Validated spectrofluorimetric and spectrophotometric methods for the determination of brimonidine tartrate in ophthalmic solutions via derivatization with NBD‐Cl. Application to stability study

Two simple, selective and accurate methods were developed and validated for the determination of brimonidine tartrate (BT) in pure state and pharmaceutical formulations. Both methods are based on the coupling of the drug with 4‐chloro‐7‐nitro‐2,1,3‐benzoxadiazole in borate buffer (pH 8.5) at 70 °C and measurement of the reaction product spectrophotometrically at 407 nm (method I) or spectrofluorimetrically at 528 nm upon excitation at 460 nm (method II). The calibration graphs were rectilinear over the concentration ranges of 1.0–16.0 and 0.1–4.0 µg/mL with lower detection limits of 0.21 and 0.03, and lower quantification limits of 0.65 and 0.09 µg/mL for methods I and II, respectively. Both methods were successfully applied to the analysis of commercial ophthalmic solution with mean recovery of 99.50 ± 1.00 and 100.13 ± 0.71%, respectively. Statistical analysis of the results obtained by the proposed methods revealed good agreement with those obtained using a comparison method. The proposed spectrofluorimetric method was extended to a stability study of BT under different ICH‐outlined conditions such as alkaline, acidic, oxidative and photolytic degradation. Furthermore, the kinetics of oxidative degradation of the drug was investigated and the apparent first‐order reaction rate constants, half‐life times and Arrhenius equation were estimated. The proposed methods are practical and valuable for routine applications in quality control laboratories for the analysis of BT. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of solution conditions on virus retention by the Viresolve® NFP filter

Virus filtration can provide a robust method for removal of adventitious parvoviruses in the production of biotherapeutics. Although virus filtration is typically thought to function by a purely size‐based removal mechanism, there is limited data in the literature indicating that virus retention is a function of solution conditions. The objective of this work was to examine the effect of solution pH and ionic strength on virus retention by the Viresolve^® NFP membrane. Data were obtained using the bacteriophage ?X174 as a model virus, with retention data complemented by the use of confocal microscopy to directly visualize capture of fluorescently labeled ?X174 within the filter. Virus retention was greatest at low pH and low ionic strength, conditions under which there was an attractive electrostatic interaction between the negatively charged membrane and the positively charged phage. In addition, the transient increase in virus transmission seen in response to a pressure disruption at pH 7.8 and 10 was completely absent at pH 4.9, suggesting that the trapped virus are unable to overcome the electrostatic attraction and diffuse out of the pores when the pressure is released. Further confirmation of this physical picture was provided by confocal microscopy. Images obtained at pH 10 showed the migration of previously captured phage; this phenomenon was absent at pH 4.9. These results provide important new insights into the factors governing virus retention using virus filtration membranes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1280–1286, 2015     

A Potent Anticancer Agent of Shikonin Derivative Targeting Tubulin

In this study, a shikonin ester derivative, compound 3g , was selected to evaluate its anticancer activities and we found that compound 3g exhibited better antitubulin activities against the human HepG2 cell line with an IC₅₀ value of 1.097 μM. Furthermore, the inhibition of tubulin polymerization results indicated that compound 3g demonstrated the most potent antitubulin activity (IC₅₀ = 13.88), which was compared with shikonin and colchicine as positive controls (IC₅₀ = 25.28 μM and 22.56 μM), respectively. Compound 3g was simulated to have good binding site with tubulin and arrested the cell cycle at G2/M phase, which also induces apoptosis in HepG2 cells, in which P53 and members of Bcl‐2 protein family were both involved in the progress of apoptosis revealed by western blot. Confocal microscopy observations revealed compound 3g targeted tubulin and altered its polymerization by interfering with microtubule organization. Based on these results, compound 3g functions as a potent anticancer agent targeting tubulin. Chirality 27:274–280, 2015.. © 2015 Wiley Periodicals, Inc.     

KNOCKDOWN OF THE IONOTROPIC γ‐AMINOBUTYRIC ACID RECEPTOR (GABAR) RDL GENE DECREASES FIPRONIL SUSCEPTIBILITY OF THE SMALL BROWN PLANTHOPPER,Laodelphax striatellus (HEMIPTERA: DELPHACIDAE)

Insect γ‐aminobutyric acid receptors (GABARs) are important molecular targets of cyclodiene and phenylpyrazole insecticides. Previously GABARs encoding rdl (resistant to dieldrin) genes responsible for dieldrin and fipronil resistance were identified in various economically important insect pests. In this study, we cloned the open reading frame cDNA sequence of rdl gene from fipronil‐susceptible and fipronil‐resistant strains of Laodelphax striatellus (Lsrdl). Sequence analysis confirmed the presence of a previously identified resistance‐conferring mutation. Different alternative splicing variants of Lsrdl were noted. Injection of dsLsrdl reduced the mRNA abundance of Lsrdl by 27–82%, and greatly decreased fipronil‐induced mortality of individuals from both susceptible and resistant strains. These data indicate that Lsrdl encodes a functional RDL subunit that mediates susceptibility to fipronil. Additionally, temporal and spatial expression analysis showed that Lsrdl was expressed at higher levels in eggs, fifth‐instar nymphs, and female adults than in third‐instar and fourth‐instar nymphs. Lsrdl was predominantly expressed in the heads of 2‐day‐old female adults. All these results provide useful background knowledge for better understanding of fipronil resistance related ionotropic GABA receptor rdl gene expressed variants and potential functional differences in insects.     

Geometric morphometric analysis of the effect of temperature on wing size and shape in Aedes albopictus

Wing geometry helps to identify mosquito species, even cryptic ones. On the other hand, temperature has a well‐known effect on insect metric properties. Can such effects blur the taxonomic signal embedded in the wing? Two strains of Aedes albopictus (laboratory and field strain) were examined under three different rearing temperatures (26, 30 and 33 °C) using landmark‐ and outline‐based morphometric approaches. The wings of each experimental line were compared with Aedes aegypti. Both approaches indicated similar associations between wing size and temperature. For the laboratory strain, the wing size significantly decreased as the temperature increased. For the field strain, the largest wings were observed at the intermediate temperature. The two morphometric approaches describing shape showed different sensibilities to temperature. For both strains and sexes, the landmark‐based approach disclosed significant wing shape changes with temperature changes. The outline‐based approach showed lesser effects, detecting significant changes only in laboratory females and in field males. Despite the size and shape changes induced by temperature, the two strains of Ae. albopictus were always distinguished from Ae. aegypti. The present study confirms the lability of size. However, it also suggests that, despite environmentally‐induced variation, the architecture of the wing still provides a strong taxonomic signal.