A novel membrane-bound esterase was purified from hepatic microsomes of male ddN mice using ω-aminooctyl Sepharose-4B, DEAE-5PW, CM-Sephadex C-50 and/or hydroxylapatite column chromatographies. The purified enzyme (ES46.5K) showed a single band on a sodium dodecylsulfate-polyacrylamide gel with a mimimum molecular weight of 46.5 Kd. The N-terminal amino acid sequence of the enzyme had no homology to those of known carboxylesterases. ES46.5K exhibited esterase activities toward 11-acetoxy derivatives of tetrahydrocannabinol (THC), whereas the enzyme did not hydrolyze phenolic acetates of cannabinoids (Δ⁸-THC, Δ⁹-THC and cannabidiol) except for 1-acetyl-cannabinol. ES 46.5K also possessed esterase activity toward p-nitrophenylacetate, 1-naphtylacetate and 2-acetylaminofluorene. Emulgen 911 and deoxycholate affected differently on the esterase activity of ES46.5K and carboxylesterase purified from porcine liver. Immuno-reactive protein to antibody against ES46.5K was present in hepatic microsomes from mice and rabbits, but not those from rats, guinea pigs and monkeys.
The present study demonstrates that mouse hepatic microsomes contain a novel type of esterase having molecular weight of 46.5 Kd and high catalytic activity toward 11-hydroxy-esters of cannabinoids.

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It is very interesting problem whether microsomal N, O-acetyltransferase (AHAT), which catalyzes the activation of carcinogenic arylacetohydroxamic acids, and carboxylesterase (CEase) are identical. Since hamsters have greater hepatic microsomal AHAT activities than other species, the microsomal AHAT in this species was investigated.
Two distinct microsomal AHATs (AT-I: pI 5.9, MW 61kDa and AT-II: pI 5.4, MW 60kDa) and a major CEase (CE-I: pI 5.9, MW 180kDa, subunit 58kDa) were purified homogeneously, as judged by SDS-PAGE. Both purified AT-I and AT-II catalyzed not only the N, O-acetyltransfer of N-hydroxy-2-acetylaminofluorene but also the hydrolysis of 4-nitrophenyl acetate, a typical substrate for CEase. The enzyme activities were strongly inhibited by paraoxon. A rabbit anti-serum raised against AT-II reacted with AT-I, but not with CE-I. The N-terminal amino acid sequences of AT-I and AT-II were highly homologous with that of the CEase form 2 purified from rabbit liver microsomes, but not with those of CE-I and major hepatic CEases of other species.
Two distinct cDNA, HmAT51 and HmCE31, were isolated from hamster liver λgt11 cDNA library using anti AT-II and anti CE-I antibodies, respectively. Amino acid sequence deduced from HmAT51 eDNA was highly homologous with that of rabbit CEase form 2 (66%), and weakly with that deduced from HmCE31 cDNA (46%) which had a high homology with those of rabbit CEase form 1 (76%) and major CEase of other species (73-88%). Also, both amino acid sequences deduced from HmAT51 and HmCE31 cDNA showed many structural features highly conserved among CEase, including active sites, C-terminal retention signal, and glycosylation sites.
The present study demonstrates that hamster liver microsomal AHAT activity is attributable to a member of the form 2 type CEase, but not to the major CEase, CE-I.

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Twenty-five forms of carboxylesterase isozymes were purified to electrophoretic homogeneity from liver microsomes of rats, mouse, hamster, guinea pig, rabbit, beagle dog, pig, cow, cynomolgus monkey, rhesus monkey and humans by the same procedure used, and their physical, enzymological and immunological properties were compared with each others. The substrate specificity and immunological reactivity of liver microsomal carboxylesterase from above animals were also examined for comparison. The twenty-five purified preparations have similar subunit weight (57, 000-64, 000), but their isoelectric point differ widely (4.3-6.5). Anti-rat RH1 IgG was found to possess high cross-reactivity with many isozymes tested by immunoblotting analysis. The amino-terminal amino acid sequences showed a striking homology, except for monkey MK2. However, differential capability of acylation/deacylation of endogenous and xenobiotic compounds by above carboxylesterase isozymes from various mammals and humans. Hepatic microsomal carboxylesterases in mammals play an important role 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 physical and immunochemical properties, but not similar in substrate specificity.

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The metabolism of arylamines was examined focusing on their N-acylation and deacylation of arylacylarmdes in mammalian species, intestinal bacteria and fish. When 2-amino fluorene (AF), 2-acetylaminofluorene (AAF) or 2-formylaminofluorene (FAF) was given orally to rats, ring-hydroxylated metabolites of AF, AAF and FAF were isolated from the urine. Formamidase and arylamine acetyltransferase purified from rat liver exhibited a significant N-formylating and N-acetylating activities toward the arylamines, respectively. The experiments with rat liver preparations showed that the deacetylating and deformylating activities are due to carboxylesterase and formamidase, respectively. Clostridium sporpgenes, Lactobacillus bifidus, Salmonella typhimurium and Escherichia coli exhibited significant N-acetyhting and N-formylating activities toward AF and 1-aminopyrene (AP), but not deacetylating activity toward AAF and FAF. When goldfish were bathed in AP solutions, 1-acetylaminopyrene (AAP) and 1-formylaminopyrene (FAP) were isolated and identified from the excetra of the fish. Liver cytosols of sea bream exhibited N-acetylating and N-formylating activities toward AP in the presence of acetyl-CoA and N-formyl-L-kynurenine, respectively, and deacylating activities toward AAP and FAP.

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The molecular basis of acetyltransferase polymorphism has been studies in hamster models of human acetylation polymorphism. We purified two forms of acetyltransferase (AT-I and AT-II) from hamster liver and reported that the acetylation polymorphism of hamster is concerned with expression level of AT-II. In this study, we investigated the genetic basis of the polymorphism. AT-I cDNA was isolated from a hamster liver cDNA library using a specific polyclonal antibody raised against AT-I. The cDNA insert consisted of 1181 nucleotides, including an open reading frame of 870 nucleotides encoding 290 amino acids (Mr 33, 503). Using AT-I cDNA as a probe, two genomic DNAs, AT-A and AT-B, were isolated from a hamster (slow acetylator) genomic library. AT-A gene located 5'-region of AT-B gene with the same orientation. Nucleotide sequence of AT-A was the same as the open reading frame of AT-I cDNA but differed in the 5'-noncoding region. Nucleotide sequence of AT-B showed high similarity with that of AT-A in the analyzed region and contained an intronless coding region encoding 290 amino acids (Mr 33, 836). The deduced amino acid sequences of AT-A (AT-I) and AT-B shared the highest similarity with those of mouse NAT1 (83.2%) and NAT2 (93.1%), respectively. The sequence of AT-B also showed high similarity (81.7%) with that of human NAT1. Expressed AT-I and AT-B showed the same migration on SDS-polyacrylamide gel electrophoresis and similar catalytic properties with purified AT-I and AT-II, respectively. These data suggest the possibility that AT-B gene encodes AT-II. Specific oligonucleotide to AT-B was used for Northern blotting analysis as a probe. A single band was observed in rapid and slow acetylator samples. Then the coding region of AT-B was directly isolated from slow acetylator genomic DNA by the polymerase chain reaction and analyzed the sequence. AT-B from slow acetylator contained 4 nucleotide changes and one of them at position 727 (C -> T) caused the deduced amino acid change (Arg -> End) at position 243. This result is consistent with the idea that the truncated protein produced in the slow acetylator is rapidly processed and consiquently is not detectable in vivo.

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Among experimental animals, Suncus murinus (suncus) which belongs to the family of Soricidae of Insectivora is usually used as an excellent animal in emesis research (5, 6). Suncus, however, have been characterized very poorly with regard to drug-metabolizing enzymes. We have recently found that N-acetyltransferase (NAT) activity could not be detected in suncus liver (1).
To examine further whether or not suncus was an NAT-deficient animal, 2-aminofluorene (2-AF) was intraperitoneally administered to suncus. After injection, blood concentration of 2-acetylaminofluorene (2-AAF) was measured. As expected, blood concentration of 2-AAF was not detected in suncus for any period. Moreover, NAT mRNA could not be detected in suncus liver.
Next, we investigated mutagenicity of various arylamines using TA98/1, 8DNP₆ in the presence of cytosols of suncus and rat. Mutagenicity of 2-AF were not observed in the use of suncus liver cytosols in contrast with those of rat. Less mutagenicities of other arylamines such as 4-aminobiphenyl, IQ, MeIQ and Glu-P-1 also increased in the use of suncus liver cytosols than those of rat. On the other hand, Trp-P-2 showed almost equal mutagenic activity when liver cytosols of rat and suncus were used. Comparison of suncus liver cytosols with those of rat is capable of informing us of carcinogens which NAT and/or OAT mainly convert into ultimate carcinogens.

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Prothiophos oxon or its S-alkyl homologs(alkyl: methyl, ethyl and butyl) was incubated with a mixture of rat liver microsome and cytosol at 37 °C for 30 min in the following conditions: (a) without NADPH and GSH, (b) with NADPH (condition for sulfoxide formation) and (c) with both NADPH and GSH. Each reaction mixture was lyophilized after adding sucrose and fed to adult female houseflies (susceptible Takatsuki strain). Their mortality was observed at intervals until 48 hr. Dose for each compound was adjusted based on its LD₅₀ value so that the time required for the residual oxon to exert its insecticidal activity was delayed. In the case of S-methyl compound, the mortality in (c) was similar to that in (a), although the remaining oxon in (c) was about half of that in (a). This suggests that the insecticidal effect in (c) may also have been given by something other than the oxon. The mortality in (c) was also higher than that in (b), although the amount of the remaining oxon was the same. Considering the unstability of the sulfoxide formed in the reaction mixture in (b), something stabilized in (c) may have contributed to the insecticidal activity. Without cytosol, the mortality in (c) decreased. The similar tendency was observed with other compounds. When bovine erythrocyte AChE was added to the above incubation mixture, the AChE was inhibited by the reaction mixture strongly in (c) and when more GSH was added regardless of the S-alkyl groups used. The reaction mixture was given to susceptible and resistant (higher glutathione S-transferase activity) strains. The death started later in the resistant strain than in the susceptible strain in (a), but was accelerated in (c) and the period of high mortality was longer in (c) than in (a). The mortality was different between (a) and (c) only during the initial period in the susceptible strain. The above result is well explained by postulating that S-alkyl phosphorothiolate compounds produce sulfoxide that inhibit AChE, while the sulfoxide give another active intermediates conjugated by the GSH-glutathione S-transferase system that also inhibit AChE. These two actions combined are to give an insecticidal effect.

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The inducing effects of musk xylene, a non-mutagenic carcinogen, on hepatic glutathione S-transferase (GST) in mice were investigated. Musk xylene posessed a potency of inducing all isozymes of mouse GSTs, whereas the effects were different between male and female mice. Induction of both GST MI (α class) and GST MII (π class) by musk xylene was observed only ip female. GST MIII (μ class) was induced up to about 5-fold by musk xylene in both sexes. The induction of MIII was higher than those of the other isozymes. There were strain differences in the induction of MII and MIII by musk xylene in female mice. The induction observed in C57BL/6N female mice was higher than that in DBA/2N female mice. These results indicate that musk xylene possesses multifunctional effects on GST induction in male and female mice.

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Induction of peroxisomal β-oxidation enzymes by dehydroepiandrosterone (DHEA) was studied in primary cultures of rat hepatocytes. Treatment of cultured hepatocytes with DHEA and its sulfate (DHEAS) for up to 5 days resulted in a progress increase in peroxisomal β-oxidation and camitine acetyltransferase activity, the magnitude of which was dose-related. Western blot analyses confirmed the induction of acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme and carnitine acetyltransferase in the treated cells. Induction of fatty acid ω-hydroxylase proteins (P4504As) was also revealed. DHEAS was a good inducer of these enzymes, and its effects coorelated well with those of DHEA; moreover, DHEAS was more potent than DHEA. Studies with structural analogues of DHEA revealed strict structural requirements for DHEA induction of peroxisomal β-oxidation. Of the steroids examined, dehydroandrosterone sulfate, androstenediol 3-sulfate, epiandrosterone sulfate and androsterone sulfate, besides DHEA and DHEAS, significantly induced the enzyme. All of the sulfated steroids were more potent than the corresponding unsulfated forms. DHEAS was the most potent inducer. The 3-sulfuric group was required for the marked induction of peroxisomal β-oxidation, and the 17-carbonyl group was also important. The presence of a double bond at position C5 was not a primary determinant for the action of DHEAS, but the relatively planar conformation of the steroidal hydrophobic backbone was crucial for inducing the enzyme. Certain metabolites, such as 16a-hydroxyDHEA, androstenedione and DHEA glucuronide, also caused enzyme induction, however the magnitude of which was lower than that seen with DHEA and DHEAS. When hepatocytes were co-treated with acetaminophen, a potent sulfate scavenger, or chlorate, an inhibitor of PAPS formation, the DHEA induction of peroxisomal β-oxidatiown as suppressed, whereas induction by DHEAS and clofibric acid was not affected. These findings support the idea that DHEA, after administration, undergoes sulfate-conjugation in the cell to gain peroxisome proliferator activity.

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Age- and gender-related changes of sulfoconjugations were studied in the rat. Alcohol and amine sulfoconjugating activities by rat hepatic soluble fractions were very low or negligible in the fetus. The activities increased after birth and attained maximal in 3-week-old males and females. Female rats maintained the maximal levels until the age of 2 years. In males, the activities lowered at 7 weeks old, were maintained at the low levels until the age of 1 year and increased thereafter but never reached the levels of 2-year-old females. Urinary excretion of sulfoconjugates of alcoholic and amine compound exhibited similar temporal changes to the in vitro activities. Phenol sulfoconjugating activity by the soluble fractions was very low in the fetus. The activity was about ten-fold higher in the neonates at birth than in the fetus and relatively constant until 3 weeks after birth. The activity in male 7-week-old rats attained the maximal level, and was about two-fold higher than females of the same age and about four-fold higher than the neonates. The activity in male and female rats at the age of 2 years were intermediate between the 7-week-old males and females, and no sex differences were observed. The results indicate that the substrate of sulfoconjugations could be divided into at least two groups, an alcohol and amine type, and a phenol type.

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Possible existence of five different subunits, designated ST-20P, ST-21P, ST-40P, ST-41P, and ST-60P, of hydroxysteroid (H) sulfotransferases (ST's) in female Sprague-Dawley rat liver cytosol was demonstrated by cloning and sequencing of cDNA's isolated from both of two rat liver cDNA libraries. The HST subunits consisted of 284 amino acid residues (Mr 33, 084-33, 256) except ST-60P (Mr 33, 535) which had an additional Met as the second amino acid residue. They shared extremely strong or very strong homology (>83%) in amino acid sequence with one another, . The HST subunits, however, shared very weak (<40%) homology with those of rat liver phenol and estrogen ST's. Of the three rat liver cytosolic HST's, STa, STb, and STc, which were separable on an anion-exchange column by direct application of the cytosol and all catalyzed sulfation of dehydroepiandrosterone and bioactivation by sulfation of the potent carcinogen, 5-hydroxymethylchrysene (5-HCR), the major enzyme STa was strongly suggested by the study on expression of cDNA's encoding ST-40P and ST-41P in Escherichia coli to consist of these microheterogeneous subunits with only one amino acid substitution. The recombinant proteins showed enzyme activities toward all of the examined 20 hydroxysteroids, 13 bile acids, and 5-HCR at rates very similar to those by STa in the presence of Tween 20. The detergent that was found to be a powerful HST stabilizer completely prevented the hepatic and recombinant HST's from facile aggregation with marked loss of their enzyme activities. In the presence of Tween 20, the recombinant HST's and STa existed as dimers and were chromatographically and electrophoretically identical. STa, STb, and STc had an equal size of subunits (30.5 kDa) which were all immunostained with anti-STa-antibody, although HST's in the rat liver cytosol had been reported to have different sizes of subunits (28-60 kDa)without immuno-homology. The present study carried out using the recombinant enzymes provides the first direct evidence for the identity of ST's catalyzing the sulfation of hydroxysteroids and bile acids and proposes that the current nomenclature system used for distinguishing HST's from bile acid STs should be improved.

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Morphine UDP-glucuronyltransferases (UGTs) inducible by phenobarbital (PB) in Sprague-Dawley (SD) rat liver were separated into two peaks by column chromatography with ω-(β-carboxypropionylamino)octyl Sepharose 4B gel. The peaks named Peak I and II exhibited different substrate specificity. An isoform of PB-inducible morphine UGT (morphine UGT_PB) was purified from Peak II by further twice chromatofocusing. The N-terminal amino acid sequence was identical to that of the UGT isoform which belongs to UGT1 family and was absent in homozygous Gunn rat. The sequence of another a morphine UGT purified partially from Peak I was different from that of morphine UGT_PB. The results suggest that at least two isoforms of PB-inducible morphine UGT are present in Sprague-Dawley rats and one of them is deficient in Gunn rats. The view was supported by comparing morphine UGT activities in liver microsomes of Sprague-Dawley, Wistar and Gunn rats treated with PB or untreated follows. That in PB-treated Gunn rats was significantly lower than that in PB-treated Wistar rats, although that in PB-treated Sprague-Dawley rats was comparable. In the column chromatography with solubilized liver microsomes of Gunn rats treated with PB, Peak II fraction which contains morphine UGT_PB disappeared. Thus the morphine UGT_PB could be an isoform belonging to UGT1 family and is deficient in Gunn rats.

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We have regionally assigned the androsterone UDP- glucuronosyltransferase(UGT2B2) gene to rat chromosome 14 at the band p21.2-p22 by fluorescence in situ hybridization. LA Wistar rats have a deficiency of UGT2B2 due to large deletion of the gene. Fischer rats and Nagase analbuminemia rats also show very low UGT2B2 activity similar to LA Wistar rats. It was clarified that the enzyme protein was absent in these strains, however, the molecular mechanism of the defect of UGT2B2 are to be elucidated.

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Pharmacokinetics of glucuronide and sulfate formed from parent drugs was studied in rats both in in vivo, and perfused liver system using a therapeutic drug for ulcerative colitis, E3040 as a model drug. The following conclusions were obtained. (i) The first-pass hepatic extraction ratio of E3040 was so high ( Eh>0.95 ) that the hepatic clearance was limited by hepatic blood flow, while those of conjugative metabolites were lower (Eh < 0.7). (ii) The glucuronide is formed more in extrahepatic organs than in the liver in vivo. (iii) The formation clearance of sulfate is 6 times as large as that of glucuronide in the liver. (iv) The glucuonide formed in the liver is transported into the bile and the perfusate equally, while the amount of sulfate effluxed into the perfusate was much larger than that excreted into the bile. (v) The biliary excretion of glucuronide is mediated by the primary active transporter, which lacks in Eisai hyperbilirubinemic rat (EHBR), while the transport carrier for the sulfate is different from the carrier for the glucuronide. (vi) Both conjugates formed in the liver undergo deconjugation to the parent drug, but the deconjugation rates are much smaller than the rates of sinusoidal efflux and biliary excretion of the conjugates.

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Permeation of ketotifen fumarate (KF), a model drug, through hairless rat full-thickness and stripped skins and two synthetic membranes (silicone membrane and polypropylene microporousm embrane) from lactic acid-ethanol-isopropyl myristate (IPM) system (LEI) and its related solvent systems was measured and compared to elucidate skin penetration-enhancing effect and its mechanism of the LEI system. Lactic acid and ethanol in the LEI system had an effect to increase permeability coefficient of KF through full-thickness skin. Those compounds increased KF flux through stripped skin, while they did not change permeability coefficient, which suggested that enhancing effect by LEI system may be realted to an increase of KF concentration in skin, especially in viable epdermis and dermis. Further experiments to measure skin permeation of two components in the LEI system, ethanol and lactic acid, and KF permeation experments using two synthetic membranes, models for lipid and pore pathway of stratum corneum, indicated that ethanol increased skin permeation of KF by increasing the drug concentration in skin and lactic acid increased KF solubility in skin containing ethanol, and that ethanol had an effect both on the pore and lipid pathway and lactic acid on pore pathway.

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Published data for skin permeation of number of compounds were analyzed based on single and parallel permeation pathway models to characterize the barrier properties of skin. Two data sets with different distribution in permeant size were used in these analyses. The permeability coefficient data were analyzed according to a single or parallel permeation pathway model with or without consideration of contribution of permeant size. The goodness-of-fit evaluated by AIC value was best for parallel pathway model considering permeant size. This suggests that the pore pathway is necessary to describe the skin permeation process as well as the dependency upon permeant size. The results of non-steady state analyses also demonstrated the necessity of pore pathway. The parallel pathway model had high predictive ability for skin permeability of a broad range of compounds.

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Nasal and pulmonal absorption of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was examined in the rat. After the intranasal and intratracheal administration, the relative bioavailability of rhG-CSF for subcutaneous administration was about 2% and about 27%, respectively, as evaluated area under the curve (AUC) of the plasma rhG-CSF concentration versus time for 8 hr. The pharmacological availability relative to subcutaneous administration was determined from the increase in the total blood leukocyte numbers. The pharmacological availability was 5-10% after the intranasal administration, determined from the AUC for the increased ratio of the total leukocyte numbers versus time for 48hr; it was slightly dependent on the pH and osmotic pressure of dosing solution. After the pulmonal absorption, on the other hand, the pharmacological availability was equal to or more than the availabilities after intravenous and subcutaneous administration; under pH6.5 and isotonic osmolarity, the availability was minimal. In comparison with the above two prenternal administration routes for rhG-CSF, the intratracheal administration was more efficient than the intranasal administartion. Laureth-9 increased the bio-availability and pharmacological availability in both nasal and pulmonary absorption of rhG-CSF, but sodium glycocholate was effective only in the pulmonary absorption. Some protease inhibitors were effective in the pulmonary absorption but not in the nasal absorption.

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The characteristics of intrapulmonary absorption of macromolecules such as biologically active peptides and FITC-dextrans (FDs) were studied using in vivo and in vitro experiment systems. The plasma concentrations and percent absorption of FDs after intrapulmonary administration decreased with increasing their molecular weights. Similar results were noted in an in vitro tissue culture system with human lung carcinoma A-549 cell line. The effects of absorption enhancers and protease inhibitors on the pulmonary absorption of insulin were also examined by means of the in vivo pulmonary experiment. The absorption of insulin from the lung was significantly increased in the presence of N-Lauryl- β-D-maltopyranoside (LM) and bacitracin. On the other hand, the effects of absorption enhancers and protease inhibitors on the stability of insulin were examined in the lung tissue homogenate. These protease inhibitors, in particular bacitracin were effective for reducing the degradation of insulin in the homogenate. These results suggested that the increase in the stability of insulin in the lung homogenate was one of the most remarkable factors for its enhanced absorption in the lung.

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Utilizing tight monolayers of rat pneumocytes cultured on tissue culture-treated polycarbonate membranes, the fate of peptides during trafficking across the alveolar epithelial barrier was determined. After instillation of peptide in the donor fluid, transepithelial flux was estimated from the appearance of peptides in the receiver fluid. Results indicated that the transport of Gly-L-Phe (as well as L-Phe) across the alveolar epithelium by a saturable transcellular mechanism operating in parallel with predominantly paracellular transport. The alveolar epithelial transport of Gly-L-Phe, AVP, and insulin appears to be vulnerable to peptidase activity located either intracellularly or apically at the epithelial cell surface. Gly-D-Phe and TRH appear to be resistant to degradation and to be transport across the RAEM by simple restricted diffusion. RAEM may be a useful model for the transport studies of peptides and other solutes in general.

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Transport of an organic anion, p-aminohippurate (PAH), was investigated in the OK kidney epithelial cell line using the cells grown on permeable supports. Specifically mediated transcellular transport of PAH across OK cell monolayers occurred only from basal side to apical side, and the accumulation of PAH in the cells was much larger from basal side than from apical side. Both the basal-to-apical transport and the accumulation from basal side were temperature dependent, saturable, and inhibited by anion transport inhibitors such as probenecid. In contrast, such a specific transport of PAH was not observed in LLC-PK₁ cells. To further characterize the transcellular transport of PAH across OK cell monolayers, PAH transport in apical and basolateral membranes of the cells was studied. Initial uptake of PAH from basal side was much higher than from apical side. In contrast, PAH efflux from the cells to apical side was higher than to basal side. Both uptake from basal side and efflux to apical side were temperature dependent and inhibited by anion transport inhibitors. In addition, basolateral uptake of PAH was inhibited by dicarboxylates. These results suggest that both basolateral and apical transports of PAH are vectorial processes, which results in vectorial, basal-to-apical transport of PAH across OK cell monolayers. PAH/dicarboxylate exchange is probably involved in PAH transport across basolateral membrane in OK cells. Thus, OK cells should provide a potent in vitro model system for the study of active secretion of organic anion in the kidney.

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To clarify the regulatory mechanisms for the tubular secretion of organic cations, we examined transcellular transport of ¹⁴C-tetraethylammonium (TEA) by monolayers of a pig kidney epithelial cell line LLC-PK₁ grown on microporous membrane filters. TEA was taken up progressively by the monolayers from the basolateral side, and was transported unidirectionally to the apical side. The basolateral-to-apical transport of TEA was stimulated by lowering pH of the apical side, resulting in a decrease in the TEA accumulation. The TEA efflux from the monolayers to the apical side caused a decrease in the intracellular pH monitored by a pH-sensitive fluorescent dye BCECF, indicating H⁺/TEA antiport. Transport studies for quinidine revealed that there may be distinct organic cation transport systems in both the basolateral and apical membranes, which can be distinguished from the systems mediating TEA transport. Basolateral uptake of TEA was decreased by lowering pH of the basolateral medium. Cell acidification by a prepulse with NH₄Cl also decreased the basolateral TEA uptake. Kinetical analysis suggested that pH sensitivity of the basolateral TEA transport was caused by changes in Km value. These results suggest that the activity of the basolateral organic cation transporter and the apical H⁺/organic cation antiporter is regulated by an environmental pH and a pH-gradient across the apical membranes, respectively.

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To construct the strategy of cell-specific targeting of proteins and low-molecular weight drugs coupled to macromolecules via sugar receptor-mediated endocytosis, pharmacokinetic characteristics of macromolecules modified with galactose and mannose residues were investigated in mice. The analysis based on a physiological model revealed that galactosylation of proteins greater than superoxide dismutase (SOD: MW = 32 kDa) led them to have extremely high affinity for the carbohydrate receptor and to be effectively delivered to the liver at lower doses less than 1 mg/kg. On the other hand, galactosylated soybean trypsin inhibitor (MW = 20 kDa) and chicken egg white lysozyme (MW = 14 kDa) showed relatively lower delivery to the liver because of their low affinity due to small numbers of galactose residues and high urinary excretion rate. Based on the obtained results, pharmocological activities of galactosylated and mannosylated SOD were examined in hepatic ischemia/reperfusion injury model in rats and showed superior effects. On the other hand, carboxymethyl-dextren with galactose or mannose residues was sythesized to develop a new carrier system for hepatic targeting of low-molecular weight drugs. Cytosine β-D-arabinosidwe as coupled to these hepatotropic carriers and successfully targeted to the liver and cultured mouse hepatoma cell lines.

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P-Glycoprotein (Pgp) actively pumps a number of antineoplastic drugs, such as etoposide, out of cancer cells and causes multidrug resistance. Pgp also expresses at the brush border membrane of the small intestine under the normal physiologic condition. We hypothesized that induction or inhibition of intestinal Pgp might increase or decrease the etoposide efflux from blood into the intestinal lumen and, therefore, decrease or increase the absorption of etoposide, respectively. Etoposide absorption was studied using an everted gut sac preparation of rat jejunum and ileum. In this study, we found that treating rats with sodium arsenite (10 μg/kg), sodium butyrate (0.5 mg/kg), or 3-methylcholanthrene (3-MC, 170 μg/kg) for 5 days decreased the intestinal absorption of etoposide in both jejunum and ileum. Western blotting with C219, a monoclonal antibody of Pgp, confirmed the increase of the intestinal Pgp in both ileum and jejunum. With reversed-transcription/polymerase chain reaction of rat mdrlb gene, we found that arsenite and butyrate induction increased mdrlb mRNA in the jejunum, whereas the inducing effect of arsenite, butyrate, or 3-MC in the ileum was not significant. Addition of C219 at 0.1 U/ml or 5'-adenylylimidodiphosphate at 0.2 N, a non-hydrolyzable ATP analog, increased the absorption of etoposide. Quinidine, an antiarrythmics, has been demonstrated to circumvent the multidrug resistance in cell lines, possibly by interfering with Pgp function. Adding quinidine at 1 mg/ml to the everted gut sac preparation also substantially increased the absorption of etoposide. In situ absorption of etoposide was also studied by intraluminal perfusion of the drug in anesthetized rats. Intravenous infusion of quinidine at either 1 or 2 mg/hr both increased serum level of etoposide. Such an increase was shown to be dose-dependent. Intravenous infusion of etoposide at 0.2 mg/hr resulted in luminal exsorption of the drug in small intestine. The intestinal clearance rate of etoposide was 41.7±7.2 ml/hr/kg. When etoposide was administered at the same infusion rate with 1 mg/hr of quinidine, the intestinal clearance of etoposide decreased to 18.4±3.9 ml/hr/kg. These in situ data confirmed that the intestinal Pgp mediated the efflux of etoposide and the use of Pgp-inhibiting agent such as quinidine may increase the intestinal absorption of etoposide. In addition to etoposide, the efflux of rhodamine 123, which is a common substrate used to measure Pgp function, in the everted gut sacs was also studied. Despite of the Pgp level increase in both ileum and jejunun shown in Western blotting, rhodamine 123 efflux increased only in the jejunum, but not in the ileum. The results suggest that there are multiple members of Pgp family in the rat small intestine. Rhodamine 123 was probably transported by a Pgp encoded by mdrlb, and etoposide was transported by another Pgp member.

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[in Japanese]

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The purpose of the present study was to understand the characteristics of in situ or in vitro experimental methods used in drug absorption study. Drug permeation parameters obtained from various methods were compared each other and their correlation to in vivo drug absorption was considered. Membrane permeabilities of cephalexin (CEX) calculated from in situ intestinal single-pass perfusion, recirculating perfusion and loop methods were coincided well and, as a whole, they showed the concentration-dependent absorption of CEX. A technique to perfuse the mesenteric blood vessel gave a new parameter for drug absorption, MPT, which represents the mean time required for drug permeation across the intestinal epithelium. Moreover, a rate-limitimg step of drug permeation was determined by the diffusion model analysis. As for the in vitro experiment, the possibility of the drug permeation study using Caco-2 monolayer to simulate in vivo drug absorption was investigated. Permeabilities of passively absorbed drugs to Caco-2 monolayer showed a good correlation with permeabilities calculated from in situ single pass perfusion experiments. However, the permeability of CEX to Caco-2 monolayer was extremely lower than that expected from in situ experiment, suggesting the inappropriate feature of Caco-2 monolayer for predicting the carrier-mediated drug absorption. This may be due to functional differences of Caco-2 monolayer compared to the epithelium of the small intestine.

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For our objectives to find ways of improving poor drug absorption from intestine, the active absorption of glucose and galactose by the glucose transport system in the intestne was so attractive that an effect of conjugating sugar to the drugs on its intestinal absorption was studied.
After absorption of 2-naphthyl α-glucoside (2-NAαglc), the major costituents appearing on the serosal side were 2-naphthol (2-NA) and its glucuronide. Meanwhile, after absorption of 2-naphthyl β-glucoside (2-NAβglc), the major constituents appearing on the serosal side were 2-NAβglc and 2-NA glucuronide. The total (glycoside + aglycone + glucuronide) absorption of 2-NAβglc and 2-NAαglc was significantly (P<0.05) decreased in the absence of Na⁺, indicating the active absorption of glucosides of 2-NA by the Na⁺-dependent glucose transport carrier (GTC). After absorption of 1-naphthyl α-galactoside (1-NAαgal) and 1-naphthyl β-galactoside (1-NAβgal), on the other hand, the major constitute appearing on the serosal side was 1-NAαgal and 1-NAβgal, respectively. A significant contribution of the GTC was observed in 1-NAβgal absorption, but not in 1-NAαgal absorption. Na⁺-dependent total absorption of 2-NAβglc was equivalent to that of 2-NAαglc, and was larger than that of 2-NAβgal. Na⁺-dependent total absorption of 1-NAβgal was larger than that of 1-NAαgal. These results, which were similar to those of p-nitrophenyl glycosides (1, 2), indicated that glucose conjugation was more effective on the active absorption by glucose transport carrier than galactose conjugation.
These results were developed to the design of sugar-conjugated tripeptide (Tyrosine-Glycine-Glycine, TGG). β-Glucose-conjugated TGG with a spacer (βglc-spacer-TGG) was transported to the serosal side, and any of the other compounds such as TGG were not determined. α-Glucose-conjugated TGG with a spacer (αglc-spacer-TGG) was also transported to the serosal side but a few amount of the desglucosylated compound (TGG possessing only spacer, spacer-TGG) was detected. However, spacer-TGG was transported to the serosal side more than β or αglc-spacer-TGG. Moreover, although the absorption of βglc-spacer-TGG, αglc-spacer-TGG and spacer-TGG were shown to be dependent on Na⁺, competitive inhibition was not observed in the absorption between βglc-spacer-TGG and spacer-TGG, indicating a contribution of a transport carrier to the absorption was unclear. Since TGG was not transported to the serosal side and about 80% of TGG on the mucosal side was degraded after 20min, these modification of TGG were indicated to protect TGG from degradation, at least, through the intestinal membrane.

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The uptake characteristics of polyamines have been investigated using rat intestinal brush-border membrane vesicles (BBMV). The uptake of these polyamines into BBMV was high and the order of uptake activity was spermine > spermidine > putrescine, and the medium pH affected remarkably the uptake of polyamines (pH 7.5 > pH 5.5). We have also examined the binding behavior of polyamines to liposome, and the similar phenomenon such as uptake activity and pH-dependency was observed. An inward Na⁺ gradient did not stimulate the initial uptake of all polyamines. The uptake rate of spermine and spermidine were saturable (Km = 30.4 and 148.1 μM, respectively), but putrescine was not saturable up to 8 mM. Spermine competitively inhibited the uptake rate of spermidine (Ki = 33.8 NM), while putrescine inhibited spermidine non-competitively (Ki = 3.28 mM). However, intravesicular spermine exhibited no trans-stimulation effect on the uptake of spermine and spermidine. Therefore, the polyamines were considered to be taken up via a passive diffusion mechanism rather than the carrier-mediated systems. The interaction between polyamine and inner-layer acidic phospholipids of BBMV might play an important role as a driving force of the polyamine uptake mechanism. Furthermore, the uptake mechanism of polyamine was different from cationic compounds because valinomycin-induced potassium diffusion potential (inside negative) did not affect the uptake of polyamines.

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In the present study, to prove the carrier-mediated transport in the intestinal absorption for orally active β-lactam antibiotics, Xenopus laevis oocytes were employed as exogenous gene expression system. It was demostrated that the uptake of zwitterionic cefadroxil (CDX) was increased by Xenopus laevis oocytes injected with poly(A)⁺RNA derived from rat, rabbit or human small intestinal mucosa. The uptake of CDX was inhibited by other orally active β-lactam antibiotics and dipeptides significantly, but not by amino acid. Furthermore at acidic extracellular pH, the uptakes of amino-β -lactam antibiotics, CDX, cephalexin and cephradine increased, suggesting that H⁺ gradient is a driving force for transports of these antibiotics. By size-fractionation of poly(A)⁺RNA on a sucrose density gradient centrifugation, poly(A)⁺RNA of which size is approximately 3 kb was identified to encode dipeptide transporter. Photoaffinity labeling of membrane protein with [³H]benzylpenicillin showed an expression of the specific binding protein with a molecular mass of 130 kDa. Stereospecific transport of ceftibuten with cis and trans-isomers was also exhibited in oocytes injectedwith poly(A)⁺RNA. From these results it was clarified that β-lactam antibiotics are absorbed in the small intestine by a H⁺ gradient dependent carrier-mediated mechanism which is common to dipeptide transporter.

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Kinetic analysis of the hepatic uptake of liposomes was performed in comparison to the degradation of liposomes in blood and liver. The degradation clearance of liposomes in blood was shown to depend on the size of liposomes. In vitro kinetic study on the degradation of liposomes suggested the mechanism of this size dependency due to the Increased affinity of liposomes for complement. There was two kinds of hepatic uptake components of liposomes, one is a size dependent opsonized pathway, and the other is a size Independent unopsonized pathway. The size dependent opsonization of liposomes was shown to result from the size dependent complement activation and this component explained the size dependended hepatic uptake clearance of liposomes in vivo. The kinetic analysis of the hepatic degradation of liposomes was also performed by labeling liposomes with both ¹²⁵I-albumin and ³H-cholesterylhexadecylether. Although the mechanism of hepatic uptake are considered to be different depending on the size of liposomes, the kinetics of hepatic degradation of liposomes was same for each size of liposomes and lay in the order of hour.

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The modified distributed model was constructed to analyze the distribution of quinolone antibiotics (NQs) in the rat brain. Eight-fold variations among NQs examined were observed for both the brain-to-plasma unbound concentration ratio (Kp, f(brain)) and the cerebrospinal fluid-to-plasma unbound concentration ratio (Kp, f(CSF)). The influx rates of NQs at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (B-CSF-B) were found to be determinant for Kp, f(brain) and Kp, f(CSF), respectively. Moreover, both the influx rates of NQs were correlated well with the octanol-water partition coefficients. Interestingly, only little effect of efflux rate at the B-CSF-B on Kp, f(CSF) was demonstrated, while in vitro uptake studies using isolated choroid plexus revealed that NQs was pumped out from cerebrospinal fluid to the circulating blood by an active organic anion transport system. In conclusion, the differential BBB permeability is suggested to a determinant for the brain interstitial fluid concentration of NQs which is believed to be responsible for toxicological effect on the central nervous system.

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