Simple And Rapid Analysis Of Plasma Protein Binding Rate And The Significance In Therapeutic Drug Monitoring

It is well known that drugs bind to plasma proteins to varying degrees in plasma,and the degree of binding can highly influence absorption,distribution,metabolism and elimination of drugs（ADME）,which is closely related to drug efficacy and side effects.Therefore,protein binding rate（PPB）can be used as an important parameter for therapeutic drug monitoring（TDM）and evaluation of ADME.However,PPB monitoring has not been given much attention in clinical practice.The main reason is that there are still some problems in the methods of characterizing PPB,which further limits the application of PPB in clinical TDM and discussion about its clinical significance.Thus,the study on the analytical method of PPB and investigation of its influencing factors and clinical significance under non-health conditions has become a difficult problem for clinical pharmacists.Two plasma samples for measuring unbound concentration（C_f）and total concentration（C_t）respectively in two systems using two different pretreatment methods are required in current methods of analyzing PPB,which means the accuracy of PPB values depends mainly on that of analytical methods for C_f and C_t.The HFCF-UF method,as a direct sampling technique,has been successfully applied for C_f analysis in clinical with excellent accuracy and precision,and the C_f value determined can be directly substituted into the formula to calculate PPB.Therefore,the accuracy of the C_t method becomes the key to the reliability of PPB.The pre-treatment methods reported for C_t in the literature are mostly protein precipitation（PPT）,liquid-liquid extraction（LLE）and solid phase extraction（SPE）.Each method has its own advantages and disadvantages.In general,the whole analysis process of C_t is tedious and time-consuming,and the multistep operation is easy to cause an increased error in the final result,which is difficult to ensure the accuracy of the C_t and PPB results.In addition,the analysis result of PPB deviates from the true value due to the influence of factors such as the acquisition time and storage conditions of the two plasma for C_f and C_t analysis.Consequently,it is difficult to monitor PPB of drugs in the clinical practice.Therefore,this study successfully improves the traditional C_t analysis method by HFCF-UF technology,and establishes a simple and accurate sample pretreatment method with only one plasma sample required for the simultaneous analysis of C_f,C_t and PPB,providing a reliable analysis platform for clinicians to make dose adjustment based on PPB and C_f.Generally,under the healthy physiological state,the PPB of the drug remains constant over the entire time period corresponding to the drug-time curve.At this time,C_f and C_t show good correlation,and C_t can reflect C_f at a certain level.However,in non-healthy conditions,such as liver and kidney dysfunction,hypoproteinemia and other conditions,PPB can be changed,and then directly affect C_f,resulting in individual differences in drug efficacy.In this case,PPB combined with C_f of the drug in the vivo can reflect the drug ADME,providing a reference for individualized treatment.Part 1:Simple and Rapid Analysis of Carbamazepine Protein Binding Rate Using Hollow Fiber Centrifugal Ultrafiltration Method and the Significance in Therapeutic Drug MonitoringObjective:A novel pre-treatment method based on hollow fiber centrifugal ultrafiltration was developed for analysis of C_f,C_t and PPB in the same plasma to investigate the significance of PPB and C_f in clinical TDM.Methods:After the accurate determination of C_f by HFCF-UF technology,the traditional C_t was reformed by this method.The type and amount of the release agent added into the plasma were optimized to ensure the bound drug was completely detached from the protein binding site.Thus,the C_f,C_t and PPB were accurately analyzed in the same plasma sample.The established method was applied for 20 patients treated with CBZ,and the results were statistically analyzed.The HPLC was used for detection of C_f and C_t.Results:The calibration curve of C_f and C_t were linear well（R²>0.999）over the range of 0.535-21.4μg/mL.The intra-day and inter-day precision（CV%）and accuracy（%bias）were all less than±3.0%.When acetone was used as a release agent and the volume was 200μL,the absolute recoveries were in the range of 100.2-100.8%（CV%<2.0%）.Preliminary clinical trials showed that the mean value of PPB in 20 clinical patients treated with CBZ was 46.3%（10.1%-68.9%）,significantly different from that of healthy volunteers（76%）.The correlation between C_f and C_t of 20 patients was poor（R²=0.470）.Conclusions:The proposed pretreatment method is simple and rapid,successfully applied for C_f and C_t analysis of CBZ in the same plasma.This method has a conspicuous predominance on the analysis of C_t and C_f with excellent accuracy and precision,improving the accuracy of the PPB results.This method provides a reliable analytical tool for clinicians to make dose adjustment based on PPB and C_f.Under non-health conditions,there is a large variation in PPB values of 20 patients.Therefore,the PPB should be monitored in clinical practice to ensure the rationality of the patient’s medication.Part 2:The Study on Analysis Method of Doxofylline Protein Binding Rate and the Investigation of Its Influencing Factors and Clinical Significance Under Non-Health StateObjective:An accurate analytical method for doxofylline protein binding rate was established,and then the relationship between PPB and physiological and pathological conditions of patients was initially discussed to provide a theoretical basis for the formulation of individualized dosing regimens.Method:C_f and C_t of DFL were accurately analyzed in the same plasma using HFCF-UF technique combined HPLC.Then the PPB was obtained by the formula.This method was applied for the determination of PPB in 48patients receiving DFL and the correlation between PPB results and patient’s gender,age,biochemical indicators,and disease status was also analyzed.Results:The intra-day,inter-day precision（CV%）and accuracy（%bias）of this method were all less than±4.5%,and the absolute recovery of C_t was99.4-100.3%（CV%<2.3%）.The average PPB of 48 patients was 39.6%（3.02%-93.2%）,significantly different from that of healthy volunteers（48%）.Correlation analysis showed that the correlation between albumin levels,total bilirubin,direct bilirubin,urea,creatinine and PPB was good（P<0.05）.Conclusions:The C_f,C_t and PPB of DFL are simultaneously analysed by this method in the same plasma,providing a reliable platform for further investigation of the influencing factors of PPB under non-healthy state.Preliminary clinical trials have demonstrated that there are individual and intra-individual differences in PPB among 48 patients receiving DFL therapy.Therefore,PPB monitoring in clinical should be used to guide physicians to optimize dosage.At the same time,PPB is susceptible to albumin,liver and kidney function.Therefore,in the formulation of individualized dosage regimens,the clinician should fully consider about the patient’s albumin level and liver and kidney function.