邵淑容　董　輝　李亞東　劉　正　曲衛(wèi)敏,4△

(1復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院藥理學(xué)系, 2 醫(yī)學(xué)神經(jīng)生物學(xué)國家重點(diǎn)實(shí)驗(yàn)室　上海　200032;3 紹興文理學(xué)院醫(yī)學(xué)院藥理學(xué)系　紹興　312000; 4 復(fù)旦大學(xué)腦科學(xué)研究院與協(xié)同創(chuàng)新中心　上?！?00032)

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紅景天苷在大鼠懸尾模型中的藥代動力學(xué)研究

邵淑容1董輝2李亞東1劉正3曲衛(wèi)敏1,4△

(1復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院藥理學(xué)系,2醫(yī)學(xué)神經(jīng)生物學(xué)國家重點(diǎn)實(shí)驗(yàn)室上海200032;3紹興文理學(xué)院醫(yī)學(xué)院藥理學(xué)系紹興312000;4復(fù)旦大學(xué)腦科學(xué)研究院與協(xié)同創(chuàng)新中心上海200032)

目的采用大鼠懸尾模型模擬失重狀態(tài),研究紅景天苷在微重力狀態(tài)下的藥代動力學(xué)變化。方法建立大鼠懸尾模擬微重力模型,分別在第1、2和5天灌胃給予紅景天苷 (100 mg/kg),HPLC法檢測紅景天苷血藥濃度。結(jié)果血漿中加紅景天苷標(biāo)準(zhǔn)品,在流動相 (甲醇:水=80∶20,流速為1 mL/min)中的保留時間為5.96 min。紅景天苷濃度在1～100 μg/mL線性相關(guān)性良好 (r=0.999 9),最低檢測限為1 μg/mL。對紅景天苷濃度為2、10、50 μg/mL的質(zhì)控樣品溶液進(jìn)行精密度和準(zhǔn)確度評價,日內(nèi)、日間誤差均≤15%。血漿樣品的平均回收率均≥90%。與對照組相比,懸尾5天的大鼠灌胃給藥后,紅景天苷的血漿峰濃度和藥時曲線下面積分別增加63%和36%;藥物清除率減少24.81%。而懸尾1天和懸尾2天的大鼠給藥后未見差異。結(jié)論懸尾模擬微重力增加紅景天苷在大鼠體內(nèi)的分布,降低清除速度,提示在太空服用該藥物時可能需要調(diào)整劑量。

藥代動力學(xué);大鼠;紅景天苷;模擬微重力;懸尾

As space flight becomes more commonplace,the influences of physiological changes associated with weightlessness environments are of interest.Exposure to weightlessness has been shown to have numerous effects on body composition and organ function in animals and humans[1-3].Also,multiple drugs have been used during manned space flight to combat various flight-related medical disorders such as space motion sickness,sleeplessness,nasal congestion,headache,back pain and constipation[4].Meanwhile,drug pharmacokinetics may differ significantly at weightlessness due to the physiological changes under these conditions, such as fluid redistribution and loss,electrolyte imbalance,and cardiovascular changes[5-9].Therefore,screening drugs by group-based model that can be used in space is becoming increasingly important.

Salidroside,an active ingredient in Rhodiola,may have potential use as an aviation medicine (structure,Fig 1).Studies of isolated organs,tissues,cells,enzymes and those in people,reveal that salidroside (Rhodiola preparations) may has neuroprotective[10],cardioprotective[11],anti-fatigue[12],antidepressant,anxiolytic,CNS stimulating[13]and life-span increasing activity[14].In addition,Rhodiola was given to astronauts in the former Soviet Union to improve endurance and adaptability and increase physical activity[15].Thus,salidroside may be useful for space flight.

Given that there are few space flights and conducting experiments during missions is inherently difficult,earthbound models for simulating weightlessness are used to study physiological and pharmacological modifications observed during flight.The most common model to simulate space flight is the rat tail-suspended model which was developed to study musculoskeletal,cardiovascular,metabolic changes[16],blood flow[17]and oxygen consumption[18]caused by weight-lessness.The physiological responses to spaceflight that this model mimics include induced cephalic fluid shifts,weight gain,and hind limb unloading without paralysis,in addition to muscle atrophy[19-22].Therefore,this model was used to study pharmacokinetics of salidroside.

Fig 1Chemical structure of salidroside

Materials and Methods

AnimalsMale Sprague-Dawley rats of (300±20) g,provided by Sino-British SIPPR/BK Lab Animal Co.,Ltd (Shanghai,China),were indivi-dually housed in 12-h light/dark cycle (light on at 07:00,illumination intensity ≈ 100 lux),ambient temperature of (24±0.5)°C and a relative humidity of 60%±2%,and had free access to food and water.The animals were fasted with free access to water for 12 h prior to salidroside administration in each experiment.All procedures were approved by the Medical Experimental Animal Administrative Committee of Shanghai.Every effort was made to minimize the number of animals used and any pain and discomfort experienced by the subjects.

Tail-suspension procedureWeightlessness was simulated using a modified version of the tail-suspended rat model[23].After anesthetic induction with diethyl ether,rats were placed supine and their tails lightly cleaned with 75% ethanol.A plastic string was fastened to each tail with adhesive tape and multiple 5 cm strips (1.25 cm wide) orthopedic paper tape (Micropore;3M Corporation,St.Paul,MN,USA).Approximately 5 cm of the distal end of the tail remained uncovered and there was a visual marker of blood circulation throughout the tail.The suspension string was attached to a swivel that was placed on the top of the cage.The arrangement allowed the rat to freely move in the cage using its forelimbs only.Hind limbs were left unsupported;hence,hind limbs were not weight-bearing.Rats were suspended at 30° from the cage floor,and monitored daily for position.Control rats were similarly prepared,but did not have a suspension apparatus attached to their tails.The start of the tail-suspension represented day 0 of the study period.

Animal surgeryAfter the tail-suspension period,catheters (inner diameter 0.6 mm,outer diameter 0.8 mm,length 20 cm) were surgically implanted during anesthesia with diethyl ether into the right external jugular vein and exteriorized at the skull base.Rats kept their position (freely moving or tail-suspended position) and were allowed to recover for more than 2 h after catheter placement prior to drug administration.

Chemicals and reagentsSalidroside was purchased from Shanghai Maicang Biological Technology Co.,Ltd (Shanghai,China).Caffeine was purchased from Alfa Aesar (London,UK).Heparin,diethyl ether,and perchloric acid were all purchased from Chinese National Medicine group (Shanghai,China).HPLC-grade methanol was obtained from Tedia Ltd (Carson city,CA,USA).All aqueous solutions including HPLC mobile phase buffer were prepared with Milli-Q (Millipore,Boston,MA) grade water.

ChromatographyThe HPLC system consisted of a series L-2130 pump,a model L-2200 auto-sampler with sample cooler,and a model L-4200 detector (Hitachi,Japan).Data were acquired and processed using D-2000 Elite Workstation Software (Hitachi,Japan).The analytical column was a Spursil C18column,150 mm×4.6 mm,5 μm particle size (Dikma,Beijing,China) fitted with a guard cartridge packed with the same stationary phase.Samples were analyzed with mobile phase contained by methanol and water (80∶20) with a flow rate of 1 mL/min,column temperature at 25 ℃.The eluate was monitored by an ultraviolet detector set at 277 nm.Injection volume was 20 μL.Caffeine was used as internal standard for plasma samples.

Sample preparation10 μL of 20 μg/mL internal standard solution was added to 100 μL of plasma and vortexed for 15 s.After the addition of 100 μL 0.4 mol/L HClO4,the mixture was vortexed for 2 min,flowed by centrifugation for 10 min at 16 000×g at 4 ℃.Finally,100 μL supernate was filtered with nylon membrane (Φ=0.45 μm) into Eppendorf tubes to assay.

Calibration curvesCalibration cures were acquired by plotting the peak area ratio of salidroside:caffeine against the nominal concent-ration of calibration standards.Concentrations used were 1,2,5,10,20,50 and 100 μg/mL.Results were fitted to linear regression analysis using 1/X as weighting factor.

Precision and accuracyIntra-assay precision and accuracy were estimated by analyzing three replicates containing salidroside at three different quality control (QC) levels,i.e.2,10 and 50 μg/mL.Inter-assay precision was determined by analyzing three levels of QC samples on three different runs.Criteria for acceptability of the data included accuracy within ±15% deviation (DEV) from the nominal values and precision within 15% relative standard deviation (RSD) (United States Pharmacopeia,1995).

Stability of experimentsThe stability of QC samples of salidroside and internal standard caffeine in rat plasma in freezer conditions,room-temperature and automatic samplers were assessed.Peak-areas of analytes obtained at the initial cycle were used as a reference to assess relative stability at subsequent points.Samples were considered to be stable if assay values were within the acceptable limits of accuracy (i.e.±15% DEV) and precision (i.e.±15% RSD) except for lowest limit of quantification (LLOQ),where it should not exceed 20% of coefficient of variation (CV) (United States Pharmacopeia,1995).

Extraction recoveryTwo sets of standards containing analytes at 3 different concentrations (2,10,50 μg/mL) were prepared.One set was prepared in rat plasma and the other was prepared in double-diluted water (neat set).The recovery was determined by comparing peak areas of spiked plasma extracts with those of unextracted neat standards prepared in Milli-Q grade water.The recovery value was calculated at the various concentrations of salidroside.

Pharmacokinetic studiesA total of 24 rats were used for the salidroside pharmacokinetic evaluation.Experiments were performed on day 1,2 or 5 of the study period for 4 groups:free suspension (FS) group,tail suspension (TS) 1 d group,TS 2 d or 5 d group.Each group included six rats.After a 12-h fasting and the post-surgery acclimation period,the rat was weighed,returned to the cage and given salidroside (100 mg/kg) by oral gavage through an 18-gauge feeding needle (10 mL/kg drug solution) on day 1,2 or 5,respectively.Serial blood samples (300 μL) were obtained from the jugular catheter before and at 5,10,15,30,60,90,120,180,240 and 300 min into microcentrifuge tubes containing heparin after salidroside administration.Plasma (100 μL) was harvested by centrifuging the blood using Biofuge (Eppendorf,German) at 3 600×g for 10 min.Plasma (100 μL) samples spiked with 10 μL of 20 μg/mL internal standard caffeine were then frozen immediately at -80 ℃ until assayed.

Data analysisPharmacokinetics were calculated by non-compartmental analysis (NCA) using the program DAS 3.0.Pharmacokinetics included area under the concentration curves to 5 h and to infinity (AUC0→5,AUC0→∞),mean retention time to 5 h and to infinity (MRT0→5,MRT0→∞),clearance rate (CLz/F),apparent volume of distribution (Vz/F),maximum measured plasma concentration (Cmax) and its time of maximum concentration (Tmax) and half-life (t1/2z).Analyses of variance (ANOVAs) were performed,assuming normal distribution,on log-transformed pharmacokinetic parameters of AUC0→∞,AUC0→5,MRT0→∞,MRT0→5,Cmax,CLz/F,Vz/F.However,Tmaxandt1/2zwere tested via the Wilcoxon signed rank test.A 5% level of significance was used for all statistical comparisons.

Results

Specificity of chromatographyUnder chosen chromatographic conditions,specificity was indicated by the absence of endogenous interference at peak of interest retention as evaluated by chromatograms of blank rat plasma and plasma spiked with salidroside and caffeine.Both analyte and caffeine were well separated (retention times 5.96 and 10.51 min,respectively;Fig 2A).System suitability parameters for methods were as follows:theoretical plates for salidroside > 4 919 and for caffeine > 15 298,asymmetry factor<1.2 and resolution between salidroside and caffeine >5.UV spectra peak of caffeine overlaped with target component around 270 nm and no interference occurred with chromatographic separation around 270 nm[24-25].Thus,caffeine was selected as an internal standard.Fig.2A shows a typical overlaid chromatogram for salidroside standard solution,control rat plasma (free of analyte and caffeine) and rat plasma spiked with salidroside and caffeine.

Calibration curvePeak area ratios of salidroside to caffeine were measured and acted as a quantitative surrogate.A representative calibration graph of peak-area ratio ρ (salidroside to caffeine)vs.salidroside concentration (C) in the range 1 to 100 μg/mL resulted in the regression equation (Fig 2B):ρ=6.54×10-2C+4.08×10-2(R2=0.999 2,n=6).The standard curve had a reliable reproducibility over the standard concentrations of analyte across the calibration range.The signal-to-noise ratio at LLOQ was > 5 (Fig 2C).

A:Salidroside standard,blank plasma,plasma spiked with 160 μg/mL salidroside (tR=5.96 min) and 200 μg/mL internal standard,caffeine(n=5).B:Calibration curve of salidroside in rat plasma.Absorbancesa/Absorbancecarepresents the ratio between the absorbance of salidroside standard and the internal standard,caffeine(n=5).C:Limit of quantification (LOQ) of salidroside in rat plasma,which shows the concentration of salidroside when S/N=10 (S:Signal,N:Noise)(n=5).

Fig 2Representative chromatograms of salidroside in rat plasma

Accuracy and precisionAccuracy and precision data for within-and between-run plasma test samples are presented in Table 1.The intra-day accuracy was 92.86%,94.31% and 95.62% at 2,10 and 50 μg/mL,respectively.The inter-day accuracy was 92.69%,92.41% and 96.76% at 2,10 and 50 μg/mL,respectively.The results indicated that accuracy and precision met the requirements of validation.

Stability of experimentsTo measure stability,salidroside was stored at -80 ℃ for 5,10,15 and 20 days at concentrations of 2,10 and 50 μg/mL.The accuracy of QC samples at 2,10 and 50 μg/mL concentrations were 92.86%,94.31% and 95.62%,respectively.The precision of QC samples at 2,10 and 50 μg/mL concentrations were 9.91%,5.55% and 5.73%,respectively.The accuracy and precision of QC samples in this evaluation were both within the assay variability of ±10% (Tab 2).In addition,the stability of salidroside was evaluated in rat plasma in room-temperature and automatic samplers.The stability of internal standard caffeine was also determined in the above three conditions mentioned above.All data were in the range of requirements (data not shown).

Tab 1　Accuracy of within-and between-run assays of salidroside in rat plasma　±s)

For each concentration,n=5.conc:Concentration;RSD:Relative standard deviation.

Tab 2　Freezer stability of salidroside in rat plasma

For each concentration,n=5.conc:Concentration;RSD:Relative standard deviation.

Extraction recoveryIn comparison of results of neat standards and plasma-extracted standards,concentrations were estimated to be 2,10 and 50 μg/mL.The absolute recoveries ranged from 94.69% to 97.95% across the concentrations (Tab 3).The absolute recovery of internal standard at 2 μg/mL was >100%.The absolute recoveries of QC samples in this evaluation were both within the assay variability of ±10%.

Tab 3　Recovery of salidroside in rat plasma　±s)

For each concentration,n=5.conc:Concentration;RSD:Relative standard deviation.

Salidroside pharmacokineticsIn order to investigate the pharmacokinetic parameters of salidroside in simulated weightless rat model,salidroside (100 mg/kg) was i.g.administered into rats on day 1,2 and 5 after tail suspension,respectively.We measured body weight,and found that there was no significant difference on body weight between control and suspended rats over the 5 days of the experiment (data not shown).Tab 4 and 5 showed that absorption,distribution and elimination phases of salidroside changed under simulated weightlessness.The mean plasma time-concentration curves of salidroside following the administration of 100 mg/kg in rats subjected to tail suspension for 1,2 or 5 days were shown in Fig 3.The analysis of variance showed a significant increase in the meanCmaxby 25.10%,28.74% and 62.55% in TS 1 d,TS 2 d,TS 5 d (Tab 4),respectively,as compared with control.Tmaxwas not significantly different over the study period (Tab 4,Fig 3).As compared with the FS group,salidroside elevated AUC(0→t)by 7.89%,20.86% and 29.70% on day 1,2,5 in the TS group,respectively.CL/F was reduced in the TS group by 8.82%,13.71% and 24.81% on day 1,2 and 5,respectively.MRT(0→t)was reduced in the TS group by 10.12%,4.81%,19.12% on day 1,2 and 5,respectively.The V/F,t1/2and kawere not significantly different among the four rat groups over the study period (Tab 5,Fig 3).These results suggested that the absorption,distribution and elimination phase of salidroside have changed under simulated weightlessness rat model.

Discussion

Tab 4　Cmax and Tmax after administration of salidroside (100 mg/kg) in control and tail-suspended rats

Tab 5　Mean plasma pharmacokinetics after salidroside (100 mg/kg) in control and tail-suspended rats

(1)P<0.05 indicates significant differences with FS group.One-way ANOVA was used to analyse PK parameters followed by the Student-Newman-Keal′s Post Hoc test.AUC:Area under the concentration curves;MRT:Mean retention time;CLz/F:Clearance rate;Vdz/F:Apparent volume of distribution;t1/2z:Half-life.

Fig3Meanplasmaconcentration-timeprofilesaftergavage

administrationofsalidroside(100mg/kg)inratssubjected

totailsuspensionfor1,2or5days

stered salidroside.The results showed that salidroside had greaterCmaxand AUC,but reduced oral clearance in the tail suspension rat model on the 5thday.Thus,simulated weightlessness alters the pharmacokinetics of salidroside.

PlasmaCmaxand AUC of salidroside increased with the duration of tail suspension and this revealed that salidroside absorption increased.An increasedCmaxmay be explained by reduced body water or greater intestinal absorption.We gave salidroside via gavage and noted decreased Vssduring tail suspension but this did not reach statistical significance.As Vssdecreased with tail-suspended time,plasma concentration increased perhaps due to cardiovascular variation in weightlessness.A lower total peripheral resistance has been observed after 10 min in the supine position compared with sitting[27]and standing[5]and after 6 h in a -5° head-down tilted position compared with a standing position[28].When total peripheral resistance decreases,blood flow increases.Salidroside is water soluble,so increased blood flow may increaseCmax.Increased plasma concentration for drugs with narrow therapeutic indices may cause unanticipated toxicity due to physiological changes during weightlessness.Use of salidroside during space flight may require dose adjustment or a novel dosage form may be created to circumvent space flight hemodynamic alterations.

In tail-suspended rats,body clearance (CL/F) of salidroside decreased relative to treatment-matched controls,especially after 5 days of tail-suspension.DecreasedCL/F occurred concomitantly with increased AUC.These findings may be explained by decreased hepatic oxidative function in tail-suspended rats,and data appear to agree with the evaluation of hepatic function in rats after actual space flight.Rats aboard the Cosmos 1887[29]and Skylab 3[30]had significant less total hepatic cytochrome P-450 content,suggesting that drug metabolism may be reduced.TotalCL/F is the fraction of volume of distribution (Vd/F),which is completely purified per unit of time.Total clearance depends on the constant of elimination (ke) and Vd/F.The constant of elimination indicates body total elimination,including the metabolism and the renal excretion rate constant.Salidroside has been reported to be extensively hepatically metabolized toP-tyrosol and urinary elimination is a major route for excretion after gavage administration[31].Therefore,less salidroside metabolism in the tail-suspended rat model may reduce total clearance.The unchangedtmaxandt1/2indicate that absorption and elimination were not modified.

In the present studies,tail suspension significantly increasedCmaxand AUC of oral salidroside,and this was likely related to decreaseCL/F in rats.These findings suggest that the dosage of salidroside should be modified when used to improve astronaut health in the space,but data should be validated during actual space flight.

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E-mail:quweimin@fudan.edu.cn

Pharmacokinetics of salidroside in a tail-suspended rat model

SHAO Shu-rong1,DONG Hui2,LI Ya-dong1,LIU Zheng3,QU Wei-min1,4 △

(1DepartmentofPharmacology,2StateKeyLaboratoryofMedicalNeurobiology,SchoolofBasicMedicalSciences,FudanUniversity,Shanghai200032,China;3DepartmentofPharmacology,MedicalCollegeofShaoxingUniversity,Shaoxing312000,ZhejiangProvince,China;4InstitutesofBrainScienceandtheCollaborativeInnovationCenterforBrainScience,FudanUniversity,Shanghai200032,China)

ObjectiveTo measure the salidroside′s pharmacokinetics in a simulated weightlessness rat model.MethodsRats were given salidroside (via gavage,100 mg/kg) on the 1st,2nd,and 5thday of tail-suspension.Pharmacokinetic parameters were measured by HPLC.ResultsPlasma retention of salidroside was 5.96 min in a methanol and water (80∶20) mobile phase (flow 1 mL/min).When salidroside concentrations ranged from 1 to 100 μg/mL,they were clinically relevant (r=0.999 9),and quantification limits were 1 μg/mL in rat plasma.The within- and between-day errors were less than 15% in quality control samples of salidroside at 2,10 and 50 μg/mL,respectively.Salidroside mean recovery exceeded 90% in rat plasma.On the 5thday of tail-suspension rats,peak concentration (Cmax) and area under the curve (AUCs) for salidroside increased by 63% and 36%,respectively,and clearance rate of salidroside decreased by 24.81%,as compared with control.While no change was found in tail-suspension rats on the 1stday and 2ndday.ConclusionsSimulated weightlessness

pharmacokinetics;rats;salidroside;simulated weightlessness;tail-suspension

R965.1, R969.1

Adoi: 10.3969/j.issn.1672-8467.2016.04.003

2016-01-13;編輯:張秀峰)

國家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃 (2011CB711000);國家自然科學(xué)基金 (31471064);紹興市科技計(jì)劃項(xiàng)目(2014A230109)

significantly increased distribution and decreased clearance rate of oral salidroside in rats,suggesting that the dosage of salidroside should be modified when used in the space flight.

*This work was supported by the National Program on Key Basic Research Project of China (2011CB711000),the National Natural Science Foundation of China (31471064),and the Science & Technology Planning Project of Shaoxing City,China (2014A230109).