反相高效液相色谱法测定茚地那韦2种制剂在健康人体的相对生物利用度(英文)_图文

1、E-mail:xyylcshyyxx.COITI .ca http:/zgxyylczz.periodicals.corn.ca。841Article ID1007-7669(200511-0841-04Relative bioavailability of two formulations of indinavir in Chinese healthy volunteers by reversed phase high performance liquid chromatographyQIAO Hailing,GAO Na,GUO Yuzhong,ZHANG Lirong,ZHANG Qit

2、ang,JIA Lin-jing(Institute of Clinical Pharmacology,Zhengzhou University,Zhengzhou HE-NAN450052,ChinaKEY WORDSindinavir;chromatography,highpressure liquid;pharmacokinetics;bioequivalenceCLC numberR969.1;R978.7Document codeAABSTRACTAIM:To establish a method to de.termine the concentration of indinavi

3、r in human plasma and study indinavir bioavailability in Chinese healthy people.METHODS:In a random two-period CROSSover study,18healthy male volunteers received a sin-gle dose of indinavir capsules800mg of two formula-tions respectively.A sensitive and specific reversed phase HPLC method was develo

4、ped to quantitate plaslna levels of indinavir.,11le drug was extracted from plasITIa with aeetonitrile.Analysis was performed on a Hypersil C18colunlnwitl a mobile phase of acetonitrile: 0.01toolL“phosphate buffer(pH5.5(43:57. The UV detector was set at210nm.The standard curve covered the concentrat

5、ion ranged from0.03to16.38mgL.RESULTS:r11le concentrationtime curves of reference and testedformulations both fitted to a onecompartment open model.r11le main pharma-cokinetic parameters of tested and reference formula-tions were(10.6±s2.4mgL“and(9.8±2.2 mgL“for c一,(0。71±0.19h and(0.8

6、±0.3hfor一,(1.30±0.24hand(1.31±0.23hfor t1.,(23±6mghL“and(22±5nag-d-i纪hLfor AUCo_10,(24±6mghL。1and(22±5mghL。for AUCo.。,respectively.Two one-side t test and variance analysis were performed in bioequivalent assessment.No statistically significant differencewas found

7、in AUCo.10,AUCo。andCmaxvalues between the tested and reference formulations.CONCLUSION: The reversed phase HPLC is a reliable method to deter-mine the concentration of indinavir in human plasma and出e two formulations of indinavir are bioequivalent.Indinavir is a member of protease ihhibitors used in

8、 the treatment of human immunodeficiency virus(HIV infection1|.It can efficiently inhibit the replication of HIV virus by blocking the activity of its protease enZylTle.HIV protease acts like a“molecular scissorstocleave the gag and gag/pol polyproteins into smaller functional proteins.thereby allow

9、ing the virion to mature.Although the precise mechanism is unknown yet, indinavir appears to prevent the virions from maturationto the infectious.Thepharmacokinetics of indinavir in westerners2has been reported,but there is no report about it on Chinese people.Th、e regimen of indinavir may not corn-

10、pletelysuitable for Chinese because of racial difference between westerners and Chinese.To have a suitable regimen in clinic,we studied the pharmacokinetics of indinavir in Chinese.Drug and chemicals The testedformulation:indinavir sulfate capsules were lot No020701from Xiamen Maike Pharmareeutical

11、Co Ltd(China.The reference Received date2005-02-28Accepted date2005-09-05BiographyQiao Hai-ling(1963一,male,native place Shandong Laiyang,doctor,professor,be engaged in pharmacokin-tics and clinic pharmacy.ConnectorQiao Hailing.Phn/Fax:86-371-6665-8190.Email:QiaohlZZU.edu.ca842Email:xyylcshyyxx.coin

12、http:/zgxyylczz.periodicals.corn.c:nformulation:indinavir sulfate capsules were lot No C1331from Merck&Co Inc.The reference substance of indinavir sulfate(content is99.8%w88generously donated by Xiamen Maike Pharmaceutical Ltd.Acetoni-trile of HPLC grade and double distilled water were used.All

13、other chemical reagents were of analytical grade.Chromatographic conditions A reversed phase HPLC method was developed to quantitate plasma levels of indinavir.The apparatus used was Agilent HPLC system(America,which is consist of an automatic injector,a computerized system controller and a UV detec

14、tor.Chromatographic separation was performed using a Hypersil C18(5pan,4.6rain x200millHPLC column. The mobilephase consistedof43%acetonitrile and 57%0.01toolLphosphate buffer(pH5.5.The flow rate was1mLmin,and the detector writs set at 210nm.Peak area of the drug was measured and takes as quantitati

15、on.Sample preparation To plasma0.5mL in a10mL test tube,0.05molL。phosphate buffer(pH9.0 300I LL was added and extracted with acetonitrite3mL vortexed for3min and centrifuged at1642x g for 15min.2mL separated organic layer was evaporated to dryness under a stream of nitrogen at50oC.The resi-due was r

16、esolved with solvent(acetonitrite:water2 43:57100止and aliquot20斗L was injected to HPLC.Calibration procedure Indinavir standard solutions was added to blank plasma0.5mL to obtain indinavir standard concentrations from0.03to16.38mgL. All calibration samples werc taken through the extraction procedure

17、.The calibration calve was plotted using peak area of indinavir versus various indinavir concen-trations.Final sample concentration was calculated by determining the peak area comparing the area with the standard curve,obtained after analysis of calibration samples.1:懈on and recovery The inter-day a

18、nd intra-day variability of the assay was determined by repeatedanal-ysis of quality control samples at concentrations0.06, 1.02,8.19mgL一1on the same dayand5different days,respectively./n vivo study design Eighteen healthy aduh male vol-unteers between21to25a,(23.0±1.0a and weigh from55to75kg,(

19、63±6kg participated in the study. On the,basis of medical history,clinical examinations and laboratory tests including hematology,blood bio-chemistry,and urine analysis,no subject had a history or evidence of hepatic,renal,gastrointestinal or hematological deviations,or any acute or chronic dis

20、eases or drug allergy.The subjects were instructed to abstain from taking any medication for at least2wk prior to and during the study period.No milk or dairy products were served during the study.Informed consents were ob-tained from the subjects after nature and purpose of the study was explained.

21、The protocol was approved by the Ethics committee and used the comrentional,two-way, split group crossover study witI nine subjects in each of group.In the first trail period,after an overnight fast, subjects were given a single dose of800mg capsules of either reference or tested formulation in a ra

22、ndomized fashion with200mL of water.Food and drinks、佗re not allowed until4h after ingestion of the capsules.Lunch and dinner were served at4and12h after dosing to all volunteers.Approximately4mL blood samples were drawn into heparinized tubes through an indwelling cannula before(0hand at0.17,0。33,0.

23、67,1.0,1.5, 2,3,4,5,6,8,10h after dosing.The blood samples were centrifuged at1207×g for15min,plasma was separatedand kept frozen at一20.After7d the study was repeated in the sanle manner to complete the crossover design.Data analysis and statistical methods Estimation and calculation of pharmac

24、okinetic parameters were per-formed using3P97software.The peal【plasma concentration(c。and the time needed to reach this maxi-mum concentration(t。were directly obtained from observed values.The relative bioavailability was calculated as foUowed:R。=面AU啄Co_二loT x100%F0.。=面AU诼Co_=T×100砺(T:tested fo

25、rmulation;R:reference formulation Cmax,AUCo.10,and AUCo_。were logarithmically transformed.bioequivalent anlysis and90%confidence intervals for the mean parameters were caculated by us-ingtwo one-side t-test and val'iance analysis.E-mail:xyylcshyyxx.corn .cil 843Quality control of皿LCassayIhe rete

26、ntion timeof indinavir was6.8min.111e peak was sharp,well separated and not interfered by the other substance in plasma(Fi91.ne calibration curve was described asfollows:c=0.005IA-0.0749(r=0.9999,Where A means the chromatographic peak area and c means the concentration(mgL“of indinavir.r11le lowest

27、limit concentration for indinavir was set at0.02mgL“(S/ N>3.nerecoveryand variation of determination were shown in Tab】.0510*r/rain t/rain t/rainFig1Ciwomatograms of indinavir A:Blank plasma; B:Blank plasma+indinavir;C:Plasma after administration of indinavir;1:indinavirTab1Recovery and variation

28、 of indinavir in plasma(露= 5Pharmacokinetic parameters The mean concentra-tiontime profile for the two formulations was shown in Fig2.The concentrationtime curves oftwo formula-tions were in accordance with a one-compartment mod-e1.At about0.8h after administration.it reached maximum concentration(c

29、。10mgL。1.During the elimination phase indinavir was rapidly eliminated with a half-life of approximately1.3h.The numerical val-ues of the pharmacokinetie parameters were listed in Tab2.The relative bioavailability(凡-10of the tested indinavir was(106±20%.Evaluation of bioequivalence There was no

30、 significant difference in c。,AUCo.10and AUCo.。between the two formulations except tm.The results showed that the two preparations were bioequivalent.Fig2Mean plasma concentrationlime curve of indinvavir after oral indinavir capusules800mg I111=18Tab2Pharmacokinetic parameters in normal volunteers a

31、f-ter oral administration of indimvir800rag capsules(/it=18Experimantal dataParameters Reference Testedformulation formulationReferencedata2+c。A。/mgL一19.8±2.210.6±2.4n5.7±1.6:。/h0.8±o.30.71±o.1981.9±o.5扣/“1.31-sO.231.30±o.24。1.0扣/“o.29:tO.24o.23士o.288",/L80

32、77;2481±30nC/:F/Lh一1AUCo.10/mghL一1AUCo一。/mghL一1凡.10/%Fo.。,%43土1222土522士543-s13823±6824±6106±20106±2055±2917±5Indinavir at800mg in healthy volunteers.A Determined value 8P>0.05Reference formulationSeveral hishperformance liquid chromatography (HPLCmethods have be

33、en reported to determine the indinavir levels in human plasma341.Solid.phase ex. traction or liquid chromatographymass spectroscopy (LCMSwere used in these methods.The assays were either timeconsuming or expensive because of the instruments,which were not available to many groups. Therefore,we tried

34、 to develop a rapid and less cosily assay that could be performed in any laboratory witll adequate HPLC instrumentation.Linear relationship was found when the peak area of indinavir were plotted versus the indinavir plasma concentrations(0.03to 16.38mgL“.This was a study about pharmacokinetics and b

35、ioavailability of indinavir in Chinese.It demonstrated that tlle concentrationtime curves of indinavir fitted to a onecompartment open model.For bioequivalence evaluation.the results indicated that there was no staffs.844Email:xyylcshyyxx.COrn hRp:/.an tieal difference in Cmax,AUCot and AUCo.。values

36、 be-tween two formulations.However,a statistically signif-icant difference was observed between the fm values of the two products.although the slight difference may not be therapeutically significant or important.HSU and his colleagues。had studied pharmaeokinetic of indinavir in healthy white people

37、.The data were shown in Tab2.Compared with westerners,Chinese have a higher value in cm“,A UC and a lower value in f.Higher Cmax has been associated with an in-creased prevalence of indinavirrelated nephrotoxicitv5|.Now.indinavir is usually administered with the sanle regimen as westerners in Chines

38、e patients,which may cause higher rate of adverse reactions and the lower compliance in clinic.It is reposed1that the protease inhibitors were mainly biotransformed by CYP 3A4.It should be studied in the future that whether the pharmacokinetic differences between Chinese and westemer were resuited f

39、rom CYP3A4or not.1KAREN TA.ne pmtease inhibitiorsJ.Prim Care Update OB/GYNS,2001,8(2:59-64.2HSU A,GRANNEMAN GR,CAO G,et以.Pharmacokinetic inter-action between ritonavir and indinavir in healthy volunteersJ.An-timlemb Agents Chemother,1998,42(11:2784-2791.3SAAH AJ,WINCHELL GA,NESsLY MN,et以.Pharmacokineticprofile and tolerability of indinavir-ritonavir combinations in healthy volunteersJ.Anfimiemb Agents Chemother,2001,45(10: 2710-2715.4MARZOLINI C,TELENTI A,BUCHN T,et a/,Simultaneous de-termination of the HIV pmtease inhibitors indinavir,amprenavir,sa-quinavir,ritonavir,nelf