The Study And Application Of Hollow Fiber Centrifugal Ultrafiltration In The Accurate Characterization Of Drug Therapeutic Indexes

Objective:We aimed to use hollow fiber centrifuge ultrafiltration（HFCF-UF）technology to solve the problem of replacing the current total drug concentration with plasma free active component concentration in clinical therapeutic drug monitoring（TDM）,and to establish an accurate,simple,and durable method for separating free drugs independent of plasma status,to accurately evaluate the efficacy and toxicity of antitumor drug-methotrexate（MTX）.It is intended to use HFCF-UF to avoid the influence of co-precipitation of proteins and the different protein concentrations on the analysis results in TDM,so as to provide a more accurate reference index for individualized drug administration plans of antiepileptic drugs-lamotrigine（LTG）and oxcarbazepine（OXC）.At the same time,we aimed to use the HFCF-UF technology to solve the problem of sample pretreatment analysis in the characterization of anti-oxidative stress（OS）ability,improve the sensitivity of analysis,reduce co-precipitation,avoid the influence of strong oxidizing reagents and unprecipitated proteins on the equilibrium system,especially when the biological samples were processed after leaving the body,avoid the influence of the excitation of redox substances and enzyme system in the samples.To accurately analyze free reduced and oxidized aminothiol concentration in vivo,and to provide a simple and accurate anti-OS index for clinical treatment of diseases,so as to evaluate the efficacy and safety of drugs and to adjust the treatment means to cure diseases and restore health.Methods:1.We use HFCF-UF technology to develop the method to analyze the free MTX concentration in human plasma.The newly developed method was successfully validated in clinical 92 plasma sample from children with HD-MTX.Combined with the results of MTX total plasma concentration,the correlations were analyzed between the free and total MTX concentrations in human plasma,as well between the MTX concentrations and renal toxicity.The HFCF-UF technology was used to develop the method of simultaneous analysis of free concentrations of LTG,OXC and Monohydroxy derivative（MHD）in human serum.A total of 43 clinical patients with epilepsy treated with LTG and/or OXC were selected for the study and the free concentrations were determined according to the established method.The relationship between free and total LTG and MHD concentrations were analyzed in order to provide accurate reference indexes for individualized drug administration plans.2.We use the HFCF-UF technology coupled with Liquid chromatography tandem mass spectrometry（LC-MS/MS）detection to develop a method for the determination of four reduced aminothiols-homocysteine（Hcy）,cysteine（Cys）,cysteine glycine（CG）and glutathione（GSH）in human blood.The developed method was successfully validated in human blood from 96 clinical samples.The influence of gender,age,and drug combination on the levels of the four reduced thiols in human blood was discussed.The relationship of the total and reduced Hcy concentrations were also discussed.Meanwhile,the SD rat with ovariectomy was chosen as the OS model with postmenopausal osteoporosis（OP）,and the hydrogen-rich saline（HRS）was antioxidant.We develop the HFCF-UF method for simultaneous determination of reduced homocysteine（Hcy）,reduced cysteine（Cys）and their own oxidized aminothiol-homocysteine（HHcy）,and cystine（Cys）in rat plasma.The ratio of reduced to oxidized（Hcy/HHcy and Cys/Cyss）were also calculated.Meanwhile,the OS indexes such as the malondialdehyde（MDA）,superoxide dismutase（SOD）and glutathione peroxidase（GSH-PX）and Mcro-CT of femoral neck were also measured.We aimed to explore a more simple and accurate biological indexes to evaluate anti-OS capacity in OP rats.Results:1.The developed HFCF-UF method was simple and time-saving which only involves a step of ordinary centrifugation.It can overcome the limitations of traditional methods and is not affected by the different plasma status of patients.The developed method was successfully validated in 92clinical children plasma samples and a linearity coefficient r² of 0.910 was obtained for the correlation between the free and total MTX plasma concentrations.However,the free and total MTX concentrations was only weakly correlated in 16 clinical plasma specimens with total MTX concentrations>2μmol·L^-1（r²=0.760）.Both the free and total MTX concentrations of the 92 samples were negatively correlated with the creatinine clearance（CCr）level.2.The developed HFCF-UF method used aqueous solution as the matrix for methodology validation.It escapes coprecipitation and avoids the effect of different serum protein concentrations in different clinical patients.The error of the method,which may not be evident in serum samples from healthy subjects,was obviously reduced.The extraction recovery was as high as 90.1～98.6%with excellent precision（RSD<6.7%）.The developed method was successfully validated in 43 with epilepsy treated with LTG or/and OXC and the linear correlation coefficients（r²）of free and total concentrations of LTG and MHD were 0.8705 and 0.7823,respectively.In clinical TDM practice,a more accurate individualized drug administration plan was developed for two epileptic patients according to the free concentration.3.We developed a sealed HFCF-UF method to separate reduced aminothiol from a complicated matrix,and the results showed great merits.The sample preparation process was simplified to a 5-min centrifugation step that did not require adding additional reagent.The initial physiological imbalance state was not disturbed and could suppress thiol-disulfide exchange,and the result was more accurate and realistic.There were no significant differences in reduced Hcy,Cys,CG or GSH for the different genders,ages,or combinations with MTX or vancomycin in clinical 96 blood samples（P>0.05）.However,there was a significant increase in reduced Hcy concentration in patients treated with valproic acid who were diagnosed with epilepsy（P=0.0007）.However,the result of the Spearman’s rank correlation for the total and reduced Hcy concentration was 0.901 which indicated there was no correlation between total and reduced Hcy levels.4.The developed HFCF-UF method as preparation process can overcome the influence of coprecipitation,strong oxidizing reagents,and unprecipitated proteins,especially,avoiding the destroy of initial balance of redox system in vivo.It was successfully used to determine the concentrations of Hcy,HHcy,Cys,Cyss in rat plasma in three groups（normal control,NC,ovariectomy with oxidative stress,OVX and Ovariectomy with hydrogen-rich saline administration to anti-oxidative stress,OVX+HRS）.There were both significant decreases of Hcy/HHcy and Cys/Cyss in OVX group.And,in OVX+HRS group,there were both significant increases compared with OVX group and tended to be NC group.The statistical analysis of MDA,SOD and GSH-PX between three groups all showed that P<0.01,which indicated that OS occurred in OVX group,and OVX+HRS could counteract OS.The results of Mcro-CT scan of the three groups showed that OP occurred in OVX group,and OVX+HRS group showed significant improvement compared with OVX group and tended to be NC group.The experimental results and theoretical equations also demonstrate the change of Hcy/HHcy is more obviously than Cys/Cyss.Conclusions:1.In present work,HFCF-UF was used to solve the problem of replacing the current total drug concentration with plasma free active ingredient concentration in clinical TDM.We developed a simple and accurate HFCF-UF method independent of plasma status was established to analyze the free MTX concentration in children plasma,and was successfully used in clinical TDM.We also developed the HFCF-UF method to determine the free concentrations of LTG and MHD in clinical patient serum.It is not affected by protein co-precipitation,and can also avoids the influence of different sample protein concentrations on the analysis results.It provides a more accurate personalized medication plan for clinical patients,so as to accurately evaluate the efficacy and toxicity of drugs.2.In present work,HFCF-UF technology was used to solve the problem of sample pretreatment analysis in anti-OS ability characterization.We developed a simple and accurate HFCF-UF method for the analysis of four reduced aminothiols（Hcy,Cys,CG and GSH）in human blood samples,and for the analysis of reducing Hcy,Cys and their respective oxidized HHcy and Cyss in rat plasma.The sample pretreatment process overcomes the influence of coprecipitation,strong oxidizing reagent and unprecipitated protein on the analysis results,and does not destroy the redox balance in vivo.The Hcy/HHcy and Cys/Cyss could be suitable biomarkers for anti-OS capacity in vivo and especially Hcy/HHcy is more sensitive.It can be used to evaluate the recovery of the body in the treatment of disease,indirectly evaluate the efficacy and safety of drugs,and can also provide a powerful means to evaluate the anti-OS ability to serve the clinical treatment of diseases in the future.