Study Conduct and Design
The protocol and the informed consent form were approved by an institutional review board operating in compliance with current regulations of local regulatory authorities and the International Conference on Harmonization Good Clinical Practice guidelines. The study was conducted in compliance with the protocol, the International Conference on Harmonization Good Clinical Practice guidelines, and Title 21 of the US Code of Federal Regulations Parts 50 (protection of human subjects), 56 (institutional review boards), and 312 (investigational new drug application).
This was a phase 1, multicenter, randomized, open-label study with the primary objectives of describing the PK and safety of repeated doses of intravenous acetaminophen during 48 hours across all pediatric age strata (www.clinicaltrials.gov study identifier: NCT00493246). A minimum of 12 neonates, 18 infants, 12 children, and 12 adolescents with evaluable PK data were to be enrolled. In response to the delay in neonate enrollment, the protocol was amended to require no minimum enrollment for this age group. Over-enrollment in each age stratum was allowed until it was confirmed that each dosing group within an age stratum was filled.
After written informed consent was obtained from the parent or guardian (with participant assent, when appropriate), screening assessments were performed. If eligible for the study, participants were then randomized to one of two dosing regimens within each age strata (see dosing section below). During the treatment period (T0 to T48 hours), the following were performed at prespecified time points: liver function tests (LFTs), PK samples, urinary collection, vital signs, recording of spontaneously reported or observed adverse drug events (ADEs) (from the time of consent/assent to study completion), and concomitant medications.
Full-term neonates (full-term to 28 days), infants (29 days to <2 years), children (2 to <12 years), and adolescents (≥12 years) with normal hepatic and renal functions who required analgesic or antipyretic therapy were eligible for enrollment. All eligible participants were hospitalized during the study period and were required to have intravenous access for the duration of the study. Participants were not eligible for study participation if they were unable to comply with the blood sampling requirements of the study, had a known hypersensitivity to acetaminophen or the inactive excipients of the formulation, had impaired liver function (total bilirubin more than 1.5 times the upper limit of the normal range [ULNR] for age or an alanine aminotransferase or aspartate aminotransferase more than 2.5 times ULNR), had renal function calculated to be less than 33% of normal for the applicable age strata, or had received any acetaminophen-containing products in the 12 hours prior to randomization. In addition, patients taking any of the following medications during the prior 48 hours were excluded: probenecid, disulfiram, isoniazid, or a number of dietary supplements (St John's wort, skullcap, chaparral, comfrey, germander, jin bu huan, kava, pennyroyal, and valerian).
Dosing Regimens Selected
Neonates were randomized to receive either 12.5 mg/kg every 6 hours or 15 mg/kg every 8 hours, and infants, children, and adolescents were randomized to receive either 12.5 mg/kg every 4 hours or 15 mg/kg every 6 hours. The maximum single dose could not exceed the lesser of 12.5 or 15 mg/kg or 1000 mg, and the maximum daily dose could not exceed the lesser of 75 mg/kg or 4000 mg. Intravenous acetaminophen was provided as a 100-mL vial containing 1000 mg (10 mg/mL). Each intravenous acetaminophen dose was delivered as a 15-minute infusion using a syringe pump.
PK Sampling (Blood and Urine)
The PK blood sampling schedule varied based on age and dosing group. Samples were obtained in neonates (n = 11-12), infants (n = 15-16), and children/adolescents (n = 18 or 20) immediately prior to the start of the intravenous infusion, at various timepoints during the infusion, and immediately at the end of infusion.
A 4-hour urine collection was performed after the first dose and a 12-hour urine collection (two aliquots: 0-4 hours and 4-12 hours) was obtained following the last dose to assess urinary excretion of free acetaminophen and key acetaminophen metabolites. The following analytes were assessed in urine samples: acetaminophen, acetaminophen glucuronide, acetaminophen sulfate, 3′–methoxyacetaminophen, and N-acetyl-p-benzoquinone imine (NAPQI)-glutathione metabolites [3′–(S– cysteinyl)] acetaminophen, acetaminophen mercapturate, and 3′–S–methyl-acetaminophen).
The concentrations of plasma and urine acetaminophen as well as urine acetaminophen metabolites were measured (MedTox Laboratories, St Paul, MN) using validated methods. Acetaminophen in plasma was analyzed using methanol-induced protein precipitation followed by high-performance liquid chromatography–tandem mass spectrometry capable of analyzing acetaminophen over a concentration range of 0.050 to 30 mg/L using d4-acetaminophen (sourced from Cerilliant, Round Rock, TX; lot no. 33378-95B) as an internal standard. The lower limit of quantitation was 0.050 mg/L. The intra-assay precision and accuracy ranged from 0.2% to 6% and 96.8% to 102.3%, respectively. The inter-assay precision and accuracy ranged from 2.1% to 5% and 95.9% to 102.3%, respectively. When sample concentrations of various analytes were found to be greater than the validated upper limit of quantitation for the assays, dilution was performed and samples were compared with high-quality control samples. The dilution process analysis was considered acceptable if within ±15% of the diluted controls.
The assay standard curve ranges for the urine analytes were: acetaminophen (0.05-5 mg/L), acetaminophen sulfate (0.25-10 mg/L), acetaminophen glucuronide (0.25-10 mg/L), 3′-methoxy-acetaminophen (0.1-5 mg/L), 3′-cysteinylacetaminophen (0.25-5 mg/L), acetaminophen mercapturate (0.10-5 mg/mL), and methylthioacetaminophen (0.1-5 mg/L).
Noncompartmental PK Analyses
Noncompartmental plasma acetaminophen PK analysis after the first and final doses included: area under the plasma concentration-time curve from time zero to time t (AUC0-t), maximum plasma concentration (Cmax), time of maximal plasma concentration (Tmax), terminal elimination rate constant (λz), and terminal elimination half-life (T1/2β).
Nonlinear Mixed-Effects PK Modeling
Data set preparation, exploration, and visualization of the data were performed using S-PLUS version 8.0.4 (TIBCO Software Inc, Palo Alto, CA), R version 2.6.1, and Microsoft Office Excel 2003 (Redmond, WA). The programming library Perl-Speaks-nonlinear mixed-effect modeling (NONMEM; PsN V2.3.0, ICON plc, Dublin, Ireland) was used to evaluate and validate the models with a visual predictive check (VPC). The results of the model validation were analyzed using S-PLUS version 8.0.4. WinNonlin Enterprise version v5.2 (Pharsight, A Certara Company, St. Louis, MO) was used to generate tables of posthoc PK parameters of acetaminophen as well as descriptive statistics for each treatment–age strata combination. Population PK analysis of acetaminophen in plasma was performed using nonlinear mixed-effect modeling (NONMEM) version VI. All models were run with the first-order conditional estimation with interaction method. The models accounted for between-subject variability, residual variability (random effects), as well as parameter differences predicted by covariates (fixed effects).
A two-compartment disposition model was deemed optimal to define the acetaminophen plasma concentration profile based on results from the model-building process. Models were parameterized by clearance (CL [L/hr]), intercompartmental clearance (Q [L/hr]), central volume of distribution (Vd1 [L]), and peripheral volume of distribution (Vd2 [L]), and were explored using various inter-individual random effect covariance structures. Additive, proportional, and combined (additive and proportional) residual-error models were considered during the model-building process. Ultimately, a combined additive and proportional error model was used to describe random residual variability.
The process of model discrimination was guided by examination of model diagnostic plots (plots of predicted vs. observed concentrations; weighted residuals vs. time; weighted residuals vs. predicted residuals), the value of the minimum objective function (MOF), the precision of parameter estimates, and the symmetry of the distributions of individual parameter estimates about the estimated median parameter.
The impact of weight on all PK parameters was investigated using an allometric model: TV
, in which TV
is the typical value of a model parameter, described as a function of individual body weight, θTV
is an estimated parameter describing the typical PK parameter value for an individual with weight equal to the reference weight represented by WTi
, 70 kg is the reference weight, and θallometric
is an allometric power parameter based on physiologic consideration of size impact on metabolic processes and is fixed at a value of 0.75 for clearances, and a value of 1 for volumes.3,6,7
The following covariates were included in the population PK data set: weight (kg), postnatal age (PNA) in weeks, height (cm), body mass index (kg/m2
), serum creatinine (mg/dL), and creatinine clearance (mL/min per 1.73 m2
). The creatinine clearance was calculated based on the formula of Schwartz et al8
The data set included also the results of LFTs measured prior to drug administration: total bilirubin, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase.
Following identification of the structural PK models, the PK parameters of acetaminophen were derived by the structural model for each patient (i.e., posthoc Bayesian PK parameters). Individual Bayesian estimates were plotted against clinical and demographic covariates to identify potential sources of variability in PK parameters of acetaminophen in pediatric patients. Covariates included but were not limited to body weight, age, and sex. Potential covariates selected from the graphical analyses were sequentially tested into the population PK model to assess the effect on between-subject variability of PK parameters of interest. A decrease in the MOF of 6.63 (p<0.01) was considered significant to include the covariate in the final model. The decision to include a covariate in the final model was based on statistical significance, clinical relevance and clinical significance. Clinical significance was evaluated based on the ability of a particular covariate to explain at least 5% of the between-subject variability of the corresponding parameter.
The effect of PNA on PK parameters was evaluated using the following age-maturation equation as proposed by van der Marel et al9
in which Xj
is the typical parameter value for the group of participants having the same covariate values (WT
is the population mean parameter value for the reference weight of 70 kg at mature age, θXage
is the constant describing age-related changes of parameter X
is the age in weeks of the participant, and Tx
represents the maturation half-lives of the age-related changes of parameter X
Model Evaluation and Validation
The appropriateness of the final model to perform simulations was evaluated using VPC on the quantiles of concentrations of acetaminophen. A total of 1000 replicates of the study were simulated with the final population PK model. The VPC examined the ability of the model to reproduce results similar to those from the original data set. A total of 1000 replicates of the study were simulated with the final population PK model. Median values as well as 25th, 75th, 90th, and 95th percentiles of the simulated data for acetaminophen exposure metrics were plotted with the actual study data overlaid. The appropriateness of the model was assessed by the ability of the simulations to accurately describe the distribution of the actual study data.
Determination of Secondary PK Endpoints
The following secondary PK endpoints were derived in the PK analysis: Cmax (mg/L), AUC0-τ (mg*hr/L), T1/2α (distribution half-life [hr]), and T1/2β (terminal elimination half-life [hr]).
Determination of Urinary Metabolites
Urinary excretion of free acetaminophen and metabolites was evaluated using noncompartmental analysis methods. The fraction of acetaminophen recovered as the free form and as metabolites was calculated taking into account the molar weight of each analyte. Individual urine concentrations and PK parameters of free acetaminophen and metabolites, determined over the 4 hour period after the first dose and at steady-state (after the last or 8th dose), and were summarized with descriptive statistics and compared when at least two observations were available for each age strata and regimen.
Safety endpoints included spontaneously reported or observed AEs, clinical laboratory testing (protocol-specified standard urine and blood tests, including LFTs), vital signs, physical examinations, and urine collections to assess the production of NAPQI/glutathione metabolites comparing fractional excretion after the first dose and at steady state (last dose day 2).
AEs were categorized based on incidence, type, expectedness, severity (i.e., mild, moderate, severe), seriousness, and relatedness. Investigators used standard definitions to determine relatedness (e.g., related, probably related, possibly related, not likely related, unrelated), presence of a serious AE, and level of severity (mild, moderate, severe). AEs occurring during or after the first dose of intravenous acetaminophen were characterized as treatment-emergent AEs (TEAEs). Preexisting AEs (prior to T0) that worsened during or after the first dose were also considered to be TEAEs. The percentage of participants withdrawn because of AEs and the percentage of participants with serious AEs were determined. Serious AEs included both serious AEs (occurring prior to T0) and serious TEAEs (occurring after T0).
Investigators determined the presence of clinically meaningful changes from baseline laboratory parameters (protocol-specified standard urine and blood tests); systolic blood pressure, diastolic blood pressure, and heart rate values immediately before and after the first intravenous study medication dosing, and at study completion/early termination; and change from the T0 predose value to after first dose, and final measurement.
The relationship between individual PK parameters and LFTs was evaluated to assess the safety of acetaminophen treatment in pediatric patients. Relative changes from baseline of LFTs between the screening and end-of-study visits were calculated. The relationships between change from baseline in LFTs and the secondary posthoc PK parameters (AUC0-τ and Cmax) were evaluated to assess the safety of acetaminophen treatment in the pediatric population. Pearson correlation coefficients (r) were calculated to identify significant linear relationship.