Discovery and druggability evaluation of pyrrolamide-type GyrB/ParE inhibitor against drug-resistant bacterial infection.

The bacterial ATP-competitive GyrB/ParE subunits of type II topoisomerase are important anti-bacterial targets to treat super drug-resistant bacterial infections. Herein we discovered novel pyrrolamide-type GyrB/ParE inhibitors based on the structural modifications of the candidate AZD5099 that was withdrawn from the clinical trials due to safety liabilities such as mitochondrial toxicity. The hydroxyisopropyl pyridazine compound 28 had a significant inhibitory effect on Gyrase (GyrB, IC50 = 49 nmol/L) and a modest inhibitory effect on Topo IV (ParE, IC50 = 1.513 µmol/L) of Staphylococcus aureus. It also had significant antibacterial activities on susceptible and resistant Gram-positive bacteria with a minimum inhibitory concentration (MIC) of less than 0.03 µg/mL, which showed a time-dependent bactericidal effect and low frequencies of spontaneous resistance against S. aureus. Compound 28 had better protective effects than the positive control drugs such as DS-2969 (5) and AZD5099 (6) in mouse models of sepsis induced by methicillin-resistant Staphylococcus aureus (MRSA) infection. It also showed better bactericidal activities than clinically used vancomycin in the mouse thigh MRSA infection models. Moreover, compound 28 has much lower mitochondrial toxicity than AZD5099 (6) as well as excellent therapeutic indexes and pharmacokinetic properties. At present, compound 28 has been evaluated as a pre-clinical drug candidate for the treatment of drug-resistant Gram-positive bacterial infection. On the other hand, compound 28 also has good inhibitory activities against stubborn Gram-negative bacteria such as Escherichia coli (MIC = 1 µg/mL), which is comparable with the most potent pyrrolamide-type GyrB/ParE inhibitors reported recently. In addition, the structure-activity relationships of the compounds were also studied.

Development and In Vitro-In Vivo Correlation Evaluation of IMM-H014 Extended-Release Tablets for the Treatment of Fatty Liver Disease.

This study aimed to develop extended-release tablets containing 25 mg IMM-H014, an original drug formulated by a direct powder pressing method based on pharmaceutical-grade hydrophilic matrix polymers such as hydroxypropyl methylcellulose, to establish an in vitro-in vivo correlation (IVIVC) to predict bioavailability. The tablets' mechanical properties and in vitro and in vivo performance were studied. The formulation was optimized using a single-factor experiment and the reproducibility was confirmed. The in vitro dissolution profiles of the tablet were determined in five dissolution media, in which the drug released from the hydrophilic tablets followed the Ritger-Peppas model kinetics in 0.01 N HCl medium for the first 2 h, and in phosphate-buffered saline medium (pH 7.5) for a further 24 h. Accelerated stability studies (40 °C, 75% relative humidity) proved that the optimal formulation was stable for 6 months. The in vivo pharmacokinetics study in beagle dogs showed that compared to the IMM-H014 immediate release preparation, the maximum plasma concentration of the extended-release (ER) preparation was significantly decreased, while the maximum time to peak and mean residence time were significantly prolonged. The relative bioavailability was 97.9% based on the area under curve, indicating that the optimal formulation has an obvious ER profile, and a good IVIVC was established, which could be used to predict in vivo pharmacokinetics based on the formulation composition.

Identification and Determination of Impurities in a New Therapeutic Agent for Fatty Liver Disease.

Methyl 7,7'-dimethoxy-5'-(morpholinomethyl)-[4,4'-bibenzo[d][1,3] dioxole]-5-carboxylate methanesulfonate (IMM) is an innovative drug for the treatment of nonalcoholic fatty liver disease (NAFLD) owing to its high efficacy and low toxicity. In this study, five minor impurities (I, II, III, IV, and V) were identified and analyzed using spectroscopic evidence, chemical synthetic methods, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The impurities included hydrolysates and oxidation by-products extracted from both the drug in its final formulation and during synthesis. Toxicity prediction revealed potential carcinogenicity of impurity V containing an N-oxygen fragment. A reliable and selective HPLC method for the quantitative analysis of impurities I-IV and a sensitive HPLC-MS/MS method for potential genotoxic impurity V were developed and optimized. The methods were validated based on the International Council for Harmonization guidelines. Satisfactory linearity was obtained for the analytes over the range of 0.1-2.0 µg/mL for impurities I-IV and 0.3-30.0 ng/mL for impurity V, and in all cases, the fitting correlation coefficients exceeded 0.999. The obtained limits of detection values were 0.05 ng/mL and 0.005 µg/mL for impurity V and impurities I-IV, respectively. The precision and repeatability of the methods were less than 1.08% and 8.72% for each impurity. The recovery percentages of all impurities were in the range of 91.18%-111.27%, with the relative standard deviation of less than 3.69%. The greenness assessment of the HPLC method and the HPLC-MS/MS method were evaluated by using AGREE software with a score value of 0.72 and 0.68, respectively. The recommended procedures that were accurate, specific, and ecofriendly were applied to the existing active pharmaceutical ingredients of IMM, and they generated satisfactory results.

Regioselective and Geometrically Controlled Heck 1,1-Diarylation of Unactivated Aliphatic Alkenes.

Pd-catalyzed double Heck reaction allows regioselective 1,1-diarylation of unactivated aliphatic alkenes. When two different aryl bromides are used, the second aryl rings are added trans to aliphatic chains in the main olefinic isomers, consistent with a mechanism of two consecutive Heck arylations at the terminal carbons.

[Effect of light intensity on growth, accumulation of ginsenosides, and expression of related enzyme genes of Panax quinquefolius].

Appropriate light intensity is favorable for the photosynthesis, biomass accumulation, key enzyme activity, and secondary metabolite synthesis of medicinal plants. This study aims to explore the influence of light intensity on growth and quality of Panax quinquefolius. To be specific, sand culture experiment was carried out in a greenhouse under the light intensity of 40, 80, 120, and 160 µmol·m~(-2)·s~(-1), respectively. The growth indexes, photosynthetic characteristics, content of 6 ginsenosides of the 3-year-old P. quinquefolius were determined, and the expression of ginsenoside synthesis-related enzyme genes in leaves, main roots, and fibrous roots was determined. The results showed that the P. quinquefolius growing at 80 µmol·m~(-2)·s~(-1) light intensity had the most biomass and the highest net photosynthetic rate. The total biomass of P. quinquefolius treated with 120 µmol·m~(-2)·s~(-1) light intensity was slightly lower than that with 80 µmol·m~(-2)·s~(-1). The root-to-shoot ratio in the treatment with 120 µmol·m~(-2)·s~(-1) light intensity was up to 6.86, higher than those in other treatments(P<0.05),and the ginsenoside content in both aboveground and underground parts of P. quinquefolius in this treatment was the highest, which was possibly associated with the high expression of farnesylpyrophosphate synthase(FPS), squalene synthase(SQS), squalene epoxidase(SQE), oxidosqualene cyclase(OSC), dammarenediol-Ⅱ synthase(DS), and P450 genes in leaves and SQE and DS genes in main roots. In addition, light intensities of 120 and 160 µmol·m~(-2)·s~(-1) could promote PPD-type ginsenoside synthesis in leaves by triggering up-regulation of the expression of upstream ginsenoside synthesis genes. The decrease in underground biomass accumulation of the P. quinquefolius grown under weak light(40 µmol·m~(-2)·s~(-1)) and strong light(160 µmol·m~(-2)·s~(-1)) was possibly attributed to the low net photosynthetic rate, stomatal conductance, and transpiration rate in leaves. In the meantime, the low expression of SQS, SQE, OSC, and DS genes in the main roots might led to the decrease in ginsenoside content. However, there was no significant correlation between the ginsenoside content and the expression of synthesis-related genes in the fibrous roots of P. quinquefolius. Therefore, the light intensity of 80 and 120 µmol·m~(-2)·s~(-1) is beneficial to improving yield and quality of P. quinquefolius. The above findings contributed to a theoretical basis for reasonable shading in P. quinquefolius cultivation, which is of great significance for improving the yield and quality of P. quinquefolius through light regulation.

Reductive-Delay Heck 1,1-Diarylation of Terminal Alkenes.

Pd-catalyzed chemo- and regiocontrollable 1,1-diarylation of unactivated aliphatic alkenes with two aryl halides was developed. Under the cationic reductive-delay Heck pathway, the first aryl insertion is followed by ß-H elimination, while the second aryl insertion is terminated by C-H bond formation.

AmMYB24 Regulates Floral Terpenoid Biosynthesis Induced by Blue Light in Snapdragon Flowers.

Floral terpenoid volatiles are impacted by light quality. In snapdragon, blue light can significantly enhance the emissions of ocimene and myrcene and the expression of ocimene synthase (AmOCS) and myrcene synthase (AmMYS). However, the mechanisms underlying the response to blue light are largely unknown. In this study, two transcription factors (TFs), AmMYB24 and AmMYB63 were screened which showed high expression level under blue light. AmMYB24 exhibited synchronous expression with AmOCS. Moreover, AmOCS transcript expression was up-regulated in response to AmMYB24 overexpression. This activation is direct and occurs through binding of AmMYB24 to MYBCORECYCATB1 sites in the AmOCS promoter. In addition, AmMYB24 interacts with the blue light signal key receptor AmCRY1 and the transcriptional activation activity of AmMYB24 was decreased in AmCRY1 silencing flowers. Taken together, our results revealed the regulatory pathway of biosynthesis of ocimene induced by blue light mediated by AmMYB24 and AmCRY1. When snapdragon flowers were exposed to blue light, AmCRY1 was first activated, the light signal is transduced to AmMYB24 through interaction with AmCRY1, and finally AmMYB24 activates AmOCS by binding to its MYBCOREATCYCB1 motif, resulting in abundant ocimene emission.

Determination of Methyl Methanesulfonate and Ethyl Methylsulfonate in New Drug for the Treatment of Fatty Liver Using Derivatization Followed by High-Performance Liquid Chromatography with Ultraviolet Detection.

A new derivatization high-performance liquid chromatography method with ultraviolet detection was developed and validated for the quantitative analysis of methanesulfonate genotoxic impurities in an innovative drug for the treatment of non-alcoholic fatty liver disease. In this study, sodium dibenzyldithiocarbamate was used as a derivatization reagent for the first time to enhance the sensitivity of the analysis, and NaOH aqueous solution was chosen as a pH regulator to avoid the interference of the drug matrix. Several key experimental parameters of the derivatization reaction were investigated and optimized. In addition, specificity, linearity, precision, stability, and accuracy were validated. The determined results of the samples were consistent with those obtained from the derivatization gas chromatography-mass spectrometry analysis. Thus, the proposed method is a reliable and practical protocol for the determination of trace methanesulfonate genotoxic impurities in drugs containing mesylate groups.

First report of root rot caused by Fusarium armeniacum on American ginseng in China.

American ginseng (Panax quinquefolius) is an important medicinal plant cultivated in China since the 1980s. Its dried roots are used for food, health care products, and medicine in China (Yuan et al. 2010). Root rot caused by Fusarium spp. was a major disease, with 33 to 41% incidence surveyed in main production areas of Wendeng County (121.80 °E, 37.09 °N) in Shandong Province, China in 2016 to 2019. Symptoms included soft, water-soaked, dark brown to black lesions on the roots. Lesions progressed and the inner parts gradually disintegrated. One-year-old diseased roots were collected in September 2016. Symptomatic tissues were surface-sterilized in 75% ethanol for 30 s and 0.8% NaOCl for 3 min, rinsed in sterile water, plated on potato dextrose agar (PDA), and incubated at 25°C in darkness. Single colonies were then obtained and transferred to carnation leaf agar (CLA) (Burgess et al. 1993) for growth at 25°C with a 12-h photoperiod. Colonies cultured on PDA for 7 days were white to light pink, turning to apricot pigmentation in color. After 30 days on CLA, the colonies produced elongate, falcate macroconidia having 3 to 5 septa, with a long, tapering and curved apical cell, and having the size ranging from 31.1 to 45.6 µm long x 4 to 4.6 µm wide. Microconidia were zero to 1septate, ellipsoid to ovoid and varied in size from 9.5 to 16.8 µm long x 3 to 3.2 µm wide. Chlamydospores formed abundantly, in chains or clusters. This fungus was identified as F. armeniacum (Burgess et al. 1993). Identification was confirmed by sequencing three DNA regions including the internal spacer ribosomal DNA (ITS), elongation factor 1α and ß-tubulin genes (Lu et al. 2019). The three DNA regions (MN417271, MG457199, and MN427653) had 100% homology to the sequences of F. armeniacum (KJ737378, HM744664 and HQ141640) (Wang et al. 2015, Yli-Mattila et al. 2011). Pathogenicity tests were conducted on 1- to 2-year-old bare roots and 2-year-old whole plants. For root inoculation, 14 healthy roots were inoculated with two mycelial PDA plugs/root. After 3 to 10 days at 25°C, all the inoculated roots showed water-soaked and root rot symptoms while no lesions were observed in the control roots. For plant inoculation, eight seedlings planted in pots filled with sterilized soil were inoculated by pouring a conidial suspension of 1×105 conidia/ml at 30 ml/pot. Eight seedlings inoculated with sterilized water served as the controls. After 90 days, only 37.5% of the roots survived with typical root rot symptoms whereas the control plants remained symptomless. F. armeniacum was re-isolated from symptomatic roots but not from the control roots. Besides F. armeniacum, F. solani and F. oxysporum that have been reported to be associated with American ginseng root rot in China and Canada (Reeleder et al. 2002; Punja et al. 2008) were also obtained from the diseased root samples in this study. However, the development of root rot caused by F. armeniacum was much more rapid and its symptoms were more severe. Moreover, F. armeniacum could directly infect American ginseng with no wound requirement. F. armeniacum was previously reported on Glycine max (Leguminosae) (Ellis et al. 2012), Platycodon grandiflorus (Campanulaceae) (Wang et al. 2015) and natural grasses (Poaceae) (Nichea et al. 2015). This is the first report of F. armeniacum causing root rot on American ginseng in China. As this species is more virulent to American ginseng, more research is needed to work on this disease.

[Effects of nutrients deficiency on growth and saponin accumulation of American ginseng].

Mineral nutrient elements are the key factors to maintain the growth and quality of American ginseng. In order to understand the comprehensive effect of different nutrient elements deficiency on American ginseng, 2-year-old American ginsengs were cultivated by Hoagland solution(CK) or 10 different nutrients deficiency solution in sand culture. During the cultivation, the deficient symptom was observed. The plant height, leaf area, biomass, photosynthetic index, root activity, ginsenoside content were measured. The results showed that N, K or Fe deficiency could lead to leaves of American ginseng yellowing. Deficiency N, K, Ca, Mg and B were the main factors that decrease plant height and leaf area. The biomass of plant decreased significantly in all the nutrient deficient treatments(P<0.05)compared with control group, and N, K, Ca or Fe deficiency groups descended over 50%. In the absence of N, K and Fe elements, the P_n, G_s, C_i, T_r and chlorophyll of leaves were decreased mostly. The first three factors decreasing root activity were N, K and Ca deficiency. The effects of nutrient deficiency on saponins of American ginseng were different.Generally, N, P, B, Zn and Cu deficiency resulted the synthesis of saponins decreased significantly(P<0.05). This study contributed to clarify the demand characteristics of American ginseng for different nutrient elements,which is of great significance for the diagnose of nutrient deficiency, the rational fertilizer and the improvement of yield and quality of American ginseng.

PEG-chitosan-coated iron oxide nanoparticles with high saturated magnetization as carriers of 10-hydroxycamptothecin: preparation, characterization and cytotoxicity studies.

A magnetic nano-sized carrier for 10-hydroxycamptothecin (HCPT) was prepared by using Fe(3)O(4) nanoparticles as cores and chitosan (CS) as a polymeric shell by a novel reverse ultrasonic emulsification method. Poly(ethylene glycol) (PEG) chains were then coupled onto the magnetic particles (CS-Fe(3)O(4)) to improve their biocompatibility (PEG-CS-Fe(3)O(4)). HCPT was loaded onto PEG-CS-Fe(3)O(4) by a subtle precipitation method. Under optimum conditions, the CS-Fe(3)O(4) was close to spherical in shape with an average size of 174 nm and a high saturated magnetization. After coupling PEG chains, the unspecific adsorption of bovine serum albumin (BSA) on PEG-CS-Fe(3)O(4) decreased significantly. The drug loading content and loading efficiency were 9.8-11.8% and 49-59% for magnetic composite nanoparticles, respectively. HCPT-loaded magnetic composite nanoparticles showed sustained release profiles up to 48 h, and the cumulative release amount of HCPT from nanoparticles at 45°C increased significantly compared to that at 37°C. Cytotoxicity assay suggests that CS-Fe(3)O(4) does not exhibit noteworthy cytotoxicity against HepG2 cells, but the antitumor activities of HCPT-loaded magnetic composite nanoparticles against HepG2 cells increased significantly in comparison with that of pristine HCPT powder. These results reveal the promising potential of PEG-CS-Fe(3)O(4) as a stable magnetic targeting drug carrier in cancer therapy.