范文一:药代动力学_药效动力学结合模型在中药研究中的应用
中国药理学通报 ChinesePharmacologicalBulletin 2008Nov;24(11):1405~8?1405?
药代动力学-药效动力学结合模型在中药研究中的应用
束 云,李连达
(中国中医科学院西苑医院,北京 100091)
中国图书分类号:R205;R282171;R96911;R972
文献标识码:A文章编号:1001-1978(2008)11-1405-04摘要:药代动力学2药效动力学(Pharmacokinetic2pharmacody2
namic,PK/PD)结合模型是研究中药体内代谢过程、药物效
确反映该药物的临床药理性质,为Ⅱ、Ⅲ期临床试验奠定基础[1]。由于在药代动力学、,、走向世界的。,,。本文就目前PK/
D,
应及二者联系的有效工具,药优化有重要的参考价值PD并就建立具有中医药特色的PK/PD结合模型提出建议,以期为今后的相关研究提供参考。
1 化学药物PK/PD研究对中药相关研究的启示 长期以
,并就中药效应物质基础的确定、效应指标的选择等关键问题进行探讨并提出建议,以期为今后的相关研究提供参考。
关键词:药代动力学;药效动力学;药代动力学-药效动力学结合模型;中药
药代动力学2药效动力学(Pharmacokinetic2pharmacody2
namic,PK/PD)结合模型是研究药物剂量与药物效应之间定
来,中药多以经验给药,至于药物中哪些成分起作用、药物的体内过程如何、药物成分与治疗作用之间有什么联系等问题常常不很清楚。这严重阻碍了中药现代化与国际化的进程。
PK/PD结合模型着眼于药物的体内过程、药物效应及二者
之间的关系,为解决以上问题提供了一种新方案。 PK/PD结合模型由Sheiner等[2]于1979年建立。自建立以来,它在化学药物理论研究及临床应用领域得到了广泛的应用,其研究内容涵盖了药物的体内代谢过程、药物与酶的对应关系和相互作用、药物毒副作用、耐受性与致敏性研究、立体异构体药物比较等众多方面[1,3]。该模型在化学药物研究中的应用不仅为解释药物的代谢机制、作用机制或耐
收稿日期:2008-07-05,修回日期:2008-09-20
基金项目:中医药科学技术研究专项资助项目(No06207ZQ16)作者简介:束 云(1980-),女,博士生,研究方向:中西医结合基
础,Tel:010262874069,E2mail:echo2cloud@163.com李连达(1934-),男,中国工程院院士,博士生导师,研究方向:中西医结合基础,通讯作者,Tel(Fax):0102
62874069,E2mail:lilianda1934@163.com
量关系的有效工具,在优选临床用药剂量、提高疗效和减少毒副反应等领域具有重要的参考价值。FDA提出,开发治疗性药物的Ⅰ期临床试验必须提供PK/PD结合模型,以便正
受机制等提供了信息,也为临床剂量调整、优化治疗方案、防治剂量相关性不良反应提供了依据。将PK/PD模型应用于中药研究,一方面可以加深研究人员对中药作用机制的认识,有利于以现代科学阐述中药组方原理,为研究古方、筛选新方提供科学依据和方法,另一方面,它还可以促使中药制剂的“给药精密化”,为拟定给药方式、剂量及间隔时间提供
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matol,2007,36(6):428-33.
αregulatoryTcellsinrheumatoidarthritisandreversalbyanti2TNF
therapy[J].JExpMed,2004,200:277-85.
[26]JiaoZ,WangW,JiaR,etal.AccumulationofFoxP32expressing
CD4+CD25+Tcellswithdistinctchemokinereceptorsinsynovial
+
+
[27]TaamsLS,SmithJ,RustinMH,etal.Humananergic/suppressive
CD4+CD25+Tcells:ahighlydifferentiatedandapoptosis2pronepopulation[J].EurJImmunol,2001,31:1122-31.
ProgressofCD4CD25regulatoryTcellsinrheumatoidarthritis
WANGTing2yu,LIJun
(SchoolofPharmacy,AnhuiMedicalUniversity,Hefei 230032,China)
Abstract:CD4
+
CD25
+
++
regulatoryTcells(CD4CD25thritis(RA).Anunderstandingoftheseissuesislikelytofacil2itatethedevelopmentofCD4CD25Tregs2cell2basedtherapiesforthetreatmentofRA.
Keywords:rheumatoidarthritis;CD25;suppressor;regulatoryTcells
+
+
Tregs),asuppressorTcellpopulation,havebeenshowntoplayanimportantroleinmaintainingperipheraltoleranceandthepreventionofautoimmunity.Thisreviewsummarizesthecharac2teristicsandmechanism,aswellastheirroleinrheumatoidar2
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ralcerebralnutrition,CBN)的研究中,以葛根素和人参皂苷Rg1为代表成分,分析二者的PK过程与抗血小板聚集、抑制
依据,从而提高临床治疗水平、减少不良反应,对于实现中药治疗学的现代化和科学化有重要意义。PK/PD模型在化学药物研究中取得的成绩,为中药PK/PD研究提供了思路和方法学参考。已有研究者就该模型在中药领域的应用进行了探讨。
2 中医药PK/PD研究现状
2.1 中药单成分提取物研究 目前,研究已发现多种具有
丙二醛(MDA)生成等药理作用的关系,却未观察到明显的全程相关性。以上结果说明,虽然以主要活性成分的药代动力学结合全方的药效动力学来探讨多成分药物(中药及复方)的PK/PD特性是可行的,但是,只有在谨慎选择了合理的测试成分及效应指标的前提下,拟合模型才有可能成功。该研究结果的可推广性有待进一步验证。中药材及其复方问题复杂,,完善. ,生物活性的中药单成分提取物,且部分提取物已开发成产品供临床使用。对这些单体成分进行PK/PD研究,可了解其代谢过程、时效关系以及二者的相互联系,有助于加深研究人员对其作用机制的认识。
,分明确,,吸收、分布、,通过PD研究阐明药物效应随时间变化的特征,然后以假想的效应室为基础,采用适当的数学模型整合这两个动力学过程,描述随药物浓度变化的药物效应动力学,研究组织、血液或其他体液中药物浓度与药物效应之间的关系。以蝙蝠葛苏林碱(daurisoline,DS)在犬体内的PK/PD研究为例,师少军等
[4]
。对于治疗窗较窄的药物(例如华法林、地高辛等)而言,这种相互作用可能导致严重的不良反应,甚至死亡[8]。目前,PK/PD研究的重点正从临床单次给药或多次给药向联合用药上发展。这种趋势在中药研究领域也得到了体现。丹参、人参、银杏、生姜、当归、川芎、黄芪等中草药对化学药物的药代动力学和药效动力学特征的影响已被广泛探讨。研究结果表明,不同的中药对同一化学药物所产生影响不同。例如,丹参会明显影响华法林在机体内的代谢过程和抗凝效应[9],而人参却只增加华法林在人体内的清除率,对其他的药代动力学参数和药效动力学过程无明显影响[10]。这类研究为联合用药情况下优化治疗方案、保障用药安全提供了依据。
目前,该类研究普遍偏重于观察中药对化学药物的影响,后者对前者的影响却很少有研究探讨。这可能与中药成分复杂、效应多样,难以选择恰当的观察指标有关。
3 建立有中医药特色的PK/PD研究体系面临的挑战
以血药浓度变化描述
药物的代谢动力学过程,并观察心率、收缩压、左室舒张末压
LVSP、Q2T间期等药效学指标随时间变化的特征,随后采用Sheiner效应室模型将各效应2血药浓度的滞后环转化为效
应2效应室浓度的正变关系。结果发现,这种中药提取物的效应滞后于血药浓度,且DS与受体的亲和力较大,药物由中央室(血液室)到效应室(心肌组织和血管平滑肌组织)平衡过程是导致效应滞后的主要原因。该研究通过建立PK/
PD结合模型成功预测DS的血浆浓度与药理效应,为未来的
临床应用提供了依据。已报道的其他中药提取物(如蝙蝠葛碱等)的PK/PD研究过程与其相似,与化学药物无明显区别[5,6]。
2.2 中药材及复方研究 中药材及复方是中医治病的主要
借鉴化学药物的研究思路与方法,研究人员已就PK/
PD结合模型在中医药研究领域的应用进行了初步尝试。他
们的工作取得了一些成绩,但也存在一些问题。针对中药及其复方复杂的成分组成和效应作用,如何建立有中医药特色的研究体系,如何有选择地进行PK和PD研究、PK和PD的研究结果怎样进行模型拟合、其模型拟合的方法与单成分药物研究相比具有何种特点,是进行中药及复方PK/PD结合模型研究需要解决的问题。
3.1 药物效应物质基础的确定 进行PK/PD研究,首先应
临床应用形式。研究中药材及复方的PK/PD规律,可以阐明和完善其作用机制及复方组方原理,为提高中药及复方制剂的质量和优化给药方案提供科学依据,同时也为发现活性代谢产物、开发新药奠定理论基础。
与化学药和中药单体提取物不同,中药材及复方组成成分复杂,常具有多靶点、整体调节的作用。虽然化学药物的相关研究为其奠定了理论和方法学基础,但是,中药的特点决定了它在研究思路和方法上会与化学药物研究存在一定的差异。杜力军等[7]就PK/PD结合模型在中药复方研究中的应用进行了探讨。在YL2000的研究中,以黄芩苷、蛇床子素和小檗碱作为黄芩、黄连、独活等中药的有效部位群的代表成分,利用PK/PD结合模型分析3者的代谢过程与发热大鼠体温变化的关系,结果发现,体温下降与黄芩苷的血药浓度变化呈正相关(r=-01864,P
PK/PD特征。但是,在有关脑血管药金森脑泰粉针剂(Natu2
确定足以代表全药生物效应的物质基础。中药单体提取物与化学药物相似,其成分明确,体内代谢过程清晰,效应物质基础较易确定。中药及复方含有多种复杂成分,确定效应物质基础是进行PK/PD研究的一大难题。
从80年代开始,国内外学者尝试用中药或复方中效应明确的某一个或某几个成分来代表全方进行研究。多年来这样的研究方法阐明了一些中药的效应物质基础,对促进中药现代化起到了一定的作用。但是,中药的药效是多种化学成分相互作用、中药与机体相互作用所产生的综合结果[11]。这种方法所获得的资料只能说明活性成分的PK/PD特点,未必能完整地反映含这种成分的中药及其方剂的体内过程。从整体和关联的角度出发重新认识中药效应的物质基础,正
中国药理学通报 ChinesePharmacologicalBulletin 2008Nov;24(11)
逐步成为国内外研究人员的共识。
目前,相关研究的发展有两种趋势。一种趋势仍以寻找中药及复方中的活性物质为研究重点。与以往研究的不同之处在于,该类研究充分考虑了中药“整体观”的特点和中药有效成分在体内的变化过程,综合运用多学科知识和现代先进的分离分析手段,构建中药及其复方吸收、分布、与靶细胞结合的体内外技术平台,通过寻找中药发挥作用的体内效应成分(群),来追溯其在中药直接提取物中的物质形式[12]。另一种趋势伴随系统生物学和代谢组学的发展而产生。相关研究人员认为,不仅中药的原型成分和代谢产物与中药的治疗效应有关,挥作用,物质基础[13]。
,的特点,。以此作为研究对象,对于全面而深刻的理解中药及复方的物质基础与生物效应之间的关系有重要意义。
3.2 药物效应指标的选择 中药及复方具有多成分、多靶
?1407?
从中药多作用、多靶点、整体调节的特点和机体对于干预措施的整体反应性出发,综合的多指标的评价体系更有利于中药疗效评价[20]。证候是中医的特色之一。目前已有研究就症候指标与效应指标的对应关系和有机结合进行了探讨[21,22]。如果能将症候指标引入效应评价系统,将使中药及复方PK/PD研究进入一个崭新的阶段。
3.3 模型的建立 以成分群或代谢物组为效应物质基础来
进行PK/PD研究,有利于从整体上把握中药及复方的代谢,但是,。成分群和代、转化、排泄的过程,,成为摆在研究人员面。
近年来在PK/PD研究中得到应用的人工神经网络为解决以上问题提供了可能。这种计算体系不需要先假定一个特定的模型,而只需从提供给它们的数据中建立输入与输出的关系,极大地简化了传统药动学数据分析所需的建模工作,而且无论是在预示能力还是预示速度方面都有显著提高。Minor等[23]的研究表明,用人工神经网络能够将给药情况与PD、给药情况与PK、PD与PK或其它与治疗相关的因素直接关联起来,获得它们之间的关系。人工神经网络因其出色的非线性拟合能力有望成为中药及复方PK/PD研究中一种有效的数据分析工具。
代谢组学研究常运用化学计量学理论和多元统计分析方法,对采集的海量多维原始信息进行压缩降维和归类分析,最终以较少的独立主成分综合体现原多维变量中蕴含的绝大部分整体信息(习惯上>85%)[24]。这种研究策略在最大化保留信息的前提下大大简化了数据分析的难度和工作量,值得中药PK/PD结合研究人员借鉴。
综上所述,中药PK/PD研究刚刚起步,在作用机制研究、临床应用、新药研发等方面具有广阔的应用前景。虽然,该类研究目前已经取得了一些成绩,但在体现中医药特色方面还存在诸多挑战。中药PK/PD研究涉及多方面的理论和技术,是集中药药理学、中药化学、分析化学、分子生物学和数学于一体的边缘学科。这一学科的发展需要上述多学科专家的共同合作与努力。近年来系统生物学及相关技术的快速发展,为多学科、多视角地认识中药效应机制提供了重要的理论基础和技术条件。将代谢组学等现代生物技术与中药PK/PD模型研究相结合,加强多学科研究人员之间交流与合作,建立有中医药特色的研究体系,对于阐明和揭示中药的作用机制和科学内涵、设计及优选中药给药方案、促进中药新药开发,推动中药现代化和国际化有重要意义。参考文献:
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点、整体调节的特点,在PK/PD模型研究中,不仅难以选定足以代表全药药代动力学特征的测试成分,而且也很难将全药的治疗作用落实到某一个或某几个药效指标。
由于对受体的选择性不高,绝大多数中药单体提取物引起的PD反应复杂多样。而中药及复方的特性也导致了生物效应多样化。因此,只有从整体、宏观的角度选择直接反映治疗效果的指标才能全面而准确的评价中药的效应,例如,降血压、降血糖、降低体温、止痛等。但是在大多数的
PK/PD研究中,药物的治疗效果难以定量,只能选用较易测
定的替代指标来反映各种疗效。如何选择合适的替代指标,中药及复方药物动力学研究中常常采用的生物效应法为
PK/PD研究提供了可借鉴的经验:(1)被观测的效应指标应
与治疗作用或毒性作用有直接关系或高度的相关性,可预测疾病的结局,并可完全解释由治疗引起的临床结局变化的净效应[14];(2)被观测的效应指标随药量的变化应是一种量反应而不是质反应。目前,已有研究就多种疾病疗效评价中替代指标的选择进行了探讨[15~17],如以血浆羟氢可待酮浓度作为替代指标评价阿片类镇痛药控释剂的止痛效果[18]、以
ATT(针对破伤风毒素的抗体效应)作为替代指标评价猴体
内CD154的单克隆抗体5c8的免疫抑制作用[19]等研究都取得了较好的效果。
有些药效指标在体内的检测受到一定的限制,且动物个体差异大,干扰因素多,结果的准确性和重现性较差;而离体实验(如离体器官、组织、细胞、受体、酶等)观测药理效应较灵敏、准确,在PK/PD研究,尤其是以作用机制为基础的
PK/PD(mechanism2basedPK/PD)研究中正发挥重要作用。
一些非创伤性技术,如核磁共振(NMR)影像和分光镜、阳电子发射断层(PET)、单光子发射计算机断层(SPECT)和生物电阻抗等,开始应用于观察健康群体和患病群体的器官功能及进行药物效应和毒性研究。这些技术进一步丰富了可检测的药物效应。
?1408?中国药理学通报 ChinesePharmacologicalBulletin 2008Nov;24(11)
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中医药大学学报,2002,19(4):245-50.
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tionalChinesemedicine[J].JGuangzhouUnivTCM,2002,19(4):245-50.
[19]郭 宾,戴仁科.代谢组学及其研究策略和分析方法进展[J].
中国卫生检验杂志,2007,17(3):554-63.
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[J].天津中医药,2003,20(6):1-5.
[11]LiuCS.ThetaskofpharmacokineticsresearchoftraditionalChi2
ApplianceofPK/PDmodelingintraditionalchinesemedicineresearch
SHUYun,LILian2da
(ChinaAcademyofChineseMedicalSciences,XiyuanHospital,Beijing 100091,China)
Abstract:Pharmacokinetic2pharmacodynamic(PK/PD)model2ing,asanavailabletoolaccountingfortheinterrelateddynamicconnectionbetweenthedosageandefficiencyofdrugs,playsanimportantroleinthefieldofstudyingthemechanismandoptimi2zingtheclinicaladministrationoftraditionalChinesemedicine(TCM).Basedonthegeneralillustrationontheforegoingappli2cationofPK/PDmodeling,thepresentworkinvestigatesthein2volvingchallengesinthefield,suchastheselectionofthetarget
chemicalcompositionandeffectmark.Inaccordancewiththeu2niquepropertiesofTCM,further,therelatedsuggestions,mainlyfocusingondeterminingthesubstancesbasedontheeffectandestablishingthemulti2indexappraisesystem,areputforwardasreferenceforthefollowingresearchwork.
Keywords:pharmacokinetic;pharmacodynamics;pharmacoki2netics2pharmacodynamicmodeling;traditionalChinesemedicine
范文二:氧氟沙星三种给药途径的药物动力学
氧氟沙星三种给药途径的药物动力学
B吼In:1SSN0253—9756Actapharma~ologieaSiniea中国琦理
1994Sep#15(5):{u一{l3
Pharmacokineticsofofloxacinthroughthreeadministrationroutes
ZHUOHal—Tong,LUJi—Zong,XIAXi—Rong,LINGShu—Sen(DepartmentofClinical
Pharmacology,JinlingHospital,Nanjing210002;FirstHospitalofWuxi,Wu
zi214002,
DepartmentofRespiratoryDiseasesofJinlingHospital,Nanjing210002,Chi
na)
ABSTRACTThispaperreportsthepharma—
cokineticcharacteristicsofofloxacin(0f1)
through3administrationroutesin42patients
withrespiratorytractinfections.Thecon
centration—timedata.werefittedwithatwo—
compartmentmodelforinfusion(inf)andim,
andaon?e—compartmentmodelforpo.The
pharmacokineticparametersofOflthrough
inf,imandpowere:了1如or丁6.0?1.3,
5.O士1.0,and5.0士O.7h;VorVd58士16,
68士27and94士25L;3.9士1.O,2.8士
0.9,and1.9士O.7g?ml,;AUC16士5,15
?4,and15士4h?g?ml,;Cf13士4,14土4,
and14士3L?h,,respectively.
KEYWORDSofloxacin;pharmacokinetics;
drugadministrationroutes;highpressure
liquidchromatography
Ofloxacin(Of1]isanantlmicrobiaIagent
withbroadantibacteriaIspectrum.When
firstintroduced,Oflwasadministeredorally,
anditspharmacokinetieswasdescribed一.
Recently,anivpreparationwasdevel
oped?.butitsimformulationwasnotmen—
tioned.Thepurposeofthisstudywastoin—
vestigatethepharmacokineticdifferencesof
Oflthrough3routesin42patientswithrespi—
ratorytractinfections.
MATERIALSANDMETH0DS
Suzhou(forinf),andNanjing3rdPharmaceutical
Factory(forira).
StudydesignForty—twopatientssufferingfrom
respiratorytractjnfectionswithnorl~laIliverandkid
neyfunctionsweredividedinto3groups:Group1for
po,14patients(allmales),aged47?17a;Group2
forinf,16patients(4femalesand12males),aged51
土17a;Group3forim,12patients(4femalesand8
males),aged47?16a.Drugdosagewas200mgof
Offforall3groups.Bloodsampleswerecollected
fromantecuhitaIveinbeforeand0.5,1,1.5,2,3,4,
8,10,and12hafterdosing.Urinesampleswere
collectedfor24hforcalculatingcumulativeexcretion
HPLCanalysisTheOff
andurineweredeterminedbyHPLCBeckman
HPLCsystemincluded114Mpump,157fluorometric
detectors(k305—395ran.k420—650nm),and427
dataprocessors.TheBeckmanUltraspheremIP
column(250mm×4.6mmID)wasused.Themo
bilephaseconsistedofmethanolandpH2.5phosphate
buffersolution(32l68,vo1?voI)andtetrahutylammo—
niumbromide5mmoI?L.Theflowratewas0.8
ral~min,.
Serumsample(0.2m1)mixedwithmethanol0.8
mlwascentrifugedat10000×gfor10min.The
supernatantwasevaporatedtodrynessat45?with
airstream.Theresiduewasdissolvedin0.4mtof
mobilephaseforHPLCanalysis.Urinesamplewas
analyzedafterbeingdiluted1000timeswithdistilled
water.
DataanalysisThedatawerefittedwithoneor
modelwiththesoftware3P87
Thepharmacokinetieparameterswereestimatedby
least—squarenonlinearregressionanalysis.
DrugandreagentsOffstandardandtabletswere
madebyKunshanPharmaceuticalFactory,Jiangsu;RESULTS
OffinjectionbyChangzhengPharmaceuticalFactory,
Received199308—31Accepted1994—06—23
QualitycontrolofHPLCassayThere-
tentiontimeofOflwas6.68rain(Fig1).
?412?BIBL1D,ISSN0253—9756Aclapharma~loalcaSmic~中田理学粗
1994Sept1S(5)
AB
l
一
Time/min
Fig1.HPLCofoflexacin.(A)blankset-oatsam-
piel(B)ofloxaetnf(C)serumsampleafterpo
oflo~n.
Thecalihrationcurvewaftlinearwithins
rangefram0.01to8g?mr(r=0.9997).
Thedetectionlimitwas5ng?_..There—
coveryratewas98士5.Coefficientsof
variationwithinadayandbetweendayswere
2.5and3.3,respectively(Tab1).
Tab1.HPLCassayofofloxadn.=S,士j
PharmaeokinetteamdysisIncasesof30
rainafterinfandimOff200rag,thepharma—
cokineticcharacteristicsshowedanopentwo.
compartmentmodel(Tab2).Thephsrnls-
cokinetieprofileafterpoOfi200mgcouldbe
Tab2.Pharmaeoldnetl~parametersofofloxadn
afterJar,lmor200咩In42patientswithmpl?
ratarytractlnfeet~ns.士=.
ParalfietersiM
(n一16)
ira
(一12)(一14)
TPO7T~K/h
ord/L
C一/?III1
一/h
6.O士l_35.0士1.05.0士0.7
58士16
3.9士1.0
AUCO--lZ/h?Pg?ml一17:1:5
a/L?h13士4
68士27
2.8士0.9
1.0士o.4
I5士4
14士4
94士25
I.9士0.7
2.8士0.9
15士4
14士3
deseribedwithanone—compartmentmodel
(Fig2).Themeancumulativeexcretionrate
ofOnin24hwas83士19forinf,73士1O
for加,and77士9forim.Themean
concentrationof0flinurinein24hwas91士
2l?,,91士34?ml,tand113士43
?ml,forinf,po,andim,respectively.
Fig2-Oflexactn~trattoathNr-d~afterhat
(0.4—16),lm(?,_=12),and(×,4=14)
铷mroll|npatkms.士f.
DIsCUSsl0N
TheHPLCmethodwerespecificityand
sensitivity.Nointerfereneeofanyendoge-
nogssubstauceorotherconcomitantlyused
drugswasdetected.Theassaywassuccess—
fullyappliedtothemeasurementofeolR~entra—
tionsofOil恐.钉tciproflbxacinn帕,andlorile—
floxacininserumandurinesamplescellected
?????
Ec0N.1—880J0jcu
BIBLID.ISSN0259—9786ActaPharmaeologicaSdnica中国琦理
1994sep|15(5)?419
frompatientsandhealthyvolunteers.
ThepharmacokineticsofOflinrespirato—
ryinfectionpatientswassimilartothatinnor—
malvolunteers.butsignificantlydifferent
fromthatinpatientswithchronicrenalfail—
ure”“
.TheAUCvaluesofOf1werefoundto
benearlythesamethrough3routesin42pa—
tientswithrespiratorytractinfection(P>
0.05).
Thebi0availabilitieswere92.5forim
and93.8forpo,similartovaluesreported
elsewhere(.Themeanserumconcentratiofls
before12hwerealIhigherthantheminimum
inhibitoryconcentration(MIC)ofmostkind
ofhacterial.Theresultsshowedthesame
biologicalequivalenceandeffectivenessiflall3
formulations.
TheconcentrationofOflwasveryhigh
(50—16Og?ml)iflurinewithin24h.The
concentrationrangewas20--100timesofMIC
1
inmOStkind0fbacteriala23.ThisimpIied牛.
thatOf1isaeftectlvedrugforthetreatmentof
urinaryinfection.
REFERENCES
1CheD.Q,LuH,XuxY.GuoDW,LiIT.Pharma—
eokineticandrelativehio~vailabilityofofloxaciaafter&
singleoraladmJnistrationinChinesehealthyvolunteers.
7. ChinJClinPhar1~laool1992f8{193—
2ZhuoHTtYaoHL,HuPtWangSJ.Thepharma—
cokinefiesandhio~vailabilltyofofloxa~ninhealthyvol-
unChinJHospPharmacy1998l13{293--7.
3MoakJPtC~npoli-RichardsDM.Ofloxacin.?review
0lit3antibacterialactivitypharmacokinetleproperties
andtherapeuticuse.Drugs1987;33:341—91.
4FI啦&P眦.neof0n0酗cm.
AmJMnd198987(Supp1)I24s一80s.
5LodeH,Hof~enG.OIB~w6kiP,SieversB,KirchA,
BornerKta1.PKarmaeokinetsofofIo~cinafter
parente~lando?Iadmlnistration,
AntimlerohAgentsC~emother198731I1838--42.
6YukJH-Nhtir培akCH,QnintilianiR,SweeneyI(R.
BioavailabilltyandpharmacokinetiesofOfl0xadnin
healthyvolunteers.
AatimierohAgentsChen~other1991?35:384—6.
7MignotAtLefehvreMA.gh一~r[orm&nceLiquid
Chromatographicdeterminationofofloxaeininplasma
andurine.
7. JChromatogrBiomndAppl1988,430}192—
8DavisRL.KoupJR.Williams—W~renJ,werAt
SmithAL.Pharra~okincticsofthaeeonlformuladon
ofciprofloxaeJn.
AntimJcrohagent~Chemother1985I28{74—7.
9ZhuoHT,L1Z.YaoHL,XiaXRtWangSJ.Pharma—
cokineticsofofloxacininpatientswithrespiratorytract
andurinarytr~tctir~ection.
ChinJaiaPharmaeol1992,8:198—208.
10ZhuoHT.YaoHL,LIHB,LiuXM.Pharmaeokinetics
ofeiprofloxacininhealthyvolunteers.
ChinJHospPharmacy1994,2:54—6.
1lLuW,XuJ,KangzQ.pharmaeokinetiesofofloxaeinin
patientswithchrorLIcrenalfailureafterasingleoral
dose.ChinJClinPharm~~ol1992Is}215—7.
12FuehsPCInvitroantimierohialactivityandsuneeptihili
tytestingofofloxacim
AmJMedl989?87(6cSupp[):10a一13s.
一4?々
曩氟沙星三种给药途径的药物动力学
圭堡堕,睦羹,夏锡荣,凌树森
?
214002
..
l,无锡第一医院.无锡,71
.南京盒陵医院呼吸辩,~210002,中国)
摘要本文报道42例呼吸道感染病人用inf,
im和户.三种途径给氧氟沙星后的药物动力学
特性.血药浓度经3P87程序拟合,inf和jm
药物动力学模型符合二房室,户.为一房室.
inf,im,户.后氧氟沙星的主要药物动力学参
数为:T6.0-4-I.3,5.0-4-1.0和Tx5.0-4-0.7
hiV58土16,68土27和d94土25l}Cm3.9土
1.0,2.8土0.9和1.9士0.7?ml一}Cl13土
4,14土4和14土3L?h,.
关键词氧氟抄星}药物动力学}给药途径
呵
7
范文三:HPLC法在中药药代动力学研究中的应用
Journal of Chromatography B, 857(2007)
32–39
Development and validation of a sensitive liquid chromatography–tandem mass spectrometry method for the determination of paeoni?orin in rat brain and its application to pharmacokinetic study
Su-Mei Xia a , b , Rong Shen b , Xue-Ying Sun b , Li-Li Shen b , Yi-Ming Yang a , Ying Ke b , Yun Wang b , Dong-Ying Chen a , ?? , Xing-Mei Han b , ?
a Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555Zu Chong Zhi Road, Shanghai 201203, PR China
b Central Research Institute, Shanghai Pharmaceutical (Group)Ltd., 555Zu Chong Zhi Road, Shanghai 201203, PR China
Received 16March 2007; accepted 25June 2007
Available online 30June 2007
Abstract
A sensitive and speci?c method was developed and validated for the determination of paeoni?orin in rat brain with liquid chromatography–tandem mass spectrometry. Sample pretreatment involved protein precipitation following solid-phase extraction. Paeoni?orin and geniposide (internal standard) were separated isocratically on a Waters Symmetry C18column (150mm ×2.1mm i.d., 5? m), using a mobile phase of methanol/water with 0.1%formic acid (50:50,v/v)at a ?ow-rate of 200–300? L/minin 4min. A Finngan LTQ tandem mass spectrometer equipped with electrospray ionization source was operated in the positive ion mode. Selective reaction monitoring was performed to quantify paeoni?orin and the internal standard at m /z transitions of 503→ 381and 411→ 231, respectively. A good linearity was found in the range of 2–500ng/mL(R 2=0.9939). The intra-and inter-batch assay precisions (coef?cient of variation, CV) at 5, 50and 400ng/mL(n =5) ranged from 6.3%to 9.7%and 1.2%to 7.2%, respectively, and the accuracies were from 95.9%to 101.6%and 99.4%to 102.9%,respectively. The mean recoveries of paeoni?orin were 81.2%, 80.9%and 82.3%at 5, 50and 400ng/mL(n =5), respectively, and the mean recovery of the internal standard was 76.7%with a concentration of 50ng/mL(n =5). Stability studies showed that paeoni?orin was stable in different conditions. Finally, the method was successfully applied to the pharmacokinetic study of paeoni?orin in rat brain following a single subcutaneous administration (10mg/kg)to rats.
?2007Elsevier B.V . All rights reserved.
Keywords:LC–MS/MS;Paeoni?orin; Brain distribution; Matrix effects; Pharmacokinetics
1. Introduction
Paeoni?orin (PF)is a monoterpene glucoside (Fig. 1) isolated from the root of Paeony Radix , one of the traditional Chinese medicines [1]. It has been reported that PF has many clinical indications such as anti-in?ammatory, anti-allergic [2], anti-hyperglycemic [3], anti-thrombosis [4], neuromuscular blocking [5–9], releasing of noradrenaline [10], enhancing glucose uptake [11], cognition-enhancing [12–18], and the neuroprotective [19,20]effects.
? Corresponding author. Tel.:+862150806623; fax:+862150806623. ?? Corresponding author. Tel.:+862150806053; fax:+862150806053. E-mail addresses:dychen@mail.shcnc.ac.cn(D.-Y. Chen),
hanxm@pharm-sh.com.cn(X.-M.Han).
Liu et al. [19,20]reported, that in vivo subcutaneous (s.c.) administration of PF (2.5and 5mg/kg)at 5min and 6h after ischemic produced a dose-dependent decrease in both neurologi-cal impairment and the histologically measured brain infarction volume in both transient and permanent rat ischemia models [19,20], which indicated its potential as an anti-stroke agent. This neuroprotective effect of PF during cerebral ischemia is related to the activation of adenosine A 1receptors [19]. The determination of PF in different biological matrices like serum [23,24], urine [25], plasma [26], and hippocampus [21,22]have been well established by using high performance liquid chromatography with ultra-violet spectroscopy detection (HPLC/UV).Although these methods present adequate linear-ity, precision and recovery, they show a series of limitations including lack of sensitivity which results in the lower limit of quanti?cation (LLOQ)ranged from 10ng/mLto 3.8? g/mL
1570-0232/$– see front matter ?2007Elsevier B.V . All rights reserved. doi:10.1016/j.jchromb.2007.06.022
S.-M. Xia et al. /J. Chromatogr. B 857(2007)32–39
33
Fig. 1. Chemical structure of PF (A)and geniposide (IS)(B).
and long chromatographic times (≥ 15min). Wang et al. [27] reported a speci?c and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS)(triple-quadrupole)method for the quantitative determination of PF in rat plasma. But to our knowledge, there were only HPLC/UVmethods described for quanti?cation in solid matrices (hippocampus)with a LLOQ at 1? g/mL,and this may not meet the requirements for tissue distribution studies if administered with a relatively low dose of PF.
This paper details a rapid (chromatographictime:4min), sensitive (LLOQ=2ng/mL)and reliable LC–MS/MS(iontrap) method for the determination of PF in rat brain using pro-tein precipitation with solid-phase extraction (SPE)for sample preparation, which was more sensitive compared with existing methods. This method was successfully applied to determine the brain concentration of PF following a single s.c. administration (10mg/kg)in rats.
2. Experimental
2.1. LC–MS/MSanalysis
2.1.1. Materials and reagents
PF and geniposide (internalstandard, IS) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing,China). HPLC-grade acetonitrile and methanol were obtained from Fisher Scienti?c (FairLawn, NJ, USA). Formic acid was from Tedia (Fair?eld, OH, USA). Ultrapure water was obtained from a Millipore (Milford,MA, USA) MilliQ apparatus.
2.1.2. Instrumentation
The HPLC–MS system consists of a Surveyor MS pump, a Surveyor auto-sampler and a Thermo Finnigan LTQ plus ion trap mass spectrometer equipped with electrospray ionization (ESI)source (ThermoElectron,San Jose, CA, USA). Xcalibur 1.4software was used for data acquisition and analysis (Thermo-Electron, San Jose, CA, USA). The data processing was carried out using Thermo Finnigan LCQquan 2.0data analysis program. 2.1.3. LC–MS/MSconditions
The LC separation was carried out on a Symmetry C18col-umn (150mm ×2.1mm i.d., 5? m; Waters, Milford, MA, USA) Table 1
LC–MS/MSmobile phase gradient program for the determination of PF and IS Time (min)Methanol%Water%(0.1%
formic acid)
Flow rate (? L/min) 05050200 2.55050200
2.65050300
3.55050300
3.65050200
4.05050200
with an Alltima RP18guard column (7.5mm ×4.6mm i.d., 5? m; Alltech, Deer?eld, IL, USA). The mobile phase consisted of methanol/waterwith 0.1%formic acid (50:50,v/v)at a ?ow rate of 200–300? L/min(Table 1), and the column temperature was maintained at 40? C. The mass spectrometer was operated in the positive ion detection mode. Nitrogen was used as the sheath gas (60L/min),auxiliary gas (30L/min),and sweep gas (1.46L/min).The I spray voltage was 4.50kV , and the spray current was 0.14? A. The voltage and the temperature of the cap-illary were 42V and 300? C, respectively, and the tube lens was 140V . The selected reaction monitoring (SRM)mode was used. PF and the IS were monitored at m /z transitions of 503→ 381 and 411→ 231, respectively. The optimized collision energy of 27V was chosen for PF and IS with argon as the collision gas.
2.1.4. Working solutions preparation
Stock solutions of PF and IS were prepared in water to result in ?nal concentrations of 256and 410? g/mL,respectively. They were stored at ? 20? C until use. A series working solutions of PF with concentrations of 24, 60, 120, 240, 600, 1200, 2400, 4800and 6000ng/mLwere obtained by further dilution of the stock solution with water. The working solution of IS with a concentration of 600ng/mLwas obtained in the same way. Cal-ibration standard samples were prepared at concentrations of 2, 5, 10, 20, 50, 100, 200and 500ng/mLby spiking 25? L of PF working solutions into 300? L blank brain homogenate, and “zero” standard sample was prepared by spiking 25? L of water. Each standard sample was also spiked with 25? L of IS working solution to give a ?nal concentration of 50ng/mL.In the same manner, quality control (QC)samples with IS at 50ng/mLand PF at low (5ng/mL),medium (50ng/mL),and high (400ng/mL) concentrations were prepared to evaluate accuracy and precision of this LC–MS/MSmethod.
2.1.5. Sample preparation procedures
For the determination of PF in brain, weighted whole brain (blankor samples) were thawed and then homogenized in precooled water (1g brain:2mL water). Each of blank brain homogenate (300? L) samples was spiked with 50? L water, and dosed brain homogenate (300? L) samples were spiked with 25? L water and 25? L IS (600ng/mL).Then, the samples were precipitated with 1200? L acetonitrile. After vortex-mixing for 60s and centrifuging at 20,000×g for 15min, the supernatant was dried under a stream of nitrogen at 45? C. And the residue was redissolved with 900? L water, then transferred into Sep-
34S.-M. Xia et al. /J. Chromatogr. B 857(2007)32–39
Pak Vac 1cc cartridges (100mg, 1mL; Waters, Milford, MA, USA). Each cartridge was pre-conditioned with 2×1mL of methanol followed by 2×1mL ultrapure water. After sample (totalredissolved sample) loaded, the cartridge was washed with 2×0.5mL of 15%methanol. Then, PF and IS were eluted with 0.5mL of 90%methanol. A 20? L aliquot of elute solvent was injected into the LC–MS/MSsystem for analysis.
2.2. LC–MS/MSmethod validation
Assays were validated according to the U.S. FDA guidance on bioanalytical method validation [28].
2.2.1. Speci?city and selectivity
The speci?city of the method was demonstrated by compar-ing chromatograms of blank brain homogenate samples (from six different drug-free rats), brain homogenate samples spiked with PF and IS, and brain homogenate samples obtained from rats administrated with PF.
2.2.2. Sensitivity
The LLOQ was determined during the evaluation of the linear range of calibration curve. LLOQ was de?ned as the lowest con-centration yielding precision with coef?cient of variation (CV) less than 20%and accuracy within 20%of the theoretical value for both intra-and inter-batch analysis.
2.2.3. Construction of calibration curve
The calibration curves for PF were constructed by plotting measured peak area ratios of analyte to IS against nominal con-centration of PF in brain homogenate using a 1/X weighted linear least-squares regression model. The minimally acceptable cor-relation coef?cient (R 2) for the calibration curve was 0.99or greater.
2.2.4. Precision and accuracy
In order to assess the intra-and inter-batch precision and accuracy of the assay, PF QC samples at low, medium, and high concentrations were prepared as described above. The intra-batch precision of the assay was assessed by calculating the CV for the analysis of QC samples in ?ve replicates, and inter-batch precision was determined by the analysis of QC samples in three batches. Accuracy was calculated by comparing the aver-aged measurements and the nominal values, and was expressed in percent. The criteria for acceptability of precision was that CV for each concentration level should not exceed ±15%with the exception of the low level, for which it should not exceed ±20%.Similarly, for accuracy, the averaged value should be ranged from within ±15%of the nominal concentration except for the low level within ±20%.
2.2.5. Recovery and matrix effect
Three sets of extraction methods were prepared to evaluate the recovery and the matrix effect (ME)[29], including both absolute and relative matrix effects.
Set 1. Analytes were added into ?ve different lots of blank brain homogenate and then extracted.
Set 2. Analytes were added into ?ve different lots of pre-extracted blank brain homogenate.
Set 3. Analytes were dissolved in matrix component-free eluent solvent.
The peak areas obtained in set 1were indicated as A , the corresponding peak areas in set 2as B , and in set 3as C . The recovery, absolute matrix effect, and relative matrix effect were calculated as follows:
Recovery(%)=
Mean A
Mean B
×100
Absolute ME(%)=
Mean B
Mean C
×100
Relative ME =(CVof B ) ? (CVof C )
2.2.6. Stability
The stability of PF was investigated. Freezing stability of PF in rat brain homogenate was assessed by analyzing QC sam-ples stored at ? 70? C for 45days. The in-autosampler (10? C) stability of PF in the elute solvent from SPE was evaluated by reinjecting QC samples 24h after the initial injection. The nor-malized concentrations of PF and IS in different QC levels were used as references to determine the stability of PF and IS in the experiments.
2.3. Pharmacokinetics of PF in the rat brain
Animal experiments were carried out according to the National Institutes of Health Guide for Care and Use of Labo-ratory Animals, and were approved by the Bioethics Committee of the Shanghai Institute of Materia Medica.
Male Sprague–Dawley rats (180–200g, n =78) were pur-chased from Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai,China). They were group housed under a 12h light/darkcycle in an environmentally con-trolled breeding room for 3days and fed with standard laboratory food and water ad libitum , and fasted overnight before the exper-iment. Rats were administered subcutaneously with PF in saline at 10mg/kgbody weight or the same volume of saline only for 0time point. Six rats for each time point were sacri?ced by cervical dislocation immediately at 0, 5, 10, 15, 20, 25, 30, 60, 90, 120, 180,and 300min (12timepoints) and the whole brain tissues samples were dissected, then stored in refrigerator at ? 70? C until use. Six rats without any treatment were sac-ri?ced and brains were collected in the same way for method development and validation.
2.4. Data analysis
Data were expressed as mean ±SD. The obtained PF brain concentrations were analyzed using noncompartmental model
S.-M. Xia et al. /J. Chromatogr. B 857(2007)32–3935
with DAS 2.0pharmacokinetic program (ChinesePharmacol-ogy Society) to obtain the relative pharmacokinetic parameters. 3. Results and discussion 3.1. Method development
3.1.1. Sample preparation
In this study, liquid–liquid extraction (LLE)by ethyl acetate and protein precipitation by acetonitrile and methanol were tested. The recoveries of PF and IS extracted by ethyl acetate were 54%and 23%,respectively, and the recovery of IS was lower than a half of PF. The signals of PF and IS in brain homogenate precipitated by acetonitrile and methanol were very low in the LC–MS/MSdetermination because of ion suppressing effect. Therefore, the SPE technique was developed. To get a higher recovery and avoid the clog-ging of the column, protein precipitation with acetonitrile was applied before loading. In previous test, the impurities were eluted out with the analytes when 50%acetonitrile
was
Fig. 2. Typical electrospray ionization mass spectra (A)and product ion mass spectra (B)of PF. The singly charged molecule plus sodium ion at m /z 503was selected as precursor ion for PF, and the possible cleavage reactions for the formation of the product ions 341+and 381+were presented, and the ion 381+was selected in SRM acquisition. used as elute solvent. It was shown that PF and IS could be eluted out by 0.5mL of 90%methanol completely instead of 0.5mL ×2of 15%methanol and both higher extraction recoveries and better selectivity were obtained with PF and IS.
3.1.2. LC–MS/MSmethod optimization
The electrospray ionization (ESI)source was chosen because better sensitivity, fragmentation and linearity were obtained for PF as compared to the atmospheric pressure ionization (APCI)source. Both the analyte and IS contain terpene and glucose moiety. Thus, they could be detected under either negative or positive electrospray ionization (ESI)conditions. However, it was found that positive ESI could offer higher sensitivity and better peak reproducibility as compared with negative ESI. In the full mass spectrum of PF and IS, molecular [M +Na]+ions (m /z 503for PF and m /z 411for IS) were the most intensive ions so they were chosen as precursor ions for the analytes. The ESI interface and mass spectrometer parameters were
opti-
Fig. 3. Typical electrospray ionization mass spectra (A)and product ion mass spectra (B)of IS. The singly charged molecule plus sodium ion at m /z 411was selected as precursor ion for IS, and the possible cleavage reactions for the formation of the product ions 203+, 217+, 231+and 249+were presented, and the ion 231+was selected in SRM acquisition.
36S.-M. Xia et al. /J. Chromatogr. B 857(2007)32–39
mized in order to obtain maximum sensitivity of [M +Na]+. In addition, methanol instead of acetonitrile was employed as mobile phase as it could provide more intensive response. It was also found that the organic additive formic acid in the mobile phase provided a higher peak response and better peak shape. In the product ion spectrum, several fragment ions were obtained, and the possible cleavage reactions for the formation of the product ions 341+and 381+of PF (m /z 503) are pre-sented in Fig. 2(B);and the possible cleavage reactions for the formation of the product ions 203+, 217+, 231+and 249+of IS (m /z 411) are presented in Fig. 3(B).And the ions 381+and 231+were selected for PF and IS in SRM acquisition, respec-tively, because they displayed best stability and intensity in all the sample analysis. The full scan and product ion spectrum of the analyte and IS are shown in Figs. 2and 3, respectively. In the developed method, the SRM mode was used to carry out the quantitative analysis, and it could detect the precur-sor ion and product ions at the same time and provided high selectivity and sensitivity. Therefore, the development of the
chromatographic system was focused on short retention times instead of chromatographic separation. Finally, methanol–water (50:50)was employed to separate and quantify the analyte in the presence of endogenous species and the matrix effect was low.
3.2. LC–MS/MSmethod validation
3.2.1. Speci?city and selectivity
Fig. 4represents chromatograms of PF and IS from rat brain homogenate after SPE. The typical retention times for PF and IS were 2.71and 2.64min, respectively. No interference of endoge-nous peaks were observed with PF or IS at their respective retention times in blank rat brain homogenate.
3.2.2. Sensitivity
The LLOQ of PF extracted from 300? L rat brain homogenate was found to be 2ng/mLafter injection of 20? L of the 500? L elute solvent from SPE. The intra and inter-batch
accuracies
Fig. 4. Representative SRM chromatograms for the determination of PF in rat brain homogenate samples by LC–MS/MSmethod:(A)a blank rat brain homogenate sample; (B)a blank rat brain homogenate sample spiked with PF at the LLOQ of 2ng/mLand IS (50ng/mL);and (C)a rat brain homogenate sample at 120min after s.c. administration of PF at dose of 10mg/kgand spiked with 50ng/mLIS. Peak I, IS; peak II, PF.
S.-M. Xia et al. /J. Chromatogr. B 857(2007)32–3937 Table 2
Precision and accuracy of the LC–MS/MSmethod for the determination of PF in rat brain homogenate (mean±SD, n =5)
Nominal concentration (ng/mL)Observed concentration (ng/mL)Accuracy (%)CV (%) Intra-batch
21.92±0.1895.99.0 55.08±0.39101.67.8 5049±3.198.56.3 400400±28100.17.0 Inter-batch
22.06±0.15102.97.2 55.07±0.06101.31.2 5050±0.999.71.8 400397±599.41.3
for brain homogenate samples were 95.9%and 102.9%,respec-tively, and precisions were 9.0%and 7.2%,which were below 20%at the LLOQ (Table 2). This limit was suf?cient to deter-mine the concentration–time pro?le of PF in rat brain following a single s.c. administration (10mg/kg)of PF.
3.2.3. Construction of calibration curve
The calibration curves for PF were linear in the con-centration range of 2–500ng/mLin rat brain homogenate. A typical equation of calibration curve was as follows: Y =0.107271+0.0505832X (R 2=0.9939), where Y is the peak area ratio of PF to IS, and X is the concentration of PF in brain homogenate sample. Concentrations of analyte in unknown sam-ples or quality control samples were subsequently determined by interpolation from these regressions.
3.2.4. Precision and accuracy
As shown in Table 2, at the concentrations of 5, 50, 400ng/mL,intra-and inter-batch accuracy ranged from 98.5%to 101.6%and 99.4%to 102.9%,respectively. The intra-and inter-day assay precisions (CV)ranged from 6.3%to 7.8%and 1.2% to 1.8%,respectively. These data suggested that this method was accurate and reproducible for the determination of PF in rat brain.
3.2.5. Extraction ef?ciency and matrix effect
The results (Table 3) showed that the recoveries of PF with protein precipitation following solid-phase extraction were 81.2%,80.9%and 82.3%at concentrations of 5, 50and 400ng/mL(n =5), respectively, while the mean recovery of the IS was 76.7%at the concentration used in the assay procedure (50ng/mL)(n =5).
A value of 100%ME indicated that the response in the mobile phase and in brain homogenate extracts was the same and no matrix effect was observed [29]. The absolute matrix effects of PF were 94.5%,82.6%and 95.4%at concentrations of 5, 50and 400ng/mL(n =5), respectively, while IS (50ng/mL)(n =5) was 90.4%.The relative matrix effects of PF were 8.55%,0.05% and 1.5%at concentrations of 5, 50and 400ng/mL(n =5), respectively, while IS (50ng/mL)(n =5) was 6.06%.Without signi?cant difference of the sets B and C, the matrix effect in the quantitative analysis could be ignored.
3.2.6. Stability
The stabilities of PF under various conditions are summa-rized in Table 4. PF stock solution (256? g/mLin water) was stable for at least 3months (datanot shown) at ? 20? C. The analyte was also shown to be stable in the elute solution of 90%methanol–water for at least 24h at 10? C and in rat brain homogenate at ? 70? C for at least 45days.
3.3. Application to pharmacokinetic study of PF
This LC–MS/MSmethod was applied to the quantitation of PF in rat brain. The mean brain concentration–time pro?le after s.c. administration of PF (10mg/kg)in the rats is shown in Fig. 5. The basic pharmacokinetic parameters of PF in the rats are summarized in Table 5.
The maximum brain concentration (C max ) of PF reached 153±26.7ng/mLat 20min after dosing, and biological half-life (t 1/2) was 271±109min. The area under brain concentration–time curve (AUC0-t ) was 11127±3091? g/L min and AUC 0-∞ was 19565±10802? g/Lmin. The mean residence time (MRT0-t ) was 96±6.5min and MRT 0-∞ was 338±152min. The total body clearance (TBCL) was 0.62±0.26L/min/kg,and the apparent volume of distribution (V d ) was 216±72L/kg.This suggests that PF was rapidly absorbed and penetrated into the brain
Table 3
Matrix effect (ME)and recovery of PF and IS in rat brain homogenate (n =5)
Nominal concentration (ng/mL)Recovery (%)Absolute ME (%)Relative ME (%) PF IS PF IS PF IS
581.2
76.7 94.5
90.4
8.55
6.06
5080.982.60.05 40082.395.41.50
38S.-M. Xia et al. /J. Chromatogr. B 857(2007)32–39 Table 4
Stability of PF at different experimental conditions (mean±SD, n =3)
Nominal concentration
(ng/mL)
Stability condition %remaining
545days storage at ? 70? C 102±10.2
24h in autosampler at 10? C 97.2±10.9
5045days storage at ? 70? C 104±12.9
24h in autosampler at 10? C 95.9±14.1
40045days storage at ? 70? C 93.7±9.2
24h in autosampler at 10? C 93.8±
10.6
Fig. 5. Brain concentration–time pro?le of PF in rat brain after a single s.c. administration of 10mg/kgboy weight of PF (mean±SD, n =6).
Table 5
Pharmacokinetic parameters of PF (10mg/kg)in rat brain after s.c. administra-tion to rats (mean±SD, n =6)
Parameters Value
t 1/2(min)271±109
C max (ng/mL)153±26.7
T max (min)20±0
AUC (0-t ) (? g/Lmin) 11127±3091
AUC (0-∞ ) (? g/Lmin) 19565±10802
TBCL (L/min/kg)0.62±0.26
V d (L/kg)216±72
MRT (0-t ) (min)96±6.5
MRT (0-∞ ) (min)338±152
with a relative low concentration after s.c. administra-tion.
4. Conclusions
A selective LC–MS/MSmethod was presented and validated for the determination of PF in rat brain. This is the ?rst report using LC–MS/MSto quantify PF in solid biological samples such as rat brain. The method was sensitive and rapid with a LLOQ of 2ng/mLusing 300? L rat brain homogenate. The short chromatographic time of 4min allowed high-throughput analy-sis and more than 300samples could be assayed daily. Indeed, the present method was successfully applied to determine the brain concentration of PF after s.c. administration of PF with a rel-atively very low concentration penetrated through blood–brain barrier. The basic pharmacokinetic parameters of PF in rat brain such as clearance, t 1/2, steady state volume of distribution, etc. were obtained, and which would be helpful to elucidate of its neuroprotection effects. The established LC–MS/MSmethod for studying the Pharmacokinetics of PF in rat brain includ-ing chromatographic conditions as well as sample preparation procedures is very useful for future study in PF related drug development. It also could facilitate, with minor modi?cation, the development and validation of LC–MS/MSanalytical assays to analyze PF in other biological matrixes such as urine, plasma and other tissue homogenates.
Acknowledgments
The authors wish to thank all members of Department of Pharmacology and Toxicology in Shanghai Institute of Tra-ditional Chinese Medicine for their technical assistance in animal experiment, especially Dr. Jia-Jun Xie and Zheng-Dong Qiao.
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范文四:中药药代动力学研究的难点和热点
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刘昌孝
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值得重视国外对人参的相关研究 , 为我们从 人参的合理应用去认识中药合理应用的重要性。 等 [41]对健康人体的研究表明 , 人参标准提取物 会改变依赖于 45026或 34代谢处置的药物的消除 , 在人肝微粒体研究人参皂苷 Rb1,Rb2, Rc,Rd,Rf,Rg1代谢 , 结果显示 , 这些成分会影响 CYP1(1A1,1A2 1B1)的活性 , 而对其他的 活性无影响 [42]。等 [43]发表的体内研究报告也表明人参提取物具有 CYP1抑制剂的作用 , 而且其作用强于对 2的抑制。作者用方法在大鼠研究了人参皂苷对 CYP1A2 CYP3A4 CYP2E1和 CYP2C19的影响 , 发现可以抑制 CYP1A2和 CYP3A4的活性 , 而不影响 CYP2E1和 CYP2C19的活性 [44]。 3.2.4 现代 科学和技术的应用 细胞转运药物的研究 由于小肠的 生理结构实用于药物吸收 , 中药大多以口服给药。药物吸收主要取决于药物能否透过肠道上皮细 胞以及与其发生生化反应 , 然后以活性形式转运到靶器官或部位 , 或与受体结合产生生物效应。 用 Caco-2细胞转运药物 , 研究表观透过系数与肠道吸收的关系 [45,46],进一步提高 Caco-2细 胞模型的科学性 , 进行模型标准化和规范化 , 在该模型上有效地对的作用底物、 抑制剂和调控剂进 行研究。已知许多药物属于 CYP P-gp的共同作用底物 , 这一技术比动物实验简便、快速、经 济。 Caco-2细胞模型已是研究中药转运、肠道吸收机制的有效工具。
用药物代谢酶研究中药的生物转化 药物代谢酶细胞色素 450系统与药物结构转化的相代谢以及结合反应的相代谢均有关。 近年来 , 随着细胞和分子生物 学技术的发展 , 可将人细胞的 450和葡糖醛酸转移酶的编码基因转染细胞 , 获得表达药物代谢的 重组酶 , 解决人肠道酶来源 , 为中药代谢研究提供模拟代谢环境。中药中含有多种糖苷类化合物 , 在口服以后 , 药物要经过胃酸、 各种消化酶和体内菌丛的作用或代谢 , 会引起这些糖苷类化学成分 的结构变化 , 进入人体以后还会在代谢酶的作用下发生进一步的代谢转化。 因此 , 真正起药效作用 的物质不一定是原来存在于中药或单味药的化学物质 , 而是经过多途径变化后的新的化学物质。
建立植物化学 -药效学 -药代动力学的三维研究体系
对于具有多成分的中药 , 从植物化学角度 , 建立中药的 化学指纹固然重要 , 也是研究药效学 (PD)和药代动力学 (PK)的基础 , 但药物进入体内后 , 其化学 成分将产生一系列变化 , 因此 , 研究三者间的关系就显得十分重要 , 而且也是发现药物作用物质基 础、 研究作用机制的关键所在。 为此 , 建立植物化学 -药效学 -药代动力学的三维研究体系 , 配合体 外 -体内实验 , 发现化学指纹 -药效指纹 -药代指纹的关系 , 鉴别指纹图谱的变化 , 研究有效部位 (成 分 ) 的指纹和药代指纹的一致性和差异性 , 进而建立新的三维模型 , 实现认识中药作用的整体观念 , 也能为现代科学阐明药物作用的物质基础提供新的研究方法。
高通量药物代谢研究系统 从药代研究的发展来看 ,20世纪 70年代 , 药代研究主要在实验动物和人体进行 , 以获得药物在体内转运的信息; 80年代药代动 力学研究与安全性评价结合 , 增加了毒代动力学 (TK)研究; 90年代将药代动力学与药物设计结
合 , 为新药发现产生了积极效果。利用酶学、受体学等实验技术 , 从药代数据中获得候选化合物 , 从高通量活性筛选获得活性化合物 , 但由于药代原因 , 难以在体内产生作用。近年来 , 发展高通量 药代动力学筛选系统已为研究者重视。但目前高通量药代动力学筛选系统尚无成熟的模型或模 式 , 一般是通过吸收速率和程度、代谢抑制和诱导以及代谢转化途径等。
3.2.5 中药方剂的药代研究
中药方剂是在漫长的历史过程中 , 通过中医师的大量临床实践 而产生的治疗方法 , 有记载的中药方剂有 6万多个 , 是国际上 “ 人种药理学 ” 的原始资料。应用现 代科学研究复方中有疗效的物质基础 , 有助于认识复方中各药的协同作用是产生药效和降低副作 用的基础。中药中的每一味药物本身就是一个复方 , 它的作用实际上是多成分、多靶点的协同作 用 , 对于复方更是如此。 复方的药理治疗作用 , 是各药的协同作用 , 其众多的活性成分可视为一 “ 活 性分子群 ” 。复方药物在制备过程中 , 化学成分发生变化会形成新的物质 (它可能是真正的活性成 分 ), 口服后 , 药物要经过胃酸、 各种消化酶和体内菌丛的作用或代谢 , 会引起化学成分的结构变化 , 进入人体以后还会在代谢酶的作用下发生进一步的代谢转化。要认识真正的有效成分的作用 , 对 于药代动力学研究来说 , 应设法和必须阐明活性成分的生成、吸收、分布、 代谢的全过程 , 包括对 制备过程、消化道运行过程、菌丛对化学物质的影响、体内转运代谢的一系列过程。近年来发 展的血清药理学研究是一种研究中药活性物质的新方法 , 直接研究用药后的血液中的有效成分 , 可能会使研究过程简化、发现有效成分的几率提高 , 对复方药物的活性物质研究更为适宜。
中药的药物代谢动力学 (简称中药药代动力学 ) 是 借助于动力学原理 , 研究中草药活性成分、组分、中药单方和复方体内吸收、分布、代谢和排泄 的动态变化规律及其体内时量 2时效关系 , 并用数学函数加以定量描述的一门边缘学科。 它是中 药药理学与药物代谢动力学相互结合、相互渗透而形成的。它借助于药代动力学的基本理论和 方法研究中草药 , 中草药药代动力学的研究也为药代动力学提出了新的课题。中药特别是中药方 剂十分复杂 , 其药代动力学的研究较通常的化学药物的药代动力学更为困难 , 中药药代动力学必 将大大促进药代动力学向更深层次的发展。与化学药物的药代动力学相比 , 中药药代动力学虽起 步较晚 , 但
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范文五:中药药代动力学研究的思与行--20161120
主要内容
一、中药药代动力学的定义
二、中药药代动力学告诉你什么
三、中药药代动力学研究的特殊性和难点
四、中药人体药代动力学研究方法
花 合片等中药人体 实例
五、双花百合片等中药人体PK实例
中药药代动力学的定义
一、中药药代动力学的定义 中药药代动力学 是借助于 动力学 原理,研究中药单方 制剂、复方制剂、中药制品、中药提取物中的活性成 分、毒性成分、或代表性成分等化学成分的体内 吸收、 分布 代谢 分布、代谢
和 排泄(ADME ) 的动态变化规律及其体 内 时 -量 或 时 -效 关系,并用数学模型提供药代动力学 参数的一门学科。
它是在西药药代动力学研究基础上发展起来的一个研
究领域 是 中药药理学 体内药物分析与药物代谢动
究领域,是 中药药理学、体内药物分析与药物代谢动 力学相互结合、相互渗透而形成的 。
中药成分复杂,成分微量,未知的东西太多,干扰因 素多,其药代动力学的研究 较通常的化学药物的药代
动力学更为困难,面临许多困难、问题和挑战 。
中 动力 意义 二、中药药代动力学研究目的和意义
中药作为一种重要的药用资源用于疾病的预防和治疗 已有几千年的历史
已有几千年的历史。
◆活性成分从体外如何吸收进入体内; 活性成分如何在体内分布 代谢和排泄; ◆活性成分如何在体内分布、代谢和排泄;
◆其动力学过程却知之甚少。。。
中药药代动力学研究
—— 正是解决这一问题的根本途径。
其研究目的和意义主要表现在以下几个
方面
方面:
1阐明和揭示中药作用的物质基础及作用机制 1、阐明和揭示中药作用的物质基础及作用机制 中药能够产生药理作用 产生疗效 其作用来自
中药能够产生药理作用、产生疗效,其作用来自: 本身存在的化学物质?
代谢产物?
在体的化学物质?
必定存在一定的物质基础及作用机制。
研究
? 中药体内过程动态变化规律
阐明 ? 药效物质基础及作用机制
提供 ? 中药传统理论的科学阐释
药动-药效相结合
研究双黄连
口服液抗菌作用 药效物质基础
思路— 药效物质基础研究 思路 药动药效相结合
大鼠灌胃给药
不同时间点取血
指纹图谱 中的各共有特征 抑菌活性试验的 峰(所对应化合物)的 体 内药动学评价
药
效动力学评价
统计分析 识别抗菌作用的有效 统计分析,识别抗菌作用的有效 成分, 揭示抗菌作用物质基础
结果 :
A
C
B
D
E
A ◆色谱图
80100120B C 152.0 2.5
4060C (n g /m l )
D E
0.5 1.0 1.5 L n I
0200
3
6
9
121518
21
24
Time (h)
0.0
3
6
9
1215
18
21
24
Time (h)
() ◆血药浓度 -时间曲线 ◆抑菌半径 -时间曲线
2为中药复方组方原理提供科学依据 2、为中药复方组方原理提供科学依据
中医药学 独特的理论体系、 精髓
药物-药物相互作用 -----
基于细胞色素P450酶代谢
药物相互作用的发生机制 X F Zh t l Id tifi ti f d th t i t t ith h b i d
X.F Zhou. et al. Identification of drugs that interact with herbs in drug development. Drug DiscoveryToday . 2007, 12: 664-673
人体主要的I II相代谢酶 人体主要的I,II相代谢酶
示例之一 诱导 示例之一:诱导
Rifampicin : a potent inducer of several
cytochrome P450 (CYP) enzymes and transporters.
Rifampicin
Pravastatin
C Kyrklund, et al. Effect of rifampicin on pravastatin pharmacokinetics in healthy subjects. Br J ClinPharmacol . 2004, 57:181-7(利福平,普伐他丁)
示例之二 抑制 示例之二:抑制
Itraconazole:a potent inhibitor of
cytochrome P450 (CYP)3A4
Itraconazole
Brotizolam
T. Osanai, et al. Effect of itraconazoleon the pharmacokinetics and
pharmacodynamics of a single oral dose of brotizolam. Br J ClinPharmacol .
示例之三:Warfarin 与传统草药
No significant interaction
X.J Jiang, et al. Effect of ginkgo(银杏) and ginger (生姜) on the
pharmacokinetics and pharmacodynamics of warfarin
(华法令) in healthy y y subjects. Br J ClinPharmacol . 2005, 59:425-32
示例之四:甘草(Liquorice)
Liquorice (Radix Glycyrrhizae, Liquiritiaeradix) is the
root of Glycyrrhizauralensis Fisch. or Glycyrrhizay y y y glabra L. or Glycyrrhizain ? ata Bat., Leguminosae.
Liquorice appears in approximately 60%of traditional Chinese medicine (TCM) prescriptions.
甘草 是中药具有代表性的 “ 使药 ” ,药物 -药物 相互作用 ,甘草酸在治疗疾病中
与其它中药配对具有协同作用。
As a unique “guide drug”
to enhance the effectiveness of other ingredientsg ;
to reduce toxicity;
to improve flavorin almost half of Chinese herbal
formulas. Herb–drug and herb–herb interactions
Herb drug and herbherb interactions Xing, P ., Wu, W ., Du, P ., Han, F., Chen, Y ., 2011. Effects of brucine combined with
g glycyrrhetinic acid or liquiritin on rat hepatic cytochrome P450activities in vivo. Acta Pharm. Sin. 46, 573–580.
The principal CYP isoforms regulated by active components of liquorice.
Xiaoying Wang, Han Zhang, XiumeiGao,et al. Journal of Ethnopharmacology, 150 (2013) 781–790. (甘草酸,甘草次酸,光甘草定 glabridin ,甘草素 liquiritin )
药有个性之特长,方有合群之妙用
。
中药各有效成分通过相同或不同的作用机制 , 或
协同或拮抗而对机体产生疗效。
从整体观点出发 研究中药的药代动力学特征,
将可以为组方配伍原理提供科学依据 。
Study on the pharmacokinetic profiles of corynoline 示例之五:双花百合片 (SBT)
y p p y and its potential interaction in traditional Chinese medicine formula Shuanghua Baihe tablets (SBT)in rats by LC-MS/MS
苦地丁 紫堇灵
黄连 小檗碱
试验结果:
试 与紫堇灵组相比,灌胃给予含有同
等剂量紫堇灵的 SBT 时,紫堇灵的
与紫堇灵组相比,紫堇灵和小檗碱 t 1/2约 延长 3倍 , C max 和 AUC 0-12分别
组中,紫堇灵的 C max 和 AUC 0-12分别 增加了 11.1倍和 5.0倍。 增加了 46.5%和 34.2%。
0-2.5 h放大
静注
紫堇灵
结果表明
结果表明:
小檗碱可 显著增加 紫堇灵在大鼠体内 血浆暴露量。 通过CYP450代谢酶产生药物-药物相互作用?
警示:两者合用时存在潜在毒性?
While, it’s interesting that…
◆SBT 组 中紫堇灵暴露量的增加量 小于 紫堇灵与小檗碱 合用组的增加量
合用组的增加量;
表明:小檗碱可以显著改变紫堇灵在大鼠体内的药动学 特征,同时 SBT中其他成分对紫堇灵药动学行为的影响也 是不可忽略的 。
◆体现了中药复方中各组分间增效减毒配伍机制的妙处。
Ruijian Liu, Li Ding*, et al . Study on the pharmacokinetic profiles of corynoline and its potential interaction in traditional Chinese medicine formula Shuanghua Baihe tablets (SBT)in rats by LC-MS/MS.DOI: 10.1016/j.jpba.2015.09.009.
为设计及优选中药给药方案提供基础和依据 3、为设计及优选中药给药方案提供基础和依据 长期以来 中药多以经验给药或辨证用药 属 长期以来,中药多以经验给药或辨证用药,属 于个人经验用药, 多数情况缺乏药代动力学研 究资料的支持 。
通过 中药药代动力学研究 可得知中药中有效成 分在体内吸收 分布 代谢 排泄等过程的动 分在体内吸收、分布、代谢、排泄等过程的动 态变化规律,获得其药动学参数,从而 可以科
学地拟定给药方式、给药剂量、给药间隔及确 定疗程,从而可提高其临床整体治疗水平 。 ,
示例:黄杨宁片
宁片
◆通过黄杨宁的药动学试验,得到其在人体 内的药动学特征;
◆选择合理的临床给药方式, 避免蓄积和毒 性的产生 。
44、 为选择合理给药途径及合适剂型提供依据 药物制剂开发
给药途径和剂型
◆较高的稳定的药效 体外质量标准 ◆体内药代研究
只有通过体内药代研究,才能提供最直接的给
药途径或剂型选择的依据
,才能真正达到可 控、高效、速效、低毒的目的。
5、推动中药的现代化和国际化 推 中药 代
◆提高药物的临
床控制程度 推动:
◆更好的指导临 床用药
中药的现代化及 国际化
揭示:
◆药效物质基础 提供依据 ◆方剂组方原理 ◆配伍规律
提供依据:◆初步筛选 中药药代动 ◆剂型设计 ◆质量评估
◆给药方案的制订
力学研究
中
动力 性
点
三、中药药代动力学研究的特殊性和难点 由于中药化学成分的复杂性、中药药效的多效性和中
医临床应用的辨证施治及复方配伍的中医特色等特 点,使得 中药药动学研究有别于化学药品的药动学研 究 , 而有其特殊性和复杂性。
中药成分及其体内过程的复杂性
1、中药成分及其体内过程的复杂性
其成分的 复杂性 ,表现在即使一味中药也含有多种成分, 复方成分更为复杂,且可能 还有配伍后的成分变化及各 种干扰因素存在 。
中药是一个复杂的体系,不论是单味中药还是中药复方, 均为含有大量化学物质的巨大复方,而且每一成分含量 极微 。这些客观存在的问题构成其药效学和药代动力学 研究的难点,其药效可能是 其中多种物质相互作用产生 难以完整地分析中药作用的物质基础。 的综合结果, 难以完整地分析中药作用的物质基础
研究方法上的难度
2、研究方法上的难度
(1)多数中药发挥药效作用的物质基础尚不确定 使得药代动力学 (1)多数中药发挥药效作用的物质基础尚不确定,使得药代动力学 研究的 目标物确定困难 ;
(2)目标化学成分在复方中的 含量低 ,其血、尿和组织中的浓度更 低,以至于难以检测;
(3)多种成分吸收入血,这些 与待测物同时吸收入血的成分及其代 这增加了生物样品测定的难度
谢物均可能干扰测定 ,这增加了生物样品测定的难度 。
(4)因中药材的特殊性,中药制剂中所含成分的含量往往不确定, 只规定含量不低于某个值, 而药动学研究是讲究剂量的,剂量不同药 动学特征有可能不同 , 这也给药代动力学研究增加了难度。
中药药动学与化药药动学相比较主要难点如下:
化 有效成分明确、单一,生 物样品(血浆 尿液等) 测定成分单一、
干扰较少 对方
临床方案设计较为
简单 般 次临
相 对
药 物样品(血浆、尿液等)
中含量较大。
干扰较少,对方
法的灵敏度要求
简单,一般一次临 床试验即可
较低 相对较低。
中 药 有效成分往往 不明确、复
杂 ,许多有效成分含量低、
测定成分 较多、
干扰较多 ,对仪
临床方案设计 较为
复杂 , 需多次临床
相 对 吸收差,导致生物样品中
浓度极低 。
器、分析方法提
出了 极高要求 。
试验 (多个成分的
半衰期差别较大时) 较高
中药的整体观难以认识
3、中药的整体观难以认识
()中药的药效往往是 多种化学成分相 作用 (1)中药的药效往往是 多种化学成分相互作用 所产 生的综合效果,其复杂性导致药动学 测定指标选择 困难 ,到底选择哪些指标能代表该中药的药效往往 是一个难以定论的问题。
是 个难以定论的问题
()中药药动学研究必须考虑 中药配伍的真 内 (2)中药药动学研究必须考虑 中药配伍的真正内 涵 ,辨证论治, 君臣佐使等原则 是中医用药的精 髓,因而 整体观思想 应该在中药药动学研究中体 现。因此, 如何从整体观研究中药药代动力学 是一 现 因此, 如何从 体观研究中药药代动力学 是 挑战。
四 中药人体药动学研究方法 四、中药人体药动学研究方法 1、药物浓度法
药物浓度法
2、生物效应法
常用方法 3、中药药代动力学研究的新理论、新方法
药物浓度法简介
1、药物浓度法简介
得到 该法通过测定在给药后不同时间 的血药浓度 ,得到一 组血药浓度-时间数据,然后通过房室分析或非房室分
方 学 算 学 阐 析方法或生理药动学模型, 计算药动学参数,从而阐 明活性成分在体内的代谢过程 。
此法适用于活性成分明确的中药或中药制剂的药动学 研究,也 是评价药代动力学特征最常用最准确的一种 该法对 新药 发 中药作 机制的阐 临 方法 。该法对于新药开发、中药作用机制的阐明及临 床合理用药具有重要的意义。
被选用于药动学研究的指标性成分应该具备以下特征:
能 代表复方的主要药效 ;
是药物的 质控指标 ;
在靶器官内有较高的分布;
其 体内浓度 与 复方药效 在时间上具有 密切联系 ; 能被 吸收入血 ;
具有 可检测性。
相关分析技术
2 相关分析技术
中药制剂中成分多种多样 且结构较为复杂 含量 中药制剂中成分多种多样,且结构较为复杂,含量一 般都很低。 进人体内后, 原药及代谢物在血浆、胆 液等 液 含 往往 汁、尿液等体液中的含量往往 只有 ng 或 pg 级水平; 一般的方法如 HPLC 法、 GC 法等难以检测,且前处理 操作繁琐 从基质中分离的难度也很大 操作繁琐,从基质中分离的难度也很大;
液质联用 (LC/MS)是中药药动
学研究的主要分析仪器 学研究的主要分析仪器。
中药人体药代动力学试验流程:
(1)文献调研;
选择具有代表 临床试验 (2)大鼠预试验考
察入血尿排成分
性的有效成分 方案设计 人体预试验确定:
临床试验 分析方法
(1)采血时间点;
(2)血药浓度范围
方案修订 方案设计 方法学确证 临床试验
分析测定生
物样品 数据统计分析
试验报告
3 临床药动学试验需考虑的问题
采样点的确定对药代动力学研究结果具有重大
的影响
的影响。 在进行中药复方制剂的药动学的临床试验时, 中药成分复杂 采样点的设计要兼 多个有 因中药成分复杂,采样点的设计要兼顾多个有 效成分,必要时需进行多个临床试验。
临床药代动力学研究技术指导原则中规定:
服药前采空白血样品,一个完整的血药浓度-时间曲线, 应包括药物各时相的采样点,即采样点应包括给药后的 吸收相、平衡相(峰浓度)、分布相和消除相 。
一般在吸收分布相至少需要 2~3个采样点,平衡相至少 个采样点 消除相至少需要 个采样点 一般不少 需要 3个采样点,消除相至少需要 6个采样点。一般不少 于 11个采样点。
应有 3~5个消除半衰期 的时间,或采样持续到血药浓度
为 C
max
的 1/10~1/20。
若中药复方中各个有效成分的半衰期相近时,可以通过 一个临床试验来完成
一个临床试验来完成。
(一)药物及研究背景
()药物及研究背景
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