Walter Jäger

Aromatherapy: Evidence for Sedative Effects of the Essential Oil of Lavender after Inhalation

The sedative properties of the essential oil of Lavender (Lavandula angustifolia Miller) and of its main constituents - linalool and linalyl acetate - were investigated in mice followed up in a series of experimental procedures. The significant decrease in the motility of female and male laboratory animals under standardized experimental conditions is found to be closely dependent on the exposure time to the drugs. Nevertheless after an injection of caffeine into mice a hyperactivity was observed which was reduced to nearly a normal motility only by inhalation of these fragrance drugs. In particular the correlation of the motility of the animals to linalool in serum is experimentally proven, thus furnishing evidence of the aromatherapeutical use of herbal pillows employed in folk medicine since ancient times in order to facilitate falling asleep or to minimize stressful situations of man.

Simultaneous determination of levofloxacin and ciprofloxacin in microdialysates and plasma by high-performance liquid chromatography

The fluoroquinolones levofloxacin and ciprofloxacin were simultaneously determined in microdialysis and plasma samples by reversed-phase high-performance liquid chromatography (HPLC) and fluorescence detection. After a simple sample preparation step the analytes were separated in the isocratic mode within 12 min. The calibration curve for levofloxacin was linear from 0.0156 to 5 gm l −1 and 0.02–12.5 gm l −1 in microdialysates and plasma samples, respectively. The limits of quantification for levofloxacin and ciprofloxacin were 0.0156 and 0.1 gm l −1 in microdialysis samples, and 0.02 and 0.1 gm l −1 in plasma samples, respectively. Some experimental details concerning microdialysate dilution and plasma protein precipitation were described to ensure accurate quantitation. The assay can be used as a routine method to analyse microdialysis and plasma samples from clinical studies.

Pgp-Mediated Interaction Between (R)-[11C]Verapamil and Tariquidar at the Human Blood–Brain Barrier: A Comparison With Rat Data

Using positron emission tomography (PET) imaging we assessed, in vivo, the interaction between a microdose of (R)‐[11C]verapamil (a P‐glycoprotein (Pgp) substrate) and escalating doses of the Pgp inhibitor tariquidar (3, 4, 6, and 8 mg/kg) at the blood–brain barrier (BBB) in healthy human subjects. We compared the dose–response relationship of tariquidar in humans with data obtained in rats using a similar methodology. Tariquidar was equipotent in humans and rats in its effect of increasing (R)‐[11C]verapamil brain uptake (expressed as whole‐brain volume of distribution (VT)), with very similar half‐maximum‐effect concentrations. Both in humans and in rats, brain VT approached plateau levels at plasma tariquidar concentrations >1,000 ng/ml. However, Pgp inhibition in humans led to only a 2.7‐fold increase in brain VT relative to baseline scans (before administration of tariquidar) as compared with 11.0‐fold in rats. The results of this translational study add to the accumulating evidence that there are marked species‐dependent differences in Pgp expression and functionality at the BBB.

METABOLISM OF THE ANTICANCER DRUG FLAVOPIRIDOL, A NEW INHIBITOR OF CYCLIN DEPENDENT KINASES, IN RAT LIVER

Flavopiridol (FLAP) is a promising novel chemotherapeutic agent currently undergoing clinical phase I trials. To examine hepatic metabolism and biliary disposition of FLAP we applied the isolated perfused rat liver system. Besides FLAP two metabolites were detected by high performance liquid chromatography in bile and perfusate. Twenty-five min after FLAP (30 microM) addition to the perfusion medium, biliary secretion of metabolite 1 and 2 reached a maximum of 1.04 +/- 0.52 and 11.34 +/- 4.72 nmol/g.liver.min, respectively. Biliary excretion of parent FLAP, however, continuously increased for 60 min up to 406 +/- 134 pmol/g liver.min. In the perfusate, metabolite 1 was below detection limit and release of metabolite 2 was low (2.8 +/- 0.7 pmol/g liver.min after 60 min). Enzymatic hydrolysis with beta-glucuronidase, mass spectroscopy and electron absorption spectroscopy revealed that both metabolites are monoglucuronides with the glucuronide in position 5 and 7 of the flavonoid core, respectively. The amount of FLAP, metabolite 1 and metabolite 2 excreted into bile during the 60 min of perfusion was 1.94 +/- 0.91, 5.15 +/- 1.95 and 57.29 +/- 23.60% of FLAP cleared from the perfusate during 60 min, respectively. In contrast to the structurally similar flavonoids genistein and daidzein, no inhibition of UDP-glucuronyltransferase with methylumbelliferone as a substrate was observed indicating that different UDP-glucuronyltransferase isoforms are involved in FLAP metabolism. In conclusion, we find that glucuronidation is the major mechanism of hepatic FLAP biotransformation. Metabolites are mainly excreted into bile but also released into systemic circulation. The pharmacological and toxicological effects of these metabolites remain to be elucidated.

Chemopreventive effects of resveratrol and resveratrol derivatives

Resveratrol is considered to have a number of beneficial effects. Recently, our group modified the molecule and synthesized a number of compounds with different biochemical effects. Polymethoxy and polyhydroxy derivatives of resveratrol were shown to inhibit tumor cell growth in various cell lines and inflammation pathways (cyclooxygenases activity), in part more effectively than resveratrol itself. One lead compound (hexahydroxystilbene, M8) turned out to be the most effective inhibitor of tumor cell growth and of cyclooxygenase 2 activity. M8 was then studied in two different human melanoma mouse models. This novel resveratrol analog was able to inhibit melanoma tumors in a primary tumor model alone and in combination with dacarbacine, an anticancer compound that is used for melanoma treatment. We also tested the development of lymph node metastasis in a second melanoma model and again M8 successfully inhibited the tumor as well as the size and weight of lymph node metastasis. Hydroxylated resveratrol analogs therefore represent a novel class of anticancer compounds and promising candidates for in vivo studies.

Resveratrol and resveratrol analogues--structure-activity relationship.

ABSTRACTResveratrol (3,4′,5-trihydroxy-trans-stilbene) is a compound found in wine and is held responsible for a number of beneficial effects of red wine. Besides the prevention of heart disease and significant anti-inflammatory effects, resveratrol might inhibit tumor cell growth and even play a role in the aging process. We here describe the structure-activity relationship of resveratrol and analogues of resveratrol regarding the free radical scavenging and antitumor effects of this exciting natural compound. In addition, we have synthesized a number of analogues of resveratrol with the aim to further improve the beneficial effects of resveratrol. Our studies were based on the analysis of structural properties, which were responsible for the most important effects of this compound. Striking in vivo effects can be observed with hexahydroxystilbene (M8), the most effective synthetic analogue of resveratrol. We could show that M8 inhibits tumor as well as metastasis growth of human melanoma in two different animal models, alone and in combination with dacarbacine.

Biliary excretion of flavopiridol and its glucuronides in the isolated perfused rat liver: role of multidrug resistance protein 2 (Mrp2)

Flavopiridol (FLAP) is a novel anticancer agent that is extensively glucuronidated in patients. Biliary excretion is the main elimination pathway of FLAP conjugates responsible for enterohepatic recirculation and for the main side effect diarrhea. To investigate the hepatic transport system for FLAP glucuronides, livers of Wistar and Mrp2-deficient TR- rats were perfused with FLAP (30 microM) in a single pass system. Biliary excretion and efflux into perfusate during a 60 min period greatly differ in TR- rats. While cumulative biliary excretion of M1 and M2 was significantly reduced to 4.3% and 5.4% efflux into perfusate was increased by 1.5 and 4.2-fold. This indicates that in control rats, M1 and M2 are almost exclusively eliminated into bile by Mrp2. Cumulative FLAP secretion into bile and perfusate, however, was non-significantly reduced by 36.7% and 43.2% in the mutant rat strain, suggesting that besides Mrp2, other transporters might also be involved in FLAP elimination. FLAP stimulates bile flow up to 24% in control rats, but secretion is nearly absent in TR- rats further supporting an efficient transport of FLAP glucuronides by Mrp2. FLAP (30 microM) also reversibly inhibited the Mrp2-mediated biliary elimination of bilirubin and bromsulphthalein in Wistar rats by 54% and 51%, respectively, indicating a competition with the elimination of Mrp2-specific substrates. In summary, we found that FLAP glucuronides are substrates of Mrp2 effectively inhibiting the biliary excretion of bilirubin. This may explain the increased serum bilirubin levels observed in cancer patients during FLAP therapy.

NF-κB mediates the 12(S)-HETE-induced endothelial to mesenchymal transition of lymphendothelial cells during the intravasation of breast carcinoma cells.

Background:The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts.Methods:To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs.Results:We show that tumour spheroids generate ‘circular chemorepellent-induced defects’ (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with ∼50% attenuation of CCID formation by treatment of cells with 10 μM Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general.Interpretation:These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell–cell contacts, migration, and the generation of CCID.

Multifactorial anticancer effects of digalloyl-resveratrol encompass apoptosis, cell-cycle arrest, and inhibition of lymphendothelial gap formation in vitro

Background:Digalloyl-resveratrol (di-GA) is a synthetic compound aimed to combine the biological effects of the plant polyhydroxy phenols gallic acid and resveratrol, which are both radical scavengers and cyclooxygenase inhibitors exhibiting anticancer activity. Their broad spectrum of activities may probably be due to adjacent free hydroxyl groups.Methods:Protein activation and expression were analysed by western blotting, deoxyribonucleoside triphosphate levels by HPLC, ribonucleotide reductase activity by 14C-cytidine incorporation into nascent DNA and cell-cycle distribution by FACS. Apoptosis was measured by Hoechst 33258/propidium iodide double staining of nuclear chromatin and the formation of gaps into the lymphendothelial barrier in a three-dimensional co-culture model consisting of MCF-7 tumour cell spheroids and human lymphendothelial monolayers.Results:In HL-60 leukaemia cells, di-GA activated caspase 3 and dose-dependently induced apoptosis. It further inhibited cell-cycle progression in the G1 phase by four different mechanisms: rapid downregulation of cyclin D1, induction of Chk2 with simultaneous downregulation of Cdc25A, induction of the Cdk-inhibitor p21Cip/Waf and inhibition of ribonucleotide reductase activity resulting in reduced dCTP and dTTP levels. Furthermore, di-GA inhibited the generation of lymphendothelial gaps by cancer cell spheroid-secreted lipoxygenase metabolites. Lymphendothelial gaps, adjacent to tumour bulks, can be considered as gates facilitating metastatic spread.Conclusion:These data show that di-GA exhibits three distinct anticancer activities: induction of apoptosis, cell-cycle arrest and disruption of cancer cell-induced lymphendothelial disintegration.

The analysis of organic anion transporting polypeptide (OATP) mRNA and protein patterns in primary and metastatic liver cancer.

Organic anion transporting polypeptides (OATP, SLCO genes) mediate the uptake of endobiotics and drugs. Thus, their expression levels and pattern could be of relevance for cancer therapy. This prompted us to investigate the expression of poorly characterized OATPs, namely OATP2A1, OATP3A1, OATP4A1 and OATP5A1 in hepatic cancer of different origin. First, mRNA levels of all eleven OATPs were determined in paired (cancerous and adjacent non-cancerous) specimens from 43 patients with primary liver cancer (hepatocellular carcinoma, HCC; cholangiocellular carcinoma, CCC) and liver metastases from colon tumors (MLT). Real-time RT-PCR analysis revealed that all OATPs, except OATP1C1 and OATP6A1, are extensively expressed in nearly all samples. In contrast to downregulated OATP1B1, OATP1B3, OATP1A2 and OATP2B1 in cancerous vs. non-cancerous samples, an increase in OATP2A1, OATP3A1, OATP4A1 and OATP5A1 mRNA levels was seen in tumors (up to 40-fold for OATP5A1 in the MLT group). Therefore, OATP2A1, OATP3A1, OATP4A1 and OATP5A1 were further investigated by immunofluorescence microscopy on paraffin-embedded cancerous and non-cancerous sections (seven per group). OATP-derived immunoreactivity was observed in plasma membranes and cytosol of hepatic tumor cells, and additionally, in various cytokeratin 19 positive bile ducts. An increased percentage of immunoreactive cells and a higher staining intensity in cancerous vs. non-cancerous paraffin sections paralleled higher mRNA levels of OATP2A1, OATP3A1, OATP4A1 and OATP5A1 in cancerous tissues of HCC, CCC and MLT patients. The extensive expression of OATP2A1, OATP3A1, OATP4A1 and OATP5A1 in hepatic tumors of different origin suggests that these transporters might be further exploited for the discovery of novel anticancer agents.