Design, Synthesis, and Characterization of a Paclitaxel Formulation Activated by Extracellular MMP9.

The concept of triggered drug release offers a possibility to overcome the toxic side effects of chemotherapeutics in cancer treatment by reducing systemic exposure to the active drug. In the present work, the concept foresees the use of the extracellular enzyme MMP9 as an enzymatic trigger for drug release in the proximity of tumor cells. METHODS: A paclitaxel-hemisuccinate-peptide conjugate as a building block for self-assembling nanoparticles was synthesized using standard conjugation approaches. The building block was purified via preparative HPLC and analyzed by LC-MS. Nanoparticles were formed using the nanoprecipitation method and characterized. For selection of a suitable in vitro model system, common bioanalytical methods were used to determine mRNA expression, enzyme amount, and activity of MMP9. RESULTS: The MMP9-labile prodrug was synthesized and characterized. Nanoparticles were formed out of MMP9-labile conjugate-building blocks. The nanoparticle's diameter averaged at around 120 nm and presented a spherical shape. LN-18 cells, a glioblastoma multiforme derived cell line, were chosen as an in vitro model based on findings in cancer tissue and cell line characterization. The prodrug showed cytotoxicity in LN-18 cells, which was reduced by addition of an MMP9 inhibitor. CONCLUSION: taken together, we confirmed increased MMP9 in several cancer tissues (cervical, esophageal, lung, and brain) compared to healthy tissue and showed the effectiveness of MMP9-labile prodrug in in vitro tests.

Thyroid Hormones Are Transport Substrates and Transcriptional Regulators of Organic Anion Transporting Polypeptide 2B1.

Levothyroxine replacement therapy forms the cornerstone of hypothyroidism management. Variability in levothyroxine oral absorption may contribute to the well-recognized large interpatient differences in required dose. Moreover, levothyroxine-drug pharmacokinetic interactions are thought to be caused by altered oral bioavailability. Interestingly, little is known regarding the mechanisms contributing to levothyroxine absorption in the gastrointestinal tract. Here, we aimed to determine whether the intestinal drug uptake transporter organic anion transporting polypeptide 2B1 (OATP2B1) may be involved in facilitating intestinal absorption of thyroid hormones. We also explored whether thyroid hormones regulate OATP2B1 gene expression. In cultured Madin-Darby Canine Kidney II/OATP2B1 cells and in OATP2B1-transfected Caco-2 cells, thyroid hormones were found to inhibit OATP2B1-mediated uptake of estrone-3-sulfate. Competitive counter-flow experiments evaluating the influence on the cellular accumulation of estrone-3-sulfate in the steady state indicated that thyroid hormones were substrates of OATP2B1. Additional evidence that thyroid hormones were OATP2B1 substrates was provided by OATP2B1-dependent stimulation of thyroid hormone receptor activation in cell-based reporter assays. Bidirectional transport studies in intestinal Caco-2 cells showed net absorptive flux of thyroid hormones, which was attenuated by the presence of the OATP2B1 inhibitor, atorvastatin. In intestinal Caco-2 and LS180 cells, but not in liver Huh-7 or HepG2 cells, OATP2B1 expression was induced by treatment with thyroid hormones. Reporter gene assays revealed thyroid hormone receptor α-mediated transactivation of the SLCO2B1 1b and the SLCO2B1 1e promoters. We conclude that thyroid hormones are substrates and transcriptional regulators of OATP2B1. These insights provide a potential mechanistic basis for oral levothyroxine dose variability and drug interactions.

Caco-2 Permeability Studies and In Vitro hERG Liability Assessment of Tryptanthrin and Indolinone.

Tryptanthrin and (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolinone (indolinone) were recently isolated from Isatis tinctoria as potent anti-inflammatory and antiallergic alkaloids, and shown to inhibit COX-2, 5-LOX catalyzed leukotriene synthesis, and mast cell degranulation at low µM to nM concentrations. To assess their suitability for oral administration, we screened the compounds in an in vitro intestinal permeability assay using human colonic adenocarcinoma cells. For exact quantification of the compounds, validated UPLC-MS/MS methods were used. Tryptanthrin displayed high permeability (apparent permeability coefficient > 32.0 × 10(-6) cm/s) across the cell monolayer. The efflux ratio below 2 (< 1.12) and unchanged apparent permeability coefficient values in the presence of the P-glycoprotein inhibitor verapamil (50 µM) indicated that tryptanthrin was not involved in P-glycoprotein interactions. For indolinone, a low recovery was found in the human colon adenocarcinoma cell assay. High-resolution mass spectrometry pointed to extensive phase II metabolism of indolinone (sulfation and glucuronidation). Possible cardiotoxic liability of the compounds was assessed in vitro by measurement of an inhibitory effect on human ether-a-go-go-related gene tail currents in stably transfected HEK 293 cells using the patch clamp technique. Low human ether-a-go-go-related gene inhibition was found for tryptanthrin (IC50 > 10 µM) and indolinone (IC50 of 24.96 µM). The analysis of compounds using various in silico methods confirmed favorable pharmacokinetic properties, as well as a slight inhibition of the human ether-a-go-go-related gene potassium channel at micromolar concentrations.

Inhibitors of plasmodial serine hydroxymethyltransferase (SHMT): cocrystal structures of pyrazolopyrans with potent blood- and liver-stage activities.

Several of the enzymes related to the folate cycle are well-known for their role as clinically validated antimalarial targets. Nevertheless for serine hydroxymethyltransferase (SHMT), one of the key enzymes of this cycle, efficient inhibitors have not been described so far. On the basis of plant SHMT inhibitors from an herbicide optimization program, highly potent inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) SHMT with a pyrazolopyran core structure were identified. Cocrystal structures of potent inhibitors with PvSHMT were solved at 2.6 Å resolution. These ligands showed activity (IC50/EC50 values) in the nanomolar range against purified PfSHMT, blood-stage Pf, and liver-stage P. berghei (Pb) cells and a high selectivity when assayed against mammalian cell lines. Pharmacokinetic limitations are the most plausible explanation for lack of significant activity of the inhibitors in the in vivo Pb mouse malaria model.

Potato (Solanum tuberosum L.) tuber ageing induces changes in the proteome and antioxidants associated with the sprouting pattern.

During post-harvest storage, potato tubers age as they undergo an evolution of their physiological state influencing their sprouting pattern. In the present study, physiological and biochemical approaches were combined to provide new insights on potato (Solanum tuberosum L. cv. Désirée) tuber ageing. An increase in the physiological age index (PAI) value from 0.14 to 0.83 occurred during storage at 4 degrees C over 270 d. Using this reference frame, a proteomic approach was followed based on two-dimensional electrophoresis. In the experimental conditions of this study, a marked proteolysis of patatin occurred after the PAI reached a value of 0.6. In parallel, several glycolytic enzymes were up-regulated and cellular components influencing protein conformation and the response to stress were altered. The equilibrium between the 20S and 26S forms of the proteasome was modified, the 20S form that recycles oxidized proteins being up-regulated. Two proteins belonging to the cytoskeleton were also differentially expressed during ageing. As most of these changes are also observed in an oxidative stress context, an approach focused on antioxidant compounds and enzymes as well as oxidative damage on polyunsaturated fatty acids and proteins was conducted. All the changes observed during ageing seemed to allow the potato tubers to maintain their radical scavenging activity until the end of the storage period as no accumulation of oxidative damage was observed. These data are interpreted considering the impact of reactive oxygen species on the development and the behaviour of other plant systems undergoing ageing or senescence processes.

Simultaneous measurement of proline and related compounds in oak leaves by high-performance ligand-exchange chromatography and electrospray ionization mass spectrometry for environmental stress studies.

A mass spectrometer was coupled to high-performance ligand-exchange liquid chromatography (HPLEC) for simultaneous analysis of stress associated solutes such as proline, hydroxyproline, methylproline, glycine betaine and trigonelline extracted from leaves of drought stressed oaks and an internal standard namely N-acetylproline. Methanol/chloroform/water extracts were analyzed using an Aminex HPX-87C column and specifically quantified by the positive ion mode of an electrospray ionisation-mass spectrometry (ESI-MS) in single ion monitoring (SIM) mode. The recovery of N-acetyl proline added to oak leaf extracts ranged from 85.2 to 122.1% for an intra-day study. Standard calibration curves showed good linearity in the measured range from 0.3125 to 10micromolL(-1) with the lowest correlation coefficient of 0.99961 for trigonelline. The advantages of this alternative procedure, compared to previously published methods using fluorescence or amperometric detections, are the simultaneous and direct detection of osmoprotectants in a single chromatographic run, a minimal sample preparation, a good specificity and reduced limits of quantification, ranging from 0.1 to 0.6micromolL(-1). Fifty-six days of water deficit exposure resulted in increased foliar free proline levels (2.4-fold, P<0.001, 155micromolg(-1) FW) and glycine betaine contents (2.5-fold, P<0.05, 175micromolg(-1) FW) of drought stressed oak compared to control.

Modification of the health-promoting value of potato tubers field grown under drought stress: emphasis on dietary antioxidant and glycoalkaloid contents in five native andean cultivars (Solanum tuberosum L.).

The effects of drought stress on dietary antioxidant and glycoalkaloid contents in potato tubers were investigated using a selection of five native Andean cultivars. Both freshly harvested and 4 month-stored tubers were analyzed. Responses to drought stress were highly cultivar-specific. The antioxidant contents of the yellow tuber-bearing cultivars (Sipancachi and SS-2613) were weakly affected by the drought treatment, whereas the pigmented cultivars demonstrated highly cultivar-dependent variations. A drastic reduction of anthocyanins and other polyphenols was revealed in the red- (Sullu) and purple-fleshed (Guincho Negra) cultivars, whereas an increase was shown in the purple-skinned and yellow-fleshed cultivar (Huata Colorada). The hydrophilic antioxidant capacity (evaluated by Folin-Ciocalteu and H-oxygen radical absorbance capacity assays) was highly correlated with the polyphenol content and followed, therefore, the same behavior upon drought. Carotenoid contents, including beta-carotene, as well as vitamin E, tended to increase or remain stable following drought exposure, except for the cultivar Sullu, in which the level of these lipophilic antioxidants was decreased. Vitamin C contents were not affected by drought with the exception of Guincho Negra, in which the level was increased. These variations of health-promoting compounds were associated with increased or stable levels of the toxic glycoalkaloids, alpha-solanine and alpha-chaconine. Storage at 10 degrees C for 4 months tended to decrease the concentrations of all dietary antioxidants, except those of vitamin E. This storage also reduced the drought-induced variations observed in freshly harvested tubers. These results were discussed in terms of their implications for human diet and health as well as in plant stress defense mechanisms.

Combining proteomics and metabolite analyses to unravel cadmium stress-response in poplar leaves.

A proteomic analysis of poplar leaves exposed to cadmium, combined with biochemical analysis of pigments and carbohydrates revealed changes in primary carbon metabolism. Proteomic results suggested that photosynthesis was slightly affected. Together with a growth inhibition, photoassimilates were less needed for developmental processes and could be stored in the form of hexoses or complex sugars, acting also as osmoprotectants. Simultaneously, mitochondrial respiration was upregulated, providing energy needs of cadmium-exposed plants.

Antioxidant profiling of native Andean potato tubers (Solanum tuberosum L.) reveals cultivars with high levels of beta-carotene, alpha-tocopherol, chlorogenic acid, and petanin.

The antioxidant profile of 23 native Andean potato cultivars has been investigated from a human nutrition perspective. The main carotenoid and tocopherol compounds were studied using high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD) and a fluorescence detector, respectively, whereas polyphenols (including anthocyanins in colored tubers) were identified by means of both HPLC-mass spectrometry and HPLC-DAD. Antioxidant profiling revealed significant genotypic variations as well as cultivars of particular interest from a nutritional point of view. Concentrations of the health-promoting carotenoids, lutein and zeaxanthin, ranged from 1.12 to 17.69 microg g(-1) of dry weight (DW) and from 0 to 17.7 microg g(-1) of DW, with cultivars 704353 and 702472 showing the highest levels in lutein and zeaxanthin, respectively. Whereas beta-carotene is rarely reported in potato tubers, remarkable levels of this dietary provitamin A carotenoid were detected in 16 native varieties, ranging from 0.42 to 2.19 microg g(-1) of DW. The amounts of alpha-tocopherol found in Andean potato tubers, extending from 2.73 to 20.80 microg g(-1) of DW, were clearly above the quantities generally reported for commercial varieties. Chlorogenic acid and its isomers dominated the polyphenolic profile of each cultivar. Dark purple-fleshed tubers from the cultivar 704429 contained exceptionally high levels of total anthocyanins (16.33 mg g(-1) of DW). The main anthocyanin was identified as petanin (petunidin-3-p-coumaroyl-rutinoside-5-glucoside). The results suggest that Andean potato cultivars should be exploited in screening and breeding programs for the development of potato varieties with enhanced health and nutritional benefits.

Andean potato cultivars (Solanum tuberosum L.) as a source of antioxidant and mineral micronutrients.

Potato tubers were evaluated as a source of antioxidants and minerals for the human diet. A genetically diverse sample of Solanum tuberosum L. cultivars native to the Andes of South America was obtained from a collection of nearly 1000 genotypes using microsatellite markers. This size-manageable collection of 74 landraces, representing at best the genetic diversity among potato germplasm, was analyzed for iron, zinc, calcium, total phenolic, total carotenoid, and total vitamin C contents. The hydrophilic antioxidant capacity of each genotype was also measured using the oxygen radical absorbance capacity (ORAC) assay. The iron content ranged from 29.87 to 157.96 microg g-1 of dry weight (DW), the zinc content from 12.6 to 28.83 microg g-1 of DW, and the calcium content from 271.09 to 1092.93 microg g-1 of DW. Total phenolic content varied between 1.12 and 12.37 mg of gallic acid equiv g-1 of DW, total carotenoid content between 2.83 and 36.21 microg g-1 of DW, and total vitamin C content between 217.70 and 689.47 microg g-1 of DW. The range of hydrophilic ORAC values was 28.25-250.67 micromol of Trolox equiv g-1 of DW. The hydrophilic antioxidant capacity and the total phenolic content were highly and positively correlated (r = 0.91). A strong relationship between iron and calcium contents was also found (r = 0.67). Principal component analysis on the studied nutritional contents of the core collection revealed that most potato genotypes were balanced in terms of antioxidant and mineral contents, but some of them could be distinguished by their high level in distinct micronutrients. Correlations between the micronutrient contents observed in the sample and the genetic distances assessed by microsatellites were weakly significant. However, this study demonstrated the wide variability of health-promoting micronutrient levels within the native potato germplasm as well as the significant contribution that distinct potato tubers may impart to the intake in dietary antioxidants, zinc, and iron.