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• 2017 Volume 48 Issue 07
  Published: 10 July 2017

    

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  Biosynthesis of Squalene by Introducing Hybrid MVA Pathway in Escherichia coli

  LIU Haiyuan, ZHANG Wei, GONG Guihua, XIE Liping, HU Youjia*

  2017, 48(07): 982. https://doi.org/10.16522/j.cnki.cjph.2017.07.004

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  Squalene is a polyunsaturated triterpene hydrocarbon (C30H50) with six double bonds produced by mevalonate (MVA) pathway or methylerythritol 4-phosphate (MEP) pathway in several organisms and has a variety of pharmacological activities. Metabolic engineering microorganism is an effective method for squalene production. One of the strategies is the introduction of heterologous MVA pathway genes into Escherichia coli and it could significantly
  improve the productivity of terpenoids. To increase the yield of squalene in E. coli, we introduced hybrid MVA pathway including upper pathway from Enterococcus faecalis and lower pathway from Saccharomyces cerevisiae into BL21(DE3). Resulted strain YSS17 can produce 0.73 mg/L squalene, 4.87-fold higher compared to the control strain YSS2 (0.15 mg/L). To further increase the production of squalene, the alanine110 of the mvaS gene was mutated to glycine so that squalene production of strain YSS18 was up to 1.0 mg/L. By substitution of T7 promoter of MVA lower pathway with trc promoter, the yield of squalene reached around 0.78 mg/L, a 22% decrease compared with YSS18. Finally, MVA upper pathway genes were moved to the low-copy-number plasmid pACY2 and engineering strain YSS20 could produce squalene up to 1.68 mg/L, which was approximately 11.2-fold higher than strain YSS2.
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  Synthesis of Oxcarbazepine via Dibromination

  DIAO Wenrui, WANG Yingli*

  2017, 48(07): 991. https://doi.org/10.16522/j.cnki.cjph.2017.07.005

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  A new synthetic route for oxcarbazepine(1) was reported. 10,11-Dihydro-5H-dibenzo[b,f]azepine(2)reacted with bis(trichloromethyl)carbonate in chlorobenzene to give 10,11-dihydro-5H-dibenzo[b,f]azepine-5-carbonyl chloride(3) with a yield of 81％ . Then compound 3 was subjected to dibromination with dibromohydantoin and benzoyl peroxide in chlorobenzene, and then dehydrobromination at 140 ℃ to afford 10-bromo-5H-dibenzo[b,f]azepine-5-
  carbonyl chloride(5), which was followed by ammonification with ammonia and hydrolysis with diluted hydrochloric acid to prepare the target compound with a purity of 99.6％ and a total yield of 39％ (based on compound 2). The synthetic method of 5 from 3 has not yet been reported in literatures.
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  Process Optimization of Chidamide

  ZHAO Jianhong, HU Hui, GUAN Yu, LIAO Fan, YU Xinhong*

  2017, 48(07): 994. https://doi.org/10.16522/j.cnki.cjph.2017.07.006

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  4-[N-[(E)-3-(3-Pyridin)acroloyl]amidomethyl]benzoic acid(5) was synthesized via a phase transfer catalysis reaction of 4-amidomethyl benzoate sodium and (E)-3-(3-pyridin)acroloyl imidazole(3) which was prepared from (E)-3-(3-pyridin)acrylic acid in a mixed solvent of dichloromethane and water, the decomposition of 3 could be avoided and the yield was increased from 62％ to 80％ . Then 5 reacted with 3,4-diaminofluorobenzene in the presence of dicyclohexylcarbodiimide and 1-hydroxybenzotriazole followed by recrystallization from ethanol to afford chidamide with an overall yield of 61.5％ and a purity of 99.5％ .
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  Improved Synthetic Process of Antiviral Drug Letermovir

  ZHANG Dajun1, ZHAI Xin2, JING Tongfei2, ZOU Bin2, WANG Chen1

  2017, 48(07): 997. https://doi.org/10.16522/j.cnki.cjph.2017.07.007

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  2-Methoxy-5-(trifluoromethyl)aniline (2) reacted with bis(trichloromethyl)carbonate to give 2-methoxy-5-(trifluoromethyl)phenyl isocyanate, which reacted with 2-bromo-6-fluoroaniline to afford N-(2-bromo-6-fluorophenyl)-N'-[2-methoxy-5-(trifluoromethyl)phenyl]urea(5). Then methyl [8-fluoro-3-[2-methoxy-5-
  (trifluoromethyl)phenyl]-2-oxo-1,2,3,4-tetrahydroquinazolin-4-yl]acetate(7) was synthesized from methyl acrylate and 5 via an “one pot” method of Heck coupling and Michael addition. In this step, dioxane and N,N-diisopropylethylamine were used instead of isobutyronitrile and triethylamine so that the reaction time was shortened from 16 h to 9 h, and the yield was increased from 68.5％ to 79.4％. Then compound 7 was chlorinated with phosphoryl chloride to prepare methyl [2-chloro-8-fluoro-3-[2-methoxy-5-(trifluoromethyl)phenyl]-3,4-dihydroquinazolin-4-yl]acetate(8). Meanwhile, 1-(3-methoxyphenyl)piperazine(9) was prepared from diethanol amine via chlorination and N-alkylation. Finally, the antiviral drug letermovir(1) was obtained via condensation of 8 and 9, followed by the chiral separation and hydrolysis with an overall yield of 12.7％ (based on 2) and a purity of 99.7％ .
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  Mutation Breeding and Optimization of Fermentation Medium of Lipstatin Producing Strain

  LIU Shunna, SHEN Xiaofang, YANG Songbai, CHEN Shaoxin*

  2017, 48(07): 1006. https://doi.org/10.16522/j.cnki.cjph.2017.07.009

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  The Streptomyces toxytricini strain LIPS-352, a producer of lipstatin of 4 125 mg/L, was mutated with UV and nitrosoguanidine. A mutant LIPS-N-27-46-42 was obtained, which yielded 8 122.9 mg/L lipstatin in shake flask cultures. The results of one-factor-at-a-time method showed that carbon sources (glycerol) and nitrogen sources (soybean meal and corn gluten) had a significant effect on the production of lipstatin. Meanwhile, it was found that sunflower oil was better than soybean oil in providing precursor and carbon source for lipstatin synthesis. Then the fermentation medium was further optimized by a Box-Behnken design with response surface methodology. The optimal medium was: glycerol 2.5％, soybean meal 3.35％, corn gluten 1.01％, and sunflower oil 7.56％. The yield of lipstatin was 9 859.8 mg/L, which was 21.4％ higher than the original fermentation condition and had a relative error of 7.27％ to the predicted value(10 633.1 mg/L). Finally, a 139.0％ increase was achieved compared to that of the original strain through mutation breeding and optimization of fermentation medium of lipstatin producing strain.
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  In vitro Evaluation of Sinapultide Liposomal Aerosols

  QIU Ruiqi, LU Weigen*

  2017, 48(07): 1017. https://doi.org/10.16522/j.cnki.cjph.2017.07.011

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  The sinapultide liposomes were prepared and then lyophilized. The average particle size, ζ potential and encapsulation efficiency of the original liposomes were 556.5 nm, -56.8 mV and 83.8％, respectively; while the above indexes of the reconstituted lyophilized liposomes were 651.2 nm, -41.6 mV and 70.4％, respectively. The average residual water of the lyophilized liposomes was 1.48％. The residual amounts of trichloromethane and methanol were below their corresponding limits in the 2015 edition of Chinese pharmacopoeia. After reconstitution by adding normal saline, the aerosols were obtained by nebulizing the sinapultide liposomes with Pari nebulizer. The deposition properties of the aerosols in vitro were determined by twin-stage impinger, next generation impactor (NGI) and time-of-flight (TOF), and the results had no significant differences. The drug amounts in the aerosols with the aerodynamic particle size
  smaller than 6.4 μm were 50.86％ measured by twin-stage impinger, and 43.87％ measured by NGI. The values of the mass median aerodynamic diameter (MMAD) were 3.09 μm measured by NGI, and 2.81 μm measured by TOF. All the results of the in vitro evaluation suggested that the nebulized sinapultide liposomal aerosol enjoyed effective pulmonary deposition and provided a promising future of development and application.
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  Investigation on Transdermal Translocation of Nanoemulsions Based on Integral Identification

  SU Rui1,2, FAN Wufa2, LI Ye3, WU Wei2, LU Yi2*

  2017, 48(07): 1022. https://doi.org/10.16522/j.cnki.cjph.2017.07.012

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  Nanoemulsions have shown excellent effects on enhancing transdermal absorption of drugs. However, whether the integral nanoemulsions can enter into the skin or not is not well understood. In this study, integral identification of nanoemulsions was achieved through labelling by an aggregation-caused quenching (ACQ) fluorescence probe (P4). The probe P4 emitted near infrared fluorescence when loaded inside nanoemulsions. However, when the
  probe was released from nanoemulsions, the fluorescence quenched immediately upon due to degradation of the carrier.Thus the fluorescence signal could be used to track the integral nanoemulsions. In addition, coumarin 6 (C6) was simultaneously entrapped into the nanoemulsions to mimic the cargos. The transdermal translocation of nanoemulsions through intact rat skin as well as rat skin treated with microneedles was thus studied. Nanoemulsions with particle size of 80 nm and 500 nm were prepared according to the optimization results of single factor experiments. At 4 h post administration on intact skin, both two nanoemulsions could not integrally and efficiently permeate into the skin. But the smaller nanoemulsions accumulated in the hair follicles. The released C6 from the nanoemulsions diffused into the surrounding dermal tissues. Microneedles significantly enhanced the transdermal translocation of the nanoemulsions. The smaller nanoemulsions entered into the deeper sites of the skin than the bigger ones. Compared with the integral nanoemulsions, the released C6 diffused into the deeper sites of the skin.
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  Preparation and in vitro Evaluation of Docetaxel-loaded Bovine Serum Albumin Nanoparticles

  Lü Peng1,4, HU Rong2, BIAN Wei3, YANG Yani3, HE Jun1,3*

  2017, 48(07): 1030. https://doi.org/10.16522/j.cnki.cjph.2017.07.013

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  Docetaxel (DCT) has been recognized as one of the most efficient anticancer drugs. However, its poor water solubility has limited the clinical applications. Albumin, a versatile protein carrier for drug delivery, has been shown to be nontoxic, non-immunogenic, biocompatible and biodegradable. Therefore, it is an ideal material to fabricate nanoparticles for drug delivery. Albumin nanoparticles have gained considerable attention owing to their high binding capacity of various drugs and being well tolerated without any serious side-effects. In this paper, the DCT-loaded bovine serum albumin nanoparticles (BSA-NPs) were prepared by NabTM-technology. Based on the results of a single factor test and an orthogonal design, the preparation and formulation of the title nanoparticles were optimized with particle size, encapsulation efficiency and drug loading as indexes. The property of drug release in vitro was investigated by the dynamic dialysis. Finally, the optimal formulation of the NPs was as follows: BSA 1％; chloroform∶ethanol in a 11∶1 ratio; oil-to-water phase ratio of 1∶49; drug to BSA ratio of 1∶14.8; pH 6.6 of water phase; and the process parameters were as follows: homogenization pressure of 151.68 MPa and for 25 cycle times. The optimized results showed that DCTBSA-NPs had small particle size of (190.10±11.91)nm, high encapsulation efficiency of (95.6±0.5)％ and drug loading of (8.1±0.1)％ as well as the characteristic of sustained-release in vitro.
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  Application of Carbonic Anhydrase Ⅸ Antibody in Preparing Anti-tumor Targeting Preparations

  WANG Lin1,2, YANG Zhijun3, XU Qiuping4

  2017, 48(07): 1035. https://doi.org/10.16522/j.cnki.cjph.2017.07.014

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  Carbonic anhydrase Ⅸ (CA Ⅸ) is a cell surface antigen characterized by hypoxia-induced expression in many solid tumors. This study investigated the feasibility of carbonic anhydrase Ⅸ antibody (anti-CAⅨ) modified nanomicelles for targeted delivery of norcantharidin (1) to human lung cancer cells A549 in vitro. The dialysis method was adopted to prepare anti-CA Ⅸ modified 1 nanomicelles. The results showed that the product had an encapsulation efficiency of (80.93±1.01)％ and the loading capacity of (5.26±0.03)％ with an average particle size of (146.5±48.9)nm and ζ potential of (-14.79±0.67)mV. In vitro antitumor activities of 1 and anti-CA Ⅸ modified 1-loaded nanomicelles were evaluated. The results showed that the inhibition effect of the anti-CA Ⅸmodified 1 nanomicelles against A549 cells was significantly higher than that of the free drug and increased in a dose- and time-dependent manner.
  The in vivo antitumor activity was evaluated in nude mice bearing A549 xenografts. According to the results of tumor growth inhibition, the anti-CA IX modified 1 nanomicelles demonstrated an active targeting effect. The highest tumor inhibition rate (75.65％) was founded in the high-dose (1 mg/kg) group of the modified namomicelles.
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  Effect of Granulation Process on Compressibility of Metformin Hydrochloride Tablets

  WANG Yueru

  2017, 48(07): 1042. https://doi.org/10.16522/j.cnki.cjph.2017.07.015

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  The effects of high shear wet granulation and fluidized bed granulation on the granule properties (particle size distribution, morphology, density and compressibility) and tablet properties (hardness and friability) of metformin hydrochloride were investigated. The granules from high shear wet granulation process were more spherical and dense compared with the irregular and porosity granules from fluidized bed granulation, but with the poor compressibility (tablet hardness only up to 32 N). The granules with the higher moisture content prepared by the fluidized bed method had the better compressibility (tablet hardness up to 120 N) and the lower friability (0.14％ ). The results showed that the granules prepared by fluidized bed granulation had significantly lower capping tendency of metformin hydrochloride tablets than the tablets from the granules by high shear wet granulation. The fluidized bed granulation process could be used for the production of the metformin hydrochloride tablets, and the moisture content of the granules should be strictly controlled during the drying process.
   
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  Influences of Different Drugs and Additives on the Glass Transition Temperature of Soluplus and Kollidon VA64

  XIE Xuemei1,2, TIAN Conghui1, GAO Yan2, WANG Bingquan2, HAN Jun2*

  2017, 48(07): 1048. https://doi.org/10.16522/j.cnki.cjph.2017.07.016

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  Hot melt extrusion (HME) is a useful technique for increasing dissolution rate of poorly watersoluble compounds. Extrusion temperature designed according to glass transition temperature (Tg) of carriers plays a most important role in the formation of desired extrudes and prevents adverse effects like drug degradation or recrystallization.The aim of this study was to evaluate the influences of different substances with different contents on Tg values of Soluplus or Kollidon VA64. Thus, the solid solutions of drugs or additives (water, griseofulvin, ritonavir, cholesterol or modified soybean phosphalipid) and carriers (Soluplus or Kollidon VA64) were prepared and the changes of Tg value were investigated by differential scanning calorimetry (DSC). It was found that the Tg value of Soluplus or Kollidon VA64 was decreased by adding all of the five substances. The Tg values of Soluplus with or without water were 63.2 and
  75.6 ℃ , and for Kollidon VA64 were 49.5 and 107.1 ℃ . The decreasing amplitude of Tg values was larger by adding ritonavir or cholesterol, compared to the solid solution with griseofulvin or modified soybean phospholipid. This study was helpful for the wide use of Soluplus and Kollidon VA64 in HME technology.
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  Process Optimization for Romidepsin Crystallization

  XIONG Lei, HU Haifeng*

  2017, 48(07): 1054. https://doi.org/10.16522/j.cnki.cjph.2017.07.017

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  In this study, the mixed solvent was used to crystallize romidepsin. The ratio of romidepsin,crystallization temperature and mixed solvent were optimized. The results showed that temperature had a great influence on the purity of romidepsin, which should be controlled at 25 ℃. When acetone and methanol were used as the mixed solvent for romidepsin crystallization, the HPLC purity of romidepsin were effectively improved, from 96.5％ before crystallization to 98.81％, with the yield of 83.07％. The mixed solvent of ethanol and methanol could help to enhance the yield of romidepsin effectively, and the final yield could reach 86.59％. In our work, romidepsin was form L. The formation of romidepsin crystal nucleus in ethanol and methanol mixed solvent was faster, and the final crystalline particles were larger. This study provided an optimized direction for dynamic crystallization control of romidepsin. The impurities of romidepsin before and after crystallization were analyzed for the first time, and impurities R1, R2, R3 were significantly reduced after crystallization. The process parameters of the mixed solvent in the crystallization of romidepsin were studied and optimized for the first time as well. As ethanol and methanol were chosen for the crystallization of romidepsin, the yield was greatly impoved. Meanwhile, the crystallization process established in our work could be
  achieved without any professional equipment.
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  Determination of p-Aminophenol in Vitamin C Yinqiao Tablets

  SUN Yuping1,2, YAN Xiangping1*, XIE Hao1

  2017, 48(07): 1060. https://doi.org/10.16522/j.cnki.cjph.2017.07.018

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  A GC method was established for the determination of p-aminophenol in vitamin C Yinqiao tablets.An HP-50+ capillary column was used, with the application of hydrogen flame ionization detector (FID), at the detector temperature of 250 ℃, the injection port temperature of 250 ℃ and the column temperature of 150 ℃. It was linear in the concentration range of 10—100 μg/ml. The average recovery of p-aminophenol was 104.8％, with RSD of 4.54％. The content of p-aminophenol in the vitamin C Yinqiao tablets from different factories varied from 0.12％ to 0.47％ of the labeled amount of acetaminophen. Vitamin C and other components were mixed with acetaminophen respectively to prepare analog preparations, in order to investigate the case of the generation of p-aminophenol. The results showed that vitamin C might be the key factor leading to the generation of p-aminophenol from acetaminophen.
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  In situ Monitoring of the Hydrogenation Process of 2,3,5-Trimethyl Benzoquinone by React Infrared Spectrum

  WANG Kanglin1, SUN Yueting2, PI Shiqin3, LIN Zirong1

  2017, 48(07): 1070. https://doi.org/10.16522/j.cnki.cjph.2017.07.020

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  The variation of the absorption peaks of the characteristic functional groups in the hydrogenation reaction of trimethyl benzoquinone was investigated by react infrared spectrum. The 1 652 cm-1 wavelength of trimethyl benzoquinone and 1 718 cm-1 wavelength of trimethyl hydroquinone were selected as the characteristic infrared absorpition peaks, respectively. The standard curves were established with determination of the characteristic infrared absorption values from the different concentrations both of reactant and product. Under four reaction parameters, the concentrations of the products and reactants in the hydrogenation reaction system were simulated by the established standard curves. The results showed that the standard curve modeling could predict the concentration change of reactant and product in the hydrogenation reaction system, so as to quickly judge the endpoint of hydrogen reaction.
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  Research on Beijing Biopharmaceutical Industrial Innovation System and Innovation Ability Evaluation

  FENG Hao1,2, LEI Ting1*, PAN Yue2, WU Shuxia1, XI Dalong3

  2017, 48(07): 1075. https://doi.org/10.16522/j.cnki.cjph.2017.07.021

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  Beijing biopharmaceutical industry is now at a critical stage of innovation-driven development, with the industrial innovation system being important support and guarantee for its development. Based on the perspective of industrial innovation system, this paper studies the innovation system of Beijing biopharmaceutical industry from the four dimensions of innovation factor, innovation environment, innovation process and innovation benefits, constructs
  its innovation ability evaluation system, and quantitatively analyzes its innovation capacity development status between 2009 and 2015. The results show that the innovation ability of Beijing biopharmaceutical industry improved steadily between 2009 and 2012, declined in 2013 and 2014, and bottomed out in 2015. A SWOT qualitative analysis conducted in combination with evaluation results shows that Beijing biopharmaceutical industry has advantages in the innovation vitality and innovation resources of enterprises, while its weaknesses lie in the areas of achievements transformation and government administration. Going forward, Beijing biopharmaceutical industry will face both opportunities and threats in its development.