| BackgroundInfants and children are far different from adults in terms of societal, psychosocial, behavioral, and medical perspectives. Functional immaturity of physiological processes and organ function predispose newborns to exhibit such disparate responses relative to the adult. With infant maturation, normal development may modify infant response to drug and toxicant exposures. As a special group, neonates’drug disposition progress is not only different from other age groups, but also shows a wide variance within the group. All these problems, together with the deficiency of neonatal therapeutically data, cause much difficult in the neonatal medication therapeutic regimen design.Due to the ethics consideration and other potential source of risk arises from the episodes when blood is sampled for PK analysis, traditional PK analysis is hard to work on in the neonatal population. That raised many problems, such as the lack of therapeutic data in newborn and the potential risk in the treating process. The Paediatric Regulations introduced in2006recognise that medicinal products for human use generally undergo extensive studies to ensure that it is safe of high quality and for use in the target population. Within the constraints of autonomy, safety and welfare it is an ethical and professional requirement to develop the information base used to guide therapeutics in neonates and infants whenever possible.Population Pharmacokinetic (PPK) analysis can be done using samples that are obtained at the same time as samples obtained in routine clinical practice, or, using samples that are obtained at study-specific sampling episodes. The benefits of using routine sampling episodes are that study-specific procedures are minimized. However, samples will be obtained at time points which may be suboptimal. If there are good reasons to prefer specific sampling times because specific time points are more informative then more participants will be required under this sampling framework in order to ensure that enough samples with informative timings are obtained. On the other hand, using study-specific time-points ensures that informative samples are obtained. This would be expected to minimize the number of participants, which is extremely suitable for neonates. Piperacillin/Tazobactam is used in neonates/infants are at risk of infection due to proven or suspected bacteria or suspected of having an infection due to proven or suspected bacteria. Piperacillin/Tazobactam will be administered by central or peripheral intravenous infusion according to the vascular access available for that child. The infusion will last30minutes. All blood sampling episodes will be undertaken by clinicians with relevant skills and experience. Efforts to minimize pain and distress will be tailored to the individual child by clinical staff with the involvement of parents. Samples will be taken from a different route to the administration route.In this study, high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) and population PK(PPK) methods were used. PPK sampling schedule was used to ensure reliable estimation of PK parameters, we choose to limit to3the number of study-specific samples per baby and per period. In order to completely cover the distribution and elimination phase in the whole population.The whole study includes the following3parts:PART One Determination of Free and Total Piperacillin/Tazobactam in Plasma by High Performance Liquid Chromatography coupled with Tandem Mass SpectrometryObjective:According to EMEA’s guideline on the investigation of medical products in the neonates, the trial-related blood loss per neonate should not exceed3%of the total blood volume during four weeks and should not exceed1%at any single time. For a3000g infant with estimated total blood volume of90mL/kg or a total blood volume of270mL,1%would be2.7mL blood. Present available methods for detecting piperacillin and tazobactam are high performance liquid chromatography (HPLC) methods or high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) methods, which have been developed for adult patients and require more volume of blood sample than recommended for neonates. Hence, a specific and highly sensitive analytical method is needed to be developed with less volume of blood samples. Moreover, this method could be applied to detect free drug concentration, since free concentration of antibiotics usually have better correlation with their clinical effects than total concentration.Methods:Total and free drugs determination were separated on a high performance liquid chromatography and quantified by mass spectrometry, with methyl-tert-butyl for extraction and pimozide as internal standard. Free drugs determination is deproteinized by a centrifugal filter device.Results:According to FDA’s guideline for bioanalytical method validation, all the criterias for validation have been met with our method. The calibration range was linear from100to 10,000ng/mL for piperacillin and from30to3,000ng/mL for tazobactam. Intra-and inter-day precision for total piperacilln was less than10%and7.3%, respectively. Intra-and inter-day precision for total tazobactam was less than5.3%and5%, respectively. Intra-and inter-day precision of dilution for piperacilln was less than14.2%and9.9%, respectively. Intra-and inter-day precision of dilution for free tazobactam was less than13.0%and9.5%, respectively. The acceptance criteria of accuracy (between85and115%) were met in all cases. A plasma volumes of50μL was required to achieve the limit of quantification of100ng/mL for piperacillin and30ng/mL for tazobactam. For free drug determination, another150μL plasma was centrifuged to get50μL ultra filtrate to achieve the same limit of quantification of total drug determination.Conclusions:The method was established and validated under guideline from FDA. It is warranted to be applied for detecting both total and free plasma concentrations of piperacillin and tazobactam with limited volume of blood samples from neonates and infants.PART Two Establishment and Authentication of Population Pharmacokinetic Model of Piperacillin/Tazobactam in NeonatesPopulation Pharmacokinetic is a new branch which develops in the field of pharmacokinetic studies in recent decades. Population pharmacokinetic is the group analysis of pharmacokinetic, which combines the classical PK model with population statistical model. It studies the distribution of the population pharmacokinetic parameters, the decisive factors of serum concentration of the quantitative analysis in patient population. It can more accurate to estimate the parameters. Traditional PK models are usually more sampling points, while the PPK can take advantage of the sparse and unbalanced datasets.The study methods in population pharmacokinetic are parametric and nonparametric methods, the most widely used method at home and abroad is the nonlinear mixed effect model (NONMEM). NONMEM method needs a few data, even if only a TDM data. It can be drawn a good correlation from the individual PPK parameters, which will be of benefit to the individual infants.Objective: To estimate the multiple dosing pharmacokinetic process of piperacillin/tazobactam in neonates and early infants, and to provide basis on the rational medication therapy.Methods:From Jan.2011to May.2011,71neonates stayed at NICU or Ordinary Neonates Units were enrolled according to the inclusion and exclusion criteria. When a clinical decision to start piperacillin/tazobactam has been made AND consent is in place AND medical eligibility has been confirmed, a neonate/infant will be recruited to the study and given a study number.In this study, Piperacillin/Tazobactam (Bangda(?), Shandong, Qilu Pharmaceutical co., Ltd) was used as the study medication. All the enrolled patients were divided into4groups, either receiving piperacillin/tazobactam in Q8h or Q12h. Several end points were estimated. Piperacillin/Tazobactam concentration and population pharmacokinetic (PK) parameters [maximum concentration, clearance, area under the curve (0-tau)], their relationship with selected covariates their interindividual variability (CV%). Covariate analysis will include postmenstrual age, gestational age, postnatal age, weight, and serum creatinine on the day of the first PK sample of each period (D1and D5). Other PK variables include apparent volume of distribution and half life. Occurrence of adverse events (AEs) and serious adverse events (SAEs) have been recorded if happened.We collected and detected drug concentrations of207blood samples from71NICU and Ordinary Neonatal Units in-patients in the first stage of research. These patients’ physiological characteristics, such as gender, gestational age, postnatal age, body weight, body surface area, hepatic and renal function, combined with pathological factors and drug-use information have been included as "fixed effect" in the process of building population pharmacokinetic model of piperacillin. Two-compartment piperacillin and tazobactam base model have been established. Estimation of inter-and intra-variability of patients helps screening fixed effects, those influence pharmacokinetic parameters statistically significant have been accepted in the final model or rejected otherwise. After establishing the model,26new samples were collected as the second stage to authenticate the model.Results:The objective function value of two-compartment piperacillin and tazobactam model are1048.746and144.293, respectively. Meanwhile, previous studies have indicated that two-compartment model has better predictive activity in population pharmacokinetics and pharmacodynamics of antibiotics. Hence, two-compartment model has been applied for estimating values of population pharmacokinetic parameters. They are depicted as population value±standard error:piperacillin CL (0.369±0.014) L/h,V,(0.742±0.069) L, Q (1.11±0.53) L/h and V2(0.269±0.062) L; and tazobactam CL (0.414±0.014) L/h, V,(0.803±0.080) L, Q (2.2±0.62) L/h and V2(0.391±0.069) L. Compared with base model, two-compartment final model has decreased inter-variability (from83.4%in base model to18.0%in final mode for inter-variability of piperacillin CL, from50.2%to20.8%for piperacillin V1; from84.7%in base model to16.1%in final mode for inter-variability of tazobactam CL, from33.5%to20.3%for tazobactam V1) and predicts drug concentration more precisely. Final model has been validated by both bootstrap method, VPC method and NPDE method, showing good stability and predictive efficacy. Based on the model, dose adjustment and individual use of piperacillin/tazobactam has been attained for another26in-patients. The final model is further validated by the fact that detected drug concentrations of these patients fit well with model-predicted values. Predictive efficacy of final model is good(Y=0.884).Conclusions:Two-compartment model of piperacillin was established by NONMEM method (piperacillin/tazobactam, piperacillin as the principal agent, and tazobactam as a reinforce agent of piperacillin). The model was authenticated to have satisfied stability and predictive efficacy, and is suitable to provide basis on the rational medication therapy. |
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