The Dark Agouti (DA) rat has been proposed as a poor metabolizer model for the human debrisoquine 4-hydroxylase polymorphism. Earlier studies suggested that the poor metabolizer phenotype in the DA rat is due to the absence of the expression of CYP2D1 mRNA. Although CYP2D 1 catalyzes debrisoquine 4-hydroxylation, we found that another P450 of CYP2D subfamily, presumably CYP2D2, which we have purified from SD rat livers by chasing bunitrolol (BTL) 4-hydroxylase activity as an indicator also has the ability to catalyze debrisoquine 4-hydroxylation. CYP 2D2 has previously been described as inactive species. Thus we obtained CYP2D1 and 2D2 cDNA from Dr. F. J. Gonzalez of NIH and using baculovirus expression system, these two isozymes were expressed in Sf9 insect cells. We found that both isozymes can catalyze debrisoquine 4-hydroxylation, but CYP 2D2 has about 10 fold higher activity than CYP 2D1. In addition, CYP 2D2 was able to catalyze BTL 4-hydroxylation and propranolol 4-, 5-, and 7-hydroxylations while CYP 2D1 was not able to catalyze BTL 4-hydroxylation and propranolol 7-hydroxylation. These results indicate that CYP isozyme responsible for PM phenotype in DA rats is not CYP2D1 as previously indicated but CYP2D2. Levels of expression of CYP proteins were assessed by Western blotting using isozyme specific peptide antibodies. The level of expression of CYP2D 1 in DA rats was about the same level as that in SD rats, but the level of expression of CYP2D2 was markedly lower in DA rats than that in SD rats. The levels of CYP2D 1 and 2D2 mRNAs were examined by Northern blotting to find that levels of both mRNAs were markedly lower in DA rats than those in SD rats. Although the discrepancy between expression levels of CYP 2D1 proteins and its mRNA remains unresolved, it is clear that the reduced level of expression of CYP2D2 is responsible for the PM phenotype of DA rats.

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Enantio and regioselectivity of adrenoceptor blocking agents [propranolol (PL) and bunitrolol (BTL)] was examined for CYP2D6 expressed in various heterologous expression systems. In human liver microsomes, propranolol was oxidized at 4 and 5-position of the naphthalene ring and at the side chain to form 4-hydroxypropranolol, 5-hydroxypropranolols and N-desisopropyl-propranolol, respectively. BTL was oxidized to 4-hydroxybunitrolol by human liver microsomes. At a lower concentration range of PL or BTL, the ring hydroxylation of the substrates is mainly mediated by CYP2D6 in human liver.
As for the enantioselectivity, CYP2D6 expressed in Sf9 insect cells showed enantioselectivity of R(+)<S(-) for PL oxidation consisting of aromatic ring 4 and 5-hydroxylations and side-chain Ndesisopropylation, which was reverse of human liver microsomes at a low PL concentration range [R(+)>S(-)]. CYP2D6 expressed in Escherichia coli also gave the same results. This was proved to be caused by the change of an amino acid residue at position 374 of CYP2D6 from valine as wildtype to methionine as allelic variant.
As for the regioselectivity, CYP2D6 expressed in lymphoblastoid cells had PL 4- and 5-hydroxylase, and BTL 4-hydroxylase activities, but did not show PL N-desisopropylase activity, on the one hand. CYP2D6 expressed in E. coli had PL 4- and 5-hydroxylase, and N-desalkylase activities but did not exhibit BTL 4-hydroxylase activity, on the other. These results suggest that different environment surrounding the CYP2D6 proteins expressed in heterologous cells or N-truncation of the enzyme to yield high efficiency in expression in E. coli. affects the properties of CYP2D6 proteins.

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The flavin-containing monooxygenases (FMOs) are classified into five isozymes (FMO1-FMO5) which are mainly expressed in the liver, the kidney and the lung. However, the function of each isozyme has not been confirmed yet. Thus, to analyze the enzyme functions we isolated cDNA encoding each isozyme and established heterologous expression systems. The cDNA encoding mouse FMO1, rat FMO1, FMO2, FMO3 or FMO5 was cloned. The rat FMO2 cDNA encoded a polypeptide chain which was 100 amino acids shorter than that encoded by macaque, rabbit or guinea-pig FMO2. The mouse and rat FMO1 expressed in yeast showed the catalytic activities toward the major substrates of FMO, while the activity of the other isozymes was not detectable. Human FMO cDNAs were also isolated. The human FMO2 cDNA had an in-frame stop codon which aligns with a Gln codon in the macaque, rabbit and guinea-pig FMO2. Thus, human FMO2 cDNA encoded a truncated polypeptide. The heterologously expressed human FMO2 was catalyticaly inactive. In contrast, when the stop codon changed to the GIn codon in human FMO2 cDNA, the modified enzyme catalyzed the oxidation of substrates. The expressed FMO1, FMO3 and FMO5 metabolized methimazole. Imipramine and n-octylamine were oxidized only by the expressed FMO1 and FMO5, respectively. Furthermore, the N-oxidation of trimethylamine (TMA) by human FMO1, FMO3 and FMO5 expressed in E. coli. was examined. TMA was metabolized only by the human FMO1 and FMO3. The Km values for FMO1 and FMO3 were 4.2 mM and 75.6 μM, respectively. The Vmax/Km value of FMO3 was about 136 times higher than that of FMO1. These results suggest that FMO3 plays a major role in the metabolism of TMA in humans.
These observations suggest that FMO1 and FMO3 have broad spectrum for substrates among FMO isozymes, though FMO show substrate specificity in general.

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Species differences and substrate specificities of liver aldehyde oxidase were examined in several mammalian species using methotrexate (MTX), benzaldehyde (BA), phthalazine (PH) and N¹-methylnicotinamide (NINA) as a substrate. Significant species differences were observed when aldehyde oxidase activities were examined in rabbits, guinea pigs, hamsters, monkeys and dogs. Monkey had the highest activities using BA or PH as a substrate, and rabbits showed a significant oxidase activity toward MTX. Five strains of rats (Slc:Wistar/ST, Sea:SD, Jcl:SD, S1c:SD and WKA/Sea) demonstrated marked strain differences. Substrate specificities, Km values and immunoblot analysis indicated that the strain differences were due to the amounts of AO. Human liver cytosols exhibited relatively high aldehyde oxidase activities, but they were individually different. The existence of AO isozymes in human was suggested from the immunoblot analysis.

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UDP-Glucuronosyltransferases UGT2B2I and UGT2B22 cDNA which encode guinea pig UGT55K and UGT59K have been cloned and characterized. It has been suggested that an active glucuronide of morphine, morphine 6-glucuronide (M6G), is responsible for the analgesia in humans. Besides humans, guinea pigs have the ability to glucuronidate morphine to M6G with great efficiency. The purification of morphine UGT in this animal species gave us a preparation of a possible hetero-oligomer of UGT55K and UGT59K. The purpose of the present work was to clone the isoforms and characterize the homo and hetero-oligomers. UGT2B21 and UGT2B22 cDNAs were cloned by screening of the Hartley guienea pig liver cDNA libraries. UGT2B2I cDNA containing full length of open reading frame were obtained by 5' and 3'-RACE, and further analysis of the full length RT-PCR product of 1821 by encoding 528 amino acids. UGT2B22 cDNA which was containing length of open reading frame was obtained by 5'-RACE, was 2508 by and deduced amino acids were 529. UGT2B21 and UGT2B22 expression vectors were constructed in pSVL. COS-1 cells were transfected with UGT2B21 and/or UGT2B22 expression vector by Trans-IT^TM, and the UGT activity in the COS I cell microsomes was determined. UGT2B21 glucuronidated the morphine 3-position, 4-hydroxybiphenyl, bomeol, testosterone, androsterone and estriol. M6G activity and chloramphenicol glucuronidation were only seen in the microsomes of co-expressed UGT2B2I and UGT2B22. As yet, we know of no substrate for UGT2B22. The present results indicate that UGT isoforms could act as hetero-oligomers by accessing a broader range of substrates than homo-oligomers.

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Two sulfotransferase (ST) cDNAs encoding amine ST-RB1 and -RB2 (AST-RB1 and AST-RB2) have been isolated by immunoscreening of a rat liver cDNA library. The sequence of AST-RB1 had less than 38% identity at the amino acid level with cytosolic STs in mammals and suggested that AST-RB1, arbitarily named ST3A1, constituted a new and third gene family of cytosolic ST. ST3A1 expressed in Escherichia coli catalyzed N-sulfation of amines, but barely O-sulfation of typical substrates for aryl and hydroxysteroid ST. AST-RB-2, arbitarily named ST2A8, was included in a subfamily of hydroxysteroid ST (ST2A) and showed high catalytic activities not only to hydroxysteroids but also to amines. However, N-sulfation of amines in rabbit was mainly catalyzed by ST3A1 rather than by ST2A8 judging from those Km values. On the contrary, ST included in ST3A subfamily was not identified inhuman and rat livers. Instead of ST3A from, ST2A form is likely to contribute the N-sulfation of amines in rats and humans. We further performed to identify for sulfating activities by recombinant enzymes. In addition, to clarify contribution for sulfating activity of the specific substrate, we compared sulfating activity of individual substrates and content of each form in human livers. As results, substrate specificities of individual STs were identified in human livers.

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Liver microsomal carboxylesterases (E.C. 3.1.1.1) 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. Multiple isozymes of liver microsomal carboxylesterases exist in various animal species, and the enzyme is involved in the metabolic activation of ester- and amide-type prodrugs. In the present study, twenty-seven carboxylesterase 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. And also we tried to compare the primary structure of carboxylesterase isozymes in various animal species and humans using cDNA cloning and analysis. The carboxylesterase isozymes from various species examined here showed considerable similarities in physicochemical and immunochemical properties, but not similar in substrate specificity. The deduced amino acid sequence of the clone possessed many structural characteristics that are highly conserved among rat RL1, RH1, RHIec, RS1, RS2, mouse NL1, MH1, MS1, beagle dog D1 and human HU1, HU2, HU3, including active site sequence (GESAGG, NKQEXG, GDNXD), and four cysteines that may be involved in the specific disulfide bond. It is well known that proteins that are retained in the endoplasmic reticulum (ER) lumen contain the retention signal at their carboxy terminal of the tetrapeptide (KDEL-COOH). The seven carboxylesterase clones (RU, RI-11, MI-11, D1, HU1, HU2, HU3) also contained an ER-retention signal of carboxylesterase (HXEL), and three clones (RS1, RS2, MS1) did not contain this peptide. When clone was expressed in COS7 and V79 cells, the plasmid-coded protein was retained. The cells expressing carboxylesterase is very high activity towards xenobiotic ester and amide. Site-specific mutation of the three amino acid residues of catalytic triad (Ser203 to Thr203, GIu335 to Ala 335, or His 448 to A1a448) greatly reduced the carboxylesterase activity. In conclusion, liver microsomal carboxylesterase 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 specificity. These reasons may be due to the variances of substrate binding site.

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Although aprindine (AP), an anti-arrhythmic drug, had been shown to follow a non-linear pharmacokinetics in human subjects with a very large inter-subject variability, the metabolic pathways contributing the non-linear pharmacokinetic disposition of AP have not been clarified yet. Pharmacokinetics of AP and its metabolites, p-OHAP, i-OHAP and DEAP, was studied in seven arrhythmic patients administering 20mg to 60mg of AP every 12 hour for 2 weeks, and was evaluated the non-linearity using a mixed effect model analysis. The partial formation clearances of i-OHAP and p-OHAP from AP showed AP dose dependency. Metabolic parameters for formation of metabolites, p-OHAP, i-OHAP and DEAP from AP were obtained using human and rat liver microsomes, and compared. Lower capacities and higher affinities of i-OHAP and p-OHAP formations shown in human microsomes reasonably described of the non-linearity of AP disposition in humans.

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Serious side-effects caused by drug interactions have become asocial problem since an interaction between sorivudine and 5-fluorouracil (5-FU) resulted in a number of deaths due to toxicity in Japan. This interaction has been found to result from mechanism-based inhibition of dihydropyrimidine dehydrogenase (DPD) which is involved in 5-FU metabolism. A mechanism-based inhibitor is one that is metabolized by an enzyme to form a metabolite which covalently binds to the same enzyme, leading to irreversible inactivation of the enzyme. In this study, we have predicted 5-FU /sorivudine and triazolam/erythromycin interactions from in vitro studies by considerating the mechanism-based inhibition of DPD and CYP3A, respectively.
Human hepatic enzyme was preincubated with the inhibitor in the presence of NADPH and the remaining metabolic activity was evaluated. The inhibitory effects of (E)-5-(2-bromovinyl)uracil (a metabolite of sorivudine) and erythromycin were found to depend on both the preincubation time and inhibitor concentration. Time-courses of 5-FU and triazolam concentrations in blood were simulated by a physiologically-based pharmacokinetic (PBPK) model, using in vitro kinetic parameters for the enzyme inactivation and pharmacokinetic parameters reported in the literature. The predicted AUC ratio of triazolam in the presence and absence of erythromycin was consistent with the AUC ratio actually observed in vivo. The AUC of 5-FU was predicted to be increased more than 5-fold by sorivudine, indicating that coadministration of these drugs is very dangerous.

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Cholesterol 7α- and 7β-hydroperoxides are mutagenic and cytotoxic steroids existing as minor components in rat skin and regarded as good aging markers in the rat. Rat liver and skin cytosols had high and very low activities of the reduction of cholesterol 7-hydroperoxides to the corresponding steroidal alcohols, respectively, in the presence of GSH. The GSH-dependent reduction of the steroid hydroperoxides in rat liver was catalyzed mainly by GSTs with the subunit A1(2) of the Alpha class, but not catalyzed by any other isoform of GST existing in rat liver. Se-containing GSH peroxidase (Se-GSH Px) also played a partial role, to a lesser extent than rat (r) GSTA1-3, in the GSH-dependent reduction of the hydroperoxides in rat liver cytosol. However, rat skin cytosol contained no detectable level of rGSTsAI-1(2) and Al-3, but contained a very low level of Se-GSH Px. The above facts strongly suggest that the age-dependent linear increase in the dermal levels of the cholesterol hydroperoxides as the aging markers in the rat is mainly responsible for the absence of rGSTs bearing the subunit A1(2) and for the very low level of Se-GSH Px in the skin.
gpGSTA1-1 existed at a concentration of 1% of cytosolic protein in the liver of guinea pigs, which are well known to lack Se-GSH Px in their tissues. A comparative study using rGSTA1-2 and Theta-class rGSTT2-2 indicated gpGSTA1-1 to have the highest GSH Px activity toward cumene hydroperoxide of all known mammalian GSTs and also to have Px activity toward polyunsaturated fatty acid hydroperoxides (PUFAOOHs) comparable to rGSTT2-2, with the highest activity toward PUFA-OOHs of rGSTs. No detectable level of the GSTA4-4 ortholog highly active toward 4-hydroxy-2(E)-nonenal (4-HNE) was found in the liver of guinea pigs. All gpGST isoforms isolated from hepatic cytosol showed modest GSH-conjugating activity toward 4-HNE.

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Dihydralazine induces immunoallergic hepatitis and anti-LM autoantibodies found in the serum of the patients have been reported to react with CYPIA2. It is thus suggested that a reactive metabolite of dihydralazine covalently binds to the P450 protein. We investigated the selectivity of inactivation of P450 enzymes during the metabolism of dihydralazine to evaluate the target protein of its reactive metabolite. Preincubation of microsomes of β-naphthoflavone-treated rats with dihydralazine in the presence of NADPH resulted in time-dependent loss of phenacetin O-deethylase activity (POD, an indicator of CYPIA2 activity), showing inactivation of CYPIA2 during the dihydralazine metabolism. The preincubation was less effective on ethoxyresorufin O-deethylase activity in microsomes of β-naphthoflavone-treated rats (CYPIAI) and pentoxyresorufin O-depentylase activity in microsomes of phenobarbital-treated rats (CYP2B). Dihydralazine metabolism in microsomes of untreated rats caused time-dependent loss of testosterone 2α-, 16α- (CYP2C11) and 6β- (CYP3A) hydroxylase activities. Preincubation of human liver microsomes with dihydralazine caused a marked loss of testosterone 6β- hydroxylase activity in addition to that of POD. These results demonstrated that dihydralazine was metabolically activated by CYPIA2, and the chemically reactive metabolite bound to the enzyme itself and inactivated it, whereas CYP2C and 3A enzymes were also suggested to be the enzymes that activate dihydralazine and lead to the target of the reactive intermediates. On the other hand, carbamazepine induces hypersensitivity reactions and autoantibodies found in the serum of the patients have been reported to react with CYP2C and 3A. However, present preincubation studies demonstrated that CYP2D (bunitrolol 4-hydroxylase) but not CYP2C or 3A was inactivated during the metabolism of the drug.

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A frequent limiting side-effect of irinotecan, CPT-11, is its gastrointestinal toxicity (diarrhea) thought to be related to the biliary excretion of CPT-11 and its metabolites. Accordingly, we have investigated their biliary excretion mechanism. Our in vivo pharmacokinetic studies in rats revealed that the biliary excretion clearance of the four anionic forms of CPT-11 and its metabolites ways much lower in Eisai hyperbilirubinemic rats (EHBR) with a genetic deficiency of the hepatic canalicular multispecific organic anion transporter (cMOAT). Detailed analysis using isolated liver bile canalicular membrane vesicles led to identify the multiplicity of transport systems. Such multiple primary active transport systems are also responsible for the biliary excretion of CPT-11 and its metabolites in humans, and the major transport systemforOPT-11 differs fromthat forthe othertwo compounds. Greater degree of inter-CMV variability in the uptake of SN-38 and SN38-Glu may imply that interindividual variability in their biliary excretion might contribute to the interpatient variability in the toxicity caused by CPT-11.

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Transepithelial transport characteristics of diphenhydramine, an antihistamine, were studied in cultured human intestinal Caco-2 cell monolayers. In the apical-to-basolateral transport studies, the flux and cellular accumulation of diphenhydramine were influenced by the apical extracellular pH values. The accumulation and transepithelial transport were saturable and inhibited by chlorpheniramine. In the basolateral-to-apical transport, the accumulation and transport of diphenhydramine were also inhibited by chlorpheniramine. Diphenhydramine accumulation from both sides was stimulated by preloading the cells with chlorpheniramine, indicating the existence of specific transport systems in the both apical and basolateral membranes. Furthermore diphenhydramine was preferentially transported and effluxed to the apical direction. Finally, it was suggested that this transport system recognized tertiary amine compounds having a N-dimethyl or -diethyl residue in their structures. These results suggest that novel pH-dependent transport systems contribute to the intestinal absorption and secretion of tertiary amine compounds such as diphenhydramine.

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cDNAs for novel organic cation transporters, OCTNI and OCTN2, were cloned from human fetal liver and adult kidney, respectively, and their transport activities were investigated. OCTN1 encodes a 551-amino-acid protein. It is strongly expressed in kidney, placenta, and several other tissues and in human cancer cell lines. When expressed in HEK293 cells, OCTN1 exhibited saturable and pH-dependent [¹⁴C]tetraethylammonium (TEA) uptake with higher activity at neutral and alkaline pH than that at acidic pH. In addition, OCTN1-transfected HEK293 cells exhibited a larger TEA efflux from the cells at an acidic extracellular pH. When OCTN1 was expressed in Xenopus oocytes, it transported TEA in a pH dependent manner with an increased activity at higher medium pH as the same as that expressed in HEK293 cells, whereas membrane potential or sodium ions had no effect. OCTN1-mediated TEA uptake was inhibited by various organic cations and OCTN1 transports cationic compounds such as [³H]pyrilamine, [³H]quinidine and [³H]verapamil. Furthermore, L-[³H]camitine was transported in a sodium dependent manner by HEK293 cells expressed with OCTN1 or OCTN2. Accordingly, OCTN1 was functionally demonstrated to be a multispecific proton/organic cation antiporter which presumably functions at the renal apical and other tissue membranes and the common physiological role of OCTN1 and OCTN2 might be transport of carnitine in several tissues.

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Drug transport mechanism across the placenta is still unknown. The trophoblast that form the placental barrier have an important role in controlling the passage of molecules from mother to fetus. In this study, we investigated that monocarboxylic acids are actively transported from mother to fetus by a monocarboxylic acid transporter on the trophoblast. The human choriocarcinoma cell line, BeWo, was cultured on tissue culture plates. When the cells reached confluence, benzoic acid uptake experiments were performed The uptake of ¹⁴C-benzoic acid by BeWo cells was saturated at higher concentrations. The metabolic inhibitors sodium azide and 2, 4-dnitrophenol significantly inhibited the uptake of ¹⁴C-benzoic acid by BeWo cells. In addition, various monocarboxylic acids inhibited the uptake of ¹⁴C-benzoic acid, whereas dicarboxylic acids dd not affect the uptake. The findings confirm that monocarboxylic acids are transported by a monocarboxylic transporter on BeWo cell monolayers.
There are no reports concerning that efflux transport system, such as P-glycoprotein (P-gp), that actively transport agents from the placenta back into the maternal circulation. We also investigated whether efflux transport system in trophoblast occurs. Calcein-AM was used to evaluate the functional activity of the P-gp efflux transport system. The calcein-AM accumulation by BeWo cells was increased significantly in BeWo cells treated with P-gp inhibitors, cyclosporin A, dipyridamole, quinidine and verapamil. The accumulation of ³H-vinblastine which is a substrate of P-gp was also increased by the P-gp inhibitors in BeWo cells. There exists an efflux transport system as P-gp in trophoblast. This efflux system act as one of a placental barrier and may protect the fetus from toxic xenobiotics from maternal circulation.

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We investigated the inhibitory effect of components in grapefruit juice (GFJ) and orange juice (OJ) on the uptake of vinblastine, a substrate of P-glycoprotein, by Caco-2 cells. Significantly increased uptake of [³H] vinblastine at the steady state by Caco-2 cells was observed in the presence of GFJ and OJ. This suggests that the both juices involve compounds inhibiting P-gp function. We examined the effect of three organic solvent extracts, ethyl acetate, diethyl ether and methylene chrolide, of GO and OJ on the uptake of [³H] vinblastine. The inhibitory effect was shown more in ethyl acetate extracts than the other two extracts, in the both case of GFJ and OJ. Furthermore, the ethyl acetate extracts of GFJ and OJ were fractionated by cosmosil column chromatography with stepwise methanol solution. Inhibitory effect of GFJ on P-gp function was maximun in the fraction eluted with 60% methanol solution, while OJ's was maximun in 70% methanol solution. Each fraction contained different component. Moreover, inhibitory effect of GFJ on CYP3A4 was maximun in the fraction eluted with 80% methanol solution. This study suggested the existence of unknown specific inhibitory compounds in GFJ and OJ for the P-gp, but not for CYP3A4 function.

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Colloidal particles are good candidates for efficient drug carriers. However, the rapid clearance by macrophages of the reticuloendothelial system (RES), mainly Kupffer cells of liver and to a lesserextent macrophages of the spleen and the bone marrow, limits their application as carriers to other tissues and/or cells. To achieve a rational design of particulate carrier system, the basic information about in vivo disposition and the uptake mechanisms by RES of particle itself should be required. Therefore, in the present study, the in vivo disposition of polystyrene microsphere (MS) with the particle size of 50 nm (MS-50) or 500 nm (MS-500) was characterized after intravenous administration to rats. To study the mechanisms of the hepatic disposition of MSs, effects of serum on their disposition were investigated for both MSs by isolated liver perfusion experiments in rats. From these studies, it was found that serum would function both as opsonin to enhance the hepatic uptake of MSs and as inhibitor by reducing non-specific interaction between MSs and the plasma membrane. Whether serum promotes or inhibits the hepatic disposition of MSs would be dependent on the particle sizes of MSs.

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We demonstrated that S-1080, a dual antagonist for the NMDA receptor glycine site and the AMPA receptor, was excreted by the kidney by the anion transport system. The accumulation of 51080 in the kidney was observed after intravenous administration of more than 25 mg/kg of S-1080 in rats. This renal accumulation of S-1080 was clearly diminished by the co-administration of N-benzoyl-β-alanine. We studied the mechanism that N-benzoyl-β-alanine ameliorates S-1080 accumulation in the kidney and found that N-benzoyl-β-alanine, which is rapidly excreted by the kidney, increases S-1080 solubility in urine.

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Powerful analytical tools were required for studies on the fate of NE-100, which undergoes extensive metabolism. An analytical method based on immunoaffinity chromatography / radioimmunoassay, using two antibodies with different specificities, was developed for the determination of unchanged NE-100 in human plasma. This tandem-type method was of high sensitivity, sufficient both precision and accuracy, and high specificity for unchanged NE-100. For the exploratory detection of potential metabolites having affinity for sigma receptors, immunoaffinity chromatography combined with liquid chromatography-mass spectrometry (/ mass spectrometry) that can recognize metabolites retaining structural features (i.e., N-dipropyl or N-propyl group) was developed. This hybrid-type method was extremely valuable for the recognition and characterization of such metabolites present in biological fluids at low concentrations. These techniques have proved to be useful for studies on the fate of NE-100.

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The concentrations of sodium valproate (VPA) in tear and plasma by a quadrapole ion trap type GC/MS (GCQ) in a negative ion/chemical ionization/electroncapture mode. VPA was converted into the pentafluorobenzyl ester and was quantified by monitoring the ion of m/z 143. The VPA concentrations [C]Ts in the tear obtained from patients by non-invasive Schirmer method correlated very well with those of the unbound form in the plasma, indicating that the [C]TS will be used as a convenient index for monitoring the unbound VPA level in the patients.

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The separation of enantiomers of dihydropyridine derivatives by high-performance liquid chromatography (HPLC) was studied using modified Pirkle-type chiral stationary phases. Enatioseparations of manidipine and nicardipine were achieved by a normal-phase mobile phase utilizing n-hexane-l, 2-dichloroethane-methanol-trifluoroacetic acid on the urea-derivative type chiral statinary phase (SUMICHIRAL OA-4500). The enantioselective HPLC methods of manidipine and nicardipine in human plasma involve a rapid and simple extraction based on a bonded-phase extraction, which were applied to an enatioselective pharmacokinetic study in healthy subjects. Also, the effects of grapefruit juice on the stereoselective dispositions of manidipine and nicardipine in healthy subjects were examined. (+)-Manidipine plasma level was 1.5 fold higher than (-)-manidipine, and both enantiomers plasma level increased to 2.3 fold with grapefruit juice. Additionally, on the study of nicardipine, subjects were received intravenous doses and oral doses. After oral administration, grapefruit juice significantly increased the plasma concentrations of (+)-nicardipine and (-)-nicardipine, but not affect after intravenous administration. It is suggested that grapefruit juice inhibited CYP3A4-mediated presystemic metabolism in the small intestine.

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We have developed the sensitive and specific detection system for S-nitrosothiols using HPLC coupled with Hg²⁺ and Griess reagent. In this study, we have tested the effect of heavy metal ions and metal proteins for the formation of S-nitrosoglutathione (GSNO) and compared this with the GSNO generation by following oxidation of NO in glutathione (GSH)/ NONOate system. In the presence of Cu ion and ceruloplasmin, GSNO formation was significantly increased. This maximun effect was observed at 0.5μM of Cu and Cp concentration in GSH (10μM)/ NONOate (10μM) system and then, the yield of reaction was 25% approximately (GSNO/ GSH, %) which showed the 25 fold higher formation of GSNO rather than control. In RAW 264 cell culture system, which expressed inducible NO syntase, Cp significantly catalzyed formation of GS-NO rather than Cu ion. These observation indicate that Cp might participate in the formation of GSNO in biological system.

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Conventional hapten immunoassays are dependent on the competitive reaction between unlabeled antigen (analyte) and labeled-antigen against a limited amount of antibody. Although noncompetitive immunoassay procedures such as two-site immunometric assays offer much higher sensitivity (attomole ?? zeptomole order) and a wider working range, this principle has not been suitable for measuring haptens. We attempted to generate monoclonal anti-idiotype and anti-metatype antibodies which may enable noncompetitive hapten immunoassays with excellent sensitivity, and succeeded in producing α-type and β-type anti-idiotype antibodies, each recognizing the framework region and paratope of a monoclonal antibody against ursodeoxycholic acid 7-N-acetylglucosaminide (UDCA 7-NAG). The combined use of the α-type and β-type antibodies together with the anti-hapten antibody provided a noncompetitive assay system for UDCA 7-NAG, whose detection limit was ca. 500 fg.

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A new chiral separation column using flavoprotein from chicken egg white was developed for HPLC. This column allow to use under the aqueous condition and can separate drug enantiomers with a broad range. And this column has both a moderate non-specific retention and a enantioselectivity, so it can separate not only enantiomers but also analogues such as metabolites. Interactions between drug enantiomers (ketoprofen as a model) and chiral discrimination region on flavoprotein was investigated using affinity capillary electrophoresis combined with circular dichroism and chemical modification of flavoprotein. The chiral discrimination region was concluded to consist of α-helix structure, and required π-π interaction of a tryptophan residue with the aromatic ring of ketoprofen and ionic interaction of the carboxyl group of ketoprofen with an amino group of the protein for chiral separation.
In order to demonstrate an application of flavoprotein column for pharmacokinetic study, a column-switching HPLC method with direct injection of a large volume of plasma was developed for simultaneous determination of E3810 and its metabolites. This method is simple, rapid, accurate, and precise, and should be very useful for enantioselective pharmacokinetic studies.

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The uniformity in β-ray sensitivity was examined by exposing IP to a ¹⁴⁷Pm planar radiation source followed by analyzing with BAS. An area dependency of appreciable extent in β-ray sensitivity was observed, which could be mainly attributed to the mechanics in BAS. A correction method for this area dependency was presented. A method for measuring directly the PSL_BG of each area and examining radioactive contamination of IP was developed, which involved exposing IPs simultaneously and assessing PSL_BG of the IP in contact with a sample from the PSL_BG of the corresponding area in the other IPs.

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