Research ArticleThe Effect of Tanreqing Injection on the Pharmacokinetics ofSirolimus in Rats

Feng Zhang Liang Sun Jianxiu Zhai Tianyi Xia Wei Jiang Mingming LiShouhong Gao Xia TaoWansheng Chen and Yifeng Chai

Department of Pharmacy Changzheng Hospital Second Military Medical University Shanghai 200003 China

Correspondence should be addressed to Wansheng Chen chenwanshengsmmueducn and Yifeng Chai yfchaismmueducn

Received 16 October 2018 Revised 18 December 2018 Accepted 20 December 2018 Published 10 March 2019

Academic Editor Wan-Liang Lu

Copyright copy 2019 Feng Zhang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

To evaluate the effect of Tanreqing injection on the pharmacokinetics of sirolimus in rats a high performance liquidchromatography tandem mass spectrometry method was developed for sirolimus assay in whole blood Calibration curve ofsirolimus was acquired over a concentration ranging from 25 to 100ngmL with r2= 09955 The matrix effects and extractionrecoveries of sirolimus ranged from 144 to 152 and from 80 to 96 respectively The inter- and intraday relative standarddeviations were both lt10 The stability investigation showed that the blood samples were stable for 30-day-storage at minus20∘Cfor 8 h storage at room temperature for 24 h storage in the auto-sampler at 4∘C and for three freeze-thaw cycle process Thepharmacokinetic results demonstrated that the 119862max AUC and AUMC of sirolimus in rats (75mgkg ig) were increased afterbeincoadministration with Tanreqing Injection at 25 50 and 75mLkg (iv) respectively or at 5min 2 h and 4 h (50mLkgiv) after SRL dosing respectively For the first time the results proved the herb-drug interaction between Tanreqing Injection andsirolimus and accordingly suggested avoiding concurrent reception of those two drugs for patients

1 Introduction

For sirolimus (SRL rapamycin) a common immunosuppres-sant its efficacy came from inhibition on the serinethreoninekinase mammalian target (mTOR) by forming a complexwith the immunophilin FK-506-binding protein (FKBP)-12[1] SRL also exhibits immunosuppressive property byblocking activity of cell cycle progression at the junctureof G1 and S phase and thus suppressing cytokine-mediatedT cell proliferation [2] SRL is metabolized extensively bycytochrome P450 3A (CYP3A) and is a substrate of thedrug efflux pump P-glycoprotein Metabolites contribute tolt10of immunosuppressive activity of the parent compound[3] As an effective immunosuppressant SRL has resulted inless nephrotoxicity compared with the calcineurin inhibitors(CNIs) like cyclosporine A (CsA) and tacrolimus (TAC) [4]However with narrow therapeutic window (recommendedtrough concentration range of 5ndash15 ngmL) the large intra-and interpatient variability in drug exposure heavily threat-ened the safety of patients in SRL treatmentWhat ismore theapplication of SRLwas also limited by its low and variable oral

bioavailability (about 14) [4] The routine therapeutic drugmonitoring (TDM) of SRL blood concentration is essentialfor patients to individualize the drug dose and therebyprevent drug toxicity or organ rejection and subsequent drugdiscontinuation

Tanreqing Injection (TRQ) a classical TCM formulationis produced from five herb raw material Scutellariae RadixFel selenarcti Cornu naemorhedi Lonicerae japonicae Flosand Forsythiae fructus InChina [5 6] it is commonly used totreat acute upper respiratory tract infection and early stage ofpneumonia in clinical practice Our previous chemical profileandmetabolism profile analyses have revealed that TRQ con-tains flavones from Scutellariae Radix and Forsythiae fructuscholic acids from Fel selenarcti amino acids from Cornunaemorhedi and phenolic acids from Lonicerae japonicaeFlos as its major constituents as well as the latest reports inother labtory [7ndash9]

Lately the safety of SRL application was concerned incombined drug administration treatment in which casedrugherb-drug interactions (DDIHDI) may result in someadverse effects (AEs) For example an article described

HindawiBioMed Research InternationalVolume 2019 Article ID 1854323 7 pageshttpsdoiorg10115520191854323

2 BioMed Research International

O

O

O

O

N

Sirolimus Cyclosporin D

O

O

O

O

OH

OH

OHH

H

H

H

H

H

H

HN

HO

O

OO

OO

O

O

O O

O

O

O

O

N

NN

N

NN

NN

N

N

Figure 1 Chemical structures of sirolimus and cyclosporin D

that a drug-drug interaction between azole antifungals ormacrolide antibiotics and SRL in five patients led to anincrease of SRL exposure [10] while another reported adecrease of SRL exposure in the presence of rifampin andphenytoin concomitantly[11] In 2011 a sudden rise of SRLlevel (235 ngmL 100) was found in a renal transplantationpatient in our hospital who was treated with SRL (2mgdayfor 10 years) and Cellcept (mycophenolate mofetil 750mgtwice a day) and then was given TRQ (30mLday for twodays) for pneumonia Under excessive exposure of SRL thepatient suffered from thrombocytopenia and leukopenia dueto high trough concentration of SRL No pharmacokineticDDI was recognized for Cellcept and SRL [12] Afterwardswith withdrawal of TRQ and constant prescription of SRLand Cellcept the trough SRL concentration was decreasedto 154 ngmL and the previous side effects for the patientdisappeared accordingly This finding not only emphasizedthe need for a close TDMof SRL concentrations in transplantpatients but also indicated a suspiciousHDI between SRL andTRQ

Immunoassay was reported as a reliable and convenientmethod for SRL blood concentration detection However itwas also known that immunoassay might lead to overestima-tion of SRL concentration as a result of cross-reactivity withmetabolites [13] Therefore several high performance liquidchromatography coupled to tandem mass spectrometry (LC-MSMS) methods have been developed for the quantitationof SRL or multiple immunosuppressants concentrations inwhole blood with improved specificity and sensitivity [14ndash18]

As above mentioned no literatures were reported aboutthe herb-drug interactions between SRL and TRQ In orderto investigate the potential HDI between SRL and TRQ a LC-MSMS method was established to evaluate the effect on thepharmacokinetics of SRL in rats after its co-administrationwithTRQ (1) on three different doses and (2) at three differenttimes

2 Materials and Methods

21 Chemicals andReagents SRL and cyclosporinD (internalstandard IS) (Figure 1) with analytical reference standards(purity ge 990)were purchased from Sigma-Aldrich Co (StLouis MO USA) SRL tablets (1mg per tablet) were obtainedfrom Wyeth Company (USA) TRQ injections (10mL pervial) were provided from Shanghai Kaibao PharmaceuticalCo Ltd (China) MS-grade acetonitrile and methanol werepurchased from Merck (Darmstadt Germany) Deionizedwater was prepared using the Milli-Q system (MilliporeBedford MA USA) and was used for all preparations Otherreagents were of analytical grade

Stock solutions (1mgmL) of SRL and IS were preparedin methanol respectively SRL stock solution was dilutedin methanol-water (5050) to obtain a series of workingsolutions at concentrations of 25 50 100 200 400 500800 and 1000 ngmL Calibration standards were preparedby dilutions of the above working solutions with appropriateblank rat blood to attain concentrations of 25 5 10 2040 50 80 and 100 ngmL QC samples were obtained withconcentrations of 5 40 and 80 ngmL All solutions werestored at minus20∘C

22 LC-MSMS Condition Analysis was carried out on anAgilent 1290 series HPLC coupled with an Agilent 6460triple-quadrupole mass spectrometer that was equipped withjet stream electrospray ionization (ESI) Data acquisitionand processing was performed on Agilent 6460 QuantitativeAnalysis version B0102 analyst data processing software(Agilent Corporation Santa Clara CA USA) A Poroshell120 SB-C18 (27120583m 21mm times 75mm ID Agilent USA)was kept at 55∘C and a flow rate of 05mLmin Mobilephases consisted of methanol with 02 formic acid and10mM ammonium acetate (A) and 02 aqueous formicacid solution with 10mM ammonium acetate (B) Gradient

BioMed Research International 3

elution program was used for analysis 0ndash3min 60 A3ndash5min 60ndash95 A 5ndash8min 95A Injection volume wasset at 10 120583L

Nitrogen (purity 999999) was applied as the collisiongas (01MPa) The detector was operated in the positivemode The conditions of the source parameters were capil-lary voltage 40 kV nebulizer 35 psi drying gas temperature225∘C drying gas flow 10 Lmin sheath gas temperature325∘C and sheath gas flow 12 Lmin The optimized multiplereaction monitoring (MRM) conditions of the analytes weremz 9316997888rarr8645 and fragmentationcollision energy 170V13 eV for SRL and mz 12339997888rarr12169 and fragmenta-tioncollision energy 190 V17 eV for IS

23 Sample Preparation 100120583L bloodwas addedwith 200 120583L04M zinc sulfate aqueous solution methanol precipitat-ing solution (14 vv containing IS 200 ngmL) followedby vortex-mixing (2min) and centrifugation (12000 rpm10min) The sample supernatant (200120583L) was obtained andthen loaded to LC-MSMS system for analysis

24 Method Validation Method validation was performedaccording to US FDA guidance [19] Selectivity of the ana-lyte was evaluated by comparing the chromatograms of sixdifferent blank rat blood with those of blood spiked withSRL and IS as well as those of the rat blood sample afterdrug administration Calibration curves were obtained byplotting the peak-area ratio between SRL and IS against thenominal concentrations Linearity was evaluated by weighted(11199092) least squares linear regression analysis The lowerlimit of quantification (LLOQ) was defined as the lowestSRL concentration with a signal-to-noise ratio of 101 andevaluated by analyzing spiked blood samples prepared in sixreplicates The intra- and interday precisions and accuracieswere obtained by analyzing five replicates of QC samples inthree levelsThe precision was defined as the relative standarddeviation (RSD) and accuracy was expressed as relative error(RE) Matrix effect was determined by comparing the peakareas of SRL and IS spiked in extractedQC samples with thoseof analytes in standard solutions at equivalent concentrationsExtraction recovery was evaluated by comparing the peakareas of SRL and IS spiked in extractedQC samples with thoseof un-extracted standard solutions containing the equivalentamount of SRL Stability evaluation was performed in thefollowing conditions Long-term stability was tested for thesamples that were kept at minus20∘C for 30 days before analysisShort-term stability was tested for the samples that were keptat room temperature for 8 h before analysis Post-preparativestability was tested when the samples were kept in an auto-sampler at 4∘C for 24 h before analysis Freeze and thawstability was tested for three freeze-thaw cycles when thesamples were stored at minus20∘C for 24 h and thawed at roomtemperature

25 Application of the Assay and HDI Investigation of SRLand TRQ All animal protocols were approved by AnimalEthics Committee of Second Military Medical UniversitySprague-Dawley rats (adult male 200 plusmn 20 g) were obtained

from Shanghai SLAC laboratory animal Co Ltd (ShanghaiChina) Rats were quarantined for one week prior to studyand were housed in well ventilated cages at 20 plusmn 1∘C and50 plusmn 10 air humidity while on a 12-h light-dark cycle Allrats were fasted with free access to water for 12 h beforeexperiment

In order to investigate the effect of different dose lev-els and dosing time of TRQ on the pharmacokinetics ofSRL analysis SD rats were divided into 6 groups of 6animals each control group-SRL (75mgkg ig) alonegroup A1-coadministration of SRL (75mgkg ig) withTRQ (25mLkg iv) group A2-co-administration of SRL(75mgkg ig) with TRQ (5mLkg iv) and group A3-co-administration of SRL (75mgkg ig) with TRQ (10mLkgiv) TRQ were given to all rats at 5min after SRL dosing ingroups A1ndashA3

In order to investigate the effect of different dosing time ofTRQ on the pharmacokinetics of SRL analysis SD rats weredivided into 4 groups of 6 animals each control group-SRL(75mgkg ig) alone and the rest rats in groups B1ndashB3 weretreated with SRL (75mgkg ig) and TRQ (5mLkg iv)TRQ were given to rats at 5min 2 h and 4 h after SRL dosingin groups B1ndashB3 respectively See the experimental protocolin supplementary Figure S1

Blood samples (05mL) were collected into heparinizedtubes via retro-orbital plexus at pre-dose (0) 20 40min 12 4 6 8 12 24 36 and 48 h post-dose Blood samples werestored at minus20∘C prior to analysis Pharmacokinetic parame-ters (including119862max119879max 11990512AUCAUMC andMRT) werecalculated on Drug and Statistics DAS 326 (MathematicalPharmacology Professional Committee of China ShanghaiChina) using noncompartmental pharmacokinetic analysisThe differences between groups were evaluated by Studentrsquost test and were considered to be significant at lowastPlt005 andlowastlowastPlt001

3 Results and Discussion

31 Method Development Cyclosporine D was served as ISas the previous report [15] As MRM mode in LC-MSMSdetection allowed simultaneous quantitative determinationof drugs with high specificity low detection limits andshort time of analysis [19] it was applied to the analysis ofSRL and IS In order to release SRL from the erythrocyteswhere SRL was predominantly distributed (about 95) zincsulphate was used to lyse the erythrocytes It was thoughtthat the further sample treatment like solid-phase extractionor liquid-liquid extraction would contribute to eliminate thephospholipids from blood and then increase the efficiency ofSRL recovery [18 19] However that step would be time- andcost-consuming hard to be applied in the high-throughputTDM Therefore a one-step PPT protocol in conjunctionwith a proper LC method was optimized to eliminate thephospholipids in the initial 3min The addition of 02formic acid and 10mM ammonium acetate in phases A andB was favorable to enhance the MS response for both SRLand IS After checking different gradients in mobile phasesand other conditions the best results were obtained with theconditions described in ldquoLC-MSMS conditionrdquo

4 BioMed Research International

Table 1 Pharmacokinetic parameters of SRL after the administration of TRQ at different doses

Parameter Control Group Group A1 Group A2 Group A3Tmax (h) 4167plusmn2563 6500plusmn2811 5167plusmn3251 6333plusmn2658Cmax (ngmL) 9487plusmn1479 15546plusmn4234lowastlowast 12201plusmn1153lowastlowast 16043plusmn1437lowastlowast

11990512 (h) 68144plusmn63013 57543plusmn46635 47119plusmn43595 118158plusmn186146AUC0997888rarrt (ngsdothmL) 219673plusmn32306 349346plusmn69514lowastlowast 305875plusmn31417lowastlowast 421518plusmn42166lowastlowast

AUC0997888rarrinfin (ngsdothmL) 493458plusmn271184 684035plusmn233463 588668plusmn339317 1456282plusmn1708762AUMC0997888rarrt (ngsdothmL) 4122178plusmn593331 6577191plusmn1205275lowastlowast 5935337plusmn819676lowastlowast 8394303plusmn820509lowastlowast

MRT0997888rarrt (h) 18772plusmn0456 18881plusmn0946 19343plusmn0976 19919plusmn0275lowastlowastlowastlowastPlt001 compared with control group

32 Method Validation Calibration curve of SRL (y=00024x-00026) exhibited effective linearity (R2= 09955) over arange of 25ndash100 ngmL with LLOQ of 25 ngmL In detailinter- and intraday precisions and accuracies for SRL hadRSD values less than 10 The extraction recovery wasbetween 80 and 96Thematrix effect of SRL was between144 and 152 Stability investigation showed that the REvalues for SRL were less than 15 in rat blood for 30-day-storage at minus20∘C below 8 for 8-h-storage at room tem-perature less than 10 for 24-h-storage in the auto-samplerat 4∘C and below 20 for three freeze-thaw cycle processwhich indicated that the blood samples were stable duringthe entire experiment All the above results were withinthe ranges requested by the FDA for bioanalytical methodvalidation and could be applied to the SRL pharmacokineticstudy in rat

33 Results of Pharmacokinetic Study and HDI InvestigationTo evaluate the effect of different dose levels of TRQ onthe pharmacokinetics of SRL SRL pharmacokinetic dataof control group and group A1ndashA3 were demonstrated inTable 1 The corresponding mean blood concentration-timeprofiles were showed in Figure 2 Several pharmacokineticparameters presented an obvious increase trend in groupsA1ndashA3 when compared with those in control groupand Cmax AUC0997888rarrt and AUMC showed significantdifference Compared with that of control group (9487plusmn1479ngml) the 119862max of A1 A2 and A3 groups weresignificantly increased by 64 (15546plusmn4234ngml plt 001) 28 (12201plusmn1153 ngml p lt 001) and 70(16043plusmn1437 ngml p lt 001) respectively In parallelthe 1198601198801198620997888rarrt of A1 A2 and A3 groups were significantlyincreased by 59 (349346plusmn69514ngsdothmL p lt 001)39 (305875plusmn31417ngsdothmL p lt 001) and 92(421518plusmn42166 ngsdothmL p lt 001) respectively whencompared to control group (219673plusmn32306ngsdothmL)As for AUMC it was significantly increased by 60(6577191plusmn1205275ngsdothmL p lt 001) 44 (5935337plusmn819676ngsdothmL p lt 001) and 104 (8394303plusmn820509 ngsdothmL p lt 001) respectively when comparedto control group (4122178plusmn593331ngsdothmL) It revealedthat coadministration of SRL with single-dose TRQ couldmarkedly increase the blood concentration of SRL In thelowest dose of TRQ group (25mLkg) the three parametersof SRL mentioned above were increased by approximately

Control GroupGroup A1Group A2Group A3

0

4

8

12

16

Con

cent

ratio

n (n

gm

l)

10 20 30 40 500Time (h)

Figure 2Mean blood concentration-timeprofiles of sirolimus afterthe administration of TRQ at different doses

1-fold suggesting that the HDI might appear even withlowest dose of TRQ coadministration However no changein119879max was observed with or without TRQ coadministrationAlso values of 11990512 and MRT were similar with or withoutTRQ co-administration The above observations suggested apotential HDI between SRL and TRQ

To evaluate the effect of different dosing time of TRQon the pharmacokinetics of SRL SRL pharmacokineticdata of control group and groups B1ndashB3 were listed inTable 2The relevant mean blood concentration-time profileswere shown in Figure 3 Among those parameters only1198601198801198620997888rarrt showed significant difference (Plt 001) in groupsB1ndashB3 when compared with those in control group and1198601198801198620997888rarrt in group B3 was smaller than that in groupsB1 and B2 119879max and AUMC presented in a similar trendwith 1198601198801198620997888rarrt (without significance) In detail comparedwith that of control group (219673plusmn32306ngsdothmL) the1198601198801198620997888rarrt of B1 B2 and B3 groups were significantlyincreased by 39 (305875plusmn31417ngsdothmL p lt 001) 37(301677plusmn73315ngsdothmL p lt 001) and 35 (296211plusmn65286 ngsdothmL p lt 001) respectively As for AUMC it wassignificantly increased by 44 (5935337plusmn819676ngsdothmL plt 001) 49 (6157161plusmn1329936ngsdothmL p lt 001) and 37(5635173plusmn1350868ngsdothmL p lt 001) respectively whencompared to control group (4122178plusmn593331ngsdothmL)

BioMed Research International 5

Table 2 Pharmacokinetic parameters of SRL after the administration of TRQ at different time

Parameter Control Group Group B1 Group B2 Group B3Tmax (h) 4167plusmn2563 5167plusmn3251 5001plusmn4472 3001plusmn2449Cmax (ngmL) 9487plusmn1479 12201plusmn1153lowastlowast 11643plusmn3648 12699plusmn347911990512 (h) 68144plusmn63013 47119plusmn43595 65408plusmn27871 33299plusmn14359AUC0997888rarrt (ngsdothmL) 219673plusmn32306 305875plusmn31417lowastlowast 301677plusmn73315lowastlowast 296211plusmn65286lowastlowast

AUC0997888rarrinfin (ngsdothmL) 493458plusmn271184 588668plusmn339317 694003plusmn91568 456371plusmn133544AUMC0997888rarrt (ngsdoth2mL) 4122178plusmn593331 5935337plusmn819676lowastlowast 6157161plusmn1329936lowastlowast 5635173plusmn1350868lowastlowast

MRT0997888rarrt (h) 18772plusmn0456 19343plusmn0976 20527plusmn0801lowastlowast 18962plusmn0716lowastlowastPlt001 compared with control group

Control GroupGroup B1Group B2Group B3

0

4

8

12

Con

cent

ratio

n (n

gm

l)

10 20 30 40 500Time (h)

Figure 3 Mean blood concentration-timeprofiles of sirolimus afterthe administration of TRQ at different time

Therefore it could be concluded that the TRQ significantlyenhanced the AUC of SRL when TRQ was administrated atthe same time with SRL or 2 hours after SRL administrationThis effect was not obvious when TRQ was administrated 4hours after SRL administration Besides the pharmacokineticprofiles of SRL also showed the similar curves in the groupsB1ndashB3 Based on the effect of different dose levels anddifferent dosing time of TRQ it could be found that theAUCsin groups A1-A3 and groups B1ndashB3 were higher than thosein control groups which indicated that TRQ might causeoverexposure of SRL in patients receiving SRL along withTRQ

SRL is a substrate for CYP3A and P-gp It is not sure thatthe five TCMs from TRQ or its main components inhibitthe hepatic and intestinal CYP3AP-gp system Howeverit have been reported by some literatures that baicalin amain component from Scutellariae Radix in TRQ couldinhibit hepatic CYP3A activity as well as P-glycoproteinefflux pump in the small intestine [20ndash22] Therefore theaffected SRL pharmacokinetics by TRQmight be mainly dueto the inhibition of CYP3A-mediated metabolism and P-gp-mediated transport by baicalin The complex mixture ofchemical constituents in herbal medicines or TCMs which

are usually believed to be medicinally efficacious are yet tobe fully characterized [23]There might be other compoundsresponsible for theHDImechanism In future further studieswill be needed to clarify this point

Actually like the pharmacokinetic profile of the meanvalue of SRL in the stable renal transplant patients [24]the control group of SRL did not show the obvious doublepeaks which were only found in the SRL-TRQ combinationtreatment groups Because of the extensive distribution in redblood cells of SRL (945) [24] zinc sulfate was applied asthe erythrolysis agent to release SRL in the circulating redblood cells In this study the absorption of SRL fluctuatedThe reason could be that the absorption rate of SRL varied indifferent parts of the intestine in rats [25] Thus the doublepeaks for SRL in the TRQ pretreatment groups might beattributed to the effect of chemical compounds from TRQ onthe elimination of SRL which should be investigated in ourfuture study

It was reported that SRL pharmacokinetics was associatedwithCYP3AandMDR1 genetic polymorphisms butwe failedto determine the relevant genetic information of the patientin the introduction If the patient expressed the CYP3A5enzyme (CYP3A5lowast1 carriers) he might need more SRL toreach target concentrations when compared with those withCYP3A5lowast3lowast3 carriers [26] It might lead to a risk of drugtoxicity especially when a potential HDI existed

4 Conclusions

A simple and accurate LC-MSMS method was developedand validated for SRL assay in rat blood following a quickPPT procedure which demonstrated good selectivity lin-earity precision and accuracy matrix effect and recoveryand stability This assay was successfully applied to evaluatethe pharmacokinetics of SRL when it was administratedalone and coadministrated with TRQ in rat The resultsshowed that different dose levels and different dosing time ofTRQ would increase SRL blood concentration and exposureindicating a potential HDI between TRQ and SRLThe studyprovided valuable information for SRL treatment from thepharmacokinetic point of view coadministration with TRQor baicalin-derived products was not recommended in clinicA systematical TDM for SRL was greatly encouraged forsafety and would help to discover a possible DDIHDI

6 BioMed Research International

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Feng Zhang and Liang Sun contributed equally to this workWansheng Chen and Yifeng Chai contributed equally to thiswork

Acknowledgments

Funding for this study was provided by National Natu-ral Science Foundation of China (81830109 and 81573793)Shanghai Key Specialty Project of Clinical Pharmacy (2016-40044-002) Important Weak Subject Construction Projectof Shanghai Health Science Education (2016ZB0303) andBeijing Medical Award Foundation (YJHYXKYJJ-122) Theauthors also greatly appreciate the kind help from Dr JunWen School of Pharmacy Second Military Medical Univer-sity

Supplementary Materials

Please fine the experimental protocol in supplementaryFigure S1 (Supplementary Materials)

References

[1] S N Sehgal ldquoRapamune (RAPA rapamycin sirolimus)Mechanism of action immunosuppressive effect results fromblockade of signal transduction and inhibition of cell cycleprogressionrdquo Clinical Biochemistry vol 31 no 5 pp 335ndash3401998

[2] N Terada J J Lucas A Szepesi R A Franklin J Domenicoand E W Gelfand ldquoRapamycin blocks cell cycle progression ofactivated T cells prior to events characteristic of the middle tolate G1 phase of the cyclerdquo Journal of Cellular Physiology vol154 no 1 pp 7ndash15 1993

[3] P R Shah V B KuteHV Patel andH L Trivedi ldquoTherapeuticdrugmonitoring of sirolimusrdquoClinical Queries Nephrology vol4 no 3-4 pp 44ndash49 2015

[4] F Shihab U Christians L Smith J R Wellen and B KaplanldquoFocus on mTOR inhibitors and tacrolimus in renal transplan-tation Pharmacokinetics exposure-response relationships andclinical outcomesrdquoTransplant Immunology vol 31 no 1 pp 22ndash32 2014

[5] Y Wang T Wang J Hu et al ldquoAnti-biofilm activity of TanRe-Qing a traditional Chinese Medicine used for the treatment ofacute pneumoniardquo Journal of Ethnopharmacology vol 134 no1 pp 165ndash170 2011

[6] P Wang X Liao YM Xie Y Chai and LH Li ldquoTanreqinginjection for acute bronchitis disease A systematic review andmeta-analysis of randomized controlled trialsrdquo ComplementTher Med vol 25 pp 143ndash158 2016

[7] L Sun H Wei F Zhang et al ldquoQualitative analysis and qualitycontrol of Traditional ChineseMedicine preparation Tanreqinginjection by LC-TOFMS and HPLC-DAD-ELSDrdquo AnalyticalMethods vol 5 no 22 pp 6431ndash6440 2013

[8] F Zhang L Sun SH Gao WS Chen and YF Chai ldquoLC-MSMS analysis and pharmacokinetic study on five bioactiveconstituents of Tanreqing injection in ratsrdquo Chinese Journal ofNature Medicines vol 14 pp 769ndash775 2016

[9] F Zhang L Sun SH Gao et al ldquoA sensitive HPLC-MSmethodfor simultaneous determination of thirteen components inrat plasma and its application to pharmacokinetic study ofTanreqing injectionrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 148 pp 205ndash213 2018

[10] B Sadaba M A Campanero E G Quetglas and J R AzanzaldquoClinical relevance of sirolimus drug interactions in transplantpatientsrdquo Transplantation Proceedings vol 36 no 10 pp 3226ndash3228 2004

[11] M Oellerich V W Armstrong F Streit L Weber and BTonshoff ldquoImmunosuppressive drug monitoring of sirolimusand cyclosporine in pediatric patientsrdquo Clinical Biochemistryvol 37 no 6 pp 424ndash428 2004

[12] T VanGelder ldquoDrug interactionswith tacrolimusrdquoDrug Safetyvol 25 no 10 pp 707ndash712 2002

[13] B C Sallustio B D Noll and R G Morris ldquoComparisonof blood sirolimus tacrolimus and everolimus concentrationsmeasured by LC-MSMS HPLC-UV and immunoassay meth-odsrdquo Clinical Biochemistry vol 44 no 2-3 pp 231ndash236 2011

[14] N Ansermot M Fathi J-L Veuthey J Desmeules SRudaz and D Hochstrasser ldquoSimultaneous quantification ofcyclosporine tacrolimus sirolimus and everolimus in wholeblood by liquid chromatography-electrospray mass spectrom-etryrdquo Clinical Biochemistry vol 41 no 9 pp 728ndash735 2008

[15] J-H Lee K-H ChaW Cho et al ldquoQuantitative determinationof sirolimus in dog blood using liquid chromatography-tandemmass spectrometry and its applications to pharmacokineticstudiesrdquo Journal of Pharmaceutical and Biomedical Analysis vol53 no 4 pp 1042ndash1047 2010

[16] D M Mueller and K M Rentsch ldquoSensitive quantification ofsirolimus and everolimus by LC-MSMS with online samplecleanuprdquo Journal of Chromatography B vol 878 no 13-14 pp1007ndash1012 2010

[17] N Mano M Sato M Nozawa et al ldquoAn accurate quantitativeLCESI-MSMS method for sirolimus in human whole bloodrdquoJournal of Chromatography B vol 879 no 13-14 pp 987ndash9922011

[18] A Navarrete M P Martınez-Alcazar I Duran et al ldquoSimul-taneous online SPE-HPLC-MSMS analysis of docetaxel Tem-sirolimus and sirolimus in whole blood and human plasmardquoJournal of Chromatography B vol 921-922 pp 35ndash42 2013

[19] Guidance for Industry Bioanalytical Method Validation USDepartment of Health and Human Services Food and DrugAdministration Center for Drug Evaluation and Research(CDER) Center for VeterinaryMedicine (CV) May 2001

[20] X Tian Z-Y Cheng J He L-J Jia and H-L QiaoldquoConcentration-dependent inhibitory effects of baicalin on themetabolism of dextromethorphan a dual probe of CYP2D andCYP3A in ratsrdquoChemico-Biological Interactions vol 203 no 2pp 522ndash529 2013

[21] Z Y Cheng X Tian J Gao L I HM L J Jia and HL Qiao ldquoContribution of baicalin on the plasma proteinbinding displacement and CYP3A activity inhibition to the

BioMed Research International 3

elution program was used for analysis 0ndash3min 60 A3ndash5min 60ndash95 A 5ndash8min 95A Injection volume wasset at 10 120583L

Nitrogen (purity 999999) was applied as the collisiongas (01MPa) The detector was operated in the positivemode The conditions of the source parameters were capil-lary voltage 40 kV nebulizer 35 psi drying gas temperature225∘C drying gas flow 10 Lmin sheath gas temperature325∘C and sheath gas flow 12 Lmin The optimized multiplereaction monitoring (MRM) conditions of the analytes weremz 9316997888rarr8645 and fragmentationcollision energy 170V13 eV for SRL and mz 12339997888rarr12169 and fragmenta-tioncollision energy 190 V17 eV for IS

23 Sample Preparation 100120583L bloodwas addedwith 200 120583L04M zinc sulfate aqueous solution methanol precipitat-ing solution (14 vv containing IS 200 ngmL) followedby vortex-mixing (2min) and centrifugation (12000 rpm10min) The sample supernatant (200120583L) was obtained andthen loaded to LC-MSMS system for analysis

24 Method Validation Method validation was performedaccording to US FDA guidance [19] Selectivity of the ana-lyte was evaluated by comparing the chromatograms of sixdifferent blank rat blood with those of blood spiked withSRL and IS as well as those of the rat blood sample afterdrug administration Calibration curves were obtained byplotting the peak-area ratio between SRL and IS against thenominal concentrations Linearity was evaluated by weighted(11199092) least squares linear regression analysis The lowerlimit of quantification (LLOQ) was defined as the lowestSRL concentration with a signal-to-noise ratio of 101 andevaluated by analyzing spiked blood samples prepared in sixreplicates The intra- and interday precisions and accuracieswere obtained by analyzing five replicates of QC samples inthree levelsThe precision was defined as the relative standarddeviation (RSD) and accuracy was expressed as relative error(RE) Matrix effect was determined by comparing the peakareas of SRL and IS spiked in extractedQC samples with thoseof analytes in standard solutions at equivalent concentrationsExtraction recovery was evaluated by comparing the peakareas of SRL and IS spiked in extractedQC samples with thoseof un-extracted standard solutions containing the equivalentamount of SRL Stability evaluation was performed in thefollowing conditions Long-term stability was tested for thesamples that were kept at minus20∘C for 30 days before analysisShort-term stability was tested for the samples that were keptat room temperature for 8 h before analysis Post-preparativestability was tested when the samples were kept in an auto-sampler at 4∘C for 24 h before analysis Freeze and thawstability was tested for three freeze-thaw cycles when thesamples were stored at minus20∘C for 24 h and thawed at roomtemperature

25 Application of the Assay and HDI Investigation of SRLand TRQ All animal protocols were approved by AnimalEthics Committee of Second Military Medical UniversitySprague-Dawley rats (adult male 200 plusmn 20 g) were obtained

from Shanghai SLAC laboratory animal Co Ltd (ShanghaiChina) Rats were quarantined for one week prior to studyand were housed in well ventilated cages at 20 plusmn 1∘C and50 plusmn 10 air humidity while on a 12-h light-dark cycle Allrats were fasted with free access to water for 12 h beforeexperiment

In order to investigate the effect of different dose lev-els and dosing time of TRQ on the pharmacokinetics ofSRL analysis SD rats were divided into 6 groups of 6animals each control group-SRL (75mgkg ig) alonegroup A1-coadministration of SRL (75mgkg ig) withTRQ (25mLkg iv) group A2-co-administration of SRL(75mgkg ig) with TRQ (5mLkg iv) and group A3-co-administration of SRL (75mgkg ig) with TRQ (10mLkgiv) TRQ were given to all rats at 5min after SRL dosing ingroups A1ndashA3

In order to investigate the effect of different dosing time ofTRQ on the pharmacokinetics of SRL analysis SD rats weredivided into 4 groups of 6 animals each control group-SRL(75mgkg ig) alone and the rest rats in groups B1ndashB3 weretreated with SRL (75mgkg ig) and TRQ (5mLkg iv)TRQ were given to rats at 5min 2 h and 4 h after SRL dosingin groups B1ndashB3 respectively See the experimental protocolin supplementary Figure S1

Blood samples (05mL) were collected into heparinizedtubes via retro-orbital plexus at pre-dose (0) 20 40min 12 4 6 8 12 24 36 and 48 h post-dose Blood samples werestored at minus20∘C prior to analysis Pharmacokinetic parame-ters (including119862max119879max 11990512AUCAUMC andMRT) werecalculated on Drug and Statistics DAS 326 (MathematicalPharmacology Professional Committee of China ShanghaiChina) using noncompartmental pharmacokinetic analysisThe differences between groups were evaluated by Studentrsquost test and were considered to be significant at lowastPlt005 andlowastlowastPlt001

3 Results and Discussion

31 Method Development Cyclosporine D was served as ISas the previous report [15] As MRM mode in LC-MSMSdetection allowed simultaneous quantitative determinationof drugs with high specificity low detection limits andshort time of analysis [19] it was applied to the analysis ofSRL and IS In order to release SRL from the erythrocyteswhere SRL was predominantly distributed (about 95) zincsulphate was used to lyse the erythrocytes It was thoughtthat the further sample treatment like solid-phase extractionor liquid-liquid extraction would contribute to eliminate thephospholipids from blood and then increase the efficiency ofSRL recovery [18 19] However that step would be time- andcost-consuming hard to be applied in the high-throughputTDM Therefore a one-step PPT protocol in conjunctionwith a proper LC method was optimized to eliminate thephospholipids in the initial 3min The addition of 02formic acid and 10mM ammonium acetate in phases A andB was favorable to enhance the MS response for both SRLand IS After checking different gradients in mobile phasesand other conditions the best results were obtained with theconditions described in ldquoLC-MSMS conditionrdquo

4 BioMed Research International

Table 1 Pharmacokinetic parameters of SRL after the administration of TRQ at different doses

Parameter Control Group Group A1 Group A2 Group A3Tmax (h) 4167plusmn2563 6500plusmn2811 5167plusmn3251 6333plusmn2658Cmax (ngmL) 9487plusmn1479 15546plusmn4234lowastlowast 12201plusmn1153lowastlowast 16043plusmn1437lowastlowast

11990512 (h) 68144plusmn63013 57543plusmn46635 47119plusmn43595 118158plusmn186146AUC0997888rarrt (ngsdothmL) 219673plusmn32306 349346plusmn69514lowastlowast 305875plusmn31417lowastlowast 421518plusmn42166lowastlowast

AUC0997888rarrinfin (ngsdothmL) 493458plusmn271184 684035plusmn233463 588668plusmn339317 1456282plusmn1708762AUMC0997888rarrt (ngsdothmL) 4122178plusmn593331 6577191plusmn1205275lowastlowast 5935337plusmn819676lowastlowast 8394303plusmn820509lowastlowast

MRT0997888rarrt (h) 18772plusmn0456 18881plusmn0946 19343plusmn0976 19919plusmn0275lowastlowastlowastlowastPlt001 compared with control group

32 Method Validation Calibration curve of SRL (y=00024x-00026) exhibited effective linearity (R2= 09955) over arange of 25ndash100 ngmL with LLOQ of 25 ngmL In detailinter- and intraday precisions and accuracies for SRL hadRSD values less than 10 The extraction recovery wasbetween 80 and 96Thematrix effect of SRL was between144 and 152 Stability investigation showed that the REvalues for SRL were less than 15 in rat blood for 30-day-storage at minus20∘C below 8 for 8-h-storage at room tem-perature less than 10 for 24-h-storage in the auto-samplerat 4∘C and below 20 for three freeze-thaw cycle processwhich indicated that the blood samples were stable duringthe entire experiment All the above results were withinthe ranges requested by the FDA for bioanalytical methodvalidation and could be applied to the SRL pharmacokineticstudy in rat

33 Results of Pharmacokinetic Study and HDI InvestigationTo evaluate the effect of different dose levels of TRQ onthe pharmacokinetics of SRL SRL pharmacokinetic dataof control group and group A1ndashA3 were demonstrated inTable 1 The corresponding mean blood concentration-timeprofiles were showed in Figure 2 Several pharmacokineticparameters presented an obvious increase trend in groupsA1ndashA3 when compared with those in control groupand Cmax AUC0997888rarrt and AUMC showed significantdifference Compared with that of control group (9487plusmn1479ngml) the 119862max of A1 A2 and A3 groups weresignificantly increased by 64 (15546plusmn4234ngml plt 001) 28 (12201plusmn1153 ngml p lt 001) and 70(16043plusmn1437 ngml p lt 001) respectively In parallelthe 1198601198801198620997888rarrt of A1 A2 and A3 groups were significantlyincreased by 59 (349346plusmn69514ngsdothmL p lt 001)39 (305875plusmn31417ngsdothmL p lt 001) and 92(421518plusmn42166 ngsdothmL p lt 001) respectively whencompared to control group (219673plusmn32306ngsdothmL)As for AUMC it was significantly increased by 60(6577191plusmn1205275ngsdothmL p lt 001) 44 (5935337plusmn819676ngsdothmL p lt 001) and 104 (8394303plusmn820509 ngsdothmL p lt 001) respectively when comparedto control group (4122178plusmn593331ngsdothmL) It revealedthat coadministration of SRL with single-dose TRQ couldmarkedly increase the blood concentration of SRL In thelowest dose of TRQ group (25mLkg) the three parametersof SRL mentioned above were increased by approximately

Control GroupGroup A1Group A2Group A3

0

4

8

12

16

Con

cent

ratio

n (n

gm

l)

10 20 30 40 500Time (h)

Figure 2Mean blood concentration-timeprofiles of sirolimus afterthe administration of TRQ at different doses

1-fold suggesting that the HDI might appear even withlowest dose of TRQ coadministration However no changein119879max was observed with or without TRQ coadministrationAlso values of 11990512 and MRT were similar with or withoutTRQ co-administration The above observations suggested apotential HDI between SRL and TRQ

To evaluate the effect of different dosing time of TRQon the pharmacokinetics of SRL SRL pharmacokineticdata of control group and groups B1ndashB3 were listed inTable 2The relevant mean blood concentration-time profileswere shown in Figure 3 Among those parameters only1198601198801198620997888rarrt showed significant difference (Plt 001) in groupsB1ndashB3 when compared with those in control group and1198601198801198620997888rarrt in group B3 was smaller than that in groupsB1 and B2 119879max and AUMC presented in a similar trendwith 1198601198801198620997888rarrt (without significance) In detail comparedwith that of control group (219673plusmn32306ngsdothmL) the1198601198801198620997888rarrt of B1 B2 and B3 groups were significantlyincreased by 39 (305875plusmn31417ngsdothmL p lt 001) 37(301677plusmn73315ngsdothmL p lt 001) and 35 (296211plusmn65286 ngsdothmL p lt 001) respectively As for AUMC it wassignificantly increased by 44 (5935337plusmn819676ngsdothmL plt 001) 49 (6157161plusmn1329936ngsdothmL p lt 001) and 37(5635173plusmn1350868ngsdothmL p lt 001) respectively whencompared to control group (4122178plusmn593331ngsdothmL)

BioMed Research International 5

Table 2 Pharmacokinetic parameters of SRL after the administration of TRQ at different time

Parameter Control Group Group B1 Group B2 Group B3Tmax (h) 4167plusmn2563 5167plusmn3251 5001plusmn4472 3001plusmn2449Cmax (ngmL) 9487plusmn1479 12201plusmn1153lowastlowast 11643plusmn3648 12699plusmn347911990512 (h) 68144plusmn63013 47119plusmn43595 65408plusmn27871 33299plusmn14359AUC0997888rarrt (ngsdothmL) 219673plusmn32306 305875plusmn31417lowastlowast 301677plusmn73315lowastlowast 296211plusmn65286lowastlowast

AUC0997888rarrinfin (ngsdothmL) 493458plusmn271184 588668plusmn339317 694003plusmn91568 456371plusmn133544AUMC0997888rarrt (ngsdoth2mL) 4122178plusmn593331 5935337plusmn819676lowastlowast 6157161plusmn1329936lowastlowast 5635173plusmn1350868lowastlowast

MRT0997888rarrt (h) 18772plusmn0456 19343plusmn0976 20527plusmn0801lowastlowast 18962plusmn0716lowastlowastPlt001 compared with control group

Control GroupGroup B1Group B2Group B3

0

4

8

12

Con

cent

ratio

n (n

gm

l)

10 20 30 40 500Time (h)

Figure 3 Mean blood concentration-timeprofiles of sirolimus afterthe administration of TRQ at different time

Therefore it could be concluded that the TRQ significantlyenhanced the AUC of SRL when TRQ was administrated atthe same time with SRL or 2 hours after SRL administrationThis effect was not obvious when TRQ was administrated 4hours after SRL administration Besides the pharmacokineticprofiles of SRL also showed the similar curves in the groupsB1ndashB3 Based on the effect of different dose levels anddifferent dosing time of TRQ it could be found that theAUCsin groups A1-A3 and groups B1ndashB3 were higher than thosein control groups which indicated that TRQ might causeoverexposure of SRL in patients receiving SRL along withTRQ

SRL is a substrate for CYP3A and P-gp It is not sure thatthe five TCMs from TRQ or its main components inhibitthe hepatic and intestinal CYP3AP-gp system Howeverit have been reported by some literatures that baicalin amain component from Scutellariae Radix in TRQ couldinhibit hepatic CYP3A activity as well as P-glycoproteinefflux pump in the small intestine [20ndash22] Therefore theaffected SRL pharmacokinetics by TRQmight be mainly dueto the inhibition of CYP3A-mediated metabolism and P-gp-mediated transport by baicalin The complex mixture ofchemical constituents in herbal medicines or TCMs which

are usually believed to be medicinally efficacious are yet tobe fully characterized [23]There might be other compoundsresponsible for theHDImechanism In future further studieswill be needed to clarify this point

Actually like the pharmacokinetic profile of the meanvalue of SRL in the stable renal transplant patients [24]the control group of SRL did not show the obvious doublepeaks which were only found in the SRL-TRQ combinationtreatment groups Because of the extensive distribution in redblood cells of SRL (945) [24] zinc sulfate was applied asthe erythrolysis agent to release SRL in the circulating redblood cells In this study the absorption of SRL fluctuatedThe reason could be that the absorption rate of SRL varied indifferent parts of the intestine in rats [25] Thus the doublepeaks for SRL in the TRQ pretreatment groups might beattributed to the effect of chemical compounds from TRQ onthe elimination of SRL which should be investigated in ourfuture study

It was reported that SRL pharmacokinetics was associatedwithCYP3AandMDR1 genetic polymorphisms butwe failedto determine the relevant genetic information of the patientin the introduction If the patient expressed the CYP3A5enzyme (CYP3A5lowast1 carriers) he might need more SRL toreach target concentrations when compared with those withCYP3A5lowast3lowast3 carriers [26] It might lead to a risk of drugtoxicity especially when a potential HDI existed

4 Conclusions

A simple and accurate LC-MSMS method was developedand validated for SRL assay in rat blood following a quickPPT procedure which demonstrated good selectivity lin-earity precision and accuracy matrix effect and recoveryand stability This assay was successfully applied to evaluatethe pharmacokinetics of SRL when it was administratedalone and coadministrated with TRQ in rat The resultsshowed that different dose levels and different dosing time ofTRQ would increase SRL blood concentration and exposureindicating a potential HDI between TRQ and SRLThe studyprovided valuable information for SRL treatment from thepharmacokinetic point of view coadministration with TRQor baicalin-derived products was not recommended in clinicA systematical TDM for SRL was greatly encouraged forsafety and would help to discover a possible DDIHDI

6 BioMed Research International

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Feng Zhang and Liang Sun contributed equally to this workWansheng Chen and Yifeng Chai contributed equally to thiswork

Acknowledgments

Funding for this study was provided by National Natu-ral Science Foundation of China (81830109 and 81573793)Shanghai Key Specialty Project of Clinical Pharmacy (2016-40044-002) Important Weak Subject Construction Projectof Shanghai Health Science Education (2016ZB0303) andBeijing Medical Award Foundation (YJHYXKYJJ-122) Theauthors also greatly appreciate the kind help from Dr JunWen School of Pharmacy Second Military Medical Univer-sity

Supplementary Materials

Please fine the experimental protocol in supplementaryFigure S1 (Supplementary Materials)

References

[1] S N Sehgal ldquoRapamune (RAPA rapamycin sirolimus)Mechanism of action immunosuppressive effect results fromblockade of signal transduction and inhibition of cell cycleprogressionrdquo Clinical Biochemistry vol 31 no 5 pp 335ndash3401998

[2] N Terada J J Lucas A Szepesi R A Franklin J Domenicoand E W Gelfand ldquoRapamycin blocks cell cycle progression ofactivated T cells prior to events characteristic of the middle tolate G1 phase of the cyclerdquo Journal of Cellular Physiology vol154 no 1 pp 7ndash15 1993

[3] P R Shah V B KuteHV Patel andH L Trivedi ldquoTherapeuticdrugmonitoring of sirolimusrdquoClinical Queries Nephrology vol4 no 3-4 pp 44ndash49 2015

[4] F Shihab U Christians L Smith J R Wellen and B KaplanldquoFocus on mTOR inhibitors and tacrolimus in renal transplan-tation Pharmacokinetics exposure-response relationships andclinical outcomesrdquoTransplant Immunology vol 31 no 1 pp 22ndash32 2014

[5] Y Wang T Wang J Hu et al ldquoAnti-biofilm activity of TanRe-Qing a traditional Chinese Medicine used for the treatment ofacute pneumoniardquo Journal of Ethnopharmacology vol 134 no1 pp 165ndash170 2011

[6] P Wang X Liao YM Xie Y Chai and LH Li ldquoTanreqinginjection for acute bronchitis disease A systematic review andmeta-analysis of randomized controlled trialsrdquo ComplementTher Med vol 25 pp 143ndash158 2016

[7] L Sun H Wei F Zhang et al ldquoQualitative analysis and qualitycontrol of Traditional ChineseMedicine preparation Tanreqinginjection by LC-TOFMS and HPLC-DAD-ELSDrdquo AnalyticalMethods vol 5 no 22 pp 6431ndash6440 2013

[8] F Zhang L Sun SH Gao WS Chen and YF Chai ldquoLC-MSMS analysis and pharmacokinetic study on five bioactiveconstituents of Tanreqing injection in ratsrdquo Chinese Journal ofNature Medicines vol 14 pp 769ndash775 2016

[9] F Zhang L Sun SH Gao et al ldquoA sensitive HPLC-MSmethodfor simultaneous determination of thirteen components inrat plasma and its application to pharmacokinetic study ofTanreqing injectionrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 148 pp 205ndash213 2018

[10] B Sadaba M A Campanero E G Quetglas and J R AzanzaldquoClinical relevance of sirolimus drug interactions in transplantpatientsrdquo Transplantation Proceedings vol 36 no 10 pp 3226ndash3228 2004

[11] M Oellerich V W Armstrong F Streit L Weber and BTonshoff ldquoImmunosuppressive drug monitoring of sirolimusand cyclosporine in pediatric patientsrdquo Clinical Biochemistryvol 37 no 6 pp 424ndash428 2004

[12] T VanGelder ldquoDrug interactionswith tacrolimusrdquoDrug Safetyvol 25 no 10 pp 707ndash712 2002

[13] B C Sallustio B D Noll and R G Morris ldquoComparisonof blood sirolimus tacrolimus and everolimus concentrationsmeasured by LC-MSMS HPLC-UV and immunoassay meth-odsrdquo Clinical Biochemistry vol 44 no 2-3 pp 231ndash236 2011

[14] N Ansermot M Fathi J-L Veuthey J Desmeules SRudaz and D Hochstrasser ldquoSimultaneous quantification ofcyclosporine tacrolimus sirolimus and everolimus in wholeblood by liquid chromatography-electrospray mass spectrom-etryrdquo Clinical Biochemistry vol 41 no 9 pp 728ndash735 2008

[15] J-H Lee K-H ChaW Cho et al ldquoQuantitative determinationof sirolimus in dog blood using liquid chromatography-tandemmass spectrometry and its applications to pharmacokineticstudiesrdquo Journal of Pharmaceutical and Biomedical Analysis vol53 no 4 pp 1042ndash1047 2010

[16] D M Mueller and K M Rentsch ldquoSensitive quantification ofsirolimus and everolimus by LC-MSMS with online samplecleanuprdquo Journal of Chromatography B vol 878 no 13-14 pp1007ndash1012 2010

[17] N Mano M Sato M Nozawa et al ldquoAn accurate quantitativeLCESI-MSMS method for sirolimus in human whole bloodrdquoJournal of Chromatography B vol 879 no 13-14 pp 987ndash9922011

[18] A Navarrete M P Martınez-Alcazar I Duran et al ldquoSimul-taneous online SPE-HPLC-MSMS analysis of docetaxel Tem-sirolimus and sirolimus in whole blood and human plasmardquoJournal of Chromatography B vol 921-922 pp 35ndash42 2013

[19] Guidance for Industry Bioanalytical Method Validation USDepartment of Health and Human Services Food and DrugAdministration Center for Drug Evaluation and Research(CDER) Center for VeterinaryMedicine (CV) May 2001

[20] X Tian Z-Y Cheng J He L-J Jia and H-L QiaoldquoConcentration-dependent inhibitory effects of baicalin on themetabolism of dextromethorphan a dual probe of CYP2D andCYP3A in ratsrdquoChemico-Biological Interactions vol 203 no 2pp 522ndash529 2013

[21] Z Y Cheng X Tian J Gao L I HM L J Jia and HL Qiao ldquoContribution of baicalin on the plasma proteinbinding displacement and CYP3A activity inhibition to the

4 BioMed Research International

Table 1 Pharmacokinetic parameters of SRL after the administration of TRQ at different doses

Parameter Control Group Group A1 Group A2 Group A3Tmax (h) 4167plusmn2563 6500plusmn2811 5167plusmn3251 6333plusmn2658Cmax (ngmL) 9487plusmn1479 15546plusmn4234lowastlowast 12201plusmn1153lowastlowast 16043plusmn1437lowastlowast

11990512 (h) 68144plusmn63013 57543plusmn46635 47119plusmn43595 118158plusmn186146AUC0997888rarrt (ngsdothmL) 219673plusmn32306 349346plusmn69514lowastlowast 305875plusmn31417lowastlowast 421518plusmn42166lowastlowast

AUC0997888rarrinfin (ngsdothmL) 493458plusmn271184 684035plusmn233463 588668plusmn339317 1456282plusmn1708762AUMC0997888rarrt (ngsdothmL) 4122178plusmn593331 6577191plusmn1205275lowastlowast 5935337plusmn819676lowastlowast 8394303plusmn820509lowastlowast

MRT0997888rarrt (h) 18772plusmn0456 18881plusmn0946 19343plusmn0976 19919plusmn0275lowastlowastlowastlowastPlt001 compared with control group

32 Method Validation Calibration curve of SRL (y=00024x-00026) exhibited effective linearity (R2= 09955) over arange of 25ndash100 ngmL with LLOQ of 25 ngmL In detailinter- and intraday precisions and accuracies for SRL hadRSD values less than 10 The extraction recovery wasbetween 80 and 96Thematrix effect of SRL was between144 and 152 Stability investigation showed that the REvalues for SRL were less than 15 in rat blood for 30-day-storage at minus20∘C below 8 for 8-h-storage at room tem-perature less than 10 for 24-h-storage in the auto-samplerat 4∘C and below 20 for three freeze-thaw cycle processwhich indicated that the blood samples were stable duringthe entire experiment All the above results were withinthe ranges requested by the FDA for bioanalytical methodvalidation and could be applied to the SRL pharmacokineticstudy in rat

33 Results of Pharmacokinetic Study and HDI InvestigationTo evaluate the effect of different dose levels of TRQ onthe pharmacokinetics of SRL SRL pharmacokinetic dataof control group and group A1ndashA3 were demonstrated inTable 1 The corresponding mean blood concentration-timeprofiles were showed in Figure 2 Several pharmacokineticparameters presented an obvious increase trend in groupsA1ndashA3 when compared with those in control groupand Cmax AUC0997888rarrt and AUMC showed significantdifference Compared with that of control group (9487plusmn1479ngml) the 119862max of A1 A2 and A3 groups weresignificantly increased by 64 (15546plusmn4234ngml plt 001) 28 (12201plusmn1153 ngml p lt 001) and 70(16043plusmn1437 ngml p lt 001) respectively In parallelthe 1198601198801198620997888rarrt of A1 A2 and A3 groups were significantlyincreased by 59 (349346plusmn69514ngsdothmL p lt 001)39 (305875plusmn31417ngsdothmL p lt 001) and 92(421518plusmn42166 ngsdothmL p lt 001) respectively whencompared to control group (219673plusmn32306ngsdothmL)As for AUMC it was significantly increased by 60(6577191plusmn1205275ngsdothmL p lt 001) 44 (5935337plusmn819676ngsdothmL p lt 001) and 104 (8394303plusmn820509 ngsdothmL p lt 001) respectively when comparedto control group (4122178plusmn593331ngsdothmL) It revealedthat coadministration of SRL with single-dose TRQ couldmarkedly increase the blood concentration of SRL In thelowest dose of TRQ group (25mLkg) the three parametersof SRL mentioned above were increased by approximately

Control GroupGroup A1Group A2Group A3

0

4

8

12

16

Con

cent

ratio

n (n

gm

l)

10 20 30 40 500Time (h)

Figure 2Mean blood concentration-timeprofiles of sirolimus afterthe administration of TRQ at different doses

1-fold suggesting that the HDI might appear even withlowest dose of TRQ coadministration However no changein119879max was observed with or without TRQ coadministrationAlso values of 11990512 and MRT were similar with or withoutTRQ co-administration The above observations suggested apotential HDI between SRL and TRQ

To evaluate the effect of different dosing time of TRQon the pharmacokinetics of SRL SRL pharmacokineticdata of control group and groups B1ndashB3 were listed inTable 2The relevant mean blood concentration-time profileswere shown in Figure 3 Among those parameters only1198601198801198620997888rarrt showed significant difference (Plt 001) in groupsB1ndashB3 when compared with those in control group and1198601198801198620997888rarrt in group B3 was smaller than that in groupsB1 and B2 119879max and AUMC presented in a similar trendwith 1198601198801198620997888rarrt (without significance) In detail comparedwith that of control group (219673plusmn32306ngsdothmL) the1198601198801198620997888rarrt of B1 B2 and B3 groups were significantlyincreased by 39 (305875plusmn31417ngsdothmL p lt 001) 37(301677plusmn73315ngsdothmL p lt 001) and 35 (296211plusmn65286 ngsdothmL p lt 001) respectively As for AUMC it wassignificantly increased by 44 (5935337plusmn819676ngsdothmL plt 001) 49 (6157161plusmn1329936ngsdothmL p lt 001) and 37(5635173plusmn1350868ngsdothmL p lt 001) respectively whencompared to control group (4122178plusmn593331ngsdothmL)

BioMed Research International 5

Table 2 Pharmacokinetic parameters of SRL after the administration of TRQ at different time

Parameter Control Group Group B1 Group B2 Group B3Tmax (h) 4167plusmn2563 5167plusmn3251 5001plusmn4472 3001plusmn2449Cmax (ngmL) 9487plusmn1479 12201plusmn1153lowastlowast 11643plusmn3648 12699plusmn347911990512 (h) 68144plusmn63013 47119plusmn43595 65408plusmn27871 33299plusmn14359AUC0997888rarrt (ngsdothmL) 219673plusmn32306 305875plusmn31417lowastlowast 301677plusmn73315lowastlowast 296211plusmn65286lowastlowast

AUC0997888rarrinfin (ngsdothmL) 493458plusmn271184 588668plusmn339317 694003plusmn91568 456371plusmn133544AUMC0997888rarrt (ngsdoth2mL) 4122178plusmn593331 5935337plusmn819676lowastlowast 6157161plusmn1329936lowastlowast 5635173plusmn1350868lowastlowast

MRT0997888rarrt (h) 18772plusmn0456 19343plusmn0976 20527plusmn0801lowastlowast 18962plusmn0716lowastlowastPlt001 compared with control group

Control GroupGroup B1Group B2Group B3

0

4

8

12

Con

cent

ratio

n (n

gm

l)

10 20 30 40 500Time (h)

Figure 3 Mean blood concentration-timeprofiles of sirolimus afterthe administration of TRQ at different time

Therefore it could be concluded that the TRQ significantlyenhanced the AUC of SRL when TRQ was administrated atthe same time with SRL or 2 hours after SRL administrationThis effect was not obvious when TRQ was administrated 4hours after SRL administration Besides the pharmacokineticprofiles of SRL also showed the similar curves in the groupsB1ndashB3 Based on the effect of different dose levels anddifferent dosing time of TRQ it could be found that theAUCsin groups A1-A3 and groups B1ndashB3 were higher than thosein control groups which indicated that TRQ might causeoverexposure of SRL in patients receiving SRL along withTRQ

SRL is a substrate for CYP3A and P-gp It is not sure thatthe five TCMs from TRQ or its main components inhibitthe hepatic and intestinal CYP3AP-gp system Howeverit have been reported by some literatures that baicalin amain component from Scutellariae Radix in TRQ couldinhibit hepatic CYP3A activity as well as P-glycoproteinefflux pump in the small intestine [20ndash22] Therefore theaffected SRL pharmacokinetics by TRQmight be mainly dueto the inhibition of CYP3A-mediated metabolism and P-gp-mediated transport by baicalin The complex mixture ofchemical constituents in herbal medicines or TCMs which

are usually believed to be medicinally efficacious are yet tobe fully characterized [23]There might be other compoundsresponsible for theHDImechanism In future further studieswill be needed to clarify this point

Actually like the pharmacokinetic profile of the meanvalue of SRL in the stable renal transplant patients [24]the control group of SRL did not show the obvious doublepeaks which were only found in the SRL-TRQ combinationtreatment groups Because of the extensive distribution in redblood cells of SRL (945) [24] zinc sulfate was applied asthe erythrolysis agent to release SRL in the circulating redblood cells In this study the absorption of SRL fluctuatedThe reason could be that the absorption rate of SRL varied indifferent parts of the intestine in rats [25] Thus the doublepeaks for SRL in the TRQ pretreatment groups might beattributed to the effect of chemical compounds from TRQ onthe elimination of SRL which should be investigated in ourfuture study

It was reported that SRL pharmacokinetics was associatedwithCYP3AandMDR1 genetic polymorphisms butwe failedto determine the relevant genetic information of the patientin the introduction If the patient expressed the CYP3A5enzyme (CYP3A5lowast1 carriers) he might need more SRL toreach target concentrations when compared with those withCYP3A5lowast3lowast3 carriers [26] It might lead to a risk of drugtoxicity especially when a potential HDI existed

4 Conclusions

A simple and accurate LC-MSMS method was developedand validated for SRL assay in rat blood following a quickPPT procedure which demonstrated good selectivity lin-earity precision and accuracy matrix effect and recoveryand stability This assay was successfully applied to evaluatethe pharmacokinetics of SRL when it was administratedalone and coadministrated with TRQ in rat The resultsshowed that different dose levels and different dosing time ofTRQ would increase SRL blood concentration and exposureindicating a potential HDI between TRQ and SRLThe studyprovided valuable information for SRL treatment from thepharmacokinetic point of view coadministration with TRQor baicalin-derived products was not recommended in clinicA systematical TDM for SRL was greatly encouraged forsafety and would help to discover a possible DDIHDI

6 BioMed Research International

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Feng Zhang and Liang Sun contributed equally to this workWansheng Chen and Yifeng Chai contributed equally to thiswork

Acknowledgments

Funding for this study was provided by National Natu-ral Science Foundation of China (81830109 and 81573793)Shanghai Key Specialty Project of Clinical Pharmacy (2016-40044-002) Important Weak Subject Construction Projectof Shanghai Health Science Education (2016ZB0303) andBeijing Medical Award Foundation (YJHYXKYJJ-122) Theauthors also greatly appreciate the kind help from Dr JunWen School of Pharmacy Second Military Medical Univer-sity

Supplementary Materials

Please fine the experimental protocol in supplementaryFigure S1 (Supplementary Materials)

References

[1] S N Sehgal ldquoRapamune (RAPA rapamycin sirolimus)Mechanism of action immunosuppressive effect results fromblockade of signal transduction and inhibition of cell cycleprogressionrdquo Clinical Biochemistry vol 31 no 5 pp 335ndash3401998

[2] N Terada J J Lucas A Szepesi R A Franklin J Domenicoand E W Gelfand ldquoRapamycin blocks cell cycle progression ofactivated T cells prior to events characteristic of the middle tolate G1 phase of the cyclerdquo Journal of Cellular Physiology vol154 no 1 pp 7ndash15 1993

[3] P R Shah V B KuteHV Patel andH L Trivedi ldquoTherapeuticdrugmonitoring of sirolimusrdquoClinical Queries Nephrology vol4 no 3-4 pp 44ndash49 2015

[4] F Shihab U Christians L Smith J R Wellen and B KaplanldquoFocus on mTOR inhibitors and tacrolimus in renal transplan-tation Pharmacokinetics exposure-response relationships andclinical outcomesrdquoTransplant Immunology vol 31 no 1 pp 22ndash32 2014

[5] Y Wang T Wang J Hu et al ldquoAnti-biofilm activity of TanRe-Qing a traditional Chinese Medicine used for the treatment ofacute pneumoniardquo Journal of Ethnopharmacology vol 134 no1 pp 165ndash170 2011

[6] P Wang X Liao YM Xie Y Chai and LH Li ldquoTanreqinginjection for acute bronchitis disease A systematic review andmeta-analysis of randomized controlled trialsrdquo ComplementTher Med vol 25 pp 143ndash158 2016

[7] L Sun H Wei F Zhang et al ldquoQualitative analysis and qualitycontrol of Traditional ChineseMedicine preparation Tanreqinginjection by LC-TOFMS and HPLC-DAD-ELSDrdquo AnalyticalMethods vol 5 no 22 pp 6431ndash6440 2013

[8] F Zhang L Sun SH Gao WS Chen and YF Chai ldquoLC-MSMS analysis and pharmacokinetic study on five bioactiveconstituents of Tanreqing injection in ratsrdquo Chinese Journal ofNature Medicines vol 14 pp 769ndash775 2016

[9] F Zhang L Sun SH Gao et al ldquoA sensitive HPLC-MSmethodfor simultaneous determination of thirteen components inrat plasma and its application to pharmacokinetic study ofTanreqing injectionrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 148 pp 205ndash213 2018

[10] B Sadaba M A Campanero E G Quetglas and J R AzanzaldquoClinical relevance of sirolimus drug interactions in transplantpatientsrdquo Transplantation Proceedings vol 36 no 10 pp 3226ndash3228 2004

[11] M Oellerich V W Armstrong F Streit L Weber and BTonshoff ldquoImmunosuppressive drug monitoring of sirolimusand cyclosporine in pediatric patientsrdquo Clinical Biochemistryvol 37 no 6 pp 424ndash428 2004

[12] T VanGelder ldquoDrug interactionswith tacrolimusrdquoDrug Safetyvol 25 no 10 pp 707ndash712 2002

[13] B C Sallustio B D Noll and R G Morris ldquoComparisonof blood sirolimus tacrolimus and everolimus concentrationsmeasured by LC-MSMS HPLC-UV and immunoassay meth-odsrdquo Clinical Biochemistry vol 44 no 2-3 pp 231ndash236 2011

[14] N Ansermot M Fathi J-L Veuthey J Desmeules SRudaz and D Hochstrasser ldquoSimultaneous quantification ofcyclosporine tacrolimus sirolimus and everolimus in wholeblood by liquid chromatography-electrospray mass spectrom-etryrdquo Clinical Biochemistry vol 41 no 9 pp 728ndash735 2008

[15] J-H Lee K-H ChaW Cho et al ldquoQuantitative determinationof sirolimus in dog blood using liquid chromatography-tandemmass spectrometry and its applications to pharmacokineticstudiesrdquo Journal of Pharmaceutical and Biomedical Analysis vol53 no 4 pp 1042ndash1047 2010

[16] D M Mueller and K M Rentsch ldquoSensitive quantification ofsirolimus and everolimus by LC-MSMS with online samplecleanuprdquo Journal of Chromatography B vol 878 no 13-14 pp1007ndash1012 2010

[17] N Mano M Sato M Nozawa et al ldquoAn accurate quantitativeLCESI-MSMS method for sirolimus in human whole bloodrdquoJournal of Chromatography B vol 879 no 13-14 pp 987ndash9922011

[18] A Navarrete M P Martınez-Alcazar I Duran et al ldquoSimul-taneous online SPE-HPLC-MSMS analysis of docetaxel Tem-sirolimus and sirolimus in whole blood and human plasmardquoJournal of Chromatography B vol 921-922 pp 35ndash42 2013

[19] Guidance for Industry Bioanalytical Method Validation USDepartment of Health and Human Services Food and DrugAdministration Center for Drug Evaluation and Research(CDER) Center for VeterinaryMedicine (CV) May 2001

[20] X Tian Z-Y Cheng J He L-J Jia and H-L QiaoldquoConcentration-dependent inhibitory effects of baicalin on themetabolism of dextromethorphan a dual probe of CYP2D andCYP3A in ratsrdquoChemico-Biological Interactions vol 203 no 2pp 522ndash529 2013

[21] Z Y Cheng X Tian J Gao L I HM L J Jia and HL Qiao ldquoContribution of baicalin on the plasma proteinbinding displacement and CYP3A activity inhibition to the

BioMed Research International 5

Table 2 Pharmacokinetic parameters of SRL after the administration of TRQ at different time

Parameter Control Group Group B1 Group B2 Group B3Tmax (h) 4167plusmn2563 5167plusmn3251 5001plusmn4472 3001plusmn2449Cmax (ngmL) 9487plusmn1479 12201plusmn1153lowastlowast 11643plusmn3648 12699plusmn347911990512 (h) 68144plusmn63013 47119plusmn43595 65408plusmn27871 33299plusmn14359AUC0997888rarrt (ngsdothmL) 219673plusmn32306 305875plusmn31417lowastlowast 301677plusmn73315lowastlowast 296211plusmn65286lowastlowast

AUC0997888rarrinfin (ngsdothmL) 493458plusmn271184 588668plusmn339317 694003plusmn91568 456371plusmn133544AUMC0997888rarrt (ngsdoth2mL) 4122178plusmn593331 5935337plusmn819676lowastlowast 6157161plusmn1329936lowastlowast 5635173plusmn1350868lowastlowast

MRT0997888rarrt (h) 18772plusmn0456 19343plusmn0976 20527plusmn0801lowastlowast 18962plusmn0716lowastlowastPlt001 compared with control group

Control GroupGroup B1Group B2Group B3

0

4

8

12

Con

cent

ratio

n (n

gm

l)

10 20 30 40 500Time (h)

Figure 3 Mean blood concentration-timeprofiles of sirolimus afterthe administration of TRQ at different time

Therefore it could be concluded that the TRQ significantlyenhanced the AUC of SRL when TRQ was administrated atthe same time with SRL or 2 hours after SRL administrationThis effect was not obvious when TRQ was administrated 4hours after SRL administration Besides the pharmacokineticprofiles of SRL also showed the similar curves in the groupsB1ndashB3 Based on the effect of different dose levels anddifferent dosing time of TRQ it could be found that theAUCsin groups A1-A3 and groups B1ndashB3 were higher than thosein control groups which indicated that TRQ might causeoverexposure of SRL in patients receiving SRL along withTRQ

SRL is a substrate for CYP3A and P-gp It is not sure thatthe five TCMs from TRQ or its main components inhibitthe hepatic and intestinal CYP3AP-gp system Howeverit have been reported by some literatures that baicalin amain component from Scutellariae Radix in TRQ couldinhibit hepatic CYP3A activity as well as P-glycoproteinefflux pump in the small intestine [20ndash22] Therefore theaffected SRL pharmacokinetics by TRQmight be mainly dueto the inhibition of CYP3A-mediated metabolism and P-gp-mediated transport by baicalin The complex mixture ofchemical constituents in herbal medicines or TCMs which

are usually believed to be medicinally efficacious are yet tobe fully characterized [23]There might be other compoundsresponsible for theHDImechanism In future further studieswill be needed to clarify this point

Actually like the pharmacokinetic profile of the meanvalue of SRL in the stable renal transplant patients [24]the control group of SRL did not show the obvious doublepeaks which were only found in the SRL-TRQ combinationtreatment groups Because of the extensive distribution in redblood cells of SRL (945) [24] zinc sulfate was applied asthe erythrolysis agent to release SRL in the circulating redblood cells In this study the absorption of SRL fluctuatedThe reason could be that the absorption rate of SRL varied indifferent parts of the intestine in rats [25] Thus the doublepeaks for SRL in the TRQ pretreatment groups might beattributed to the effect of chemical compounds from TRQ onthe elimination of SRL which should be investigated in ourfuture study

It was reported that SRL pharmacokinetics was associatedwithCYP3AandMDR1 genetic polymorphisms butwe failedto determine the relevant genetic information of the patientin the introduction If the patient expressed the CYP3A5enzyme (CYP3A5lowast1 carriers) he might need more SRL toreach target concentrations when compared with those withCYP3A5lowast3lowast3 carriers [26] It might lead to a risk of drugtoxicity especially when a potential HDI existed

4 Conclusions

A simple and accurate LC-MSMS method was developedand validated for SRL assay in rat blood following a quickPPT procedure which demonstrated good selectivity lin-earity precision and accuracy matrix effect and recoveryand stability This assay was successfully applied to evaluatethe pharmacokinetics of SRL when it was administratedalone and coadministrated with TRQ in rat The resultsshowed that different dose levels and different dosing time ofTRQ would increase SRL blood concentration and exposureindicating a potential HDI between TRQ and SRLThe studyprovided valuable information for SRL treatment from thepharmacokinetic point of view coadministration with TRQor baicalin-derived products was not recommended in clinicA systematical TDM for SRL was greatly encouraged forsafety and would help to discover a possible DDIHDI

6 BioMed Research International

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Feng Zhang and Liang Sun contributed equally to this workWansheng Chen and Yifeng Chai contributed equally to thiswork

Acknowledgments

Funding for this study was provided by National Natu-ral Science Foundation of China (81830109 and 81573793)Shanghai Key Specialty Project of Clinical Pharmacy (2016-40044-002) Important Weak Subject Construction Projectof Shanghai Health Science Education (2016ZB0303) andBeijing Medical Award Foundation (YJHYXKYJJ-122) Theauthors also greatly appreciate the kind help from Dr JunWen School of Pharmacy Second Military Medical Univer-sity

Supplementary Materials

Please fine the experimental protocol in supplementaryFigure S1 (Supplementary Materials)

References

[1] S N Sehgal ldquoRapamune (RAPA rapamycin sirolimus)Mechanism of action immunosuppressive effect results fromblockade of signal transduction and inhibition of cell cycleprogressionrdquo Clinical Biochemistry vol 31 no 5 pp 335ndash3401998

[2] N Terada J J Lucas A Szepesi R A Franklin J Domenicoand E W Gelfand ldquoRapamycin blocks cell cycle progression ofactivated T cells prior to events characteristic of the middle tolate G1 phase of the cyclerdquo Journal of Cellular Physiology vol154 no 1 pp 7ndash15 1993

[3] P R Shah V B KuteHV Patel andH L Trivedi ldquoTherapeuticdrugmonitoring of sirolimusrdquoClinical Queries Nephrology vol4 no 3-4 pp 44ndash49 2015

[4] F Shihab U Christians L Smith J R Wellen and B KaplanldquoFocus on mTOR inhibitors and tacrolimus in renal transplan-tation Pharmacokinetics exposure-response relationships andclinical outcomesrdquoTransplant Immunology vol 31 no 1 pp 22ndash32 2014

[5] Y Wang T Wang J Hu et al ldquoAnti-biofilm activity of TanRe-Qing a traditional Chinese Medicine used for the treatment ofacute pneumoniardquo Journal of Ethnopharmacology vol 134 no1 pp 165ndash170 2011

[6] P Wang X Liao YM Xie Y Chai and LH Li ldquoTanreqinginjection for acute bronchitis disease A systematic review andmeta-analysis of randomized controlled trialsrdquo ComplementTher Med vol 25 pp 143ndash158 2016

[7] L Sun H Wei F Zhang et al ldquoQualitative analysis and qualitycontrol of Traditional ChineseMedicine preparation Tanreqinginjection by LC-TOFMS and HPLC-DAD-ELSDrdquo AnalyticalMethods vol 5 no 22 pp 6431ndash6440 2013

[8] F Zhang L Sun SH Gao WS Chen and YF Chai ldquoLC-MSMS analysis and pharmacokinetic study on five bioactiveconstituents of Tanreqing injection in ratsrdquo Chinese Journal ofNature Medicines vol 14 pp 769ndash775 2016

[9] F Zhang L Sun SH Gao et al ldquoA sensitive HPLC-MSmethodfor simultaneous determination of thirteen components inrat plasma and its application to pharmacokinetic study ofTanreqing injectionrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 148 pp 205ndash213 2018

[10] B Sadaba M A Campanero E G Quetglas and J R AzanzaldquoClinical relevance of sirolimus drug interactions in transplantpatientsrdquo Transplantation Proceedings vol 36 no 10 pp 3226ndash3228 2004

[11] M Oellerich V W Armstrong F Streit L Weber and BTonshoff ldquoImmunosuppressive drug monitoring of sirolimusand cyclosporine in pediatric patientsrdquo Clinical Biochemistryvol 37 no 6 pp 424ndash428 2004

[12] T VanGelder ldquoDrug interactionswith tacrolimusrdquoDrug Safetyvol 25 no 10 pp 707ndash712 2002

[13] B C Sallustio B D Noll and R G Morris ldquoComparisonof blood sirolimus tacrolimus and everolimus concentrationsmeasured by LC-MSMS HPLC-UV and immunoassay meth-odsrdquo Clinical Biochemistry vol 44 no 2-3 pp 231ndash236 2011

[14] N Ansermot M Fathi J-L Veuthey J Desmeules SRudaz and D Hochstrasser ldquoSimultaneous quantification ofcyclosporine tacrolimus sirolimus and everolimus in wholeblood by liquid chromatography-electrospray mass spectrom-etryrdquo Clinical Biochemistry vol 41 no 9 pp 728ndash735 2008

[15] J-H Lee K-H ChaW Cho et al ldquoQuantitative determinationof sirolimus in dog blood using liquid chromatography-tandemmass spectrometry and its applications to pharmacokineticstudiesrdquo Journal of Pharmaceutical and Biomedical Analysis vol53 no 4 pp 1042ndash1047 2010

[16] D M Mueller and K M Rentsch ldquoSensitive quantification ofsirolimus and everolimus by LC-MSMS with online samplecleanuprdquo Journal of Chromatography B vol 878 no 13-14 pp1007ndash1012 2010

[17] N Mano M Sato M Nozawa et al ldquoAn accurate quantitativeLCESI-MSMS method for sirolimus in human whole bloodrdquoJournal of Chromatography B vol 879 no 13-14 pp 987ndash9922011

[18] A Navarrete M P Martınez-Alcazar I Duran et al ldquoSimul-taneous online SPE-HPLC-MSMS analysis of docetaxel Tem-sirolimus and sirolimus in whole blood and human plasmardquoJournal of Chromatography B vol 921-922 pp 35ndash42 2013

[19] Guidance for Industry Bioanalytical Method Validation USDepartment of Health and Human Services Food and DrugAdministration Center for Drug Evaluation and Research(CDER) Center for VeterinaryMedicine (CV) May 2001

[20] X Tian Z-Y Cheng J He L-J Jia and H-L QiaoldquoConcentration-dependent inhibitory effects of baicalin on themetabolism of dextromethorphan a dual probe of CYP2D andCYP3A in ratsrdquoChemico-Biological Interactions vol 203 no 2pp 522ndash529 2013

[21] Z Y Cheng X Tian J Gao L I HM L J Jia and HL Qiao ldquoContribution of baicalin on the plasma proteinbinding displacement and CYP3A activity inhibition to the

6 BioMed Research International

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Feng Zhang and Liang Sun contributed equally to this workWansheng Chen and Yifeng Chai contributed equally to thiswork

Acknowledgments

Funding for this study was provided by National Natu-ral Science Foundation of China (81830109 and 81573793)Shanghai Key Specialty Project of Clinical Pharmacy (2016-40044-002) Important Weak Subject Construction Projectof Shanghai Health Science Education (2016ZB0303) andBeijing Medical Award Foundation (YJHYXKYJJ-122) Theauthors also greatly appreciate the kind help from Dr JunWen School of Pharmacy Second Military Medical Univer-sity

Supplementary Materials

Please fine the experimental protocol in supplementaryFigure S1 (Supplementary Materials)

References

[1] S N Sehgal ldquoRapamune (RAPA rapamycin sirolimus)Mechanism of action immunosuppressive effect results fromblockade of signal transduction and inhibition of cell cycleprogressionrdquo Clinical Biochemistry vol 31 no 5 pp 335ndash3401998

[2] N Terada J J Lucas A Szepesi R A Franklin J Domenicoand E W Gelfand ldquoRapamycin blocks cell cycle progression ofactivated T cells prior to events characteristic of the middle tolate G1 phase of the cyclerdquo Journal of Cellular Physiology vol154 no 1 pp 7ndash15 1993

[3] P R Shah V B KuteHV Patel andH L Trivedi ldquoTherapeuticdrugmonitoring of sirolimusrdquoClinical Queries Nephrology vol4 no 3-4 pp 44ndash49 2015

[4] F Shihab U Christians L Smith J R Wellen and B KaplanldquoFocus on mTOR inhibitors and tacrolimus in renal transplan-tation Pharmacokinetics exposure-response relationships andclinical outcomesrdquoTransplant Immunology vol 31 no 1 pp 22ndash32 2014

[5] Y Wang T Wang J Hu et al ldquoAnti-biofilm activity of TanRe-Qing a traditional Chinese Medicine used for the treatment ofacute pneumoniardquo Journal of Ethnopharmacology vol 134 no1 pp 165ndash170 2011

[6] P Wang X Liao YM Xie Y Chai and LH Li ldquoTanreqinginjection for acute bronchitis disease A systematic review andmeta-analysis of randomized controlled trialsrdquo ComplementTher Med vol 25 pp 143ndash158 2016

[7] L Sun H Wei F Zhang et al ldquoQualitative analysis and qualitycontrol of Traditional ChineseMedicine preparation Tanreqinginjection by LC-TOFMS and HPLC-DAD-ELSDrdquo AnalyticalMethods vol 5 no 22 pp 6431ndash6440 2013

[8] F Zhang L Sun SH Gao WS Chen and YF Chai ldquoLC-MSMS analysis and pharmacokinetic study on five bioactiveconstituents of Tanreqing injection in ratsrdquo Chinese Journal ofNature Medicines vol 14 pp 769ndash775 2016

[9] F Zhang L Sun SH Gao et al ldquoA sensitive HPLC-MSmethodfor simultaneous determination of thirteen components inrat plasma and its application to pharmacokinetic study ofTanreqing injectionrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 148 pp 205ndash213 2018

[10] B Sadaba M A Campanero E G Quetglas and J R AzanzaldquoClinical relevance of sirolimus drug interactions in transplantpatientsrdquo Transplantation Proceedings vol 36 no 10 pp 3226ndash3228 2004

[11] M Oellerich V W Armstrong F Streit L Weber and BTonshoff ldquoImmunosuppressive drug monitoring of sirolimusand cyclosporine in pediatric patientsrdquo Clinical Biochemistryvol 37 no 6 pp 424ndash428 2004

[12] T VanGelder ldquoDrug interactionswith tacrolimusrdquoDrug Safetyvol 25 no 10 pp 707ndash712 2002

[13] B C Sallustio B D Noll and R G Morris ldquoComparisonof blood sirolimus tacrolimus and everolimus concentrationsmeasured by LC-MSMS HPLC-UV and immunoassay meth-odsrdquo Clinical Biochemistry vol 44 no 2-3 pp 231ndash236 2011

[14] N Ansermot M Fathi J-L Veuthey J Desmeules SRudaz and D Hochstrasser ldquoSimultaneous quantification ofcyclosporine tacrolimus sirolimus and everolimus in wholeblood by liquid chromatography-electrospray mass spectrom-etryrdquo Clinical Biochemistry vol 41 no 9 pp 728ndash735 2008

[15] J-H Lee K-H ChaW Cho et al ldquoQuantitative determinationof sirolimus in dog blood using liquid chromatography-tandemmass spectrometry and its applications to pharmacokineticstudiesrdquo Journal of Pharmaceutical and Biomedical Analysis vol53 no 4 pp 1042ndash1047 2010

[16] D M Mueller and K M Rentsch ldquoSensitive quantification ofsirolimus and everolimus by LC-MSMS with online samplecleanuprdquo Journal of Chromatography B vol 878 no 13-14 pp1007ndash1012 2010

[17] N Mano M Sato M Nozawa et al ldquoAn accurate quantitativeLCESI-MSMS method for sirolimus in human whole bloodrdquoJournal of Chromatography B vol 879 no 13-14 pp 987ndash9922011

[18] A Navarrete M P Martınez-Alcazar I Duran et al ldquoSimul-taneous online SPE-HPLC-MSMS analysis of docetaxel Tem-sirolimus and sirolimus in whole blood and human plasmardquoJournal of Chromatography B vol 921-922 pp 35ndash42 2013

[19] Guidance for Industry Bioanalytical Method Validation USDepartment of Health and Human Services Food and DrugAdministration Center for Drug Evaluation and Research(CDER) Center for VeterinaryMedicine (CV) May 2001

[20] X Tian Z-Y Cheng J He L-J Jia and H-L QiaoldquoConcentration-dependent inhibitory effects of baicalin on themetabolism of dextromethorphan a dual probe of CYP2D andCYP3A in ratsrdquoChemico-Biological Interactions vol 203 no 2pp 522ndash529 2013

[21] Z Y Cheng X Tian J Gao L I HM L J Jia and HL Qiao ldquoContribution of baicalin on the plasma proteinbinding displacement and CYP3A activity inhibition to the

BioMed Research International 7

pharmacokinetic changes of nifedipine in rats in vivo and invitrordquo PLoS One vol 9 article e87234 2014

[22] Y-A Cho J-S Choi and J-P Burm ldquoEffects of the antioxidantbaicalein on the pharmacokinetics of nimodipine in rats Apossible role of P-glycoprotein and CYP3A4 inhibition bybaicaleinrdquo Pharmacological Reports vol 63 no 4 pp 1066ndash1073 2011

[23] E F Oga S Sekine Y Shitara and T Horie ldquoPharmacoki-netic Herb-Drug Interactions Insight into Mechanisms andConsequencesrdquo European Journal of Drug Metabolism andPharmacokinetics vol 41 no 2 pp 93ndash108 2016

[24] K Mahalati and B D Kahan ldquoClinical pharmacokinetics ofsirolimusrdquoClinical Pharmacokinetics vol 40 no 8 pp 573ndash5852001

[25] J Wang H T Song X Zhou and Q Zhang ldquoIn Situ intestinalabsorption of Sirolimus in ratsrdquo China Pharmacy vol 20 pp27ndash30 2009

[26] D AnglicheauD LeCorre S Lechatonet al ldquoConsequences ofgenetic polymorphisms for sirolimus requirements after renaltransplant in patients on primary sirolimus therapyrdquo AmericanJournal of Transplantation vol 5 no 3 pp 595ndash603 2005

Medicinal ChemistryInternational Journal of

Hindawiwwwhindawicom Volume 2018

ToxicologyJournal of

Hindawiwwwhindawicom Volume 2018

PainResearch and TreatmentHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Arthritis

Neurology Research International

Hindawiwwwhindawicom Volume 2018

StrokeResearch and TreatmentHindawiwwwhindawicom Volume 2018

Drug DeliveryJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawiwwwhindawicom Volume 2018

AddictionJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Emergency Medicine InternationalHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Anesthesiology Research and Practice

Journal of

Hindawiwwwhindawicom Volume 2018

Pharmaceutics

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Infectious Diseases and Medical Microbiology

Hindawiwwwhindawicom Volume 2018

Canadian Journal of

Hindawiwwwhindawicom Volume 2018

Autoimmune DiseasesScientica

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

Submit your manuscripts atwwwhindawicom

Medicinal ChemistryInternational Journal of

Hindawiwwwhindawicom Volume 2018

ToxicologyJournal of

Hindawiwwwhindawicom Volume 2018

PainResearch and TreatmentHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Arthritis

Neurology Research International

Hindawiwwwhindawicom Volume 2018

StrokeResearch and TreatmentHindawiwwwhindawicom Volume 2018

Drug DeliveryJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawiwwwhindawicom Volume 2018

AddictionJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Emergency Medicine InternationalHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Anesthesiology Research and Practice

Journal of

Hindawiwwwhindawicom Volume 2018

Pharmaceutics

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Infectious Diseases and Medical Microbiology

Hindawiwwwhindawicom Volume 2018

Canadian Journal of

Hindawiwwwhindawicom Volume 2018

Autoimmune DiseasesScientica

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

Submit your manuscripts atwwwhindawicom