Quantitative Analysis, Preclinical Pharmacokinetics And Tissue Distribution Of Rhychophylline In Biological Matrix

Uncaria rhynchophylla, is one of the most popular traditional Chinese medicine （TCM）has been used more than one thousand years. The pharmacologically active indole alkaloidsare isolated from the Uncariar rhynchophylla. The content of rhychophylline （Rhy） was thehighest among the dominant alkaloids, and this alkaloid exhibit antihypertension andneuroprotective properties, such as antihypertensive, cerebral ischemia protection, antianxietyand sedation. Our previous experiments show that Rhy has anti-anxiety effect. If new TCMpreparation can be designed with reliable curative effect and few less advers effect, it not onlybrings evangel to patients, but also bring a great societal and economic benefit.In order to explore the in vivo pharmacokinetic profiles of Rhy after oral administration,a well-developed ultra-performance liquid chromatography tandem mass spectrometry（UPLC-MS/MS） method was applied for the quantitative determination of Rhy in rabbitplasma and rat tissues. The experimental results can help us understand its in vivo actions andexplain the efficacy and toxicity of the relevant herbs or herbal preparations in which thisingredient is involved. It also provide the theoretical bases for the clinical use.The main research contents：1. Development and validation of UPLC-MS/MS method for the detection anddetermination of Rhy in biological samplesThe establishment of UPLC-MS/MS for the determination of Rhy in biologicalsamples.The chromatography and mass spectrometry conditions for chromatographic wereoptimized, and the method was fully validated with selectivity, sensitivity, linearity, precision,accuracy, stability and matrix effect （ME）. This developed method was suitable and durablefor the study on pharmacokinetics of Rhy.2. Pharmacokinetic study of Rhy following oral administration to rabbitsEighteen male New Zealand healthy rabbits were randomly divided into three groups（six per group）. Then, three doses （20,40,80mg/kg） were choose as the oral administration.Blood samples were collected into heparinized tubes from the ear vein at times of0,0.25, 0.50,0.75,1.0,1.5,2.0,3.0,4.0,6.0,8.0and12.0h. The drug concentration in plasma wasdetermined by the validated UPLC-MS/MS method. Pharmacokinetic parameters, includingthe area under the blood concentration-time curve （AUC0-12）, elimination half-life （T1/2β）,apparent volume of distribution （Vd/F） and oral clearance （CL/F） were calculated by anon-compartmental method using the BECS1.0software.3. The distribution of Rhy in miceForty-two male Kunming healthy mice were randomly divided into seven groups （six pergroup）. One dose （40mg/kg） was choose to investigate the distribution of Rhy in mice. Thetissues or organs, including heart, liver, spleen, lung, kidney and brain were excised at timesof10min,20min,30min,45min,75min,105min,135min. The drug concentration in tissuesor organs were determined by the validated UPLC-MS/MS method. Then study thedistribution characteristics （targeting performance and blood-brain barrier） of Rhy.The main results：1. Development and validation of UPLC-MS/MS method for the detection anddetermination of Rhy in biological samplesA reliable, novel and sensitive UPLC-MS/MS following a one-step protein precipitationfor biological matrix removal was developed and fully validated for the determination of Rhyin rabbit plasma. The mobile phase consisted of acetonitrile （solution A） and0.01mol/Lammonium acetate （pH5.0adjusted with acetic acid,solution B）. Gradient elution with A:Bfrom20:80to30:70within5min at a flow rate of0.3mL/min. A Waters Acquity UPLCsystem coupled to the TQ detector was used （Waters, Milford, USA）. The separation wasperformed on an Acquity UPLC BEH C18column （2.1×50mm,1.7μm）. The column andautosampler temperature were maintained at30℃and4℃, respectively. The injectionvolume was3μL. The eluent was detected by mass spectrometry with electrospray ionization（ESI） in positive scan mode. The TQ detector parameters were optimized as following:positive electrospray ionization with capillary voltage of3.5kV, cone voltage of50V,desolvation temperature of350℃, source temperature of150℃, desolvation gas flow of600L/h, and cone gas flow of10L/h. Selected multiple reaction monitoring （MRM） using theprecursor to product ion combinations of m/z385.3â†'159.8and m/z237.1â†'194.0was usedto detect Rhy and the IS, respectively. The method was specific, and its linearity was superb（r2>0.99） for Rhy in the biological matrices. The precision value was <15%and recoveryvalue was89.82%-110.77%. It was suitable for preclinical pharmacokinetic studies on Rhy. 2. Pharmacokinetic study of Rhy following oral administration to rabbitsAfter oral administration of20,40and80mg/kg of Rhy, the mean C_maxin plasma ofrabbits calculated in this study were1.02μg/mL at0.96h,1.92μg/mL at0.92h and3.50μg/mL at1.25h, respectively. All doses of the drug were quickly eliminate from plasmawith elimination half-life less than1.50h. The CL/F was about7.59mL/h/kg （ranging from6.75to8.93mL/h/kg）. Further, the Vd/F was about14.74mL/h/kg （ranging from13.06to15.80mL/h/kg）. The parameters of AUC0-12and C_maxwere very significantly correlated withthree dosages, and the correlation coefficients were0.9939（P<0.01） and0.9989（P<0.01）.In addition, there was no significant difference for other parameters such as CL/F, Vd/F andT1/2βamong the three dose levels （P>0.05）.3.The distribution of Rhy in miceTissue distribution of Rhy was investigated following a single oral administration tomice at40mg/kg dosage. Concentrations of Rhy were determined in various tissues of mousesuch as heart, liver, spleen, lung, kidney and brain. Tissue distribution showed that theconcentration was highly in liver, followed by the kidney and spleen; the lowest levelappeared in the heart and brain.Conslusion：1. A simple, rapid and reliable UPLC-MS/MS method for the determination of Rhy inbiological specimen was developed and validated.2. Rhy has rapid absorption and elimination from plasma with half-life was less than1.5h, and it might lead to drug accumulation in plasma with low CL/F. The results of this studysupport dose-independent pharmacokinetic characteristics of Rhy across the investigateddosage range in rabbits （20-80mg/kg）.3. Tissue distribution showed that the concentration was highly in liver and kidneyindicated that Rhy was mainly metabolized in the liver and renal excretion of mice. Itconsistented with channel entry theory of Rhy. The detectable level in brain suggested thatRhy does cross the blood-brain barrier.