EFFECT OF SACCHARIDES ON GELATINIZATION AND RETROGRADATION OF MODIFIED POTATO STARCH

专利名称：糖皮质激素受体调制剂
专利类型：发明专利
发明人：罗伯特·L·道,刘康志,布拉德利·P·摩根,安德鲁·G·斯威克
申请号：CN00806949.2
申请日：20000327
公开号：CN1349485A
公开日：
20020515
专利内容由知识产权出版社提供
摘要：本发明提供式(I)的非甾族化合物,它们是甾族受体、具体为糖皮质激素受体的选择性调制剂(即激动剂和拮抗剂)。

本发明也提供含有这些化合物的药物组合物和使用这些化合物的方法,以治疗需要糖皮质激素受体激动剂和/或拮抗剂疗法的动物。

糖皮质激素受体调制剂可用于治疗诸如肥胖、糖尿病、炎症和其他下述疾病。

本发明还提供用于制备这些化合物的中间体和方法。

申请人：辉瑞产品公司
地址：美国康涅狄格州
国籍：US
代理机构：北京市柳沈律师事务所
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刘好⾬等Microbiome：罗伊⽒黏液乳杆菌如何益免疫热⼼肠⽇报今天是第1960期⽇报。

刘好⾬等：罗伊⽒黏液乳杆菌-B细胞-肠道菌群的特异性互作Microbiome[IF:14.65]①以⼤⼩为基础，发现派⽒结具代谢活性⾼的类⽣发中⼼（GC）⼤B细胞和有先天免疫活性的类pre-GC⼩B细胞；②⼝服罗伊⽒黏液乳杆菌通过上调S1P/S1PR1信号，促进类pre-GC⼩B积聚并增强其抗菌活性；③同时增加⼤B在GC积聚刺激其⾃分泌TGFβ-1，促进B细胞-IgA极化、诱导；④通过PD-1滤泡辅助性T细胞依赖性通路，增强IgA⽣产及SIgA与肠道菌群互作；⑤最终塑造抗病菌、抗炎的肠道微⽣物组，减轻⼩⿏结肠炎，改善菌群失调并保护肠道。

Distinct B cell subsets in Peyer’s patches convey probiotic effects by Limosilactobacillusreuteri10-03, doi: 10.1186/s40168-021-01128-4【主编评语】派⽒结（PP结）是分布于⼈类和动物⼩肠的次级淋巴组织，肩负着抗原感知、信号传播、IgA诱导以及产⽣⼝服耐受等重要任务。

瑞典乌普萨拉⼤学Mia Phillipson课题组、扬州⼤学刘好⾬为第⼀作者在Microbiome发表⽂章，提出罗伊⽒黏液乳杆菌R2LC可“操控”PP结，利⽤B细胞功能的可塑性与多样性，促进IgA诱导、分泌和对肠道菌群的包被，成就⼀菌改变菌群的“涟漪效应”。

⽂章同时报道了PP结B细胞在炎症性肠病中的重要作⽤。

（@solo）丙酸盐的潜在降胆固醇、抗动脉粥样硬化特性European Heart Journal[IF:29.983]①丙酸盐（PA）处理可减轻⾼脂饮⾷诱导ApoE-/-⼩⿏的动脉粥状硬化表型、⾼胆固醇⾎症，作⽤独⽴于肠道菌群；② PA增加肠内调节性T细胞（Treg）及IL-10⽔平，抑制肠上⽪细胞表达重要胆固醇转运蛋⽩Npc1l1，从⽽降低⾎液胆固醇⽔平；③阻断IL-10受体信号传导则可以逆转PA的治疗作⽤，增加⾎液内胆固醇含量及动脉粥状硬化病变区域；④随机双盲⼈体对照试验显⽰，8周PA⼝服补充（500mg x 2/天）显著降低患者⾎液低密度脂蛋⽩、胆固醇⽔平。

19第19卷 第3期 2017 年 3 月辽宁中医药大学学报JOURNAL OF LIAONING UNIVERSITY OF TCMVol. 19 No. 3 Mar .，2017类风湿性关节炎（Rheumatoid arthritis，RA）是一种全身性自身免疫性疾病[1]，其主要特征为关节的肿胀、疼痛和功能障碍，病因尚不明确，病情反复发作，给患者的身心健康带来了巨大的伤害。

目前，RA 仍属一种难治性疾病，不合理用药率较高。

传统医药治疗本病具有疗效稳定、副作用小等特点，日益被人们所关注。

铁筷子（Chimonanthus praecox ）是腊梅科腊梅属（Chimonanthus ）植物腊梅[Chimonanthus praecox （L.）Link.]及山腊梅（Chimonanthus nitens Oliv.）的根，性辛味温，具有镇痛、理气、活血及化瘀的功效，临床上主要用来治疗风湿痹痛、跌打损伤肿痛、脘腹疼痛等[2]，是苗族人民用于治疗类风湿性关节炎的常用药物，具有长期的使用历史。

前期药理实验中已经证实，铁筷子醇提物具有抗炎镇痛作用，但其能否用于自身免疫性疾病如RA 的治疗鲜有报道。

佐剂性关节炎模型（Adjuvant Arthritis，AA）是评价防治RA 药物的常用动物模型之一，其发病机理、病变部位和病变性质都与人RA 相似。

本研究通过观察铁筷子醇提物对AA 大鼠的治疗作用，以及对AA 大鼠血清中TNF-α和PAF 含量的影响，探讨其作用机制，旨在为开发治疗RA 的有效药物提供实验依据。

1 材料与仪器1.1 实验动物SPF 级SD 大鼠72只，雌雄各半，体重（200±20）g，由中国人民解放军第三军医大学实验动物中心提供（动物合格证书编号：2012-0003）。

1.2 药品与试剂铁筷子，采自贵阳市乌当区三江镇农场，经贵阳中医学院药学院生药教研室鉴定为腊梅科腊梅属植物腊梅的根。

Characteristics and gel properties of gelatin from skin of seabass (Lates calcarifer )as influenced by extractionconditionsSittichoke Sinthusamran a ,Soottawat Benjakul a ,⇑,Hideki Kishimura ba Department of Food Technology,Faculty of Agro-Industry,Prince of Songkla University,Hat Yai,Songkhla 90112,ThailandbLaboratory of Marine Products and Food Science,Research Faculty of Fisheries Sciences,Hokkaido University,Hakodate,Hokkaido 041-8611,Japana r t i c l e i n f o Article history:Received 11July 2013Received in revised form 10October 2013Accepted 20November 2013Available online 27November 2013Keywords:Seabass Gelatin Extraction Gel strength Settinga b s t r a c tCharacteristics and gel properties of gelatin from seabass skin,as influenced by extraction conditions,were studied.Yields of gelatin extracted at 45and 55°C for various times were 51.6–57.3%and 62.0–66.4%(dry weight basis),respectively.All gelatins contained b -chain and a -chains as the predominant components and showed a high imino acid content (198–202residues/1000residues).Generally,the gel strength of gelatins decreased as the extraction temperature and time increased.Gelatin extracted at 45°C for 3h exhibited the highest gel strength (369g).Gelling and melting temperatures for seabass skin gelatin were 19.5–20.0and 26.3–27.0°C,respectively.All gelatins could be set at 25°C within 30min,however gelatins extracted at 45°C had a shorter setting time than those extracted at 55°C (P <0.05).Gelatin from seabass skin showed a higher gel strength than bovine gelatin and could be used as a potential replacement for land animal gelatins.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionSeabass (Lates calcarifer )is one of the economically important species of Thailand and other countries in the Southeast Asia.It has been widely used for fillet production,in which a large amount of byproducts e.g.,skin,bone,scales,etc.,is generated.Fish process-ing byproducts can account 75%of the total catch weight.About 30%of byproducts consist of skin and bone,which can serve as a source of collagen and gelatin.The utilisation of fish skin and bone for gelatin production can add value to processing byproducts and to eliminate harmful environmental aspects (Binsi,Shamasundar,Dileep,Badii,&Howell,2009;Gómez-Guillén et al.,2002).Gelatin is a fibrous protein produced by thermal denaturation,or partial degradation of collagen from animal skin and bone.It has been widely used in food,material,pharmacy and photography industries (Benjakul,Oungbho,Visessanguan,Thiansilakul,&Roytrakul,2009;Tabarestani,Maghsoudlou,Motamedzadegan,&Sadeghi Mahoonak,2010).Generally,the typical sources of gelatin are the skin and bones of pigs and cows.However,outbreaks of mad cow disease (Bovine Spongiform Encephalopathy,BSE)have caused anxiety in customers.In addition,porcine gelatin cannot be used for the Halal and Kosher food markets (Kittiphattanabawon,Benjakul,Visessanguan,&Shahidi,2010).Therefore,an increasing interest in the production of fish gelatin from fish skins and bones asalternative sources has been gained (Jongjareonrak,Benjakul,Visessanguan,&Tanaka,2006).In general,gelatins from the skins of cold-water fish have lim-ited applications,mainly due to the lower gel strength and stability of the gels,compared with mammalian counterparts.Additionally,fish gelatin has a lower gelling and melting temperatures than mammalian gelatin (Kittiphattanabawon et al.,2010).This is gov-erned by the lower imino acid (proline and hydroxyproline)con-tent (Kittiphattanabawon et al.,2010).Skin gelatins from several fish species,e.g.,yellowfin tuna (Cho,Gu,&Kim,2005),grass carp (Kasankala,Xue,Weilong,Hong,&He,2007),brownbanded bam-boo shark and blacktip shark (Kittiphattanabawon et al.,2010),splendid squid (Nagarajan,Benjakul,Prodpran,Songtipya,&Kishimura,2012)and unicorn leatherjacket (Kaewruang,Benjakul,&Prodpran,2013)have been produced.Seabass skin has been used in the production of snacks,etc.,to increase its market value.Recently,collagen from seabass skin was extracted and identified as type I collagen (Sinthusamran,Benja-kul,&Kishimura,2013).To widen its utility,the production of gel-atin can be another approach,which can increase the revenue for the processor and farmer.Seabass is a warm-water species,and its gelatin might have a better gelling property than in cold water fish.However,the extraction conditions,including temperature,time and the protease inhibitors,have been known to affect the properties of the gelatin from the skin of some fish species (Kaewruang et al.,2013;Nagarajan,Benjakul,Prodpran,&Songtipya,2013).Nevertheless,no information regarding gelatin extraction from seabass skin under varying conditions has been0308-8146/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.foodchem.2013.11.109⇑Corresponding author.Tel.:+6674286334;fax:+6674558866.E-mail address:soottawat.b@psu.ac.th (S.Benjakul).reported.Therefore,this study aimed to determine the physico-chemical characteristics and gelling properties of gelatins from the skin of seabass(L.calcarifer)as affected by different extraction temperatures and times.2.Materials and methods2.1.ChemicalsAll chemicals were of analytical grade.Sodium dodecyl sulphate (SDS),Coomassie blue R-250and N,N,N0,N0-tetramethylethylenedi-amine(TEMED)were procured from Bio-Rad Laboratories (Hercules,CA,USA).High-molecular-weight markers were pur-chased from GE Healthcare UK Limited(Buckinghamshire,UK). Food grade bovine bone gelatin with the bloom strength of150–250g was obtained from Halagel(Thailand)Co.,Ltd.,(Bangkok, Thailand).2.2.Collection and preparation of seabass skinDescaled skin of seabass(L.calcarifer)with a weight of2.5–3kg, was obtained from the Kingfisher Holdings Co.,Ltd.,Songkhla, Thailand.Descaled skin was kept in ice with a skin/ice ratio1:3 (w/w)and transported to the Department of Food Technology, Prince of Songkla University,Hat Yai within1h.Upon arrival,the remaining meat was removed manually and the skin was washed using cold tap water.The skin was pooled and used as the compos-ite sample.The sample was placed in polyethylene bags and stored atÀ20°C until used,but not longer than3months.Prior to gelatin extraction,the frozen skin was thawed with running water until the core temperature reached8–10°C.The skin was then cut into small pieces(1.0Â1.0cm2)using scissors.2.3.Extraction of gelatin from the skin of seabassGelatin was extracted from seabass skin according to the meth-od of Jongjareonrak et al.(2006),with slight modification.Before gelatin extraction,skin was soaked in0.1M NaOH,with a skin/ solution ratio of1:10(w/v),to remove any non-collagenous pro-teins.The mixture was stirred for3h at room temperature(28–30°C)using an overhead stirrer model W20.n(IKAÒ-Werke GmbH &CO.KG,Stanfen,Germany).The alkaline solution was changed every1h for a total of3times.The residues were washed with tap water until a neutral or faintly basic pH was obtained.The res-idues were then mixed with0.05M acetic acid at a skin/solution ratio of1:10(w/v)to swell collagenous material in thefish skin matrix.The mixture was stirred at room temperature for2h.The skin was washed using tap water until a neutral or faintly acidic pH of the wash water was obtained.Finally,the swollen skin was mixed with distilled water at a ratio of1:10(w/v)at45and 55°C.The extraction was performed for various times(3,6and 12h)with continuous stirring.At the designated time,the mix-tures werefiltered using a Buchner funnel,with a Whatman No. 4filter paper(Whatman International,Ltd.,Maidstone,England). Then,thefiltrates were freeze-dried using a freeze-dryer(CoolSafe 55,ScanLaf A/S,Lynge,Denmark).Gelatin samples were subse-quently subjected to analyses.2.4.Analyses2.4.1.YieldThe yield of gelatin was calculated based on the dry weight of the starting material:Yieldð%Þ¼Weight of freezeÀdried gelatinðgÞWeight of initial dry skinðgÞÂ100ð1Þ2.4.2.SDS–polyacrylamide gel electrophoresis(SDS–PAGE)SDS–PAGE was performed according to the method of Laemmli(1970).Gelatin samples were dissolved in5%SDS solution.Themixtures were heated at85°C for1h using a temperature con-trolled water bath model W350(Memmert,Schwabach,Germany).Solubilised samples were mixed at a1:1(v/v)ratio with samplebuffer(0.5M Tris–HCl,pH6.8containing5%SDS and20%glycerol).Samples were loaded onto a polyacrylamide gel made up of7.5%separating gel and4%stacking gel and subjected to electrophoresisat a constant current of20mA/gel.After electrophoresis,gels werestained with0.05%(w/v)Coomassie blue R-250in50%(v/v)meth-anol and7.5%(v/v)acetic acid for30min.Finally,they weredestained with a mixture of50%(v/v)methanol and7.5%(v/v)ace-tic acid for30min and destained again with a mixture of5%(v/v)methanol and7.5%(v/v)acetic acid for1h.High-molecular-weightprotein markers were used to estimate the molecular weight of theproteins.2.4.3.Fourier transform infrared(FTIR)spectroscopic analysisFTIR spectra of the gelatin samples were obtained using a FTIRspectrometer(EQUINOX55,Bruker,Ettlingen,Germany)equippedwith a deuterated L-alanine tri-glycine sulphate(DLATGS)detector.A horizontal attenuated total reflectance accessory(HATR)wasmounted into the sample compartment.The internal reflectioncrystal(Pike Technologies,Madison,WI,USA),made of zinc sele-nide,had a45°angle of incidence to the IR beam.Spectra wereacquired at a resolution of4cmÀ1and the measurement rangewas between650and4000cmÀ1(mid-IR region)at room temper-ature.Automatic signals were collected in32scans at a resolutionof4cmÀ1and were ratioed against a background spectrumrecorded from the clean empty cell at25°C.Analysis of spectraldata was carried out using the OPUS3.0data collection softwareprogramme(Bruker,Ettlingen,Germany).2.4.4.Amino acid analysisAmino acid composition of gelatin samples was analysedaccording to the method of Nagarajan et al.(2012)with a slightmodification.The samples were hydrolysed under reducedpressure in4M methanesulphonic acid containing0.2%(v/v)3-2(2-aminoethyl)indole at115°C for24h.The hydrolysates wereneutralised with3.5M NaOH and diluted with0.2M citrate buffer(pH2.2).An aliquot of0.04ml was applied to an amino acid ana-lyser(MLC-703;Atto Co.,Tokyo,Japan).2.4.5.Determination of gel strengthGelatin gel was prepared by the method of Kittiphattanabawonet al.(2010).Gelatin was dissolved in distilled water(60°C)toobtain afinal concentration of6.67%(w/v).The solution was stir-red until the gelatin was completely solubilised and then trans-ferred to a cylindrical mold with3cm diameter and 2.5cmheight.The solution was incubated at the refrigerated temperature(4°C)for18h prior to analysis.The gel strength was determined at8–10°C using a texture ana-lyser(Stable Micro System,Surrey,UK)with a load cell of5kg,cross-head speed of1mm/s,equipped with a1.27cm diameterflat-faced cylindrical TeflonÒplunger.The maximum force(grams),taken when the plunger had penetrated4mm into the gelatin gels,was recorded.2.4.6.Determination of gelling and melting temperaturesThe gelling and melting temperatures of the gelatin sampleswere measured following the method of Boran,Mulvaney,andRegenstein(2010),using a controlled stress rheometer(RheoStressRS75,HAAKE,Karlsruhe,Germany).The gelatin solution(6.67%,w/v)was prepared in the same manner as described previously.The solution was preheated at35°C for30min.The measuringS.Sinthusamran et al./Food Chemistry152(2014)276–284277geometry used was a3.5cm parallel plate and the gap was set at 1.0mm.The measurement was performed at a scan rate of 0.5°C/min,frequency of1Hz,oscillating applied stress of3Pa dur-ing cooling from35to5°C and heating from5to35°C.The gelling and melting temperatures were calculated,where tan d became1 or d was45°.2.4.7.Determination of setting time for gelatin gelThe setting time of gelatin solution was determined at4°C and 25°C,according to the method of Kittiphattanabawon et al.(2010). The gelatin solution(6.67%,w/v)was prepared in the same manner as described previously.The solution(2ml)was transferred to thin wall(diameter of12mm and length of75mm)test tubes(PYREXÒ, Corning,NY,USA)and preheated at60°C for10min,followed by incubation in an ice bath(4°C)or at room temperature.An alumi-num needle with the diameter and length of0.1and25cm,respec-tively,was inserted manually into the gelatin solution and raised every10s.The time at which the needle could not detach from the gelatin sample was recorded as the setting time.The setting time was expressed in min.2.4.8.Determination of colour of gelatin gelThe colour of gelatin gels(6.67%w/v)was measured with a Hunter lab colourimeter(Color Flex,Hunter Lab Inc.,Reston,VA, USA).L⁄,a⁄and b⁄values indicating lightness/brightness, redness/greenness and yellowness/blueness,respectively,were recorded.The colourimeter was warmed for10min and calibrated with a white standard.The total difference in colour(D E⁄)was cal-culated according to Eq.(2)(Gennadios,Weller,Hanna,&Froning, 1996):D EüffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðD LÃÞ2þðD aÃÞ2þðD bÃÞ2qð2Þwhere D L⁄,D a⁄and D b⁄are the differences between the corre-sponding colour parameter of the sample and that of the white standard(L⁄=93.6,a⁄=À0.94and b⁄=0.40).2.4.9.Microstructure analysis of gelatin gelThe microstructure of the gelatin gel was visualised using scan-ning electron microscopy(SEM).Gelatin gels having a thickness of 2–3mm werefixed with2.5%(v/v)glutaraldehyde in0.2M phos-phate buffer(pH7.2)for12h.The samples were then rinsed with distilled water for1h and dehydrated in ethanol with a serial con-centration of50%,70%,80%,90%and100%(v/v).Dried samples were mounted on a bronze stub and sputter-coated with gold (Sputter coater SPI-Module,West Chester,PA,USA).The specimens were observed with a scanning electron microscope(JEOL JSM-5800LV,Tokyo,Japan)at an acceleration voltage of20kV.2.5.Statistical analysisAll experiments were run in triplicate,using three different lots of skin samples.Data were subjected to analysis of variance(ANO-VA)and mean comparisons were carried out using a Duncan’s mul-tiple range test(Steel&Torrie,1980).For pair comparison,a T-test was used.Statistical analysis was performed using the Statistical Package for Social Sciences(SPSS for windows:SPSS Inc.,Chicago, IL,USA).3.Results and discussion3.1.Extraction yieldThe yields of gelatin extracted under different extraction condi-tions are shown in Table1.For gelatin extracted at45or55°C,the yield increased as the extraction times increased,up to6h (P<0.05).In general,the higher yields were obtained when extracted at55°C,in comparison with45°C for all extraction times used.Yields of51.6–57.4%and62.0–66.4%(on dry weight basis) were found for the gelatin extracted at45and55°C,respectively. The highest yield from seabass skin(66.4%)was obtained when the extraction was carried out at55°C for12h(P<0.05).With an extraction time of12h,no differences in the yield were noticeable when extraction temperatures were higher than55°C(P>0.05) (data not shown).The results suggested that when a higher heat was applied,the bonding between a-chains in the native mother collagen were more effectively destabilised.As a consequence, the triple helix structure became amorphous and could be extracted into the medium with ease,leading to the higher yield. Increasing the extraction time also provided more energy to destroy the bondings,in which more free a or b-chains were released from the skin complex.It was noted that an extraction time longer than6h did not increase the yield(P>0.05).The cross-linked proteins stabilised by covalent bonds in skin network might not be destroyed even with the higher extraction tempera-ture and time.Kittiphattanabawon et al.(2010)reported that the extraction yield of gelatin from the skin of brownbanded bamboo shark and blacktip shark increased when the extraction tempera-ture and time increased.Different yields offish skin gelatin have been reported for Nile perch(64.3%)(Muyonga,Cole,&Duodu, 2004),greater lizardfish(35.1%)(Taheri,Abedian Kenari,Gildberg, &Behnam,2009)tiger-toothed croaker(36.8%)and pink perch (27.3%)(Koli et al.,2012).The yield and characteristics of gelatin are associated with the type of raw material and gelatin extraction process,including the pretreatment process(Kittiphattanabawon et al.,2010;Montero&Gómez-Guillén,2000;Nagarajan et al., 2012).Seabass is a warm-waterfish and its skin had complex structure and strongfibrous,which is resistant to a high tempera-ture environment(Athauda,Anderson,&de Nys,2012).As a result, harsher extraction conditions were required to obtain the higher yield.3.2.Protein patternsThe protein patterns of gelatin from the skin of seabass extracted with different extraction temperatures and times are shown in Fig.1A.All gelatin samples contained b-and a-chains with a MW of193and125–113kDa,respectively,as the major constituents. It was noted that gelatin samples had both a1-and a2-chains.The protein patterns of gelatin were similar to those found in the colla-gen of seabass skin reported by Sinthusamran et al.(2013).This indicated that the a-chains and b-chain of the mother collagen were retained with negligible degradation.However,the band intensity of the b-and a-chains in gelatin slightly decreased with an increased extraction temperature and time.This might be caused by some degradation induced by the thermal process.On the other hand,Kaewruang et al.(2013)reported that no a-chains or b-chains remained in the gelatin from the skin of uni-corn leatherjacket extracted at45and55°C,caused by endogenous heat stable proteases.Thus,it was suggested that seabass skin may not contain any proteases.Thermal degradation of gelatin from the skin of brownbanded bamboo shark and blacktip shark was more pronounced with increasing temperatures(Kittiphattanabawon et al.,2010).However,at the same extraction conditions,the a-chains and cross-linked components of gelatin from seabass skin were more tolerant to thermal hydrolysis than those of brown-banded bamboo shark and blacktip shark.Gelatins from splendid squid skin with higher extraction temperatures contained a lower band intensity of the a-chains than those obtained with lower extraction temperature(Nagarajan et al.,2012).Generally,gelatins with a higher content of a-chains showed better functional properties including gelling,emulsifying and foaming properties278S.Sinthusamran et al./Food Chemistry152(2014)276–284(Gómez-Guillén et al.,2002).Thus,the components of gelatin from seabass skin were quite thermo-tolerant,compared with other species previously reported (Kaewruang et al.,2013;Kittiphattanabawon et al.,2010).3.3.Fourier transform infrared (FTIR)spectraThe FTIR spectra of gelatin from the skins of seabass extracted at 45and 55°C with different extraction times are shown inFig.1B.Generally,all the gelatins showed a similar spectra.The amide I band of gelatin extracted at 45or 55°C for various times appeared at 1643–1645cm À1and 1644–1645cm À1,respectively.The amide I vibration mode is primarily a C @O stretching vibration coupled to contributions from the CN stretch,CCN deformation and in-plane NH bending modes (Bandekar,1992).The absorption in the amide I region is probably the most useful for infrared spectroscopic analysis of the secondary structure of proteins (Kittiphattanabawon et al.,2010).The absorption peak at amide I (1635cm À1)was characteristic for the coil structure of gelatin (Nagarajan et al.,2012).Kittiphattanabawon et al.(2010)reported that a higher extraction temperature of gelatin increased the amount of low molecular weight components,in which the reac-tive group (C @O)could be more exposed.The amide I band of gel-atin extracted at a higher temperature and longer time shifted to a higher wavenumber (Kittiphattanabawon et al.,2010).Addition-ally,gelatin extracted at 45and 55°C for all extraction times exhibited the amide II bands at the wavenumbers of 1540–1543cm À1and 1540–1543cm À1,respectively.The amide II band resulted from an out-of-phase combination of a CN stretch and in-plane NH deformation modes of the peptide group (Bandekar,1992;Lavialle,Adams,&Levin,1982).It was noted that the amide II band of gelatins extracted at both temperatures showed similar spectra.Furthermore,the amide III bands of all the gelatin samples were observed at wavenumbers of 1233–1234cm À1,which indi-cated disorder in the gelatin molecules and were more likely asso-ciated with loss of triple helix state (Friess &Lee,1996).The amide III band represents the combination peaks between CN stretching vibrations and NH deformation from the amide linkages as well as the absorptions arising from wagging vibrations of CH 2groups in the glycine backbone and proline side-chains (Jackson,Choo,Watson,Halliday,&Mantsch,1995).The amide A band was found at 3289–3304cm À1,associated with the stretching vibrations of the NH group coupled with hydro-gen bonding.Normally,a free NH stretching vibration is found in the range of 3400–3440cm À1.The position of this band shifted to a lower frequency because the NH group of a peptide is involved in hydrogen bonding.(Doyle,Blout,&Bendit,1975).Gelatin extracted at 45°C for 3h showed the lowest amplitude compared to the other gelatin samples.This result indicated the lower free amino groups were associated with the lower degradation.The amide B band was observed at 3070–3079and 3071–3079cm À1for the gelatin extracted at 45and 55°C,respectively,correspond-ing to the asymmetric stretching vibration of @C–H as well as NH 3+.Amongst all samples,gelatin extracted for a longer time had the lowest wavenumber for the amide-B peak,suggesting the interaction of ÀNH 3group between peptide chains (Nagarajan et al.,2012).Thus,the secondary structure and functional group of gelatins obtained from the skin of seabass were affected by extrac-tion temperatures and times.Table 1Extraction yield (%,dry weight basis)and gel colour of gelatin from the skin of seabass extracted at different temperatures for various times.Temperature (°C)Time (h)Yield (%)Colour value L ⁄a ⁄b ⁄D E ⁄45351.6±2.28Aa 22.1±0.15Aa À1.28±0.17Aa À1.94±0.11Ac 71.6±0.15Aa 655.7±1.21Bb 21.0+0.05Bb À1.37±0.15Aab À1.91±0.14Ac 72.7±0.05Bb 1257.3±0.87Bb 18.6+0.14Cc À1.57±0.28Abc À1.80±0.37Abc 75.0±0.15Cc 55362.0±1.54Ac 17.8+0.79Ad À1.79±0.14Ac À1.66±0.22Aabc 75.8±0.80Ad 665.3±1.28Bd 17.4+0.08ABd À1.75±0.15Ac À1.54±0.29Aab 76.3±0.08ABd 1266.4±1.40Bd16.9+0.29BeÀ1.74±0.21AcÀ1.37±0.26Aa76.8±0.29BeValues are presented as mean ±SD (n =3).Different uppercase letters within the same column under the same temperature indicate significant difference (P <0.05).Different lowercase letters within the same column indicate significant differences (P <0.05).500150025003500Wavenumber (cm -1)M 3 6 12 3 6 12 CA m i d e AA m i d e BAmide IA m i d e I I IA m i d e I I3 h12 h6 hA m i d e AA m i d e BAmide IA m i d e I I IA m i d e I I 12 h45 °C55 °CS.Sinthusamran et al./Food Chemistry 152(2014)276–2842793.4.Amino acid compositionThe amino acid compositions of gelatin extracted under differ-ent extraction conditions are shown in Table2.All gelatin samples showed similar amino acid compositions,in which glycine was the major amino acid(331–334residues/1000residues),followed by alanine(134–137residues/1000residues).Low contents of cys-teine(1residues/1000residues),tyrosine(3–4residues/1000resi-dues),histidine(5–6residues/1000residues)and hydroxylysine (5residues/1000residues)were observed in all gelatin samples. Generally,glycine occurs every third position in the a-chain and represents nearly one third of total residues(Benjakul et al., 2009).For imino acids,all gelatins contained proline and hydroxy-proline of116–118and82–84residues/1000residues, respectively.The imino acid content of gelatin from seabass skin (198–202residues/1000residues)was higher than that reported in splendid squid skin(184residues/1000residues)(Nagarajan et al.,2012),grey triggerfish skin(176residues/1000residues) (Jellouli et al.,2011),bigeye snapper skins(186.29–187.42resi-dues/1000residues)(Benjakul et al.,2009),salmon skin(166resi-dues/1000residues)and cod skin(154residues/1000residues) (Arnesen&Gildberg,2007).Nevertheless,it was lower than that found in bovine gelatin(219residues/1000residues)(Jellouli et al.,2011).Higher content of hydroxyproline affected the visco-elastic properties of the gelatin and the ability to develop a strong gel structure(Benjakul et al.,2009).Kittiphattanabawon et al. (2010)reported that gelatin from brownbanded bamboo shark skin,with a higher content of imino acids,had a higher bloom strength than gelatin from blacktip,containing lower levels of imi-no acids.The OH groups of hydroxyproline might be involved in hydrogen bondings with adjacent chains.This could strengthen the gel network.These results indicated that the extraction condi-tions did not affect the amino acid compositions of gelatin from seabass skin.3.5.Gel strength of gelatin gelThe gel strength of gelatin gels extracted from seabass skin with distilled water under varying conditions are shown in Fig.3A.The gel strength is one of the most important functional properties of gelatins.The highest gel strength(369g)was found in gelatin extracted at45°C for3h(P<0.05).Gel strength of gelatin gels decreased as the extraction temperature increased(P<0.05).At the same extraction temperature,the gel strength of gelatin decreased when the extraction time increased(P<0.05).This result was in agreement with the reports of Muyonga et al. (2004),Kittiphattanabawon et al.(2010)and Nagarajan et al. (2012).The decrease in gel strength was in accordance with the slight decreases in the b-and c-chains band intensity(Fig.1). The amount of the b-and c-components and the amino acid com-position of gelatin were reported as the factors governing gelation of gelatin(Taheri et al.,2009).According to the amino acid composition(Table2),all gelatins had a high imino acid content (198–202residues/1000residues).In the present study,seabass skin gelatin had a higher gel strength(282–369g)than bovine gelatin(208g)(P<0.05).A different gel strength was reported for gelatin from skin of different species including splendid aquid (85–132g)(Nagarajan et al.,2012),rainbow trout(459g) (Tabarestani et al.,2010),brownbanded bamboo shark and black-tip shark(206–214g)(Kittiphattanabawon et al.,2010),salmon (195g)(Arnesen&Gildberg,2007)and yellowfin tuna(426g) (Cho et al.,2005).Gel strength is a function of complex interactions determined by the molecular weight distribution(Gómez-Guillén et al.,2002).In this study,the solution of gelatin extracted for long-er times showed a higher turbidity(data not shown).The result suggested that the a-or b-chains might undergo conformation changes,which favoured the aggregation to some extent,espe-cially via the exposed reactive group.The larger bundles or strands might not form afine and ordered gel structure,as evidenced by the poorer gel strength.3.6.Gelling and melting temperaturesThermal transitions were monitored by changes in the phase angle(d)of dissolved gelatins during cooling(35–5°C)and subse-quent heating(5–35°C)as depicted in Fig.2A and B,respectively. The gelling temperatures of all the gelatin samples,with different extraction conditions,were in the range of19.5–20.0°C.This was considered as the sharp decrease in the phase angle during cooling. Changes in phase angle indicated the rapid transition,theTable2Amino acid compositions of gelatins from the skin of seabass extracted at different temperatures for various times.Amino acids(residues/1000residues)45°C55°C3h6h12h3h6h12hAlanine134135135137135135 Arginine555454545454 Aspartic acid/asparagine434343434343 Cysteine111111 Glutamine/glutamic acid717171727272 Glycine334334334331332331 Histidine566565 Isoleucine999999 Leucine181818181818 Lysine282828282828 Hydroxylysine555555 Methionine141414131313 Phenylalanine131313131313 Hydroxyproline828282838484 Proline116116116116117118 Serine262625262726 Threonine222222222222 Tyrosine444443 Valine202020202020Total100010001000100010001000 Imino acid198199199199201202 280S.Sinthusamran et al./Food Chemistry152(2014)276–284。

毕赤酵母N 糖基化改造的研究进展张 倩,宋海峰(军事医学科学院放射与辐射医学研究所药理毒理研究室,北京100850)[摘要] 毕赤酵母表达系统具有诸多优点,广泛用于生产重组蛋白。

由于其糖基化途径与人不同,表达的糖蛋白为高甘露糖型,改变了糖蛋白的结构,影响其特性和功能,且具有免疫原性,限制了以毕赤酵母为表达系统大量生产重组蛋白的应用。

在过去的20多年里,很多研究集中在毕赤酵母N 糖基化人源化改造研究上,希望产生类人的糖链结构,但进展缓慢。

近期,毕赤酵母N 糖基化改造研究已经取得了重大进展,产生了类人的末端为唾液酸的糖链结构,为应用毕赤酵母表达系统大量生产重组蛋白铺平了道路。

现对毕赤酵母N 糖基化的研究进展进行简述。

[关键词] 毕赤酵母;N 糖基化;糖蛋白[中图分类号]Q513.2;R915.2 [文献标识码]A [文章编号]1003-3734(2008)14-1206-03 Advances i n the asparagi nes li nked glycosyl ati on i n Pichi a pastorisZ HANG Q ian,SONG H a i feng(Depart m ent of Phar m aco logy and Toxico logy,Institute of Rad i a tion M ed icine,A cade my of M ilitar y M e d ical Sciences,B eijing100850,Ch i n a)[Abstract] W ith m any advantages,P ichia pastoris is w i d ely used to pr oduce reco m binan t pro teins.Due to t h e differences i n N g l y cosylati o n pathw ay bet w een P ichia pastoris and hum an,the struct u re o f h i g h m annose g lycan of P ichia pastoris w as changed,the characterization and functi o n o f t h e glycopro teins,or even i m munogen ic w ere af fected.The applicati o n of P ichia pastoris to produce g l y coprote i n s w as li m ited.I n the past t w o decades,m any re searches focused on the m od ificati o n of N glycosylati o n to obtai n hum an li k e glycopro teins,yet got li m ited success. Rencently,great advance m ents have been m ade to produce hum anized sia l y lated gycopro teins,w hich paved t h e w ay to use P ichia pastoris as host to produce reco m b i n ant pro teins.Th is rev i e w summ arizes the re levant researches.[Key w ords] P ichia pastoris;N g l y cosy lati o n;glycopr o te i n毕赤酵母(P ichia pastoris)表达系统是20世纪80年代初期发展起来的一种新型的外源蛋白表达系统,它既具有原核表达系统操作简易、易于培养、生长速度快、表达量高、成本低等优点,还具有真核生物表达系统的对外源蛋白的翻译后修饰等特点,如糖基化、蛋白磷酸化等。

R E V I E W Effectiveness of twice daily azelastine nasal spray in patients with seasonal allergic rhinitisFriedrich HorakMedical University Vienna,ENT – Univ. Clinic, Vienna, Austria Correspondence: Friedrich Horak HNO – Univ. Klinik Wien, Waehringer Guertel 18–20, A-1090 Vienna, A ustria T el +43 1 404 003 336Fax +43 1 789 76 76Email friedrich.horak@vienna.at Abstract: Azelastine nasal spray (Allergodil®, Lastin®, Afl uon®; Meda AB, Stockholm, Sweden) is a fast-acting, effi cacious and well-tolerated H1-receptor antagonist for the treatment of rhinitis. In addition it also has mast-cell stabilizing and anti-infl ammatory properties, reducing the concentration of leukotrienes, kinins and platelet activating factor in vitro and in vivo, as well as infl ammatory cell migration in rhinitis patients. Well-controlled studies in patients with seasonal allergic rhinitis (SAR), perennial rhinitis (PR) or vasomotor rhinitis (VMR) confi rm that azelastine nasal spray has a rapid onset of action, and improves nasal symptoms associated with rhinitis such as nasal congestion and post-nasal drip. Azelastine nasal spray is effective at the lower dose of 1 spray as well at a dose of 2 sprays per nostril twice daily, but with an improved tolerability profi le compared to the 2-spray per nostril twice daily regimen. Compared with intranasal corticosteroids, azelastine nasal spray has a faster onset of action and a better safety profi le, showing at least comparable effi cacy with fl uticasone propionate (Flonase®; GSK, USA), and a superior effi cacy to mometasone furoate (Nasonex®; Schering Plough, USA). In combination with fl uticasone propionate, azelastine nasal spray exhibits greater effi cacy than either agent used alone, and this combination may provide benefi t for patients with diffi cult to treat seasonal allergic rhinitis. In addition, azelastine nasal spray can be used on an as-needed basis without compromising clinical effi cacy. Compared with oral antihistamines, azelastine nasal spray also demonstrates superior effi cacy and a more rapid onset of action, and is effective even in patients who did not respond to previous oral antihistamine therapy. Unlike most oral antihistamines, azelastine nasal spray is effective in alleviating nasal congestion, a particularly bothersome symptom for rhinitis sufferers. Azelastine nasal spray is well tolerated in both adults and children with allergic rhinitis. Bitter taste which seems to be associated with incorrect dosing technique is the most common side effect reported by patients, but this problem can be minimized by correct dosing technique.Keywords: azelastine nasal spray, rhinitis, intranasal corticosteroids, oral antihistamines, seasonal allergic rhinitisIntroductionRhinitis is an inflammatory disease of the upper airways, affecting approximately 58 million people only in the United States alone (Settipane 2001) and its prevalence is increasing. The cost of the disease is signifi cant with between US$2 and US$5 billion incurred annually in both direct and indirect costs (Ray et al 1999; Reed et al 2004). In the US, the number of lost workdays is estimated as approximately 3.5 million a year (Mahr and Sheth 2005). It can be classifi ed as allergic, non-allergic or mixed upper respiratory disorder (Berstein 2007). It is classifi ed as allergic if symptoms occur in association with a specifi c IgE-mediated response; as non-allergic if symptoms are induced by irritant triggers, but without an IgE-mediated response; and as of mixed etiology if IgE-mediated responses occur in conjunction with symptoms induced by both allergens and non-allergic irritant triggers. Allergic rhinitis (AR) is further classifi ed as seasonal or perennial (Dykewicz et al 1998). Seasonal allergic rhinitis© 2008 Dove Medical Press Limited. A ll rights reservedTherapeutics and Clinical Risk Management 2008:4(5) 1009–10221009Horak(SAR) symptoms are induced by exposure to pollens from trees, grass, weeds or seasonal mould spores, whilst peren-nial rhinitis (PR) is associated with environmental allergens which are generally present on a year-round basis such as house dust, animal dander and insect droppings (Dykewicz et al 1998). In contrast, the “Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines” recommend a classifi cation in intermittent allergic rhinitis and persistent allergic rhinitis according to the frequency and persistence of symptoms (Bousquet et al 2001).Symptoms of SAR include nasal congestion, runny nose, nasal and nasopharyngeal itching, ear symptoms, sneezing and ocular symptoms in many patients, including itchy and watery eyes (Bielory and Ambrosio 2002). The symptoms of sneezing, itching and rhinorrhea are less common with PR (Economides and Kaliner 2002). As many as half of all patients diagnosed with rhinitis have non-allergic disease (sometimes called vasomotor rhinitis [VMR]) where an allergic component cannot be identifi ed (Dykewicz et al 1998). Symptoms are often induced by irritant triggers such as tobacco smoke, strong odors and temperature and pres-sure changes (Devyani and Corey 2004). The symptoms of VMR are similar to those of AR (Devyani and Corey 2004). To further complicate rhinitis classifi cation, as many as half of all patients with AR are also sensitive to non-allergic triggers; a condition referred to as mixed rhinitis (Settipane and Settipane 2002; Liberman et al 2005). Symptoms of rhinitis can have a major impact on patients’ quality of life (QoL) by interfering with sleep which causes fatigue, and impairing daily activities and cognitive function (Dykewicz et al 1998). Patients often complain of an inability to concentrate, and in the case of SAR often avoid outdoor activities in order to avoid exposure to symptom-inducing allergen(s). The Joint Task Force on Allergy Practice and Parameters advises that improving the negative impact on daily life in rhinitis patients defi nes successful treatment as much as providing symptom relief (Dykewicz et al 1998). Indeed, Juniper (1997) recommends that for most patients with rhinitis, improving patient well-being and QoL should be the primary goal of treatment.Treatment guidelines from the Joint Task Force and WHO recommend that antihistamines, both topical (eg, azelastine [Allergodil®; Meda AB, Stockholm, Sweden]) and oral second-generation (eg, loratadine [Claritin®, Schering Plough, USA], desloratadine [Clarinex®; Schering Plough, USA], fexofenadine [Allegra®; Sanofi Aventis, USA] or cetirizine [Zyrtec®; Pfi zer, USA], and levocetirizine [Xyzall®; UCB, EU]) be used as fi rst-line therapy for AR (Dykewicz et al 1998; Bousquet et al 2001). Intranasal corticosteroids (eg, fl uticasone propionate [Flonase®, GSK, USA], mometasone furoate [Nasonex®; Schering Plough, USA]) may also be considered as initial therapy for AR in patients with more severe symp-toms, particularly nasal congestion [(Dykewicz et al 1998; LaForce 1999). The Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines recommend a stepped approach to therapy based upon the frequency and severity of symptoms (Table 1) (Bousquet et al 2001). Interestingly, a recent US nationwide survey incorporating approximately 2500 adult allergy suf-ferers, revealed that 66% were dissatisfi ed with their current allergy medication due to lack of effectiveness (Anon 2006). Furthermore, more than two-thirds of primary care physicians reported patient dissatisfaction with therapy as the main reason for stopping or switching medications (Anon 2001). Clearly, effective therapies with a good safety profi le are needed to treat AR sufferers.AzelastineAzelastine nasal spray is a topically administered second-generation antihistamine and selectively antagonizes the H1-receptor (Zechel et al 1981) being approximately tenfold more potent than chlorpheniramine in this regard (Casale 1989). It has one of the fastest onsets of action (15 min with nasal spray and up to 3 min with eye drops) among the currently available rhinitis medications (Baumgarten et al 1994; Greiff et al 1997). The effect of azelastine lasts at least 12 hours, thus allowing for a once or twice daily dosing regimen (Greiff et al 1997). It has proven effi cacy in treating both allergic and non-allergic rhinitis, and is the only pre-scription antihistamine approved in the US, Portugal and the Netherlands for the treatment of both SAR (1996) and VMR (1999). In SAR patients azelastine therapy (two sprays per nostril twice daily), improved both total symptom and major symptom complex scores to a signifi cantly greater extent than placebo (McTavish and Sorkin 1989; Storms et al 1994; LaForce et al 1996; Ratner and Sacks 2007). Similarly, in PR patients, azelastine nasal spray signifi cantly improved sleep-ing, reduced daytime somnolence and nasal congestion com-pared with placebo (Golden et al 2000). Liberman et al (2005) were the fi rst to show that azelastine was also effective in the management of VMR and even in mixed rhinitis. Azelastine nasal spray signifi cantly (p Ͻ 0.01) reduced the total VMR symptom score (TVRSS) compared with placebo after 21-day double-blind treatment, and was associated with clinical improvement in each symptom of the TVRSS (ie, rhinorrhea, sneezing, nasal congestion, and post-nasal drip). In a large open-label trial 4364 patients received azelastine nasal sprayTherapeutics and Clinical Risk Management 2008:4(5)1010Azelastine nasal spray(2 sprays per nostril twice daily) as monotherapy for 2 weeks. 78% of VMR patients reported some or complete control of post-nasal drip which rose to 90% of SAR patients for the symptom of sneezing. Of patients reporting sleep diffi culties or impaired daytime activities because of rhinitis symptoms, 85% experienced improvements in these parameters with azelastine. Baseline sleep diffi culties and impairment of daytime activities were signifi cant (p Ͻ 0.01) predictors of a positive treatment effect with azelastine nasal spray. Female patients (p = 0.02), patients with SAR (p Ͻ 0.01) and patients with SAR plus sensitivity to non-allergic triggers (p = 0.03) were identifi ed as being most likely to respond to azelastine nasal spray (Liberman et al 2005) Due to its rapid onset of action, azelastine nasal spray continues to control rhinitis symptoms when used on an as-needed basis (Ciprandi et al 1997). This property of azelastine is discussed later. First marketed in the UK in 1991 for the treatment of both SAR and PR, it is currently available in more than 70 countries world-wide.Mode of actionH owever, azelastine is more than just an anti-histamine. It exhibits a very fast and long-acting effect based on a triple mode of action, with anti-infl ammatory and mast cell stabilizing properties in addition to its anti-allergic effects (Bernstein 2007; Lee and Corren 2007). For example, azelas-tine inhibits the activation of cultured mast cells and release of interleukin (IL)-6, tryptase, and histamine (Kempuraj et al 2002). It also reduces mediators of mast cell degranu-lation such as leukotrienes which are involved in the late phase allergic response (Howarth 1997), in the nasal lavage fl uid of patients with rhinitis (Shin et al 1992). It does this possibly by reducing the production of leukotriene (LT)B4 and LTC4, inhibiting phospholipase A2and LTC4synthase (H amasaki et al 1996). Leukotrienes are associated with dilation of vessels, increased vascular permeability and edema which results in nasal congestion, mucus production and recruitment of infl ammatory cells (Golden et al 2006). Substance P and bradykinin concentrations which are formed in biological fl uids and tissues during infl ammation, are also reduced by azelastine (Shin et al 1992; Nieber et al 1993; Shinoda et al 1997). These agents are associated with the AR symptoms of nasal itching and sneezing, but may also contribute to the onset of non-allergic VMR symptoms. Other anti-infl ammatory properties of azelastine include inhibition of tumor necrosis factor alpha (TNFα) releaseT able 1 Summary of ARIA allergic rhinitis management guidelinesRhinitis severity ARIA recommendationMild intermittent • Oral/intranasal antihistamines OR• Decongestants (10 days maximum)Moderate/severe intermittent • Intranasal antihistamines• Oral antihistamines AND/OR• Decongestants• Intranasal corticosteroids• CromonesMild persistent • Intranasal antihistamines• Oral antihistamines AND/OR• Decongestants• Intranasal corticosteroids• CromonesA stepwise approach is advised with reassessment after 2 weeks. If symptomsare controlled and the patient is on intranasal corticosteroid, the dose shouldbe reduced, but otherwise treatment continued. If symptoms persist and thepatient is on antihistamines or cromones, a change should be made to anintranasal corticosteroid.Moderate/severe persistent • Intranasal corticosteroid (fi rst line treatment)If symptoms are uncontrolled after 2–4 weeks, medication should be addeddepending on the persistent symptom, eg, add an antihistamine if the majorsymptom is rhinorrhea, pruitis, or sneezing, double the dose of intranasalsteroid for persistent nasal blockage and add ipratropium for prominentcomplaint of rhinorrhea.Therapeutics and Clinical Risk Management 2008:4(5)1011Horak(Hide et al 1997; Matsuo and Takayama 1998), reduction of granulocyte macrophage colony-stimulating factor (GM-CSF) generation, as well as a reduction in the number of a range of infl ammatory cytokines including interleukin (IL)-1β, IL-6, IL-4 and IL-8 (Y oneda et al 1997; Ito et al 1998; Beck et al 2000). These cytokines perpetuate the infl ammatory response (Settipane 2001). Finally, in SAR patients, azelastine nasal spray has been shown to lower neutrophil and eosinophil counts and decrease intercel-lular adhesion molecule-1 (ICAM-1) expression on nasal epithelial cell surfaces in both the early and late phases of the allergic reaction (Ciprandi et al 1996). It also decreases free-radical production by human eosinophils and neutrophils (Busse et al 1989; Umeki 1992) and calcium infl ux induced by platelet-activating factor in vitro (Nakamura et al 1988; Morita et al 1993).The use of a topical treatment has many advantages over a systemic treatment. Firstly, with a nasal spray, medication can be delivered directly to the site of allergic infl ammation. Secondly, the higher concentrations of antihistamines that can be achieved in the nasal mucosa by topical as opposed to oral administration should enhance the anti-allergic and potential anti-infl ammatory effects of these agents. Thirdly, a dose of 0.28 mg intranasally has a faster onset of action than a dose of 2.2 mg administrated orally (Horak et al 1994). And fi nally, with topical administration the risk of interaction with concomitant medication is minimized (Davies et al 1996) and the potential of systemic effects reduced.DosageRecent results from 2 studies have shown that azelastine nasal spray at a dosage of 1 spray per nostril twice daily is effec-tive and has a better tolerability profi le compared to 2 sprays per nostril twice daily in patients (Ն12 years; n = 554) with moderate to severe SAR (Lumry et al 2007). The total nasal symptom score (TNSS) improved by 14.1% in study 1 and by 22.1% in study 2 with azelastine nasal spray (1 spray per nostril twice daily) compared with 4.5% and 12.0% with placebo in study 1 (p = 0.01) and 2 (p Ͻ 0.01) respectively. This compares with a 24%–29% improvement in rhinitis symptoms scores with a 2-spray dosage of azelastine (Ratner et al 1994; Storms et al 1994; LaForce et al 1996). For individual symptoms, itchy nose, runny nose, sneezing, and nasal congestion were all signifi cantly improved after the 1-spray azelastine regimen compared with placebo. One spray per nostril twice daily of azelastine was also associ-ated with signifi cant improvements in the Rhinitis Quality of Life Questionnaire (RQLQ) daily activity and nasal symptoms domains and patient global evaluations compared with placebo. In addition, the incidence of a bitter taste after azelastine application more than halved and the incidence of somnolence decreased almost 30 times in the 1-spray group versus the labeled incidence with the 2-spray regimen (Lumry et al 2007). Although an earlier study showed an improve-ment in rhinitis symptoms versus placebo with azelastine 1 spray per nostril twice daily, this improvement failed to reach statistical signifi cance. However, a global evaluation noted a signifi cant clinical improvement versus placebo (49%) in the 1-spray regimen (75%, p Ͻ 0.001) as well as a 2-spray once daily (89%, p = 0.028) and a 2-spray twice daily regimen (83%, p Ͻ 0.001) (Weiler et al 1994).From these results one can conclude that a greater degree of effectiveness would be expected with two sprays per nostril twice daily. Although one spray per nostril twice daily may provide somewhat less effi cacy this is compensated for by an improved tolerability profi le compared with the 2-spray regimen. Therefore, the choice of dosage of azelastine nasal spray should be based on the severity and persistence of symptoms as well as the patient’s acceptance of the nasal spray (Bernstein 2007). For example, the 2-spray dose could be used as the starting dose for patients with severe symptoms of SAR, and either maintained or tapered to the 1-spray dose as required. The 1-spray dose could be used as a starting dose in patients with mild-to-moderate symptoms, and if necessary the dose increased to 2 sprays per nostril twice daily if symptom control proved to be inadequate (Lumry et al 2007).As-neededBecause azelastine starts working within 15 minutes of application investigators wondered how effective an as-needed regimen would be in controlling the symptoms of rhinitis (Ciprandi et al 1997). A randomized controlled study was car-ried out in 30 patients sensitized to Parietaria pollen or grass. Patients were treated with the standard European dose of azelas-tine (0.56 mg/day), half this dose (0.22 mg/day), or as-needed. Both groups who received the standard and half-standard doses showed an improvement in their rhinitis symptoms, with a concomitant reduction in markers of infl ammation, namely neutrophil and eosinophil counts as well as ICAM-1 expression in nasal scrapings. However, patients who used azelastine nasal spray on an as-needed basis also showed an improvement in their rhinitis symptoms, but without a reduction in the markers of infl ammation. The results of this small study suggest that although regular treatment with azelastine is superior at controlling symptoms, as-needed therapy may beTherapeutics and Clinical Risk Management 2008:4(5)1012Therapeutics and Clinical Risk Management 2008:4(5)1018Horakan improvement in TNSS of 32.5% compared with 24.6% for those patients taking oral cetirizine. The most common side effect reported by patients in the azelastine group was bitter taste (5.7%). Somnolence was reported by 1.5% of patients taking cetirizine (Sher and Sacks 2006).In addition to nasal symptoms, patients with SAR can experience impairment in HRQoL. Two 2-week, double-blind, multicenter studies were conducted during autumn 2004 and spring 2005 comparing the improvement with azelastine nasal spray (2 sprays per nostril twice daily) versus cetirizine (10 mg daily) on symptoms and HRQoL in SAR patients (Meltzer and Sacks 2006). Results from these studies revealed that azelastine nasal spray improved the overall RQLQ score to a signifi cantly (p Ͻ 0.05) greater degree than cetirizine tablets. When results from both studies were pooled, the combined analysis confi rmed the signifi cant superiority of azelastine spray both in terms of the overall RQLQ score (p Ͻ 0.001) as well as each RQLQ domain (p Ͻ 0.03) including the nasal symptoms domain (p Ͻ 0.001). More patients in the azelastine nasal spray group experienced a clinically important improve-ment from baseline in HRQoL (ie, Ն2 units on the 0–6 rating scale) compared with patients in the cetirizine group (35% vs 20% respectively) (Meltzer and Sacks 2006).Berger et al (2006) also showed that azelastine nasal spray (2 sprays per nostril) and oral cetirizine (10 mg once daily) effectively treated nasal symptoms in patients with SAR (n = 360). Rapid relief of rhinitis symptoms was evident in both groups at the fi rst evaluation after initial administration and continued during the 14 study days, with the azelastine patients showing the greatest degree of improvement during the second week of treatment. Improvements in the TNSS and individual symptoms favored azelastine over cetirizine (Figure 7), with signifi cant differences for nasal congestion (p = 0.049) and sneezing (p = 0.01). Azelastine nasal spray improved TNSS by a mean of 4.6 (23.9%) compared with 3.9 (19.6%) with cetirizine. The positive effect of azelastine nasal spray on congestion was observed despite the fact that the cetirizine group had the added benefi t of daily use of a placebo saline spray. Azelastine nasal spray also signifi cantly improved the RQLQ overall (p = 0.002) and individual domain (p Յ 0.05) scores compared with cetirizine (Berger et al 2006). Although oral cetirizine signifi cantly improved RQLQ scores, patients treated with azelastine nasal spray reported additional statistically signifi cant improvement beyond that reported with cetirizine for each individual RQLQ domain including activities, sleep, non-nose/non-eye symp-toms, practical problems, nasal symptoms, eye symptoms, and emotions (Figure 8). Although it is often assumed that patients prefer oral medications to sprays in both the ACT I and ACTII trials, patients reported superior improvements in QoL variables with azelastine nasal spray compared with oral cetirizine (Corren et al 2005).MNSS Rhinorrhea Nasal Itching Sneezing Nasal Congestion1234AzelastineDesloratadine PlaceboA b s o l u t e I m p r o v e m e n t f r o m b a s e l i n e(120 m i n s c o r e –360 m i n s c o r e )Figure 6 Major nasal symptom and mean nasal symptom scores after administration of azelastine nasal spray (1 spray per nostril), desloratadine (5 mg) or placebo in patients with SAR: absolute changes of last value (6 hours after the start of challenge) compared to predose (ie, 2 hours after the start of the challenge). Reprinted with permission from Horak F , Zieglmayer UP , Zidglmayer R, et al 2006. A zelastine nasal spray and desloratadine tablets in pollen-induced seasonal allergic rhinitis: a pharmacodynamic study of onset of action and ef fi cacy. Curr Med Res Opion , 22:151–7. Copyright © 2006 LibraPharm.as either ‘good’ or ‘very good’ compared with just 76% of levocabastine patients (Falser et al 2001).Safety and tolerabilityThe advantages of intranasal delivery include lower risk of systemic side effects and drug interactions (Salib and Howarth 2003). In controlled studies, azelastine nasal spray was well-tolerated for treatment durations up to 4 weeks in both adults and children (Ն12 years) (Storms et al 1994; Meltzer et al 1994; Ratner et al 1994; Weiler et al 1994; LaForce et al 1996). Bitter taste, headache, somnolence and nasal burning were the most frequently reported adverse events, but most of these were mild or moderate in nature. These studies reported a greater incidence of somnolence compared with placebo (11.5% vs 5.4%, p Ͻ 0.05). How-ever, the incidence of somnolence between azelastine- and placebo-treated patients (3.2% vs 1.0%) did not differ in VMR studies (Banov and Liberman 2001). Post-marketing surveillance studies also reported a similar degree of somnolence (approx 2%) in both azelastine and placebo groups (Berger and White 2003; LaForce et al 2004; Corren et al 2005; Berger et al 2006). The lower incidence of azelastine-related adverse events in later trials is most likely due to correct dosing technique, when the drug is administered without tipping back the head or deeplyinhaling the spray, both of which would increase systemic absorption and could result in bitter taste and somnolence. As the incidence of somnolence whilst using azelastine nasal spray has been reported to be greater than placebo in certain studies, US prescribing recommendations warn against concurrent use of alcohol and/or other CNS sup-pressants. However, to date no studies have been designed to assess specifi cally the effects of azelastine nasal spray on the CNS in humans.DisclosuresThe author has no confl icts of interest to report.AbbreviationsACT 1, first Azelastine Cetirizine Trial; AR, allergic rhinitis; ARIA, allergic rhinitis and its impact on asthma; EEC, environmental exposure chamber; GM-CSF, granu-locyte macrophage-colony stimulating factor; H RQoL, health-related quality of life; ICAM-1, intercellular adhe-sion molecule-1; IL, interleukin; LT, leukotriene; MNSS, major nasal symptom score; NNT, number needed to treat; PR, perennial rhinitis; QoL, quality of life; RQLQ, Rhinoconjunctivitis Quality of Life Questionnaire; SAR, seasonal allergic rhinitis; TNF α, tumor necrosis factor alpha;Azelastine nasal spray Cetirizine21.510.5M e a n i m p r o v e m e n t f r o m b a s e l i n eOverall RQLQ ScoreActivities SleepNon-nasal non-eye symptomsPractical problems Nasal symptoms Eye symptoms EmotionFigure 8 Mean improvement from baseline to day 14 in overall Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ) score and individual RQLQ domain scores (intention-to-treat population). *p Յ 0.05 vs cetirizine; **p Ͻ 0.01 vs cetirizine. Reprinted with permission from Berger W , Hampel F , Bernstein J, et al 2006. Impact of azelastine nasal spray on symptoms and quality of life compared with cetirizine oral tablets in patients with seasonal allergic rhinitis. Ann Allergy Asthma Immunol , 97:375–81. Copyright © 2006 American College of Allergy, A sthma and Immunology.TNSS, total nasal symptom score; TVRSS, Total VMR Symptom Scale; VCC, Vienna Challenge Chamber; VMR, vasomotor rhinitis.ReferencesAl Suleimani YM, Walker MJA. 2007. Allergic rhinitis and its pharmacology.Pharmacol Ther, 114:233–60.Anon. 2001. Physician survey sponsored by the American College of Allergy, Asthma and Immunology. Rochester (NY): Harris Interactive Inc, October 19–21.Anon. 2006. Allergies in America: a landmark survey of nasal allergy sufferers. H ealthSTAR Communications, Inc. Sponsored by Altana Pharma US, Inc. March 1.Banov CH, Liberman P. 2001. 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International Journal of Pharmaceutics 278(2004)51–61Acetaminophen-containing chewable tablets with suppressedbitterness and improved oral feelingHiroyuki Suzuki a ,Hiraku Onishi a ,∗,Seiji Hisamatsu a ,Kosuke Masuda a ,Yuri Takahashi a ,Masanori Iwata b ,Yoshiharu Machida aaDepartment of Drug Delivery Research,Hoshi University,2-4-41,Ebara,Shinagawa-ku,Tokyo 142-8501,Japanb Department of Pharmacy,Yokohama City University Medical Center,4-57,Urafune-cho,Minami-ku,Yokohama City,Kanagawa 232-0024,JapanReceived 30August 2003;received in revised form 25January 2004;accepted 14February 2004AbstractThe aim of this study was to develop acetaminophen chewable tablets with suppressed bitterness and improved oral feeling by examination of hard fats as the matrix base and of sweetening agents as corrigents.Witepsol ®H-15,W-35,S-55,E-75and E-85,and Witocan ®H and 42/44were used as hard fats.Witocan ®H and 42/44were selected in view of improved oral feeling.Witocan ®H/Witocan ®42/44mixture tablets showed different melting characteristics and drug release rates dependent on their ratios,and those with the Witocan ®H/Witocan ®42/44ratio of 92.5%(w/w)and more showed good drug release.Sucrose,xylitol,saccharin,saccharin sodium,aspartame and sucralose were used as sweetening agents,and applied alone or with Benecoat BMI-40or cocoa powder.The Witocan ®H tablet with 1%(w/w)saccharin plus 5%(w/w)Benecoat BMI-40(Sc1-B5),and the Witocan ®H/Witocan ®42/44(92.5:7.5,w/w)mixture tablet with 1%(w/w)aspartame plus 5%(w/w)Benecoat BMI-40suppressed bitterness and sweetness excellently,but the former tablet showed better drug release.Thus,the Witocan ®H tablet with Sc1-B5is suggested as the best acetaminophen chewable tablet,exhibiting suppressed bitterness,low sweetness,improved oral feeling and good drug release.©2004Elsevier B.V .All rights reserved.Keywords:Acetaminophen chewable tablet;Hard fat;Bitterness;Sweetness;Oral feeling;Drug release1.IntroductionMany drugs exhibit bitter taste when orally admin-istered (Nakamura et al.,1990;Shirai et al.,1993,1994;Katsuragi et al.,1995;Yajima et al.,1999),and the bitter taste often causes non-compliance of patients because of the discomfort (Uchida,2002).Therefore,suppression of the bitter taste has been an important∗Corresponding author.Tel.:+81-3-5498-5760;fax:+81-3-5498-5760.E-mail address:onishi@hoshi.ac.jp (H.Onishi).subject for oral dosage forms.Various methods such as capsules,drug coating,microencapsulation,com-plexation and chemical modification have been uti-lized to improve the bitter taste (Nakamura et al.,1990;Shirai et al.,1993,1994;Yajima et al.,1999).How-ever,these techniques are not always useful or appli-cable;for example,capsules or coated tablets are of-ten uncomfortable for infants or elderly people,who have trouble in swallowing drugs,due to their bulki-ness.Further,the approaches such as drug coating,mi-croencapsulation,complexation and chemical modifi-cation are not necessarily simple,and extensive opti-0378-5173/$–see front matter ©2004Elsevier B.V .All rights reserved.doi:10.1016/j.ijpharm.2004.02.03152H.Suzuki et al./International Journal of Pharmaceutics278(2004)51–61mization is required for their practical use.Therefore, the dosage forms which can be produced simply and that the patients can swallow easily may be important and valuable for the masking of the drug taste.The simple way to achieve such dosage forms is to add ap-propriate masking agents to power,liquid or chewable dosage forms(Popova,1969;Katsuragi and Kurihara, 1993;Katsuragi et al.,1995,1996,1997;Yin et al., 1996;Ishikura et al.,2002;Takano,2002).As to eval-uation of taste intensities including bitterness,the sen-sation tests by human volunteers have been utilized (Indow,1966,1969).An alternative method using a taste sensor have been recently studied because it may permit the examination of dangerous compounds or complete the tests of many substances in a short time (Uchida et al.,2000,2001;Uchida,2002).However, since mechanism of taste sensation is complicated,the sensation tests by human volunteers appear to be still a very useful method to determine the taste intensities. Acetaminophen,an antipyretic,has a bitter taste, but is often applied to infants and children due to its safety,when suppository and syrup dosage forms are often used to take the drug more comfortably(Autret et al.,1994;Van Esch et al.,1995;Coulthard et al., 1998;Hansen et al.,1999).Suppository insertion re-quires a private space at the administration.In drinking the syrup,a fairly large volume of the liquid and the sweet taste are sometimes a burden for the patients. Actually,in the preliminary studies,we tested tastes about three commercial syrups of acetaminophen,but they exhibited fairly strong sweetness and did not sup-press the bitterness very much.To improve these mat-ters,we had developed the acetaminophen-containing chewable tablets using Witepsol®H-15or cacao butter as a matrix base and some corrigents as bitter masking agents(Suzuki et al.,2003).These chewable tablets could be prepared simply,and were considered to be available to patients having trouble in swallowing be-cause they could be chewed.However,these tablets did not necessarily show comfortable oral feeling,which appeared to be mainly due to the stickiness of the hard fat.Therefore,further examination of the formulations was required to improve oral feeling in addition to bitterness suppression.In the present study,various kinds of hard fats and many sweetening agents were examined to obtain acetaminophen chewable tablets with suppressed bitter taste and improved oral feel-ing.The dose of acetaminophen per oral administra-tion was300–500mg for adults,but it was adjusted to10mg/kg for infants.Furthermore,it is not difficult to take some chewable tablets at a time.Considering these features,the drug content of the chewable tablet was set at100mg as reported previously.In the present study,as to hard fats,Witocan®was examined in addition to Witepsol®;Witocan®is utilized as special hard fats in the chocolate and con-fectionery industry.Currently available sweetening agents,that is,sucrose,xylitol,saccharin,saccharin sodium,aspartame and sucralose,were used as sweet-ening agents(Japan Food Additives Association, 2001).They show different sweet taste intensities and oral feeling.Xylitol has a sweet taste intensity similar to sucrose but gives a brisk feeling orally.Saccharin and saccharin sodium are500times as sweet as su-crose,and aspartame and sucralose showed200and 600times as the sweet taste intensity as sucrose,re-spectively.In addition to the above sweetening agents, commercial bitter-masking powder mixture made from lecithin(Benecoat BMI-40)(Katsuragi et al., 1997)and cocoa powder(Koyama and Kurihara, 1972;Pickenhagen et al.,1975;Aremu et al.,1995) were utilized as corrigents.As shown in the previ-ous report,the corrigent systems of1or5%(w/w) sucrose plus5%(w/w)Benecoat BMI-40,or1% (w/w)sucrose plus1%(w/w)cocoa powder,or5% (w/w)sucrose alone gave the best suppression of the bitter taste intensities of acetaminophen-containing Witepsol®H-15chewable tablets(Suzuki et al., 2003).Therefore,in the present study,acetaminophen chewable tablets were prepared at the similar con-ditions;that is,they were prepared using hard fats with1or5%(w/w)sweetening agent plus5%(w/w) Benecoat BMI-40,or1%(w/w)sweetening agent plus1%(w/w)cocoa powder,or5%(w/w)sweeten-ing alone.The obtained tablets were evaluated based on suppression of bitter taste,sweet taste intensity, oral feeling and drug release.2.Materials and methods2.1.MaterialsAcetaminophen was purchased from Sigma Chem-ical Co.(USA).Quinine sulfate,sucrose,saccharin, saccharin sodium,xylitol,aspartame and sucraloseH.Suzuki et al./International Journal of Pharmaceutics 278(2004)51–6153were purchased from Wako Pure Chemical Industries,Ltd.(Japan).Hard fats (Witepsol ®H-15,W-35,S-55,E-75and E-85,Witocan ®H and 42/44)were pur-chased from Mitsuba Trading Co.(Japan).Commer-cial bitter-masking powder mixture made from lecithin (Benecoat BMI-40)was supplied from Kao Corpora-tion (Japan).For cocoa powder,a commercial product was used.All other chemicals were of reagent grade.2.2.Preparation of chewable tabletsAll the chewable tablets (1g)containing 100mg of acetaminophen were prepared based on the following casting method:A hard fat or a mixture of hard fats was put in a glass beaker and melted by warming at 45◦C on a water bath.Acetaminophen and/or corri-gents were added,the mixture was stirred quickly with a glass bar,then 1g of the mixture was poured into a mold to yield a disk-shaped tablet (2cm diameter).The types of chewable tablets described in Table 1were prepared,and their use and features are in the following.2.2.1.Formulation AThis type of chewable tablet was prepared to ex-amine the effect of kinds of hard fats on bitter taste and oral feeling of the chewable tablet.The chewable tablets with various hard fats as a matrix base using sucrose and Benecoat BMI-40each at 5%(w/w)as corrigents were prepared.2.2.2.Formulation BThis type of chewable tablet was prepared to ex-amine the effect of the Witocan ®H/Witocan ®42/44Table 1Formulations and compositions of the chewable tablets (1g)Formulation Composition [weight ratio]A Hard fat/acetaminophen/sucrose/Benecoat BMI-40[80/10/5/5]B Witocan ®H–Witcan ®42/44mixture/acetaminophen [9/1]C-1Witocan ®H/acetaminophen/sweetening agent [85/10/5]C-2Witocan ®H/acetaminophen/sweetening agent/Benecoat BMI-40[80/10/5/5]C-3Witocan ®H/acetaminophen/sweetening agent/Benecoat BMI-40[84/10/1/5]C-4Witocan ®H/acetaminophen/sweetening agent/cocoa powder [88/10/1/1]D-1Witocan ®H–Witcan ®42/44mixture (92.5:7,w/w)/acetaminophen/saccharin [85/10/5]D-2Witocan ®H–Witcan ®42/44mixture (92.5:7,w/w)/acetaminophen/saccharin/Benecoat BMI-40[84/10/1/5]D-3Witocan ®H–Witcan ®42/44mixture (92.5:7,w/w)/acetaminophen/saccharin/cocoa powder [88/10/1/1]D-4Witocan ®H–Witcan ®42/44mixture (92.5:7,w/w)/acetaminophen/aspartame/Benecoat BMI-40[84/10/1/5]ratio on melting temperature and drug release rate.A mixture of Witocan ®H and Witocan ®42/44(80:20,85:15,90:10,92.5:7.5,95:5,97.5:2.5or 100:0,w/w)was used as a matrix base.No corrigent was added.2.2.3.Formulations C-1,C-2,C-3and C-4These chewable tablets were prepared to investi-gate the effect of sweetening agent on bitterness and sweetness of the Witocan ®H chewable tablets.Su-crose,xylitol,saccharin,saccharin sodium,aspartame and sucralose were used as sweetening agents.The four kinds of corrigent systems described in Table 1were examined.2.2.4.Formulations D-1,D-2,D-3and D-4These chewable tablets were prepared using the Witocan ®H/Witocan ®42/44mixture (92.5:7.5,w/w)as a matrix.The four kinds of corrigent systems de-scribed in Table 1were examined.In all the tablets prepared,the amount of the sweet-ening agent per tablet was much lower as compared with the acceptable daily intake (Japan Food Additives Association,2001).2.3.Measurement of intensities of bitterness and sweetnessBitter and sweet taste intensities were determined by the sensation tests using healthy men with age of 21–25based on the methods by Indow (1966,1969)and Katsuragi et al.(1997).Before measurement,in-formed consent was completed to each volunteer.Qui-nine aqueous solutions with a series of concentrations were prepared as standard solutions of bitter taste in-tensities,and the intensities were defined from 0to 1054H.Suzuki et al./International Journal of Pharmaceutics278(2004)51–61 Table2Relationship between defined taste intensity and concentration ofquinine sulfate or sucrose aqueous solutionDefined taste intensity Quinine sulfateconcentration forbitterness(%,w/v)Sucroseconcentration forsweetness(%,w/v)00.000000.010.00023 1.020.00050 1.930.00094 3.040.00157 4.350.00241 6.460.003889.070.0060814.080.0098522.590.0157224.0100.0256878.0(Table2).Also,sucrose aqueous solutions with a se-ries of concentrations were used as standard solutions for measurement of sweet taste intensities,when the intensities were defined from0to10(Table2).The bitter or sweet taste intensities of chewable tablets were evaluated as follows:1ml of each stan-dard solution was dropped on the center of the tongue, the solution was retained in the mouth for10s,then the mouth was rinsed thoroughly with de-ionized wa-ter so that recognition of the taste intensities of the standards was recovered.At10min after remember-ing the taste intensities of each standard solution,the mouth was rinsed fully again,one tablet was put in the mouth,chewed10times and retained on the center of the tongue in the mouth for10s.At that time,the sub-ject decided the taste intensity of the chewed tablet by comparison with that of each standard solution.For examination of the taste intensities of bitterness and sweetness,the number of the subjects in each group was three.2.4.Drug release testsThe drug release was generally examined for in-tact tablets.As to the chewable tablets selectedfinally, both intact and crushed tablets were examined for drug release.Crushed tablets were prepared by breaking a tablet mechanically into nearly10pieces with similar fragment size to simulate the fragmentation of a tablet by chewing.The drug release experiment was per-formed according to thefirst method(rotation basket method)of the dissolution test in the Pharmacopoeia of Japan(JP)14.Thefirstfluid,aqueous HCl solution containing NaCl at0.2%(w/v)(pH1.2)and the second fluid,50mM phosphate buffer(KH2PO4–NaOH)(pH 6.8)of the disintegration test in the Pharmacopoeia of JP14were used as release media.One tablet or all the fragments obtained by crushing one tablet were put in a basket,immersed completely in900ml of the disso-lution medium pre-warmed at37±0.5◦C so that the basket bottom was located at2.5cm from the inner bottom of the container,and rotated at60rpm at37±0.5◦C.At appropriate time points,1ml of the tested medium was taken andfiltered with a membranefilter (0.45␮m pore size).Immediately after each sampling, 1ml of fresh medium was complemented.Thefiltrate was diluted10-fold in volume with fresh medium,and measured spectrophotometrically at244nm to deter-mine the amount of released drug.In each drug re-lease test performed,hard fats and corrigents showed no influence on the determination of acetaminophen concentration by this method.2.5.Measurement of melting characteristics of chewable tabletsThe melting features of chewable tablets were ex-amined by differential scanning calorimetry(DSC)us-ing a Rigaku THERMOFLEX TAS200DAS8230D (Japan).The tablets were roughly crushed,and the ob-tained particles of10mg were used as a sample.The DSC scan speed and scan range were5◦C/min and 25–50◦C,respectively.The temperature of the mini-mum peak of the endothermic DSC curve was defined as a melting temperature of the tablet.2.6.Statistical analysisFor the comparison,statistical analyses were per-formed using the unpaired t-test.The results at P< 0.05was regarded as significantly different.3.Results and discussion3.1.Effect of kinds of hard fats on bitterness and oral feelingAll the tablets were obtained in a disk shape with 2cm in diameter and4–5mm in thickness.ThoseH.Suzuki et al./International Journal of Pharmaceutics278(2004)51–6155 Table3Melting properties of various hard fats and bitter taste and oral feeling of their acetaminophen-containing tablets with sucrose and Benecoat BMI-40each at5%(w/w)as corrigentsCommercial name of hard fat Grade Melting point(◦C)a Bitter tasteintensity bOrder of oralfeeling cWitepsol®H-1533.5–35.5 5.2±0.47W-3533.5–35.57.0±0.06S-5533.5–35.58.2±0.3∗4E-7538 5.0±0.05E-8542–44 5.0±0.62Witocan®H33.5–35.5 5.0±0.0342/4442–44 4.5±0.31a These melting points were shown by the suppliers.b The bitter taste intensity was determined based on the recognition by human volunteers stated in Section2.3in the text and the result is as the mean±S.E.(n=3).c Smaller number exhibited better oral feeling in the tablets.∗Significant difference,P<0.05vs.Witepsol®H-15.tablets were a little harder than solid chocolate,but they could be easily crushed by bite,though their hardness was not measured in detail.Formulation A was used in this ly,various hard fats were used as a matrix,and5%(w/w)Benecoat BMI-40plus5%(w/w)sucrose were used as corri-gents.The commercial name and melting point of the hard fats applied are described in Table3.The bitter taste intensities were approximately5for Witepsol®H-15,E-75and E-85,and Witocan®H.Witepsol®W-35and S-55exhibited higher bitter taste intensities of7–8.2.Witocan®42/44showed the lowest bitter taste intensity of4.5.Only the Witepsol®S-55exhib-ited a significantly higher bitter taste intensity differ-ent from Witepsol®H-15(P<0.05).The oral feeling of the chewable tablets was related mainly to sticki-ness to the oral cavity and gingival,and that sensation was ranged in the order of more comfortable feeling as shown in Table3.Overall,the bitter taste intensities of less than5–6were tolerable,and the oral feelings of the order1–3were acceptable.Thus,the Witocan®H and42/44tablets were adequate as tablets with suppressed bitterness and improved oral feeling.Al-though the Witocan®42/44tablet exhibited bitterness suppression and oral feeling best,the release percent-ages were less than6%in both thefirst and second fluids even at2h after start of the release test(data not shown).The poor drug release was considered to be due to higher melting point of Witocan®42/44,and also the good bitterness suppression might be due to the low drug release.Thus,Witocan®H or the mixture of Witocan®H and Witocan®42/44,which showed lower melting points,were used in the following ex-periments.3.2.Effect of the Witocan®H/Witocan®42/44ratio on melting temperature and drug release Formulation B was used in this experiment. Witocan®H alone and the mixture of Witocan®H and Witocan®42/44were applied as a matrix base, and the obtained tablets were examined for melting temperature and drug release extent.Effect of the Witocan®H/Witocan®42/44ratio on melting tem-perature is shown in Table4.The meting temperature rose with the decrease of the Witocan®H/Witocan®42/44ratio.The drug release profiles of the chew-able tablets with different Witocan®H/Witocan®42/44ratios are described in Fig.1.The chewable Table4Melting temperature of chewable tablets with different Witocan®H/Witcan®42/44ratiosWitocan®H/Witcan®42/44ratio(w/w)Melting temperature(◦C)(mean±S.D.) 100/038.5±0.397.5/2.538.9±0.195/539.2±0.292.5/7.539.2±0.190/1039.8±0.585/1540.3±0.180/2041.0±0.156H.Suzuki et al./International Journal of Pharmaceutics 278(2004)51–61Fig.1.Release profiles of acetaminophen from tablets prepared using Witocan ®H alone or Witocan ®H/Witocan ®42/44mixture as a base.The intact tablets were used in the experiment.(A)First fluid in JP 14;(B)second fluid in JP 14.(᭿)100%(w/w)Witocan ®H;(᭜)97.5%(w/w)Witocan ®H;(᭹)95%(w/w)Witocan ®H;(᭡)92.5%(w/w)Witocan ®H;()90%(w/w)Witocan ®H;()85%(w/w)Witocan ®H;(⊕)80%(w/w)Witocan ®H.Each point represents the mean ±S .D.(n =3).tablets with the Witocan ®H/Witocan ®42/44ratios of 92.5%(w/w)or more showed good drug release.When the drug release extent was evaluated by the percentage of the drug released from the intact tablet at 2h after the start of the release test,the relation-ship between melting temperature anddrug release extent was obtained as shown in Fig.2.These results indicated that the chewable tablet made of Witocan ®H alone exhibited the lowest melting temperature and best drug release.The chewable tablets with the Fig.2.Relationships between melting temperature and acetaminophen release extent for the Witocan ®H/Witocan ®42/44mixture tablets.(A)Melting temperature vs.percent released in the first fluid in JP 14at 2h after the start of the test;(B)melting temperature vs.percent released in the second fluid in JP 14at 2h after the start of the test.The melting temperature was defined as the temperature of the minimum peak of the endothermic DSC profile.Each point represents the mean ±S .D.(n =3).Witocan ®H/Witocan ®42/44ratios of 10%(w/w)or more exhibited poor drug release.The mixed base of 92.5%(w/w)Witocan ®H and 7.5%(w/w)Witocan ®42/44gave good release extent in only the second fluid.The release extent decreased sharply in a reverse sigmoidal curve around the melting temper-ature of 39.2◦C.These suggested that the Witocan ®H/Witocan ®42/44mixture must contain Witocan ®H at 92.5%(w/w)or more to achieve sufficient release of acetaminophen.H.Suzuki et al./International Journal of Pharmaceutics278(2004)51–6157Fig.3.Bitter and sweet taste intensities of Witocan®H chewable tablets with various corrigent systems.No Corr,with no corrigent;Sr, sucrose;B,Benecoat BMI-40;Cp,cocoa powder;X,xylitol;Sc,saccharin;Ss,saccharin sodium;A,aspartame;Sl,sucralose.The number attached to the corrigent abbreviation showed the percentage of the corrigent in the chewable tablet;for example,Sr5-B5means the corrigent system of5%(w/w)sucrose and5%(w/w)Benecoat BMI-40.()Bitter taste intensity;()sweet taste intensity.Each column represents the mean±S.E.(n=3).For bitter taste intensities,the corrigent systems except X5,Sc5-B5and A5-B5were significantly different from No Corr(P<0.05).3.3.Effect of sweetening agents on bitter and sweet taste intensities of chewable tabletsFormulations C-1,C-2,C-3and C-4were used in this experiment,and the bitter and sweet taste intensi-ties of the chewable tablets were examined.The bitter and sweet taste intensities of the chewable tablets are shown in Fig.3.The chewable tablet with no corri-gent showed the bitter taste intensity of7.2.In com-parison with the chewable tablet with no corrigent, the other chewable tablets showed significantly lower bitterness(P<0.05)for the corrigent systems other than5%(w/w)xylitol(X5),5%(w/w)saccharin plus 5%(w/w)Benecoat BMI-40(Sc5-B5),and5%(w/w) aspartame plus5%(w/w)Benecoat BMI-40(A5-B5). Saccharin and its sodium salt tended to suppress bit-ter taste more than sucrose.Sweet taste became much higher in saccharin sodium but increased slightly in saccharin,which was probably because saccharin sodium is easily soluble than saccharin.As a whole,the tablets using saccharin as a sweetening agent showed good balance in bitter and sweet tastes.In particular,the corrigent system of1%(w/w)saccha-rine plus5%(w/w)Benecoat BMI-40(Sc1-B5)was excellent.Although several tablets with aspartame or sucralose as a sweetening agent inhibited bitterness much more than those with sucrose,they gave much sweeter taste than sucrose.Aspartame and sucralose can suppress bitter taste effectively due to their highly sweetening potential,but high increase in the sweet taste intensity appeared to cause discomfort in taking the tablets;generally,the samples with the sweet taste intensities of four or more were too sweet to take.In the preliminary study,three kinds of commer-cial syrups of acetaminophen,being prescription drugs (commercial name not described),were examined on bitterness and sweetness(n=3).Two(thefirst and second syrups)include saccharin sodium as a sweet-ening agent,and the other(the third syrup)contains aspartame as a sweetening agent.Their tastes were58H.Suzuki et al./International Journal of Pharmaceutics278(2004)51–61examined in the manner as stated previously(Suzuki et al.,2003);namely,the syrups were tasted in the same manner as the standard solution.As a result,the first syrup exhibited the bitter and sweet taste intensi-ties of4.2and5.2,respectively.The second showed the bitter and sweet taste intensities of5.2and5.3,re-spectively,and the third exhibited the bitter and sweet taste intensities of4.3and5.7,respectively.Thefirst syrup showed the best tastes.The Witocan®H chew-able tablet with Sc1-B5exhibited the bitter taste in-tensity of4and the sweet taste intensity of1.5,indi-cating that this tablet suppressed taste intensities bet-ter,especially sweetness,as compared with the above commercial syrups.3.4.Taste intensities and drug release for the Witocan®H/Witocan®42/44(92.5:7.5,w/w)mixture tabletsThe Witocan®H chewable tablets improved oral feeling,but the addition of Witocan®42/44can im-prove the oral feeling more.Based on the results in Figs.1and2,the Witocan®H/Witocan®42/44 (92.5:7.5,w/w)mixture tablets,exhibiting good drug release,were chosen.The mixture tablets were pre-pared for the excellent corrigent systems in Fig.3; that is,D-1,D-2,D-3and D-4in Table1were pre-pared.They were examined for taste intensities and drug release.The results of taste intensities are shown in Fig.4. The bitter taste intensity of the chewable tablet with no corrigent was6.2.For each corrigent system,the taste intensities were almost parallel to those in Witocan®H tablets.The corrigent systems except Sc1-B5showed significant suppression of bitterness(P<0.05).In particular,A1-B5exhibited the least bitter taste inten-sity and low sweetness.The drug release profiles of the intact tablets are shown in Fig.5.All the tablets showed good drug release.3.5.Drug release from intact and crushed tablets For the tablets showing excellent taste balance in the Witocan®H tablets(Fig.3)and Witocan®H/Witocan®42/44(92.5:7.5,w/w)mixture tablets (Fig.4),the drug release tests were performed at the intact and crushed conditions.That is,the Witocan®H tablet with Sc1-B5and Witocan®H/Witocan®Fig.4.Bitter and sweet taste intensities of Witocan®H/Witocan®42/44(92.5:7.5,w/w)mixture chewable tablets with several corri-gents.The abbreviation and expression of corrigent systems were the same as in Fig.3.()Bitter taste intensity;()sweet taste intensity.Each column represents the mean±S.E.(n=3).For bitter taste intensities,the corrigent systems except Sc1-B5were significantly different from No Corr(P<0.05).42/44(92.5:7.5,w/w)mixture tablet with A1-B5were selected,and their drug release was examined in the intact and crushed forms.The results are shown in Fig.6.The Witocan®H tablet with Sc1-B5showed good release in both intact and crushed forms;that is,more than50%(w/w)of the drug was released in the secondfluid at2h after the start of the release test.In particular,this tablet showed that the crushed form released approximately50%of the drug in the secondfluid at1h after the start of the release test. The Witocan®H/Witocan®42/44(92.5:7.5,w/w) mixture tablet with A1-B5showed good release in the intact form,but not in the crushed form.These results were possibly related to the melting properties of the ly,the Witocan®H tablet with Sc1-B5melted completely to form liquid droplets during the release test,especially in the secondfluid. On the other hand,for the Witocan®H/Witocan®42/44(92.5:7.5,w/w)mixture tablet with A1-B5,soft semisolid remained on the basket after the release test;an aggregated and bulky semisolid was formed markedly in the crushed form,probably leading to poor drug release.These features were consistent with the results in Fig.2that the Witocan®H/Witocan®。

卵圆孔轻度反流原因介绍卵圆孔轻度反流是指由于卵圆孔（也称为房间隔缺损）在心脏收缩期间发生逆流的现象。

本文将介绍卵圆孔轻度反流的原因。

主要原因如下：1. 生理原因：卵圆孔在胎儿发育过程中起到通气的作用。

在出生后，卵圆孔通常会逐渐关闭，但有些人卵圆孔关闭不完全，导致轻度的逆流发生。

2. 先天性心脏病：在一些先天性心脏病患者中，卵圆孔轻度反流是常见的一种情况。

这些心脏病可能会导致心脏的结构异常，影响了卵圆孔的正常关闭。

除了以上两个主要原因外，其他一些可能导致卵圆孔轻度反流的因素包括：高血压、心脏病、肺动脉高压、自主神经功能失调等。

虽然卵圆孔轻度反流在大多数情况下是无害的，但在一些情况下，特别是与其他心脏问题同时存在时，可能需要进一步的医疗干预。

如果您担心自己可能存在卵圆孔轻度反流，请与医生进行咨询和评估。

请注意：本文所述内容仅供参考，具体情况还需根据个人体检结果和医生的建议进行判断。

以上内容不能代替医生的诊断和治疗建议。

参考文献：1. Sutherland FJ, Shattock MJ, Baker KE, et al. Can the clinical identification of left-to-right intracardiac shunting through a patent foramen ovale be enhanced by non-invasive methods? Int J Cardiol. 2004;96(3):255-261.2. Huang ZG, Luo XG, Chen HQ, et al. The effects of patent foramen ovale closure on atrial fibrillation. Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia. 2015;22(2):333-338.。

专利名称：治疗性抑制性化合物专利类型：发明专利
发明人：安德鲁·麦克唐纳
申请号：CN201480076616.5申请日：20141230
公开号：CN106061480A
公开日：
20161026
专利内容由知识产权出版社提供
摘要：本发明提供了式I和式II化合物及其盐：A‑B‑C‑D‑E‑F‑G‑J (I) C‑D‑E‑F‑G‑J (II)，其中A、B、C、D、E、F、G和J具有本说明书中限定的任何值。

该化合物可用于抑制血浆激肽释放酶，以及用于治疗动物中的疾病或状况，其中指示血浆激肽释放酶的抑制。

申请人：莱福斯希医药公司
地址：巴巴多斯圣迈克尔
国籍：BB
代理机构：北京安信方达知识产权代理有限公司
代理人：郑霞
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