BNCT for primary synovial sarcoma.

Synovial sarcoma is a rare tumor requiring new treatment methods. A 46-year-old woman with primary monophasic synovial sarcoma in the left thigh involving the sciatic nerve, declining surgery because of potential dysfunction of the affected limbs, received two courses of BNCT. The tumor thus reduced was completely resected with no subsequent recurrence. The patient is now able to walk unassisted, and no local recurrence has been observed, demonstrating the applicability of BNCT as adjuvant therapy for synovial sarcoma. Further study and analysis with more experience accumulation are needed to confirm the real impact of BNCT efficacy for its application to synovial sarcoma.

Feasibility studies towards future self-sufficient supply of the (99)Mo-(99m)Tc isotopes with Japanese accelerators.

In order to establish a self-sufficient supply of (99m)Tc, we studied feasibilities to produce its parent nucleus, (99)Mo, using Japanese accelerators. The daughter nucleus, (99m)Tc, is indispensable for medical diagnosis. (99)Mo has so far been imported from abroad, which is separated from fission products generated in nuclear reactors using enriched (235)U fuel. We investigated (99m)Tc production possibilities based on the following three scenarios: (1) (99)Mo production by the (n, 2n) reaction by spallation neutrons at the J-PARC injector, LINAC; (2) (99)Mo production by the (p, pn) reaction at Ep = 50-80 MeV proton at the RCNP cyclotron; (3) (99m)Tc direct production with a 20 MeV proton beam from the PET cyclotron. Among these three scenarios, scenario (1) is for a scheme on a global scale, scenario (2) works in a local area, and both cases take a long time for negotiations. Scenario (3) is attractive because we can use nearly 50 PET cyclotrons in Japan for (99m)Tc production. We here consider both the advantages and disadvantages among the three scenarios by taking account of the Japanese accelerator situation.

Cytochrome P450 2D6 (CYP2D6) inhibitory constituents of Catharanthus roseus.

The MeOH-soluble fraction of the water extract of Catharanthus roseus from Indonesia, having shown potent inhibitory activity on the metabolism mediated by CYP2D6, was subjected to activity-guided isolation to yield two triterpenes, ursolic acid (1) and oleanolic acid (2), and three alkaloids, vindoline (3), ajmalicine (4), and serpentine (5). The isolated compounds were tested for their inhibitory activity on the metabolism mediated by CYP3A4 or CYP2D6 using [N-methyl-14C]erythromycin or [O-methyl-14C]dextromethorphan as a substrate, respectively. Ajmalicine (4) and serpentine (5) showed very potent inhibitory activity against CYP2D6 with IC50 values of 0.0023 and 3.51 microM, respectively. All isolated compounds showed weak or no inhibition against CYP3A4. On time-, concentration-, and NADPH-dependent assay, serpentine (5) appear to be the mechanism-based inhibitor for CYP2D6 enzyme in which the inhibition was irreversible and driven by catalytic process. K(I) and k(inact) values for serpentine (5) were 0.148 microM and 0.090 min-1, respectively. On the other hand, ajmalicine (4) showed no time-dependent inhibition or reversible inhibition, and thus appear to be not mechanism-based inhibitor.

Mechanism-based inactivation of human liver microsomal CYP3A4 by rutaecarpine and limonin from Evodia fruit extract.

Evodia fruit (Evodiae Fructus) is used as a herbal medicine prepared from the matured fruit of the Evodia rutaecarpa Bentham or Evodia officinalis Dode, of the Rutaceae plant family. An extract of Evodia fruit in the presence of NADPH was shown to inhibit human liver microsomal erythromycin N-demethylation activity, mediated by cytochrome P450 3A4 (CYP3A4), in a preincubation-time dependent manner. The present study was conducted to identify components of Evodia fruit extract having preincubation-time dependent inhibitory effects on CYP3A4 by analyzing human liver microsomal erythromycin N-demethylation activity. Rutaecarpine, a major component of Evodia fruit, and limonin caused the most dramatic decrease in residual CYP3A4 activity (IC50 before and after 20 min preincubation with: rutaecarpine, >100 microM and 1.4 microM; limonin, 23.5 microM and 1.8 microM, respectively). Furthermore, rutaecarpine and limonin were identified as mechanism-based inhibitors of CYP3A4 from the following observations: 1) The inhibitory effects of rutaecarpine and limonin on CYP3A4 activity were dependent on the preincubation time, 2) The inhibition required NADPH, 3) The inhibition was depressed in the presence of the competitive CYP3A4 inhibitor, ketoconazole, 4) Dialysis resulted in no recovery of CYP3A4 activity. The kinetic parameters for inactivation k(inact) and K(I) were: 0.387 min-1 and 107.7 microM for rutaecarpine, 0.266 min-1 and 23.2 microM for limonin, respectively. These results indicate that rutaecarpine and limonin are mechanism-based inhibitors of CYP3A4.

Metabolite-cytochrome P450 complex formation by methylenedioxyphenyl lignans of Piper cubeba: mechanism-based inhibition.

Five methylenedioxyphenyl lignans, (-)-clusin (1), (-)-dihydroclusin (2), (-)-yatein (3), (-)-hinokinin (4), and (-)-dihydrocubebin (5), were isolated from Piper cubeba as potent and selective inhibitors against cytochrome P450 3A4 (CYP3A4). In this study, we investigated the mechanism of inhibition of CYP3A4 by these lignans and the possibility of their mechanism-based inhibition. Using [N-methyl-14C]erythromycin as a substrate, all lignans appear to be showed mixed-type of inhibition with apparent Ki of 1.96-4.07 microM. Furthermore, all lignans (1-5) inhibited CYP3A4 in a time-, concentration-, and NADPH-dependent manners and thus appear to be the mechanism-based inhibitors of CYP3A4. The apparent inactivation parameter, K(I) for these compounds were in the range of 0.054-0.373 microM, whereas the k(inact) values were 0.225-0.320 min-1. Among them, (-)-clusin (1) and (-)-dihydroclusin (2) were found to be the most potent CYP3A4 inactivator with apparent K(I) and k(inact) values of 0.082, 0.054 microM and 0.253, 0.310 min-1, respectively. Spectral scanning of microsomes with these lignans yielded an absorbance at 455 nm, suggesting that all of them appear to inactivate the cytochrome P450 via the formation of a metabolite intermediate complex. This pattern is consistent with the metabolism of the methylenedioxyphenyl compounds. These results indicate that (-)-clusin (1), (-)-dihydroclusin (2), (-)-yatein (3), (-)-hinokinin (4), and (-)-dihydrocubebin (5) are effective mechanism-based inhibitors of CYP3A4.

Mechanism-based inhibition of CYP3A4 by constituents of Zingiber aromaticum.

Sixteen compounds isolated from Zingiber aromaticum and showing concentration-dependent inhibition with IC50 values less than 100 microM, were analyzed for their possibility of time-, concentration-, and NADPH-dependent inhibition of CYP3A4 and four were analyzed for CYP2D6. All seven kaempferol glycosides and two kaempferol derivatives (4, 5, 8-14) appear to be the mechanism-based inhibitors of CYP3A4 enzyme in which the inhibition is irreversible and driven by the catalytic process. The other compounds showed no NADPH-dependent inhibition or reversible inhibition, and thus do not appear to be mechanism-based inhibitors. K(I) values for compounds 4, 5, 8-14 were in the range of 2.21-27.01 microM, whereas the k(inact) values were 0.23-0.65 min(-1). Kaempferol-3-O-(2,3,4-tri-O-acetyl-alpha-L-rhamnopyranoside) (5) was found to be the most potent CYP3A4 inactivator with K(I) and k(inact) values of 2.21 microM and 0.45 min(-1), respectively.

Potent CYP3A4 inhibitory constituents of Piper cubeba.

The EtOAc-soluble fraction of the water extract of Piper cubeba, having shown potent inhibitory activity on the metabolism mediated by CYP3A4, was subjected to activity-guided isolation to yield two new lignans, (8R,8'R)-4-hydroxycubebinone (1) and (8R,8'R,9'S)-5-methoxyclusin (2), and two new sesquiterpenes, (5 alpha,8 alpha)-2-oxo-1(10),3,7(11)-guaiatrien-12,8-olide (3) and (1 alpha,2 beta,5 alpha,8 alpha 10 alpha)-1,10-epoxy-2-hydroxy-3,7(11)-guaiadien-12,8-olide (4), along with 16 known compounds (5-20). The structures of the isolated compounds were elucidated on the basis of spectroscopic and chemical analyses. The isolated compounds were tested for their inhibitory activity on the metabolism mediated by CYP3A4 or CYP2D6 using [N-methyl-(14)C]erythromycin or [O-methyl-(14)C]dextromethorphan as a substrate, respectively. The compounds (8R,8'R,9'S)-5-methoxyclusin (2), (-)-clusin (10), (-)-yatein (13), ethoxyclusin (15), and (-)-dihydroclusin (17), having one methylenedioxyphenyl moiety in their structures, showed very potent and selective inhibitory activity against CYP3A4 with IC(50) values (0.44-1.0 microM) identical to that of the positive control, ketoconazole (IC(50), 0.72 microM).

Regioselective monosulfation and disulfation of the phytoestrogens daidzein and genistein by human liver sulfotransferases.

Regioselective sulfation of the phytoestrogens daidzein (DZ, 7,4'-dihydroxyisoflavone) and genistein (GS, 5,7,4'-trihydroxyisoflavone) was investigated using human liver cytosol and purified recombinant human sulfotransferase (SULT) isoforms, SULT1A1, SULT1A3, SULT2A1, and SULT1E1. 7-Position-preferential sulfation of DZ and GS was observed in human hepatic cytosols from 3 male and 3 female subjects. Average ratios for 7- to 4'-sulfate formation were 4.5:1 from DZ and 8.4:1 from GS in these human liver cytosols. Apparent K(m) values for the 7- and 4'-sulfation of DZ and GS by these cytosols were similar and in a range from 0.46 to 0.66 microM. All recombinant human SULTs had activity for 7- and 4'-sulfation of these phytoestrogens except for 7-sulfating activity of SULT1A3. SULT1A1 and SULT1E1 exhibited much higher catalytic efficiency, k(cat)/K(m), for 7- and 4'-sulfation of these substrates than did the other two, SULT1A3 and SULT2A1. SULT1A1 showed K(m) values of 0.47 and 0.52 microM for the mono-sulfation of DZ and GS, respectively, which were very similar to those of human cytosol. The observed k(cat)/K(m) indicated that SULT1A1 catalyzed 7-sulfation of DZ and GS at rates 4.4- and 8.8-fold higher, respectively, than such 4'-sulfation. However, with SULT1E1, catalytic efficiency was very similar for the sulfation of both positions. These data strongly suggest that SULT1A1 plays a major role in monosulfation of the phytoestrogens and determines the regioselectivity of sulfation in human hepatic cytosol. A kinetic study for 7,4'-disulfate formation of DZ and GS from their 7- and 4'-monosulfates indicated that SULT1E1 most efficiently catalyzed both reactions among human SULTs.

Identification and characterization of potent CYP3A4 inhibitors in Schisandra fruit extract.

Schisandra fruit, a Schisandraceae family herb, is used as a component in Kampo medicines (developed from Chinese medicines, but established in Japan). It can act as a sedative and antitussive, improve hepatic function, and give a general tonic effect. An extract of Schisandra fruit has been shown with a potent inhibitory effect on human liver microsomal erythromycin N-demethylation activity mediated by cytochrome P450 3A4 (CYP3A4). The present study was conducted to identify Schisandra fruit components having inhibitory effects on CYP3A4 by surveying the effect on human liver microsomal erythromycin N-demethylation activity. Known components of Schisandra fruit, gomisins B, C, G, and N and gamma-shizandrin, showed inhibitory effects on N-demethylation activity. Among these components, gomisin C displayed the most potent and competitive inhibitory effect, with a Ki value of 0.049 microM. Furthermore, the inhibitory effect of gomisin C was stronger than that of ketoconazole (Ki = 0.070 microM), a known potent CYP3A4 inhibitor. Gomisin C, however, inhibited CYP1A2-, CYP2C9-, CYP2C19-, and CYP2D6-dependent activities only to a limited extent (IC50 values >10 microM). Moreover, gomisin C inactivated human liver microsomal erythromycin N-demethylation activity in a time- and concentration-dependent manner. The inactivation kinetic parameters k(inact) and K(I) were 0.092 min(-1) and 0.399 microM, respectively. The human liver microsomal erythromycin N-demethylation activity inactivated by gomisin C did not recover on dialysis of the microsomes. Spectral scanning of CYP3A4 with gomisin C yielded an absorbance at 455 nm, suggesting that gomisin C inactivated the cytochrome P450 via the formation of a metabolite intermediate complex. This pattern is consistent with the metabolism of the methylenedioxy substituent in gomisin C. These results indicate that gomisin C is a mechanism-based inhibitor that not only competitively inhibits but irreversibly inactivates CYP3A4.

Sesquiterpenes and flavonol glycosides from Zingiber aromaticum and their CYP3A4 and CYP2D6 inhibitory activities.

Three new sesquiterpenes, (2R,3S,5R)-2,3-epoxy-6,9-humuladien-5-ol-8-one (1), (2R,3R,5R)-2,3-epoxy-6,9-humuladien-5-ol-8-one (2), and (5R)-2,6,9-humulatrien-5-ol-8-one (3), and two new flavonol glycosides, kaempferol-3-O-(2,3-di-O-acetyl-alpha-l-rhamnopyranoside) (4) and kaempferol-3-O-(2,3,4-tri-O-acetyl-alpha-l-rhamnopyranoside) (5), were isolated from the EtOAc-soluble fraction of the water extract of Zingiber aromaticum, along with 13 known compounds (6-18). The structures of the isolated compounds were elucidated on the basis of spectroscopic and chemical analyses. The isolated compounds were tested for their inhibitory activity on the metabolism mediated by CYP3A4 or CYP2D6 using [N-methyl-(14)C]erythromycin or [O-methyl-(14)C]dextromethorphan as a substrate, respectively. Kaempferol-3-O-(2,3,4-tri-O-acetyl-alpha-l-rhamnopyranoside) (5) showed the most potent inhibitory activity (IC(50), 14.4 microM) on the metabolism mediated by CYP3A4, and kaempferol-3-O-methyl ether (14) inhibited CYP2D6 most potently (IC(50), 4.63 microM).