Ombrabulin_hydrochloride_COA_19830_MedChemExpress
产品说明(阿米迪)
• 妥洛特罗贴剂（阿米迪®）治疗6-8天后，患儿症状体征评 分显著下降
* * *P<0.05 *
仅供内部培训学习使用
阿米迪®显著改善喘息性或毛细支气管炎 患儿的生活质量
一项在299例喘息性或毛细支气管炎患儿中进行的为期8天的研究显示：
口服或静脉给药困难的患者也可使用。
无肝脏首过效应，不增加肝脏负担。 适用年龄范围广（半岁以上儿童患者至高龄患者）。
毛细支气管炎
仅供内部培训学习使用
研究设计
• 多中心、随机、开放标签、阳性药物平行对照研究 • 共入299例6个月~5岁喘息性支气管炎或毛细支气管炎患儿：
治疗组： n=140
皮肤 (角质层)
给药中
给药结束
日本药学会（2001年）发表资料
药物结晶储存系统
• 溶解的妥洛特罗分子与均匀分散的妥洛特罗结晶共存于膏
体中，妥洛特罗结晶具有作为药物贮藏槽的功能。
• 伴随着皮肤吸收，减少的妥洛特罗分子从妥洛特罗结晶中 逐步得到补充，可保持膏体及接触皮肤表面的妥洛特罗浓 度长时间稳定，从而使持续的药物释放成为可能。
使血药浓度达峰时间（Tmax）与“晨降” 的发作时间带相吻合
抑制血药峰浓度（Cmax）过度上升从而 减轻全身性副作用
1日只需给药1次，提高患者依从性
浜松CPT研究所中岛光好（浜松医科大学名誉教授）资料（改编）
6
阿米迪®贴剂的构成示意图
有效控制药物持续释放
：妥洛特罗晶体 ：妥洛特罗分子
支持体 给药开始 膏体
经皮吸收型长效支气管扩张剂
妥洛特罗贴剂（阿米迪® ）
今晚一贴 明朝呼吸舒畅 平喘止咳 “剂”高一筹
大肠杆菌高产L丙氨酸
APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGYProduction of L-alanine by metabolically engineered Escherichia coliXueli Zhang&Kaemwich Jantama&J.C.Moore&K.T.Shanmugam&L.O.IngramReceived:23May2007/Revised:13August2007/Accepted:16August2007/Published online:15September2007 #Springer-Verlag2007Abstract Escherichia coli W was genetically engineered to produce L-alanine as the primary fermentation product from sugars by replacing the native D-lactate dehydroge-nase of E.coli SZ194with alanine dehydrogenase from Geobacillus stearothermophilus.As a result,the heterolo-gous alanine dehydrogenase gene was integrated under the regulation of the native D-lactate dehydrogenase(ldhA) promoter.This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation.Strain XZ111accumulated alanine as the primary product during glucose fermentation.The methyl-glyoxal synthase gene(mgsA)was deleted to eliminate low levels of lactate and improve growth,and the catabolic alanine racemase gene(dadX)was deleted to minimize conversion of L-alanine to D-alanine.In these strains,re-duced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth.This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production.The resulting strain, XZ132,produced1,279mmol alanine from120g l−1 glucose within48h during batch fermentation in the mineral salts medium.The alanine yield was95%on a weight basis(g g−1glucose)with a chiral purity greater than99.5%L-alanine.Keywords Alanine.Fermentation.E.coli.Evolution. GlycolysisIntroductionWorldwide production of L-alanine has been estimated at 500tons per year(Ikeda2003).In pharmaceutical and veterinary applications,L-alanine is used with other L-amino acids as a pre-and postoperative nutrition therapy(Hols et al.1999).Alanine is also used as a food additive because of its sweet taste(Lee et al.2004).The use of L-alanine is limited in part by the current high cost.L-Alanine is pro-duced commercially by the enzymatic decarboxylation of L-aspartic acid using immobilized cells or cell suspensions of Pseudomonas dacunhae as a biocatalyst with a yield greater than90%(Shibatani et al.1979).The substrate for this enzymatic production process,L-aspartate,is usually pro-duced from fumarate by enzymatic catalysis with aspartate ammonia-lyase.Fumaric acid is produced primarily from petroleum,a nonrenewable feedstock.An efficient fermen-tative process with a renewable feedstock such as glucose offers the potential to reduce L-alanine cost and facilitate a broad expansion of the alanine market into other products.Alanine is a central intermediate(Fig.1)and an essential component of cellular proteins.Most microorganisms produce alanine only for biosynthesis using a glutamate–pyruvate transaminase(Hashimoto and Katsumata1998). Some organisms such as Arthrobacter oxydans(Hashimoto and Katsumata1993;Hashimoto and Katsumata1998; Hashimoto and Katsumata1999),Bacillus sphaericus (Ohashima and Soda1979),and Clostridium sp.P2Appl Microbiol Biotechnol(2007)77:355–366DOI10.1007/s00253-007-1170-yElectronic supplementary material The online version of this article (doi:10.1007/s00253-007-1170-y)contains supplementary material, which is available to authorized users.X.Zhang:J.C.Moore:K.T.Shanmugam:L.O.Ingram(*) Department of Microbiology and Cell Science,University of Florida,Box110700,Gainesville,FL32611,USAe-mail:ingram@K.JantamaDepartment of Chemical Engineering,University of Florida, Gainesville,FL32611,USA(Orlygsson et al.1995)produce alanine from pyruvate and ammonia using an reduced nicotinamide adenine dinucleo-tide (NADH)-linked alanine dehydrogenase (ALD).How-ever,fermentations are slow,and yields from the best natural producers are typically 60%or less because of coproduct formation (Hashimoto and Katsumata 1998;Table 1).Plasmid-borne genes encoding NADH-linked ALD have been tested as an approach to develop improved biocatalysts with varying degrees of success (Table 1).Engineered strains of Zymomonas mobilis CP4expressing the B.sphaericus alaD gene produced low levels of racemic alanine during the anaerobic fermentation of 5%glucose (Uhlenbusch et al.1991).A native chromosomal lactate dehydrogenase gene (ldhA )-deleted strain of Lactococcus lactis containing a mutation in alanine racemase was engineered in a similar fashion and produced 12.6g l −1L -alanine from 1.8%glucose (Hols et al.1999).An Escherichia coli aceF ldhA double mutant containing pTrc99A-alaD plasmid produced 32g l −1racemic alanine in 27h during a two-stage (aerobic and anaerobic)fermentation with a yield of 0.63g alanine g −1glucose (Lee et al.2004).With further gene deletions and process optimization,the racemic alanine titer wasincreasedFig.1Alanine pathway in recombinant E.coli .a Native and recom-binant fermentation pathways.The foreign gene,G.stearothermophilus alaD ,is shown in bold .G.stearothermophilus alaD coding region and transcriptional terminator were integrated into the native ldhA gene under transcriptional control of the ldhA promoter.Solid stars represent deletions of native genes in XZ132.Note that the native biosynthetic route for alanine production is omitted for simplicity.ackA Acetate kinase,adhE alcohol/aldehyde dehydrogenase,alaD alanine dehydro-genase (Geobacillus stearothermophilus XL-65-6),aldA aldehyde dehydrogenase A,aldB aldehyde dehydrogenase B,alr alanine race-mase 1,dadX alanine racemase 2,frd fumarate reductase,gloA glyoxalase I,gloB glyoxalase II,gloC glyoxalase III,ldhA D -lactate dehydrogenase,mdh malate dehydrogenase,mgsA methylglyoxal synthase,pflB pyruvate –formate lyase,ppc phosphoenolpyruvate carboxylase,pta phosphate acetyltransferase.b Coupling of ATP production and growth to NADH oxidation and L -alanine production.Glucose is metabolized to pyruvate,ATP,and NADH.Energy conserved in ATP is utilized for growth and homeostasis,regenerating ADP.NADH is oxidized by alanine formation allowing glycolysis and ATP production to continueT a b l e 1C o m p a r i s o n o f a l a n i n e -p r o d u c i n g s t r a i n sO r g a n i s m s M o d i f i e d p r o p e r t yM e d i a ,s u b s t r a t e a n d p r o c e s s c o n d i t i o n sT i m e (h )A l a n i n e (g l −1)Y i e l d (%)L -A l a n i n ep u r i t y (%)R e f e r e n c e E .c o l i X Z 132I n t e g r a t e d G .s t e a r o t h e r m o p h i l u s a l a D ;Δp f l ,Δa c k A ,Δa d h E ,Δl d h A ,Δm g s A ,Δd a d XM i n e r a l m e d i u m ,b a t c h ,g l u c o s e 120g l −148.0114.095>99T h i s s t u d yA r t h r o b a c t e r o x y d a n s H A P -1M i n e r a l m e d i u m ,t w o -s t a g e f e d -b a t c h ,g l u c o s e 150g l −1120825560.0H a s h i m o t o a n d K a t s u m a t a 1998A .o x y d a n s D A N 75A l a n i n e r a c e m a c e d e f i c i e n tM i n e r a l m e d i u m ,t w o -s t a g e f e d -b a t c h ,g l u c o s e 150g l −1,0.2g l −1D -a l a n i n e120775198H a s h i m o t o a n d K a t s u m a t a 1998E c o l i A L 1(p O B P 1)P l a s m i d w i t h A .o x y d a n s H A P -1a l a D M i n e r a l m e d i u m ,g l u c o s e 20g l −1,l i m i t e d o x y g e n40841N o t r e p o r t e dK a t s u m a t a a n d H a s h i m o t o 1996C o r y n e b a c t e r i u m g l u t a m i c u m A L 107(p O B P 107)P l a s m i d w i t h A .o x y d a n s H A P -1a l a DC o r n s t e e p l i q u o r ,g l u c o s e 200g l −1,4g l −1D L -a l a n i n e ,l i m i t e d o x y g e n 707136>99K a t s u m a t a a n d H a s h i m o t o 1996Z y m o m o n a s m o b i l i s C P 4(p Z Y 73)P l a s m i d w i t h B .s p h a e r i c u s I F O 3525a l a D M i n e r a l s a l t s m e d i u m ,s i m p l e b a t c h ,g l u c o s e 50g l −126816N o t r e p o r t e dU h l e n b u s c h e t a l .1991L a c t o c o c c u s l a c t i s N Z 3950(p N Z 2650)P l a s m i d w i t h B .s p h a e r i c u s I F O 3525a l a D Δl d h AR i c h m e d i u m (M 17),g l u c o s e 18g l −117137085–90H o l s e t a l .1999L .l a c t i s P H 3950(p N Z 2650)P l a s m i d w i t h B .s p h a e r i c u s I F O 3525a l a D Δl d h A ,Δa l rR i c h m e d i u m (M 17),g l u c o s e 18g l −1,0.2g l −1D -a l a n i n e 17N o t k n o w nN o t k n o w n >99H o l s e t a l .1999E .c o l i A L S 887(p T r c 99A -a l a D )P l a s m i d w i t h B .s p h a e r i c u s I F O 3525a l a D Δl d h A ,Δa c e FY e a s t e x t r a c t ,t w o -s t a g e b a t c h ,g l u c o s e 50g l −1,a e r o b i c a i r 1l m i n −1273263N o t r e p o r t e d L e e e t a l .2004E .c o l i A L S 929(p T r c 99A -a l a D )P l a s m i d w i t h B .s p h a e r i c u s I F O 3525a l a D Δp f l ,Δp p s ,Δp o x B ,Δl d h A ,Δa c e E FY e a s t e x t r a c t a n d c a s a m i n o a c i d s ,t w o -s t a g e b a t c h (a e r o b i c c e l l g r o w t h a n d a n a e r o b i c f e r m e n t a t i o n )223486N o t r e p o r t e d S m i t h e t a l .2006E .c o l i A L S 929(p T r c 99A -a l a D )P l a s m i d w i t h B .s p h a e r i c u s I F O 3525a l a D Δp f l ,Δp p s ,Δp o x B ,Δl d h A ,Δa c e E FY e a s t e x t r a c t a n d c a s a m i n o a c i d s ,t w o -s t a g e f e d -b a t c h (a e r o b i c c e l l g r o w t h a n d a n a e r o b i c f e r m e n t a t i o n )4888100N o t r e p o r t e d S m i t h e t a l .2006to88g l−1in a more complex process with yields ap-proaching the theoretical maximum(Smith et al.2006). However,this strain produced only racemic alanine,utilized multicopy plasmids requiring antibiotic selection,and required complex media with a complex multistage fermen-tation process(Smith et al.2006).In this study,we developed novel biocatalysts that pro-duce chirally pure L-alanine in batch fermentations without using plasmid-containing biocatalysts,antibiotics,or com-plex nutrients.The resulting strains are based on a deriva-tive of E.coli W(strain SZ194)that produces D-lactate (Zhou et al.2006b).The ldhA gene in SZ194was replaced with a single,chromosomally integrated copy of the ALD gene from the thermophile,Geobacillus stearothermophilus XL-65-6(formerly B.stearothermophilus;Lai and Ingram 1993).After additional deletions of alanine racemase (dadX)and methylglyoxal synthase(mgsA)and metabolic evolution,the resulting strain produced L-alanine at high titers(over1M)and yields in batch fermentations using the mineral salts medium.Materials and methodsStrains,plasmids,media,and growth conditionsThe strains and plasmids used in this study are listed in Table2.Strain SZ194was previously engineered from a derivative of E.coli W(ATCC9637)and served as a starting point for constructions(Zhou et al.2006b).G. stearothermophilus XL-65-6(Lai and Ingram1993)was used for cloning the ALD gene.During sequencing of chro-mosomal genes,we discovered a20-year-old error in culture labeling.Strain SZ194,the parent used to construct the alanine strains,is a derivative of E.coli W(ATCC9637). Other constructs for ethanol production and lactate produc-tion that have been reported previously as derivatives of E. coli B are now known to be derivates of E.coli W(ATCC 9637).Primers used in this study are listed in Table3.During strain construction,cultures were grown aerobi-cally at30,37,or39°C in Luria broth(10g l−1Difco tryptone,5g l−1Difco yeast extract,and5g l−1NaCl) containing2%(w/v)glucose or5%(w/v)arabinose. Ampicillin(50mg l−1),tetracycline(12.5mg l−1), kanamycin(50mg l−1),or chloramphenicol(40mg l−1) were added as needed.For initial tests of fermentative alanine production,strains were grown without antibiotics at37°C in NBS mineral salts medium(Causey et al.2004) supplemented with100mM ammonia sulfate,1mM betaine,and2%(w/v)glucose.Fermentation experiments (2–12%sugar)were carried out in NBS medium and AM1 medium(Martinez et al.2007).Broth was maintained at pH 7by the automatic addition of5M NH4OH.Genetic methodsStandard methods were used for genomic deoxyribonucleic acid(DNA)extraction(Qiagen,Valencia,CA),polymerase chain reaction(PCR)amplification(Stratagene,La Jolla CA,and Invitrogen,Carlsbad,CA),transformation,plas-mid extration(Qiagen),and restriction endonuclease diges-tion(New England Biolabs,Ipswich,MA).Methods for foreign gene(alaD)integration and for chromosomal gene (mgsA and dadX)deletion are described below.DNA sequencing was provided by the University of Florida Interdisciplinary Center for Biotechnology Research.The Biocyc and Metacyc databases(Karp et al.2005)were instrumental in the design and completion of these studies. Cloning the alanine dehydrogenase gene alaD from G. stearothermophilus XL-65-6and detection of the enzyme activityThe primers for amplifying alaD from G.stearothermophilus XL-65-6were designed based on the alaD sequence of G. stearothermophilus strain10.The forward primers(5′–3′GGAAAAA GGAGGAAAAAGTG ATGAAGATCGG CATT)included the ribosomal-binding region(bold)and the amino terminus(italicized).The reverse primer(5′–3′GAA GGAGTTGATCATTGTTTAACGAGAGAGG)was down-stream from the putative transcriptional terminator region (Table3).ALD was verified in clones using an activity stain (Kuroda et al.1990).E.coli TOP10F′harboring plasmids containing alaD was grown on Luria–Bertani(LB)plates at 37°C,then transferred to a Whatman7.0-cm filter paper. The filter was immersed in10mM potassium phosphate buffer(pH7.2)and incubated for20min at80°C for lysis of the cells and denaturation of the E.coli proteins.The dried filter paper was assayed in a reaction mixture containing50mM L-alanine,50mM Tris–HCl buffer (pH9.0),0.625mM NAD+,0.064mM phenazine metho-sulfate,and0.24mM nitro blue tetrazolium.The cells with ALD appeared as blue spots on the filter.Integration of alaD into E.coli SZ194The alaD gene was integrated into the chromosomal ldhA gene of SZ194.The fragment(Sma I–Kpn I,1.7kb)con-taining a tet gene flanked by two FRT sites was isolated from pLOI2065and cloned into pLOI4211between a unique Bam HI site(Klenow-treated)and Kpn I site to produce plasmid pLOI4213(6.0kb).In this plasmid,transcription of alaD and tet are oriented in the same direction.The Apa I(treated with T4DNA polymerase to produce a blunt end)–Kpn I fragment(2.2kb)containing alaD and tet was isolated from pLOI4213and cloned into pLOI2395Table2 E.coli strains and plasmids used in this studyRelevant characteristics Source or referenceStrainsSZ194plfB frd adhE ackA deletions Zhou et al.2006bXZ103-110SZ194,ldhA::FRT-tet-FRT::This studyG.stearothermophilus alaDXZ111XZ105,ldhA::G.stearothermophilus alaD This studyXZ112XZ111,metabolic evolution in NBS medium with2%glucose This studyXZ113XZ112,metabolic evolution in NBS medium with5%glucose This studyXZ115XZ113,metabolic evolution in NBS medium with8%glucose This studyXZ121XZ115,mgsA deletion This studyXZ123XZ121,metabolic evolution in NBS medium with8%glucose This studyXZ126XZ123,dadX deletion This studyXZ129XZ126,metabolic evolution in NBS medium with8%glucose This studyXZ130XZ129,metabolic evolution in AM1medium with8%glucose This studyXZ131XZ130,metabolic evolution in AM1medium with10%glucose This studyXZ132XZ131,metabolic evolution in AM1medium with12%glucose This studyPlasmidspCR2.1-TOPO bla kan;TOPO TA cloning vector InvitrogenDatsenko and Wanner2000pKD46Blaγβexo(Red recombinase),temperature conditionalpSC101repliconpFT-A Bla flp,temperature conditional pSC101replicon Posfai et al.1997pEL04cat-sacB targeting cassette Lee et al.2001;Thomason et al.2005 pLOI2224kan;R6K conditional integration vector Martinez-Morales et al.1999pLOI2065bla;FRT-tet-FRT cassette Zhou et al.2003bpLOI2395bla;ldhA franked by two Asc I site Zhou et al.2003apLOI3421 1.8kbp SmaI fragment containing aac Wood et al.2005pLOI4151bla cat;cat-sacB cassette This studyalaD integrationThis studypLOI4211bla kan alaD;alaD(PCR)from G.stearothermophilus XL-65-6cloned into pCR2.1-TOPO vectorpLOI4213bla kan;alaD-FRT-tet-FRT Kpn I-Sma I fragment(FRT-tet-FRT)This studyfrom pLOI2065cloned into Kpn I-BamH I(blunted)site of pLOI4211This studypLOI4214bla kan;ldhA’-alaD-FRT-tet-FRT-ldhA”Apa I(blunted)-Kpn I fragment(alaD-FRT-tet-FRT)from pLOI4213cloned into ldhA at Hinc II-Kpn Isites of pLOI2395This studypLOI4215kan;ldhA’-alaD-FRT-tet-FRT-ldhA”Asc I fragment(ldhA’-alaD-FRT-tet-FRT-‘ldhA)from pLOI4214cloned into Asc I sites of pLOI2224mgsA deletionThis studypLOI4228bla kan;yccT’-mgsA-helD’(PCR)from E.coli W clonedinto PCR2.1-TOPO vectorThis studypLOI4229cat-sacB cassette PCR amplified from pLOI4151(Eco RV digested)cloned into mgsA in pLOI4228This studypLOI4230PCR fragment amplified from pLOI4228(using mgsA-1/mgsA-2primers),kinase treated,and self-ligateddadX deletionThis studypLOI4216bla kan;dadA’-dadX-cvrA’(PCR)from E.coli W clonedinto PCR2.1-TOPO vectorpLOI4218cat-sacB cassette PCR amplified from pLOI4151(Eco RV digested)This studycloned into dadX in pLOI4216This studypLOI4220PCR fragment amplified from pLOI4216(using dadX-4/dadX-5primers),kinase treated,and self-ligated(Hinc II to Kpn I sites)to produce pLOI4214(6.5kb).In this plasmid,ldhA ,alaD ,and tet genes are transcribed in the same direction.The Asc I fragment (4.3kb)containing these three genes was isolated from pLOI4214and cloned into the R6K integration vector pLOI2224to produce pLOI4215(6.2kb).Plasmid pLOI4215contains resistance genes for both tetracycline and kanamycin (Fig.2).The Asc I fragment (4.3kb)containing ldhA ,alaD ,and tet genes was isolated from pLOI4215,further cut by Xmn I to eliminate any remaining uncut plasmid DNA,and electroporated into SZ194containing the Red recombinase plasmid pKD46(Datsenko and Wanner 2000).Integrants were selected for tetracycline resistance,confirmed by sensitivity to kanamycin and ampicillin and by PCR analysis using the primers of ldhA and its neighboring genes ydbH and hslJ (Table 3).Deletion of mgsA and dadX genesA modified method for deleting E.coli chromosomal genes was developed using two steps of homologous recom-bination (Thomason et al.2005).With this method,no antibiotic genes or scar sequences remain on the chromo-some after gene deletion.In the first recombination,part of the target gene was replaced by a DNA cassette containing a chloramphenicol resistance gene (cat )and levansucrase gene (sacB ).In the second recombination,the cat –sacBcassette was removed by selection for resistance to sucrose.Cells containing the sacB gene accumulate levan during incubation with sucrose and are killed.Surviving recombi-nants are highly enriched for loss of the cat –sacB cassette.A new cassette was constructed as a template to facilitate gene deletions.The cat –sacB region was amplified from pEL04(Lee et al.2001;Thomason et al.2005)by PCR using the JM catsacB up Nhe I and JM catsacB down Nhe I primers (Table 3),digested with Nhe I,and ligated into the corresponding site in pLOI3421to produced pLOI4151.The cat –sacB cassette was amplified by PCR using pLOI4151as a template with the cat -up2and sacB -down2primers (Eco RV site included in each primer),digested with Eco RV ,and used in subsequent ligations.The mgsA gene and neighboring 500-bp regions (yccT ′–mgsA –helD ′,1,435bp)were amplified using the mgsA -up and mgsA -down primers and cloned into the pCR 2.1-TOPO vector (Invitrogen)to produce plasmid pLOI4228.A 1,000-fold diluted plasmid preparation of this plasmid served as a template for inside-out amplification using the mgsA -1and mgsA -2primers (both within the mgsA gene and facing outward).The resulting 4,958-bp fragment containing the replicon was ligated to the Eco RV-digested cat –sacB cassette from pLOI4151to produce pLOI4229(Fig.3a).This 4,958-bp fragment was also used to construct a second plasmid,pLOI4230(Fig.3b),by phosphorylation and self-ligation.In pLOI4230,the central region of mgsA is deleted (yccT ′–mgsA ′–mgsA ″–helD ′).After digestion of pLOI4229and pLOI4230with Xmn I (within the vector),each served as a template for amplifica-tion using the mgsA -up and mgsA -down primers to produce linear DNA for integration step 1(yccT ′–mgsA ′–cat –sacB –mgsA ″–helD ′)and step II (yccT ′–mgsA ′–mgsA ″–helD ′),respectively.After electroporation of the step 1fragment into XZ115containing pKD46(Red recombinase)and 2h ofTable 3Primers used in this study Primers SequencealaD -forward GGAAAAAGGAGGAAAAAGTGATGAA GATCGGCATTalaD -reverse GAAGGAGTTGATCATTGTTTAACGA GAGAGGldhA -forward AGTACCTGCAACAGGTGAAC ldhA -reverse CAGGCGACGGAATACGTCAT ldhA -up (ydbH )CTGATAACGCAGTTGCTGGA ldhA -down (hslJ )TTCATTAAATCCGCCAGCTTJM catsacB up NheI TTAGCTAGCATGTGACGGAAGATC ACTTCGJM catsacB down NheI CCGCTAGCATCAAAGGGAAAACTGT CCATATcat -up2AGAGAGGATATCTGTGACGGAAGAT CACTTCGsacB -down2AGAGAGGATATCGAATTGATCCGGT GGATGACmgsA -up CAGCTCATCAACCAGGTCAA mgsA -down AAAAGCCGTCACGTTATTGG mgsA -1AGCGTTATCTCGCGGACCGT mgsA -2AAGTGCGAGTCGTCAGTTCC dadX -up AGGCTACTCGCTGACCATTC dadX -down GGTTGTCGGTGACCAGGTAG dadX -4TGGGCTATGAGTTGATGTGC dadX -5CTGTATCGGACGGGTCATCTFig.2Integration vector used for chromosomal insertion of G.stearothermophilus alaD into E.coli ldhA .Sequence encoding the N-terminal and C-terminal regions are designated ldhA ′and ldhA ″,respectivelyincubation at 30°C to allow expression and segregation,recombinants were selected for chloramphenicol (40mg l −1)and ampicillin (50mg l −1)resistance in Luria broth at 30°C (18h).Three clones were selected,grown in Luria broth containing ampicillin and 5%(w/v)arabinose (to induce expression of red recombinase),and prepared for electro-poration.After electroporation with the step 2fragment,cells were incubated at 30°C for 4h and then transferred into a 250-ml flask containing 100ml of modified LB (100mM 3-(N -morpholino)propanesulfonic acid [MOPS]buffer added and NaCl omitted)containing 10%sucrose.After overnight incubation (30°C),clones were selected on modified LB plates (no NaCl;100mM MOPS added)containing 6%sucrose (39°C,16h).Resulting clones were tested for loss of ampicillin and chloramphenicol resistance.Construction was confirmed by PCR using the mgsA-up/down primer set.A clone containing a deletion in the central region of mgsA was selected and designated XZ121.The dadX gene was deleted in a manner analogous to that used to delete the mgsA gene.Primers for dadX deletion are shown in Table 3,and the corresponding plasmids are shown in Table 2.FermentationNBS mineral salts medium (Causey et al.2004)with 1mM betaine (Zhou et al.2006a )was used in the initial fermentation (pH 7.0).Preinoculum was grown by inocu-lating three colonies into a 250ml flask (100ml NBS medium,2%glucose,and 100mM ammonium sulfate).After 16h (37°C,120rpm),this preinoculum was diluted into 500-ml fermentation fleakers containing 300ml NBS medium (2–8%glucose,100mM ammonium sulfate,and 1mM betaine)with 33mg cell dry weight (CDW)l −1.In early experiments,pH was maintained at 7.0by automat-ically adding 2M potassium hydroxide.In later experi-ments,5M ammonium hydroxide was used to maintain pH,and a low salt medium,AM1(Martinez et al.2007),was used to replace the NBS medium for fermentation (8–12%glucose).AM1medium contains much less salt and has been optimized for E.coli .Metabolic evolutionCells from pH-controlled fermentations were serially transferred at 24-h intervals to facilitate metabolic evolution through competitive,growth-based selection (Fig.1b).At the beginning,sequentially transferred cultures were inoc-ulated with an initial density of 33mg CDW l −1.As growth increased,the inoculum was changed to a 1:100dilution and subsequently to a 1:300dilution.Periodically,clones were isolated from these experiments,assigned new strain designations,and frozen for storage.AnalysesCell mass was estimated by measuring the optical density at anic acids and glucose concentrations were mea-sured by high-performance liquid chromatography (HPLC,Underwood et al.2002).Analysis of fermentation products by mass spectroscopy and amino acid analyzer were provided by the University of Florida Interdisciplinary Center for Bio-technology Research.Alanine was found to be the predominant product.The alanine concentration and isomeric purity were further measured by HPLC using the Chiralpak MA(+)chiral column (Chiral Technologies,West Chester,PA).ResultCloning of the alanine dehydrogenase geneALD is found in Bacillus (and Geobacillus )species where it plays a pivotal role in energy generation during sporulation (Ohashima and Soda 1979;Kuroda et al.1990).ALD from B.sphaericus IFO3525has beenwidelyFig.3Plasmids used to delete mgsA .Plasmid pLOI4229(a )was used to delete the mgsA gene and insert the cat-sacB cassette in the first recombina-tion step.Plasmid pLOI4230(b )was used to remove the cat-sacB cassette to create a deletion devoid of foreign sequence.Se-quence encoding the N-terminal and C-terminal regions are des-ignated mgsA ′and mgsA ″,respectivelyused with varying degrees of success to engineer alanine production in recombinant bacteria(Uhlenbusch et al. 1991;Hols et al.1999;Lee et al.2004;Smith et al.2006). Selection of the B.sphaericus IFO3525is presumed to be due in part to the high specific activity(Ohashima and Soda 1979).In contrast,we have selected a thermostable ALD from the thermophile,G.stearothermophilus XL-65-6, based on our prior experience in expressing genes from this organism in recombinant E.coli(Burchhardt and Ingram 1992;Lai and Ingram1993;Lai and Ingram1995).The ribosomal-binding region,coding region,and tran-scriptional terminator of alaD were amplified from G. stearothermophilus XL-65-6and sequenced(EF154460in GenBank).The deduced amino acid sequence was identical to that reported for Geobacillus kaustophilus HTA426and very similar to G.stearothermophilus strain10(99%iden-tity)and G.stearothermophilus strain IFO12550(94% identity).The nucleotide sequence(65%identity)and the deduced ALD amino acid sequence(74%identity)were quite different from the B.sphaericus IFO3525gene,the gene pre-viously used for alanine production in recombinant bacteria.Modification of E.coli W for homoalanine productionE.coli W strain SZ194(pflB frdBC adhE ackA)was previously constructed to produce only D-lactic acid.All major fermentation pathways except lactate have been blocked in this strain by gene deletions(Fig.1a).To convert this strain to the production of alanine,part of the native ldhA-coding region was replaced by a DNA fragment containing the ribosomal-binding region,coding region,and transcriptional terminator of alaD from G. stearothermophilus XL-65-6.The promoterless alaD was oriented in the same direction as ldhA to allow expression from the native ldhA promoter(Fig.2).After electroporation,approximately500colonies were recovered with tetracycline resistance and sensitivity to kana-mycin,consistent with a double-crossover event.These colo-nies were further examined by PCR using ldhA forward and reverse primer set(Table3).Only eight colonies of the500 tested were correct based on an analysis of PCR fragments. These eight colonies were further verified using primer sets for alaD,ldhA forward and alaD reverse,alaD forward and ldhA reverse,and ldhA outside primers(Table3)and de-signated XZ103,XZ104,XZ105,XZ106,XZ107,XZ108, XZ109,and XZ110,respectively.These eight strains were initially tested in15-ml screw-cap tubes containing NBS medium with2%glucose and100mM ammonium sulfate, which were filled to the brim.Strain XZ105appeared to grow faster than the other strains(37°C for48h)and was selected for further development.XZ105was transformed with pFT-A,which contains an inducible flippase(FLP)recombinase(Martinez-Morales et al.1999;Posfai et al.1997).The chromosomal FRT-flanked tet gene in XZ105was removed by inducing the FLP recombinase.After growing in39°C to eliminate the temperature-sensitive plasmid pFT-A,resulting strain was designated XZ111.Expression of G.stearothermophilus alaD in XZ111is transcriptionally regulated by the ldhA promoter,the same promoter that regulates the production of lactate dehydrogenase(dominant fermentation pathway) in native E.coli.pH-controlled batch fermentation for alanine production Alanine production by strain XZ111was tested in500-ml fermentation vessels containing300ml NBS medium, 20g l−1glucose,100mM ammonium sulfate,and1mM betaine.Broth pH was automatically controlled by adding 2N potassium hydroxide.After96h,181mM alanine was produced.The alanine yield from total glucose was 81%(g/g),and84%based on glucose that had been metabo-lized.The chiral purity of L-alanine was96.1%(Table4). Very low levels of other products(lactate,succinate,ace-tate,ethanol)were present,typically below1mM.This result demonstrated that the integrated G.stearothermophilus alaD gene as a single chromosomal copy under the control of the native ldhA promoter can provide sufficient levels of ALD to support E.coli growth from the production of alanine as the sole fermentation product.Metabolic evolution of strain XZ111Although XZ111could accumulate alanine as the primary product,incubation times were long,and volumetric productivity was limited.When using a high-glucose concentration(80g l−1),growth and alanine productivity were further reduced(Table4).In this strain,adenosine triphosphate(ATP)production and growth are tightly coupled to NADH oxidation and alanine production by ALD(Fig.1b).This coupling provided a basis for strain improvement by selecting for increased growth during serial cultivation,i.e.,metabolic evolution.Cells with increased growth because of spontaneous mutations will successively displace their parents while coselecting for increased alanine productivity.Serial transfers of XZ111were carried out at24-h intervals in NBS mineral salts medium with1mM betaine.Cultures were first transferred in the medium containing20g l−1 glucose,and the pH was controlled by automatically adding 2N potassium hydroxide.However,after ten transfers to strain XZ112,little improvement was observed(data not shown).Because ammonia is essential for alanine pro-duction,it was thought that ammonia may be limiting for fermentation.Two normals potassium hydroxide containing 1N ammonia carbonate and5N ammonia hydroxide alone。
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationFLUOXETINE HClC17H18F3NO•HClM.W. = 345.79CAS — 59333-67-4STABILITY INDICATINGA S S A Y V A L I D A T I O NMethod is suitable for:ýIn-process controlþProduct ReleaseþStability indicating analysis (Suitability - US/EU Product) CAUTIONFLUOXETINE HYDROCHLORIDE IS A HAZARDOUS CHEMICAL AND SHOULD BE HANDLED ONLY UNDER CONDITIONS SUITABLE FOR HAZARDOUS WORK.IT IS HIGHLY PRESSURE SENSITIVE AND ADEQUATE PRECAUTIONS SHOULD BE TAKEN TO AVOID ANY MECHANICAL FORCE (SUCH AS GRINDING, CRUSHING, ETC.) ON THE POWDER.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationTABLE OF CONTENTS INTRODUCTION........................................................................................................................ PRECISION............................................................................................................................... System Repeatability ................................................................................................................ Method Repeatability................................................................................................................. Intermediate Precision .............................................................................................................. LINEARITY................................................................................................................................ RANGE...................................................................................................................................... ACCURACY............................................................................................................................... Accuracy of Standard Injections................................................................................................ Accuracy of the Drug Product.................................................................................................... VALIDATION OF FLUOXETINE HCl AT LOW CONCENTRATION........................................... Linearity at Low Concentrations................................................................................................. Accuracy of Fluoxetine HCl at Low Concentration..................................................................... System Repeatability................................................................................................................. Quantitation Limit....................................................................................................................... Detection Limit........................................................................................................................... VALIDATION FOR META-FLUOXETINE HCl (POSSIBLE IMPURITIES).................................. Meta-Fluoxetine HCl linearity at 0.05% - 1.0%........................................................................... Detection Limit for Fluoxetine HCl.............................................................................................. Quantitation Limit for Meta Fluoxetine HCl................................................................................ Accuracy for Meta-Fluoxetine HCl ............................................................................................ Method Repeatability for Meta-Fluoxetine HCl........................................................................... Intermediate Precision for Meta-Fluoxetine HCl......................................................................... SPECIFICITY - STABILITY INDICATING EVALUATION OF THE METHOD............................. FORCED DEGRADATION OF FINISHED PRODUCT AND STANDARD..................................1. Unstressed analysis...............................................................................................................2. Acid Hydrolysis stressed analysis..........................................................................................3. Base hydrolysis stressed analysis.........................................................................................4. Oxidation stressed analysis...................................................................................................5. Sunlight stressed analysis.....................................................................................................6. Heat of solution stressed analysis.........................................................................................7. Heat of powder stressed analysis.......................................................................................... System Suitability stressed analysis.......................................................................................... Placebo...................................................................................................................................... STABILITY OF STANDARD AND SAMPLE SOLUTIONS......................................................... Standard Solution...................................................................................................................... Sample Solutions....................................................................................................................... ROBUSTNESS.......................................................................................................................... Extraction................................................................................................................................... Factorial Design......................................................................................................................... CONCLUSION...........................................................................................................................ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationBACKGROUNDTherapeutically, Fluoxetine hydrochloride is a classified as a selective serotonin-reuptake inhibitor. Effectively used for the treatment of various depressions. Fluoxetine hydrochloride has been shown to have comparable efficacy to tricyclic antidepressants but with fewer anticholinergic side effects. The patent expiry becomes effective in 2001 (US). INTRODUCTIONFluoxetine capsules were prepared in two dosage strengths: 10mg and 20mg dosage strengths with the same capsule weight. The formulas are essentially similar and geometrically equivalent with the same ingredients and proportions. Minor changes in non-active proportions account for the change in active ingredient amounts from the 10 and 20 mg strength.The following validation, for the method SI-IAG-206-02 , includes assay and determination of Meta-Fluoxetine by HPLC, is based on the analytical method validation SI-IAG-209-06. Currently the method is the in-house method performed for Stability Studies. The Validation was performed on the 20mg dosage samples, IAG-21-001 and IAG-21-002.In the forced degradation studies, the two placebo samples were also used. PRECISIONSYSTEM REPEATABILITYFive replicate injections of the standard solution at the concentration of 0.4242mg/mL as described in method SI-IAG-206-02 were made and the relative standard deviation (RSD) of the peak areas was calculated.SAMPLE PEAK AREA#15390#25406#35405#45405#55406Average5402.7SD 6.1% RSD0.1ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::PRECISION - Method RepeatabilityThe full HPLC method as described in SI-IAG-206-02 was carried-out on the finished product IAG-21-001 for the 20mg dosage form. The method repeated six times and the relative standard deviation (RSD) was calculated.SAMPLENumber%ASSAYof labeled amountI 96.9II 97.8III 98.2IV 97.4V 97.7VI 98.5(%) Average97.7SD 0.6(%) RSD0.6PRECISION - Intermediate PrecisionThe full method as described in SI-IAG-206-02 was carried-out on the finished product IAG-21-001 for the 20mg dosage form. The method was repeated six times by a second analyst on a different day using a different HPLC instrument. The average assay and the relative standard deviation (RSD) were calculated.SAMPLENumber% ASSAYof labeled amountI 98.3II 96.3III 94.6IV 96.3V 97.8VI 93.3Average (%)96.1SD 2.0RSD (%)2.1The difference between the average results of method repeatability and the intermediate precision is 1.7%.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationLINEARITYStandard solutions were prepared at 50% to 200% of the nominal concentration required by the assay procedure. Linear regression analysis demonstrated acceptability of the method for quantitative analysis over the concentration range required. Y-Intercept was found to be insignificant.RANGEDifferent concentrations of the sample (IAG-21-001) for the 20mg dosage form were prepared, covering between 50% - 200% of the nominal weight of the sample.Conc. (%)Conc. (mg/mL)Peak Area% Assayof labeled amount500.20116235096.7700.27935334099.21000.39734463296.61500.64480757797.52000.79448939497.9(%) Average97.6SD 1.0(%) RSD 1.0ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::RANGE (cont.)The results demonstrate linearity as well over the specified range.Correlation coefficient (RSQ)0.99981 Slope11808.3Y -Interceptresponse at 100%* 100 (%) 0.3%ACCURACYACCURACY OF STANDARD INJECTIONSFive (5) replicate injections of the working standard solution at concentration of 0.4242mg/mL, as described in method SI-IAG-206-02 were made.INJECTIONNO.PEAK AREA%ACCURACYI 539299.7II 540599.9III 540499.9IV 5406100.0V 5407100.0Average 5402.899.9%SD 6.10.1RSD, (%)0.10.1The percent deviation from the true value wasdetermined from the linear regression lineHPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::ACCURACY OF THE DRUG PRODUCTAdmixtures of non-actives (placebo, batch IAG-21-001 ) with Fluoxetine HCl were prepared at the same proportion as in a capsule (70%-180% of the nominal concentration).Three preparations were made for each concentration and the recovery was calculated.Conc.(%)Placebo Wt.(mg)Fluoxetine HCl Wt.(mg)Peak Area%Accuracy Average (%)70%7079.477.843465102.27079.687.873427100.77079.618.013465100.0101.0100%10079.6211.25476397.910080.8011.42491799.610079.6011.42485498.398.6130%13079.7214.90640599.413080.3114.75632899.213081.3314.766402100.399.618079.9920.10863699.318079.3820.45879499.418080.0820.32874899.599.4Placebo, Batch Lot IAG-21-001HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::VALIDATION OF FLUOXETINE HClAT LOW CONCENTRATIONLINEARITY AT LOW CONCENTRATIONSStandard solution of Fluoxetine were prepared at approximately 0.02%-1.0% of the working concentration required by the method SI-IAG-206-02. Linear regression analysis demonstrated acceptability of the method for quantitative analysis over this range.ACCURACY OF FLUOXETINE HCl AT LOW CONCENTRATIONThe peak areas of the standard solution at the working concentration were measured and the percent deviation from the true value, as determined from the linear regression was calculated.SAMPLECONC.µg/100mLAREA FOUND%ACCURACYI 470.56258499.7II 470.56359098.1III 470.561585101.3IV 470.561940100.7V 470.56252599.8VI 470.56271599.5(%) AverageSlope = 132.7395299.9SD Y-Intercept = -65.872371.1(%) RSD1.1HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSystem RepeatabilitySix replicate injections of standard solution at 0.02% and 0.05% of working concentration as described in method SI-IAG-206-02 were made and the relative standard deviation was calculated.SAMPLE FLUOXETINE HCl AREA0.02%0.05%I10173623II11503731III10103475IV10623390V10393315VI10953235Average10623462RSD, (%) 5.0 5.4Quantitation Limit - QLThe quantitation limit ( QL) was established by determining the minimum level at which the analyte was quantified. The quantitation limit for Fluoxetine HCl is 0.02% of the working standard concentration with resulting RSD (for six injections) of 5.0%. Detection Limit - DLThe detection limit (DL) was established by determining the minimum level at which the analyte was reliably detected. The detection limit of Fluoxetine HCl is about 0.01% of the working standard concentration.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::VALIDATION FOR META-FLUOXETINE HCl(EVALUATING POSSIBLE IMPURITIES)Meta-Fluoxetine HCl linearity at 0.05% - 1.0%Relative Response Factor (F)Relative response factor for Meta-Fluoxetine HCl was determined as slope of Fluoxetine HCl divided by the slope of Meta-Fluoxetine HCl from the linearity graphs (analysed at the same time).F =132.7395274.859534= 1.8Detection Limit (DL) for Fluoxetine HClThe detection limit (DL) was established by determining the minimum level at which the analyte was reliably detected.Detection limit for Meta Fluoxetine HCl is about 0.02%.Quantitation Limit (QL) for Meta-Fluoxetine HClThe QL is determined by the analysis of samples with known concentration of Meta-Fluoxetine HCl and by establishing the minimum level at which the Meta-Fluoxetine HCl can be quantified with acceptable accuracy and precision.Six individual preparations of standard and placebo spiked with Meta-Fluoxetine HCl solution to give solution with 0.05% of Meta Fluoxetine HCl, were injected into the HPLC and the recovery was calculated.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::META-FLUOXETINE HCl[RECOVERY IN SPIKED SAMPLES].Approx.Conc.(%)Known Conc.(µg/100ml)Area in SpikedSampleFound Conc.(µg/100mL)Recovery (%)0.0521.783326125.735118.10.0521.783326825.821118.50.0521.783292021.55799.00.0521.783324125.490117.00.0521.783287220.96996.30.0521.783328526.030119.5(%) AVERAGE111.4SD The recovery result of 6 samples is between 80%-120%.10.7(%) RSDQL for Meta Fluoxetine HCl is 0.05%.9.6Accuracy for Meta Fluoxetine HClDetermination of Accuracy for Meta-Fluoxetine HCl impurity was assessed using triplicate samples (of the drug product) spiked with known quantities of Meta Fluoxetine HCl impurity at three concentrations levels (namely 80%, 100% and 120% of the specified limit - 0.05%).The results are within specifications:For 0.4% and 0.5% recovery of 85% -115%For 0.6% recovery of 90%-110%HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::META-FLUOXETINE HCl[RECOVERY IN SPIKED SAMPLES]Approx.Conc.(%)Known Conc.(µg/100mL)Area in spikedSample Found Conc.(µg/100mL)Recovery (%)[0.4%]0.4174.2614283182.66104.820.4174.2614606187.11107.370.4174.2614351183.59105.36[0.5%]0.5217.8317344224.85103.220.5217.8316713216.1599.230.5217.8317341224.81103.20[0.6%]0.6261.3918367238.9591.420.6261.3920606269.81103.220.6261.3920237264.73101.28RECOVERY DATA DETERMINED IN SPIKED SAMPLESHPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::REPEATABILITYMethod Repeatability - Meta Fluoxetine HClThe full method (as described in SI-IAG-206-02) was carried out on the finished drug product representing lot number IAG-21-001-(1). The HPLC method repeated serially, six times and the relative standard deviation (RSD) was calculated.IAG-21-001 20mg CAPSULES - FLUOXETINESample% Meta Fluoxetine % Meta-Fluoxetine 1 in Spiked Solution10.0260.09520.0270.08630.0320.07740.0300.07450.0240.09060.0280.063AVERAGE (%)0.0280.081SD 0.0030.012RSD, (%)10.314.51NOTE :All results are less than QL (0.05%) therefore spiked samples with 0.05% Meta Fluoxetine HCl were injected.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::Intermediate Precision - Meta-Fluoxetine HClThe full method as described in SI-IAG-206-02 was applied on the finished product IAG-21-001-(1) .It was repeated six times, with a different analyst on a different day using a different HPLC instrument.The difference between the average results obtained by the method repeatability and the intermediate precision was less than 30.0%, (11.4% for Meta-Fluoxetine HCl as is and 28.5% for spiked solution).IAG-21-001 20mg - CAPSULES FLUOXETINESample N o:Percentage Meta-fluoxetine% Meta-fluoxetine 1 in spiked solution10.0260.06920.0270.05730.0120.06140.0210.05850.0360.05560.0270.079(%) AVERAGE0.0250.063SD 0.0080.009(%) RSD31.514.51NOTE:All results obtained were well below the QL (0.05%) thus spiked samples slightly greater than 0.05% Meta-Fluoxetine HCl were injected. The RSD at the QL of the spiked solution was 14.5%HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSPECIFICITY - STABILITY INDICATING EVALUATIONDemonstration of the Stability Indicating parameters of the HPLC assay method [SI-IAG-206-02] for Fluoxetine 10 & 20mg capsules, a suitable photo-diode array detector was incorporated utilizing a commercial chromatography software managing system2, and applied to analyze a range of stressed samples of the finished drug product.GLOSSARY of PEAK PURITY RESULT NOTATION (as reported2):Purity Angle-is a measure of spectral non-homogeneity across a peak, i.e. the weighed average of all spectral contrast angles calculated by comparing all spectra in the integrated peak against the peak apex spectrum.Purity Threshold-is the sum of noise angle3 and solvent angle4. It is the limit of detection of shape differences between two spectra.Match Angle-is a comparison of the spectrum at the peak apex against a library spectrum.Match Threshold-is the sum of the match noise angle3 and match solvent angle4.3Noise Angle-is a measure of spectral non-homogeneity caused by system noise.4Solvent Angle-is a measure of spectral non-homogeneity caused by solvent composition.OVERVIEWT he assay of the main peak in each stressed solution is calculated according to the assay method SI-IAG-206-02, against the Standard Solution, injected on the same day.I f the Purity Angle is smaller than the Purity Threshold and the Match Angle is smaller than the Match Threshold, no significant differences between spectra can be detected. As a result no spectroscopic evidence for co-elution is evident and the peak is considered to be pure.T he stressed condition study indicated that the Fluoxetine peak is free from any appreciable degradation interference under the stressed conditions tested. Observed degradation products peaks were well separated from the main peak.1® PDA-996 Waters™ ; 2[Millennium 2010]ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationFORCED DEGRADATION OF FINISHED PRODUCT & STANDARD 1.UNSTRESSED SAMPLE1.1.Sample IAG-21-001 (2) (20mg/capsule) was prepared as stated in SI-IAG-206-02 and injected into the HPLC system. The calculated assay is 98.5%.SAMPLE - UNSTRESSEDFluoxetine:Purity Angle:0.075Match Angle:0.407Purity Threshold:0.142Match Threshold:0.4251.2.Standard solution was prepared as stated in method SI-IAG-206-02 and injected into the HPLC system. The calculated assay is 100.0%.Fluoxetine:Purity Angle:0.078Match Angle:0.379Purity Threshold:0.146Match Threshold:0.4272.ACID HYDROLYSIS2.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as in method SI-IAG-206-02 : An amount equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent was added and the solution sonicated for 10 minutes. 1mL of conc. HCl was added to this solution The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with NaOH 10N, made up to volume with Diluent and injected into the HPLC system after filtration.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 98.8%.SAMPLE- ACID HYDROLYSISFluoxetine peak:Purity Angle:0.055Match Angle:0.143Purity Threshold:0.096Match Threshold:0.3712.2.Standard solution was prepared as in method SI-IAG-206-02 : about 22mg Fluoxetine HCl were weighed into a 50mL volumetric flask. 20mL Diluent were added. 2mL of conc. HCl were added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with NaOH 10N, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 97.2%.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSTANDARD - ACID HYDROLYSISFluoxetine peak:Purity Angle:0.060Match Angle:0.060Purity Threshold:0.099Match Threshold:0.3713.BASE HYDROLYSIS3.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as per method SI-IAG-206-02 : An amount equivalent to 20mg Fluoxetine was weight into a 50mL volumetric flask. 20mL Diluent was added and the solution sonicated for 10 minutes. 1mL of 5N NaOH was added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with 5N HCl, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 99.3%.SAMPLE - BASE HYDROLYSISFluoxetine peak:Purity Angle:0.063Match Angle:0.065Purity Threshold:0.099Match Threshold:0.3623.2.Standard stock solution was prepared as per method SI-IAG-206-02 : About 22mg Fluoxetine HCl was weighed into a 50mL volumetric flask. 20mL Diluent was added. 2mL of 5N NaOH was added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH=5.5 with 5N HCl, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease - 99.5%.STANDARD - BASE HYDROLYSISFluoxetine peak:Purity Angle:0.081Match Angle:0.096Purity Threshold:0.103Match Threshold:0.3634.OXIDATION4.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as per method SI-IAG-206-02. An equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent added and the solution sonicated for 10 minutes.1.0mL of 30% H2O2 was added to the solution and allowed to stand for 5 hours, then made up to volume with Diluent, filtered and injected into HPLC system.Fluoxetine peak intensity decreased to 95.2%.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSAMPLE - OXIDATIONFluoxetine peak:Purity Angle:0.090Match Angle:0.400Purity Threshold:0.154Match Threshold:0.4294.2.Standard solution was prepared as in method SI-IAG-206-02 : about 22mg Fluoxetine HCl were weighed into a 50mL volumetric flask and 25mL Diluent were added. 2mL of 30% H2O2 were added to this solution which was standing for 5 hours, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity decreased to 95.8%.STANDARD - OXIDATIONFluoxetine peak:Purity Angle:0.083Match Angle:0.416Purity Threshold:0.153Match Threshold:0.4295.SUNLIGHT5.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as in method SI-IAG-206-02 . The solution was exposed to 500w/hr. cell sunlight for 1hour. The BST was set to 35°C and the ACT was 45°C. The vials were placed in a horizontal position (4mm vials, National + Septum were used). A Dark control solution was tested. A 2%w/v quinine solution was used as the reference absorbance solution.Fluoxetine peak decreased to 91.2% and the dark control solution showed assay of 97.0%. The difference in the absorbance in the quinine solution is 0.4227AU.Additional peak was observed at RRT of 1.5 (2.7%).The total percent of Fluoxetine peak with the degradation peak is about 93.9%.SAMPLE - SUNLIGHTFluoxetine peak:Purity Angle:0.093Match Angle:0.583Purity Threshold:0.148Match Threshold:0.825 ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSUNLIGHT (Cont.)5.2.Working standard solution was prepared as in method SI-IAG-206-02 . The solution was exposed to 500w/hr. cell sunlight for 1.5 hour. The BST was set to 35°C and the ACT was 42°C. The vials were placed in a horizontal position (4mm vials, National + Septum were used). A Dark control solution was tested. A 2%w/v quinine solution was used as the reference absorbance solution.Fluoxetine peak was decreased to 95.2% and the dark control solution showed assay of 99.5%.The difference in the absorbance in the quinine solution is 0.4227AU.Additional peak were observed at RRT of 1.5 (2.3).The total percent of Fluoxetine peak with the degradation peak is about 97.5%. STANDARD - SUNLIGHTFluoxetine peak:Purity Angle:0.067Match Angle:0.389Purity Threshold:0.134Match Threshold:0.8196.HEAT OF SOLUTION6.1.Sample solution of IAG-21-001-(2) (20 mg/capsule) was prepared as in method SI-IAG-206-02 . Equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent was added and the solution was sonicated for 10 minutes and made up to volume with Diluent. 4mL solution was transferred into a suitable crucible, heated at 105°C in an oven for 2 hours. The sample was cooled to ambient temperature, filtered and injected into the HPLC system.Fluoxetine peak was decreased to 93.3%.SAMPLE - HEAT OF SOLUTION [105o C]Fluoxetine peak:Purity Angle:0.062Match Angle:0.460Purity Threshold:0.131Match Threshold:0.8186.2.Standard Working Solution (WS) was prepared under method SI-IAG-206-02 . 4mL of the working solution was transferred into a suitable crucible, placed in an oven at 105°C for 2 hours, cooled to ambient temperature and injected into the HPLC system.Fluoxetine peak intensity did not decrease - 100.5%.ED. N0: 04Effective Date:APPROVED::。
艾贝宁说明书
艾贝宁说明书 Revised by Jack on December 14,2020【药品名称】通用名称：盐酸右美托咪定注射液商品名称：艾贝宁英文名称：Dexmedetomidine Hydrochloride Injection汉语拼音：Yansuan Youmeituomiding Zhusheye【成份】本品主要成份为盐酸右美托咪定，其化学名为：(+)-4-(S)-[1-(2,3-二甲基苯基)乙基]-1H-咪唑盐酸盐。
分子式：C13H16N2·HCl分子量：本品辅料为氯化钠。
【性状】本品为无色或几乎无色的澄明液体。
【适应症】用于行全身麻醉的手术患者气管插管和机械通气时的镇静。
【规格】2ml∶200mg(按右美托咪定计)【用法用量】成人剂量：配成4mg /ml浓度以1mg/kg剂量缓慢静注，输注时间超过10分钟。
本品在给药前必须用%的氯化钠溶液稀释达浓度4mg /ml，可取出2mL本品加入%的氯化钠注射液中形成总的50ml溶液，轻轻摇动使均匀混合。
操作过程中必须始终维持严格的无菌技术。
一般地，静脉用药前应该肉眼检查药品有无颗粒物质和颜色是否改变。
剂量调整：由于可能的药效学相互作用，当本品与其他麻醉剂、镇静剂、安眠药或阿片类药物同时给药时可能需要减少给药剂量(见药物相互作用)。
肝、肾功能损伤的患者和老年患者可能需要考虑减少给药剂量。
药品相容性：因为物理相容性尚不确定，本品不应与血液或血浆通过同一静脉导管同时给予。
当本品与以下药物同时给予时显示不相容：两性霉素B，地西泮。
当本品与以下静脉液体和药物同时给予时已经显示了相容性：%的氯化钠水溶液，5%的葡萄糖水溶液。
已经证实一些类型的天然橡胶可能吸收本品，建议使用合成的或有涂层的橡胶垫给药装置。
【不良反应】由于临床试验是在多种不同情况下进行的，因此一种药物在临床试验中观察到的不良反应发生率不能与另一种药物进行直接比较，而且可能无法反映实际临床应用中观察到的不良反应情况。
CAS1204313-51-8_Icotinib Hydrochloride_MedBio相关资料
在体外激酶测定中，将2.4 ng /μLEGFR蛋白与32 ng /μLCrk在含有1μM冷ATP和1μCi32P-γ-ATP的25μL激酶反应缓冲液中混合。将混合物与Icotinib在0,0.5,2.5,12.5或62.5nM下在冰上温育10分钟，然后在30℃温育20分钟。用SDS样品缓冲液在100℃猝灭4分钟后，通过在10％SDS-PAGE凝胶中电泳分离蛋白质混合物。然后暴露干燥的凝胶以检测放射性。量化由软件[1]执行。
CAS
1、产品物理参数：
常用名
凯美纳
英文名
Icotinib Hydrochloride
CAS号
1204313-51-8
分子量
427.881
密度
无资料
沸点
无资料
分子式
C22H22ClN3O4
熔点
无资料
闪点
无资料
2、技术资料：
体外研究
与Iconitib在0.5μM孵育导致激酶活性抑制分别为91％，99％，96％，61％和61％。 Iconitib抑制A431和BGC-823 A549，H460和KB细胞系的增殖，IC50分别为1,4.06,12.16,16.08,40.71μM。当用88种激酶进行分析时，Icotinib仅对EGFR及其突变体显示出有意义的抑制活性。 Icotinib阻断人表皮样癌A431细胞系中EGFR介导的细胞内酪氨酸磷酸化（IC50 = 45 nM）并抑制肿瘤细胞增殖[1]。
10mM (in 1mL DMSO)
≥98%
1172133-28-6
5mg
≥98%
品牌
货号
中文名称
英文名称
CAS
包装
纯度
MedBio
美索巴莫注射液标准
美索巴莫注射液标准美索巴莫注射液是一种中枢性肌肉松弛剂，主要用于治疗急性骨骼肌疼痛或不适症状。
以下是关于美索巴莫注射液的一些标准信息：1. 中文通用名：美索巴莫注射液2. 英文通用名：Methocarbamol Injection3. 标准号：ws-10001-(hd-0269)-20024. 药品名称：美索巴莫注射液5. 药品英文名：Methocarbamol Injection6. 主要成分：本品为美索巴莫的灭菌聚乙二醇，400水溶液。
含美索巴莫（c11h15no5）应为标示量的95.0%~105.%。
7. 处方：具体处方应根据医生建议和患者病情来确定。
8. 性状：本品为无色或几乎无色略带黏稠的澄明液体。
9. 鉴别：通过化学分析和物理测试等方法来鉴别美索巴莫注射液的真伪和质量。
10. 作用类别：中枢性肌肉松弛剂11. 药理毒理：本品对中枢神经系统有选择作用，特别对脊椎中神经元作用明显。
抑制与骨骼肌痉挛有关的神经突触反射，有抗士的宁和电刺激所致惊厥的作用，并有解痉、镇痛和抗炎作用。
其作用机制主要是阻断脊髓内中枢神经元从而使骨骼肌松弛。
12. 药代动力学：美索巴莫注射液在体内的吸收、分布、代谢和排泄过程。
13. 适应症：主要用于急性骨骼肌疼痛或不适症状的辅助治疗。
14. 用法用量：美索巴莫注射液的用法主要是采用静脉滴注或者是静脉推注的方式给药。
如果是用于静脉推注时，患者在静卧的条件下缓慢推注，给药的速度每分钟不可以超过3ml，注射之后应该至少休息10~15分钟。
如果是用于静脉滴注时，将药品配在9%的氯化钠注射液或者是5%的葡萄糖注射液当中，滴注的速度不宜过快。
使用剂量和次数根据病情和治疗效果来决定。
成人一次使用剂量为1.0g，一日最大剂量为3.0g，连续使用不得超过3天。
轻度病例静注后应改为口服给药以维持治疗。
15. 不良反应：使用美索巴莫注射液可能出现的不良反应，如头痛、恶心、呕吐、皮疹等。
16. 禁忌症：对美索巴莫过敏者、肝肾功能不全者、哺乳期妇女禁用。
Bupropion hydrochloride_多巴胺去甲肾上腺素抑制剂_31677-93-7_Apexbio
产品名: Cas No.: 分子量: 分子式: 化学名: SMILES: 溶解性: 储存条件: 一般建议:
运输条件:
Bupropion hydrochloride
31677-93-7 276.2
C13H19Cl2NO
2-(tert-butylamino)-1-(3-chlorophenyl)propan-1-one;hydrochloride
生物活性
靶点 :
信号通路:
产品描述:
Bupropion hydrochloride 是去甲肾上腺素和多巴胺神经递质的双重高效抑制剂,IC50 值分别为 6.5 ± 0.6 μM 和 3.4 ± 0.4 μM [1]. Bupropion hydrochloride 是一种氨基酮,作为非尼古丁以帮助戒烟.Bupropion hydrochloride 也 是经典的抗抑郁药,能够干扰去甲肾上腺素\血清素和多巴胺的摄取[2].此外,Bupropion hydrochloride 能够降低多巴胺生物合成中的限速酶—酪氨酸羟化酶的表达[3].体外实验 中,Bupropion hydrochloride 能够显著降低 SH-SY5Y 细胞的活力.而且,Bupropion hydrochloride 能选择性激活 ER 应激通路.此外,这些结果也表明 Bupropion hydrochloride 能通过 JNK 和 ER
ApexBio Technology
bupropionhydrochloride是去甲肾上腺素和多巴胺神经递质的双重高效抑制剂ic50值分别为6506m和34bupropionhydrochloride是一种氨基酮作为非尼古丁以帮hydrochloride 修订日期: 6/30/2016
应激导致 caspase 3 活化,从而诱导细胞死亡[2]. 参考文献: [1] Dr. R. M. Ferris*, H. L. White,B. R. Cooper, R. A. Maxwell, F. L. M. Tang,O. J. Beaman and A. Russell .Some neurochemical properties of a new antidepressant, bupropion hydrochloride (Wellbutrin). Drug Development Research Volume 1, Issue 1, pages 21–35, 1981 [2] Jang EH1, Park CS, Kang JH. Bupropion, an atypical antidepressant, induces endoplasmic reticulum stress and caspase-dependent cytotoxicity in SH-SY5Y cells. Toxicology. 2011 Jul 11;285(1-2):1-7. [3] Nestler, E. J., McMahon, A., Sabban, E.L., Tallman, J.F., Duman, R.S., 1990. Chronicantidepressant administration decreases the expression of tyrosine hydroxylase in the rat locus coeruleus. Proc. Natl. Acad. Sci. U. S. A. 87, 7522–7526.
海松树皮提取物(碧萝芷)中的低聚原花青素可有效抑制α-葡萄糖苷酶
出， 故该 化合 物 为有效 成 分之一 。 无菜 蓟苦 素而 富含 保 护作 用 ， 完 全抑制 胶 原蛋 白和 ４ＨＰ的增 加 （ 并 － ＱＣ 酮类 也是 有效 成 分之一 。 化合 物 １ ２与 ３做对 比 ， 将 、
ＱＣ脂 质 体 对 星形 细 胞 、 半乳 糖 化 ＱＣ脂 发 现化合 物 １ 量浓 度 为 ５１ ／ 质 、０ｍｇ Ｌ使胆 汁流 量分 作 用 最 强 ；
维普资讯
３ ２
国 外 医药 ・ 物 药 分 册 植
２０ ０ ８年 第 ２ ３卷 第 １期
（） ２ 的作 用 ， 与 具促 胆 汁分 泌作 用 的朝 鲜蓟 酸 （ ） 并 ３ 做 了比较 。
组 单 次量 Ｓ 理盐水 ； 型组 每 隔 １ Ｃ生 模 ０Ｓ于腹 部 不 同
部位 Ｓ Ｃ溶于 生理 盐水 的 Ｎａ Ｏ２ ０ ． ６ｂ ｋ ， Ａｓ ０ ０ ｔ ｇ 共 １ ｍ／
制 备 了该 植 物 的 水 浸 剂 （ 取 物 １ ； 将 该 植 提 ）另 物 粉 末 依 次 用 醋 酸 乙酯 （ 取 物 ２ 、 醇 （ 取 物 提 ）甲 提
３、 （ ） 水 提取物 ４ 提取 。 提取 物 １ ） 将 ～４用 于实 验 。 肝
Ｎａ Ｏ。 射 液 ２ Ａｓ 注 ４ｈ后 ， 醉 所 有 大 鼠 ， 脏 采 血 ， 麻 心
灌 流 实验 采 用 Ｊｇ ｒ等 曾报 道 过 的 方法 。提 取物 １ ａｅ
用 克一 氏缓 冲 液 （ 汉 ＫＨＢ） 释 至终 质量 浓 度 为 ２ ～ 稀 Ｏ
１０ ０ｍｇ Ｌ； ０ ／ 提取 物 ２ ～４稀 释后 的质 量浓 度 为 １ ～ ５ ０ｍｇ Ｌ ０ ／ 。在 每次 实验 开始 进行 预灌 流 ３ ｉ ， ０ｒ ｎ后 ａ 开始 灌 流 １ ｎ（ 断 增 加 药 物 质 量 浓 度 ） 隔 １ ０ｍｉ 不 ， Ｏ
黄檗提取物对阿尔茨海默病的神经保护作用研究
黄檗提取物对阿尔茨海默病的神经保护作用研究黄檗提取物在阿尔茨海默病的神经保护作用研究中的重要性阿尔茨海默病（Alzheimer's Disease，AD）是一种常见的神经退行性疾病，其主要特征是记忆力的丧失、认知功能的衰退以及行为和人格的改变。
目前，阿尔茨海默病的治疗主要是通过药物控制病情，但还没有找到完全治愈的方法。
因此，寻找新的治疗策略对于缓解患者症状和改善生活质量非常重要。
黄檗（Scutellaria baicalensis）是一种中草药，因其具有多种生物活性成分而被广泛应用于传统中医。
最近的研究表明，黄檗提取物可能具有对阿尔茨海默病的神经保护作用。
首先，黄檗提取物含有丰富的黄酮类化合物，如黄芩素、黄酮素等。
这些黄酮类化合物具有抗炎、抗氧化和抗神经炎症的特性，可以减轻炎症和氧化应激导致的神经细胞损伤。
实验证明，黄檗提取物可以抑制炎症因子的释放，降低神经细胞的细胞毒性，从而减轻阿尔茨海默病患者的症状。
其次，黄檗提取物可以改善神经递质的失衡情况。
阿尔茨海默病患者常常伴随着乙酰胆碱能神经元的损失，导致乙酰胆碱水平的下降。
黄檗提取物中的活性成分可以促进乙酰胆碱的合成和释放，从而改善乙酰胆碱缺乏引发的神经功能障碍。
研究还发现，黄檗提取物可影响阿尔茨海默病患者大脑中多巴胺和谷氨酸的水平，进一步调节神经递质的平衡。
此外，黄檗提取物还可以抑制β-淀粉样蛋白（β-amyloid）的沉积和聚集，这是阿尔茨海默病的典型特征之一。
β-淀粉样蛋白的沉积会引发炎症反应和氧化应激，导致神经细胞的损伤和死亡。
黄檗提取物中的黄酮类化合物可以减少β-淀粉样蛋白的沉积，保护神经细胞免受损伤。
最后，黄檗提取物还具有促进神经细胞再生和修复的作用。
阿尔茨海默病患者的大脑中存在神经细胞损伤和死亡，因此恢复和重新生长神经细胞对于病情的改善至关重要。
研究表明，黄檗提取物可以促进神经前体细胞的分化和成熟，增强神经细胞的生长和再生能力。
莫迪司(钆贝葡胺注射液)
云芝胞内糖肽胶囊【药品名称】通用名称：云芝胞内糖肽胶囊英文名称：Polystictus Glycopeptide Capsules【成份】本品成份为云芝胞内糖肽。
【适应症】用于慢性乙型肝炎、肝癌及老年免疫功能低下者的辅助治疗。
【用法用量】口服。
一次0．5～1．0g，一日3次。
【不良反应】尚不明确。
【禁忌】对本品过敏者禁用。
【注意事项】1 尿病患者慎用或遵医嘱。
2 药品性状发生改变时禁止使用。
【药物相互作用】尚不明确。
【药理作用】1 本品能通过对单核-巨噬细胞的增殖增强及对每个吞噬细胞本身功能的激活从而增强网状内皮系统（RES）功能。
可使小鼠诱生血清干扰素浓度明显增高；可在不增加白细胞数的情况下，通过对白细胞本身功能的激活而使中性白细胞吞噬活性增强；可促使SRBC单次免疫所致615小鼠特异性溶血素抗体的生成；可拮抗环磷酰胺、氨甲蝶呤、5-FU、丝裂霉素C等所致细胞免疫强烈抑制，使细胞免疫功能改善，恢复正常。
2 对于D-氨基半乳糖、四氯化碳所致大鼠和小鼠急性肝损伤，本品能显著抑制SGP的升高，肝细胞的脂肪性变出现率较对照组低，并能完全避免灶性坏死的出现。
3 本品能使小鼠腹腔巨噬细胞对艾氏腹水癌细胞杀死率达41%～54%；能增强抗癌药物的作用；本品还能通过降低化生长因子TGF-β1的水平，有效抑制肿瘤血管生成和移植性乳腺癌生长。
4 本品能强烈地激活机体本身防御免疫系统，具有继发性的非特异性抗感染和抗休克作用，能明显降低黄金色葡萄球菌、大肠杆菌、痢疾杆菌感染所致的死亡率，对于流感病毒感染所致小鼠死亡，本品也有明显保护作用，保护率达50%～60%，平均生存时间可延长153%～188%。
【贮藏】密闭，在阴凉干燥处保存。
【批准文号】国药准字H20053490【生产企业】企业名称：江苏神华药业有限公司生产地址：江苏省金湖县神华大道188号。
美索巴莫注射液标准
性状
本品为无色或几乎无色略带黏稠的澄明液体。
鉴别
（1）取本品5ml，缓缓加入硫酸3ml，小心加热，即分解并放出二氧化碳气体，通人氢氧化钙试液中，即生成白色沉淀。
（2）取本品5ml，加氢氧化钠试液3ml，加热，即发生氨臭，能使湿润的红色石蕊试纸变蓝色。
（3）取本品，加乙醇稀释制成每1ml中约含50g的溶液，照分光光度法（中国药典2000年版二部附录IV A）测定，在274nm的波长处有最大吸收。
注意
1．肝肾功能障碍者慎用。
2．孕妇及哺乳期妇女禁用。
3．对本品过敏者禁用。
规格
（1）20ml︰0.2g（2）5ml︰0.5g
贮藏
遮光，密闭保存。
有效期
暂定2年
曾用名
舒筋灵注射液
起草单位
重庆药友Байду номын сангаас药有限公司
复核单位
重庆市药品检验所
出处
化学药品地方标准上升国家标准（第三册）
其他应符合注射剂项下有关的各项规定（中国药典2000年版二部附录I B）。
含量测定
照高效液相色谱法（中国药典2000年版二部附录V D）测定。
色谱条件与系统适用性试验用十八烷基硅烷键合硅胶为填充剂；以甲醇-磷酸盐缓冲溶液（取磷酸二氢钾6.8g，加水溶解并稀释至1000ml，用磷酸或10mol/L氢氧化钾溶液调节pH值至4.500.05）（30︰70）为流动相；检测波长为274nm。理论板数按美索巴莫峰计算应不低于3000，美索巴莫峰与相邻杂质峰的分离度应符合要求。
测定法取本品，用流动相制成每1mg中含1mg的溶液，取续滤液20l注入液相色谱仪，记录色谱图，另精密称取美索巴莫对照品50mg，同法测定。按外标法以峰面积计算，即得。
灵芝α-(1→3)-D-葡聚糖羧甲基化衍生物及其用途和制备方法[发明专利]
![灵芝α-(1→3)-D-葡聚糖羧甲基化衍生物及其用途和制备方法[发明专利]](https://img.taocdn.com/s3/m/b92ac702551810a6f4248608.png)
专利名称：灵芝α－(1→3)－D－葡聚糖羧甲基化衍生物及其用途和制备方法
专利类型：发明专利
发明人：张俐娜,陈敬华
申请号：CN99116590.X
申请日：19990805
公开号：CN1283633A
公开日：
20010214
专利内容由知识产权出版社提供
摘要：本发明公开了具有抗肿瘤活性的灵芝α－(1→3)－D一葡聚糖羧甲基化衍生物的制备方法。
该方法用碱水溶液首次从灵芝子实体中提取出水不溶性线型α－(1→3)－D－葡聚糖,该多糖不具有抗肿瘤活性。
将α－(1→3)－D－葡聚糖溶解在NaOH水溶液中,用氯乙酸钠水溶液在80℃对其羧甲基化6-8小时制备了该多糖的羧甲基衍生物。
这种衍生物是水溶性的,对植入小鼠体内的爱氏腹水瘤(EAC)的生长具有明显的抑制作用,并且无毒副作用。
申请人：武汉大学
地址：430072 湖北省武汉市武昌珞珈山
国籍：CN
代理机构：武汉大学专利事务所
代理人：康俊明
更多信息请下载全文后查看。
Ombrabulin_hydrochloride_LCMS_19830_MedChemExpress
=====================================================================Acq. Operator : Su Xiao Ying(LCMS-02) Seq. Line : 98Acq. Instrument : HY-LCMS-02 Location : P1-B-01Injection Date : 3/21/2016 5:24:45 PM Inj : 1Inj Volume : 3.000 µl Different Inj Volume from Sample Entry Actual Inj Volume : 10.000 µlAcq. Method : D:\AGLIENT 1260\DATA\20160321\20160321 2016-03-21 09-08-05\100-1000MS+3MIN- 1.5_(0.02%FA).MLast changed : 3/21/2016 9:08:05 AM by Su Xiao Ying(LCMS-02)Analysis Method : D:\AGLIENT 1260\DATA\20160321\20160321 2016-03-21 09-08-05\100-1000MS+3MIN- 1.5_(0.02%FA).M (Sequence Method)Last changed : 3/21/2016 5:36:20 PM by Su Xiao Ying(LCMS-02) (modified after loading)M ethod Info : HY-365_5HO1RS,M,A-RP-108，210nm,23minCatalog No : HY-18256 Batch#19830 A-RP-134Additional Info : Peak(s) manually integratedmin0.511.52 2.53mAU 02505007501000125015001750 DAD1 B, Sig=214,4 Ref=off (D:\AGLIENT...\DATA\20160321\20160321 2016-03-21 09-08-05\BIZ2016-321-WJ11-3.D)1.6241.9082.607===================================================================== Area Percent Report =====================================================================Sorted By : Signal Multiplier : 1.0000Dilution : 1.0000Do not use Multiplier & Dilution Factor with ISTDsSignal 1: DAD1 B, Sig=214,4 Ref=offPeak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.624 MM 0.0733 9352.80469 2126.61353 99.5686 2 1.908 MM 0.0717 20.52279 4.77074 0.2185 3 2.607 MM 0.0751 20.00430 4.43991 0.2130Totals : 9393.33177 2135.82417===================================================================== *** End of Report ***=====================================================================Acq. Operator : Su Xiao Ying(LCMS-02) Seq. Line : 98Acq. Instrument : HY-LCMS-02 Location : P1-B-01Injection Date : 3/21/2016 5:24:45 PM Inj : 1Inj Volume : 3.000 µl Different Inj Volume from Sample Entry Actual Inj Volume : 10.000 µlAcq. Method : D:\AGLIENT 1260\DATA\20160321\20160321 2016-03-21 09-08-05\100-1000MS+3MIN- 1.5_(0.02%FA).MLast changed : 3/21/2016 9:08:05 AM by Su Xiao Ying(LCMS-02)Analysis Method : D:\AGLIENT 1260\DATA\20160321\20160321 2016-03-21 09-08-05\100-1000MS+3MIN- 1.5_(0.02%FA).M (Sequence Method)Last changed : 3/21/2016 5:35:01 PM by Su Xiao Ying(LCMS-02) (modified after loading)M ethod Info : HY-365_5HO1RS,M,A-RP-108，210nm,23minCatalog No : HY-18256 Batch#19830 A-RP-134Additional Info : Peak(s) manually integratedmin0.511.522.53100000200000300000400000500000600000700000 MSD1 TIC, MS File (D:\AGLIENT 1260\DATA\20160321\20160321 2016-03-21 09-08-05\BIZ2016-321-WJ11-3.D) ES-API, Pos, S1.632MS Signal: MSD1 TIC, MS File, ES-API, Pos, Scan, Frag: 50 Spectra averaged over upper half of peaks. Noise Cutoff: 1000 counts.Reportable Ion Abundance: > 10%.Retention Mol. Weight Time (MS) MS Area or Ion1.632 5043901 404.20 I 403.20 Im/z10020030040050060070080020406080100*MSD1 SPC, time=1.598:1.689 of D:\AGLIENT 1260\DATA\20160321\20160321 2016-03-21 09-08-05\BIZ2016-321-WJ11-3.D ES-AP Max: 458475404.2403.2*** End of Report ***。
银耳多糖-壳寡糖纳米复合体系构建及其对BSA释放特性的影响
银耳多糖-壳寡糖纳米复合体系构建及其对BSA释放特性的影响王欢;赵永亮;安星亮;姚启悦;李飞寰;任军华;赵春杰【期刊名称】《食品研究与开发》【年(卷),期】2022(43)14【摘要】功能性蛋白质因其在疾病预防与健康改善方面的功效突出,从而受到广泛关注,但其生物利用率较低,使用范围被极大限制。
研究银耳多糖(Tremella fuciformis polysaccharide,TFP)和壳寡糖(chitooligosaccharides,COS)通过聚电解质络合法构建制备负载牛血清蛋白(bovine serum protein,BSA)的COS-TFP纳米复合体系,并以复合物粒径、多分散指数、电位为指标,探究复合物稳定性,以牛血清蛋白(bovine serum protein,BSA)为模型,研究COS-TFP复合物的载药与释药特性。
结果表明:当COS与TFP总浓度为2 mg/mL、COS与TFP浓度比为1∶5~1∶3、NaCl浓度为20 mmol/L~50 mmol/L、pH值为4.0时,纳米粒具有良好的稳定性;当COS-TFP∶BSA浓度比为1∶0.6时,包埋率达到91%;同时,BSA 在pH值为4.0(饱食胃液环境)时释药量低且速率缓慢,pH值为7.0(肠道环境)时释药迅速,可作为潜在的蛋白质类控释运载体系。
【总页数】8页(P125-132)【作者】王欢;赵永亮;安星亮;姚启悦;李飞寰;任军华;赵春杰【作者单位】河南工业大学生物工程学院【正文语种】中文【中图分类】G63【相关文献】1.壳聚糖-海藻酸盐纳米粒子的制备及其对BSA的载药与释放特性2.甘草酸二铵半乳糖化壳寡糖纳米粒的制备及体外释放考察3.两步法制备核壳结构Fe3O4@PDA@BSA纳米复合材料4.果胶、BSA及壳寡糖相互作用及对BSA负载效果的影响因版权原因，仅展示原文概要，查看原文内容请购买。