Formulation and sterilization assessment of kanamycin sulfate injection based on impurity profiles / 药学学报

The compatibility of kanamycin with sodium citrate for the formulation of kanamycin sulfate injection was determined, including optimization of the amount of sodium citrate in the injection and the sterilization process. An HPLC coupled with an evaporative light scattering detector (ELSD) was used to measure the amount of sodium citrate and the impurity profiles. A validated post-column derivatization HPLC coupled with a fluorescence detector (FLD) was used to determine the correlation between specific impurities in a domestic factory and sodium citrate, and then the formulation was evaluated by HPLC coupled with mass detector (MS) characterization of degradation products. The results show that the amount of sodium citrate in kanamycin sulfate injection from a domestic factory is about 40 times higher than that of the Meiji formulation. Several specific impurities can be detected in solutions heated under simulated sterilization conditions (121 ℃), which were correlated with the amount of sodium citrate. Impurities were characterized by HPLC-MS/MS, and data showed that the identified impurities were interaction products of kanamycin and sodium citrate. These results indicate that greater attention should be directed at formula optimization in domestic factories, as it is crucial to the safety and efficacy of the preparations. Drug-excipient chemical compatibility should also be evaluated in the development of pharmaceutical dosages forms especially when the active pharmaceutical ingredients have a primary amine group.

Construction of rat beta defensin-2 eukaryotic expression vector and expression in the transfected rat corneal epithelial cell / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2), transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 <i>in vitro </i>and <i>in vivo</i> and to assess the probability of defensins as a new application for infectious corneal diseases in the future. <p>METHODS: The synthetic rBD-2 DNA fragment was inserted between the <i>Xho</i>I and <i>BamHI</i> restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into <i>E.coli DH5α</i>, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. <p>RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. <p>CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in the transfected cells.

High-throughput determination of preservatives in eye drops and antimicrobial effectiveness assessment / 中国药学杂志

OBJECTIVE: To establish an HPLC method for rapid screening and simultaneous determination of 10 preservatives in eye drops and assess their antimicrobial effectiveness. METHODS: A Waters XBridge C18 column (4.6 mm × 150 mm, 5 μm) was used, and the mobile phase was 0.1% phosphoric acid-methanol-THF eluted in gradient mode at a flow rate of 1.0 mL·min-1. The detection wavelength was set at 214 nm. The antimicrobial effectiveness of the preservatives was assessed according to Ch. P 2015. RESULTS: The calibration curves were linear in the range of the corresponding test concentrations (r=0.999 3 -1.000 0). The recoveries were between 96.1%-101.8%. One of the eye drops products did not meet the requirement of Ch. P 2015. CONCLUSION: The established method is rapid and inexpensive. And it ensures excellent simplicity, sensitivity, specificity, and reproducibility and can be used for rapid screening and determination of the contents of preservatives in eye drops. The amount of preservatives should be established during the R&D period or determined according to the results of antimicrobial effectiveness test rather than using empirical values.

The damage effects of hypothermia combined with hypoxia on rat pulmonary microvascular endothelial cells / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To study the synergistic effects of hypothermia and hypoxia on the damage of pulmonary microvascular endothelial cells (PMVEC) in rat.</p><p><b>METHODS</b>Primary PMVECs were obtained by complex phosphoesterasum digesting from isolated lung tissues of Wistar rats, the PMVECs were identified by phase contrast microscope and immunofluorescence studies for CD31 antigen and bandeiraea simplicifolia isolectin (BSI) binding test. Factorial design was adopted in trial according to hypothermia and hypoxia existing or not. Using corresponding kit measured the levels of lactate dehydrogenase (LDH) activity in cell medium. Level of nitric oxide (NO) concentration was measured by Griess Assay. RT-PCR was used to examine the expression of vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) mRNA in PMVECs.</p><p><b>RESULTS</b>The monolayer of cultured PMVECs displayed the shape of pavingstone. CD31 antigen and binding BSI results by fluorescence microscope identified the cultured cells were PMVECs. Compared to the control group, LDH activity and VEGF, ET-1 expression levels were significantly increased in hypothermia group, hypoxia group and hypoxia combined with hypothermia group. And the levels of NO concentration were reduced in these three groups. The results of One-way ANOVA showed that there was a synergistic effect between hypothermia and hypoxia.</p><p><b>CONCLUSION</b>Hypothermia and hypoxia both have an effect on PMVECs whether in altering the cell permeability or in releasing of vasoactive substances including NO and ET-1. In addition, there is a synergistic effect between hypothermia and hypoxia.</p>

Effects of low temperature exposure on dermal microvascular endothelial cells function / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To explore the damage effects and expression of vascular endothelial growth factor (VEGF) exposed with different low-temperatures on rat dermal microvascular endothelial cells (DMVECs).</p><p><b>METHODS</b>Primary DMVECs were obtained by discontinuous Percoll gradient centrifugation. The DMVECs were identified by phase contrast microscope and immunofluorescence studies for CD31 antigen. Applied 28 degrees C, 12 degrees C and 0 degrees C to interfere with rat DMVECs as cold-exposure model. The changes of cells morphology were observed under invert microscope. The membrane integrity was determined by lactate dehydrogenase (LDH) activity. RT-PCR was used to examine the expression of vascular endothelial growth factor mRNA in cells.</p><p><b>RESULTS</b>The monolayer of cultured PMVECs displayed the shape of pavingstone. CD31 antigen and binding BSI results by fluorescence microscope identified the cultured cells were DMVECs. After 24 h cold exposure, the cell morphology of 0 degrees C group was shrunken, the other groups were "Fibroblast-like". The LDH activity (U/L) in the medium of 28 degrees C, 12 degrees C and 0 degrees C groups was 54.17 +/- 3.02, 64.66 +/- 3.03, 82.13 +/- 10.91 respectively, which was significantly higher than that of 37 degrees C group (12.23 +/- 3.0, P < 0.01). The VEGF mRNA expression level was up-regulated in 28 degrees C group and 12 degrees C group versus control group (P < 0.05), but unchanged in 0 degrees C group.</p><p><b>CONCLUSION</b>The rat DMVECs injury severity are deteriorated with temperature decreasing, and VEGF might be involved in the regulation of membrane permeability in this period.</p>

Protective effects of SUR2B/Kir6.1 potassium channel opener natakalim against RAVECs injuries induced by hypoxia / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the protective effects of natakalim against rat aortic vascular endothelial cells (RAVECs) injuries induced by hypoxia and its mechanisms.</p><p><b>METHODS</b>Selecting RAVECs as a cell model injured by hypoxia, these RAVECs were divided into 5 groups: i.e. control group, hypoxia group, natakalim low, medium and high group. The cell survival rate was determined by MTT assay, con was measured using Griess Assay, RT-PCR was used to examine t he expression of intercellular adhesion molecule-1 (ICAM-1), vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) mRNA in RAVEC.</p><p><b>RESULTS</b>Natakalim could reverse hypoxia-induced changes in endothelial cell function, including increased endothelial cell survival rate and level of NO concentration, significantly inhibited the hypoxia-induced endothelial ICAM-1, ET-1, VEGF mRNA expression levels increased.</p><p><b>CONCLUSION</b>Natakalim have protective effects on hypoxia-induced changes in endothelial cell function, increasing of permeation, excess expression of cell adhesion molecules.</p>

Changes of protein kinase A expressions in central amygdaloid nuclei during the process of chronic morphine-induced conditioned place aversion in rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To explore neurobiological mechanisms of the withdrawal-induced aversion. The changes of protein kinase A were measured in central amygdaloid nucleic (CeA) of conditioned place aversion (CPA) model rats.</p><p><b>METHODS</b>(1) All 72 male SD rats were divided into three groups, model group (MN group), and control group (MS group and SN group). MN group was injected with morphine,6.5 days, 10 mg/kg, intraperitoneally (ip), twice per day, naloxone injection, 0.3 mg/kg, ip, along with conditioned place aversion training, to develop the CPA model. The MS group was administrated equivalent volume of morphine and saline. Also the SN group was injected with equivalent volume of saline and naloxone. (2) During the process of morphine-induced CPA, the expression of protein kinase A was assayed with immunohistochemistry in the CeA.</p><p><b>RESULTS</b>In the MN group, protein kinase A expressions in the CeA occurred adaptive changes at different points of CPA (P < 0.05). Protein kinase A expressions after establishment(Day7,134.43 +/- 4.481, P < 0.05), and after extinction (Day 13, 141.01 +/- 3.360, P < 0.01), and after reinstatement (Day 14,137.18 +/- 40.330, P < 0.05) were also lower than those before the establishment of the CPA (Day 5, 124.48 +/- 6.722). However, PKA expressions were not significantly different both in MS group (P > 0.05)and SN group (P > 0.05).</p><p><b>CONCLUSION</b>(1) Protein kinase A expression, in turn regulating the aversion expression, in the CeA probably is a key pathway contributing to the development of CPA. (2) The neuroadaptation mediated by protein kinase A may be one of the important molecular underpinnings of CPA.</p>

Relationship between the expression of uncoupling protein 2 and the damage by oxygen free radicals in acute liver failure rats / 中华肝脏病杂志

To investigate the relationship between uncoupling protein 2 (UCP2) expression and the damage caused by oxygen free radicals in acute liver failure rat models. Thirty-five male Sprague-Dawley rats were randomly divided into two groups: the control group (15 rats) and liver failure group (20 rats). The rats were injected intraperitoneally with thioacetamide (TAA) to induce models of acute liver failure. The levels of endotoxin (ET) were detected by double antibody sandwich enzyme-linked immunosorbent assay. The expression of liver UCP2 mRNA was detected by reverse transcription polymerase chain reaction. The superoxide dismutase (SOD) and malonaldehyde (MDA) were detected by spectrophotometry. The expression of UCP2 protein was observed by immunohistochemistry. The data of the two groups were compared using Mann-Whitney U test or ANOVA. The expression of UCP2 mRNA in liver failure group was higher as compared to the control group (P value is less than 0.01); the level of MDA and endotoxin of liver failure group were higher than that of the control group (P value is less than 0.01). SOD of the liver failure group was lower (P value is less than 0.01). There was a certain correlation between UCP2 mRNA expression and ET, SOD and MDA (r = 0.952, -0.667, 0. 634 respectively, P value is less than 0.05 or 0.01). UCP2 is highly expressed in the livers of liver failure rats. A certain correlation perhaps existed between the expression of UCP2 mRNA and the serous SOD, MDA and ET.