Target-mediated disposition population pharmacokinetics model of erythropoietin in premature neonates following multiple intravenous and subcutaneous dosing regimens

European Journal of Pharmaceutical Sciences 138 (2019) 105013

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Target-mediated disposition population pharmacokinetics model of erythropoietin in premature neonates following multiple intravenous and subcutaneous dosing regimens

T

Ronilda D'Cunhaa, Robert Schmidtb, John A. Widnessb, Donald M. Mockc, Xiaoyu Yand, ⁎ Gretchen A. Cressb, Denison Kuruvillaa,e, Peter Veng-Pedersena, Guohua Ana, a

Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA Department of Pediatrics Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, USA d School of Pharmacy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong e MedImmune, LLC., San Francisco, CA, USA b c

ARTICLE INFO

ABSTRACT

Keywords: Erythropoietin Population pharmacokinetics Target-mediated drug disposition Premature neonates

Routine erythropoietin (Epo) therapy for neonatal anemia is presently controversial due to its modest response. We speculate that an important contributor to this modest response is that previous clinical study designs were not driven by rigorous mechanistic and kinetic insights into the complex pharmacokinetics (PK) and pharmacodynamics (PD) of Epo in this population. To address this therapeutic opportunity, we conducted a prospective clinical study to investigate the PK of Epo in very-low-birth-weight (VLBW) premature neonates using a unique Epo dosing algorithm that accounts for complex neonatal erythropoietic physiology. Twenty-seven subjects received up to 10 intravenous or subcutaneous exogenous doses of Epo (600 or 1200 U/kg) during the first 4 weeks of life. Subjects were administered two doses of Epo 1200 U/kg on days 2 and 16, and eight doses of Epo 600 U/kg on days 4, 5, 6, 7, 9, 14, 15, and 28 following birth. We have developed for the first time a mechanistic, target-mediated disposition model that provides novel insights into the mechanisms driving Epo PK in VLBW neonates. Epo association rate, kon, was estimated to be 0.00610 pM-1h-1, and the dissociation rate koff was 0.112 h-1. Internalization of the Epo-target complex (kint) and the total receptor concentration (Rmax) were estimated to be 0.118 h-1 and 133 pM, respectively. Following s.c. administration, the absorption rate (ka) of Epo was 0.0738h-1 and bioavailability was 78.0%. Our mechanism-based population pharmacokinetic analysis provided quantitative insight into Epo kinetics in VLBW neonates; the information gained will assist in deriving dosing strategies for neonatal anemia and for neuroprotection efficacy studies.

1. Introduction Neonatal anemia is the most common hematological problem in the neonatal intensive care unit (Widness, 2008). This anemia is a particularly important clinical issue for critically ill, very low birth weight (VLBW) infants weighing < 1500 g at birth. For this patient population, neonatal anemia develops rapidly during the first few weeks of life when the neonatal cardiorespiratory system is most compromised and blood sampling required for laboratory monitoring to enhance survival rates is most intense. Approximately 80% of premature VLBW infants receive at least one red blood cell transfusion (RBCTx) for the treatment of anemia (Carroll and Widness, 2012). Because RBCTx is associated

with life-altering complications in preterm infants including infections, retinopathy of prematurity, and electrolyte disturbances (Ohlsson and Aher, 2006), alternate therapies for neonatal anemia such as the administration of recombinant human erythropoietin (Epo) are needed. Since Epo therapy in VLBW infants has only modestly reduced the number of RBCTxs, routine Epo therapy for neonatal anemia is presently controversial (Ohlsson and Aher, 2006; Wilimas and Crist, 1995). We speculate that an important contributor to this modest response to Epo is that study designs in previous clinical studies were not driven by rigorous mechanistic and kinetic insights into the complex pharmacokinetics (PK) and pharmacodynamics (PD) of Epo determining the erythropoiesis in this population. Specifically, because Epo clearance is

⁎ Corresponding author at: Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa, S227 Phar, 115 S. Grand Ave, Iowa City, IA 52242, USA. E-mail address: [email protected] (G. An).

https://doi.org/10.1016/j.ejps.2019.105013 Received 9 April 2019; Received in revised form 16 June 2019; Accepted 18 July 2019 Available online 21 July 2019 0928-0987/ © 2019 Elsevier B.V. All rights reserved.

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faster in infants compared to adults and is dynamically changing, empirical Epo dosing schedules used in infants so far have been inadequate. To address this therapeutic opportunity, we developed an optimal Epo dosing algorithm for preterm infants; the algorithm is based on multidose optimization simulations of endogenous plasma Epo levels (Rosebraugh et al., 2012). This algorithm accounts for the complexity of neonatal erythropoietic physiology and Epo's PK/PD (Rosebraugh et al., 2012). Although our long term goal is to evaluate whether an optimized Epo dosing algorithm can reduce (or even eliminate) the number of RBCTxs in premature neonates, prior to testing this hypothesis it is critically important to first evaluate the PK of Epo in this difficult to study, vulnerable population. The aim of the current study was to evaluate the population PK of Epo when our optimized dosing algorithm (Rosebraugh et al., 2012) is used and apply non-linear mixed effect modeling to evaluate the potential impact of covariates on Epo's PK parameters. The PK information obtained will assist in the development of more appropriate optimized dosing strategies based on exogenously administered Epo in premature neonates.

Fig. 1. Epo dosing schedule applied based on endogenous plasma Epo in prior study of VLBW infants (Rosebraugh et al., 2012). Each infant was scheduled to receive 10 Epo doses in the first 4 weeks of life. Two different doses were administered; the 1st and 9th doses were 1200 U/kg, while the remaining eight were 600 U/kg.

2. Materials and methods 2.1. Patients and study design

samples were analyzed for Epo using the following selection criteria: 1) all samples from the 1st day of life, 2) all samples within 48 h post each Epo dosing, 3) daily samples after the 1st 48 h post Epo dosing. When multiple samples were collected at the same time, only one was selected for analysis. Studies in infants—particularly those born prematurely—are often hampered by the limited blood volumes available as determined primarily by ethical restrictions on investigational blood sampling. In the current Epo study, we employed a population-based approach that does not require collecting samples at predetermined times. Instead, plasma Epo was measured in opportunistic blood samples (samples collected from remaining blood after routine laboratory tests have been performed).

The study was conducted at the University of Iowa Hospitals and Clinics following protocol approval by the University of Iowa Institutional Review Board. Written consent was obtained from the parents of neonatal subjects. Because Epo is not approved for use in infants < 4 months of age, the study was performed under FDA IND protocol 118057. The study was also registered with ClinicalTrials.gov (NCT00731588). 2.1.1. Subjects Entry criteria included: 1) gestational age at birth < 37 weeks; 2) birth weight between 1000 and 1500 g; and 3) postnatal age at study entry < 48 h. Exclusion criteria included: 1) hemolytic anemia; 2) lifethreatening major anatomic anomalies; 3) clinical seizures; 4) congenital thrombotic or hemorrhagic conditions; 5) positive bacterial or fungal infection from blood or spinal fluid culture, 6) TORCH infection; 7) hematocrit > 60%; or 8) systolic blood pressure > 100 mmHg.

2.1.3. Safety evaluation As required by the FDA, serious adverse events and adverse events anticipated in VLBW infants potentially related to Epo treatment were examined in all study participants for one month after the last dose of Epo.

2.1.2. Protocol The previously published Epo dosing schedule based on endogenous Epo during neonatal anemia is provided in Fig. 1 (Rosebraugh et al., 2012). Study neonates received 10 Epo doses within the first 4 weeks of life. This optimized regimen contrasts with the commonly employed regimens in which Epo has been administered either only once per week or three times per week (Juul et al., 2008; Locatelli et al., 2002; Ohls et al., 2001). Two different doses of Epo (Epoetin Alfa, Janssen Pharmaceuticals of Johnson & Johnson, Raritan, NJ USA) were administered; the 1st and 9th doses were 1200 U/kg, and the remaining eight were 600 U/kg. Epo was given more frequently in early weeks because phlebotomy loss is greatest during this period. When an intravenous (i.v.) line was available, the Epo dose was administered by bolus injection over less than 1 min. When an i.v. line was not in use, Epo was administered subcutaneously (s.c.). For the neonates enrolled in this study, i.v. catheters were typically in use for 7 to 14 days. Consequently, some neonates received their 7th though the 10th doses subcutaneously. Whole blood samples from study subjects were drawn in EDTA tubes or in sodium heparin blood gas tubes. Exact blood sampling times were entered into the subject's electronic health record by the clinical bedside nurse or by the NICU laboratory phlebotomist. These times were used in the final population PK analysis. The collected blood samples were centrifuged at 1000g for 5 min to separate plasma, which was stored at −20 °C until Epo analysis. Almost all available plasma

2.2. Chemiluminescence assay for Epo measurement The plasma concentration of Epo was determined using a commercial electrochemiluminescence detection plate assay (Human Hypoxia Serum/Plasma Kit #K15122C, Meso Scale Discovery, Rockville, MD) following the manufacturer's recommended procedure (MESO SCALE DIAGNOSTICS L, 2014). Briefly, the plate was activated with the manufacturer's blocking solution, washed, 25 μL buffer and 25 μL sample or standard added and then incubated for 2 h allowing the Epo to bind to the capture antibody attached to the plate. After rinsing, 25 μL of the detection antibody was added and incubated for 2 h. After a final rinse, 150 μL of read buffer was added and the plate was analyzed on a Sector Imager 6000 (Meso Scale Discovery, Rockville, MD, USA). The assay covered an expansive dynamic range of 2.4 to 10,000 mU/ mL, with an LLOQ of 2.0 mU/mL. In addition to the calibration standard samples, quality control (QC) samples were also included in each plate. QC samples were prepared by pooling de-identified plasma samples with low, medium and high levels. QC plasma samples were assayed in triplicate on each assay plate. Plasma pool Epo (mU/mL) and inter-assay variation (% CV) for these three Epo levels was 17.4 (15.7%), 424 (12.3%) and 5024 (14.0%), respectively. Intra-assay % CV for the triplicates averaged 4.6%. None of the clinical samples were pooled for Epo analysis. Serial dilutions of high Epo plasma samples 2

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were shown to have the same slope as the Epo standard providing evidence against a plasma matrix effect. The concentrations of Epo in therapeutic Epo vials were also analyzed using our MSD assay and we observed that the apparent EPO dose detected using our assay was approximately 60% of the labeled EPO dose. This is not surprising since Janssen may have used a different EPO assay and/or EPO reference preparations of different origin. Therefore, this difference is essentially an assay artifact and thus does not have any impact on actual healthcare. The detailed explanation on the phenomenon of assay-dependent result of protein drugs can be found in a brief note that has been accepted very recently (An et al., 2018). Therefore, in the population PK analysis, an assay calibration factor of 0.6 was employed. Additional information about the obtained evidence for the use of an assay calibration factor has been included in the Supplementary Material.

include Epo receptor zero-order synthesis rate (ksyn) and Epo receptor first-order degradation rate constant (kdeg) in our TMDD model. Instead, we incorporated an approximation using the total receptor concentration (Rmax) (Mager and Krzyzanski, 2005). Molar Epo concentrations and doses were used for this model. Based on a literature report, 1000 U of Epo is equivalent to 7.7 μg, and Epo's molecular weight is 30,400 Da (Jelkmann, 1992). The key equations used to characterize the TMDD model are provided as follows:

2.3. Population PK modeling

b) The equation for the peripheral compartment was:

Population PK data from the multiple i.v. and s.c. doses of Epo were analyzed simultaneously using the nonlinear mixed effect modeling approach with NONMEM (Version 7.3; Icon Development Solutions, Ellicott City, Maryland) interfaced with Pirana (version 2.9.5b, http:// www.pirana-software.com/). The first-order conditional estimation method with interaction (FOCEI) and a user-defined subroutine (ADVAN6) were used to estimate the population mean values of the PK parameters, inter-individual variability (IIV), inter-occasion variability (IOV), and residual variability (RV) between observed and individually predicted plasma Epo concentrations. RStudio (version 1.0.143, https://www.rstudio.com/) was used for graphical analysis and data handling. Because endogenous Epo cannot be distinguished by the assay from exogenously administered recombinant human Epo, baseline-corrected Epo concentrations were used in our analysis.

dCt Q = × Cp dt Vt

a) The equation for the central compartment is: dCp dt

= ka ×

Asc Vp

k on × Cp × (Rmax

RC ) + k off × RC

CL × Cp Vp

Q Q × Cp + × Ct Vp Vp

(1)

Q × Ct Vt

(2)

c) The equation for the binding and internalization with the Epo receptor:

dRC = kon × Cp × (Rmax dt

RC ) + koff × RC

kint × RC

(3)

2.3.1.2. Stochastic models evaluated 2.3.1.2.1. Inter-individual variability (IIV). IIV was evaluated using an exponential model which is assumed to be normally distributed with a mean of 0 and a variance of ω2. 2.3.1.2.2. Residual variability (RV). Different RV models were explored including additive error on log-scale, proportional and additive error on normal scale, and proportional error on normal scale. The residual error is assumed to be normally distributed with a mean of 0 and a variance of σ2. 2.3.1.2.3. Inter-occasion variability (IOV). The variation in different structural PK parameters within subject was explored by incorporating IOV. Each dosing event (that may or may not be followed by plasma Epo concentrations) was defined as an “occasion”. To evaluate the improvement in the model performance for selecting the final model, the log-likelihood ratio test was used to compare rival hierarchical models where a decrease in the NONMEM objective function (−2 log likelihood) of 3.84 was necessary to consider the improvement in model performance statistically significant at α = 0.05. The Akaike information criterion was used to compare rival nonhierarchical models. Other selection criteria included improved goodness of fit plots, successful estimation of the parameters precision, plausibility of the estimated parameters, and reduced variance of intersubject, inter-occasion, and residual errors. Covariate analysis was performed using the standard forward addition and backward elimination method. Forward addition was applied first to determine significant covariates. Only covariates that decreased the objective function value by > 3.84 (i.e., p < 0.05) when compared to the base model were considered for the full covariate selection. Backward elimination was then applied to remove covariates from the model with an increase in the objective function value > 6.63 corresponding to 1 df at p ≤ 0.01. Only covariates that produced this magnitude of increase were retained in the model.

2.3.1. Model building process 2.3.1.1. Structural models evaluated 2.3.1.1.1. Linear PK model. The linear PK models that we tested included a 1-, 2-, and 3-compartment models, all of which include a linear elimination pathway from the central compartment. 2.3.1.1.2. Nonlinear Michaelis-Menten (M-M) PK model. We tested a) 1- and 2-compartment models with central nonlinear M-M elimination only; and b) 1- and 2-compartment models with both nonlinear M-M elimination and linear central elimination pathways. The M-M kinetics was characterized by maximum rate of elimination (Vmax) and the Michaelis constant (Km). 2.3.1.1.3. Target-mediated drug disposition model (TMDD). In addition to the nonlinear M-M PK model, another nonlinear PK model that we tested was the TMDD model, which is a model that has been commonly used to describe the PK of protein drugs having a disposition influenced by a receptor interaction (Jin and Krzyzanski, 2004; Abraham et al., 2010; Mager, 2006). In this model, there is a dynamic distribution of exogenous Epo between a central compartment (Cp, Vp), and a peripheral compartment (Ct, Vt) determined by a distribution clearance (Q). The elimination takes place from the central compartment via a linear pathway (CL), and/ or via an interaction with Epo receptors (Epo-R) with a second-order association rate constant (kon) to form an Epo-R complex (RC). This complex can then dissociate to release the bound Epo with a first-order dissociation rate constant (koff) or Epo can undergo elimination via internalization of the Epo-R complex (kint). Additional model parameters include bioavailability (F1, for s.c. data only) and firstorder absorption rate constant (ka, for s.c. data only). Initially, attempts were made to employ a full TMDD model with the endogenous production rate of Epo (kEpo) being included in the model. Despite numerous attempts, the TMDD model with kEpo component failed to converge. Efforts were also made to fit the TMDD quasi-equilibrium model (Mager and Krzyzanski, 2005), which did not work either. In addition, to avoid the issue of over-parameterization, we did not

2.3.2. Model evaluation The adequacy of the final TMDD population Epo PK model was evaluated using the visual predictive check (VPC). The time course of the Epo concentrations was simulated 1000 times through Monte Carlo simulations using the original dataset and the final model. The 95% confidence intervals of the 5th, 50th, and 95th percentiles of the simulated Epo concentrations were compared with all observed Epo 3

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concentrations and the 5th, 50th, and 95th percentiles of the observed Epo concentrations.

Table 1 Demographic features of the study subjects.

3. Results 3.1. Subject participation and demographics Among the 33 subjects enrolled, 2 subjects were transferred to other hospitals before study completion and 4 additional subjects had insufficient doses of Epo to include. Data from these six subjects were completely excluded. A summary of subject demographics for the remaining 27 study neonates is presented in Table 1. The disposition of Epo was evaluated in these 27 neonates over the first 28 days of life (Fig. 1). Exogenous Epo was given via i.v. bolus or s.c. administration, and the maximum number of exogenous Epo doses was 10. Most subjects received 1200 U/kg at the 1st dose and 9th doses and 600 U/kg at the remaining doses. Among the total of 260 Epo doses received by these 27 neonates, 210 were given through i.v. bolus and 50 were administered via s.c. injections. In total, 495 Epo plasma concentration samples from the 27 subjects were available for pharmacokinetic analysis (Fig. 2). An additional figure (Supplementary Fig. 1) illustrates baseline-corrected Epo plasma concentrations after i.v. or s.c. administration in all patients.

Variable

Mean ± SD (Range)

Birth weight (g) Gender Gestational age at birth (wk) Age at first Epo dose (h) Weight at first Epo dose (g) Hemoglobin at first Epo dose (g/dL)

1256 ± 154 (1025–1500) 18 males/9 females 28.4 ± 1.2 (27.0–31.0) 45.1 ± 15.5 (27.8–104) 1206 ± 163 (940–1475) 16.7 ± 2.4 (11.3–21.5)

which is a capacity limited elimination pathway, we tested different nonlinear models involving M-M kinetics. However, these models either did not converge or resulted in poor model fitting. Additional information with figures and a tabular comparison showing poor model fitting have been included in the Supplementary Material. Ultimately, we evaluated a mechanistic TMDD model. In the TMDD model, we included two elimination pathways- a linear elimination and a nonlinear target-mediated elimination. 3.3.1.2. Stochastic model. IIV was investigated for all structural parameters and IOV was assessed in kint, Rmax, Vp and Vt, after IIV was found to be insufficient in explaining the variability in PK parameters. IIV in kint and IOV in kint were found to be statistically significant. The final residual model was described using a proportional error component on the normal scale.

3.2. Safety results The only serious adverse event was a Grade 4 intraventricular hemorrhage that developed in one study participant. No subject developed Stage 3 or 4 retinopathy of prematurity. Adverse events observed in participants included: anemia (n = 11), fever (n = 1), bacteremia (n = 4), Grade 1 or 2 intraventricular hemorrhage (n = 4), thrombocytopenia (n = 1), thrombocytosis (n = 1), and Stage 1 retinopathy of prematurity (n = 1).

3.3.1.3. Covariate model. Subject covariates evaluated include both continuous covariates (birthweight, hemoglobin level at birth, gestational age at birth) that were collected at baseline and categorical variables (gender, dose group (600 U/kg vs. 1200 U/kg), administration route (i.v. vs s.c.)). No significant covariates were identified. 3.3.1.4. Final model. Based on the known mechanism of Epo action and available data, a TMDD model (Fig. 3) was deemed to be the most appropriate to describe the PK of Epo in VLBW infants; this model also provided the best fit. The final model included IIV and IOV in kint. A proportional error model on the normal scale was used to describe the RV. Table 2 summarizes the parameter estimates obtained from the final TMDD population PK model.

3.3. Population PK modeling 3.3.1. Model development 3.3.1.1. Structural model. During the model development process, we first tested a number of simple linear compartmental PK models and had poor model fitting results. Since Epo is known to be primarily eliminated by Epo receptors (Nalbant et al., 2010; Chapel et al., 2001a),

3.3.2. Model evaluation The goodness of fit of the final model was assessed using diagnostic plots. As shown in Fig. 4a and b, the population- and individual-predicted concentrations versus the observed concentrations were evenly distributed around the line of identity without bias, indicating that the

Fig. 3. Final TMDD model describing the PK of Epo. Cp = Epo concentration in the central compartment; Vp = central volume of distribution; Ct = Epo concentration in the peripheral compartment; Vt = peripheral volume of distribution; Q = inter-compartmental clearance; CL = linear Epo clearance; kon = Epo association rate; koff = Epo dissociation rate and kint = internalization of the Epo-target complex.

Fig. 2. Observed Epo plasma concentrations collected during the study and available for the population pharmacokinetic analysis. The triangles and circles represent Epo plasma concentrations after subjects received a 1200 U/kg and 600 U/kg, respectively. Arrows represent dosing events using the Epo dosing regimen presented in Fig. 1. 4

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incorporated a two-compartment model, a linear elimination pathway, and a nonlinear target-mediated elimination process. Our work is of importance because Epo PK in this population is particularly difficult to study due to study constraints and because the prediction of neonatal PK by extrapolation from adult studies is highly problematic. The concept of TMDD was originally proposed by Levy more than two decades ago to describe a special type of nonlinearity caused by saturable binding of the drug to a high-affinity, low-capacity pharmacological target such as an enzyme or a receptor (Levy, 1994). The concept of TMDD is now widely recognized and utilized in PK modeling of peptide and protein based pharmaceuticals (Jin and Krzyzanski, 2004; Abraham et al., 2010; Mager, 2006; Meijer et al., 2002). As such, TMDD is highly appropriate for modeling Epo protein PK behavior. In 2001, Mager and Jusko developed a general PK model framework for drugs exhibiting TMDD (known as TMDD model) (Mager and Jusko, 2001); their TMDD model framework serves as the basis for the final model employed in the current study. Because TMDD model employs the mechanism of receptor binding and degradation, this mechanismbased modeling approach has advantages over the traditional M-M models. Among the various models we evaluated, the TMDD model was most appropriate because it provides the best fit and because the model is consistent with current knowledge concerning the mechanism of Epo elimination. Although the mechanisms of Epo elimination in humans are not completely understood, there are strong lines of evidence that Epo is mainly degraded by its pharmacological target, the Epo-R, which is in greatest abundance on erythroid progenitors located primarily in bone marrow (Chapel et al., 2001a; Chapel et al., 2001b; Stohlman Jr, 1959). Consistent with this mechanism, we have previously demonstrated that bone marrow ablation can cause substantial reduction in Epo clearance (up to 80% decrease) in both sheep and humans (Chapel et al., 2001a; Widness et al., 2007). Based on our model prediction, the linear pathway accounts for 14.7% of the overall elimination when Epo was given at 600 U/kg, and 21.5% when Epo was given at 1200 U/kg. This indicated that Epo was eliminated mainly through the nonlinear target-mediated pathway (79%–85%) in premature neonates. This result is in line with our previous observation of an 80% Epo clearance reduction by bone marrow ablation (Chapel et al., 2001a). Most studies on Epo population PK analysis in various human populations employed either a non-mechanistic M-M model or a linear PK model (Frymoyer et al., 2017; Olsson-Gisleskog et al., 2007; Krzyzanski and Wyska, 2008). In contrast, our mechanism-and physiology-based TMDD model arose naturally from our analysis of the Epo PK data collected in this study. Two strengths of this study are 1) a unique multidose optimization Epo dosing regimen and 2) an opportunistic sampling strategy. Most subjects received 10 Epo doses within the first 4 weeks of life; two different doses and two different routes of administration were employed for each subject. Because we used opportunistic blood samples that were left over from laboratory testing, timing of such plasma Epo samples relative to Epo dosing are randomly spread out, which is an advantage. Blood sampling also included frequent early sampling (especially during the first week of life) where frequent phlebotomies were performed due to numerous clinical laboratory testing, and a sparse later sampling (e.g., week 4 in which clinical lab testing was about 7-fold less frequent compared with that in week 1) because the severity of illness typically decreased. Because of the above reasons, the plasma Epo data we obtained provided comprehensive information about the Epo kinetics. The nonlinear PK of Epo observed in the 27 VLBW premature neonates in our study is consistent with literature reports. Juul et al. evaluated the PK of Epo in extremely low birth weight infants following three i.v. doses of 500, 1000, or 2500 U/kg at 24 h intervals beginning on day 1 of age (Juul et al., 2008). They found that the clearance of Epo reduced with increasing Epo doses, and consequently the exposure to Epo increased more than dose proportionally (Juul et al., 2008). Similarly, Wu et al. also reported nonlinear PK of Epo when they evaluated Epo PK in 24 full-term newborns (Wu et al., 2012). In addition to

Table 2 Population pharmacokinetic parameter estimates for the final model. Parameters

Estimate

% RSE

-1

0.0738 0.780 0.0803 0.00066 0.201 0.00370 0.00610 0.112 133 0.118 0.0677 0.640 0.146

9.5 8.3 16.2 16.2 30.9 32.4 24.9 20.4 17.3 9.5 45.1 22.7 9.1

ka (h ) F Vp (L) Q (L/h) Vt (L) CL (L/h) kon (pM-1h-1) koff (h-1) Rmax (pM) kint (h-1) IIV, ωkint2 IOV, Πkint2 σ2 (proportional residual error)

95% CI (0.0600, 0.0880) (0.654, 0.906) (0.0550, 0.106) (0, 0.00100) (0.0790, 0.323) (0.00100, 0.00600) (0.00300, 0.00900) (0.0670, 0.157) (87.9, 178) (0.0960, 0.140) NA NA NA

ka = first-order absorption rate constant, F = bioavailability, Vp = central volume of distribution, Q = distribution clearance, Vt = peripheral volume of distribution, CL = clearance, kon = second-order association rate constant, koff = first-order dissociation rate constant, Rmax = total receptor concentration, kint = Epo-receptor complex internalization constant, IIV = inter-individual variability assumed to be normally distributed with mean 0 and variance ω2, ωkint2 = variance of inter-individual variability on kint, IOV = interoccasion variability assumed to be normally distributed with mean 0 and variance Π2, Πkint2 = variance of inter-occasion variability on kint, σ2 = variance of residual variability, RSE = relative standard error, CI = confidence interval, NA = not available.

final model describes Epo PK adequately at both the population and individual levels. Additionally, the conditional weighted residuals appear distributed uniformly around the zero line when plotted either by population-predicted concentrations (Fig. 4c) or by time (Fig. 4d); further indicating the absence of significant bias in the model fit. Fig. 5 shows the time-course of observed versus individual predicted plasma Epo concentrations from 4 representative VLBW neonates. The proposed PK model captures the concentration-time profiles of Epo adequately for both i.v. doses and s.c. doses, and for both 1200 U/kg and 600 U/kg doses. The result of the VPC of the final model is presented in Fig. 6. As shown, the 5th, 50th, and 95th percentiles of the observed Epo concentrations at binned time periods were within the 95% confidence interval of the corresponding prediction percentiles in VPC plot for the most part, indicating that the final model is appropriately in agreement with the observed data. 3.4. Epo dose-exposure relationship Simulations were performed to assess Epo exposure within the first 4 weeks of life on the administration of Epo 600 U/kg i.v., 600 U/kg s.c., 1200 U/kg i.v. and 1200 U/kg s.c. using the dosing times shown in Fig. 1. Fig. 7 presents these predicted Epo concentration-time courses in a typical study VLBW premature infant showing that Epo exposure is the highest following week 2 per the dosing times of the proposed dosing schedule. To obtain the 95% prediction intervals as shown in Fig. 7, the dosing information and sampling points were set up in a 1000 subject simulation dataset. Using the PK parameter, IIV, IOV and RV estimates from the final TMDD model, NONMEM was used to simulate the Epo concentration-time profiles for those 1000 subjects. Summary statistics, which included the median, 2.5th and 97.5th percentiles, were calculated and then plotted for each simulated dosing regimen. 4. Discussion Here we report for the first time the model-based population pharmacokinetics analysis of Epo in VLBW premature neonates. In this study, neonatal Epo PK was best characterized by a TMDD model that 5

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Fig. 4. Goodness-of-fit plots for the final population pharmacokinetic model of Epo: a) observed versus population-predicted Epo plasma concentrations; b) observed versus individual-predicted Epo plasma concentrations; c) conditional weighted residuals versus population-predicted Epo plasma concentrations; and d) conditional weighted residuals versus time. Solid lines represent the lines of identity from panels a and b and the zero line from panels c and d.

infants, nonlinear PK of Epo has also been reported in children and adults (Widness et al., 1996; Freeman 3rd et al., 2006). Thus, the use of a TMDD model that captures the mechanism of this nonlinear PK behavior is very suitable for modeling Epo. The currently used model is a simplified TMDD model. We think it is sufficient as it well characterized the observed Epo concentration time data. Efforts to employ a full TMDD model and TMDD quasi-equilibrium model with endogenous Epo production rate kEpo did not work. Although we were unable to include the endogenous production rate of Epo in the model, we do not expect the PK parameters to be significantly affected since this study used large doses of Epo, 600 U/kg and 1200 U/kg, and the resulting Epo plasma concentrations were up to three orders of magnitude higher than the endogenous Epo level which averaged 8.83 mU/mL (2.2–30.0 mU/mL). More importantly, since the model is likely going to be used to predict the PK/PD following large Epo doses to get a sufficient effect in this population, inaccuracies at low concentration where the baseline effect is most pronounced, appears clinically insignificant. Because both s.c. and i.v. doses of Epo were evaluated in the current study, we were able to estimate the bioavailability of Epo. Our model estimated that the bioavailability of Epo was 78.7%. Olsson-Gisleskog et al. reported that, following s.c. administration, the bioavailability of Epo increased from 30% at low doses to 71% at the highest does (i.e., 160,000 IU) in healthy adults (Olsson-Gisleskog et al., 2007). These investigators speculated that the increase in Epo bioavailability results from saturable pre-systemic processes. We did not identify such dose-

dependence in Epo bioavailability in our analysis and this is perhaps because we evaluated two high doses of Epo that only differed by twofold. When accounting for body weight, the PK of Epo was generally consistent among our neonatal subjects with very small inter-subject variability. We infer that, for a given dose across VLBW neonates, very similar Epo exposure will be obtained whenever weight-based Epo dosing is employed. This finding is consistent with Frymoyer's report in which small inter-patient variability on Epo PK parameters was found, when corrected for bodyweight as needed, in 47 full term neonates with HIE who received a wider range of Epo doses (Frymoyer et al., 2017). In contrast to the small inter-subject variability, we found a larger intrasubject variability in Epo's elimination parameter kint. Given that Epo-R is located on bone marrow erythroid progenitors and that erythroid progenitors expand in response to increased Epo concentrations (VengPedersen et al., 1999), we speculate that the larger intra-subject variability in Epo PK is attributable to dynamic changes in Epo-R following multiple Epo doses in each neonate that subsequently lead to time-dependent changes in Epo elimination. The current study used multidose Epo dosing (600 or 1200 U/kg) to achieve optimization of Epo efficacy. These doses were considerably greater than those used in treating anemia (100–500 U/kg/dose). We recommend that these higher doses be employed for therapy of neonatal anemia because our data indicate that empirical Epo dosing schedules may not be adequate since Epo clearance is faster in infants 6

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Fig. 5. Time courses of observed (symbols) and model predicted (lines) Epo plasma concentrations in four representative neonates receiving multiple i.v. and s.c. dose regimens.

5. Conclusion In summary, we report for the first time the population PK of Epo in VLBW premature neonates following multiple i.v. and s.c. Epo doses administered within the first 4 weeks of life. Because of the unique multidose optimization Epo dosing regimen administered and the powerful sampling strategy used in the analysis, the PK data provided comprehensive information about Epo kinetics. These data allowed us to characterize the time course of Epo concentrations using a mechanistic TMDD population PK model that provided novel insights into the mechanisms driving Epo PK in VLBW neonates. We found that variation in EPO PK among premature neonates was low; and no significant covariates were identified. Our mechanism-based population PK analysis provides quantitative insight into Epo kinetics in infants. The Epo PK information gained here will assist in improving Epo dosing strategies for future therapeutic efficacy studies for the prevention and treatment of neonatal anemia and neuroprotection. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejps.2019.105013.

Fig. 6. Visual predicted check of plasma Epo data. The open black circles represent the observed Epo plasma concentrations, the solid lines represent the median of the observed data, the dashed line represent the 5th and 95thpercentiles of the observed data, the shaded areas represent the 95% confidence intervals surrounding the 5th, 50th, and 95th percentiles of the simulated Epo concentrations.

Declaration of Competing Interest None. Acknowledgements

compared to adults. Adoption of these greater doses for therapy of anemia may also have additional therapeutic benefits. Some studies have also indicated that Epo may exhibit a beneficial neuroprotective effect when given at high doses (Wu et al., 2012; Kumral et al., 2003; Kellert et al., 2007). The neuroprotective potential of Epo could be particularly effective in neonates considering the high incidence of HIE and cognitive delay in premature infants.

The authors acknowledge Sysmex for the loan of their hematology analyzer. We also appreciate the many contributions of the University of Iowa clinical laboratory staff led by Mitchell J. Owen, MT (ASCP), and Mary Capper, MT (ASCP)SH, and overseen by Matthew D. Krasowski, MD, PhD, and with special thanks to the Investigational Drug Services staff, Kristine Johnson, Theresa Hobbs, Angela Merris, 7

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Funding This work was supported in part by National Institutes of Health (NIH) US Public Health Service Program Project Grant P01 HL046925 and the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number U54TR001356. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. References Abraham, A.K., Kagan, L., Kumar, S., Mager, D.E., 2010. Type I interferon receptor is a primary regulator of target-mediated drug disposition of interferon-beta in mice. J. Pharmacol. Exp. Ther. 334 (1), 327–332. https://doi.org/10.1124/jpet.110.167650. An, G., Schmidt, R.L., Mock, D.M., Veng-Pedersen, P., Widness, J.A., 2018. Overlooked issues on pharmacokinetics data interpretation of protein drugs – a case example of erythropoietin. AAPS J. 21 (1), 6. https://doi.org/10.1208/s12248-018-0269-7. Carroll, P.D., Widness, J.A., 2012. Nonpharmacological, blood conservation techniques for preventing neonatal anemia–effective and promising strategies for reducing transfusion. Semin. Perinatol. 36 (4), 232–243. https://doi.org/10.1053/j.semperi. 2012.04.003. Chapel, S., Veng-Pedersen, P., Hohl, R.J., Schmidt, R.L., McGuire, E.M., Widness, J.A., 2001a. Changes in erythropoietin pharmacokinetics following busulfan-induced bone marrow ablation in sheep: evidence for bone marrow as a major erythropoietin elimination pathway. J. Pharmacol. Exp. Ther. 298 (2), 820–824. Chapel, S.H., Veng-Pedersen, P., Schmidt, R.L., Widness, J.A., 2001b. Receptor-based model accounts for phlebotomy-induced changes in erythropoietin pharmacokinetics. Exp. Hematol. 29 (4), 425–431. Freeman 3rd, B.B., Hinds, P., Iacono, L.C., Razzouk, B.I., Burghen, E., Stewart, C.F., 2006. Pharmacokinetics and pharmacodynamics of intravenous epoetin alfa in children with cancer. Pediatr. Blood Cancer 47 (5), 572–579. https://doi.org/10.1002/pbc. 20685. 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Fig. 7. Predicted Epo concentration-time course during the first 4 weeks of life in a typical study VLBW infant (solid or dashed lines) receiving a) 600 U/kg intravenously or 600 U/kg subcutaneously, b) 1200 U/kg intravenously or 1200 U/kg subcutaneously, and c) 600 U/kg or 1200 U/kg as per the proposed dosing regimen, at the dosing times as illustrated in Fig. 1. The shaded areas represent 95% prediction intervals surrounding the predicted Epo plasma concentrations.

and Joanna Nohr, and the satellite pharmacy staff at University of Iowa Hospitals for procuring, storing, monitoring, and filling Epo prescriptions 24/7 for the duration of this study. This work would not have been possible without the outstanding clinical research contributions of Iowa's neonatal research nurse team that included Karen Johnson, R.N., Laura Knosp, R.N., Ruthann Schrock, R.N., Jin Zhou R.N. and Jan Jeter, R.N. We also acknowledge the research laboratory teams in University of Iowa and University of Arkansas for Medical Sciences. We are grateful to the families of study subjects in allowing their neonates to participate. The human Epo used in this study, Epoetin Alfa, was provided by Janssen Research & Development, LLC. 8

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e520. Widness, J.A., Veng-Pedersen, P., Peters, C., Pereira, L.M., Schmidt, R.L., Lowe, L.S., 1996. Erythropoietin pharmacokinetics in premature infants: developmental, nonlinearity, and treatment effects. J. Appl. Physiol. 80 (1), 140–148. https://doi.org/10. 1152/jappl.1996.80.1.140. Widness, J.A., Schmidt, R.L., Hohl, R.J., Goldman, F.D., Al-Huniti, N.H., Freise, K.J., et al., 2007. Change in erythropoietin pharmacokinetics following hematopoietic transplantation. Clin. Pharmacol. Ther. 81 (6), 873–879. https://doi.org/10.1038/sj. clpt.6100165. Wilimas, J.A., Crist, W.M., 1995. Erythropoietin–not yet a standard treatment for anemia of prematurity. Pediatrics 95 (1), 9–10. Wu, Y.W., Bauer, L.A., Ballard, R.A., Ferriero, D.M., Glidden, D.V., Mayock, D.E., et al., 2012. Erythropoietin for neuroprotection in neonatal encephalopathy: safety and pharmacokinetics. Pediatrics 130 (4), 683–691. https://doi.org/10.1542/peds.20120498.

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