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Objective:To investigate the inhibitory effect and killing mechanism of Bcl-2 BH4 selective inhibitor BDA-366 on NK/T cell lymphoma (NK/TCL) .Methods:Human NK cell leukemia cell line YT and human NK/TCL cell line NK92 cells were treated with 0, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50 μmol/L BDA-366. CCK-8 assay was used to calculate the half inhibitory concentration (IC 50) value of BDA-366 on these cells. The apoptosis levels of cells in control group and IC 50 BDA-366 treated group were detected by flow cytometry. Western blotting was used to detect the expression levels of apoptosis-related proteins in cells of control group and 1/2 IC 50, IC 50, 2× IC 50 BDA-366 treated groups. TMRE and Fluo-3 fluorescent probe were used to detect mitochondrial membrane potential of control group and IC 50 BDA-366 treated group, and the intracellular Ca 2+ concentration of control group, IC 50, 2× IC 50 BDA-366 treated groups. NOD-SCID mice in control group and 10 mg/kg BDA-366 intraperitoneal injection group were weighed and HE staining was performed to evaluate the toxicity of BDA-366 in vivo. Results:The IC 50 of BDA-366 for YT and NK92 cells were 0.065 and 0.086 μmol/L respectively. The apoptosis rates of YT cells in the control group and 0.065 μmol/L BDA-366 group were (6.62±1.59) % and (34.60±3.06) % respectively. The apoptosis rates of NK92 cells in the control group and 0.086 μmol/L BDA-366 group were (5.57±0.88) % and (29.18±0.90) % respectively, both with statistically significant differences ( t=14.05, P<0.001; t=32.58, P<0.001). The relative expression of Bax in NK92 cells of the control group, 0.043, 0.086 and 0.172 μmol/L BDA-366 groups were 0.85±0.00, 1.26±0.04, 1.51±0.18, 1.15±0.10 ( F=20.70, P<0.001), the relative expression of Bax in BDA-366 groups were higher than that in the control group (all P<0.05). The fluorescence intensity of TMRE of YT cells in the control group and 0.065 μmol/L BDA-366 group were 8 372.00±330.47 and 6 419.67±311.34, and that of NK92 cells in the control group and 0.086 μmol/L BDA-366 group were 9 169.00±535.72 and 7 311.67±295.52 respectively, and there were statistically significant differences ( t=7.45, P=0.002; t=5.26, P=0.006). In YT cells, the intracellular Ca 2+ concentrations of 0.065 and 0.130 μmol/L BDA-366 groups were significantly higher than that of the control group (5 791.67±220.45, 6 729.33±585.39, 4 874.67±112.61, F=19.16, P=0.003) ( P=0.039; P=0.002). In NK92 cells, the intracellular Ca 2+ concentrations of 0.086 and 0.172 μmol/L BDA-366 groups were significantly higher than that of the control group (4 553.67±17.62, 4 740.33±254.50, 4 185.67±17.67, F=10.96, P=0.010) ( P=0.039; P=0.007). There was no statistically significant difference in body weight change on day 12 compared with day 0 of NOD-SCID mice between BDA-366 group and control group [ (3.18±0.01) g vs. (2.73±0.58) g, t=0.60, P=0.570], and HE staining showed no abnormal morphology of heart, liver, spleen, lung and kidney in BDA-366 group. Conclusion:BDA-366 promotes NK/TCL cells apoptosis in vitro, but does not cause weight loss and morphological changes of organs by HE staining in vivo. The inhibitory effect of BDA-366 on NK/TCL cells may be achieved by increasing Bax expression, inducing Ca 2+ release and reducing mitochondrial membrane potential.
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@#An HPLC pre-column derivatization detection method was established to detect and analyze the formaldehyde and acetaldehyde in polysorbate 80 and polysorbate 20 from different manufacturers.The effects of aldehyde and acetaldehyde on the aggregation of adalimumab under different conditions were monitored.Based on the control of genotoxic impurities and the influence on the stability of monoclonal antibody preparations, the control limits of the two chemicals were preliminarily obtained.2, 4-dinitrophenylhydrazine (2, 4-DNPH) was applied as the derivatization reagent in HPLC pre-column derivatization; acetonitrile and water were used as mobile phase to perform a gradient elution on a C8 (4.6 mm × 150 mm, 5 μm) chromatographic column.The detection wavelength was 360 nm, and the external standard method was used for quantification.Verification results showed that the method was suitable for the quantitative analysis of trace formaldehyde and acetaldehyde in polysorbate 80 and polysorbate 20 . The detection and analysis of formaldehyde or acetaldehyde in different batches of polysorbate 80 and polysorbate 20 from different manufacturers showed that the content of formaldehyde and acetaldehyde were quite different. The content of formaldehyde and acetaldehyde in polysorbate 80 were significantly higher than those of polysorbate 20. After monitoring the changes of adalimumab aggregates treated by formaldehyde and acetaldehyde by size exclusion chromatography (SEC), it was found that the effect of formaldehyde on adalimumab aggregation was significantly higher than that of acetaldehyde.According to the requirements of ICH M7 (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, M7: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk), the impurity limits of formaldehyde and acetaldehyde in polysorbate 80 and polysorbate 20 for monoclonal antibody preparations were calculated from the perspective of risk assessment.Combined with the influence on the aggregation stability of monoclonal antibodies, the preliminary limis for acetaldehyde and acetaldehyde were recommended to be ≤ 7 μg/g and ≤ 765 μg/g, respectively.
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Objective To investigate the clinical features of polymyositis and dermatomyositis ( PM/DM) with lym-phoma .Methods The clinical data , treatment and prognosis of PM/DM with lymphoma from 2000 to 2015 in Pe-king Union Medical College Hospital were retrospectively collected and analyzed .Results Ten cases of PM/DM patients complicated with lymphoma were recruited .6 were female and 4 was male.The mean ages of the diagnosis of PM/DM and lymphoma were 44.5 and 44.9 years old respectively .The average disease course from PM/DM to lymphoma was 4.7months.Six cases were dermatomyositis and 4 cases were polymyositis.Three cases had pulmo-nary fibrosis and 2 cases had arthritis .No case had a positive result of anti-Jo-1 antibody .The most frequent symp-toms complicated with lymphoma included as following:enlargement of lymph nodes (9 cases), fever (8 cases), splenomegaly(5 cases).The top 3 lymphoma biopsy specimen included lymph node (4 cases), skin(2 cases)and local mass(2 cases).All the 10 patients were diagnosed as non-Hodgkin lymphoma.4 cases were derived from T cell, 2 cases from B cell, and 4 cases with unknown origin .The clinical staging showed III B (4 cases) and IV B (6 cases).Immunosuppressant agents were given to treat PM/DM.During the follow-up period, 5 patients died, 4 patients received chemotherapy and 1 patient was lost .Conclusions The coincidence of PM/DM and lymphoma is not rare .The course between PM/DM and lymphoma is short , and the mortality is significant , which should arise the attention of clinicians .
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To explore the establishment of a scientific and innovative working mode of the national drug reference standards. Methods:Project management was adopted. Results and Conclusion: The project management mode can effectively ar-range the work of the national drug reference standards including raw material collection, research, review, subpackage, package and so on, which exhibits guiding significance for the development direction and improving products supplement.
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Objective:To investigate the growth-promoting properties and applicability of the mycoplasma test culture media pre-scribed in Chinese pharmacopoeia, and provide reference for the standardization of the drug mycoplasma test method. Methods: Re-spectively using four quantitative detection methods including sensitivity, degree of color change, colony counts and colony diameter, and mycoplasma media widely used in the world as the reference media, the growth-promoting properties of 4 batches of mycoplasma broth and 4 batches of arginine mycoplasma broth from four domestic manufacturers were investigated. Results:The results of sensitivity assay and absorbance detection showed that all the media inoculated with below 100 CFU test microorganisms exhibited visible color change. Furthermore, the results of color change degree and colony diameter showed that there were significant differences among the media products from different manufacturers(P<0. 01). Conclusion:Mycoplasma broth and arginine mycoplasma broth both can sup-port the growth of below 100 CFU test microorganisms. Due to the difference in the growth-promoting properties among the media prod-ucts from different manufacturers, the drug mycoplasma test workers should use more sensitive methods to examine the applicability of the media.
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Microbiological data deviations ( MDD) investigation differs from out of the standard ( OOS) investigation in chemical inspection items. Investigating Out-of-Specification( OOS) Test Results for Pharmaceutical Production issued by FDA specifically ex-cludes microbiological and other biological assays. Until recently, there is not a definitive guideline for MDD investigation in the world. Some institutions and experts have explored the practice and theory of MDD investigation. Referred to the existing practical experience and theoretical achievement and based on the provisions of Chinese GMP (2010 revision) and the requirements of Chinese Pharmaco-poeia Ⅱ(2010 edition) Appendix XIX Q, the article discussed the methods for MDD laboratory investigation.
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Recently, there are many prominent problems about the quality ofIndigo Naturalis. To explore the underlying reasons, both ancient and modern herbal literatures were reviewed. Through the organization of recordings onIndigo Naturalis, the traditional process method required soaking and fermentation, stiring and depositing, foaming and shade drying. The currently usedIndigo Naturalis was mostly isolated and purified from crude deposit -“Cu-Dian.” The modern process technique ofIndigo Naturalis was gradually deviated from the traditional processing method. This paper also discussed the quality standards ofIndigo Naturalis. The author considered that the current standard on descriptions ofIndigo Naturalis characteristics was deviated from the traditional morphological features ofIndigo Naturalis, which should be further researched and verified. Therefore, this study provided certain references for the quality control and market supervision ofIndigo Naturalis.
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Objective:To provide the basic data for the further revision of mycoplasma test method described in Chinese Pharma-copoeia and some references for the operation standardization of drug mycoplasma test. Methods:Two incubation conditions,namely aerobic conditions and microaerophilic conditions,were compared with respect to the growth status of mycoplasma in liquid and solid media. Results:The growth of mycoplasma was obvious difference between the two incubation conditions,and the microaerophilic con-ditions were better than the aerobic conditions. Conclusion:The microaerophilic conditions can be used in the incubation of mycoplas-ma test,which should be defined and standardized in the future Chinese Pharmacopoeia.
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Recently,thesituationofchemicalsubstancesillegallyaddedinTCMandhealthproductsismoreandmorecomplex and subtle, which not only results in latent danger and harm to customers, but also induce supervision difficulty and challenge for su-pervision departments. The variety and composition of chemical substances added illegally, analytical methods and relative state laws and regulations were illustrated in detail in the paper, which can provide the basis for the rational and safe use of TCM and health prod-ucts, and provide the reference and technology support for the supervision departments.
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<p><b>OBJECTIVE</b>To observe the effect of Euphorbia kansui (E. KS) alcohol extracts on urination and kidney-related expressions of mice injected with normal saline and to discuss its impact on kidney.</p><p><b>METHOD</b>Mice intraperitoneally injected with normal saline were observed for urination and changes in kidney-related histiocytic factors of after intragastrical administration of E. KS and compared with normal mice.</p><p><b>RESULT</b>E. KS alcohol extracts can promote urination of mice injected with normal saline and enhance peripheral serum creatinine, with no obvious pathological change showed in tissue sections. It had a certain effect on reducing AQP2 expression and enhancing TNF-alpha expression.</p><p><b>CONCLUSION</b>Euphorbia kansui in large dose has a remarkable effect on kidney but may be accompanied with pathological reactions to some extent, especially the dose of 1.2 g x kg(-1). The pathological reactions may be related with increased serum creatinine and TNF-alpha expression.</p>