The flux of β-blocking agents increased by an ion-pair formation in the presence of fatty acids. In case of oxprenolol-caprate, it was found that the contribution of this ion-pair to the flux was about 98 % and it had a high permeability coefficient almost close to that of oxprenolol base. We compared the enhancing effect of the ion-pair formation both in the water vehicle and in the lipophilic vehicle. We analyzed the penetration enhancing effects on the basis of the two-layer skin model. In case of water vehicle, we found this enhancing effect was caused by the increase of partition coefficient between the vehicle and stratum corneum, and that ion-pair formation didn't increase the diffusion coefficient in the skin. In water vehicle only a small amount of drug could be absorbed through the skin, because of the low solubility of the lipophilic ion-pair, although it had high permeability coefficient. Therefore IPM was used as lipophilic vehicle. We experimented using IPM saturated with ion-pair. We found that the maximum flux value of propranololcaprylate and oxprenolol-caprate were about 3 and 4 times respectively higher than that of their bases, being caused by high solubility in IPM and high permeability coefficient. The permeability coefficient, however, decreased with the increase of lipophilicity of ion-pair. With the use of two-layer skin model it was proved that the decrease of permeability coefficient was caused by the decrease of its partition coefficient between the stratum corneum and the viable epidermis. It can be said that ion-pair formation bring about different effects in penetration process dependent upon what vehicle it is dissolved in.

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Barrier function of intestinal mucosa in the rat colitis models was assessed by electrophysiological parameters and membrane permeability of FITC-dextran 4000. In the trinitrobenzenesulfonic acid-induced colitis and acetic acid-induced colitis, potential difference, short circuit current and resistance tended to decrease with the increase in damage score, but the correlation was not so excellent that morphological assessment did not always reflect the changes in the mucosal function. Increase in the permeability was observed in these colitis models. In the dextran sulfate sodium-induced model, morphological change was found but significant difference in parameters was not. In acetic acid-induced model, the recovery of inflammatory sympton was observed with the recovery of parameters and permeability to the control values. These results suggest that the above assesment of membrane inflammation is more accurate and objective than macroscopic visible criteria which have been commonly used.

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Recently, Caco-2 monolayer has recieved considerable attention from the pharmaceutical industry because it may serve as a model for predicting oral drug absorption. In this report, Caco-2 monolayer was used to describe the oral absorption of (1) drugs which are absorbed via a carrier-mediated system, (2) an ester-type prodrug of anitibiotics, (3) peptide drugs which undergo the extensive degradation in the intestine. The result of each study was as follows: (1) The contribution of carrier-mediated transport to the total permeability of cephalexin to Caco-2 monolayer was much smaller than that to the rat jejunum obserbed in vivo. It was indicated that the prediction from the result of Caco-2 experiment might underestimate the oral absorption of cephalexin. (2) The enhanced oral absorption of cefotiam by its ester-type prodrug, cefotiam-hexetil, was well demonstrated in the in vitro experiment using Caco-2monolayer. Caco-2 monolayer was shown to have the similar esterase activity with that of a small intestine which cleaves the ester-bond of prodrug during the absorption. (3) The degradtion pattern of three peptides in the homogenate of Caco-2 cells are different from that in rat intestinal one, suggesting the inter-species difference of the mucosal peptid ase activity. The permeability of those peptides to Caco-2 monolayer was very low, thus, it was considered that the low pemeability, as well as the rapid degradation, was a main reason for their poor oral absorption. Finally, the effect of emulsion which solubilizes the high lipophlic drug on its oral absorption was studied using Caco-2 monolayer. From those results, the possibility and the limit of in vitro system using Caco-2 monolayer to predict the oral drug absorption were discussed.

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We have isolated a cDNA clone from a rat small intestinal library which encodes a functional monocarboxylate transporter (ratMCTl). The cDNA of MCT1 in chinese hamster ovary (CHO) cells was used to screen the cDNA library. The ratMCT1 cDNA predicted a protein of 494 amino acids with twelve potential transmembrane domains. The amino acid sequence showed 93.1% and 84.6% identity to the hamster and human monocarboxylate transporters, respectively. When transfected into Xenopus laevis oocytes, the ratMCT1 cRNA caused a significant increase in the uptake of monocarboxylic acids. The mRNA transcripts hybridizing to the ratMCT1 cDNA are detectable in the brain, heart, kidney, lung and muscle and also the rabbit intestinal enterocytes and Caco-2 cells. The size of the principal transcript is 3.3kb. [¹⁴C]Lactic acid uptake was pH dependent, saturable, stereospecific by using the rat intestinal brush-border membrane vesicles. In addition, several monocarboxylic acids were transported in Caco-2 cells in pH-dependent and saturable manners. These results suggest that the intestinal MCT1-related protein contributes to a carriermediated absorption for organic weak acid compounds.

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Function and tissue and intra-cellular distributions of rat intestinal oligopeptide transporter, rat PepT1 cloned as a homologue of rabbit PepT1, were studied. Functional characteristics of a rat PepT1 were evaluated by measuring transport activities expressed in Xenopus laevis oocytes injected with complementary RNA for PepT1 synthesized by in vitro transcription. Rat PepT1 exhibited a pH-dependent transport of [14C]glycylsarcosine and the transport was specifically inhibited by di- and tripeptides but not by their constitutive amino acids or tetra- and pentapeptides. Rat PepT1 also transported β-lactam antibiotics with pH-dependent and stereoselective manners. Furthermore, transport activities expressed in oocytes injected with intestinal total mRNA were abolished by the hybridization of the mRNA with an antisense oligonucleotide against PepT1. Polyclonal antibody against PepT1 was prepared and used for immunohistochemical analysis of PepT1. PepTi was strongly detected in intestinal epithelium, especially in the brush-border side. Accordingly, PepT1 was shown to function in the uptakes of oligopeptides and peptide-like drugs from intestinal lumen to epithelial cells.

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A complementary DNA (cDNA) encoding the rat H⁺-coupled peptide transporter, designated PEPT1, was isolated, and the transport characteristics of orally active β-lactam antibiotics were assessed by measuring uptake into Xenopus oocytes expressing the rat PEPT1. The rat PEPT1 cDNA encoded a 710-amino acid protein with 77% and 83% identity to the rabbit PEPT1 and human PEPT1, respectively. The message for rat PEPT1 was 2.9 kilobases and was found predominantly in the small intestine, whereas reverse transcription-polymerase chain reaction amplification revealed that the message was expressed both in the small intestine and in the kidney cortex. The 75-kDa protein was identified by translation of in vitro synthesized transcript of rat PEPT1 cDNA using rabbit reticulocyte lysates and by Western blot analysis using a specific antibody against the rat PEPT1. When expressed in oocytes, rat PEPT1 stimulated the uptake of ceftibuten (anion) and cephradine (zwitterion) in the presence of an inward H⁺ gradient, and the expressed upake was inhibited by excess dipeptides. Kinetical analysis revealed that ceftibuten has 14-fold higher affinity for the rat PEPT1 than cephradine. Thus, the rat PEPT1 mediates H⁺-coupled uphill transport of the oral β-lactam antibiotics across the brush-border membranes of intestinal and renal proximal tubular cells. We also isolated a cDNA encoding another member of peptide transporter family, rat PEPT2. The rat PEPT2 consists of 729 amino acids with 48% amino acid identity to the rat PEPT1. In contrast to the rat PEPT1, mRNA expression of rat PEPT2 was predominant in the kidney medulla, and was also detected in the brain and lung tissues, but not in the small intestine. These peptide transporters should be responsible for intestinal absorption and/or for renal handling of peptide-like drugs such as β-lactam antibiotics.

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The objective of this study was to demonstrate that main factor of improvement of intestinal drug absorption by protease inhibitors was opening the Tight Junction (TJ), and to investigate the effectiveness of protease inhibitors and the mechanism by which these inhibitors expand TJ. In vitro electro-physiological study showed that all of the inhibitors tested (Bacitracin, FK448, Camostat, Gabexate) increased membrane permeability of FITC-Dextran4000 (FD-4) and decreased the membrane resistance dose-dependently in rat colon.In situ experiment, concentration of FD-4 in plasma from colonic absorption was increased at the presence of protease inhibitors. These results show that promoting effects induced by these inhibitors in colon are based on opening the TJ.Molecular weight dependency was investigated with FDs of various molecular weights.The protease inhibitors increased the permeability of the high molecular weight compounds up to 20, 000 Da.This indicates that the protease inhibitors can be used to improve poly-peptide absorption in colon. Investigating the TJ opening mechanism, we observed the effect of Gabexate was reduced by acalmodulin antagonist, W7, which indicates the contraction of actin-myosin ring work for opening. The other inhibitors, however, was unaffected by the antagonist. these findings suggest that there are at least two mechanism of the protease inhibitors to open TJ.

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Recently, there is increasing interest in the targeting of these drugs to the colon because of the low activity of proteolytic enzymes in the colon. Therefore, many dosage forms such as a time controlled-release dosage form and a pH-sensitive coating dosage form were examined for the specific drug delivery to the colon. However, these approaches have recently been shown to lack site specificity, since the variability of pH and small intestinal transit time of these dosage forms were observed. On the other hand, chitosan, which is one of the polysaccharides widely distributed in nature, is known to be specifically degraded by microorganisms distributed in the colon. In this study, therefore, we prepared chitosan capsules containing insulin and examined the effectiveness of these capsules to colon-specific delivery of insulin.
The chitosan capsules containing CF or insulin were obtained from Aicello Chemical Co. Ltd (Toyohashi, Japan). The mean diameter and weight of these capsules were 3.5 x 1.6 mm and 1.2-1.5 mg, respectively. The surface of these capsules were coated with hydroxypropyl methylcellulose phthalate-4 and rosephthalate as enteric coating materials. The release studies of drug from the chitosan capsules were carried out using Japan Pharmacopoeia (J.P.) rotating basket method 6-Carboxyfluorescein (CF), which was encapsulated in the chitosan capsules was used as a water soluble model compound No release of CF from the capsules was observed in liquid 1 as an artificial gastric juice (pH 1) and liquid 2 as an artificial intestinal juice (pH 7). However, the release of CF was markedly increased in the presence of rat decal contents. These findings suggested that the chitosan capsules were degraded by the microorganisms in rat cecal contents. The effectiveness of the chitosan capsules to the colon specific delivery of insulin was investigated by an in vivo absorption experiment. A marked decrease in plasma glucose levels was observed following oral administration of these capsules containing 20 IU insulin and Na-glycodtolate, as compared with the capsules containing lactose or insulin only. In addition, the chitosan capsules containing insulin and Na-glycocholate were more effective for reducing the plasma glucose levels than the gelatin capsules containing the same components. Thus, this capsule may be a useful carrier for colon-specific delivery of peptides including insulin.

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Disposition of micronized cyclosporine A (CyA) dry powder in rats administered by inhalation route was characterized by physiologically-based pharmacokinetic (PBPK) model. At the end of one hour inhalation (10 mg/kg), 30 % of dose was deposited in the lung, while the rest (70 %) was recovered in digestive organs (mouth, stomach, and intestine). PBPK model analyses on the blood and tissue measurements post-inhalation suggested only 5 % of dose was absorbed via pulmonary route; the rest in the lung (25%) was considered slowly (t_1/2>20h) transfer to mouth and swallowed into digestive route. These kinetic information help assessing efficacy (when applied to bronchial asthma) and toxicity (systemic exposure) in a dynamic fashion.

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We examined the in vitro penetration characteristics of FITC-dextrans (FDs) with various molecular weights and peptides such as calcitonin and insulin across human lung carcinoma A-549 cell monolayer. The permeability coefficients of FDs through the A-549 monolayers (type-II like cells) were much larger than those in the rat alveolar epithelial cell monolayers (RAEMs: type-I like cells), which were established by Kim and coworkers. The high permeability characteristics of A-549 monolayers may be attributed to their morphological differences as compared with the RAEMs.
On the other hand, we studied the degradation characteristics of peptide drugs such as calcitonin and insulin using rat lung and A-549 cell homogenates. Calcitonin was rapidly disappeared than insulin in both homogenates. The apparent degradation clearance (Km/Vmax) values of these peptides were higher in the cytosol fraction than membrane fraction. In both homogenates, chymotrypsin activity was the highest among the six protease activities such as trypsin, elastase, chymotrypsin, Leu-aminopeptidase, aminopeptidase B and aminopeptidase N examined in this study. These results confirmed our previous report that the degradation of insulin in the rat lung homogenate was suppressed by the addition of soybean trypsin inhibitor.
Furthermore, the penetration characteristics of water soluble compounds, such as phenol red and FDs across the Xenopus isolated pulmonary membrane were examined with the modified Ussing chamber system. The permeability coefficients of FDs with various molecular weights through the Xenopus isolated pulmonary membrane were similarto those through the small and large intestine. A good correlation was observed between the penetrated amounts of drugs across the Xenopus isolated pulmonary membrane with the in vitro Ussing chamber system and their absorbed amounts calculated by deconvolution method in an in situ rat intrapulmonary absorption experiment. Three types of absorption enhancers, such as laurylmaltoside, mixed micelle and Na-caprate, increased the penetration of phenol red across the Xenopus isolated pulmonary membrane, while EDTA and Na-salicylate did not affect its penetration across the Xenopus isolated pulmonary membrane. These results were in agreement with our previous report of an in situ rat intrapulmonary experiment.

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In order to establish the strategy for targeted delivery systems for protein drugs by chemical modification, cellular uptake characteristics and cytotoxicity of model proteins and macromolecules were systematically studied using cell culture systems. In uptake studies, it was demonstrated that succinylated proteins were taken up by macrophages and brain microvessel endothelial cells via scavenger receptor mediated pathway whereas cationized proteins were taken up by these cells via adsorptive endocytosis. Efficient cellular uptake was observed for mannosylated proteins by macrophages based on mannose receptor mediated endocytosis. In cytotoxicity studies, neutral polymer and polyanions showed no significant cytotoxicity while polycations showed diversity in cytotoxic effect. On the basis of these findings, we studied the inhibitory effect of superoxide dismutase (SOD) derivatives, mannosylated SOD and cationized SOD, on superoxide anion release from macrophages. These SOD derivatives showed remarkable effects compared with unmodified SOD, indicating mannosylation and cationization are useful methods for targeted delivery of SOD.

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The uptake of fractionated [³H]heparin was examined to elucidate the uptake mechanism in isolated rat Kupffer cells. The equilibrium binding of high molecular weight fractionated [³H]heparin (HMWFH; 23000 Da.) to Kupffer cells was concentration-dependent with the dissociation constant of 5.7 nM and the maximum binding capacity of 1.5 pmol/ 10⁶ cells. Several ligands of scavenger receptors inhibited the binding of HMWFH to Kupffer cells competitively and also the internalization of HMWFH, suggesting the involvement of scavenger receptors in the uptake of HMWFH. HMWFH was also suggested to be internalized according to first order kinetics with the apparent internalization rate constant of 0.010 min^-1. Lowering temperature from 37° to 4°C reduced the fraction internalized from 33% to 6% without affecting the total association, while the fraction internalized at 25° was comparable with that at 37°C. Metabolic inhibitors (2, 4-dinitrophenol and rotenone), an inhibitor of receptor-mediated and absorptive endocytosis of polypeptides (phenylarsine oxide) and phagocytosis inhibitors (cytocalasine B and colchicine) did not inhibit the internalization of HMWFH. These results suggested the scavenger receptor-mediated uptake of HMWFH is ATP-independent and different from the receptor-mediated and absorptive endocytosis of polypeptides and phagocytosis, although, as for temperature dependency, it showed the typical characteristics of receptor-mediated endocytosis. Low molecular weight fractionated [³H]heparin (LMWFH; 10000 Da.) was also suggested to be taken up by scavenger receptors. The binding affinity to scavenger receptors and apparent internalization rate constant were reduced with the decrease in molecular weight, suggesting that the molecular weight of fractionated heparin is one of the major factors determining the uptake by scavenger receptors.

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Among the HMG-CoA reductase inhibitors, pravastatin which is hydrophilic in nature, has been shown to exhibit relatively specific inhibition of cholesterol synthesis in the liver. As one of the reasons for this relatively specific pharmacological activity, we demonstrated that the tissue distribution of pravastatin is limited because of its high hydrophilicity, while hepatic uptake by active transport mechanism via multispecific anion transporter takes place at the liver surface. We examined the hepatic elimination of HMG-CoA reductase inhibitor, pravastatin, at steady-state by measuring the plasma concentration in both arterial (Ca, ss) and hepatic venous (Chv, ss) blood. The hepatic extraction ratio at steady-state showed a clear decrease with increasing pravastatin infusion rate. The total hepatic elimination velocity (V) at steady-state exhibited the Michaelis-Menten type saturation with the mean logarithmic concentration (C), which is used in the parallel-tube model, and the values of Km and Vmax estimated from the plot of V vs. C were comparable with those obtained from the analysis of the initial uptake velocity using isolated hepatocytes. This result indicates that overall intrinsic clearance (CLint, all) was regulated by the uptake process, since pravastatin was efficiently taken up by an active transport mechanism followed by rapid metabolism and/or biliary excretion. Pravastatin is a typical drug whose clearance in the liver, which is the target organ in terms of its pharmacological effect as well as the major organ for its pharmacokinetics in the whole body, is governed mainly by an uptake process mediated by active transport.

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Recently, for many endogenous and exogenous compounds(including drugs), it has been reported that carrier-mediated primary active transport contributes to biliary excretion. Compounds known to be excreted by this mechanism are anticancer drugs, endogenous bile acids and organic anions including glutathione and glucuronic anid conjugates. Primary active excretion into bile means the positive removal of xenobiotics from the body, and the elimination process is now designated as “Phase III” (T.Ishikawa, Trends Biochem.Sci., 17, 1992) in the detoxification mechanisms for xenobiotics in addition to Phase I by P-450 and Phase II by conjugation. There exist multiplicities in the biliary excretion mechanisms. Clarification of these multiplicities in transport is necessary not just from a biochemical point of view, but for our understanding of the physiological adaptability of the living body in terms of the removal (detoxification) of xenobiotics. This may provide a lot of important informations for studying the pharmacokinetics of new drugs. In this presentation, I would like to summarize the latest experimental results on carrier-mediated biliary excretion systems and the multiplicities in the biliary excretion of xenobiotics, including small peptides.

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Male F344 rats were treated with a chemical (aniline, nitrobenzene, 2-methoxy-p-phenylenediamine, 2-methoxy-4-nitroaniline or 2-methoxy-4-nitroazobenzene) produced by the azo-reduction and/or N-oxidation of 2-methoxy-4-aminoazobenzene, a selective inducer of CYP1A2, and effects of their chemicals on CYP1A induction in the liver were examined at the levels of mRNA, protein and enzyme activity. 2-Methoxy-4-nitroaniline and 2-methoxy-4-nitroazobenzene, but not other chemicals used, induced CYP1A enzymes, mainly CYP1A2, as assessed by Western and Northern blots. It is noteworthy that 2-methoxy-4-nitroaniline was more selective than 2-methoxy-4-aminoazobenzene for induction of CYP1A2, and it has the smallest molecular size among known CYP1A2 inducers. Although in induction of CYP1A1 by a polycyclic aryl hydrocarbon (Ah) is thought to be regulated though Ah receptor, cellular component(s) other than Ah receptor would play an important role in the induction of CYP1A2. Presence of a structural unit such as “bay-region” in benzo[a]pyrene molecule or “polyhalogenated aromatic ring” in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin molecule is reportedly requisite for binding of the chemical to Ah receptor, but such structural unit is absent in 2-methoxy-4-nitroaniline molecule.

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Six lines of mice transgenic for the human fetus-specific CYP3A7 have been established using microinjection method. The CYP3A7 transgene was found to be integrated into mouse genome randomly with different copy numbers, ranging from 1 to several hundreds. Northern blot analysis revealed that the transgene expressed in livers only in line M10, whereas all except line M10 expressed the transgene in kidneys. In line M10 mice, increases of weight were observed in the liver, kidney, and uterus while the testis was found to decrease in weight, comparing to their non-transgenic littermates. In addition, serum total testosterone was significantly elevated in line M10 mice, indicating the involvement of CYP3A7 in the metabolism of steroid hormones.
The CYP3A7 expressed in the transgenic mice activated AFB₁ in vivo resulting in significant increases of DNA damage in the livers and kidneys in line M10 and M2 mice, respectively. The data of midazolam metabolism using line M 10 mice also revealed that CYP3A7 mainly catalyzed hydroxylation at 1' position, similar to CYP3A4 which is an adult-specific isozyme in the CYP3A subfamily.

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Levels and catalytic activities of cytochrome P450 (P450) enzymes involved in the oxidation of drugs and carcinogens were determined in human adult lungs and fetal livers and compared with those in microsomes from adult livers. P450s immunoreactive with anti-human P450 1A1 and anti-human P450 3A antibodies were detected in fetal liver microsomes by immunoblotting analysis, and P450s related to P450 1A1, 2A6, 2C9, 2E 1, and 3A4 were determined in adult lung microsomes; all of these P450 enzymes were detected in much higher amounts in adult liver microsomes except that P450 1A2 was only the 1A subfamily of P450 found in adults. Drug oxidation activities with several substrates were determined in these microsomes, and we found that none of the activities were higher in microsomes of adult lungs and fetal livers than in adult livers. Activation of procarcinogens by P450 enzymes was also examined and it was found that activities with (+)- and (-)-enantiomers of 7, 8-dihydroxy-7, 8-dihydrobenzo[a]pyrene were higher in fetal liver microsomes than adult lung or liver microsomes. Anti-human P450 1A1 and 1A2 and a-naphthoflavone, known to inhibit P450 IA-related activities, did not affect these procarcinogen activation in fetal liver microsomes. Fetal liver microsomes catalyzed activation of aflatoxin B₁ and sterigmatocystin, although activation of carcinogenic arylamines was much lower in microsomes of fetal livers and adult lungs than of adult livers. These results suggest that in human fetal livers at least two P450 enzymes, namely P450 IA-related enzyme and P450 3A7, are actually expressed and these enzymes are involved in the activation of the (+)- and (-)-enantiomers of 7, 8-dihydroxy-7, 8-dihydrobenzo[a]pyrene and the carcinogenic mycotoxins, respectively. In adult human lungs, several P450 enzymes are expressed, though the precise roles of these enzymes in the oxidation of xenobiotics were not determined due to the low level of expression of these P450s.

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A new P450 responsible for mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) which is a potent procarcinogen was purified from renal microsomes of male mice using an index of umu gene expression. The purified P450 had high bioactivation toward 2-aminofluorene (2-AF), 2-aminoanthracene (2-AA), and 3, 3'-dichlorobenzidine (DCB) as well as 3-MeO-AAB. The antibody against this P450 completely inhibited mutagenic activation of 3-MeO-AAB, 2-AF, 2-AA, and DCB by mouse renal microsomes. With immunoblotting, this form was present abundantly in renal microsomes of male mice but not in those of female mice. This P450 was also present in mouse pulmonary and bladder microsomes but not in hepatic microsomes. The NH₂-terminal amino acid sequence analysis indicated that this form belonged to the CYP4B subfamily. Thus, mouse kidney cDNA library was screened with rat CYP4B1 probe. The cDNA-deduced amino acid sequence of isolated cDNA consisted of 511 amino acids and the NH₂-terminal amino acid sequence of the purified renal P450 agreed with the cDNA-deduced amino acid sequence, indicating that renal P450 purified in this study is mouse CYP4B1. Human pulmonary and renal cDNA library were screened with mouse CYP4B1 cDNA probe. Two full-length cDNAs were isolated and they had two or three amino acid changes to human CYP4B1 reported previously.

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In the design of effective and safe drugs for oral administration several ‘biological aspects’ are crucial. These include the extent to which the parent drug is absorbed from the gut and the nature and extent of metabolism either in the gut contents and/or during transport across the gut wall into blood. The most unambiguous method of measuring both the absorption of drugs from the gut lumen and, in particular, the activation or deactivation of drugs by the gut, is using the isolated vascular perfused intestine. In the basic model, a segment of intestine extending from the pyloric sphincter to the caecum is isolated and perfused via the mesenteric artery with diluted oxygenated heparinised blood at 37°C and 60-80 mmHg pressure. Venous blood from the portal vein is re-oxygenated and returned to the blood reservoir. Bile is infused into the gut lumen via a cannula inserted into the bile duct. For absorption/metabolism studies the drugs are administered directly into the gut lumen. However, when the contribution of the gut to the metabolism of blood-borne drugs is required, the compounds are added directly into the blood perfusate. After dosing blood samples are removed, at time intervals, up to 4 hours for quantitative and qualitative analysis. Alternatively, all the effluent perfusate from the portal vein may be collected for first pass studies. This presentation will deal with the transport and metabolism in the gut of a variety of compounds of disparate structures including low molecular weight, organics, steroids and peptides.

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Liver microsomal carboxylesterases (CES) function in the hydrolysis of a wide variety of endogenous and xenobiotic compounds, and play an important role in drug and lipid metabolism in many mammalian species. In the present study, twenty-seven CES isozymes were purified to electrophoretic homogeneity from liver microsomes of ten mammalian species and humans, and their physical, enzymological and immunological properties were compared with each other. The CES isozymes from various species examined here showed considerable similarities in physicochemical and immunochemical properties, but not similar in substrate specificities. λZap II library from mouse liver, and λgt11 libraries from human and rat liver were screened with antibodies raised to a purified liver CES isozymes. The deduced amino acid sequence of the clone possessed many structural characteristics that are highly conserved among rat RL1, RH1, RHlec, RS1, RS2, mouse ML1, MH1, MS1 and human HU1, including active site sequence(GESAGG, NKQEXG, GDHXD), and four cysteines which may be involved in the specific disulfide bond. It is well known that proteins which are retained in the endoplasmic reticulum (ER) lumen contain the retention signal at their carboxy terminal of the tetrapeptide. The five CES clones (RL1, RH1, RHlec, MH1, and HU1) also contained an ER-retention signal (HXEL). When clone was expressed in COS cells, the plasmid-coded protein was retained. The cells expressing CES is very high activity towards xenobiotic ester and amide. In conclusion, liver microsomal CES in mammals and humans are closely involved in drug and lipid metabolism in the endoplasmic reticulum, and it is noteworthy that the isozymes from various species examined here showed considerable similarities in amino acid sequences, but not similar in substrate specificities. These reasons may be, at least in part, due to the variances of substrate binding site, although the sequence is not clarified yet.

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Cytochrome P450 isozymes involvement in the metabolic activation of tetrahydrocannabinol (THC) have been investigated. CYP2C29 was a major isozyme responsible for 11-hydroxylation as well as 7α- and 8 α-hydroxylation of THC in the mouse liver. In rats, CYP2C11 and CYP2C6 were major isozymes involvement in the 11-hydroxylation of THC in the male and female rat liver, respectively. P450G-A belonging to CYP2B subfamily was a major isozyme for metabolizing THC in the guinea pig liver. In monkey liver, P450RM-A (CYP2A) and P450JM-D (CYP2C), and P45OJM-C (CYP2B) were responsible for the 11-hydroxylation and 3'-hydroxylation of TUC, respectively. the 11-hydroxylation of TUC In human liver was also catalyzed by P450 isozyme belonging to CYP2C, weheras 8 β-hydroxylation of Δ⁹-TIC and 7 α-hydroxylation of Δ⁸-THC were catalyzed by CYP3A. The present results indicate that CYP2C Is mainly responsible for the metabolic activation of 1HC in most of animals including humans, while CYP2A has some role in the metabolic activation of 1W in the monkey liver.

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We examined the mechanisims of inhibition andelelvation of CYP enzyme activities in liver microsomes (Ms) from propranolol (PL)-pretreated rats. In the studies on the inhibition, we measured radioactivities derived from side chain-[14C]-labeled and aromatic ring-[14C]-labeled 4-hydroxypropranolols (4-OH-PL) after incubation with Ms and NADPH. Radioactivities were significantly higher in aromatic ring-labeled 4-OH-PL than in side chain-labeled one. A strain difference was observed in radioactivities derived from the aromatic ring-labeled 4-OH-PL(Wistar>DA), but not in those from the side chain-labeled one. We speculated that CYP2D enzymes converted PL to 4-OH-PL, which was then converted to 1, 4-naphthoquinone (1, 4-NO), causing the enzyme inhibition by covalently binding to apoprotein. However, 1, 4-NQ or 1, 4-dihydroxynaphthalene, when added to the incubation medium containing Ms and NADPH, did not inhibit CYP2D activities. The results suggest that some other metabolite(s) together with 1, 4-NQ could contribute to the enzyme inhibition. In the studies on the elevation of enzyme activities, we observed that elevated PL N-desisopropylase activity highly correlated with O-deethylase activities of phenacetin (PA) and ethoxyresorufin (ER) in liver microsomes from PL-pretreated rats. After repeated administrations of PLrecemate and enantiomers to rats, the potencies in elevation of CYP1A1 protein and activities (ER oxidation and PL N-desalkylation) were RS>S>R, whereas those in elevation of CYPIA2 protein and activities (PA oxidation and PLN-desalkylation) were RS>R>S. In the primary culture system of rat hepatocytes, potencies in elevation of CYP1A1 protein and the activities were R>RS>S, and no effect was observed from CYP1A2. These results indicate that elevation of PLN-desisopropylase activities could be caused by induction of CYPIA enzyme by repeated administration of PL.

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Inhibitory effect of E4080 on the cytochrome P450 was studied using laboratory animals in vitro and in vivo.
In vitro study E4080 has a high affinity for P450 heme in rat, dog, monkey and human liver microsomes. In rat microsomes, E4080 inhibited testosterone 6β and 16β-hydroxylase which are involved in CYP2B and CYP3A. Using human microsomes, E4080 inhibited metabolism of 7-benzyloxyresolfin, tolbutamide, bufuralol and testosterone. These results suggest that E4080 inhibits CYP 2B6, 2C9, 2D6 and 3A4 in human liver microsomes.
In vivo study E4080 prolonged antipyrine half-life and decreased antipyrine clearance in rats, dogs and monkeys at the doses of 10mg/kg which exert pharmacological effect postulated from the minimal effective plasma concentrations(1-2 μg/ml ). The results may be suggested that E4080 interacts with antipyrine metabolism which is thought to be carried out by CYP 1A, 2C and 3A.
In the clinical trial E4080 (100mg/man) decreased the excretion of urinary 6β-hydroxycortisol which is a marker of human hepatic cytochrome P4503A induction and inhibition.
In conclusion, it is suspected that E4080 inhibits CYP3A not only in animals, but also in human and should be noticed drug-drug interaction in clinical use.

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The relationships between plasma or brain concentrations and analgesic effects of both buprenorphine (BN) and its metabolite, norbuprenorphine (NBN), were investigated in rats. Maximal analgesic effects were obtained at 10 and 30 min after iv administration of BN (8 μg/kg) and NBN (100 μg/kg) administration. No correlation was observed between analgesic effect and the early plasma BN or NBN concentrations. However, a good correlation was found between the estimated specific binding concentration of BN in the brain and the analgesic effects. Further, it was suggested that the intrinsic analgesic activity of NBNwas one-fourth that of BN. We concluded that the remarkabley weak analgesic effect of NBN after iv administration may be due not only to the low permeability of NBN into the brain but also to its small intrinsic analgesic effect.
Further, the toxicodynamics of BN and NBN on respiratory depression was studied. Although the respiratory rate after iv bolus administration of BN did not change within the range of 1-3 mg/kg dose, that after administration of NBN was decreased dose-dependently. The action of respiratory depression of NBN was approximately 10 times more potents than that of the parent drug. The respiratory rate was not influenced after directly injection of NBN into 3 different brain regions. The respiratory depression induced by NBN was rapidly reduced after supplement of naloxon and β-FNA. These results suggest that the respiratory depression by NBN may be mediated by opioid μ-receptors in the peripheral tissue, probably in the lung, rather than in the brain.

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Ebselen, 2-phenyl-1, 2-benzisoselenazol-3(2H)-one, is a selenoorganic antioxidant with glutathione peroxidase (GSH-Px)-like activity. After oral administration, ebselen reacted with thiol group of plasma proteins (albumin) in rat, forming a selenosulfide complex (Se-S complex) and unchanged ebselen was hardly found. We focused on the GSH-Px-like activity of the complex itself. Se-S complex prepared from ebselen and BSA showed GSH-Px-like activity towards hydroperoxides. Other selenosulfide and diselenide derivatives as well as ebselen showed similar GSH-Px-like activity to that of the complex. Ebselen is extensively metabolized in vivo, and authentic samples of main urinary and biliary metabolites have no antioxidant activities. We therefore investigated metabolic patterns in tissues and found two polar unknown metabolites in rat fat. These metabolites were isolated, analyzed by HPLC/MS, and identified as ebselen Se-oxide (SeOx) and 2-(methylseleninyl)benzanilide (Se(O)Me). SeOx can be reduced back to ebselen by reductant such as GSH. Interestingly, the isolated Se(O)Me afforded ebselen and its Se-oxide in a solution left standing. Either metabolite can revert to ebselen. Se-S complex as well as these metabolites are possibly responsible for the pharmacological activity shown by oral dose of ebselen in vivo.

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An attempt was made to elucidate the mechanism for accumulation and retention of imidazole derivatives in the connective tissue by using the radio-labeled imidazoles, [2-¹⁴C]imidazole and 2-methyl[2-¹⁴C]imidazole. Distribution studies in rats revealed that these compounds were retained in the connective tissue, probably with the irreversible binding to elastin. The binding formation could not be ascribed to spontaneously occurring noncatalytic reaction or to P450-mediated activation. Although pretreatment of rats with a xanthine oxidase (XOD) inhibitor allopurinol (ALP) resulted in decreased levels of tissue-bound aortic radioactivity from the imidazoles, in vitro binding to aortic tissue was scarcely observed in the presence of purified XOD. On the other hand, certain oxidation products generated in copper(II)/ascorbate system (Cu/VC) were shown to bind firmly to aortic tissue. Under this condition, addition of ALP decreased the tissue binding. ALP can form the complex with both Cu and VC in vitro as reported previously. In view of this information, in vivo ALP-treatment effect described above may be well explained by inhibition of Cu/VC-catalyzed oxidation. Moreover, from LC/MS/MS analysis of the oxidation products in Cu/VC, it was conceivable that imidazolones bind to α-aminoadipic-δ-semialdehyde (allysine) residues in elastin or collagen. Based on these findings, the nonenzymatic oxidation process is considered to be one of the major causative elements in the metabolic activation of imidazole derivatives. Further studies using LEC rat, an animal model of Wilson's disease, are currently being carried out.

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Sixteen deaths occurred among patients orally co-administered the new antiviral, sorivudine, with fluoropyrimidine anticancer agents in Japan 1993. The objective of our present research is to investigate the mechanism for the severe toxicity exerted by simultaneous administration of sorivudine and the typical fluoropyrimidine, tegafur (FT) in rats. When rats were co-administered FT with sorivudine for 6 days, a remarkable decrease in body weight and death of the animals were induced. A marked suppression of bone marrow, marked decreases in white blood cells and platelets and marked atrophy in jejunal mucosa were observed in the co-administered rats. Administration of FT alone did not induce such toxicological changes in the rat. The toxic effects were demonstrated to be caused by the accumulation of 5-fluorouracil (5-FU) in the co-administered rats, i.e. AUC and Cmax of 5-FU in co-administered rat plasma were 10 and 17.5 times higher than those in FT-treated rats, respectively. Hepatic dihydropyrimidine dehydrogenase (DPD), a rate-limiting factor for the catabolism of 5-FU, was significantly inactivated in rats repeatedly administered with sorivudine as well as with 5-bromovinyluracil (BVU), a major metabolite of the antiviral in gut flora, resulting in a marked accumulation of 5-FU from FT co-administered. An in vitro study using DPD purified from rat liver indicated that in the presence of NADPH the radioactivity of [¹⁴C]BVU was incorporated into the DPD protein in manner of covalent binding stoichiometrically with complete loss of its activity. Sorivudine had no activity to inactivate purified DPD under the same conditions. These results demonstrated that the lethal toxicity exerted by co-administration of FT and sorivudine was due to the marked accumulation of 5-FU, which resulted from inactivation of DPD by its modification with a reactive metabolite formed from BVU.

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