The Analysis Of Free Drug Concentration Related Parameters In Human Plasma And Their Application In TDM

Clinical therapeutic drug monitoring(TDM)has been an important work to ensure the safety and efficacy of clinical medication for a long time.At present,using total drug concentration(C_t)to evaluate efficacy and to guide the clinical medication is a common in clinical TDM.However,free drug concentration(C_f)is considered to evaluate the drug efficacy and safety directly,because free drugs are the real part to exert drug efficacy.C_f and C_t are instantaneous blood drug concentration,which can't reflect the drug concentration of each time point during drug metabolism cycle.They are susceptible to the effect of sampling time.Plasma protein binding(PPB),as an important parameter,can characterize the binding extent between drugs and plasma proteins.It is constant during stable disease condition,which can characterize the whole condition during drug-time curve.However,because disease and physiological conditions are extremely different between different patients,PPB of patients is very different between individuals.The changes of PPB can lead to the changes of C_f,which directly influence the efficacy and side effect of drugs.Therefore,the PPB monitoring of patients is vital for the safety and efficacy of clinical medication.What's more,using PPB and C_f to guide individual dose adjustment simultaneously is more accurate and more scientific than using single parameter(C_t or C_f).Plasma protein precipitation(PPT),liquid liquid extraction(LLE),solid phase extraction(SPE)and so on are usually used for the determination of C_t.These methods are tedious,time-consuming and can cause the loss of the target compounds.With the development of analytical techniques,direct injection technology become hotspot and the dream of analysts increasingly.Hollow fiber centrifugal ultrafiltration(HFCF-UF),as a direct injection technique,can be used to analyze C_t of cefaclor by changing the pH.While the changes of pH only can be used for the analysis of water soluble plasma protein binding drugs but for the analysis of liposoluble plasma protein binding drugs.This may because drugs combine to plasma proteins in liposoluble area,the changes of the pH can't act on the region.Besides,although the changes of pH can make the drugs released from the plasma proteins,the released liposoluble drugs can't redissolve in the water soluble matrix.What's more,the released drugs may gather together or attach on the surface of proteins,which can't enter inside the hollow fiber for tha analysis.Therefore,there is no choice but to choose PPT,LLE or SPE to analyze C_t of liposoluble plasma protein binding drugs.This research studies the direct injection technique,which can make the liposoluble plasma protein binding drugs released.Finally,this reseach make the HFCF-UF possible for the C_t analysis of liposoluble plasma protein binding drugs.This technique not only provides an easier and more accurate technique for the monitoring of C_t in TDM,but also laies the foundation of the development of PPB analysis.However,there is no research about these.This subject use carbamazepine(CBZ)and Phenobarbital(PB)as examples to develop and validate a simple,accurate,reliable HFCF-UF for the C_t analysis of liposoluble plasma protein binding drugs for the first time.The technique was developed by the addition of liposolubel release agent,which not only can make the drugs released from drug-plasma combines,but also can redissolve the realeased drugs avoiding gatheration and adsorption.Finally,acetone was used as release agent,absolute recovery is about 100%,and calibration curves can be established in plasma-free solution.The results indicate that HFCF-UF can be used for the C_t analysis of liposoluble plasma protein binding drugs,which not only provides a reliable technique for TDM,but also laies the foundation of the development of PPB analysis.As we all know,free drugs are the trule part to exert pharmacological effect,so the analysis of C_f in TDM can reflect the relationship between plasma drug concentration and pharmacological effect or side effect well.The key to susccessfuly analyze C_f is keep the drug-protein balance unchanged.Centrifugal ultrafiltration(CF-UF)is wide applied in the analysis of C_f.However,the ultrafiltrate volume in CF-UF is susceptible to experiment conditions and plasma conditions.It is uncontrolled and has a significant effect on C_f.The therapeutic range of theophilline(ThP)is so narrow that side effect occured frequently.Therefore,the TDM of ThP is the vital mean to ensure pharmacological eficasy and avoid side effect.But there is a few of reports on the C_f analysis of ThP,and the reported methods haven't been well validated.It is unknown whether these methods can analyze C_f accurately.In order to ensure the efficacy and safety of ThP,to develop an accurate and reliable analytical technique of C_f become badly in need.A simple and reliable HFCF-UF method was developed to analyze C_f of ThP,and was applied for the TDM of ThP successfully in this study.Accuracy and precision of the developed method was evaluated during a cross-validation involving centrifugal ultrafiltration(CF-UF)currently considered as a gold standard for analysis of unbound drug.CF-UF method was also well validated in this study.The results indicate that the accuracy of two methods is comparable,while the precision of HFCF-UF is better than CF-UF.What's more,HFCF-UF is less affected by the experiment conditions and plasma conditions.Consequently,using HFCF-UF to analyze C_f of ThP is more accurate,and this method was applied in TDM.HFCF-UF provides a simple and reliable analytical platform for the TDM of ThP and laies the foundation of the development of PPB analysis.Both C_f and C_t just reflect the concentration at a specific time rather than the whole process of drug metabolism,which is susceptible to sampling time.While plasma protein binding(PPB)don't vary over time and it is constant in the whole process of concentration-time curve.Thus,PPB can reflect the whole process of drug metabolism changes in a relatively long time,when the patients' physiological indexes only change tinily.There are a lot of literatures reported that the change of albumin concentration,drug-drug interaction and the metabolites of the therapeutic drugs have a significant influence on C_f.The root of the changes of C_f is the changes of PPB.Therefore,the monitoring of PPB can understand the changes of C_f fundermentally.PPB between different patients and between patients and healthy people are extremely different.Using PPB of healthy people to calculate C_f based on C_t is unscientific and unaccurate.Besides,a moment of C_f is unable to predict the plasma drug concentration of other time.What's more,C_f can't guide how much drug dose should increase or decrease.Individualized drug delivery is increasingly becoming an important means to improve the effect of clinical treatment.How to conduct a comprehensive and accurate individualization has not been reported in resent reserches.The monitoring of PPB in patients and using PPB and C_f simultaneously to adjust drug dose were first carried out in this research.The monitoring of PPB in patients is hampered by the tedious,time-consuming methods.We use ThP as example drug to develop a simple HFCF-UF technique for the analysis of C_t and C_f simultaneously,and then PPB can be calculated accurately.The proposed method provides a simple and reliable analytical platform.