Brigitte Brem

Antitumor Activity and Mechanism of Action of the Cyclopenta[b]benzofuran, Silvestrol

Background Flavaglines are a family of natural products from the genus Aglaia that exhibit anti-cancer activity in vitro and in vivo and inhibit translation initiation. They have been shown to modulate the activity of eIF4A, the DEAD-box RNA helicase subunit of the eukaryotic initiation factor (eIF) 4F complex, a complex that stimulates ribosome recruitment during translation initiation. One flavagline, silvestrol, is capable of modulating chemosensitivity in a mechanism-based mouse model. Methodology/Principal Findings Among a number of flavagline family members tested herein, we find that silvestrol is the more potent translation inhibitor among these. We find that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. We show that silvestrol exhibits significant anticancer activity in human breast and prostate cancer xenograft models, and that this is associated with increased apoptosis, decreased proliferation, and inhibition of angiogenesis. We demonstrate that targeting translation by silvestrol results in preferential inhibition of weakly initiating mRNAs. Conclusions/Significance Our results indicate that silvestrol is a potent anti-cancer compound in vivo that exerts its activity by affecting survival pathways as well as angiogenesis. We propose that silvestrol mediates its effects by preferentially inhibiting translation of malignancy-related mRNAs. Silvestrol appears to be well tolerated in animals.

Activity of selected phytochemicals against Plasmodium falciparum

According to the WHO, in 2008, there were 247 million reported cases of malaria and nearly one million deaths from the disease. Parasite resistance against first-line drugs, including artemisinin and mefloquine, is increasing. In this study the plant-derived compounds aglafolin, rocaglamid, kokusaginine, arborine, arborinine and tuberostemonine were investigated for their anti-plasmodial activity in vitro. Fresh Plasmodium falciparum isolates were taken from patients in the area of Mae Sot, north-western Thailand in 2008 and the inhibition of schizont maturation was determined for the respective compounds. With inhibitory concentrations effecting 50%, 90% and 99% inhibition (IC(50), IC(90) and IC(99)) of 60.95 nM, 854.41 nM and 7351.49 nM, respectively, rocaglamid was the most active of the substances, closely followed by aglafoline with 53.49 nM, 864.55 nM and 8354.20 nM. The activity was significantly below that of artemisinin, but moderately higher than that of quinine. Arborine, arborinine, tuberostemonine and kokusaginine showed only marginal activity against P. falciparum characterized by IC(50) and IC(99) values higher than 350 nM and 180 μM, respectively, and regressions with relatively shallow slopes S>14.38. Analogues of rocaglamid and aglafoline merit further exploration of their anti-plasmodial activity.

High antitrypanosomal activity of plant-derived sulphur-containing amides

Chagas disease, caused by Trypanosoma cruzi, represents an important public health problem in endemic geographic regions in Middle and South America, affecting 15 million infected people. Treatment options are still limited due to the toxicity of available drugs, parasite resistance and poor drug activity during the chronic phase of the disease. In this study, we investigated the in vitro antitrypanosomal activity of 15 tropical plant-derived compounds with the aim of finding new drug candidates. Three novel sulphur-containing amides (methyldambullin, methylgerambullin and sakambullin) showed promising antitrypanosomal activities, with 50% effective concentrations (EC₅₀ values) after 72 h exposure of 1.7, 1.23 and 5.18 μM, respectively, compared with EC₅₀ values for amphotericin B and benznidazole of 0.71 μM and 30.89 μM, respectively.

Development of a pharmacodynamic screening model with Crithidia fasciculata

ZusammenfassungDas Genus Crithidia gehört zur Familie der Trypanosomatidae, welche u.a. auch die Genera Leishmania und Trypanosoma umfasst. Mit diesen Genera bestehen Gemeinsamkeiten hinsichtlich biochemischer Vorgänge, wie der Polyaminsynthese und dem Methioninstoffwechsel. Daher wurde Crithidia fasciculata als Modellorganismus für die Untersuchung der antiparasitären Aktivität verschiedener Substanzen herangezogen. C. fasciculata kommt in Insekten und Amphibien natürlich vor, ist aber nicht humanpathogen. Zunächst wurden verschiedene Kulturmedien ausgewertet, von welchen sich TPS am besten für die Stammpassage eignet und E-Medium – eine neue Formulierung – für die eigentliche Sensibilitätsprüfung. Optimales Wachstum von C. fasciculata wurde unter mikro-aerophiler Atmosphäre beobachtet. Schließlich wurde ein System für die Sensibilitätstestung ausgearbeitet und in der Auswertung von Extrakten aus höheren tropischen Pflanzen der Genera Stemona und Aglaia eingesetzt. Extrakte mit signifikanter Crithidienhemmung wurden für die chromatographische Fraktionierung ausgewählt, mit nachfolgender Isolierung, Strukturanalyse und weiterer Aktivitätsbestimmung der reinen Inhaltsstoffe. Ermutigende Ergebnisse wurden mit Extrakten von Aglaia odorata (Blättern), A. elaeagnoidea (Borke) und A. edulis (Blättern) erzielt, mit EC90-Werten von 1213 ng/ml, 1606 ng/ml bezw. 1462 ng/ml.SummaryThe genus Crithidia is a member of the family Trypanosomatidae and is related to the genera Leishmania and Trypanosoma with which it shares a variety of biochemical mechanisms, such as polyamine synthesis and methionin salvage. In consequence, a screening system for antiparasitic candidate material has been developed with Crithidia fasciculata, a parasite naturally occurring in insects and amphibians, but devoid of pathogenicity for humans. Initially a variety of culture media were evaluated of which TPS was best suited for the maintenance of stock cultures, and E-medium – a newly developed formula – for sensitivity testing. Optimal growth of C. fasciculata was observed under microaerophilic conditions. A system for sensitivity testing was developed and applied to the investigation of extracts from higher tropical plants of the genera Stemona and Aglaia for anticrithidial activity. Extracts with significant anti-crithidial activity were scheduled for chromatographic fractionation and the subsequent isolation, purification and structural identification of individual compounds for further sensitivity testing. Encouraging results were obtained with extracts from Aglaia odorata leaves, A. elaeagnoidea stem bark and A. edulis leaves, with EC90 values of 1213 ng/ml, 1606 ng/ml, and 1462 ng/ml, respectively.

Anti-Leishmanial Activity of Plant-Derived Acridones, Flavaglines, and Sulfur-Containing Amides

Visceral and cutaneous leishmaniases are an important public health problem in endemic geographic regions in 88 countries worldwide, with around 12 million infected people. Treatment options are limited due to toxicity and teratogenicity of the available drugs, response problems in HIV/Leishmania co-infections, and upcoming resistances. In this study, we investigated the anti-leishmanial activity of 13 plant-derived compounds in vitro aiming to find new drug candidates. Toxicity of the compounds was evaluated in human primary hepatocytes, and hemolytic activity was examined in freshly isolated erythrocytes. Two acridones, 5-hydroxynoracronycine and yukocitrine, two flavaglines, aglafoline and rocaglamide, and the sulfur-containing amide methyldambullin showed promising anti-leishmanial activities with 50% effective concentrations (EC50s) of 34.84, 29.76, 7.45, 16.45, and 6.29 μM, respectively. Hepatotoxic activities of 5-hydroxynoracronycine, yukocitrine, and methyldambullin were significantly lower compared to miltefosine and lower or equal compared to artesunate, whereas the ones of rocaglamide and aglafoline were slightly higher compared to miltefosine and significantly higher compared to artesunate. None of the compounds showed hemolytic activity.

Development of a pharmacodynamic screening model with Entamoeba histolytica

ZusammenfassungDurch Entamoeba histolytica verursachte menschliche Amöbiasis ist in den Tropen und Subtropen weit verbreitet und kommt auch in benachbarten Ländern der gemäßigten Breiten vor. Invasive Amöbiasis löst Dysenterie aus. Durch hämatogene Streuung entstehende Leber-, Lungen- und Hirnabszesse führen nicht selten zum Tod. Die Wirksamkeit und Verträglichkeit der verfügbaren spezifischen Medikamente ist mangelhaft. Um die Prüfung neuer Wirkstoffe zu erleichtern wurde ein in vitro System entwickelt, welches die Ermittlung der spezifischen Aktivität gegen E. histolytica gestattet. PYE Medium mit Zusatz von Rinderserum eignet sich zur Erhaltung der Dauerkultur von Entamoeba histolytica Stamm HM1:1MSS. Für die Sensibilitätsprüfung erwiesen sich Waymouth Medium und aerobe Kultur als zuverlässig. Nach Adaptation des Systems an 96-Loch- (8 × 12) Gewebekulturplatten wurden Sensibilitätsprüfungen mit Metronidazol, Dehydroemetin und Dihydroartemisinin als aktive Kontrollsubstanzen und 7 verschiedenen Extrakten von Stemona tuberosa, Aglaia edulis, Aglaia elaeagnoidea und Aglaia odorata durchgeführt. Rindenextrakt von Aglaia elaeagnoidea war am wirksamsten mit IC99 = 496 ng/ml und Steigung S = 1,1325, gefolgt von einem Blattextrakt aus Stemona tuberosa mit IC99 = 638 ng/ml und Steigung S = 1,1325. Sämtliche geprüften Pflanzen-extrakte zeigten volle Aktivität bei Konzentrationen unter 4000 ng/ml und eignen sich daher zu weiterer Aufarbeitung.SummaryHuman amoebiasis caused by Entamoeba histolytica is widely distributed in the tropics and subtropics, but also occurring in neighbouring parts of the temperate zones. Invasive amoebiasis causes dysentery and, by haematogenous spread, also extra-intestinal hepatic, pulmonary or cerebral abscesses, not rarely fatal conditions. The available anti-amoebic drugs have shortcomings regarding tolerability and efficacy. To facilitate the screening of candidate material, an in vitro system has been developed that permits the determination of specific anti-amoebic activity. PYE medium, supplemented with bovine serum, proved to be suitable for the maintenance of the stock cultures of Entamoeba histolytica strain HM1:1MSS. For sensitivity testing, Waymouth medium and cultivation under aerobic conditions were most reliable. After adapting the system to the use of 96-well (8 × 12) tissue culture plates, sensitivity tests were carried out with metronidazole, dehydroemetine and dihydroartemisinin as active control drugs, and seven extracts from Stemona tuberosa, Aglaia edulis, Aglaia elaeagnoidea and Aglaia odorata. Stem bark extract from Aglaia elaeagnoidea was the most active material with an IC99 of 496 ng/ml and a slope S of 1.1325, followed by leaf extract from Stemona tuberosa with an IC99 of 638 ng/ml and a slope S of 1.5648. All seven extracts showed full activity at concentrations <4000 ng/ml and qualified for further investigation.