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Clinical Pharmacokinetics of Lyophilized Recombinant Human Erythropoietin-[Alpha] Following Single-Dose Subcutaneous Administration in Premature Newborns

Posted on: Tuesday, 26 April 2005, 03:00 CDT

Abstract

Objective: The aim of this study was to determine pharmacokinetic parameters after subcutaneous administration of a single dose (400 IU/kg) of lyophilized recombinant human erythropoietin-α (rhEPOα) to preterm newborns. The parameters determined were: maximum concentration (C^sub max^), time to reach maximum concentration (T^sub max^), absorption half-life (t^sub 1/2abs^), volume of distribution (V^sub d^), elimination half-life (t^sub 1/ 2el^), clearance (C^sub L^), constant of elimination (k^sub el^) and area under the 0-72 h curve (AUC^sub 0-72^).

Methods: The study group comprised 20 premature newborns (eight males and 12 females) delivered in the Teaching Hospital, University of So Paulo. The inclusion criteria were birth weight < 1500 g or gestational age ≤ 34 weeks, and clinical and hemodynamic stability. Serum erythropoietin (EPO) concentration was determined before and 1, 4, 6, 12, 24, 48 and 72 h after subcutaneous administration of 400 IU/kg rhEPOα, and the pharmacokinetic parameters were calculated.

Results: There was a significant difference in serum EPO concentration between t^sub 72^ and t^sub 0^ (p = 0.001). Mean values (range) of the pharmacokinetic parameters were as follows: C^sub max^, 739.8 (188.0-1390.0) mIU/ml; T^sub max^, 7.7 (4.0-12.0) h; t^sub 1/2abs^, 2.9 (0.8-4.8) h, V^sub d^, 0.705 (0.23-1.73) l/ kg; t^sub 1/2el^, 14.9 (8.7-36.1) h; C^sub L^, 0.032 (0.014-0.066) l/ h; k^sub el^, 0.0475 (0.0200-0.0700); and AUC^sub 0-72^, 19 058.2 (7648.0-34 701.5) mIU/ml per h. The Spearman test showed no correlation between the pharmacokinetic parameters analyzed and the characteristics of the population studied.

Conclusions: Studies evaluating the effectiveness of therapy with recombinant human erythropoietin in premature newborns have used various doses, administered at intervals between 24 and 48 h. The kinetics of absorption measured in our study supports the use of 400 IU/kg within an interval of no less than 72 h, together with therapeutic control of the drug and evaluation of the erythropoietic response.

Keywords: Preterm newborns, anemia of prematurity, recombinant erythropoietin, pharmacokinetics, subcutaneous administration, radioimmunoassay

Introduction

Newborns generally present a gradual decrease in hemoglobin levels between 8 and 12 weeks of life. Preterm neonates develop this hematological manifestation earlier than term newborns owing to their immaturity and insult caused by the clinical necessity of performing laboratory examinations [1,2]. These factors contribute to the development of anemia of prematurity. Furthermore, preterm newborns also exhibit inadequate erythropoietin (EPO) production, leading to a reduction in maturation of marrow erythroid progenitors [3-5].

Recombinant human erythropoietin (rhEPO) has been studied as an alternative to blood transfusion in the premature newborn with anemia of prematurity. Although some researchers have demonstrated the response and tolerance of the premature neonate to rhEPO, the optimal age, dose, route and frequency with which this drug should be administered remain to be established.

We report here the results of a prospective study, aimed to determine the effectiveness of a single-dose (400 IU/kg) subcutaneous administration of lyophilized recombinant human erythropoietin (rhEPOα) in premature newborns. The following pharmacokinetic parameters were evaluated: maximum concentration (C^sub max^), time to reach the maximum concentration (T^sub max^), absorption half-life (t^sub 1/2abs^), volume of distribution (V^sub d^), elimination half-life (t^sub 1/2el^), clearance total (C^sub L^), constant of elimination (k^sub el^) and area under the 0-72 h curve (AUC^sub 0-72^).

Patients and methods

The study group comprised 20 premature newborns (eight males and 12 females) delivered in the Teaching Hospital, University of So Paulo. The inclusion criteria were birth weight ≤ 1500 g or gestational age ≤ 34 weeks, and clinical and hemodynamic stability. The maximum quantity of blood collected for laboratory examinations was < 10% of total blood volume, including any collected in the week preceding the evaluation or during the study for other tests. There were no cases of clinical or hemodynamic events.

Newborns were excluded that presented with congenital cardiopathy, heart failure, arrhythmia, myocardiopathy, renal insufficiency, hepatic insufficiency, lethal congenital malformation, infection, hemolytic disease, seizure, persistence of ductus arteriosus, hypothermia, hyperthermia and hypoxic-ischemic disease. Further criteria for exclusion were use of vasoactive catecholamines, methylxanthines and glucocorticoids, and transfusion of blood component(s) up to 7 days prior to or during the study.

Free and informed consent was obtained from the parents or legal guardians for the participation of their newborn in the study. The research was approved by the institution's ethics committee.

Serum EPO concentration was determined before (t^sub 0^) and 1, 4, 6, 12, 24, 48 and 72 h (t^sub 1^, t^sub 4^, t^sub 6^, t^sub 12^, t^sub 24^, t^sub 48^ and t^sub 72^) after subcutaneous administration of a single 400 IU/kg dose of rhEPOα. The samples were obtained by the micro-method and stored in tubes for capillary micro-collection.

After centrifugation, serum was frozen at -20 C for subsequent detection of serum EPO level. A total of 200 l of serum was necessary for each sample.

Detection of serum EPO was performed by radioimmunoassay (RIA). The kit used for this quantitative determination was EPO-Trac(TM) ^sup 125^I, INCSTAR.

The Wilcoxon non-parametric test was used to evaluate the difference in serum EPO levels between t^sub 0^ and t^sub 72^. The Spearman correlation test was applied to evaluate the relationship between pharmacokinetic parameters and characteristics of the studied population. Statistical significance was considered to be p ≤ 0.05.

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Table I summarizes the characteristics of the studied population. There was a significant difference in serum EPO concentrations between t^sub 72^ and t^sub 0^ (p = 0.001). There was no correlation (Spearman test) between the pharmacokinetic parameters analyzed and the characteristics of the studied population (Table II). Pharmacokinetic data obtained following subcutaneous administration of rhEPOα are given in Table III and mean levels of serum EPO are shown in Figure 1.

Discussion

The inclusion of premature newborns in pharmacokinetic studies is difficult, especially very-low-birth-weight newborns who in their first weeks of life present with a series of clinical problems due to immaturity of their organs and tissues. Consequently, in our study the age of the premature newborns at the time of rhEPOα administration was not constant. Other authors have also included newborns at various postnatal ages in their study populations. In the first pharmacokinetic study of rhEPO in this group, Brown and colleagues [6] included in 12 preterm newborns between 2 and 29 days of life. Krishnan and associates [7] included 12 newborns between 2 weeks and 2 months of life, while Widness and co-workers [8] included newborns between 7 and 42 days of life.

Drug absorption following subcutaneous or intramuscular administration can be altered in premature newborns because of differences related to muscular mass, subcutaneous tissue, changes in blood perfusion, peripheral vasomotor instability and insufficient muscular contraction [9]. Studies evaluating the pharmacokinetics of intravenously administered rhEPO demonstrated fast achievement of maximum plasma concentrations and short elimination half-life, which varied between 6 and 8 h. On the other hand, subcutaneous administration resulted in lower serum concentrations but longer elimination half-life [10-13].

In our study, peak serum EPO concentration, which ranged from 188.0 to 1390.0 mIU/ml, was reached between 4 and 12 h after administration and the absorption half-life was between 0.8 and 4.8 h. After subcutaneous administration of 200 IU rhEPO/kg, Brown and colleagues [6] observed that the maximum concentration varied from 65 to 253.9 IU/ 1 and was reached between 2 and 11.9 h. Krishnan and associates [7] administered rhEPO at 300 lU/kg subcutaneously, finding the maximum concentration to vary between 172.7 and 499.6 mIU/ml and to be reached between 5.8 and 11.7h. The absorption half- life varied between 1.6 and 6.6 h.

In our research, the elimination half-life varied from 8.7 to 36.1 h. These are different from the values found by Brown's group (1.8-13.9 h) [6] and Krishnan's group (5.6-11.7 h) [7]. This variation in results may be related to the characteristics of the individual premature newborns as well as to differences in the doses used by the researchers.

Table I. Basic characteristics of the 20 infants studied.

Table II. Correlation of pharmacokinetic data with clinical variables.

Table III. Pharmacokinetic data obtained after administration of lyophilized recombinant human erythropoietin-α (400 IU/kg) to 20 preterm infants

Figure 1. Time profile of serum erythropoietin (EPO) concentration after subcutaneous administration of lyophilized recombinant human erythropoietin-α (400 IU/kg) to 20 preterm \infants. Data are expressed as means with the standard deviation (SD) represented by error bars. Mean baseline value is at time zero. Serum EPO concentrations are plotted as log^sub 10^.

In this study, EPO clearance varied from 0.014 to 0.066 1/h, similar to previous results (11-46 ml/h [6]; 19.4-65.2 ml/h [7]).

Knowledge regarding the elimination of EPO is still incomplete. Initially the kidneys and the liver were considered to be the main organs responsible. However, more recent studies suggest that the main path for the metabolism and elimination of EPO is via binding to receptors on the surface of erythroid progenitors of the bone marrow, with endocytosis and lysosomal degradation occurring later [14]. In a study in which sheep were submitted to bone marrow ablation with busulfan, Chapel and coworkers demonstrated an important reduction in the clearance and half-life of elimination, thereby confirming the important role of the bone marrow in EPO elimination [15].

The presence has also been described of EPO receptors in a variety of cells, such as endothelial cells, cardiomyocytes and enterocytes, and in various tissues, such as the placenta, testicle, smooth muscle and the central nervous system, where EPO performs various functions. The EPO receptors localized in the endothelium can induce the angiogenic response. Receptors present in enterocytes seem to be functional, since cultivation of enterocytes with rhEPO leads to an increase in cellular migration and a decrease in cellular death following exposure to toxic cytokines [16-18].

Developmental stage also appears to influence the pharmacokinetics of EPO. Veng-Pedersen and colleagues [19] compared EPO pharmacokinetics in newly born and adult sheep, submitted to ablation of the bone marrow with busulfan. They observed that total clearance was significantly faster in newly born animals, and therefore concluded that clearance from non-erythropoietic tissues was greater in the newborn animals than in the adult animals. They suggested that the elimination of EPO does not depend on the erythropoietic receptor alone but also on a population of receptors existing in other tissues, thus demonstrating a significant difference in the proportion of these receptors in newly born and adult animals.

As stated by those authors [19], it is very difficult to extrapolate the results of work done with animal models to humans. Nevertheless, such studies have made a great contribution towards our understanding of the pharmacokinetics of EPO in man, given the similarity between the pharmacokinetics in sheep and humans.

The serum EPO concentration necessary for the successful treatment of anemia of prematurity has not been fully defined. Ideally one should use a dosage regimen that provides the highest serum level with the lowest number of rhEPO doses [11]. However, as previously observed, there are remarkable singularities in the pharmacokinetics of premature newborns. These aspects must be considered carefully when therapeutic strategies are developed for neonates.

Meister and associates [20] performed a multi-center study aimed to determine the association between serum EPO levels and the erythropoietic response to rhEPO. After 1 month's use of this drug, a reduction in the serum levels of EPO was observed, as well as a decrease in erythropoietic activity. In view of these results, the authors concluded that there is a need to monitor EPO efficiency during its and that adjustments to the dose may become necessary after several weeks of treatment. According to OhIs and co- investigators [11] the reduction in serum EPO levels may have a pharmacokinetic or a pharmacodynamic origin, or even be related to inherent characteristics of prematurity.

In summary, previous studies investigating the effectiveness of rhEPO in preterm infants have used various doses administered at intervals between 24 and 48 h [19-25]. The kinetics of absorption demonstrated in our study indicate the use of a single dose of 400 lU/kg at an interval of not less than 72 h, with therapeutic control of the drug associated to an evaluation of the erythropoietic response.

Acknowledgements

This work was supported by Biosinttica Laboratory. We thank Dr Sandra Gualandro of the hematology laboratory of Fundao Pr-Sangue de So Paulo for performing the erythropoietin assay.

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A. M. A. G. P. MELO1, M. T. Z. COSTA1, V. PORTA2, & F. A. C. VAZ3

1 Neonatal Unit, Teaching Hospital, University of So Paulo Medical School, So Paulo, Brazil, 2 School of Pharmaceutic Sciences, University of So Paulo, So Paulo, Brazil, and 3 Department of Pediatrics, Department of Neonatology, School of Medicine, University of So Paulo, So Paulo, Brazil

Correspondence: Dr A. M. A. G. Pereira de Melo, R: Cayowa 560 apt 92, CEP 05018-000, So Paulo, SP, Brazil

Copyright CRC Press Jan 2005

Source: Journal of Maternal - Fetal & Neonatal Medicine

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