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Study_on_the_pharmacological_activities_and_chemic

ReviewStudy on the pharmacological activities and chemicalstructures of Viburnum dilatatumZhiheng Gao, Yufei Xi, Man Wang, Xiaoxiao Huang*, Shaojiang Song*Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research &Development, Liaoning Province, School of Traditional Chinese Materia Medica, ShenyangPharmaceutical University, Shenyang 110016, ChinaAbstractViburnum dilatatum (jiami in Chinese), belonging to the Caprifollaceae family, is widely distributed in Japan and China. Phytochemical investigations of Viburnum dilatatum (V. dilatatum) have resulted in the isolation of triterpenoids, phenolic glycosides essential oil, norisoprenoids, etc. Research results have shown that the chemical constituents of V. dilatatum possess various pharmacological activities, including antihyperglycemic, antioxidant activity and antiulcer effects. This study reviewed the chemical constituents and pharmacological activities of V. dilatatum to provide practical and useful information for further research and development of this plant.Keywords: Viburnum dilatatum; pharmacological activity; chemical structures1 IntroductionViburnum dilatatum (called jiami in Chinese, gamazumi in Japanese and snowball tree in English), beloinging to family Caprifoliaceae, is a deciduous low tree distributed widely in the hills of northern China and Japan [1]. There are many types of chemical constituents in Viburnum dilatatum (V. dilatatum), including triterpenoids, * Author to whom correspondence should be addressed. Address:School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Rd., Shenyang 110016, China; Tel.: +86-24-43520793 (Xiaoxiao Huang); +86-24-43520707 (ShaojiangSong);E-mail:*******************(XiaoxiaoHuang); ****************(ShaojiangSong).Received: 2021-04-16 Accepted: 2022-08-28phenolic glycosides and norisoprenoids [2-4]. The leaves have been utilized as a traditional Chinese medicine, and phenolic compounds have been reported as the main active chemical component of the leaves. Many researchers have analyzed the functions of these medicinal components and found that these components have good antioxidant antihyperglycemic and antiulcer effects. For example, the gamazumi crude extract obtained from the squeezed juice of the fruit prevented oxidative injury in rats [5]. This review described the chemical structures and pharmacological activities of V. dilatatum, so as to help readers understand comprehensively the research progress of V. dilatatum and provide help for the development of V. dilatatum.2 Chemical constituents and structuresPrevious reports have indicated that the main chemical constituents of V. dilatatum are phenolic glycosides and triterpenoids.2.1 Phenolic glycosidesThirteen phenolic glycosides were isolated and identified from V. dilatatum by extensive spectroscopic methods, namely p -hydroxyphenyl-6-O -trans-caffeoyl-β-D -glucoside (1) [6], p -hydroxyphenyl-6-O -trans-caffeoyl-β-D -alloside (2) [6], 4-allyl-2-methoxyphenyl-6-O -β-D -apiosyl(1→6)-β-D -glucoside (3) [6], 1-(4’-hydroxy-3’-methoxypheny1)-2-[2’’-hydroxy-4’’-(3’’’-hydroxypropyl)]-1,3-propanediol-l-O -β-D -glucopyranoside (erythro isomer) (4-7) [7], neochlorogenic acid methyl ester (8-9) [7], cryptochlorogenic acid methyl ester (10-11) [7], cyanidin-3-sambubioside (Cy-3-sam) (12) [8], cyanidin-3-glucoside (Cy-3-glc) (13) [8], 5-O -caffeoyl-4-methoxyl quinic acid (4-MeO-5-CQA) (14) [8], chlorogenic acid (5-CQA) (15) [8], quercetin (16) [8], 2-(glucopyranosyloxy)-benzyl-3-(glucopyranosyloxy)-benzoate (17) [9] and jiamizioside E (18) [10]. These structures are shown in Fig. 1.Fig. 1 Phenolic glycosides isolated from V . dilatatumContinued fig. 12.2 TriterpenoidsThere were about seventeen triterpenoids isolated and characterized from V. dilatatum , such as viburnols A (19) [11], viburnols B (20) [11], viburnols C (21) [11], viburnols D (22) [11], viburnols E (23) [11], viburnols F (24) [12], viburnols G (25) [12], viburnols H (26) [12], viburnols I (27) [12], viburnols J (28) [12],viburnols K (29) [12], viburnudienone B 2methyl ester (30) [13], viburnenone H 2 (31) [13],v i b u r n e n o n e B 2 m e t h y l e s t e r (32) [13], viburnudienone B 1 methyl ester (33) [13], viburnenone H 1 (34) [13], and viburnenone B 2 methyl ester (35) [13]. The structures are shown in Fig. 2.Continued fig. 23 Pharmacological activities3.1 Antioxidant activityOxidative stress caused by free radicals and their derivatives leads to disturbances in redox homeostasis. Reactive oxygen species (ROS) are not only endogenously produced during intracellular metabolic processes but also generated by exogenous stimuli such as UV radiation, pollutants, smoke and drugs. The cell triggers its defense systems or undergoes apoptosis when intracellular oxidative status increases. It influences numerous cellular processes including core signaling pathways, which are associated with development of systematic and chronic disorders, such as aging and cancer. Therefore, it is critical to remove cellular oxidants and restore redox balance.solution of V. dilatatum (GSS) had strong antioxidant activity in vivo and prevent stress-induced oxidative damage by the XYZ-dish method and the澳electron spin resonance (ESR) method [14]. The experimental result showed that the concentrations of lipid peroxide in plasma, liver and stomach in the GSS group were reduced. Furthermore, the activities of plasma lactic dehydrogenase, amylase and creatine phosphokinase are ordinarily increased by stress. However, these activities in the GSS group decreased to that in the control group. It was concluded that gastric ulcer formation, increase of lipid peroxidation in plasma and tissues and elevation of plasma enzymatic activities were confirmed in rats with water immersion restraint stress. It was also found that intake of GSS could protect the stomach and other tissues from oxidative damage.Kim et al. identified and isolated two major anthocyanins by NMR and LC-ESI-MS/MS, namely, cyanidin 3-sambubioside (I) and kuromanin (II) [15]. By the electron spin resonance method, the superoxide anion radical scavenging activities of I and II were evaluated with the IC 50 values of 17.3 and 69.6 µM, and their activities on hydroxyl radicals were evaluated with the IC 50 values of 4.3 and 53.2 mM. As the positive control, the IC 50 values of ascorbic acid were 74.2 µM on superoxide anion radicals and 3.0 mM on hydroxyl radicals, respectively. The above results suggested that these anthocyanins with radical scavenging properties might be the key compounds contributing to the antioxidant activity and physiological effects of V . dilatatum fruits.Woo et al. determined the free radical scavenging capacity of VD (the leaves of V. dilatatum ) [16]. Anti-oxidant activity of the extracts was assessed by the ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) or 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals. Butylated hydroxytoluene (BHT), a synthetic antioxidant, or α-tocopherol, was used as the positive control in these assays. The experimental result showed that VD inducedincrease in radical scavenging activity. In addition, lipid peroxidation inhibitory activity was determined via measurement of MDA (Malondialdehyde) levels using mouse liver tissue homogenate treated with various concentrations of the extracts. The concentration-dependent decrease in MDA levels observed was consistent with radical scavenging activities of the extracts. To examine whether VD extracts could protect mam-malian cells from oxidative stress, cultures of a human mammary gland-derived epithelial cell line MCF-7 were treated with each extract prior to challenging them with tBHP. The intracellular ROS (Reactive oxygen species) production was determined with the relative intensity of dichlorofluorescein fluorescence. While intracellular ROS formation was significantly promoted by tBHP treatment, the augmented ROS level was significantly reduced after the treatment with VD extracts.3.2 Antihyperglycemic effectIwai et al. used an oral glucose tolerance test on the diabetic rats [17]. They found that the elevation of plasma glucose level after oral administration of 2 g/kg glucose was suppressed by the repeated administration of the freeze-dried powder of V. dilatatum fruit juice (CEV). The α-glucosidase inhibitory activities of isolated compounds from CEV were also measured. Cyanidin 3-sambubioside and 5-caffeoyl quinic acid A showed inhibitory activity. These results suggested that V. dilatatum fruit had the antihyperglycemic effects.4 ConclusionV. dilatatum is distributed widely in the hills of northern China and Japan. Currently, the studies on V. dilatatum have been conducted at home and abroad, but few studies focus on its chemical components and pharmacological activities. Previousphytochemical investigations showed that the constituents of V. dilatatum included triterpenoids, phenolic glycosides, norisoprenoids and other compounds. This study describes thirteen phenolic glycosides and seventeen triterpenoids and their different degrees of antihyperglycemic, antioxidant activity and antiulcer effects, aiming to provide a reference for further studies on V. dilatatum and pharmaceutical development.References[1] Jeffrey B, Harborne A. Colour atlas of medicinal plantsof Japan. Phytochemistry, 1981, 20: 1467.[2] Miyazawa M, Hashidume S, Takahashi T, et al. Aromaevaluation of gamazumi (Viburnum dilatatum) by aroma extract dilution analysis and odour activity value.Phytochem Anal, 2012, 23: 208-213.[3] Kurihara T, Kikuchi M. Studies on the constituentsof flowers. IV. On the components of the flower of Viburnum dilatatum Thunb. J Health Sci, 1975, 95: 1098-1102.[4] Machida K, Kikuchi M. Norisoprenoids from Viburnumdilatatum. Phytochemistry, 1996, 41: 1333-1336. [5] Iwai K, Onodera A, Matsue H. Mechanism of preventiveaction of Viburnum dilatatum Thunb (gamazumi) crude extract on oxidative damage in rats subjected to stress. J Sci Food Agric, 2010, 83: 1593-1599.[6] Machida K, Nakano Y, Kikuchi M. Phenolic glycosidesfrom Viburnum dilatatum. Phytochemistry, 1991, 30: 2013-2014.[7] Machida K, Kikuchi M. Phenolic compounds fromViburnum dilatatum. Phytochemistry, 1992, 31: 3654-3656.[8] Kim MY, Iwai K, Matsue H. Phenolic compositions ofViburnum dilatatum Thunb. fruits and their antiradical properties. J Food Compos Anal, 2005, 18: 789-802. [9] Lu D, Yao S. Phenolic glycoside from the roots ofViburnum dilatatum. Nat Prod Commun, 2009, 4: 945-946.[10] Wu B, Zeng X, Zhang Y. New metabolite fromViburnum dilatatum. Nat Prod Commun, 2010, 5: 1097-1098.[11] Machida K, Kikuchi M. Viburnols: Novel triterpenoidswith a rearranged dammarane skeleton from Viburnum dilatatum. Tetrahedron Lett, 1996, 37: 4157-4160. [12] Machida K, Kikuchi M. Viburnols: Six noveltriterpenoids from Viburnum dilatatum. Tetrahedron Lett, 1997, 38: 571-574.[13] Machida K, Kikuchi M. Studies on the Constituents ofViburnum Species. XIX. Six New Triterpenoids from Viburnum dilatatum Thunb. Chem Pharm Bull, 1999, 47: 692-694.[14] Iwai K, Onodera A, Matsue H, et al. Antioxidant activityand inhibitory effect of Gamazumi (Viburnum dilatatum THUNB.) on oxidative damage induced by water immersion restraint stress in rats. Int J. Food Sci Nutr, 2001, 52: 443-451.[15] Kim MY, Iwai K, Onodera A, et al. Identification andAntiradical Properties of Anthocyanins in Fruits of Viburnum dilatatum Thunb. J Agric Food Chem, 2003, 51: 6173-6177.[16] Woo YJ, Lee HJ, Jeong YS, et al. Antioxidant Potentialof Selected Korean Edible Plant Extracts. Bio Med Res Int, 2017, 2017: 1-9.[17] Iwai K, Kim MY, Akio O, et al. Alpha-glucosidaseinhibitory and antihyperglycemic effects of polyphenols in the fruit of Viburnum dilatatum Thunb. J Agric Food Chem, 2006, 54: 4588-4592.。

高效液相色谱-荧光检测器法检测单克隆抗体注射液中吐温80的含量

Vol.7 No.1Feb. 2021生物化工Biological Chemical Engineering第 7 卷第 1 期2021 年 2 月高效液相色谱-荧光检测器法检测单克隆抗体注射液中吐温80的含量张博慧，贾戴辉，许俊彦*（宝船生物医药科技（上海）有限公司药物分析部门，上海 201203）摘要：目的：采用高效液相色谱-荧光检测器法（HPLC-FLD）测定单克隆抗体注射液中吐温80的含量，并进行方法学验证。

方法：按设定的色谱条件对检测单克隆抗体注射液中吐温80含量的HPLC-FLD 法进行方法学验证，包括专属性、精密度、准确度、线性和范围、耐用性，并采用该方法对不同单克隆抗体注射液进行检测。

结果：经验证，该方法具有专属性；重复性验证试验中，保留时间和含量的RSD 值分别为0.5%和0.4%；中间精密度验证试验中，保留时间和含量的RSD 值分别为0.4%和2.2%；准确度验证试验中，回收率分别为105%、98%和108%；线性相关系数为0.999 1，检测范围为0.05~0.80 mg/mL；不同批次反应线圈对检测结果无影响，不同品种单克隆抗体注射液吐温80含量检测结果偏差较小。

结论：该方法具有专属性，线性关系良好，精密度和准确度较好，测定结果稳定可靠，适用于单克隆抗体注射液中吐温80含量的检测。

关键词：高效液相色谱-荧光检测器；吐温80；单克隆抗体注射液中图分类号：R927.2 文献标识码：ADetermination of Tween80 Content in Monoclonal Antibody Injection by HPLC-FLDZHANG Bohui, JIA Daihui, XU Junyan *(Drug Analysis Department, Dragonboat Biopharmaceutical, Co., Ltd, Shanghai 201203)Abstract: Objective: To establish a method for the determination of Tween 80 in monoclonal antibody injection byHigh-performance liquid chromatography fluorescence detector. Methods: Under designed condition, the method was verified for specificity, precision, accuracy, linearity, range and durability. The method was also used to determination of Tween 80 content of different monoclonal antibody injections. Results: The HPLC-FLD method showed good specificity. In reproducibility test, the RSD of retention time and content were 0.5% and 0.4% respectively. In intermediate-precision test, the RSD of retention time and content were 0.4% and 2.2% respectively. The recovery rates were 105%, 98% and 108% respectively. The correlation Coefficient was 0.999 1 and the range was from 0.05 to 0.8 mg/mL. The different batches of reaction coils had no effect on the detection results. The Tween 80 contents in different mAb injections showed little deviation. Conclusion: The HPLC-FLD method showed good specificity, precision, accuracy and linearity, and the test result was stable and reliable, which was suitable for determination of Tween 80 content of different monoclonal antibody injections.Keywords: High-performance Liquid Chromatography-Fluorescence Detector; Tween 80; monoclonal antibody injection吐温80又名聚山梨酯80，其化学名为聚氧乙烯20山梨醇酐单油酸酯，作为助溶剂、乳化剂和稳定剂，常用于治疗性单克隆抗体注射液制剂中[1]。

东洋纺反转录试剂盒

11-05
高效率逆转录试剂盒 First Strand cDNA Synthesis Kit
ReverTra Ace -α-
(Code No. FSK-100,FSK-101)
使用说明书
科研用
TOYOBO CO., LTD. Life Science Department OSAKA JAPAN
目录
※ 除Positive Control RNA外，所有组分请均保存在-20℃条件下；Positive Control RNA请保存于-80℃条件下。
※ Positive Control RNA 由于运输过程中不能一直保证 -80℃条件，在运输过程中可能降解。建议用户选择人、鼠来源的 Total RNA 作为 Control RNA。
G3PDH mRNA
Primer F 559
intron
Size(bases)
1634
1237
1110 Primer R
90 129 90 92 k-193 104
图 2. Control Primer F，R 的 location
[Positive Control RNA]
本试剂盒作为 Positive Control 用 RNA ，添附有 Human G3PDH (Glyceraldehyde 3-Phosphate Dehydrogenase)遗传基因的 in vitro 转录产物。（在 3’末端添加了 22mer 的 Poly（A）tail）（请参照图 3）。
※ 组分中的 5×RT Buffer 在溶解时，可能会出现白色沉淀现象，但不影响其品质。此时，请使用振荡器等仪器使其混合均匀，等完全溶解后再使用。
[各引物的序列]

Gateway LR Clonase II Enzyme Mix

Gateway®LR Clonase II Enzyme MixCat. No. 11791-020 Size: 20 reactionsCat. No. 11791-100 Size: 100 reactionsStore at -20°C (non-frost-free freezer) Gateway® TechnologyThe Gateway® Technology is a universal cloning method that takes advantage of the site-specific recombination properties of bacteriophage lambda (1) to provide a rapid and highly efficient way to move DNA sequences into multiple vector systems. The Gateway® Technology is schematically represented below.att B1-gene-att B2 u att P1-ccd B-att P2 att L1-gene-att L2 u att R1-ccd B-att R2(expression clone) (pDONR™) (entry clone) (destination vector) The att B u att P reaction is mediated by Gateway® BP Clonase II enzyme mix; the att L u att R reaction is mediated by Gateway® LR Clonase II enzyme mix. ccd B is the F plasmid-encoded gene that inhibits growth of E. coli (2,3) and “gene” represents any DNA segment of interest (e.g. PCR product, cDNA, genomic DNA). DescriptionGateway® LR Clonase™ II enzyme mix is a proprietary enzyme and buffer formulation containing the bacteriophage lambda recombination proteins Integrase (Int) and Excisionase (Xis), the E. coli-encoded protein Integration Host Factor (IHF) (1), and reaction buffer provided in a single mix for convenient reaction set up. Gateway® LR Clonase™ II enzyme mix catalyzes in vitro recombination between an entry clone (att L-flanked “gene”) and an att R-containing destination vector to generate an att B-containing expression clone. Store Gateway® LR Clonase™ II enzyme mix at -20ºC (non-frost-free freezer) for up to 6 months. For long-term storage, store at -80ºC.Components Supplied20 rxns 100 rxnsP lGateway® LR Clonase II Enzyme Mix 40 P l 200P lP l 200 Proteinase K Solution (2 P g/P l) 40P lpENTR™-gus Positive Control (50 ng/P l) 20P l 20 Quality ControlLR Clonase II enzyme mix is functionally tested in a 1 hour recombination reaction followed by a transformation assay.Part No. 11791.II.pps Rev. Date: 10 Jun 2004 This product is distributed for laboratory research only. CAUTION: Not for diagnostic use. The safety and efficacy of this product in diagnostic orother clinical uses has not been established.For technical questions about this product, call the Invitrogen Tech-Line SM U.S.A. 800 955 6288Page 2 General Recommendations and Guidelinesx pENTR™-gus is provided for use as a positive control in the LR reaction and is an entry clone containing the Arabidopsis thaliana E-glucuronidase (gus)gene (4). Refer to our Web site () for a map andsequence of pENTR™-gus.x We recommend using plasmid DNA purified with the PureLink™ HQ Mini Plasmid Purification Kit (Catalog no. K2100-01). Mini-prep (alkaline lysis) DNA preparations are adequate for Gateway® cloning reactions; however, in general, such DNA cannot be quantitated by UV absorbance due tocontaminating RNA and nucleotides. Estimate concentrations by gelelectrophoresis in comparison with standard DNA (e.g. DNA Mass Ladder, Catalog no. 10068-013 or 10496-016).x For LR recombination reactions, the most efficient substrates are supercoiled att L-containing entry vectors and supercoiled att R-containing destination vectors. For large (>10 kb) entry clones or destination vectors, linearizing the entry clone or destination vector may increase the efficiency by up to 2-fold.x To increase the number of colonies containing the desired expression clone, increase the incubation time from the recommended 1 hour to 2 hours-overnight. Longer incubations are recommended for plasmids t10 kb toincrease the yield of colonies.x We recommend using 50-150 ng entry clone per 10 P l reaction. Highest colony yields are typically obtained using 150 ng entry clone and 150 ngdestination vector. Do not use >150 ng entry clone as you may obtaincolonies containing multiple DNA molecules (often with an associated“small colony” phenotype). Using <50 ng entry clone will generate fewer colonies.Page 3 ProceduresLR ReactionLR Clonase II enzyme mix is supplied as a 5X solution. If you wish to scale the reaction volume, make sure the LR Clonase II enzyme mix is at a final concentration of 1X. For a positive control, use 100 ng (2 P l) of pENTR™-gus.1. Add the following components to a 1.5 ml microcentrifuge tube at roomtemperature and mix:Entry clone (50-150 ng) 1-7 P lP lDestination vector (150 ng/P l) 1TE buffer, pH 8.0 to 8 P l2. Thaw on ice the LR Clonase II enzyme mix for about 2 minutes. Vortex theLR Clonase™ II enzyme mix briefly twice (2 seconds each time).3. To each sample (Step 1, above), add 2 P l of LR Clonase II enzyme mix to thereaction and mix well by vortexing briefly twice. Microcentrifuge briefly.4. Return LR Clonase II enzyme mix to -20q C or -80q C storage.5. Incubate reactions at 25q C for 1 hour.6. Add 1 P l of the Proteinase K solution to each sample to terminate thereaction. Vortex briefly. Incubate samples at 37q C for 10 minutes. Transformation1. Transform 1 P l of each LR reaction into 50 P l of One Shot® OmniMAX™ 2 T1Phage-Resistant Cells (Catalog no. C8540-03). Incubate on ice for 30 minutes.Heat-shock cells by incubating at 42q C for 30 seconds. Add 250 P l of S.O.C.Medium and incubate at 37q C for 1 hour with shaking. Plate 20 P l and 100 P l of each transformation onto selective plates. Note: Any competent cells witha transformation efficiency of >1.0 u 108 transformants/P g may be used.2. Transform 1 P l of pUC19 DNA (10 ng/ml) into 50 P l of One Shot®OmniMAX™ 2 T1 Phage-Resistant Cells as described above. Plate 20 P l and 100 P l on LB plates containing 100 P g/ml ampicillin.Expected ResultsAn efficient LR recombination reaction will produce >5000 colonies if the entire LR reaction is transformed and plated.Page 4 References1. Landy, A. (1989) Ann. Rev. Biochem. 58, 913.2. Bernard, P. and Couturier, M. (1992) J. Mol. Biol.226, 735.3. Miki, T., Park, J.A., Nagao, K., Murayama, N., and Horiuchi, T. (1992) J. Mol. Biol. 225, 39.4. Kertbundit, S., Greve, H.D., Deboeck, F., Montagu, M.V., and Hernalsteens, J.P. (1991)Proc. Natl. Acad. Sci. USA, 88, 5212.Limited Use Label License No. 19: Gateway® Cloning ProductsThis product and its use is the subject of one or more of U.S. Patent Nos. 5,888,732, 6,143,557, 6,171,861, 6,270,969, 6,277,608, and 6,720,140 and/or other pending U.S. and foreign patent applications owned by Invitrogen Corporation. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for profit entity). The purchase of this product does not convey a license under any method claims in the foregoing patents or patent applications, or to use this product with any recombination sites other than those purchased from Invitrogen Corporation or its authorized distributor. The right to use methods claimed in the foregoing patents or patent applications with this product for research purposes only can only be acquired by the use of Clonase™ purchased from Invitrogen Corporation or its authorized distributors. The buyer cannot modify the recombination sequence(s) contained in this product for any purpose. The buyer cannot sell or otherwise transfer (a) this product, (b) its components, or (c) materials made by the employment of this product or its components to a third party or otherwise use this product or its components or materials made by the employment of this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the employment of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) not to transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Notwithstanding the preceding, any buyer who is employed in an academic or government institution may transfer materials made with this product to a third party who has a license from Invitrogen under the patents identified above to distribute such materials. Transfer of such materials and/or information to collaborators does not convey rights to practice any methods claimed in the foregoing patents or patent applications. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that none of (i) this product, (ii) any of its components, or (iii) a method claim of the foregoing patents, was used in the manufacture of such product. Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the use of this product to manufacture a protein for sale, provided that no method claim in the above patents was used in the manufacture of such protein. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license to use this product for purposes other than those permitted above, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200.Limited Use Label License No. 23: GUS Control VectorThe GUS positive control vector in these products is claimed in patents and patent applications (See U.S. Patent No. 5,599,670 and Great Britain Patent No. 2,197,653) licensed to Invitrogen by Cambia Biosystems, L.L.C. ("CBL"). Use of the GUS gene is restricted to use as a positive control. Any other use may require a license from CBL.©2004 Invitrogen Corporation. All rights reserved.。

Roche_Xtreme GeneHP_protocal

0910.06479774001ቢ

The recommended starting concentration is a 3:1. For most cell types, these X-tremeGENE HP DNA Transfection Reagent to DNA ratios provide excellent transfection efficiency. Further optimization may increase transfection efficiency in your particular application. In addition to varying the ratio, other parameters may also be evaluated, such as the amount of transfection complex added. For additional optimization guidelines, see Section 3, Troubleshooting and visit . Plasmid DNA • For best results, accurately determine the plasmid DNA concentration using 260-nm absorption; estimates of DNA by measuring gel band density are not recommended. Determine DNA purity using a 260 nm/280 nm ratio (the optimal ratio is 1.8). • Prepare the plasmid DNA solution using sterile TE (Tris/EDTA) buffer or sterile water at a concentration of 0.1 to 2.0 µg/µl. • Use high quality DNA preparation kits to obtain endotoxin-free DNA. Cell Culture Conditions • Minimize intra- and inter-experimental variance in transfection efficiency using cells that are regularly passaged, proliferating well in a log-growth phase, and plated at a consistent density. • For best results, accurately quantify cell concentration using a hematocytometer or automated system. • Cells must be healthy and free of Mycoplasma. • Cells should have a low passage number to achieve best results. Other Media Additives In some cell types, antimicrobial agents (e.g., antibiotics and fungicides) commonly included in cell-culture media may adversely affect the transfection efficiency of X-tremeGENE HP DNA Transfection Reagent. If possible, exclude additives in initial experiments. Once high-efficiency conditions have been established, these components can be added back while monitoring transfection results. Cell growth and/or transfection efficiency may be affected by variations in serum quality and medium formulations. Verification of Vector Function Optimize transfection conditions using a known positive-control reporter gene construct before transfecting cells with a new vector construct: • Determine transfection efficiency using a reporter gene assay, such as ␤-Gal*, Luciferase*, or SEAP*. • Sequence flanking vector insert regions to verify the integrity of your new construct. 2.2 Transfection Procedure Adherent Cells: Plate cells approximately 24 hours before transfection making sure cells are at the optimal concentration in the appropriate cell culture vessel. Suspension Cells: Plate freshly passaged cells at optimal concentration.

三羟甲基氨基甲烷盐酸盐质谱裂解碎片

三羟甲基氨基甲烷盐酸盐质谱裂解碎片1. 引言1.1 背景介绍三羟甲基氨基甲烷盐酸盐（简称TMAH）是一种广泛应用于化学分析领域的试剂，其在有机物质质谱分析中具有重要的作用。

TMAH可以将有机物质中的酯类、醇类、酚类等化合物甲基化，使其具有更适合在质谱仪中进行分析的性质。

TMAH的盐酸盐形式是其常见的实验室试剂，广泛用于有机物质的分析化学实验中。

TMAH盐酸盐的质谱裂解碎片特征对于分析化学领域起着至关重要的作用。

了解TMAH盐酸盐在质谱中的裂解碎片特征可以帮助分析师更准确地识别未知化合物、推断其结构以及进行定量分析。

对TMAH盐酸盐质谱裂解碎片特征进行深入研究具有重要的理论和应用意义。

本文将从TMAH盐酸盐的合成与性质、质谱技术在TMAH盐酸盐质谱裂解中的应用、TMAH盐酸盐质谱裂解碎片特征分析、相关反应机制研究以及质谱技术在TMAH盐酸盐质谱裂解中的局限性等方面展开详细讨论，旨在深入探讨TMAH盐酸盐在质谱分析中的重要性以及相关研究领域存在的问题和挑战。

【2000字】.1.2 研究目的本研究旨在通过对三羟甲基氨基甲烷盐酸盐质谱裂解碎片进行详细分析，揭示其分子结构特征及相关反应机制，为进一步探索其生物活性和药理作用奠定基础。

具体研究目的包括：1. 探究三羟甲基氨基甲烷盐酸盐质谱裂解碎片的形成机理，揭示其分子内部的键合情况和反应路径；2. 分析三羟甲基氨基甲烷盐酸盐质谱裂解碎片的碎片特征，解析其质谱图谱中各离子峰的起源及关联；3. 探讨质谱技术在三羟甲基氨基甲烷盐酸盐分子结构鉴定中的优势和局限性，为其质谱分析提供科学依据；4. 为进一步深入研究三羟甲基氨基甲烷盐酸盐在药物开发和医学应用中的潜在作用提供理论支持和实验基础。

1.3 研究意义三羟甲基氨基甲烷盐酸盐是一种重要的有机化合物，具有广泛的应用价值。

其在药物合成、生物医药和化学工业领域具有重要的地位。

对三羟甲基氨基甲烷盐酸盐进行质谱分析，可以更深入地了解其结构和性质，为其应用提供重要的信息和参考。

FuGENE HD转染试剂说明书

FuGENE ®HD Transfection Reagent1.What this Product DoesNumber of Transfection ExperimentsIn a typical experiment using HeLa or COS-1 cells, 1 ml of FuGENE ®HD Transfection Reagent can be used to perform up to three hundred transfections in 35-mm tissue-culture dishes, using 3 ␮l of reagent combined with 1 – 2 ␮g DNA per well. This is equivalent to over 6,000wells in a 96-well plate or 1,000 wells in a 24-well plate.L Optimal expression depends upon experimental conditions includ-ing cell type, passage history, confluence, seeding protocol, com-plex incubation time, serum batch, etc. The above amounts of reagents work well with HeLa or COS-1 cells. In other test systems,two- to three-fold higher amounts of reagent yield optimal levels of expression.FormulationFuGENE ® HD Transfection Reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterile-filtered, and pack-aged in glass vials. It does not contain any ingredients of human or animal origin.Storage and Stability•FuGENE ® HD Transfection Reagent is shipped at +15 to +25°C.•Store FuGENE ® HD Transfection Reagent at +2 to +8°C, with the lid very tightly closed. The reagent is stable through the expiration date printed on the label when stored under these conditions.L FuGENE ® HD Transfection Reagent remains fully functional evenafter repeatedly opening the vial (at least five times over a two-month period) as long as the vial is tightly recapped and stored at +2 to +8°C between uses.L Do not store FuGENE ® HD Transfection Reagent below 0°C.Components may precipitate and alter results. If you accidently place the reagent at Ϫ20°C, briefly warm it to 37°C to dissolve any precipitate. It should function normally; however, do not return it to the freezer.Special HandlingN Always bring to room temperature and mix FuGENE ® HD Transfec-tion Reagent prior to use (vortex for one second or use inversion).N Do not aliquot FuGENE ® HD Transfection Reagent from the originalglass vials. Chemical residues in plastic vials can significantly decrease the biological activity of the reagent. Minimize the contact of undiluted FuGENE ® HD Transfection Reagent with plastic sur-faces.N Always dilute the reagent by pipetting directlyinto serum-free medium. Do not allow the FuGENE ® HD Transfection Reagent to contact the plastic walls of the tube containing the serum-free medium during the dilution step.N Do not use siliconized pipette tips or tubes.Additional Equipment and Reagents RequiredAdditional reagents and equipment required to perform transfection assays using FuGENE ® HD Transfection Reagent, but not provided,include:General Laboratory Equipment•standard cell culture equipment (e.g., biohazard hoods, incubators,microscope)•standard pipetters and micropipetters •vortex mixerFor Plasmid Preparation•purified plasmid stock (0.1 ␮g/␮l – 2.0 ␮g/␮l) in sterile TE (10 mM Tris, 1 mM EDTA, pH 8.0) buffer or sterile water•Genopure Plasmid Midi Kit*, Genopure Plasmid Maxi Kit*, or High Pure Plasmid Isolation Kit* can be used to prepare plasmid.For Verification of Vector Function •assay appropriate for transfected gene•G-418* or Hygromycin B* (optional; for stable transfection experi-ments)For Transfection-Complex Formation•Opti-MEM I Reduced Serum Medium, water, or serum-free medium •24-well plate to serve as test tube rack for FuGENE ® HD Transfec-tion Reagent vial•sterile polystyrene tubes or round-bottom 96-well platesCells Growing in Log Phase•select subconfluent cultures in log phase for preparation of the cell cultures for transfection•method to quantify cell number to reproducibly plate the same number of cellsApplicationFuGENE ® HD Transfection Reagent is a multi-component reagent that forms a complex with DNA, then transports the complex into animal or insect cells. Benefits of FuGENE ® HD Transfection Reagent include:•High transfection efficiency in many common cell types, including HeLa, NIH/3T3, COS-1, COS-7, CHO-K1, Hep G2, HEK-293, MCF7,and some insect cell lines. In addition, you will achieve excellent transfection efficiency in some cell lines (e.g., RAW) that are not transfected well by other reagents. Detailed transfection protocols and sample results are available at .•Demonstrates minimal cytotoxicity or changes in morphology when adequate numbers of cells are transfected, and eliminates the requirement to change media after the addition of transfection complex.•Suitable for transient and stable transfection.•Functions exceptionally well in the presence or absence of serum;eliminates the need to change media.To ensure the quality of cells to be transfected, Roche recommends using freshly-obtained, low-passage cell sines form ATCC ®. For more information please visit and bookmark .For the transient and stable transfection of animal and insect cellsCat. No. 04 709 691 0010.4 ml (up to 120 transfections)Version November 2007Cat. No. 04 709 705 001 1 ml (up to 300 transfections)Store at +2 to +8°CCat. No. 04 709 713 001 1)Mega-pack 5 × 1 ml (up to 1,500 transfections)Cat. No. 05 061 369 00110 ml (up to 3,000 transfections)Cat. No. 04 883 560 001Trial-pack1)The five vials are packaged together in one box with one pack insertFor life science research only. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY.2.How to Use this Product2.1Before you BeginRequired Amount of FuGENE® HD Transfection ReagentFor initial optimization experiments, transfect a monolayer of cells that is 80 – 90% confluent in a six-well culture dish, using 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2 ratios of FuGENE® HD Transfection Reagent (␮l) to DNA (␮g), respectively. For most cell types, these FuGENE® HD Transfection Reagent:DNA ratios provide excellent transfection levels.L Subsequent optimization may further increase efficiency in your particular application. In addition to varying the volume of FuGENE® HD Transfection Reagent, other parameters may be evaluated (see section 2.6, Parameters for Optimization, and sec-tion 3, Troubleshooting).Plasmid DNA•It is critical to accurately determine the plasmid DNA concentration using 260-nm absorption (estimates of DNA content based on the intensity of gel bands are not sufficiently accurate). Determine the DNA purity using a 260 nm/280 nm ratio; the optimal ratio is 1.8.•Prepare the plasmid DNA solution in sterile TE (Tris/EDTA) buffer or sterile water at a concentration between 0.1 ␮g/␮l and 2.0 ␮g/␮l. Cell Culture ConditionsMinimize both intra- and inter-experimental variance in transfection efficiency by using cells that are regularly passaged, proliferating well (best when in a log-growth phase), and plated at a consistent den-sity. FuGENE® HD Transfection Reagent is different from FuGENE® 6 Transfection Reagent regarding the optimal density of cells required for maximal expression with minimal negative effect; FuGENE®6 Transfection Reagent is formulated to work at low cell densities, whereas FuGENE® HD Transfection Reagent is formulated to work at higher cell densities. Cells must be healthy and free of mycoplasma and other contaminants.L If you have used FuGENE® 6 Transfection Reagent in the past, we suggest that you increase the plating density for initial tests using FuGENE® HD Transfection Reagent. For most cell lines, use the reagent at cell-plating densities at least twice that used with FuGENE® 6 Transfection Reagent to yield maximum protein expression. For most cell lines, cultures should be 80 – 90% con-fluent at the time of transfection. For contact-inhibited cell lines such as NIH/3T3, optimal results are obtained when cells are plated at lower densities.Other Media AdditivesIn some cell types, antimicrobial agents (e.g., antibiotics and fungicides) that are commonly included in cell-culture media may adversely affect the transfection efficiency of FuGENE® HD Transfection Reagent. If possible, exclude additives for initial experi-ments. Once high-efficiency conditions have been established, these components can be added back while monitoring your transfection results. Cell growth and/or transfection efficiency may be affected by variations in sera quality or media formulations.Verification of Vector FunctionOptimize transfection conditions with a known positive-control reporter gene construct prior to transfecting cells with a new vector construct:•Determine transfection efficiency with a reporter gene assay (CAT*,␤-Gal*, Luciferase*, SEAP*, or hGH*).•Sequence across the flanking vector insert regions to verify the integrity of your new construct.2.2Preparation of Cells for Transfection2.3Overview of Initial Transfection ExperimentAdherent and Suspension Cells in a Six-well Plate or 35-mm Culture DishFor initial optimization, test FuGENE® HD Transfection Reagent:DNA ratios of 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2 (␮l for FuGENE® HD Transfection Reagent, and ␮g for DNA, respectively). The preparation of the com-plex for a single well of a six-well plate, or a 35-mm culture dish, is described in section 2.4. These ratios will function very well for com-monly used adherent cells and suspension cells. For your particular cell line and culture conditions you may find that ratios of 9:2, 10:2, 11:2, or 12:2 result in even greater expression. Try these ratios if you find the highest expression levels in the 8:2 ratio well.N Prepare the transfection complex in diluent that does not contain serum (e.g., Opti-MEM I Reduced Serum Medium), even if the cells are transfected in the presence of serum. For some cell lines, the complex may be formed in DMEM or sterile water.L For additional optimization tips, see section 2.6 and visit /fugene/hdRatio OverviewPreparation of a transfection complex that is sufficient for a 35-mm culture dish, or one well of a six-well plate, at six different ratios: Tab. 1: Preparation of transfection complex for a 35-mm culture dish.Cell Type ProcedureAdherentcellsOne day before the transfection experiment, trypsinizethe monolayer, adjust cell concentration, and plate thecells in the chosen cell-culture vessel. For most celltypes, plating 3 – 6 × 105 cells in 2 ml of medium in a35-mm culture dish (or six-well plate) overnight willachieve the desired density of Ͼ80% confluency atthe time of transfection. For cell lines with specialcharacteristics, such as contact-inhibited NIH/3T3cells, a lower plating density should be used. If usingculture plates of a different size, adjust the total num-ber of cells, starting volume of FuGENE® HD Transfec-tion Reagent, and the starting mass of DNA inproportion to the relative surface area (Table 2). SuspensioncellsUse freshly passaged cells at a concentration of 5×105/ml to 1 × 106/ml in 2 ml of medium in a 35-mmculture dish (or six-well plate). If using culture platesof a different size, adjust the total number of cells,starting volume of FuGENE® HD TransfectionReagent, and the starting mass of DNA in proportionto the relative volume (Table 2).Tubelabel(ratio)Diluent(␮l)FuGENE® HDTransfectionReagent (␮l)DNA(␮g)Comments3:210032Add the entire volume toa 35-mm culture dish oreach well of a six-wellplate, or 2 – 15 ␮l to eachwell of a 96-well plate.Suggested volumes fordifferent culture vesselsare included in Table 2. 4:2100425:2100526:2100627:2100728:2100822.4Transfection ProcedureNotes:L As with any experiment, include appropriate controls. Preparewells with cells that remain untransfected, cells with transfection reagent alone, and cells with DNA alone.L For stable transfection experiments, the complex-containingmedium should be left unchanged until the cells need to be pas-saged. At that time, include the appropriate selection antibiotics (G 418* or Hygromycin B*).L To prepare transfection complexes for different-sized containers orparallel experiments, proportionally change the quantity of all components according to the total surface area of the cell culture vessel being used (Table 2).L For ease-of-use when transfecting small volumes, as in 96-wellplates containing 0.1 ml culture medium per well, prepare 100 ␮l of transfection complex and add 2 – 15 ␮l to each well depending upon the cell type.L The optimal ratio of transfection reagent:DNA and the optimaltotal amount of complex may vary with cell line, cell density, day of assay, and gene expressed.L After performing the optimization experiment where several ratioswere tested, select a ratio in the middle of the plateau for future experiments.2.5Cotransfection ExperimentsSuggestionsFor cotransfection experiments with FuGENE ® HD Transfection Reagent, maintain the same total reagent:total DNA ratio as that used for a single plasmid in your system. Thus, the total amount of the plas-mid DNA should be equal to the amount of plasmid used in a single plasmid transfection.ᕡAllow FuGENE ® HD Transfection Reagent, DNA, anddiluent to adjust to +15 to +25°C. Vortex for one second or invert the FuGENE ® HD Transfection Reagent vial to mix.ᕢDilute DNA with appropriate diluent, for example, Opti-MEM IReduced Serum Medium, serum-free medium (without anti-biotics or fungicides), or sterile water to a concentration of 2␮g plasmid DNA/100 ␮l Opti-MEM (0.02␮g/␮l).L For insect cells, use sterile water as diluent. For other celllines, try sterile water or serum-free medium as an alterna-tive diluent.ᕣPlace 100 ␮l diluent, containing 2 ␮g DNA into each of six ster-ile tubes labeled 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2.Recommendation : Use sterile polystyrene tubes or round-bottom, 96-well plates to form the transfection complex.L Due to manufacturer variability with release agents for96well plates, we suggest using tissue culture treated 96well plates to reduce variablity.ᕤForm the transfection complex by adding FuGENE ® HDTransfection Reagent to tubes containing diluted DNA :Pipet the FuGENE ® HD Transfection Reagent (3, 4, 5, 6, 7, or 8␮l) directly into the medium containing the diluted DNA with-out allowing contact with the walls of the plastic tubes.N To avoid adversely affecting transfection efficiency, do notallow undiluted FuGENE ® HD Transfection Reagent to come into contact with plastic surfaces (such as the walls of the tube that contains the serum-free medium) other than pipette tips. Do not use siliconized pipette tips or tubes.ᕥMix and incubate the transfection complex :Vigorously tap the tube or vortex for one to two seconds to mix the contents. If using a 96-well plate, place the plate on a rotat-ing shaker for 5 – 10 seconds. Incubate the transfection reagent:DNA complex for 15 minutes at room temperature.For some ratios and cell types, incubation is not necessary for optimal complex formation, while a longer incubation time is better for other cell types. Determine this for your particular cell line and the ratio you use.ᕦAdd the transfection complex to cell s:Remove culture vessel from the incubator. Removal of growth medium is not necessary. Add the transfection complex to the cells in a drop-wise manner or add below the surface of the medium. Swirl the wells or flasks to ensure distribution over the entire plate surface. Use of a rotating platform shaker for 30 seconds at low speed provides adequate mixing for 96-well plates.Once the FuGENE ® HD Transfection Reagent:DNA complex has been added to the cells, there is no need to remove and replace with fresh medium (as is necessary with some other transfection reagents).L In our experience, the exposure of most common laboratorycell types (COS-1, CHO-K1, HEK-293, HeLa, Hep G2, MCF-7) to the transfection complex until performance of the gene expression assay (24–48 hours later) does not affect the results. If you desire to transfect cells that are in serum-free medium during the transfection process, then replace the medium with serum-containing medium 3 – 8 hours after transfection, unless the cells normally grow in serum-free medium. ᕧIncubate cells and assay the results :Following transfection, incubate the cells for 18 – 72 hours prior to measuring protein expression. The length of incubation depends upon the transfected vector construct, the cell type being transfected, the cell medium, cell density, and the type of protein being expressed. After this incubation period, measure protein expression using an assay that is appropriate for your system.L If you observe low transfection levels or more than10 – 30% cell death, refer to section 3, Troubleshooting and /fugene/hd2.6Parameters for Optimization2.7Transfection of Adherent Cells Adapted for Suspension Growth•In some cases, adherent cells may be adapted for suspension growth, thus enabling the production of transiently transfected cells on a very large scale.•HEK-293 cells grown in suspension in serum-free medium that did not contain heparin or dextran sulfate produced significant amounts of protein following transfection.2.8Guidelines for Preparing FuGENE® HD Transfection Reagent:DNA Complex for Various Culture Vessel SizesThe starting volume and mass to add to the different culture vessels is based upon preparing a 100-␮l transfection complex as described in sec-tions 2.3 and 2.4. For best results, prepare a 100-␮l complex at different ratios and add varying amounts of each ratio when optimizing. The amounts below are based on the 100-␮l complex as prepared in sections 2.3 and 2.4.Suggested seeding density for adherent cells = 30,000 – 70,000 cells per cm2Suggested seeding density for suspension cells = 250,000 – 500,000 cells per mlTab. 2: Refer to the table below when setting up your transfection reac-tions. T hese are suggested seeding densities and are media, passage level, laboratory, and cell-line dependent. It is critical that log phase cultures are selected for subculture for the transfection experiments, and that cultures are seeded at the proper density for the transfection experiment. Observe cultures and plate them so that the monolayer is 80–90% confluent at the time of trans-fection. This must be determined empirically. For some cell lines, 60–80% con-fluency is sufficient. However, a contact-inhibited cell line, such as NIH/3T3, should be plated at lower confluence due to its growth characteristics.1) Scale up total volume for larger vessels.3.TroubleshootingParameter to beoptimizedProcedureFuGENE® HD Transfection Reagent:DNA ratio Form the transfection complex at several ratios: 3:2, 4:2, 5:2, 6:2, 7:2, 8:2, 10:2, and 12:2 (␮l FuGENE® HD Transfec-tion Reagent: ␮g DNA).In some systems, altering the ratio of FuGENE® HD Transfection Reagent to DNA can increase the level of protein expression.L It has been reported that for some plasmid preparations, a ratio of 2:2 yielded optimal results. This is unusual and may reflect some property of the plasmid preparation rather than a characteristic of the FuGENE® HD Transfec-tion Reagent.Amount of transfectioncomplex addedTry adding 200%, 150%, 75%, 50%, and 25% of the amount of 100-␮l transfection complex suggested in Table 2.Number of cells plated Plating more cells will overcome negative growth effects of excess transfection complex. For cells with special growth characteristics, such as NIH/3T3 cells, do not use this as the first parameter for optimization.Incubation time for the transfection complex to form Vary the length of incubation time for transfection-complex formation: add the complex to the cells immediately after the components are combined and mixed, and then at several intervals up to 40 minutes (i.e., 0, 15, 25, and 40 min-utes). We have observed that in some cell lines, the transfection-complex incubation time tends to have no effect on results when using higher ratios; however, results using lower-ratio-complexes varied depending on the incubation time for complex formation.Special tips for sensitive cell lines •Reduce the time of exposure to the transfection complex (2–3 hours maximum), then replace the medium.•Use the lower ratios, and allow the complex to form for a longer period of time (determine empirically for your cell line), then add lower amounts of the complex (50% or less of what was originally tested).Culture vessel Surfacearea(cm2)TotalvolumeofmediumSuggested seeding densitySuggested amount ofthe 100-␮l trans-fection complex toadd to each well (␮l)Final amount of FuGENE® HDTransfection Reagent (␮l) ineach well following addition ofsuggested amount of 100-␮ltransfection complexCells/wellAdherent cellsCells/wellSmall or suspensioncellsUsing the3:2 ratioUsing the8:2 ratio totalvolumevolume forlargerlow high low high96-well plate(1 well)0.30.110,00020,00025,00050,00050.150.424-well plate(1 well)1.90.550,000125,000250,000500,000250.752.012-well plate(1 well)3.8 1.0100,000250,000375,000750,00050 1.54.0 35-mm dish82200,000500,000500,0001,000,000100 3.08.06-well plate(1 well)9.42200,000600,000500,0001,000,000100 3.08.0 60-mm dish215500,0001,400,0001,250,0002,500,000250 1)7.520.0 10-cm dish55101,500,0003,500,0002,500,0005,000,000500 1)15.040.0 T-25 flask256700,0001,700,0001,500,0003,000,000300 1)9.024.0 T-75 flask75202,000,0005,000,0005,000,00010,000,000900 1)27.072.0Low trans-fection efficiency Poor quality orinsufficient quantityof nucleic acidsVerify the amount, purity, and sequence of nucleic acid.Perform a control transfection experiment with a commercially available transfection-grade plasmidpreparation.Chemical contaminants may be in the plasmid preparation. Avoid phosphate buffers until you havetested them in your system.L Endotoxins are reported to be cytotoxic to some very sensitive cell lines.Insufficient numberof cellsUse adherent cells that are at least 80% confluent. Low cell density results in fewer cells available totake up transfection complex, and excess complex may be cytotoxic; in addition, fewer cells yieldless protein.Too many cells orcells post log phaseWhen confluent cultures are subcultured, or cells are plated at too high a density, the cells fail to dividein the culture being transfected. This results in suboptimal expression.Suboptimal FuGENE®HD TransfectionReagent:DNA ratio,complex incubationtime, total amount oftransfection complexadded, or cell densityOptimize the FuGENE® HD Transfection Reagent:DNA ratio, complex incubation time, amount ofcomplex added to cells, and cell density, according to the following procedure:Day before transfection:Prepare two 96-well plates of cells at high and low seeding densities (see Table 2 for suggestions).Day of transfection:•Form 200 ␮l of transfection complex at ratios of 2:2, 3:2, 4:2, 5:2, 6:2. 7:2, and 8:2 (␮l transfectionreagent:␮g DNA) following the protocol in this pack insert (sections 2.3, 2.4) and doubling theamounts of all components.•As soon as the complexes are combined and mixed, add 10, 5, or 2.5 ␮l of each complex to one of3columns of cells in each 96-well plate (i.e., columns 2, 3, and 4). Leave all outer wells empty ascontrols.•Continue to incubate the complexes at room temperature. After an additional 10 – 15 minutes, add 10,5, or 2.5 ␮l of each complex to the next 3 columns (5, 6, and 7) of cells in each 96-well plate.•Continue to incubate the complexes at room temperature. After an additional 10 – 15 minutes, add 10,5, or 2.5 ␮l of each complex to the next 3 columns (8, 9, and 10) of cells in each 96-well plate.•Assay the plates 1–2 days later. Select the ratio, amount of complex, and time of transfection-complexincubation that resulted in optimal expression.•If optimal transfection occurs at the higher ratios, repeat this process using ratios of 6:2, 7:2, 8:2, 10:2,12:2, and 14:2. Add 5, 10, and 15 ␮l of complex. We have never successfully transfected cells using theratio of 2:2, but it has been reported that some plasmid preparations transfect at this ratio.See section 2.6, Optimization of FuGENE® HD Transfection Reagent:DNA ratio, for more information andvisit /fugene/hdFuGENE® HD Trans-fection Reagent wasaliquotedCheck that FuGENE® HD Transfection Reagent is stored in the original container. If the reagent wasaliquoted into plastic containers, there is a high chance of inactivation. Make sure the reagent isimmediately mixed with the dilute DNA either by vortexing or pipetting up to 10 – 15 times.FuGENE® HD Trans-fection Reagent cameinto contact withplastic or wasinadequately mixedRepeat transfection, carefully pipetting FuGENE® HD Transfection Reagent directly into the serum-freemedium, being careful not to touch the sides of the container while adding the FuGENE® HD Transfec-tion Reagent to the diluted DNA. If the FuGENE® HD Transfection Reagent is added too gently, it maylayer on top of the medium, thus making contact with the plastic.Transfection complexwas formed in serum-containing mediumCheck original bottle of medium used for complex formation. Repeat experiment using new bottle ofOpti-MEM that does not contain any additives (e.g., serum, antibiotics, growth enhancers, heparin,dextran sulfate, etc.). Try forming the complex in sterile water or plain DMEM.Media and mediacomponentsDifferent media and media components may influence the level of transfection efficiency andsubsequent growth of the transfected cells, as well as expression of the recombinant protein. Some lotsof sera have been reported to interfere with optimal transfection.Quality and/or lot-to-lot differences that affect transfection experiments have been noted in both seraand media. Check that the medium and/or serum is from the same lot that worked previously. Try newlots or a different vendor.Culture may becontaminated withmycoplasmaCultures contaminated with mycoplasma have been shown to have decreased transfection efficacy.Determine if culture is contaminated with mycoplasma; use the Mycoplasma Detection Kit* orMycoplasma PCR ELISA* to assess contamination.Inconsistent results Ratio or amount oftransfection complexis at the edge ofperformance plateauInitial experiments should be completed to determine the ratios, amount of complex to be added, andlength of time for complex formation for optimal performance. In our experience, we have found the pla-teau to be relatively broad. We recommend that future experiments be performed with ratios, incubationtime, and amounts of complex that were in the middle of the plateau. If conditions are selected at theedge of the plateau, very small procedural differences may cause large differences in the resulting pro-tein expression. Increased consistency may be achieved by shifting parameters away from the edge ofthe plateau to the middle of the plateau.Transfection complexformation:timing,amounts, and ratioFormation of the complex involves a multifaceted interaction between the transfection reagent and DNAas well as biological parameters. Differences in any of the components or techniques may result ininconsistencies. If results do not meet your expectations, then repeat the optimization experimentselecting areas near the plateau found in previous experiments. For current experiments, determine ifyou should use a different ratio, length of time, or amount of complex for more consistent transfectionresults.Extensive testing of the FuGENE® HD Transfection Reagent is performed on two cell lines: one easy totransfect and one very difficult to transfect. All reagent lots must pass this rigorous testing before wemake it available to you. However, we cannot test all cell lines, media, sera, and vectors; in your labora-tory, you may find slight differences in the optimal ratio, amount of complex, or time for complex forma-tion for some lots of FuGENE® HD Transfection Reagent.Cells For consistent results, cells must be properly maintained. Cells change with passage level, passage conditions, media, and sera. For some cell lines, these changes have little to no effect on transfectionexperiments, but for other cell lines, these changes have profound effects. Each cell type may have a dif-ferent optimal transfection condition. Optimal values for a single cell type may also change slightly withvector construct and type of protein expressed.Observation Possible cause RecommendationSigns of cytotoxicity Transfected protein iscytotoxic or isproduced at highlevelsReduced viability or slow growth rates may be the result of high levels of protein expression, as the cell’smetabolic resources are directed toward production of the heterologous protein. The expressed proteinmay also be toxic to the cell at the level expressed.To analyze cytotoxicity, prepare experimental controls as described below.Prepare extra control wells containing:ቢ Cells that are not transfectedባ Cells treated with DNA alone (e.g., without FuGENE® HD Transfection Reagent)ቤ Cells treated with FuGENE® HD Transfection Reagent alone (no DNA added)ብ Cells transfected with a non-toxic or secreted protein.Compare experimental transfected cells to cells in the control wells (described above). Considerrepeating the experiment with a secreted reporter gene such as SEAP, hGH, or a standard ␤-gal controlvector. Cells expressing SEAP should show little to no evidence of cytotoxicity.Too much transfec-tion complex fornumber of cellsIncrease the number of cells plated, and/or decrease the total amount of complex added to the cells. Trydifferent ratios and allow the complexes to form for different time intervals. Add different amounts ofcomplex; for example, make the complex as usual but add 75%, 50%, or 25% of the usual amounts toeach well. See Suboptimal FuGENE® HD:DNA Ratio in "Low transfection efficiency" section of this tablefor details or optimization protocol.Culture may becontaminated withmycoplasmaDetermine if culture is contaminated with mycoplasma; use the Mycoplasma Detection Kit* orMycoplasma PCR ELISA* to assess contamination.Cells may not behealthyAssess physiological state of cells and the incubation conditions (e.g., check incubator CO2, humidity,and temperature levels). Observe cells prior to each passage for morphology and absence of contami-nants. Make sure cells do not overgrow. Routinely passage cells prior to reaching confluency. Make surethat culture media and additives are within expiration date and have been stored properly.Diluent is toxic to thecellsDMEM is toxic to some insect cell lines. For these cells, prepare the transfection complex in sterilewater. You may also try forming the complex in the medium in which the cells are growing, providingthat the medium does not contain serum, heparin, or dextran sulfate.Plasmid preparationcontaminated withendotoxinEndotoxin is reported to be cytotoxic to sensitive cell lines.If above tests provenegative, FuGENE®HD TransfectionReagent may not beoptimal for your cells.Try FuGENE® 6 Transfection Reagent*, DOTAP Liposomal Transfection Reagent*, DOSPER LiposomalTransfection Reagent*, or X-tremeGENE Q2 Transfection Reagent*.High protein-expression levelsHigh expression levels of certain intracellular proteins (e.g., Green Fluorescent Protein [GFP]) may becytotoxic to some cell types. Cell proliferation, toxicity, and cell death may be monitored using Apoptosisand Cell Proliferation products from Roche Applied Science (visit /apoptosis for more information).Media and mediacomponentsTest different media and optimize the level of each medium component for these cytotoxic effects.Although it is not usually necessary to remove the transfection complex following the transfection step,it may be necessary to feed your cells with fresh media for extended growth periods. This is particularlyimportant if the transfected cells are allowed to continue to grow for 3 – 7 days to provide maximalprotein expression.。

WHO_TRS_937__annex8_eng

© World Health OrganizationWHO Technical Report Series, No. 937, 2006Annex 8Proposal to waive in vivo bioequivalence requirements for WHO Model List of Essential Medicines immediate-release, solid oral dosage forms Introduction1. Background2. WHO revisions to the criteria for Biopharmaceutics Classiﬁ cation Systemclassiﬁ cation3. WHO extensions to the scope of application of the biowaiver4. WHO additional criteria for application of the biowaiver procedure5. Explanation of the tables6. Biowaiver testing procedure according to WHOIntroductionThis proposal is closely linked to the Multisource (generic) pharmaceutical products: guidelines on registration requirements to establish interchange-ability (WHO Technical Report Series, No. 937, Annex 7). It aims to give national authorities sufﬁ cient background information on the various orally administered active pharmaceutical ingredients (APIs) on the WHO Model List of Essential Medicines (EML), also taking into account local usage of the API, to enable them to make an informed decision as to whether generic formulations should be subjected to in vivo bioequivalence (B E) studies or whether a biowaiver can be granted. In light of scientiﬁ c work and dis-cussion in the last decade, some of the criteria used to evaluate the API in terms of potential for a biowaiver have been revised to allow a broadened scope of application. The result is that many APIs on the EML can now be considered for the biowaiver procedure, subject to the usage and risks in the national setting.1. Background1.1Initiatives to allow biowaivers based on the BiopharmaceuticsClassiﬁ cation SystemIn 1995 the American Department of Health and Human Services, US Food and Drug Administration (HHS-FDA) instigated the B iopharmaceutics391Classiﬁ cation System (BCS), with the aim of granting so-called biowaiv-ers for scale-up and post-approval changes (SUPAC) (/cder/ guidance/cmc5.pdf). A biowaiver means that in vivo bioavailability and/or bioequivalence studies may be waived (i.e. not considered necessary for product approval). Instead of conducting expensive and time-consuming in vivo studies, a dissolution test could be adopted as the surrogate basis for the decision as to whether two pharmaceutical products are equivalent. At that time the biowaiver was only considered for SUPAC to pharmaceutical products.More recently, the application of the biowaiver concept has been extended to approval of certain orally administered generic products (/ cder/guidance/3618fnl.htm).Within the context of the documents cited above, only APIs with high solu-bility and high permeability and which are formulated in solid, immediate-release (IR) oral formulations can be approved on the basis of the biowaiver procedure. A major advantage of the biowaiver procedure is the simpliﬁ ca-tion of the product approval process and the reduction of the time required, thus reducing the cost of bringing new products to market.1.2What is the Biopharmaceutics Classiﬁ cation System?The Biopharmaceutics Classiﬁ cation System (BCS) was proposed in 1995 by Amidon et al.1 It is a scientiﬁ c framework which divides APIs into four groups, according to their solubility and permeability properties.1.3 Classiﬁ cation of active pharmaceutical ingredients accordingto the Biopharmaceutics Classiﬁ cation SystemAccording to the HHS-FDA deﬁ nitions in the documents cited above, the four possible categories for an API according to the BCS are:•BCS class I: “high” solubility – “high” permeability•BCS class II: “low” solubility – “high” permeability•BCS class III: “high” solubility – “low” permeability•BCS class IV: “low” solubility – “low” permeability.Depending on the classiﬁ cation, the oral availability of the API may be expected to range from being heavily dependent on the formulation and manufacturing method (e.g. Class II APIs: poorly soluble yet highly perme-able) to being mostly dependent on the API permeability properties (e.g.Class III APIs: highly soluble yet poorly permeable).1Amidon GL, Lennemas H, Shah VP, Crison JR. A theoretical basis for a biopharmaceutic drug classiﬁ cation: the correlation of in vitro drug product dissolution and in vivo bioavailability. Phar-maceutics Research, 1995, 12:413–420.3921.4How is high or low solubility currently deﬁ ned by the Departmentof Health and Human Services, US Food and Drug Administration?The aqueous solubility of a drug substance is considered as high according to the HHS-FDA BCS criteria when:• the ratio of the highest orally administered dose (in mg) to the solubility (mg/ml) is 250 ml or lower.—This criterion is met over the pH range 1–7.5 at 37 °C.According to HHS-FDA guidances, the determination of the equilibrium solubility should be carried out with the shake-ﬂ ask method (other methods such as acid or base titration are permitted when their ability to predict the equilibrium solubility is justiﬁ ed). The experiments should be carried out at a temperature of 37 ± 1°C. Further, a sufﬁ cient number of pH conditions should be chosen to cover the pH range of 1–7.5 and each determination should be carried out at least in triplicate. The buffer solutions given in the United States Pharmacopeia (USP) are appropriate for the tests, but other buffers are also allowed for these experiments. The pH value of each buffer solution should be checked before and after each experiment. Degradation of the API due to pH or buffer composition should be reported together with other stability data.The reason for the 250-ml cut-off criterion for the dose:solubility ratio is that in pharmacokinetic bioequivalence studies, the API formulation is to be ingested with a large glass of water (8 ounces corresponds to about 250 ml). If the highest orally administered dose can be completely dissolved in this amount of water, independent of the physiological pH value (hence the determination over the pH range 1–7.5), solubility problems are not expected to hinder the uptake of the API in the small intestine.The other important parameter for the BCS is the intestinal permeability of the API.1.5How is high or low permeability currently deﬁ ned by the Departmentof Health and Human Services, US Food and Drug Administration?According to HHS-FDA a drug is considered highly permeable, when 90 % or more of the orally administered dose is absorbed in the small intestine.Permeability can be assessed by pharmacokinetic studies (for example, mass balance studies), or intestinal permeability methods, e.g. intestinal perfusion in humans, animal models, Caco 2 cell lines or other suitable, validated cell lines. In vivo or in situ animal models or in vitro models (cell lines) are only considered appropriate by HHS-FDA for passively trans-ported drugs. It should be noted that all of these measurements assess the fraction absorbed (as opposed to the bioavailability, which can be reduced substantially by ﬁ rst-pass metabolism).393HHS-FDA suggests use of two different methods for determining the per-meability classiﬁ cation if results with one method are inconclusive.1.6Which pharmaceutical formulations can currently be consideredfor a biowaiver according to the Department of Health andHuman Services, US Food and Drug Administration?To be considered bioequivalent according to the HHS-FDA biowaiver pro-cedure, a pharmaceutical product:• should contain a Class I API;• should be rapidly dissolving, meaning it should release at least 85% of its content in 30 minutes in three different media (pH 1.2, pH 4.5 and pH6.8, composition see “Multisource document”)1 in a paddle (50 rpm) orbasket (100 rpm) apparatus at 37 °C and a volume of 900 ml;• should not contain excipients which could inﬂ uence the absorption of the API;• should not contain an API with a narrow therapeutic index; and• should not be designed to be absorbed from the oral cavity.The reasoning for the above-mentioned dissolution restrictions is that whena highly soluble, highly permeable API dissolves rapidly, it behaves like asolution in the gastrointestinal tract. If this is the case, the pharmaceutical composition of the product is insigniﬁ cant, provided that excipients which inﬂ uence the uptake across the gut wall are excluded from the formulation.The API is not prone to precipitation after its dissolution due to its good solu-bility under all pH conditions likely to be found in the upper gastrointestinal tract. The high permeability ensures the complete uptake (> 90%) of the API during its passage through the small intestine. The rapid dissolution of the product guarantees that the API is available long enough for the uptake in the small intestine (the passage time in the small intestine is approximately four hours) and negates any slight differences between the formulations.Pharmaceutical products containing an API with a narrow therapeutic index should always be tested with in vivo methods, because the risk to the patient resulting from a possible incorrect bioequivalence decision using the bio-waiver procedure is considered too high with these kinds of APIs.As the BCS is only applicable to APIs which are absorbed from the small intestine; drugs absorbed from other sites (e.g. from the oral cavity) are not eligible for a biowaiver.It is clear that the HHS-FDA requirements for the classiﬁ cation of APIs and eligibility criteria for the biowaiver are very strict. During the last decade,1Multisource (generic) pharmaceutical products: guidelines on registration requirements to establish interchangeability (WHO Technical Report Series, No. 937, Annex 7).394several publications and continuing scientiﬁ c discussions have suggested that the original HHS-FDA criteria for application of the biowaiver pro-cedure could be relaxed without substantially increasing the risk to public health or to the individual patient. On the basis of these publications and dialogue, WHO has proposed revised BCS criteria and additional consid-erations for the eligibility of a pharmaceutical product for the biowaiver procedure in the “Multisource document”.12.WHO revisions to the criteria for BCS classiﬁ cationWHO revisions to the BCS criteria are as follows:•WHO high-solubility deﬁ nitionWhen an API shows a dose:solubility ratio of 250 ml or lower at 37 °C over a pH range of 1.2–6.8, it can be classiﬁ ed as “highly soluble”. The decrease in pH from 7.5 in the FDA guidances to 6.8 reﬂ ects the need to dissolve the drug before it reaches the mid-jejunum to ensure absorption from the gastrointestinal tract.• Furthermore, the dose that is to be used for the calculation is the highestdose indicated in the Model List of Essential Medicines (EML). In some countries, products may be available at doses exceeding the highest dose on the EML. In such cases, the classiﬁ cation given in the tables at the end of this Annex may no longer be appropriate and the dose:solubil-ity ratio and the permeability will have to be reassessed at the product dose.•WHO permeability deﬁ nitionWhen an API is absorbed to an extent of 85% or more, it is considered to be “highly permeable”. The permeability criterion was relaxed from 90% in the FDA guidance to 85% in the WHO “Multisource document”.Some examples of APIs now included in BCS Class I that were previ-ously considered to be in Class III are paracetamol, acetylsalicylic acid, allopurinol, lamivudine and promethazine.Application of these revised criteria has changed the classiﬁ cation of some APIs in the list. Thus, the classiﬁ cations in the tables attached to this docu-ment supersede those in previous publications. As new APIs appear on the EML, it will be necessary to classify them according to the revised BCS;so it is therefore anticipated that the tables will be revised regularly. In addition, some APIs have not yet been sufﬁ ciently characterized to assign them a BCS classiﬁ cation. As the tables evolve, it is anticipated that more concrete information will be generated for these APIs as well.1Multisource (generic) pharmaceutical products: guidelines on registration requirements to establish interchangeability (WHO Technical Report Series, No. 937, Annex 7).395the basket apparatus (applies to pharmaceutical products containingClass III APIs);—rapidly dissolving (release of > 85% of the labelled amount of drug in 30 minutes) in standard media at pH 1.2, 4.5 and 6.8, at a rota-tional speed of 75 rpm in the paddle apparatus or 100 rpm in the bas-ket apparatus (applies to pharmaceutical products containing Class IAPIs and/or Class II APIs which are weak acids and meet the 250 mldose:solubility requirement at pH 6.8).(4)Considerations relating to excipientsThe national authority should be aware that some excipients can inﬂ uencemotility and/or permeability in the gastrointestinal tract. Therefore, the ex-cipients used in the multisource product formulation should be scrutinized.In this regard, the national authority can draw on the experience relat-ing to formulations which have been approved on the basis of humanbioequivalence studies in their own or in other jurisdictions.If the multisource product under consideration contains excipients thathave been used before in similar amounts in other formulations of thesame API, it can be reasonably concluded that these excipients will haveno unexpected consequences for the bioavailability of the product. If,however, the formulation contains different excipients, or amounts ofthe same excipients that are very different from usual, the national au-thority may choose to declare the biowaiver procedure inapplicable.A list of usual and acceptable excipients can be found at the following website: /cder/iig/iigfaqWEB.htm; formulations of some productscan be found on the web sites of some national drug regulatory authorities.5.Explanation of the tablesThe decision of a national authority to allow a biowaiver based on the BCS should take into consideration the solubility and permeability char-acteristics as well as the therapeutic use and therapeutic index of the API, its pharmacokinetic properties, the similarity of the dissolution proﬁ les of the multisource and the comparator products in standard buffers with a pH of 1.2, pH 4.5 and pH 6.8 at 37 °C. Data related to the excipients compo-sition in the multisource product are also required. A systematic approach to the biowaiver decision has been established by the International Pharma-ceutical Federation (FIP) and published in the Journal of Pharmaceutical Sciences (/cgi-bin/jhome/68503813).The relevant documents can also be downloaded from the FIP web site at: http://www.ﬁ/. These monographs provide detailed information which should be taken into account whenever available in the biowaiver consideration.3985.1Which active pharmaceutical ingredients are included in thetables?The substances listed in the 14th WHO Model List of Essential Medicines (EML) of March 2005 have been evaluated and classiﬁ ed according to the revised criteria given above.5.2Where do the data come from?The solubility and permeability values were found in the publicly available literature, such as Martindale’s, the Merck Index and scientiﬁ c journals.Please note that the doses used for the calculation of the dose:solubility ratio are those stated in the EML.The indications given in the tables are reproduced directly from the EML. If the EML speciﬁ es the dosage form (e.g. sublingual tablet) this is indicated under “comments”.5.3“Worst case” approach to the Biopharmaceutics Classiﬁ cationSystemThe drugs listed in the EML were classiﬁ ed according to the criteria explained above. Where no clear classiﬁ cation could be made, the “worst case” was as-sumed. For example if a substance is highly soluble, but absolute bioavailability data were not available, the test conditions for BCS Class III substances have been proposed. The same procedure was adopted for ﬁ xed combinations, for example amoxicillin and clavulanic acid, the testing procedure was always ﬁ xed according to the “worst” BCS classiﬁ cation, in this example clavulanic acid (BCS Class III/1), because amoxicillin is a BCS Class I drug. This com-bination would therefore be tested according to BCS Class III requirements.The results of the revised classiﬁ cation can be found in Tables 1–3.5.4Why are there three Tables?Table 1 lists all APIs on the EML that are administered orally, with the excep-tion of the APIs listed as complementary. Table 2 summarizes the APIs listed as complementary in the EML and Table 3 lists the APIs for which no classiﬁ cation had previously been assigned, or that had been introduced with the 14th EML (March 2005), together with a more detailed explanation of their classiﬁ cation.5.5 Risk assessmentTo minimize the risks of an incorrect biowaiver decision in terms of public health and risks to individual patients, the therapeutic indications of the API, known pharmacokinetic variations, food effects, etc. should be evalu-ated based on local clinical experience, taking into account the indications399for which the API is prescribed in that country as well as speciﬁ c pharmaco-kinetic population variations (for example CYP polymorphisms). Known potential risks are listed under “potential risks” in the tables. The absence of an entry under “potential risks” should not, however, be misconstrued as meaning that there are no risks associated with the use of the medicine. 6.Biowaiver testing procedure according to WHODepending on the BCS classiﬁ cation of the API, based on solubility and permeability characteristics listed in the accompanying tables, the testing procedure is deﬁ ned in section 9.2.1 of the “Multisource document”1:6.1For pharmaceutical products containing BiopharmaceuticsClassiﬁ cation System Class I (highly soluble, highlypermeable) APIsFor rapidly dissolving (as deﬁ ned above) pharmaceutical products contain-ing BCS Class I APIs, more than 85% dissolution of the labelled amount is required within 30 minutes in standard media at pH 1.2, 4.5 and 6.8 using the paddle apparatus at 75 rpm or the basket apparatus at 100 rpm. The dis-solution proﬁ les of the comparator and the multisource products should be compared by an f> 50 or an equivalent statistical criterion.2If within 15 minutes more than 85% of the API are released from the compar-ator and the multisource formulation under the above-mentioned conditions the products will be considered very rapidly dissolving. In this case the prod-ucts are deemed to be equivalent and a proﬁ le comparison is not required.6.2For pharmaceutical products containing BiopharmaceuticsClassiﬁ cation System Class III (highly soluble, lowpermeability) APIsA biowaiver can be considered only if both the multisource and the com-parator product are very rapidly dissolving. Eighty-ﬁ ve per cent or more dissolution of the labelled amount of the API should be achieved within15 minutes in standard media at pH 1.2, 4.5 and 6.8 using the paddle ap-paratus at 75 rpm or the basket apparatus at 100 rpm.Generally, the risks of an inappropriate biowaiver decision should be more critically reviewed (e.g. site-speciﬁ c absorption, induction/competition at the absorption site, excipient composition and therapeutic risks) for prod-ucts containing BCS Class III APIs than for BCS Class I drugs.1Multisource (generic) pharmaceutical products: guidelines on registration requirements to establish interchangeability (WHO Technical Report Series, No. 937, Annex 7).4006.3For pharmaceutical products containing APIs with highsolubility at pH 6.8 but not at pH 1.2 or 4.5 and with highpermeability (by deﬁ nition, BCS Class II compoundswith weak acidic properties)These are eligible for a biowaiver provided that the multisource product:• is rapidly dissolving, i.e. 85% or more dissolution of the labelled amount of the API should be achieved within 30 minutes in standard media at pH 6.8 using the paddle apparatus at 75 rpm or the basket apparatus at 100 rpm; and• the multisource product exhibits similar dissolution proﬁles, as deter-mined with the f2 value or equivalent statistical evaluation, to those ofthe comparator product in buffers at all three pH values (pH 1.2, 4.5 and6.8).For multisource products containing BCS Class II APIs with dose:solubility ratios of 250 ml or less, at pH 6.8, the excipients should also be critically evaluated in terms of type and amounts of surfactants in the formulation.Further details of eligibility for the biowaiver and appropriate test proce-dures can be found in sections 5 and 9 of the “Multisource document”.11Multisource (generic) pharmaceutical products: guidelines on registration requirements to establish interchangeability (WHO Technical Report Series, No. 937, Annex 7).401405c h l o r p h e n a -m i n e h yd r o ge n m a l e a t e 4 m g h i g hB A 25-59%, ﬁ r s t p a s s 3/19.2.1.2C Y P 2D 6 p o l y -m o r p h i s m a n t i a l l e r g i ce x t e n t of ﬁ r s t -p a s s m e t a b o l i s m u n c e r t a i nc h l o r p r o m a z i n e h yd r o c h l o r i de 100 m gh i g hl o w39.2.1.2 p s y c h o t h e r a p e u -t i c m e d i c i n e c i p r o ﬂ o x a c i nh y d r o c h l o r i d e 250 m g h i g hB A 70–82%, p o s s i b l e ﬁ r s t p a s s , h i g h i nC a c o -2 c e l l s3/19.2.1.2 a n t i b a c t e r i a le x t e n t of ﬁ r s t - p a s s m e t a b o l i s m u n c e r t a i nc l o f a z i m i n e100 m gi n s u f ﬁ c i e n t l i t e r a t u r e l o w 4/3N o t e l i g i b l e f o r b i o w a i v e r a t p r e s e n t a n t i l e p r o s y m e d i c i n ec l o m i f e n e c i t r a t e50 m g h i g h i n s u f ﬁ c i e n t l i t e r a t u r e 3/19.2.1.2o v u l a t i o n i n d u c e rc l o m i p r a m i n e h yd r o c h l o r i de 25 m g h i g h66% e x c r e t e d i n t h e u r i n e , t h e r e m a i n d e r b e i n g e l i m i -n a t e d i n t h e f a e c e s 3/19.2.1.2p s y c h o t h e r a p e u -t i c m e d i c i n el a c k o f a b s o l u t e b i o a v a i l a b i l i t y d a t ac l o x a c i l l i n (a s s od i u m s a l t )1000 m g h i g hl o w 39.2.1.2a n t i b a c t e r i a lc ode i n e p h o s p h a t e 30 m g h i g h l o w39.2.1.2r i s k o f a b u s eo p i o i d a n a l g e s i c ,d i a r r h oe a i n a d u l t sd a p s o n e100 m gl o w (w e a k b a s e ) h i g h 2N o t e l i g i b l e f o r b i o w a i v e rG 6P D d e ﬁ -c i e n c ya n t i l e p r o s y m e d i c i n ed i a ze p a m 5 m g h i g hh i g h19.2.1.1p s y c h o t h e r a p e u -t i c m e d i c i n e s c o r e d t a b l e tB A , B i o a v a i l a b i l i t y ; G 6P D , g l u c o s e -6-p h o s p h a t e d e h y d r o g e n a s e .409g l y c e r y l t r i n i t r a t e 500 μgh i g hs u b l i n g u a l a p p l i c a t i o n ,p e r m e a b i l -i t y i n t h e o r a l c a v i t y m o r e i m p o r t a n t t h a n G I p e r m e a b i l i t y3/1N A hl o c a l a b s o r p t i o n a n t i a n g i n a l m e d i c i n e s u b l i n g u a l a p p l i c a t i o ng r i s e o f u l v i n 250 m gl o w (n e u t r a l ) h i g h2N o t e l i g i b l e f o r b i o w a i v e r a n t i f u n g a lh a l o p e r i d o l2 m gb o r d e r l i n e < 0.01 m g /m l 2l o w 4/3N o t e l i g i b l e f o r b i o w a i v e rp s y c h o t h e r a p e u -t i c m e d i c i n eh y d r a l a z i n e h y d r o c h l o r i d e50 m g h i g hl o w 39.2.1.2a n t i h y p e r t e n s i v e m e d i c i n eh y d r o c h l o r o -t h i a z i d e 25 m g h i g h l o w 39.2.1.2a n t i h y p e r t e n s i v e m e d i c i n e , d i u r e t i c a n d u s e d i n h e a r t f a i l u r es c o r e d t a b l e ti b u p r o f e n 400 m gl o w , w e a k a c i d (p K a 4.4,5.2)h i g h 29.2.1.3N S A I D , a n t i m i -g r a i n e m e d i c i n ei n d i n a v i r s u l f a t e 400 m g l o w l o w (?)4/2N o t e l i g i b l e f o r b i o w a i v e r C Y P 450 3A 4, f o o d e f f e c t (–)a n t i r e t r o v i r a lu n k n o w n w h e t h e r p o o r B A i s d u e t o p o o r s o l u b i l i t y o r p o o r s o l u b i l i t y a n d p o o r p e r m e a b i l i t yD :S , D o s e :s o l u b i l i t y r a t i o ; B A , b i o a v a i l a b i l i t y .426T a b l e 3C o m p o u n d s i n t r o d u c e d t o t h e W H O M o d e l L i s t o f E s s e n t i a l M e d i c i n e s s i n c e M a r c h 2005 f o r w h i c h n o c e r t a i n c l a s s i ﬁ c a t i o n h a d b e e n p r e v i o u s l y r e p o r t e d (t h e s e c o m p o u n d s a l s o a p p e a r i n T a b l e 1 a n d T a b l e 2)M e d i c i n e aH i g h e s t o r a l s t r e n g t h a c c o r d i n g t o W H O E s s e n t i a l M e d i c i n e s L i s t a S o l u b i l i t y bP e r m e a b i l i t y c B C S c l a s s dD i s s o l u t i o n t e s t (f o r b i o w a i v e r )e P o t e n t i a l r i s k s fI n d i c a t i o n (s )a c c o r d i n g t o W H O E s s e n t i a l M e d i c i n e s L i s t (E M L )aC o m m e n t s a n d s p e c i a l d o s a g e f o r m i n d i c a t i o n s aa m l o d i p i n e 5 m gs l i g h t l y s o l u b l e (1),D :S 5 m lB A a b s60–65%,e x c r e t i o n o f d r u g m e t a b o -l i t e s i n u r i n e 90–95% (2)19.2.1.1a n t i h y p e r t e n s i v e m e d i c i n eB A a b s < 85% a s c r i b e d t o ﬁ r s t -p a s s m e t a b o l i s ma m o d i a q u i n e(b a s e )200 m g45 m g /m l 2,D :S 4.4 m lB A > 75% (3)3/19.2.1.2C Y P 2C 8p o l y m o r p h i s m ,i n c r e a s e d r i s k f o r a g r a n u l o c y -t o s i s a n d h e p a -t o t o x i c i t y (4)a n t i m a l a r i a la m o x i c i l l i n + c l a v u l a n i c a c i d 500 m g + 125 m gf r e e l y s o l u b l e i n w a t e r (1),D :S 1.25 m la b s o r p t i o n > 73% (5)1 + 3/19.2.1.2a n t ib ac t e r i a lt e s t s b a s e d o n c l a v u l a n i c a c i d c l a s s i ﬁ c a t i o na r t e s u n a t e 50 m gv e r ys l i g h t l y s o l u b l e (6),D :S 500 m l ;(w e a k a c i d ,p K a ~ 6.4)B A a b s 82% (1),B A a b s 88% (7),B A a b s 61% (8)4/2N o t e l i g i b l e f o r b i o w a i v e ra n t i m a l a r i a lp e r m e a b i l i t y d e p e n d s o n s e v e r i t y o f d i s e a s eD :S , D o s e : s o l u b i l i t y ; B A , B i o a v a i l a b i l i t y .427a z i t h r o m y c i n 500 m gp r a c t i c a l l y i n s o l u b l e i n w a t e r (1)< 0.01m g /m l , D :S 50 000 m lB A a b s 16% (9);B A 37%(10, 11); 4/2N o t e l i g i b l e f o r b i o w a i v e ra n t ib ac t e r i a l u n k n o w n w h e t h e r p o o r B A i sd ue t o p o o r s o l u b i l i t y o r p o o r s o l u b i l i t y a n d p o o r p e r m e a b i l i t yc a l c i u m f o l i n a t e 15 m gs p a r i n g l y s o l u b l e i n w a t e r (P h . E u r . 5.2); v e r y s o l u b l e (U S P 28); D :S 15 m l a n d 0.015 m l , r e s p e c -t i v e l yB A a b s 92% 25 m g (12, 13);B A a b s 73.4%(15 m g ) (14);f u l l y a b s o r b e d ;A UC a n d t 1/2s i m i l a r a f t e r i.v . & p .o (15)19.2.1.1 a n t i c y t o t o x i c m e d i c i n el e v o d o p a (l ) + c a r b i d o p a (c )(l ) 250 m g + (c ) 25 m g(l ) h i g h +(c ) s o l u b l e 1 i n 500 o f w a t e r , f r e e l y s o l u b l e i n 3 M H C l (1)(l ) h i g h +(c ) B A 58% (16); B A a b s88% (d o g s ) (17)(l ) 1 +(c ) 3/19.2.1.2n a r r o w t h e r a p e u t i c i n d e xa n t i p a r k i n s o n m e d i c i n et e s t s b a s e d o n c a r b i d o p a c l a s s i ﬁ c a t i o nc e ﬁ x i m e 400 m gs l i g h t l ys o l u b l e (2),D :S 400 m l22–54% (2)4N o t e l i g i b l e f o r b i o w a i v e ra n t ib ac t e r i a lD :S , D o s e : s o l u b i l i t y ; B A : B i o a v a i l a b i l i t y ; P h .E u r ., E u r o p e a n P h a r m a c o p o e i a ; U S P , U n i t e d S t a t e s P h a r m a c o p o e i a ; A U C , a r e a u n d e r t h e c u r v e ; i.v ., i n t r a v e n o u s .。

骨源性细胞因子在激素性股骨头坏死发生发展中的研究进展

-综述•骨源性细胞因子在激素性股骨头坏死发生发展中的研究进展蒋捷,黄林科，胡峰△(广西医科大学第二附属医院，广西南宁530007)［摘要］股骨头坏死是骨外科常见的难治性疾病,其机制仍有待研究。

目前为止,医源性糖皮质激素是非创伤性股骨头坏死的主要原因。

激素的长期使用可导致股骨头骨细胞凋亡、血液循环障碍所致缺血缺氧，最终导致股骨头塌陷。

激素性股骨头坏死的发生发展与骨组织中细胞直接接触和其间接分泌的细胞因子调控相关。

本文综述了骨组织中成骨细胞分泌的核因子k B受体结合配体，骨保护素,骨碱性磷酸酶，骨细胞表达硬化素，破骨细胞分泌骨形态发生蛋白2等因子在SONFH的研究进展，骨源性细胞因子在SONFH中扮演重要作用。

［关键词］骨源性细胞因子;激素性股骨头坏死;临床分型;病理表现;综述［中图分类号］R681［文献标识码］A［文章编号］961-5639(2621)61-482-04doi:16.3969/E R w x961-5639.402000025Resevrch prggress of bone derived cytokines in developmenS of steaid induced osteynecrgsis of the femorai hecdJIANG Jia,HUANG Lia-Ue,HU Feng'(Department of OrtUopebic trauma,the Second Affiliated Hospital of Guanypi Medical University?Nanning Citp,Guangpi Zhuang Autonomous Repiox530007,China)J Abstrach]Avascular necrosis of the femoral heah is a common refracton disease in ortUopebic shraeru/whose mechanism remains to be studied.So far,iatngenic glucocorncoids are the main cause of nox-EaumaWe necrosis of the femoral heah.Long term treatment of glucocorncoids leafs to osteocyte apoptosis of femoral heah,ischemia and hypoxia caused bp blood cinulaWon OisorUer,and eventuaLy leafs to coXapse of femoral heah.The occurrence and Oevelopment of steroid induced osteonecrosis of the femoral heah are related to the direct contact of cells in bone tissue and the repulaLox of cypdines secreted indirectly.We reviewed the research progress of Nuclear factor kaypa B receptor binding ligand,RANKL,OsPoproPpeEn；OPG and Bone aldaLne phosphamse；BAP secreted bp osteoblasts, eceeeohnt(SOST)peoducedbsoeheocsheeatdBotemoepeogetehncpeohent-2BMP-2eeceehedbsoeheoceaehenteheeondntducedoeheote-eeoeneoeheeeemoeaeeead0Botedeeneedeshoenteepeasatnmpoehatheoeenteheeondntdueedoeheoteeeoeneoeheeeemoeaeeead0J Key woras]Bone PeEved cytodines;2proid induced necrosis of the femoral heah；Clinical classification;PatUologicai manifestation；Reenew股骨头坏死(OWewecrwis of femoral heah,ONFH)是骨外科常见的难治性疾病,严重影响患者的生活质量。

Toyobo ReverTra Ace qPCR RT Master Mix技术手册说明书

Ｆ1173ＫReverTra Ace® qPCR RT Master MixFSQ-201 200 reactionsStore at -20°C Contents[1] Introduction[2] Components[3] Protocol1. RNA template for reverse transcription2. Reverse transcription[4] Application data[5] Related protocol1. DNase I treatment of total RNA[6] Troubleshooting[7] Related productsC AUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnostic or clinical purposes. Please observe general laboratory safety precautions while using this kit.-ReverTra Ace® is a registered trademark of Toyobo Co., Ltd., Japan.JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio 1JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************1[ 1 ] Introduction [ 2 ] Components DescriptionReverTra Ace® qPCR RT Master Mix is an efficient and convenient reagent to synthesize high quality cDNAs for real-time PCR. The master mix reagent (5x) contains the highly efficient reverse transcriptase “ReverTra Ace®”, primers and buffer optimized for highly efficient synthesis of short-chain cDNAs suitable for real-time PCR. The protocol is simple, and the reaction can be completed in 15 min.ReverTra Ace® is a mutant M-MLV reverse transcriptase that shows excellent efficiency. Features-5x Master Mix reagent contains all components for reverse transcription.The Master Mix reagent will not freeze at -20°C.-No reverse transcription control experiments (no RT-Control) can be performed.-The master mix reagent contains random and oligo dT primers optimized for efficient reverse transcription.-The reaction can be completed in 15 min. The protocol does not contain an additional RNase H treatment step to remove residual RNA after reverse transcription (Patent Pending).-Since the RT buffer is optimized for real-time PCR, the addition of 20% (v/v) of the synthesized cDNA solution to the PCR solution does not inhibit the PCR reaction.Therefore, this kit is suitable for the detection of low abundance mRNAs.The kit includes the following reagents, which can be used for 200 (FSQ-201) and 40 (FSQ-201S) 10 µl reactions. All reagents should be stored at -20°C. For extended storage, -30°C is recommended.FSQ-201FSQ-201S (SAMPLE) 5x RT Master Mix 400 μl80 μl5x RT Maser Mix no RT-Control 40 μl8 μlNuclease-free water 1000 μl x 2400 μl5× RT Maser MixThis reagent is a 5x master mix that contains highly efficient reverse transcriptase “ReverTra Ace®”, RNase inhibitor, oligo dT primer, random primer, MgCl2 and dNTPs .NotesBe aware that “5x RT Master Mix” and “5x RT Master Mix II” in ReverTra Ace® qPCR RT Master Mix with gDNA remover (Code No. FSQ-301) are not compatible.5× RT Maser Mix no-RT ControlThe composition of “5x RT Master Mix no-RT Control” is identical to that of “5x RT Master Mix” except that the reverse transcriptase (RT) is omitted. This master mix can be used in control experiments due to the absence of reverse transcriptase.Nuclease-free waterThis nuclease-free water has been prepared without DEPC treatment.JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio********************2[ 3 ] Protocol1. Template RNA for reverse transcriptionThe following RNAs are appropriate for highly efficient reverse transcription.(1)Total RNATotal RNA usually contains 1-2% mRNA. Total RNA can be used directly as template with this kit. RNA prepared using acid guanidium-phenol-chloroform (AGPC) or the spin-column method contains genomic DNA, so total RNA should be treated with DNase I before transcription.(2)Poly(A)+ RNA (mRNA)Poly(A)+ RNA is useful to detect low abundance mRNAs. However, poly(A)+ RNA should be treated carefully because it is more sensitive to RNase than total RNA.2. Reverse transcription(1) Denaturation of RNA [optional]Incubate the RNA solution at 65°C for 5 min, and then keep on ice.Notes-This step increases the efficiency of reverse transcription of RNA templates that form secondary structures.-This step should be performed before adding 5x RT Master Mix.(2) Preparation of the reaction solutionPrepare the following reagents on ice.Notes-The master mix reagent contains oligo dT and random primers. Do not use with specific primers.-For control experiments, “5x RT Master Mix no RT-Control” should be used instead of 5x RT Master Mix. A control experiment without reverse transcription is useful to prove whether amplicons originate from cDNA and/or genomic DNA.-The reaction volume can be increased according to need.-Master mix reagents should be spun-down prior to use due to high viscosity.5x RT Master Mix2 μl RNA template 1 pg – 1 μgNuclease-free Water X μl Total V olume10 μlJAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************3-This kit contains nuclease-free water for 200 reverse transcription reactions. The kit does not contain sufficient nuclease-free water for the dilution of RNA samples.Nuclease-free water prepared without DEPC-treatment is recommended for the dilution of RNA samples.(3)Incubate at 37°C for 15 min(4)Incubate at 50°C for 5 min [optional](5)Heat to 98°C for 5 min(6)Store the reacted solution* at 4°C or – 20°C*This solution can be used directly or after dilution for real-time PCR.Notes-The reaction time at 37°C can be prolonged up to 1 hr.-ReverTra Ace® excels at high reaction temperatures (up to 50°C). This step may increase the efficiency of the reverse transcription.-Up to 20% of the synthesized cDNA solution can be added to the PCR reaction solution.JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio********************4[ 4 ] Application data<Materials and Methods>cDNA synthesis Reagent: ReverTra Ace ® qPCR RT Master Mix (Code No.FSQ-201) Template: HeLa total RNA 2 pg-2 μg /20 μl reactionReal-time PCRReagent: THUNDERBIRD ® SYBR ® qPCR Mix (Code No.QPS-201) Template: cDNA 2 μl/20 μl reaction (cDNA solution: 10%) Targets: Typical house-keeping genes Real-time cycler: Applied Biosystems 7900HT<Results>Template RNA (pg)Log (RNA amount)Ct of qPCRATP5F TFRC RPLP1 RPLP2 RPS182 0.301 33.76 31.16 32.89 32.5420 1.301 31.43 30.73 27.70 30.05 28.65 200 2.301 28.64 27.29 24.44 26.72 25.22 2,000 3.301 25.41 23.79 21.12 23.31 21.98 20,000 4.301 21.86 20.43 17.69 19.88 18.42 200,000 5.301 18.65 17.09 14.14 16.59 15.10 1,000,000 6.000 16.03 15.03 11.63 14.37 13.09 2,000,000 6.301 15.42 14.28 11.11 13.53 12.28/20μl Slope -3.280 -3.303 -3.384 -3.284 -3.368R20.999 0.999 1.000 1.000 0.999 Eff.101.8%100.8%97.5% 101.6% 98.1%High linearity and no crossing over of the standard curves of five housekeeping genes suggest that the reagent shows high performance in a broad concentration range.JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************5[ 5 ] Related Protocol1. DNase I treatment of total RNATotal RNA prepared by general methods contains genomic DNA. Genomic DNA can beeliminated by the following method.(1)Mix the following reagents.Nuclease-free water X μlTotal RNA (<10 μg)Yμl10 x DNase I Buffer[e.g. 100 mM Tris-Cl, 20 mM MgCl2(pH 7.5)]1 μlRNase-free DNase I (10 U/μl)0.5μlTotal volume 10 μl(2)Incubate on ice for 10-30 min.(3)Purify the treated RNA according to the following step.DNase I-treated RNAAdd nuclease-free water (adjust volume to 100 μl)Add 100 μl TE-saturated phenolVortexKeep on ice for 5 minCentrifuge at 12,000 rpm for 5 minSupernatantAdd 100 μl chloroform: isoamyl alcohol (24:1), V ortexCentrifuge at 12,000 rpm for 5 min at 4 °CSupernatantAdd 100 μl 5 M ammonium acetate + 200 μl isopropanol+ [5 μl 2 mg/ml glycogen* (for coprecipitation) : optional]VortexIncubate at - 20 °C for 30 minCentrifuge at 12,000 rpm for 10-15 min at 4 °CDiscard supernatantPrecipitateAdd 1 ml 70% ethanolCentrifuge at 12,000 rpm for 5 minDiscard supernatantPrecipitateDissolve in appropriate volume of nuclease-free waterRNA solution*Molecular biology gradeJAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************6[ 6 ]TroubleshootingSymptom Cause SolutionLow signal afterreal-time PCRLow purity of RNA Repurify the RNA sample.Degradation of RNA Prepare fresh RNA sample. Diluted RNA templates havea tendency to degrade and to adsorb on the vessel walls.RNA template for the reaction should be prepared from ahighly concentrated stock prior to use.Excess or small amount of RNA The recommended RNA concentration range for reverse transcription is from 1 pg to 1 μg in a 10 μl reaction. However, the optimal concentration of RNA template should be determined for each case.Secondary structure of RNA template The efficiency of reverse transcription of RNAs that form secondary structures tends to be low. Incubation at 65°C for 5 min and quenching prior to the reaction is usually effective on such templates. Also, the additional step of 50°C for 5 min after the reaction at 37°C for 15 min might be effective for such difficult templates.Inappropriate temperature conditions Perform the reaction according to this instruction manual.Excess amount of cDNAsolution compared to thetotal PCR reaction volumeReduce the cDNA solution to less than 10%.Amplification in no-RT control reaction Contamination of genomicDNA in RNA templateRedesign the primers to prevent amplification fromgenomic DNA. Or treat the template RNA with DNase Iprior to reverse transcription.Primer dimer formation Optimize the PCR conditions or redesign the primers.HPLC-grade primers sometimes improve PCRspecificity.JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************7[ 7 ] Related productsProduct name Package Code No.High efficient revers transcriptaaseReverTra Ace®10,000 U TRT-101RNase inhibitor (Recombinant type) 2,500USIN-201 Real-time PCR master mix for probe assayTHUNDERBIRD® Probe qPCR Mix 1.67 mL x 3 QPS-101Real-time PCR master mix for SYBR® Green assayTHUNDERBIRD® SYBR® qPCR Mix 1.67 mL x 3 QPS-201High efficient cDNA synthesis kit for Real-time PCRReverTra Ace® qPCR RT Kit200 reactions FSQ-101High efficient cDNA synthesis master mix Real-time PCR with gDNA removerReverTra Ace® qPCR RT Master Mixwith gDNA remover200 reactions FSQ-301。

肝病专业英语词汇

3α-羟类固醇脱氢酶(Y' 蛋白) γ -谷氨酰转移酶 γ-氨基丁酸甲胎蛋白人兽共患病异种肝移植脂肪性纤维瘤,黄色瘤黄嘌呤氧化酶黄斑瘤全球移植中心名录窗口期肝豆状核变性肥达反应外斐反应韦克斯勒成人智力测验呕吐视觉诱发电位病毒学应答病毒复制病毒性肝炎静脉-静脉转流 VOD 肝小静脉闭塞病静脉-动脉转流血管活性肽静脉曲张胆管消失综合征疫苗熊去氧胆酸尿胆素原尿胆素二磷酸尿苷异构酶尿素生成鸟氨酸循环,尿素循环上消化道出血粗纤维调节素肝未分化肉瘤充盈不足学说非结合高胆红素血症游离胆红素,非结合胆红素超声(波)检查法
短潜伏期肝炎移动性浊音腹水白蛋白浓度梯度血清肝炎血清诊断血清胆红素血清白蛋白血清学应答乙肝血清学检查血清转换败血症相关胆汁淤积正链,有义链镇静剂次级胆酸海蓝组织细胞增多症硬化疗法硬化性胆管炎日本血吸虫病湄公血吸虫曼氏血吸虫日本血吸虫间插血吸虫埃及血吸虫血吸虫瘢痕形成期粗面内质网滚环机制(环状DNA复制的机理) RNA干扰核酶核糖体胆固醇逆向转运
伤寒 Ⅳ型胶原 Ⅲ型前胶原甲肝和乙肝疫苗抑癌基因肿瘤坏死因子肝结核滋养体甘油三酯颠换效应经颈静脉肝内门腔分流转换输血传染的病毒输血性肝炎转化生长因子
transcatheter arterial chemoembolization 肝动脉化疗栓塞 trans-activation toxic hepatitis total cholesterin total bilirubin tomor necrosis factor tocopherol TNF-related apoptosis inducing ligand tissue inhibitor of metalloproteinase thymosin Thymopolypeptides for Injection thromboxane the core promoter element tentative diagnosis tension of muscle tenderness Telbivudine taurocholic acid taurochenodeoxycholate acid systemic inflammatory response syndrome syncytial giant-cell hepatitis sustained virus response sustained response 反式激活中毒性肝炎总胆固醇总胆红素肿瘤坏死因子生育酚,维生素E 肿瘤坏死因子相关凋亡诱导配体基质金属蛋白酶组织抑制物胸腺肽胸腺肽血栓素核心启动子元件,启动子核心元件暂时的(假定的)诊断,试验性诊断肌张力压痛 LdT 牛(磺)胆酸牛磺鹅(去氧)胆酸盐,牛磺鹅(脱氧)胆酸盐全身炎症反应综合症融合巨细胞性肝炎持续病毒应答持久应答

01 APIC清洗验证指南(APIC,中文)

活性药物成分清洁验证指南APIC(Active Pharmaceutical Ingredients Committee)2000.12目录1.0 前言 (1)2.0 目标 (1)3.0 范围 (1)4.0 可接受标准 (1)4.1 简介 (1)4.2 可接受标准的计算方法 (1)4.2.1 基于治疗日剂量 (1)4.2.2 基于毒性数据 (3)4.2.3 一般限度 (4)4.2.4 擦拭限度 (5)4.2.5 淋洗限度 (11)5.0 清洁水平 (12)5.1 简介 (12)5.2 程序 (12)6.0 分类和最坏情况评估（WCR） (14)6.1 简介 (14)6.2 分类程序 (15)6.3 清洁程序 (17)6.4 调查及最坏情况评估 (18)6.5 最坏情况评估 (21)7.0 残留量确定 (24)7.1 简介 (24)7.2 验证要求 (24)7.3 取样方法 (29)7.4 分析方法 (31)8.0 清洁验证方案 (32)8.1 背景 (33)8.2 目的 (33)8.3 范围 (33)8.4 职责 (34)8.5 取样程序 (34)8.6 检测程序 (34)8.7 可接受标准 (35)8.8 与方案的偏差 (37)9.0 验证相关的问题 (37)10.0 参考书目 (40)11.0 术语 (40)12.0 版权和声明 (43)1.0 前言本指南由CEFIC的原料药委员会(APIC)清洁验证特别工作组所编写。

近年来，原料药厂的清洁验证这个主题受到了药政官员、公司与顾客的众多关注。

重要的是，对制剂生产公司的要求没有反馈到原料药制造企业的工艺中，而没有考虑在本阶段不同的工艺。

本文件的主要内容是原料药委员会的成员公司关于如何满足清洁要求以及如何在日常操作中执行清洁验证的讨论结果。

本文件应当与原料药委员会发布的名为“Cleaning Validation in Active Pharmaceutical Ingredient Manufacturing Plants”的文件一道阅读。

北京阿匹斯生物技术

L(+)-LACTIC ACID LITHIUM SALT
乳酸锂
AC0051A
TEST ASSAY SOLUBILITY(1%;H2O) WATER APPEARANCE WATER SPECIFICATIONS >99% CLEAR,COLORLESS <2% WHITE POWDER MAX. 2% VALUE 100.3% CONFORM 0.02% CONFORM 0.04%
2-Nitrophenyl β-D-galactopyranoside（ONPG）
邻硝基苯-β-D-半乳糖苷 AD0024B

Formula: C12H15NO8 Molecular Mass: 301.25 CAS Number: 369-07-3
HO O OH O OH OH
NO2
Appearance (color): Off-white Assay (HPLC): ≥ 98% Solubility (color): Light Yellow Specific rotation: -67~-70 degree (C-1% in water at 20 degree centigrade) Valid period: 3 years. (-20 degree)
p-Nitropheyl Phosphate disodium hexahydrate 对硝基苯磷酸二钠 AC0091A
TEST SPECIFICATIONS
APPEARANCE
WHITE TO YELLOW CRYSTALLINE SOLID
P-NITROPHENOL
<0.005%
WATER(KARL FISHER)
3 5-Dichloro-2-hydroxybenzene sulfonic acid sodium salt 3 5-二氯-2-羟基苯磺酸钠 AD0010B

The Soprophor Series_201007

杀虫剂的乳化剂; 乳化剂和分散剂, 用于较难的配方和强电解质系统中；悬浮剂的稳定剂。
Multipurpose dispersing, wetting agent recommended for low pH formulations. 作为低 pH 值体系的多功能分散剂、润湿剂。
Soprophor FLK
具有独特的大体积疏水基团的表面活性剂对大多数有效成分具有强大的亲和力全面提高制剂的各项性能
surfactant with unique physicochemical properties well suit and highly recommended for AG formulations
具有独特的物化性能的表面活性剂良好的配伍性, 非常值得推荐应用于农药制剂中
Versatile emulsifiers and dispersants for diverse applications Co-emulsifiers and co-dispersants
通用的乳化剂和分散剂, 适用于各种应用场合。助乳化剂和助分散剂两者兼而有之。
3
Why choose Soprophor®? 为ܼ么选择 Soprophor® 产໬?
降低药害
植
Lower phytotoxic profile making them well
物
suited for different agricultural formulation
毒害
types even at high dosages
性
更低的植物毒害性，适用于各种不同类型
指数
的农药剂型中，甚至是在高用量的情况下
表面活性剂水溶液浓度（Wt%）

化学工作者网站

化学工作者看过来，可以在线查找化学结构式！1./cgi-bin/hsrun/Distributed/HahtShop/HAHTShop.htx;start=HS_SearchCenter中，选择Search Structures，然后，就可以分别根据：Product Name 和CAS登录号，还有Molecular Formula进行查找。

在这里查到的化合物基本都有一些参数和它的结构式（Structure Image），然后将图片保存成Gif即可！当然，如果你只知道化合物的结构式，不知道叫什么，可以选择下面的Sub Structure Search ，然后 Step 1 Choose a Drawing tool. I want to Download Kekule (requires download of Kekv214.exe) Step 2 Install the plug-in you downloaded in step 1 Close this browser. Return to the directory where you saved the plug-in and double click on the file name given in step 1. Step 3 Return to this page and click the "Structure Search"button. 安装好软件即可将画好的图片粘贴上即可查找，非常方便，我用它查了一些结构式，给了我一大堆，呵呵，本人化学方面英语不是很好，一个个查看才找到，不过还好是宽带，呵呵：－）2./大家去这儿看看，查询非常简单3.【推荐】【原创】能查几万个化合物的NMR IR谱图的网址 [精华]4.能查几万个化合物的NMR IR谱图的网址 SDBS Integrated Spectral Data BaseSystem for Organic Compounds http://www.aist.go.jp/RIODB/SDBS/sdbs/owa/sdbs_sea.cre_frame_sea NIST WebBook /5.美国专利pdf全文下载的好地方 6.免费在线查合成路线/depts/chemistry/courses/toolkits/247/practice/m edialib/data/7.中科院上海有机化学研究所化学专业数据库http://202.127.145.134/tf123456/6543218.中科院上海有机化学研究所数据库（/lccdb/sjk.htm），免费注册使用，现在已经改版，性能提高许多。

ApisTransfector-D(CN)

APIS Transfector-D 使用手册（用于将核酸转染入动物细胞）Cat. No. BJ0001D-05 Size:0.5mlCat. No. BJ0001D-10 Size:1.0mlCat. No. BJ0001D-15 Size:1.5ml注意：常温运输，储存于4℃一产品介绍ApisTransfector TM是阿匹斯生物公司研发合成的新型转染试剂，由于纳米技术的应用，该试剂在保持常规脂质体转染试剂高效的基础上，更加低毒、稳定。

本品既可用于普通动物细胞DNA的转染，更可用于siRNA的转染，尤其对转染siRNA，一些难以转染的细胞，效果也非常理想，并且毒性很低。

二转染前重要提示1.本转染试剂在转染过程中可以添加抗生素，抗生素的添加与否不影响转染效率和毒性。

2.本试剂用于DNA转染时细胞密度(汇合度，confluent)一般为60-70%，用于siRNA转染时细胞密度为20-30%。

3.优化的必要性。

由于核酸和转染试剂的用量比值是决定转染效率的重要因素，初次应用时，建议先进行优化。

最优条件确定后，实验的结果将非常稳定。

初次应用时，建议核酸(μg)和转染试剂(μl)的比例在0.5:2与1:3之间进行优化。

4.对转染的DNA的要求：建议转染的DNA采用纯水溶解，采用纯水溶解会更好与转染试剂作用从而提高转染效率。

三实验方法下面以6孔板为例，其他培养容器按培养容器的底面积按比例增减。

1.提前一天细胞铺板提前一天将适量细胞种植在6孔板中。

2.转染复合物的制备a.将核酸用50μl的Opti-MEM或无血清DMEM稀释，充分混匀，制成核酸稀释液。

b.将转染试剂用50μl Opti-MEM或无血清DMEM稀释，充分混匀，制成转染试剂稀释液。

室温静置5分钟。

c.将转染试剂稀释液与对应核酸稀释液中充分混匀（可用振荡器振荡或用加样器吹吸10次以上），室温静置20分钟。

转染复合物制备完成。

3.转染⑴将转染复合物加入到1.9ml 的细胞和培养基(可含血清和抗生素)上，前后晃动混匀。

化学网址大全

查询物质结构性质等的网站：//chemidplus/chemidlite.jsp/cn/psearch.cgi?terms=552-66-9+&sel=dict化工资源网：/fj/上海枫林店/scr/化学论坛：/bbsxp/ShowForum.asp?forumid=16（）中国科学院上海有机所:http://202.127.145.69/chub/chub.htm http://202.127.145.69/index.htm免费图谱网站：www.aist.go.jp/RIODB/SDBS/menu-e.html不同压力条件下的沸点转换工具/magnus/boil.html化合物英文缩写查询网站http://www.chemie.fu-berlin.de/cgi-bin/abbscomp有机合成（org syn）网络版：/1991-2004年J.Mater Chem全文查阅/is/journals/current/jmc/jmcon.htm可以免费查询化合物的物化性质。

合成资料部分收费：CAS和性质等查询：/Chem/ChemMain.htmlSigma公司网站：https:///cgi-bin/hsrun/Suite7/Suite/HAHTpage/Suite.HsAdvanc edSearch.formAction电子期刊：http://210.64.24.31/maillist/maillist_browse.cgi?show_old_epaper=pisa&filename= 20031129014425.html免费有机杂环期刊、杂环数据库。

http://www.heterocycles.jp/heterohtml/JournalMenu.html2000年以后可以直接看到原文。

（很好的英文网站）全部专利连接/sipo/xglj/gwzscqwz/default.htm1997-2004年Angew.Chem.Int.Ed.Engl全文查阅/cgi-bin/jtoc/26737/中国和外国的化学化工网站集合：/hxgcx/service/link_wenzi.htm外文文献服务网首页：/book/化学信息资源：数据库资源1.综合性数据库STN系统, /stn.html, http://www.fiz-karlsruhe.de/DIALOG系统, Entrez系统, /Entrez/中国科学院科学数据库, http://159.226.2.20:1205(上海有机所站点)/lccdb/sjk.htm中国科技信息中心数据库检索服务，http://168.160.24.11/htbin/tripnet2.化学信息数据库剑桥结构数据库(CSD), /布鲁克海文(Brookhaven)蛋白质数据库, or/pdb/Rutgers大学的核酸数据库, :80有机化合物数据库(Organic CompoundsDatabase), /chemistry/cmp/cmp.html化学危险品数据库(Hazardous Cehmicals Database),/erd/纳米技术数据库(Nanotechnology Database), /nanobase/生物大分子晶体结构数据库(The Biological Macromolecule Crystallization Database , BMCD) ,:4400/bmcd/bmcd.html澳大利亚蛇毒和毒素数据库(Australia Venom & ToxinDatabase), .au/~ddbfry/化学结构数据库(The WWW Chemical Structures Database),c.uni-erlangen.de/services/webmol.htm化合物基本性质数据库(CS ChemFinder), /SDBS有机化合物谱图库，http://www.aist.go.jp/RIODB/SDBS/上海有机化学研究所红外光谱数据库，http://202.127.145.67/ows-doc/irdoc/irlogin.htm3.物理化学常数美国国家标准和技术研究所物理实验室主页/物理化学参数搜索或查找/cuu/Constants/index.html/cuu/Constants/links.html(其它数据站点的连接点)http://www.chemie.fu-berlin.de/chemistry/general/constants_en.html/~sanders/constants.h (C语言header file)/~sanders/constants.h (FORTRAN语言参数文件)4.化学元素周期表/chemistry/web-elements//web-elements/web-elements-home.html这个网站物性查找极方便/chemacx/index.asp网上化学资源/istl/98-winter/interne1.html一个查询药物理化性质的网站化学文献网/丁香园论坛：/bbs/中国科学（B辑）(免费,全文) /scienceinchina_b_cn.htm 中国化学快报（英文版）(免费,全文) /ccl.asp美国专利商标局网站/国际网上化学学报(免费,全文，中国编辑) /default.htm中华人民共和国知识产权局：/sipo/default.htmBasic Proton and C-13 NMR：/web1/ocol/spec/NMR.htm美国化学文摘查阅法/book.html中国实验室/安徽药品信息网: /chem-lett 的网站：http://www.chemistry.or.jp/gakujutu/chem-lett/index.html（可以看近几年的全文）j.a.c.s全文（1993年后）：/subscribe/journals/jacsat/supmat/提供一个合成方法的数据库：/1965-2004Chem. Commun全文文献查阅/is/journals/current/chemcomm/cccon.htm/is/journals/j1.htm化学领域多来米站：/weblink.html一个提供各种英文网站：http://www.abc.chemistry.bsu.by/current/fulltextj.htm有机反应机理大全chembio.ethz.ch/links/en/orgchem_reaktionen_lehr.html一个学术论坛ChIN论坛：/ChINBB/index.php论坛地址: 各国新药信息网站USA /cder/approval/index.htmEurope http://www.emea.eu.int/htms/human/epar/a-zepar.htmJapanese http://www.pharmasys.gr.jp/shinyaku/shinyaku_index.html Netherlands http://www.cbg-meb.nl/uk/prodinfo/index.htmSweden http://www.mpa.se/eng/index.shtml奇迹文库/eprint/75/index.htmlInternet网络上有着丰富的化学化工资料，从Internet网络上检索资料方便迅捷。

RNase I商品说明书

PRODUCT INFORMATIONRNase IPub. No. MAN0012009Rev. Date 09 January 2017 (Rev. B.00)#_Lot: _ Expiry Date: _ Store at -20 °CFor Research Use Only. Not for use in diagnostic procedures. DescriptionRNase I, an endoribonuclease, preferentially hydrolyzes single-stranded RNA to nucleoside 3'-monophosphates via nucleoside 2'-, 3'-cyclic monophosphate intermediates (1). The enzyme does not require any metal ions for activity. This product is the periplasmic form of RNase I.Applications∙Removal of RNA from DNA solutions (2).∙Removal of RNA from recombinant protein preparations. ∙Ribonuclease protection assays (3).SourceE.coli cells with a cloned rna gene of E.coli. Definition of Activity UnitOne unit of the enzyme catalyzes degradation of 100 ng of E.coli ribosomal RNA per second into acid-soluble nucleotides at 37 °C.Enzyme activity is assayed in the following mixture:20 mM Tris-acetate (pH 8.0), 100 mM NaCl,0.1 mM EDTA, 0.01% Triton X-100,40 µg/mL E.coli ribosomal [3H]-RNA.Storage BufferThe enzyme is supplied in:50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.01% Triton X-100 and 50% (v/v) glycerol.Inhibition and Inactivation∙Inhibitors: SDS at 0.1% concentration irreversibly inactivates the enzyme.∙Inactivated by heating at 100 °C for 30 min, reliably removed by spin column or phenol/chloroform extraction. Note∙Mammalian ribonuclease inhibitors have no effect on RNase I.∙RNase I binds to DNA, but does not degrade it.∙RNase I amino acid sequence is typical for the RNase T2 family.∙Non-ionic detergents (e.g., Triton X-100) do not inhibit RNase I, and may even slightly stimulate its activity and stabilize it against heat inactivation. Triton X-100 or BSA (at 0.1 mg/mL) may prevent RNase I from sticking to glass vessels when working with dilute solutions.∙Polyamines stimulate the activity of RNase I. CERTIFICATE OF ANALYSIS Endodeoxyribonuclease AssayNo detectable degradation was observed after incubation of supercoiled plasmid DNA with RNase I.Labeled Oligonucleotide (LO) AssayNo detectable degradation after incubation of single-stranded or double-stranded radiolabeled oligonucleotides with RNase I.Quality authorized by:Jurgita Zilinskiene(continued on back page)Reference1.Shen, V., Schlessinger, D., RNase I of Escherichia coli, The Enzymes (Boyer, P.D., ed), Academic Press Inc., New York, vol. 15B, 503-506, 1982.2. Zhu, L., Deutscher, M.P., The Escherichia coli rna geneencoding RNase I: sequence and unusual promoterstructure, Gene, 119, 101-106, 1992.3. Sambrook, J., Russell, D.W., Molecular Cloning:A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001. LIMITED USE LABEL LICENSE: Internal Research and Development Use Only.The purchase of this product conveys to the buyer the limited, non-exclusive, non-transferable right (without the right to resell, repackage, or further sublicense) to use this product for internal research and development purposes. No other license is granted to the buyer whether expressly, by implication, by estoppel or otherwise. In particular, the purchase of the product does not include or carry any right or license to use, develop, or otherwise exploit this product commercially and no rights are conveyed to the buyer to use the product or components of the product for purposes including but not limited to provision of services to a third party, generation of commercial databases or clinical diagnostics. This product is sold pursuant to authorization from Thermo Fisher Scientific and Thermo Fisher Scientific reserves all other rights. For information on purchasing a license for uses other than internal research and development purposes, please contact***************************************,LifeTechnologiesInc.,5781Van Allen Way, Carlsbad, California 92008.PRODUCT USE LIMITATIONThis product is developed, designed and sold exclusively for research purposes and in vitro use only. The product was not tested for use in diagnostics or for drug development, nor is it suitable for administration to humans or animals.Please refer to for Material Safety Data Sheet of the product. © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.。