Study design and setting
This was a prospective observational population pharmacokinetic study of posaconazole administered intravenously to critically ill patients with presumed or confirmed fungal infections. The setting was a quaternary referral intensive care unit (ICU) at the Royal Brisbane and Women’s Hospital, Australia. The Hospital’s Human Research Ethics Committee (HREC/16/QRBW/377) and that of The University of Queensland (2016001354) granted ethical clearance.
Patients
All patients admitted to the ICU at the study hospital during the study period were screened for eligibility. The inclusion criteria were age ≥ 18 years, admission for ICU care, the presence of suspected or confirmed fungal infection requiring systemic antifungal therapy, and the presence of central venous access for drug administration. The exclusion criteria were age < 18 years, pregnancy, prescription of drugs known to interact with posaconazole, use of posaconazole within the last 2 weeks prior to enrolment and any documented history of a drug reaction to triazole antifungals. Informed consent was obtained from participants or their next of kin.
Posaconazole administration
A single dose of 300 mg intravenous posaconazole solution, diluted with 0.9% sodium chloride or 5% dextrose in water, was administered to each study participant by slow infusion over 90 min through a central venous catheter. The drug was infused through a 0.22 μm polyethersulfone (PES) or polyvinylidene difluoride (PVDF) filter. The single dose of posaconazole was administered for the study purpose, as an add-on to a full course of another antifungal drug prescribed for therapeutic purpose as part of the usual care and at the discretion of the attending physician.
Sample collection
Fourteen blood samples (each 2 mL) were collected over 48 h from an arterial catheter. The sampling scheme was the first sample immediately before the commencement of the posaconazole infusion, then during infusion at 15 min, 45 min, 75 min and 90 min and subsequently at 3 h, 5 h, 8 h, 12 h, 18 h, 24 h, 30 h, 36 h and 48 h after the commencement of infusion. Lithium heparin tubes were used for sample collection. The plasma was separated by centrifugation (3000 rpm for 10 min) and frozen under − 80 °C for storage until assay of total and unbound concentrations.
Clinical data collection
Clinical data were collected for each patient using an electronic case report form, which included patient demographics, diagnosis, clinical microbiology data (isolated organism, susceptibility and the minimum inhibitor concentration, MIC, when available), clinical chemistry (makers of renal and hepatic function and serum albumin level), illness severity scores (Acute Physiology and Chronic Health Evaluation II [APACHE II] score on ICU admission and the Sequential Organ Failure Assessment [SOFA] score), renal replacement therapy modality and settings (if any) and concomitant medications.
Posaconazole assay
Total and unbound posaconazole concentrations were measured using a validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. The total concentration assay methodology, using a Shimadzu 8030+ mass spectrometer (Kyoto, Japan) with a range of measurement of 0.02 to 5 mg/L (precision of 4.7, 2.7 and − 5.3% and accuracy of 8.7, 0.2 and − 2.5% at concentrations of 0.06, 0.4 and 4 mg/L) has been summarised elsewhere [6]. The unbound assay method was based on the chromatography and detection of the total assay, but used a Shimadzu 8050 mass spectrometer (Kyoto, Japan) to achieve a calibration range of 0.0005 to 0.1 mg/L (precision of 6.8, 3.3 and 5.4% and accuracy of − 3.9, 5.2 and 8.2% at concentrations of 0.0015, 0.01 and 0.08 mg/L spiked in ultracentrifugated plasma). Sample preparation involved ultracentrifugation of plasma using Centrifree devices (Merck Millipore, Tullagreen, Ireland) to separate the unbound fraction. The ultracentrifuged plasma (30 μL) was spiked with internal standard (posaconazole-[d4]) and mixed with methanol. An aliquot of 2 μL of the supernatant was injected onto the UHPLC-MS/MS. The assay method met the Food and Drug Administration (FDA) validation criteria for bioanalysis, with stability and storage conditions covering the sample conditions prior to receipt at the analytical laboratory, as well as during receipt and analysis at the analytical laboratory [10].
Population pharmacokinetic modelling
The Pmetrics user interface in R for non-parametric adaptive grid (NPAG) algorithm was used to develop a population pharmacokinetic model. Both total and unbound posaconazole concentration-time data were included in the model building together with available covariates.
Structural base model and binding model
Initially, one- and two-compartment, linear and capacity-limited protein-binding models were fitted to total and unbound posaconazole concentrations simultaneously. Elimination from the central compartment and intercompartmental distribution were modelled as first-order processes.
For the linear binding models, unbound posaconazole concentrations were related to total concentrations by Eq. 1.
$$ {C}_{\mathrm{free}}={C}_{\mathrm{total}}\times \mathrm{FF}\times \frac{{\mathrm{Alb}}_{\mathrm{median}}}{\mathrm{Alb}} $$
(1)
where Cfree and Ctotal are the unbound and total posaconazole concentrations (mg/L) respectively, FF is the free fraction of posaconazole, Alb is the plasma albumin concentration (g/L) and Albmedian is the median Alb for the study population.
For the capacity-limited (Michaelis-Menten type) binding models, unbound posaconazole concentrations were related to total concentrations by Eqs. 2–5, assuming that albumin is the sole binding protein for posaconazole in the plasma and that all binding sites have the same affinity for posaconazole.
$$ {C}_{\mathrm{bound}}=\frac{B_{\mathrm{max}}\times {C}_{\mathrm{free}}}{K_D+{C}_{\mathrm{free}}} $$
(2)
$$ {B}_{\mathrm{max}}=\mathrm{Alb}\times N\times \frac{M_{\mathrm{posa}}}{M_{\mathrm{Alb}}}\times 1000 $$
(3)
$$ {K}_D=\frac{1}{K_A}=\frac{k_{\mathrm{off}}}{k_{\mathrm{on}}} $$
(4)
$$ {C}_{\mathrm{free}}={C}_{\mathrm{total}}-{C}_{\mathrm{bound}} $$
(5)
where Ctotal, Cbound and Cfree are the total, bound and free plasma posaconazole concentrations (mg/L), respectively, Bmax is the maximum binding concentration of posaconazole (mg/L), N is the number of posaconazole binding sites per molecule of albumin, Mposa is the molecular weight of posaconazole, MAlb is the molecular weight of albumin, KD is the equilibrium dissociation constant (mg/L), KA is the equilibrium affinity constant (L/mg), koff is the first-order dissociation rate constant (h−1) and kon is the second-order association rate constant (L/mg/h). N was assumed to be 1.
Error model
Based on the standard deviation (SD) of observations ([obs]), either a multiplicative (Error = SD*γ) or an additive (Error = [SD2 + λ2]0.5) error model was tested with each of the structural base models. In addition, assay error was modelled as a linear function (Error = C0 + C1*[obs]) starting with a generic set of coefficients, followed by iterative optimization.
Development of the covariate model
Available clinical covariates were tested on structural model parameters of volume of distribution (V) and total clearance (CL). Tested covariates included age, gender, height, weight, body mass index (BMI), albumin, serum creatinine, creatinine clearance (urinary), presence of renal replacement therapy, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, bilirubin, gamma-glutamyltransferase and SOFA score. If the inclusion of the covariate resulted in a statistically significant improvement in the log-likelihood value (p < 0.05) and/or improved the goodness-of-fit plots, it was supported for inclusion in the final model.
Model evaluation
Model evaluation was performed by visual inspection and statistical evaluation of goodness of fit via the combination of diagnostic plots and objective functions metrics. Scatter plots of observed-versus-predicted concentrations were examined together with model bias and imprecision metrics. Bias was defined as the mean weighted error of predicted minus observed concentrations, Σ (predicted-observed/standard deviation)/N, and imprecision was defined as the bias-adjusted, mean weighted squared error of predicted minus observed concentration, i.e. Σ[(predicted-observed)2/(standard deviation)2]/N - Σ (predicted-observed)/standard deviations/N, where N is the number of observations/predictions. Scatter and histogram plots of residuals versus predicted-concentration or time were also examined. Normality of residual distribution was evaluated with D’Agostino test. The objective functions examined were the log-likelihood ratio (LLR) test for the nested models, Akaike information criterion (AIC) and Bayesian information criterion (BIC). The LLR chi-squared test within Pmetrics was used for statistical comparison of nested models with p < 0.5 considered as significant.
Dosing simulations
Monte-Carlo dosing simulations (n = 1000) were performed using the final covariate model over the same period as the original sample collection, 48 h. Given the previously recommended targets of total steady-state concentration ≥ 0.7 mg/L for prophylaxis and ≥ 1 mg/L for treatment [11], and studies suggesting that posaconazole trough concentrations on day 2 post dose-commencement are approximately half of steady-state concentrations [12, 13], trough concentrations of 0.35 mg/L and 0.5 mg/L at 48 h were considered as a surrogate targets for prophylaxis and treatment, respectively. These total trough concentration targets correspond to unbound trough concentration targets of 0.0023 mg/L (prophylaxis) and 0.0033 mg/L (treatment) at 48 h, respectively, based on the mean free fraction for posaconazole of 0.65% in study patients. These trough targets were chosen to determine the probability of target attainment (PTA) for various simulated posaconazole dosage regimens. PTA was also determined based on the area under the total concentration-time cure (AUC) and the area under the free-concentration-time curve (fAUC) from 24 to 48-h post dose, normalised to the MIC. Considering previously recommended total AUC/MIC ratios for posaconazole of 100 for prophylaxis and 200 for treatment of fungal infections [13], fAUC/MIC targets of 0.65 (prophylaxis) and 1.3 (treatment) were used based on the mean free fraction of 0.65%. Simulated dosage regimens included loading doses of 300 to 800 mg given either hourly or 12 hourly for 24 to 48 h.