Evaluation of Polyphenolic Profile and Antioxidant Activity of Pistacia lentiscus L. Leaves and Fruit Extract Obtained by Optimized Microwave-Assisted Extraction.

Pistacia lentiscus L. is a Mediterranean shrub known for its health promoting effects attributed to a large extent to polyphenols accumulated in all parts of the plant. Microwave-assisted extraction is a green extraction technique enabling fast and effective isolation of plant polyphenols. Therefore, the aim of this research was to optimize the microwave-assisted extraction of polyphenols from Pistacia lentiscus L. leaves and fruit in terms of temperature, extraction time and microwave power and to evaluate their polyphenolic profile by UPLC/ESI-MS2 and antioxidant capacity by ORAC assay. Optimal extraction conditions for leaf polyphenols were 69 °C, 512 W and 12 min, while for fruit were slightly more intensive-75 °C, 602 W and 15 min. Obtained total phenolic content in leaves and fruit was similar to that obtained after 30 min of the heat-reflux method. The polyphenolic profile of extracts included 34 compounds, with myricetin glycosides being the most abundant compounds among flavonoids in Pistacia lentiscus L. leaves and fruit and gallic acid and its derivates among the phenolic acids. ORAC assay showed higher antioxidant capacity for Pistacia lentiscus L. leaves extract than for fruit, which is in correlation with their respective phenolic content.

Essential Oil Composition of Different Plant Parts from Croatian Petasites albus (L.) Gaertn. and Petasites hybridus (L.) G.Gaertn., B.Mey. & Scherb. (Asteraceae).

Essential oil compositions of fresh leaves, flower stems and rhizomes obtained by hydrodistillation from different Croatian populations of Petasites albus (L.) Gaertn. and Petasites hybridus (L.) G.Gaertn., B.Mey. & Scherb. (four of each species) were investigated using gas chromatography-flame ionization detection and gas chromatography-mass spectrometry. Altogether, 118 constituents were identified, accounting for 81.19-96.81 % of total oil composition. All essential oils were characterized by oxygenated sesquiterpenes, with distinct compounds recorded for both investigated species. Clear separation between the two species was confirmed by principal component analysis and hierarchical cluster analysis. This is the first study that recorded intraspecific variations of essential oil constituents from P. albus and P. hybridus. Obtained results could contribute to the understanding of their medicinal and nutritional value.

Long-term seizure outcome following epilepsy surgery in the parietal lobe: a meta-analysis.

Due to a limited number of patients with drug-resistant parietal lobe epilepsy in surgical series, there are insufficient data about long-term seizure outcome following surgery restricted to the parietal lobe. We performed a meta-analysis to asses long-term outcomes in patients with parietal lobe epilepsy who underwent surgery confined to the parietal lobe or resection with major involvement of the parietal cortex. An English language literature search for studies on parietal lobe surgery and outcome was conducted using the MEDLINE database, followed by a manual search based on specific criteria. An inverse variance random effect meta-analysis model was used to estimate the pooled proportion of Engel Class I. Meta-regression models were used to examine the association between outcome and potential predictors. The search yielded seven retrospective studies with a total sample size of 253 patients (mean follow-up: 104.9±74.8 months). Following surgery, Engel Class I surgical outcome was achieved in 62.4% (95% CI: 0.492-0.755). Two independent predictors were identified for positive long-term outcome: interictal EEG localized to the parietal region (p=0.007) and the presence of tumour (p=0.022). Following surgery confined to the parietal lobe or resection with major involvement of the parietal cortex, the long-term prognosis of patients with parietal lobe epilepsy is favourable.

Effect of human glutathione S-transferase hGSTP1-1 polymorphism on the detoxification of reactive metabolites of clozapine, diclofenac and acetaminophen.

Recent association studies suggest that genetically determined deficiencies in GSTs might be a risk factor for idiosyncratic adverse drug reactions resulting from the formation of reactive drug metabolites. hGSTP1-1 is polymorphic in the human population with a number of single nucleotide polymorphisms that yield an amino acid change in the encoded protein. Three allelic variants of hGSTP1-1 containing an Ile105Val or Ala114Val substitution, or a combination of both, have been the most widely studied and showed different activity when compared to wild-type hGSTP1-1*A (Ile105/Ala114). In the present study, we studied the ability of these allelic variants to catalyze the GSH conjugation of reactive metabolites of acetaminophen, clozapine, and diclofenac formed by bioactivation in in vitro incubations by human liver microsomes and drug metabolizing P450 BM3 mutants. The results show that effects of the change of amino acid at residue 105 and 114 on conjugation reactions were substrate dependent. A single substitution at residue 105 affects the ability to catalyze GSH conjugation, while when both residue 105 and 114 were substituted the effect was additionally enhanced. Single mutation at position 114 did not show a significant effect. The different hGSTP1-1 mutants showed slightly altered regioselectivities in formation of individual GSH conjugates of clozapine which suggests that the binding orientation of the reactive nitrenium ion of clozapine is affected by the mutations. For diclofenac, a significant decrease in activity in GSH-conjugation of diclofenac 1',4'-quinone imine was observed for variants hGSTP1-1*B (Val105/Ala114) and hGSTP1-1*C (Val105/Val114). However, since the differences in total GSH conjugation activity catalyzed by these allelic variants were not higher than 30%, differences in inactivation of reactive intermediates by hGSTP1-1 are not likely to be a major factor in determining interindividual difference in susceptibility to adverse drug reactions induced by the drugs studied.

Effect of human glutathione S-transferases on glutathione-dependent inactivation of cytochrome P450-dependent reactive intermediates of diclofenac.

Idiosyncratic adverse drug reactions due to the anti-inflammatory drug diclofenac have been proposed to be caused by the generation of reactive acyl glucuronides and oxidative metabolites. For the oxidative metabolism of diclofenac by cytochromes P450 at least five different reactive intermediates have been proposed previously based on structural identification of their corresponding GSH-conjugates. In the present study, the ability of four human glutathione S-transferases (hGSTs) to catalyze the GSH-conjugation of the different reactive intermediates formed by P450s was investigated. Addition of pooled human liver cytosol and recombinant hGSTA1-1, hGSTM1-1, and hGSTP1-1 to incubations of diclofenac with human liver microsomes or purified CYP102A1M11 L437N as a model system significantly increased total GSH-conjugation. The strongest increase of total GSH-conjugation was observed by adding hGSTP1-1, whereas hGSTM1-1 and hGSTA1-1 showed lower activity. Addition of hGSTT1-1 only showed a minor effect. When considering the effects of hGSTs on GSH-conjugation of the different quinoneimines of diclofenac, it was found that hGSTP1-1 showed the highest activity in GSH-conjugation of the quinoneimine derived from 5-hydroxydiclofenac (5-OH-DF). hGSTM1-1 showed the highest activity in inactivation of the quinoneimine derived from 4'-hydroxydiclofenac (4'-OH-DF). Separate incubations with 5-OH-DF and 4'-OH-DF as substrates confirmed these results. hGSTs also catalyzed GSH-conjugation of the o-iminemethide formed by oxidative decarboxylation of diclofenac as well as the substitution of one of the chlorine atoms of DF by GSH. hGSTP1-1 showed the highest activity for the formation of these minor GSH-conjugates. These results suggest that hGSTs may play an important role in the inactivation of DF quinoneimines and its minor reactive intermediates especially in stress conditions when tissue levels of GSH are decreased.

Human precision-cut liver slices as an ex vivo model to study idiosyncratic drug-induced liver injury.

Idiosyncratic drug-induced liver injury (IDILI) is a major problem during drug development and has caused drug withdrawal and black-box warnings. Because of the low concordance of the hepatotoxicity of drugs in animals and humans, robust screening methods using human tissue are needed to predict IDILI in humans. According to the inflammatory stress hypothesis, the effects of inflammation interact with the effects of a drug or its reactive metabolite, precipitating toxic reactions in the liver. As a follow-up to our recently published mouse precision-cut liver slices model, an ex vivo model involving human precision-cut liver slices (hPCLS), co-incubated for 24 h with IDILI-related drugs and lipopolysaccharide (LPS), was developed to study IDILI mechanisms related to inflammatory stress in humans and to detect potential biomarkers. LPS exacerbated the effects of ketoconazole and clozapine toxicity but not those of their non-IDILI-related comparators, voriconazole and olanzapine. However, the IDILI-related drugs diclofenac, carbamazepine, and troglitazone did not show synergistic toxicity with LPS after incubation for 24 h. Co-incubation of ketoconazole and clozapine with LPS decreased the levels of glutathione in hPCLS, but this was not seen for the other drugs. All drugs affected LPS-induced cytokine release, but interestingly, only ketoconazole and clozapine increased the level of LPS-induced TNF release. Decreased levels of glutathione and cysteine conjugates of clozapine were detected in IDILI-responding livers following cotreatment with LPS. In conclusion, we identified ketoconazole and clozapine as drugs that exhibited synergistic toxicity with LPS, while glutathione and TNF were found to be potential biomarkers for IDILI-inducing drugs mediated by inflammatory stress. hPCLS appear to be suitable for further unraveling the mechanisms of inflammatory stress-associated IDILI.

Characterization of human cytochrome P450s involved in the bioactivation of clozapine.

Clozapine is known to cause hepatotoxicity in a small percentage of patients. Oxidative bioactivation to reactive intermediates by hepatic cytochrome P450s (P450s) has be proposed as a possible mechanism. However, in contrast to their role in formation of N-desmethylclozapine and clozapine N-oxide, the involvement of individual P450s in the bioactivation to reactive intermediates is much less well characterized. The results of the present study show that 7 of 14 recombinant human P450s were able to bioactivate clozapine to a glutathione-reactive nitrenium ion. CYP3A4 and CYP2D6 showed the highest specific activity. Enzyme kinetical characterization of these P450s showed comparable intrinsic clearance of bioactivation, implicating that CYP3A4 would be more important because of its higher hepatic expression, compared with CYP2D6. Inhibition experiments using pooled human liver microsomes confirmed the major role of CYP3A4 in hepatic bioactivation of clozapine. By studying bioactivation of clozapine in human liver microsomes from 100 different individuals, an 8-fold variability in bioactivation activity was observed. In two individuals bioactivation activity exceeded N-demethylation and N-oxidation activity. Quinidine did not show significant inhibition of bioactivation in any of these liver fractions, suggesting that CYP2D6 polymorphism is not an important factor in determining susceptibility to hepatotoxicity of clozapine. Therefore, interindividual differences and drug-drug interactions at the level of CYP3A4 might be factors determining exposure of hepatic tissue to reactive clozapine metabolites.

AMAP, the alleged non-toxic isomer of acetaminophen, is toxic in rat and human liver.

N-acetyl-meta-aminophenol (AMAP) is generally considered as a non-toxic regioisomer of the well-known hepatotoxicant acetaminophen (APAP). However, so far, AMAP has only been shown to be non-toxic in mice and hamsters. To investigate whether AMAP could also be used as non-toxic analog of APAP in rat and human, the toxicity of APAP and AMAP was tested ex vivo in precision-cut liver slices (PCLS) of mouse, rat and human. Based on ATP content and histomorphology, APAP was more toxic in mouse than in rat and human PCLS. Surprisingly, although AMAP showed a much lower toxicity than APAP in mouse PCLS, AMAP was equally toxic as or even more toxic than APAP at all concentrations tested in both rat and human PCLS. The profile of proteins released into the medium of AMAP-treated rat PCLS was similar to that of APAP, whereas in the medium of mouse PCLS, it was similar to the control. Metabolite profiling indicated that mouse PCLS produced the highest amount of glutathione conjugate of APAP, while no glutathione conjugate of AMAP was detected in all three species. Mouse also produced ten times more hydroquinone metabolites of AMAP, the assumed proximate reactive metabolites, than rat or human. In conclusion, AMAP is toxic in rat and human liver and cannot be used as non-toxic isomer of APAP. The marked species differences in APAP and AMAP toxicity and metabolism underline the importance of using human tissues for better prediction of toxicity in man.

Role of residue 87 in the activity and regioselectivity of clozapine metabolism by drug-metabolizing CYP102A1 M11H: application for structural characterization of clozapine GSH conjugates.

In the present study, a site-saturation mutagenesis library of drug-metabolizing CYP102A1 M11H with all 20 amino acids at position 87 was applied as a biocatalyst for the production of stable and reactive metabolites of clozapine. Clozapine is an atypical antipsychotic drug in which formation of reactive metabolites is considered to be responsible for several adverse drug reactions. Reactive intermediates of clozapine can be inactivated by GSH to multiple GSH conjugates by nonenzymatic and glutathione transferase (GST)-mediated conjugation reactions. The structures of several GST-dependent metabolites have not yet been elucidated unequivocally. The present study shows that the nature of the amino acid at position 87 of CYP102A1 M11H strongly determines the activity and regioselectivity of clozapine metabolism. Some mutants showed preference for N-demethylation and N-oxidation, whereas others showed high selectivity for bioactivation to reactive intermediates. The mutant containing Phe87 showed high activity and high selectivity for the bioactivation pathway and was used for the large-scale production of GST-dependent GSH conjugates by incubation in the presence of recombinant human GST P1-1. Five human-relevant GSH adducts were produced at high levels, enabling structural characterization by (1)H NMR. This work shows that drug-metabolizing CYP102A1 mutants, in combination with GSTs, are very useful tools for the generation of GSH conjugates of reactive metabolites of drugs to enable their isolation and structural elucidation.

Diclofenac inhibits tumor necrosis factor-α-induced nuclear factor-κB activation causing synergistic hepatocyte apoptosis.

UNLABELLED: Drug-induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor α (TNF-α). HepG2 cells were treated with diclofenac followed by TNF-α challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF-α. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF-α-mediated enhancement involved activation of caspase-3 through a caspase-8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c-Jun N-terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF-α-induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF-α-mediated nuclear factor kappaB (NF-κB) translocation oscillation in association with reduced NF-κB transcriptional activity. This was associated with inhibition by diclofenac of the TNF-α-induced phosphorylation of the inhibitor of NF-κB alpha (IκBα). Finally, inhibition of IκB kinase ß (IKKß) with BMS-345541 as well as stable lentiviral short hairpin RNA (shRNA)-based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNF-α-induced cytotoxicity. CONCLUSION: Together, our data suggest a model whereby diclofenac-mediated stress signaling suppresses TNF-α-induced survival signaling routes and sensitizes cells to apoptosis.

Role of human glutathione S-transferases in the inactivation of reactive metabolites of clozapine.

The conjugation of reactive drug metabolites to GSH is considered an important detoxification mechanism that can be spontaneous and/or mediated by glutathione S-transferases (GSTs). In case GSTs play an important role in GSH conjugation, genetically determined deficiencies in GSTs may be a risk factor for adverse drug reactions (ADRs) resulting from reactive drug metabolites. So far, the role of GSTs in the detoxification of reactive intermediates of clozapine, a drug-causing idiosyncratic drug reactions (IDRs), has not been studied. In the present study, we studied the ability of four recombinant human GSTs (hGST A1-1, hGST M1-1, hGST P1-1, and hGST T1-1) to catalyze the GSH conjugation of reactive metabolites of clozapine, formed in vitro by human and rat liver microsomes and drug-metabolizing P450 BM3 mutant, P450 102A1M11H. Consistent with previous studies, in the absence of GSTs, three GSH conjugates were identified derived from the nitrenium ion of clozapine. In the presence of three of the GSTs, hGST P1-1, hGST M1-1, and hGST A1-1, total GSH conjugation was strongly increased in all bioactivation systems tested. The highest activity was observed with hGST P1-1, whereas hGST M1-1 and hGST A1-1 showed slightly lower activity. Polymorphic hGST T1-1 did not show any activity in catalyzing GSH conjugation of reactive clozapine metabolites. Interestingly, the addition of hGSTs resulted in major changes in the regioselectivity of GSH conjugation of the reactive clozapine metabolite, possibly due to the different active site geometries of hGSTs. Two GSH conjugates found were completely dependent on the presence of hGSTs. Chlorine substitution of the clozapine nitrenium ion, which so far was only observed in in vivo studies, appeared to be the major pathway of hGST P1-1-catalyzed GSH conjugation, whereas hGST A1-1 and hGST M1-1 also showed significant activity. The second GSH conjugate, previously also only found in in vivo studies, was also formed by hGST P1-1 and to a small extent by hGST A1-1. These results demonstrate that human GSTs may play a significant role in the inactivation of reactive intermediates of clozapine. Therefore, further studies are required to investigate whether genetic polymorphisms of hGST P1-1 and hGST M1-1 contribute to the interindividual differences in susceptibility to clozapine-induced adverse drug reactions.

Column selection for pharmaceutical analyses based on a column classification using four test parameters.

This paper focuses on the usability of a previously developed column classification system, applied to pharmaceutical analyses. The separation of two drugs from their respective related substances was investigated on 65 new reversed-phase liquid chromatographic columns. The chromatographic procedure for fluoxetine hydrochloride was performed according to the method prescribed in the European Pharmacopoeia monograph while the separation of gemcitabine hydrochloride was carried out according to the United States Pharmacopeia monograph. It was shown that the column ranking system is a helpful tool in the selection of a suitable column.