Pharmacokinetics of Midazolam in Neonates Undergoing Extracorporeal Membrane Oxygenation

After local research and ethics committee approval, neonates undergoing ECMO were recruited in the study with written informed parental consent. Twenty neonates were randomized into two groups: group 1 (n = 10) received midazolam extracorporeally, prereservoir, via  a pigtail catheter, whereas group 2 received the drug via  a central or peripheral venous catheter. Patients were excluded from the study if midazolam was administered to them before initiation of ECMO.

Midazolam was administered as a continuous infusion, at a rate usually between 50–250 μg · kg⁻¹· h⁻¹, initiated as soon as cannulation was achieved and extracorporeal blood flow was established. Infusion rates were titrated according to the unit sedation scoring guidelines: 1 = wide awake, 2 = awake but sleepy, 3 = asleep but moved spontaneously, 4 = asleep, but responded to stimulation, and 5 = hard to rouse. The target sedation score in most cases was 4. When levels of sedation were not satisfactory, rates of infusion were increased or decreased as indicated and additional bolus injections of 50–100 μg/kg were given if necessary. Infusions continued for the duration of ECMO, and weaned postdecannulation and before extubation. Infusions were stopped during ECMO when deemed clinically necessary by the responsible clinician (for assessment of neurologic status or to avoid oversedation).

Along with continuous heparinization to activated clotting times of 160–200 s, various drugs such as antibiotics, inotropes, diuretics, and H²blockers were coadministered during the study as indicated. ECMO circuits used in all neonates consisted of Tygon® S-65-HL (Norton Performance Plastics, Corby, Northants., U.K.) and a silicone membrane oxygenator (0800, Avecor, Cardiovascular Inc., Minneapolis, MN). The priming volume of the circuit was approximately 500 ml. The clear prime consisted of 100 ml of human albumin solution, 20%, and 400 ml Plasmalyte A, whereas the blood prime consisted of 500 ml donor blood, 120 units heparin, 15 ml sodium bicarbonate, 8.4%, and 2.5 ml calcium chloride, 10%.

Blood samples (1 ml) for assay were collected from indwelling arterial lines. Samples were drawn at baseline (before cannulation for ECMO), at 2, 4, 6, 12, 18, and 24 h and every 12 h thereafter. Samples were immediately stored at −20°C until analysis. Concentrations of midazolam and the free unconjugated form of its major metabolite 1-hydroxy midazolam were determined using gas chromatography-mass spectrometry. 10The assay was validated over the concentration range 10–10,000 ng/ml. The within and between day coefficient of variation was less than 10%.

Population pharmacokinetics of midazolam were estimated using WinNonMix, a nonlinear mixed effects regression program (WinNonMix, Pharsight Corporation, Mountain View, CA). This approach estimates not only the structural parameters of the model but also the interindividual (population) variability in parameters and residual error (difference between observed and predicted midazolam concentrations). 11In a preliminary analysis, the parameters of one-, two-, and three-compartment models were fitted to the data by use of this approach. The models were parameterized in terms of volume of distribution and clearance. The interindividual variability in parameters was modeled as a proportional deviation from the “true” parameter values (assuming a log normal distribution). Residual error was estimated with a combined proportional and additive model.

The influence of demographic and clinical variables on parameters of the model were also analyzed and, when significant, were added to the model to determine whether the overall variability in the model could be reduced. These included gestational and postnatal age, weight, gender, creatinine, urine output, continuous veno-venous hemofiltration, liver function tests, plasma albumin concentrations, comedication, cannulation mode, duration of ECMO, and ECMO pump-flow rates.

Because it was not practical to collect new data prospectively, the ability of our final model to perform in prospective tests was estimated using the cross-validation technique. 12Although cross-validation is not a truly prospective validation method, it is a recognized and established approach to estimating model performance, assuming identical experimental conditions. 13–15 

The study group was divided into eight smaller groups of two patients each and one group of three patients. The pharmacokinetic model was fitted to the data while excluding one group. The estimated structural parameters from the submodels were then used to predict the concentrations in the excluded group. This process was repeated nine times, excluding each group in turn. A measure of the predictive performance of models can be determined by calculating the median prediction error, i.e. , observed minus predicted concentrations, and the absolute median prediction error. 16The median prediction error describes the bias and absolute median prediction error, the precision (variability) of the predictions. Because the excluded group is not used to develop the model, the median prediction error and the absolute median prediction error are almost unbiased estimates of the predictive capabilities of the model. 14In addition, the area under the curve plasma metabolic ratio (MR) (1-hydroxymidazolam/midazolam MR) was determined using the following equation 17:

where C^{(t1)1-hydroxy MDZ}, C^{(t2)1-hydroxy MDZ}, and C^{(t1) MDZ}, C^{(t2) MDZ}are the concentrations of 1-hydroxy midazolam and midazolam, respectively, at two time points. This ratio has been used as a marker of CYP3A activity. 17 

Data are expressed as mean ± SD, mean ± standard error for population parameter estimates, and median (range). The MR was determined as the median MR from all patients. Selection of the optimal model was based on examination of graphic residuals plots and the objective function (−2 times the logarithm of the likelihood of the results). WinNonMix minimizes the objective function in performing nonlinear regression analysis. A model with a lower objective function value (OFV) offers an improvement in the goodness of fit. The difference in the OFV obtained before and after the addition of parameters is approximately chi-square distributed with degrees of freedom equal to the number of parameters that are set to the null hypothesis value. A change in the OFV greater than 7.88 (P < 0.005) was considered statistically significant. The impact of significant covariates was also evaluated using this approach.

Demographic data of the study population are shown in table 1. There were no significant differences in characteristics between the two randomized groups. Three patients died during ECMO, one patient postdecannulation. In three of the cases, deaths were as a result of pulmonary or cardiopulmonary failure, whereas myophosphorylase deficiency was the cause in another. From a total of 210 midazolam plasma concentrations analyzed, 45 (21%) observations were excluded from the pharmacokinetic analysis. Excluded were 13 observations from the patient with myophosphorylase deficiency who exhibited no decrease in plasma concentrations despite no drug being administered for 50 h; 15 observations collected during the post-ECMO period; and 17 observations that confounded normal physiologic principles (e.g. , increase in plasma midazolam concentrations when no drug had been administered).

The one-compartment model was selected for further analysis of midazolam because it demonstrated a more appropriate structural model on examination of the graphical residual plots. Furthermore, the OFV was not significantly lower with the two- and three-compartment models and estimates of the pharmacokinetic parameters were less precise, which is probably a reflection of our limited sampling (particularly in the initial phase of ECMO) and inability to discern peripheral compartments. Plots of model parameters versus  clinical and demographic covariates revealed no significant influences apart from randomization allocation (i.e. , whether the drug was administered extracorporeally or intravenously) with volume of distribution. The latter suggested that those patients administered midazolam extracorporeally had a higher volume of distribution (mean ± SD: 13.8 ± 5.2 vs.  10.9 ± 5.9 L) (fig. 1). Although this improved the model by reducing the variability on the volume and the OFV by 3.1 U, this was not significant at the P = 0.005 level (table 2) and thus was excluded from the final model.

A closer inspection of the residuals against time plot (fig. 2, top ) suggested that the model underpredicted concentrations in the early phase of ECMO. Therefore, the influence of various time covariates added categorically to both clearance and volume (range, 2–24 h) were tested in the model. The model improved measurably with the inclusion of a 12-h time covariate on volume, producing a drop in the OFV of 16.6 U (table 2). This time-dependent change in volume was attributed to reversible sequestration of midazolam by components of the circuit. Thus, after detecting time dependency as a significant covariate for volume, the phenomenon was incorporated into the structural model. Midazolam uptake by the circuit was accommodated by allowing the initial value of volume (V⁰) to increase mono-exponentially to an asymptotic value, V^max. This approach was analogous to that previously described by Rostami-Hodjegan et al.  for time-dependent change in clearance due to enzyme induction 18(Volume (L) = V^max− (V^max− V⁰)e^kseq.time, where K^seq= the rate constant for uptake of midazolam into the circuit and time = duration of infusion).

The revised model was considerably better at fitting plasma midazolam concentrations, abolishing the early trend in residuals (fig. 2, bottom ), and reducing OFV by 21 U, although it explained only 4% of the variance. The mean individual Bayesian posterior volumes increased approximately fivefold from the onset of ECMO to the steady-state value (median [range]): 3.1 (1.0–8.0) versus  14.2 (4.7–35.8) L, respectively (fig. 3). These changes were accompanied by a significant increase in the initial to steady-state half-life (median [range]): 6.8 (2.2–39.8) versus  33.3 (7.4–178) h, respectively (fig. 3). Interpatient variability in clearance and volume was 73 and 53%, respectively, whereas residual error corresponded to a proportional error of 26% and an additive error of 0.17 μg/ml (table 3). The relationship between observed and model predictions and selected individual patient profiles before and after the addition of the time dependency model are shown in figures 4 and 5, respectively. The individual Bayesian posterior parameters estimated from the model described the data well with bias of −0.3% and precision of 20%. The cross-validation exercise revealed an overall bias and predictive precision for the submodels of −0.3 and 19.3%, respectively, indicating that the full model is likely to perform well in a truly prospective trial during similar conditions (table 2).

Plasma concentrations of the free unconjugated 1-hydroxy midazolam were assayed in 12 patients (106 observations with no exclusions). The median MR was 0.17 with a large interindividual variability (range, 0.03–0.9). Seven neonates (35%) exhibited biochemical signs of liver dysfunction with raised alkaline phosphatase (ALP) and alanine transaminase (ALT), though no correlation was observed with MR. However, the lowest MR (0.03) was observed in a patient with significantly deranged liver function tests (ALT 281, ALP 922, bilirubin 100 mm), suggesting impaired metabolism of midazolam. The highest MR (0.9) was observed in the patient with highest estimate of midazolam clearance (3.8 ml · kg⁻¹· min⁻¹). Midazolam and 1-hydroxy midazolam pharmacokinetics are summarized in table 3.

The most significant observations of this study are the exhibition of markedly altered midazolam pharmacokinetics by neonates treated with ECMO compared with previously reported values for such patients. Previous investigators have reported that a two-compartment model is optimal for describing the pharmacokinetics of midazolam, in contrast to our one-compartment model. 2–6This may be a reflection of our study design, which used continuous infusions of midazolam with limited sampling in the distribution phase and few samples captured in the elimination phase.

Our parameter estimates are based on a model in which volume of distribution expands with time as ECMO support progresses. In this population, the mean initial volume on ECMO was estimated at 0.8 l/kg, similar to that previously reported by Burtin et al.  in non-ECMO neonates of gestational ages of 26–42 weeks (1 l/kg). 5This initial volume is perhaps lower than expected considering the expanded circulating volume for a child on ECMO. The normal intravascular volume for a neonate of 80 ml/kg would be significantly diluted at cannulation with the addition of a circuit volume of 500 ml. However, this parameter was imprecisely estimated with a coefficient of variation of almost 63%. A time-dependent increase in volume produced a maximum mean population value of 4.1 l/kg, three to four times greater than previously reported in neonates. 2,3,5,6The mechanism for this change may be attributable to sequestration of midazolam by the ECMO circuit. The uptake of drugs by polymeric components of the ECMO circuit has previously been demonstrated through in vitro  studies. 1,9This is an exponential, concentration-driven process eventually reaching a state of equilibrium. Many physicochemical factors will affect the time to equilibrium and the extent of drug loss, including lipid solubility (octanol:water coefficient or log P  values) of drugs, dosing rate, plasma concentration of drug, presence of codrugs, and ECMO flow rates. Inflation in volume resulted in a mean terminal half-life of 33.3 h, substantially longer than previous reports in neonates (12 ± 4.9 h), 2older infants, children and adults (range, 1–7 h). 19–22This accounts for raised plasma concentrations of midazolam observed during the later phases of ECMO in our study population (fig. 6) with levels far in excess of those required for adequate sedation (< 1 μg/ml). 10Similar increases in plasma fentanyl levels and the development of tolerance during ECMO have been reported. 23–25Analogous to midazolam (log P , 2.7; Roche FH-L: Midazolam (base) Safety Data Sheet. Basel, Switzerland, 2000), fentanyl, a highly lipophilic molecule with a log P  value of 2.9 26has frequently shown to be sequestered by polymeric components of the cardiopulmonary bypass circuit. 27–31Interestingly, the group that received midazolam extracorporeally had a tendency toward a higher volume of distribution. Although the addition of this covariate did not significantly improve the fit, it is plausible that a higher volume of distribution during extracorporeal drug administration exists because of greater distribution of the drug into the polymeric components of the circuit. This implies that midazolam should ideally be administered directly via  peripheral or central venous access.

For ECMO neonates, the metabolic clearance of midazolam is similar to that previously reported in non-ECMO neonates. Population estimates of clearance in our cohort of term or near-term neonates (1.4 ml · kg⁻¹· min⁻¹) was comparable with values previously reported in population studies of neonates by Burtin et al.  (1.2 ml kg⁻¹· min⁻¹) and Lee et al.  (0.9 ml · kg⁻¹· min⁻¹), but significantly lower than those reported in older infants and children (5.8–13.6 ml · kg⁻¹· min⁻¹) and adults (6.4–11.1 ml · kg⁻¹· min⁻¹). 5,6,32,33Reduced midazolam elimination in our population was not unexpected, reflecting the pattern for the ontogeny of CYP3A4. 34In adults, plasma clearance of midazolam has been delineated and correlated with CYP3A4/5 activity. 35Hepatic CYP3A4 is abundantly expressed in adults being responsible for catalyzing the biotransformation of approximately 50% of currently used drugs. CYP3A5 expression shows large interindividual differences and has been shown to be present in only 10–30% of liver samples tested. 36In contrast, CYP3A7 is the major isoform expressed in the fetal liver, with probably only marginal contribution to midazolam clearance in the postnatal period. CYP3A4 expression is activated during the first few weeks after birth, increasing thereafter to reach adult values at 1 yr of age, with a simultaneous decrease in CYP3A7 activity. 34 

We determined the 1-hydroxy midazolam/midazolam MR as a surrogate marker for CYP3A4/5 activity. The median (range) values in our patients (0.17 [0.03–0.9]) appear to be significantly higher than previously reported in preterm infants (0.09) and similar to those in older children and adults (0.13–0.25). 37,38Reports indicate that CYP3A4 expression is activated irrespective of gestational age 39and that enzyme activity increases only marginally during the first 2 weeks of life. Though gestational age range in our group was higher than previous reports (33–42 vs.  26–34 weeks), postnatal age ranges were similar (0.5–18 vs.  3–11 days) (reference ranges). Thus, the higher MR in our group does not necessarily represent higher CYP3A activity. Rather, it may be indicative of reduced glucuronidation of 1-hydroxy midazolam by uridine-diphosphate glucuronosyl transferases and thus reduced renal elimination. The developmental ontogeny of uridine-diphosphate glucuronosyl transferases has not yet been delineated. The wide interpatient variability in elimination of midazolam and metabolite and MR observed in our population corresponds with previous reports 3and reflects not only the heterogeneous and critically ill nature of the ECMO population but also indicates that CYP3A and glucuronidation activity in the neonate exhibits large variability.

The clinical implications of our findings are illustrated in figure 7. The figure depicts simulated concentrations for a hypothetical neonate on ECMO receiving continuous infusions of midazolam at various dose rates, using the estimated population parameters. These simulations closely resemble the mean observed concentration profile in this population (fig. 6) revealing attenuation of plasma levels during the early phase of ECMO followed by a significant rise. The results also suggest that a higher initial dose rate in the early phase of ECMO needs to be followed by a significant decrease in the dose rate to prevent excessive plasma concentrations. For optimum sedation, plasma concentrations of midazolam up to 1.0 μg/ml are required. 10This was simulated using an initial infusion rate of 350 μg · kg⁻¹· h⁻¹)for 6 h followed by a drop in the dose rate to 50 μg · kg⁻¹· h⁻¹(fig. 8). Cross-validation provides a measure of the predictive ability of the model in the absence of prospectively collected test data. The exercise revealed an insignificant change in bias or precision, suggesting that the estimated model is likely to perform well in truly prospective trials.

In summary, we have analyzed the pharmacokinetics of midazolam in neonates undergoing ECMO, deriving parameters and their variability using a population approach. The midazolam model reveals a significantly altered volume of distribution in ECMO patients compared with reports in non-ECMO patients, probably through reversible binding to the circuit. Consequently, half-life was significantly prolonged and hence midazolam must not be considered a sedative with a short half-life in neonatal ECMO patients. We also determined the MR, a surrogate measure of CYP3A activity, as being higher than previous reports in preterm neonates and attribute this to a reduced clearance of the metabolite. Using final model parameters, we simulated plasma midazolam concentration profiles at various dose rates and a strategy to achieve adequate plasma levels. An understanding of the pharmacokinetics of midazolam during ECMO helps promote the development of rational dosing approaches and titration to sedation scoring systems. Appropriate sedation levels reduce the incidence of hemodynamic side effects associated with oversedation, expediting return to normal mental status following cessation of the drug.