for endocytosis of aminoglycosides. We have studied the possible role of megalin in in vivo renal accumulation of aminoglycosides. The positive relationship between megalin expression and amikacin accumulation in renal cortex, either under normal or disease condition, strongly suggests that megalin acts as an endocytic receptor for aminoglycosides. There are two important functional proteins for endosomal acidification, which is important for overall endocytic process; these are vacuolar H⁺-ATPase (V-H⁺-ATPase) and chloride channel, ClC-5. Cisplatin, an anticancer drug, often induces nephrotoxicity, which is accompanied by albumin(protein) uria. In order to clarify the mechanisms underlying albuminuria by cisplatin, we examined the effect of cisplatin treatment on V-H⁺-ATPase activity in the kidney. Proton transporting activity as well as ATP hydrolysis activity of V-H⁺-ATPase was markedly inhibited by cisplatin treatment. Similarly, receptormediated endocytosis of albumin was inhibited by cisplatin in OK cells and in vivo in rats. Reabsorption of a megalin substrate, vitamin D binding protein, was also inhibited and excreted into the urine. These findings suggest that albumin (protein) uria induced by cisplatin would be due to inhibition of V-H⁺-ATPase. Receptor-mediated endocytosis in the kidney is physiologically and pathophisiologically important process. Also, understanding the receptor-mediated endocytosis would help to consider drug delivery systems, especially those for proteins and genes with large molecular weights.

View full abstract