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My research focused on the effects of various drugs on (1) dopamine formation from p-tyramine catalyzed by polymorphic cytochrome P450 (CYP or P450) 2D6 variants and (2) endogenous steroid hormone hydroxylation catalyzed by CYP3A subfamily members (CYP3A4, CYP3A5, CYP3A7). The activation (cooperativity) of metabolic reactions catalyzed by P450s was especially emphasized. The effects of various psychotropic agents on dopamine formation from p-tyramine, catalyzed by wild-type CYP2D6.1 and CYP2D6 variants, including CYP2D6.2 (Arg296Cys;Ser486Thr), CYP2D6.10 (Pro34Ser;Ser486Thr), and CYP2D6.39 (Ser486Thr) were compared. Michaelis (K m ) and inhibition (K i ) constants of the psychotropic agents in the presence of CYP2D6.10 were higher than those observed in the presence of other CYP2D6 variants. Fluvoxamine, fluoxetine, milnacipran, and haloperidol activated CYP2D6-catalyzed dopamine formation [decreasing the K m and/or increasing the maximal velocity (k cat )], and this activation was CYP2D6 variant-dependent. Regarding the CYP3A subfamily, the effects of various compounds including endogenous steroid hormones on the 6β-hydroxylation of steroid hormones, such as testosterone, progesterone, and cortisol, were determined; it was found that testosterone, dehydroepiandrosterone, and/or α-naphthoflavone activated 6β-hydroxylation of cortisol and/or progesterone, but the effects varied in the presence of different CYP3A subfamily members. Further studies are required to confirm the mechanisms and therapeutic relevance of these activation phenomena.

We have developed regio- and chemoselective heteroelement-introducing reactions based on ate complex formation. This review is focused on novelly designed reactive species for efficient access to perfluoroalkylated aliphatic and aromatic compounds as well as Csp 2 -borylated compounds.

Naturally occurring polyhydroxylated amines such as (+)-1-deoxynojirimycin, polyoxamic acid, anisomycin, (-)swainsonine, and alexine stereoisomers, which have interesting biological activities including glucosidase- and mannosidase-inhibitory activity, immunoregulatory activity, and antibacterial effects, were synthesized stereoselectively starting from (S)-pyroglutamic acid derivatives. α,β-Unsaturated lactams ((S)-5-hydroxymethyl-2-oxo-3-pyrroline derivatives), α,β-unsaturated δ-lactone ((S)-4-amino-2-penten-5-olide derivative), and E-olefin ((S,E)-methyl-4-amino-5-hydroxypent-2-enoate derivative) from (S)-pyroglutamic acid derivatives were dihydroxylated using OsO4 in the presence of N-methyl morpholine N-oxide (NMO) to afford various chiral building blocks with different configurations. The stereoselectivity of cis-dihydroxylation for α,β-unsaturated lactams and α,β-unsaturated δ-lactone was very high, while the stereoselectivity was low for E-olefin. Therefore, the double asymmetric induction of E-olefin using K2OsO4 with chiral ligands was successively applied to yield high stereoselectivity. (2R,3S)-2-Hydroxymethyl-3-hydroxypyrrolidine and Gaissman-Weiss lactone, important intermediates for the preparation of pyrrolizidine alkaloids, were synthesized from a (3R,4R,5R)-3,4-dihydroxy-5-hydroxymethyl-2-pyrrolidinone derivative derived from α,β-unsatulated lactam. (+)-1-Deoxynojirimycin was synthesized from a (2S,3R,4R)-methyl 4-amino-2,3,5-trihydroxypentanoate derivative of E-olefin. (-)-Swainsonine and its stereoisomers were synthesized from (2R,3S,4R)- or (2R,3R,4R)-2-hydroxymethyl-3,4-dihydroxypyrrolidine derivatives of α,β-unsaturated δ-lactone or α,β-unsaturated lactam. The key reaction was diastereoselective allylation of the aldehyde derived from the corresponding 2-hydroxymethylpyrrolidine derivatives with various allylation reagents. The high diastereoselectivity could be explained by cyclic chelate formation between metals and the α-aminocarbonyl group or β-alkoxycarbonyl group, in which the nucleophile approaches from the less hindered face. Four alexine stereoisomers were synthesized from (2R,3R,4S,5R)- and (2R,3R,4S,5S)-2,3-dihydroxymethyl-3,4-dihydroxyl pyrrolidine derivatives of α,β-unsaturated lactam.

Density functional theory (DFT) is now extensively used as a research tool for the investigation of structure and reactivity of biological systems; however, its high computational demands still restrict the applicability of DFT to systems of a few tens up to one hundred atoms. A combined quantum mechanical/molecular mechanical (QM/MM) approach is applicable as an important method to study whole enzyme systems more than ten thousands atoms. We have investigated methane monooxygenase, dopamenie β-monooxygenase, tyrosinase, B12 dependent diol dehydratase, etc. using DFT and QM/MM calculations. In particular, we have done some computational mutation analyses about the amino acid residues at the active site of diol dehydratase. Our DFT and QM/MM calculations can correctly describe the structures and activation barriers of intermediates and transition states in the protein environment, and therefore, we successfully revealed the catalytic role of amino acid residues at the active site of diol dehydratase. Predicted relative activities of mutants are consistent with experimentally observed reaction rates. These results encourage us to apply QM/MM research to enzymatic reactions, functional analysis of active-site residues, and rational design of enzymes with new catalytic functions.

The antioxidant 2-hydroxyestradiol 17-sulfate (2-OH-E2-17-S) was found to be present in the placenta and to prevent the onset of preeclampsia. From experiments using rats, 2-OH-E2-17-S was confirmed to be a highly functional compound with stronger antioxidant activity than alpha-tocopherol and to sustain its antioxidant activity. 2-OH-E2-17-S was confirmed to be produced in the placenta from its precursor, estradiol 17-sulfate (E2-17-S), which is derived from fetal testosterone sulfate (TS). Since the fetal adrenal gland has been shown to convert testosterone (T) into TS, the following metabolic pathway may exist during pregnancy: T-->TS-->E2-17-S-->2-OH-E2-17-S. This fetoplacental pathway may contribute to the maintenance of healthy pregnancy. Details and the experimental outline of these discoveries are reported in this review.

The estrogenic activities of several hydroxylated metabolites of PCBs and PCDFs were investigated by yeast two-hybrid assay based on the ligand-dependent interaction of estrogen receptor with coactivator. For the hydroxylated PCBs, the order of estrogenic potency was 4-OH-2',4',6'-triCB > 4-OH-4'-monoCB, 4-OH-biphenyl. These compounds were evaluated as 10(3) to 10(4) less potent than 17 beta-estradiol based on the concentrations of test compounds showing 10% activity of 10(-7) M 17 beta-estradiol. 2-OH-3',4,4'-triCB, 4-OH-2',3,4'-triCB and 3-OH-/4-OH-2,2',5,5'-tetraCB, the metabolites of 2,2',5,5'-tetraCB were inactive as estrogens at the highest concentrations used in this study (10(-5) M). Also 4-OH-3,3',4',5-tetraCB, the metabolite of 3,3',4,4'-tetraCB was inactive as estrogen, indicating that this hydroxylated metabolite did not take part in the estrogenic activity of 3,3',4,4'-tetraCB. OH group at 4-position of biphenyl was necessary for the expression of estrogenicity, but one or two chloro-substitution adjacent to OH group inhibited the activity. For the hydroxylated PCDFs, 8-OH-2-monoCDF, 7-OH-3,4-diCDF, 8-OH-3,4-diCDF, 8-OH-3,4,6-triCDF and 3,8-(OH)2-2-monoCDF exhibited estrogenic activity. The estrogenic activity of 3,8-(OH)2-2-monoCDF was comparable to those of 4-OH-2',4',6'-triCB and 4-nonylphenol (mixture of compounds with branched sidechain). The order of activity was 3,8-(OH)2-monoCDF > 8-OH-3,4-diCDF, 7-OH-3,4-diCDF > 8-OH-2-monoCDF, 8-OH-3,4,6-triCDF. These compounds were evaluated as 2.5 x 10(3) to 3 x 10(4) less potent than 17 beta-estradiol. On the other hand, no estrogenic activity was observed for 2-OH-dibenzofuran, 3-OH-2,8-diCDF, 6-OH-3,4-diCDF and 9-OH-3,4-diCDF at concentrations as high as 10(-4) M. Substitution of OH group at 2(8)- or 3(7)-position of dibenzofuran and no chloro-substitution adjacent to OH group was required for the estrogenic activity.

In vitro metabolism of 3,5,3',4'-, 3,5,3',5'- and 2,4,3',4'-tetrachlorobiphenyls (TCBs) was studied using liver microsomes from rats, guinea pigs and hamsters. 3,5,3',4'-TCB was metabolized to 4-hydroxy-3,5,3',4'-TCB with liver microsomes of 3-methyl-cholanthrene (MC)- and 3,4,5,3',4'-pentachlorobiphenyl (PenCB)-treated rats but not of phenobarbital (PB)-treated ones. This result suggests that a MC-inducible cytochrome P450 isoform, probably CYP1A1, is more important in the in vitro metabolism of 3,5,3',4'-TCB in rat liver and that the isoform attacks the 3,5-dichloro-substituted phenyl ring more predominantly than 3,4-dichloro-substituted one. In 3,5,3',5'-TCB metabolism, liver microsomes from MC- and 3,4,5,3',4'-PenCB-treated hamsters formed 4-hydroxy-3,5,3',5'-TCB to a similar extent to rats reported previously. Guinea pig liver microsomes formed no metabolite. In 2,4,3',4'-TCB metabolism, PB accelerated 3-, 5- and 4-hydroxylations in guinea pigs and also 3- and 5-hydroxylations in hamsters, suggesting the involvement of a PB-inducible P450 isoform, presumably P450GP-1 and P450HPB-1, respectively. On the other hands, MC- and 3,4,5,3',4'-PenCB-treatment resulted in the marked increase of 4-hydroxylation in hamsters, but in the suppression of 4-hydroxylation in guinea pigs. From these results, it is suggested that the hydroxylation of coplanar TCBs such as 3,5,3',4'- and 3,5,3',5'-TCB is catalyzed by a MC-inducible P450 in rats and hamsters, whereas non-coplanar TCBs such as 2,4,3',4'-TCB which possesses both PB- and MC-like inducing ability of liver enzymes are metabolized by one or more kinds of P450 isoform induced by PB and MC.

We recently reported that certain hydroxylated PCB (OH-CB) metabolites were selectively retained in blood of rats experimentally dosed with PCB (Aroclor 1254), and also in blood of seals and humans environmentally exposed to PCBs. We also showed that metabolism in vivo of polychlorinated dibenzofurans (PCDFs) gave rise to a large number of hydroxylated PCDF (OH-CDF) metabolites, excreted in faeces, after oral administration to rats of PCDFs mixture (1, 2, 7, 8-tetraCDF 14%, 2, 3, 7, 8-tetraCDF 35%, 1, 2, 3, 7, 8-pentaCDF 48%). These results suggest that OH-CDFs could be present in blood. In the present study, potential retention of OH-CDF metabolites in blood (serum) was investigated in a female Wistar rat exposed to the PCDFs mixture. Serum was analyzed for the methylated OH-CDFs by gas chromatography (GC) and GC-mass spectrometry. Two major metabolites were determined in serum 1 day after oral administration. These were identified to be 3-OH-2, 4, 7, 8-tetraCDF (A) and 3-OH-2, 4, 7, 8, 9-pentaCDF (B) by comparison with synthesized reference compounds. 3-OH-2, 4, 7, 8-tetraCDF and 1, 2, 3, 7, 8-pentaCDF, respectively, via 3, 4(6, 7)-epoxide intermediate and subsequent NIH-shift of the 3(7)-chlorine to the 4(6)-position. The amounts of 3-OH-2, 4, 7, 8-tetraCDF and 3-OH-2, 4, 7, 8, 9-pentaCDF in the serum (5.72 g) analyzed accounted for 0.018% of 2, 3, 7, 8-tetraCDF and 0.003% of 1, 2, 3, 7, 8-pentaCDF dosed, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthetic steroids, having delta 4-3-ketone structure, are commonly metabolised to the corresponding tetrahydro-metabolites. The reduction of the 20-ketone group is also essential for pregnan series progestogens. When these metabolic pathways are obstructed in some way, steroids are hydroxylated at proper positions. Hydroxylation of a steroid is primarily directed to form the inactive form, namely, conjugated steroid. However, in some cases, it induces activation or activity change to the original compound. Activation of lynestrenol, as a progestogen and the conversion of norethindrone to ethynylestradiol, are examples. Metabolism of some new steroids and factors affecting the effect of oral contraceptive pills are discussed.

Concentrations of clomipramine and its metabolites, N-desmethylclomipramine (DC), 8-hydroxy-N-desmethylclomipramine (HDC) and 8-hydroxyclomipramine (HC), in plasma were determined in 99 patients treated with clomipramine hydrochloride. Doses patients received were not fixed but titrated according to their clinical severity and response to the treatment. Large interindividual variations were present in the concentrations of parent or each metabolic compounds in plasma, however, strong correlations existed between these drug concentrations and daily doses of clomipramine (0.40-5.10 mg/kg of body weight) (r = 0.62-0.80). Metabolic ratios for desmethylation and those for hydroxylation were calculated from these data, and revealed very large inter-individual variations of 30-36 fold and 10-96 fold, respectively. Pharmacological data from 65 of these patients with DSM-III-R mood disorders were analyzed by discriminant analysis using the scores of Global Assessment of Functioning. The overall prediction value for responder or nonresponder was calculated as 70% 2 wk after the initiation of pharmacotherapy. Concentrations of hydroxylated metabolites in plasma does contribute as much to the prediction of clinical response to clomipramine as desmethylated metabolites.

This study was conducted to investigate some biochemical characteristics of compound odontoma obtained from three cases, all found in the mandible. The results were compared with data obtained from deciduous teeth (3) and permanent teeth (3). The mole ratios of calcium to inorganic phosphorus (Ca/P) among three groups were deviated between 1.67 and 1.68 in the enamel extracts and between 1.65 and 1.69 in the dentine extracts. No significant differences were found in each case. Amino acid analysis of dentine showed apparent differences among these groups in the degrees of hydroxylation of proline (Hyp/Pro + Hyp) and of lysine (Hyl/Lys + Hyl). Hydroxylation rates of proline were 43.1 +/- 0.98% for the permanent teeth, 44.4 +/- 0.47% for the deciduous teeth and 40.9 +/- 0.44% for the odontoma. Hydroxylation rates of lysine were 32.0 +/- 0.67% for the permanent teeth, 27.3 +/- 0.0% for the deciduous teeth and 26.5 +/- 0.46% for the odontoma. T-test confirmed the significance of the differences between the permanent teeth and odontoma in the case of lysine (p less than 0.5%) and between the deciduous teeth and odontoma in the case of proline (p less than 0.5%). These results suggest the differential organization of dentine matrix between human permanent teeth, deciduous teeth and compound odontoma.

There is a world-wide demand for a clinically usable sensitizer for radio-resistant hypoxic cells. After unsuccessful clinical trials of misonidazole, many efforts have been made to develop new sensitizers. In the U.S., Brown and others reported a new drug named SR-2508 (ethanidazole), which is now in phase II and III clinical trials. In Japan, many drugs were synthesized and tested with the screening systems using EMT6 and SCCVII tumor. Since the failure with misonidazole is due to its neurotoxicity, two methods have been applied to find new sensitizers. The first one is to increase the sensitizing effects and the other is to decrease the neurotoxicity. KU-2285 is fluorinated nitroimidazole, and it has a higher sensitizing effect than misonidazole or SR-2508. Its sensitizing effect is 1.65 at 200 mg/kg, and the LD50 value is 2.3 g/kg. Hoping for less neurotoxicity, RK-28, RP-170 and KIH-801 were synthesized. RP-170 demonstrated less toxicity than Miso, and KIH-801 demonstrated an unexpectedly high sensitizing effect especially is in vivo experiments. Although RK-28 has a low LD50 value, it shows rapid clearance rate from serum and is supposed to have less cumulative neurotoxicity. KU-2285, RK-28, RP-170 and KIH-801 all await further clinical trials.

Fluorine modification of nitroazoles has been characterized as a useful method of molecular design of hypoxic cell sensitizers. In vitro sensitizing activities were correlated to the promotion efficiency of radiolytic hydroxylation of thymine to thymine glycol and to the one-electron reduction potential of a variety of fluorinated and non-fluorinated nitroazole derivatives. The absolute activity of sensitizers, which was evaluated from the enhancement ratio in vivo (SERvivo) and the tumor affinity, showed a linear relationship with the in vitro sensitizing activity. The sensitizer dose required to achieve an SERvivo of 1.5 decreased as the tumor affinity became greater. The fluorine modification of nitroazole sensitizers could enhance both the absolute activity and the tumor affinity.

Liver cirrhosis (LC) is often associated with osteomalacia and osteoporosis. Since it has been shown that serum levels of 25 hydroxy vitamin D (25-OH-D) are reduced in LC, defective hepatic hydroxylation of vitamin D has been postulated to be responsible for the low serum 25-OH-D levels and skeletal demineralization. This study was designed, therefore, to determine serum 25-OH-D and 1 alpha, 25-(OH)2-D levels in patients with LC. Further, the response of serum 1 alpha, 25-(OH)2-D to a single oral dose of 1 alpha-OH-D3 (2 micrograms) was investigated. In 5 patients with severe decompensated LC and 3 patients with compensated LC, serum 25-OH-D and 1 alpha, 25-(OH)2-D levels were respectively measured by the modified method of Belsey and by that of Eisman. Serum 25-OH-D in patients with compensated and decompensated LC was significantly higher than that in normals. Serum levels of 1 alpha, 25-(OH)2-D in patients with decompensated LC were significantly lower than those in patients with compensated LC and normals. After a single oral administration of 1 alpha-OH-D3 at a dose of 2 micrograms, the 1 alpha, 25-(OH)2-D rose in each patient within 6h, reaching the maximum levels at 12h. The percent increase over the basal value in decompensated LC was similar to that in compensated LC.(ABSTRACT TRUNCATED AT 250 WORDS)