Study On Individualized Medication Of Meropenem In Neonates Based On DBS And UPLC-MS/MS Analysis

Objective:Meropenem,as a carbapenem antibacterial agent,shows strong antibacterial activity against a variety of gram-positive and gram-negative bacteria,and is often used to the anti-infection treatment for neonates with severe diseases in clinical.Previous studies have shown that the physiological characteristics of meropenem may cause individual differences during the development of neonates,which may lead to treatment failure and adverse reactions during treatment.Therefore,there is an urgent need to explore the individualized administration of meropenem in neonatal patients.Therapeutic drug monitoring（TDM）evaluates drug use by measuring plasma concentration of patients,and provides basis for clinical adjustment of drug dose,so as to realize individualized medication.However,there are controversies about whether TDM can bring clinical benefits for hydrophilic antimicrobial.Based on this,this study firstly analyzed the clinical efficacy,the occurrence of adverse reactions and pharmacoeconomic evaluation of severe infection patients treated with meropenem after TDM intervention,and explored the significance of application of TDM to the use of meropenem.Then,based on the limitation of TDM work in neonatal care unit,a new method was developed for the determination of meropenem plasma concentration using dried blood spot（DBS）sampling technique.Finally,a population pharmacokinetic model of meropenem in neonates was established to provide evidence for further clinical optimization of meropenem use.Methods:1.Efficacy,safety analysis and pharmacoeconomic evaluation of meropenem guided by TDM in the treatment of critical ill patients:Patients with severe infection who were admitted to Zhongda Hospital affiliated to Southeast University from 2019to 2020 and received meropenem treatment were collected as research objects.The patients were selected by frequency matching method to match the general condition data,past medical history,severity of infection and main treatment plans of the patients.Patients were divided into study group（TDM intervention）and control group（non-TDM intervention）according to whether the patients underwent meropenem plasma concentration detection.The clinical efficacy of meropenem after treatment was determined according to the Guiding Principles for Clinical Application of Antimicrobial Clinical Trial Technology issued by the former Ministry of Health,and the adverse reactions were determined according to the association evaluation criteria for adverse drug reactions formulated by the National Center for Adverse Drug Reaction Monitoring.Cost identification was carried out according to the Chinese Pharmacoeconomic Evaluation Guidelines,and the direct medical costs of patients during hospitalization were collected.Pharmacoeconomic evaluation was conducted using cost-effectiveness analysis and sensitivity analysis.χ² test,Mann-Whitney U test and t test were used for univariate analysis,while logistic regression model was used for multivariate analysis in order to explore the factors affecting the efficacy and safety of the study group.The threshold of C_min associated with meropenem adverse reactions was determined using the receiver operating characteristic curve.2.Establishment and application of plasma concentration quantification method of meropenem based on dried blood spot combined with UPLC-MS/MS:Using meropenem-d6 as internal standard,the chromatography was performed using Kinetex XB-C18 column（50 mm×2.1 mm ID,1.7μm）with 8 mmol·L^-1 ammonium acetate,0.2%formic acid and acetonitrile as mobile phases（gradient elution）at a flow rate of0.4m L·min^-1.Positive ion mode multiple reaction monitoring scanning was used for analysis,and investigated selectivity,standard curve and lower limit of quantification,accuracy and precision,recovery,matrix effect,residual effect and stability.Furthermore,ratio method,general linear regression and Deming regression were used for the conversion of DBS concentration to plasma concentration,and the Deming transformation was applied to TDM in meropenem in neonatal intensive care unit.3.Establishment of a population pharmacokinetic model of meropenem in neonates:Steady-state plasma concentrations of meropenem in neonates with severe infection were collected,and population pharmacokinetic model of meropenem in neonates was established based on NONMEM method.On the basis of the basic model,the final model was determined by selecting covariables through the forward inclusion and the back elimination,and the goodness of fit graph,bootstrap and visual predictive check were used to verify the goodness of fit,stability and predictability of the model.Based on the final model,monte-carlo simulation was used to compare the area under the curve and C_min of typical drugs in subjects with different characteristics at different doses.Results:1.The clinical effective rate of TDM intervention group was 12.78%higher than that of non-intervention group,but there was no statistical significance（P>0.05）.The incidence of adverse reactions was decreased in TDM intervention group,and there was significant difference in the incidence of eosinophilia（P<0.05）,but no significant difference in the incidence of nephrotoxicity（P>0.05）.Cost-effectiveness analysis showed that TDM intervention could save an average cost of 2292.57 yuan per patient,which was more economical.C_min level was the factor affecting the occurrence of renal toxicity and eosinophilia of meropenem（P<0.05）.ROC analysis showed that when C_minof meropenem reached 6.95μg·m L^-1 or above,patients were more prone to nephrotoxicity.APACHEⅡscore and the number of days from drug administration to concentration detection were risk factors for clinical efficacy（P<0.05）.Higher APACHEⅡscore before treatment and longer days from drug administration to the concentration detection may lead to low clinical response rate.2.An accurate,rapid UPLC-MS/MS method for the quantitative analysis of meropenem in dried blood spot（DBS）samples was developed in this study.m/z384.2→141.2（meropenem）and m/z 390.3→147.2（meropenem-d6）were selected as the detection ion reaction pairs.The analysis time of the method was 2 min.Meropenem showed good linearity（R²>0.99）in the concentration range of 1μg·m L^-1-100μg·m L^-1,and the lower limit of quantification was 1μg·m L^-1.This method has good specificity,with RSD of meropenem and internal standard（meropenem-d6）less than 15%between and within batches.Matrix effect,extraction recovery,residual effect and stability meet the requirements of biological sample determination.The plasma concentration of meropenem based on DBS concentration predicted by Deming regression was in good agreement with the measured plasma concentration.3.A total of 49 patients in the neonatal care unit of Zhongda Hospital affiliated to Southeast University with 145 plasma concentration points were enrolled in this study.One-compartment model was used for the structure model,and the final model expressed as:/=[0.265*（/34.3）^4.81*（/278）^0.347]*xp（₁）(1/=[1.09*(（2/2.09）^0.904]*xp（₂）.The results indicated that body weight had significant influence on Vd（P<0.005）,gestational age and platelet count had significant influence on CL（P<0.005）.The Bootstrap method showed that the robustness of the model was 100%,and the 90%confidence interval of covariate parameters did not include 0.The median of parameter estimation obtained by Bootstrap is basically consistent with that of the original data set.The goodness-of-fit diagram showed that the predicted values of individuals and populations were well correlated with the observed values,and the residual of conditional weight residuals were evenly distributed between±4.The visual predictive check of the model showed that most of the measured values fell within 90%CI predicted by the model.The verification results suggested that the established PPK model could well reflect the distribution characteristics of the measured values of meropenem plasma concentration,and the parameter estimation of the model was relatively robust.Monte-carlo simulation showed that gestational age had the most significant effect on meropenem PK among the three covariables.Conclusion:1.It is necessary for patients to use TDM during the treatment of meropenem.There is no statistical difference in the clinical effective rate between the intervention group and the non-intervention group,but the risk of adverse drug reactions is significantly reduced after intervention,and the medical expenditure of patients is reduced,which has more economic advantages.In clinical practice,it is recommended to use TDM as early as possible to monitoring the plasma concentration of meropenem in patients and develop individualized medication.2.For neonatal patients,the UPLC-MS/MS method developed in this study can accurately and rapidly detect the concentration of meropenem in DBS,and the concentration of DBS can be converted into plasma concentration through Deming regression,providing a new strategy for the development of TDM in NICU.3.In this study,a population pharmacokinetic model of meropenem in neonatal patients was established,suggesting that gestational age,platelet count and body weight had significant influence on meropenem PK,and gestational age had the greatest influence on meropenem plasma concentration.When meropenem is used in clinical therapy for neonatal patients,more attention should be paid to premature infants and low birth weight infants,and individualized medication should be formulated.