Lower EC50 Values Research Articles

Design, Synthesis, and SAR of Novel 2-Glycinamide Cyclohexyl Sulfonamide Derivatives against Botrytis cinerea

N-(2-trifluoromethyl-4-chlorophenyl)-2-oxocyclohexyl sulfonamide (chesulfamide) is in the limelight as a novel fungicide, and has fungicidal activity against Botrytis cinerea. For exploring more novel structures, 33 new compounds were synthesized by N-alkylation and acid–amine coupling reactions with chesulfamide as the core moiety, and their structures were characterized and established by 1H-NMR, 13C-NMR, MS, and elemental analysis. The structure of (1R,2S)-2-(2-(N-(4-chloro-2-trifluoromethylphenyl)sulfamoyl)-cyclohexylamino)-N-(2-trifluoromethylphenyl) acetamide (II-19) was defined by X-ray single crystal diffraction. The in vivo and in vitro fungicidal activities against B. cinerea were evaluated. The bioassay results of mycelial growth demonstrated that most compounds exhibited excellent inhibitory activity against B. cinerea at 50 μg mL−1, and 7 compounds showed lower EC50 values than boscalid (EC50 = 4.46 μg mL−1) against B. cinerea (CY-09). In cucumber pot experiment, the inhibitory rates of four compounds (II-4, II-5, II-12, and II-13) against B. cinerea were 90.48, 93.45, 92.86, and 91.07, which were better than cyprodinil (88.69%), the best performing of all controls. In tomato pot experiment, the control efficacy of two analogs (II-8 and II-15) were 87.98 and 87.97% at 200 μg mL−1, which were significantly higher than boscalid (78.10%). Most compounds have an excellent fungicidal effect on B. cinerea, with potential as a lead compound for developing new pesticides.

The discovery of novel diarylpyri(mi)dine derivatives with high level activity against a wide variety of HIV-1 strains as well as against HIV-2

By means of structure-based molecular hybridization strategy, a series of novel diarylpyri(mi)dine derivatives targeting the entrance channel of HIV-1 reverse transcriptase (RT) were designed, synthesized and evaluated as potent non-nucleoside reverse transcriptase inhibitors (NNRTIs). Encouragingly, all the tested compounds showed good activities against wild-type (WT) HIV-1 (IIIB) with EC50 in the range of 1.36 nM–29 nM, which is much better than those of nevirapine (NVP, EC50 = 125.42 nM) and azidothymidine (AZT, EC50 = 11.36 nM). Remarkably, these compounds also displayed effective activity against the most of the single and double-mutated HIV-1 strains with low EC50 values, which is comparable to the control drugs. Besides, these compounds were also exhibited favorable enzymatic inhibitory activity. Moreover, preliminary structure-activity relationships (SARs) and molecular modeling study were investigated and discussed in detail. Unexpectedly, four diarylpyrimidines yielded moderate anti-HIV-2 activities. To our knowledge, this is rarely reported that diarylpyrimidine-based NNRTIs have potent activity against both HIV-1 and HIV-2 in cell culture.

Isocyanides Derived from α,α-Disubstituted Amino Acids: Synthesis and Antifouling Activity Assessment.

Herein, we contribute to the development of environmentally friendly antifoulants by synthesizing eighteen isocyanides derived from α,α-disubstituted amino acids and evaluating their antifouling activity/toxicity against the cypris larvae of the Balanus amphitrite barnacle. Almost all isocyanides showed good antifouling activity without significant toxicity and exhibited EC50 values of 0.07-7.30μg/mL after 120-h exposure. The lowest EC50 values were observed for valine-, methionine-, and phenylalanine-derived isocyanides, which achieved >95% cypris larvae settlement inhibition at concentrations of less than 30μg/mL without exhibiting significant toxicity. Thus, the prepared isocyanides should be useful for further research focused on the development of environmentally friendly antifouling agents.

Functional screening of FGFR4-driven tumorigenesis identifies PI3K/mTOR inhibition as a therapeutic strategy in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and outcomes have stagnated, highlighting a need for novel therapies. Genomic analysis of RMS has revealed that alterations in the receptor tyrosine kinase (RTK)/RAS/PI3K axis are common and that FGFR4 is frequently mutated or overexpressed. Although FGFR4 is a potentially druggable receptor tyrosine kinase, its functions in RMS are undefined. This study tested FGFR4-activating mutations and overexpression for the ability to generate RMS in mice. Murine tumor models were subsequently used to discover potential therapeutic targets and to test a dual PI3K/mTOR inhibitor in a preclinical setting. Specifically, we provide the first mechanistic evidence of differential potency in the most common human RMS mutations, V550E or N535K, compared to FGFR4wt overexpression as murine myoblasts expressing FGFR4V550E undergo higher rates of cellular transformation, engraftment into mice, and rapidly form sarcomas that highly resemble human RMS. Murine tumor cells overexpressing FGFR4V550E were tested in an in vitro dose-response drug screen along with human RMS cell lines. Compounds were grouped by target class, and potency was determined using average percentage of area under the dose-response curve (AUC). RMS cells were highly sensitive to PI3K/mTOR inhibitors, in particular, GSK2126458 (omipalisib) was a potent inhibitor of FGFR4V550E tumor-derived cell and human RMS cell viability. FGFR4V550E-overexpressing myoblasts and tumor cells had low nanomolar GSK2126458 EC50 values. Mass cytometry using mouse and human RMS cell lines validated GSK2126458 specificity at single-cell resolution, decreasing the abundance of phosphorylated Akt as well as decreasing phosphorylation of the downstream mTOR effectors 4ebp1, Eif4e, and S6. Moreover, PI3K/mTOR inhibition also robustly decreased the growth of RMS tumors in vivo. Thus, by developing a preclinical platform for testing novel therapies, we identified PI3K/mTOR inhibition as a promising new therapy for this devastating pediatric cancer.

Metal-chelating 3-hydroxypyrimidine-2,4-diones inhibit human cytomegalovirus pUL89 endonuclease activity and virus replication

Human cytomegalovirus terminase complex cleaves the concatemeric genomic viral DNA into unit lengths during genome packaging and particle assembly. Terminase complex ATPase and endonuclease activity is provided by the viral protein pUL89. pUL89 is an attractive drug target because its activities are required for infectious virus production. A domain located in the C-terminus of pUL89 has an RNase H/integrase-like fold and endonuclease activity that can be inhibited by compounds featuring a chelating triad motif. Previously, we developed a novel ELISA approach to screen for pUL89 inhibitors. In this report, we used the ELISA to identify 3-hydroxypyrimidine-2,4-dione as a promising scaffold for pUL89 inhibitor development. Several potent pUL89 inhibitors yielded low micromolar IC50 values in the enzymatic assay and low micromolar EC50 values for inhibition of HCMV replication. Two representative compounds inhibitory effects depended upon metal ions and occurred late in virus replication consistent with pUL89 inhibitors in infected cells.

Abstract B06: Targeting PI3K/mTOR in mouse rhabdomyosarcoma models driven by FGFR4 activation

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue sarcoma and while survival rates have increased over the past few decades, outcomes for intermediate and high-risk patients remain dismal. Genomic analyses show that 93% of RMS have RTK/RAS/PI3K alterations and that fibroblast growth factor receptor 4 (FGFR4) is frequently mutated or overexpressed. The point mutation V550E constitutively activates FGFR4, stimulating downstream signaling pathways. In this translational study, we demonstrate that FGFR4V550E is a potent oncogene in mouse models of RMS and that secondary tumor models can be generated following transplantation of FGFR4V550E overexpressing tumor cells into immunocompetent host mice. These models uncovered mechanisms of transformation in FGFR4V550E-driven RMS and helped identify novel compounds that inhibit RMS growth. Specifically, AKT and mTOR signaling pathways were activated in myoblasts overexpressing FGFR4V550E and similarly, FGFR4V550E overexpressing tumors and tumor-derived cells exhibited increased AKT and mTOR phosphorylation by immunoblot. Murine tumor cells overexpressing FGFR4V550E were tested in an in vitro dose-response drug screen along with human RMS cell lines. Compounds were grouped by target class, and potency was determined using average percent area under the dose response curve (AUC). Using this technique, FGFR4V550E overexpressing tumor cells were highly sensitive to PI3K/mTOR inhibitors. In particular, GSK2126458 (omipalisib) was a potent inhibitor of FGFR4V550E tumor-derived cell and human RMS cell viability. FGFR4V550E overexpressing myoblasts and tumor cells had low nanomolar GSK2126458 EC50 values. Mass cytometry using mouse and human RMS cell lines validated GSK2126458 specificity at single cell resolution, decreasing the abundance of phosphorylated Akt as well as decreasing phosphorylation of the downstream mTOR effectors 4ebp1, Eif4e, and S6. In a preclinical study, GSK2126458 inhibited tumor growth in vivo. A statistically significant increase in disease-specific survival was observed in mice treated with GSK2126458 compared to mice treated with vehicle alone (p<0.001) or standard of care, vincristine (p<0.05). In summary, RMS driver mutations were validated in vivo and our model system was effectively used as a preclinical tumor model to identify and test therapeutic agents. Importantly, these results suggest a role for PI3K/mTOR inhibition in precision therapy regimens for RMS with FGFR4 mutations. This study provides further evidence for RMS clinical studies involving mTOR inhibitors (e.g., temsirolimus). Citation Format: Timothy McKinnon, Rosemarie Venier, Marielle Yohe, Berkley E. Gryder, Brendan Dickson, Krista Schleicher, Dariush Davani, Winnie Wei, Cynthia Guidos, Abha Gupta, Javed Khan, Rebecca Gladdy. Targeting PI3K/mTOR in mouse rhabdomyosarcoma models driven by FGFR4 activation [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B06.

Chemical Management of Invasive Shot Hole Borer and Fusarium Dieback in California Sycamore (Platanus racemosa) in Southern California.

Fusarium dieback (FD) is a new vascular disease of hardwood trees caused by Fusarium spp. and other associated fungal species which are vectored by two recently introduced and highly invasive species of ambrosia beetle (Euwallacea spp. nr. fornicatus). One of these ambrosia beetles is known as the polyphagous shot hole borer (PSHB) and the other as the Kuroshio shot hole borer (KSHB). Together with the fungi that they vector, this pest-disease complex is known as the shot hole borer-Fusarium dieback (SHB-FD) complex. Mitigation of this pest-disease complex currently relies on tree removal; however, this practice is expensive and impractical given the wide host range and rapid advancement of the beetles throughout hardwoods in southern California. This study reports on the assessment of various pesticides for use in the management of SHB-FD. In vitro screening of 13 fungicides revealed that pyraclostrobin, trifloxystrobin, and azoxystrobin generally have lower effective concentration that reduces 50% of mycelial growth (EC50) values across all fungal symbionts of PSHB and KSHB; metconazole was found to have lower EC50 values for Fusarium spp. and Paracremonium pembeum. Triadimefon and fluxapyroxad were not capable of inhibiting any fungal symbiont at the concentrations tested. A 1-year field study showed that two insecticides, emamectin benzoate alone and in combination with propiconazole, and bifenthrin, could significantly reduce SHB attacks. Two injected fungicides (tebuconazole and a combination of carbendazim and debacarb) and one spray fungicide (metconazole) could also significantly reduce SHB attacks. Bioassays designed to assess fungicide retention 1 year postapplication revealed that six of the seven fungicides exhibited some level of inhibition in vitro and all thiabendazole-treated trees sampled exhibiting inhibition. This study has identified several pesticides which can be implemented as part of an integrated pest management strategy to reduce SHB infestation in low to moderately infested landscape California sycamore trees and potentially other landscape trees currently affected by SHB-FD.

Discovery of biphenyl-substituted diarylpyrimidines as non-nucleoside reverse transcriptase inhibitors with high potency against wild-type and mutant HIV-1

A novel series of diarylpyrimidine (DAPY) derivatives bearing the biphenyl motif with multiple substituted groups was synthesized as human immunodeficiency virus (HIV)-1 non-nucleoside reverse transcriptase inhibitors. All of the target compounds were evaluated for their in vitro activity against HIV in MT-4 cells. Most of the compounds exhibited excellent activity with low nanomolar EC50 values against wild-type, single and double mutant HIV-1 strains. Compound 4b displayed an EC50 value of 1 nM against HIV-1 IIIB, 1.3 nM against L100I, 0.84 nM against K103 N, 1.5 nM against Y181C, 11 nM against Y188L, 2 nM against E138K, 10 nM against K103 N + Y181C, and almost 110 nM against F227L + V106. The improvement in the selectivity and potency of the target molecules against the wild-type and mutant HIV-1 strains validated our hypothesis. The biphenyl ring in the DAPY derivatives could strengthen the π-π stacking effect between the target molecule and the non-nucleoside inhibitor-binding pocket in the reverse transcriptase by extending the conjugating systems. This research represented a significant step toward the discovery of novel therapeutic DAPYs for treating acquired immunodeficiency syndrome in patients infected with HIV-1.

Antioxidant potential and HPLC-DAD profile of phenolic compounds from leaves and culms of Merostachys pluriflora

Brazil has the highest diversity of bamboo species in the New World. Knowledge about the medicinal applications of native Brazilian bamboos is extremely underdeveloped in comparison to Asian species. This study aimed to evaluate different antioxidant activities of extracts and phases from leaves and culms of Merostachys pluriflora Munro ex. C. G. Camus and to correlate this data with the amount of total phenolic and flavonoid content. Dried leaves and culms were macerated in 70% ethanol for 7days at room temperature, in the dark. Extracts were freeze-dried and crude ethanol extracts (EE) were fractionated by partition to obtain hexane (HP), dichloromethane (DP), ethyl acetate (EAP) and hydromethanol (HMP) phases. Characterization and quantification of phenolic compounds was performed using a HPLC-DAD. Antioxidant capacity was evaluated using the DPPH, FRAP, ABTS, ICR and ORAC assay, using trolox as a positive control. The yield of crude ethanol extract from leaves (EE-L) and culms (EE-C) was 7.68% and 3.22%, respectively. In total 33 peaks were integrated in the chromatograms considering both organs (leaves and culms), but only seven peaks presented UV–Vis spectra characteristic of flavonoids, indicating the presence of phenolic acids and/or hydroxycinnamic acid derivatives as major constituents of crude ethanol extracts of M. pluriflora. Dichloromethane phase from culms (DP-C) showed the highest amount of hydroxycinnamic acids derivative (64.52mg/g) while ethyl acetate phase from leaves (EAP-L) presented the highest contents of flavonoids (8.31mg/g). For antioxidant assays, EAP-L (117.68μg/mL) showed lower EC50 values in DPPH; for ABTS and ICR, hydromethanolic phase from leaves (HMP-L) showed lower EC50 values, 16.30μg/mL and 20.46μg/mL, respectively. DP-C presented the lowest EC50 values in FRAP (27.92μg/mL) and ORAC (1.22μg/mL) assays. M. pluriflora proved to be a promising species for prospecting studies, presenting a large amount of phenolic compounds with antioxidant activity. The antiradical potential seems to be related to the presence of flavonoids while iron-reducing capacity could be correlated to other phenolic constituents.

Antioxidant and neuroprotective effects of gossypitrin, a flavonoid from Talipariti elatum, against chemical hypoxia-induced PC12 cell death

Context: Some flavonoids have been described as neuroprotectors. Gossypitrin (Gos) is a natural occurring flavonoid and the main bioactive substance from the flowers of Talipariti elatum Sw. (Majagua azul), traditionally used in Cuba as expectorant and antiasthmatic. Only few reports have documented its antioxidant properties. Aims: To evaluate the antioxidant and cytoprotective effects of Gos against cyanide-induced oxidative stress and cell death in PC12 cells. Methods: Gos effects on DPPH/ ABTS radical scavenging, ferric reducing power and lipid peroxidation were examined. The ischemia/reperfusion neuronal damage was produced by exposing PC12 cells to KCN in glucose-free medium. Gos was added to the incubation medium 30 min before chemical hypoxia induction. The neuroprotective potential of Gos was assessed by measuring cell viability by the MTT assay, the antioxidant enzymes SOD and CAT, and GSH and lipid peroxidation levels. Rutin (Rut), the well-known antioxidant, was used as a reference compound. Results: Gos showed a potent intrinsic antioxidant capacity evidenced by low IC50 and EC50 values for DPPH/ABTS/malondialdehyde and ferric reducing power, respectively. Pre-treatment of PC12 cells with Gos, significantly increased their survival against KCN, restored the levels of GSH and the SOD and CAT enzymes activities, as well as reduced the level of lipid peroxidation. Its antioxidant effects were higher than those elicited by Rut. Conclusions: The results show for the first time the neuroprotective potential of Gos against hypoxic cell damage, probably associated to its antioxidant effects.

The antioxidant activity and organoleptic properties of soursoup leaf tea (Annona Muricata L.) and moringa leaf (Moringa Oliefera L.) in combination with guava leaf (Psidium guajava)

Soursop leaf tea (Annona muricata L.) and drumstick tree leaf tea (Moringa oliefera L) in combination with guava leaves (Psidium guajava) are innovative herbal drinks from Indonesia. Soursop, drumstick and guava leaves contain antioxidant compounds such as tannin, saponin, flavonoids, alkaloids, triterpene and quercetin. This study aims to compare the antioxidant activity and organoleptic properties between soursop leaf tea and drumstick tree leaf tea in combination with guava leaves. The study was conducted through analysing the production of tea with an overall drying temperature at 50 oC, 55 oC, 60 oC and 65 oC respectively. The antioxidant activity was measured using the UV-Vis spectrophotometric method (λ 517 nm), while the organoleptic properties were measured using the parameters of taste, colour aroma, and viscosity. The highest antioxidant activity was performed at a temperature of 50 oC, which measured the lowest EC50 value while having the lowest level of organoleptic properties.

GI-530159, a novel, selective, mechanosensitive two-pore-domain potassium (K2P ) channel opener, reduces rat dorsal root ganglion neuron excitability.

Background and PurposeTREK two‐pore‐domain potassium (K2P) channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study, we describe a new, selective opener of TREK channels, GI‐530159.Experimental ApproachThe effect of GI‐530159 on TREK channels was demonstrated using 86Rb efflux assays, whole‐cell and single‐channel patch‐clamp recordings from recombinant TREK channels. The expression of K2P2.1 (TREK1), K2P10.1 (TREK2) and K2P4.1 (TRAAK) channels was determined using transcriptome analysis from single dorsal root ganglion (DRG) cells. Current‐clamp recordings from cultured rat DRG neurons were used to measure the effect of GI‐530159 on neuronal excitability.Key ResultsFor recombinant human TREK1 channels, GI‐530159 had similar low EC50 values in Rb efflux experiments and electrophysiological recordings. It activated TREK2 channels, but it had no detectable action on TRAAK channels nor any significant effect on other K channels tested. Current‐clamp recordings from cultured rat DRG neurones showed that application of GI‐530159 at 1 μM resulted in a significant reduction in firing frequency and a small hyperpolarization of resting membrane potential.Conclusions and ImplicationsThis study provides pharmacological evidence for the presence of mechanosensitive TREK K2P channels in sensory neurones and suggests that development of selective K2P channel openers like GI‐530159 could aid in the development of novel analgesic agents.Linked ArticlesThis article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc

Peramivir conjugates as orally available agents against influenza H275Y mutant

Peramivir is an efficacious neuraminidase (NA) inhibitor for treatment of influenza by intravenous administration. However, the efficacy of peramivir toward the H275Y mutant is appreciably reduced. To address this drawback, conjugation of peramivir with caffeic acid is devised in this study to enhance the binding affinity with neuraminidases. The C2-OH group of peramivir is elaborated to link with caffeate derivatives, giving the desired conjugates 8 and 9 that possess potent NA inhibitory activity against both wild-type and H275Y viruses with the IC50 values in nanomolar range. The molecular modeling reveals that the caffeate moiety of conjugate 9 prefers to reside in the 295-cavity of H275Y neuraminidase, thus providing additional hydrogen bonds and hydrophobic interactions to compensate the reduced binding affinity of the peramivir moiety due to Glu-276 dislocation in H275Y mutant. In comparison with peramivir, the lipophilicity of conjugates 8 and 9 also increases by incorporation of the caffeate moiety. Thus, conjugates 8 and 9 offer better effect to protect MDCK cells from infection of H275Y virus with low EC50 value (∼17 nM). Administration of conjugates 8 or 9 by oral gavage is effective in treatment of mice that are infected by lethal dose of wild-type or H275Y influenza viruses. Considering drug metabolism, since the ester linkage in conjugate 8 is susceptible to hydrolysis in plasma, conjugate 9 with robust amide linkage may be a better candidate for development into orally available anti-influenza drug that is also active to mutant viruses.

In vitro sensitivity of Corynespora cassiicola isolated from soybean to fungicides and field chemical control of target spot

ABSTRACT Soybean target spot (Corynespora cassiicola) has become an important disease in most soybean growing regions in Brazil. The sensitivity of 34 isolates of C. cassiicola to 11 fungicides was evaluated based on mycelial growth inhibition (boscalid, carbendazim, cyproconazole, fluopyram, fluxapyroxad, prothioconazole and thiophanate-methyl) or spore germination inhibition (azoxystrobin, picoxystrobin, pyraclostrobin and trifloxystrobin). In addition, the efficacy of five fungicides to control target spot was tested in four field trials carried out during three crop seasons: 2011/2012, 2012/2013 and 2013/2014. Fungal isolates were collected from soybean plants in several soybean growing areas in Brazil. The effective concentration of each fungicide to inhibit fungal growth or spore germination by 50% (EC50) was calculated for all isolates. Fluxapyroxad and prothioconazole provided the greatest mycelial growth inhibition and pyraclostrobin led to the lowest spore germination percentage, with the lowest EC50 values. At field experiments, cyproconazole and carbendazim showed target spot control ranging from 26% to 29%. On the other hand, fluxapyroxad and prothioconazole prevented an epidemic of the disease by 45% to 55%, respectively. In general, the efficacy of fungicides in the field reflected the in vitro sensitivity averages. Large sensitivity reduction was detected to benzimidazoles (MBC), indicating that this group of fungicides should no longer be used for target spot control. There was a negative and significant correlation (-0.265) between target spot severity and soybean yield. The pathogen showed variability in sensitivity to the fungicide groups carboxamides (SDHI), triazoles (DMI) and strobilurins (QoI), which denotes a high risk of selection for resistance.

Discovery of a New Fungicide Candidate through Lead Optimization of Pyrimidinamine Derivatives and Its Activity against Cucumber Downy Mildew.

Downy mildew is one of the most highly destructive of the diseases that cause damage to fruits and vegetables. Because of the continual development of resistance, it is important to discover new fungicides with different modes of action from existing fungicides for the control of downy mildew. This study is a continuation of our previous work on the novel pyrimidinamine lead compound, 9, and includes field trials for the identification of the optimal candidate. A new compound, 1c, was obtained, which gave a lower EC50 value (0.10 mg/L) against downy mildew than lead compound 9 (0.19 mg/L) and the commercial fungicides diflumetorim, dimethomorph, and cyazofamid (1.01-23.06 mg/L). Compound 1c displayed similar broad-spectrum fungicidal activity to compound 9 but better field efficacy than compound 9, cyazofamid, and flumorph. The present work indicates that pyrimidinamine compound 1c is a candidate for further development as a commercial fungicide for the control of downy mildew.

Antifungal activity of pinosylvin fromPinus densifloraon turfgrass fungal diseases

The objective was to examine the antifungal activity of Pinus densiflora extract for the control of turfgrass fungal diseases. Antifungal activities of the various fractions of n-hexane, methylene chloride (Ch), ethyl acetate (EtOAc), and n-butanol from P. densiflora were evaluated against Rhizoctonia solani AG1-1B, R. solani AG2-2IV, Sclerotinia homoeocarpa, R. cerealis, Pythium spp., and Colletotrichum graminicola. The Ch and EtOAc fractions showed antifungal activity against Pythium sp. and C. graminicola in paper disc assay. The effective concentration to produce 50% mycelial inhibition (EC50) using five discriminatory concentrations of pinosylvin (1) from the Ch fraction of P. densiflora was evaluated on R. solani AG1-1B, R. solani AG2- 2IV, R. cerealis, and S. homoeocarpa. S. homoeocarpa showed the highest sensitivity with the lowest mean EC50 value (8.426 μg/ mL) among the four pathogens. Among the three Rhizoctonia pathogens, R. cerealis had the highest mean EC50 value (99.832 μg/mL) and R. solani AG2-2IV, with the lowest sensitivity, had the lowest EC50 value (39.696 μg/mL). These results suggested that pinosylvin (1) from P. densiflora could be a valuable lead compound in the improvement of a novel antifungal agent.

Drug repurposing strategy against Trypanosoma cruzi infection: In vitro and in vivo assessment of the activity of metronidazole in mono- and combined therapy

Metronidazole (Mtz) is a commercial broad-spectrum nitroimidazolic derivative with relevant antimicrobial activity and relative safety profile. Therefore, it is fair to consider Mtz a candidate for drug repurposing for other neglected conditions such as Chagas disease (CD), a parasitic pathology caused by Trypanosoma cruzi. CD is treated only with benznidazole (Bz) and nifurtimox, both introduced in clinics decades ago despite important limitations, including low efficacy on the later disease stage (chronic form) and severe side effects. New cheap and fast alternative treatments for CD are needed, thus the repurposing of Mtz was assessed in vitro and in vivo in mono- and combined therapy. In vitro assays demonstrated EC50>200µM for Mtz, while for Bz the values ranged from 2.51µM (intracellular forms) to 11.5µM (bloodstream trypomastigotes). When both drugs were combined in fixed-ratio proportions, Mtz promoted Bz potency (lower EC50 values). In vivo toxicity assays for Mtz in mice showed no adverse effects neither histopathological alterations up to 2000mg/kg. Regarding experimental T. cruzi infection, Bz 100mg/kg suppressed parasitemia while Mtz (up to 1000mg/kg) in monotherapy did not, but prolonged animal survival at 250 and 500 regimen doses. The combination of both drugs (Bz 10+Mtz 250) prevented mortality (70%) besides protected against electric cardiac alterations triggered by the parasite infection. Although not able to reduce parasite load, the combination therapy prevented animal mortality; this was possibly due to a protection of the electric cardiac physiology that is normally altered in experimental infection of T. cruzi. It also suggested that the interaction with Mtz could have improved the pharmacokinetics of Bz. Our study emphasizes the importance of drug repurposing and combined therapy for CD to contribute to alternative therapies for this neglected and silent pathology.

ID: 1008 Quercus infectoria semi-purified fractions promoted BMP-2, Runx2 and osteopontin expression in human fetal osteoblastic cell line

Quercus infectoria (QI) is traditional medicines in Asia which contain many active phytochemical compounds that have the potential to stimulate bone formation. However, the precise mechanism of the therapeutic effect of QI on osteoblast has not been elucidated. The present study was carried out to extract the active compound of QI through a series of purification in order to obtain semi-purified fractions of QI followed by examining its phytochemical profile through liquid chromatography mass spectrometry (LC-MS) as well as to delineate the molecular mechanism of QI semi-purified fraction-enhanced bone formation by investigating the protein expression of bone morphogenic protein-2 (BMP-2), Runx2 and osteopontin (OPN) in the hFOB 1.19 human fetal osteoblastic cell line. The semi-purified fractions of QI were produced in series of column chromatography and thin layer chromatography (TLC) technique. The obtained fractions were then tested in a series of bio-guided assay through MTT assay. Three most potent fractions with lowest EC50 value (Fraction A-10.85 µg/ml, Fraction B-12.00 µg/ml and Fraction C-11.60 µg/ml) were selected to be treated with hFOB1.19 cell followed by quantification of BMP-2, Runx2 and OPN protein expression at day 1, 3 and 7. Compound profiling through LC-MS analysis showed that the chosen fractions consist of main active component known as gallic acid that has been showed previously to promote bone formation. Results show that QI semi-purified fractions increased the activity of BMP-2, Runx2 and OPN at day 1 and day 3. Meanwhile as the expression of BMP-2 and Runx2 continue to increase at day 7, the expression of OPN shows a down regulation at day 7. Furthermore, the study showed that combination of Fraction A, B and C wit osteoporotic drug (pamidronate) further increase the expression of BMP-2 and Runx2 contrary to OPN expression. These finding demonstrated that semi-purified fraction of QI enhanced bone formation through expression of investigated bone-related marker.

Co-composting of Green Waste Mixed with Unprocessed and Processed Food Waste: Influence on the Composting Process and Product Quality

Green waste (GW) represents a large fraction of municipal solid waste (MSW) and has increased significantly with the rapid development of urban green areas in some countries. Composting is an appropriate method for the treatment and recovery of these wastes. However, the high content of lignocellulosic substances in GW is known to slow down the decomposition process. This research evaluated the effect of introducing processed food waste (PFW) and unprocessed food waste (UPFW) on composting of GW, it monitored the process itself and assessed the end-product quality. A field-scale experiment was developed using three treatments: Treatment A (100%GW), Treatment B (40%UPFW + 60%GW) and Treatment C (50%GW + 30%UPFW + 20%PFW). Treatment C reached thermophilic temperatures in a shorter time than the other treatments and maintained those temperatures for longer periods. In addition, treatment C reached ambient temperatures during curing in a shorter time compared to treatments A and B. An improved product quality was observed in treatment C compared to other treatments. For example, products from treatment C had lower ash content, higher concentrations of TOC and PTotal and lower EC values compared to treatments A and B. The final fertility index of the end-product from treatment C was 4.8–5.0, which indicates that the product can be suitable for agricultural use.

Model based population PK-PD analysis of furosemide for BP lowering effect: A comparative study in primary and secondary hypertension

Though numerous reports have demonstrated multiple mechanisms by which furosemide can exert its anti-hypertensive response. However, lack of studies describing PK-PD relationship for furosemide featuring its anti-hypertensive property has limited its usage as a blood pressure (BP) lowering agent. Serum concentrations and mean arterial BP were monitored following 40 and 80mgkg−1 multiple oral dose of furosemide in spontaneously hypertensive rats (SHR) and DOCA-salt induced hypertensive (DOCA-salt) rats. A simultaneous population PK-PD relationship using Emax model with effect compartment was developed to compare the anti-hypertensive efficacy of furosemide in these rat models. A two-compartment PK model with Weibull-type absorption and first-order elimination best described the serum concentration-time profile of furosemide. In the present study, post dose serum concentrations of furosemide were found to be lower than the EC50. The EC50 predicted in DOCA-salt rats was found to be lower (4.5-fold), whereas the tolerance development was higher than that in SHR model. The PK-PD parameter estimates, particularly lower values of EC50, Ke and Q in DOCA-salt rats as compared to SHR, pinpointed the higher BP lowering efficacy of furosemide in volume overload induced hypertensive conditions. Insignificantly altered serum creatinine and electrolyte levels indicated a favorable side effect profile of furosemide. In conclusion, the final PK-PD model described the data well and provides detailed insights into the use of furosemide as an anti-hypertensive agent.