Lignans from the bark of Machilus thunbergii and their DNA topoisomerases I and II inhibition and cytotoxicity

Activity-guided fractionation based on topoisomerase I inhibitory activity lead to the isolation of ten lignans (1-10) from the methylene chloride extract of the bark of Machilus thunbergii SIEB. et ZUCC. (Lauraceae). These were identified as machilin A (1), erythro-austrobailignan-6 (2), meso-monomethyl dihydroguaiaretic acid (3), meso-dihydroguaiaretic acid (4), galbacin (5), machilin F (6), nectandrin A (7) nectandrin B (8), (-)-acuminatin (9) and (7S,8S)-7-(4-hydroxy-3-methoxyphenyl)-1'-formyl-3'-methoxy-8-methyldihydrobenzofuran (10) by spectral evidence. In DNA topoisomerase I and II assays in vitro at a concentration of 100 microM, 4 showed the most potent inhibitory activity, 93.6 and 82.1% inhibition, respectively, and 8 showed 79.1 and 34.3% inhibition, respectively. All of these compounds exhibited weak or no cytotoxicities against either the human colon carcinoma cell line (HT-29) or the human breast carcinoma cell line (MCF-7).     

Cytotoxic and DNA topoisomerases I and II inhibitory constituents from the roots of<Emphasis Type="Italic">Aralia cordata</Emphasis>

Bioactivity-guided fractionation, based on the DNA topoisomerase inhibitory activity, lead to the isolation of five compounds (1-5) from the methylene chloride extract of the roots of Aralia cordata Thunb. (Araliaceae). These compounds were identified as ent-pimara-8(14), 15-dien-19-oic acid (1), ent-pimara-8(14), 15-dien-18-oic acid (2), 16alpha-hydrogen-17-isovaleryloxy-ent-kauran-19-oic acid (3), 16alpha-hydroxy-17-isovaleryloxy-ent-kauran-19-oic acid (4) and dehydrofalcarindiol-8-acetate (5) from their spectral data. Compound 3 was isolated for the first time from this plant, and also showed the strongest inhibition of both DNA topoisomerase I and II activities, with 53 and 96% inhibitions, respectively, at a concentration of 20 microM. However, all the compounds exhibited either weak or no cytotoxicities against the human colon carcinoma cell line (HT-29), the human breast carcinoma cell line (MCF-7) and human hepato blastoma cell line (HepG-2).     

L-proline analogues of anthraquinone-2-carboxylic acid: cytotoxic activity in breast cancer MCF-7 cells and inhibitory activity against topoisomerase I and II.

A series of proline analogues of anthraquinone-2-carboxylic acid (1-3) were synthesized and evaluated for cytotoxic activity in the cultured breast cancer MCF-7 cells. The concentrations of 1, 2 and 3 needed to inhibit [3H]thymidine incorporation into DNA by 50% (IC50) were found to be 107 +/- 6 microM, 185 +/- 5 microM and 87 +/- 6 microM, respectively. To test whether cytotoxic properties were related to topoisomerase action, the most potent compounds 1 and 3 were evaluated in a cell-free system. Compound 3, which contains a basic substituent at C terminus of the amino acid such as (dimethylamino)propyl inhibited the catalytic activity of both topoisomerases I and II at a concentration of 30 and 60 microM, respectively. However, compound 1 containing an electrostatically neutral moiety, such as methyl ester did not inhibit topoisomerase I or topoisomerase II. In summary, compound 3 is a promising lead compound for a further structural variation in the design of new antitumour drugs.     

Two new benzofurans from Gastrodia elata and their DNA topoisomerases I and II inhibitory activities

Two new benzofurans, 1-furan-2-yl-2-(4-hydroxyphenyl)-ethanone (1) and 5-(4-hydroxybenzyloxymethyl)-furan-2-carbaldehyde (2), together with five known compounds, 4-hydroxybenzyl methyl ether (3), 5-(hydroxymethyl)-furfural (4), gastrodin (5), beta-sitosterol (6) and beta-sitosterol glucoside (7) were isolated from the rhizome of Gastrodia elata Blume. In DNA topoisomerase I and II inhibition assays in vitro at a concentration of 20 microM, 1 showed potent inhibitory activity, 66% and 71% inhibition, respectively, compared to the positive control compounds, camptothecin and etoposide, 71% and 22% inhibition, respectively. All of these compounds exhibited weak or no cytotoxicities against human colon carcinoma cell line (HT-29), human breast carcinoma cell line (MCF-7) and human hepatocellular carcinoma cell line (HepG-2).     

Effect of minor groove binding drugs on mammalian topoisomerase I activity

Three minor groove binding drugs, distamycin A, bisbenzimide (Hoechst 33258) and 4',6-diamidino-2-phenylindole (DAPI), were examined for their abilities to modulate the activity of topoisomerase I purified from L1210 cells. At 0.5 and 1.0 microM, distamycin stimulated topoisomerase I relaxation of supercoiled DNA by 38 and 13%, respectively, while increasing the drug concentration above 2.0 microM resulted in inhibition. Inhibition was reversible. Complete relaxation could be achieved even in the presence of inhibitory concentrations of distamycin if the incubation time with topoisomerase I was increased from 7.5 to 120 min. The velocity of topoisomerase I mediated relaxation was reduced by 2 microM distamycin at DNA levels ranging from 350 to 2000 ng/reaction. Hoechst 33258 and DAPI inhibited topoisomerase I relaxation in a concentration-dependent manner. Hoechst 33258 and distamycin were equivalent in their abilities to inhibit topoisomerase I, whereas DAPI had a lesser effect (e.g. relaxation was reduced by 50% with 2.7 microM distamycin and 2.8 microM Hoechst 33258 compared to 5 microM DAPI). This study suggests that ligand binding in the minor groove can be a factor in the regulation of topoisomerase I activity.     

Inhibition of ribonucleotide reductase and growth of human colon carcinoma HT-29 cells and mouse leukemia L1210 cells by N-hydroxy-N'-aminoguanidine derivatives

A series of N-hydroxy-N'-aminoguanidine (HAG) derivatives were studied and compared for their effects on ribonucleotide reductase activity in cell-free extracts; on nucleic acid synthesis and the growth of human colon carcinoma HT-29 cells; and on mouse leukemia L1210 cells in culture. The HAG derivatives [RCH=NNHC(=NH)NHOH-tosylate] studied could be grouped as: (1) hydroxybenzylidines; (2) methoxybenzylidines; and (3) nitrobenzylidines substituted at the R position. 2'-Hydroxybenzylidine-HAG, the lead compound, was relatively active in both HT-29 cells and L1210 cells (20 +/- 5 and 13 +/- 4 microM for 50% inhibition of HT-29 and L1210 cell growth respectively). The monohydroxybenzylidene compounds were generally more active than the dihydroxy- and trihydroxybenzylidene-HAG derivatives. The methoxybenzylidene-HAGs were as active as the monohydroxybenzylidene-HAGs. 2'-Hydroxy-4'-methoxybenzylidene-HAG was much more active than 2',4'-dihydroxybenzylidene-HAG. The mononitrobenzylidene-HAGs were more active than the dinitrobenzylidene-HAG compound. In general, L1210 cells were more sensitive to the effects of the HAG compounds than were HT-29 cells. There was good agreement between the concentration of drug required to inhibit the growth of HT-29 cells and that required to inhibit the growth of L1210 cells. There was also good correlation between the ability of HAG derivatives to inhibit ribonucleotide reductase activity and to inhibit tumor cell growth. Some derivatives, such as 2',3',4'- and 3',4',5'-trihydroxybenzylidene-HAG inhibited L1210 cell growth by 50% at lower concentrations (7.8 and 11.9 microM respectively) than the concentrations needed for 50% inhibition of HT-29 cell growth (196 and 234 microM respectively) and ribonucleotide reductase activity (122 and 188 microM respectively). The studies of nucleic acid synthesis in L1210 cells using [3H]cytidine as a precursor showed that 2',3',4'-trihydroxybenzylidine-HAG inhibited DNA synthesis at a lower concentration (29 microM for 50% inhibition) than was needed for the inhibition of RNA synthesis and formation of [3H]deoxycytidine nucleotides in the acid-soluble fraction (320 and 820 microM for 50% inhibition respectively). These results indicate that 2',3',4'-trihydroxybenzylidine-HAG inhibits DNA synthesis in L1210 cells through other mechanisms rather than exclusively through the inhibition of ribonucleotide reductase activity.     

Cudraflavanone A purified from Cudrania tricuspidata induces apoptotic cell death of human leukemia U937 cells, at least in part, through the inhibition of DNA topoisomerase I and protein kinase C activity

A chloroform extract of the root bark of Cudrania tricuspidata showed an inhibitory effect on mammalian DNA topoisomerase I. The topoisomerase I inhibitory compound was purified and identified as 2S-2',5,7-trihydroxy-4',5'-(2,2-dimethylchromeno)-6-prenyl flavanone (cudraflavanone A). Cudraflavanone A was shown to inhibit the activity of topoisomerase I with approximately 0.4 mmol/l 50% inhibitory concentration. A concentration of 6 micromol/l cudraflavanone A caused a 50% growth inhibition of human cancer cell U937. Cudraflavanone A-induced cell death was characterized by the cleavage of poly(ADP-ribose) polymerase and pro-caspase-3. Furthermore, cudraflavanone A induced the fragmentation of DNA into multiples of 180 bp (an apoptotic DNA ladder), indicating that the inhibitor triggered apoptosis. This induction of apoptosis by cudraflavanone A was also confirmed using flow-cytometry analysis. In addition, this compound inhibited protein kinase C activity with approximately 150 micromol/l 50% inhibitory concentration. Taken together, these results suggest that cudraflavanone A may function by inhibiting oncogenic disease, at least in part, through the inhibition of protein kinase C and topoisomerase I activity.     

Aromatic extended bisamidines: synthesis, inhibition of topoisomerases, and anticancer cytotoxicity in vitro

A series of four aromatic extended bisamidines (12-15) differing in the nature of their terminal basic side chains were synthesized and evaluated for cytotoxic activity in MCF-7 cultured breast cancer cells. The concentrations of 12, 13, 14, and 15 needed to inhibit [3H]thymidine incorporation into DNA by 50% (IC50) were found to be 63 microM, 85 microM, 77 microM, and 97 microM, respectively. To test whether cytotoxic properties were related to DNA-binding and topoisomerase action, the bisamidines 12-15 were evaluated in a cell-free system. Data from the ethidium displacement assay showed that bisamidines 12-15 have significant affinity for DNA and show moderate specificity for AT base pairs. In the topoisomerase II assay, the relaxation of DNA was inhibited with all four drugs and the extent of inhibition was directly proportional to the drug concentration. This suggests that DNA binding may be implicated in the cytotoxicity of these bisamidines, possibly by inhibiting interactions between topoisomerase II and their DNA targets.     

Calcium influx modulates DNA synthesis and proliferation in A7r5 vascular smooth muscle cells

In A7r5 vascular smooth muscle cultures basal Ca2+ influx was higher in growing versus quiescent cells (P less than 0.05), due primarily to a five-fold increase in dihydropyridine-inhibitable Ca2+ influx (P less than 0.005). Verapamil decreased [3H]thymidine incorporation in a concentration dependent fashion with a significant 6 +/- 2% inhibition at 0.1 microM and a maximal inhibition of 67 +/- 2% at 100 microM. Similarly, nitrendipine inhibited fetal calf serum-stimulated [3H]thymidine incorporation with a threshold concentration of 1 nM and a maximal inhibition of 79 +/- 12% at 10 microM. In quiescent cells, verapamil (10 microM) inhibited the increases in [3H]thymidine incorporation stimulated by fetal calf serum, serotonin, vasopressin or 12-0-tetradecanoyl phorbol-13-acetate by 37-43% (P less than 0.001 vs. control for all). Finally, verapamil (100 microM) and nitrendipine (10 microM) inhibited proliferation by 39 +/- 10 and 20 +/- 6%, respectively (P less than 0.01 and 0.02 vs. control, respectively). Thus in A7r5 cells, proliferation is associated with increased Ca2+ influx via dihydropyridine-sensitive Ca2+ channels and organic Ca2+ channel antagonists inhibit DNA synthesis and cell proliferation.     

Human DNA topoisomerase inhibitors from Potentilla argentea and their cytotoxic effect against MCF-7

Two polyphenolics, kaempferol 3-O-beta-D-(6"-E-p-coumaroyl)-glucopyranoside (tiliroside) (1) and methyl brevifolincarboxylate (2) isolated from aerial parts of Potentilla argentea L. (Rosaceae) were evaluated for their cytotoxicities against human breast carcionoma cell line (MCF-7) and their DNA-binding ability. The DNA-binding ability of these compounds was studied by means of the human DNA topoisomerase I and II inhibition assay and ethidium displacement assay using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2. Compound 2 was much more active and showed a higher level of cytotoxic potency than compound 1, with IC50 values of 1.11 +/- 2 microM and 21.60 +/- 2 microM, respectively. In DNA topoisomerase I and II inhibition in vitro assays both investigated compounds 1 and 2 were more effective against topoisomerase II than I. The results of DNA binding studies reveal that methyl brevifolincarboxylate had a greater DNA binding affinity that tiliroside, which correlates with its greater potency as a topoisomerase I/II inhibitor.     

Modulation of topoisomerase II catalytic activity by DNA minor groove binding agents distamycin, Hoechst 33258, and 4',6-diamidine-2-phenylindole

The effects of distamycin, Hoechst 33258, and 4',6-diamidine-2-phenylindole (DAPI) on the catalytic activity of topoisomerase II from L1210 cells were determined. These compounds were used as model agents capable of AT-specific binding in the minor groove of DNA while producing no profound long-range alterations to the DNA structure. Two types of reactions catalyzed by topoisomerase II were examined, relaxation of supercoiled DNA and decatenation of highly catenated DNA. Distamycin at low concentrations (0.2-2 microM) substantially stimulated relaxation of supercoiled pBR322 DNA. Higher drug levels (25-50 microM) resulted in a potent inhibition of relaxation. At the stimulatory concentrations of distamycin, only completely relaxed reaction products were observed, as in the absence of the drug. The onset of inhibition (caused by 5-10 microM distamycin) was accompanied by the appearance of partially relaxed intermediates. Similar inhibition of relaxation was observed for Hoechst 33258 and DAPI but, unlike distamycin, these agents produced only marginal stimulation of relaxation when added in low noninhibitory concentrations. Another reaction of topoisomerase II, decatenation of catenated kinetoplast DNA, was also inhibited by distamycin, Hoechst 33258, and DAPI at concentrations similar to those inhibiting the relaxation reaction. This study demonstrates that agents binding to the minor groove of DNA represent a new class of drugs interfering with topoisomerase II and provides possibilities for modulation of this important enzyme.     

In-vitro trypanocidal activity evaluation of crude extract and isolated compounds from Baccharis dracunculifolia D.C. (Asteraceae)

We have performed a trypanocidal bioactivity-guided study of Baccharis dracunculifolia (Asteraceae), the main botanical origin of Brazilian green propolis. The leaf rinse extract of B. dracunculifolia, at a concentration of 3.0 mg mL(-1), displayed 100% lysis of trypomastigote forms of the Y strain of Trypanosoma cruzi (2 x 10(6) parasites mL(-1)). The chromatographic fractionation of the leaf rinse, using several techniques, afforded the isolation of the compounds isosakuranetin (1), aromadendrin-4'-methylether (2), baccharis oxide (3), ferulic acid (4), dihydrocinnamic acid (5), 3-prenyl-4-(dihydrocinnamoyloxy)-cinnamic acid (6), and friedelanol (7). The chemical structures of all compounds were established by UV-vis, (1)H and (13)CNMR data analysis in comparison with the literature. Compounds 1 and 3 were the most active in the trypanocidal assay, showing IC50 values (inhibitory concentration required for 50% inhibition) of 247.6 and 249.8 microM, respectively. Compounds 2, 4, and 6 displayed moderate activity, whilst compounds 5 and 7 were inactive.     

Effects of tramadol and O-demethyl-tramadol on human 5-HT reuptake carriers and human 5-HT3A receptors: a possible mechanism for tramadol-induced early emesis

([3H]5-HT)-uptake and patch-clamp techniques were used to study the actions of (+) and (-) tramadol and the active metabolites of tramadol, (+) and (-) O-demethyl-tramadol on the human serotonin (5-HT) transporter and the human 5-HT3A receptor, stably expressed in HEK-293 cells. The (+) and (-) enantiomers of tramadol suppressed the human 5-HT transporter concentration-dependently (IC50=1.0 and 0.8 microM, respectively), resulting in 97% and 87% transport inhibition at their respective initial plasma concentrations (9.5 microM). The (+) and (-) enantiomers of the active tramadol metabolite were less potent than tramadol in inhibiting the human 5-HT transporter (IC50=15 and 44 microM, respectively), resulting in 19.2% and 4.8% transport inhibition at their highest plasma concentrations (2.5 microM). In contrast to their potent suppression of the 5-HT transporter, both, (+) and (-) tramadol inhibited 5-HT (30 microM)-induced currents only at substantially higher concentrations (IC50=199 and 251 microM, respectively), resulting in only 6% and 4% inhibition at the initial maximum plasma concentration. A similar low potent inhibition of human 5-HT(3A) receptors was found for (+) and (-) O-demethyl-tramadol (IC50=158 and 63 microM, respectively). In conclusion, at clinical plasma concentrations tramadol potently suppresses the human 5-HT transporter, whereas it has only a slight effect on the human 5-HT3A receptor. The results are compatible with a possible mechanism for tramadol-induced early emesis involving the serotonergic system.     

Studies on the interactions between drugs and estrogen. III. Inhibitory effects of 29 drugs reported to induce gynecomastia on the glucuronidation of estradiol

To determine the inhibition effects of drugs on the glucuronidation of estradiol (E2), 29 drugs that have been reported to induce gynecomastia were examined in the presence of UDP-glucuronic acid using human hepatic microsomes (pooled) as the enzyme source. The percentage inhibition of the E2 glucuronidation was determined at drug concentrations of 1 microM (approximate therapeutic concentration) and 100 microM (non-clinical overdose concentration) based on the rate constants for the 3- and 17-glucuronidation of E2 (11.2 and 2.52 pmol/min/mg protein, respectively). The only drug that exhibited 50% or higher inhibition of the 3-glucuronidation at a concentration of 1 microM was manidipine (54.4%). When the concentration was 100 microM, manidipine exhibited 100% inhibition of the 3-glucuronidation, and other drugs that exhibited 50% or higher inhibition of the 3-glucuronidation were nicardipine (92%), nisoldipine (90%), nifedipine (84%), domperidone (81%), tacrolimus (80%), nitrendipine (77%) and ketoconazole (69%). Conversely, ipriflavone accelerated the formation of estradiol 3-glucuronide in the activity of 165% at the concentration of 100 microM. On the 17-glucuronidation, all of the drugs showed less than 50% inhibition at the concentration of 1 microM, but at the concentration of 100 microM, drugs that exhibited 50% or higher inhibition consisted of manidipine (79%), chlormadinone acetate (74%), nisoldipine (66%), nitrendipine (60%) and ketoconazole (55%). Although IC(50) values of these drugs were all lower than the K(m) value (285 microM) for the 3-glucuronidation of E2, they were higher than the K(m) value for the 17-glucuronidation (18.8 microM). Thus, the effect of the drugs on the E2 glucuronidation should be greater for hydroxy group at the C-3 than that at the C-17 of E2 molecule. On the other hand, metabolic clearances (V(max)/K(m)) of the 3- and 17-glucuronidation were about 1/14th and 1/18th of that of the 2-hydroxylation of E2, respectively. The result implies that, when the contribution of the glucuronidation to enterohepatic circulation is taken into consideration, the effect of this metabolic inhibition in the estrogen pool cannot be ignored.     

Role of prostanoids in the contraction induced by a tachykinin NK2 receptor agonist in the hamster urinary bladder

In isolated strips of the hamster urinary bladder the selective tachykinin NK2 receptor agonist [betaAla8]NKA(4-10) induced a concentration-dependent (1 nM-10 microM) contraction (EC50 104 nM) associated with significant release of prostaglandin E2 (PGE2, 50+/-17 pg/mg tissue). In mucosa-free bladder strips [betaAla8]NKA(4-10) was as potent as in the presence of mucosa (EC50 46 nM), although the evoked PGE2 release was significantly less than in controls (6+/-1.7 pg/mg tissue). Dexketoprofen (10 microM) produced a significant but limited rightward shift of the concentration/response curve to [betaAla8]NKA(4-10) both in the presence and absence of the mucosal layer: the EC50 for [betaAla8]NKA(4-10) was increased five- and threefold, respectively. The evoked PGE2 release was abolished in both cases. The selective tachykinin NK2 receptor antagonist, nepadutant (10 nM-1 microM) produced a concentration-dependent and even inhibition of both contraction and PGE2 release induced by [betaAla8]NKA(4-10). The L-type calcium channel blocker, nifedipine (1 microM) and the non-selective cationic channel blocker SKF 96365 (30 microM) both inhibited the contractile response to [betaAla8]NKA(4-10) (89+/-2 and 83+/-2% inhibition, respectively). The evoked PGE2 release was not affected by nifedipine but was almost abolished by SKF 96365. Arachidonic acid (100 microM) induced a contractile response (5.9+/-0.7 mN) associated with a large production of PGE2 (383+/-78 pg/mg tissue). The contractile response to arachidonic acid was inhibited by both nifedipine (1 microM) and SKF 96365 (30 microM) (83+/-5 and 79+/-3% inhibition, respectively). The PGE2 production induced by arachidonic acid was markedly inhibited by SKF 96365 only (about 94% inhibition). Exogenous PGE2 contracted hamster bladder strips in the presence of dexketoprofen (EC50 1 microM) and strongly potentiated the contractile response to a submaximal concentration of [betaAla8]NKA(4-10). In anaesthetized hamsters the administration of [betaAla8]NKA(4-10) (10 nmol/kg, i.v.) produced a contractile response of the urinary bladder (13+/-0.4 mmHg) that was inhibited partly by dexketoprofen (25+/-3 and 35+/-4% inhibition for 0.2 and 2 mg/kg, i.v. dexketoprofen, respectively). We conclude that activation of tachykinin NK2 receptors determines prostanoid synthesis/release in the hamster urinary bladder and that this effect is largely ascribable to structures present in the bladder mucosa. Prostanoids generated following NK2 receptor activation amplify the direct contractile effect of NK2 receptor agonists. This latter response is largely due to activation of L-type calcium channels (nifedipine-sensitive) although this source of calcium apparently is not essential for activation of prostanoid synthesis.     

Interaction of DNA intercalator 3-nitrobenzothiazolo (3,2-a)quinolinium with DNA topoisomerases: a possible-mechanism for its biological activity

There is multiple evidence linking the inhibition of DNA topoisomerases I and II with the cytotoxic effects of antitumor drugs such as camptothecin and the DNA intercalators, 4-(9-acridinylamino)methanesulfon-m-anisidine) (mAMSA) and Adriamycin. We have assessed the effect of the DNA intercalator 3-nitrobenzothiazolo(3,2-a)quinolinium (NBQ) on the DNA topoisomerase I and II activities. NBQ has no effect on the activity of purified topoisomerase I, whereas it induced purified topoisomerase II binding to DNA without inducing DNA scission. Above 30 microM, NBQ stimulated formation of double- and single-strand breaks mediated by topoisomerase II in plasmid DNA. Using the alkaline elution technique we determined that NBQ induced single-strand and DNA-protein-associated breaks in the human promyelocytic leukemia cell line HL-60. At sublethal concentrations (less than or equal to 1 microM), NBQ induce HL-60 cells to differentiate. Topoisomerase II-mediated DNA cleavage induced by mAMSA was substantially reduced in NBQ-differentiated cells. Our data suggest that topoisomerase II could play a major role in the biological activity of NBQ in vivo.     

Regulation of human basophil function by phosphatase inhibitors.

1. Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-mediated release of the pre-formed mediator, histamine from human basophils in a time- and dose-dependent manner. Optimal inhibition (77 +/- 4%, P < 0.0001) of histamine release was observed following a 2 h incubation with 1 microM okadaic acid. 2. Okadaic acid and two analogues of okadaic acid were also studied and were found to inhibit the IgE-dependent release of histamine. Concentrations required to inhibit release by 50% (IC50) were 0.6 microM for okadaic acid and 7.5 microM for okadaol, whereas okadaone was inactive. 3. The structurally-unrelated PP inhibitor, calyculin A, also inhibited IgE-dependent histamine release from basophils dose-dependently and was approximately six fold more potent than okadaic acid. 4. The IgE-mediated generation of sulphopeptidoleukotrienes (sLT) from basophils was inhibited by okadaic acid and related analogues with the following rank order of potency; okadaic acid (approx. IC50 0.3 microM) > okadaol (3 microM) > okadaone (inactive). 5. Okadaic acid, okadaol and okadaone (all at 3 microM) inhibited the IgE-mediated generation of the cytokine interleukin 4 (IL4) from human basophils by 67 +/- 9% (P < 0.002), 48 +/- 14% (P < 0.05) and 8 +/- 7% (P = 0.31), respectively. 6. Extracts of purified human basophils liberated 32P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by 17 +/- 3% (P < 0.0005) by a low (2 nM) concentration of okadaic acid and was inhibited by 96 +/- 1% (P < 0.0001) by a higher (5 microM) concentration of okadaic acid. Because a low (2 nM) concentration of okadaic acid inhibits PP2A selectively whereas a higher (5 microM) concentration inhibits both PP1 and PP2A, these findings suggest that both PP1 and PP2A are present in basophils. 7. In total these data suggest that PPs are resident in human basophils and that PPs may be important in the regulation of basophil function.     

Selective serotonin reuptake inhibitors and theophylline metabolism in human liver microsomes: potent inhibition by fluvoxamine.

1. Fluvoxamine and seven other selective serotonin reuptake inhibitors (SRRI) were tested for their ability to inhibit a number of human cytochrome P450 isoforms (CYPs). 2. None of the drugs showed potent inhibition of CYP2A6 (coumarin 7-hydroxylase) or CYP2E1 (chlorzoxazone 6-hydroxylase), while norfluoxetine was the only potent inhibitor of CYP3A having IC50 values of 11 microM and 19 microM for testosterone 6 beta-hydroxylase and cortisol 6 beta-hydroxylase, respectively. 3. Norfluoxetine, sertraline and fluvoxamine inhibited CYP1A1 (7-ethoxyresorufin O-deethylase) in microsomes from human placenta (IC50 values 29 microM, 35 microM and 80 microM, respectively). Fluvoxamine was a potent inhibitor of CYP1A2-mediated 7-ethoxyresorufin O-deethylase activity (IC50 = 0.3 microM) in human liver. 4. In microsomes from three human livers fluvoxamine potently inhibited all pathways of theophylline biotransformation, the apparent inhibitor constant, Ki, was 0.07-0.13 microM, 0.05-0.10 microM and 0.16-0.29 microM for inhibition of 1-methylxanthine, 3-methylxanthine and 1,3-dimethyluric acid formation, respectively. Seven other SSRIs showed either weak or no inhibition of theophylline metabolism. 5. Ethanol inhibited the formation of 1,3-dimethyluric acid with K(i) value of 300 microM, a value which is consistent with inhibition of CYP2E1. Ethanol and fluvoxamine both inhibited 8-hydroxylation by about 45% and, in combination, the compounds decreased the formation of 1,3-dimethyluric acid by 90%, indicating that CYP1A2 and CYP2E1 are equally important isoforms for the 8-hydroxylation of theophylline. 6. It is concluded that pharmacokinetic interaction between fluvoxamine and theophylline is due to potent inhibition of CYP1A2.     

Monoamine oxidase inhibitory components from<Emphasis Type="Italic">Cayratia japonica</Emphasis>

Seven flavonoids were isolated from the whole plants and fruits of Cayratia japonica through the activity-guided isolation of a methanol extract using a monoamine oxidase (MAO) inhibition assay as a monitor. The chemical structures of the isolates were assigned as apigenin-7-O-beta-D-glucuronopyranoside (1), apigenin (2), luteolin (3), luteolin-7-O-beta-D-glucopyranoside (4), (+)-dihydroquercetin (taxifolin) (5), (+)-dihydrokaempferol (aromadendrin) (6) and quercetin (7). Among the isolated compounds, flavones such as apigenin (2) and luteolin (3), as well as the flavonol, quercetin (7) showed potent inhibitory effects against the MAO activity with IC50 values of 6.5, 22.6, and 31.6 microM, respectively. However, the flavone glycosides, apigenin-7-O-beta-D-glucuronopyranoside (1) and luteolin-7-O-beta-D-glucopyranoside (4), showed mild MAO inhibition (IC50 values: 81.7 and 118.6 microM, respectively). The flavanonol derivatives, taxifolin (5) and aromadendrin (6), also showed weak inhibition (IC50 values: 154.7 and 153.1 microM, respectively). Furthermore, quercetin (7) had a more potent inhibitory effect on MAO-A (IC50 value: 2.8 microM) than MAO-B (IC50 value: 90.0 microM). Apigenin (2) and luteolin (3) also preferentially inhibited MAO-A (IC50 values: 1.7 and 4.9 microM, respectively) compared with MAO-B (IC50 values: 12.8 and 59.7 microM, respectively).     

Interaction of buprenorphine and its metabolite norbuprenorphine with cytochromes p450 in vitro.

Buprenorphine is a thebaine derivative used in the treatment of heroin and other opiate addictions. In this study, the selective probe reactions for each of the major hepatic cytochromes P450 (P450s) were used to evaluate the effect of buprenorphine and its main metabolite norbuprenorphine on the activity of these P450s. The index reactions used were CYP1A2 (phenacetin O-deethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (diclofenac 4'-hydroxylation), CYP2C19 (omeprazole 5-hydrxoylation), CYP2D6 (dextromethorphan O-demethylation), CYP2B6 (7-ethoxy-4-trifluoromethyl-coumarin 7-deethylation), CYP2E1 (chlorzoxazone 6-hydroxylation), and CYP3A4 (omeprazole sulfoxidation). Buprenorphine exhibited potent, competitive inhibition of CYP2D6 (Ki 10 +/- 2 microM and 1.8 +/- 0.2 microM) and CYP3A4 (Ki 40 +/- 1.6 microM and 19 +/- 1.2 microM) in microsomes from human liver and cDNA-expressing lymphoblasts, respectively. Compared with buprenorphine, norbuprenorphine demonstrated a lower inhibitory potency with CYP2D6 (22.4% inhibition at 20 microM norbuprenorphine) and CYP3A4 (13.6% inhibition at 20 microM) in microsomes from human cDNA-expressing lymphoblast cells. Furthermore, buprenorphine was shown to be a substrate of CYP2D6 (Km = 600 microM; Vmax = 0.40 nmol/min/mg protein) and CYP3A4 (Km = 36 microM; Vmax = 0.19 nmol/min/mg protein). The present in vitro study suggests that buprenorphine and its major metabolite norbuprenorphine are inhibitors of CYP2D6 and CYP3A4; however, at therapeutic concentrations they are not predicted to cause potentially clinically important drug interactions with other drugs metabolized by major hepatic P450s.