Synthesis and Comparative Evaluation of Novel Cationic Amphiphile C12-Man-Q as an Efficient DNA Delivery Agent In Vitro.

New amphiphilic 1,4-DHP derivative C12-Man-Q with remoted cationic moieties at positions 2 and 6 was synthesised to study DNA delivery activity. The results were compared with data obtained for cationic 1,4-DHP derivative D19, which is known to be the most efficient one among the previously tested 1,4-DHP amphiphiles. We analysed the effects of C12-Man-Q concentration, complexation media, and complex/cell contact time on the gene delivery effectiveness and cell viability. Transmission electron microscopy data confirms that lipoplexes formed by the compound C12-Man-Q were quite uniform, vesicular-like structures with sizes of about 50 nm, and lipoplexes produced by compound D19 were of irregular shapes, varied in size in the range of 25⁻80 nm. Additionally, confocal microscopy results revealed that both amphiphiles effectively delivered green fluorescent protein expression plasmid into BHK-21 cells and produced a fluorescent signal with satisfactory efficiency, although compound C12-Man-Q was more cytotoxic to the BHK-21 cells with an increase of concentration. It can be concluded that optimal conditions for C12-Man-Q lipoplexes delivery in BHK-21 cells were the serum free media without 0.15 M NaCl, at an N/P ratio of 0.9. Compound D19 showed higher transfection efficiency to transfect BHK-21 and Cos-7 cell lines, when transfecting active proliferating cells. Although D19 was not able to transfect all studied cell lines we propose that it could be cell type specific. The compound C12-Man-Q showed modest delivery activity in all used cell lines, and higher activity was obtained in the case of H2-35 and B16 cells. The transfection efficiency in cell lines MCF-7, HeLa, and Huh-7 appears to be comparable to the reference compound D19 and minimal in the HepG2 cell line.

Involvement of H2O2 in fluazifop-P-butyl-induced cell death in bristly starbur seedlings.

In order to understand the action mechanism of fluazifop-P-butyl (FB) in bristly starbur (Acanthospermum hispidum D.C.), a susceptible plant, the role of active oxygen species (ROS) in herbicide-induced cell death in shoots was investigated. FB-induced phytotoxicity was not reduced by the antioxidants, 1,4-diazabicyclooctane (dabaco), sodium azide, l-tryptophan, d-tryptophan, hydroquinone and dimethyl pyridine N-oxide (DMPO). The activities of superoxide dismutase (SOD) and catalase (CAT), in bristly starbur seedlings were significantly increased by FB at 12 HAT and 24 HAT, while ascorbate peroxidase (APX) and glutathione reductase (GR) activities increased only at 12 HAT. The contents of H2O2 in FB-treated bristly starbur seedlings were significantly higher to that of control between 8 and 24 HAT. According to the analysis of potassium iodide - starch or 3,3-diaminobenzidine, the accumulation of hydrogen peroxide was observed in the apical growing point, stem, petiole and veins of FB-treated bristly starbur seedlings at 24 HAT. The cell viability of bristly starbur seedlings treated by 10µM FB decreased at 18 HAT. These results suggested that FB-induced cell death in bristly starbur shoots may be caused by ROS (O2- and H2O2) generation and lipid peroxidation.

Fluazifop- p-butyl, an aryloxyphenoxypropionate herbicide, diminishes renal and hepatic functions and triggers testicular oxidative stress in orally exposed rats.

Fluazifop- p-butyl (FPB) is a selective aryloxyphenoxypropionate herbicide. Its phytotoxicity mechanism involves inhibition of lipid biosynthesis, free-radical generation, and oxidative stress in vulnerable plants. This study evaluates the impact of orally administered FPB on selected tissues in non-target animal model. Twenty-four male wistar rats (160-180g) were randomized into groups (I-IV). Group-I served as control, while animals in groups II, III, and IV received FPB at 18.75, 37.5, and 75 mg/kg body weight/day p.o., respectively, for 21 days. FPB caused significant ( p < 0.05) increase in plasma biomarkers of renal and hepatic function (urea, creatinine, bilirubin, alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase) when compared to control. Significant reductions in testicular ascorbic acid, glutathione, and activities of glutathione-S transferase, superoxide dismutase, and catalase were observed in FPB-treated animals when compared to control, in a dose-dependent manner. This was accompanied by increased testicular lipid peroxidation in the treated groups. Furthermore, a significant decrease in testicular acid phosphatase and γ-glutamyl transferase activities was also observed in the FPB-treated groups in a dose-dependent manner compared to control. However, testicular lactate dehydrogenase activity was significantly increased in the FPB-treated rats when compared to control. Additionally, histopathological studies revealed severe interstitial oedema and congestion of testicular blood vessels in the FPB-treated groups. Overall, data from this study suggest that FPB induced hepatotoxicity, nephrotoxicity, and oxidative stress-mediated alteration of testicular functions in rat.

Subacute Toxicity Profile of Lacidipine Nanoformulation in Wistar Rats.

The present study was aimed at investigating the safety of Lacidipine (LCDP) loaded nanostructured lipid carriers (NLCs) in Wistar rats. NLCs were formulated using ultrasound dispersion technique. Animals were orally treated once daily with NLCs containing 0.140 mg, 0.350 mg, and 0.875 mg of LCDP as low, medium, and high dose per kg body weight, respectively, during 28 days along with blank formulation and pure LCDP. Control rats were fed with water. Animals were observed throughout experiment period and their body weight was recorded once weekly. Overnight fasted rats were sacrificed on the 29th day. Study revealed no signs or symptoms of toxicity or morbidity. No significant changes in the body weight were observed between treated and control group. Significant increase in left testis weight and liver weight was observed in male and female rats, respectively. Haematological estimation revealed significant decrease in haemoglobin count in male rats while female rats showed significant increase in granulocyte count. All the serum clinical parameters were within the normal range and no gross histopathological changes were observed. No delayed effect was noted in satellite group. The results indicate that developed LCDP loaded NLCs are safe when administered orally in rats.

Genotoxic and antigenotoxic assessment of four newly synthesized dihydropyridine derivatives.

The current study aims to determine the genotoxic and antigenotoxic potential of four newly synthesized dihydropyridine derivatives using Escherichia coli WP2 and Ames/Salmonella bacterial reversion assay systems. The bacterial mutant tester strains, E. coli WP2uvrA with a point mutation and Salmonella typhimurium TA1537 with a frameshift mutation, were used to determine genotoxic potentials of the test compounds. To determine antigenotoxic potentials of the test compounds, the same strains were also used together with positive mutagens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for E. coli WP2uvrA and 9-aminoacridine (9-AA) for S. typhimurium TA1537. According to the results, neither of the test compounds showed significant genotoxic activity on both tester strains at the tested concentrations. However, except compound 4, all the test compounds showed significant antigenotoxic activity on MNNG- or/and 9-AA-induced mutations. The inhibition rates of mutagenesis ranged from 27.0% (compound 2: 2.5 mM/plate) to 65.0% (compound 2: 0.5 mM/plate) for MNNG and from 30.6% (compound 2: 2 mM/plate) to 58.5% (compound 1: 1 mM/plate) for 9-AA genotoxicity. According to these results, it is concluded that all the test compounds do not have a mutagenic potential on the bacterial strains at the tested concentrations, and some of them have antigenotoxic potentials against MNNG- and 9-AA-induced mutagenesis.

Changing Gly311 to an acidic amino acid in the MATE family protein DTX6 enhances Arabidopsis resistance to the dihydropyridine herbicides.

In modern agriculture, frequent application of herbicides may induce the evolution of resistance in plants, but the mechanisms underlying herbicide resistance remain largely unexplored. Here, we report the characterization of rtp1 (resistant to paraquat 1), an Arabidopsis mutant showing strong resistance to the widely used herbicides paraquat and diquat. The rtp1 mutant is semi-dominant and carries a point mutation in the gene encoding the multidrug and toxic compound extrusion family protein DTX6, leading to the change of glycine to glutamic acid at residue 311 (G311E). The wild-type DTX6 with glycine 311 conferred weak paraquat and diquat resistance when overexpressed, while mutation of glycine 311 to a negatively charged amino acid (G311E or G311D) markedly increased the paraquat and diquat resistance of plants, whereas mutation to a positively charged amino acid (G311R or G311K) compromised the resistance, suggesting that the charge property of residue 311 of DTX6 is critical for the paraquat and diquat resistance of Arabidopsis plants. DTX6 is localized in the endomembrane trafficking system and may undergo the endosomal sorting to localize to the vacuole and plasma membrane. Treatment with the V-ATPase inhibitor ConA reduced the paraquat resistance of the rtp1 mutant. Paraquat release and uptake assays demonstrated that DTX6 is involved in both exocytosis and vacuolar sequestration of paraquat. DTX6 and DTX5 show functional redundancy as the dtx5 dtx6 double mutant but not the dtx6 single mutant plants were more sensitive to paraquat and diquat than the wild-type plants. Collectively, our work reveals a potential mechanism for the evolution of herbicide resistance in weeds and provides a promising gene for the manipulation of plant herbicide resistance.

SAMe prevents the induction of the immunoproteasome and preserves the 26S proteasome in the DDC-induced MDB mouse model.

Mallory-Denk bodies (MDBs) form in the liver of alcoholic patients. This occurs because of the accumulation and aggregation of ubiquitinated cytokeratins, which hypothetically is due to the ubiquitin-proteasome pathway's (UPP) failure to degrade the cytokeratins. The experimental model of MDB formation was used in which MDBs were induced by refeeding DDC to drug-primed mice. The gene expression and protein levels of LMP2, LMP7 and MECL-1, the catalytic subunits in the immunoproteasome, as well as FAT10, were increased in the liver cells forming MDBs but not in the intervening normal hepatocytes. Chymotrypsin-like activity of the UPP was decreased by DDC refeeding, indicating that a switch from the UPP to the immunoproteasome had occurred at the expense of the 26S proteasome. The failure of the UPP to digest cytokeratins would explain MDB aggregate formation. SAMe prevented the decrease in UPP activity, the increase in LMP2, LMP7, and MECL-1 protein levels and MDB formation induced by DDC. DDC refeeding also induced the TNFalpha and IFNgamma receptors. SAMe prevented the increase in the TNFalpha and IFNgamma receptors, supporting the idea that TNFalpha and IFNgamma were responsible for the up regulation of LMP2, LPM7, and FAT10. These results support the conclusion that MDBs form in FAT10 over-expressing hepatocytes where the up regulation of the immunoproteasome occurs at the expense of the 26S proteasome.

SAMe prevents the up regulation of toll-like receptor signaling in Mallory-Denk body forming hepatocytes.

Mallory-Denk body (MDB) formation is a component of alcoholic and non alcoholic hepatitis. In the present study, the role of the toll-like receptor (TLR) signaling pathway was investigated in the mechanism of MDB formation in the DDC-fed mouse model. Microarray analysis data mining, performed on the livers of drug-primed mice refed DDC, showed that TLR2/4 gene expression was significantly up regulated by DDC refeeding. SAMe supplementation prevented this up regulation and prevented the formation of MDBs. qRT-PCR analysis confirmed these results. TLR2/4 activates the adapter protein MyD88. The levels of MyD88 were increased by DDC refeeding. The increase of MyD88 was also prevented by SAMe supplementation. Results showed that MyD88-independent TLR3/4-TRIF-IRF3 pathway was not up regulated in the liver of DDC refed mice. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is the downstream protein recruited by the MyD88/IRAK protein complex, and is involved in the regulation of innate immune responses. Results showed a significant increase in the levels of TRAF-6. TRAF-6 activation leads to activation of NFkB and the mitogen-activated protein kinase (MAPK) cascade. The TRAF-6 increase was ameliorated by SAMe supplementation. These results suggest that DDC induces MDB formation through the TLR2/4 and MyD88-dependent signaling pathway. In conclusion, SAMe blocked the over-expression of TLR2/4, and their downstream signaling components MyD88 and TRAF-6. SAMe prevented the DDC-induced up regulation of the TLR signaling pathways, probably by preventing the up regulation of INF-gamma receptors by DDC feeding. INFgamma stimulates the up regulation of TLR2. The ability of SAMe feeding to prevent TLR signaling up regulation has not been previously described.

Anti-leishmanial and anti-trypanosomal activities of 1,4-dihydropyridines: In vitro evaluation and structure-activity relationship study.

Leishmaniasis and Chagas' disease constitute a relevant health and socio-economic problem in Latin America, Africa, and Asia. The therapeutic interventions rely on inefficient and highly toxic drugs with systemic side effects in patients. Considering the multiple biological activities of the calcium channel blockers and the high versatility of 1,4-dihydropyridines, eight clinically used 1,4-dihydropyridines (azelnidipine, amlodipine, cilnidipine, lercanidipine, nicardipine, nifedipine, nimodipine and nitrendipine) were in vitro tested against Leishmania and Trypanosoma cruzi parasites, and their cytotoxicity was tested against mammalian cells. In addition, a QSAR study was performed in order to delineate further structural requirements for the anti-protozoan activity and to predict the biological potency of 1,4-dihydropyridines. The tested compounds were effective against Leishmania (L.) amazonensis, Leishmania (V.)braziliensis, Leishmania (L.) chagasi, and Leishmania (L.) major promastigotes, L. (L.) chagasi intracellular amastigotes and T. cruzi trypomastigotes with 50% inhibitory concentration (IC(50)) values in the range of 2.6-181µM. The QSAR provided useful information about the structural features of the anti-protozoan activities, including diphenylpropyl and diphenylmethylazetidin groups at position 4 of the 1,4-dihydropyridine ring, allowing the prediction of two novel potential anti-protozoan analogs.

Investigating the potential of utilizing glycerosomes as a novel vesicular platform for enhancing intranasal delivery of lacidipine.

Lacidipine is a potent dihydropyridine calcium channel blocker used for management of hypertension and atherosclerosis. The drug has low and fluctuating oral bioavailability owing to its extensive hepatic first-pass metabolism and reduced water solubility. Accordingly, this work aimed at overcoming the aforementioned challenges through the formulation of intranasal nano-sized lacidipine glycerosomes. Box-Behnken was successfully employed for the formulation and in vitro optimization of the glycerosomes. Statistical analysis revealed that cholesterol concentration exhibited a significant effect on the vesicle size, while Phospholipon® 90G and glycerol concentrations exhibited significant effects on both entrapment efficiency and deformability index. The optimized formulation showed spherical shape, good deformability, vesicular size of 220.25 nm, entrapment efficiency of 61.97%, and enhanced ex vivo permeation by 3.65 fold compared to lacidipine suspension. Confocal laser scattering microscope revealed higher penetration depth via nasal mucosa for rhodamine labelled glycerosomes (up to 60 µm) in comparison to rhoadamine dye solution (26 µm). In addition, the optimized lacidipine glycerosomes caused significant reduction in methylprednisolone acetate-induced hypertension in rats for up to 24 h in comparison to oral drug suspension. Histopathological assessment showed intact nasal mucosal epithelial lining with no signs of inflammation or necrosis confirming the safety and tolerability of the proposed glycerosomes. The declared results highlights the potential of utilizing the proposed glycerosomes as safe and effective platform for intranasal delivery of lacidipine.

Dose-related shortening of ventricular tachycardia cycle length after administration of the KATP channel opener bimakalim in a 4-day-old chronic infarct anesthetized pig model.

Potassium channel openers are known to act on potassium ATP-dependent channels in cardiac tissue. Such agents may exacerbate acceleration of acute ischemia-induced ventricular repolarization and aggravate arrhythmias. To test whether activation of K( ATP) channels during the healing period of myocardial infarction (MI) can still influence the electrophysiologic properties and the type of inducible arrhythmias, we investigated the effects of bimakalim (BIM) on sustained ventricular tachycardia (VT) 4 days after ligation of the left anterior descending (LAD) coronary artery in pigs. Programmed stimulation was performed to elicit VT prior to and after intravenous (IV) BIM. Combination monophasic action potential (MAP)/PACING catheters were used to enable simultaneous ventricular MAP recording and pacing. Ventricular effective refractory period (ERP) and MAP duration determined at 50% and 90% repolarization were measured prior to and after BIM. After completion of baseline measurements, BIM was consecutively given at 0.5, 1, and 3 mg/kg bolus followed by 0.025, 0.05, and 0.1 mg/kg per minute maintenance infusion, respectively. From a total of 23 pigs subjected to LAD ligation, 4 animals succumbed to infarction and the remaining 19 animals were studied by programmed stimulation. Only animals that exhibited reproducible and hemodynamically stable monomorphic VTs during control stimulation were selected for evaluation (n = 14). After the first, second, and third dose of BIM, the mean VT rate was increased by 6%, 14% (P <. 01), and 47% (P < .001) compared to control values, respectively. Ventricular ERP and repolarization were significantly shortened only by the second and third dose of BIM. Of 14 pigs receiving the highest BIM dosage, 3 revealed polymorphic VTs degenerating into ventricular fibrillation (VF). Our data suggest that high BIM doses may lead to faster and more aggressive pacing-induced reentrant VTs after subacute MI. This is consistent with the drug-induced acceleration of ventricular repolarization with shortening of MAP duration and refractoriness.

Metal ions modify DNA-protecting and mutagen-scavenging capacities of the AV-153 1,4-dihydropyridine.

1,4-Dihydropyridines (1,4-DHP) possess important biochemical and pharmacological properties, including antioxidant and antimutagenic activities. AV-153-Na, an antimutagenic and DNA-repair enhancing compound was shown to interact with DNA by intercalation. Here we studied DNA binding of several AV-153 salts to evaluate the impact of AV-153 modifications on its DNA binding capacity, the ability to scavenge the peroxynitrite, to protect HeLa and B-cells cells against DNA damage. Affinity of the AV-153 salts to DNA measured by a fluorescence assay was dependent on the metal ion forming a salt in position 4 of the 1,4-DHP, and it decreased as follows: Mg > Na > Ca > Li > Rb > K. AV-153-K and AV-153-Rb could not react chemically with peroxynitrite as opposed to AV-153-Mg and AV-153-Ca, the latter increased the decomposition rate of peroxynitrite. AV-153-Na and AV-153-Ca effectively reduced DNA damage induced by peroxynitrite in HeLa cells, while AV-153-K and AV-153-Rb were less effective, AV-153-Li did not protect the DNA, and AV-153-Mg even caused DNA damage itself. The Na, K, Ca and Mg AV-153 salts were also shown to reduce the level of DNA damage in human B-cells from healthy donors. Thus, metal ions modify both DNA-binding and DNA-protecting effects of the AV-153 salts.

In vitro phototoxicity of dihydropyridine derivatives: a photochemical and photobiological study.

Some photosensitizing drugs can cause phototoxic skin responses even after systemic administration; therefore, avoidance of undesired side-effects is a key consideration in drug discovery and development. As a prediction tool for phototoxic risk, we previously proposed the monitoring of reactive oxygen species (ROS) generated from compounds irradiated with UVA/B, which can be effective for understanding photochemical/photobiological properties. In this investigation, we evaluated the photosensitizing properties of a novel dihydropyridine derivative, with bradykinin B(2) receptor antagonist activity (compound A) using our ROS assay and several analytical/biochemical techniques. Exposure of compound A, and several dihydropyridine-type calcium channel antagonists to simulated sunlight resulted in the significant production of singlet oxygen, superoxide, or both, which indicates their photosensitive/phototoxic potential. This is consistent with the observation that compound A under UVA/B light exposure caused significant photodegradation and even peroxidation of fatty acid, which could lead to phototoxic dermatitis. Interestingly, the addition of radical scavengers, especially GSH, MPG and BHA, could attenuate the lipid peroxidation, suggesting the involvement of ROS generation in the phototoxic pathways of compound A. In the 3T3 neutral red uptake phototoxicity test, compound A also showed a phototoxic effect on 3T3 mouse fibroblast cells. These findings also support the usefulness of the ROS assay for the risk assessment studies on the drug-induced phototoxicity even at the early stages of pharmaceutical development.

Reactive oxygen species assay-based risk assessment of drug-induced phototoxicity: classification criteria and application to drug candidates.

We have previously demonstrated that the phototoxic potential of chemicals could be partly predicted by the determination of reactive oxygen species (ROS) from photo-irradiated compounds. In this study, ROS assay strategy was applied to 39 marketed drugs and 210 drug candidates in order to establish provisional classification criteria for risk assessment of drug-induced phototoxicity. The photosensitizing properties of 39 model compounds consisting of phototoxic and non-phototoxic chemicals, as well as ca. 210 drug candidates including 11 chemical series were evaluated using ROS assay and the 3T3 neutral red uptake phototoxicity test (NRU PT). With respect to marketed drugs, most phototoxic drugs tended to cause type I and/or II photochemical reactions, resulting in generation of singlet oxygen and superoxide. There seemed to be a clear difference between phototoxic drugs and non-phototoxic compounds in their abilities to induce photochemical reactions. A plot analysis of ROS data on the marked drugs provided classification criteria to discriminate the photosensitizers from non-phototoxic substances. Of all drug candidates tested, 35.2% compounds were identified as phototoxic or likely phototoxic on the basis of the 3T3 NRU PT, and all ROS data for these phototoxic compounds were found to be over the threshold value. Furthermore, 46.3% of non-phototoxic drug candidates were found to be in the subthreshold region. These results verify the usefulness of the ROS assay for understanding the phototoxicity risk of pharmaceutical substances, and the ROS assay can be used for screening purposes in the drug discovery stage.

Design and synthesis of new symmetrical derivatives of dihydropyridine containing a pyridyl group on the 3, 5-positions and evaluation of their cytotoxic and multidrug resistance reversal activity.

Today, chemotherapy is an important part in the treatment of several kinds of cancer; however, the development of drug resistance remains one of the major obstacles in successful chemotherapy. Several types of agents have been recognized as multidrug resistance (MDR) inhibitors, among which the 1,4-dihydropyridines (DHPs) have been investigated the most. P-glycoprotein inhibition has been reported as the main MDR reversal mechanism of DHPs, whilst other mechanisms such as inhibition of topoisomerase II have received less attention. Therefore, in this study new derivatives of DHP have been synthesized. Their cytotoxic activity and their effects in reversing atypical MDR have been evaluated. The results confirmed the appropriate effect of these compounds on atypical MDR. Although it was observed that these compounds had a moderate cytotoxic effect, the cytotoxicity of one compound on the K562 cell line (IC50 = 6.61 microM) was comparable with that of doxorubicin (IC50 = 4.17 microM). Finally, the Ca(2+)-channel antagonistic activity, an undesired effect for these compounds, was evaluated.

Biological evaluation of bishydroxymethyl-substituted cage dimeric 1,4-dihydropyridines as a novel class of p-glycoprotein modulating agents in cancer cells.

A series of N-substituted cage dimeric 1,4-dihydropyridines 3a-e was evaluated as inhibitors of membrane efflux pump P-glycoprotein (P-gp) in multidrug resistant (mdr) cancer cells. Structure-activity relationships (SAR) and cytotoxic properties are discussed. Effective concentrations for overcoming mdr have been demonstrated in competition studies with the P-gp substrate epirubicin.

Tumour-specific cytotoxicity and MDR-reversal activity of dihydropyridines.

The ability of 41 1,4-diphenyl-1,4-dihydropyridine derivatives to inhibit the transport activity of P-glycoprotein were studied by flow cytometry in a multidrug-resistant human colon cancer cell line (COLO320) and in human mdr1 gene-transfected mouse lymphoma cells (L 5178 Y). The cytotoxicities of these compounds were also examined against human normal and cancer cell lines. The majority of the tested compounds proved to be effective inhibitors of rhodamine 123 outward transport, but their cytotoxicities were not negligible. Some dihydropyridine derivatives displayed cytotoxic activity against four human oral tumour cell lines and against three normal human oral cell lines. There was no clear-cut relationship between the multidrug-resistance activity or cytotoxicity and the chemical structures of the compounds. New ring substituents could prevent the oxidation of the ring of the aromatic compound.

Activities of newly synthesized dihydropyridines in overcoming of vincristine resistance, calcium antagonism, and inhibition of photoaffinity labeling of P-glycoprotein in rodents.

Newly synthesized 1,4-dihydropyridine derivatives (NK-compounds) were screened to determine whether they could overcome vincristine (VCR) resistance in VCR-resistant (P388/VCR) leukemia-bearing mice. Among the 57 NK-compounds examined, six compounds had strong reversing ability (Grade A), 18 partially overcame the resistance (Grade B), and 33 did not reverse the resistance (Grade C). The ability to overcome resistance varied considerably with the nature of substituents at positions 3.5 of the 1,4-dihydropyridine, and the most suitable substituents were the pyridylalkyl-including esters. Calcium antagonistic activity of NK-compounds having pyridylalkyl-including esters at positions 3.5 and dithiene ring at position 4 of the 1,4-dihydropyridine was greater than in those compounds having the dioxene ring at position 4. NK-242, which was assessed at Grade A and had no calcium antagonistic activity, improved therapeutic effects in both VCR-sensitive (P388/S) leukemia- and P388/VCR leukemia-bearing mice when combined with VCR. Fourteen NK-compounds were screened to determine whether they could inhibit photoaffinity labeling of the P-glycoprotein (Mr 170,000 glycoprotein) in a multidrug-resistant cell line by [3H]azidopine. All six compounds of Grade A and two of the three compounds of Grade B almost completely inhibited the labeling of Mr 170,000 glycoprotein at 1 to 10 microM. Thus there was a good correlation between the ability to reverse VCR resistance in vivo and the inhibition of photoaffinity labeling of Mr 170,000 glycoprotein.

Comparison and evaluation of pesticide monitoring programs using a process-based mixture model.

A number of European countries run large-scale pesticide monitoring schemes in watersheds aimed at identifying and evaluating the presence of pesticide residues in the environment. These schemes provide national and regional scale assessments of pesticide concentrations within the context of environmental quality assessment, aiming to ensure some degree of ecological protection. The present study is aimed at evaluating the joint effects of the pesticide mixtures detected in monitoring programs, using a process-based mixture model that was parameterized for Daphnia magna. In total, over 15 000 samples containing over 1 million individual measurements were evaluated for effects. It was found that there are only a small number of places where one can expect to have effects on daphnids, based on measured concentrations. The most polluted samples would cause extinction of a daphnid population within only 30 h. The results show that effects are mostly triggered by a limited number of pesticide residues at locations with high emissions. It was also shown that the analytical detection limits are basically too high to exclude mixture effects. So, despite all the effort that is put into chemical monitoring programs, it remains a challenge to make statements on whether or not the environment is protected. Recommendations are offered for a different setup of monitoring programs to improve this situation. Environ Toxicol Chem 2016;35:3113-3123. © 2016 SETAC.

Development of tridentate iron chelators: from desferrithiocin to ICL670.

Successful treatment of beta-thalassemia requires two key elements: blood transfusion and iron chelation. Regular blood transfusions considerably expand the lifespan of patients, however, without the removal of the consequential accumulation of body iron, few patients live beyond their second decade. In 1963, the introduction of desferrioxamine (DFO), a hexadentate chelator, marked a breakthrough in the treatment of beta-thalassemia. DFO significantly reduces body iron burden and iron-related morbidity and mortality. DFO is still the only drug for general use in the treatment of transfusion dependent iron overload. However, its very short plasma half-life and poor oral activity necessitate special modes of application (subcutaneous or intravenous infusion) which are inconvenient, can cause local reactions and are difficult to be accepted by many patients. Over the past four decades, many different laboratories have invested major efforts in the identification of orally active iron chelators from several hundreds of molecules of synthetic, microbial or plant origin. The discovery of ferrithiocin in 1980, followed by the synthesis of the tridentate chelator desferrithiocin and proof of its oral activity raised a lot of hope. However, the compound proved to be toxic in animals. Over a period of about fifteen years many desferrithiocin derivatives and molecules with broader alterations led to the discovery of numerous new compounds some of which were much better tolerated and were more efficacious than desferrithiocin in animals, however, none was safe enough to proceed to the clinical use. The discovery of a new chemical class of iron chelators: The bis-hydroxyphenyltriazoles re-energized the search for a safe tridentate chelator. The basic structure of this completely new chemical class of iron chelators was discovered by a combination of rational design, intuition and experience. More than forty derivatives of the triazole series were synthesized at Novartis. These compounds were evaluated, together with more than 700 chelators from various chemical classes. Using vigorous selection criteria with a focus on tolerability, the tridentate chelator 4-[(3,5-Bis-(2-hydroxyphenyl)-1,2,4)triazol-1-yl]-benzoic acid (ICL670) emerged as an entity which best combined high oral potency and tolerability in animals. ICL670 is presently being evaluated in the clinic.